Lysosomal Acid Lipase Deficiency

ABSTRACT

Lysosomal acid lipase deficiency (LAL-D) is an autosomal recessive genetic disease with variable presentation which often leads to severe morbidity and mortality. More than 100 LIPA loss of function mutations have been identified, the most common reported mutation being a splice junction mutation in exon 8. The true prevalence of the disease is unknown, but is estimated to be between 1:40,000 to 1:300,000. Infantile-onset LAL-D is generally fatal within the first 12 months of life. Common presenting symptoms in the late-onset form include dyslipidemia (elevated LDL-C, low HDL-C), elevated liver transaminases, hepatomegaly, and splenomegaly. Prior to the availability of enzyme-replacement therapy, individuals with LAL-D were treated with lipid lowering medication, liver transplant, and stem cell transplant, none of which corrected the multisystem nature of the disorder. Sebelipase alfa (Kanuma^®), a recombinant human lysosomal acid lipase, was approved by the FDA in 2015 to treat LAL-D. Phase 3 studies have shown an improvement in lipid parameters and liver enzymes. Long term studies demonstrating the safety and efficacy of sebelipase alfa in infants, children and adults are ongoing.

INTRODUCTION

Lysosomal acid lipase deficiency (LAL-D) is a rare, heterogeneous, autosomal recessive genetic disease, the manifestations of which include a clinical continuum. LAL-D is characterized by accumulation of cholesteryl esters and triglycerides primarily in the liver and spleen, but with involvement of other organs as well. Clinically, LAL-D is under-recognized, leading to a delay in diagnosis. It is often mistaken for more common conditions with similar clinical and laboratory findings, such as heterozygous familial hypercholesterolemia (FH) and non-alcoholic fatty liver disease (NAFLD) (1,2). Correct diagnosis and timely intervention are critical to prolonging life and improving outcomes.

Similar to other lysosomal storage disorders, LAL-D presents across a clinical spectrum from infancy to adulthood. Historically, affected infants who presented within the first year of life were known as Wolman Disease while those who symptoms were delayed until childhood were referred to as cholesteryl ester storage disease [CESD]. Wolman disease, which has a rapidly progressive course, was first described in 1956. Affected infants have severe malnutrition, adrenal calcifications, hepatosplenomegaly, and death within the first few months of life (3). In contrast, CESD is seen as having a variable clinical spectrum with recognition of the disorder occurring from childhood into adulthood. Fredrickson, Schiff, Langeron, and Infante were the first to describe CESD in individuals with presentation from the first to fourth decades of life, and noted them to be less severe than those described by Wolman (4-6).

INHERITANCE AND GENETICS

LAL-D is an autosomal recessive disease that arises from mutations at the LAL locus on chromosome 10q23.2. Affected individuals are either homozygous or compound heterozygous for LIPA mutations, with more than 100 LIPA mutations having been identified (7).

Lysosomal acid lipase (LAL) plays a central role in intracellular lipid metabolism (8,9). LAL is the only lipase contained within lysosomes that hydrolyzes cholesteryl esters and triglycerides. After cleavage by LAL, free cholesterol and fatty acids exit the lysosome to enter the cytosol (Figure 1). These cleaved products play an important role in cholesterol homeostasis. Free cholesterol interacts with transcription factors (sterol regulatory element binding proteins [SREBPs]) to modulate production of intracellular cholesterol. As intracellular free cholesterol increases, there is a down regulation of LDL receptors mediated by SREBP-2, resulting in less LDL entering the cell. Additionally, there is inhibition of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase, resulting in decreased cholesterol production, as well as stimulation of acyl-cholesterol acyltransferase (leading to increased cholesterol esterification). Finally, increased intracellular fatty acid leads to inhibition of triglyceride and phospholipid production and decreased fatty acid synthesis (10-12).

Deficiency of LAL results in diminished or absent hydrolysis of cholesteryl esters and triglycerides, trapping cholesterol esters and TG within the lysosome. This results in a decrease in cytosolic free cholesterol and a compensatory, upregulation in the cholesterol synthetic pathway (HMG CoA reductase activity) and endocytosis via increased LDL receptors. There is increased production of apolipoprotein B and very low-density lipoprotein (VLDL-C) (13-15). The dysregulated expression of the LDL-cholesterol-dependent ATP binding cassette transporter 1 (ABCA1), similar to that seen in Niemann-Pick type C1, results in decreased levels of HDL-C (16). The characteristic dyslipidemia seen in individuals with LAL-D includes elevated total cholesterol, elevated LDL-C, and low HDL-C (2).

Figure 1. Cellular Cholesterol Homeostasis in Heathy Individuals and Patients with LAL-D

The true incidence of LAL-D is unknown. Estimates suggest overall disease prevalence between 1:40,000 to 1:300,000, depending on ethnicity and geographical location (1,2,17). The most commonly inherited defect is a splice junction mutation in exon 8, E8SJM (c.894G>A). It is assumed that 50-70% of adults and children with LAL-D have E8SJM (17,18). Studies in the general population have shown that the estimated frequency of E8SJM allele is 0.0013 in Caucasians, 0.0017 in US Hispanics, 0.0010 in US Ashkenazi Jews, and 0.0005 in Asians (19). Population screening for E8SJM among healthy West German individuals reveal a heterozygote frequency of ~ 1:200 individuals. Jewish infants of Iraqi or Iranian origin appear to be at high risk for LAL-D with an estimated incidence of 1:4,200 in the Los Angeles community (20).

A study attempting to identify the prevalence of LAL-D from patients with abnormal results in laboratory databases (elevated LDL-C and abnormalities on liver tests) identified a total of 1825 patients who subsequently underwent a dried blood spot sample for determination of LAL enzyme activity. No cases of LAL-D were identified. The results of this study demonstrate the potential of databases in helping to identify patients with specific patterns of results to allow targeted testing for possible causes of disease. Biochemical screening suggested that the gene frequency of LAL deficiency in adults is less than 1:100 (21). Additionally, histopathology databases of liver biopsies were analyzed searching for patients with features of 'microvesicular cirrhosis' or 'cryptogenic cirrhosis'. DNA was available from six patients and two were homozygous for LAL c.46A>C;p.Thr16Pro, an unclassified variant in exon 2 (21). The results of these studies suggest the potential of databases in helping to identify patients with specific laboratory results or those who had certain biopsy findings to allow targeted testing for possible causes of disease.

PRESENTATION

The symptoms of LAL-D are quite varied, and are related to the age that clinical manifestations first appear (Figure 2). Individuals who present within the first few days to first month of life often have vomiting, diarrhea, hepatosplenomegaly, abdominal distention, and severe failure to thrive. The first symptom observed is usually vomiting, which has been described as forceful and persistent. Accompanying these symptoms are usually watery diarrhea and low-grade fever. Symptoms generally persist despite multiple medical interventions and may lead to severe malnutrition. A hallmark of infantile-onset LAL-D is adrenal enlargement and calcification, often seen on imaging, but not required for diagnosis. Calcifications of the adrenal gland as well as adrenal insufficiency have been documented. Few patients survive beyond 12 months of age (2,3,22), with those that have growth failure often dying by four months of age (23).

In contrast, the clinical presentation and progression of LAL-D can be variable in older children and adults. However, there are common clinical manifestations that have been reported in this group of patients. In a review of 71 patients two thirds presented with their first symptoms before the age of 5 years. Hepatomegaly was present in all the patients; 86% had splenomegaly. Gastrointestinal symptoms were present in 30% and included vomiting and diarrhea [18%], failure to thrive [16%], abdominal pain [10%], gastrointestinal bleeding [8%], and gallbladder disease [4%]. Elevation of cholesterol was present in 90% (24). In a separate review of 135 patients, the median age of onset of symptoms was 5 years with a range from birth to 68 years. Hepatomegaly was present in 99.3% of patients. The most common extrahepatic findings were steatorrhea, poor growth, gallbladder dysfunction, and cardiovascular disease. Total cholesterol was elevated in all 110 patients (1).

The disease severity is likely dependent on the efficiency of alternative pathways, but not on the level of residual enzyme activity (25). In adults, the most frequent symptoms are abdominal pain, hepatomegaly, and laboratory abnormalities that include increased levels of transaminases and cholesterol. Differential diagnostic considerations include autoimmune hepatitis, NASH, alpha1-antitrypsin deficiency, and Wilson disease. Of concern is the potential for premature atherosclerosis in affected individuals. Although the occurrence of cardiovascular events has not been extensively studied, case reports and observational studies have documented the presence of arterial plaque and atheroma at a very early age (26-28). As a result, many patients with this disease have been prescribed lipid-lowering medications (1). While lipid lowering in the setting of LAL-A has been variable, statins increase hepatic uptake of LDL and, as a consequence, may worsen the lipid overload (29). It is important to note that seven asymptomatic adults, diagnosed in the third to sixth decade of life, have been reported. All were coincidently found to have confirmed LAL-D, yet none had detectable hepatomegaly (28).

The most consistent biochemical abnormalities seen in late onset LAL-D include elevated liver transaminases and plasma lipids. In a study of 49 patients designed to characterize clinical manifestations of LAL-D, mean ALT, AST, and GGT were 92.4, 87.8, and 52.2 U/L at the first measurement. In this study elevated GGT levels were uncommon (only 20% had values > 40 U/L) (30). In another study, liver dysfunction occurred in 100% of 135 patients and 73% of the 11 reported deaths were due to liver failure (1). Mean LDL-C at the time of first measurement was 202.9 mg/dL, and reported as abnormal in 64.4% of patients. Mean total cholesterol was 269.5 mg/dL and was abnormal in 62.5%. Mean HDL-C was 37.5 mg/dL and abnormal in 43.5% of patients (30). The lipid abnormalities seen most closely resemble type II-b dyslipidemia (31). Although elevated LDL-C seems to be a feature of LAL-D, it remains unclear whether or not LAL-D is a cause of early atherosclerosis. Case reports and several autopsy studies have noted aortic stenosis and found narrowing of the coronary artery secondary to atheromatous plaque in patients with LAL-D (2,32).

Figure 2. Clinical Presentation of LAL-D

On gross examination, the liver of patients with LAL-D is enlarged and appears greasy. Liver biopsies in paraffin sections have a predominance of microvesicular steatosis, which is uniform. Microvesicular steatosis, per se, is not pathognomonic of LAL-D, being found in other liver diseases as well. Foamy macrophages, containing lipid and ceroid, are present in the sinusoids and portal tracts (Figure 3). Staining for LAMP1, LAMP2, and LIMP2, or with a lysosomal luminal protein (cathepsin D), can assist identifying lipid accumulation as lysosomal, may help differentiate LAL-D from other causes of microvesicular steatosis. Another pathognomonic feature of LAL-D is birefringent cholesterol ester crystals in hepatocytes and Kupffer cells, using polarized light on electron microscopy. The liver disease generally progresses to fibrosis followed by micronodular cirrhosis (1,2,33).

Figure 3. Liver Biopsies in Patients with LAL-D. A) Image of the portal tract and hepatocytes with mainly microvesicular steatosis. With microvesicular steatosis, the fat does not cause the nucleus to be pushed out to the side. B) Larger magnification of the portal tract. FM points to the foamy appearing cytoplasm, these are macrophages with something being stored in them. GC is pointing to a giant cell.

DIAGNOSTIC TESTS

LAL-D can be diagnosed by demonstrating deficient LAL enzyme activity, as well as by genetic testing identifying mutations of the LIPA gene. Historically, enzyme activity was measured in cultured fibroblasts, peripheral leukocytes, or liver tissue. Various lipase substrates, which were not specific for LAL, were used. In the review by Bernstein, enzyme activities were reported in 114 patients and ranged from undetectable to 16% of normal, with values for most patients being between <1%-10%. However, given assay variability, residual enzyme activity is not predictive of disease severity nor can it be compared from one lab to another (1).

A newer method has been developed to determine LAL activity. This method measures LAL activity in dried blood spots (DBS), and uses Lalistat 2, a highly specific inhibitor of LAL. LAL activity is determined by comparing total lipase activity to lipase activity with Lalistat 2. This method is able to differentiate normal from affected individuals. This DBS technique has advantages over the fibroblast/peripheral leukocyte based test including small sample size, the ability to transport the specimen to the testing facility at ambient temperature, and sample stability (34). This blood test is available at a number of academic and commercial labs around the world.

LIPA gene analysis is also helpful in the diagnosis of LAL-D, with over 100 LIPA mutations having been identified in patients with LAL deficiency (7). Gene panels for associated diagnoses are becoming available and may allow diagnosis of LAL-D even when clinical awareness is low.

DIFFERENTIAL DIAGNOSIS

Given the clinical presentation of LAL-D, it is important to consider it in the differential diagnosis of patients presenting with characteristic lipid findings and liver disease. The lipid abnormalities of LAL-D are similar to patients with heterozygous familial hypercholesterolemia (HeFH) and familial combined hypercholesterolemia. A detailed family history may help differentiate the autosomal dominant HeFH from recessive LAL-D. Expert opinion recommends checking liver transaminases in all children and adults before initiating statin therapy (35). LAL-D should be considered in patients with elevated liver enzymes and lipid abnormalities.

LAL-D is often mistaken for non-alcoholic fatty liver disease (NAFLD); however, LAL-D is associated with mainly microvesicular steatosis and NAFLD with macrovesicular steatosis. LAL-D should be included the differential diagnosis of any non-obese patient with hepatic steatosis, as well as patients with unexplained ALT elevations.

MANAGEMENT

Disease specific therapy is now available to treat patients with LAL-D. However, prior to the approval of sebelipase alfa (Kanuma^â, Alexion Pharmaceuticals, New Haven, CT), lipid lowering therapy, liver transplant, and stem cell transplant were often tried.

HMG-CoA reductase inhibitors have been used to lower LDL-C as well as reduce the risk of atherosclerotic heart disease. The first reported use in a patient with LAL-D was in a 9-year-old girl with elevated LDL-C, low HDL-C, and hepatomegaly with a liver biopsy that showed fibrosis and cirrhosis. During therapy with lovastatin, lipid parameters improved and the authors showed a reduction in cholesterol synthesis and decreased secretion of apo B-containing lipoproteins (36). However, in a report of three patients treated with lovastatin for 12 months, no significant changes were seen in lipid parameters and liver histology (37). In a review of cases in the literature, 12 patients with LAL-D were treated with HMG CoA reductase inhibitors with multiple liver biopsies. None of the 12 patients had improvement on liver histology, with all 12 patients having progressive liver disease (1).

Both hematopoietic stem cell transplant and liver transplant have been attempted to treat LAL-D, however, neither address the multi-system nature of the disease. Limited information is available about the long-term outcome of patients who have undergone liver transplant (1).

Sebelipase alfa, a recombinant human enzyme-replacement, is FDA approved for the treatment of LAL-D (38). The amino acid sequence for sebelipase alfa is the same as that of human LAL. A multicenter, double-blind, placebo controlled, randomized study in 66 patients analyzed the safety and effectiveness of sebelipase alfa (39). By week 20, patients treated with sebelipase alfa demonstrated a decrease in LDL-C of 28% versus 6% in the placebo group. The treatment group also demonstrated improvement in triglyceride and HDL-C level. Normalization of ALT occurred in 31% of patients in the treatment group versus 7% in the placebo group. This was accompanied by reduction in hepatic fat content assessed by multi-echo gradient echo MRI of 32% in the treatment group versus 4% in the placebo group

Table 1. Clinical Trials of Sebelipase Alfa
Study	Subjects	Age	Dose (per kg body weight)	Duration	Reference
LAL-CL01	9	31.6 ± 10.7 yrs (mean ± SD):	Escalating doses: 0.35, 1, or 3 mg weekly (given to cohort of 3 patients each)	4 wks	(38)
LAL-CL02	66	50 <18 yrs, age range at randomization: 4-58 years	1 mg every other week	Initial 20 wks, followed by an open-label treatment phase for 65 patients	(39)
LAL-CL03	9	3.0 months (median)	Weekly infusions: 0.35 mg x 2 weeks; then 1 mg, with dose increase to 3 mg*	12 months	(40)
LAL-CL04	8	18 to 65 yrs	1 or 3 mg every other week	Through to 52 wks	(41)
*Two infants had dose subsequently increased to 5 mg/kg weekly Modified from Pastores GM, Hughes DA. Lysosomal Acid Lipase Deficiency: Therapeutic Options. Drug Des Devel Ther. 2020 Feb 11;14:591-601.

The frequency and distribution of adverse events were similar in the treatment and placebo group, and most adverse events were considered unrelated to the study drug (Table 2) (39). Clinical trials have shown that 3/106 patients experienced reactions consistent with anaphylaxis during infusion, occurring as early as the sixth infusion and as late as 1 year. Twenty percent (21/106) of patients experienced symptoms consistent with hypersensitivity reaction during or within 4 hours of completion of the infusion (38-41). The current dosing recommendation from the manufacturer for infantile-onset LAL-D is 1mg/kg IV weekly with escalation to 3mg/kg weekly in those who do not achieve appropriate clinical response. For child and adults presenting with LAL-D, the recommended dose is 1 mg/kg every other week (38). Further long-term follow-up studies are needed.

Table 2. Adverse Events with Sebelipase Alfa
Event	Sebelipase Alfa (N=36)	Placebo (N=30)
Any adverse event	31 (86%)	28 (93%)
Gastrointestinal events¹	18 (50%)	12 (40%)
Headache	10 (28%)	6 (20%)
Fever	7 (19%)	6 (20%)
Oropharyngeal pain	6 (17%)	1 (3%)
Upper respiratory tract infection	6 (17%)	6 (20%)
Epistaxis	4 (11%)	3 (10%)
Asthenia	3 (8%)	1 (3%)
Cough	3 (8%)	3 (10%)

Adapted from Burton, et al., NEJM 2015

¹Gastrointestinal adverse events (diarrhea, abdominal pain, constipation, nausea, vomiting)

In contrast to survival rates of <12 months in infants with rapidly progressive LAL-D, results of two open-label studies of enzyme replacement therapy with sebelipase alfa, VITAL (NCT01371825) and CL08 (NCT02193867), in 19 infants reported prolonged survival to 12 months (79%) and 5 years of age (68%) in the combined population. The median age of surviving patients was 5.2 (VITAL) and 3.2 years (CL08). In both studies, median weight-for-age, length-for-age, and mid-upper arm circumference-for-age Z-scores increased from baseline to end of study, and decreases in median liver and spleen volume were observed. No patient discontinued treatment because of treatment-emergent adverse events. Infusion-associated reactions (94% in VITAL and 88% in CL08) were mild or moderate in severity (42).

In older children (>4 years) and adults with LAL-D, a phase III randomized study of sebelipase alfa (RISE, NCT01757184) included a 20-week, double-blind, placebo-controlled period; a 130-week, open-label, extension period; and a 104-week, open-label, expanded treatment period. 59/66 patients completed the study. The study found that early and rapid improvements in markers of liver injury and lipid abnormalities with sebelipase alfa were sustained, with no progression of liver disease, for up to 5 years (43).

CONCLUSION

Consensus recommendations for the initial assessment and ongoing monitoring of children and adults with LAL deficiency have been published to help improve the management of infants, children and adults with confirmed LAL-D (Figures 4 and 5) (44).

Figure 4. Recommendations for Baseline Assessment of Children and Adults with LAL Deficiency.

Figure 5. Schedule of Ongoing Monitoring of Adults and Children with LAL Deficiency.

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Lysosomal Acid Lipase Deficiency

ABSTRACT

INTRODUCTION

INHERITANCE AND GENETICS

Figure 1. Cellular Cholesterol Homeostasis in Heathy Individuals and Patients with LAL-D

PRESENTATION

Figure 2. Clinical Presentation of LAL-D

DIAGNOSTIC TESTS

DIFFERENTIAL DIAGNOSIS

MANAGEMENT

Table 1. Clinical Trials of Sebelipase Alfa
Study	Subjects	Age	Dose (per kg body weight)	Duration	Reference
LAL-CL01	9	31.6 ± 10.7 yrs (mean ± SD):	Escalating doses: 0.35, 1, or 3 mg weekly (given to cohort of 3 patients each)	4 wks	(38)
LAL-CL02	66	50 <18 yrs, age range at randomization: 4-58 years	1 mg every other week	Initial 20 wks, followed by an open-label treatment phase for 65 patients	(39)
LAL-CL03	9	3.0 months (median)	Weekly infusions: 0.35 mg x 2 weeks; then 1 mg, with dose increase to 3 mg*	12 months	(40)
LAL-CL04	8	18 to 65 yrs	1 or 3 mg every other week	Through to 52 wks	(41)
*Two infants had dose subsequently increased to 5 mg/kg weekly Modified from Pastores GM, Hughes DA. Lysosomal Acid Lipase Deficiency: Therapeutic Options. Drug Des Devel Ther. 2020 Feb 11;14:591-601.

Table 2. Adverse Events with Sebelipase Alfa
Event	Sebelipase Alfa (N=36)	Placebo (N=30)
Any adverse event	31 (86%)	28 (93%)
Gastrointestinal events¹	18 (50%)	12 (40%)
Headache	10 (28%)	6 (20%)
Fever	7 (19%)	6 (20%)
Oropharyngeal pain	6 (17%)	1 (3%)
Upper respiratory tract infection	6 (17%)	6 (20%)
Epistaxis	4 (11%)	3 (10%)
Asthenia	3 (8%)	1 (3%)
Cough	3 (8%)	3 (10%)

Adapted from Burton, et al., NEJM 2015

¹Gastrointestinal adverse events (diarrhea, abdominal pain, constipation, nausea, vomiting)

CONCLUSION

Figure 4. Recommendations for Baseline Assessment of Children and Adults with LAL Deficiency.

Figure 5. Schedule of Ongoing Monitoring of Adults and Children with LAL Deficiency.

REFERENCES

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Anatomy and Ultrastructure of Bone – Histogenesis, Growth and Remodeling

ABSTRACT

Bones have three major functions: to serve as mechanical support, sites of muscle insertion and as a reserve of calcium and phosphate for the organism. Recently, a fourth function has been attributed to the skeleton: an endocrine organ. The organic matrix of bone is formed mostly of collagen, but also non-collagenous proteins. Hydroxyapatite crystals bind to both types of proteins. Most components of the bone matrix are synthesized and secreted by osteoblasts. Resorption of the bone matrix is required for adaptation to growth, repair and mineral mobilization. This process is performed by the macrophage-related osteoclast. Bone is remodeled throughout life through a coordinated sequence of events which involve the sequential actions of osteoclasts and osteoblasts, replacing old bone with new bone. In the normal adult skeleton, remodeling is coupled such that the level of resorption is equal to the level of formation and bone density remains constant. Intramembranous ossification is the process by which flat bones are formed. For this process, osteoblasts differentiate directly from mesenchymal cells to form the bone matrix. Long bones are formed by endochondral ossification, which is characterized by the presence of a cartilaginous model in which chondrocytes differentiate and mineralized cartilage is replaced with bone through remodeling.

INTRODUCTION

Bone, a specialized and mineralized connective tissue, makes up, with cartilage, the skeletal system, which serves three main functions: A mechanical function as support and site of muscle attachment for locomotion; a protective function for vital organs and bone marrow; and finally a metabolic function as a reserve of calcium and phosphate used for the maintenance of serum homeostasis, which is essential to life. Recently, a fourth important function has been attributed to bone tissue – that of an endocrine organ. Bone cells produce fibroblast growth factor 23 (FGF23) and osteocalcin. FGF23 regulates phosphate handling in the kidney and osteocalcin regulates energy and glucose metabolism (see below) (1,2).

In this chapter the anatomy and cell biology of bone is described as well as the mechanisms of bone remodeling, development, and growth. Remodeling is the process by which bone is turned-over, allowing the maintenance of the shape, quality, and amount of the skeleton. This process is characterized by the coordinated actions of osteoclasts and osteoblasts, organized in bone multicellular units (BMUs) which follow an Activation-Resorption-Formation sequence of events. During embryonic development, bone formation occurs by two different means: intramembranous ossification and endochondral ossification. Bone Growth is a term used to describe the changes in bone structure once the skeleton is formed and during the period of skeletal growth and maturation.

BONE AS AN ORGAN: MACROSCOPIC ORGANIZATION

Two types of bones are found in the skeleton: flat bones (skull bones, scapula, mandible, and ileum) and long bones (tibia, femur, humerus, etc.). These are derived by two distinct types of development: intramembranous and endochondral, respectively, although the development and growth of long bones actually involve both cellular processes. The main difference between intramembranous and endochondral bone formation is the presence of a cartilaginous model, or anlage, in the latter.

Long bones have two wider extremities (the epiphyses), a cylindrical hollow portion in the middle (the midshaft or diaphysis), and a transition zone between them (the metaphysis). The epiphysis on the one hand and the metaphysis and midshaft on the other hand originate from two independent ossification centers, and are separated by a layer of cartilage, the epiphyseal cartilage (which also constitutes the growth plate) during the period of development and growth. This layer of proliferative cells and expanding cartilage matrix is responsible for the longitudinal growth of bones; it progressively mineralizes and is later remodeled and replaced by bone tissue by the end of the growth period (see section on Skeletal Development). The external part of the bones is formed by a thick and dense layer of calcified tissue, the cortex (compact bone) which, in the diaphysis, encloses the medullary cavity where the hematopoietic bone marrow is housed. Toward the metaphysis and the epiphysis, the cortex becomes progressively thinner and the internal space is filled with a network of thin, calcified trabeculae forming the cancellous or trabecular bone. The spaces enclosed by these thin trabeculae are also filled with hematopoietic bone marrow and are continuous with the diaphyseal medullary cavity. The outer cortical bone surfaces at the epiphyses are covered with a layer of articular cartilage that does not calcify.

Bone is consequently in contact with the soft tissues along two surfaces: an external surface (the periosteal surface) and an internal surface (the endosteal surface). These surfaces are lined with osteogenic cells along the periosteum and the endosteum, respectively.

Cortical and trabecular bone are made up of the same cells and the same matrix elements, but there are structural and functional differences. The primary structural difference is quantitative: 80% to 90% of the volume of compact bone is calcified, whereas only 15% to 25% of the trabecular volume is calcified (the remainder being occupied by bone marrow, blood vessels, and connective tissue). The result is that 70% to 85% of the interface with soft tissues is at the endosteal bone surface, including all trabecular surfaces, leading to the functional difference: the cortical bone fulfills mainly a mechanical and protective function and the trabecular bone mainly a metabolic function, albeit trabeculae definitively participate in the biomechanical function of bones, particularly in bones like the vertebrae.

Recently, more attention has been given to cortical bone structure since cortical porosity is intimately linked to the remodeling process as well as to bone strength. Indeed, an increase in cortical porosity is associated with an increase in fragility fractures (3).

BONE AS A TISSUE: BONE MATRIX AND MINERAL

Bone matrix consists mainly of type I collagen fibers (approximately 90%) and non-collagenous proteins. Within lamellar bone, the fibers are forming arches for optimal bone strength. This fiber organization allows the highest density of collagen per unit volume of tissue. The lamellae can be parallel to each other if deposited along a flat surface (trabecular bone and periosteum), or concentric if deposited on a surface surrounding a channel centered on a blood vessel (cortical bone Haversian system). Spindle- or plate-shaped crystals of hydroxyapatite [3Ca ₃ (PO ₄) ₂ ·(OH) ₂] are found on the collagen fibers, within them, and in the matrix around. They tend to be oriented in the same direction as the collagen fibers.

When bone is formed very rapidly during development and fracture healing, or in tumors and some metabolic bone diseases, there is no preferential organization of the collagen fibers. They are then not as tightly packed and found in somewhat randomly oriented bundles: this type of bone is called woven bone, as opposed to lamellar bone. Woven bone is characterized by irregular bundles of collagen fibers, large and numerous osteocytes, and delayed, disorderly calcification which occurs in irregularly distributed patches. Woven bone is progressively replaced by mature lamellar bone during the remodeling process that follows normally development or healing (see below).

Numerous non-collagenous proteins present in bone matrix have been purified and sequenced, but their role has been only partially characterized (Table 1) (4). Most non-collagenous proteins within the bone matrix are synthesized by osteoblasts, but not all: approximately a quarter of the bone non-collagenous proteins are plasma proteins which are preferentially absorbed by the bone matrix, such as a 2-HS-glycoprotein, which is synthesized in the liver. The major non-collagenous protein produced is osteocalcin, which makes up 1% of the matrix, and may play a role in calcium binding and stabilization of hydroxyapatite in the matrix and/or regulation of bone formation, as suggested by increased bone mass in osteocalcin knockout mice. Another negative regulator of bone formation found in the matrix is matrix gla protein, which appears to inhibit premature or inappropriate mineralization, as demonstrated in a knockout mouse model. In contrast to this, biglycan, a proteoglycan, is expressed in the bone matrix, and positively regulates bone formation, as demonstrated by reduced bone formation and bone mass in biglycan knockout mice. Osteocalcin has recently been shown to have an important endocrine function acting on the pancreatic beta cell. Its hormonally active form (undercarboxylated osteocalcin, stimulates insulin secretion and enhances insulin sensitivity in adipose tissues and muscle, improving glucose utilization in peripheral tissues (2).

