A conserved NR5A1-responsive enhancer regulates SRY in testis-determination.

The Y-linked SRY gene initiates mammalian testis-determination. However, how the expression of SRY is regulated remains elusive. Here, we demonstrate that a conserved steroidogenic factor-1 (SF-1)/NR5A1 binding enhancer is required for appropriate SRY expression to initiate testis-determination in humans. Comparative sequence analysis of SRY 5' regions in mammals identified an evolutionary conserved SF-1/NR5A1-binding motif within a 250 bp region of open chromatin located 5 kilobases upstream of the SRY transcription start site. Genomic analysis of 46,XY individuals with disrupted testis-determination, including a large multigenerational family, identified unique single-base substitutions of highly conserved residues within the SF-1/NR5A1-binding element. In silico modelling and in vitro assays demonstrate the enhancer properties of the NR5A1 motif. Deletion of this hemizygous element by genome-editing, in a novel in vitro cellular model recapitulating human Sertoli cell formation, resulted in a significant reduction in expression of SRY. Therefore, human NR5A1 acts as a regulatory switch between testis and ovary development by upregulating SRY expression, a role that may predate the eutherian radiation. We show that disruption of an enhancer can phenocopy variants in the coding regions of SRY that cause human testis dysgenesis. Since disease causing variants in enhancers are currently rare, the regulation of gene expression in testis-determination offers a paradigm to define enhancer activity in a key developmental process.

Analysis of genetic variability in Turner syndrome linked to long-term clinical features.

Background: Women with Turner syndrome (TS) (45,X and related karyotypes) have an increased prevalence of conditions such as diabetes mellitus, obesity, hypothyroidism, autoimmunity, hypertension, and congenital cardiovascular anomalies (CCA). Whilst the risk of developing these co-morbidities may be partly related to haploinsufficiency of key genes on the X chromosome, other mechanisms may be involved. Improving our understanding of underlying processes is important to develop personalized approaches to management. Objective: We investigated whether: 1) global genetic variability differs in women with TS, which might contribute to co-morbidities; 2) common variants in X genes - on the background of haploinsufficiency - are associated with phenotype (a "two-hit" hypothesis); 3) the previously reported association of autosomal TIMP3 variants with CCA can be replicated. Methods: Whole exome sequencing was undertaken in leukocyte DNA from 134 adult women with TS and compared to 46,XX controls (n=23), 46,XX women with primary ovarian insufficiency (n=101), and 46,XY controls (n=11). 1) Variability in autosomal and X chromosome genes was analyzed for all individuals; 2) the relation between common X chromosome variants and the long-term phenotypes listed above was investigated in a subgroup of women with monosomy X; 3) TIMP3 variance was investigated in relation to CCA. Results: Standard filtering identified 6,457,085 autosomal variants and 126,335 X chromosome variants for the entire cohort, whereas a somatic variant pipeline identified 16,223 autosomal and 477 X chromosome changes. 1) Overall exome variability of autosomal genes was similar in women with TS and control/comparison groups, whereas X chromosome variants were proportionate to the complement of X chromosome material; 2) when adjusted for multiple comparisons, no X chromosome gene/variants were strongly enriched in monosomy X women with key phenotypes compared to monosomy X women without these conditions, although several variants of interest emerged; 3) an association between TIMP3 22:32857305:C-T and CCA was found (CCA 13.6%; non-CCA 3.4%, p<0.02). Conclusions: Women with TS do not have an excess of genetic variability in exome analysis. No obvious X-chromosome variants driving phenotype were found, but several possible genes/variants of interest emerged. A reported association between autosomal TIMP3 variance and congenital cardiac anomalies was replicated.

An integrated single-cell analysis of human adrenal cortex development.

