Mutations involving the SRY-related gene SOX8 are associated with a spectrum of human reproductive anomalies.

© The Author(s) 2018. Published by Oxford University Press. All rights reserved. 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 < 0.05) and POI (5.06%; P=4.5×10 -5 ) as compared with fertile/normospermic control populations (0.74%). The mutant proteins identified altered SOX8 biological activity as compared with the wild-type protein. These data demonstrate that SOX8 plays an important role in human reproduction and SOX8 mutations contribute to a spectrum of phenotypes including 46, XY DSD, male infertility and 46, XX POI.Link_to_subscribed_fulltex

New Technologies for the Identification of Novel Genetic Markers of Disorders of Sex Development (DSD)

Although the genetic basis of human sexual determination and differentiation has advanced considerably in recent years, the fact remains that in most subjects with disorders of sex development (DSD) the underlying genetic cause is unknown. Where pathogenic mutations have been identified, the phenotype can be highly variable, even within families, suggesting that other genetic variants are influencing the expression of the phenotype. This situation is likely to change, as more powerful and affordable tools become widely available for detailed genetic analyses. Here, we describe recent advances in comparative genomic hybridisation, sequencing by hybridisation and next generation sequencing, and we describe how these technologies will have an impact on our understanding of the genetic causes of DSD

Mutations involving the SRY-related gene SOX8 are associated with a spectrum of human reproductive anomalies

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 analyzed 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<0.05) and POI (5.06%; p=4.5x10-5) as compared to fertile/normospermic control populations (0.74%). The mutant proteins identified altered SOX8 biological activity as compared to the wild-type protein. These data demonstrate that SOX8 plays an important role in human reproduction and SOX8 mutations contribute to a spectrum of phenotypes including 46,XY DSD, male infertility and 46,XX POI

A recurrent p.Arg92Trp variant in steroidogenic factor-1 (NR5A1) can act as a molecular switch in human sex development

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

Biallelic and monoallelic ESR2 variants associated with 46,XY disorders of sex development

Purpose: Disorders or differences of sex development (DSDs) are rare congenital conditions characterized by atypical sex development. Despite advances in genomic technologies, the molecular cause remains unknown in 50% of cases. Methods: Homozygosity mapping and whole-exome sequencing revealed an ESR2 variant in an individual with syndromic 46, XY DSD. Additional cases with 46, XY DSD underwent whole-exome sequencing and targeted next-generation sequencing of ESR2. Functional characterization of the identified variants included luciferase assays and protein structure analysis. Gonadal ESR2 expression was assessed in human embryonic data sets and immunostaining of estrogen receptor-beta (ER-beta) was performed in an 8-week-old human male embryo. Results: We identified a homozygous ESR2 variant, c.541_543del p. (Asn181del), located in the highly conserved DNA-binding domain of ER-beta, in an individual with syndromic 46, XY DSD. Two additional heterozygous missense variants, c.251G>T p.(Gly84Val) and c.1277T>G p.(Leu426Arg), located in the N-terminus and the ligand-binding domain of ER-beta, were found in unrelated, nonsyndromic 46, XY DSD cases. Significantly increased transcriptional activation and an impact on protein conformation were shown for the p.(Asn181del) and p.(Leu426Arg) variants. Testicular ESR2 expression was previously documented and ER-beta immunostaining was positive in the developing intestine and eyes. Conclusion: Our study supports a role for ESR2 as a novel candidate gene for 46, XY DSD

Genetics In Endocrinology: Approaches to molecular genetic diagnosis in the management of differences/disorders of sex development (DSD): position paper of EU COST Action BM 1303 "DSDnet"

The differential diagnosis of differences or disorders of sex development (DSD) belongs to the most complex fields in medicine. It requires a multidisciplinary team conducting a synoptic and complementary approach consisting of thorough clinical, hormonal and genetic workups. This position paper of EU COST (European Cooperation in Science and Technology) Action BM1303 "DSDnet" was written by leading experts in the field and focuses on current best practice in genetic diagnosis in DSD patients. Ascertainment of the karyotpye defines one of the three major diagnostic DSD subclasses and is therefore the mandatory initial step. Subsequently, further analyses comprise molecular studies of monogenic DSD causes or analysis of copy number variations (CNV), or both. Panels of candidate genes provide rapid and reliable results. Whole exome and genome sequencing (WES and WGS) represent valuable methodological developments that are currently in the transition from basic science to clinical routine service in the field of DSD. However, in addition to covering known DSD candidate genes, WES and WGS help to identify novel genetic causes for DSD. Diagnostic interpretation must be performed with utmost caution and needs careful scientific validation in each DSD case

Mutation Analysis of NR5A1 Encoding Steroidogenic Factor 1 in 77 Patients with 46, XY Disorders of Sex Development (DSD) Including Hypospadias

