ABSTRACT

Congenital adrenal hypoplasia is a rare cause of primary adrenocortical failure, which was first described in 1948. During the last two decades, the genetic basis for several forms of familial adrenal insufficiency syndromes has been elucidated. The molecular mechanisms for these disorders involve a broad spectrum of cellular and physiologic processes, including metabolism, nuclear protein import, oxidative stress defense-mechanisms, and regulation of cell cycle. Adrenal hypoplasia can occur: 1) secondary to defects in transcription factors involved in pituitary development or (2) defects in ACTH synthesis and secretion; 3) as a primary defect in the development of the adrenal gland; 4) as part of rare syndromes associated with adrenal hypoplasia/aplasia, which are inherited in an autosomal recessive or autosomal dominant manner; and 5) in the context of chromosomal abnormalities. Early diagnosis and management are crucial because of the life-threatening nature of the condition. Depending on the etiology, adrenal crisis may occur in early infancy or could insidiously develop over the course of childhood or adolescence. Moreover, some of these conditions previously thought to occur only in childhood, may also be diagnosed later in adulthood and present with variable phenotypes, including isolated infertility or disorders of sex differentiation. The clinical manifestations of primary adrenal insufficiency (PAI) result from deficiency of all adrenocortical hormones (aldosterone, cortisol, androgens). The acute presentation can be precipitated by physiologic stress, such as surgery, trauma, or an intercurrent infection. Patients may present with signs and symptoms of complete adrenal insufficiency, usually early in life, including hypoglycemic convulsions, hyponatremia, hyperkalemia, metabolic acidosis or later with hyperpigmentation, vomiting and poor weight gain. It should be remembered, that the most common cause of PAI in children is congenital adrenal hyperplasia due to 21-hydroxylase deficiency and can be excluded by measuring baseline or ACTH-stimulated 17-hydroxyprogesterone levels in serum. Screening for autoimmune Addison disease includes detection of 21-hydroxylase antibodies. Males with negative 21-hydroxylase antibodies should be tested for adrenoleukodystrophy measuring very–long-chain fatty acids concentrations in plasma. The presence of alacrima in patients with PAI should raise suspicion for Triple A syndrome, whereas the combination of PAI and hypogonadotropic hypogonadism in a male patient point towards X-linked adrenal hypoplasia congenita. To date, molecular genetic testing is commercially available for the identification of several genes involved in adrenal hypoplasia syndromes. The early identification of these diseases can have important prognostic and therapeutic implications for patients with respect to surveillance for associated conditions, initiation of early treatment or screening of family members who are at risk. Adrenal insufficiency is potentially life threatening, thus treatment should be initiated as soon as the diagnosis is confirmed, or sooner if the patient presents in adrenal crisis. Therapy consists of life-long replacement therapy with glucocorticoids and mineralocorticoids. Hypogonadism or other associated disorders should be treated appropriately. Screening of family members for the disease or carrier status may also be indicated and can be critical for family planning. When a monogenic cause of adrenal failure is identified, genetic counseling is indicated. For complete coverage of all related areas of Endocrinology, please visit our on-line FREE web-text, WWW.ENDOTEXT.ORG.

INTRODUCTION

The adrenal glands consist of two anatomically and functionally distinct subunits, the cortex and the medulla. The adrenal cortex secretes glucocorticoids, mineralocorticoids and androgens. The glucocorticoid, cortisol, is secreted by the cells of the intermediate zona fasciculata. Its secretion is tightly regulated by the hypothalamic corticotropin-releasing hormone (CRH) and vasopressin (AVP) and by the pituitary adrenocorticotropic hormone (ACTH) (1). Glucocorticoids regulate a broad spectrum of physiologic functions essential for life and play an important role in the maintenance of basal and stress-related homeostasis. The mineralocorticoid, aldosterone, is produced by the outer adrenal zona glomerulosa. This steroid regulates water and electrolyte homeostasis and its secretion is primarily under the control of the renin-angiotensin system, although it may be weakly influenced by ACTH. The adrenal androgens, dehydroepiandrosterone (DHEA), its sulfate (DHEA-S) and androstenedione, are secreted by the inner zona reticularis under the control of ACTH.

CONGENITAL ADRENAL HYPOPLASIA

Congenital adrenal hypoplasia is a rare cause of primary adrenocortical failure, which was first described in 1948. It has an estimated frequency of 1:12,500 live births (2). During the last decade there have been significant advances in our understanding of the genetic etiology of several forms of adrenal insufficiency with a presentation in infancy or childhood. Several of these conditions affect adrenal development and are commonly known as adrenal hypoplasia. Adrenal hypoplasia may be due to (3-9):

1. Secondary to defects in transcription factors involved in pituitary development (e.g. HESX1, LHX4, SOX3) or defects in ACTH synthesis (TPIT), processing and release (e.g. POMC or PC1);
2. Part of an ACTH resistance syndrome [MC2R/ACTH receptor, MRAP, AAAS (triple A syndrome), StAR, CYP11A1, MCM4, NNT, TXNRD2, GPX1, PRDX3 mutations];
3. A primary defect in the development of the adrenal gland itself (primary/congenital adrenal hypoplasia; X-linked form/DAX1 gene mutations or deletions, autosomal recessive form/SF-1 gene mutations or deletions, autosomal recessive form of uncertain etiology, IMAGe syndrome, MIRAGE syndrome, Familial steroid-resistant nephrotic syndrome with adrenal insufficiency due to SGPL1 deficiency);
4. Part of rare syndromes associated with adrenal hypoplasia/aplasia, which are inherited in an autosomal recessive (Meckel-Gruber syndrome, Pena-Shokeir syndrome, Pseudotrisomy 13, Hydrolethalus syndrome, Galloway-Mowat syndrome) or autosomal dominant (Pallister-Hall syndrome) manner; and
5. In the context of chromosomal abnormalities (tetraploidy, triploidy, trisomy 18, trisomy 21, 5p duplication, monosomy 7 and the 11q syndrome), which are often associated with central nervous system (CNS) abnormalities.

There are two distinct histological patterns of the adrenal cortices in this rare syndrome, the miniature adult and cytomegalic forms. In the miniature adult form of adrenal hypoplasia congenital (AHC), the small amount of residual adrenal cortex is composed primarily of permanent adult cortex with normal structural organization. The miniature adult form is either sporadic or inherited in an autosomal recessive manner, and is frequently associated with abnormal CNS development, including anencephaly or pituitary gland abnormalities.

In the cytomegalic form of AHC, the residual adrenal cortex is structurally disorganized with scattered irregular nodular formations of eosinophilic cells, with the adult permanent zone absent or nearly absent. Enlarged cells are present, some with abundant vacuolated cytoplasm. The cytomegalic form is generally considered to be X-linked, but there may be one or more autosomal genes associated with this phenotype (6, 10, 11).

Genetic causes of adrenal hypoplasia and aplasia syndromes are summarized in Table 1. However, this review focuses on ACTH resistance syndromes and disorders of adrenal gland development.

AAAS=achalasia, adrenocortical insufficiency, alacrima syndrome. CDKN1C= Cyclin-dependent kinase inhibitor 1C (p57, Kip2). CLAH=Congenital Lipoid Adrenal Hyperplasia. CYP11A1= Cytochrome P450, family 11, subfamily A, polypeptide 1. DAX1= Dosage sensitive sex reversal, Adrenal hypoplasia congenita, critical region on X chromosome, gene-1. DOX7=Docking protein 7. FGD: familial glucocorticoid deficiency. FSH: Follicle stimulating hormone. GLI3=gene responsible for Greig cephalopolysyndactyly syndrome (GCPS), Pallister-Hall syndrome (PHS), Preaxial polydactyly type IV and Postaxial polydactyly type-A1 and B. GPX1= Glutathione Peroxidase 1. HYLS1= Hydrolethalus syndrome protein 1. IMAGe=Intrauterine growth restriction (IUGR), Metaphyseal dysplasia, Adrenal hypoplasia congenita, and Genitourinary abnormalities.  MC=Mineralocorticoids.  MC2R=Melanocortin 2 receptor. MCM4= Minichromosome maintenance complex component 4. MIRAGE=Myelodysplasia, Infection, Restriction of growth, Adrenal hypoplasia, Genital phenotypes and Enteropathy. MKS1=gene responsible for Meckel syndrome, type 1 and Bardet-Biedl syndrome type 13. MRAP=Melanocortin 2 receptor accessory protein.  NNT= Nicotinamide nucleotide transhydrogenase. NR0B1= Nuclear Receptor subfamily 0, group B, member 1. NR5A1= Nuclear receptor subfamily 5 group A member 1. PRDX3=Peroxiredoxin 3. RAPSN=Receptor-associated protein of the synapse. SAMD9=Sterile Alpha Motif Domain-Containing 9. SF-1=Steroidogenic factor 1. SGPL1= Sphingosine-1-Phosphate Lyase 1. Shh= Sonic hedgehog. StAR= Steroidogenic acute regulatory protein. TXNRD2= Thioredoxin reductase 2. WDR73= WD repeat domain 73.

* To date, nine StAR mutations have been reported in patients with NCLAH (30).

**Leydig cell adenoma identified in one case (40).

¶ Heterozygous loss of function mutations in NNT gene (42).

‡ TXNRD2 mutations have been detected in 3 out of 227 patients with a diagnosis of dilated cardiomyopathy, however, no data are available on their adrenal function (15).

ADRENAL HYPOPLASIA AS PART OF AN ACTH RESISTANCE SYNDROME   

ACTH resistance syndromes include two distinct genetic disorders, both of which are inherited in an autosomal recessive manner and are characterized by ACTH insensitivity:

1. Familial Glucocorticoid Deficiency (FGD)
2. Allgrove syndrome or Triple A syndrome

Familial Glucocorticoid Deficiency (FGD)

Familial (isolated) glucocorticoid deficiency (FGD), which is also known as hereditary unresponsiveness to ACTH, is a rare autosomal recessive disorder characterized by glucocorticoid deficiency (12, 13).  The underlying genetic defect is known in approximately 70% of patients with FGD.

CLINICAL AND LABORATORY FEATURES OF FGD

Patients with FGD are usually diagnosed during the neonatal period or in early childhood. However, the oldest affected member of the kindred, carrying MCM4 and TXNRD2 mutations (see Genetics below), presented at the age of 8.5 years and 10.8 years, respectively (14, 15). Patients with FDG may present with hypoglycemic seizures, hyperpigmentation, recurrent infections, transient neonatal hepatitis, failure to thrive, collapse and coma. The long-term neurological sequelae of FGD can vary from learning difficulties to spastic quadriplegia, which may reflect the severity and number of hypoglycemic episodes in childhood. There may be a family history of unexplained neonatal death, history of other family member(s) affected with FGD and/or parental consanguinity (12, 16).

The clinical manifestations of FGD reflect resistance to ACTH. The typical hormonal profile in FGD is a combination of low cortisol but high plasma ACTH concentrations, in the presence of normal plasma renin activity and aldosterone concentrations. Most patients with FGD have markedly elevated ACTH concentrations, which correlate with the degree of ACTH resistance. Hyperpigmentation is often observed during the first months of life owing to the effect of ACTH on the melanocortin-1 receptors in melanocytes (12).

ADRENAL IMAGING

In the MRI or CT scans, the adrenal glands appear small in size.

HISTOPATHOLOGY

Absence of fasciculata or reticularis cells and disorganization of glomerulosa cells have been observed (17).

GENETICS

FGD was first described by Shepard et al. (18) in 1959, when he reported two siblings with “familial Addison’s disease”. It took 30 years for the first inactivating ACTH receptor mutations to be detected (19, 20). To date, FGD has been associated with mutations in seven genes: MC2R (ACTH receptor/melanocortin 2 receptor) (OMIM 202200), MRAP (MC2R accessory protein) (OMIM 607398), StAR (steroidogenic acute regulatory protein) (OMIM 201710), CYP11A1 (cytochrome P450, family 11, subfamily A, polypeptide 1) (OMIM 613743), NNT (nicotinamide nucleotide transhydrogenase) (OMIM 614736), MCM4 (the mini chromosome maintenance-deficient 4 homolog gene) (OMIM 609981), TXNRD2 (thioredoxin reductase 2) (OMIM 617825), GPX1 (Glutathione Peroxidase 1) and PRDX3 (peroxiredoxin 3) (9, 21,). Mutations in the MC2R and MC2R accessory protein (MRAP) account for approximately 50% of all cases.

The ACTH receptor MC2R is a 7-membrane G-protein coupled receptor located almost exclusively in the adrenocortical cells. To date, more than 50 mutations have been described in the MC2R gene (Human Gene Mutations Database, www.hgmd.cf.ac.uk) and represent the most common cause of FGD (25% of cases, FGD type 1) (8, 16). Some of them are shown in Table 2. FGD type 1 patients usually present in early childhood. Tall stature has been observed in some cases (22).

In 2005, a second gene was identified, located at 21q22.1 and encoding MC2R accessory protein (MRAP), a 19-kDa single-transmembrane domain protein. In humans, MRAP is expressed in the adrenal cortex, pituitary, brain, testis, ovary, breast, thyroid, lymph node, skin, and fat. This protein serves as an essential cofactor of MC2R to promote its trafficking from the endoplasmic reticulum to the cell surface and subsequent signaling in response to ACTH (16, 23-25). Mutations in MRAP are responsible for a further 15-20% of FGD cases (FGD type 2). Most patients with FGD type 2 present in the neonatal period or in very early infancy. However, missense MRAP mutations are associated with a milder phenotype and late onset adrenal insufficiency (AI) (26). Interestingly, obesity has been reported in a patient harboring homozygous MRAP mutations and his heterozygous family members, whereas the only unaffected member of the family had normal weight (25). Studies on Mrap-/- mice demonstrated the important role of MRAP plays in both steroidogenesis and the regulation of adrenal cortex zonation. Mrap-/- mice were shown to have isolated GC deficiency with normal aldosterone and catecholamine production and small adrenal glands with gross impairment of the adrenal capsular morphology and cortex zonation. Furthermore, progenitor cell differentiation was significantly impaired, with dysregulation of WNT4/b-catenin and sonic hedgehog pathways (27). MRAP mutations are summarized in Table 3.

Interestingly, mutations in steroidogenic acute regulatory protein (StAR) and more rarely cytochrome P450 family 11 subfamily A member 1 (CYP11A1) have also been detected in patients with FGD (StAR:  approximately 5% of FGD patients). Mutations in these two enzymes usually result in Congenital Lipoid Adrenal Hyperplasia (CLAH), a severe disorder with both adrenal and gonadal steroid insufficiencies. However, certain, partial loss-of-function mutations may be associated with a milder phenotype with no gonadal derangement, termed non-classic CLAH (NCLAH). To date, nine StAR mutations have been reported in patients with NCLAH. Of note, affected individuals require life-long monitoring of both adrenal and gonadal function because their disorder may evolve. Hypogonadism and infertility may occur in adulthood. (28-31).