Table 1. Non-Collagenous Proteins in Bone (4)
PROTEIN	MW	ROLE
Osteonectin (SPARC)	32K	Calcium, apatite and matrix protein binding Modulates cell attachment
α-2-HS-Glycoprotein	46-67K	Chemotactic for monocytes Mineralization via matrix vesicles
Osteocalcin (Bone GLA protein)	6K	Involved in stabilization of hydroxyapatite Binding of calcium Chemotactic for monocytes Regulation of bone formation
Matrix-GLA-protein	9K	Inhibits matrix mineralization
Osteopontin (Bone Sialoprotein I)	50K	Cell attachment (via RGD sequence) Calcium binding
Bone Sialoprotein II	75K	Cell attachment (via RGD sequence) Calcium binding
24K Phosphoprotein (α-1(I) procollagen N-propeptide)	24K	Residue from collagen processing
Biglycan (Proteoglycan I)	45K core	Regulation of collagen fiber growth Mineralization and bone formation Growth factor binding
Decorin (Proteoglycan II)	36K core + side chains	Collagen fibrillogenesis Growth factor binding
Thrombospondin & Fibronectin		Cell attachment (via RGD sequence) Growth factor binding Hydroxyapatite formation
Others (including proteolipids		Mineralization
Growth Factors IGFI & IGFII TGFβ Bone morphogenetic proteins (BMPs)		Differentiation, proliferation and activity of osteoblasts Induction of bone and cartilage in osteogenesis and fracture repair

CELLULAR ORGANIZATION WITHIN THE BONE MATRIX: OSTEOCYTES

The calcified bone matrix is not metabolically inert, and cells (osteocytes) are found embedded deep within the bone in small lacunae (Figure 1). All osteocytes are derived from bone forming cells (osteoblasts) which have been trapped in the bone matrix that they produced and which became calcified. Even though the metabolic activity of the osteoblast decreases dramatically once it is fully encased in bone matrix, now becoming an osteocyte, these cells still produce matrix proteins.

Figure 1. Wnt signaling determines the cell fate of mesenchymal progenitor cells and regulates bone formation and resorption. The Wnt canonical pathway represses adipocyte differentiation and chondrocyte differentiation from progenitor cells, whereas it is required for the transition of chondrocytes to hypertrophy. In contrast, Wnt pathway activation promotes the osteoblast cell lineage by controlling proliferation, maturation, terminal differentiation, and bone formation. Differentiated osteoblasts and/or osteocytes produce Wnt inhibitors such as Dickkopf (Dkk1) and sclerostin (Sost) proteins as a negative feedback control of osteoblast differentiation and function. Wnt signaling also induces osteoblasts to produce more osteoprotegerin (OPG), increasing the ratio of OPG to receptor activator of NF-κB ligand (RANKL) to decrease osteoclast differentiation and bone resorption.

Osteocyte morphology varies according to cell age and functional activity. A young osteocyte has most of the ultrastructural characteristics of the osteoblast from which it was derived, except that there has been a decrease in cell volume and in the importance of the organelles involved in protein synthesis (rough endoplasmic reticulum, Golgi). An older osteocyte, located deeper within the calcified bone, shows a further decrease in cell volume and organelles, and an accumulation of glycogen in the cytoplasm. These cells synthesize small amounts of new bone matrix at the surface of the osteocytic lacunae, which can subsequently calcify. Osteocytes express, in low levels, a number of osteoblast markers, including osteocalcin, osteopontin, osteonectin and the osteocyte marker E11.

Osteocytes have numerous long cell processes rich in microfilaments, which are in contact with cell processes from other osteocytes (there are frequent gap junctions), or with processes from the cells lining the bone surface (osteoblasts or flat lining cells). These processes are organized during the formation of the matrix and before its calcification; they form a network of thin canaliculi permeating the entire bone matrix. Osteocytic canaliculi are not distributed evenly around the cell, but are mainly directed toward the bone surface. Between the osteocyte's plasma membrane and the bone matrix itself is the periosteocytic space. This space exists both in the lacunae and in the canaliculi, and it is filled with extracellular fluid (ECF), the only source of nutrients, cytokines and hormones for the osteocyte. ECF flow through the canalicular network is altered during bone matrix compression and tension and is believed not only to allow exchanges with the extracellular fluids in the surrounding tissues but also to create shear forces that are directly involved in mechanosensing and regulation of bone remodeling. Current understanding of mechanotransduction is based upon the presence of a mechanosensing cilium at the level of the osteocyte’s cell body, capable of detecting the changes in fluid flow determined by mechanical loading of bone. In turn, the activation of the mechanosensing cilium may determine the local concentration of cytokines capable of regulating bone formation and/or bone resorption, such as RANKL, OPG or sclerostin (see below).

Indeed, given the structure of the network and the location of osteocytes within lacunae where ECF flow can be detected, it is likely that osteocytes respond to bone tissue strain and influence bone remodeling activity by recruiting osteoclasts to sites where bone remodeling is required. Osteocyte cellular activity is increased after bone loading; studies in cell culture have demonstrated increased calcium influx and prostaglandin production by osteocytes after mechanical stimulation, but there is no direct evidence for osteocytes signaling to cells on the bone surface in response to bone strain or microdamage to date. Osteocytes can become apoptotic and their programmed cell death may be one of the critical signals for induction of bone remodeling. Ultimately, the fate of the osteocyte is to be phagocytosed and digested together with the other components of bone during osteoclastic bone resorption. The recent ability to isolate and culture osteocytes, as well as the creation of immortalized osteocytic cell lines now allows the study of these cells at the molecular level and this is expected to significantly further our understanding of their role in bone biology and disease.(5) In particular, the discoveries that osteocytes can secrete the Wnt antagonist sclerostin and that this secretion is inhibited both by PTH treatment and by mechanical loading establishes the first direct link between biomechanics, endocrine hormones, bone formation and osteocytes. Similarly, osteocytes can secrete RANKL and OPG, contributing also to the regulation of bone resorption. Thus, osteocytes are emerging as the critical cell type linking mechanical forces in bone to the regulation of bone mass and shape through remodeling.

THE OSTEOBLAST AND BONE FORMATION

The osteoblast is the bone lining cell responsible for the production of the bone matrix constituents, collagen and non-collagenous proteins (Figure 2). Osteoblasts never appear or function individually but are always found in clusters of cuboidal cells along the bone surface (~100–400 cells per bone-forming site).

Figure 2. Osteocyte. Electron micrograph of an osteocyte within a lacuna in calcified bone matrix. The cell has a basal nucleus, cytoplasmic extensions, and well-developed Golgi and endoplasmic reticulum.

Osteoblasts do not operate in isolation and gap junctions are often found between osteoblasts working together on the bone surface. Osteoblasts also appear to communicate with the osteocyte network within the bone matrix (see above), since cytoplasmic processes on the secreting side of the osteoblast extend deep into the osteoid matrix and are in contact with processes of the osteocytes dwelling there.

At the light microscope level, the osteoblast is characterized morphologically by a round nucleus at the base of the cell (away from the bone surface), an intensely basophilic cytoplasm, and a prominent Golgi complex located between the nucleus and the apex of the cell. Osteoblasts are always found lining the layer of bone matrix that they are producing, but before it is calcified (osteoid tissue). Osteoid tissue exists because of a time lag of approximately 10 days between matrix formation and its subsequent calcification. Behind the osteoblast can usually be found one or two layers of cells: activated mesenchymal cells and preosteoblasts (see below). A mature osteoblast does not divide.

At the ultrastructural level, the osteoblast is characterized by the presence of a well-developed rough endoplasmic reticulum with dilated cisternae and a dense granular content, and the presence of a large circular Golgi complex comprising multiple Golgi stacks. These organelles are involved in the major activity of the osteoblast: the production and secretion of collagenous and non-collagenous bone matrix proteins, including type I collagen. Osteoblasts also produce a range of growth factors under a variety of stimuli, including the insulin-like growth factors (IGFs), platelet-derived growth factors (PDGFs), basic fibroblast growth factor (bFGF), transforming growth factor-beta (TGFb), a range of cytokines, the bone morphogenetic proteins (BMPs and Wnts.(3) Osteoblast activity is regulated in an autocrine and paracrine manner by these growth factors, whose receptors can be found on osteoblasts, as well as receptors for a range of endocrine hormones. Classic endocrine receptors include receptors for parathyroid hormone/ parathyroid hormone related protein receptor, thyroid hormone, growth hormone, insulin, progesterone and prolactin. Osteoblastic nuclear steroid hormone receptors include receptors for estrogens, androgens, vitamin D ₃ and retinoids. Receptors for paracrine and autocrine effectors include those for epidermal growth factor (EGF), IGFs, PDGF, TGFb, interleukins, FGFs, BMPs and Wnts (LRP5/6 and Frizzled) (6,7) Osteoblasts also have receptors for several adhesion molecules (integrins) involved in cell attachment to the bone surface.

Among the cytokines secreted by the osteoblast are the main regulators of osteoclast differentiation, i.e. M-CSF, RANKL and osteoprotegerin (OPG) (8,9). M-CSF is essential in inducing the commitment of monocytes to the osteoclast lineage whereas RANKL promotes the differentiation and activity of osteoclasts (see below).

Osteoblasts originate from local pluripotent mesenchymal stem cells, either bone marrow stromal stem cells (endosteum) or connective tissue mesenchymal stem cells (periosteum). These precursors, with the right stimulation, undergo proliferation and differentiate into preosteoblasts, at which point they are committed to differentiate into mature osteoblasts.

The committed preosteoblast is located in apposition to the bone surface, and usually present in layers below active mature osteoblasts. They are elliptical cells, with an elongated nucleus, and are still capable of proliferation. Preosteoblasts lack the well-developed protein synthesizing capability of the mature osteoblast, and do not have the characteristically localized, mature rough endoplasmic reticulum or Golgi apparatus of the mature cell.

The development of the osteoblast phenotype is gradual, with a defined sequence of gene expression and cell activity during development and maturation, controlled by a sequence of transcription factors and cytokines (Figure 3).

Figure 3. Osteoblasts and Osteoid Tissue. A: Light micrograph of a group of osteoblasts producing osteoid; note the newly embedded osteocyte. B: Electron micrograph of 3 osteoblasts covering a layer of mineralizing osteoid tissue. Note the prominent Golgi and endoplasmic reticulum characteristic of active osteoblasts. The black clusters in the osteoid tissue are deposits of mineral. C: Osteoblast Lineage. Osteoblasts originate from undifferentiated mesenchymal cells which are capable of proliferation and which may differentiate into one of a range of cell types. The preosteoblast is also capable of proliferation and may be already committed to an osteoblast phenotype. The mature osteoblast no longer proliferates, but can differentiate further into an osteocyte once embedded in the bone matrix, or to a lining cell on the bone surface.

Two transcription factors, Runx2 and Osterix (Osx), which is downstream of Runx2, are absolutely required for osteoblast differentiation. Runx2 is expressed in mesenchymal condensations and chondrocytes, in addition to osteoblasts. Runx2 target genes include several genes expressed by the mature osteoblast including osteocalcin, bone sialoprotein, osteopontin and collagen a1(I), as well as the Runx2 gene itself. Osx may be mostly important for pushing precursors cells away from the chondrocyte and into the osteoblast lineage.

The most important breakthrough in the understanding of the regulation of bone formation in recent years, is the finding of a clear link between LRP5, a co-receptor for Wnts, and bone mass in humans and in mice. Loss of function in LRP5 leads to the Osteoporosis Pseudo-Glioma syndrome (OPPG), with extremely low bone mass, whereas gain of function leads to the High Bone Mass (HBM) phenotype in humans. In addition, deletion mutations in the gene encoding sclerostin (Sost), another endogenous inhibitor of the Wnt pathway, also lead to osteosclerotic phenotypes (Sclerosteosis, Van Buchem syndrome).(7) These findings have opened a whole new field of investigation both in terms of understanding the mechanism that regulate osteoblasts and their bone-matrix secreting activity and in terms of drug discovery in the hope to target one component of the Wnt signaling pathway and thereby increase bone mass in osteoporotic patients. Of note, in 2019, the FDA approved romosozumab, a monoclonal antibody to sclerostin, for the treatment of postmenopausal women with osteoporosis at high risk for fracture.

Toward the end of the matrix secreting period, a further step is involved in osteoblast maturation. Approximately 15% of the mature osteoblasts become encapsulated in the new bone matrix and differentiate into osteocytes. In contrast, some cells remain on the bone surface, becoming flat lining cells.

Mechanism of Bone Formation

Bone formation occurs by three coordinated processes: the production of osteoid matrix, its maturation, and the subsequent mineralization of the matrix. In normal adult bone, these processes occur at the same rate, so that the balance between matrix production and mineralization is equal. Initially, osteoblasts deposit collagen rapidly, without mineralization, producing a thickening osteoid seam. This is followed by an increase in the mineralization rate to equal the rate of collagen synthesis. In the final stage, the rate of collagen synthesis decreases, and mineralization continues until the osteoid seam is fully mineralized. This time lag (termed the mineralization lag time or osteoid maturation period) appears to be required for osteoid to be modified so it is able to support mineralization. While this delay is not yet understood, it is likely that either collagen cross-linking occurs or an inhibitor of mineralization, such as matrix gla protein, is removed during this time, thus allowing mineralization to proceed.

To initiate mineralization in woven bone, or in growth plate cartilage, high local concentrations of Ca²⁺ and PO₄^3- ions must be reached in order to induce their precipitation into amorphous calcium phosphate, leading to hydroxyapatite crystal formation. This is achieved by membrane-bound matrix vesicles, which originate by budding from the cytoplasmic processes of the chondrocyte or the osteoblast and are deposited within the matrix during its formation. In the matrix, these vesicles are the first structure wherein hydroxyapatite crystals are observed. The membranes are very rich in alkaline phosphatases and in acidic phospholipids, which hydrolyze inhibitors of calcification in the matrix including pyrophosphate and ATP allowing condensation of apatite crystals. Once the crystals are in the matrix environment, they will grow in clusters which later coalesce to completely calcify the matrix, filling the spaces between and within the collagen fibers. In adult lamellar bone, matrix vesicles are not present, and mineralization occurs in an orderly manner through progression of the mineralization front into the osteoid tissue.

THE OSTEOCLAST AND BONE RESORPTION

The osteoclast is the bone lining cell responsible for bone resorption (Figure 4). The osteoclast is a giant multinucleated cell, up to 100mm in diameter and containing four to 20 nuclei. It is usually found in contact with a calcified bone surface and within a lacuna (Howship's lacunae) that is the result of its own resorptive activity. It is possible to find up to four or five osteoclasts in the same resorptive site, but there are usually only one or two. Under the light microscope, the nuclei appear to vary within the same cell: some are round and euchromatic, and some are irregular in contour and heterochromatic, possibly reflecting asynchronous fusion of mononuclear precursors. The cytoplasm is "foamy" with many vacuoles. The zone of contact with the bone is characterized by the presence of a ruffled border with dense patches on each side (the sealing zone).

Figure 4. Osteoclasts and the Mechanism of Bone Resorption. A: Light micrograph and B: electron micrograph of an osteoclast, demonstrating the ruffled border and numerous nuclei. C: Osteoclastic resorption. The osteoclast forms a sealing zone via integrin mediated attachment to specific peptide sequences within the bone matrix, forming a sealed compartment between the cell and the bone surface. This compartment is acidified such that an optimal pH is reached for lysosomal enzyme activity and bone resorption.

Characteristic ultrastructural features of this cell are abundant Golgi complexes around each nucleus, mitochondria, and transport vesicles loaded with lysosomal enzymes. The most prominent features of the osteoclast are, however, the deep foldings of the plasma membrane in the area facing the bone matrix (ruffled border) and the surrounding zone of attachment (sealing zone). The sealing zone is formed by a ring of focal points of adhesion (podosomes) with a core of actin and several cytoskeletal and regulatory proteins around it, that attach the cell to the bone surface, thus sealing off the subosteoclastic bone-resorbing compartment. The attachment of the cell to the matrix is performed via integrin receptors, which bind to specific RGD (Arginine-Glycine-Aspartate) sequences found in matrix proteins (see Table 1). The plasma membrane in the ruffled border area contains proteins that are also found at the limiting membrane of lysosomes and related organelles, and a specific type of electrogenic vacuolar proton ATPase involved in acidification. The basolateral plasma membrane of the osteoclast is specifically enriched in Na+, K+-ATPase (sodium pumps), HCO ₃ ^- /Cl ^-exchangers, and Na+/H+ exchangers and numerous ion channels (10).

Lysosomal enzymes such as tartrate resistant acid phosphatase and cathepsin K are actively synthesized by the osteoclast and are found in the endoplasmic reticulum, Golgi, and many transport vesicles. The enzymes are secreted, via the ruffled border, into the extracellular bone-resorbing compartment where they reach a sufficiently high extracellular concentration because this compartment is sealed off. The transport and targeting of these enzymes for secretion at the apical pole of the osteoclast involves mannose-6-phosphate receptors. Furthermore, the cell secretes several metalloproteinases such as collagenase (MMP-13) and gelatinase B (MMP-9) which appear to be involved in preosteoclast migration to the bone surface as well as bone matrix digestion. Among the key enzymes being synthesized and secreted by the osteoclast is cathepsin K, an enzyme capable or degrading collagen at low pH and a target for inhibition of bone resorption. (11)

Attachment of the osteoclast to the bone surface is essential for bone resorption. This process involves transmembrane adhesion receptors of the integrin. Integrins attach to specific amino acid sequences (mostly RGD sequences) within proteins in or at the surface of the bone matrix. In the osteoclast, avb3 (vitronectin receptor), a2b1 (collagen receptor) and avb5 integrins are predominantly expressed. Without cell attachment the acidified microenvironment cannot be established and the osteoclast cannot be highly mobile, a functional property associated with the formation of podosomes.

After osteoclast adhesion to the bone matrix, avb3 binding activates cytoskeletal reorganization within the osteoclast, including cell spreading and polarization. In most cells, cell attachment occurs via focal adhesions, where stress fibers (bundles of microfilaments) anchor the cell to the substrate. In osteoclasts, attachment occurs via podosomes. Podosomes are more dynamic structures than focal adhesions, and occur in cells that are highly motile. It is the continual assembly and disassembly of podosomes that allows osteoclast movement across the bone surface during bone resorption. Integrin signaling and subsequent podosome formation is dependent on a number of adhesion kinases including the proto-oncogene src, which, while not required for osteoclast maturation, is required for osteoclast function, as demonstrated by osteopetrosis in the src knockout mouse. Pyk2, another member of the focal adhesion kinase family is also activated by avb3 during osteoclast attachment, and is required for bone resorption.(10) Several actin-regulatory proteins have also been shown to be present in podosomes and required for bone resorption, again pointing to the importance of integrin signaling and podosome assembly and disassembly in the function of osteoclasts. (12)

Osteoclasts resorb bone by acidification and proteolysis of the bone matrix and hydroxyapatite crystals encapsulated within the sealing zone. Carbonic anhydrase type II produces hydrogen ions within the cell, which are then pumped across the ruffled border membrane via proton pumps located in the basolateral membrane, thereby acidifying the extracellular compartment. The protons are highly concentrated in the cytosol of the osteoclast; ATP and CO₂ are provided by the mitochondria. The basolateral membrane activity exchanges bicarbonate for chloride, thereby avoiding alkalization of the cytosol. K+ channels in the basolateral domain and Cl ^- channels in the apical ruffled border ensure dissipation of the electrogenic gradients generated by the vacuolar H+-ATPase The basolateral sodium pumps might be involved in secondary active transport of calcium and/or protons in association with a Na ⁺ /Ca ²⁺ exchanger and/or a Na+/H+ antiport. Genetic mutations in several of these components of the acidification and ion transport systems have been shown to be associated with osteopetrosis (defective bone resorption by osteoclasts) in humans and in mice.

The first process during bone matrix resorption is mobilization of the hydroxyapatite crystals by digestion of their link to collagen via the non-collagenous proteins and the low pH dissolves the hydroxyapatite crystals, exposing the bone matrix. Then the residual collagen fibers are digested by cathepsin K, now at optimal pH. The residues from this extracellular digestion are either internalized, or transported across the cell and released at the basolateral domain. Residues may also be released during periods of sealing zone relapse, as probably occurs during osteoclast motility, and possibly induced by a calcium sensor responding to the rise of extracellular calcium in the bone-resorbing compartment.

The regulation of bone resorption is mostly mediated by the action of hormones on stromal cells, osteoblasts and osteocytes. For example, PTH can stimulate osteoblastic production of M-CSF, RANKL, OPG or IL-6, which then act directly on the osteoclast (5,6).

Origin and Fate of the Osteoclast (6)

The osteoclast derives from cells in the mononuclear phagocyte lineage (Figure 5). Their differentiation requires the transcription factors PU-1 and MiTf at early stages, committing the precursors into the myeloid lineage. M-CSF is then required to engage the cells in the monocyte lineage and ensure their proliferation and the expression of the RANK receptor. At that stage, the cells require the presence of RANKL, a member of the TNF family of cytokines produced by stromal cells, to truly commit to the osteoclast lineage and progress in their differentiation program. This step also requires expression of TRAF6, NFκB, c-Fos and NFAT c1, all downstream effectors of RANK signaling. Although this differentiation occurs at the early promonocyte stage, monocytes and macrophages already committed to their own lineage might still be able to form osteoclasts under the right stimuli. Despite its mononuclear phagocytic origin, the osteoclast membrane express distinct markers: it is devoid of Fc and C ₃ receptors, as well as of several other macrophage markers; like mononuclear phagocytes, however, the osteoclast is rich in nonspecific esterases, synthesizes lysozyme, and expresses CSF-1 receptors. Monoclonal antibodies have been produced that recognize osteoclasts but not macrophages. The osteoclast, unlike macrophages, also expresses, millions of copies of the RANK, calcitonin, and vitronectin (integrin avb3) receptors. Whether it expresses receptors for parathyroid hormone, estrogen, or vitamin D is still controversial. Dendritic cell-specific transmembrane protein (DC-STAMP) is currently considered to be the master regulator of osteoclastogenesis. Knock out of DC-STAMP completely abrogates cell-cell fusion during osteoclastogenesis; osteoclasts isolated from DC-STAMP knock-out mice are mononucleated. (13) Another important factor involved in cell fusion is Pin 1, an enzyme that specifically recognizes the peptide bond between phosphorylated serine or threonine and proline. Pin 1 regulates cell fusion during osteoclastogeneis by suppressing DC-STAMP. (14,15) Recent evidence suggest that the osteoclast undergoes apoptosis after a cycle of resorption, a process favored by estrogens, possibly explaining the increased bone resorption after gonadectomy or menopause.

Figure 5. Osteoclast Life Cycle. The osteoclast is derived from a mononuclear hematopoietic precursor cell which, upon activation, fuses with other precursors to form a multinucleated osteoclast. The osteoclast first attaches to the bone surface then commences resorption. After a cycle of bone resorption, the osteoclast undergoes apoptosis.

Relations to the Immune System (Osteoimmunology)

In the last few years it has been recognized that, in part due to the link between the osteoclast, macrophages and dendritic cells (all three belong to the same cell lineage), osteoclasts are regulated by and share regulatory mechanisms with cells of the immune system. For instance, T cells can produce locally RANKL, activating osteoclastogenesis. B cells may share a common precursor with and regulate osteoclast precursors. RANKL signaling and “immunoreceptor tyrosine-based activation motif” (ITAM) signals cooperate in osteoclastogenesis (16).

BONE REMODELING

Bone remodeling is the process by which bone is turned over; it is the result of the activity of the bone cells at the surfaces of bone, mainly the endosteal surface (which includes all trabecular surfaces). Remodeling is traditionally classified into two distinct types: Haversian remodeling within the cortical bone and endosteal remodeling along the trabecular bone surface. This distinction is more morphological than physiological because the Haversian surface is an extension of the endosteal surface and the cellular events during these two remodeling processes follow exactly the same sequence.

The Remodeling Sequence

Bone formation and bone resorption do not occur along the bone surface at random: they are coordinated as part of the turnover mechanism by which old bone is replaced by new bone, providing an opportunity to change the shape, architecture or density of the skeleton. In the normal adult skeleton, bone formation only occurs, for the most part, where bone resorption has already occurred. This basic principle of cellular activity at the remodeling site constitutes the Activation-Resorption-Reversal-Formation (ARRF) sequence (Figure 6).

Figure 6. The Bone Remodeling Sequence. The Activation-Resorption-Reversal-Formation cycle of bone remodeling as it occurs in trabecular bone. See text for details.

Under some signal, today considered to emanate from osteocytes, a locally acting factor released by lining cells, osteocytes, marrow cells, or in response to bone deformation or fatigue-related microfracture, a group of preosteoclasts are activated. These mononuclear cells attach to the bone via avb3 integrins and fuse to form a multi-nucleated osteoclast which will, in a definite area of the bone surface, resorb the bone matrix. After resorption of the bone, and osteoclast detachment, uncharacterized mononuclear cells cover the surface and a cement line is formed. The cement line marks the limit of bone resorption, and acts to cement together the old and the new bone. This is termed the reversal phase, and is followed by a period of bone formation. Preosteoblasts are activated, proliferate and differentiate into osteoblasts, which move onto the bone surface, forming an initial matrix (osteoid), which becomes mineralized after a time lag (the osteoid maturation period). The basic remodeling sequence is therefore Activation-Resorption-Formation; it is performed by a group of cells called the Basic Multicellular Unit (BMU). The complete remodeling cycle takes about 3 months in humans (Figure 7).

Figure 7. Bone Growth and Remodeling at the Growth Plate. The light micrograph demonstrates the zones of chondrocyte differentiation, as well as mineralization (black). The schematic representation shows the cellular events occurring at the growth plate in long bones. Note that bone formation in this process occurs by repeated Activation-Resorption-Formation cycles of bone remodeling beginning with the calcified cartilage matrix.

For decades, the reversal phase of the remodeling cycle was the least well understood. It was recognized that during this phase, the resorption cavity was occupied by mononucleated cells, but the nature of these cells was unknown (17). Recent work by Delaisse and colleagues (18) has definitively identified the reversal cells as belonging to the osteogenic lineage, expressing classic osteoblast markers: Runx2, ALP, and Col3. By applying immunocytochemistry and histomorphometry to femur and fibula samples harvested from teenagers and adults, these investigators have provided a much more complete picture of the temporal sequence of cellular events that occur between the start of resorption and the onset of formation. In order to visualize the entire sequence of events, they analyzed longitudinal sections of evolving Haversian systems. They observed osteoclasts at two distinct locations: at the cutting cone (referred to as primary osteoclasts) and close to the reversal cells (referred to as secondary osteoclasts). The presence of secondary osteoclasts in the reversal phase suggests that bone resorption continues during this phase, which has been renamed the resorption-reversal phase. The authors have concluded that the primary osteoclasts are responsible for drilling the tunnel (initial resorption) and the secondary osteoclasts work to increase its diameter by radial resorption. This radial resorption was shown to be a major contributor to the overall amount of bone resorbed in each BMU. This new and more complete model of the resorption-reversal phase will lead to enhanced understanding of the delicate and all-important balance between resorption and formation (Figure 8).

Figure 8. Cartoon of a bone remodeling unit in cortical bone, showing the change in the designation of the reversal phase as a result of recent new findings. IR = initial resorption; RR = radial resorption; Og = osteoprogenitor cell; Oc = osteoclast. (17)

For many years it has been accepted that bone resorption and formation are coupled in the same way that bone matrix formation and calcification are linked. In other words, in the normal adult skeleton, the coupling of bone resorption and formation in remodeling results in equal levels of cellular activity so that bone turnover is balanced: the volume of bone resorbed is equal to the volume formed. This paradigm implies that, for example, a reduction in osteoblast activity would affect a similar reduction in osteoclast activity such that bone volume is maintained. Conversely, an increase in osteoclast activity should be compensated by an increase in osteoblasts and bone formation, resulting in a maintained bone mass with a high turnover, as in hyperparathyroidism for instance. Similarly, decreased osteoclast numbers or bone resorption activity should be associated with a decrease in bone formation, maintaining bone mass but with a decreased turnover rate.

