ABSTRACT

X-linked adrenoleukodystrophy (X-ALD) is a rare inherited neurodegenerative disorder, involving mainly the white matter and axons of the central nervous system and the adrenal cortex and is a frequent but under-recognized cause of primary adrenocortical insufficiency. X-ALD is caused by a defect in the gene ABCD1 that maps to Xq 28 locus. The primary biochemical disorder is the accumulation of saturated very long chain fatty acids (VLCFA) secondary to peroxisomal dysfunction. The incidence in males is estimated to be 1:14,700 live births, without any difference among different ethnicities. X-ALD presents with a variable clinical spectrum that includes primary adrenal insufficiency, myelopathy, and cerebral ALD; however, there is no correlation between X-ALD phenotype and specific mutations in the ABCD1 gene. When suspected, the diagnosis is established biochemically with the gold standard for diagnosis being genetic testing (ABCD1 analysis). Currently, there is no satisfying treatment to prevent the onset or modify the progression of the neurologic or endocrine components of the disease. Allogeneic hematopoietic stem cell (HSC) transplantation is the treatment of choice for individuals with early stages of the cerebral form of the disease. An alternative option for patients without HLA-matched donors is autologous HSC-gene therapy with lentivirally corrected cells. Once adrenal insufficiency is present, hormonal replacement therapy is identical to that of autoimmune Addison’s disease.

INTRODUCTION

Leukodystrophies are inherited neurodegenerative disorders, primarily affecting the brain myelin. X-linked adrenoleukodystrophy (X-ALD; OMIM:300100) is the most common leukodystrophy usually presenting as chronic myelopathy and peripheral neuropathy, a clinical entity called adrenomyeloneuropathy (AMN), frequently accompanied by adrenocortical insufficiency (1). The pattern of inheritance is X- linked and the disease is clinically evident in almost all male patients and in more than 80% of female carriers older than 60 years, though with milder clinical presentation. Occasionally, male patients and very rarely female carriers may develop a rapidly progressive, devastating cerebral form of the disease known as Cerebral Adrenoleukodystrophy (CALD). The pathophysiological basis of the disease is peroxisome dysfunction and accumulation of very long chain fatty acids (VLCFA) due to impaired VLCFA degradation (2).

In the early 20th century, patients with signs and symptoms belonging to the Leukodystrophies spectrum were grouped under the name “Addison–Schilder disease”. It was not until the 1960s that Blaw introduced the term “adrenoleukodystrophy” as a distinct disease entity with X-linked inheritance (3). In 1976 it was shown that the principal biochemical disorder in X-ALD was the accumulation of VLCFA (4). In 1993, the gene responsible for the disease was identified at the Xq28 locus and it was subsequently shown to be the ABCD1 gene, which encodes the Adrenoleukodystrophy Protein (ALDP) (5).

This chapter summarizes the latest data in the literature regarding the progress made in elucidating the pathogenesis of the disease, the strategies for early diagnosis, and the results of established as well as newer experimental therapies.

GENETICS & PATHOPHYSIOLOGY

ALD is a rare progressive neurodegenerative disorder with an annual incidence of 1:14,700 live births (considering both hemizygous males and heterozygous females), and no marked difference between males and females (6).  

X-ALD is caused by mutations in the ABCD1 gene located on the X chromosome (Xq28), which covers 19.9 kb and contains 10 exons (7) with approximately 900 different mutations reported (8). Mutations in the ABCD1 gene include missense, nonsense, frameshift, and splice-site variants (9). However, identical variants can result in highly diverse clinical phenotypes, suggesting the presence of unknown additional factors that have an impact on the expression of the disease (2). Thus, there is a lack of a genotype-phenotype correlation in ALD (10, 11).

The ABCD1 gene encodes a peroxisomal trans-membrane protein of 745 amino acids, ALDP, a member of the ATP binding cassette (ABC) transport protein family, which helps to form the channel through which VLCFAs move into the peroxisome as VLCFA-CoA (12). ALDP deficiency leads to an impaired peroxisomal β-‑oxidation of saturated straight-chain very long-chain fatty acids (VLCFA) (13) resulting in the accumulation of VLCFAs in plasma and tissues, including the white matter of the brain, spinal cord, and adrenal cortex (14). Chronic accumulation of cholesterol with saturated VLCFA in the zona fasciculata and reticularis of the adrenal cortex is believed to result in cytotoxic effects, apoptosis and ultimately atrophy of the adrenal cortex and with loss of cortisol production (15, 16). The pathogenesis of X-ALD is summarized in Figure 1.

Figure 1. The pathogenesis of ALD. Adapted with permission from www.adrenoleukodystrophy.info.

Figure 2. Adapted with permission from www.adrenoleukodystrophy.info.

Significant intra-familiar phenotype variability has been observed as different clinical phenotypes can occur even among monozygotic twins (17). Fifty percent of ABCD1 mutations lead to a truncated ALDP, whereas many missense mutations result in the formation of an unstable protein (18). The complete absence of a functional ALDP does not necessarily lead to the severe form of X-ALD, implicating the existence of additional factors that could modify the disease’s clinical expression. Factors, such as moderate head trauma, have been shown to trigger the progression of the disease to the severe central nervous system (CNS) form (19), but other unknown genetic and environmental factors are likely required for the development of CALD. In contrast, mutations with residual transporter activity or over-expression of ALDP-related protein (ALDRP, ABCD2), the closest homolog of ALDP, might prevent this progression (20). Variations in methionine metabolism have also been associated with the wide phenotypic spectrum of X-ALD (21).

CLINICAL MANIFESTATIONS OF X-ALD

The range of clinical expression of X-ALD varies widely. The main phenotypes of X-ALD are primary adrenal insufficiency (Addison’s disease), myelopathy, and cerebral ALD (CALD), either alone or in any combination.

The most devastating form of ALD is CALD which presents early in life between 4-12 years of age, affecting 1/3 of boys with X-ALD and is rare after 15 years of age (22). It is characterized by inflammatory demyelination mainly of the supratentorial and infratentorial white matter and brain magnetic resonance imaging (MRI) findings usually precede clinical symptomatology (23). The onset of CALD is insidious, with symptoms at school age such as learning, behavioral, and cognitive disabilities often being attributed to Attention Deficit/Hyperactivity Disorder that delay the diagnosis. As the disease progresses, overt neurologic deficits become apparent, including cortical blindness, central deafness, hemiplegia, and quadriparesis. Progression of the disease is often rapid, leading to death within 5 – 10 years following diagnosis (24). Most men who do not develop CALD during childhood develop myelopathy in adult life.

Myelopathy manifests later in life, typically presents in adult males between 20 and 40 years of age, with a median age at onset of 28 years (25). The primary clinical presentation is spinal cord and peripheral nerve dysfunction, leading to progressive spastic paraparesis, abnormal sphincter control, sensory ataxia, and sexual dysfunction. Symptoms are progressive over years or decades, with most patients losing unassisted functionality by the 5^th – 6^th decade of life. Brain MRI is usually normal but spinal cord atrophy can be detected by conventional T2-weighted MRI sequences. Although myelopathy is a milder form of ALD, cerebral involvement can occur in 27% to 63% of patients (26). Cerebral involvement leads to rapid neurologic deterioration with disabilities and early death in 10% to 20% of adult males (26). Adrenal insufficiency is often present at the time of myelopathy diagnosis, a clinical entity called adrenomyeloneuropathy (AMN).

Incidence Of Primary Adrenal Deficiency In X-ALD

The natural history of adrenal insufficiency in ALD is largely unknown because large prospective natural history studies are lacking. However, the loss of adrenal function evolves gradually and initially starts with elevated plasma corticotropin hormone (ACTH) levels before overt adrenal insufficiency with an abnormal cortisol response after cosyntropin stimulation and endocrine symptoms become apparent (10, 27). The average time to adrenal insufficiency or time from initial plasma ACTH elevation to the onset of endocrine symptoms is unknown (27, 28).

The estimated lifetime prevalence of adrenal insufficiency in ALD is considered to be approximately 80% (27, 29, 30). Addison’s disease is reported to be the initial clinical manifestation of ALD in 38% of cases, representing the most common presenting symptom of ALD in childhood (10, 29). ALD has been reported to account for 4% to 35% of cases of idiopathic primary adrenal insufficiency with no detectable steroid-21-hydroxylase antibodies or other obvious cause (31, 32, 33).

Therefore, all boys must be tested for ALD upon diagnosis of adrenal insufficiency if the cause is otherwise not clear. The risk for adrenal insufficiency varies throughout the lifetime and peaks during the first decade of life between 3 and 10 years of age (27, 29). The youngest patients suffering from adrenal insufficiency and ALD have been reported to be as young as 3, 5 and 7 months of age (27, 29, 34). it has therefore been recommended to start adrenal testing in the first six months of life (29).

In a large natural history study of adrenal insufficiency in ALD (29) the cumulative probability of adrenal insufficiency was highest until the age of 10 years, remained prominent until 40 years of age, and decreased substantially thereafter. A timeframe for adrenal testing has been suggested as follows: Besides on-demand testing if endocrine symptoms are present, screening for adrenal insufficiency should be initiated in the first 6 months of life, then routine adrenal testing should be performed every 3 to 6 months until 10 years of age, annual testing thereafter until 40 years of age, and solely on-demand testing in case of endocrine symptoms from age 41 years onward (29).

In this context, International Recommendations for the Diagnosis and Management of Patients with Adrenoleukodystrophy have been recently issued emphasizing the need for early and regular adrenal testing (35, figure 3).

Figure 3. Screening and management overview in ALD. Adapted with permission from: International Recommendations for the Diagnosis and Management of Patients with Adrenoleukodystrophy. Neurology 2022.

The most recent Endocrine Society and Pediatric Endocrine Society clinical practice recommendations for the evaluation of adrenal insufficiency are used as guides for establishing cutoff values for ACTH and cortisol (36).

An ACTH value of > 100 pg/mL and a cortisol value of < 10 mcg/dL is suggestive of adrenal insufficiency. Children with normal ACTH and cortisol levels (<100 pg/mL and ≥5 mcg/dL respectively) do not require immediate treatment and should be retested in 3 to 4 months. Children with clearly abnormal ACTH (> 300 pg/mL) and inappropriately low cortisol levels should begin daily and stress-dose glucocorticoid replacement. ACTH and cortisol values of 100 - 299 pg/mL and < 10 mcg/dL respectively should prompt high-dose ACTH (cosyntropin) stimulation testing (34). The median time to transition from stress to maintenance dose has been reported as short as 1.46 years (30). The recommended hormonal workup is depicted in figure 4.

