Identification of De Novo Copy Number Variants Associated with Human Disorders of Sexual Development

Disorders of sexual development (DSD), ranging in severity from genital abnormalities to complete sex reversal, are among the most common human birth defects with incidence rates reaching almost 3%. Although causative alterations in key genes controlling gonad development have been identified, the majority of DSD cases remain unexplained. To improve the diagnosis, we screened 116 children born with idiopathic DSD using a clinically validated array-based comparative genomic hybridization platform. 8951 controls without urogenital defects were used to compare with our cohort of affected patients. Clinically relevant imbalances were found in 21.5% of the analyzed patients. Most anomalies (74.2%) evaded detection by the routinely ordered karyotype and were scattered across the genome in gene-enriched subtelomeric loci. Among these defects, confirmed de novo duplication and deletion events were noted on 1p36.33, 9p24.3 and 19q12-q13.11 for ambiguous genitalia, 10p14 and Xq28 for cryptorchidism and 12p13 and 16p11.2 for hypospadias. These variants were significantly associated with genitourinary defects (P = 6.08×10−12). The causality of defects observed in 5p15.3, 9p24.3, 22q12.1 and Xq28 was supported by the presence of overlapping chromosomal rearrangements in several unrelated patients. In addition to known gonad determining genes including SRY and DMRT1, novel candidate genes such as FGFR2, KANK1, ADCY2 and ZEB2 were encompassed. The identification of risk germline rearrangements for urogenital birth defects may impact diagnosis and genetic counseling and contribute to the elucidation of the molecular mechanisms underlying the pathogenesis of human sexual development

Copy Number Variation in Patients with Disorders of Sex Development Due to 46,XY Gonadal Dysgenesis

Disorders of sex development (DSD), ranging in severity from mild genital abnormalities to complete sex reversal, represent a major concern for patients and their families. DSD are often due to disruption of the genetic programs that regulate gonad development. Although some genes have been identified in these developmental pathways, the causative mutations have not been identified in more than 50% 46,XY DSD cases. We used the Affymetrix Genome-Wide Human SNP Array 6.0 to analyse copy number variation in 23 individuals with unexplained 46,XY DSD due to gonadal dysgenesis (GD). Here we describe three discrete changes in copy number that are the likely cause of the GD. Firstly, we identified a large duplication on the X chromosome that included DAX1 (NR0B1). Secondly, we identified a rearrangement that appears to affect a novel gonad-specific regulatory region in a known testis gene, SOX9. Surprisingly this patient lacked any signs of campomelic dysplasia, suggesting that the deletion affected expression of SOX9 only in the gonad. Functional analysis of potential SRY binding sites within this deleted region identified five putative enhancers, suggesting that sequences additional to the known SRY-binding TES enhancer influence human testis-specific SOX9 expression. Thirdly, we identified a small deletion immediately downstream of GATA4, supporting a role for GATA4 in gonad development in humans. These CNV analyses give new insights into the pathways involved in human gonad development and dysfunction, and suggest that rearrangements of non-coding sequences disturbing gene regulation may account for significant proportion of DSD cases

Disorders of sex development: effect of molecular diagnostics

Disorders of sex development (DSDs) are a diverse group of conditions that can be challenging to diagnose accurately using standard phenotypic and biochemical approaches. Obtaining a specific diagnosis can be important for identifying potentially life-threatening associated disorders, as well as providing information to guide parents in deciding on the most appropriate management for their child. Within the past 5 years, advances in molecular methodologies have helped to identify several novel causes of DSDs; molecular tests to aid diagnosis and genetic counselling have now been adopted into clinical practice. Occasionally, genetic profiling of embryos prior to implantation as an adjunct to assisted reproduction, prenatal diagnosis of at-risk pregnancies and confirmatory testing of positive results found during newborn biochemical screening are performed. Of the available genetic tests, the candidate gene approach is the most popular. New high-throughput DNA analysis could enable a genetic diagnosis to be made when the aetiology is unknown or many differential diagnoses are possible. Nonetheless, concerns exist about the use of genetic tests. For instance, a diagnosis is not always possible even using new molecular approaches (which can be worrying for the parents) and incidental information obtained during the test might cause anxiety. Careful selection of the genetic test indicated for each condition remains important for good clinical practice. The purpose of this Review is to describe advances in molecular biological techniques for diagnosing DSDs

INSL3/RXFP2 signaling in testicular descent.