Recently, mutations in the mini chromosome maintenance-deficient 4 (MCM4) homolog gene have been identified in an Irish travelling community presenting with a variant of FGD. These patients had short stature, chromosomal breakage, natural killer cell deficiency and progressive primary adrenal insufficiency (PAI) characterized by ACTH resistance with glucocorticoid deficiency and normal mineralocorticoids (MC) levels. Typically, patients started with normal adrenal function and developed PAI over time. The MCM4 gene, mapped on 8q11.2 chromosome, is part of a heterohexameric helicase complex, which is important for DNA replication and genome integrity. MCM4 deficiency leads to genomic instability and is associated with increased incidence of cancer and developmental defects. Therefore, it is recommended that patients carrying this mutation are followed-up closely. The c.71-1insG splice site mutation found in the Irish travelling community was predicted to lead to a frameshift with a prematurely terminated translation product (p.Pro24ArgfsX4) (32, 33).

ΝΝΤ (nicotinamide nucleotide transhydrogenase), a highly conserved gene, encodes a redox-driven proton pump of the inner mitochondrial membrane. This enzyme uses energy from the mitochondrial proton gradient to produce high concentrations of NADPH. Detoxification of reactive oxygen species (ROS) in mitochondria by glutathione peroxidases (GPX) depends on this NADPH for regeneration of reduced glutathione (GSH) from oxidized glutathione (GSSG) to maintain a high GSH/GSSG ratio (Figure 1). The adrenal cortex contains high amounts of P450 steroid enzymes, which use NADPH for their catalytic activity. Its function is therefore very sensitive to ROS (34). ROS may suppress StAR protein synthesis and thus inhibit steroidogenesis (9). In addition, the peroxiredoxin system (PRDX), another antioxidant defense mechanism which removes H₂O₂ and lipid peroxides also requires NADPH (9, 34).  PRDX3 is a mitochondrial protein highly expressed in human adrenals. Inactivation of PRDX3 results in accumulation of H₂O₂, activation of p38 MAPK signaling pathways, suppression of StAR protein synthesis and inhibition of steroidogenesis (9, 35). Mutations in GPX1 and PRDX3 have been rarely identified in patients with FGD (9, 36).

Figure 1. Detoxification of reactive oxygen species in the mitochondria. ΝΝΤ (nicotinamide nucleotide transhydrogenase) is a key enzyme, located in the inner mitochondrial membrane, that plays an important role in maintaining the mitochondrial redox balance. It utilizes the electrochemical proton gradient to generate NADPH from NADH and NADP. NNT provides high concentrations of NADPH for detoxification of H2O2 by the glutathione and thioredoxin pathways. Manganese superoxide dismutase catalyzes the conversion of the superoxide radical Ο2.- to H2O2. The peroxiredoxin system (PRDX), another antioxidant defense mechanism, which removes H2O2 and lipid peroxides also requires NADPH. NNT loss would result in compromised NADPH production, thereby rendering the mitochondria more susceptible to oxidative stress. Modified by Prasad et al (34) and Flück (9).

StAR: Steroidogenic acute regulatory protein; CYP11A1= Cytochrome P450, family 11, subfamily A, polypeptide 1; GSR: glutathione reductase; GSH:  reduced glutathione; TXNRD2: thioredoxin reductase 2; TXN2: thioredoxin 2; GLRX2: glutaredoxin 2; NNT: nicotinamide nucleotide transhydrogenase; PRDX3: peroxiredoxin 3; GPX: glutathione peroxidase; MnSOD: manganese superoxide dismutase.

NNT mutations account for 5–10% of FGD patients. The first mutations in the NNT gene were identified six years ago in 20 patients with FGD (candidate region localized on chromosome 5p13–q12), in whom mutations of MC2R, MRAP and StAR had not been detected. A novel homozygous missense mutation at exon 5 of the NNT gene was subsequently reported in a Japanese patient and was predicted to have a loss-of-function effect (c.644T>C, p.Phe215Ser) (37, 38). In mice with Nnt loss, higher levels of adrenocortical cell apoptosis and impaired glucocorticoid production were observed. NNT knockdown in a human adrenocortical cell line resulted in impaired redox potential and increased ROS levels.

It is of great interest, that two patients from non-consanguineous parents of East Asian and South African origin were diagnosed with FGD at the ages of 21 and 8 months respectively, caused by compound heterozygous mutations in NNT, i.e. a heterozygous intron 20 mutation (pseudoexon activation) in combination with a heterozygous stop-gain mutation in exon 3 of NNT gene (p.Arg71) (21).

Recent studies provide new insights into the effects of NNT deletion. Altered mitochondrial morphology, lower ATP content and increased ROS levels have been observed in fibroblasts derived from a patient harboring biallelic NNT mutations (35). Most recently, it was shown that both NNT loss and overexpression can negatively affect steroidogenesis and cause redox imbalance, resulting in reduced protein levels of two mitochondrial antioxidant enzymes (Prdx3 and thioredoxin reductase 2/Txnrd2) and CYP11A1. Transcriptomic analysis of Nnt−/− mice demonstrated upregulation of heat shock proteins, alpha- and beta-hemoglobins, possibly reflecting activations of compensatory mechanisms to cope with oxidative stress (39).

To date, more than 40 pathogenic variants of NNT gene have been identified. They are scattered throughout the gene, including abolishment of the initiating methionine, and splice, missense and nonsense mutations (35, 40, www.hgmd.cf.ac.uk). Phenotypic heterogeneity has been observed among patients carrying the same mutation or within the same family. Unlike “classic FGD”, adrenal dysfunction is not restricted to glucocorticoid deficiency, but may include mineralocorticoid deficiency as well (35, 40). AI is usually diagnosed around the first year of life, may be severe and present with hypoglycemic seizures

Although, NNT mutations have been known to affect preferentially the adrenal glands, all tissues rich in mitochondria may be affected. Extra-adrenal features have been first demonstrated in Nnt-mutant mice, which had reduced insulin secretion and high-fat diet-induced diabetes mellitus, in addition to adrenal dysfunction (27). More recently, extra-adrenal manifestations were also noted in patients harboring homozygous or compound heterozygous NNT mutations, including: precocious puberty associated with testicular nodules (Leydig cell adenoma identified in one case), hypothyroidism, hypertrophic cardiomyopathy, azoospermia associated with testicular adrenal rests and elevated FSH levels and mild plagiocephaly (40, 41).

Of note, heterozygous loss of function mutations in NNT have been recently identified in two patients presenting with left cardiac ventricular noncompaction, an autosomal-dominant cardiomyopathy, which is frequently associated with mitochondrial disorders and cardiac hypertrophy (42).

In 2014, Prasad et al described the first homozygous mutation in the thioredoxin reductase 2 (TXNRD2) gene in an extended consanguineous Kashmiri kindred presenting with FGD (stop gain mutation, c.1341T>G; p.Y447X within exon 15). The selenoprotein TXNRD2, one of three thioredoxin reductases, is mitochondria specific and contributes to the maintenance of redox homeostasis. Particularly high TXNRD2 mRNA levels have been noted in the adrenal cortex compared with the other human tissues investigated, suggesting a susceptibility of the adrenal cortex and especially zona fasciculata to oxidative stress. Given that the final step of cortisol production, which is catalyzed by CYP11B1 in the mitochondria, accounts for approximately 40% of the total electron flow from NAPDH directed at reactive oxygen species production during steroidogenesis, individuals with TXNRD2 and NNT mutations primarily develop glucocorticoid deficiency. Extra-adrenal manifestations, associated with TXNRD2 mutations have also been reported. Txnrd2 deletion in mice is embryonically lethal, resulting in fatal cardiac and hematopoietic defects. In humans, two novel heterozygous mutations in TXNRD2 were identified in 3 of 227 patients with a diagnosis of dilated cardiomyopathy, however, no data are available on their adrenal function (15, 34).

Oxidative stress has been implicated in other causes of adrenal insufficiency, including triple A syndrome and X-linked adrenoleukodystrophy (ALD). In ALD, mutations in ABCD1 (encoding the peroxisomal ABCD transporter) result in the accumulation of very long-chain fatty acids in the tissues and plasma, the toxic effects of which are thought to result from an increase in steady-state ROS production, depletion of glutathione and dysregulation of the cell redox homeostasis. The adrenal and CNS are most susceptible to the disease process (34).

Triple A Syndrome

Triple A syndrome (OMIM 231550) is an autosomal recessive disorder characterized by ACTH-resistant adrenal insufficiency, achalasia of the esophagus, alacrima (absence of tears) and a variety of progressive central, peripheral and autonomic neurological defects (62). It was first described by Jeremy Allgrove in 1978 (63). It has been estimated that Triple A accounts for approximately 1% of all cases of primary adrenal insufficiency (PAI) with a prevalence of 1 per 1,000,000 individuals (64, 65).

CLINICAL FEATURES OF TRIPLE A SYNDROME

The spectrum of clinical manifestations is unique and encompasses a range of phenotypic abnormalities that vary even within families. Alacrima is the most consistent sign, and is attributed to both autonomic dysregulation and structural abnormalities of the lacrimal glands. Achalasia usually presents within the first two decades of life and may precede the adrenal failure by several years (62, 66). Older children/adults usually complain of dysphagia especially for liquids (67). The pathogenesis of achalasia includes a decrease in non-adrenergic and non-cholinergic neurons, as well as a lack of neuronal nitric oxide synthase in autonomic plexus (68). Adrenal failure does not occur in the immediate postnatal period. It usually presents during the first, or more rarely, the second decade of life, suggesting progressive adrenal destruction or degeneration. However, in some cases it may be the presenting symptom leading to the diagnosis of the condition. AI in Triple A syndrome typically manifests as isolated glucocorticoid deficiency, with less than 15% of patients having evidence of mineralocorticoid deficiency (69, 70).

Neurodegenerative disease may include progressive central, peripheral, autonomic neuropathy (pupillomotor, lacrimotor, erectile dysfunction), sensory and motor defects, hyperreflexia, cerebellar dysfunction, bulbospinal syndrome, distal amyotrophy, amyotrophic lateral sclerosis, spastic paraparesis, syringomyelia, atrophy and myofasciculations of the tongue, epilepsy, pyramidal syndrome, dystonia, dysarthria, ataxia, optic atrophy chorea, deafness, mental retardation, Parkinsonism and dementia (64, 65, 67-69).

Based on data of 133 index cases, alacrima was present in all but one patient (99.2%), achalasia in 93.2%, AI in 90.1% and ND in 79.4%. The most common presenting features were AI and achalasia, followed by neurological dysfunction and alacrima. Eight percent of patients developed clinical features of the syndrome in the 3rd to 5th decade of life, however, none presented with AI (70). The above data support previous recommendations, that in cases of presence of alacrima and at least one more symptom of triple A syndrome, adrenal function testing and molecular analysis should be performed (71).

Moreover, a number of associated features have been described in association with Triple A syndrome, including palmo‐plantar and punctate hyperkeratosis, short stature, osteoporosis, xerostomia, nasal speech, angular cheilitis, glossitis and fissured tongue, enamel defect, poor wound healing, hypolipoproteinemia type IIb, scoliosis, pes cavus, long QT syndrome,   microcephaly and dysmorphic features, such as long narrow face, long philtrum, down-turned mouth, thin upper lip, and lack of eyelashes. Premature loss of permanent teeth has also been reported (62, 64-70, 72-74).

DIAGNOSIS

The diagnosis should be confirmed by the Schirmer test, basal and dynamic endocrine testing, genetic analysis and detailed gastroenterological and neurological evaluation (75). The diagnosis may be extremely challenging, given that the clinical manifestations may evolve at a variable time. Therefore, patients who undergo surgery for achalasia may be at risk of life-threatening adrenal crisis during anesthesia.

GENETICS

The first step towards in identifying the genetic etiology of triple A syndrome was the chromosomal localization by linkage analysis of the gene responsible for this condition to an 6cM area in chromosome 12

(76). Subsequently, homozygote or compound heterozygote mutations were found in the AAAS gene on 12q13 in families with triple A syndrome (77). This gene encodes a 60-kDa nuclear pore protein, termed ALADIN (alacrima-achalasia-adrenal insufficiency, neurologic disorder) (62). AAAS belongs to WD-repeat regulatory protein family, which exhibits wide functional diversity, in that they are involved in signal transduction, RNA processing, vesicular trafficking, cytoskeleton assembly and cell division control. WD-repeat proteins are characterized by the presence of four or more repeating units containing a conserved core of approximately 40 amino acids that usually end with tryptophan-aspartic acid (WD). AAAS mRNA and the ALADIN protein are ubiquitously expressed with predominance in the adrenal and CNS structures in humans and rats (34, 77). ALADIN is the only nucleoporin to be associated with hereditary adrenal disease and the first to be associated with hereditary neurodegenerative disease.

Screening of patients with triple A syndrome worldwide revealed that the IVS14+1G A splice donor mutation is the most common AAAS mutation. In the Puerto Rican and Middle Eastern/southern European populations, the frequent presence of this mutation is the result of a founder effect. A variety of disease-associated missense, nonsense, splice-site and frameshift mutations have been shown to result in either ALADIN deficiency or mis-localization of the abnormal protein, found predominantly into the cytoplasm, suggesting that correct targeting of ALADIN to the nuclear pore complex is required. Splice-site, indel, intronic region, regulatory element and 5′ UTR mutations have been also detected in affected individuals (70). Over 75 different mutations have been described in the literature (www.hgmd.cf.ac.uk), some of which are shown in Table 4 (62, 64, 77-85). However, there is little phenotype/genotype correlation, even between affected siblings, suggesting that other factors may be involved in disease progression (86). A recent review of the literature, showed that AI was more prevalent and diagnosed at a younger age in patients harboring truncating mutations. On the other hand, neurological dysfunction was more prevalent, with an older age at onset, in patients carrying non-truncating mutations (70). In addition, patients with truncating mutations were more likely to present with symptomatic AI, while those with non-truncating mutations with neurological dysfunction.

Oxidative stress may play a role in the pathogenesis of this complex disorder. Data derived from experimental in vitro models of the disease, have shown that dermal fibroblasts of patients with triple A syndrome have higher basal intracellular ROS and are more sensitive to oxidative stress than wild-type fibroblasts. It has been suggested, that the failure of the nuclear accumulation of DNA repair proteins, aprataxin, and DNA ligase I together with the antioxidant protein ferritin heavy chain in skin fibroblasts of patients with triple A syndrome may render these cells more susceptible to oxidative stress. A disruption in redox homeostasis is suggested in the ALADIN-deficient adrenal cells with a depletion of reduced GSH, a major endogenous antioxidant and a cofactor of the antioxidant enzyme glutathione peroxidase. Moreover, AAAS knockdown results in cell cycle arrest and an increase in cell death by apoptosis. Increased chromosomal fragility has also been reported (34, 87). ALADIN protein has been shown to localize around the mitotic spindle and at spindle poles in Drosophila and human cells. It interacts with the microsomal protein progesterone receptor membrane component 2 (PGRMC2), regulator of cell cycle and activity regulator of CYP P450 enzymes, as well as with the inactive form of Aurora A, a serine/threonine kinase involved in various mitotic events. Recent studies suggest that ALADIN protein has functions in cell division. Interestingly, mitotic spindle assembly errors have been observed in cultured fibroblasts of patients with Triple A syndrome (88, 89). Finally, AAAS gene deficiency affects steroidogenesis and results in a reduction in StAR and P450c11β protein expression, and consequently in a significant reduction of cortisol production, an effect that is partially reversed with antioxidant N-acetylcysteine treatment (87). In addition, AAAS knock-down induces downregulation of genes coding for 17α-hydroxylase/17,20-lyase (CYP17A1), 21-hydroxylase (CYP21A2) and their electron donor cytochrome P450 oxidoreductase (POR), resulting in decreased production of glucocorticoid and androgen precursors (90).

