Additional evidence for the role of chromosomal imbalances and SOX8, ZNRF3 and HHAT gene variants in early human testis development.

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.

Anomalies in Human Sex Determination: Usefulness of a Combined Cytogenetic Approach to Characterize an Additional Case with Xp Functional Disomy Associated with 46,XY Gonadal Dysgenesis

Objective: Disorders of sexual development (DSD) are a heterogeneous group of genital defects affecting chromosomal, gonadal and anatomical sex. 46,XY DSD is a subset of DSD which covers a wide range of phenotypes in which 46,XY gonadal dysgenesis (GD) is the most severe form. In this study, we report on the clinical and molecular cytogenetic findings of a study on a Tunisian girl with the syndromic form of 46,XY DSD. Methods: This case was a phenotypic female patient having several congenital anomalies including growth retardation. Karyotype, fluorescence in situ hybridization and array Comparative Genome Hybridization (array CGH) were performed. Results: The proband exhibited a de-novo 46,X,der(Y) karyotype. Array CGH revealed a pathogenic 27.5Mb gain of an Xp21.2 chromosome segment leading to Xp functional disomy. No deletion was observed in the Y-chromosome. The duplicated region encompassed the NR0B1 (DAX1) and MAGEB genes, located within the dosage sensitive sex (DSS) reversal locus, known as promote genes responsible for human sex reversal and testis repression. The extra-dosage and interactions of these genes with different specific genes could result in the impairment of the male sex pathway. Over-dosage of KAL1 and IL1RAPL1 genes fall within the somatic features observed in the patient. Conclusion: To the best of our knowledge, we report on the fourth case of Xp21.2-pter duplication within Xp;Yp translocation associated with XY GD. Our findings suggest that when duplicated, the NR0B1 and MAGEB genes could be a major cause of XY GD. Therefore, we emphasize the usefulness of a combined cytogenetic approach in order to provide an accurate genetic diagnosis for those patients having syndromic XY DSD in a clinical setting.

Disorders of sex development in Wolf-Hirschhorn syndrome: a genotype-phenotype correlation and MSX1 as candidate gene.

BACKGROUND: Wolf-Hirschhorn (WHS) is a set of congenital physical anomalies and mental retardation associated with a partial deletion of the short arm of chromosome 4. To establish a genotype-phenotype correlation; we carried out a molecular cytogenetic analysis on two Tunisian WHS patients. Patient 1 was a boy of 1-year-old, presented a typical WHS phenotype while patient 2, is a boy of 2 days presented an hypospadias, a micropenis and a cryptorchidie in addition to the typical WHS phenotype. Both the array comparative genomic hybridization and fluorescence in situ hybridization techniques were used. RESULTS: Results of the analysis showed that patient 2 had a greater deletion size (4.8 Mb) of chromosome 4 than patient 1 (3.4 Mb). Here, we notice that the larger the deletion, the more genes are likely to be involved, and the more severe the phenotype is likely to be. If we analyze the uncommon deleted region between patient1 and patient 2 we found that the Muscle Segment Homeobox (MSX1) gene is included in this region. MSX1 is a critical transcriptional repressor factor, expressed in the ventral side of the developing anterior pituitary and implicated in gonadotrope differentiation. Msx1 acts as a negative regulatory pituitary development by repressing the gonadotropin releasing hormone (GnRH) genes during embryogenesis. We hypothesized that the deletion of MSX1 in our patient may deregulate the androgen synthesis. CONCLUSION: Based on the MSX1 gene function, its absence might be indirectly responsible for the hypospadias phenotype by contributing to the spatiotemporal regulation of GnRH transcription during development.

Small supernumerary marker chromosomes (sSMC) and male infertility: characterization of five new cases, review of the literature, and perspectives.

PURPOSE: To characterize small supernumerary marker chromosomes (sSMC) in infertile males RESEARCH QUESTION: Are molecular cytogenetic methods still relevant for the identification and characterization of sSMC in the era of next-generation sequencing? METHODS: In this paper, we report five males with oligoasthenozoospermia or azoospermia with a history of recurrent pregnancy loss in partnership in four cases. R-banding karyotyping and fluorescence in situ hybridization (FISH) analysis were performed and showed sSMC in all five cases. Microdissection and reverse-FISH were performed in one case. RESULTS: One sSMC, each, was derived from chromosome 15 and an X-chromosome; two sSMC were derivatives of chromosome 22. The fifth sSMC was a ring chromosome 4 complemented by a deletion of the same region 4p14 to 4p16.1 in one of the normal chromosomes 4. All markers were mosaics except one of sSMC(22). CONCLUSION: Through this study, we emphasize the necessity of a proper combination of high-throughput techniques with conventional cytogenetic and FISH methods. This could provide a personalized diagnostic and accurate results for the patients suffering from infertility or RPL. We also highlight FISH analyses, which are essential tools for detecting sSMC in infertile patients. In fact, despite its entire composition of heterochromatin, sSMC can have effects on spermatogenesis by producing mechanical perturbations during meiosis and increasing meiotic nondisjunction rate. This would contribute to understand the exact chromosomal mechanism disrupting the natural and the assisted reproduction leading to offer a personalized support.

Clinical and molecular findings in nine new cases of tetrasomy 18p syndrome: FISH and array CGH characterization.

BACKGROUND: Small Supernumerary Marker Chromosomes (sSMC) are rare chromosomal abnormalities, which have abnormal banding arrangement and take many shapes. Several disorders have been correlated with sSMC presence. The aim of this study is to characterize the sSMC derived from chromosome 18 by Fluorescence in situ hybridization (FISH) and Array Comparative Genomic Hybridization (aCGH). RESULTS: Nine children with dysmorphic features have been investigated. They have these features in common: a triangular face, low-set ears, a large mouth with a thin upper lip, and a horizontal palpebral fissure. Epicanthus and strabismus were present in two patients. In addition, we have noticed microcephaly and mental and/or developmental delay with low birth weight. However, two patients had standard birth weight; one patient had hypospadias; two had skin problems; and three showed different congenital heart defects. One patient had corpus callosum hypoplasia. Systematic karyotype analysis revealed a de novo supernumerary chromosome. Array CGH showed a gain in copy number on the short arm of chromosome 18 in the nine cases. In one case, the sSMC seemed to be in mosaic. The breakpoints of the marker were identified using aCGH and FISH. Thus, the sSMC led to 18p tetrasomy with approximately 14 Mb lengths, between 364344 and 14763575 based on the human genome version 18. CONCLUSIONS: These results have been completed by FISH in order to ascertain the shape of the sSMC. Our results confirm the uniqueness and particularity of the iso18p syndrome on the phenotypic as well as on the genetic level.