The adrenal glands synthesize and release essential steroid hormones such as cortisol and aldosterone, but many aspects of human adrenal gland development are not well understood. Here, we combined single-cell and bulk RNA sequencing, spatial transcriptomics, IHC, and micro-focus computed tomography to investigate key aspects of adrenal development in the first 20 weeks of gestation. We demonstrate rapid adrenal growth and vascularization, with more cell division in the outer definitive zone (DZ). Steroidogenic pathways favored androgen synthesis in the central fetal zone, but DZ capacity to synthesize cortisol and aldosterone developed with time. Core transcriptional regulators were identified, with localized expression of HOPX (also known as Hop homeobox/homeobox-only protein) in the DZ. Potential ligand-receptor interactions between mesenchyme and adrenal cortex were seen (e.g., RSPO3/LGR4). Growth-promoting imprinted genes were enriched in the developing cortex (e.g., IGF2, PEG3). These findings reveal aspects of human adrenal development and have clinical implications for understanding primary adrenal insufficiency and related postnatal adrenal disorders, such as adrenal tumor development, steroid disorders, and neonatal stress.

Investigating ultrastructural morphology in MIRAGE syndrome-derived fibroblasts using transmission electron microscopy.

Background: Heterozygous de novo variants in the gene SAMD9 cause the complex multisystem disorder, MIRAGE syndrome. Patients are characterised by myelodysplasia, infections, growth restriction, adrenal insufficiency, gonadal dysfunction and enteropathies. Pathogenic variants in SAMD9 are gain-of-function and enhance its role as a growth repressor, leading to growth restriction of many tissues. Two studies have reported changes in skin fibroblasts derived from MIRAGE patients, more specifically identifying enlarged endosomes. We have also previously shown subtle changes in endosome size in patients' fibroblasts compared to controls. However, these variations in endosomes were not as marked as those described in the literature. Methods: We have performed an observational study using transmission electron microscopy (TEM) in a larger number of cells derived from three patients' fibroblasts to assess ultrastructure morphology compared to control images. Results: Consistent changes were observed in cell organelles in all patient samples. In particular, increased endosomal activity was detected, characterised by augmented pinocytosis and vesicle budding, increased endosome number, as well as by large lysosomes and endosomes. Endoplasmic reticulum was also prominent. Mitochondria appeared enlarged in selected cells, possibly due to cellular stress. Cell nuclei did not display major differences compared to controls. Conclusions: TEM is a powerful tool to investigate morphological features of tissues and cell organelles, although TEM data could be affected by sample preparation methodology, therefore potentially explaining the variability between independent studies, and its analysis can be dependent on the experience of the researcher. The increased endosomal activity we have observed in patients' fibroblasts could indicate that SAMD9 regulates endocytosis of receptors, acting as an endosome fusion facilitator, or in lysosomal activation. However, the precise mechanism(s) by which SAMD9 regulates cell growth is still not fully understood, and further studies are needed to elucidate its pathogenic pathway and develop therapeutic approaches to support patients.

Emerging phenotypes linked to variants in SAMD9 and MIRAGE syndrome.

Background: Heterozygous de novo variants in SAMD9 cause MIRAGE syndrome, a complex multisystem disorder involving Myelodysplasia, Infection, Restriction of growth, Adrenal hypoplasia, Genital phenotypes, and Enteropathy. The range of additional clinical associations is expanding and includes disrupted placental development, poor post-natal growth and endocrine features. Increasingly, milder phenotypic features such as hypospadias in small for gestational age (SGA) boys and normal adrenal function are reported. Some children present with isolated myelodysplastic syndrome (MDS/monosomy 7) without MIRAGE features. Objective: We aimed to investigate: 1) the range of reported SAMD9 variants, clinical features, and possible genotype-phenotype correlations; 2) whether SAMD9 disruption affects placental function and leads to pregnancy loss/recurrent miscarriage (RM); 3) and if pathogenic variants are associated with isolated fetal growth restriction (FGR). Methods: Published data were analyzed, particularly reviewing position/type of variant, pregnancy, growth data, and associated endocrine features. Genetic analysis of SAMD9 was performed in products of conception (POC, n=26), RM couples, (couples n=48; individuals n=96), children with FGR (n=44), SGA (n=20), and clinical Silver-Russell Syndrome (SRS, n=8), (total n=194). Results: To date, SAMD9 variants are reported in 116 individuals [MDS/monosomy 7, 64 (55.2%); MIRAGE, 52 (44.8%)]. Children with MIRAGE features are increasingly reported without an adrenal phenotype (11/52, 21.2%). Infants without adrenal dysfunction were heavier at birth (median 1515 g versus 1020 g; P < 0.05) and born later (median 34.5 weeks versus 31.0; P < 0.05) compared to those with adrenal insufficiency. In MIRAGE patients, hypospadias is a common feature. Additional endocrinopathies include hypothyroidism, hypo- and hyper-glycemia, short stature and panhypopituitarism. Despite this increasing range of phenotypes, genetic analysis did not reveal any likely pathogenic variants/enrichment of specific variants in SAMD9 in the pregnancy loss/growth restriction cohorts studied. Conclusion: MIRAGE syndrome is more phenotypically diverse than originally reported and includes growth restriction and multisystem features, but without adrenal insufficiency. Endocrinopathies might be overlooked or develop gradually, and may be underreported. As clinical features including FGR, severe infections, anemia and lung problems can be non-specific and are often seen in neonatal medicine, SAMD9-associated conditions may be underdiagnosed. Reaching a specific diagnosis of MIRAGE syndrome is critical for personalized management.

