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Abstract NR2F2 encodes COUP-TFII, an orphan nuclear receptor required for the development of the steroidogenic lineages of the murine fetal testes and ovaries. Pathogenic variants in human NR2F2 are associated with testis formation in 46,XX individuals, however, the function of COUP-TFII in the human testis is unknown. We report a de novo heterozygous variant in NR2F2 (c.737G > A, p.Arg246His) in a 46,XY under-masculinized boy with primary hypogonadism. The variant, located within the ligand-binding domain, is predicted to be highly damaging. In vitro studies indicated that the mutation does not impact the stability or subcellular localization of the protein. NR5A1, a related nuclear receptor that is a key factor in gonad formation and function, is known to physically interact with COUP-TFII to regulate gene expression. The mutant protein did not affect the physical interaction with NR5A1. However, in-vitro assays demonstrated that the mutant protein significantly loses the inhibitory effect on NR5A1-mediated activation of both the LHB and INSL3 promoters. The data support a role for COUP-TFII in human testis formation. Although mutually antagonistic sets of genes are known to regulate testis and ovarian pathways, we extend the list of genes, that together with NR5A1 and WT1, are associated with both 46,XX and 46,XY DSD.

Abstract 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.

Abstract Squamous cell carcinoma antigen recognized by T cells 3 (SART3) is an RNA-binding protein with numerous biological functions including recycling small nuclear RNAs to the spliceosome. Here, we identify recessive variants in SART3 in nine individuals presenting with intellectual disability, global developmental delay and a subset of brain anomalies, together with gonadal dysgenesis in 46,XY individuals. Knockdown of the Drosophila orthologue of SART3 reveals a conserved role in testicular and neuronal development. Human induced pluripotent stem cells carrying patient variants in SART3 show disruption to multiple signalling pathways, upregulation of spliceosome components and demonstrate aberrant gonadal and neuronal differentiation in vitro. Collectively, these findings suggest that bi-allelic SART3 variants underlie a spliceosomopathy which we tentatively propose be termed INDYGON syndrome (Intellectual disability, Neurodevelopmental defects and Developmental delay with 46,XY GONadal dysgenesis). Our findings will enable additional diagnoses and improved outcomes for individuals born with this condition.

Abstract Background Forty-six ,XY Differences/Disorders of Sex Development (DSD) are characterized by a broad phenotypic spectrum ranging from typical female to male with undervirilized external genitalia, or more rarely testicular regression with a typical male phenotype. Despite progress in the genetic diagnosis of DSD, most 46,XY DSD cases remain idiopathic. Methods To determine the genetic causes of 46,XY DSD, we studied 165 patients of Tunisian ancestry, who presented a wide range of DSD phenotypes. Karyotyping, candidate gene sequencing, and whole-exome sequencing (WES) were performed. Results Cytogenetic abnormalities, including a high frequency of sex chromosomal anomalies (85.4%), explained the phenotype in 30.9% (51/165) of the cohort. Sanger sequencing of candidate genes identified a novel pathogenic variant in the SRY gene in a patient with 46,XY gonadal dysgenesis. An exome screen of a sub-group of 44 patients with 46,XY DSD revealed pathogenic or likely pathogenic variants in 38.6% (17/44) of patients. Conclusion Rare or novel pathogenic variants were identified in the AR, SRD5A2, ZNRF3, SOX8, SOX9 and HHAT genes. Overall our data indicate a genetic diagnosis rate of 41.2% (68/165) in the group of 46,XY DSD.