Figure 4. Suggested hormonal work up for glucocorticoid deficiency in ALD.

Of note, the mineralocorticoid function often remains intact, reflecting the relative sparing of the zona glomerulosa in the adrenal cortex (10). Mineralocorticoid deficiency, leading to salt wasting, is not typically described in patients with ALD, consistent with the preservation of aldosterone production and the lack of VLCFA accumulation (37, 38). As VLCFAs mainly accumulate in the zona fasciculata and reticularis, the relative preservation of the zona glomerulosa aligns with the observation that mineralocorticoid function remains functional in approximately 50% of the patients (29). Therefore, mineralocorticoid replacement therapy should not be initiated unless abnormal signs/plasma renin activity and electrolyte levels become evident.

Once the diagnosis of glucocorticoid deficiency has been made, further evaluation of aldosterone production should be considered in case of symptoms, such as salt craving and hypotension. Because symptoms are difficult to assess in infancy, it is recommended that serum plasma renin activity and electrolytes be tested every 6 months (34). Fludrocortisone should be started when there is evidence of mineralocorticoid deficiency. Infants would also require additional salt supplementation.

Mineralocorticoid deficiency is reported to be present in 40% of patients with ALD with the vast majority presenting in adulthood (30). Given that mineralocorticoid deficiency is less common and generally follows glucocorticoid deficiency, evaluation with plasma renin activity and electrolytes is recommended every 6 months starting after diagnosis of glucocorticoid deficiency (34). The median time until mineralocorticoid replacement therapy has been reported to be 56 years of age in contrast to a much shorter time for glucocorticoid replacement therapy which was 16 years of age (29).

Female Patients

As ALD is an X-linked disease, women were previously considered to be asymptomatic carriers. It is now known that even though adrenal insufficiency and cerebral disease are rare in women, more than 80% eventually develop progressive spinal cord disease (39, 40); however, the progression rate of myeloneuropathy remains slow (29). Female patients with ALD typically remain asymptomatic in childhood and adolescence, while, myeloneuropathy symptoms usually arise in adulthood.

Fewer than 1% of female patients are reported to develop adrenal insufficiency (30, 35, 39, 41). Therefore, routine monitoring for adrenal insufficiency and MRI of the brain in women are not recommended (34). Only a few females have been reported to develop CALD and this has been attributed to skewed inactivation of the X-chromosome carrying the mutated ABCD1 gene (42).

Primary Hypogonadism

Gonadal function can also be affected in ALD. Abnormal hormone levels indicating gonadal insufficiency have been described in boys and men with ALD (35, 43, 44). Levels of testosterone in men with ALD are usually in the low-normal range with elevation of luteinizing hormone in some patients (45). These findings indicate primary hypogonadism, possibly due to the toxicity of VLCFA in Leydig cells, but tissue androgen receptor resistance has also been suggested as an alternative hypothesis to explain this finding (46).

To date, no trials have been performed to test the outcome of testosterone supplementation in men with ALD. In most men with ALD, fertility seems to be normal (29, 47). No data exists on fertility in women with ALD.

Tables 1 and 2 summarize the clinical phenotypes in male and female patients.

DIAGNOSIS OF X-ALD

In patients highly suspected of having ALD, measurement of very long chain fatty acids (VCLFA) in the blood is diagnostic, with high specificity and sensitivity (48). VLCFA levels are already increased on the day of birth and in untreated patients remain stable throughout life. Testing typically includes three VLCFA parameters: the level of hexacosanoic acid (C26:0) and tetracosanoic acid (C24:0), and the ratio of these two compounds to docosanoic acid (normal values of C24:0/C22:0 ratio <1.0 and C26:0/C22:0 ratio <0.02). Hexacosanoic acid is the one most consistently elevated and is therefore considered to be diagnostic of the disease. Of note, VLCFA levels are also elevated in other peroxisomal disorders whereas they can be falsely elevated in patients with liver insufficiency or on ketogenic diets (49). False negative results may occur in approximately 20% of female patients, thus, any woman with symptoms of myelopathy with or without a family history of ALD should undergo further genetic testing (48). Plasma C26:0/C22:0 and C24:0/C22:0 ratios, although diagnostic for ALD, are not associated with the (age-dependent) risk of developing adrenal insufficiency, spinal cord disease, or cerebral disease (50, 51).  

However, genetic testing (ABCD1 analysis) is the gold standard for diagnosis.

The diagnosis of X-ALD should be sought (35):

1. In boys and men with confluent white matter abnormalities on brain MRI in a pattern suggestive of ALD with or without cognitive and neurologic symptoms
2. In adult men and women with symptoms and signs of chronic myelopathy with a normal MRI;
3. In boys and men with primary adrenal insufficiency with no detectable steroid-21-hydroxylase antibodies or other organ-specific antibodies;
4. In all at-risk patients with a relative diagnosed with ALD.

Genetic Testing

To date, more than 800 ABCD1 mutations have been described in the X-ALD database (52). Mutations include missense mutations (49%), large deletions (3%), frameshifts (24%), amino acid insertions/ deletions (6%), and nonsense mutations (12%), leading to decreased or absent ABCD1 protein expression. De novo mutation rate is reported to range from 5% to 19% (53). Importantly all clinical phenotypes of X-ALD can occur within the same nuclear family and there is no correlation between ABCD1 mutation and clinical phenotype except for rare cases such as all reported cases of translation initiation mutations in ABCD1 have presented with an AMN-only phenotype (54, 55).

Newborn Screening

Newborn screening (NBS) is justified for a disorder, provided that therapy is available, and that early diagnosis allows timely implementation. This is particularly relevant for X-ALD as early diagnosis at birth would allow for the early detection of adrenal insufficiency for timely initiation of adrenal steroid replacement therapy and early detection of cerebral ALD would permit hematopoietic stem cell transplantation (HSCT) before severe neurologic impairment is established. Important improvements towards this target were the development of mass spectrometry methods to assess the presence of VLCFA in dried-blood spots as well as a combined liquid chromatography/tandem mass spectrometry (LC-MS/MS) high-throughput assay that could measure VLCFA enriched lysophosphatidylcholine (lysoPC), thus providing the technical background for NBS (56).

New York State (NYS) in 2013 was the first authority to include screening for X-ALD in the NBS program and since February 2016, X-ALD has been added to the United States Recommended Uniform Screening Panel (RUSP) (57).

NYS NBS for X-ALD is used by most states in the United States (US) and is based on a 3-tier algorithm: the first tier is tandem mass spectrometry (MS/MS) of C26:0-lysophosphatidylcholine (LPS); the second tier is a confirmatory HPLC-MS/MS; and the third tier is Sanger DNA sequencing of the ABCD1 gene (58). If ABCD1 mutation analysis is negative, then other peroxisomal disorders which are also C26:0-HLPC positive should be sought, such as Zellweger Spectrum Disorders, ACOX1, HSD1B4, ACBD5 deficiency, and CADDS (Contiguous ABCD1 DXS1357/BCAP31 Deletion Syndrome) (57).

As of January 2023, thirty-five US states have successfully added ALD to the conditions screened via NBS with plans to expand to all states (59, 60, 61, 62, 63, 64, 65, 66). Globally, the Netherlands is the only other country that is actively screening for ALD through the Screening for ALD in the Netherlands (SCAN) pilot study, a sex-specific newborn screen for boys (67). Since the implementation of Newborn screening for ALD, data show a rise in the diagnosis of ALD up to ~1 in 10,500 births as well as an earlier diagnosis of adrenal insufficiency (30).

Genetic Counseling

As soon as an index case is detected either as a consequence of symptoms or as a result of NBS, genetic counseling should be offered to the family. If the index case is male, testing should be offered to his mother and female offspring.  If the mother is confirmed to be a carrier for an ABCD1 mutation, testing should also include all the male siblings of the index case. If the index case is female, initial testing should include both parents. Regarding mutation testing of minor females of an affected family, there is no consensus on whether it should be performed on a routine base. (57).

Imaging

All individuals with confirmed ALD/AMN complex should undergo neuroimaging to determine if cerebral involvement is present. Brain MRI abnormalities precede symptoms in patients with the cerebral forms of X-ALD (23). Findings are always abnormal in symptomatic patients, demonstrating cerebral white matter demyelination (Figure 5). The lesions typically begin in the splenium of the corpus callosum before gradually expanding to the occipito-parietal region and they are usually bilateral but occasionally can be limited to only one side, particularly if previous head trauma has triggered CALD (19). The presence of contrast enhancement just behind the outermost edge of the lesions as seen in T1-weighted images (WI), heralds the progression to inflammatory devastating form of CALD (68). A grading system to assess the degree of MRI abnormalities in X-ALD has been proposed by Loes et al. (69). This is a 32-point scale score (0: normal, 32: most severe) that assesses the degree and extent of hyperintense lesions on FLAIR or T2W images as well as the degree of regional atrophy and has proven to have predictive value for the response to allogenic hematopoietic stem cell transplantation (70).

Regarding AMN, MRI of the spinal cord is unremarkable on standard sequences, it can however show atrophy in advanced cases (71). Contrast enhancement is not observed in AMN, since inflammation is not a feature of extra-cerebral lesions.

Brain F18 fludeoxy-glucose positron emission tomography (PET) may reveal hypometabolic regions particularly in the cerebellum and temporal lobe areas, before lesions emerge in MRI (72). In contrast, hypermetabolism may be evident in the frontal lobes, related to the clinical severity of the disease (73).

Figure 5. MRI of a patient with cerebral ALD, showing reduced volume and increased signal intensity of the white matter localized mainly at the parieto-occipital regions. The anterior white matter is spared. (http://en.wikipedia.org/w/index.php?title=Adrenoleukodystrophy&oldid=506277486).

THERAPY

Dietary Treatment

Τherapeutic options include dietary therapies with restriction of fat intake and particularly of VLCFAs and saturated fats to avoid their accumulation. In order to achieve this, total fat intake is restricted to 15% of the total calorie supply and a maximum of 5-10 mg of C26:0 is allowed on a daily basis (Table 3).

However, since the majority of VLCFA are of endogenous origin (74), this approach is not sufficient. A mixture of oleic acid [C18:1] and erucic acid [C22:1], also referred to as Lorenzo's Oil (LO), has also been applied (75). LO has been shown to halt the elongation of VLCFA by inhibiting ELOVL1, the primary enzyme responsible for VLCFA synthesis.