Although this “coupling” may indeed function in most cases, there are multiple examples of dysfunctions, such as in osteoporosis or osteopetrosis for instance. It now appears that the number of osteoclasts rather than their strict activity is a key determinant of subsequent bone formation. This suggests that factors generated locally by the osteoclast, either directly or through resorption of the bone matrix, are capable of stimulating bone formation (19).

Haversian vs Endosteal Bone Remodeling

As previously mentioned, although cortical bone is anatomically different to trabecular bone, its remodeling occurs following the same sequence of events. The major difference is that while the average thickness of a trabecula is 150-200 microns, the average thickness of the cortex is of the order of 1-10 mm. There are no blood vessels in the trabeculae but the bone envelope system and the osteocyte network are able to carry out enough gaseous exchange, being always relatively close to the surface and the highly vascularized marrow. Consequently, bone remodeling in the trabecular bone will take place along the trabecular surface. On the other hand, the cortical bone itself needs to be vascularized. Blood vessels are first embedded during the histogenesis of cortical bone; the blood vessel and the bone which surrounds it is then called a primary osteon. Later, cortical bone remodeling will be initiated either along the surface of these vascular channels, or from the endosteal surface of the cortex. The remodeling process in cortical bone also follows the ARF sequence. Osteoclasts excavate a tunnel, creating a cutting cone. Again, there is a reversal phase, where mononuclear cells attach and lay down a cement line. Osteoblasts are then responsible for closing the cone, leaving a central canal, centered on blood vessels and surrounded by concentric bone lamellae. For mechanical reasons, all these Haversian systems are oriented along the longitudinal axis of the bone.

Bone Turnover and Skeletal Homeostasis

In a normal young adult, about 30% of the total skeletal mass is renewed every year (half-life = 20 months). In each remodeling unit, osteoclastic bone resorption lasts about 3 days, the reversal 14 days, and bone formation 70 days (total = 87 days). The linear bone formation rate is 0.5mm/day. During this process, about 0.01mm of bone is renewed in one given remodeling unit. Theoretically, with balanced matrix deposition and calcification as well as a balance between osteoclast and osteoblast activity, the amount of bone formed in each remodeling unit (and therefore in the total skeleton) equals the amount of bone which was previously resorbed. Thus, the total skeletal mass remains constant. This skeletal homeostasis relies upon a normal remodeling activity. The rate of activation of new remodeling units would then determine only the turnover rate.

SKELETAL DEVELOPMENT-HISTOGENESIS

Bone development is achieved through the use of two distinct processes, intramembranous and endochondral bone formation. In the first, mesenchymal cells differentiate directly into osteoblasts whereas in the second mesenchymal cells differentiate into chondrocytes and it is only secondarily that osteoblasts appear and form bone around the cartilage model. Through a process that involves bone resorption by osteoclasts, vascular invasion and resorption of calcified cartilage, the cartilage model is progressively replaced by osteoblast-derived bone matrix. Bone is then remodeled through continuous cycles of bone resorption and formation, thereby allowing shape changes and adaptation to the local and systemic environment.

Intramembranous Ossification

During intramembranous ossification, a group of mesenchymal cells within a highly vascularized area of the embryonic connective tissue proliferates, forming early mesenchymal condensations within which cells differentiate directly into osteoblasts. Bone Morphogenetic Proteins, as well as FGFs appear to be essential in the process of mesenchymal cell condensation. The newly differentiated osteoblasts will synthesize a woven bone matrix, while at the periphery, mesenchymal cells continue to differentiate into osteoblasts. Blood vessels are incorporated between the woven bone trabeculae and will form the hematopoietic bone marrow. Later this woven bone will be remodeled through the classical remodeling process, resorbing woven bone and progressively replacing it with mature lamellar bone.

Endochondral Ossification

Development of long bones begins with the formation of a cartilage anlage (model) from a mesenchymal condensation, as in intramembranous ossification. (Figure 9). But here, under the influence of a different set of factors and local conditions, mesenchymal cells undergo division and differentiate into prechondroblasts and then into chondroblasts rather than directly into osteoblasts. These cells secrete the cartilaginous matrix, where the predominant collagen type is collagen type II. Like osteoblasts, the chondroblasts become progressively embedded within their own matrix, where they lie within lacunae, and they are then called chondrocytes. Unlike osteocytes however, chondrocytes continue to proliferate for some time, this being allowed in part by the gel-like consistency of cartilage. At the periphery of this cartilage (the perichondrium), the mesenchymal cells continue to proliferate and differentiate through appositional growth. Another type of growth is observed in the cartilage by cell proliferation and synthesis of new matrix between the chondrocytes (interstitial growth).

Figure 9. Duration and depth of the phases of the normal cancellous bone remodeling sequence, calculated from histomorphometric analysis of bone biopsy samples from young individuals (Adapted from: Eriksen EF, Axelrod DW, Melsen F. Bone Histomorphometry. Raven Press, New York, pp13-20, 1994).

Beginning in the center of the cartilage model, at what is to become the primary ossification center, chondrocytes continue to differentiate and become hypertrophic. During this process, hypertrophic cells deposit a mineralized matrix, where cartilage calcification is initiated by matrix vesicles. Once this matrix is calcified, it is partially resorbed by osteoclasts. After resorption and a reversal phase, osteoblasts differentiate in this area and form a layer of woven bone on top of the remaining cartilage. This woven bone will later be remodeled into lamellar bone.

Chondrocyte differentiation is regulated by a number of factors which have recently been described. The first factor shown to control chondrocyte differentiation was parathyroid hormone related peptide (PTHrP) acting on PTH receptors mostly found in prehypertrophic chondrocytes. This factor prolongs chondrocyte proliferation, and in PTHrP knockout mice, the main phenotype is bone shortening caused by premature chondrocyte hypertrophy. Targeted overexpression of PTHrP results in the opposite phenotype, with prolonged delay in chondrocyte maturation. PTHrP is part of a genetic signaling cascade, where not only is it regulated by factors expressed earlier in chondrocyte differentiation, such as Indian hedgehog (Ihh), but it also regulates chondrocyte differentiation itself, and alters gene expression in more mature chondrocytes. Other factors which regulate chondrocyte differentiation include the FGFs and bone morphogenetic proteins (BMPs). The transcription factors Runx2 and Sox9, together with the Wnt signaling pathway, control the commitment and differentiation within the chondrocytic lineage (20).

The embryonic cartilage is avascular. During its early development, a ring of woven bone is formed, the bone collar, at the periphery by intramembranous ossification in the future midshaft area under the perichondrium (which becomes periosteum). Following calcification of this woven bone, blood vessels, preceded by osteoclasts enter the primary ossification center, penetrate the bone collar and the calcified cartilage, to form the blood supply and allow seeding of the hematopoietic bone marrow. The osteoclast invasion and its concomitant wave of resorbing activity leads to the removal of the calcified cartilage and its replacement by woven bone in the primary spongiosa, as described above.

Secondary ossification centers begin to form at the epiphyseal ends of the cartilaginous model, and by a similar process, trabecular bone and a marrow space are formed. Between the primary and secondary ossification centers, epiphyseal cartilage (growth plates) remain until adulthood. The continued differentiation of chondrocytes, cartilage mineralization and subsequent remodeling cycles allow longitudinal bone growth to occur, such that as new bone is formed the bone will reach its final adult shape. There is, however, a progressive decrease in chondrocyte proliferation so that the growth plate becomes progressively thinner, allowing mineralization and resorption to catch up. It is at this point that the growth plates are completely remodeled and longitudinal growth is arrested.

The growth plate demonstrates, from the epiphyseal area to the diaphyseal area, the different stages of chondrocyte differentiation involved in endochondral bone formation (Figure 10). Firstly, a proliferative zone, where the chondroblasts divide actively, forming isogenous groups, and actively synthesizing the matrix. These cells become progressively larger, enlarging their lacunae in the pre-hypertrophic and hypertrophic zones. Lower in this area, the matrix of the longitudinal cartilage septa selectively calcifies (zone of provisional calcification). The chondrocytes become highly vacuolated and then die through programmed cell death (apoptosis). Once calcified, the cartilage matrix is resorbed, but only partially, by osteoclasts, leaving the calcified longitudinal septae and blood vessels appear in the zone of invasion. After resorption, osteoblasts differentiate and form a layer of woven bone on top of the cartilaginous remnants of the longitudinal septa. Thus, the first remodeling sequence is complete: the cartilage has been remodeled and replaced by woven bone. The resulting trabeculae are called the primary spongiosa. Still lower in the growth plate, this woven bone is subjected to further remodeling (a second ARF sequence) in which the woven bone and the cartilaginous remnants are replaced with lamellar bone, resulting in the mature trabecular bone called secondary spongiosum.

Figure 10. Bone Development. Schematic diagram showing the initial stages of endochondral ossification. Bone development begins with mesenchymal condensation to form a cartilage model of the bone to be formed. Following chondrocyte hypertrophy and cartilage matrix mineralization, osteoclast activity and vascularization result in the formation of the primary, and then secondary ossification centers. In mature adult bones, the growth plate is fully resorbed, so that one marrow cavity extends the full length of the bone. See text for details.

GROWTH IN BONE SHAPE AND DIAMETER (MODELING)

During longitudinal growth, and due to the fact that the midshaft is narrower than the metaphysis, the growth of a long bone progressively destroys the lower part of the metaphysis and transforms it into a diaphysis, a process accomplished by continuous resorption by osteoclasts beneath the periosteum.

In contrast, growth in the diameter of the metaphysis is the result of a deposition of new membranous bone beneath the periosteum that will continue throughout life. In this case, resorption does not immediately precede formation. Recently, more attention has been focusing on this type of bone formation inasmuch as periosteal bone formation seems to respond differently and/or independently from endosteal bone formation activity to different stimuli such as PTH or biomechanical loading. This is particularly important in the context of osteoporosis where it has been demonstrated that growth in diameter in the midshaft is a more important contributor to the decrease in the fracture risk than trabecular bone density and/or cortical thickness.

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Sitosterolemia

ABSTRACT

Sitosterolemia is a rare autosomal recessive disorder of non-cholesterol sterol metabolism, caused by mutations of the ABCG5 or ABCG8 transporter genes. This results in hyperabsorption and decreased biliary excretion of non-cholesterol sterol, especially sitosterol, from the gastrointestinal tract. Affected individuals have excessive accumulation of plant sterols and 5 alpha-saturated stanols in plasma and tissues, resulting in premature cardiovascular disease. The condition is often clinically confused with familial hypercholesterolemia. This article provided overview of this rare condition, including diagnostic evaluation and treatment.

BACKGROUND

Sterols are waxy insoluble substances and are synthesized from acetyl coenzyme A (CoA). Perhaps the most familiar example is cholesterol. In addition to cholesterol, over forty non-cholesterol sterols are also present in the human diet. Non-cholesterol sterols are contained in plants, fungi, and yeast. Instead of converting squalene to cholesterol, non-cholesterol sterols occur when squalene is converted to stigmasterol, sitosterol, campesterol, ergosterol, etc., while shellfish produce fucosterol.

In a typical Western diet, plant sterols, or phytosterols, are often consumed in nuts, seeds, legumes, and vegetable oils. They are present in amounts equal to cholesterol and processed by the intestine in a similar manner (Figure 1). While most individuals absorb, on average, 40-50% of dietary cholesterol, less than 5% of dietary plant sterols are absorbed (1-3).

Figure 1. Enterocyte Trafficking of Cholesterol and Plant Sterols. From Phytoserolemia by Thomas Daysring, MD in Therapeutic Lipidology, Michael H Davis in, MD, Peter P Toth, MD and Kevin C Maki, PhD, Editors. 2007 Humana Press, Incorp. Totowa, New Jersey.

Phytosterols have no role in human metabolism. Therefore, except in inherited disorders of metabolism, there is limited systemic absorption of phytosterols, as their entry into the plasma is highly regulated by the intestine and liver. Concentrations of phystosterols in plasma are normally less than 0.5% that of cholesterol.

Stanols, i.e., saturated sterols, also exist in the diet, primarily from plant sources. Stanols are not normally absorbed from the GI tract. Both stanols and sterols interfere with the absorption of cholesterol. Therefore, both have been used as dietary supplements for over 5 decades to help reduce plasma cholesterol levels.

Phytosterols and free cholesterol are normally absorbed by the Niemann-Pick C1-Like 1 (NPC1L1) protein expressed on enterocytes (Figure 1) (4). Almost all of the absorbed plant phytosterols are excreted back into the intestinal lumen by the ABCG5 or ABCG8 transporters. The normal body is thus able to discriminate between cholesterol and non-cholesterol sterols (5). The function of ABCG5 or ABCG8 transporter genes, found at the STSL locus of human chromosome 2p21, is to limit intestinal absorption and promote biliary excretion (6, 7) (Figure 2).

Figure 2. Normal Intestinal and Hepatic Transport of Cholesterol and Phytosterols. T. Plösch, A. Kosters, A.K. Groen, F. Kuipers. The ABC of Hepatic and Intestinal Cholesterol Transport. Chapter. Atherosclerosis: Diet and Drugs. Volume 170 of the series Handbook of Experimental Pharmacology pp 465-482.

SITOSTEROLEMIA

Sitosterolemia (also known as phytosterolemia) is a rare autosomal recessive disease of non-cholesterol sterol metabolism. It is characterized chemically by the accumulation of plant sterols and 5 alpha-saturated stanols in plasma and tissues. The condition occurs when either ABCG5 or ABCG8 are defective, leading to hyperabsorption of sitosterol from the gastrointestinal tract. The problem is compounded by decreased biliary excretion, resulting in accumulation of dietary phytosterols in different tissues (8, 9).

HISTORY AND ETHNICITY

Sitosterolemia was first reported in 1974 when two sisters with extensive tendon xanthomas were found to have normal plasma cholesterol levels and elevated levels of plant sterols (10). Several hundred cases have since been reported but the condition is thought to be substantially underdiagnosed (11). The disorder has been found in a wide range of diverse populations, including the Old-Order Amish, Chinese, Finnish, Japanese, Norwegian, Indian and Caucasian South Africans, as well as others. The condition is transmitted as an autosomal recessive trait (12, 13)

CLINICAL FEATURES

Signs and Symptoms

Phenotypically, sitosterolemia is very heterogeneous in its presentation. The disorder is characterized by premature coronary artery disease (14-18) although the degree of atherosclerosis present varies significantly (19-24). Presenting signs and symptoms of sitosterolemia, such as lipid deposition in cutaneous and subcutaneous structures (xanthomas), can occur in the first decade of life, but sitosterolemia has been diagnosed in asymptomatic adults as well. Typical xanthomas occur most prominently in the extensor tendons of the hands and Achilles tendon, but can occur in the knees, elbows and buttocks. Xanthomas have been reported in children as young as one to two years of age (25-31). Spinal xanthomas, causing spinal cord compression, have also been reported (32)

The phenotype of sitosterolemia includes abnormal liver function tests, arthralgia, splenomegaly, and hematologic findings (hemolytic anemia, abnormally shaped erythrocytes and large platelets) (33-37). Occasionally, hematologic findings appear as isolated findings (11, 38-41), and there is a case report of an infant with cholestatic jaundice who was ultimately diagnosed with sitosterolemia (42). Aortic stenosis has also been reported (21, 43), as have arthralgias and arthritis (44, 45).

Occasionally, the diagnosis of sitosterolemia is made after an individual with total cholesterol and LDL-cholesterol in the range of familial hypercholesterolemia fails to achieve expected reductions with statin therapy (46). A recent study of 220 hypercholesterolemic children found that 6.4% had elevated and 1.4% had markedly elevated sitosterol levels, with 2 children ultimately diagnosed with genetically confirmed sitosterolemia (47). This has been demonstrated in other publications as well (48, 49). This reaffirms that sitosterolemia is likely underdiagnosed, and high clinical suspicion is warranted. This is particularly important as most genetic testing panels for familial hypercholesterolemia test for pathogenic variants in LDLR, APOB, PCSK9, and LDLRAP1; therefore, individuals with sitosterolemia will frequently have negative genetic testing results.

Although sitosterolemia is a recessive disorder, there is some data suggesting that heterozygous carriers of loss of function mutations can have higher sitosterol levels, higher LDL-cholesterol levels, and a 2-fold higher risk of ASCVD (50).

Differential Diagnosis

Besides sitosterolemia, other disorders that cause tendon xanthomas in children and adults include:

Heterozygous familial hypercholesterolemia (HeFH) - most commonly caused by a co-dominantly inherited disorder of the LDL-C receptor, presents with high total serum and LDL-cholesterol, normal plasma levels of plant sterols and at least one parent with hypercholesterolemia.

Homozygous familial hypercholesterolemia (HoFH) - in which hypercholesterolemia is present in both parents of an affected child. In addition, individuals with HoFH have normal rather than enlarged platelets (macrothrombocytopenia).

Cerebrotendinous xanthomatosis (CTX) - can be distinguished by increased concentrations of plasma cholestanol, protracted diarrhea starting in childhood, and juvenile cataracts. Adults with CTX typically have neurologic involvement (cerebellar ataxia, cognitive decline, and dementia).

Alagille Syndrome, is accompanied by a characteristic syndromic facial appearance, high rates of congenital heart disease, and signs of liver cholestasis (51).

Sitosterolemia should be considered in a child or adult with tendon xanthomas and unexplained hemolysis and/or macrothrombocytopenia, as these hematologic abnormalities are not present in FH, CTX or Alagille syndrome.

Testing

Routine laboratory methods do not always distinguish plant sterols from cholesterol. Detection of plant sterol levels in blood requires gas-liquid chromatography (GLC), gas chromatography/mass spectrometry (GC/MS), or high-pressure liquid chromatography (HPLC).

Plant sterols, especially sitosterol, and the 5-alpha derivatives of plant sterols, are dramatically elevated in patients with sitosterolemia. Plasma concentrations of sitosterol above 1 mg/dL (10µg/mL) are considered to be diagnostic, although a recent study suggested a cutoff value of 15µg/mL had higher positive predictive value (52). Levels typically range from 8-60 mg/dL, 10-25 times higher than normal individuals. Age-dependent reference intervals for phytosterols have also been proposed (53). Molecular genetic testing of mutations in ABCG5 and ABCG8 can help confirm the diagnosis and direct clinical care (54).

In contrast to the very high levels of plant sterols in adults and adolescents with sitosterolemia, total cholesterol levels are sometimes normal or only moderately elevated (34). However, at least three cases of breastfed infants with sitosterolemia presenting with very elevated serum cholesterol levels have been reported. The mechanism of exceptionally high cholesterol levels in sitosterolemic children is unclear (25, 26, 55).

Increased plasma concentrations of plant sterols (especially sitosterol, campesterol, and stigmasterol) are only observed once foods with plant sterols are included in the diet and accumulate in the body. Care must be taken when evaluating infants, since commercial formula feedings with large amounts of vegetable oil may result in elevated sitosterol levels (56).

Children with parenteral nutrition associated cholestasis may have plasma concentrations of plant sterols as high as those seen in patients with hereditary sitosterolemia (i.e., total plant phytosterols of 1.3-1.8 mmol/L). Intralipid typically contains cholesterol, sitosterol, campesterol, and stigmasterol, the latter three of which are plant sterols. Adults receiving parenteral nutrition may also have elevated plasma plant sterol levels (57).

MANAGEMENT OF SITOSTEROLEMIA

Dietary Treatment

Treatment includes dietary restriction of non-cholesterol sterols, limiting intake of shellfish (clams, scallops, oysters), plant foods that contain high fats, such as olives, margarine, nuts, seeds, avocados, and chocolate, and avoidance of vegetable fats and oils (10, 58-61). Fruits, vegetables and cereal products without germ may be used, however (62).

In homozygotes, plasma sterol levels may not improve significantly despite significant dietary sitosterol restriction (63, 64). Margarines and other products containing stanols (e.g., campestanol and sitostanol), which are recommended for use by individuals with hypercholesterolemia, are contraindicated in those with sitosterolemia as they can exacerbate plant stanol accumulation (65).

Medical Treatments

Ezetimibe (Zetia^®), inhibits NPC1L1 and decreases the absorption of sterols. It is the first-line drug therapy, lowering plant sterols by 10 to 50% and may stabilize xanthomas (66-69). Hemolytic anemia and platelet abnormalities have been reported to improve as well (66).

Bile acid sequestrants, such as cholestyramine (8-15 g/d), may be considered in those with an incomplete response to ezetimibe(26) Regression of xanthomas has been reported in an 11-year-old after treatment with diet and cholestyramine (70). A 60-year-old man with compound heterozygous mutations in ABCG5 responded to a combination of ezetimibe and alirocumab (71).

Sitosterolemic patients do not have expected clinical responses to statins, which can help to distinguish these patients with elevated plasma sterols and xanthomas from those with familial hypercholesterolemia (64). As stated above, sitosterolemia should be suspected in individuals with hypercholesterolemia who fail to respond as expected to a statin treatment.

Surgical Treatments

Partial ileal bypass surgery (i.e., shortening of the ileum) has been used to increase intestinal bile acid loss. Partial or complete ileal bypass surgery in persons with sitosterolemia has resulted in at least 50% reduction of plasma and cellular sterol and stanol levels (72-74).

Surgical treatments for complications of sitosterolemia have been reported. Liver cirrhosis has been observed at least once in a patient with the ABCG8 mutation. The patient underwent successful treatment by liver transplant, which led to a dramatic improvement in the sitosterolemia. It is possible that restoration of the ABCG8 function in the liver alone may be sufficient to correct the biochemical abnormality (22).

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Sitosterolemia

ABSTRACT

BACKGROUND

SITOSTEROLEMIA

HISTORY AND ETHNICITY

CLINICAL FEATURES

Signs and Symptoms

Differential Diagnosis

Besides sitosterolemia, other disorders that cause tendon xanthomas in children and adults include:

Alagille Syndrome, is accompanied by a characteristic syndromic facial appearance, high rates of congenital heart disease, and signs of liver cholestasis (51).

Testing

MANAGEMENT OF SITOSTEROLEMIA

Dietary Treatment

Medical Treatments

Surgical Treatments

REFERENCES

Gould RG, Jones RJ, LeRoy GV, Wissler RW, Taylor CB. Absorbability of beta-sitosterol in humans. Metabolism. 1969;18(8):652-62.
Salen G, Ahrens EH, Jr., Grundy SM. Metabolism of beta-sitosterol in man. J Clin Invest. 1970;49(5):952-67.
Salen G, Tint GS, Shefer S, Shore V, Nguyen L. Increased sitosterol absorption is offset by rapid elimination to prevent accumulation in heterozygotes with sitosterolemia. Arterioscler Thromb. 1992;12(5):563-8.
Davis HR, Jr., Zhu LJ, Hoos LM, Tetzloff G, Maguire M, Liu J, et al. Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis. J Biol Chem. 2004;279(32):33586-92.
Patel SB, Honda A, Salen G. Sitosterolemia: exclusion of genes involved in reduced cholesterol biosynthesis. J Lipid Res. 1998;39(5):1055-61.
Patel SB, Salen G, Hidaka H, Kwiterovich PO, Stalenhoef AF, Miettinen TA, et al. Mapping a gene involved in regulating dietary cholesterol absorption. The sitosterolemia locus is found at chromosome 2p21. The Journal of clinical investigation. 1998;102(5):1041-4.
Patel SB. Plant sterols and stanols: their role in health and disease. J Clin Lipidol. 2008;2(2):S11-S9.
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Thyroid Hormone Serum Transport Proteins

ABSTRACT

Thyroid hormone (TH) effects are dependent on the quantity of the hormone that reaches the tissues, hormone activation, and the availability of unaltered TH receptors in the cell’s nuclei and cytoplasm. Since TH enters the cell unbound, the concentration of free rather than total hormone reflects more accurately the activity level of TH-dependent processes. Under normal conditions, changes in free hormone level are adjusted by appropriate stimulation or suppression of hormone secretion and disposal. Total TH concentration in serum is normally kept at a level proportional to the concentration of carrier proteins, and appropriate to maintain a constant free hormone level.

INTRODUCTION

Most carrier protein dependent alterations in total hormone concentration in serum are due to quantitative changes in the hormone-binding proteins and less commonly to changes in affinities for the hormone. Since wide fluctuations in the concentration of TH carrier proteins does not alter the hormonal economy or metabolic status of the subject (1), their function is open to speculation. They are responsible for the maintenance of a large extrathyroidal pool of TH of which only the minute, <0.5 % fraction of free hormone is immediately available to tissues. It can be estimated that in the absence of binding proteins the small extrathyroidal T4 pool would be significantly reduced, if not completely depleted in a matter of hours following a sudden cessation of hormone secretion. In contrast, in the presence of normal concentrations of T4-binding serum proteins, and in particular thyroxine-binding globulin (TBG), a 24-h arrest in hormonal secretion would bring about a decrease in the concentration of T4 and T3 in the order of only 10 and 40 per cent, respectively. Thus, it seems logical to assume that one of the functions of T4-binding proteins in serum is to safeguard the body from the effects of abrupt fluctuations in hormonal secretion. The second likely function of T4-binding serum proteins is to serve as an additional protection against iodine wastage by imparting macromolecular properties to the small iodothyronine molecules, thus limiting their urinary loss (2). The lack of high affinity T4-binding proteins in fish (3), for example, may be teleologically attributed to the greater iodine abundance in their natural habitat. Liver perfusion studies suggest a third function, that facilitating the uniform cellular distribution of T4, allowing for changes in the circulating thyroid hormone level to be rapidly communicated to all cells within organ tissues (4). A fourth function, modeled after the corticosteroid-binding globulin (5), is targeting the amount of hormone delivery by site specific, enzymatic, alteration of TBG. Indeed neutrophil derived elastase transforms TBG into a heat resistant, relaxed, form with reduced T4-binding affinity (6). TBG was found to have a putative role on the testicular size of the boar. In fact, Meishan pigs with histidine rather than an asparagine in codon 226 have a TBG with lower affinity for T4, smaller testes and earlier onset of puberty (7, 8).

In normal man, approximately 0.03 per cent of the total serum T4, and 0.3 per cent of the total serum T3 are present in free or unbound form (3, 9). The major serum thyroid hormone-binding proteins are thyroxine-binding globulin [TBG or thyropexin], transthyretin [TTR or thyroxine-binding prealbumin (TBPA)], and albumin (HSA, human serum albumin)(10). Several other serum proteins, in particular high density lipoproteins, bind T4 and T3 as well as rT3 (9, 11) but their contribution to the overall hormone transport is negligible in both physiological and pathological situations. In term of their relative abundance in serum, HSA is present at approximately 100-fold the molar concentration of TTR and 2,000-fold that of TBG. However, from the view point of the association constants for T4, TBG has highest affinity, which is 50-fold higher than that of TTR and 7,000-fold higher that of HSA. As a result, TBG binds 75% of serum T4, while TTR and HSA binds only 20% and 5%, respectively (Table 1). The distribution of the iodothyronine metabolites among the three serum binding proteins is distinct (12). According to their affinity, T4 > tetraiodothyroacetic acid (TETRAC or T4A) = 3,3’,5’-triiodothyronine (reverse T3 or rT3) > T3 > triiodothyroacetic acid (TRIAC or T3A) = 3,3’-diiodothyronine (T2) > 3-monoipdothyronine (T1) = 3,5-T2 > thyronine (T0) for TBG (IC50-range: 0.36 nM to >100 lM) and T4A > T4 = T3A > rT3>T3 > 3,3’-T2 > 3-T1 > 3,5-T2 > T0 for transthyretin (IC50-range: 0.94 nM to >100 lM). TBG, transthyretin, and albumin were not associated with T0, 3-T1, 3,3-T2, rT3, and T4A. From evolutionary point of view, the three iodothyronine-binding serum proteins developed in reverse order of their affinity for T4, HSA being the oldest (13).

Table 1. Some Properties and Metabolic Parameters of the Principal TH-Binding Proteins in Serum
	TBG	TTR	HSA
Molecular weight (K daltons)	54*	55	66.5
Structure	Monomer	Tetramer	Monomer
Carbohydrate content (%)	20
Number of binding sites for T4 and T3	1	2	4
Association constant, Ka (M-1)
For T4	1 x 1010	2 x 108**	1.5 x 106**
For T3	1 x 109	1 x 106	2 x 105
Concentration in serum
(mean normal, mg/liter)	16	250	40,000
Relative distribution of T4 and T3 in serum (%)
For T4	75	20	5
For T3	75	<5	20
In-Vivo Survival
Half‑life (days)	5***	2	15
Degradation rate (mg/day)	15	650	17,000

*Apparent molecular weight on acrylamide gel electrophoresis 60 K daltons.