Mutations in the AAAS gene have been identified in 90-95% of patients with a clinical diagnosis of Triple A syndrome (69, 70). The remaining cases may result from unidentified large deletions, mutations in uncharted intronic or regulatory regions, or mutations in two novel genes that may produce a “triple-A-like” phenotype without AI. GMPPA (guanosine diphosphate (GDP)-mannose pyrophosphorylase A) mutations were reported to cause an autosomal-recessive disorder characterized by achalasia, alacrima, and neurological deficits. Very recently, a homozygous splice mutation in TRAPPC11 gene, encoding for trafficking protein particle complex subunit 11, has been detected in patients presenting with achalasia, alacrima, myopathy and neurological symptoms (91, 92).

PRIMARY/CONGENITAL ADRENAL HYPOPLASIA

Five forms of AHC have been identified: 1) The X-linked form (OMIM 300200) caused by a mutation or deletion of the DAX1 gene (Dosage-sensitive sex reversal Adrenal hypoplasia congenita critical region of the X chromosome gene-1; NR0B1) on the X chromosome; 2) The autosomal recessive form owing to a mutation or deletion of the gene that encodes for the steroidogenic factor 1 (SF-1)/NR5A1 on chromosome 9q33 (OMIM 184757); 3) An autosomal recessive form of uncertain etiology (OMIM 240200); and 4) The IMAGe syndrome  (Intrauterine growth restriction, Metaphyseal dysplasia, Adrenal hypoplasia congenita, and Genital abnormalities) (OMIM 614732) 5) The MIRAGE syndrome (Myelodysplasia, Infection, Restriction of growth, Adrenal hypoplasia, Genital phenotypes and Enteropathy) (OMIM 617053).

Most recently, mutations in the gene encoding sphingosine-1-phosphate (S1P) lyase 1 (SGPL1), located on chromosome 10q22.1 have been associated with a syndrome comprising primary adrenal insufficiency and steroid-resistant nephrotic syndrome 9, 10) (OMIM: 617575).

X-linked Adrenal Hypoplasia Congenita (AHC)

The incidence of X-linked AHC is unknown. The latest reports estimate it to be less than 1:70,000 live male births (5, 93). X-linked AHC is characterized by infantile-onset acute adrenal insufficiency at an average age of 3 weeks in approximately 60% of affected individuals. Onset in childhood accounts for 40% of the cases, whilst only a few individuals are diagnosed in adulthood due to infertility.

CLINICAL FEATURES OF X-LINKED AHC

Adrenal insufficiency typically presents acutely with vomiting, feeding difficulties, dehydration and shock owing to salt-wasting. Hypoglycemia, frequently presenting with seizures, may be the first symptom. If untreated, adrenal insufficiency may lead to hyperkalemia, metabolic acidosis, hypoglycemia, hypovolemic shock and death. Cryptorchidism may be present. Affected males typically present with delayed puberty due to hypogonadotropic hypogonadism and are infertile. Carrier females may occasionally have symptoms of adrenal insufficiency or hypogonadotropic hypogonadism (5, 94). Imaging studies may reveal small, ectopic, or normal in size adrenal glands (5).

DIAGNOSIS

Primary adrenal insufficiency, as evidenced by hyponatremia, hyperkalemia, metabolic acidosis, low aldosterone and elevated ACTH concentrations in the presence of normal or low 17-hydroxyprogesterone concentrations, in a male infant strongly suggests X-linked AHC (5). Serum cortisol concentrations in the first weeks of life vary from very low to high (95). An ACTH test would detect cortisol deficiency, whilst a GnRH test would most possibly reveal impaired gonadotropin secretion (94, 96, 97).

Elevated 11-deoxycortisol concentrations have been documented in kindreds with DAX1 mutations, but only when determined very early in life. A mouse model that displays elevated 11-deoxycorticosterone concentrations and evidence of hyperplasia of the zona glomerulosa has recently been described. DAX1 testing may be considered in patients with evidence of 11β-hydroxylase deficiency, especially in those with severe salt-wasting (98).

GENETICS

Males with the above manifestations should undergo genetic analysis for the DAX1 gene. The DAX1 gene also known as NR0B1, (Nuclear Receptor subfamily 0, group B, member 1) is located on chromosome Xp21.2 and is responsible for the X-linked AHC (93, 97, 99). The NR0B1 gene (MIM#300473) encodes an orphan member of the nuclear receptor superfamily that is expressed in the hypothalamus, the anterior pituitary, the adrenal glands and the gonads. Nuclear receptors are thought to play a functional role in the establishment and maintenance of steroidogenic tissues. They are transcription factors that regulate gene networks important for reproduction, development and homeostasis in response to various extracellular and intracellular signals. The DAX1 carboxy-terminal domain (CTD) shares high similarity to the ligand-binding domain (LBD) of other nuclear receptors. The amino-terminal region is an atypical DNA binding domain, consisting of 3.5 repeats of 66–67 amino acid repeat motifs (100). At this time, DAX1 lacks a known ligand and is therefore named an orphan nuclear receptor.

The molecular mechanism of DAX1 action during development remains unclear. However, many studies have shown that DAX1 functions as a transcriptional repressor of steroid biosynthesis pathways regulated by other nuclear receptors, such as the SF1-mediated transactivation of genes StAR, 3β-hydroxysteroid dehydrogenase and cholesterol side-chain cleavage enzyme (P450scc). In addition to SF1, it acts as a repressor to other nuclear receptors, such as the estrogen receptor (ER) (101), progesterone receptor (PR), glucocorticoid receptor (GR) (102), androgen receptor (AR) (103) and the liver receptor homologue-1 (LRH-1) (104). DAX1 has also been proposed to act as a shuttling RNA binding protein associated with ribonucleoprotein structures in the nucleus and polyribosomes in the cytoplasm, raising the possibility that it plays an additional regulatory role in post-transcriptional processes (105). Other studies have demonstrated that DAX-1 may activate gene transcription (5, 100). It has been suggested that DAX-1 represses adrenal stem cell differentiation during organ development so that a pool of progenitor stem cells can be expanded before these cells differentiate into mature steroidogenic cells. Loss of DAX-1 function, would lead to premature differentiation of progenitor cells into mature cells before expansion of cell number takes place, resulting in a transient overactivity of the gland followed by adrenal hypoplasia.

To date, more than 200 mutations of the DAX1 gene have been reported (www.hgmd.cf.ac.uk). These include large and small deletions, insertions, missense, nonsense, frameshift and splice site mutations (93, 106-111). Most missense mutations tend to cluster within the C-terminal region of the DAX-1 gene, indicating the essential role of the ligand-binding domain for the biological function of DAX1 protein (112). Gross deletions usually occur as a continuous gene deletion including the genes of glycerol kinase (GK) and Duchene muscular dystrophy (DMD). Of note, some of the patients with the contiguous gene syndrome also present with mental retardation.

DAX1 mutations have been detected in 58% of males with primary adrenal insufficiency of unknown etiology, in which common causes of adrenal failure, such as 21-hydroxylase deficiency, ALD or autoimmune disease had been excluded (93). A family history of AI (or unexplained death) or hypogonadism in male relatives is highly suggestive of X-linked AHC. Of note, positive adrenal (21-hydroxylase) antibodies and normal adrenal imaging have been recently reported in a male patient presenting with adrenal insufficiency who had a DAX-1 mutation (113). Two thirds of the patients have point mutations. Small deletions and insertions causing frameshift mutations, as well as nonsense mutations are mutations scattered throughout exons 1 and 2, whereas missense mutations are detected in exon 2 (encoding the putative ligand binding domain in the carboxyl-end of the protein).

It has been estimated that isolated and contiguous NR0B1 gene deletions account for 22 and 5% of all NR0B1 mutations, respectively. Mental retardation (MR) associated with AHC cannot be explained with GK deficiency or DMD in every case. Deletions extending to the IL1RAPL1 gene have been shown to be responsible for MR in several cases. Moreover, female carriers of NR0B1, as well as of GK or DMD mutations are at risk of developing symptoms, due to non-random X inactivation. Furthermore, in case of a contiguous gene deletion, the manifestation of the symptoms depends on the pattern of X inactivation in different tissues. Multiplex ligation-dependent probe amplification (MLPA) analysis is a valuable tool to detect NR0B1 and contiguous gene deletions in patients with AHC, showing a good genotype-phenotype correlation. It is especially helpful for the detection of IL1RAPL1 deletions causing MR, as no clinical markers for MR are available. Furthermore, MLPA has the advantage of identifying female carriers manifesting milder symptoms (114).

Patients with AHC harboring DAX1 mutations present with variable phenotypes. Typically, they develop primary adrenal failure during infancy but also later in childhood, adolescence or early adulthood. Of note, a milder form of AHC, presenting with isolated mineralocorticoid deficiency was described in an 11-yr-old boy carrying a W105C missense mutation in the amino-terminal region of DAX1 (115).

The hypogonadotropic hypogonadism may manifest as delayed puberty or pubertal arrest at about Tanner stage 3. Hypogonadotropic hypogonadism seems to involve combined hypothalamic and pituitary defects, as reflected by an impaired gonadotropin response to gonadotropin-releasing hormone (GnRH) stimulation. However, normal mini-puberty of infancy has been observed in affected boys, implying that hypothalamic-pituitary-gonadal axis defects may develop after early infancy. In addition, patients with normal puberty, gonadotropin-independent precocious puberty, central precocious puberty (5, 95, 116, 117), and impaired spermatogenesis with low inhibin B levels (5, 107, 118) have also been reported. Gonadotropin-independent precocious puberty in affected individuals may be due to a) enhanced stimulation of human melanocortin 1 receptors (MC1R) on Leydig cells by ACTH and b) an increased expression of testicular steroidogenesis activators secondary to a reduction of DAX1 repression activity. The above mechanisms may result in an increased testicular testosterone production, despite prepubertal gonadotropin levels.

Isolated infertility with normal pubertal development and normal integrity of the hypothalamic–pituitary–gonadal axis has been recently reported in a patient with adrenal insufficiency owing to a DAX1 mutation. The severely impaired spermatogenesis in this patient suggests that DAX1 mutations may lead to progressive deterioration of testicular function, independently of gonadotropin and testosterone production. The DAX1 represses aromatase production and therefore the production of estrogen in Leydig cells. It has been recently suggested that the deletion of the second exon of DAX1 may abolish the aforementioned repressor effect, resulting in aromatase overexpression and increased estrogen production. Consequently, this DAX1 dysfunction, through an indirect effect, may be able to disrupt spermatogenesis even in the presence of normal testosterone concentrations (119). Hence, semen preservation should be offered to young men with DAX1 mutations (120). Patients with oligo- or azoospermia usually fail to respond to gonadotropin treatment. Frapsauce et al reported a unique case of an infertile azoospermic patient harboring a nonsense mutation in DAX1, who was treated with FSH/hCG for 20 months and fathered a healthy boy following testicular sperm extraction-intracytoplasmic sperm injection (TESE-ICSI) (100, 121).There is no clear phenotype – genotype correlation, and the phenotypes are heterogeneous even within families, with respect to the age of onset of adrenal insufficiency, the severity of the disease and the occurrence (or not) of hypogonadotropic hypogonadism (95, 122-126). It is noteworthy, however, that adult-onset adrenal insufficiency and hypogonadotrophic hypogonadism have been linked to eight DAX1 mutations (127, 128). Interestingly, a novel non-sense p.Gln208X mutation in the amino terminal domain of the DAX-1 gene has been associated with both precocious puberty and hypogonadotropic hypogonadism in different members of a large pedigree, who had all presented with adrenal manifestations at different ages (129). This heterogeneity within families may be explained by the unique structure of the DAX-1 gene. It is also indicative of the presence of modifier genes or environmental effects on the expression of clinical manifestations (94, 130, 131). Although this is an X-linked condition, females carrying homozygous or heterozygous mutations may present with isolated hypogonadotropic hypogonadism or extreme pubertal delay, respectively. Moreover, adrenal insufficiency, moderate developmental delay and mild muscular dystrophy was reported in a girl with deletion at Xp21.2 on the maternal chromosome and skewed X inactivation (5, 108, 132-134).

Other phenotypic features such as attention deficit disorder, short stature and growth hormone deficiency have been noted in a few patients (135, 136). Inappropriate tall stature and renal ectopy associated with a DAX-1 missense mutation was reported in a single case (137). Macrophalia in infancy may be a rare feature of X-linked AHC (31). Hepatic iron deposition was documented in a male infant presenting with adrenal insufficiency as part of Xp21 deletion (138).

It is worth noting that DAX1 has anti-testis properties and antagonizes SRY (sex-determining gene region of the Y chromosome) action, required for male sex determination. NR0B1 locus duplications have been associated with 46,XY DSD/testicular dysgenesis (100).

Congenital Adrenal Hypoplasia Due to SF1 Mutations

The steroidogenic factor 1 (SF1) protein, encoded by the nuclear receptor subfamily 5 group A member 1 (NR5A1) gene, is also an orphan member of the nuclear receptor family. It was first recognized in 1992 as an element that regulates the proximal promoter region of the cytochrome p450 21-hydroxylase enzyme (139). The NR5A1 gene is located on chromosome 9q33 and encodes a protein of 461 amino acids, which is expressed in the adrenal gland, gonads, hypothalamus, anterior pituitary and spleen during development and postnatal life (140, 141). SF1 is considered the main regulator of enzymes involved in adrenal and gonadal steroidogenesis (142, 143). It is essential not only for adrenal and gonadal development and sex differentiation, but also for CNS function and metabolic homeostasis (144, 145). Among others, SF1 regulates the expression of luteinizing hormone/choriogonadotropin receptors (LHCGR), StAR, CYP11A1, and CYP17A1 in Leydig cells, SRY and SOX9 (testis-determining genes), anti-Müllerian hormone (AMH) and its receptor AMHR2 in Sertoli cells, insulin-like peptide 3 (INSL3), which is involved in testicular descent, and T-cell leukemia homeobox-11 (HOX11-TLX1), a transcription factor essential for spleen development (146, 147). SF1 expression in the hypothalamus and pituitary gland contributes to the differentiation of pituitary primordial cells into gonadotrophs (140).

CLINICAL CASES AND MUTATIONAL ANALYSIS

Targeted deletion of NR5A1 gene in mice resulted in adrenal and testicular agenesis, retained Mullerian structures and partial hypogonadotropic hypogonadism in males, as well as hyposplenism and late onset obesity (141, 144, 148-150). In the adrenals, SF1 represses the CYP11B2 (aldosterone synthase) gene (151) and facilitates CYP17 (cytochrome P450 family 17) transcription under the control of ACTH (152).