LO in combination with a low-fat diet nearly normalizes plasma VLCFA levels within four weeks and in a study involving asymptomatic X-ALD patients with normal brain MRI, dietary treatment with LO resulted in a two-fold or greater reduction in the risk of developing the childhood cerebral form of X-ALD (76). However, in patients who are already symptomatic, controlled clinical trials failed to show improved neurological or endocrine function, nor did it arrest the progression of the disease (35, 77, 78). Treatment with LO may be continued for an indefinite time until disease progression and/or severe side effects occur. It is not recommended in children under one year of age, as it causes a decrease in the levels of other fatty acids, particularly docosahexaenoic acid, which is essential for neurocognitive development.

Allogenic Hematopoietic Stem Cell Transplantation (HSCT)

Allogeneic HSCT is the treatment of choice for individuals with early stages of cerebral involvement of X-ALD, which may increase disease-specific survival and can lead to long-term stabilization and improvement of neurological status (77, 79, 80). Stem cells can be harvested from peripheral blood, bone marrow, and umbilical cord blood of immune-compatible donors. Although the mechanism of this effect is still unclear, bone marrow cells do express the ABCD1 gene and plasma VLCFA levels are reduced after bone marrow transplantation, offering a useful biomarker for the assessment of engraftment, graft failure, or rejection (81). It has been shown that bone marrow-derived cells do enter the brain-blood barrier and that a portion of perivascular microglia is gradually replaced by donor-derived cells (82).

Allogeneic HSCT has been shown to increase 5-year survival compared to no transplant (95% versus 54%) and arrest the progression of the neurologic disease when undertaken early in the course of cerebral disease (44). In contrast, hematopoietic stem cell transplantation is not effective in patients with advanced cerebral ALD, therefore the general criteria for eligibility are a genetically and/or clinically confirmed diagnosis of ALD and the presence of cerebral disease that is not advanced, based on neurological symptoms and brain MRI findings (83). Eligibility of a patient for transplantation can be assessed using the ALD-specific Neurologic Function Scale (NFS) and the Loes MRI severity score (54). The NFS scale is a 25-point, ALD-specific tool that assesses the severity of neurological disability according to the severity of symptoms, but no score absolutely determines the decision for HSCT. HSCT affects not only survival, but also the long-term functional status of patients. Studies have shown that post-transplant survival and major functional disability (MFD)-free survival are superior in patients with lower NFS and Loes score (84, 85). A recent multi-center analysis showed that in early-stage transplanted patients the overall survival at 5 years from CALD diagnosis was 94% and the MFD -free survival was 91%, whereas in patients with advanced disease the overall survival and the MFD-free survival were 90% and 10% respectively (83).

Allogeneic HSCT has its limitations. Transplantation is not effective in patients with advanced disease. Neurologic findings present at the time of HSCT do not reverse and symptoms can progress after HSCT as cerebral disease stabilization is not achieved before 3 to 24 months after stem cell infusion (54). Furthermore, the identification of an acceptable donor for HSCT could be very challenging. Significant risks associated with HSCT include acute mortality (10% at day 100 from transplant), failure of donor cell engraftment (5% risk), and graft-versus-host disease (GVHD) (10-40% risk of acute GVHD and 20% risk of chronic GVHD) (85).

HSCT has not been tested systematically in AMN because of concerns that the risk-benefit ratio may not be favorable: up to 50% of AMN patients will never develop cerebral involvement, whereas it is highly unlikely that HSCT will affect the non-inflammatory distal axonopathy which is the main pathological feature in AMN (86). Moreover, in retrospective series of patients who successfully underwent HSCT for CALD in childhood, it was shown that it could not prevent the onset of AMN in adulthood (87).

Although data are limited, HSCT is unlikely to affect adrenal insufficiency (35). The proposed underlying mechanism is that VLCFAs accumulation in the adrenal cortex has already reached a critical point that is irreversible by the time of transplant, whereas cerebral ALD has a considerable progressive inflammatory component that is stabilized by the transplant (88).

Gene Therapy

In case of patients without HLA-matched donors or adult patients with CALD (given the higher mortality risk of allogeneic HSCT compared to children), an alternative option is autologous HSC-gene therapy with lentivirally corrected cells (89). In this procedure, CD34+ cells from X-ALD patients are transfected ex vivo using a lentiviral vector encoding the wild-type ABCD1 cDNA. As a result of this therapy, 7-14% of granulocytes, monocytes, T and B lymphocytes express the lentivirally encoded ALDP. In a recent phase 2-3 study including 17 boys, short-term clinical outcomes were reported to be comparable to that of allogeneic HSCT (90). The procedure called Lenti-D gene therapy resulted in clinical disease and imaging stabilization according to neurological symptoms and brain MRI findings in the vast majority of enrolled patients. An ongoing study recruiting for a phase III trial that has been recently opened across the US and Europe (NCT03852498) will further evaluate the efficacy and safety of Lenti-D gene therapy in participants with CALD.Nevertheless, concerns regarding long-term efficacy, biosafety of lentiviral vectors, as well as the high cost of this therapy need to be taken into account (91, 92). An alternative approach is performing allogeneic HSCT from healthy siblings conceived after preimplantation HLA matching which offers the possibility of selecting unaffected embryos that are HLA compatible with the sick child (93).

Regarding adrenal function, similarly to HSCT, there is no evidence for the reversal of adrenal failure after autologous HSC gene therapy (88). Advances in gene therapy could offer new treatment options for ALD. Potential therapies include a) antisense oligonucleotides which target specific mutations to exclude pathogenic variants or to establish a normal reading frame shift, b) gene editing through the use of endonucleases that allows permanent modifications to specific DNA segments and c) targeted viral vector therapy that could deliver a normal copy of the ABCD1 gene to steroidogenic and to microglial cells to prevent adrenal disease and neurological dysfunction respectively (54).

Treatment Of Adrenal Insufficiency and Hypogonadism

For those patients with X-ALD who have impaired adrenal function, glucocorticoid replacement therapy is mandatory. Glucocorticoid replacement requirements are generally the same as in other forms of PAI, whereas most patients may not require mineralocorticoid replacement.

 Male patients who present clinical manifestations of hypogonadism and confirmed low serum testosterone levels, should be treated with testosterone. Nevertheless, careful evaluation should be warranted, since impotence, in most instances may imply spinal cord involvement or neuropathy, rather than testosterone deficiency.

Experimental Therapies

Experimental treatment options include a) agents that bypass the defective ALDP by inducing alternative pathways for VLCFA degradation, b) combinations of antioxidants that diminish oxidative stress, c) agents that halt VLCFA elongation and d) the use of neurotrophic factors.

 Apart from ALDP, three additional closely related ABC half-transporters exist: ALDRP, PMP70, and PMP69, which are located on the membrane of peroxisomes. ALDP must dimerize with one of these half-transporters to form a functional full transporter (94). Over-expression of ABCD2, the gene producing ALDRP has been shown to compensate for ABCD1 deficiency and ameliorate VLCFA production from X-ALD cell series (95). Valproic acid (VPA), a widely used anti-epileptic drug, 4-phenylbutyrate, and other histone deacetylase inhibitors, are known inducers of the expression of ALDRP. In a 6-month pilot trial of VPA in X-ALD patients marked correction of the protein oxidative damage was observed (96). Other agents known to evoke induction of the ABCD2 gene are ligands to several nuclear receptors: fibrates for PPAR alpha, thyroid hormones and thyromimetics, retinoids, and lately LXR antagonists, which are being tested in vitro and in vivo for the treatment of X-ALD (97, 98, 99). Lately, it has been shown that AMP-activated protein kinase (AMPKα1) is reduced in X-ALD, raising the question if metformin, a well-known AMPKα1inducer, may have a therapeutic role for X-ALD (100).

 Regarding the use of antioxidative treatments, experimental data show that treatment of ABCD1 null mice with a combination of antioxidants containing α-tocopherol, N-acetyl-cysteine and α-lipoic acid reversed oxidative damage, axonal degeneration, and locomotor impairment (101). Similar results have been observed with the oral administration of pioglitazone, an agonist of the PPAR gamma receptor, which restored oxidative damage to mitochondrial proteins and DNA, and reversed bioenergetic failure. Lately, bezafibrate, a PPAR pan agonist has been demonstrated to reduce VLCFA levels in X-ALD fibroblasts (102). The mechanism for this action is by decreasing the synthesis of C26:0 through a direct inhibition of ELOVL-1 and subsequent fatty acid elongation activity. Unfortunately, these actions could not be confirmed in vivo as in a recent clinical trial, bezafibrate was unable to lower VLCFA levels in plasma or lymphocytes of X-ALD patients (103).

The options for treatment of the advanced progressive form of CALD remain limited. Even though the presence of inflammatory lesions is well recognized, trials of immunosuppressive therapies have yielded poor results. Cyclophosphamide, interferon, IVIG, and other immunomodulators have been used without success (104, 105). Promising results have been extracted by the use of the antioxidant N-acetyl-L-cysteine as adjunctive therapy to HSCT in patients with advanced CALD (106, 107).

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ABSTRACT

Primary generalized glucocorticoid resistance syndrome is a rare genetic disorder characterized by resistance of entire tissues to glucocorticoids. Affected subjects demonstrate elevation of serum cortisol without Cushingoid manifestations, as the hypothalamic-pituitary-adrenal (HPA) axis is upregulated to compensate for the reduced action of this steroid in local tissues. Instead, these patients develop hypertension and/or signs of hyperandrogenism, because hyper-secreted adrenocorticotropic hormone (ACTH) stimulates production of adrenal mineralocorticoids and/or androgens in addition to the glucocorticoid cortisol. At the molecular level, this syndrome is caused by inactivating mutations in the NR3C1 gene that encodes the human glucocorticoid receptor (hGR) protein. Biochemical, molecular and structural exploration on pathologic mutant receptors revealed a variety of functional defects, such as reduced affinity to glucocorticoids or target DNA, inability to transactivate glucocorticoid-responsive genes, and slowing of the cytoplasmic to nuclear translocation. The clinical spectrum of this syndrome is thus broad, ranging from asymptomatic to severe cases of mineralocorticoid and/or androgen excess depending on the severity of genetic defects and resulting dysfunction of the mutated receptors. When this syndrome is suspected, a detailed personal and family history should be obtained. Physical examination should include an assessment for signs of mineralocorticoid and/or androgen excess. In neonates and young children, severe hypoglycemia and loss of consciousness due to reduced actions of glucocorticoids in the liver may be present as initial manifestations in addition to hypertension and/or genital abnormalities. Suspected subjects should undergo a detailed endocrinologic evaluation with particular emphasis on the measurement of diurnal serum cortisol and plasma ACTH concentrations and determination of the 24-hour urinary free cortisol excretion to identify upregulation of the HPA axis with preservation of the normal circadian rhythmicity. The diagnosis of this syndrome should be confirmed by sequencing of the NR3C1 gene including exon/intron junctions and subsequent validation of functional defects of the mutated receptors. Treatment involves administration of high doses of mineralocorticoid activity-sparing pure glucocorticoids like dexamethasone, which stimulate the mutant and/or the wild-type hGR, and suppress the endogenous secretion of ACTH and adrenal steroids in the affected subjects.

