Introduction

Primary adrenal insufficiency (PAI) is defined by cortisol deficiency in humans due to disorders of the adrenal cortex affecting its production. Cortisol is produced in the zona fasciculata (zF, middle zone) of the adrenal cortex. This glucocorticosteroid hormone is responsible for the acute and chronic stress response and thus the regulation and maintenance of the energy homeostasis of the human body. Other steroid hormones produced by the adrenal cortex are mineralocorticoids in the zona glomerulosa (zG) for maintaining water and electrolyte balance, and adrenal androgens in the zona reticularis (zR), which contribute to the sex steroid pool ⁽¹⁾. Cortisol production of the adrenal cortex is controlled by the hypothalamic-pituitary-adrenal (HPA) axis. Essential players comprised in this axis are the hormones corticotropin releasing hormone (CRH), adrenocorticotropic hormone ACTH, the ACTH receptor (MC2R), as well as the GPCR/cAMP/MAPK signaling pathway and cortisol. Cortisol then acts predominantly on the glucocorticoid (GC) receptor NR3C1 to exert its action on multiple biological processes and organs. The HPA axis also co-regulates adrenal androgen production, while mineralocorticoid production (mainly aldosterone) is controlled by the renin-angiotensin-aldosterone system.

Genetic variants causing PAI either disrupt cortisol production and steroidogenesis only leading to isolated PAI or they cause additional organ malfunctions as part of a syndrome in syndromic PAI. In isolated PAI, genetic disorders may affect the structure and function of the adrenal cortex or adrenal steroidogenesis specifically, but often also lead to overall disturbances of steroidogenesis affecting other steroid organs (mainly the gonads or the placenta). This can then result in different disorders of sex development (DSD), as steroid hormone biosynthesis, regulation and metabolism rely on a common gene network. Nevertheless, cell- and tissue-specific expression and regulation of steroidogenic genes leads to organ-specific steroid production. Genetic variants in core genes of steroid hormone biosynthesis may therefore be recognized by characteristic clinical phenotypes and changes in steroid profiles assessed in biosamples such as plasma and urine. However, considerable overlap exists so that a characteristic clinical and biochemical profile may be caused by variants in more than one gene, and different variants in one gene may manifest as phenotypically variable.

Although syndromic forms of PAI seem easier to recognize through their broader range of typically involved organ systems, this remains just a theory in many cases. Syndromes with PAI can manifest similar, oligosymptomatic or atypical mainly because the typical spectrum may only develop over time or simply because the phenotype is only recognized when searched for.

However, disorders leading to PAI may not only be grouped according to whether they affect just the adrenal structure and function or lead to defects in other organ systems. They may also be characterized by their suggested molecular disease mechanism. Table 1 gives an overview of all monogenetic disorders causing PAI reported in the literature to date.

PAI often manifests (very) early in life or even goes undiagnosed when embryonic or neonatal lethal, although late-onset manifestation in adulthood is also seen. Clinical signs of adrenal insufficiency are non-specific, but with severe stress such as major illness, trauma or surgery an acute adrenal crisis may be triggered. Signs and symptoms of an acute crisis include abdominal pain, fever, hypoglycemic seizures, weakness, apathy, nausea, vomiting, anorexia, hyponatremia, hypochloremic acidemia, hyperkalemia, hypotension, shock, cardiovascular collapse, and sudden death.

Milestones in medicine concerning PAI were the first clinical description of a patient with PAI by Thomas Addison in 1849, the first description of a patient with congenital adrenal hyperplasia (CAH) by Luigi De Crecchio in 1865, the isolation of cortisol from adrenal extracts by Edward Calvin Kendall and its synthesis for medical use by Lewis H. Sarett in 1946, and the identification of the first genes causing CAH (CYP21A2) by Perrin White in 1984 and familial glucocorticoid deficiency (MC2R) by Adrian J.L. Clark and Constantine Tsigos in 1993.

Several comprehensive reviews have recently been published on the topic of genetic disorders of the adrenal cortex and steroidogenesis ^(2-11). This update summarizes the genetic causes of PAI with an emphasis on the most recent findings and a perspective on current diagnostic yield and future therapeutic options.