To date, more than 100 pathogenic SF1 mutations have been reported (153). A genotype-phenotype correlation cannot be observed and diverse clinical presentations even among family members carrying the same mutation may be attributed to incomplete penetrance, pathogenic variants in other testis/ovarian-determining genes, polymorphisms, environmental and epigenetic factors. The first mutation was detected in a patient with adrenal failure and complete 46,XY sex reversal, who presented during the first weeks of life with low circulating cortisol, low aldosterone and high ACTH concentrations. Although the karyotype of the patient was 46,XY, normal Müllerian structures and streak-like gonads containing poorly differentiated seminiferous tubules and connective tissue were detected (154). The patient had a de novo, heterozygous loss-of-function missense mutation (p.G35E) causing substitution of glycine at amino acid 35 by glutamate in the DNA-binding domain of the protein, abolishing its DNA-binding activity. Pituitary gonadotropins responded to GnRH stimulation, but testosterone did not respond to exogenous hCG administration, suggesting defective gonadal function. After introduction of estrogen and progesterone, the uterus grew and regular menstruation ensued. This case was the first to indicate that SF1 is essential for sex determination, steroidogenesis and reproduction.

The second patient was a phenotypically female infant, who presented with hypoglycemic convulsions, progressive hypotonia, weight loss, hyponatremia and hypokalemia. Genetic testing revealed homozygosity for the p.R92Q mutation, whilst her consanguineous parents and her sister were heterozygous for the mutation. Although DHEA concentrations were detectable, 17-hydroxyprogesterone concentrations were low. The abdominal CT scan demonstrated left adrenal hypoplasia and right adrenal agenesis. The patient’s karyotype was 46,XY and a uterus was seen on pelvic ultrasound and confirmed by magnetic resonance imaging (155).

A phenotypically and genotypically normal girl (46,XX), with adrenal failure and no apparent defect in ovarian maturation was described in 2000 (156). The patient had a heterozygous G to T transversion in exon 4 of the NR5A1 gene, resulting in the missense p.R255L mutation. The inability of the mutant NR5A1/SF1 to bind canonical DNA sequences offered a possible explanation for the failure of the mutant protein to transactivate target genes. This was the first report of a mutation in the NR5A1 gene in a genotypically female patient, suggesting that SF1 is not necessary for female gonadal development, although it plays a crucial role in adrenal gland formation in both sexes.

Since then, only two cases of isolated adrenal insufficiency (AI) have been reported (31, 157). One of them, a 46 XX female, with early-onset primary AI, was homozygous for the p.R92Q mutation, previously associated with 46XY DSD (31).

In contrast, there have been several reports of various types of NR5A1 mutations (including missense, nonsense, and frameshift), affecting the DNA binding domain of the protein in individuals with different forms of 46,XY disorders of sex differentiation (DSD) and associated adrenal insufficiency (93, 158, 159) or without an adrenal phenotype (160-165). Pathogenic NR5A1 variants have been identified in 10-20% of all 46 XY DSD cases. They usually arise de novo, but can be maternally inherited in a sex-limited dominant manner in 30% of cases (100). Phenotypic features include: female or ambiguous genitalia with inguinal or labial testes and remnant or no Müllerian structures (present in 24% of patients) (147), clitoral hypertrophy, labioscrotal folds, labioscrotal testes, bilateral anorchia (166), micropenis and hypospadias (164, 167-169). Biochemical evidence of hypogonadotrophic hypogonadism along with testicular dysfunction and borderline adrenal dysfunction was observed in a case of 46XY DSD dizygotic twins, harbouring a heterozygous frameshift mutation in the C-terminal region of NR5A1 (170). Of note, there are several reports of affected individuals, presenting with female external genitalia in the neonatal period followed by spontaneous and progressive virilization in adolescence. However, FSH levels remained persistently elevated in all cases, suggesting that Leydig cell function may be preserved while Sertoli cells are more severely affected (171).

Splenic anomalies may be an additional feature of patients with 46 XY DSD harboring SF1 mutations. A homozygous SF1 mutation, R103Q was found in a 46 XY patient presenting with complete sex reversal, asplenia and mildly elevated ACTH levels but no evidence of an AI. The SF1 R103Q mutant was shown to decrease the transcriptional activity of the spleen development gene TLX1, and impair the transcriptional activation of steroidogenic enzymes, without disrupting the synergistic effect of SF-1 with either SRY or SOX9 (146). Moreover, the de novo heterozygous deletion of 143 bp (c.616_758del) was identified in 6-week-old 46,XY female with complete sex reversal, AI and splenic hypoplasia. Finally, polysplenia was reported in a phenotypically female 46,XY-DSD patient carrying a heterozygous SF1 mutation, p.Tyr409* in the ligand-binding domain. The same mutation was found in her father, who had asplenia and hypospadias (172).

The phenotypic spectrum of SF1 mutations has been further expanded to include 46,XX ovotesticular/testicular DSD associated with the p.Arg92Trp and p.Arg92Gln variants. Affected patients may present with ambiguous genitalia with a uterus/hemi-uterus or as phenotypic males with testes (173-175). It has been suggested that p.Arg92Trp mutation results in downregulation of the pro-ovarian Wnt4/β-catenin pathways, thus leading to increased expression of SOX9 and other pro-testis genes at the gonadal level, switching organ fate from ovary to testis.

In addition, missense changes, in-frame deletions, frameshift, and nonsense mutations in NR5A1 have been found in 46,XX females with isolated ovarian insufficiency and account for about 1.4–1.6% of women presenting with sporadic primary ovarian insufficiency (POI) of unknown origin (100, 165, 176).  Mothers or sisters who are heterozygous carriers may experience menstrual irregularities, decreased ovarian reserve, early menopause and rarely absence of puberty (100, 175).

Furthermore, NR5A1 mutations mostly located in the hinge region (100) may be found in 1.6-4% of men with otherwise unexplained severe impairment in spermatogenesis (177, 178). Gonadoblastoma and Germ Cell Neoplasia In Situ (GCNIS) have also been reported (179). Recent data indicate, that patients carrying NR5A1 mutations show distinct testicular histological features, i.e. reduced number of thin seminiferous tubules and focal aggregations of Leydig cells, containing cytoplasmic lipid droplets. Hence, testicular histology may be useful in identifying NR5A1 mutations in 46,XY patients with DSD before puberty. More recently, studies in mice indicate that lipid accumulation in the Leydig cells in 46 XY DSD is associated with decreased expression of StAR and CYP11A1, resulting in an increase in unmetabolized cholesterol (180, 181).

The above data indicate that SF1 mutations may lead to a wide range of endocrine phenotypes, which are only rarely related to adrenal insufficiency.

To date, microdeletions of chromosome 9q33.3, involving the NR5A1 gene have been reported in three patients with DSD. The first is a 3 Mb deletion in a 46,XY female, presenting with clinical features of Genitopatellar syndrome, developmental delay and ovotestes (182). The second is a unique 970kb microdeletion encompassing NR5A1, and resulting in XY sex reversal with clitoromegaly, neonatal male testosterone and AMH levels and a normal urine steroid profile (183). The third is a de novo 1.54 Mb microdeletion in a patient with 46,XY DSD and mild developmental delay (184).

Recently, a novel heterozygous p.Cys65Tyr mutation in NR5A1 gene has been identified in three 46,XY siblings of a Brazilian family, who presented with ambiguous genitalia without Müllerian derivatives and apparently normal Leydig function after birth and at puberty, respectively. Their mother, who reported symptoms suggestive of primary ovarian insufficiency was also heterozygous for this mutation. Basal ACTH and cortisol concentrations were slightly elevated and normal, respectively, in all three patients. After 1 mcg ACTH stimulation test, only the older sibling showed subnormal cortisol response. The above data indicate that NR5A1 analysis should be performed in 46,XY DSD patients with normal testosterone concentrations without AR mutations. Furthermore, a long-term follow-up for adrenal function is important for those patients (185).

IMAGE SYNDROME

CLINICAL FEATURES AND LABORATORY FINDINGS

The acronym IMAGe indicates the presence of Intrauterine growth restriction, Metaphyseal dysplasia, Adrenal hypoplasia congenita, and Genital anomalies (10, 186).

The life-threatening components of the adrenal insufficiency in this syndrome generally develop in the neonatal period. It usually manifests in the first few days of life with adrenal crises and may be the first sign of the disease. In some patients it may present later in childhood with failure to thrive and recurrent vomiting or in early adulthood. Hypoaldosteronism without evidence of glucocorticoid deficiency was also reported in one case (187). On imaging studies, the adrenal glands may appear small or normal in size.  Radiologic identification of metaphyseal dysplasia is often crucial for the diagnosis, but this could be very mild and identifiable only in late infancy or in childhood and then progress with age. Additional radiographic features may include: epiphyseal dysplasia, mesomelia, osteopenia, gracile long bones, and delayed bone age (188).

A more precocious sign, i.e. delayed endochondral ossification associated with osteopenia, hypercalcemia, and/or hypercalciuria of unclear aetiology and of variable degree can be encountered in patients with this syndrome. Abnormalities in serum calcium concentrations may be present at birth and resolve later in infancy. Soft tissue calcifications have been occasionally reported (188).

Another endocrine involvement in these patients is GH deficiency and early substitution therapy could improve linear growth.

Specific dysmorphic craniofacial features in IMAGe syndrome include nonspecific signs, such as prominent forehead, macrocephaly, low-set ears, ear dysplasia, flat nasal bridge, and short nose, short arms and legs. micrognathia or retrognathia, cleft palate or cleft uvula, craniosynostosis, short palpebral fissures, smooth philtrum, microglossia, arachnodactyly, and bilateral 2–3 toe syndactyly (187-189).

Genital abnormalities seem to be confined to males and include micropenis, undescended testes, chordee and hypospadias of variable severity. Two female patients were reported to give birth to children. Labor may be complicated by cephalopelvic disproportion.

Additional features associated with the syndrome include:

• Skeletal abnormalities: progressive and severe scoliosis with onset before age five years, ovoid-shaped vertebral bodies, short first metatarsals, hallux valgus, hip dysplasia, fractures of the humerus and tibia present at birth
• Renal abnormalities: hydronephrosis, hypercalciuria-associated nephrocalcinosis
• Other: oligohydramnios (187-188).

GENETICS

IMAGe syndrome (OMIM 614732) is exclusively related to mutations of CDKN1C gene [cyclin-dependent kinase inhibitor 1C (p57, Kip2)] (190). Notably, familial analysis demonstrated de novo mutations or an imprinted mode of inheritance, exclusively with maternal transmission of the mutation. The responsible gene lies on 11p15, contains three exons and encodes p57 (KIP2), a potent tight-binding inhibitor of several G1 cyclin/Cdk complexes (cyclin E-CDK2, cyclin D2-CDK4, and cyclin A-CDK2). It is a negative regulator of cell proliferation, playing a role in the maintenance of the non-proliferative state throughout life, probably acting as a tumour suppressor gene. CDKN1C is expressed in the placenta, heart, brain, lung, skeletal muscle, kidney, pancreas, testis, eye, and in the subcapsular or developing definitive zone of the adrenal gland. To date, clinical manifestations suggestive of IMAGe syndrome have been described in 28 individuals. Six missense mutations have been documented in 17 out of 28 patients, all of which occur in the PCNA-binding domain in the carboxy-terminal region of CDKN1C (186, 188). Recently, Hamajima et al (191) demonstrated that the IMAGe-associated mutations cause a dramatically increased stability of the CDKN1C proteins, which probably results in a functional gain of growth inhibition properties. Further studies have shown that mutations in the PCNA-binding site of CDKN1C lead to a block in the G1 phase and impaired S-phase entry resulting in decreased cell proliferation (192).  In contrast, loss-of-function CDKN1C mutations are associated with the Beckwith-Wiedemann syndrome (BWS), which represents an additional imprinting disorder with a mirror phenotype of IMAGe syndrome. BWS mutations are not clustered within a single domain and promote cell proliferation (186).

A novel CDKN1C mutation (c.842G>T, p. R281I) that did not entirely abrogate proliferating cell nuclear antigen binding has been recently associated with features of IMAGe syndrome, however, without adrenal insufficiency or metaphyseal dysplasia, but with early-adulthood-onset diabetes (189). A novel missense variant of CDKN1C (c.836G>[G;T], p.Arg279Leu) was also identified in a familial case of Russell Silver syndrome (193). Of note, both mutations were located within the PCNA-binding site of CDKN1C gene and were maternally inherited, thus producing phenotypic overlaps of IMAGe syndrome.

MIRAGE Syndrome

MIRAGE syndrome (OMIM 617053) is a rare form of syndromic adrenal hypoplasia, associated with high mortality rates during the first years of life. First described in 2016, MIRAGE stands for Myelodysplasia, Infection, Restriction of growth, Adrenal hypoplasia, Genital phenotypes and Enteropathy. The genetic basis of the syndrome has been linked to germline, mostly de novo, gain-of-function, heterozygous mutations in SAMD9 (sterile alpha motif domain-containing protein 9) gene. Homozygous loss-of-function SAMD9 mutations have been shown to result in normophosphatemic familial tumoral calcinosis (194).

GENETICS

SAMD9 gene resides on the long arm of chromosome 7 (7q21.2) and encodes a 1,589-amino acid protein that regulates cell proliferation and exhibits wide tissue expression, including in adrenal glands, colon, bone marrow, liver, immune system, lung, and testis (195, 196). SAMD9 facilitates endosome fusion and is likely to function as a growth repressor. It has been shown that expression of the wild-type SAMD9 resulted in decreased cell proliferation, whereas expression of mutants resulted in profound growth inhibition. At the cellular level, patient-derived fibroblasts displayed increased size of early endosomes, intracellular accumulation of giant vesicles and decreased plasma membrane epidermal growth factor receptor (EGFR) expression, likely due to defects in receptor recycling (194).

CLINICAL FEATURES AND LABORATORY FINDINGS

To date, heterozygous SAMD9 mutations associated with two or more components of MIRAGE syndrome have been reported in 24 patients (194-198).

Genital abnormalities may range from micropenis, cryptorchidism and hypospadias to ambiguous genitalia and completely feminized external genitalia in 46XY affected individuals. Of note, only 25% of reported cases were females, indicating that the syndrome may be underdiagnosed in girls. Histologically, the ovaries were markedly hypoplastic and dysgenetic in two patients, containing few primordial follicles (194, 195, 199).

Neonatal severe adrenal insufficiency is a common manifestation. Adrenal imaging may reveal hypoplasia or even absence of adrenal glands. Histologic studies have shown very small, highly disorganized, dysgenetic adrenal glands (194,195).

Thrombocytopenia and/or anemia, requiring transfusions may manifest within the first week of life, however spontaneous resolution has been reported in many cases (196, 197).

Myelodysplastic syndrome (MDS) associated with monosomy 7 or monosomy 7q was reported in 6 out of 24 MIRAGE-affected individuals. The researchers demonstrated that the preferential loss of the allele harboring the gain-of-function SAMD9 mutation, through the development of monosomy 7 (–7), deletions of 7q (7q–) or secondary somatic loss-of-function provide a survival advantage in affected hematopoietic cells. This is an example of an “adaptation by aneuploidy” mechanism, relieving the growth-restricting effect of the mutated gene, however at the expense of an increased risk for MDS (194, 195, 197, 199). Interestingly, two patients harboring two de novo SAMD9 mutations on the same allele, one activating SAMD9 mutation, and one second-site reversion nonsense mutation in the haematopoietic cells, exhibited no haematologic manifestations (198).

