A 46,XY female DSD patient with bilateral gonadoblastoma, a novel SRY missense mutation combined with a WT1 KTS splice-site mutation

Patients with Disorders of Sex Development (DSD), especially those with gonadal dysgenesis and hypovirilization are at risk of developing malignant type II germ cell tumors/cancer (GCC) (seminoma/dysgerminoma and nonseminoma), with either carcinoma in situ (CIS) or gonadoblastoma (GB) as precursor lesion. In 10-15% of 46,XY g

Severe sex differentiation disorder in a boy with a 3.8 Mb 10q25.3-q26.12 microdeletion encompassing EMX2

International audienceThe molecular basis of male disorders of sex development (DSD) remains unexplained in a large number of cases. EMX2 has been proposed to play a role in the masculinization process for the past two decades, but formal evidence for this causal role is scarce. The aim of this study is to yield additional support to this hypothesis by reporting on a male patient who presented with 46,XY DSD, a single kidney, intellectual disability, and the smallest microdeletion including EMX2 reported to date. EMX2 haploinsufficiency is likely to explain the masculinization defect observed in our patient, similar to what has been described in the mouse. In the case of cytogenetically diagnosed cases, deletions of EMX2 have been associated with a wide range of DSD, ranging from hypospadias to complete sex reversal

A 46,XY Female DSD Patient with Bilateral Gonadoblastoma, a Novel SRY Missense Mutation Combined with a WT1 KTS Splice-Site Mutation

Patients with Disorders of Sex Development (DSD), especially those with gonadal dysgenesis and hypovirilization are at risk of developing malignant type II germ cell tumors/cancer (GCC) (seminoma/dysgerminoma and nonseminoma), with either carcinoma in situ (CIS) or gonadoblastoma (GB) as precursor lesion. In 10-15% of 46,XY gonadal dysgenesis cases (i.e., Swyer syndrome), SRY mutations, residing in the HMG (High Mobility Group) domain, are found to affect nuclear transport or binding to and bending of DNA. Frasier syndrome (FS) is characterized by gonadal dysgenesis with a high risk for development of GB as well as chronic renal failure in early adulthood, and is known to arise from a splice site mutation in intron 9 of the Wilms' tumor 1 gene (WT1). Mutations in SRY as well as WT1 can lead to diminished expression and function of SRY, resulting in sub-optimal SOX9 expression, Sertoli cell formation and subsequent lack of proper testicular development. Embryonic germ cells residing in this unfavourable micro-environment have an increased risk for malignant transformation. Here a unique case of a phenotypically normal female (age 22 years) is reported, presenting with primary amenorrhoea, later diagnosed as hypergonadotropic hypogonadism on the basis of 46,XY gonadal dygenesis with a novel missense mutation in SRY. Functional in vitro studies showed no convincing protein malfunctioning. Laparoscopic examination revealed streak ovaries and a normal, but small, uterus. Pathological examination demonstrated bilateral GB and dysgerminoma, confirmed by immunohistochemistry. Occurrence of a delayed progressive kidney failure (focal segmental glomerular sclerosis) triggered analysis of WT1, revealing a pathogenic splice-site mutation in intron 9. Analysis of the SRY gene in an additional five FS cases did not reveal any mutations. The case presented shows the importance of multi-gene based diagnosis of DSD patients, allowing early diagnosis and treatment, thus preventing putative development of an invasive cance

STAG3 homozygous missense variant causes primary ovarian insufficiency and male non-obstructive azoospermia

International audienceInfertility, a global problem affecting up to 15% of couples, can have varied causes ranging from natural aging to the pathological development or function of the reproductive organs. One form of female infertility is premature ovarian insufficiency (POI), affecting up to 1 in 100 women and characterised by amenorrhea and elevated follicle stimulating hormone before the age of 40. POI can have a genetic basis, with over 50 causative genes identified. Non-obstructive azoospermia (NOA), a form of male infertility characterised by the absence of sperm in semen, has an incidence of 1% and is similarly heterogeneous. The genetic basis of male and female infertility is poorly understood with the majority of cases having no known cause. Here, we study a case of familial infertility including a proband with POI and her brother with NOA. We performed whole-exome sequencing (WES) and identified a homozygous STAG3 missense variant that segregated with infertility. STAG3 encodes a component of the meiosis cohesin complex required for sister chromatid separation. We report the first pathogenic homozygous missense variant in STAG3 and the first STAG3 variant associated with both male and female infertility. We also demonstrate limitations of WES for the analysis of homologous DNA sequences, with this variant being ambiguous or missed by independent WES protocols and its homozygosity only being established via long-range nested PCR