INTRODUCTION

Organisms are exposed continuously to internal and external stressors, and live through them by maintaining the internal equilibrium called homeostasis (1). In order to respond adequately to such stressors through coordinating various body activities, we humans are equipped with a highly sophisticated stress responsive system, the hypothalamic-pituitary-adrenal (HPA) axis, which consists of the brain hypothalamus, the anterior pituitary gland, and the adrenal cortex, and employs glucocorticoids as its end-effector hormones. Actions of glucocorticoids, which are essential for life, can be determined by a balance between circulating levels of these hormones and local tissue sensitivity (2, 3). Exceeding appropriate ranges of tissue sensitivity to glucocorticoids may present either as glucocorticoid resistance or glucocorticoid hypersensitivity with their specific manifestations (3, 4). Such alterations in tissue glucocorticoid actions can occur in general (that is, throughout the body) or in tissue-specific manner (restricted in some organs and tissues; e.g., immune organs/cells, central nervous system (CNS), liver and fat tissues) (3). They are caused primarily by genetic defects of the molecules involved in the glucocorticoid signaling pathway or secondary through modulation of this pathway by other pathologic conditions, such as infectious, inflammatory and autoimmune diseases, obesity, and insulin resistance/overt diabetes mellitus. One such condition is the primary generalized glucocorticoid resistance syndrome, which is caused by inactivating mutations in the glucocorticoid receptor gene (5). Affected subjects develop partial glucocorticoid resistance observed in entire organs and tissues of the affected subjects (5). In recognition of Professor George P. Chrousos' novel and extensive research work in this field, the term “Chrousos Syndrome” may be used for this syndrome (6, 7).

GLUCOCORTICOIDS

Glucocorticoids (cortisol in humans and corticosterone in rodents) are produced from cholesterol through multiple enzymatic reactions in the zona fasciculata of the adrenal cortex in response to the adrenocorticotropic hormone (ACTH) released from the pituitary gland (1). Glucocorticoids regulate a broad spectrum of physiologic functions essential for life, such as growth, reproduction, immunity, intermediary metabolism, cardiovascular tone, and CNS functions, playing essential and indispensable roles in the maintenance of resting and stress-related homeostasis (1, 7, 8). In addition, glucocorticoids exert potent anti-inflammatory and immunomodulatory effects particularly with their stress-equivalent or pharmacologic doses, thus they are widely used in the treatment of inflammatory, autoimmune, and lymphoproliferative diseases (8).

GLUCOCORTICOID RECEPTOR PROTEINS, ISOFORMS AND ITS ENCODING GENE, NR3C1

Circulating cortisol freely passes through the cytoplasmic membrane and enters into the cytoplasm of its target cells, and binds to an intracellular protein, the glucocorticoid receptor (GR) (9, 10). The human (h) GR is one of the steroid/thyroid/retinoic acid nuclear hormone receptor superfamily proteins, which consist of over 600 members in the animal kingdom (11). Many of them mediate extracellular signals transduced mainly by lipophilic hormones/compounds into the cell nucleus by binding them as ligands and by acting as ligand-dependent transcription factors (12, 13). hGR influences transcription rates of numerous glucocorticoid-responsive genes (up to 3~5% of the entire protein-coding genes) in a positive or a negative fashion by interacting directly or indirectly with promoter/enhancer regions of these genes (14). The hGR gene (NR3C1: nuclear receptor subfamily 3, group C, member 1) consists of 9 exons and is located at chromosome 5q31.3. Exons 2-9 constitute the protein-coding sequence, whereas exon 1 encodes an untranslated region (12, 14, 15). The human NR3C1 gene has multiple exon 1s (see below) that harbor specific promoters containing a respective transcription start site for conferring tissue-specific expression of the receptor protein (15). Alternative splicing of the NR3C1 gene in exon 9s generates two highly homologous receptor isoforms, the hGRα and the hGRβ (16). They share amino (N)-terminal 727 common amino acids, but then diverge, with hGRα having an additional 50 amino acids and hGRβ having an additional, nonhomologous 15 amino acids at their carboxyl (C)-termini (17). hGRα resides primarily in the cytoplasm of cells and represents the classic GR that binds natural and synthetic glucocorticoids and mediates most of the actions of these hormones (15). On the other hand, hGRβ does not bind glucocorticoids, has intrinsic, gene-specific transcriptional activity, and exerts a dominant negative effect on the transcriptional activity of hGRa (18). Although physiologic and pathologic roles of hGRβ are still largely unknown (19, 20), recent studies demonstrated that this isoform is implicated in modulation of the insulin signaling and participates in the pathogenesis of brain gliomas (21-23).  

The hGRα mRNA expresses not only the classic, full-length hGRα, but also multiple translational isoforms by using at least eight alternative amino-terminal translation initiation sites (24). All these hGRα isoforms are differentially distributed in the cytoplasm and/or the nucleus in the absence of ligand, have different transcriptional activity, and display distinct transactivating or transrepressing activities on various glucocorticoid-responsive genes (24). Since hGRβ shares with hGRα a common amino-terminal domain that contains the same translation initiation sites, the hGRβ variant mRNA might also be translated through the same translation initiation sites to a similar host of hGRβ isoforms (14).

The human NR3C1 has 11 different promoters with their alternative first exons (1A1, 1A2, 1A3, 1B, 1C, 1D, 1E, 1F, 1H, 1I and 1J) (25, 26). Therefore, it can produce 11 different hGRa mRNA transcripts from different promoters that encode the same hGRa protein, as these transcripts share common exon 2 to exon 9a that contains the same translation initiation codon. 1A1, 1A2, 1A3 and 1I are located in the distal promoter region spanning ~32,000-36,000 bps upstream of the translation initiation site, while 1B, 1C, 1D, 1E, 1F, 1H and 1J position in the proximal promoter region located up to ~5,000 bps upstream of this site (25). Through differential use of these promoters, expression levels of hGRa can vary among tissues in different physiologic and pathologic conditions, as each tissue has specific expression profiles of local transcription factors and epigenetic modification of chromatin-associated molecules bound on these exon 1-associated promoters (25, 27, 28). Again, differential tissue-specific expression of hGRb through the use of these promoters appears to be present. The above-indicated marked complexity in transcription/translation of the human NR3C1 gene enables target tissues to respond differently to circulating cortisol and accounts for stochastic, but still a highly organized nature of tissue glucocorticoid actions, in order to fulfil specific local needs of glucocorticoid hormonal effects (14). Such complexity of the glucocorticoid signaling at the receptor level also indicates that the proper biologic action of glucocorticoids in every target tissue is extremely important.

The hGRα protein consists of three major domains and one region, namely the N-terminal (NTD), DNA-binding (DBD) and ligand-binding domain (LBD), and the hinge region (HR) (15). Exact amino acid location of these domains/region in the hGRa protein explained below is based on the data retrieved from the Pfam source of the Ensembl database (www.ensembl.org). NTD is encoded by exon 2 and represents the largest domain of the receptor, spanning over amino acids 1 to 401. It contains an unstructured acidic transactivation surface called activation function (AF) -1, which is used as a molecular platform for modulating the transcription of glucocorticoid-responsive genes (10). This domain also undergoes several post-translational modifications particularly at AF-1.  DBD is expressed from exons 3 and 4, and lies between amino acids 417 and 494. This domain consists of two 4C (cysteine)-type zinc fingers and support the interaction between the receptor and its target DNA sequences known as glucocorticoid response elements (GREs) (10, 29). LBD is encoded by exons 5-9 and positions at the C-terminal end of the receptor corresponding to amino acids 531 to 777. This domain is structurally formed with 12 a-helices and four b-sheets, and contains two functional structures, the ligand-binding pocket (LBP) and the second transactivation surface called AF-2, as well as several other molecular platforms including the one responsible for nuclear translocation of the receptor (29, 30). Most of the protein surfaces of LBD that mediate these LBD-specific functions are formed upon binding of the receptor to a ligand and following conformational changes of this domain (15). Finally, HR lies between DBD and LBD, is encoded by 5’ part of exon 5, and spans between amino acids 495 and 530. This region provides appropriate structural flexibility to the receptor and allows the dimerized receptors to interact with different classic/alternative tandem GREs with various length of spacing nucleotides (the classic tandem GREs has three spacing nucleotides)  (15).

MOLECULAR ACTIONS OF hGRa

Intracellular Shuttling of hGRa and its Regulators

At target cells, hGRα in the absence of glucocorticoids resides primarily in the cytoplasm as part of the hetero-oligomeric complex consisting of chaperone heat shock proteins (HSPs) 90, 70 and 50, immunophilins (e.g., FK506-binding protein (FKBP)), and possibly other proteins (31) (Figure 1). Binding of HSP90 to hGRα induces a conformational change in receptor’s LBD, and confers its ligand-friendly state, exposing the LBP to glucocorticoids and masking two nuclear localization signals (NLS), NL1 and NL2. Upon binding to a ligand, hGRα dissociates from the complex, exposes NL1 and NL2 to their counterpart molecular machinery, and translocates into the nucleus through the nuclear pore. NL1 harbors a classic NLS and is located between the C-terminal portion of DBD and the N-terminal part of HR (32). The function of NL1 is dependent on the importin a, a protein component of the nuclear pore-associated nuclear import system, which transports a liganded GRa as a cargo from the cytoplasm to the nucleus through the nuclear pore in an ATP-dependent fashion (33). NL2 spans over most of the LBD whose molecular mechanism(s) for supporting nuclear translocation of the receptor has(ve) not yet been elucidated (31, 34). Inside the nucleus, ligand-bound hGRα dimerizes and modulates transcription rates of glucocorticoid-responsive genes by associating with promoter/enhancer regions of their encoding genes (15) (Figure 1). The receptor subsequently liberates the ligand and is dissociated from its target genes and slowly translocates back to the cytoplasm with the molecular mechanisms described below (15). The ubiquitin-proteasomal pathway degrades some of the liganded hGRa in the nucleus, facilitating clearance of the receptor from GREs; thus this system negatively regulates the transcriptional activity of hGRa (35).

