A Novel Intronic Splice—Site Mutation of the CYP11A1 Gene Linked to Adrenal Insufficiency with 46,XY Disorder of Sex Development

A novel CYP11A1: c.1236 + 5G > A was identified, expanding the mutation spectrum of the congenital adrenal insufficiency with 46,XY sex reversal. In a now 17-year-old girl delivered full-term (G2P2, parents unrelated), adrenal failure was diagnosed in the first year of life based on clinical picture of acute adrenal crisis with vomiting, dehydration, weight loss, hypotension, and electrolyte disturbances. At the time, hormonal tests revealed primary adrenocortical insufficiency and steroid profiles showed lack of products of steroidogenesis, and since then the patient has been treated with substitution doses of hydrocortisone and fludrocortisone. At the age of 14, considering the absence of puberty symptoms, extended diagnostic tests revealed elevated LH levels (26.5 mIU/mL) with pre-puberty FSH levels (4.9 mIU/mL), low estradiol (28 pmol/L), testosterone (<2.5 ng/mL), and extremely high levels of ACTH (4961 pg/mL). A cytogenetic study revealed a 46 XY karyotype. A molecular examination confirmed the missense mutation and a novel splice-site mutation of CYP11A1 gene. Compound heterozygosity for the CYP11A1 gene with a known pathogenic variant in one allele and a novel splice site mutation in the second allele is most probably responsible for congenital adrenal insufficiency with 46,XY sex reversal. We discuss the necessity of cytogenetic test in the case of early onset of adrenal failure in the absence of steroidogenesis metabolites in the steroid profile.

Molecular screening of XY SRY-negative sex reversal cases in horses revealed anomalies in amelogenin testing

Male-to-female sex reversal in horses is a developmental disorder in which phenotypic females have a male genetic constitution. Male-to-female sex reversal is the second most common genetic sex abnormality, after X chromosome monosomy. All male-to-female sex reversal cases studied to date have been found to be infertile. Therefore, a screening test is particularly useful in laboratories doing DNA genotyping in horses. Our laboratory has tested > 209,000 horses for parentage using a panel of microsatellite markers and the sex marker gene amelogenin ( AMEL). Suspect XY sex reversal cases are reported females with a male profile by AMEL testing. After routine genotyping, 49 cases were detected and further tested using the sex-determining region Y ( SRY) gene, confirming the XY SRY-negative genotype of suspect sex reversal cases. When some inconsistencies arose in the initial result, a molecular panel of X- and Y-linked markers was analyzed for these samples. Of the 49 cases, 33 were confirmed as XY SRY-negative. The remaining 16 cases were identified as false-positives as a result of anomalies of AMEL testing in horses.

Ovotesticular disorders of sex development in FGF9 mouse models of human synostosis syndromes

In mice, male sex determination depends on FGF9 signalling via FGFR2c in the bipotential gonads to maintain the expression of the key testis gene SOX9. In humans, however, while FGFR2 mutations have been linked to 46,XY disorders of sex development (DSD), the role of FGF9 is unresolved. The only reported pathogenic mutations in human FGF9, FGF9S99N and FGF9R62G, are dominant and result in craniosynostosis (fusion of cranial sutures) or multiple synostoses (fusion of limb joints). Whether these synostosis-causing FGF9 mutations impact upon gonadal development and DSD etiology has not been explored. We therefore examined embryonic gonads in the well-characterized Fgf9 missense mouse mutants, Fgf9S99N and Fgf9N143T, which phenocopy the skeletal defects of FGF9S99N and FGF9R62G variants, respectively. XY Fgf9S99N/S99N and XY Fgf9N143T/N143T fetal mouse gonads showed severely disorganized testis cords and partial XY sex reversal at 12.5 days post coitum (dpc), suggesting loss of FGF9 function. By 15.5 dpc, testis development in both mutants had partly recovered. Mitotic analysis in vivo and in vitro suggested that the testicular phenotypes in these mutants arise in part through reduced proliferation of the gonadal supporting cells. These data raise the possibility that human FGF9 mutations causative for dominant skeletal conditions can also lead to loss of FGF9 function in the developing testis, at least in mice. Our data suggest that, in humans, testis development is largely tolerant of deleterious FGF9 mutations which lead to skeletal defects, thus offering an explanation as to why XY DSDs are rare in patients with pathogenic FGF9 variants.