A retrospective analysis of endocrine disease in sphingosine-1-phosphate lyase insufficiency: case series and literature review.

Sphingosine-1-phosphate lyase (SGPL1) insufficiency syndrome (SPLIS) is an autosomal recessive multi-system disorder, which mainly incorporates steroid-resistant nephrotic syndrome and primary adrenal insufficiency. Other variable endocrine manifestations are described. In this study, we aimed to comprehensively annotate the endocrinopathies associated with pathogenic SGPL1 variants and assess for genotype-phenotype correlations by retrospectively reviewing the reports of endocrine disease within our patient cohort and all published cases in the wider literature up to February 2022. Glucocorticoid insufficiency in early childhood is the most common endocrine manifestation affecting 64% of the 50 patients reported with SPLIS, and a third of these individuals have additional mineralocorticoid deficiency. While most individuals also have nephrotic syndrome, SGPL1 variants also account for isolated adrenal insufficiency at presentation. Primary gonadal insufficiency, manifesting with microphallus and cryptorchidism, is reported in less than one-third of affected boys, all with concomitant adrenal disease. Mild primary hypothyroidism affects approximately a third of patients. There is paucity of data on the impact of SGPL1 deficiency on growth, and pubertal development, limited by the early and high mortality rate (approximately 50%). There is no clear genotype-phenotype correlation overall in the syndrome, with variable disease penetrance within individual kindreds. However, with regards to endocrine phenotype, the most prevalent disease variant p.R222Q (affecting 22%) is most consistently associated with isolated glucocorticoid deficiency. To conclude, SPLIS is associated with significant multiple endocrine disorders. While endocrinopathy in the syndrome generally presents in infancy, late-onset disease also occurs. Screening for these is therefore warranted both at diagnosis and through follow-up.

Can Digenic, Tri-Allelic Inheritance of Variants in STAR and CYP11A1 Give Rise to Primary Adrenal Insufficiency? A Case Report.

An eight-year old South Asian boy presenting with progressive hyperpigmentation was found to have primary adrenal insufficiency (PAI) in the form of isolated glucocorticoid deficiency. Follow up of this boy for nine years, until the age of 17 years showed normal pubertal onset and progression. Molecular evaluation, by targeted next generation sequencing of candidate genes linked to PAI revealed changes in two genes that are intricately linked in the early stages of steroid biosynthesis: compound heterozygous variants in STAR, c.465+1G>A and p.(E99K), plus a heterozygous rs6161 change in CYP11A1. No variants in other known causal genes were detected. The proband's mother was heterozygous for the c.465+1G>A STAR and rs6161 CYP11A1 variants, while the father was homozygous for the p.(E99K) alteration in STAR but wild-type for CYP11A1. Both parents had normal adrenal cortical function as revealed by short Synacthen tests. The STAR variant c.465+1G>A will lead to abnormal splicing of exon 4 in mRNA and the addition of the p.(E99K) variant, predicted damaging by SIFT and CADD, may be sufficient to cause PAI but this is by no means certain given that the unaffected father is homozygous for the latter change. The rs6161 CYP11A1 variant [c.940G>A, p.(E314K)] has recently been demonstrated to cause PAI in conjunction with a severe rare disruptive change on the other allele, however sequencing of the coding region of CYP11A1 revealed no further changes in this subject. We wondered whether the phenotype of isolated glucocorticoid deficiency had arisen in this child due to tri-allelic inheritance of a heterozygous CYP11A1 change along with the two STAR variants each of which contribute a partial loss-of-function burden that, when combined, is sufficient to cause PAI or if the loss-of-function c.465+1G>A combined with the presumed partial loss-of-function p.(E99K) in STAR could be causative.