Additional features of the disorder include (194-196, 198-199):

-           Moderate-to-severe growth restriction during both the prenatal and postnatal periods, premature delivery, fetal death

-           Severe bacterial and viral infections, including sepsis, meningitis, and fungal infections thymus hypoplasia

-           Chronic diarrhea with colonic dilation, feeding difficulties frequently requiring surgical feeding tube placement

-           Dysmorphic features: frontal bossing, low-set ears, ptosis, down-turned corners of the mouth, round face, sparse hair, small feet and hands, tapered fingers, short phalanges, abnormal nails

-           Bronchopulmonary dysplasia

-           Neurologic abnormalities: dysautonomia hypolacrima, hyperhidrosis and blood pressure

dysregulation, syringomyelia, hypoplastic pons and cerebellum, hydrocephalus, bilateral auditory neuropathy, developmental delay

-           Skeletal abnormalities: scoliosis, joint contracture in wrists and ankles

-           Renal defects: renal tubular acidosis, glucosuria, defects in phosphate reabsorption and urinary concentration

-           Apneas

-           Reduced body fat

The majority of patients reported to date died within the first two years of life.

Familial Steroid-Resistant Nephrotic Syndrome with Adrenal Insufficiency

Most recently, in 2017, three study groups unraveled concurrently the genetic basis of a syndrome encompassing steroid-resistant nephrotic syndrome (SRNS) and primary adrenal insufficiency (PAI). Using whole exome sequencing analysis on patient cohorts with PAI or SRNS the researchers identified novel genetic mutations in the gene encoding sphingosine-1-phosphate (S1P) lyase 1 (SGPL1), located on chromosome 10q22.1 (200-202).

GENETICS

SGPL1 is an important endoplasmic reticulum (ER) enzyme that catalyzes the irreversible cleavage of the lipid molecule S1P to trans-2-hexadecenal and ethanolamine phosphate. S1P exhibits extracellular actions by activating a family of five differentially expressed extracellular G-protein-coupled receptors (G protein-coupled receptors (S1PRs) and intracellular functions via S1PR-independent mechanisms as well. S1P regulates multiple biological processes including cell migration, differentiation, angiogenesis, vascular maturation, cardiac development and immunity (200-202).

A total of 13 SGPL1 variants in 14 families have been reported so far (203). These were recessive loss-of-function mutations (homozygous or compound heterozygous) resulting in decreased or absent SGPL1 expression and/or enzyme activity, subcellular mis-localization of SGPL1 and altered levels of sphingolipid metabolism intermediates (200-202).

The pathogenesis of the syndrome may involve an excess of intracellular S1P, an imbalance of other sphingoid bases, S1P signaling through the S1P receptors or a lack of phosphoethanolamine production (201, 202).

SGPL1 is expressed in several mammalian tissues, among which in the adrenals and testes. Sgpl1–/– mice were shown to have impaired testicular and ovarian steroidogenesis and infertility.  Recent studies have documented several histologic abnormalities in the adrenal glands of Sgpl1–/– mice, including compromised cortical zonation with less definition between zona glomerulosa (ZG) and zona fasciculata (ZF) and between ZF and X-zone as well as loss of vacuolization in the ZF. Furthermore, Sgpl1–/– adrenals displayed decreased cytochrome P450 side-chain cleavage (CYP11A1), reflecting impaired steroidogenesis. These data may indicate the potential role of SGPL1 on adrenal development (200).

CLINICAL FEATURES AND LABORATORY FINDINGS

Human SGPL1 mutations cause a multisystemic disorder, with the main components being PAI and SRNS (200-204).

PAI is manifested in almost all cases, usually during infancy and less frequently during childhood or later. Most patients exhibit an FDG phenotype, necessitating treatment with hydrocortisone only. However, in some cases additional mineralocorticoid treatment may be required. Of note, markedly low adrenal androgen levels were reported in one affected postpubertal patient. Adrenal imaging (U/S or MRI) performed in some cases revealed i) normal findings ii) calcifications in the adrenals and iii) bilateral enlarged adrenal glands in one case (200-202).

Most affected patients suffer from nephrotic syndrome (NS), which is typically manifested as congenital NS (clinical symptoms occurring during the 3 months after birth) or within the first year of life and is steroid-resistant, leading rapidly to end-stage renal disease requiring renal transplantation. Histologic examinations have shown mainly focal segmental glomerulosclerosis, but diffuse mesangial sclerosis and foci of calcification have also been reported (200-203).

The phenotypic spectrum of this syndrome is broad and associated features other than SRNS and PAI may include (200-204):

-           Adrenal calcifications

-           Dermatologic abnormalities: ichthyosis, acanthosis, hyperpigmentation, scaly lesions, calcinosis cutis

-           Neurologic abnormalities: developmental delay, ptosis, strabismus, abnormal gait, ataxia, sensorineural deafness, seizures, microcephaly, cortical, cerebellar or corpus callosum hypoplasia, peripheral neuropathy, contrast enhancement of cerebellar structures and bilateral globus pallidus, medial thalamic nucleus and central pons, FLAIR-hyperintensity in hippocampus and brainstem.

-           Ophthalmologic abnormalities: “salt and pepper” retinopathy, amblyopia

-           Immunodeficiencies: lymphopenia, deficiency of cellular immunity, multiple bacterial infections, hypogammaglobulinemia, thrombocytopenia and anemia

-           Genital abnormalities: micropenis, cryptorchidism, hypergonadotropic hypogonadism, microorchidism associated with low serum anti-Müllerian hormone

-           Skeletal abnormalities: craniotabes, rachitic rosary, asymmetric skull, scoliosis, short stature

-           Hypothyroidism

-           Muscular hypotonia

-           Fetal demise, fetal hydrops

-           Other: facial dysmorphism (microstomia, hypertelorism, down-slanting palpebral fissures, epicanthus, dysplastic ears), hypocalcemia, mild dilated cardiomyopathy, intestinal malrotation, capillary leak syndrome.

Lovric et al have proposed the term Nephrotic syndrome, type 14 (NPHS14) to describe this syndromic form of SRNS associated with SGPL1 gene mutations (OMIM: 617575) (201).

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ABSTRACT

Glucocorticoids are steroid hormones produced by the adrenal cortex. They have pleiotropic effects and contribute substantially to the maintenance of resting and stress-related homeostasis. Although the molecular mechanisms of their actions are not fully understood, most of glucocorticoid effects are mediated by a ubiquitously expressed transcription factor, the glucocorticoid receptor. The latter influences the transcription rate of several glucocorticoid-target genes or interact physically with other transcription factors regulating their transcriptional activity in a positive or negative fashion. We present the molecular mechanisms of glucocorticoid action, and we discuss glucocorticoid treatment in endocrine and non-endocrine disorders, the side effects of glucocorticoids, their concomitant use and interactions with other drugs, and the risk factors for adrenal suppression. We suggest regimens for weaning patients from long-term glucocorticoid therapy, describe the glucocorticoid withdrawal syndrome, and provide some future perspectives on glucocorticoid treatment. For complete coverage of all related areas of Endocrinology, please visit our on-line FREE web-text, WWW.ENDOTEXT.ORG.

INTRODUCTION

Glucocorticoids are steroid hormones produced by the zona fasciculata of the adrenal cortex. These molecules are secreted into the peripheral blood under the control of the hypothalamic-pituitary-adrenal (HPA) axis in an ultradian, circadian and stress-related fashion (1). Glucocorticoids influence a myriad of physiologic functions contributing substantially to the maintenance of resting and stress-related homeostasis. At the cellular level, glucocorticoids regulate proliferation, differentiation and programmed cell death (apoptosis) of various cell types and may change the methylation status of cytosine-guanine dinucleotides (CpG) located in the regulatory regions of many genes, leading to important epigenetic alterations (1, 2).

Although glucocorticoids have been introduced in the treatment of rheumatoid arthritis since 1949, their molecular mechanisms of actions remain an evolving field of molecular and cellular endocrinology. Their anti-inflammatory and immunosuppressive effects are mediated mostly by their cognate receptor, the glucocorticoid receptor (GR), a transcription factor that belongs to the steroid receptor subfamily of the nuclear receptor superfamily (3). The therapeutic applications of synthetic glucocorticoids have been greatly broadened to encompass a large number of non-endocrine and endocrine diseases. Indeed, the prevalence of long-term glucocorticoid use worldwide is estimated at between 1% and 3% of adults (4).

When glucocorticoids are used at supraphysiologic doses, glucocorticoid-induced Hypothalamic-pituitary-adrenal (HPA) axis suppression renders the adrenal glands unable to generate sufficient cortisol if glucocorticoid treatment is abruptly stopped. In addition to adrenal suppression, a growing list of glucocorticoid adverse effects have been documented.

Glucocorticoid resistance has become another limitation in the therapeutic use of glucocorticoids. Our ever-increasing and deeper understanding of the molecular mechanisms of glucocorticoid actions might provide the basis for designing selective GR agonists that will optimize the therapeutic outcome, while minimizing undesired side effects.

MOLECULAR MECHANISMS OF GLUCOCORTICOID ACTION

Within the glucocorticoid target cell, the human (h) GR interacts with heat shock proteins (HSP90, HSP70) and immunophillins (FKBP51 and FKBP52), forming a multiprotein complex. Upon glucocorticoid-binding, the hGR dissociates from its protein partners, translocates into the nucleus, and forms homo- or hetero-dimers that bind to specific DNA sequences, termed “glucocorticoid response elements” (“GREs”), influencing the transcription of several glucocorticoid target genes in a positive or negative fashion (3, 5). For additional information please see the

Endotext chapter on Glucocorticoid receptors (6). Many anti-inflammatory genes are trans-activated by glucocorticoids, while pro-inflammatory genes are trans-repressed by these hormones. Aside from the genomic actions, accumulating evidence suggests that glucocorticoids may exert some effects in a very short time frame, independently of gene transcription and/or translation (7). These nongenomic glucocorticoid effects are believed to be mediated by membrane-bound hGRs that trigger specific kinase signaling pathways (8).

In addition to the above-mentioned actions, glucocorticoids can influence gene expression independently of hGR binding to DNA. These actions are mediated by physical interaction between the monomeric hGR with other transcription factors, such as the nuclear factor κB (NF-κB), the activator protein 1 (AP-1), and the signal transducers and activators of transcription (STATs), influencing the transcription rate of target genes of the latter (3, 5, 6).

SYNTHETIC GLUCOCORTICOIDS

Since the introduction of glucocorticoids (GCs) in the treatment of rheumatoid arthritis in 1949, intense efforts have been made by science and industry to maximize the beneficial effects and minimize the side effects of glucocorticoids. Thus, many synthetic compounds with glucocorticoid activity were manufactured and tested (9). The pharmacologic differences among these chemicals result from structural alterations of their basic steroid nucleus and its side groups. These changes may affect the bioavailability of these compounds - including their gastrointestinal or parenteral absorption, plasma half-life, and metabolism in the liver, fat, or target tissues - and their abilities to interact with the glucocorticoid receptor and to modulate the transcription of glucocorticoid - responsive genes (10, 11). In addition, structural modifications diminish the natural cross-reactivity of glucocorticoids with the mineralocorticoid receptor, eliminating their undesirable salt-retaining activity. Other modifications increase glucocorticoids' water solubility for parenteral administration or decrease their water solubility to enhance topical potency (11).

Synthetic GCs' clinical efficacy depends on their pharmacokinetics and their pharmacodynamics. Pharmacokinetic parameters such as the elimination half-life and pharmacodynamic parameters such as the concentration producing the half-maximal effect determine the duration and intensity of GC effects (12). It is known that the presence of an 11β-hydroxyl group is essential for the anti-inflammatory and immunosuppressive effects of GCs and for the sodium retaining effects of the mineralocorticoids (MCs). The most important pharmacokinetic systems for GCs and MCs are the 11β- hydroxysteroid dehydrogenases (11β-HSDs) because they regulate the target cell adjustment between the active hydroxy- and the inactive oxo- form of a steroid (13, 14). 11β- hydroxysteroid dehydrogenase type 2 (11β-HSD2, oxidizing enzyme) catalyzes the conversion of cortisol to cortisone, the inactive metabolite, whereas 11β- hydroxysteroid dehydrogenase type 1 (11β-HSD1, reductase) converts cortisone to cortisol. Thus, 11β-HSD1, which is expressed in a wide range of tissues, mainly in the liver, facilitates GC hormone action whereas the major role of 11β-HSD2 is to prevent cortisol from gaining access to high-affinity MC receptors and, therefore, the enzyme is predominantly expressed in the MC responsive cells of the kidney and other MC target tissues (colon, salivary glands) and the placenta (13).

The main structural features determining GC potency are the size and the polarity of the substituent in position 6 or 16. A hydrophobic residue increases GC activity (statistically significant enhancement with 6-α methyl and 16-methylene substitution). The more polar 16-hydroxy substitution decreases GC potency. The 6α and 9α-fluorination (such as in 6α and 9α fluorocortisol respectively) leads to increased GC and MC activity and double fluorination in the same positions augments this shift. Moreover, the Δ1-dehydro-configuration (in prednisolone) enhances GC activity but opposite to that effect it attenuates MC potency. The same effect is observed with the 16-methylene, 16α-methyl (dexamethasone) and 16β-methyl (betamethasone) groups. Thus, the more selective GC transactivation activity of GCs with a 16α-methyl or 16β-methyl group and a Δ1-dehydro-configuration, results from a significantly decreased activity via the mineralocorticoid receptor (MR) and an enhanced activity via the glucocorticoid receptor (GR) (15). Moreover, whereas GC selectivity can be improved by hydrophobic substituents in position 16 and the Δ1-dehydro-configuration, maximal GC activity needs additional fluorination in position 9α (such as in dexamethasone) (16). Figure 1 presents the chemical structures of cortisol and the most commonly used synthetic GCs.

Figure 1: Chemical Structures of the Most Commonly Used Synthetic GCs.

Protein binding is another pharmacokinetic property that influences GCs biological activity because only the unbound GC fraction is biologically active (14). In humans, endogenous cortisol binding to cortisol binding globulin (CBG) ranges between 67% and 87%, whereas a further 7-19% of total cortisol is bound to albumin, leading to about 95% of cortisol being protein-bound in the plasma. Except for prednisolone, synthetic GCs bind predominantly to albumin and only marginally to CBG. Plasma binding e.g. of dexamethasone and betamethasone is 75% and 60% respectively, and this is quite constant across a wide concentration rate (17). Thus, CBG binding is not a major determinant of plasma and biological half-lives of synthetic GCs.