SOX8 is an HMG-box transcription factor closely related to SRY and SOX9. Deletion of the gene encoding Sox8 in mice causes reproductive dysfunction but the role of SOX8 in humans is unknown. Here, we show that SOX8 is expressed in the somatic cells of the early developing gonad in the human and influences human sex determination. We identified two individuals with 46, XY disorders/differences in sex development (DSD) and chromosomal rearrangements encompassing the SOX8 locus and a third individual with 46, XY DSD and a missense mutation in the HMG-box of SOX8. In vitro functional assays indicate that this mutation alters the biological activity of the protein. As an emerging body of evidence suggests that DSDs and infertility can have common etiologies, we also analysed SOX8 in a cohort of infertile men (n = 274) and two independent cohorts of women with primary ovarian insufficiency (POI; n = 153 and n = 104). SOX8 mutations were found at increased frequency in oligozoospermic men (3.5%; P

In a majority of individuals with disorders/differences of sex development (DSD) a genetic etiology is often elusive. However, new genes causing DSD are routinely reported and using the unbiased genomic approaches, such as whole exome sequencing (WES) should result in an increased diagnostic yield. Here, we performed WES on a large cohort of 125 individuals all of Algerian origin, who presented with a wide range of DSD phenotypes. The study excluded individuals with congenital adrenal hypoplasia (CAH) or chromosomal DSD. Parental consanguinity was reported in 36% of individuals. The genetic etiology was established in 49.6% (62/125) individuals of the total cohort, which includes 42.2% (35/83) of 46, XY non-syndromic DSD and 69.2% (27/39) of 46, XY syndromic DSD. No pathogenic variants were identified in the 46, XX DSD cases (0/3). Variants in the AR, HSD17B3, NR5A1 and SRD5A2 genes were the most common causes of DSD. Other variants were identified in genes associated with congenital hypogonadotropic hypogonadism (CHH), including the CHD7 and PROKR2. Previously unreported pathogenic/likely pathogenic variants (n = 30) involving 25 different genes were identified in 22.4% of the cohort. Remarkably 11.5% of the 46, XY DSD group carried variants classified as pathogenic/likely pathogenic variant in more than one gene known to cause DSD. The data indicates that variants in PLXNA3, a candidate CHH gene, is unlikely to be involved in CHH. The data also suggest that NR2F2 variants may cause 46, XY DSD.

Objective: Disorders of sex development (DSD) are defined as congenital co nditions in which the development of chromosomal, gonadal and anatomical sex is atypical. Despite wide laboratory and imaging investigations, the etiology of DSD is unknown in over 50% of patients. Methods: We evaluated the etiology of DSD by whole-exome sequencing (WES) at a mean age of 10 years in nine patients for whom extensive evaluation, including hormonal, imaging and candidate gene approaches, had not identified an eti ology. Results: The eight 46,XY patients presented with micropenis, cryptorchidism and hypospadias at birth and the 46,XX patient presented with labia majora fusion. In seven patients (78%), pathogenic variants were identified for RXFP2, HSD17B3, WT1, BMP4, POR, CHD7 and SIN3A. In two atients, no causative variants were found. Mutations in three genes were reported previously with different phenotypes: an 11- year-old boy with a novel de novo variant in BMP4; such variants are mainly associated with microphthalmia and in few cases with external genitalia anomalies in males, supporting the role of BMP4 in the development of male external genitalia; a 12-year-old boy with a known pathogenic variant in RXFP2, encoding insulin-like 3 hormone receptor, and previously reported in adult men with cryptorchidism; an 8-year-old boy with syndromic DSD had a de novo deletion in SIN3A. Conclusions: Our findings of molecular etiologies for DSD in 78% of our patie nts indicate a major role for WES in early DSD diagnosis and management – and highlights the importance of rapid molecular diagnosis in early infancy for sex of rearing decisions.