New insights into the genetic basis of premature ovarian insufficiency: Novel causative variants and candidate genes revealed by genomic sequencing

International audienceOvarian deficiency, including premature ovarian insufficiency (POI) and diminished ovarian reserve (DOR), represents one of the main causes of female infertility. POI is a genetically heterogeneous condition but current understanding of its genetic basis is far from complete, with the cause remaining unknown in the majority of patients. The genes that regulate DOR have been reported but the genetic basis of DOR has not been explored in depth. Both conditions are likely to lie along a continuum of degrees of decrease in ovarian reserve. We performed genomic analysis via whole exome sequencing (WES) followed by in silico analyses and functional experiments to investigate the genetic cause of ovarian deficiency in ten affected women. We achieved diagnoses for three of them, including the identification of novel variants in STAG3, GDF9, and FANCM. We identified potentially causative FSHR variants in another patient. This is the second report of biallelic GDF9 and FANCM variants, and, combined with functional support, validates these genes as bone fide autosomal recessive “POI genes”. We also identified new candidate genes, NRIP1, XPO1, and MACF1. These genes have been linked to ovarian function in mouse, pig, and zebrafish respectively, but never in humans. In the case of NRIP1, we provide functional support for the deleterious nature of the variant via SUMOylation and luciferase/β-galactosidase reporter assays. Our study provides multiple insights into the genetic basis of POI/DOR. We have further elucidated the involvement of GDF9, FANCM, STAG3 and FSHR in POI pathogenesis, and propose new candidate genes, NRIP1, XPO1, and MACF1, which should be the focus of future studies

Mutational analysis of <i>SRY</i> and <i>WT1</i>.

<p>(A) wild type (upper panel, control) and mutated sequence (lower panel, patient) of <i>SRY</i>. (B) schematic representation of the SRY protein. The K128R mutation resides in the HMG domain, just before the cNLS. (C) <i>In vitro</i> luciferase assays of SRY-WT (wild-type) and SRY-K128R (mutant) in HEK293T cell line. Cells were co-transfected with TESCO-E1b-<i>Luc</i>, SF1 and WT or mutant SRY to assess for activation of TESCO. The mean percentages of fold change of luciferase activity of TESCO-E1b-<i>luc</i> over the empty vector, relative to WT SRY levels from six independent assays (each performed in triplicate) are shown. Error bars represent standard error of the mean (SEM). (D) pcDNA3-FLAG-SRY wild-type (WT, 2 µg) or pcDNA3-FLAG-SRY mutant (K128R, 2 µg) were transiently transfected into HEK293T cells using Fugene 6. Exogenous SRY (WT or K128R) expression was detected using a FLAG antibody and a green fluorescent Alexa-488 dye coupled secondary antibody. Nuclei were stained with 4′,6-diamino-2-phenylindole (DAPI). Both wild type and mutant SRY show strong nuclear staining. (E) SRY fluorescence was quantified as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040858#pone.0040858-Argentaro1" target="_blank">[35]</a>. Nuclear accumulation of SRY (WT or K128R) expressed as fluorescence in the nucleus over that in the cytoplasm (Fn/c) were background fluorescence has been subtracted. Measurements represent the average of 3 independent transfections. Results are relative to WT transfected cells (Fn/c given value of 100%). The number of cells analysed is n = 111 (WT) and n = 121 (K128R). Error bars represent the standard error of mean values. Two-tail t-Test of unpaired sample means was performed between WT transfected cells and mutant transfected cells and showed no significant differences. P = 0.49. (F) mutated sequence (upper panel, patient) and wild type sequence (lower panel, control) of <i>WT1</i>, showing the heterozygous +4C&gt;T change.</p

Immunohistochemical staining of the left GB lesion.

<p>(A) representative hematoxylin and eosin (HE) staining. The germ cells present in the GB stain positive for OCT3/4 (B, brown staining), TSPY (C, red staining), and SCF (D, red staining). Supportive cells in the GB stain positive for FOXL2 (E, brown staining), while SOX9 (F) is negative. All slides are counterstained with hematoxylin. Magnification 100x for all.</p