Genetic study of a family segregating Shah-Waardenburg syndrome

Session: Genetics of Hirschsprung’s Disease, no. E28Poster presentationBackground: Type IV Waardenburg syndrome (WS4, MIM_277580), also known as Shah-Waardenburg syndrome or Waardenburg-Hirschsprung disease, is a congenital developmental disorder characterized by pigmentary abnormalities of the skin, eyes and hair, sensorineural deafness and Hirschsprung disease. The WS4 is caused by mutations in any of the following three genes: endothelin-3(EDN3), endothelin-B receptor (EDNRB), or SOX10. Materials and methods: Exons and exon/intron boundaries of the three candidate genes (EDN3, EDNRB, SOX10) were screened for mutations by direct sequencing of PCR products in a three-generation family (14 individuals) whose members were affected with WS4 or only HSCR. The family members were also genotyped for 2.5 million genetic markers (single nucleotide polymorphisms -SNP- and copy number variation probes -CNVs-) using Illumina Human Omni2.5-quad BeadChip. After quality control and pruning of SNPs in high linkage-disequilibrium, the Merlin software was used for parametric and non-parametric linkage. To test the effect of the mutation identified, wild-type and mutated mammalian expression vectors encoding for EDNRB isoforms (with green fluorescent protein -GFP-) were/will (isoform 3) transiently transfected in Human Embryonic Kidney 293 cells. Results: Direct sequencing revealed a heterozygous novel nonsense mutation in EDNRB (Chromosome 13). This mutation corresponds to M1V of the EDNRB isoforms 1 and 2 and M91V of the EDNRB isoform 3. It was present in four affected and two unaffected family members. The PolyPhen and SIFT prediction for M1V (isoforms 1&2) and for M91V (isoform 3) was damaging (abolishes the ATG start site) and benign respectively. Importantly, the 3 EDNRB transcripts are expressed in the human new-born gut. HEK 293 cells transfected with vector with wild-type EDNRB-GFP showed mainly a cell membrane localization, while the M1V mutated EDNRB-GFP revealed a clustered localization in cytoplasm indicating that EDNRB receptor cannot translocate to cell membrane. Analysis of M91V is underway. Our linkage analysis indicated that additional genes may be contributing to the phenotype. Conclusion: We found and characterized a novel nonsense mutation. The EDNRB mutation is deleterious in isoforms 1 and 2 (M1V) but predicted benign in isoform 3 (M91V) (in vitro is study underway). The variable manifestation of the disease may depend on the ratio between the EDNRB isoforms expressed in gut or/and additional genetic factors involved in the pathogenesis of the disease