A multi-exon deletion within WWOX is associated with a 46,XY disorder of sex development

Abstract

Disorders of sex development (DSD) are congenital conditions where chromosomal, gonad or genital development is atypical. In a significant proportion of 46,XY DSD cases it is not possible to identify a causative mutation, making genetic counseling difficult and potentially hindering optimal treatment. Here, we describe the analysis of a 46,XY DSD patient that presented at birth with ambiguous genitalia. Histological analysis of the surgically removed gonads showed bilateral undifferentiated gonadal tissue and immature testis, both containing malignant germ cells. We screened genomic DNA from this patient for deletions and duplications using an Illumina whole-genome SNP microarray. This analysis revealed a heterozygous deletion within the WWOX gene on chromosome 16, removing exons 6-8. Analysis of parental DNA showed that the deletion was inherited from the mother. cDNA analysis confirmed that the deletion maintained the reading frame, with exon 5 being spliced directly onto exon 9. This deletion is the first description of a germline rearrangement affecting the coding sequence of WWOX in humans. Previously described Wwox knockout mouse models showed gonadal abnormalities, supporting a role for WWOX in human gonad development. Keywords: disorders of sex development; copy number; WWOX; gonad; microarrays INTRODUCTIONIn the early stages of human embryogenesis the developing gonads are bipotent, being capable of forming either testes or ovaries. In males the expression of the Y chromosomal SRY gene initiates testis development, whereas ovarian development in principle occurs only in the absence of SRY (reviewed in Wilhelm et al 1 ). Following the establishment of sex-specific expression of key regulatory genes in the gonad, gonadal differentiation results in development of the external genitalia. As a result there are two main stages in gonad formation, and disruption of either can lead to disorders of sex development (DSD). DSD are surprisingly common, with ambiguous genitalia estimated to occur with an incidence of 1 in 4500 live births. 2 A number of genes important in the regulation of sex determination have been identified, yet in as many as 70% of 46,XY DSD cases no genetic cause has been identified. We have previously demonstrated the power of whole-genome copy number analysis with high-density microarrays to identify causative mutations in DSD. 3,4 Here, we describe the use of this approach to identify a multi-exon heterozygous deletion in the WWOX gene of a 46,XY DSD patient.

Parent-of-origin-specific signatures of de novo mutations

“…Notably, the two 1-Mb windows that exhibit the highest maternal mutation rates are spanned by one large gene each, CSMD1 and WWOX (Supplementary Tables 21-23). WWOX is at a well-known fragile site in the genome 13 and is known to be involved in human gonad development 14 .…”

The Role of Copy Number Variants in Disorders of Sex Development

“…Indeed, CNV arrays and MLPA are often used sequentially; MLPA is frequently utilized to confirm CNV array results, determine the mode of inheritance, and screen patient cohorts for known CNVs which in turn are then fine-mapped using CNV arrays [White et al, , 2012Kim et al, 2015;Ohnesorg et al, 2017]. MLPA can also be used to detect CNVs of single exons in genes of interest [Barbaro et al, 2011]; however, due to the expense of CGD, complete gonadal dysgenesis; GD, gonadal dysgenesis; OT, ovotesticular DSD the probes, this would only be cost-effective if large cohorts were screened for particular well-known candidate genes.…”

“…These smaller-scale rearrangements are more often detected using high density CGH and microarrays that are designed to have targets at the resolution of exons [Harrison et al, 2014]. For example, White et al [2012] identified a deletion in the WWOX gene affecting exons 6-8, while Ledig et al [2012] found a deletion of exons 3 and 4 in the DMRT1 gene. Other groups have also utilized specific MLPA probes to increase the density of their CNV analysis by focusing on each individual exon to increase the resolution [Barbaro et al, 2011].…”

Disorders of sex development: advances in genetic diagnosis and challenges in management

“…7 In addition, a probable regulatory element lying downstream of the GATA4 gene was identified when a 35 kb deletion was detected in a person with 46,XY complete gonadal dysgenesis. 17 Similarly, in those with 46,XY DSD, microarray analysis detected an intragenic deletion of WWOX 18 and a deletion at least 42 kb upstream of the DMRT1 gene. 19 In fact, array analyses have now identified many DNA duplications and deletions in people with DSD, several of which are located close to known sex development genes and are suspected to have caused disruption of gene regulatory regions.…”