BackgroundThe clinical profile and genetics of individuals with Disorders/Differences of Sex Development (DSD) has not been reported in Ukraine.Materials and MethodsWe established the Ukrainian DSD Register and identified 682 DSD patients. This cohort includes, 357 patients (52.3% [303 patients with Turner syndrome)] with sex chromosome DSD, 119 (17.5%) with 46,XY DSD and 206 (30.2%) with 46,XX DSD. Patients with sex chromosome DSD and congenital adrenal hyperplasia (CAH, n=185) were excluded from further studies. Fluorescence in situ hybridization (FISH) was performed for eight 46,XX boys. 79 patients underwent Whole Exome Sequencing (WES).ResultsThe majority of patients with 46,XY and 46,XX DSD (n=140), were raised as female (56.3% and 61.9% respectively). WES (n=79) identified pathogenic (P) or likely pathogenic (LP) variants in 43% of the cohort. P/LP variants were identified in the androgen receptor (AR) and NR5A1 genes (20.2%). Variants in other DSD genes including AMHR2, HSD17B3, MYRF, ANOS1, FGFR11, WT1, DHX37, SRD5A1, GATA4, TBCE, CACNA1A and GLI2 were identified in 22.8% of cases. 83.3% of all P/LP variants are novel. 35.3% of patients with a genetic diagnosis had an atypical clinical presentation. A known pathogenic variant in WDR11, which was reported to cause congenital hypogonadotropic hypogonadism (CHH), was identified in individuals with primary hypogonadism.ConclusionsWES is a powerful tool to identify novel causal variants in patients with DSD, including a significant minority that have an atypical clinical presentation. Our data suggest that heterozygous variants in the WDR11 gene are unlikely to cause of CHH.

Cell lineages of the early human gonad commit to one of the two mutually antagonistic organogenetic fates, the testis or the ovary. Some individuals with a 46,XX karyotype develop testes or ovotestes (testicular or ovotesticular disorder of sex development; TDSD/OTDSD), due to the presence of the testis-determining gene, SRY Other rare complex syndromic forms of TDSD/OTDSD are associated with mutations in pro-ovarian genes that repress testis development (e.g. WNT4); however, the genetic cause of the more common non-syndromic forms is unknown. Steroidogenic factor-1 (known as NR5A1) is a key regulator of reproductive development and function. Loss-of-function changes in NR5A1 in 46,XY individuals are associated with a spectrum of phenotypes in humans ranging from a lack of testis formation to male infertility. Mutations in NR5A1 in 46,XX women are associated with primary ovarian insufficiency, which includes a lack of ovary formation, primary and secondary amenorrhoea as well as early menopause. Here, we show that a specific recurrent heterozygous missense mutation (p.Arg92Trp) in the accessory DNA-binding region of NR5A1 is associated with variable degree of testis development in 46,XX children and adults from four unrelated families. Remarkably, in one family a sibling raised as a girl and carrying this NR5A1 mutation was found to have a 46,XY karyotype with partial testicular dysgenesis. These unique findings highlight how a specific variant in a developmental transcription factor can switch organ fate from the ovary to testis in mammals and represents the first missense mutation causing isolated, non-syndromic 46,XX testicular/ovotesticular DSD in humans.

The role of the prokineticin 2 pathway in human reproduction, olfactory bulb morphogenesis, and gonadotropin-releasing hormone secretion is well established. Recent studies have highlighted the implication of di/oligogenic inheritance in this disorder. In the present study, we aimed to identify the genetic mechanisms that could explain incomplete penetrance in hypogonadotropic hypogonadism (HH). This study involved two unrelated Tunisian patients with HH, which was triggered by identifying a homozygous p.(Pro290Ser) mutation in the PROKR2 gene in a girl (HH1) with Kallmann syndrome (KS). The functional effect of this variant has previously been well demonstrated. Unexpectedly, her unaffected father (HH1P) and brother (HH1F) also carried this genetic variation at a homozygous state. In the second family, we identified a heterozygous p.(Lys205del) mutation in PROKR2, both in a male patient with normosmic idiopathic IHH (HH12) and his asymptomatic mother. Whole-exome sequencing in the three HH1 family members allowed the identification of additional variants in the prioritized genes. We then carried out digenic combination predictions using the oligogenic resource for variant analysis (ORVAL) software. For HH1, we found the highest number of disease-causing variant pairs. Notably, a CCDC141 variant (c.2803C > T) was involved in 18 pathogenic digenic combinations. The CCDC141 variant acts in an autosomal recessive inheritance mode, based on the digenic effect prediction data. For the second patient (HH12), prediction by ORVAL allowed the identification of an interesting pathogenic digenic combination between DUSP6 and SEMA7A genes, predicted as “dual molecular diagnosis.” The SEMA7A variant p.(Glu436Lys) is novel and predicted as a VUS by Varsome. Sanger validation revealed the absence of this variant in the healthy mother. We hypothesize that disease expression in HH12 could be induced by the digenic transmission of the SEMA7A and DUSP6 variants or a monogenic inheritance involving only the SEMA7A VUS if further functional assays allow its reclassification into pathogenic. Our findings confirm that homozygous loss-of-function genetic variations are insufficient to cause KS, and that oligogenism is most likely the main transmission mode involved in Congenital Hypogonadotropic Hypogonadism.

