Primary adrenal insufficiency: New genetic causes and their long‐term consequences

Abstract Primary adrenal insufficiency (PAI) is a potentially life‐threatening condition that requires urgent diagnosis and treatment. Whilst the most common causes are congenital adrenal hyperplasia (CAH) in childhood and autoimmune adrenal insufficiency in adolescence and adulthood, more than 30 other physical and genetics cause of PAI have been reported. Reaching a specific diagnosis can have implications for management and for monitoring associated features, as well as for counselling families about recurrence risk in siblings and relatives. Here, we describe some recent insights into the genetics of adrenal insufficiency and associated molecular mechanisms. We discuss (a) the role of the nuclear receptors DAX‐1 (NR0B1) and steroidogenic factor‐1 (SF‐1, NR5A1) in human adrenal and reproductive dysfunction; (b) multisystem growth restriction syndromes due to gain‐of‐function in the growth repressors CDKN1C (IMAGE syndrome) and SAMD9 (MIRAGE syndrome), or loss of POLE1; (c) nonclassic forms of STAR and P450scc/CYP11A1 insufficiency that present with a delayed‐onset adrenal phenotype and represent a surprisingly prevalent cause of undiagnosed PAI; and (d) a new sphingolipidosis causing PAI due to defects in sphingosine‐1‐phosphate lyase‐1 (SGPL1). Reaching a specific diagnosis can have life‐long implications for management. In some situations, milder or nonclassic forms of these conditions can first present in adulthood and may have been labelled, “Addison's disease.”

some of these conditions may only first present in teenage years or adulthood.
Here, we review some recent insights into the genetics and molecular mechanisms of rare forms of PAI and show how reaching a specific diagnosis can have implications for management and longterm care. We will not discuss forms of CAH (including POR-related syndromes), or well-established adrenal insufficiency syndromes such as Triple A syndrome (achalasia, Addison's, alacrima) or X-linked adrenoleukodystrophy.
Humans have 41 different nuclear receptors, 20 of which currently have associated clinical conditions. 4 DAX-1 and SF-1 are the two nuclear receptors that regulate both adrenal and reproductive development and function. 5

| DAX-1 (NR0B1)
Pathogenic variants in NR0B1/DAX-1 were first reported as a cause of X-linked adrenal hypoplasia congenita (AHC) in 1994. 6 Efforts to localize the gene were helped by the co-existence of adrenal hypoplasia with Duchenne muscular dystrophy due to a contiguous gene deletion syndrome on the short arm of the X chromosome (Xp21).
More than 300 individuals and families with X-linked AHC due to loss of NR0B1/DAX-1 have been reported. 5,7 The classic clinical features of X-linked AHC include primary salt-losing adrenal insufficiency, hypogonadotropic hypogonadism (HH) and infertility. Boys may also present with predominantly either mineralocorticoid or glucocorticoid insufficiency, or may have paradoxical features such as macrophallia or early puberty. 8,9 One report of fertility in a man with X-linked AHC using testicular sperm extraction-intracytoplasmic sperm injection (TESE-ICSI) has been published. 10 Making the specific diagnosis is important so that associated features can be monitored and treated. The risk of presymptomatic adrenal insufficiency in brothers and males in the maternal family needs to be considered. 11 Since 2000, several reports of late-onset X-linked AHC in men with PAI have emerged. [12][13][14][15][16] Usually, this condition is associated with partial HH but infertility might be the main feature. Often the genetic change involves a 5' (aminoterminal) stop variant in the gene and translation of a shorter protein with partial function, or a partial loss-of-function variant in the ligand-like binding domain of NR0B1 ( Figure 1A, 1). 12 One recent review of adult men with PAI in a single UK centre identified two patients with X-linked AHC due to partial loss-of-function variants in DAX-1, suggesting that lateonset X-linked AHC may be underdiagnosed in the adult population ( Figure 1B & 1
Targeted deletion of the gene encoding Nr5a1 in the mouse causes adrenal and gonadal dysgenesis; children with a similar phenotype of adrenogonadal dysfunction were first reported in 1999 and 2002. [17][18][19] These individuals had variants affecting key DNA-binding regions of SF-1 (P-box and A-box). 20 Since these first publications, more than 250 individuals with pathogenic variants in NR5A1/SF-1 have been reported. 5 These changes are usually heterozygous de novo variants but can occur in a "sex-limited dominant" pattern; in this situation, an unaffected woman carries a heterozygous change and passes it to affected 46,XY children, thereby resembling an X-linked condition.
Pathogenic SF-1 variants are associated with a spectrum of phenotypes in 46,XY subjects including testicular dysgenesis/dysfunction (46,XY differences/disorders in sex development), severe hypospadias (accounting for approximately 5%-7% of cases) and male factor infertility (1%-2%). [21][22][23][24][25][26][27][28] Although data are limited, it has been proposed that this subset of infertile men could develop hypogonadism and low testosterone with time, so this may represent a group who need longer term endocrine follow-up. 25 Analysis of larger pedigrees where individuals with 46,XY testicular dysfunction were found together with 46,XX women with ovarian insufficiency (POI) has revealed that defects in SF-1 can affect human ovary function too. 29 Loss-of-function variants in NR5A1 are now well-established in familial POI, but occur less commonly in sporadic (nonfamilial) POI or secondary amenorrhoea (1%-2%). 22,[28][29][30][31][32] Of note, variants in a specific amino acid in the A-box of SF-1 (p.R92) are found in 46,XX ovotesticular DSD (ovotestes or testis), suggesting that very localized alterations in this key transcriptional regulator can "switch" ovary development into a testis development pathway in humans. [33][34][35] Despite these wide-ranging effects on reproductive function, SF-1 variants causing adrenal dysfunction are comparatively rare. Only six children have been reported to date to have SF-1-associated adrenal dysfunction, usually with variants in p.G35 or p.R92. 18,19,[36][37][38] It remains to be seen whether adrenal dysfunction will occur progressively in individuals with reproductive dysfunction due to defects in SF-1; available insights currently suggest that this is not the case, although longer term systematic follow-up studies are needed. Therefore, it seems that human gonadal function is more sensitive to haplo-insufficiency or partial loss of SF-1 activity than adrenal function.