**Value given is for the high affinity binding site only.

***Longer under the influence of estrogen.

The existence of inherited TH-binding protein abnormalities was recognized 1959, with the report of a family with TBG-excess (14) but it took 30 years before the first mutation in the TBG (serine protease inhibitor, SERPIN A7) gene was identified (15). Genetic variants of TH-binding proteins having different capacity or affinity for their ligands than the common type protein result in euthyroid hyper- or hypo-iodothyroninemia. The techniques of molecular biology have traced these abnormalities to polymorphisms or mutations in genes encoding TBG and TTR and HSA (see Chapter on Defects of Thyroid Hormone Transport in Serum).

THYROXINE-BINDING GLOBULIN (TBG)

The Molecule, Structure and Physical Properties

TBG is a 54 kD acidic glycoprotein migrating in the inter-α-globulin zone on conventional electrophoresis, at pH 8.6. The term, thyroxine-binding globulin, is a misnomer since the molecule also binds T3 and reverse T3. It was first recognized to serve as the major thyroid hormone transport protein in serum in 1952 (16). Since TBG binds 75% of serum T4 and T3, quantitative and qualitative abnormalities of this protein have most profound effects on the total iodothyronine levels in serum. Its primary structure was deduced in 1989 from the nucleotide sequence of a partial TBG cDNA and an overlapping genomic DNA clones (17). However, it took 17 years to characterize its three dimensional structure by crystallographic analysis (18) (Fig. 1).

Figure 1. Structure of the TBG molecule: Reactive loop (in yellow). Insertion occurs following its cleavage by proteases to give an extra strand in the main sheet of the molecule but the T4-binding site can still retain its active conformation. This is in keeping with other findings showing that the binding and release of T4 is not due to a switch from an on to an off conformation but rather results from an equilibrated change in plasticity of the binding site. So, the S-to-R change in TBG results in a 6 -fold decrease but not a total loss of affinity. The important corollary is that that the release of thyroxine is a modulated process as notably seen in response to changes in temperature (19). (Courtesy of Dr, R.W. Carrell),

TBG is synthesized in the liver as single polypeptide chain of 415 amino acids. The mature molecule, minus the signal peptide, is composed of 395 amino acids (44 kD) and four heterosaccharide units with 5 to 9 terminal sialic acids. The carbohydrate chains are not required for hormone binding but are important for the correct post-translational folding and secretion of the molecule (20, 21) and are responsible for the multiple TBG isoforms (microheterogeneity) present on isoelectric focusing (22, 23). The isoelectric point of normal TBG ranges from pH 4.2 to 4.6, however, this increases to 6 when all sialic acid residues are removed.

The protein is very stable when stored in serum, but rapidly loses its hormone binding properties by denaturation at temperatures above 55°C and pH below 4. The half-life of denaturation at 60°C is approximately 7 min but association with T4 increases the stability of TBG (24-26). TBG can be measured by immunometric techniques or saturation analysis using one of its iodothyronine ligands (26-28).

The tertiary structure of TBG was solved by co-crystallizing the in-vitro synthesized non-glycosylated molecule with T4 and speculations regarding the properties of TBG and its variants have been confirmed (18, 19). The molecule caries T4 in a surface pocket held by a series of hydrophobic interactions with underlying residues and hydrogen bonding of the aminoproprionate of T4 with adjacent residues (Figure 1). TBG differs from other members of the SERPIN family in having the upper half of the main ß-sheet completely opened. This allows the reactive center peptide loop to move in and out of the sheet, resulting in binding and release of the ligand without cleavage of TBG. Thus the molecule can assume a high-affinity and a low-affinity conformation, a model proposed earlier by Grasberger et al (29) and confirmed crystallographically (18). This reversibility is due to the unique presence of P8 proline in TBG, rather than a threonine in all other SERPINs, limiting loop insertion. The coordinated movements of the reactive loop, hD, and the hormone-binding site allow the allosteric regulation of hormone release.

Gene Structure and Transcriptional Regulation

The molecule is encoded by a single gene copy located in the long arm of the human X-chromosome (Xq22.2) (30, 31). The gene consists of 5 exons spanning 5.5kbp (Fig. 2). The first exon is a small and non-coding. It is preceded by a TATAA box and a sequence of 177 nucleotides containing an hepatocyte transcription factor-1 (HNF-1) binding motif that imparts to the gene a strong liver specific transcriptional activity (32). The numbers and size of exons, their boundaries and types of intron splice junctions as well as the amino acid sequences they encode are similar to those of other members of the SERPIN family, to which TBG belongs (32). These include cortisol-binding globulin and the serine protease inhibitors, α1-antitrypsin (α1AT) and α1-antichymotrypsin (α1ACT).

Figure 2. A. Genomic organization and chromosomal localization of thyroid hormone serum binding proteins. Filled boxes represent exons. Location of initiation codons and termination codons are indicated by arrows. B. Structure of promoter regions with the location of cis-acting transcriptional regulatory elements. Reproduced with permission from Hayashi and Refetoff, Molecular Endocrinology: Basic concepts and clinical correlations, Raven Press Ltd. 1995.

Biological Properties

The TBG molecule has a single iodothyronine binding site with affinity slightly higher for T4 than for T3 (33) (Table 1). Optimal binding activity requires the presence of the L-alanine side chain, an unsubstituted 4'-hydroxyl group, a diphenyl ether bridge, and halogen (I or Br) constituents at the 3,5,3' and 5' positions (34). Compared to L-T4, 3,3’,5’-triiodothyronine (rT3) binds to TBG with ~40% higher affinity, D-T4 with half that of the L-isomer and tetraiodothyroacetic acid with ~25%. A number of organic compounds compete with thyroid hormone-binding to TBG. Most notable are: 5,5‑diphenylhydantoin (35), 1,8-anilinonaphthalenesulfonic acid, and salicylates (36). While reversible flip-flop conformational changes of TBG allow for binding and release of the hormone ligand, cleavage of the molecule by leukocyte elastase produces a permanent change in the properties of the molecule. This modified form has reduced T4-binding and increased heat stability (6).

Denatured TBG does not bind iodothyronines but can be detected with antibodies that recognize the primary structure of the molecule (26). In euthyroid adults with normal TBG concentration, about one-third of the molecules carry thyroid hormone, mainly T4. When fully saturated, it carries about 20 µg of T4/dl of serum. The biologic half-life is about 5 days, and the volume of distribution is similar to that of albumin (37, 38) (Table 1). TBG is cleared by the liver. Loss of sialic acid accelerates its removal through interaction with the asialo-glycoprotein receptors reducing the half live by 500-fold (24). However, it is unknown whether desialylation is a required in the normal pathway of TBG metabolism.

Physiology

TBG concentration in the serum of normal adults ranges from 1.1 to 2.1 mg/dl (180 - 350 nM), 14 - 26 µg T4/dl in terms of maximal T4-binding capacity. The protein is present in serum of the 12th week old fetus and in the newborn until 2-3 years of age it is about 1.5 times the normal adult concentration (39-41). TBG levels decline slightly reaching a nadir during mid-adulthood and tend to rise with further advance in age (42). Variable amounts of TBG, though much smaller than those in serum, have been detected in amniotic fluid (43), cerebrospinal fluid (44) and urine (45).

Estrogen excess, either from an endogenous source (hydatidiform mole, estrogen-producing tumors, etc.) or exogenous (therapeutic or birth control use) is the most common cause of increased serum TBG concentration. The level of several other serum proteins such as corticosteroid-binding globulin, testosterone-binding globulin, ceruloplasmin, and transferrin, are also increased (46). This effect of estrogen is mediated through an increase in the complexity of the oligosaccharide residues in TBG together with an increase in the number of sialic acids resulting in prolonged biological half-life (47, 48). Androgens and anabolic steroids produce an opposite effect (49, 50). Although sex hormones affect the serum level of TBG, gender differences are small except during pregnancy during which concentrations are on the average 2.5-fold the normal value (28, 51). Extreme changes in TBG concentration (low or high) alters the accuracy of immunometric measurements of free iodothyronines and particularly that of T3 (52).

Acquired TBG Abnormalities

Altered synthesis, degradation, or both are responsible for the majority of acquired TBG abnormalities (38). Severe terminal illness is undoubtedly the most common cause for acquired decrease in TBG concentration. Interleukin-6, a stimulator of acute phase reactants, is a candidate for mediation of this effect (53). In vivo studies in man showed a reduction in the turnover of TBG in hypothyroidism and an increase in hyperthyroidism (37, 38). Thus, alterations in the degradation rate, rather than changes in the rate of synthesis, may be responsible for the changes of TBG concentration observed in these two conditions.

Partially desialylated TBG, has slow electrophoretic mobility (sTBG, not to be confused with the variant TBG-S), and was found in the serum of some patients with severe liver disease (54) and may be present in relatively higher proportion than TBG in serum of patients with a variety of non-thyroidal illnesses and particularly those with compromised hepatocellular function (55). This is not surprising considering that sTBG is removed by the asialoglycoprotein receptors present in abundance on liver cells (24, 56).

Patients with the carbohydrate-deficient glycoprotein (CDG) syndrome show a characteristic cathodal shift in the relative proportion of TBG isoforms compatible with diminished sialic acid content (57). This inherited syndrome presenting psychomotor retardation, cerebellar hypoplasia, peripheral sensorimotor neuropathy, and variably, retinitis pigmentosa, skeletal abnormalities and lipodystrophy (58), manifests also abnormalities of charge and mass in a variety of serum glycoproteins (59).

TRANSTHYRETIN (TTR)

The Molecule, Structure and Physical Properties

TTR is a 55kD homotetramer which is highly acidic although it contains no carbohydrate. Formerly known as thyroxine-binding prealbumin (TBPA), for its electrophoretic mobility anodal to albumin, was first recognized to bind T4 in 1958 (60). Subsequently it was demonstrated that TTR also forms a complex with retinol-binding protein and thus plays a role in the transport of vitamin A (retinol, or trans retinoic acid) (61, 62).

TTR circulates in blood as a stable tetramer of identical subunits, each containing 127 amino acids (63). Although the tetrameric structure of the molecule was demonstrated by genetic studies (64, 65), detailed structural analysis is available through X-ray crystallography (66, 67) (Fig. 3). Each TTR subunit has 8 ß-strands four of which form the inner sheet and four the outer sheet. The four subunits form a symmetrical ß-barrel structure with a double trumpeted hydrophobic channel that traverses the molecule forming the two iodothyronine binding sites. Despite the apparent identity of the two iodothyronine binding sites, TTR usually binds only one T4 molecule because the binding affinity of the second site is greatly reduced through a negative cooperative effect (69). The TTR tetramer can bind four molecules of RBP that do not interfere with T4-binding, and vice versa (70). TTR can be measured by densitometry after its separation from the other serum proteins by electrophoresis, by hormone saturation, and by immunoassays.

Figure 3. X-ray structure of TTR. The molecule is a homotetrameric protein composed of four monomers of 127 amino acids. Structurally, in its native state, TTR contains eight stands (A-H) and a small α-helix. The contacts between the dimers form two hydrophobic pockets where T4 binds (T4 channel). As shown in the magnified insert, each monomer contains one small α-helix and eight β-strands (CBEF and DAGH). Adapted from a model; PDB code 1DVQ (68).

Gene Structure and Transcriptional Regulation

TTR is encoded by a single gene copy located on human chromosome 18 (18q11.2-12.1) (63, 71) (Fig. 2). The gene consists of 4 exons spanning for 6.8kbp. Knowledge about the transcriptional regulation of the human TTR gene comes from studies of the mouse gene structural and sequence homology which extends to the promoter region (72, 73). In both species a TATAA box and binding sites for HNF-1, 3 and 4 are located within 150 bp from the transcription start site.

Although TTR in serum originates from the liver (74), TTR mRNA is also found in kidney cells, the choroid plexus, meninges, retina, placenta, pancreatic islet cells and fetal intestine (75-78). TTR constitutes up to 25% of the total protein present in ventricular cerebrospinal fluid where it binds 80% of T4 (79).

Biological Properties

Despite the 20-fold higher concentration of TTR in serum relative to that of TBG, it plays a lesser role in iodothyronine transport. In the presence of normal levels of TBG, wide fluctuations in TTR concentration or its removal from serum by specific antibodies has little influence on the concentration of free T4 (80). Some of the properties of TTR are summarized in Table 1.

The first T4 molecule binds to TTR with a Ka of about 100‑fold higher than that for HSA and about 100-fold lesser than that for TBG. Properties necessary for optimal binding activity include iodines at the 3' and 5' positions and a desamino acid side chain which explain the lower T3 and higher T4A affinities relative to that of T4 (34, 81). Non-iodothyronine ligands are also differentially bound, the most notable example being the flavonoid compounds which have a markedly higher binding affinity for TTR than for TBG (82). Among drugs that compete with T4-binding to TTR are ethacrynic acid, salicylates, 2,4-dinitrophenol, penicillin (83, 84) and perﬂuoroalkyl substances (85). The latter have with near equal affinity to TTR and TBG. Barbital also inhibits iodothyronine binding to TTR.

Only 0.5% of the circulating TTR is occupied by T4. TTR has a relatively rapid turnover (t1/2 = 2 days) and a distribution space similar to that of HSA and TBG (86, 87) except that it also exists in CSF. Hence, acute diminution in the rate of synthesis is accompanied by a rapid decrease of its concentration in serum.

Physiology

Normal average concentration in serum is 25 mg/dl, and corresponds to a maximal binding capacity of approximately 300 µg T4/dl. Changes in TTR concentration have relatively little effect on the serum concentration of serum iodothyronines (80, 88). There is a distinct reciprocal relationship between acquired changes in TBG and TTR concentration related to gender, age, glucocorticoids, estrogen and androgens (42, 51, 89-91).

Acquired TTR Abnormalities

The reduction or serum TTR concentration surpasses that of TBG in major illness, nephrotic syndrome, liver disease, cystic fibrosis, hyperthyroidism, and protein-calorie malnutrition (10, 92-94). Increased serum TTR concentration can occur in some patients with islet cell carcinoma (95). Studies on the metabolism of TTR in man, utilizing radioiodinated purified human TTR, indicate that diminished TTR concentration associated with severe illness or stress is due to a decrease in the rate of synthesis or an increase in the rate of degradation, or both (86, 87).

HUMAN SERUM ALBUMIN (HSA)

The Molecule, Structure and Physical Properties

HSA is a 66.5 kD protein synthesized by the liver. It is composed of 585 amino acids with high content of cystines and charged amino acids but no carbohydrate (96). The three domains of the molecule can be conceived as three tennis balls packaged in a cylindrical case.

Gene Structure and Transcriptional Regulation

HSA is encoded by a single gene copy located on human chromosome 4 (4q11-q13) (97). The gene contains 15 exons, 14 of which are coding (98) (Fig. 2). The promoter region of the HSA gene has been most intensive studied. The transcriptional regulation has been best characterized in rodents that share 90% sequence homology with the corresponding human gene, including a distal enhancer element 10 kbp upstream from the promoter region (99). Binding sites for hepatocyte enriched nuclear proteins, such as HNF-1, C/EBP, and DBP have been identified (100-102).

Biological Properties

HSA associates with a wide variety of substances including hormones and drugs possessing a hydrophobic region, and thus the association of TH to HSA can be viewed as nonspecific. Of the several iodothyronine-binding sites on the HSA molecule, only one has a relatively high affinity for T4 and T3. Yet these are 10,000-fold inferior to those of TBG (27). Fatty acids and chloride ions decrease their binding to HSA (27). The biologic t1/2 of HSA is relatively long (103). Some of its properties are summarized in Table 1.

More than half of the total protein content in serum is HSA. As a result, it is the principal contributor to the maintenance of the colloid osmotic pressure (96). It has been suggested that HSA synthesis may be, in part, regulated by a feedback mechanism involving alteration in the colloid osmotic pressure. Indeed, down-regulation of HSA gene expression has been recently observed during the infusion of macromolecules in the rat (104).

Physiology

Because of the low affinity and despite the high capacity of HSA for iodothyronines, its contribution to thyroid hormone transport is relatively minor. Thus, even the most marked fluctuations of serum HSA concentration, including analbuminemia, have no significant effects on thyroid hormone levels (105).

LIPOPROTEINS

Lipoproteins bind T4, and to some extent T3 (9, 106). The affinity for T4-binding is similar to that of TTR. These proteins are estimated to transport roughly 3% of the total T4 and perhaps as much as 6% of the total T3 in serum. The binding site of apolipoprotein A1 is a region of the molecule that is distinct from that portion which binds to the cellular lipoprotein receptors, and the physiological role of such binding is still unclear.

ACKNOWLEDGMENTS

Supported in part by grants DK-15070 from the National Institutes of Health (USA).

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Cerebrotendinous Xanthomatosis

ABSTRACT

Cerebrotendinous xanthomatosis is a rare autosomal recessive disorder caused by homozygous or compound heterozygous mutations in the CYP27A1 gene. These patients lack mitochondrial sterol 27-hydroxylase enzyme, which is responsible for conversion of cholesterol to cholic acid and chenodeoxycholic acid (CDCA) in bile acid synthesis pathway. CYP27A1 mutation leads to decreased synthesis of bile acid, excess production of cholestanol, and consequent accumulation of cholestanol in tissues, including brain, leading to progressive neurological dysfunction marked by dementia, spinal cord paresis, and cerebellar ataxia. Deposition in other tissues causes tendon xanthomas, premature atherosclerosis, and cataracts. The clinical manifestations usually start at infancy and develop during the first and second decades of life. The diagnosis of CTX is based on clinical findings, biochemical testing, and neuroimaging. Molecular genetic analysis although not necessary for initiation of treatment, provides definitive confirmation of CTX. Early initiation of CDCA is the treatment of choice for neurological and non-neurological symptoms of CTX and treatment with cholic acid has also been shown to be effective for non-neurological symptoms.

INTRODUCTION

Cerebrotendinous xanthomatosis (CTX; OMIM#213700) is a rare autosomal recessive disorder of bile acid metabolism and lipid storage characterized by abnormal deposition of cholestanol and cholesterol in multiple tissues. It is caused by homozygous or compound heterozygous mutation in the CYP27A1 gene located on chromosome 2q33-qter. CYP27A1 gene encodes sterol 27-hydroxylase which is involved in bile acid synthesis. Over 100 different mutations (missense, deletions, insertions, splice site, and nonsense mutations) of the CYP27A1 gene have been reported worldwide in patients of different ethnic origin (1,2). About 50% of these mutations were found in the region of exons 6–8 of the CYP27A1 gene, however here is no genotype phenotype correlation (3,4). The metabolic pathway for bile acids synthesis is shown in the figure 1.

Figure 1. Bile acids are synthesized from cholesterol in the liver through two pathways: the classic pathway and the alternative pathway. The bile acid synthesis mainly produces two primary bile acids, cholic acid and chenodeoxycholic acid (CDCA). In the classic pathway, the mitochondrial sterol 27-hydroxylase (CYP27A1) catalyzes the steroid side-chain oxidation in both CA and CDCA synthesis. In the alternative pathway, CYP27A1 catalyzes the first step to convert cholesterol to 27-hydroxycholesterol which eventually is converted to CDCA.

CTX was first described by Van Bogart, Scherer and Epstein in 1937 as a condition characterized by early development of cataracts and large tendon xanthomas and later by progressive neurologic impairment (5,6). The incidence of CTX is estimated to be 3 to 5 per 100,000 people (1,7). The prevalence is estimated to be highest in Asians (1:44,407-93,084), intermediate in Europeans, Americans, and Africans/African Americans (1:70,795-233,597) and lowest in the Finnish population (1:3,388,767) (8).

ETIOLOGY AND PATHOGENESIS

Patients with CTX lack mitochondrial sterol 27-hydroxylase, which is an important enzyme in both the alternative and classic bile acid synthesis pathways (9). The biochemical defect prevents the synthesis of chenodeoxycholic acid (CDCA) and cholic acid (10).The absence of the negative feedback mechanism of CDCA on 7a-hydroxylase, the rate limiting step, leads to the accumulation of cholestanol and its precursor 7α-hydroxy-4-cholesten-3-one (Figure 1) (9-11). Bile acid alcohols are glucoronidated and can be found in significantly increased amounts in blood, urine, and feces of untreated CTX subjects. It has been hypothesized that the increased bile alcohol levels may lead to disruption of the blood–brain barrier (BBB), and that increased permeability of BBB may be caused by circulating bile alcohol glucuronides (12). Cholestanol and cholesterol accumulate in many tissues, including brain (primarily white matter), leading to progressive neurological dysfunction marked by dementia, spinal cord paresis, and cerebellar ataxia. Deposition in other tissues causes tendon xanthomas, premature atherosclerosis, and cataracts (13).

CLINICAL FEATURES

Patients with CTX present diverse manifestations with multi-organ involvement and a broad range of neurological and non-neurological symptoms (Table 1). The clinical manifestations usually start at infancy and develop during the first and second decades of life. Neonatal cholestatic jaundice and infantile-onset diarrhea with failure to thrive may be the earliest clinical manifestation (14-16). Childhood-onset cataracts are a common early symptom, described in 92% patients with CTX. Cataracts precede neurological signs and tendon xanthoma, and if present, are considered useful for early diagnosis (14,16,17). Tendon xanthomas have been documented in 71% patients with CTX and can appear in first, second or third decade. They are common in Achilles tendon, but can also be seen in fingers, tibial tuberosity, and triceps (17,18). Pes cavus deformity has also been described (19,20). Neurological dysfunction is almost always present in patients with CTX and usually occurs in late adolescents and early adulthood. The range of symptoms are wide and include intellectual disability; dementia; psychiatric symptoms (i.e., behavioral changes, depression, agitation, hallucination, and suicide attempts); pyramidal signs (spasticity, hyperreflexia, extensor plantar responses); cerebellar signs (progressive ataxia, dysarthria); movement disorder (parkinsonism, dystonia, myoclonus, postural tremor); seizures; and peripheral neuropathy (1,4,21-23). Osteoporosis, heart involvement, and premature atherosclerosis have also been described later in life (19,24-26).

Table 1. Clinical Features of Cerebrotendinous Xanthomatosis

Early childhood

Neonatal cholestatic jaundice

Chronic diarrhea

Cataracts

Developmental Delay

Late childhood

Tendon xanthomas

Psychiatric disorders

Adulthood

Neurological dysfunction

Pyramidal signs (spasticity, hyperreflexia, extensor plantar responses)

Cerebellar signs (ataxia, dysarthria)

Movement disorder (parkinsonism, dystonia, myoclonus, postural tremor)

Seizures

Dementia

Pes Cavus

Osteoporosis

Premature atherosclerosis

DIAGNOSIS

The diagnosis of CTX is mainly based on clinical features, biochemical testing, neuroimaging, and molecular genetic analysis. Since clinical presentation can be variable in type, severity, and timing, diagnosis is often delayed. Patients with CTX include having high plasma cholestanol concentration (five- to ten-fold greater than normal), normal-to-low plasma cholesterol concentration, elevated urine bile alcohol concentration, elevated plasma bile alcohol concentration, decreased CDCA level, and increased levels of cholestanol and apolipoprotein B in cerebrospinal fluid (10,17). Cholesterol concentration in tissue is increased but plasma cholesterol levels are low to normal (27). Brain MRI features are strongly suggestive of diagnosis and include bilateral hyperintensity of the dentate nuclei, diffuse cerebral and cerebellar atrophy, and white matter signal abnormalities. Brain MRI spectroscopy shows decreased n-acetylaspartate and increased lactate signals, suggestive of widespread axonal damage and cerebral mitochondrial dysfunction (20). Although not necessary for initiation of treatment, molecular testing provides definitive confirmation of CTX. Various mutations in all nine exons and in introns 2,4,6,7 and 8 of the CYP27A1 gene have been described worldwide (8). Mignarri et al (22) developed a suspicion index to be used by clinicians to calculate CTX prediction score. They proposed using family history, systemic and neurologic features as diagnostic indicators, classified as very strong (score of 100; e.g., tendon xanthoma or, sibling with CTX); strong (score of 50; e.g., juvenile cataract, chronic diarrhea, prolonged neonatal cholestasis, ataxia, MRI alterations, intellectual disability and/or psychiatric disturbances); or moderate (score of 25; e.g., early osteoporosis, epilepsy, parkinsonism or polyneuropathy. A total score ≥ 100 warrants serum cholestanol assessment, and elevated cholestanol or total score ≥ 200, with one very strong or four strong indicators, warrants CYP27A1 gene analysis (22). Quantification of bile acid precursor 7α-hydroxy-4-cholesten-3-one has been proposed to be a rapid, convenient diagnostic test for CTX (28). Early detection of ketosterol bile acid precursors can play important role in early detection through newborn screening (29).

MANAGEMENT

Early initiation of oral chenodeoxy-cholic-acid (CDCA) therapy at a dose of 250 mg given 3 times daily for adults and 15 mg/kg/d for children is the treatment of choice for neurological and non-neurological symptoms such as diarrhea (30-32), however it still does not have U.S. Food and Drug Administration approval for CTX. FDA has granted it an orphan drug designation for use in CTX. CDCA has been approved in the European Union to treat adults and children with CTX over 1 month of age. This treatment suppresses synthesis of cholesterol, cholestanol, bile alcohols, and bile acids and alleviates clinical symptoms if started at an early age (33,34); however it has not been shown to improve bone mineral density in affected patients (35). The treatment is most effective when started early (31), and patients diagnosed later in life with significant neurological disease may progress despite CDCA therapy. A retrospective cohort study in 79 Dutch patients with CTX showed that the MRI brain remained normal even after 25 year of follow-up treatment if therapy was started in young patients with a normal MRI at diagnosis. Treatment with cholic acid has also been shown to be effective, especially in patients with side effects to CDCA therapy (36,37). HMG-CoA-reductase-inhibitors along with CDCA can improve lipoprotein metabolism, inhibit cholesterol synthesis, and reduce plasma levels of cholestanol (38). Surgical excision of bilateral tendon may worsen the gait imbalance and cannot prevent the deterioration of neurologically affected patients (39). Lumbreras et al (40) showed that adeno-associated virus (AAV) vectors expressing CYP27A1 restored bile acid metabolism and normalized the concentration of most bile acids in plasma in a CTX mouse model, making gene therapy a feasible option.

SCREENING

Screening of all first-degree relatives, when feasible, should be considered as early detection and treatment are the most important aspects for preventing morbidity and mortality (41). A recent pilot study has also shown promising option of screening for CTX as part of newborn screening (42). A recent study on de-identified dried blood spots of 20,076 newborns from the 2019 cohort of the biobank of the Dutch newborn screening program (Dutch National Institute for Puublic Health and the Environment, Bilthoven, The Netherlands) showed that metabolite ratios bile alcohol cholestanetetrol glucuronide (GlcA-tetrol)/ tauro-chenodeoxycholic acid (t-CDCA) to be informative biomarker, paving way for introduction of CTX newborn screen (43).

CONCLUSIONS

Cerebrotendinous xanthomatosis is a rare autosomal recessive disorder of bile acid metabolism and lipid storage characterized by abnormal deposition of cholestanol and cholesterol in multiple tissues. Clinical manifestations include neonatal cholestatic jaundice, infantile-onset diarrhea with failure to thrive, childhood-onset cataracts, tendon xanthomas, and progressive neurological dysfunction including intellectual disability, dementia, and psychiatric symptoms. Treatment with CDCA is treatment of choice, however cholic acid, HMG-Co-A reductase inhibitors, and surgical excision also play a role in management. Timely detection and treatment are the key to prevent severe morbidity and mortality in these patients.