Update on more recent findings on the topic of monogenetic disorders causing PAI

In the group of steroid biosynthetic defects (Table 1), we have learned from affected persons that autosomal recessive gene variants coding for enzymes and cofactors involved in adrenal steroidogenesis (and beyond) may manifest with variable phenotype. This has long been known for variants in CYP21A2, where loss of function mutations cause classic CAH and milder enzyme defects lead to non-classic, late-onset CAH ⁽¹⁰⁾. Likewise, less severe autosomal recessive genetic variants in STAR ⁽¹²⁾ and CYP11A1 ⁽¹³⁾ have been identified in patients with an adrenal phenotype only mimicking familial GC deficiency (FGD) ⁽¹⁴⁾ and not typical lipoid CAH, in which adrenal and gonadal steroidogenesis is affected leading to PAI and 46,XY DSD ⁽¹⁵⁾. Similarly, different variants in P450 oxidoreductase can manifest with a vast range of phenotypes ranging from the severest Antley Bixler skeletal and genital malformation syndrome to moderate forms with a CAH phenotype, and to mild forms overlapping with polycystic ovary syndrome in females or lyase deficiency syndrome in males ^(16-18).

In the group of structural developmental defects of the adrenal cortex, X-linked adrenal hypoplasia congenita due to pathogenic variants in the DAX1/NROB1 gene remains the most frequently identified genetic defect in males with PAI ⁽⁸⁾. By contrast, genetic variants in SF1/NR5A1 are rarely identified in PAI, but they typically manifest with a broad range of DSDs in both sexes ⁽¹⁹⁾. Conversely, very rare syndromic forms of PAI affecting adrenal development and function and affecting fetal growth have been identified more recently through the efforts of next-generation genetic work-up. These include the IMAGe and MIRAGE syndromes (Table 1).

In IMAGe syndrome specific heterozygous gain-of-function mutations in the CDKN1C (cyclin-dependent kinase inhibitor 1C) gene were found in patients manifesting with intrauterine growth retardation, metaphyseal dysplasia, adrenal hypoplasia congenita, and genital anomalies ⁽²⁰⁾. In addition, biallelic POLE mutations causing DNA polymerase epsilon deficiency with a variable degree of immunodeficiency were discovered in another 15 patients of 12 families with IMAGe syndrome ⁽²¹⁾. POLe is one of the leading polymerases for DNA replication essential for the correct transmission of genetic information. To date, all CDKN1C mutations identified in IMAGe syndrome cluster in the proliferating cell nuclear antigen (PCNA) binding domain. At the initiation of replication, PCNA loads with POLe, and thus the phenotypic overlap of CDKN1C with POLE-associated IMAGe syndrome suggests a mechanistic link ⁽²¹⁾. Together with MCM4 mutations, CDKN1C and POLE mutations share a common profile of PAI including growth restriction and immunodeficiency due to impaired replisome function ^{(21, 22)}.

MIRAGE syndrome is characterized by myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes, enteropathy and early death. In patients with MIRAGE syndrome, SAMD9 (sterile alpha motif domain-containing protein 9) mutations have been discovered ^{(23, 24)}. SAMD9 is involved in endosome fusion and is reported to play a role in growth factor signaling transduction. Thus, heterozygote SAMD9 mutations seem to enhance its intrinsic endosome-fusing activity and may thereby lead to abnormal tissue development including dysgenetic and hypoplastic adrenal glands, ovaries and thymus, and result in deleterious overall growth-restricting effects and short survival. In some MIRAGE patients, longer survival seems possible through genetic escape mechanisms such as somatic mutations and progressive monosomy resulting in loss of mutated SAMD9 effects in the bone marrow ⁽²³⁾. However, this may come at the cost of increased risk for myelodysplastic syndromes in the long run. Interestingly, SAMD9 mutations were recently also found in a severely undervirilized 46,XY DSD child born SGA without PAI, but otherwise typical features of MIRAGE syndrome ⁽²⁵⁾. And another recent report of two unrelated patients carrying novel and known SAMD9 mutations expanded the typical phenotypic spectrum by including PAI by CNS anomalies and global developmental delay, dysautonomy, hearing loss and chronic lung disease ⁽²⁶⁾.