In addition to translocating into the nucleus, some liganded hGRas migrate to the cytoplasmic membrane where they modulate the activity of cell surface receptors by associating with their intracellular signaling molecules, such as classic and small GTP-binding (G) proteins, and several serine/threonine and tyrosine kinases (36-38). The ligand-bound hGRais also known to translocate into the mitochondria and to modulate the activity of this intracellular organelle (39). After modulating transcription rates of glucocorticoid-responsive genes in the nucleus, ligand-liberated hGRα is exported back to the cytoplasm and is re-incorporated into the HSP-containing multiprotein complex to function again as a ligand-binding competent receptor (31, 40) (Figure 1). Several mechanisms are postulated for mediating the GRa export from the nucleus to the cytoplasm. The Ca²⁺-binding protein calreticulin plays a role in this process, directly binding to DBD of the receptor (41-43). The chromosomal maintenance 1 (CRM1, also known as exportin 1)- and the classic nuclear export signal (NES)-mediated nuclear export machinery does not appear to function directly on hGRa (32, 42). Rather, NES-harboring and phospho-serine/threonine-binding proteins 14-3-3s can bind hGRa, and shift its intracellular localization toward the cytoplasm (44, 45). This action of 14-3-3s on hGRa appears to be independent to the ligand-induced nuclear translocation of the receptor, which is mediated in part by the NL1/importin a-associated nuclear pore complex. Numbers of serine and threonine residues of hGRa are phosphorylated by several serine/threonine kinases at their specific target residues, some of which function as phosphorylation-dependent binding sites of 14-3-3 proteins (46). For example, the v-akt murine thymoma viral oncogene homolog 1 (AKT1) (or the protein kinase B a) phosphorylates serine (S) 134 of the hGRa, and 14-3-3 binds to phosphorylated S134. Binding of 14-3-3 on hGRa at this site shifts subcellular localization of the latter to the cytoplasm and downregulates its transcriptional activity inside the nucleus (45, 47). The misshapen-like kinase 1 (MINK1) and the Rho-associated protein kinase (ROCK) respectively phosphorylate threonine (T) 524 and S617 (48). 14-3-3s bind phosphorylated forms of these residues as a dimer (48), possibly modulating subcellular localization and transcriptional activity of the hGRa.

Figure 1. Intracellular circulation and actions of hGRα. hGRα resides in the cytoplasm in the absence of ligand by forming a heterocomplex with several heat shock proteins (HSPs), immunophilins (e.g., FKBP), and some other proteins. Upon binding to ligand cortisol, hGRα dissociates from the complex and translocates into the nucleus through the nuclear pore. Inside the nucleus, hGRα binds directly to glucocorticoid response elements (GREs) located in promoter/enhancer regions of glucocorticoid-responsive genes. DNA-bound hGRα then stimulates transcription rates of glucocorticoid-responsive genes by attracting the regulatory regions the transcription regulatory complex including the RNA polymerase II (RNPII) and its ancillary components through bridging coactivators, such as p300/CBP and p160 proteins. Promoter/enhancer-bound hGRα also recruits in collaboration with these coactivators various chromatin remodeling molecules, including the DRIP/TRAP complex (DRIP/TRAP), the SWI/SNF chromatin modulator (SWI/SNF), and the Mediator complex (MED). In addition to binding directly to DNA and regulating transcription, hGRα interacts indirectly with regulatory regions of glucocorticoid-responsive genes via protein-protein interaction with other transcription factors (TFs) and/or attracted cofactor molecules, ultimately modulating positively and negatively the transcriptional activity of GRE- and non-GRE-containing glucocorticoid-responsive genes. hGRα then moves back to the cytoplasm to re-form a heterocomplex with HSPs for regaining a ligand-friendly status or is cleared from DNA by proteasomal degradation. Further, hGRα can influence the action of cell surface receptors by associating with their intracellular signaling molecules, such as classic and small G-proteins, and several serine/threonine and tyrosine kinases (known as non-genomic actions of glucocorticoids). Accumulating evidence suggests that liganded hGRα also influences the transcription of mitochondrial genes by translocating into this intracellular organelle. CBP: cAMP-responsive element-binding protein (CREB)-binding protein; DRIP/TRAP: vitamin D receptor-interacting protein/thyroid hormone receptor-associated protein complex; FKBPs: FK506-binding proteins; GREs: glucocorticoid response elements; GR: glucocorticoid receptor; HSPs: heat shock proteins; MED: Mediator complex; p160: p160-type nuclear receptor coactivator; RNPII: RNA polymerase II; SWI/SNF: switching/sucrose non-fermenting complex; TFs: transcription factors; TREs: transcription factor response elements.

Genomic and Non-genomic Actions of hGRα

After binding to glucocorticoids and translocating into the nucleus, hGRα binds as a dimer to a tandem GREs located in promoter/enhancer regions of glucocorticoid-responsive genes, and regulates their mRNA expression positively or negatively, depending on the GRE sequence and the promoter/enhancer context (15, 49, 50) (Figure 1). GRE-bound hGRα stimulates transcription of responsive genes by facilitating formation of the transcription regulatory complex, which includes the RNA polymerase II (RNPII) and its ancillary components (51). Mechanically, hGRα uses its two transactivation domains, AF-1 and AF-2, as protein surfaces for interacting with and attracting nuclear receptor coactivators (51). These proteins then act as bridges between the DNA-bound hGRα and the RNPII-containing transcription initiation complex (52, 53) (Figure 1). In addition, they act in themselves as histone acetyltransferases (HAT) as well as attract other enzymatic proteins, and loosen tightly packed chromatin DNA by chemically modulating specific amino acid residues of histones and other chromatin-associated molecules (54). Representatives of these HAT coactivators include p300 and its homologous cAMP-responsive element-binding protein (CREB)-binding protein (CBP), and the p160 family of nuclear receptor coactivators (NCoAs). The former proteins serve as macromolecular docking “platforms” for many transcription factors, including nuclear hormone receptors, CREB, activator protein-1 (AP-1), nuclear factor-κB (NF-κB), p53, and signal transducers and activators of transcription (STATs), and thus, are called co-integrators (55). On the other hand, the p160 family of nuclear receptor coactivators (NCoAs) is more specific to nuclear hormone receptors including hGRa, and play a central role in the initiation of transcription by hGRa, as they are first attracted to the DNA-bound receptor molecule (55, 56). For physical interaction with hGRa, p160-type coactivators employ the LxxLL motif in which “L” is leucine and “x” is any amino acids. They harbor in their nuclear receptor-binding domain (NRB) multiple LxxLL motifs, each of which have different affinity to respective nuclear hormone receptors (55-58). The LxxLL motif forms the a-helical structure and is deeply buried into the molecular cleft formed by the AF-2 surface of the liganded hGRa (58). Interestingly, p160 family proteins also serve as transcriptional coactivators for some other transcription factors (e.g., NF-kB) (59, 60). In collaboration with these transcriptional coactivators and promoter/enhancer-bound other transcription factors, hGRα interacts with and attracts several distinct chromatin remodeling complexes (e.g., the mating-type switching/sucrose non-fermenting (SWI/SNF) complex, the vitamin D receptor-interacting protein/thyroid hormone receptor-associated protein (DRIP/TRAP) complex, and the Mediator (MED) complex) as well as various enzymatic molecules, scaffold proteins, and long non-coding RNAs (e.g., the steroid receptor RNA coactivator (SRA) and the growth arrest-specific 5 (Gas5)), ultimately forming a huge transcriptional regulatory complex for initiating transcription of the downstream coding sequence though the attracted RNPII (61-64). These newly identified functional oligonucleotides exert their transcriptional regulatory activity in part by modulating the liquid-liquid phase separation among various proteins inside the transcription regulatory complex formed on the DNA-bound hGRa (65).

Similar to the transcription factors incorporated in the transcriptional regulatory complex recruited by GREs-bound hGRα, liganded hGRα is also attracted to the transcription regulatory complex formed by DNA-bound other transcription factors (e.g., AP-1, NF-κB, p53, STATs, and forkhead transcription factors: FOXOs). This incorporation of hGRα can be independent to its physical association with DNA GREs, and the recruited hGRα modulates their transcriptional activity positively or negatively (15, 66) (Figure 1). The interaction between hGRα and these transcription factors are mediated by mutual protein-protein interactions between these proteins or indirectly through bridging coactivators, such as p300/CBP and p160-type coactivators (15). This GRE-independent activity of hGRα may be more important than the GRE-mediated one, given that the mice harboring a mutant GR defective in the dimerization surface, and thus, active in protein-protein interaction but inactive in transactivation via tandem GREs, survive and procreate, in contrast to the mice with Nr3c1 gene knock-out, which die immediately after birth due to respiratory failure (67). Suppression of transactivation of other transcription factors through such protein-protein interactions appears to be important particularly in the suppression of immune functions and inflammation by glucocorticoids (68-70).

Mounting evidence suggests that glucocorticoids also signal within seconds or minutes. These effects are called “non-genomic”, since they do not require the transcriptional activity of hGRα (15). Representative examples of these actions are: (i) the immediate suppression of ACTH release from the anterior pituitary gland by glucocorticoids (71); (ii) the increased frequency of excitatory post-synaptic potentials by glucocorticoids in the brain hippocampus (72); (iii) the cardioprotective role of glucocorticoids through nitric oxide-mediated vasorelaxation (73); and (iv) some immunomodulatory effects of glucocorticoids via inhibition of the T-cell receptor signaling (74). Some of the molecular mechanisms underlying these actions of hGRα have been proposed. For example, ligand-activated hGRα physically interacts with the classic G protein b through its NTD, and may modulate the action of G protein-coupled receptors located at the cytoplasmic membrane (36). Recent studies also demonstrated that hGRα influences the activity of kinase-mediated signaling, such as of the mitogen-activated protein kinase and the phosphatidylinositol 3-kinase through interacting with their key signaling molecules residing under the cytoplasmic membrane or in the cytoplasm (71-75)(Figure 1).

These non-genomic effects of hGRα modulate the action of some intracellular signaling pathways, whereas the latter can influence the activity of hGRα through post-translational modifications (PTMs) of this receptor protein. Such PTMs include phosphorylation, ubiquitination, acetylation, and sumoylation (15). These covalent changes may influence receptor stability, subcellular localization, as well as its interaction with other proteins including transcription factors and transcriptional cofactors/regulators (10). Thus, enzymes catalyzing these PTMs act as molecular effectors of their upstream intracellular signaling pathways for modulating the biologic effects of glucocorticoids by targeting the hGRaprotein.