Background: Peripheral precocious puberty of ovarian origin is a very rare condition compared to central form. It may be associated with an isolated ovarian cyst (OC). The causes of OC in otherwise healthy prepubertal girls is currently unknown.Methods: Exome sequencing was performed on a cohort of 18 unrelated girls presenting with prenatal and/or prepubertal OC at pelvic ultrasonography. The presenting symptom was prenatal OC in 5, breast development in 7 (with vaginal bleeding in 3) and isolated vaginal bleeding in 6. All had OC ≥ 10 mm. The girls had no other anomalies. Four patients had a familial history of ovarian anomalies and/or infertility.Results: In 9 girls (50%), candidate or known pathogenic variants were identified in genes associated with syndromic and non-syndromic forms of hypogonadotropic hypogonadism including PNPLA6, SEMA3A, TACR3, PROK2, KDM6A, KMT2D, OFD1, GNRH1, GNRHR, GLI3, INSR, CHD7, CDON, RNF216, PROKR2, GLI3, LEPR. Basal plasma concentrations of gonadotropins were undetectable and did not increase after gonadotropin-releasing hormone test in 3 of them whilst 5 had prepubertal values. The plasma estradiol concentrations were prepubertal in 6 girls, high (576 pmol/L) in one and not evaluated in 2 of them.Conclusions: In the first study reporting exome sequencing in prepubertal OC, half of the patients with OC carry either previously reported pathogenic variants or potentially pathogenic variants in genes known to be associated with isolated or syndromic forms of congenital hypogonadotropic hypogonadism. Functional studies and studies of other cohorts are recommended to establish the causality of these variants.

Pituitary stalk interruption syndrome is a rare disorder characterized by an absent or ectopic posterior pituitary, interrupted pituitary stalk and anterior pituitary hypoplasia, as well as in some cases, a range of heterogeneous somatic anomalies. A genetic cause is identified in only around 5% of all cases. Here, we define the genetic variants associated with PSIS followed by the same pediatric endocrinologist. Exome sequencing was performed in 52 (33 boys and 19 girls), including 2 familial cases single center pediatric cases, among them associated 36 (69.2%) had associated symptoms or syndromes. We identified rare and novel variants in genes (37 families with 39 individuals) known to be involved in one or more of the following-midline development and/or pituitary development or function (BMP4, CDON, GLI2, GLI3, HESX1, KIAA0556, LHX9, NKX2-1, PROP1, PTCH1, SHH, TBX19, TGIF1), syndromic and non-syndromic forms of hypogonadotropic hypogonadism (CCDC141, CHD7, FANCA, FANCC, FANCD2, FANCE, FANCG, IL17RD, KISS1R, NSMF, PMM2, SEMA3E, WDR11), syndromic forms of short stature (FGFR3, NBAS, PRMT7, RAF1, SLX4, SMARCA2, SOX11), cerebellum atrophy with optic anomalies (DNMT1, NBAS), axonal migration (ROBO1, SLIT2), and agenesis of the corpus callosum (ARID1B, CC2D2A, CEP120, CSPP1, DHCR7, INPP5E, VPS13B, ZNF423). Pituitary stalk interruption syndrome is characterized by a complex genetic heterogeneity, that reflects a complex phenotypic heterogeneity. Seizures, intellectual disability, micropenis or cryptorchidism, seen at presentation are usually considered as secondary to the pituitary deficiencies. However, this study shows that they are due to specific gene mutations. PSIS should therefore be considered as part of the phenotypic spectrum of other known genetic syndromes rather than as specific clinical entity.