BACKGROUND: Mutations of the NR5A1 gene encoding steroidogenic factor-1 have been reported in association with a wide spectrum of 46,XY DSD (Disorder of Sex Development) phenotypes including severe forms of hypospadias. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the frequency of NR5A1 gene mutations in a large series of patients presenting with 46,XY DSD and hypospadias. Based on their clinical presentation 77 patients were classified either as complete or partial gonadal dysgenesis (uterus seen at genitography and/or surgery, n = 11), ambiguous external genitalia without uterus (n = 33) or hypospadias (n = 33). We identified heterozygous NR5A1 mutations in 4 cases of ambiguous external genitalia without uterus (12.1%; p.Trp279Arg, pArg39Pro, c.390delG, c140_141insCACG) and a de novo missense mutation in one case with distal hypospadias (3%; p.Arg313Cys). Mutant proteins showed reduced transactivation activity and mutants p.Arg39Pro and p.Arg313Cys did not synergize with the GATA4 cofactor to stimulate reporter gene activity, although they retained their ability to physically interact with the GATA4 protein. CONCLUSIONS/SIGNIFICANCE: Mutations in NR5A1 were observed in 5/77 (6.5%) cases of 46,XY DSD including hypospadias. Excluding the cases of 46,XY gonadal dysgenesis the incidence of NR5A1 mutations was 5/66 (7.6%). An individual with isolated distal hypopadias carried a de novo heterozygous missense mutation, thus extending the range of phenotypes associated with NR5A1 mutations and suggesting that this group of patients should be screened for NR5A1 mutations

Association of the MTHFR A1298C Variant with Unexplained Severe Male Infertility

The methylenetetrahydrofolate reductase (MTHFR) gene is one of the main regulatory enzymes involved in folate metabolism, DNA synthesis and remethylation reactions. The influence of MTHFR variants on male infertility is not completely understood. The objective of this study was to analyze the distribution of the MTHFR C677T and A1298C variants using PCR-Restriction Fragment Length Polymorphism (RFLP) in a case group consisting of 344 men with unexplained reduced sperm counts compared to 617 ancestry-matched fertile or normozoospermic controls. The Chi square test was used to analyze the genotype distributions of MTHFR polymorphisms. Our data indicated a lack of association of the C677T variant with infertility. However, the homozygous (C/C) A1298C polymorphism of the MTHFR gene was present at a statistically high significance in severe oligozoospermia group compared with controls (OR = 3.372, 95% confidence interval CI = 1.27–8.238; p = 0.01431). The genotype distribution of the A1298C variants showed significant deviation from the expected Hardy-Weinberg equilibrium, suggesting that purifying selection may be acting on the 1298CC genotype. Further studies are necessary to determine the influence of the environment, especially the consumption of diet folate on sperm counts of men with different MTHFR variants

IVF for premature ovarian failure: first reported births using oocytes donated from a twin sister

BACKGROUND: Premature ovarian failure (POF) remains a clinically challenging entity because in vitro fertilisation (IVF) with donor oocytes is currently the only treatment known to be effective. METHODS: A 33 year-old nulligravid patient with a normal karyotype was diagnosed with POF; she had a history of failed fertility treatments and had an elevated serum FSH (42 mIU/ml). Oocytes donated by her dizygotic twin sister were used for IVF. The donor had already completed a successful pregnancy herself and subsequently produced a total of 10 oocytes after a combined FSH/LH superovulation regime. These eggs were fertilised with sperm from the recipient\u27s husband via intracytoplasmic injection and two fresh embryos were transferred to the recipient on day three. RESULTS: A healthy twin pregnancy resulted from IVF; two boys were delivered by caesarean section at 39 weeks\u27 gestation. Additionally, four embryos were cryopreserved for the recipient\u27s future use. The sister-donor achieved another natural pregnancy six months after oocyte retrieval, resulting in a healthy singleton delivery. CONCLUSION: POF is believed to affect approximately 1% of reproductive age females, and POF patients with a sister who can be an oocyte donor for IVF are rare. Most such IVF patients will conceive from treatment using oocytes from an anonymous oocyte donor. This is the first report of births following sister-donor oocyte IVF in Ireland. Indeed, while sister-donor IVF has been successfully undertaken by IVF units elsewhere, this is the only known case where oocyte donation involved twin sisters. As with all types of donor gamete therapy, pre-treatment counselling is important in the circumstance of sister oocyte donation

Addressing gaps in care of people with conditions affecting sex development and maturation

Differences of sex development are conditions with discrepancies between chromosomal, gonadal and phenotypic sex. In congenital hypogonadotropic hypogonadism, a lack of gonadotropin activity results primarily in the absence of pubertal development with prenatal sex development being (almost) unaffected in most patients. To expedite progress in the care of people affected by differences of sex development and congenital hypogonadotropic hypogonadism, the European Union has funded a number of scientific networks. Two Actions of the Cooperation of Science and Technology (COST) programmes - DSDnet (BM1303) and GnRH Network (BM1105) - provided the framework for ground-breaking research and allowed the development of position papers on diagnostic procedures and special laboratory analyses as well as clinical management. Both Actions developed educational programmes to increase expertise and promote interest in this area of science and medicine. In this Perspective article, we discuss the success of the COST Actions DSDnet and GnRH Network and the European Reference Network for Rare Endocrine Conditions (Endo-ERN), and provide recommendations for future research