In addition to the above-explained diverse actions, glucocorticoids can modulate expression of the mitochondrial genes by translocating into this cytoplasmic organelle, and by binding to the classic GREs located in some regulatory sites (D-loop) of these genes (76-78) (Figure 1). This action of hGRα in the mitochondria appears to play a role in the glucocorticoid-mediated modulation of apoptosis, a well-known process of the programmed cell death, and may contribute to the therapeutic effects of glucocorticoids on hematologic and other malignancies (79).

PRIMARY GENERALIZED GLUCOCORTICOID RESISTANCE SYNDROME

Pathophysiology and Clinical Manifestations

This syndrome is a condition first described by Chrousos, et.al., as a rare, familial or sporadic, genetic disorder characterized by generalized, partial target tissue insensitivity to glucocorticoids (80). Because of glucocorticoid insensitivity in the central components of the HPA axis, glucocorticoid-mediated negative feedback inhibition on the brain hypothalamus and the anterior pituitary gland is decreased (5, 81) (Figure 2). These changes result in compensatory elevation of the corticotropin-releasing hormone (CRH) and the arginine-vasopressin (AVP) at the hypothalamus and systemic release of the ACTH from the anterior pituitary gland. Excess ACTH secretion then causes bilateral adrenocortical hyperplasia and increased production/secretion of cortisol, which compensates for its reduced actions in target tissues. However, elevated circulating ACTH also stimulates production of other adrenal steroids, such as mineralocorticoids (e.g., deoxycorticosterone (DOC) and corticosterone) and/or adrenal androgens (e.g., androstenedione, dehydroepiandrosterone (DHEA), and DHEA-sulfate (DHEA-S)), leading to the development of excess manifestations of these hormones, because tissue sensitivity to these steroids is not altered. Increased mineralocorticoids may cause hypertension and/or hypokalemic alkalosis, whereas elevated adrenal androgens may develop manifestations (see below) through their direct effects on target tissues and/or indirect actions via modulation of the hypothalamic-pituitary-gonadal axis.

Figure 2. Pathophysiologic mechanisms and clinical manifestations of primary generalized glucocorticoid resistance syndrome (PGGRS). The HPA axis consists of the brain hypothalamus, the anterior pituitary gland, and the adrenal cortex with their secreting hormones/peptides, CRH/AVP, ACTH and cortisol, respectively. In patients with this syndrome, their HPA axis is re-set to upward with preservation of circadian rhythmicity due to generalized, partial insensitivity to glucocorticoids in entire tissues. Thus, hypothalamic CRH/AVP, pituitary ACTH and adrenal cortisol are all hyper-secreted in order to compensate for the reduced actions of cortisol in both CNS and peripheral tissues. In addition to augmenting production of cortisol in the adrenal glands, elevated ACTH stimulates secretion of mineralocorticoids (e.g., deoxycorticosterone and corticosterone) and androgens (e.g., androstenedione, dehydroepiandrosterone (DHEA) and DHEA-sulfate(S)), which in turn cause a variety of manifestations associated with excess secretion of these hormones. In contrast, manifestations associated with overproduction of cortisol are rare in adult patients but neonates/young children may develop hypoglycemia and associated seizures due to reduced actions of cortisol in the liver. Elevated CRH/AVP in CNS may precipitate anxiety and depression in some patients. Solid lines indicate positive effects, whereas dashed lines show negative effects. Manifestations associated with elevation of the indicated molecules/compounds are shown with red letters. ACTH: adrenocorticotropic hormone; AVP: arginine vasopressin; CNS: central nervous system; CRH: corticotropin-releasing hormone; DHEA: dehydroepiandrosterone; DHEA-S: DHEA-sulfate; PGGRS; primary generalized glucocorticoid resistance syndrome.

Manifestations associated with excess adrenal androgens observed in patients with this syndrome include acne, hirsutism (more common in females), decreased fertility in both sexes, male-pattern hair loss, menstrual irregularities and oligo-anovulation in females, and oligospermia in males. Affected children may develop advanced bone age and subsequent short stature in their adulthood. In female new born babies, clitoromegaly/ambiguous genitalia may be seen (5, 82, 83).

Clinical manifestations of glucocorticoid deficiency are rare in adult patients but are reported in neonates/young children as severe hypoglycemia and associated seizures/coma, because gluconeogenesis depends on the proper action of glucocorticoids in the liver during early childhood (84-86). Some adult patients develop anxiety and/or chronic fatigue, which appear to be caused by elevated hypothalamic CRH and/or AVP (87-92). Increased circulating ACTH may cause bilateral adrenal hyperplasia (5). Some patients harbor adrenal incidentalomas (93, 94). Although this adrenal neoplasm is very common in general population (95), elevated circulating ACTH may facilitate tumor development and/or its growth. Further, one patient with this syndrome harbored an ACTH-producing pituitary adenoma, which might have been caused/facilitated by the elevated CRH/AVP (96).

Finally, the clinical spectrum of this syndrome is broad, ranging from severe to mild forms, and a number of patients may even be asymptomatic, displaying biochemical alterations only (5, 93, 97, 98). This heterogeneity is mainly due to variable impact of the patients’ genetic changes in the receptor protein, but other factors, such as their genetic backgrounds and/or epigenetic and biochemical changes, for example, associated with their ageing and lifestyles, may also contribute to variability of disease expression.

NR3C1 Gene Mutations That Cause Primary Generalized Glucocorticoid Resistance Syndrome

The molecular basis of this syndrome is ascribed to inactivating mutations in the NR3C1 gene, which impair molecular actions of hGRα and hence decrease tissue sensitivity to glucocorticoids. Currently, 36 pathologic mutations that cause this syndrome have been reported (Table 1 and Figure 3). Chrousos, et. al., reported the first family of this syndrome who carried a homozygous miss-sense mutation, which replaces adenine by thymine at nucleotide position 1,922 (80). The NR3C1 gene harboring this mutation expresses the hGRαD641V mutant receptor, which has valine (V) instead of aspartic acid (D) at amino acid position 641 in the LBD (80). Since then, numbers of patients were reported whose pathologic mutations were identified mostly as heterozygous in the coding sequence of LBD (90, 96, 99-102). Most of these patients demonstrated characteristic manifestations, such as those of mineralocorticoid and androgen excess, similar to the original case of Chrousos, et. al., thus they may be considered as “classic cases”. More recently, technological progress in the genome sequencing including the use of capillary or high through-put next generation sequencers enabled clinical researchers to conduct large studies with recruitment of the subjects with conventional/unconventional manifestations, (e.g., obesity and bilateral adrenal incidentalomas, as evident in the French Muta-GR study (ClinicalTrials.gov Identifier: NCT02810496) (97)). Clinicians are now able to obtain much easier and faster than before the data of patients’ genome sequence around the NR3C1 gene. Together with growing acknowledgement of this syndrome among clinicians and clinical researchers, such technological progress appears to have facilitated the discovery of new cases with classic symptoms, as well as those with much milder and/or alternative manifestations or even with biochemical changes only. Further, the identified mutations tend to distribute over the entire NR3C1 gene including coding areas of all three major domains and intronic sequences (Table 1 and Figure3).

Among 36 pathologic NR3C1 mutations, only three are homozygous mutations, while the other 33 are heterozygous (Table 1 and Figure 3). One patient harbors two different NR3C1 mutations each of which are identified in different alleles (thus, compound heterozygous) (86). Among 34 mutations found in the NR3C1 coding sequence, 24 are miss-sense mutations, which replace one amino acid with another (thus, point mutations), five are non-sense mutations, which introduce a stop codon and generate truncated receptor proteins, and another five are frame-shift mutations, which also develop truncated receptors but with additional unrelated amino acids after the mutation point. At the receptor protein level, 22 mutations are located in LBD, two in HR, seven in DBD, and four are in NTD (Figure 3). In addition to these coding sequence mutations, two mutations are identified in the intronic sequence, located in intron F (between exon 5 and 6) and in intron I (between exon 7 and 8), respectively (91, 103).

*: The case also harbors a heterozygous mutation in the 21-hydroxylase gene.

^†:  The 1201G>T D401H mutation causes mild glucocorticoid hypersensitivity.

N.D.; not determined。

Figure 3. Location of the NR3C1 gene mutations that cause primary generalized glucocorticoid resistance syndrome†. Currently, 36 independent mutations are reported. The mutations identified in the coding sequence of LBD, HR, DBD and NTD are shown in a light green, green, yellow and red box, respectively. Miss-sense mutations, non-sense mutations and frame-shift mutations are shown with black, purple and blue letters, respectively. Two mutations identified in the intronic sequence are shown with red letters. Homozygous mutations are shown with underlines. †: The 1201G>T D410H mutation causes mild glucocorticoid hypersensitivity; *: The same miss-sense mutation but found in unrelated subjects/families; $: Prediction only (the mutated hGR protein was not biologically identified); #: These two mutations were found as compound heterozygous in one affected subject. Numbers of nucleotides and amino acids are based on the transcription initiation site and the first methionine of the hGR protein, respectively. DBD: DNA-binding domain; HR: hinge region; LBD: ligand-binding domain; NTD: N-terminal domain

Molecular Defects of Pathologic hGRa Mutants

Molecular defects of pathologic mutant receptors have been extensively investigated by focusing on their defects in ligand-association, transactivation of glucocorticoid-responsive genes, cytoplasmic to nuclear translocation, and others (5). Recently, computer-based in silico structural simulation has also been used for estimating the structural impact of mutations to hGRa LBD and DBD (88, 114).

Pathologic mutant receptors generally cause inactivation/reduction of one or some of the receptor functions, whereas they are in most cases heterozygous mutations that enables affected subjects to harbor both mutated and intact hGRaprotein in their tissues (5, 6). Thus, affected subjects of this syndrome demonstrate partial loss of glucocorticoid actions in their tissues, consistent with the experimental evidence that genetic knock-out (inactivation) of the Nr3c1 gene in mice (thus, complete abbreviation of the GR protein and its actions) is lethal (115). However, one homozygous case who only expresses a mutant receptor with complete loss of the ligand-binding activity was reported (2318-2319delTG F774SfsX24) (85). Given that the ligand-binding is essential for subsequent receptor activation, this mutant receptor might have residual activities including minimal association to glucocorticoids or other steroids, enabling the patient to survive even though he only expresses this highly damaged receptor.