Insights From Long-term Follow-up of a Girl With Adrenal Insufficiency and Sphingosine-1-Phosphate Lyase Deficiency.

Introduction: Sphingosine-1-phosphate lyase (SGPL1) insufficiency syndrome (SPLIS) is a multisystemic disorder which, in the main, incorporates steroid-resistant nephrotic syndrome and primary adrenal insufficiency (PAI). Case Presentation: We present a young girl with a novel homozygous variant in SGPL1, p.D350G, with PAI in the absence of nephrotic syndrome. In the course of 15 years of follow-up she has further developed primary hypothyroidism and while she has progressed through puberty appropriately, ovarian calcifications were noted on imaging. The p.D350G variant results in reduced protein expression of SGPL1. We demonstrate that CRISPR engineered knockout of SGPL1 in human adrenocortical (H295R) cells abrogates cortisol production. Furthermore, while wild-type SGPL1 is able to rescue cortisol production in this in vitro model of adrenal disease, this is not observed with the p.D350G mutant. Conclusion: SGPL1 deficiency should be considered in the differential diagnosis of PAI with close attention paid to evolving disease on follow-up.

Pathogenic variants in the human m6A reader YTHDC2 are associated with primary ovarian insufficiency.

Primary ovarian insufficiency (POI) affects 1% of women and carries significant medical and psychosocial sequelae. Approximately 10% of POI has a defined genetic cause, with most implicated genes relating to biological processes involved in early fetal ovary development and function. Recently, Ythdc2, an RNA helicase and N6-methyladenosine reader, has emerged as a regulator of meiosis in mice. Here, we describe homozygous pathogenic variants in YTHDC2 in 3 women with early-onset POI from 2 families: c. 2567C>G, p.P856R in the helicase-associated (HA2) domain and c.1129G>T, p.E377*. We demonstrated that YTHDC2 is expressed in the developing human fetal ovary and is upregulated in meiotic germ cells, together with related meiosis-associated factors. The p.P856R variant resulted in a less flexible protein that likely disrupted downstream conformational kinetics of the HA2 domain, whereas the p.E377* variant truncated the helicase core. Taken together, our results reveal that YTHDC2 is a key regulator of meiosis in humans and pathogenic variants within this gene are associated with POI.

Pathogenic variants in RNPC3 are associated with hypopituitarism and primary ovarian insufficiency.

PURPOSE: We aimed to investigate the molecular basis underlying a novel phenotype including hypopituitarism associated with primary ovarian insufficiency. METHODS: We used next-generation sequencing to identify variants in all pedigrees. Expression of Rnpc3/RNPC3 was analyzed by in situ hybridization on murine/human embryonic sections. CRISPR/Cas9 was used to generate mice carrying the p.Leu483Phe pathogenic variant in the conserved murine Rnpc3 RRM2 domain. RESULTS: We described 15 patients from 9 pedigrees with biallelic pathogenic variants in RNPC3, encoding a specific protein component of the minor spliceosome, which is associated with a hypopituitary phenotype, including severe growth hormone (GH) deficiency, hypoprolactinemia, variable thyrotropin (also known as thyroid-stimulating hormone) deficiency, and anterior pituitary hypoplasia. Primary ovarian insufficiency was diagnosed in 8 of 9 affected females, whereas males had normal gonadal function. In addition, 2 affected males displayed normal growth when off GH treatment despite severe biochemical GH deficiency. In both mouse and human embryos, Rnpc3/RNPC3 was expressed in the developing forebrain, including the hypothalamus and Rathke's pouch. Female Rnpc3 mutant mice displayed a reduction in pituitary GH content but with no reproductive impairment in young mice. Male mice exhibited no obvious phenotype. CONCLUSION: Our findings suggest novel insights into the role of RNPC3 in female-specific gonadal function and emphasize a critical role for the minor spliceosome in pituitary and ovarian development and function.