REFERENCES

Salen G, Steiner RD. Epidemiology, diagnosis, and treatment of cerebrotendinous xanthomatosis (CTX). J Inherit Metab Dis 2017; 40:771-781
Chen C, Zhang Y, Wu H, Sun YM, Cai YH, Wu JJ, Wang J, Gong LY, Ding ZT. Clinical and molecular genetic features of cerebrotendinous xanthomatosis patients in Chinese families. Metab Brain Dis 2017; 32:1609-1618
Gallus GN, Dotti MT, Federico A. Clinical and molecular diagnosis of cerebrotendinous xanthomatosis with a review of the mutations in the CYP27A1 gene. Neurol Sci 2006; 27:143-149
Verrips A, Hoefsloot LH, Steenbergen GC, Theelen JP, Wevers RA, Gabreels FJ, van Engelen BG, van den Heuvel LP. Clinical and molecular genetic characteristics of patients with cerebrotendinous xanthomatosis. Brain2000; 123 ( Pt 5):908-919
van Bogaert L, Scherer, H.J. and Epstein, E. . Une forme cérébrale de la cholestérinose généralisée. Paris: Masson et Cie.
Swanson PD. Cerebrotendinous xanthomatosis. N Engl J Med 1968; 278:857
Lorincz MT, Rainier S, Thomas D, Fink JK. Cerebrotendinous xanthomatosis: possible higher prevalence than previously recognized. Arch Neurol 2005; 62:1459-1463
Pramparo T, Steiner RD, Rodems S, Jenkinson C. Allelic prevalence and geographic distribution of cerebrotendinous xanthomatosis. Orphanet J Rare Dis 2023; 18:13
Russell DW. The enzymes, regulation, and genetics of bile acid synthesis. Annu Rev Biochem 2003; 72:137-174
Lorbek G, Lewinska M, Rozman D. Cytochrome P450s in the synthesis of cholesterol and bile acids--from mouse models to human diseases. FEBS J 2012; 279:1516-1533
Bjorkhem I, Hansson M. Cerebrotendinous xanthomatosis: an inborn error in bile acid synthesis with defined mutations but still a challenge. Biochem Biophys Res Commun 2010; 396:46-49
Salen G, Berginer V, Shore V, Horak I, Horak E, Tint GS, Shefer S. Increased concentrations of cholestanol and apolipoprotein B in the cerebrospinal fluid of patients with cerebrotendinous xanthomatosis. Effect of chenodeoxycholic acid. N Engl J Med 1987; 316:1233-1238
Cali JJ, Hsieh CL, Francke U, Russell DW. Mutations in the bile acid biosynthetic enzyme sterol 27-hydroxylase underlie cerebrotendinous xanthomatosis. J Biol Chem 1991; 266:7779-7783
Cruysberg JR. Cerebrotendinous xanthomatosis: juvenile cataract and chronic diarrhea before the onset of neurologic disease. Arch Neurol 2002; 59:1975
van Heijst AF, Verrips A, Wevers RA, Cruysberg JR, Renier WO, Tolboom JJ. Treatment and follow-up of children with cerebrotendinous xanthomatosis. Eur J Pediatr 1998; 157:313-316
Clayton PT, Verrips A, Sistermans E, Mann A, Mieli-Vergani G, Wevers R. Mutations in the sterol 27-hydroxylase gene (CYP27A) cause hepatitis of infancy as well as cerebrotendinous xanthomatosis. J Inherit Metab Dis 2002; 25:501-513
Moghadasian MH. Cerebrotendinous xanthomatosis: clinical course, genotypes and metabolic backgrounds. Clin Invest Med 2004; 27:42-50
Keren Z, Falik-Zaccai TC. Cerebrotendinous xanthomatosis (CTX): a treatable lipid storage disease. Pediatr Endocrinol Rev 2009; 7:6-11
Berginer VM, Shany S, Alkalay D, Berginer J, Dekel S, Salen G, Tint GS, Gazit D. Osteoporosis and increased bone fractures in cerebrotendinous xanthomatosis. Metabolism 1993; 42:69-74
Berginer VM, Abeliovich D. Genetics of cerebrotendinous xanthomatosis (CTX): an autosomal recessive trait with high gene frequency in Sephardim of Moroccan origin. Am J Med Genet 1981; 10:151-157
Nie S, Chen G, Cao X, Zhang Y. Cerebrotendinous xanthomatosis: a comprehensive review of pathogenesis, clinical manifestations, diagnosis, and management. Orphanet J Rare Dis 2014; 9:179
Mignarri A, Gallus GN, Dotti MT, Federico A. A suspicion index for early diagnosis and treatment of cerebrotendinous xanthomatosis. J Inherit Metab Dis 2014; 37:421-429
Stelten BML, van de Warrenburg BPC, Wevers RA, Verrips A. Movement disorders in cerebrotendinous xanthomatosis. Parkinsonism Relat Disord 2019; 58:12-16
Bjorkhem I, Andersson O, Diczfalusy U, Sevastik B, Xiu RJ, Duan C, Lund E. Atherosclerosis and sterol 27-hydroxylase: evidence for a role of this enzyme in elimination of cholesterol from human macrophages. Proc Natl Acad Sci U S A 1994; 91:8592-8596
Valdivielso P, Calandra S, Duran JC, Garuti R, Herrera E, Gonzalez P. Coronary heart disease in a patient with cerebrotendinous xanthomatosis. J Intern Med 2004; 255:680-683
Fujiyama J, Kuriyama M, Arima S, Shibata Y, Nagata K, Takenaga S, Tanaka H, Osame M. Atherogenic risk factors in cerebrotendinous xanthomatosis. Clin Chim Acta 1991; 200:1-11
Salen G. Cholestanol deposition in cerebrotendinous xanthomatosis. A possible mechanism. Ann Intern Med1971; 75:843-851
DeBarber AE, Connor WE, Pappu AS, Merkens LS, Steiner RD. ESI-MS/MS quantification of 7alpha-hydroxy-4-cholesten-3-one facilitates rapid, convenient diagnostic testing for cerebrotendinous xanthomatosis. Clin Chim Acta 2010; 411:43-48
DeBarber AE, Luo J, Star-Weinstock M, Purkayastha S, Geraghty MT, Chiang JP, Merkens LS, Pappu AS, Steiner RD. A blood test for cerebrotendinous xanthomatosis with potential for disease detection in newborns. J Lipid Res 2014; 55:146-154
Sekijima Y, Koyama S, Yoshinaga T, Koinuma M, Inaba Y. Nationwide survey on cerebrotendinous xanthomatosis in Japan. J Hum Genet 2018; 63:271-280
Duell PB, Salen G, Eichler FS, DeBarber AE, Connor SL, Casaday L, Jayadev S, Kisanuki Y, Lekprasert P, Malloy MJ, Ramdhani RA, Ziajka PE, Quinn JF, Su KG, Geller AS, Diffenderfer MR, Schaefer EJ. Diagnosis, treatment, and clinical outcomes in 43 cases with cerebrotendinous xanthomatosis. Journal of clinical lipidology2018; 12:1169-1178
Stelten BML, Huidekoper HH, van de Warrenburg BPC, Brilstra EH, Hollak CEM, Haak HR, Kluijtmans LAJ, Wevers RA, Verrips A. Long-term treatment effect in cerebrotendinous xanthomatosis depends on age at treatment start. Neurology 2019; 92:e83-e95
Salen G, Meriwether TW, Nicolau G. Chenodeoxycholic acid inhibits increased cholesterol and cholestanol synthesis in patients with cerebrotendinous xanthomatosis. Biochem Med 1975; 14:57-74
Berginer VM, Salen G, Shefer S. Long-term treatment of cerebrotendinous xanthomatosis with chenodeoxycholic acid. N Engl J Med 1984; 311:1649-1652
Zubarioglu T, Bilen IP, Kiykim E, Dogan BB, Enver EO, Cansever MS, Zeybek ACA. Evaluation of the effect of chenodeoxycholic acid treatment on skeletal system findings in patients with cerebrotendinous xanthomatosis. Turk Pediatri Ars 2019; 54:113-118
Koopman BJ, Wolthers BG, van der Molen JC, Waterreus RJ. Bile acid therapies applied to patients suffering from cerebrotendinous xanthomatosis. Clin Chim Acta 1985; 152:115-122
Mandia D, Chaussenot A, Besson G, Lamari F, Castelnovo G, Curot J, Duval F, Giral P, Lecerf JM, Roland D, Pierdet H, Douillard C, Nadjar Y. Cholic acid as a treatment for cerebrotendinous xanthomatosis in adults. J Neurol 2019; 266:2043-2050
Kuriyama M, Tokimura Y, Fujiyama J, Utatsu Y, Osame M. Treatment of cerebrotendinous xanthomatosis: effects of chenodeoxycholic acid, pravastatin, and combined use. J Neurol Sci 1994; 125:22-28
Moghadasian MH, Salen G, Frohlich JJ, Scudamore CH. Cerebrotendinous xanthomatosis: a rare disease with diverse manifestations. Arch Neurol 2002; 59:527-529
Lumbreras S, Ricobaraza A, Baila-Rueda L, Gonzalez-Aparicio M, Mora-Jimenez L, Uriarte I, Bunuales M, Avila MA, Monte MJ, Marin JJG, Cenarro A, Gonzalez-Aseguinolaza G, Hernandez-Alcoceba R. Gene supplementation of CYP27A1 in the liver restores bile acid metabolism in a mouse model of cerebrotendinous xanthomatosis. Mol Ther Methods Clin Dev 2021; 22:210-221
Wilson DP, Patni N. Should children with chronic diarrhea be referred to a lipid clinic? Journal of clinical lipidology 2018; 12:1099-1101
DeBarber AE, Kalfon L, Fedida A, Fleisher Sheffer V, Ben Haroush S, Chasnyk N, Shuster Biton E, Mandel H, Jeffries K, Shinwell ES, Falik-Zaccai TC. Newborn screening for cerebrotendinous xanthomatosis is the solution for early identification and treatment. J Lipid Res 2018; 59:2214-2222
Vaz FM, Jamal Y, Barto R, Gelb MH, DeBarber AE, Wevers RA, Nelen MR, Verrips A, Bootsma AH, Bouva MJ, Kleise N, van der Zee W, He T, Salomons GS, Huidekoper HH. Newborn screening for Cerebrotendinous Xanthomatosis: A retrospective biomarker study using both flow-injection and UPLC-MS/MS analysis in 20,000 newborns. Clin Chim Acta 2023; 539:170-174

Cerebrotendinous Xanthomatosis

ABSTRACT

INTRODUCTION

ETIOLOGY AND PATHOGENESIS

CLINICAL FEATURES

Table 1. Clinical Features of Cerebrotendinous Xanthomatosis

Early childhood

Neonatal cholestatic jaundice

Chronic diarrhea

Cataracts

Developmental Delay

Late childhood

Tendon xanthomas

Psychiatric disorders

Adulthood

Neurological dysfunction

Pyramidal signs (spasticity, hyperreflexia, extensor plantar responses)

Cerebellar signs (ataxia, dysarthria)

Movement disorder (parkinsonism, dystonia, myoclonus, postural tremor)

Seizures

Dementia

Pes Cavus

Osteoporosis

Premature atherosclerosis

DIAGNOSIS

MANAGEMENT

SCREENING

CONCLUSIONS

REFERENCES

Salen G, Steiner RD. Epidemiology, diagnosis, and treatment of cerebrotendinous xanthomatosis (CTX). J Inherit Metab Dis 2017; 40:771-781
Chen C, Zhang Y, Wu H, Sun YM, Cai YH, Wu JJ, Wang J, Gong LY, Ding ZT. Clinical and molecular genetic features of cerebrotendinous xanthomatosis patients in Chinese families. Metab Brain Dis 2017; 32:1609-1618
Gallus GN, Dotti MT, Federico A. Clinical and molecular diagnosis of cerebrotendinous xanthomatosis with a review of the mutations in the CYP27A1 gene. Neurol Sci 2006; 27:143-149
Verrips A, Hoefsloot LH, Steenbergen GC, Theelen JP, Wevers RA, Gabreels FJ, van Engelen BG, van den Heuvel LP. Clinical and molecular genetic characteristics of patients with cerebrotendinous xanthomatosis. Brain2000; 123 ( Pt 5):908-919
van Bogaert L, Scherer, H.J. and Epstein, E. . Une forme cérébrale de la cholestérinose généralisée. Paris: Masson et Cie.
Swanson PD. Cerebrotendinous xanthomatosis. N Engl J Med 1968; 278:857
Lorincz MT, Rainier S, Thomas D, Fink JK. Cerebrotendinous xanthomatosis: possible higher prevalence than previously recognized. Arch Neurol 2005; 62:1459-1463
Pramparo T, Steiner RD, Rodems S, Jenkinson C. Allelic prevalence and geographic distribution of cerebrotendinous xanthomatosis. Orphanet J Rare Dis 2023; 18:13
Russell DW. The enzymes, regulation, and genetics of bile acid synthesis. Annu Rev Biochem 2003; 72:137-174
Lorbek G, Lewinska M, Rozman D. Cytochrome P450s in the synthesis of cholesterol and bile acids--from mouse models to human diseases. FEBS J 2012; 279:1516-1533
Bjorkhem I, Hansson M. Cerebrotendinous xanthomatosis: an inborn error in bile acid synthesis with defined mutations but still a challenge. Biochem Biophys Res Commun 2010; 396:46-49
Salen G, Berginer V, Shore V, Horak I, Horak E, Tint GS, Shefer S. Increased concentrations of cholestanol and apolipoprotein B in the cerebrospinal fluid of patients with cerebrotendinous xanthomatosis. Effect of chenodeoxycholic acid. N Engl J Med 1987; 316:1233-1238
Cali JJ, Hsieh CL, Francke U, Russell DW. Mutations in the bile acid biosynthetic enzyme sterol 27-hydroxylase underlie cerebrotendinous xanthomatosis. J Biol Chem 1991; 266:7779-7783
Cruysberg JR. Cerebrotendinous xanthomatosis: juvenile cataract and chronic diarrhea before the onset of neurologic disease. Arch Neurol 2002; 59:1975
van Heijst AF, Verrips A, Wevers RA, Cruysberg JR, Renier WO, Tolboom JJ. Treatment and follow-up of children with cerebrotendinous xanthomatosis. Eur J Pediatr 1998; 157:313-316
Clayton PT, Verrips A, Sistermans E, Mann A, Mieli-Vergani G, Wevers R. Mutations in the sterol 27-hydroxylase gene (CYP27A) cause hepatitis of infancy as well as cerebrotendinous xanthomatosis. J Inherit Metab Dis 2002; 25:501-513
Moghadasian MH. Cerebrotendinous xanthomatosis: clinical course, genotypes and metabolic backgrounds. Clin Invest Med 2004; 27:42-50
Keren Z, Falik-Zaccai TC. Cerebrotendinous xanthomatosis (CTX): a treatable lipid storage disease. Pediatr Endocrinol Rev 2009; 7:6-11
Berginer VM, Shany S, Alkalay D, Berginer J, Dekel S, Salen G, Tint GS, Gazit D. Osteoporosis and increased bone fractures in cerebrotendinous xanthomatosis. Metabolism 1993; 42:69-74
Berginer VM, Abeliovich D. Genetics of cerebrotendinous xanthomatosis (CTX): an autosomal recessive trait with high gene frequency in Sephardim of Moroccan origin. Am J Med Genet 1981; 10:151-157
Nie S, Chen G, Cao X, Zhang Y. Cerebrotendinous xanthomatosis: a comprehensive review of pathogenesis, clinical manifestations, diagnosis, and management. Orphanet J Rare Dis 2014; 9:179
Mignarri A, Gallus GN, Dotti MT, Federico A. A suspicion index for early diagnosis and treatment of cerebrotendinous xanthomatosis. J Inherit Metab Dis 2014; 37:421-429
Stelten BML, van de Warrenburg BPC, Wevers RA, Verrips A. Movement disorders in cerebrotendinous xanthomatosis. Parkinsonism Relat Disord 2019; 58:12-16
Bjorkhem I, Andersson O, Diczfalusy U, Sevastik B, Xiu RJ, Duan C, Lund E. Atherosclerosis and sterol 27-hydroxylase: evidence for a role of this enzyme in elimination of cholesterol from human macrophages. Proc Natl Acad Sci U S A 1994; 91:8592-8596
Valdivielso P, Calandra S, Duran JC, Garuti R, Herrera E, Gonzalez P. Coronary heart disease in a patient with cerebrotendinous xanthomatosis. J Intern Med 2004; 255:680-683
Fujiyama J, Kuriyama M, Arima S, Shibata Y, Nagata K, Takenaga S, Tanaka H, Osame M. Atherogenic risk factors in cerebrotendinous xanthomatosis. Clin Chim Acta 1991; 200:1-11
Salen G. Cholestanol deposition in cerebrotendinous xanthomatosis. A possible mechanism. Ann Intern Med1971; 75:843-851
DeBarber AE, Connor WE, Pappu AS, Merkens LS, Steiner RD. ESI-MS/MS quantification of 7alpha-hydroxy-4-cholesten-3-one facilitates rapid, convenient diagnostic testing for cerebrotendinous xanthomatosis. Clin Chim Acta 2010; 411:43-48
DeBarber AE, Luo J, Star-Weinstock M, Purkayastha S, Geraghty MT, Chiang JP, Merkens LS, Pappu AS, Steiner RD. A blood test for cerebrotendinous xanthomatosis with potential for disease detection in newborns. J Lipid Res 2014; 55:146-154
Sekijima Y, Koyama S, Yoshinaga T, Koinuma M, Inaba Y. Nationwide survey on cerebrotendinous xanthomatosis in Japan. J Hum Genet 2018; 63:271-280
Duell PB, Salen G, Eichler FS, DeBarber AE, Connor SL, Casaday L, Jayadev S, Kisanuki Y, Lekprasert P, Malloy MJ, Ramdhani RA, Ziajka PE, Quinn JF, Su KG, Geller AS, Diffenderfer MR, Schaefer EJ. Diagnosis, treatment, and clinical outcomes in 43 cases with cerebrotendinous xanthomatosis. Journal of clinical lipidology2018; 12:1169-1178
Stelten BML, Huidekoper HH, van de Warrenburg BPC, Brilstra EH, Hollak CEM, Haak HR, Kluijtmans LAJ, Wevers RA, Verrips A. Long-term treatment effect in cerebrotendinous xanthomatosis depends on age at treatment start. Neurology 2019; 92:e83-e95
Salen G, Meriwether TW, Nicolau G. Chenodeoxycholic acid inhibits increased cholesterol and cholestanol synthesis in patients with cerebrotendinous xanthomatosis. Biochem Med 1975; 14:57-74
Berginer VM, Salen G, Shefer S. Long-term treatment of cerebrotendinous xanthomatosis with chenodeoxycholic acid. N Engl J Med 1984; 311:1649-1652
Zubarioglu T, Bilen IP, Kiykim E, Dogan BB, Enver EO, Cansever MS, Zeybek ACA. Evaluation of the effect of chenodeoxycholic acid treatment on skeletal system findings in patients with cerebrotendinous xanthomatosis. Turk Pediatri Ars 2019; 54:113-118
Koopman BJ, Wolthers BG, van der Molen JC, Waterreus RJ. Bile acid therapies applied to patients suffering from cerebrotendinous xanthomatosis. Clin Chim Acta 1985; 152:115-122
Mandia D, Chaussenot A, Besson G, Lamari F, Castelnovo G, Curot J, Duval F, Giral P, Lecerf JM, Roland D, Pierdet H, Douillard C, Nadjar Y. Cholic acid as a treatment for cerebrotendinous xanthomatosis in adults. J Neurol 2019; 266:2043-2050
Kuriyama M, Tokimura Y, Fujiyama J, Utatsu Y, Osame M. Treatment of cerebrotendinous xanthomatosis: effects of chenodeoxycholic acid, pravastatin, and combined use. J Neurol Sci 1994; 125:22-28
Moghadasian MH, Salen G, Frohlich JJ, Scudamore CH. Cerebrotendinous xanthomatosis: a rare disease with diverse manifestations. Arch Neurol 2002; 59:527-529
Lumbreras S, Ricobaraza A, Baila-Rueda L, Gonzalez-Aparicio M, Mora-Jimenez L, Uriarte I, Bunuales M, Avila MA, Monte MJ, Marin JJG, Cenarro A, Gonzalez-Aseguinolaza G, Hernandez-Alcoceba R. Gene supplementation of CYP27A1 in the liver restores bile acid metabolism in a mouse model of cerebrotendinous xanthomatosis. Mol Ther Methods Clin Dev 2021; 22:210-221
Wilson DP, Patni N. Should children with chronic diarrhea be referred to a lipid clinic? Journal of clinical lipidology 2018; 12:1099-1101
DeBarber AE, Kalfon L, Fedida A, Fleisher Sheffer V, Ben Haroush S, Chasnyk N, Shuster Biton E, Mandel H, Jeffries K, Shinwell ES, Falik-Zaccai TC. Newborn screening for cerebrotendinous xanthomatosis is the solution for early identification and treatment. J Lipid Res 2018; 59:2214-2222
Vaz FM, Jamal Y, Barto R, Gelb MH, DeBarber AE, Wevers RA, Nelen MR, Verrips A, Bootsma AH, Bouva MJ, Kleise N, van der Zee W, He T, Salomons GS, Huidekoper HH. Newborn screening for Cerebrotendinous Xanthomatosis: A retrospective biomarker study using both flow-injection and UPLC-MS/MS analysis in 20,000 newborns. Clin Chim Acta 2023; 539:170-174

Defects of Thyroid Hormone Transport in Serum

ABSTRACT

Inherited abnormalities of thyroid hormone-binding proteins are not uncommon and can predominate in some ethnic groups. They alter the number of iodothyronines present in serum and, although the concentration of free hormones remains unaltered, routine measurement can give erroneous results. With a single exception, inherited defects in thyroxine-binding globulin (TBG), are X-chromosome linked and thus, the full phenotype is expressed mostly in males. Partial TBG deficiency is more common than complete deficiency. High frequency of variants TBGs have been identified in African Blacks, Australian Aborigine, and Eskimos. Most defects producing TBG deficiency are caused by mutations in the structural gene. However, inherited X-linked partial deficiency can occur as the consequence of mutations of a gene enhancer. Inherited forms of TBG excess are all caused by gene duplication or triplication. Mutations in the transthyretin (TTR) gene producing a molecule with increased affinity for T4 are relatively rare. A variant TTR produces transient hyperthyroxinemia during non-thyroidal illness. Mutations of the human serum albumin (HSA) gene produce increased concentration of serum T4, a condition known as familial dysalbuminemic hyperthyroxinemia (FDH). They are relatively more common in individuals of Hispanic origin. They cause an increase in serum T4 owing to increased affinity for this iodothyronine but high concentrations in free T4 observed in direct measurement by some commercial methods are erroneous. A variant with increased affinity for T3 has been also identified.

INTRODUCTION

Abnormalities in the serum proteins that transport thyroid hormone do not alter the metabolic state and do not cause thyroid disease. However, they do produce alterations in thyroid hormone concentration in serum and when unrecognized have led to inappropriate treatment. When the abnormality is the consequence of altered synthesis, secretion or stability of the variant serum protein, the free thyroid hormone level estimated by most of the clinically available techniques remains within the range of normal. In contrast, when the defect results in a significant alteration of the affinity of the variant protein for the hormone, estimates of the free thyroid hormone level often give erroneous results and thus, it is prudent to measure the free hormone concentration by more direct methods such as equilibrium dialysis or ultrafiltration. This is also true in cases of complete TBG deficiency, in whom the estimation of free thyroid hormone level in serum by indirect methods, or using iodothyronine analogs as tracers, can also give erroneous results.

The existence of inherited defects of serum transport of thyroid hormone was first recognized in 1959 with the report of TBG-excess by Beierwaltes and Robbins (1). Genetic variants for each of the three major thyroid hormone transport proteins have since been described and in subsequent years, the molecular basis of a number of these defects has been identified (2). Clinically, these defects usually manifest as either euthyroid hyperthyroxinemia or hypothyroxinemia and more rarely, isolated hypertriiodothyroninemia (3). Associated abnormalities such as thyrotoxicosis, hypothyroidism, goiter, and familial hyperlipidemia are usually coincidental (4). However, individuals with thyroid disorders are more likely to undergo thyroid testing leading to the fortuitous detection of a thyroid hormone transport defect.

THYROXINE-BINDING GLOBULIN (TBG) DEFECTS

Familial TBG abnormalities are inherited as X-chromosome linked traits (5, 6), compatible with the location of the TBG (SERPINA7) gene on the long arm of the X-chromosome (Xq22.2) (7, 8). This mode of inheritance also suggests that the defects involve the TBG gene proper, rather than the rate of TBG disposal, as long ago postulated (5). The normal, common type TBG (TBG-N or TBG-C), has a high affinity for iodothyronines [affinity constants (Ka): 10-10 M-1 for T4 and 10-9 M-1 for T3] and binds 75% of the total T4 and T3 circulating in blood. Thus, with a single exception [HSA R218P and R218S (9-11), see below], among the inherited abnormalities of thyroid hormone transport proteins, those involving the TBG molecule produce usually more profound alterations of thyroid hormone concentration in serum.

Clinically TBG defects are classified according to the level of TBG in serum of affected hemizygotes (XY males or XO females, that express only the mutant allele): complete TBG deficiency (TBG-CD), partial TBG deficiency (TBG-PD) and TBG excess (TBG-E). In families with TBG-CD, affected males have no detectable TBG and carrier females (mothers or daughters) have on the average half the normal TBG concentration (4). In families with partially TBG deficient males, the mean TBG concentration in heterozygous females is usually above half the normal. Serum TBG concentration in males with TBG-E is 2 to 4-fold the normal mean and that in the corresponding carrier females, is slightly higher than half that of the affected males. These observations indicate an equal contribution of cells expressing the normal and mutant TBG genes. On rare occasions, selective inactivation of the X-chromosome has been the cause of manifestations of the complete defect (hemizygous phenotype) in heterozygous females (12, 13).

Inherited TBG defects can be further characterized by the level of denatured TBG (dnTBG) in serum and the physicochemical properties of the molecule. The latter can be easily determined without the need of purification. These properties are: (a) immunologic identity; (b) isoelectric focusing (IEF) pattern; (c) rate of inactivation when exposed to various temperatures and pH; and (d) affinity for the ligands, T4 and T3. More precise identification of TBG defects requires sequencing of the TBG gene.

MiP a Subject With TBG-CD

The proposita, a phenotypic female, was 13 years old when first seen because of retarded growth, amenorrhea and absence of secondary sexual traits. She was the first sibling of a second marriage for both parents. The family included a younger brother and four older half-siblings, two maternal and two paternal. The proposita was born to her 30-year-old mother after full-term, uncomplicated pregnancy. Infancy and early childhood development were normal until 4 years of age when it became apparent that she was shorter than her peers. She was 12 years of age when a low protein bound iodine (PBI, then a measure of T4) of 2.2 µg/dl (normal range 4.0-8.0) was noted and treatment with 120 mg of desiccated thyroid (equivalent to 200µg L-T4) daily was initiated. Since, during the ensuing 6 months, no change in her growth rate occurred and because PBI remained unchanged (2.0 µg/dl), the dose of desiccated thyroid was increased to 180 mg/day. This produced restlessness, perturbed sleep and deterioration of school performance necessitating discontinuation of thyroid hormone treatment. No family history of thyroid disease or short stature was elicited and the parents’ denied consanguinity.

On physical examination, the patient appeared younger than her chronological age, was short (137 cm) and showed no signs of sexual development. She had a webbed neck, low nuchal hairline, bilateral eyelid ptosis, shield-shaped chest, increased carrying angle and short 4th metacarpals and metatarsals. The thyroid gland was normal in size and consistency.

Buccal smear was negative for Barr bodies and karyotyping revealed 45 chromosomes consistent with XO Turner's syndrome. No chromosomal abnormalities were found in lymphocytes from the mother and father. Bone age was 12 years and X-ray of the scull showed a mild degree of hypertelorism. PBI and butanol extractable iodine were low at 2.0 and 1.8 µg/dl, respectively. Resin-T3 uptake was high at 59.9% (normal range 25-35%) indicating reduced TBG-binding capacity. A 24-hour thyroidal radioiodide uptake was normal at 29%, basal metabolic rate was +20% (normal range -10 to +20) and TG autoantibodies were not present. Serum cortisol was normal as were the responses to ACTH and metyrapone. Basal growth hormone concentration was normal at 8.0 ng/ml which rose to 32 ng/ml following insulin hypoglycemia.

Studies were carried out in all first-degree relatives and the proposita was treated cyclically with diethylstilbestrol which produced withdrawal uterine bleeding and gradual breast development.

Five family members, in addition to the proposita had thyroid function tests abnormalities. Two were males and three females. The two males (maternal grandfather and maternal half-brother) and the proposita had the lowest PBI levels and undetectable T4-binding to serum TBG. In contrast, the three females (mother, maternal aunt and maternal half-sister) had a lesser reduction of their PBI and T4-binding capacity to TBG approximately one-half the normal mean value. The two sons of the affected grandfather (maternal uncles to the proposita) had normal PBI and T4-binding to TBG. No interference with T4-binding to TBG or other serum protein abnormalities were found in affected members of the family. In vivo T4 kinetic studies revealed a rapid extrathyroidal turnover rate but normal daily secretion and degradation, compatible with their eumetabolic state.