LBD MUTATIONS

There are 22 pathologic mutations whose amino acid changes are identified in the LBD. Among them, 17 are miss-sense mutations (see Table 1 and Figure 3 for details), one is a non-sense mutation (1992A>T Y660X) (115), and four are frame-shift mutations (1762-1765insTTAC>G H588LfsX5, 1835delC S612YfsX15, 2317-2318delCT L773VfsX25 and 2318-2319delTG F774SfsX24) (85, 92, 109, 113). Since LBD is the domain harboring a majority of receptor functions with established evaluation means (15), molecular defects of these mutant receptors have been most extensively and systemically investigated. These molecular examinations include: i) the affinity of the mutant receptors for the ligand (the synthetic pure glucocorticoid dexamethasone was used in most cases, thus the method is called “dexamethasone binding assay”); ii) the transcriptional activity of the mutant receptors on endogenous glucocorticoid-responsive genes and/or transiently introduced exogenous GRE-driven reporters; iii) the ability of in vitro physical interaction of the mutant receptors with p160-type nuclear receptor coactivators, such as the glucocorticoid receptor-interacting protein 1 (GRIP1 or NCoA2); iv) the subcellular localization of the mutant receptors and their nuclear translocation in response to glucocorticoids (in most cases, dexamethasone was used as a ligand); v) the ability of the mutant receptors to bind endogenous DNA GREs (using the chromatin-immunoprecipitation (ChIP) assay); vi) the structural analysis on the mutant receptors’ LBDs by employing the computer-based in silico three-dimensional (3D) simulation using as a template crystallographic data of the LBD peptide; vii) the motility of the mutant receptors inside the nucleus using the fluorescence recovery after photobleaching (FRAP) analysis.

Molecular defects in two major functions of the hGRa, the ability to bind glucocorticoids and the transactivation of glucocorticoid-responsive genes are summarized in Table 1. Compared with the wild-type receptor, all mutant receptors demonstrate variable reduction in their affinity to dexamethasone, and attenuate their transactivation of GREs-driven genes following exposure to this steroid, with the most severe impairment observed in the cases of I559N, V571A, D641V, L672P, R714Q, I747M, L773P, L773VfsX25 and F774fsX24 mutations (80, 82, 84, 85, 96, 99, 100, 102, 110, 113). In the in silico 3D structural simulation analysis on LBD of the miss-sense point mutant receptors, most of the replaced amino acids are located outside the molecular structures, which directly mediate these two major functions, LBP and the AF-2 surface, respectively (114). The latter is used for physical interaction with the LxxLL motif of p160-type coactivators (58). Further analysis revealed that these point mutations damage and/or alter multiple intramolecular amino acid interactions necessary for maintaining the proper structural conformation of LBD, resulting in the alteration in these two protein surfaces indirectly but simultaneously (114). More detailed structural analysis revealed that the amino acid replacements damage LBP by indirectly reducing the electrostatic interaction between key residues of LBP and those of the dexamethasone molecule (especially, the interaction formed against the carbonyl oxygen of carbon (C) 3 of this steroid) (114). Their impact on the interaction between the AF-2 surface and the LxxLL motif of the p160-type coactivator GRIP1 protein is variable, but tends to damage the ionic interaction (or salt bridge) of non-core leucines of this motif as well as the noncovalent interaction of its core leucine residues formed against key amino acids of the AF-2 surface, ultimately reducing the affinity of this motif to the hydrophobic cleft of the AF-2 surface (114).

The C-terminal portion of the hGRa LBD that follows the a-helix-12 of this domain is one of the hot spots of pathologic hGRa mutations, as evident in the accumulation of three independent mutations to this region (L773P, L773VfsX25 and F774fsX24) (85, 90, 113). Indeed, this molecular area is particularly important for creating the AF-2 surface and for maintaining the ligand-bound LPB conformation through its dramatic intramolecular shift upon binding to a ligand (30). Arginine (R) at amino acid position 714 is another hot spot of the point mutations, as three patients independently harbor this mutation that replaces this amino acid to glutamine (Q) (84, 86, 112). In the structural simulation analysis on the R714Q mutant receptor, substitution of R for Q in LBD causes a rearrangement of the side chains resulting in forming a new salt bridge between R704 and D662 and displacing Q714 (84). This relaxes some constraint on the helix-10 and results in structural changes throughout the LBD, indirectly damaging conformation of both LBP and the AF-2 surface (84). Interestingly, the third case with the R714Q mutation harbors another point mutation (592G>T E198X) in the other allele (compound heterozygous), which generates a truncated receptor at E198 (E198X) (86). Thus, the patient expresses both R714Q and E198X mutant receptors but no intact receptor in her tissues.

The LBD mutant receptors frequently demonstrate delay of their translocation from the cytoplasm to the nucleus compared to the wild-type receptor, consistent with the fact that the ligand-binding “turns on” the nuclear translocation of the receptor by inducing the conformational change that allows the receptor to expose NL1 and NL2 surfaces to their counterpart nuclear import systems (7, 84, 85, 89, 90, 98-100, 102, 116). Although detailed molecular mechanisms underlying this defect have not been examined yet, it is likely that the mutations interrupt proper functions of these domains (32). Some mutant receptors, such as hGRaV729I and hGRaF737L, shift their subcellular localization toward the nucleus in the absence of ligand (7, 100), possibly by their defective intracellular circulation, such as through defective NL1 activity and/or altered interaction with14-3-3 proteins, calreticulin or others.

All LBD mutant receptors tested for their interaction with DNA GREs preserve their ability to bind this recognition sequences, because they have intact DBD, which can function independently to LBD (7, 84, 85, 89, 90, 98-100, 102, 116). Further, many of these mutant receptors demonstrate a dominant negative effect on the transcriptional activity of the wild-type receptor, because they are in most cases partially active mutants, and thus, can interfere with the full activity of the wild-type receptor, such as by competing for the molecules mediating the latter’s transcriptional activity (e.g., by squelching transcriptional cofactors including p160-type coactivators) (5, 6, 102). Finally, the LBD point mutant receptors tested in the FRAP analysis demonstrate dynamic motility defects inside the nucleus of living cells, possibly due to their reduced affinity to ligand and/or inability to interact properly with key cofactors and/or chromatin molecules (117).

Molecular characterization of the LBD mutants explained above have been performed mostly by employing cell-based bioassays. However, Kaziales, et. al., recently performed in vitro biochemical assays on the L773P mutant receptor by employing its purified peptide consisting of DBD, HR, and intact or mutated LBD (118). The “wild-type” receptor peptide (called GRm) employed for their assays harbors multiple amino acid replacements for conferring its peptide stability. Thus, the authors compared GRm and GRmL773P, and found that the latter has altered physical interaction with HSP90 (118). They suggested that this molecular defect underlies the reduced interaction of the receptor peptide to dexamethasone, the LxxLL motif, and further, DNA GREs, although exact molecular evidence and associated mechanisms were not demonstrated.

HR MUTATIONS

Two pathologic mutations were identified in HR (Table 1 and Figure 3). One is a non-sense mutation (1471 C>T R491X) and the other is a miss-sense mutation (1503 G>T Q501H) (97). Both are located in exon 5. The patient harboring R491X developed typical manifestations of Chrousos syndrome, as the mutant receptor lacks the entire LBD (97). On the other hand, the subject harboring Q501H demonstrated biochemical changes only, while the mutant receptor showed weakly reduced transactivation of the exogenous glucocorticoid-responsive gene (97).

DBD MUTATIONS

Currently, seven pathologic mutations were identified in DBD (Table 1 and Figure 3). Among them, five are miss-sense (point) mutations. The other two are a non-sense mutation and a frame-shift mutation. All five point mutant receptors reduce or lose their affinity to DNA GREs  (88, 92, 93, 97, 107). In contrast, they retain intact affinity for ligand dexamethasone, because DBD and LBD function independently with each other (88, 92, 93, 97, 107). Among these point mutations, three (1430G>A R477H, 1429C>T R477S and 1429C>T R477C) replace arginine (R) at amino acid position 477 to other amino acids (histidine (H), serine (S) and cysteine (C), respectively), while one targets tyrosine (Y) at position 478 and changes it to cysteine (C) (1433A>G Y478C). Thus, the area around R477 and Y478 appears to be a hot-spot of DBD mutations. These two amino acids are located just C-terminally to the fourth cysteine residue of the second zinc finger of DBD, which participates in holding a zinc ion together with the other three cysteines of this finger motif. R477 is critical for maintaining the ability of the receptor to bind GREs by providing the hydrophobicity required for its interaction with the backbone chain of the GRE DNA. Thus, replacement of either of these two amino acids seems to reduce the affinity of the mutant receptors to the GRE DNA through damaging this local hydrophobicity.

The point mutation 1268T>C V423A replaces valine (V) at amino acid position 423 to alanine (A) (88). V423 is located just N-terminally to the second cysteine of the first zinc finger of DBD. Replacement of this valine to alanine at amino acid position 423 permits water molecules to diffuse into the zinc-binding region of the receptor and indirectly damages the hydrophobicity maintained by R477, leading to the reduction in the affinity of this mutant receptor to the GRE DNA (88).

Interestingly, the mutant receptors V423A, R477S and Y478C demonstrate delayed cytoplasmic to nuclear translocation upon exposure to dexamethasone (88, 93). Molecular defect(s) underlying this impairment have(s) not been elucidated, but these mutations appear to affect indirectly the function of NL1, because this molecular surface spans over the second zinc finger of DBD, while these mutations damage the hydrophobic circumstance around this finger (88, 107). The second zinc finger of the DBD is also critical for receptor homodimerization, which is a prerequisite for the receptor to bind a tandem GREs and subsequent transactivation of glucocorticoid-responsive genes harboring this DNA sequence (119). Thus, defective homodimerization may also contribute to the reduced transcriptional activity of these DBD mutant receptors.

NTD MUTATIONS

Four independent point mutations are reported in NTD. These include 26C>G P9R, 367G>T Q123X, 592G>T E198X and 1201G>T D401H (86, 87, 104, 105). The 367G>T Q123X and the 592G>T E198X are non-sense mutations generating truncated receptors, respectively at amino acid position 123 and 198. Because both receptors appear to be highly damaged as they lack the entire DBD and LBD, the affected subjects demonstrated clear-cut manifestations of Chrousos syndrome (86, 87).

The patient harboring the 26C>G P9R mutation demonstrated mild clinical manifestations with slight increase in ACTH and cortisol secretion (104). Molecular characterization was not performed for this mutant receptor (104), thus there is a possibility that the identified nucleotide change is not pathologic. Indeed, NTD (exon 2) is the domain most harboring single nucleotide polymorphisms (SNPs) among all three major domains throughout the nuclear hormone receptor genes (13), thus this domain can well tolerate to nucleotide replacements and tends to maintain its proper functions compared to the other domains.