Impact of COVID-19 lockdown on the perception of home meals and meal-related variables: A large-scale study within the Italian population during the acute phase of the pandemic.

Italy was the first European country struck by the COVID-19 epidemic and experienced a national lockdown. This study explored the effect of lockdown on the perception of any meals prepared and/or conducted at home (home meals) and investigated which variables played a role in this. A group of Italians (n = 3,060) not suspected/diagnosed as having COVID-19 (18-91 years old; 33% males) completed an online survey during the first lockdown (April 2020). Liking for home meals either increased (51% of the population) or did not vary (43%), while it decreased for only 6% of respondents. Total meal intake similarly either increased (51%) or remained unchanged (33%). Core variables describing meal perception (Liking for meal, Pleasure in meal preparation, Meal duration, Meal Time, Overall food intake, Snack intake) were positively associated with each other. Two clusters with different perceptions of home meals were found, characterised by an increased appreciation (Cl1, 61%) and an unchanged appreciation (Cl2, 39%), respectively. In the acute phase of lockdown, increased meal pleasure was associated with home togetherness (not living alone), cooking with others more often, having high cooking dynamism (use of different kitchen tools, engaging in online food-related activities like using online recipe/website for cooking, use of ready-to-eat meal delivery), and being young, a student or a worker (Cl1). Conversely, Cl2 showed an unchanged meal pleasure, and it was mostly associated with living alone (before and during lockdown), being elderly, retired, widowed, having a low degree of cooking-related activities and dedicating a small weekly budget to food. Variables strictly describing the meal were discussed. Lockdown did not homogenously affect the population in terms of meal pleasure, and high enjoyment of meals was related to high meal involvement. Younger subjects seemed to be more resilient and appreciated meals more due to high cooking dynamism, food-related activities and togetherness. Public health policies could consider these outputs to set up interventions that use meal-dedication activities to increase meal pleasure in vulnerable targets or in subjects experiencing poorly appreciated diets in similar future stressful situations.

Missplicing due to a synonymous, T96= exonic substitution in the T-box transcription factor TBX19 resulting in isolated ACTH deficiency.

SUMMARY: Congenital isolated ACTH deficiency (IAD) is a rare condition characterised by low plasma ACTH and serum cortisol with normal production of other pituitary hormones. TBX19 (also known as TPIT) is a T-box pituitary restricted transcription factor important for POMC gene transcription and terminal differentiation of POMC-expressing cells. TBX19 gene mutations have been shown to cause neonatal-onset congenital IAD. We report a neonate of Romanian origin, who presented at 15 h of life with respiratory arrest and hypoglycaemia which recurred over the following 2 weeks. Biochemical investigations revealed IAD, with undetectable serum cortisol (cortisol < 1 µg/dL; normal range (NR): 7.8-26.2) and plasma ACTH levels within the normal range (22.1 pg/mL; NR: 4.7-48.8). He responded to hydrocortisone treatment. Patient DNA was analysed by a HaloPlex next-generation sequencing array targeting genes for adrenal insufficiency. A novel homozygous synonymous mutation p.Thr96= (Chr1:168260482; c.288G>A; rs376493164; allele frequency 1 × 10-5, no homozygous) was found in exon 2 of the TBX19 gene. The effect of this was assessed by an in vitro splicing assay, which revealed aberrant splicing of exon 2 giving rise to a mutant mRNA transcript whereas the WT vector spliced exon 2 normally. This was identified as the likely cause of IAD in the patient. The predicted protein product would be non-functional in keeping with the complete loss of cortisol production and early presentation in the patient. LEARNING POINTS: Synonymous variants (a nucleotide change that does not alter protein sequence) usually thought to be benign may still have detrimental effects on RNA and protein function causing disease. Hence, they should not be ignored, especially if very rare in public databases. In vitro splicing assays can be employed to characterise the consequence of intronic and exonic nucleotide gene changes that may alter splicing. Establishing a diagnosis due to a TBX19 mutation is important as it defines a condition of isolated ACTH deficiency not associated with additional pituitary deficiencies.