Mutations of the insulin-like peptide 3 (INSL3) hormone or its receptor, RXFP2, cause intraabdominal cryptorchidism in male mice. Specific RXFP2 expression in mouse gubernacula was detected at embryonic day 14.5 and markedly increased after birth in the developing cremaster muscle, as well as in the epididymis and testicular Leydig and germ cells. INSL3 treatment stimulated cell proliferation of embryonic gubernacular and Leydig cells, implicating active INSL3-mediated signaling. The transcription factor SOX9, a known male sex determination factor, upregulated the activity of the RXFP2 promoter. INSL3 is sufficient to direct the first transabdominal phase of testicular descent in the absence of hypothalamic-pituitary-gonadal axis signaling or Hoxa10, although these factors are important for inguinoscrotal testicular descent. Similarly, conditional ablation of the androgen receptor gene in gubernacular cells resulted in disruption of inguinoscrotal descent. We performed mutation screening of INSL3 and RXFP2 in human patients with cryptorchidism and control subjects from different populations in Europe and the USA. Several missense mutations were described in both the INSL3 and RXFP2 genes. A novel V39G INSL3 mutation in a patient with cryptorchidism was identified; however, the functional analysis of the mutant peptide did not reveal compromised function. In more than 2000 patients and controls analyzed to date, the T222P RXFP2 mutation is the only one strongly associated with the mutant phenotype. The T222P mutant receptor, when transfected into 293T cells, had severely decreased cell membrane expression, providing the basis for the functional deficiency of this mutation

INSL3/RXFP2 signaling in testicular descent.

Mutations of the insulin-like peptide 3 (INSL3) hormone or its receptor, RXFP2, cause intraabdominal cryptorchidism in male mice. Specific RXFP2 expression in mouse gubernacula was detected at embryonic day 14.5 and markedly increased after birth in the developing cremaster muscle, as well as in the epididymis and testicular Leydig and germ cells. INSL3 treatment stimulated cell proliferation of embryonic gubernacular and Leydig cells, implicating active INSL3-mediated signaling. The transcription factor SOX9, a known male sex determination factor, upregulated the activity of the RXFP2 promoter. INSL3 is sufficient to direct the first transabdominal phase of testicular descent in the absence of hypothalamic-pituitary-gonadal axis signaling or Hoxa10, although these factors are important for inguinoscrotal testicular descent. Similarly, conditional ablation of the androgen receptor gene in gubernacular cells resulted in disruption of inguinoscrotal descent. We performed mutation screening of INSL3 and RXFP2 in human patients with cryptorchidism and control subjects from different populations in Europe and the USA. Several missense mutations were described in both the INSL3 and RXFP2 genes. A novel V39G INSL3 mutation in a patient with cryptorchidism was identified; however, the functional analysis of the mutant peptide did not reveal compromised function. In more than 2000 patients and controls analyzed to date, the T222P RXFP2 mutation is the only one strongly associated with the mutant phenotype. The T222P mutant receptor, when transfected into 293T cells, had severely decreased cell membrane expression, providing the basis for the functional deficiency of this mutation

Prevalence of endocrine and genetic abnormalities in boys evaluated systematically for a disorder of sex development

STUDY QUESTION What is the likelihood of identifying genetic or endocrine abnormalities in a group of boys with 46, XY who present to a specialist clinic with a suspected disorder of sex development (DSD)? SUMMARY ANSWER An endocrine abnormality of the gonadal axis may be present in a quarter of cases and copy number variants (CNVs) or single gene variants may be present in about half of the cases. WHAT IS KNOWN ALREADY Evaluation of 46, XY DSD requires a combination of endocrine and genetic tests but the prevalence of these abnormalities in a sufficiently large group of boys presenting to one specialist multidisciplinary service is unclear. STUDY, DESIGN, SIZE, DURATION This study was a retrospective review of investigations performed on 122 boys. PARTICIPANTS/MATERIALS, SETTING, METHODS All boys who attended the Glasgow DSD clinic, between 2010 and 2015 were included in the study. The median external masculinization score (EMS) of this group was 9 (range 1–11). Details of phenotype, endocrine and genetic investigations were obtained from case records. MAIN RESULTS AND THE ROLE OF CHANCE An endocrine abnormality of gonadal function was present in 28 (23%) with a median EMS of 8.3 (1–10.5) whilst the median EMS of boys with normal endocrine investigations was 9 (1.5–11) (P = 0.03). Endocrine abnormalities included a disorder of gonadal development in 19 (16%), LH deficiency in 5 (4%) and a disorder of androgen synthesis in 4 (3%) boys. Of 43 cases who had array-comparative genomic hybridization (array-CGH), CNVs were reported in 13 (30%) with a median EMS of 8.5 (1.5–11). Candidate gene analysis using a limited seven-gene panel in 64 boys identified variants in 9 (14%) with a median EMS of 8 (1–9). Of the 21 boys with a genetic abnormality, 11 (52%) had normal endocrine investigations. LIMITATIONS, REASONS FOR CAUTION A selection bias for performing array-CGH in cases with multiple congenital malformations may have led to a high yield of CNVs. It is also possible that the yield of single gene variants may have been higher than reported if the investigators had used a more extended gene panel. WIDER IMPLICATIONS OF THE FINDINGS The lack of a clear association between the extent of under-masculinization and presence of endocrine and genetic abnormalities suggests a role for parallel endocrine and genetic investigations in cases of suspected XY DSD