SUN-034 Combined CNV, Haplotyping and Whole Exome Sequencing Implicates Inherited Novel SRY Mutations Not Account for Familial 46,XY Sex Reversal

SRY is one of the important genes involved in the process of human sex determination. The disturbed sex determination caused by SRY mutation accounts for 10-15% cases with complete gonadal dysplasia (CGD), also known as 46, XY sex reversal. Recently, three distal enhancers are disclosed in the upstream of SOX9 gene. In an inherited 46, XY sex reversal pedigree with 5 patients, p.Arg76Leu mutation of SRY and p.G212S mutation of NR5A1were identified from the proband who present with primary amenorrhea and lack of puberty development. The missense mutation of NR5A1was found to be derived from the mother. Interestingly, the paternal inherited p.Arg76Leu mutation of SRY was revealed from other 2 CGD patients, as well as from apparent normal male family members with fertility. P.Arg76Leu variation was found have no effect to the transcriptional activity of target gene SOX9, neither alteration of the nuclear translocation of SRY. Whole exome sequencing also found SRY mutation, FGF10 mutation, GJB4 gene mutation, etc. with no segregation in the family, which suggested SNVs are not main cause of the disease in this pedigree. By copy number variation and SNP haplotype analysis, SOX9 gene far upstream deletion of 68kb was disclosed from the 3 patients in this family, containing one of the enhancers of SOX9. Real-time PCR confirmed that the heterozygous deletion of the region result in loss of SR-XY, but not eSR-B and eALDI. Therefore, single nucleotide variation (SNV) of SRY and NR5A1 are not main causes of severe phenotype of CGD, the enhancers of SOX9 should be investigated carefully in such patients.

Inherited Missense Mutation Occurring in Arginine76 of the SRY Gene Does Not Account for Familial 46, XY Sex Reversal

Background SRY (sex determining region of Y) is one of the important genes involved in the process of human sex determination. The disturbed sex determination caused by an SRY mutation accounts for 10% to 15% of cases with 46, XY sex reversal. Recently, 3 distal enhancers were identified upstream of the SOX9 gene. Objectives The purpose of this study was to investigate the molecular etiology of 46, XY sex reversal in 3 familial patients and a sporadic patient. Design Next-generation sequencing was used to reveal the genotype and inherited pattern. Copy number variations and single nucleotide polymorphism haplotyping were analyzed to observe the alteration of enhancers of SOX9. Transcriptional activity of SRY mutation were assessed by a dual luciferase reporting system, and nuclear translocation was observed by confocal microscopy. Results Two novel SRY gene mutations, p.Arg76Leu and p.Glu89flx15, were identified. In the pedigree with multiple patients, p.Arg76Leu mutation in SRY and p.Gly212Ser mutation in NR5A1 were identified in the proband. The heterozygous deletion far upstream of the SOX9 gene in chromosome 17 was identified in the 3 patients in this family, containing the distal enhancer eSR-A of SOX9 but not eSR-B and eALDI. The frameshift mutation p.Glu89flx15 was revealed to inhibit the transcriptional activity of the target gene, whereas the missense mutation p.Arg76Leu barely showed an effect. Conclusion In contrast to sporadic cases, inherited single nucleotide variations of SRY are not the main cause of the severe phenotype of 46, XY sex reversal, and the enhancers of SOX9 should be investigated carefully in such patients.

Sex reversal following deletion of a single distal enhancer of Sox9

Cell fate decisions require appropriate regulation of key genes. Sox9, a direct target of SRY, is pivotal in mammalian sex determination. In vivo high-throughput chromatin accessibility techniques, transgenic assays, and genome editing revealed several novel gonadal regulatory elements in the 2-megabase gene desert upstream of Sox9. Although others are redundant, enhancer 13 (Enh13), a 557–base pair element located 565 kilobases 5′ from the transcriptional start site, is essential to initiate mouse testis development; its deletion results in XY females with Sox9 transcript levels equivalent to those in XX gonads. Our data are consistent with the time-sensitive activity of SRY and indicate a strict order of enhancer usage. Enh13 is conserved and embedded within a 32.5-kilobase region whose deletion in humans is associated with XY sex reversal, suggesting that it is also critical in humans.