46,XY DSD in 2 sisters due to compound heterozygous mutations in the LH/CG receptor gene involving cryptic Exon 6A identified by exome sequencing

We report on a familiar case of two sisters with 46,XY DSD and non-consanguineous parents. Both sisters have a complete inconspicuous female appearance. Testes were detected in the elder sister at 10yrs during herniotomy. Chromosomal analysis revealed a normal 46,XY karyotype. A labial fusion was detected in her younger sister and a familiar case of 46,XY DSD was suspected and confirmed by SRY-PCR in both. Testing showed no increase in testosterone synthesis after 3 consecutive doses of hCG, indicating a testosterone synthesis defect or Leydig cell hypoplasia. Gonadal dysgenesis was excluded because of absence of a uterus in combination with complete female external genitalia. Hormone profiling and Sanger sequencing excluded a defect in SRD5A1, HSD17B3, NR5A1 or AR. Further CGH-array analysis also revealed no common CNVs between both sisters.

Because all previous sequence analysis of investigated genes failed to identify a causative mutation, we used exome sequencing to screen for common mutations in both sisters. The only DSD candidate gene mutation found in common in both sisters was a heterozygous missense mutation in Exon 5 of the LHCGR gene, predicted to be possible damaging by PolyPhen 2. Visual inspection of the bam file revealed further mutations in cryptic exon 6A of the LHCGR gene affecting splicing and mRNA accumulation. This exon is enriched in the SureSelect XT Human All Exon V4 enrichment kit (Agilent) used and variants are called, but not further filtered as possible pathogenic variants because this exon is not annotated in the reference sequences of GRCh37/hg19. Long range PCR and subcloning confirmed a compound heterozygous inheritance of the mutation. Loss of LHCGR function is in good agreement with previous data and phenotype. Functional analysis to confirm the deleterious effect of the newly identified LHCGR mutation in exon 5 is currently on the way.