INTERPRETATION

The incidental identification of thyroid tests abnormalities in the proposita is typical for most subjects with TBG deficiency as well as TBG excess. So is the initial unnecessary treatment; though less frequent with the routine measurement or estimation of free T4. The inherited nature of the defect is suspected by exclusion of factors known to cause acquired TBG abnormalities and should be confirmed by the presence of similar abnormalities in members of the family. The absence of male-to-male transmission and the carrier state of all female offspring of the affected males is a typical pattern of X-chromosome linked inheritance. This is further supported by the complete TBG deficiency in individuals having a single X chromosome (males and the XO female) and only partial TBG deficiency in carrier XX females.

Since the publication of this family in 1968 (14), the cause of the TBG defect was identified. From the mutation identified in the TBG gene of this family [TBG Harwichport (TBG-CD H)], it can be deduced that the molecule is truncated, missing 12 amino acids at the carboxyl terminus (15).

Fifty six TBG variants have been so far identified and in 50 the precise defect has been determined by gene analysis. Their primary structure defect, some of their physical and chemical properties and the resulting serum T4 concentrations are summarized in Table 1 and figure 1.

Figure 1. Properties of some TBG variants causing partial TBG deficiency (TBG-PD). The TBG variants are: -SD, San Diego; -G, Gary; -M, Montreal, -S, slow; -A, Aborigine; -Poly, polymorphic; -Cgo, Chicago; and -Q, Quebec. For detailed description, see (1) Sarne et al (39) and Bertenshaw et al (37); (2) Murata et al (34), Mori et al (43) and Kambe et al (60); (3) Takamatsu et al (44) and Janssen et al (45); (4) Takamatsu et al (55) and Waltz et al (56); (5) Murata et al (61) and Takeda et al (47); (6) Mori et al (26) and Takeda et al (57); (7) Takamatsu et al (59) and (58); (8) Takamatsu et al (44) and Bertenshaw et al (49). [Modified from Refetoff et al (62)].

Table 1. TBG Variants and Gene Mutations
TBG NAME	Abbreviated name	Intron Exon	CODON*	AMINO ACID		NUCLEOTIDE		References
TBG NAME	Abbreviated name	Intron Exon	CODON*	WT	Variant	WT	Variant	References
Complete Deficiency (CD)
Milano (fam A)	CDMi†	IVS 1	fs	5' DSS	unknown	gtaagt	gttaagt	(16)
Andrews	CDAN	IVS 1	fs	5' DSS	unknown	gtaagt	gcaagt	(17)
Portuguese 1 (pt A)	CDP1	1	23	S (Ser)	X (OCH)	TCA	TAA	(18)
Yonago	CDY	1	28-29fs-51	D F	X (OPA)	GA(CT)TT	GAATT	(19)
Negev (Bedouin	CDN	1	38fs-51	T (Thr)	X (OPA)	ACT	T del	(20, 21)
Nikita (fam B)	CDNi	1	50fs-51	P (Pro)	X (OPA)	CCT	T del	(16)
Taiwanese 1	CDT1†	1	52	S (Ser)	N (Asn)	AGC	AAC	(22)
Parana	CDPa†	1	61	S (Ser)	C (Cys)	TCC	TGC	a
No name	CD6	1	165fs-168	V (Val)	X (OCH)	GTT	T del	(23)
Kankakee	CDK	IVS 2	188fs-195	3' ASS	X (OPA)	agCC	ggCC	(24)
Poland	CDPL	2	201fs-206	D (Asp)	X (OCH)	GAC	G del	(25)
Portuguese 2(pt B)	CDP2	2	223	Q (Gln)	X (OCH)	CAA	TAA	(18)
No name	CD5†	2	227	L (Leu)	P (Pro)	CTA	CCA	(26)
Portuguese 3§	CDP3	2	233	N (Asn)	I (Ile)	ACC	ATC	(27)
Berlin	CDBn	IVS 3+3	fs	intronic	unknown	intronic	79nt del	(17)
Houston	CDH	IVS 3	279fs-374	3’ ASS	X (OPA)	agAT	aaAT	(28)
Buffalo	CDB	3	280	W (Trp)	X (AMB)	TGG	TAG	(29)
Taiwanese 2	CDT2	3	280	W (Trp)	X (OPA)	TGG	TGA	(22)
Lisle	CDL	IVS 4	280fs-325	5' DSS	X (OPA)	gtaaa	ggaaa	b
Jackson (fam K)	CDJa	IVS 4	280fs-325	5' DSS	X (OPA)	gtaaa	gtaag	(30)
No name	CD7	3	283fs-301	L (Leu)	X (OPA)	TGT	G del	(15)
No name	CD8†	4	329fs-374	A (Ala)	X (OPA)	GCT	G del	(15)
Japan	CDJ	4	352fs-374	L (Leu)	X (OPA)	CTT	C del	(31, 32)
Penapolis	CDPe	4	332fs-374	K (Lys)	X (OPA)	AAG	A del	a
Kyoto§	CDKo	4	370	S (Ser)	F (Phe)	TCT	TTT	(33)
Harwichport	CDH	4	381fs-396	Y (Tyr)	X (OPA)	AGG	19nt del	(14, 15, 34)
NeuIsenburg	CDNl	4	384fs-402	L (Leu)	7 aa add	CTC	TC del	(35)
Partial Deficiency (PD)
Allentown	PDAT	1	-2	H (His)	Y (Tyr)	CAC	TAC	(36)
San Diego	PDSD†	1	23	S (Ser)	T (Thr)	TCA	ACA	(37-39)
Brasilia	PDB	1	35	R (Arg)	W (Trp)	CGG	TGG	(40)
Wanne-Eickel	PDWE	1	35	R (Arg)	E (Glu)	CGG	CAG	(41)
No name		1	35	R (Arg)	Q (Gln)	CGG	TGG	(41)
No name	?	1	52	S (Ser)	R (Arg)	AGT	AGA	c
No name	?	1	64	A (Ala)	D (Asp)	GCC	GAC	(42)
Korea	PDKa	1	74	E (Glu)	K (Lys)	GAG	AAG	a
Gary	PDG	1	96	I (Ile)	N (Asn)	ATC	AAC	(43)
No name		1	112	N (Asn)	L (Lys)	AAT	AAG	c
Montréal	PDM	1	113	A (Ala)	P (Pro)	GCC	CCC	(44, 45)
Berlin2	Bn2	1	161	T (Thr)	N (Asn)	ACC	AAC	a.
Aborigine	PDA†	2	191	A (Ala)	T (Thr)	GCA	ACA	(46, 47)
Glencoe	PDGe	2	215	V (Val)	G (Gly)	GTG	GGG	(48)
Quebec	PDQ†	4	331	H (His)	Y (Tyr)	CAT	TAT	(44, 49)
Japan (Kumamoto)	PDJ	4	363	P (Pro)	L (Leu)	CCT	CTT	(50, 51)
Heidelberg	PDHg	4	368	D (Asp)	G (Gly)	GAT	GGT	(52)
No name	?	4	381	R (Arg)	G (Gly)	AGG	GGG	c
No name	?	4	382	S (Ser)	R (Arg)	AGT	CGT	c
No name		enhancer		-	-	G	A	(53)
Other Variants
Slow	S	1	171	D (Asp)	N (Asn)	GAC	AAC	(54-56)
Polymorphism	Poly	3	283	L (Leu)	F (Phe)	TTG	TTT	(26, 57)
Chicago	CH or Cgo	3	309	Y (Tyr)	F (Phe)	TAT	TTT	(58, 59)

* Codon numbering from fist amino acid of the mature protein. The 20 amino acids of the signal peptide are numbered -1 to -20, from N- to C-terminus. The codon at the site of mutation is followed by the codon at the site of termination of translation.

† Coexistence of TBG Poly

complete deficiency is uncertain as the TBG assay used was unable to detect values <10% the mean normal

¶ Also a silent mutation at codon 55: GCA -> GCG

a Personal communication

b Personal observation

c Communicated by Pia Hermanns and Joachim Pohlenz, University of Mainz, Germany

del, delete; add, addition; aa, amino acid; fs, frame shift

Pt, patent; fam, family

IVS, intervening sequence or intron; ASS, acceptor splice site; DSS, donor splice site

Complete Deficiency of TBG (TBG-CD)

TBG-CD is defined as undetectable TBG in serum of affected hemizygous subjects or a value lesser than 0.03% the normal mean; the current limits of detection using the most sensitive radioimmunoassay (RIA) being 5ng/dl (26). The prevalence is approximately 1:15,000 newborn males. Twenty-seven TBG variants having this phenotype have been characterized at the gene level. These are shown in table 1 that also contains references to the original publications. Twenty two of the 27 TBG-CDs have truncated molecules. Early termination of translation of these variants is caused in 4 by a single nucleotide substitution (TBG-CDP1, TBG-CDP2, CD5, TBG-CDB and TBG-CDT2) or by a frame shift due to one nucleotide deletion (TBG-CDY, TBG-CDN, TBG-CDNi, TBG-CD6, CD-PL, TBG-CD7, TBG-CD8, and TBG-CDJ, TBG-CDPe) or deletion of 19 nucleotides (TBG-CDH). In 7 variants mutations occurred in introns, 6 of which are close to splice sites (TBG-CDMi, TBG-CDAN, TBG-CDK, TBGBn, TBG-CDH, TBG-CDL and TBG-CDJa). A mutation at the acceptor splice junction caused also a frame shift producing early termination of translation in TBG-CDK (24). In contrast a nucleotide substitutions in the 5' donor splice site of intron IV (TBG-CDL and TBG-CDJa), resulted in a complete splicing of exon 3, also producing a truncated molecule (30) and personal observation. A similar mechanism is likely responsible for CD in TBG-CDMi, though direct experimental prove was not provided (16). Single amino acid substitution was the cause of CD in five families (TBG-CDT1, TBG-CDPa, TBG-CD5, TBG-CDP3 and TBG-CDKo). In TBG-CD5 Leucine-227 with a proline was shown to cause aberrant post-translational processing (45). One TBG variant (TBG-CDNI), with two nucleotides deleted close to the carboxyl terminus, the resulting frame shift predicts an extension of the molecule by the addition of 7 nonsense residues (35). TBG-CDJ has been so far identified only in Japanese but its allele frequency in the population remains unknown (32, 57) (Table. 1).

Partial Deficiency of TBG (TBG-PD)

This is the most common form of inherited TBG deficiency having a prevalence of 1:4,000 newborn. Identification of heterozygous females by serum TBG measurement may be difficult because levels often overlap the normal range. In contrast to variants with complete TBG deficiency, all TBG-PDs have missense mutations. It is possible that three of the five variants with single amino acid substitutions included in the category of TBG-CD have also partial deficiency which was not identified owing to the low sensitivity of routine assays for the measurement of TBG. Twenty-one different mutations, producing a variable degree of reduction of TBG concentration in serum, have been identified, 20 of which involve mutations in the TBG gene proper. They are listed in table 1. In addition, some of these variants are unstable (TBG-PDG, TBG-PDA, TBG-PDSD, TBG-PDM TBG-PDQ and TBG-PDJ) or have lower binding affinity for T4 and T3 (TBG-PDG, TBG-PDA, TBG-PDS TBG-PDSD, TBG-PDM and TBG-PDQ), impaired intracellular transport and secretion (TBG-PDJ and TBG-CDJ) and some exhibit an abnormal migration pattern on IEF electrophoresis (TBG-PDG, TBG-PDM, and TBG-PDQ) (Fig. 1). Variants with decreased affinity for T4 and T3 have a disproportionate reduction in hormone concentration relative to the corresponding serum TBG level (Fig. 2) and estimations of the free hormone levels by any of the index methods gives erroneous results (39, 64). One of these variants, TBG-PDA, is found with high frequency (allele frequency of 51%) in Australian Aborigines (47).

Figure 2. Serum T4-bound to TBG and the concentration of TBG and denatured TBG (dnTBG) in hemizygous subjects expressing the different TBG variants. Results, graphed as mean ± SD, were normalized by expressing them as % of those for the common type TBG (TBG-C). For abbreviations used in the nomenclature of the TBG variants, see legend to figure 1. [Adapted from Janssen et al (63)].

A unique family with TBG-PD has been described in which inheritance of the partial deficiency was autosomal dominant with transmission of the phenotype from father to son (65). The concentration of TBG in affected males and females was about one half the normal mean value. The TBG had normal affinity for T4. normal IEF and heat lability. No sequence changes were found in the entire coding arias of the gene or in the promoter region. Although the mechanism of TBG-CD in this family is unknown an abnormality in one of the factors regulating TBG gene transcription is a distinct possibility. Further studies to determine the genetic defect have been hampered by lack of subjects’ cooperation.

In 5% (4 or 74) families with X-chromosome lined TBG deficiency, studied in the author’s laboratory, no mutations were identified in the entire TBG gene, including all exons, introns, untranslated regions and the promoter region of the gene, covering a total of 9.2 kb. Next-generation sequencing identified a novel single nucleotide substitution 20 kb downstream of the TBG gene in all four families. In silico analysis predicted that the variant resides within a liver-specific enhancer. In vitro studies confirmed the enhancer activity of a 2.2-kb fragment of genomic DNA containing the novel variant and showed that the mutation reduces the activity of this enhancer. The affected subjects share a haplotype of 8 Mb surrounding the mutation. Three were of known Arab ethnicity and in all four families the most recent common ancestor was estimated to be 19.5 generations ago (95% confidence intervals). This is first report of an inherited endocrine disorder caused by a mutation in an enhancer region (53).

TBG Excess (TBG-E)

TBG-E has a lower prevalence than TBG deficiency, with values obtained from neonatal screening programs from 1:6,000 to 1:40,000 (66, 67). Considering that some newborn may have non-inherited, transient TBG excess, a conservative overall estimate of inherited TBG-E would be 1:25,000 (68). Early sequencing of the coding and promoter regions of subjects with TBG-E failed to show any defects (69). However, in 1995, Mori et al (70) found that gene amplification was the cause of TBG-E in two families. Gene triplication and duplication were demonstrated by gene dosage studies using HPLC measurements of the PCR -amplified product. As expected, hemizygous affected males had approximately 3- and 2-fold the average normal serum TBG concentration, respectively. The presence of multiple TBG gene copies in tandem was confirmed by in situ hybridization of prometaphase and interphase chromosomes from an affected male (Fig. 3).

Figure 3. Hybridization in prometaphase chromosomes of cultured skin fibroblasts obtained from an affected male with TBG-E. A complete TBG cDNA was used as a probe. Three TBG gene copies are seen in tandem with each exon clearly identified in the starched chromosome shown in panel B.

TBG Variants with Unaltered TBG Concentrations in Serum

Five TBG variants have been identified that present with normal or slight and clinically insignificant alterations in their concentration in serum. Four occur with high frequency in some population groups and thus, can be considered as polymorphic. TBG-Poly (Fig. 1), with no alterations of its physical or biological properties, has been detected in 16% and 20% of the French Canadian and Japanese populations, respectively (26, 57). TBG-S exhibits a slower mobility on polyacrylamide gel electrophoresis and cathodal shift on IEF (54, 55), owing to the loss of a negative change due to the replacement of the normal Asp171 by Asn (56) (Figs. 1 and 4). It has an allele frequency of 5 to 16% in Black populations of African origin and 2 to 10% in Pacific Islanders. The molecular structure of two other polymorphic TBG variants has not been identified. TBG-F has an allele frequency of 3.2% in Eskimos residing on the Kodiac and St. Lawrence islands. It has a slight anodal (fast) mobility on IEF (71). TBG-C1 has been identified in subjects inhabiting two Mali village (72). It has a small cathodal shift on IEF and an allele frequency of 5.1%. TBG-Cgo, resistant to high temperatures (59), has normal affinity for T4 and T3. All SERPINs except human TBG have a Phe at a position corresponding to Tyr309. Structure modeling suggests that the replacement of the normal Tyr309 by Phe in TBG-Cgo, ties the internal α-helix hI1 to the molecule, thus stabilizing its tertiary structure (58). Studies using recombinant TBG–Cgo showed that the molecule exists in loop expelled conformation. However, when exposed at 37°C, the protein readily converts to a more stable loop inserted conformation explaining its subsequent enhanced heat stability, as observed in vivo (73).

Figure 4. Microheterogeneity of TBG. Tracer amounts of l25I T4 were added to serum prior to submission to isoelectric focusing and radioautography. TBG C (common type) exhibits 6 bands spanning from pH 4.18 to 4.58. Three of the six are major and shown here between pH 4.35 and 4.50. TBG-Slow (TBG S) from a hemizygous male shows a cathodally shifted pattern. A mixed pattern occurs in heterozygous females expressing both TBG-C and TBG-S. [Reproduced from Waltz et al (56)].

Biological Consequences of Structural Changes Caused by Mutations in the TBG Gene

The mechanisms whereby structural abnormalities of the TBG molecule produce the variant phenotypes have been investigated by expression of some of these molecules in living cells. Contrary to earlier speculation, increased extracellular degradation due to instability is a rare cause reduced concentration of the variant TBG in serum (38). More commonly, intracellular retention and degradation of the defective TBG molecules is responsible for their presence in low concentrations in serum (45, 51, 60, 74). Of note is the full intracellular retention of TBG-CD5 despite synthesis in normal quantities. A single amino acid substitution in TBG-CD5 is sufficient to alter its tertiary structure and thus prevent export. The same finding in the case of TBG-CDJ has been traced to its retention within the endoplasmic reticulum. Furthermore, the increased amount of GRP78 mRNA in cells transfected with TBG-PDJ suggests that association of this TBG variant with the GRP78 molecular chaperon is responsible for its impaired secretion (51). The variant TBG-AL is unique and important as it provides information about the function of the signal peptide. The resulting variable decrease in the serum TBG concentration associated with diminished in vitro secretion is compatible with impaired cotranslational processing (36).

Several speculations regarding the properties of variant TBGs have been confirmed based on the elucidation of the TBG structure by X-ray crystallography (75). The reduced ligand-binding of TBG-SD (38, 39) can be explained by the direct proximity of the amino acid substitution to the binding pocket. Indeed, the methyl group of the side chain of Thr23, replacing the normal Ser, will sterically hinder the binding of T4. Similarly, in TBG-A, the replacement of Ala191 by Thr (47) perturbs the H-bounds that stabilizes the binding pocket, leading to the reduced T4 binding. In contrast, the loss of His331 in TBG-Q (H331Y) (44, 49) allows unrestricted loop insertion in the upper half of the A-sheet, accounting for the increased in serum denatured (dn)TBG and reduced T4 binding.

TBG deficiency was found to coexist in the same family with resistance to thyroid hormone beta (RTHß) (76). Both TBG (P50fs51X) and THRB (P453A) gene mutations have been previously described in unrelated families (16, 77) but not in the same family. The mother harbored both gene mutations, whereas the proband and his sister had only the THRB gene mutation and a brother only the TBG gene mutation. This family illustrates the difficulty that might be encountered in the interpretation of thyroid function tests when different genetic defects, having opposite effect on thyroid function tests, coexist in the same family, and especially the same individual.

TRANSTHYRETIN (TTR) DEFECTS

Sequencing of the TTR gene, formerly known as thyroxine-binding prealbumin (TBPA) on chromosome 18 (18q11.2-q12.1), has uncovered mutations that produce variant TTR molecules with or without alterations in the binding affinity for iodothyronines (2, 78). Only those known to affect iodothyronine binding are listed in table 2. Some of the TTR variants are responsible for the dominantly inherited familial amyloidotic polyneuropathy (FAP), causing multiple organ failure and death in early adulthood.(78). Because TTR has a relatively lower affinity for T4 (about 100-fold lesser than that of TBG), it plays a minor role in thyroid hormone transport in blood. Accordingly, changes in the TTR concentration in serum and variant TTRs with reduced affinity for T4 have little effect on the concentration of serum T4 (79, 91). Only variant TTRs with a substantially increased affinity for iodothyronines produce significant elevation in serum T4 and rT3 concentrations and account for 2% of subjects with euthyroid hyperthyroxinemia (90).

Table 2. TTR Variants with Altered Affinity for T4 and Potentially an Effect on Tests of Thyroid Function in Serum
AFFINITY FOR T4 Mutant / Normal		TTR CONCENTRATION	CODON Number	AMINO ACID (Normal - Variant)	REFERENCES
HOMO*	HETERO*
DECREASED
<0.1	0.17 - 0.41	N	30	Val - Met	(79, 80)
	0.54		58	Leu - His	(80)
	0.45		77	Ser - Tyr	(80)
	0.19 – 0.46	N	84	Ile - Ser	(79, 80)
~1.0	0.44			Val - Ile	(80)
INCREASED
	3.5†	N	6	Gly - Ser	(81-83)
8.3-9.8	3.2 - 4.1‡	N	109	Ala - Thr	(80, 84-86)
		N	109	Ala - Val	(85)
		Inc or N	119	Thr - Met	(87-90)

* HOMO, homozygous; HETERO, heterozygous.

† Probably overestimated since the subjects harboring this TTR variant have normal serum TT4 concentrations.

‡ Affinity of recombinant TTR Thr109 is 9-fold that of the normal TTR (86).

Variant TTR tested and shown not to have altered affinity to T4 are: Ala60, (hetero) (79, 80).

N, normal; Inc, increased

Endonucleases useful in the identification of TTR variants: Msp I -ve for Ser6 in exon 2 associated PHA; BsoFI -ve and Fnu 4H +ve for Thr109; BsoFI -ve for Val109 and Nco I +ve for Met119, all in exon 4.

A family with elevated total T4 concentration which was predominantly bound to TTR was first described in 1982 by Moses et al (92). The inheritance was autosomal dominant and affected members were clinically euthyroid with normal free T4 levels measured by equilibrium dialysis. The variant TTR has a single nucleotide substitution replacing the normal Ala109 with a Thr which increases its affinity for T4, rT3 and tetraiodothyroacetic acid and to a lesser extend T3 and triiodothyroacetic acid (84, 86). Crystallographic analysis of this variant TTR revealed an alteration in the size of the T4-binding pocket (93). Another TTR gene mutation involving the same codon has been subsequently described (85). This mutant TTR with Val109 has an increased affinity for T4 that is of similar magnitude as TTR Thr109, about 10-fold higher than that of wild-type TTR.

A more common defect found in subjects with prealbumin associated hyperthyroxinemia (PAH) is a point mutation in exon 4 of the TTR gene replacing the normal Thr119 with Met (90). First described in a single individual with normal serum total and free T4 levels (89), the majority of subsequently identified heterozygous subjects harboring the TTR Met119 had an increase in the fraction of T4 and rT3 associated with TTR, but only few had serum T4 levels above the upper limit of normal. Furthermore, their hyperthyroxinemia appears to be transient, usually in association with non-thyroidal illness (90). The variant TTRs associated with PAH are not amyloidogenic.

The unique occurrence in an Argentinian family of TTR A109T, known to have increased affinity for T4, in association with TGB-PD A64D mitigated the phenotype of the latter mutation (42)

Variant TTRs without Known Biological Effects

Several TTR variants have been found that do not alter the properties of the molecule, nor cause FAP, and are thus of no clinical significance. Of interest is a TTR variant found in the rhesus monkey, Macaca mulatta, but not in man (94, 95)[. This variant has a slower electrophoretic mobility resulting in three phenotypes which exhibit: (a) a single rapidly migrating band similar to that found in human and other primates (PAFF); (b) a single slowly migrating band cathodal to albumin (PASS); and (c) a five banded form corresponding to the various tetrameric recombinants present in the heterozygous state possessing the two different subunits (PAFS). This finding was important because the variant rhesus PA-S could be hybridized in vitro with human TTR yielding a five-banded pattern hence, demonstrating that human TTR is also a tetramer. All naturally occurring and hybrid polymorphic variants show no detectable alteration in the binding of either T4 or retinol binding protein (96).

HUMAN SERUM ALBUMIN (HSA) DEFECTS

Another form of dominantly inherited euthyroid hyperthyroxinemia, later to be linked to the albumin gene on chromosome 4 (4q11-q13), was first described in 1979 (97, 98). Known as familial dysalbuminemic hyperthyroxinemia (FDH) (99), it is the most common cause of inherited increase in total T4 in serum in the Caucasian population, producing on the average a 2-fold increase in the serum total T4 concentration. In a study of 430 subjects suspected of having euthyroid hyperthyroxinemia 12% were proven to have FDH (90). The prevalence varies from 0.01 to 1.8%, depending on the ethnic origin, with the highest prevalence in Hispanics (100-103). This form of FDH has not been reported in subjects of African origin and the isolated occurrence in a Chinese (104) was possibly brought by Hispanic travelers (see below). The euthyroid status of subjects with FDH has been confirmed by normal TSH response to TRH, normal free T4 concentration measured by equilibrium dialysis using appropriate buffer systems, normal T4 production rate and normal serum sex hormone-binding globulin concentration (97, 99, 105, 106). Nevertheless, the falsely elevated free T4 values, when estimated by standard clinical laboratory techniques, has often resulted in inappropriate thyroid gland ablative or drug therapy (107-109). A recent survey of commonly used commercial tests for measurement of free T4 indicates that equilibrium or symmetric dialysis are the only tests that will consistently provide accurate values in subjects with FDH (110), in particular one using dialysis in combination with tandem mass spectrometry (111).

FDH is suspected when serum total T4 concentration is increased without proportional elevation in total T3 level and non-suppressed serum TSH. Half of affected subjects have also rT3 values above the normal range (112) (Table 3). Since the same combination of test results are found in subjects with the Thr109 TTR variant, the diagnosis of FDH should be confirmed by the demonstration that an increased proportion of the total serum T4 migrates with HSA on non-denaturing electrophoresis or precipitates with anti-HSA serum.

Table 3. Albumin Variants with Increased Affinities for Iodothyronines, Their Effect on the Serum Concentrations of and Affinities to these Hormones
VARIANT	SERUM CONCENTRATION				BINDING AFFINITY (Ka) of the variant albumins		Reference
VARIANT	T4 µg/dl	T3 ng/dl	rT3 ng/dl	N	T4	T3
	(Fold the normal mean)				(Fold the normal mean)
WT	8.0 ± 0.2	125 ± 4	22.5 ± 0.9	83	1	1	A
R218H	16.0 ± 0.5 (2.0)	154 ± 3 (1.2)	33.1 ± 1.1 (1.5)	83	(10 – 15)	(4)	a,(113, 114)
R218P	135 ± 17 (16.8)	241 ± 25 (1.9)	136 ± 13 (6.1)	8	(11-13*)	(1.1*)	(9, 10)
R218S	70 (8.8)	159 (1.3)	55.7 (2.6)	1	NM	NM	(11)
R222I	21±1.4 (2.6)	135±18 (1.2)	1417±107 (86)	8	NM	NM	a, (115)
L66P	8.7 (1.1)	320 (3.3)	22.3 (1)	6	(1.5)	(40)	(3)

Values reported are means ± standard error, and the number of subjects per genotype are indicated under ‘‘N.’’

* Determined at saturation. Affinities are higher at the concentrations of T4 and T3 found in serum.

NM, not measured

a, Personal observation

All data were generated in the Chicago laboratory except for 4 of the 8 individuals with ALB R218P and hose with ALB R222I, provided by Nadia Schoenmakers, University of Cambridge, UK.

A tight linkage between FDH and the HSA gene (lod score 5.25) was found in a large Swiss-Amish family using two polymorphic markers (112). This was followed by the identification of a missense mutation in codon 218 of the HSA gene replacing the normal arginine with a histidine (R218H) (113, 116). Furthermore, the same mutation was present in all subjects with FDH from 11 unrelated families. Its association with a Sac I+ polymorphism, suggest a founder effect and is compatible with ethnic predilection of FDH (113). The coexistence of FDH and a TTR variant with increased affinity for T4 in the same individual (82, 83) and FDH with TBG-PD in another (117) have been reported. In both instances these individuals were the product of parents each heterozygous for of one of the two defects.

Another mutation in codon 218, with increased affinity to iodothyronines, was first identified by Wada et al (9). The mutation, a replacement of the normal Arg218 with a Pro (R218P), initially believed to be unique for Japanese was also identified in a Swiss family with no Asian ancestry (Fig. 5) (10). In this form of FDH, serum total T4 levels are 14-20-fold the normal mean, a level confirmed by measurements in serum extracts by HPLC. Total rT3 and T3 concentrations are 7- and 2-fold above the mean, respectively. Thus, in order to maintain a normal free T4 level, the calculated affinity constant (Ka) of HSA R218P should be about 16-fold higher than that of HSA R218H. Surprisingly, the Kas measured at saturation were similar, 5.4 x 106 M-1 and 6.4 x 106 M-1 for HSA R218H, respectively (10, 118, 119) (Table 3). However, at T4 concentrations equivalent to those found in subjects with HSA R218P, the dialyzable FT4 concentration was 11-fold higher in serum of subjects with HSA R218H and 49-fold higher in serum with the common type HSA only (10).