| COMPLE X MULTISYS TEM G ROW TH D ISORDER S: CDKN1C , SAMD9 AND P OLE1
Adrenal insufficiency has also been reported as part of three recently described multisystem growth restriction disorders: IMAGe syndrome, MIRAGE syndrome and POLE1. Although relatively rare, these conditions are associated with interesting pathogenic mechanisms and may be underdiagnosed.

Late-onset X-linked Adrenal Hypoplasia Congenita
Adrenal insufficiency Delayed puberty (Partial HH) Impaired fertility

| CDKN1C: IMAGe syndrome
IMAGe syndrome is characterized by intrauterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia and genitourinary anomalies (often mild hypospadias) and was first described in 1999. 39 The adrenal dysfunction can be variable, including both glucocorticoid and mineralocorticoid insufficiency, and diabetes mellitus has been reported in some members of a large kindred. 40 IMAGe syndrome is usually caused by heterozygous missense variants in the key negative cell cycle regulator, cyclin-dependent kinase inhibitor 1C (CDKN1C). 41 These changes are localized to the PCNA-binding domain and cause a gain-of-function and growth repression (Figure 2A). [41][42][43][44] The mechanism is unclear but may involve decreased degradation of CDKN1C, allowing prolonged cell cycle repression and delayed S-phase progression.
CDKN1C is an imprinted gene, which is only expressed from the maternal allele, so inheritance can mimic an X-linked condition (although both boys and girls are affected). Interestingly, loss-offunction of this growth repressor, CDKN1C, is found in approximately 10% of patients with Beckwith-Wiedemann syndrome (BWS), an "overgrowth" syndrome. 41,45 Children with BWS are at risk of adrenal tumours highlighting how developmental hypoplasia and cancer can sometimes be at opposite ends of a molecular spectrum.

| SAMD9: MIRAGE syndrome
Another multisystem growth restriction disorder occurs due to gain-of-function variants in the growth repressor, Sterile Alpha Motif Domain Containing 9 (SAMD9) ( Figure 2B). 46

SAMD9 model (B) (A)
Most gain-of-function SAMD9 variants occur de novo, although some germline inheritance and variable penetrance has been described. 46,48 SAMD9 may be involved in recycling growth factor receptors (eg EGFR) through endosome trafficking, so that gain-offunction reduces availability of these receptors and leads to growth restriction.
SAMD9 is located on the long arm of chromosome 7 (7q21). One fascinating feature of this condition is that children with SAMD9 mutations who survive early infancy often develop monosomy 7, partial 7q deletions or somatic nonsense (stop gain) changes in SAMD9 in haematopoietic cell lines, which "remove" the mutant SAMD9 allele and confer a clonal growth advantage on those cells ( Figure 2B). 46,47,[49][50][51] This phenomenon can rescue the blood phenotype in the short term. However, loss of 7q21 (including SAMD9 and SAMD9L) can result in myelodysplastic syndrome in the bone marrow (the "M" in MIRAGE) and further somatic "hits" can sometimes lead to the development of leukaemia. 52 In contrast, some children have different forms of revertant mosaicism, such as gene conversion or uniparental disomy, which replace the mutant SAMD9 allele with a wild-type allele. 51,53 In these situations, the bone marrow features reverse and no haematopoietic issues develop.
It is hypothesized that such dynamic changes may modify the phenotype in different organs, explaining why some children have a mild or even no adrenal features. 47 Indeed, somatic modulation and revertant mosaicism could play a wider role in the phenotypic of endocrine disorders than is currently recognized.