Genetic Analysis of Pediatric Primary Adrenal Insufficiency of Unknown Etiology: 25 Years' Experience in the UK.

CONTEXT: Although primary adrenal insufficiency (PAI) in children and young people is often due to congenital adrenal hyperplasia (CAH) or autoimmunity, other genetic causes occur. The relative prevalence of these conditions is poorly understood. OBJECTIVE: We investigated genetic causes of PAI in children and young people over a 25 year period. DESIGN SETTING AND PARTICIPANTS: Unpublished and published data were reviewed for 155 young people in the United Kingdom who underwent genetic analysis for PAI of unknown etiology in three major research centers between 1993 and 2018. We pre-excluded those with CAH, autoimmune, or metabolic causes. We obtained additional data from NR0B1 (DAX-1) clinical testing centers. INTERVENTION AND OUTCOME MEASUREMENTS: Genetic analysis involved a candidate gene approach (1993 onward) or next generation sequencing (NGS; targeted panels, exomes) (2013-2018). RESULTS: A genetic diagnosis was reached in 103/155 (66.5%) individuals. In 5 children the adrenal insufficiency resolved and no genetic cause was found. Pathogenic variants occurred in 11 genes: MC2R (adrenocorticotropin receptor; 30/155, 19.4%), NR0B1 (DAX-1; 7.7%), CYP11A1 (7.7%), AAAS (7.1%), NNT (6.5%), MRAP (4.5%), TXNRD2 (4.5%), STAR (3.9%), SAMD9 (3.2%), CDKN1C (1.3%), and NR5A1/steroidogenic factor-1 (SF-1; 0.6%). Additionally, 51 boys had NR0B1 variants identified through clinical testing. Although age at presentation, treatment, ancestral background, and birthweight can provide diagnostic clues, genetic testing was often needed to define the cause. CONCLUSIONS: PAI in children and young people often has a genetic basis. Establishing the specific etiology can influence management of this lifelong condition. NGS approaches improve the diagnostic yield when many potential candidate genes are involved.

Current Insights Into Adrenal Insufficiency in the Newborn and Young Infant.

Adrenal insufficiency (AI) is a potentially life-threatening condition that can be difficult to diagnose, especially if it is not considered as a potential cause of a child's clinical presentation or unexpected deterioration. Children who present with AI in early life can have signs of glucocorticoid deficiency (hyperpigmentation, hypoglycemia, prolonged jaundice, poor weight gain), mineralocorticoid deficiency (hypotension, salt loss, collapse), adrenal androgen excess (atypical genitalia), or associated features linked to a specific underlying condition. Here, we provide an overview of causes of childhood AI, with a focus on genetic conditions that present in the first few months of life. Reaching a specific diagnosis can have lifelong implications for focusing management in an individual, and for counseling the family about inheritance and the risk of recurrence.

Long-term outcome of partial P450 side-chain cleavage enzyme deficiency in three brothers: the importance of early diagnosis.