Figure 5. A Swiss family with HSA R218P: genotyping, pedigree and thyroid function tests. A, Genotyping for the mutation HSA R218P. Results are aligned with each subject depicted on the pedigree in B. Amplification of a segment of the HSA gene containing the mutation with a mismatched oligonucleotide primer creates a new restriction site for Ava II only in the presence of the mutant nucleotide (CGC -> CCC). Affected subjects expressing proline 218 (CCC) show a 122 bp DNA fragment produced by enzymatic digestion of the mutant allele. Note that all affected subjects are heterozygous and that the 153 bp fragment amplified from DNA of the two normal subjects, expressing arginine 218 (CGC) only, resists enzymatic digestion. B, Pedigree of the family. Roman numerals indicate each generation and numbers below each symbol identify the subject. Individuals expressing the FDH phenotype are indicated by half-filled symbols. C, Thyroid function tests. Results are aligned with each symbol. Values outside the normal range are in bold numbers. Note the disproportionate increase in serum T4 concentration as compared to that of T3 in the affected individuals. Subject I-1, a year old man, had diabetes mellitus with multiple organ complications and mild subclinical hypothyroidism, explaining the relatively lower serum T4 and T3 but not rT3 levels. [Adapted from Pannain et al (10)].

More recently two additional HSA gene mutations have been identified. One in the same codon resulting in a different amino acid substitution (R218S) (11) and another in a different amino acid (R222I) (115) in the proximity of the same iodothyronine-binding pocket (Fig. 6). While both manifest increased affinity for T4 and rT3, it is considerably higher for T4 in the former and for rT3 in the latter (Table 3). It is of note that the two amino acids, 218 and 222, involved in the gain-of-function mutations are located in the main predominantly hydrophobic pocket where T4 is bound in a cisoid conformation (120).

Figure 6. The structures of HSA in the presence of T4 as modeled on the structures 1BM0, 1HK1, 1HK3 in the Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). Top panel shows on the left the entire WT HSA molecule (in green) with its four T4 binding sites [T4(1) to T4(4)] according to Petitpas et al (120) and to the right a close up of the binding pocket, T4 (1) containing arginine’s 218 and 222 along with the T4 molecule (carbons are in white, nitrogen’s in blue, oxygens in red and iodine in magenta). In the bottom panel are represented the structures of the T4 (1) binding pockets of the four mutant HSA showing, a better accommodation of T4 than in the WT HSA and thus, resulting in enhanced binding (From Erik Schoenmakers, University of Cambridge, UK).

A fifth gain-of-function mutation, a replacement of the normal Leu66 with a Pro (L66P) has been identified in a single Thai family (3). It produces a 40-fold increase in the affinity for T3 but only 1.5-fold increase in the affinity for T4 (Table 3). As a consequence, patients have hypertriiodothyroninemia but not hyperthyroxinemia. In this FDH-T3, serum T3 concentrations are falsely low, or even undetectable, when T3 is measured using an analog of T3 as a tracer rather than a radioisotope. It has resulted in the inappropriate treatment with thyroid hormone (3).

Bisalbuminemia and Analbuminemia

Variant albumins, with altered electrophoretic mobility produce "bisalbuminemia" in the heterozygotes (121). T4 binding has been studied in subjects from unrelated families with a slow HSA variant. In two studies only the slow moving HSA bound T4 (122, 123) and in another, both (124). The differential binding of T4 to one of the components of bisalbumin may be due to enhanced binding to the variant component with charged amino acid sequence. Bisalbuminemia does not seem to be associated with gross alterations in thyroid hormone concentration in serum.

Analbuminemia is extremely rare, occurring in less than 1 in a million individuals (125). The first case was reported in 1954 (126) but the HSA gene mutation was identified 56 years later (127). The less than 50 cases so far reported have nonsense mutations causing premature termination of translation or splicing defects (128). Despite the complete lack of such an important substance, symptoms are remarkably mild owing to the compensation by an increase in non-albumin serum proteins. Studies with respect to T4-transport showed no clear effect or slight increase total serum iodothyronines, associated with increased levels of TBG and TTR. (128, 129). The latter two normalized when serum HSA was restored to normal by multiple transfusions (129).

ACKNOWLEDGMENTS

Supported in part by grants DK-15070 from the National Institutes of Health (USA).

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Effect of Pregnancy on Lipid Metabolism and Lipoprotein Levels

ABSTRACT

Lipoprotein lipid physiology in pregnancy has important implications for the developing fetus and newborn as well as the mother. Cholesterol and essential fatty acids are essential for normal fetal development. In pregnancy, multiple physiological changes occur that contribute to the alterations in lipid profiles of healthy, gestating women. Initially, there is an anabolic phase with an increase in lipid synthesis and fat storage in preparation for the increases in fetal energy needs in late pregnancy. During the third trimester, lipid physiology transitions to a net catabolic phase with a breakdown of fat deposits. The catabolism increases substrates for the growing fetus. Overall, the changes in lipid physiology throughout the course of pregnancy allow for proper nutrients for the fetus and they reflect increasing insulin resistance in the mother. In a normal pregnancy, total cholesterol levels increase by approximately 50%, LDL-C by 30-40%, HDL-C by 25%, and triglycerides by 2- to 3-fold. Our understanding and appreciation of the full scope and implications of dyslipidemia in pregnancy on both maternal and fetal outcomes is not complete; however, it is well known that dyslipidemia in pregnancy is associated with adverse pregnancy outcomes affecting both maternal and fetal health. There are direct implications of dyslipidemia on perinatal outcomes as well as intricate relationships between dyslipidemia and other comorbid intrauterine conditions. There is also developing research indicating that the in-utero environment influences susceptibility to chronic diseases later in life, a concept known as “developmental programming.” Given all of these implications of dyslipidemia in pregnancy on maternal and fetal health, it is prudent to screen women for lipid disorders. The ideal time for this is before conception; if a woman has not been screening before pregnancy, the initial obstetrical visit is ideal. Abnormal lipids should be followed through pregnancy. The treatment of dyslipidemia in pregnancy is multifactorial, including diet, exercise, and weight management. Medical management is complicated by FDA classifications for medication risks to the fetus, however some evidence indicates there may be permissible pharmacological treatments for dyslipidemia in pregnancy. In certain instances, plasmapheresis or lipoprotein apheresis can be employed.

INTRODUCTION

Lipoprotein lipid physiology before and during pregnancy has important implications for the mother, the developing fetus, the newborn, and their future health. Cholesterol is important for normal fetal development. It is provided to the fetus via both endogenous and exogenous mechanisms. As our understanding of normal and abnormal lipid metabolism in pregnancy improves, it is clear that abnormal lipid metabolism reflected as dyslipidemia is associated with adverse perinatal outcomes. Dyslipidemia has profound associations with other pathologies in pregnancy, most notably hypertensive disorders and gestational diabetes. There is accumulating evidence of the impact of hyperlipidemia in pregnancy on the epigenetic programming of a fetus and the subsequent risk for atherogenesis for the mother and her offspring.

CHOLESTEROL AND OTHER LIPIDS IN FETAL DEVELOPMENT

Cholesterol plays a key role in the formation of cell membranes. It is essential for the formation of cell membranes, maintaining membrane integrity, and preserving cholesterol-rich domains essential for most membrane-associated signaling cascades, including sonic hedgehog signaling (1). It is also the precursor for many important hormones, such as steroids, vitamin D, and bile acids.

There are multiple sources of fetal cholesterol. A significant portion is produced de novo by the fetus. Defects affecting cholesterol biosynthesis are associated with many, sometimes lethal, birth defects (2,3). Both endogenous and exogenous sources are important to fetal cholesterol homeostasis, as illustrated by a number of lines of evidence. Cholesterol in the maternal circulation, which similarly has endogenous and exogenous sources, contributes significantly to the fetal cholesterol pool in animals and in humans (4,5). Interestingly, Vuorio and colleagues noted that concentrations of plant stanols in the cord blood of healthy newborns were 40% to 50% lower than the maternal levels (6). Because the plant stanols evaluated can only be derived from the maternal diet, placental transfer is illustrated. Fetuses with null-null mutations of 7-dehydrocholesterol reductase (Smith-Lemli-Opitz syndrome), a disorder characterized by an inability to synthesize endogenous cholesterol at normal rates, have measurable amounts of cholesterol in their bodies. This also illustrates maternal derivation (7,8). The umbilical vein, which carries blood to the fetus, has higher levels of LDL-C than the umbilical artery (9).

For exogenous cholesterol to be available for fetal use, it must be transported across the tissues separating the mother and fetus. Early in pregnancy, the yolk sac is the site of the transport system between the two (10). Approaching 8 weeks of gestation, the placenta becomes fully functional and takes over as the nutrient transporter (10). The transfer of lipids across the placenta and yolk sac under normal and abnormal circumstances is complex and is still incompletely understood. Cholesterol is taken up on trophoblasts’ apical or maternal side via receptor-mediated and receptor-independent transport processes. Apolipoprotein lipids are then transported across cellular barriers and delivered into the fetal circulation on the basolateral, or fetal, side of trophoblasts (4,10,11). Cultured trophoblast cells express low-density lipoprotein (LDL) receptors (LDLRs), and LDLR-cholesterol taken up by endothelial cells is well understood. How placental endothelial cells transport and deliver substantial amounts of cholesterol to the fetal microcirculation and regulate the efflux of cholesterol is undergoing intense study.

As opposed to adults, high-density lipoprotein (HDL) is the main cholesterol-carrying lipoprotein in fetal circulation. It differs from adult HDL by its higher proportion of apolipoprotein (Apo) E (12), but lower proportion of Apo A1 (13). The major HDL receptor, scavenger receptor class B type I (SR-BI), contributes to local cholesterol homeostasis. Arterial endothelial cells (ECA) from the human placenta are enriched with cholesterol compared to venous endothelial cells (ECV). Moreover, umbilical venous and arterial plasma cholesterol levels differ markedly. There is elevated SR-BI expression and protein abundance in endothelial cell arteries compared to veins in situ and in vitro. Immunohistochemistry demonstrated that SR-BI is mainly expressed on the apical side of placental endothelial cells in situ, allowing interaction with mature HDL circulating in the fetal blood (14). This was functionally linked to a higher increase of selective cholesterol ester uptake from fetal HDL in endothelial arteries than in endothelial veins and resulted in increased cholesterol availability in ECA. SR-BI expression on endothelial veins tended to decrease with shear stress, which, together with heterogeneous immunostaining, suggests that SR-BI expression is locally regulated in the placental vasculature (15).

Changes in maternal vasculature enable an increased uterine blood flow, placental nutrient, and oxygen exchange, and subsequent fetal development. Potassium (K+) channels seem to be important modulators of vascular function, promoting vasodilation, inducing cell proliferation, and regulating cell signaling (16). Different types of K(+) channels, such as Ca(2+)-activated, ATP-sensitive, and voltage-gated, have been implicated in the adaptation of maternal vasculature during pregnancy. Conversely, K(+) channel dysfunction has been associated with vascular-related complications of pregnancy, including intrauterine growth restriction and pre-eclampsia. It is thought that vascular ischemia may lead to inflammation important in pregnancy complications. Abnormalities in these pregnancy-associated vascular dilation and remodeling processes are associated with the pregnancy complications of intrauterine growth restriction (IUGR) and pre-eclampsia, in which, the normal vasodilatory effects of acetylcholine (ACh), bradykinin, Nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF), and thromboxane-mediated responses are impaired (16).

Placental lipid metabolism may influence pregnancy outcomes, fetal growth, development, and life-long health (17). The placenta converts circulating maternal lipids to free fatty acids (FFAs) for uptake and processing by trophoblast cells, for metabolic demands, to produce hormones for pregnancy maintenance, and to transfer them to the developing fetus. Robust lipid uptake and metabolism early in gestation are vital to meeting the high energetic demands needed to simultaneously grow the placenta and develop embryonic organ systems. Late in gestation the human fetus requires lipids for neurodevelopment and growth, so as pregnancy progresses, metabolic adaptations in the mother and placenta uniquely support increasing lipid transport and biomagnification of essential long-chain polyunsaturated fatty acids in the last trimester (18). These fatty acids serve as local mediators of metabolism, inflammation, immune function, platelet aggregation, signal transduction, neurotransmission, and neurogenesis for the developing fetal brain and retina (19). Because these fatty acids cannot be synthesized de novo, the fetus relies on increasing placental transport, especially during the last trimester when the peak in utero accretion can surpass maternal intake to support rapid fetal brain growth. Placental lipid uptake and metabolism is a critical, highly-regulated, and surprisingly dynamic process as gestation proceeds.

Lipids are crucial structural and bioactive components that sustain embryo, fetal, and placental development, and growth. Intrauterine development can be disturbed by several diseases that impair maternal lipid homeostasis and lead to abnormal lipid concentrations in fetal circulation. Deficiency in essential fatty acids can lead to congenital malformations and visual and cognitive problems in the newborn. Either deficient mother-to-fetus lipid transfer or abnormal maternal-fetal lipid metabolism can cause fetal growth restriction. On the other hand, excessive mother-to-fetus fatty acid transfer can induce fetal overgrowth and lipid overaccumulation in different fetal organs and tissues. The placenta plays a fundamental role in the transfer of lipid moieties to the fetal compartment and is affected by maternal diseases associated with impaired lipid homeostasis. Studies investigating the relationship between gestational dyslipidemia and small for gestational age (SGA) have reported differing results. A recent meta-analysis found that gestations complicated with lower concentrations of TC, TG, and LDL-C, were at significantly higher risk of delivery of SGA (20). Postnatal consequences may be evident in the neonatal period or later in life. Indeed, both defects and excess of different lipid species can lead to the intrauterine programming of metabolic and cardiovascular diseases in the offspring (21)

NORMAL LIPID CHANGES IN A PREGNANT MOTHER

Figure 1 shows the average values of total cholesterol, triglycerides, low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) measured in normal women from pre-conception through several months postpartum. These values were from measurements in a cohort of women proceeding through normal pregnancy and delivery in the U.S. Circulating levels may differ depending upon the nutritional environment. It is reported that most lipoprotein concentrations increase throughout pregnancy in Gambian women for example yet are lower vs. U.S. women, the exception being medium-sized LDL and HDL particle concentrations which decrease during gestation and are similar in both cohorts (22). In a careful evaluation of normal pregnant women in Oklahoma traversing through pregnancy and delivering normal viable infants, we found that non-HDL particles, very small highly atherogenic LDL particles (small dense LDL), and total and active pcsk9 levels increase as pregnancies progress (23). BMI, an indicator of obesity, was associated with higher levels of atherogenic lipoproteins during each trimester. Most of the women in the Wiznitzer and Wild cohorts were of young reproductive age at the time of sampling and thus their values overlap values before pregnancy and are considered in the normal range for a nonpregnant woman. In the first trimester, there is a discernible decrease in levels during the first 6 weeks of pregnancy. As pregnancy progresses, there is a noticeable increase by the third month or at the end of the first trimester. There is a steady increase throughout pregnancy. By the third trimester or near the end of pregnancy (term), levels peak (24,25). Levels of lipoprotein particles and lipids, particularly in the later part of pregnancy, are in the atherogenic range when compared to non-pregnant levels in women of comparable ages without medical conditions. After delivery, lipid and lipoprotein levels rapidly return to normal. The changes in lipid metabolism throughout pregnancy allow for proper nutrients for the fetus and the normal, steady increase throughout pregnancy is associated with increased insulin resistance in the mother. Regardless of dietary differences in cholesterol, by late pregnancy, plasma cholesterol levels are approximately 50% higher than routinely seen pre-pregnancy while triglyceride levels are increased 2-3 times (see figure 1 and table 1) (25). These changes can be viewed as important for the enhanced availability of substrates for the fetus (26,27). However, derangements in lipids are associated with adverse pregnancy outcomes and likely are associated with residual vascular damage after some key obstetrical adverse events which may put the mother at risk for later cardiovascular disease.

Figure 1. Lipid and Lipoprotein Levels During Pregnancy (Adapted from Wiznitzer A, Mayer A, Novack V, et al. Association of lipid levels during gestation with preeclampsia and gestational diabetes mellitus: a population-based study. Am J Obstet Gynecol 2009; 201(5):482.e1–8;)

For understanding clinical management including healthy targets, maximum plasma cholesterol values usually do not exceed 250 mg/dL during normal pregnancy, even with the marked increases in triglyceride levels that occur normally as pregnancy progresses. If abnormal pregnancies are included in cross-sectional evaluations, cholesterol levels are commonly 300 mg/dL or higher. Higher levels are consistent with a variety of maternal adverse pregnancy conditions. In normal pregnant women, the atherogenic index, LDL/HDL, remains essentially unchanged during pregnancy. This suggests that while the total lipoprotein levels increase, the cholesterol-containing lipoprotein fractions are evenly distributed (28). Physiological hyperlipidemia/hypertriglyceridemia is distinguished from pathological dyslipidemias by a paralleled increase in HDL-C in normal women as they progress through pregnancy. During pregnancy LDL and HDL are enriched in triglycerides (29). There is an increase in large HDL late in gestation and a decrease in medium HDL (30).

Table 1. Increase in Lipid, Lipoprotein, and Apolipoprotein Levels (from (25))
Triglycerides	2.7-fold increase
Total Cholesterol	43% increase
LDL Cholesterol	36% increase
HDL Cholesterol	25% increase
Lipoprotein (a)	190%*
Apolipoprotein B	56% increase
Apolipoprotein AI	32% increase

All increases are from 3rd trimester except the increase in HDL cholesterol

*from reference (31).

Small dense LDL levels increase during pregnancy, particularly in individuals who have a large increase in triglyceride levels (32,33). As one would expect given the increase in triglycerides, LDL-C, and HDL-C, apolipoprotein B, and A-I levels are also increased (table 1). In most cross-sectional studies Lp(a) levels are not elevated in pregnancy (33-35). However, in some studies that measured Lp(a) serially throughout pregnancy an increase in Lp(a) levels was observed near term (table 1) (31,36,37) (23). The failure of cross-sectional studies to find a difference in Lp(a) levels is likely due to the wide variation in Lp(a) levels between individuals. Values in individuals range from 1mg/dL to over 200mg/dL and are largely determined by genetic factors.

MECHANISMS ACCOUNTING FOR THE CHANGES IN LIPIDS DURING PREGNANCY

Multiple physiological changes occur during pregnancy (27,38). Hormonal and metabolic changes that occur in the mother contribute to changes in the lipid profile in healthy, gestating women. It is useful to think of two phases of lipid metabolism in normal pregnancy. During the first two trimesters, lipid metabolism is 'primarily anabolic. There is an increase in lipid synthesis and fat storage in preparation for the exponential increases in fetal energy needs in late pregnancy. This increase in lipid synthesis between 10 and 30 weeks of pregnancy is promoted by maternal hyperphagia in early pregnancy as well as an increase in insulin sensitivity. The increase in insulin sensitivity stimulates fatty acid synthesis in adipocytes and stimulates the expression of lipoprotein lipase, which results in increased uptake of fatty acids from circulating triglyceride-rich lipoproteins. Additionally, the increased production of progesterone, cortisol, leptin, and prolactin contributes to increased fat storage (24,26,27,38). There is also significant hypertrophy of the adipocytes to accommodate increased fat storage (26,27,38).

Lipid metabolism in the third trimester is in a ‘net catabolic phase’, associated with a decrease in insulin sensitivity (i.e., insulin resistance) (27,38). This decrease in insulin sensitivity is associated with enhanced lipolysis of stored triglycerides in adipocytes. The third-trimester elevation of human placental lactogen (HPL) also stimulates lipolysis in adipocytes. In addition, insulin resistance results in a decrease in lipoprotein lipase in adipocytes leading to a decrease in the uptake of fatty acids from plasma triglyceride-rich lipoproteins. These changes result in a reduction in fat stored in adipocytes.

The hypertriglyceridemia during pregnancy is due to both the increased production and the decreased clearance of triglyceride-rich lipoproteins (27). The increased production of triglyceride-rich lipoproteins by the liver is due to the increased lipolysis of triglycerides that occurs in adipocytes, which increases free fatty acids transported to the liver. These free fatty acids are then packaged into VLDL and secreted by the liver. The high estrogen levels in the third trimester stimulate liver lipogenesis and VLDL production. Insulin resistance may also play a role in the increase in fatty acid synthesis in the liver as inhibition of glucose production can be resistant to insulin while lipogenesis is not. The increase in insulin levels that occur can stimulate hepatic fatty acid synthesis as shown in a mouse model (39).The decrease in clearance of triglyceride-rich lipoproteins is due to a decrease in lipoprotein lipase and hepatic lipase (29). The decrease in hepatic lipase is due to elevated estrogen levels (40). The decrease in lipoprotein lipase is believed to be due to a combination of factors including insulin resistance and elevated estrogen levels. The triglyceride enrichment of LDL and HDL is due to an increase in CETP activity (29) resulting in the transfer of triglyceride from VLDL to LDL and HDL and a decrease in hepatic lipase, which decreases the removal of triglycerides from these lipoprotein particles.

At term, LPL activity increases in the mammary glands, which will enhance the uptake of fatty acids to increase the formation of triglycerides for lactation (26). The increase in plasma cholesterol levels is likely due to increased hepatic cholesterol synthesis (41,42). The increase in PCSK9 during pregnancy suggests an additional mechanism. The PCSK9 could result in a decrease in hepatic LDL receptors leading to increased LDL-C levels (23).

Table 2. Role of Hormones in Inducing Hyperlipidemia in the Third Trimester
Estrogen increase	Inhibits Hepatic Lipase
	Stimulates VLDL production
	Stimulates lipogenesis in the liver
Human Placental Lactogen increase	Induces insulin resistance
	Increases lipolysis
Insulin Resistance	Decreases LPL activity
	Increases lipolysis
	Increase CETP
	Stimulates lipogenesis in the liver

IMPLICATIONS FOR THE FETUS AND MOTHER

Our understanding and appreciations of the full scope and implications of dyslipidemia in pregnancy on both maternal and fetal outcomes are not complete. Maternal dyslipidemia, particularly, high triglyceride and low HDL-C levels, are associated with several adverse perinatal outcomes. To demonstrate that lipid abnormalities are causative, intervention studies to lower lipid levels demonstrating a reduction in adverse perinatal outcomes are needed. This is difficult because the study of pregnant women is protected, underfunded, and understudied, in part because of medical legal concerns and the first rule of do no harm.

Dyslipidemia, while asymptomatic, is an integral factor in the metabolic syndrome (MetS). Having the MetS has clear implications for maternal vascular health, and this portends a multitude of other health concerns for the mom and her fetus.

Gestational Diabetes

Pregnancy is an insulin resistance state and gestational diabetes (GDM) is thought to be unmasked due to the stress test of insulin resistance of pregnancy. Risks to the fetus from GDM include brachial plexus injuries, hypoglycemia, respiratory distress, hyperbilirubinemia, and cardiomyopathy. Women with GDM are at increased risk of pre-eclampsia and after pregnancy, a very high risk of developing overt diabetes.

A meta-analysis of thirteen cohorts and three nested case‐control studies found that high triglycerides early in pregnancy were associated with an increased risk of the development of GDM (43). Similarly, increasing triglycerides during pregnancy was also associated with an increased risk of developing GDM (43). An HDL‐C of <51mg/dL was associated with higher odds of GDM (43). Another meta-analysis similarly found a link between GDM and high triglyceride levels and low HDL-C levels (44). Total cholesterol levels and LDL-C levels during pregnancy were not associated with an increased risk of GDM (44).

In women with a history of GDM, triglyceride, total cholesterol, and LDL-C levels are increased and HDL-C levels are decreased signaling the need to closely follow lipid levels in women with a history of GDM (45). Additionally, women with GDM have an increased risk of developing cardiovascular events later in life even in the absence of developing diabetes (46)

Several high-risk groups may have derangements in lipid levels that put them at risk before pregnancy. In the PPCOS II study, conducted by the Reproductive Medicine Network, having the metabolic syndrome before ovulation induction for fertility enhancement was associated with a lower rate of live birth success, independent of obesity, and it was also a risk factor for pregnancy complications, in particular gestational diabetes and macrosomia (47). Efforts to reduce weight before fertility treatments have been challenging. In an obese population with unexplained infertility, although weight loss was not associated with an improvement in healthy live birth success, it was associated with a reduction in the risk of preeclampsia (48)

High Birth Weight

A review of 46 publications with 31,402 pregnancies reported that maternal high triglycerides and low HDL-C levels during pregnancy were associated with increased birthweight, a higher risk of large-for-gestational-age, macrosomia, and a lower risk of small-for-gestational-age (49). Another meta-analysis also found a link between high triglycerides and low HDL-C levels and large birthweight (50). Elevations in triglyceride levels in the first trimester are associated with increased birth weight (51). The concentration of triglycerides in the third trimester is a stronger predictor of birth weight than glucose parameters (52-54). Additionally, in women with a normal glucose tolerance test during pregnancy triglyceride levels are still predictive of birthweight (55). Elevated levels of maternal triglycerides predict macrosomia independently of other maternal factors, such as BMI and glucose levels (52-54). In contrast, total cholesterol and LDL-C levels were not predictive of large birthweight (50). Some studies suggest that high levels of maternal HDL-C are significantly associated with a decreased risk for macrosomia, perhaps indicating that HDL might have protective qualities (56,57).

Pre-Eclampsia

Pre-eclampsia is a rapidly progressive condition that affects 5-8% of pregnancies and is characterized by hypertension and proteinuria. Risks to the fetus with preeclampsia include poor fetal growth and sometimes devastating consequences of preterm birth, whether spontaneous or induced. These can manifest as cerebral palsy, epilepsy, small size, and even death.

In a meta-analysis of 74 studies, pre-eclampsia was associated with elevated total cholesterol, non-HDL-C, and triglyceride levels, regardless of gestational age at the time of blood sampling, and with lower levels of HDL-C in the third trimester (58). Other meta-analyses have confirmed the linkage of elevated triglyceride levels with pre-eclampsia (59,60). LDL-C levels were not associated with pre-eclampsia in one meta-analysis (58) but in another meta-analysis, it was (60). Enquobahrie et. al followed a cohort of women from early pregnancy onward and found that women who developed pre-eclampsia had significantly higher concentrations of LDL-C and triglyceride levels as early as 13 weeks of gestation compared to women who remained normotensive (61). They also found that HDL-C was 7.0% lower in pre-eclamptic women than in the control group. They noted a 3.6-fold increase in risk for pre-eclampsia in women with total cholesterol >205 mg/dL, compared to women whose total cholesterol levels were <172 mg/dL, even after adjusting for confounders. Not all recent studies have found an increase in LDL-cholesterol concentration with preeclampsia during pregnancy however (62). In a secondary analysis of the FIT-PLESE randomized controlled trial, we found that elevated highly atherogenic very small LDL particles were elevated in those persons who developed pre-eclampsia after ovulation induction (63).

In a meta-analysis comparing women with a past history of eclampsia/pre-eclampsia vs. without, there was an increase in total cholesterol (Mean Difference = 4.6 mg/dL, CI 1.5 to 7.7), LDL-C (MD = 4.6 mg/dL; 95%CI 0.2 to 8.9), and triglycerides (MD = 7.7 mg/dL, 95%CI 3.6 to 11.7) and a decrease in HDL-C (MD = -2.15 mg/dL, 95%CI -3.46 to -0.85) (64). It is now well recognized that women who have a history of having had eclampsia/pre-eclampsia have approximately twice the risk of cardiovascular disease later in life (65,66).

Preterm Birth

In a meta-analysis of three nested case-control studies and eight cohort studies of 13,025 pregnant women, women with elevated lipid levels were at increased risk of preterm birth (OR 1.68; 95% CI 1.25-2.26) (67). The increased risk was seen for elevated levels of total cholesterol (OR 1.71), triglycerides (OR 1.55), LDL-C (OR 1.19 not significant), and lower levels of HDL (OR 1.33). A study by Vrijkotte et al that was not included in the meta-analysis also found that elevated triglycerides but not elevated total cholesterol were associated with an increased risk of preterm birth (51).