| POLE1
Recently, biallelic loss-of-function variants in polymerase epsilon-1 (POLE1, Pol ε) have been reported to cause an IMAGe-like syndrome, in children with growth restriction and adrenal hypoplasia (with variable salt-loss), together with variable immune dysfunction and distinctive facial features. 54

| NON CL A SS I C S TEROIDOG ENI C D ISORDER S: S TAR AND C YP11 A1
Steroidogenic acute regulatory protein (STAR) and cytochrome P450 side-chain (P450scc, encoded by CYP11A1) are two key factors involved in the initial stages of steroidogenesis in the adrenal gland and gonads. 55 STAR is located on the outer mitochondrial membrane and facilitates transfer of cholesterol from the cytoplasm to the mitochondrial inner membrane ( Figure 3A). P450scc is the limiting step in steroidogenesis and catalyses the three steps needed for conversion of cholesterol to pregnenolone ( Figure 3A). 55 Severe disruption of STAR or P450scc/CYP11A1 causes a block in all aspects of adrenal and gonadal steroid synthesis. [56][57][58] For STAR defects this is known as congenital lipoid adrenal hyperplasia (CLAH). 56  female-typical genitalia due to a lack of testosterone biosynthesis in utero. Ovarian insufficiency can occur variably in 46,XX girls at adolescence.
In addition to these "classic" conditions, it has now become apparent that partial defects in these enzymes cause nonclassic disorders; patients present with a predominant adrenal phenotype, usually affecting glucocorticoid synthesis and often resembling familial glucocorticoid deficiency (FGD).

| Nonclassic CYP11A1 deficiency
Similar to NCLAH, it is now emerging that partial loss of function of CYP11A1 (encoding P450scc) also presents with a predominant adrenal phenotype, affecting glucocorticoids and sometimes mineralocorticoid synthesis. 62,63 Patients can present at different ages throughout childhood, often with hyperpigmentation, hypoglycaemia or prolonged illness with infections.
Genetic analysis has revealed that many European individuals and families of European ancestry with this condition are compound (double) heterozygous for a c.940G > A variant (rs6161) on one allele of CYP11A1 and a severely disruptive change on the other allele ( Figure 3A). 64 Although the c.940G>A variant is predicted to cause a benign protein change (p.E314K), detailed molecular studies have shown that it generates a novel splice site so that missplicing occurs. 64 insufficiency can also occur in other populations, such as due to the p.R451W variant common in central Turkey. 37,63 As with NCLAH, long-term monitoring of sex hormone production and fertility is warranted at puberty and in adulthood. Fertility has been reported in men, although raised gonadotrophins are sometimes seen, and testicular adrenal rest tumours (TART) can occur if glucocorticoid insufficiency is poorly controlled. 66 Sperm banking might also be considered.

| A NE W S PHING OLIPIDOS IS: SG PL1
Another recent discovery is the association of PAI with steroid-resistant nephrotic syndrome, due to homozygous or compound heterozygous variants in sphingosine-1-phosphate lyase-1 (SGPL1). [67][68][69] SGPL1 is an enzyme that catalyses the breakdown of sphingolipids by cleaving sphingosine-1-phosphate. 67   As well as the known causes described here, gene discovery approaches using genome wide analysis, better understanding of human adrenal development and function, and newer genetic approaches should help in the discovery of additional causes of PAI in the future. 71 Where no genetic cause is identified, other physical causes may have been overlooked. Finally, in a small but important group, adrenal insufficiency resolves and no specific cause is found.

| CON CLUS IONS
The genetics of many rare forms of primary adrenal insufficiency is gradually being elucidated. Making a specific diagnosis can have implications for immediate management and for monitoring long-term care. Milder or nonclassic forms of adrenal dysfunction can sometimes first present in teenage years or adulthood. In some situations, these individuals may have been labelled as having "Addison's" disease and more detailed genetic investigations to find a specific cause have not been undertaken.

CO N FLI C T O F I NTE R E S T
Nothing to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data in this review are derived from published sources and are acknowledged or referenced accordingly.