Purpose:We aimed to identify the genetic cause in a cohort of 11 unrelated cases and two sisters with 46,XX SRY-negative (ovo)testicular disorders of sex development (DSD).Methods:Whole-exome sequencing (n = 9), targeted resequencing (n = 4), and haplotyping were performed. Immunohistochemistry of sex-specific markers was performed on patients' gonads. The consequences of mutation were investigated using luciferase assays, localization studies, and RNA-seq.Results:We identified a novel heterozygous NR5A1 mutation, c.274C>T p.(Arg92Trp), in three unrelated patients. The Arg92 residue is highly conserved and located in the Ftz-F1 region, probably involved in DNA-binding specificity and stability. There were no consistent changes in transcriptional activation or subcellular localization. Transcriptomics in patient-derived lymphocytes showed upregulation of MAMLD1, a direct NR5A1 target previously associated with 46,XY DSD. In gonads of affected individuals, ovarian FOXL2 and testicular SRY-independent SOX9 expression observed.Conclusions:We propose NR5A1, previously associated with 46,XY DSD and 46,XX primary ovarian insufficiency, as a novel gene for 46,XX (ovo)testicular DSD. We hypothesize that p.(Arg92Trp) results in decreased inhibition of the male developmental pathway through downregulation of female antitestis genes, thereby tipping the balance toward testicular differentiation in 46,XX individuals. In conclusion, our study supports a role for NR5A1 in testis differentiation in the XX gonad.Genet Med 19 4, 367–376.

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BackgroundOne in 4500 children is born with ambiguous genitalia, milder phenotypes occur in one in 300 newborns. Conventional time-consuming hormonal and genetic work-up provides a genetic diagnosis in around 20-40% of 46,XY cases with ambiguous genitalia. All others remain without a definitive diagnosis. The investigation of milder cases, as suggested by recent reports remains controversial.MethodsIntegrated clinical, hormonal and genetic screening was performed in a sequential series of 46, XY children, sex-assigned male, who were referred to our pediatric endocrine service for atypical genitalia (2007–2013).ResultsA consecutive cohort of undervirilized 46,XY children with external masculinization score (EMS) 2–12, was extensively investigated. In four patients, a clinical diagnosis of Kallmann syndrome or Mowat-Wilson syndrome was made and genetically supported in 2/3 and 1/1 cases respectively. Hormonal data were suggestive of a (dihydro)testosterone biosynthesis disorder in four cases, however no HSD17B3 or SRD5A2 mutations were found. Array-CGH revealed a causal structural variation in 2/6 syndromic patients. In addition, three novel NR5A1 mutations were found in non-syndromic patients. Interestingly, one mutation was present in a fertile male, underlining the inter- and intrafamilial phenotypic variability of NR5A1-associated phenotypes. No AR, SRY or WT1 mutations were identified.ConclusionOverall, a genetic diagnosis could be established in 19% of non-syndromic and 33% of syndromic cases. There is no difference in diagnostic yield between patients with more or less pronounced phenotypes, as expressed by the external masculinisation score (EMS). The clinical utility of array-CGH is high in syndromic cases. Finally, a sequential gene-by-gene approach is time-consuming, expensive and inefficient. Given the low yield and high expense of Sanger sequencing, we anticipate that massively parallel sequencing of gene panels and whole exome sequencing hold promise for genetic diagnosis of 46,XY DSD boys with an undervirilized phenotype.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-014-0209-2) contains supplementary material, which is available to authorized users.

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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-β (ER-β) was performed in an 8-week-old human male embryo.

Genetic studies in Disorders of Sex Development (DSD), representing a wide spectrum of developmental or functional conditions of the gonad, have mainly been oriented towards the coding genome. Application of genomic technologies, such as whole‐exome sequencing, result in a molecular genetic diagnosis in ∼50% of cases with DSD. Many of the genes mutated in DSD encode transcription factors such as SRY, SOX9, NR5A1, and FOXL2, characterized by a strictly regulated spatiotemporal expression. Hence, it can be hypothesized that at least part of the missing genetic variation in DSD can be explained by non‐coding mutations in regulatory elements that alter gene expression, either by reduced, mis‐ or overexpression of their target genes. In addition, structural variations such as translocations, deletions, duplications or inversions can affect the normal chromatin conformation by different mechanisms. Here, we review non‐coding defects in human DSD phenotypes and in animal models. The wide variety of non‐coding defects found in DSD emphasizes that the regulatory landscape of known and to be discovered DSD genes has to be taken into consideration when investigating the molecular pathogenesis of DSD.

The Coupled Particle Light Scattering technique was evaluated for serological diagnosis of Epstein-Barr Virus (EBV) infection. Two hundred ninety-six patient sera selected from several clinical categories (acute infection, non-primary infection, interfering non-EBV infection, non-infected) were tested for IgM and IgG antibodies (anti-VCA, anti-EBNA and anti-EA). Determination of EBV IgG with Copalis multiplex was accurate when compared with Enzygnost Anti-EBV/IgG ELISA. Although the sensitivity of Copalis IgM for acute infections was 100% a positive IgM result did not always indicate an acute infection. Strong reactivity to IgG EA (ratio 3, 1) and IgG VCA (ratio 13, 3) correlated with persistent infection or reactivation. The CopalisI has many advantages over the existing methods, such as the possibility to measure three semi-quantitative IgG responses to three different EBV antigens simultaneously.