Overall, it is remarkable how few variants in genes that are critically involved in adrenal development, as revealed by basic research studies, have been identified in humans with dysgenetic, syndromic PAI ⁽²⁷⁾. One of these genes is WNT4. But so far a homozygous mutation in WNT4 has been identified in only one family with the SERKAL syndrome (female sex reversal and dysgenesis of kidneys, adrenals, and lungs) ⁽²⁸⁾. It is therefore very likely that pathogenic variants in genes essential for adrenal development and beyond are embryonic lethal.

In the early era of genetics, first two genes causing familial glucocorticoid deficiency (FGD) were identified, namely mutations in the ACTH receptor (MC2R) and its accessory protein (MRAP) ^{(29, 30)}. In addition, the syndromic variant of FGD, triple A or Allgrove syndrome, was solved genetically by identifying mutations in the AAAS gene ⁽³¹⁾. AAAS is a nucleoporin component thought to be involved in cellular stress response ⁽³²⁾. However, mutations in the aforementioned genes were identified in less than a third of patients with FGD. More recently, newer genetic methods discovered a novel group of genes located in the mitochondria responsible for oxidative stress homeostasis ⁽³³⁾. Very quickly, many mutations in the nicotinamide nucleotide transhydrogenase (NNT) gene of the energy transfer system of the respiratory chain have been found in a fairly large number of unsolved patients with non-syndromic FGD ^{(34, 35)}. Nnt deficient mice show disorganized adrenal gland zonation, high apoptosis, and diminished steroid production, while NNT knockdown in adrenal cells revealed impaired redox potential through elevated reactive oxygen species and decreased glutathione (GSH) to GSH-disulfide (GSSG) ratio ⁽³⁵⁾. Meanwhile, mutations in other genes associated with the mitochondrial redox system have been found in FGD patients including variants in TXNRD2, GPX1 and PRDX3 ^{(34, 36)}.

It seems important to be aware that NNT is widely expressed in adrenal, heart, kidney, thyroid, and adipose tissues. Accordingly, the latest reports of older patients with PAI due to NNT mutations describe a broader phenotype with mineralocorticoid deficiency and extra-adrenal anomalies predominantly affecting the gonads (e.g., cryptorchidism, testis adenoma, precocious puberty, and azoospermia) ⁽³⁷⁾. In addition, hypothyroidism, hypertrophic cardiomyopathy and insulin-dependent diabetes mellitus have been reported ^{(37, 38)}. Phenotypical variability was seen with the same NNT mutations and within families informing that careful follow-up of each individual is warranted.

Several metabolic disorders can lead to PAI (Table 1). Most of them manifest with other characteristic anomalies. However, the X-linked form of adrenoleukodystrophy (X-ALD) due to ABCD1 gene mutations can sometimes present first with an adrenal-only phenotype. The latest syndromic form of PAI due to a metabolic disorder is sphingosine-1-phosphate lyase (SGPL1) deficiency ^(39-41). It manifests typically as a syndrome comprising steroid-resistant nephrotic syndrome and PAI, optionally associated with ichthyosis, primary hypothyroidism, cryptorchidism, immunodeficiency, and neurological anomalies. This novel sphingolipidosis results from impaired breakdown of sphingosine 1-phosphate (S1P). S1P regulates cell migration, differentiation, survival as well as angiogenesis and development. Identified human SGPL1 mutations were shown to behave as recessive loss-of-function mutations affecting protein expression and localization, enzyme activity, and thus degradation of long-chain sphingoids ⁽⁴⁰⁾. The pathomechanism of PAI in SGPL1 deficiency includes both compromised adrenal development as well as disrupted steroidogenesis ⁽⁴¹⁾. Recent reports of patients carrying SGPL1 mutations have broadened the clinical spectrum when finding individuals without PAI, without renal manifestations or without a central nervous phenotype ^{(42, 43)}. Long-term follow-up might be necessary to understand the whole spectrum of disease. Of 31 patients with reported SGPL1 mutations, 27 and 26 had PAI and steroid-resistant nephrotic syndrome, respectively (80%), most of them had skin anomalies and immunodeficiency (80%), and 60% had a neurological phenotype ^{(39-41, 44, 45)}.