However, especially for hydrocortisone and prednisone, pharmacokinetics are non-linear due to protein binding. As a result, higher doses result in more rapid clearance rates. It has to be mentioned that prednisone itself is biologically inactive and its 11-keto group must be reduced by hepatic 11βHSD1 to form the active drug, prednisolone. Moreover, clearance rate depends on age and is more rapid in children than adults (18) and also depends upon individual variability. Finally, certain diseases may influence synthetic GCs' pharmacokinetics. Thus, clearance is reduced particularly in renal and hepatic diseases and hypothyroidism and increased in hyperthyroidism. The concomitant use of other drugs influences synthetic GCs' half-lives and, thus, their final effect in target tissues (18, 19). Classic bioassays measure synthetic GC potency by testing the ability to suppress eosinophils and inhibit inflammation and the ability to stimulate hepatic glycogen deposition. The biologic effective half-life of glucocorticoids divides them into short-, intermediate-, or long-acting, based on the duration of corticotropin suppression after a single dose of the compound. The main corticosteroids used in clinical practice together with their relative biologic potencies and their plasma and biological half-lives are listed in Table 1.

SYSTEMIC GLUCOCORTICOID ADMINISTRATION

Therapeutic Indications

GCs are used in both endocrine and non-endocrine disorders (11, 22). First of all, they are administered as replacement therapy in patients with primary or secondary adrenal insufficiency, and as adrenal suppression therapy in congenital adrenal hyperplasia and glucocorticoid resistance (11). They are also used in patients with Grave's opthalmopathy and for some diagnostic purposes such as in establishing Cushing's syndrome (11). Moreover, due to their immunosuppressive and anti-inflammatory properties they are used in a broad range of non-endocrine disorders affecting many different systems (22, 23). Thus, they are given to treat skin disorders such as dermatitis and pemphigus, rheumatologic diseases such as systemic lupus erythematosus, polyarteritis and rheumatoid arthritis, and also polymyalgia rheumatica and myasthenia gravis. In hematology, they are used, along with chemotherapy, for the treatment of lymphomas and leukemias (24) and in hemolytic anemias and idiopathic thrombocytopenic purpura. In addition, they are administered in gastrointestinal diseases such as inflammatory bowel disease, in liver diseases (chronic active hepatitis) and in respiratory diseases (angioedema, anaphylaxis, asthma, sarcoidosis, tuberculosis, obstructive airway disease). Moreover, GCs are used in nephrotic syndrome and vasculitis and also in the suppression of the host-versus-graft and graft-versus-host reaction in cases of organ transplantation. In nervous disorders such as cerebral edema and raised intracranial pressure the use of GCs is also beneficial (25, 26).

Acute administration of pharmacologic doses of glucocorticoids is advocated in a small number of nonendocrine diseases, such as for patients suffering from acute traumatic spinal cord injury, although two recent meta-analyses support that the use of methylprednisolone should be limited (27, 28). Moreover, steroid administration should be considered as a post-operative additional therapy for cases with severe neurological deficits even after surgery (29). Glucocorticoids are also used for postoperative pain relief after severe bone operations (30). In addition, as it is known that premature birth is associated with an increased risk of neonatal mortality and morbidity, including respiratory distress syndrome (RDS), and because 7-10% of all pregnancies in North America are under such risk, in 1994 the National Institutes of Health (NIH) Consensus Developmental Conference on the Effects of Corticosteroids for Fetal Maturation on Perinatal Outcomes concluded that all fetuses between 24 and 34 week gestation at risk of preterm delivery should be considered as candidates for antenatal treatment with GCs. Recommended treatment consisted of 2 doses of 12mg betamethasone given IM 24 hours apart or 4 doses of 6mg dexamethasone given 12 hours apart. In 2001 the NIH Consensus Developmental Panel recommended that repeat courses should not be used routinely until insightful findings are available. However, the Australian Collaborative Trial (ACTORDS), that has been completed, reported that repeat course synthetic GCs improved short-term neonatal outcome compared to single course therapy (31).

Acute administration of pharmacologic doses of glucocorticoids is also necessary in some types of acute illness. For years it is known that any type of acute illness or trauma results in loss of the diurnal variation in cortisol secretion. In the early phase of critical illness cortisol levels frequently rise and levels of CBG and albumin are substantially depleted. In the chronic phase of critical illness, however, high ACTH and cortisol levels are generally sustained and CBG levels gradually increase. Both very high and very low cortisol levels have been associated with increased mortality from critical illness. High cortisol levels reflect severe stress, whereas low levels reflect an inability to sufficiently respond to stress (32). The term "critical illness-related cortisol insufficiency" (CIRCI) defines a state of both the inadequate production of GCs as well as a corticosteroid tissue resistance. It has been estimated that the overall incidence of adrenal insufficiency in critically ill patients is approximately 20%, with an incidence as high as 60% in patients with severe sepsis and septic shock. It is possible that CIRCI is an epiphenomenon and a marker of illness severity (33).

According to the current recommendations, CIRCI should be suspected in hypotensive patients who respond poorly to fluids and vasopressor agents, particularly in the setting of sepsis. To diagnose CIRCI, the clinician may use a delta serum cortisol <9 μg/dl after cosyntropin (250μg) administration or a random plasma cortisol <10μg/dl. The authors suggest that clinicians should not use plasma-free cortisol or salivary cortisol level over plasma total cortisol. For patients with septic shock that is not responsive to fluid and moderate- to high-dose vasopressor therapy, the authors suggest IV hydrocortisone < 400 mg/day for ≥ 3 days at full dose. They, however, do not suggest using corticosteroids in adult patients with sepsis without any evidence of shock. The dose regimen in patients with early moderate to severe ARDS is methylprednisolone 1mg/kg/day for at least 14 days. Finally, glucocorticoids are not suggested for cases of major trauma (34). In a second part of the guidelines, the authors formulated statements for or against the use of synthetic corticosteroids for other common pathologic conditions, including community-acquired pneumonia, influenza, meningitis, and non-septic systemic inflammatory response syndrome (SIRS) that may be associated with shock, namely burns, cardiac arrest and cardiopulmonary bypass surgery (35).

Benefits of GCs replacement has been demonstrated in a number of other patient populations including low cardiac output syndrome after cardiac surgery (36), acute exacerbation of chronic obstructive pulmonary disease (37), and cirrhosis (38).

Adverse Effects (AEs)

Although synthetic GCs remain an important component of therapy for many conditions, in recent years there are arguments against their use based mainly on the concern of toxicity. Nowadays, GCs toxicity is one of the commonest causes of iatrogenic illness associated with chronic inflammatory disorders. Despite the fact that the adverse effects of GCs have been known for decades, the actual risk-benefit ratio is incomplete and/or inconsistent. This happens because it is in general difficult to separate the effects of GCs from the outcome of the underlying disease, other comorbidities, or the use of other medications. Moreover, toxicity reports usually concern patients using high doses of GCs, different types of GCs with different relative drug potencies, for a heterogeneous group of related diseases, and for different periods of time (39, 40).

Only recently there has been intense effort by scientists and clinicians to explore and quantify the incidence and severity of the AEs of GC therapy. Generally, it is known that GCs' toxicity is related to both the average and cumulative dose during their use (41). The question that arises is whether or not patterns relating the frequency of AEs to GC dosage and/or length of GC treatment exist (39).

Historically, GCs at a prednisone equivalent of 5-10mg/day are considered low dose. However, a review of "the 4 extensively reviewed trials on low dose GCs in rheumatoid arthritis" led to the conclusion that definitive association of low dose GCs with many AEs such as osteoporosis, myopathy, cardiovascular disease, glaucoma, increased incidence of any kind of infection, and behavior disturbances remains elusive, and that the fear of GCs toxicity is probably overestimated based on extrapolation from observations with higher dose treatment. However, according to the same analysis, the use of 5-10mg/day of prednisolone (or equivalent) for over 2 years is associated with an increase of mean body weight in the range of 4-8% (40).

The prevalence of GC associated AEs was identified in a large survey of 2167 long term (≥60 days) users of GCs with mean prednisone equivalent dose of 16±14mg/day. The AE with the greatest prevalence was weight gain, experienced by 70% of the individuals, followed by skin bruising/thinning, and sleep disturbances. Cataracts (15%) and fractures (12%) were among the most serious AEs. All AEs demonstrated a strong dose-dependent association with cumulative GC use. Acne, skin bruising, weight gain and cataracts were significantly associated with longer duration (>90 days) of low-dose GCs (≤7.5mg/day of prednisolone), while fractures and sleep disturbances were more strongly associated with small increments in daily dosage (within the 0-7.5mg/d range). In conclusion, this survey adds further evidence that more GC associated AEs are dependent on both the average dose and the duration of therapy and that even low dose GC therapy could lead to serious AEs (42).

As in more severe cases of chronic inflammatory diseases long-term (≥ 1month) dosage of GCs is medium to low (≤30mg/d prednisolone or equivalent), a systematic review of 28 studies (2382 patients) concerning patients with rheumatoid arthritis (RA), polymyalgia rheumatica, and inflammatory bowel disease was the first to present a pooled analysis of the commonest reported AEs associated with this pattern of administration. The AE rate depends both on the quality of the study and primarily- on the disease in the study population. The overall mean rate of AEs was 150 per 100 patient-years, varying from 43/100patient years in rheumatoid arthritis and 80/100patient years in polymyalgia rheumatica to 555/100 patient years in inflammatory bowel disease. Psychological and behavioral disturbances (e.g. minor mood disturbances) were most frequently reported, followed by gastrointestinal events (e.g. dyspepsia, dysphagia) (43).

A recent retrospective population-based cohort study and self-controlled case series aimed to assess the risk of sepsis, venous thromboembolism, and fracture in 327,452 adults aged 18 to 64 years who received at least one prescription for less than 30 days over a three-year period (44). The authors found increased rates of sepsis (incidence rate ratio 5.30, 95% confidence interval 3.80 to 7.41), venous thromboembolism (3.33, 2.78 to 3.99), and fracture (1.87, 1.69 to 2.07), which decreased within the next 90 days. The increased risk for these adverse effects was observed at prednisolone doses lower than 20mg per day (44).

An important observational study aimed to identify patterns of self-reported health problems relating to dose and duration of GCs in 1066 unselected patients with RA (39). The study identified 2 distinct dose-related patterns of AEs. A continuous, approximately linear rising with increasing dose was found for cushingoid phenotype, ecchymosis, leg edema, mycosis, parchment-like skin, shortness of breath, and sleep disturbance. The most clearly attributable adverse drug reaction to GCs, Cushing syndrome, becomes evident after at least one month of treatment and was observed in 2.7, 4.3, 15.8, 24.6% of patients with no GCs in the past 12 months, and <5, 5-7,5 and >7,5mg/d of prednisolone or equivalent for >6 months, respectively. The second pattern identified describes an elevation in the frequency of health problems beyond a certain threshold value and is defined as a "threshold pattern". The threshold for the increase in glaucoma, depression, and an increase in blood pressure was observed at dosages of greater than 7.5mg/d. Dosages of 5mg/d or more were associated with epistaxis and weight gain. A very low threshold was observed for eye cataract (<5mg/d). All the associations found are in agreement with biological mechanisms and clinical observations (39). However, more extensive research on the risk-benefit ratio of long-term GCs is needed and could help to create new targets for drug development.

An overview of the most common and most serious AEs associated with GC therapy is discussed below.

ADRENAL INSUFFICIENCY (AI)

Iatrogenic, tertiary adrenal insufficiency induced by chronic administration of high doses of GCs is the most common cause of adrenal insufficiency (45). Physiologically, the hypothalamus secretes CRH which stimulates the release of ACTH from the anterior pituitary. ACTH leads to the release of cortisol from the zona fasciculata of the adrenal gland, which in turn exerts negative feedback on CRH and ACTH release. Administration of exogenous GCs even in small doses for only few days leads to a measurable suppression of the HPA axis by decreasing CRH synthesis and secretion and by blocking the trophic and ACTH-releasing actions of CRH on the anterior pituitary. This leads to suppressed synthesis of POMC, ACTH and other POMC derived peptides and later, to the atrophy of the corticotrophin cells of the anterior pituitary. As a result, in the absence of ACTH, the adrenal cortex loses the ability to produce cortisol. Nevertheless, the adrenal cortex retains the ability to secrete enough cortisol for some period of time and also mineralocorticoids, as this latter function depends mainly on the renin-angiotensin system rather than on ACTH.

The association between AI and treatment with oral GCs has been recognized for decades, although the magnitude of the risk has not been determined until recently. It has also been reported that the inhibition of the HPA axis function induced by exogenous GCs may persist for 6 to 12 months after treatment is withdrawn (46). Based on the literature the absolute risk of adrenal crisis after cessation of oral and inhaled GCs might be considered rare, but it is likely to be substantially underreported in clinical practice (10, 47).

The first study that quantified the increased risk of AI in people prescribed oral and inhaled GCs in the general population was published in 2006 (48). This case-control study, that used data from a cohort of 2.4 million people, found a strong dose-response relationship between oral GCs exposure and the risk of AI with an OR of 3.4 (95% CI, 1.6-2.5) per course of treatment per year. Furthermore, the study indicated, that administration of inhaled GCs within 90 days of diagnosis is associated with an increased risk of AI (OR 3.4, 95% CI 1.9-5.9) and this effect was dose related. However, after adjustment for oral GCs exposure, this association was reduced (OR 1.6, 95% CI 0.8-3.2) although the dose relation remains. The largest increase in risk occurred in association with a recent prescription for fluticasone proprionate (48). These findings were confirmed by more recent studies that aimed to investigate the prevalence of AI in patients treated either with inhaled (49) or with oral GCs (47, 50). Interestingly, in a recently published systematic review the authors found that the percentage of patients with glucocorticoid-induced AI had a median (IQR) of 37.4%, ranging from 13%-63% (51). Three years after glucocorticoid withdrawal, AI persisted in 15% of retested patients. AI occurred in patients receiving <5mg prednisolone equivalent dose/day, for less than 4 weeks, and with a cumulative dose <0.5g (51).

CARDIOVASCULAR DISEASE

A population-based study that compared the risk for CVD in 68,781 patients using GCs versus 82,202 nonusers identified that the relative risk for a cardiovascular event in patients receiving high-dose GCs (≥7,5mg/d prednisolone) was 2.56 (CI 2.18-2.99) after adjustment for known covariates (52). Similar associations were noted in another observational study that included 50,656 patients and an equal number of matched controls. According to this study, current use of GCs was associated with an increased risk of heart failure (OR 2.66, 95% CI 2.46-287) and a smaller risk of ischemic heart disease (OR 1.20, 95% CI 1.11-1.29) (53). However, the previous results are not confirmed by other studies (54, 55). Additionally, an association of GCs use and the risk for atrial fibrillation and flutter has been established by several studies (56-58).

GLUCOSE HOMEOSTASIS

The alterations in glucose homeostasis induced by GCs are multifactorial and could be explained by several potential mechanisms including the induction of enzymes involved in hepatic gluconeogenesis, the decrease in glucose uptake in peripheral tissues, the stimulation of lipolysis, the prevention of insulin production, and the induction of ceramide biosynthesis leading to insulin resistance (59). An interesting review of the existing literature published between 1950-2009 shows that GC-induced hyperglycemia is common among patients with and without diabetes mellitus. The OR for new onset diabetes mellitus ranges from 1.5 to 2.5 and the induction of the disease is strongly predicted by GC accumulative dose and duration of therapy (60).