Abstract Background Several studies have shown a high rate of consanguinity and endogamy in North African populations. As a result, the frequency of autosomal recessive diseases is relatively high in the region with the co‐occurrence of two or more diseases. Methods We report here on a consanguineous Libyan family whose child was initially diagnosed as presenting Fanconi anemia (FA) with uncommon skeletal deformities. The chromosome breakage test has been performed using mitomycin C (MMC) while molecular analysis was performed by a combined approach of linkage analysis and whole exome sequencing. Results Cytogenetic analyses showed that the karyotype of the female patient is 46,XY suggesting the diagnosis of a disorder of sex development (DSD). By looking at the genetic etiology of FA and DSD, we have identified p.[Arg798*];[Arg798*] mutation in FANCJ (OMIM #605882) gene responsible for FA and p.[Arg108*];[Arg1497Trp] in EFCAB6 (Gene #64800) gene responsible for DSD. In addition, we have incidentally discovered a novel mutation p.[Gly1372Arg];[Gly1372Arg] in the ERCC6 (CSB) (OMIM #609413) gene responsible for COFS that might explain the atypical severe skeletal deformities. Conclusion The co‐occurrence of clinical and overlapping genetic heterogeneous entities should be taken into consideration for better molecular and genetic counseling.

Introduction: Disorders of gonadal development represent a clinically and genetically heterogeneous group of DSD, and the etiology in many cases remains unknown, indicating that our knowledge of factors controlling sex determination is still limited. Methods: We describe a 46,XY DSD patient from Egypt. The patient was reared as female, born to consanguineous parents, and was referred to us at the age of 5 years because of ambiguous genitalia. On examination, the girl was microcephalic (head circumference –3 SD), but her height and weight were normal for her age and sex. Results: Exome sequencing identified a homozygous variant in the hedgehog acyltransferase (HHAT) gene, which encodes an enzyme that is required for multimerization and signaling potency of the hedgehog secreted proteins. The variant is a novel homozygous missense change c.1329C>A (p.N443K), located within transmembrane domain 9, which segregated with the phenotype in the family. Discussion/Conclusion: Our results expand the phenotypic spectrum associated with HHAT variants to include 46,XY gonadal dysgenesis and reinforce the role of exome sequencing in unraveling new genes that play a pivotal role in sexual development. [ABSTRACT FROM AUTHOR]

Background: DSD encompass a wide range of pathologies that impact gonad formation, development, and function in both 46,XX and 46,XY individuals. The majority of these conditions are considered to be monogenic, although the expression of the phenotype may be influenced by genetic modifiers. Although considered monogenic, establishing the genetic etiology in DSD has been difficult compared to other congenital disorders for a number of reasons including the absence of family cases for classical genetic association studies and the lack of evolutionary conservation of key genetic factors involved in gonad formation. In recent years, the widespread use of genomic sequencing technologies has resulted in multiple genes being identified and proposed as novel monogenic causes of 46,XX and/or 46,XY DSD. Summary: In this review, we will focus on the main genomic findings of recent years, which consists of new candidate genes or loci for DSD as well as new reproductive phenotypes associated with genes that are well established to cause DSD. For each gene or loci, we summarize the data that are currently available in favor of or against a role for these genes in DSD or the contribution of genomic variants within well-established genes to a new reproductive phenotype. Key Messages: Based on this analysis, we propose a series of recommendations that should aid the interpretation of genomic data and ultimately help to improve the accuracy and yield genetic diagnosis of DSD. [ABSTRACT FROM AUTHOR]