Role of the underlying genetic diagnosis for treatment and outcome of PAI

Phenotype-genotype correlation in PAI can be difficult in both syndromic and non-syndromic forms. Generally, the clinical and biochemical phenotype of PAI and family history (including consanguinity and ancestral background) may be hinting at the underlying genetic defect. Comprehensive biochemical testing may be specifically informative for steroidogenesis disorders or metabolic diseases (e.g., X-ALD). Thus, the neonatal screening program includes testing for elevated 17-hydroxyprogesterone as a marker for 21-hydroxylase deficiency CAH (CYP21A2) in many countries ⁽¹⁰⁾. More recently, screening for X-ALD has been recommended in the USA to allow early diagnosis for considering hematopoietic stem cell transplantation as a treatment option ⁽⁴⁶⁾. This screening in dried blood spots is based on the elevation of a lysophosphatidylcholine derivative C26:0-LPC of a very-long-chain fatty acid marker. However, even in these disorders a specific diagnosis at the molecular genetic level is advised to provide detailed information for personalized patient care and family counseling.

Next-generation sequencing (NGS) methods, including whole exome (WES) and genome sequencing (WGS) and hybridization techniques have revolutionized the diagnostic yield for genetic disorders over the past decade. While the gene candidate approach for defining CYP21A2 mutations is still valid in clinical routine, gene panels comprising a selected group of genes are nowadays used for the genetic work-up of PAI offering a molecular genetic diagnosis in 60-80% of cases ^{(34, 47, 48)}. For the remaining cases, TRIO WES or WGS are used to search for novel genetic defects. NGS may also reveal thus far missed genetic hits in genes known to cause PAI. For instance, predicted benign synonymous variants in the CYP11A1 gene (Thr330= and Ser391=) were recently reported to cause PAI in 19 individuals of 13 families when occurring in a compound heterozygous state with a rare disruptive mutation ⁽⁴⁹⁾; corresponding mutations were then identified in a large number of European people with PAI.

Current and future therapeutic aspects and perspectives

Lack of corticosteroids will lead to life-threatening conditions in the short or long run. Treatment of all forms of PAI consists in immediate replacement of steroid hormones including glucocorticoids and mineralocorticoids as needed ⁽¹⁾. This can prevent affected individuals from suffering a deadly adrenal crisis. But hormonal replacement treatment is not trivial with current drugs bearing a considerable risk for adverse effects due to over- and undertreatment. Sex hormone replacement may be added, if gonadal steroidogenesis is affected. In addition, adrenal androgen excess as a consequence of negative feedback overstimulation through the HPA axis in CAH (mainly due to CYP21A2 deficiency) is often difficult to control with GC replacement only, and may therefore require additional anti-androgenic treatments, especially in females ^{(10, 11)}.

Overall, there is a need for improved therapy of PAI including newer GC formulations with near-physiological, pharmacokinetic properties, and novel adrenal regenerative strategies ^{(9-11, 50)}. While newer drugs and treatment modalities mimicking the circadian rhythm of physiologic cortisol production are well on their way into routine clinical care, regenerative approaches are still in the research phase. Treatment or even a cure for some monogenic forms of PAI might be feasible in the future through gene therapeutic options, but they will only become possible when the underlying genetic defect is solved. In the research setting, enzyme replacement therapy using adenoviral gene carrier vectors has been successful in transiently restoring 21-hydroxylase activity in CYP21A2 deficient mice, and may be used in humans in the near future ⁽⁵¹⁾. On the other hand, permanent correction of pathogenic variants would be desirable. Gene therapy directed at patients’ own stem cells could theoretically cure steroid disorders. Early studies aimed at achieving cell-based therapies have shown promising results ⁽⁵⁰⁾, when patient-derived mesenchymal cells were reprogrammed to steroidogenic cells with functional activity ⁽⁵²⁾. The additional use of gene-editing technology may be an option for a future cure for the disease.

Conclusion

Genetic studies conducted to understand adrenal cortex development and function in health and disease are fundamental to improve diagnostic and therapeutic options for individuals with PAI, and to offer informed counseling for affected families. To date, comprehensive genetic work-up of individuals with PAI yields a diagnosis in up to 80%. This may not only have short-term consequences, but also informs on possible long-term effects that might be prevented.

Conflicts of interest

Authors declare no potential Conflicts of Interest.

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