Developmental Programming

Preclinical models demonstrate that interventions that reduce maternal cholesterol during pregnancy, that decrease oxidative stress associated with gestational dyslipidemia, or that enhance active immune defenses against oxidative stress in offspring protect against developmental programming (68-70). This is thought to be because of excess maternal cholesterol during pregnancy, all factors collectively provide evidence for causality. Early atherogenic processes in the human aorta begin during fetal development and are accelerated by dyslipidemia during pregnancy.Maternal hypercholesterolemia is associated with greatly accelerated atherogenesis in normocholesterolemic children, as shown by the FELIC study (71). In experimental models lacking the genetic and dietary variability of humans, postnatal atherosclerosis increases in proportion to the maternal cholesterol levels well into adult ages (69,72). A molecular mechanism explaining the transfer of maternal cholesterol to the fetus has been elucidated (73) and involvement of increased oxidative stress has been established (68-70).

The absence of routine cholesterol determinations during gestation in most countries has limited investigations of the impact of elevated maternal cholesterol during pregnancy on the clinical

manifestations of dyslipidemia in adult offspring. In the Framingham Heart Study gestational dyslipidemia in mothers was predictive of dyslipidemia in their offspring (74). Adults who had been exposed to elevated maternal LDL-C levels had 3.8 times higher odds of having elevated LDL-C levels. They found that this explained 13% of the variation in adult offspring LDL-C levels beyond common genetic variants and classic risk factors for elevated LDL-C levels. A positive association has also been reported between maternal cholesterol and newborn HDL cholesterol and subclasses (75).

From studies of 78 fetal aortas, maternal cholesterol explained 61% of the variance of early lesion sizes by multivariate analysis, independent of HDL-C, triglycerides, glucose, and body mass index (BMI). Maternal total cholesterol and LDLC levels were positively associated with methylation of SREBP2 in fetal aortas, suggesting a role of maternal cholesterol levels during pregnancy on epigenetic signature in offspring as reported by Napoli et al (71). SREBP2 methylation has been mapped (71). The long-term effects of maternal dyslipidemia on the progression of atherosclerosis and, its clinical manifestations are understudied. In several cohort studies, whole blood DNA methylation signatures of diet were associated with cardiovascular disease (76). Dyslipidemia can persist well into adult age and affect clinically relevant outcomes (72). In studies where elevated maternal cholesterol during pregnancy is associated with atherogenesis in childhood, maternal lipid levels during pregnancy have been associated with adult BMI, atherosclerosis-related risk, and the severity of anterior myocardial infarctions as reported by Cacciatore et al (72).

Pre-eclampsia (77) and gestational diabetes (46) are linked to having a greater risk for early maternal cardiovascular events.

LIPID SCREENING

The National Lipid Association (NLA) supports checking lipids routinely if there is no normal current pre-pregnancy lipid profile (78). Screening for reproductive-aged women, in general, remains deficient in part due to disparities in health services (79). Identifying pregnant women with prior atherosclerotic cardiovascular disease (ASCVD), familial hypercholesterolemia, or hypertriglyceridemia is important to allow for multi-disciplinary collaborative care. Increased knowledge and awareness are needed at both the patient and provider levels. In a survey study of 200 pregnant women within the University of Pennsylvania Health System, 59% self-reported previous lipid screening; non- Hispanic Black women were less likely to report screening (43% vs. 67%) and they had lower awareness of high cholesterol as a risk factor for ASCVD (66% vs 92%) (80). The perinatal period, when a woman sees a physician most regularly, is an opportunity to screen for lipid disorders and facilitate prevention by bringing lipid values to normal age specific target ranges. Hypertensive disorders of pregnancy are among the leading causes of maternal morbidity and mortality in the US. Pre-eclampsia, which includes hypertension and proteinuria during pregnancy, is thought to result from placental ischemia. Risk factors for pre-eclampsia parallel those for cardiovascular disease and recent studies point to hyperlipidemia, specifically hypertriglyceridemia. Current practice does not routinely include lipid testing pre-conception or during pregnancy. Professional and societal recommendations should advocate for hyperlipidemia screening, followed by appropriate management, pre-conception, and during pregnancy as an important evaluation for risk of preeclampsia during pregnancy (48).

A recent review recommended measuring lipids at the first visit and if normal at the beginning of third trimester (81). High risk patients should have lipids measured at first visit, beginning of second trimester, and monthly during the 3rd trimester. If triglyceride levels are greater than 250mg/dl at any time the lipid panel should be measured monthly. When and if more specialized lipid testing such as NMR or ion mobility to measure small dense LDL levels, apo B and apo A1 levels, and Lp(a) levels is needed is not defined and further studies are required. Measuring Lp(a) levels at the first visit is reasonable in patients who have not had their Lp(a) levels determined previously. If lipid abnormalities are noted during pregnancy follow-up lipid panels post-pregnancy should be obtained.

TREATMENT OF DYSLIPIDEMIA DURING PREGNANCY

Lifestyle Modifications

Addressing lifestyle modifications is vital in the management of any lipid disorder regardless of pregnancy status. Counseling patients regarding a heart-healthy dietary pattern that includes vegetables, fruits, whole grains, legumes, healthy protein sources, and limiting intake of sweets, sweetened beverages, and red meats along with an emphasis on weight management and exercise is essential (82). To counsel patients with elevated lipids to lower their intake of saturated fats and increase dietary fiber is important. Approaches to Stop Hypertension (DASH) diet or Mediterranean diet are well described diets that are beneficial in reducing cardiovascular risk (83). Given that women report increased motivation to enact dietary changes during pregnancy, this is an ideal time to intervene with lifestyle modifications. Diet is a critical pillar of management for hypertriglyceridemia. A very low-fat diet is recommended to mitigate the risk of pancreatitis particularly when triglyceride levels are >500 mg/dL. Severe gestational hypertriglyceridemia can lead to acute pancreatitis and the maternal mortality rate is approximately 20%.

Dietary interventions have benefits beyond LDL lowering due to their effects on the placenta. A low-cholesterol low-saturated fat diet in a trial of 290 pregnant patients led to a decrease in the umbilical artery pulsatility index, a method for fetal surveillance in high-risk pregnancies (less vascular resistance) (84). Increased vascular resistance is associated with adverse pregnancy outcomes such as preeclampsia, preterm delivery, and small for gestational age infants.

Drug Therapy

STATINS

While statins are the first-line treatment of hypercholesterolemia in the general population, their use is not recommended during pregnancy in several guidelines. The 2018 AHA/ACC/multi-society cholesterol guidelines give a class 1 recommendation that women of childbearing age with hypercholesterolemia who plan to become pregnant should stop statins 1 to 2 months before pregnancy is attempted, or if they become pregnant while on a statin, should have the statin stopped as soon as possible (85). Similarly, the European Society of Cardiology (ESC) 2019 guidelines have a class III recommendation that statin therapy is not recommended in premenopausal patients with diabetes who are considering pregnancy or are not using adequate contraception (86). Historically, statins became contraindicated in pregnancy as a result of a case series in 2004 that demonstrated an association between first-trimester statin exposure and fetal malformations. Other cohort studies of statin exposure in pregnancy have not shown an increase in teratogenic risk (87). Of all the statins, hydrophilic statins, such as pravastatin, have not been associated with anomalies (88). Meta-analyses of studies of pregnant women exposed to statins showed no increased risk of birth defects (RR 1.15) but did reveal an increased risk of miscarriage (RR 1.35) (87,89). This increased risk of miscarriage may be due to confounders such as older age and ASCVD risk factors.

In a retrospective review of 39 pregnancies including 20 patients with FH and 18 patients on statins, miscarriage rates were not higher in statin-exposed patients as compared to the healthy population; there was also no difference in birth weights between statin-exposed and not-exposed (90). Given the lack of clear evidence on the teratogenicity of statins, in July 2021 the FDA requested the removal of the strongest recommendation against using statins during pregnancy. They continue to advise against the use of statins in pregnancy given the limited data and quality of information of studies. The decision of whether to continue a statin during pregnancy requires shared decision-making between the patient and clinician, and healthcare professionals need to discuss the risks versus the benefits in high-risk women, such as those with homozygous FH or prior ASCVD events, that may benefit from statin therapy (91).

Discontinuation of statins in patients with FH allows cholesterol levels to increase even beyond pre-treatment levels due to higher physiologic levels during pregnancy. This period is a vulnerable time as interruptions of treatment can increase the lifelong risk of ASCVD. In women with familial hypercholesterolemia (FH), the percent increase in LDL-C levels during pregnancy is similar to that observed in women with a normal lipid profile before pregnancy even though the baseline LDL-C levels are much higher in women with familial hypercholesterolemia (92). Despite the markedly higher LDL-C levels in women with FH, the incidence of prematurity, low birth weight, and congenital malformations did not differ, however, maternal hypertension incidence was higher than for women without dyslipidemia before pregnancy who became pregnant (92,93).

In a retrospective review of women with homozygous familial hypercholesterolemia of which 18/39 were exposed to statins with or without ezetimibe,1 was treated with a statin, ezetimibe, and a PCSK9 inhibitor, and 5 patients were exposed to cholestyramine, and only 14 patients were not exposed to lipid-lowering therapy, complications associated with pregnancy included 3 premature infants, one preeclampsia related, the other two because of chorioamnionitis and maternal cardiac disease (90). One Intrauterine death was because of intrauterine infection.

More studies examining outcomes in pregnant women with FH are needed to assess short-term and long-term outcomes on the mother and the offspring. The FDA has taken a wait and see approach. Certain statins, such as pravastatin, are being investigated for use in the prevention of preeclampsia. Given the impact of statins on endothelial dysfunction and their potential ability to mediate pathways of inflammation and oxidative stress, statins are promising agents for preeclampsia treatment in persons at high risk for a severe disease which centers on vascular dysfunction. Pravastatin is the most hydrophilic statin with a short half-life and is also a substrate for multiple efflux transporters, which leads to lower transplacental transfer. The safety of pravastatin 10 mg (low-intensity) was evaluated in a very small 20-patient randomized controlled trial for the prevention of preeclampsia in high-risk pregnant women, in which the primary outcome was maternal-fetal safety. There were no differences between groups in rates of drug side effects, congenital anomalies, or other adverse events. In a larger trial, 1120 women at high risk of pre-eclampsia were randomized to either pravastatin 20 mg (low-intensity) or placebo. There was no significant reduction in the incidence of preeclampsia or differences in other biomarkers such as soluble fms-like tyrosine-kinase-1 in either treatment arm (94). Hirsch et all reviewed cohort studies assessing the effects of pravastatin on placental insufficiency disorders and found that pravastatin treatment prolonged pregnancy duration and improved associated obstetrical outcomes in pregnancies complicated with uteroplacental insufficiency disorders in cohort studies (95). Additional studies will help ascertain the efficacy of statins, such as pravastatin, during pregnancy for preeclampsia prevention.

BILE ACID SEQUESTRANTS

Bile acid sequestrants such as cholestyramine and colestipol can be used for LDL-C lowering during pregnancy (96). Since bile acid sequestrants are not absorbed they do not pass into the systemic circulation and are safer than other lipid-lowering agents. However, they do decrease the absorption of fat-soluble vitamins. Patients should be monitored for vitamin D and K deficiency (96). According to the 2011 NLA recommendations, Colesevelam was classified as a Class B pregnancy category medication, as there are no adequate studies in pregnant women and animal studies have failed to demonstrate a risk to the fetus (97). Thus, bile acid sequestrants are considered safe for use in treating LDL-C elevations during pregnancy and breastfeeding. However, there is a lack of controlled trial data during pregnancy. Additionally, bile acid sequestrants are well known to increase triglyceride levels. They can be associated with constipation in pregnancy.

EZETIMIBE

Ezetimibe was classified as Class C pregnancy category; Class C meant that there is a lack of adequate studies in pregnant women, but animal studies have demonstrated a risk to the fetus (78). Animal studies have found that ezetimibe crosses the placenta. At levels higher than those achieved with human doses, there appears to be a slightly increased risk of skeletal abnormalities in rats and rabbits. Therefore, this agent is not recommended for use during pregnancy. If used prior to pregnancy, ezetimibe should be discontinued prior to attempting to become pregnant (98).The 2011 NLA Familial Hypercholesterolemia guidelines state that ezetimibe should be stopped at least 4 weeks before discontinuing contraception for women with familial hypercholesterolemia who are planning on conceiving and should not be used during pregnancy and lactation (97).

PCSK9 INHIBITORS (EVOLOCUMAB, ALIROCUMAB, AND INCLISIRAN)

Evolocumab and alirocumab, have not been tested for safety during pregnancy so their role in dyslipidemia treatment in pregnancy is unclear. While older medications were labeled with pregnancy categories, such as A, B, C, D, and X, the FDA has removed these labels for all prescription medications approved after 2015 so there is no pregnancy classification for PCSK-9 inhibitors. The FDA drug package insert for evolocumab and alirocumab describe that monoclonal antibodies are unlikely to cross the placenta in the first trimester, but may cross

the placenta near term, in the second and third trimester.

Inclisiran, a small interfering RNA that targets hepatic PCSK9 synthesis, has been shown to significantly lower LDL-C levels. Given its infrequent dosing regimen, it could hypothetically be used before conception and immediately afterward though further trials and outcome data are needed (99). The product labeling states that there is no available data on its use in pregnant patients, although animal reproduction studies have shown no adverse developmental effects.

PCSK9 inhibitors are not approved for use in pregnancy nor currently recommended.

BEMPEDOIC ACID

Bempedoic acid, an inhibitor of ATP citrate lyase (an enzyme in the cholesterol synthesis pathway), is a lipid-lowering therapy shown to reduce the levels of LDL-C. Per the product labeling by the FDA, there is no available data on its use in pregnant women though animal reproduction studies did not show teratogenicity in rat and rabbit models (package insert). It is not recommended that bempedoic acid be taken during breastfeeding. They suggest discontinuing bempedoic acid when pregnancy is recognized, unless the benefits of therapy outweigh the potential risks to the fetus. Based on the mechanism of action of bempedoic acid may cause fetal harm.

EVINACUMAB

No data are available on use during pregnancy (96). Based on animal studies, exposure during pregnancy may lead to fetal harm. Evinacumab is a monoclonal antibody and human immune globulin are known to cross the placental barrier (96). Therefore, evinacumab could be transmitted from the mother to the developing fetus.

LOMITAPIDE

This drug is contraindicated during pregnancy due to the risk of fetal toxicity (formerly Class X pregnancy category) (100).

FIBRATES

While fibrates were classified as the Class C pregnancy category, they can be considered later on in pregnancy depending on the risk vs. benefit discussion. The AHA Scientific Statement for Cardiovascular Considerations in Caring for Pregnant Patients proposes the consideration of fenofibrate or gemfibrozil in the second trimester if triglycerides are >500 mg/dL despite lifestyle modifications (101). The AHA/ American College of Obstetricians and Gynecologists (ACOG) Presidential Advisory states that pregnant patients with a history of pancreatitis may benefit from the use of fenofibrate when triglyceride levels are >1000 mg/dL (66). The use of fibrates during the second trimester is after embryogenesis occurs reducing the risk. Studies in animals have found no increased risk of congenital malformations (98).

NIACIN

Niacin was classified in the Class C pregnancy category. Niacin should not be used during pregnancy and lactation.

OMEGA-3-FATTY ACIDS

Omega-3 fatty acids are widely used albeit without controlled clinical trials during pregnancy.

Studies are limited on the use of omega-3 fatty acids for dyslipidemia management during pregnancy. In one study of 341 pregnant women, omega-3 fatty acids in the form of 10 mL cod liver oil given daily until 3 months after delivery increased docosahexaenoic acid (DHA) levels in both maternal and infant plasma while also reducing maternal plasma lipid levels. Most other medications used to treat hypertriglyceridemia are not considered safe during pregnancy, but omega-3 fatty acids are considered safe as most prenatal vitamins and baby formula

contain DHA. Omega-3 fatty acids can potentially be utilized for their triglyceride-lowering effect, but evidence is only based on a small number of case reports (102). Prescription omega-3-fatty acids are not approved for use during pregnancy.

VOLANESORSEN

No data are available on use of volanesorsen during pregnancy. If used prior to pregnancy, volanesoren should be discontinued one month before attempting conception (103). This drug is approved in Europe but not in the U.S.

SUMMARY

Several recent reviews have provided information on the use of lipid lowering drugs during pregnancy and breast feeding (96,104,105). The class of evidence and levels of evidence for lipid lowering drugs are not strong for any of the options given a lack of definitive studies regarding efficacy and safety.

Plasmapheresis and Lipoprotein Apheresis

In patients with severe elevations in triglyceride levels with pancreatitis or who are at high risk for pancreatitis plasmapheresis has been employed to rapidly and safely decrease triglyceride levels (81,106). Plasmapheresis should be considered early in asymptomatic pregnant women with fasting triglyceride levels >1000 mg/dL or in pregnant women with clinical signs and symptoms of pancreatitis and triglyceride levels >500 mg/dL despite maximal lifestyle changes and pharmacologic therapy.

Lipoprotein apheresis can be safely used during pregnancy and may be beneficial for some women with severely elevated LDL-C levels (85,106,107). An expert American College of Cardiology expert committee suggests consideration of lipoprotein apheresis in pregnant patients with homozygous familial hypercholesterolemia and patients with severe heterozygous familial hypercholesterolemia and an LDL-C ≥ 300 mg/dL despite lifestyle therapy (96). In patients with familial hypercholesterolemia, ASCVD, and pregnancy, lipoprotein apheresis may be considered when the LDL-C ≥190 mg/dL.

MANAGEMENT OF PREGNANT WOMEN WITH PRE-EXISTING LIPID ABNORMALITIES

Patients with the following disorders are best managed using a team approach that includes a lipid and maternal-fetal specialist. Persons with these disorders should be provided with genetic counseling in addition to multi-specialty team management and pregnancy offers an important opportunity to address the impact in families and how to recognize, prevent, and treat these disorders.

Elevated LDL-C Levels Including Homozygous and Heterozygous Familial Hypercholesterolemia

In women with familial hypercholesterolemia the percent increase in LDL-C levels during pregnancy is similar to that observed in women with a normal lipid profile prior to pregnancy even though the baseline LDL-C levels are much higher in women with familial hypercholesterolemia (92). Despite the markedly higher LDL-C levels in women with familial hypercholesterolemia the prevalence of hypertension, duration of gestation, and fetal body weight were similar between patients with familial hypercholesterolemia and women without dyslipidemia before pregnancy (92).

Decisions to use lipid-lowering medications involve a risk-benefit decision. In patients at very high risk of a heart attack or stroke, such as individuals with homozygous familial hypercholesterolemia and those who have clinical ASCVD the benefits of therapy may outweigh the risks of therapy. If the patient and physician elect to continue or add LDL-C lowering medications it is recommended that these be used after the first trimester of pregnancy if possible. Additionally, the hydrophilic statin, pravastatin, would be a good choice if one elects to use a statin (108). If the patient is on lipoprotein apheresis this can be continued during pregnancy or initiated if available.

Familial Chylomicronemia Syndrome (TG> 500mg/dL)

This is managed primarily with a very low-fat diet (<20 g total fat/d or <15% total calories) that requires consultation with an expert in nutritional advice to ensure adequate caloric intake during pregnancy and sufficient vitamins. Medium-chain triglycerides can help provide calories and make the very low-fat diet tolerable. In the third trimester as triglyceride levels increase hospitalization with parenteral feeding has been employed. In patients with familial chylomicronemia syndrome drug therapy often is not beneficial but one can consider omega-3 fatty acids in high doses. In patients with episodes of pancreatitis or with very high triglyceride levels at high risk for pancreatitis plasmapheresis can be beneficial.

Multifactorial Chylomicronemia Syndrome (TG> 500mg/dL)

This disorder is typically due to a genetic predisposition to high triglyceride levels combined with secondary factors that increase triglyceride levels into the range that causes pancreatitis. Therefore, one should try to control secondary disorders (Table 3) and if possible, stop drugs that increase triglyceride levels (Table 4). Additionally, a low-fat diet, avoidance of simple sugars and alcohol, and exercise can be helpful.

Table 3. Disorders Associated with an Increase in Triglyceride Levels

Obesity

Alcohol intake

High simple carbohydrate diet

Diabetes

Metabolic syndrome

Polycystic ovary syndrome

Hypothyroidism

Chronic renal failure

Nephrotic syndrome

Inflammatory diseases (Rheumatoid arthritis, Lupus, psoriasis, etc.)

Infections

Acute stress (myocardial infarctions, burns, etc.)

HIV

Cushing’s syndrome

Growth hormone deficiency

Lipodystrophy

Glycogen Storage disease

Acute hepatitis

Monoclonal gammopathy

Table 4. Drugs That Increase Triglyceride Levels

Alcohol

Oral Estrogens

Tamoxifen/Raloxifene

Glucocorticoids

Retinoids

Beta-blockers

Thiazide diuretics

Loop diuretics

Protease Inhibitors

Cyclosporine, sirolimus, and tacrolimus

Atypical antipsychotics

Bile acid sequestrants

L-asparaginase

Androgen deprivation therapy

Cyclophosphamide

Alpha-interferon

Propofol

If these are not successful in keeping triglyceride levels in a safe range treatment with omega-3- acids and the addition of fenofibrate later in pregnancy is indicated. If despite therapy there is an episode of pancreatitis due to poorly controlled triglyceride levels or very high triglyceride levels at high risk for pancreatitis plasmapheresis can be employed.

Patients with Moderate Hypertriglyceridemia (TG 200-500mg/dL)

These patients should be treated the same as patients with the multifactorial chylomicronemia syndrome described above. Diet therapy, treatment of secondary disorders, and stopping, if possible, drugs that increase triglyceride levels can frequently prevent an increase in triglycerides into the range that causes pancreatitis. If this approach is not successful treatment with omega-3- acids and the addition of fenofibrate later in pregnancy is indicated if the triglyceride levels are greater than 500mg/dL.

Patients with Very High Lp(a) Levels

High Lp(a) may promote atherosclerosis as well as thrombosis by affecting fibrinolysis, inflammation, endothelial function, macrophage lipid uptake, and oxidative stress. Lipoprotein(a) level correlates with the severity of preeclampsia and Lp(a) may be involved in the pathogenesis of preeclampsia (109). Further studies are required to determine if Lp(a) has a detrimental role during pregnancy and whether therapies that lower Lp(a) levels are beneficial. To prevent pre-eclampsia aspirin therapy is recommended and can be considered for persons with high Lp(a).

Patients with Monogenic Disorders Causing Hypobetalipoproteinemia (i.e., Low LDL-C Levels)

There are a number of causes of low LDL-C including secondary factors such as a strict vegan diet, malnutrition, malabsorption, hyperthyroidism, malignancy, and chronic liver disease, polygenic disorders due to small effect variants in a number of genes, and several monogenic disorders (110). Amongst the monogenic disorders only the most severe are associated with pregnancy issues including bi-allelic FHBL-SD1 due to mutations in microsomal triglyceride transfer protein (abetalipoproteinemia), bi-allelic FHBL-SD2 due to mutations in Apo B, and bi-allelic FHBL-SD3 due to mutations in SAR1B (chylomicron retention disorder) (111). These disorders are characterized by very low LDL-C levels and malabsorption (111). The dietary treatment of these individuals is summarized in table 5 (111). Other monogenic disorders causing low LDL-C levels, such as mono-allelic FHBL-SD2 due to mutations in Apo B (familial hypobetalipoproteinemia), bi-allelic FHBL-EC1 due to mutations in ANGPTL3 (combined hypolipidemia), and bi-allelic FHBL-EC2 due to mutations in PCSK9 do not cause major issues during pregnancy because they do not lead to malabsorption.

Table 5. Treatment of Patients with Severe Monogenic Hypobetalipoproteinemia (Low LDL-C)

Low-fat diet- Less than 10-15% (<15 g/day) of total daily calories. Can be adjusted depending upon the tolerance

Essential fatty acids- 2-4% daily caloric intake (alpha-linolenic acid/linoleic acid)

DHA and EPA- Supplementation may be considered depending on the diet

Vitamin E- 100-300 IU/kg/day

Vitamin A- 100-400 IU/kg/day

Vitamin D- 800-1200 IU/day

Vitamin K- 5-35 mg/week

Dosing of vitamins A, D, and K can be tailored to plasma vitamin A/β-carotene levels, 25-hydroxy vitamin D levels, and INR reference intervals.

Preconception and pregnancy medical counseling, including genetic counseling, should be provided. Because patients with these disorders are very uncommon ideally these patients should be cared for in specialty clinics. During pregnancy, one must balance the need to limit fat intake and the need to increase caloric intake. Consultation with a dietician is important. Supplementation with medium-chain triglycerides can be used to increase caloric intake if needed (111). Because maternal serum DHA concentrations have been linked to neurocognitive and anti-inflammatory benefits supplementation with DHA (1-3 g/day) can be utilized to increase plasma concentrations (111). Additionally, to prevent neural tube defects in pregnancy, a daily supplement of 400-800 µg folic acid is also recommended. The dosing of vitamins A, D, and K should be adjusted as indicated by plasma vitamin A/β-carotene levels, 25-hydroxy vitamin D levels, and INR reference intervals (111). In normal individuals, excess vitamin A can cause toxicity during pregnancy and therefore it is recommended to set the vitamin A goal at the lower limit of normal levels, which may decrease the dose by 50% during pregnancy (111). Vitamin E supplements should be continued during pregnancy as vitamin E deficiency has been shown to increase miscarriages (111)

Progesterone production during pregnancy may be reduced during pregnancy and it is recommended to monitor progesterone levels throughout pregnancy and consider the use of exogenous progesterone if levels are low (111).

CONSIDERATIONS FOR BREASTFEEDING

The postpartum period is a critical time requiring proactive counseling and shared decision-making regarding plans for lactation. Enabling women to breastfeed is a public health priority because, on a population level, interruption of lactation is associated with adverse health outcomes for the woman and her child, including higher maternal risks of breast cancer, ovarian cancer, diabetes, hypertension, and heart disease, and greater infant risks of infectious disease, sudden infant death syndrome, and metabolic disease. Contraindications to breastfeeding are few. Most medications and vaccinations are safe for use during breastfeeding, with few exceptions. Breastfeeding confers medical, economic, societal, and environmental advantages; however, each woman is uniquely qualified to make an informed decision surrounding infant feeding.

Risks vs. benefits need to be considered to determine the optimal management of lipid disorders, as all lipid-lowering medications are contraindicated during pregnancy and remain contraindicated during breastfeeding. Breastfeeding has enormous benefits and is encouraged. Medications taken by the mother can transfer into breast milk through passive diffusion or active transport by membrane proteins and therefore expose the baby to the medication. Therefore, continuing breastfeeding given its benefits in metabolic health needs to be balanced with stopping breastfeeding to resume lipid-lowering therapies and the progression of atherosclerosis (112). Shared decision-making and discussions of risks and benefits of both time without statin treatment and breastfeeding vs. treatment during pregnancy and lactation are best initiated before delivery and every visit in the fourth-trimester period (after delivery) (113,114). The LactMed database (National Institute of Child Health and Development) contains information on drugs and other chemicals to which breastfeeding mothers may be exposed. It includes information on the levels of such substances in breast milk and infant blood and the possible adverse effects in the nursing infant. Suggested therapeutic alternatives to those drugs are provided, where appropriate. All data are derived from the scientific literature and fully referenced. A peer review panel reviews the data to assure scientific validity and currency. The consensus opinion is that women taking a statin should not breastfeed because of a concern with disruption of infant lipid metabolism. However, others have argued that children homozygous for familial hypercholesterolemia are treated with statins beginning at 1 year of age, that statins have low oral bioavailability, and that risks to the breastfed infant are low, especially with pravastatin and rosuvastatin.

In a recent systematic review of 33 articles from 15 randomized controlled trials limited evidence suggests that omega-3 fatty acid supplementation during pregnancy may result in favorable cognitive development in the child. There was insufficient evidence to evaluate the effects of omega-3 fatty acid supplementation during pregnancy and/or lactation on other developmental outcomes (115).

CONCLUSION

There is an increase in lipid levels in normal gestation. Dyslipidemia in pregnancy beyond physiologic levels is associated with adverse pregnancy outcomes. Adverse pregnancy events enhance the risk of clinical ASCVD events in later life. Screening for and adequately addressing atherogenic dyslipidemia before and during pregnancy is a priority. Major barriers to the management of hyperlipidemia during pregnancy and the postpartum period include limited studies in pregnant patients. Many therapeutic agents are categorized as contraindicated without adequate evidence. Future research is needed to allow for evidence-based decisions to guide therapeutic options. Pregnancy is a unique opportunity for multidisciplinary and collaborative care across various specialties to improve rates of screening and optimize the long-term cardiovascular health of women. Many women are overwhelmed post-partum. They need to be encouraged to prioritize ASCVD risk reduction as integral to their care. Improving access to quality preventive care is still in need of improvement.

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