The patient harboring the 1201G>T D401H mutation demonstrated mild hypersensitivity to glucocorticoids in contrast to the other pathologic mutations that cause glucocorticoid resistance (105). Compared to the wild-type receptor, the D401H mutant receptor demonstrated ~2-fold stronger transcriptional activity in a reporter assay, which is equivalent to the activity of the N363S mutant receptor in a side-by-side assay. The nucleotide change causing the N363S replacement is a well-known polymorphism associated with mild glucocorticoid hypersensitivity (120-122). Thus, the 1201G>T D401H may be another weakly functional polymorphism causing mild tissue hypersensitivity to glucocorticoids.

The 3-year-old girl with the 592G>T E198X mutation additionally harbors the 2141G>A R714Q mutation in the other allele as explained in the section of “LBD mutations” (86). She developed severe manifestations of glucocorticoid resistance, such as uncontrollable hypertension, brain micro-infarctions, and hypoglycemic coma, because both mutant receptors she harbored are highly damaged. The family study revealed that the 592G>T E198X mutation is maintained among her family, while the 2141G>A R714Q mutation is de novo in the affected girl (86).

INTRONIC MUTATIONS

So far, only two intronic mutations are reported. One is the 1891-1894 delGAGT NR, which deletes four nucleotides (GAGT) at the nucleotide position 1891-1894 (in intron F located between exon 4 and 5) that destroys the intron-acceptor site located 5’ terminally to exon 6 (91, 103). The mutated mRNA expressed from the affected allele loses its biological stability, therefore the mutation functionally “knocks-out” NR3C1 of this allele (103). The amount of patient’s tissue hGRa is thus 50% of the healthy subjects as the receptor protein is only expressed from the intact allele (103). The other mutation is the 2024G>T, which replaces G with T at the position one nucleotide 5’ terminally to exon 8 (thus, located at the 3’-terminal portion of intron I) (91). Although biochemical characterization on the mutant receptor was not performed, the computer-based prediction indicated that the mutation appears to cause a skip of the entire exon 8 and to generate the V675GfsX10 truncated receptor whose molecular function appears to be highly damaged (91). It is also possible that the mutation reduces stability of its mRNA, leading to functional “knock-out” of NR3C1 of the affected allele similar to the 1891-1894 delGAGT NR mutation (103). Thus, biochemical evaluation on the mutated mRNA and hGRaprotein is needed.

Clinical Evaluation of Patients with Primary Generalized Glucocorticoid Resistance Syndrome

Key for evaluating patients with this syndrome is to identify the manifestations suggesting upregulation of the HPA axis without Cushingoid features (5) (Table 2). Circadian rhythmicity of circulating ACTH and cortisol should be preserved, in contrast to the patients with Cushing syndrome (5). In addition, any evidence suggesting psychiatric problems (e.g., anxiety and depression), possibly through upregulation of brain CRH and/or AVP may be noted (5).

Physical examination should include an assessment for signs of hypertension and associated metabolic alkalosis caused by elevated levels of adrenal mineralocorticoids (5). Arterial blood pressure should be recorded and should be monitored over a 24-hour period. Signs of hyperandrogenism and/or virilization caused by over-production of the adrenal androgens, such as acne, hirsutism, pubic and axillary hair development, male-pattern hair loss, and clitoromegaly, should be evaluated. Hirsutism should be assessed using the Ferriman-Gallwey score (123), while pubic hair development should be classified according to the Tanner scale (124, 125). All subjects should be screened for signs associated with Cushing syndrome or therapeutic use of high-dose glucocorticoids.

Endocrinological Evaluation of Patients with Primary Generalized Glucocorticoid Resistance Syndrome

The aim of the endocrinological evaluation is to demonstrate up-regulation of the HPA axis with preservation of its normal circadian rhythmicity and blunted responsiveness to exogenous glucocorticoids (5). Concentrations of plasma ACTH, renin activity and aldosterone, as well as serum cortisol, corticosterone, deoxycorticosterone, testosterone, androstenedione, DHEA, and DHEA-S should be measured. Determination of 24-hour UFC excretion on 2 or 3 consecutive days is important to access the presence of hypercortisolism. Diurnal fluctuation of plasma ACTH and serum cortisol should be evaluated, for example, by monitoring them both in the morning and in the evening.

Responsiveness of the HPA axis to exogenous glucocorticoids should be examined using the dexamethasone suppression test (5). Increasing doses of dexamethasone (e.g., 0.3, 0.6, 1.0, 1.5, 2.0, 2.5, and 3.0 mg) should be given orally at midnight every other day, and a serum sample should be drawn at 0800h the following morning for determining serum cortisol concentrations. Affected subjects demonstrate resistance of the HPA axis to administered dexamethasone but can respond to higher doses. Concurrent measurement of serum dexamethasone concentrations is recommended in order to exclude the possibility of increased metabolic clearance or decreased absorption of this compound (83). Pituitary and adrenal imaging studies should be performed, because patients with this syndrome frequently harbor hypertrophy of these organs or may develop their benign tumors. 

Cellular and Molecular Studies on Patients with Primary Generalized Glucocorticoid Resistance Syndrome

The purpose of cellular studies is to identify the presence of tissue resistance to glucocorticoids in actual tissues of the affected subjects. The thymidine incorporation assay and the dexamethasone binding assay employing subjects’ peripheral blood mononuclear cells (PBMCs) are generally employed (5, 126) (Table 2). In the former assay, dexamethasone administration strongly suppresses phytohemagglutinin-stimulated thymidine incorporation of PBMCs in normal subjects. However, this response is significantly blunted in the affected subjects due to reduced affinity/actions of this steroid in these cells. The dexamethasone binding assay can address reduction in the affinity of patients’ tissue hGRa to dexamethasone, because mutant receptors harboring their defects in LBD almost always show reduced affinity for this steroid.

As part of the molecular examination for verifying pathologic causes and their molecular mechanisms, sequencing of the coding region of the NR3C1 gene including exon/intron junctions should be performed (126). Identification of mutations in the NR3C1 gene is critical for diagnosing this syndrome. Once mutations are identified, the next step is to prove that the identified mutations have biologic impact. Because the NR3C1 gene harbors so many neutral polymorphisms (13), there is always a possibility that the identified nucleotide changes are just coincidental but not pathologic. Population incidence of the identified nucleotide changes is important if available, as pathologic mutations generally have a very low allele frequency. Molecular studies can be started by constructing the mutant hGRa-expressing plasmids. Then, molecular actions of mutant receptors can be examined by transfecting the created plasmids (transiently or stably) to appropriate cell lines (e.g., GR-negative African green monkey kidney CV1 and COS7 cells, and GR-positive human cervical cancer HeLa cells). Using mutant receptor-expressing cultured cells, reporter transactivation assays using the GREs-driven luciferase gene can be performed to address the reduced transcriptional activity of mutant receptors. The dexamethasone binding assay can also be performed in the COS7 cells transiently expressing mutant receptors to evaluate their affinity to dexamethasone in the absence of the wild-type GR. In microscope-based imaging studies on the cells transfected with plasmids expressing mutant receptors, their abnormal subcellular localization and delayed nuclear translocation in response to dexamethasone can be evaluated.

Management of Patients with Primary Generalized Glucocorticoid Resistance Syndrome

The aim of the treatment for patients with this syndrome is to suppress the excess ACTH secretion in order to reduce production of the adrenal steroids with mineralocorticoid and/or androgenic activity to minimize their pathologic effects (5). Treatment involves the administration of high doses of mineralocorticoid activity-sparing pure glucocorticoids (e.g., dexamethasone), which activate mutated and/or wild-type hGRα in the hypothalamus/pituitary gland of the affected subjects and suppress their ACTH secretion. Adequate suppression of the HPA axis is of particular importance, given that the treatment is virtually life-long, thus any side effects of exogenous glucocorticoids should be avoided as much as possible. Long-term dexamethasone treatment should be titrated carefully according to the clinical manifestations and biochemical profiles of the affected subjects.

CONCLUSIVE REMARKS AND FUTURE PERSPECTIVES

Primary generalized glucocorticoid resistance syndrome is characterized by hypercortisolism without Cushingoid features but with manifestations caused by upregulation of the HPA axis, such as hypertension (by mineralocorticoid excess) and signs of hyperandrogenism (by adrenal androgen excess) (81). The pathologic cause of this syndrome is ascribed to mutations in the NR3C1 gene, which decrease the action of its encoding protein hGRa, a ligand-dependent transcription factor (15, 81). In honor to Professor George P. Chrousos who discovered the first case and significantly contributed to the progress of this field, this syndrome may be called “Chrousos syndrome”, particularly for the cases who demonstrate classic and characteristic manifestations of this syndrome (6, 80). Recent progress in genome technology including high through-put sequencing has enabled clinical researchers to handle large patient cohorts and clinicians can get access to the NR3C1 gene sequencing much easier and faster than before. Consequently, 35 cases/families of this syndrome are currently reported world-wide who harbor pathologic mutations in the NR3C1 gene. It is of note that some of the recent cases tend to demonstrate much milder manifestations compared to the classic cases of Chrousos syndrome (97, 110). Further, some of them even lack obvious manifestations but show biochemical or imaging abnormalities only (93, 97). For these cases with very mild or no manifestations, their genetic changes may be considered as rare polymorphisms rather than pathologic mutations. Further discussion is needed for distinguishing pathologic mutations and mildly functional polymorphisms based on their clinical manifestations and allele frequency of the nucleotide changes. 

In some reported cases, molecular defects of the mutated receptors were not evaluated. Testing them in tandem with the wild-type receptor is crucial for avoiding false-diagnosis, because the NR3C1 gene harbor substantial numbers of biologically silent polymorphisms (13). On the other hand, there are patients who demonstrate characteristic manifestations of Chrousos syndrome but do not harbor pathologic mutations in the NR3C1 gene. These “mutation-silent” subjects might carry their genetic defects not in NR3C1 but in other genes whose encoding proteins function in the glucocorticoid signaling pathway. For example, there was a boy who demonstrated manifestations compatible with multiple steroid hormone resistance (127). He harbored a small gene segmental deletion around one zinc finger protein (ZNF) gene, and Its encoding protein ZNF764 turned out to function as a coactivator of several steroid hormone receptors including the hGRa (127). As our knowledge of the glucocorticoid signaling pathway increases, including new players like long non-coding RNAs (15, 128, 129), we hope that genetic cause(s) of undiagnosed cases with Chrousos syndrome will soon be identified, by employing classic genetic methods (e.g., the linkage analysis) as well as cutting-edge genome-related methodologies including the whole genome/exome sequencing and sophisticated bioinformatical/statistical analysis tools.

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