ESPE Abstracts

Background: 46,XX DSD (Disorder of Sex Development) includes individuals with ovotestes (ovotesticular DSD (OTDSD)) or testes (testicular DSD (TDSD)). Most individuals with 46,XX TDSD carry the SRY gene. Other known causes of TDSD/OTDSD include chromosomal rearrangements involving SOX9 or SOX3 and mutations of WNT4 and a WNT regulator, R-SPONDIN 1. However, our understanding of the molecular causes of TDSD and OTDSD remain incomplete.

Objective and hypotheses: To identify novel genes/mutations associated with 46,XX TDSD/OTDSD.

Method: Paired-end sequencing was performed on the Illumina HiSeq2000 platform using TruSeq v3 chemistry at an average coverage of x50. Reads were mapped using the Burrows-Wheeler Aligner and their local realignment were carried out with the GATK version 1.6. SNP novelty was determined against public databases. Potentially pathogenic mutations were verified by Sanger sequencing. The effect of mutation on the biological activity of the protein was assessed using an array of in-silico and in-vitro methods.

Results: We identified a de novo missense mutation of a highly conserved arginine residue in the fourth zinc-finger of WT1 (p.Arg495Gly) in a patient with 46,XX TDSD. Normal ploidy was established by high resolution aCGH and qPCR indicated two copies of the RevSex SOX9 enhancer. The p.Arg495Gly mutation is not present in public databases. The patient of Egyptian origin presented with dysgenic testis, microcephaly, a small uterus and no kidney tumour. Transient gene expression assays and protein-protein interaction studies showed that the mutant protein abnormally regulated/interacted with genes/proteins involved in both male and female gonadal development.

Conclusion: Mutations in WT1 have been previously reported in anomalies of testis formation in 46,XY individuals. This is the first time that a mutation has been identified in WT1, in a patient presenting with 46,XX TDSD. This raises the intriguing possibility that specific mutations in WT1 may be associated with testis formation in 46,XX chromosomal context.