INFECTIOUS EVENTS

GC therapy is associated with an increased risk of infectious complications, as GCs are known to have suppressive effects upon both innate and acquired immunity. This is confirmed by several studies. According to a large observational study of 16,788 patients with RA, prednisone use, even at doses of 5mg/kg, increased the risk of hospitalization for pneumonia. Furthermore, there was a dose related relationship between prednisone use and pneumonia risk in RA (61). Another study of 15,597 patients with RA found that GC use doubled the rate of serious bacterial infections compared with methotrexate with a dose response relationship for doses greater than 5mg/d (62). The latter results were confirmed by additional studies that have identified GCs as an independent risk factor for infections (63, 64). Moreover, a more recent study demonstrated that patients receiving 5mg prednisolone continuously for the last 3 months, 6 months or 3 years, had a 30%, 46% or 100% increased risk of serious infection, respectively (65). Also, caution about GC use in patients with active or dormant TB is well accepted as these individuals are susceptible to contract or to sustain activation of the disease (66). An epidemiological study of patients with TB showed that they were nearly 5 times more likely to have been using GCs at the time of their diagnosis (67).

OSTEOPOROSIS

The effects of GCs on bone homeostasis are both systemic and local. Systemic effects include a reduction in calcium absorption from the intestine and a reduction in calcium reabsorption in the kidney, both enhancing PTH secretion and thus bone loss. Furthermore, the attenuation of sex steroids and growth hormone by GCs enhances bone loss. The direct effects of GCs on bone cells include induction of osteoblast and osteocyte apoptosis through activation of pro-apoptotic molecules, impairment of Wnt signaling, and induction of RANKL, a potent stimulator of osteoclastogenesis produced by osteoblasts (68). As a result, GCs induced osteoporosis is the most common type of iatrogenic osteoporosis. This has been confirmed by several studies. One of them showed that therapy with high-dose oral GCs caused significant decrease in BMD even in the first 2 months of therapy (69). As a result, there is an increased risk of osteoporotic fractures (70) and it has been estimated that fractures may occur in up to 30-50% of patients on GC therapy (71) but fortunately there is a rapid decrease of the risk on cessation of therapy (70, 72). Similar findings were observed by a more recent study showing that low daily dose prednisone (≤7,5mg/d) with high cumulative doses increases the risk for fractures. Intermittent high-dose regimens with cumulative doses less than 1gr, however, did not show an increased risk. Risk declines rapidly, the decrease beginning 3 months after cessation of therapy (73). For additional details please see the Endotext chapter on Glucocorticoid-induced osteoporosis (74).

NEUROPSYCHIATRIC EVENTS

Despite a slight increase in their overall sense of well-being independent of improvement in disease activity, it has been established that synthetic GC treatment may induce behavioral, psychic, and cognitive disturbances (75). These disturbances can be detected by structural, functional, and spectroscopic imaging. Behavioral changes in feeding and sleeping are commonly observed. Among psychic AEs, hypomania and mania are the most common during acute GC therapy and depression during long-term treatment. Suicides have also been reported (76). These AEs are usually mild/moderate but are severe in 5-10% of cases. Cognitive changes affect mostly declarative and working memory. All these AEs are generally dose and time dependent (infrequent at prednisone equivalent doses <20mg/d) and usually reversible. There has to be greater concern for pediatric patients. Several medications such as lithium, phenytoin, lamotrigine, memantine and other anti-seizure, anti-psychotics, and anti-depressants could be useful for treating such disorders (77, 78).

PEDIATRIC EVENTS

Prolonged GC treatment of children with chronic illnesses impairs their longitudinal growth (79). GCs exert multiple growth suppressing effects, such as inhibition of GH secretion and IGF-1 expression, reduction of bone and collagen formation, bone mineralization, and vascularization. These effects are more pronounced with daily oral GCs than alternate day oral GC therapy (80). According to a study of 224 children with cystic fibrosis who have received alternate day treatment with prednisone, boys but not girls, had persistent growth impairment (mean final height 4cm less than children who were treated with placebo) after discontinuation of treatment (81).

Apart from growth retardation, children may also be more susceptible to other AEs associated with GCs such as osteoporosis, glaucoma and cataracts. Moreover, fracture risk seems to be higher in GC-treated children (82).

Intrauterine exposure to GCs is able to affect fetal HPA axis development causing reduction in fetal and, in some cases newborn and infant HPA axis activity under basal conditions and more consistently after pain-related stress. Although baseline HPA axis function seems to recover within the first 2 weeks postpartum, there is initial evidence that blunted HPA axis reactivity to pain-related stress persists throughout the first 4 months of life. These effects are dose dependent and vary with the time between GC exposure and HPA assessment. It seems that programming of the HPA axis involves interaction with other endocrine systems such as the Hypothalamus-Pituitary-Gonadal axis (HPG). Moreover, exposure to GCs during pregnancy has been linked to impaired fetal growth and modulated fetal immune functions, indicators of compromised cognitive, neurological and psychological functions, and increased blood pressure into adolescence. Furthermore, there is some evidence that reduced HPA axis activity early in life will switch to a hyperactive state later in life due to over-adjustment and because of that, affected infants may be vulnerable to stress related disorders associated with hypercortisolemia such as depression and cardiovascular disease. Finally, it seems that changes in HPA axis function following antenatal exposure to GCs are trans-generational and likely involve epigenetic mechanisms (17).

In addition, according to a recent meta-analysis, early postnatal GC treatment (≤7 days), particularly with dexamethasone, causes short term AEs including gastrointestinal bleeding, intestinal perforation, hyperglycemia, hypertension, hypertrophic cardiomyopathy and growth failure (83, 84). Long term follow-up studies report an increased risk of abnormal neurological examination and cerebral palsy (85).

PHEOCHROMOCYTOMA CRISIS

Severe isolated cases of pheochromocytoma crisis have been reported after administration of exogenous GCs (86, 87). Thus, GCs should be avoided or administered only if absolutely necessary in patients with known or suspected pheochromocytomas.

The most common AEs of GC therapy are summarized in Table 2.

COMPARTMENTAL GLUCOCORTICOID ADMINISTRATION

Topical Glucocorticoids

Glucocorticoids are the first line of treatment for various skin disorders such as atopic dermatitis, vitiligo, psoriasis, etc. (10, 89-94). They are quite effective when applied topically and nontoxic to the skin in the short term. The factors that determine local penetration are the structure of the compound employed, the vehicle, the basic additives, occlusion versus open use, normal skin versus diseased skin, and small areas versus large areas of application. Fluorinated steroids (e.g. dexamethasone, triamcinolone acetonide, betamethasone, and beclomethasone) penetrate the skin better than nonfluorinated steroids, such as hydrocortisone. However, fluorinated steroids also produce more local complications and may be associated with systemic absorption and side effects.

The most frequent AEs are local and include atrophy, striae, rosacea, perioral dermatitis, acne, and purpura. Less frequently, hypertrichosis, pigmentation alterations, delayed wound healing, and exacerbation of skin infections occur. Furthermore, the rate of contact sensitization against GCs is greater than previously believed. Systemic reactions such as hyperglycemia, glaucoma and adrenal insufficiency are less frequent (95). Some cases of Cushing's syndrome following overuse of topical GCs have also been described (96). The frequency of systemic effects by topical corticosteroids is increased in newborns and small children compared to adolescents and adults, because GCs penetrate the skin of newborns and small children more easily and in larger proportional amounts. Infants, especially, have a greater risk for Cushing's syndrome or adrenal insufficiency and also hepatosteatosis. An infant's death due to generalized CMV infection following administration of topical GCs has been reported (97). Based on the Body Surface Area, a simple guideline for how much topical GC to prescribe for a child has been proposed. Roughly, infants require one fifth of adults' doses, children two fifths and adolescents two thirds of adults' doses (98). Finally, the use of skin lightning cosmetics used in most African countries includes corticosteroids and may have many serious and sometimes fatal complications, including adrenal insufficiency (99).

Opthalmic Glucocorticoids

In the past 10 years intravitreal GCs injections have been increasingly used for patients with a variety of posterior segment diseases, including diabetic macular edema, branch and central retinal vein occlusion, pseudophakic cystoid macular edema, and uveitic macular edema (100). Currently, novel agents including preservative-free and sustained-release intravitreal implants are being studied in clinical trials. Potential complications of intravitreal steroid treatment are divided into steroid-related and injection-related side effects. Steroid-related side effects include cataract formation and glaucoma (101). Injection related side effects include retinal detachment, vitreous hemorrhage, and bacterial and sterile endopthalmitis.

Inhaled Glucocorticoids

GC inhalation therapy is widely used in patients with asthma and chronic obstructive pulmonary disease. Their relative topical to systemic effect ratio or therapeutic index depends upon the pharmacokinetic differences for inhaled GCs. Factors that enhance the therapeutic index are: decreased oral absorption retention in the lung and rapid systemic clearance once the drug is absorbed into the systemic circulation. More recently, it has been posited that the therapeutic index is also enhanced by high plasma protein binding. Inhaled GCs have important pharmacokinetic differences (102).

In general, inhaled GCs have fewer and less severe AEs than oral and systemic GCs. However, systemic AEs may be observed and this risk is influenced by the dose, the period of treatment, the delivery system used, the site of delivery (i.e. gastrointestinal tract, lung), the concomitant use of other medications, and the altered steroid metabolism due to individual's differences in the patient's response to GCs.

As far as growth deceleration in children is concerned, the results are somewhat contradictory. Although inhaled GCs seem to cause a dose-dependent reduction in height velocity (103), these changes are not significantly associated with final adult height (104). However, a study of 1041 asthmatic children treated with budesonide, nedocromil, and placebo for 4.3 years, a decrease in growth velocity was observed in the budesonide group which was most evident in the first year of treatment (105). When 90% of these children were followed-up for an additional 4.8 years, a lower mean height was found in the budesonide group and this was more pronounced in girls than in boys (106).

Moreover, as GCs effect on bone metabolism is of great concern, some studies have shown that inhaled GC therapy is associated with increased fracture risk (107, 108) but this has not been confirmed by a meta-analysis (109). Nevertheless, several studies confirm a negative relation between total accumulative dose of inhaled GCs and bone mineral density (110). The loss in BMD is of concern, especially in early postmenopausal women and boys during puberty (111, 112). Again, these results have been argued (113).

It has been shown that adrenal insufficiency is also associated with inhaled GCs, although with lower prevalence (114). The newest inhaled GC, Ciclesonide, appears to have different pharmacokinetics enhancing its therapeutic index. It is administered as a pro-drug converted to the active metabolite des-Ciclesonide in the lung. Thus, it has low oral bioavailability and also rapid clearance and high protein binding, factors that reduce pharmacologically relevant systemic exposure (115). Furthermore, Ciclesonide appears to have less suppressive effects on HPA axis function (116, 117).

Nasal Glucocorticoids

Intranasal GCs are effectively used for the treatment of allergic rhinitis, rhinosinusitis, rhinoconjunctivitis, and nasal polyposis (118, 119). Topical steroid drops are used for the treatment of sinus ostia stenosis in the postoperative period (120). Interestingly, molecules designed specifically to achieve potent localized activity with minimum risk of systemic exposure such as mometasone furoate, fluticasone proprionate, and fluticasone furoate may be preferable. Studies in children have not found any adverse effects including HPA axis suppression or growth retardation (118). Yet, some studies suggest a relationship between intranasal steroids and increased intraocular pressure (119). Generally, frequent and chronic use should be avoided to prevent local and systemic complications (121).

Intraarticular Glucocorticoids

The main beneficial effect of intraarticular GC injection is pain relief. Most favorable results are seen in juvenile idiopathic arthritis patients. Local AEs are either rare or insignificant and include joint infection, intraarticular and periarticular calcifications, cutaneous atrophy, cutaneous depigmentation, avascular necrosis, rapid destruction of the femoral head, acute synovitis, Charcot's arthropathy, tendinopathy, Nicolau's syndrome, and joint dislocation (122). Moreover, some systemic AEs have also been reported. These include a transient HPA axis suppression, a transient increase in blood glucose in diabetic patients, and other metabolic, hematologic, vascular, allergic, visual and psychological AEs (123). The most used intraarticular glucocorticoids are triamcinolone hexacetonide, triamcinolone acetonide, and methylprednisolone acetate (124).

MONITORING OF PATIENTS ON GLUCOCORTICOID TREATMENT

As osteoporosis, with resultant fractures, constitutes one of the most serious morbid complications of GC use, worsening patients quality of life, recently, the American College of Rheumatology (ACR) updated the 2010 recommendations for patients receiving oral GC therapy (125). In this systematic review, the authors addressed the initial assessment in patients that began or continue glucocorticoid treatment for longer than 3 months, and discussed the advantages and disadvantages of lifestyle modification, as well as for calcium, vitamin D and pharmaceutical treatment, including bisphosphonates, raloxifene, teriparatide, and denosumab. According to the ACR guidelines, there are three categories of fracture risk. High risk criteria include patients aged over 40 years, previous osteoporotic fracture, hip or spine BMD T-score ≤ −2.5, or 10-year fracture probability of ≥20% (major osteoporotic fracture) or ≥3% (hip fracture) (125, 126). Moderate and low risk criteria are based only on FRAX-derived fracture probability (10–19% and >1 to ≤3% respectively for moderate

risk, and <10% and ≤1% respectively for low risk) (125, 126). In patients aged less than 40 years, a previous osteoporotic fracture is considered as a high-risk criterion, whereas the criteria of moderate and low risk are based on the BMD. Patients at low fracture risk are recommended to receive only calcium and vitamin D, whereas adults at moderate-to-high fracture risk should be treated with calcium and vitamin D plus an oral bisphosphonate. However, adults in whom oral bisphosphonates are not appropriate, are recommended to continue calcium plus vitamin D but switch from an oral bisphosphonate to another anti-fracture medication (125). Finally, adults who complete a planned regimen with oral bisphosphonates and continue glucocorticoid treatment, are recommended to continue oral bisphosphonate treatment or switch to another anti-fracture medication (125). Recommendations were also suggested for children, women of childbearing potential, and people with organ transplants, as well as patients receiving very high doses of glucocorticoids (125). For additional details please see the Endotext chapter on Glucocorticoid-induced osteoporosis (74).

CONCOMITANT USE OF GLUCOCORTICOIDS WITH OTHER DRUGS

Special attention is required in the concomitant use of glucocorticoids with other drugs because of potential interactions, and because some drugs may affect the metabolism of the steroids, which may lead to a decreased or increased glucocorticoid effect on their target tissues (20). Such interactions and effects are shown in Tables 3, 4 and 5.

PREDICTING GLUCOCORTICOID-INDUCED HPA AXIS SUPPRESSION

Several predictors of glucocorticoid-induced HPA axis suppression have been discussed, the major of which are the following:

Kind of Steroid Used and GC Potency

As shown in Table 1 long acting preparations have a longer tissue life which induces a chronic state of tissue hypercortisolism, making HPA axis suppression more likely. Thus, hydrocortisone and cortisone acetate are the least potent and, therefore, least suppressive agents. Prednisone, prednisolone, methylprednisolone and triamcinolone are moderately suppressive, and dexamethasone suppresses ACTH the longest.

