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.

Figure 1

Representative histological and immunohistochemical findings for the left gonad: (a) total overview of the gonad histology (H&E); (b) higher magnification ( × 2.5 magnification, indicated by square in (a); immunohistochemical detection of (c) SOX9, positive in the Sertoli cells; (d) FOXL2, positive in the granulosa cells; (e) OCT3/4; (f) TSPY; (g) KITL, all positive in the transformed germ cells. All immunohistochemical images are at the magnification of × 100, except G, being × 50.

Figure 2

Molecular analysis of DNA from the 46,XY DSD patient. (A). The deletion identified by microarray analysis, removing 767 kb of genomic DNA on chromosome 16. Data are plotted along the chromosome, with each point representing the copy number estimate of an individual probe. The breakpoints of the deletion are shown by the broken vertical lines. (B) PCR analysis of the deletion. cDNA was derived from lymphocytes of the index patient, and primers were designed to amplify a PCR product across the predicted breakpoint. Sequence analysis shows that exon 5 is spliced directly onto exon 9. (C) Effect of the deletion on the WWOX protein. The full-length WWOX protein has two WW domains at the N-terminal, and a SDR domain at the C-terminal. A deletion of exons 6–8 is predicted to result in an in-frame but shortened product, with the SDR domain largely missing.