OBJECTIVE: CYP11A1 mutations cause P450 side-chain cleavage (scc) deficiency, a rare form of congenital adrenal hyperplasia with a wide clinical spectrum. We detail the phenotype and evolution in a male sibship identified by HaloPlex targeted capture array. FAMILY STUDY: The youngest of three brothers from a non-consanguineous Scottish family presented with hyperpigmentation at 3.7 years. Investigation showed grossly impaired glucocorticoid function with ACTH elevation, moderately impaired mineralocorticoid function, and normal external genitalia. The older brothers were found to be pigmented also, with glucocorticoid impairment but normal electrolytes. Linkage studies in 2002 showed that all three brothers had inherited the same critical regions of the maternal X chromosome suggesting an X-linked disorder, but analysis of NR0B1 (DAX-1, adrenal hypoplasia) and ABCD1 (adrenoleukodystrophy) were negative. In 2016, next-generation sequencing revealed compound heterozygosity for the rs6161 variant in CYP11A1 (c.940G>A, p.Glu314Lys), together with a severely disruptive frameshift mutation (c.790_802del, K264Lfs*5). The brothers were stable on hydrocortisone and fludrocortisone replacement, testicular volumes (15-20 mL), and serum testosterone levels (24.7, 33.3, and 27.2 nmol/L) were normal, but FSH (41.2 µ/L) was elevated in the proband. The latter had undergone left orchidectomy for suspected malignancy at the age of 25 years and was attending a fertility clinic for oligospermia. Initial histology was reported as showing nodular Leydig cell hyperplasia. However, histological review using CD56 staining confirmed testicular adrenal rest cell tumour (TART). CONCLUSION: This kinship with partial P450scc deficiency demonstrates the importance of precise diagnosis in primary adrenal insufficiency to ensure appropriate counselling and management, particularly of TART.

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

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."

Pathogenic variants in the DEAH-box RNA helicase DHX37 are a frequent cause of 46,XY gonadal dysgenesis and 46,XY testicular regression syndrome.

PURPOSE: XY individuals with disorders/differences of sex development (DSD) are characterized by reduced androgenization caused, in some children, by gonadal dysgenesis or testis regression during fetal development. The genetic etiology for most patients with 46,XY gonadal dysgenesis and for all patients with testicular regression syndrome (TRS) is unknown. METHODS: We performed exome and/or Sanger sequencing in 145 individuals with 46,XY DSD of unknown etiology including gonadal dysgenesis and TRS. RESULTS: Thirteen children carried heterozygous missense pathogenic variants involving the RNA helicase DHX37, which is essential for ribosome biogenesis. Enrichment of rare/novel DHX37 missense variants in 46,XY DSD is highly significant compared with controls (P value = 5.8 × 10-10). Five variants are de novo (P value = 1.5 × 10-5). Twelve variants are clustered in two highly conserved functional domains and were specifically associated with gonadal dysgenesis and TRS. Consistent with a role in early testis development, DHX37 is expressed specifically in somatic cells of the developing human and mouse testis. CONCLUSION: DHX37 pathogenic variants are a new cause of an autosomal dominant form of 46,XY DSD, including gonadal dysgenesis and TRS, showing that these conditions are part of a clinical spectrum. This raises the possibility that some forms of DSD may be a ribosomopathy.

Next-Generation Sequencing Reveals Novel Genetic Variants (SRY, DMRT1, NR5A1, DHH, DHX37) in Adults With 46,XY DSD.

CONTEXT: The genetic basis of human sex development is slowly being elucidated, and >40 different genetic causes of differences (or disorders) of sex development (DSDs) have now been reported. However, reaching a specific diagnosis using traditional approaches can be difficult, especially in adults where limited biochemical data may be available. OBJECTIVE: We used a targeted next-generation sequencing approach to analyze known and candidate genes for DSDs in individuals with no specific molecular diagnosis. PARTICIPANTS AND DESIGN: We studied 52 adult 46,XY women attending a single-center adult service, who were part of a larger cohort of 400 individuals. Classic conditions such as17ß-hydroxysteroid dehydrogenase deficiency type 3, 5α-reductase deficiency type 2, and androgen insensitivity syndrome were excluded. The study cohort had broad working diagnoses of complete gonadal dysgenesis (CGD) (n = 27) and partially virilized 46,XY DSD (pvDSD) (n = 25), a group that included partial gonadal dysgenesis and those with a broad "partial androgen insensitivity syndrome" label. Targeted sequencing of 180 genes was undertaken. RESULTS: Overall, a likely genetic cause was found in 16 of 52 (30.8%) individuals (22.2% CGD, 40.0% pvDSD). Pathogenic variants were found in sex-determining region Y (SRY; n = 3), doublesex and mab-3-related transcription factor 1 (DMRT1; n = 1), NR5A1/steroidogenic factor-1 (SF-1) (n = 1), and desert hedgehog (DHH; n = 1) in the CGD group, and in NR5A1 (n = 5), DHH (n = 1), and DEAH-box helicase 37 (DHX37; n = 4) in the pvDSD group. CONCLUSIONS: Reaching a specific diagnosis can have clinical implications and provides insight into the role of these proteins in sex development. Next-generation sequencing approaches are invaluable, especially in adult populations or where diagnostic biochemistry is not possible.