Systemic Versus Compartmental Therapy

Systemic GC therapy, particularly parenterally, is more likely to suppress the HPA axis. However, other routes of administration such as inhalation, topical, intra-ocular cause HPA axis suppression as well as other systemic AEs and this depends on the systemic bioavailability of the drug (19, 48, 49, 95, 114, 123).

Daily Therapy

There is evidence that patients are at lower risk for adrenal insufficiency if they can take glucocorticoids on alternate days from the outset or if they can convert to alternate-day therapy before the HPA axis is suppressed (21, 129).

Split Doses and Night Doses

Administering GCs in several different doses during the day imposes a greater risk for HPA axis suppression. In the same way, evening doses of glucocorticoids tend to suppress the normal early morning surge of ACTH secretion, resulting in greater adrenal suppression. Whenever possible, it is better to treat patients with a single morning dose. Once-a-day dosing is usually feasible for intermediate or long acting GCs e.g. prednisone, triamcinolone and dexamethasone. The short-acting hydrocortisone and cortisone acetate are usually given twice a day, at waking and around 5 PM. To mimic normal diurnal cortisol rhythms, the morning dose is two thirds, and the afternoon dose one third of the total daily dose (19, 130, 131).

Duration and Cumulative Dose of Glucocorticoid Treatment

Although traditionally the duration of glucocorticoid therapy and the cumulative dose of glucocorticoid received have been considered as predictive of the likelihood of HPA axis suppression, several studies suggest that they only roughly predict HPA axis suppression (132-134). Adrenal insufficiency is extremely rare in patients treated for 1 week or less (135, 136). Nevertheless, with a so called "short-term" 14 day course of systemic GCs, generally considered safe, in patients with acute exacerbation of chronic obstructive pulmonary disease, suppression of the HPA axis has been defined (47).

Cushingoid Features

Patients with Cushing's syndrome symptoms due to GC therapy are more likely to have a suppressed HPA axis and adrenal atrophy (19).

It has been suggested that the best predictor of HPA axis suppression is the patient's current glucocorticoid dosage. A strong correlation has been found between prednisone maintenance doses above 5 mg/d and a subnormal ACTH-stimulation test result (137). Finally, it can be assumed that patients who are more likely to develop HPA axis suppression are those who receive high doses (>20-30mg prednisolone or equivalent) of systemic GCs for long periods (>3weeks) and those who appear to have Cushingoid features. As the HPA axis function in patients treated with synthetic GCs cannot be reliably estimated from the above parameters several tests are commonly used in order to assess the axis' recovery.

WEANING PATIENTS FROM GLUCOCORTICOID THERAPY

Besides their multiple therapeutic uses, GC withdrawal is indicated when their use is no longer recommended as the maximum therapeutic benefit has been obtained or when significant side effects appear and become uncontrollable, such as GC induced psychosis, diabetes mellitus, severe hypertension, and incapacitating osteoporosis. The goal of a successful GC withdrawal regimen can be described as the rapid transition from a state of tissue hypercortisolism to a state of total exogenous GC deprivation without resurgence of the underlying disease and without adrenal insufficiency or any other GC dependency. Although there are no consensus documents, several tapering regimens have been published so far. In clinical practice, the majority of physicians develop their own withdrawal regimens. The common point is that GC withdrawal should never be abrupt (19).

A systematic review published in 2002 found 9 randomized, controlled clinical trials, 7 of which investigated bronchial asthma and chronic obstructive pulmonary disease, which compared different GC tapering regimens. According to this review there was no significant difference between rapid or slow tapering, regarding the diseases' exacerbation and relapse rates, suggesting that prolonged withdrawal may not be necessary for a better outcome of the underlying disease. However, the same review highlighted the uncertainty about the safety and efficacy of GC withdrawal in many chronic diseases, emphasizing the need for further research in this area (131).

In general, patients taking any steroid dose for less than 2 weeks are not likely to develop HPA axis suppression and can stop therapy suddenly without tapering. The possible exception to this is the patient who receives frequent "short" steroid courses e.g. in asthma. Where there has been chronic therapy, the objective is to rapidly reduce the therapeutic dose to a physiological level (equivalent to 7.5mg/d prednisolone) e.g. by reducing 2.5mg every 3-4 days over a few weeks, and then proceed with slower withdrawal in order to permit the HPA axis to recover (19, 21).

As far as patients with underlying disease are concerned it is recommended that all available clinical, biochemical and laboratory data on the activity status of the disease be collected in order to easily identify signs of recurrence. In such a case prescribed doses should be increased (19).

After the initial reduction to physiological levels, doses should be reduced by 1mg/d of prednisolone or equivalent every 2-4 weeks depending upon patient's general condition, until the medication is discontinued. Alternatively, after the initial reduction to 5-7.5mg of prednisolone, the clinician can switch the patient to HC 20mg/d and reduce by 2.5mg/d every week until the dose of 10mg/d is achieved. After 2-3 months on the same dose, the HPA axis function should be assessed through a Corticotropin (ACTH-Synachten) test or through an Insulin Tolerance test (ITT). A pass response to these tests indicates adequate function of the axis and GCs can be safely withdrawn. If the axis has not fully recovered, treatment should be continued and the axis function should be reassessed (21).

Other tapering regimens have been published some of them dealing with switching the patient to an alternate dosage of GC before discontinuation (138).

Irrespectively of the tapering regimen used, if GC withdrawal syndrome, adrenal insufficiency's symptomatology, or exacerbation of the underlying disease appears, the dose being given at the time should be elevated or maintained for a longer period of time. Moreover, in the absence of evidence of HPA axis full recovery in patients who have been treated with GCs for prolonged periods, supplementation equivalent to 100-150mg of HC is recommended during situations of severe stress such as major surgery, fractures, severe systemic infections, major burns, etc.

Finally, it has become obvious, that all patients treated long-term with GCs should be treated in a similar fashion to patients with chronic ACTH deficiency, thus, they should be instructed to carry some type of identification (worn around the neck or wrist or carried as a card) (19, 21).

ACUTE ADRENAL CRISIS

Full HPA axis recovery after cessation of GC therapy may take as long as 1 year or more (11, 139). Abrupt cessation of glucocorticoid treatment or quick tapering can precipitate an acute adrenal insufficiency crisis. The main symptoms range from anorexia, fatigue, nausea, vomiting, dyspnea, fever, arthralgia, myalgia, and orthostatic hypotension to dizziness, fainting, and circulatory collapse. Hypoglycemia is occasionally observed in children and very thin adult individuals. The diagnosis is a medical emergency, and treatment should be immediate administration of fluids, electrolytes, glucose, and parenteral glucocorticoids.

GLUCOCORTICOID WITHDRAWAL SYNDROME (GWS)

Chronic administration of high doses of GCs and also other hormones such as estrogens, progestins, androgens and growth hormone induce varying degrees of tolerance, resulting in a progressively decreased response to the effect of the drug, followed by dependence and rarely "addiction". Traditionally, the term "Endocrine Withdrawal Syndromes" has been used to describe symptoms and signs of specific hormone deficiency after discontinuation of hormonal therapy or removal of an endocrine gland. However, discontinuation of hormonal therapy frequently results in a mixed picture of two different syndromes: a typical hormone deficiency syndrome and a generic withdrawal syndrome. Four aspects of GCs withdrawal after cessation of pharmacological high-dose therapy are important: 1) relapse of the underlying disease for which the drug was prescribed 2) HPA axis suppression which can persist for a long time 3) psychological dependence 4) a non-specific withdrawal syndrome despite normal HPA axis function and even while patients are receiving physiological replacement doses of GCs (140, 141).

Amatruda et al. first defined the steroid withdrawal syndrome as a symptom complex resembling true adrenal insufficiency, with nonspecific symptoms like weakness, nausea, and arthralgias, occurring in patients who have finished a dosage reduction of glucocorticoid therapy and who respond normally to HPA axis testing (142). Thus, after cessation of GC therapy patients may develop anorexia, nausea, emesis, weight loss, fatigue, myalgias, arthralgias, weakness, headache, abdominal pain, lethargy, postural hypotension, fever, skin desquamation, tachycardia, emotional lability, and even delirium, and psychotic states even if the response of the HPA axis to stimuli has returned to normal (140). Children and adolescents may experience signs and symptoms of GWS even when GCs are still being administered in supraphysiological doses (19). Biochemical evidence related to the GWS includes hypercalcemia and hyperphosphatemia (140).

The GWS has been considered a withdrawal reaction due to established physical dependence on supraphysiological GC levels (140). It has also been described as a state of relative GC resistance in these patients, effectively rendering them hypoadrenal (141). The mechanisms responsible for GWS have not been fully elucidated. Nevertheless, several mediators should be considered and include CRH, vasopressin, POMC, several cytokines such as IL-1β, IL-6, TNF-α, prostaglandins such as E2, I2, phospholipase A2 and also alterations of the noradrenergic and dopaminergic systems (19, 140).

The severity of GWS depends on the genetics and developmental history of the patient, on his environment, and on the phase and degree of dependence the patient has reached (140). The syndrome is self-limited with a median duration of 10 months. Its management should include a temporary increase in the dose of GCs followed by gradual, slow tapering to a maintenance dose (141).

BIOCHEMICAL DIAGNOSIS OF ADRENAL INSUFFICIENCY

Glucocorticoid treatment may not suppress the HPA axis at all, or it may cause central suppression and adrenal gland atrophy of varying degrees. Several endocrine tests have been used to define progression of glucocorticoid-induced adrenal insufficiency. The insulin tolerance test and the metyrapone test have been employed in the diagnosis of adrenal suppression and are quite sensitive, however, the risks involved with both tests do not justify their use when a rapid ACTH stimulation test can distinguish clinically significant adrenal suppression.

To evaluate the adequacy of hypothalamic-pituitary-adrenal axis recovery, the rapid Synachten (or high-dose ACTH stimulation test) is mostly used. An intravenous bolus of 250 ug of corticotropin 1-24 is administered and cortisol is measured after 30 or 60 minutes or both. A plasma cortisol concentration > 18 - 20 μg/ dL at these times indicates adequate recovery of the hypothalamic-pituitary-adrenal axis (139).

The low-dose Synachten test (1ug or 500 ng ACTH(1-24)/1.73 m2) is also being used for the assessment of the HPA axis after prolonged use of GC medication (143-145). It is unclear if the low-dose test is superior to the high-dose test for the detection of secondary adrenal insufficiency. Some studies have shown that the low-dose Synachten test is more sensitive in detecting partial secondary adrenal insufficiency (as can occur in chronic use of GCs), which is not detected by the standard high-dose test because the latter provides a supraphysiologic stimulus able to stimulate a partially damaged adrenal (146-149). A meta-analysis of 28 studies evaluated the utility of the high and low-dose ACTH test. At a specificity of 95% the sensitivity of the high-dose test for primary adrenal insufficiency was 97%, greater than that for secondary adrenal insufficiency (57%). The sensitivities for secondary adrenal insufficiency were similar between the high-dose (57%) and the low-dose Synachten test (61%) (150). In contrast, a review of the literature published between 1965 and 2007 suggests that the low-dose test is the best test currently available for establishing the diagnosis of secondary AI (151). Further studies are needed to establish if the low-dose Synachten test is preferable for the diagnosis of secondary AI.

The Corticotropin Releasing Hormone (CRH) test can also be used in patients taking GC treatment for prolonged periods, as it can assess both the ACTH and cortisol responses and can distinguish between secondary and tertiary adrenal insufficiency (133, 152).

The Dexamethasone Suppression Test has been shown to predict the later development of an impaired adrenal function after a 14-day course of prednisone in healthy volunteers and this information may allow a more targeted approach for the patients after cessation of steroid therapy (153).

FUTURE PERSPECTIVES ABOUT GLUCOCORTICOID THERAPY

Although hydrocortisone (HC) is the most commonly used regimen for replacement in patients with primary and secondary adrenal insufficiency, it is evident that this conventional therapy cannot provide the physiological rhythm of cortisol release. Moreover, with current replacement therapy, the majority of patients with adrenal insufficiency report impaired health-related quality of life, early morning fatigue, socioeconomic health problems and, finally, increased mortality (154). Circadian infusions of HC delivered by a programmable pump can mimic the normal rhythm of cortisol secretion and improve biochemical control and quality of life in patients with adrenal insufficiency and congenital adrenal hyperplasia. Because such infusions are not a practical solution, new formulations of oral HC, which mimic cortisol physiology have been evaluated. A dual-release hydrocortisone tablet with an immediate-release outer layer covering a sustained-release core, has been used in patients with Addison’s disease showing improvements in cardiovascular risk factors, including body weight, hemoglobin A1C and blood pressure, as well as a significant improvement in fatigue (154-156). In the long-term, this once-daily formulation was well-tolerated with a small number of adverse effects (157). Another modified-release multi-particulate hydrocortisone capsule formulation has been developed recently (158). This formulation was well tolerated and very effective in controlling disease biomarkers of congenital adrenal hyperplasia, such as androstenedione and 17-hydroxyprogesterone, with a lower hydrocortisone dose equivalency (154).

Apart from their use for hormonal replacement, the clinical success of synthetic GCs as anti-inflammatory agents is largely attributed to their ability to reduce the expression of proinflammatory genes, via activation of the GR and the concomitant inhibition of the activity of proinflammatory transcription factors, including NF-κB and AP-1, through a mechanism called trans-repression. On the other hand, the appearance of their AEs mainly arise from their ability to activate, after induction of the GR, target genes involved in the metabolism of sugar, protein, fat, muscle and bone via a mechanism called trans-activation (159, 160). There is a plethora of recent work dealing with the characteristics of novel selective GR ligands with equal efficacy and improved side-effects profiles, in other words ligands that show an improved therapeutic index (159-162). These efforts have resulted in a number of different terminologies: Selective GR modulators, selective GR agonists, gene-selective compounds, dissociated compounds, etc. (161, 163), which have been developed and are still being developed mainly focusing on the trans-repression mechanism and stimulating the side-effect pathway to a lesser extent, at least in specific tissues. Nevertheless, the likelihood of finding a compound that actually separates all activated genes from all repressed genes is highly unlikely mainly because the transactivation vs trans-repression characteristics are highly cell-type and gene specific. Moreover, it is also unclear whether such a compound would be truly desirable, as upregulation of anti-inflammatory genes may also play a role in the treatment of many diseases (159-161). In addition, many non-steroidal dissociated GR modulators, some of which do not support trans-activation, have shown promising benefit to side-effect ratios (e.g. ZK216348, CpdA) (159).

Considering the complexity of pathways regulated by GR, it is clearly too naive to assume that an ideal exogenous GR modulator only eliciting the beneficial anti-inflammatory effects without any trace of side-effects will ever be found. Complementing genome-wide gene profiling studies and transcription factor/DNA binding patterns on various target tissues at once will become an adamant strategy for the future (159). However, recent reports of Selective GR modulators provide fertile ground for additional efforts and it is obvious that any progress in this area would be a major benefit for thousands of patients receiving GC therapy (161).

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