Predicted Benign and Synonymous Variants in CYP11A1 Cause Primary Adrenal Insufficiency Through Missplicing.

Primary adrenal insufficiency (PAI) is a potentially life-threatening condition that can present with nonspecific features and can be difficult to diagnose. We undertook next generation sequencing in a cohort of children and young adults with PAI of unknown etiology from around the world and identified a heterozygous missense variant (rs6161, c.940G>A, p.Glu314Lys) in CYP11A1 in 19 individuals from 13 different families (allele frequency within undiagnosed PAI in our cohort, 0.102 vs 0.0026 in the Genome Aggregation Database; P < 0.0001). Seventeen individuals harbored a second heterozygous rare disruptive variant in CYP11A1 and two had very rare synonymous changes in trans (c.990G>A, Thr330 = ; c.1173C>T, Ser391 =). Although p.Glu314Lys is predicted to be benign and showed no loss-of-function in an Escherichia coli assay system, in silico and in vitro studies revealed that the rs6161/c.940G>A variant, plus the c.990G>A and c.1173C>T changes, affected splicing and that p.Glu314Lys produces a nonfunctional protein in mammalian cells. Taken together, these findings show that compound heterozygosity involving a relatively common and predicted "benign" variant in CYP11A1 is a major contributor to PAI of unknown etiology, especially in European populations. These observations have implications for personalized management and demonstrate how variants that might be overlooked in standard analyses can be pathogenic when combined with other very rare disruptive changes.

Analysis of CDKN1C in fetal growth restriction and pregnancy loss.

Background: Cyclin-dependent kinase inhibitor 1C (CDKN1C) is a key negative regulator of cell growth encoded by a paternally imprinted/maternally expressed gene in humans. Loss-of-function variants in CDKN1C are associated with an overgrowth condition (Beckwith-Wiedemann Syndrome) whereas "gain-of-function" variants in CDKN1C that increase protein stability cause growth restriction as part of IMAGe syndrome ( Intrauterine growth restriction, Metaphyseal dysplasia, Adrenal hypoplasia and Genital anomalies). As three families have been reported with CDKN1C mutations who have fetal growth restriction (FGR)/Silver-Russell syndrome (SRS) without adrenal insufficiency, we investigated whether pathogenic variants in CDKN1C could be associated with isolated growth restriction or recurrent loss of pregnancy. Methods: Analysis of published literature was undertaken to review the localisation of variants in CDKN1C associated with IMAGe syndrome or fetal growth restriction. CDKN1C expression in different tissues was analysed in available RNA-Seq data (Human Protein Atlas). Targeted sequencing was used to investigate the critical region of CDKN1C for potential pathogenic variants in SRS (n=66), FGR (n=37), DNA from spontaneous loss of pregnancy (n= 22) and women with recurrent miscarriages (n=78) (total n=203). Results: All published single nucleotide variants associated with IMAGe syndrome are located in a highly-conserved "hot-spot" within the PCNA-binding domain of CDKN1C between codons 272-279. Variants associated with familial growth restriction but normal adrenal function currently affect codons 279 and 281. CDKN1C is highly expressed in the placenta compared to adult tissues, which may contribute to the FGR phenotype and supports a role in pregnancy maintenance. In the patient cohorts studied no pathogenic variants were identified in the PCNA-binding domain of CDKN1C. Conclusion: CDKN1C is a key negative regulator of growth. Variants in a very localised "hot-spot" cause growth restriction, with or without adrenal insufficiency. However, pathogenic variants in this region are not a common cause of isolated fetal growth restriction phenotypes or loss-of-pregnancy/recurrent miscarriages.