Analysis Of Molecular Mechanism About A Chinese Family With Waardenburg Syndrome

Waardenburg syndrome(WS)is a genetically and clinically heterogeneous disorder characterized by sensorineural deafness and pigmentation defects of the hair,skin,and iris.According to the different clinical symptoms of patients,it can be divided into four types:WS1,WS2,WS3,and WS4.The PAX3 mutation is the main cause of WS1 and WS2,and mutations in MITF and SNAI2 were found to induce WS2,while the mutations of SOX10,EDNRB,and EDN3 usually cause WS2 and WS4.A large Chinese family with autosomal dominant WS spans five generations was collected and identified in this study,the affected individuals main characterized with sensorineural deafness,dystopia canthorum,and pigmentation defects of the hair and iris,which was diagnosed as type 1 WS.Firstly,the microsatellite D2S126 located 1.07 Mb away from the physical map of PAX3 was selected as a landmark for linkage analysis,and the polymorphism of D2S126 was found closely linked with the patients in this family,indicating a possible role of PAX3 in the generation of Waardenburg syndrome in this family.The exons and the junctions of exon and intron of PAX3 were screened by Sanger sequencing,and a novel missense mutation c.272A>C(p.Gln91Pro)in exon 2 was found in the proband.The c.272A>C mutation lead to the destruction of restriction endonuclease site of Bcn I,and restriction fragment length polymorphism(RFLP)assay showed that the c.272A>C mutation was fully segregateed with the disease phenotype in the family,not found in 200 normal control samples.Further bioinformatics analysis revealed that the mutation is probably deleterious and the corresponding glutamine is highly conserved from zebrafish(Denio rerio)to humans(Homo sapiens).PAX3 encodes the paired box protein PAX3 that acts as a transcription factor.The structural analysis showed that the Gln91 locates at the C-terminal of α-helix 3 of the PD domain,and the p.Gln91 Pro mutation resulted in the conversion of this residue from the C-terminus of α-helix 3 to aβ-turn between α-helix 3 and α-helix 4 in the PD domain.Based on these results,we speculated that this mutation might disrupt the DNA binding ability of PAX3 and thereby affect its transcriptional regulation on the downstream genes.To examine the pathogenicity of PAX3 mutation,the eukaryotic expression vectors of pc DNA3.1(+)-PAX3-WT-HA and pc DNA3.1(+)-PAX3-Mut-HA were constructed to investigate whether the mutation affects the function of the protein.Western blot and immunofluorescence assays showed that the mutation did not alter the expression nor the subcellular localization of PAX3.Finally,the effect of PAX3 mutant on the activation of MITF promoter transcription was analyzed by the Dual-Luciferase Reporter Assay,while the MITF is an important target gene downstream of PAX3.The analysis showed that wild type PAX3 significantly increased the transcriptional activity of MITF promoter by about6.3 times,while the mutant displayed a slight promotion by about only 1.4 times.These results indicated that p.Gln91Pro mutation of PAX3 reduced the transcriptional activity on MITF promoter,which may be the cause of WS1 in this family.Taken together,we identified a novel PAX3 mutation(c.272A>C,p.Gln91Pro)in a large Chinese family with autosomal dominant WS1.Further functional analysis revealed that the p.Gln91 Pro mutation changed the original structure of the C-terminus of-helix 3of PD,and the mutant PAX3 protein was significantly reduced the transcriptional activation ability on the MITF promoter,which may be the pathological mechanisms of WS1 in this family.This study will provide a genetic basis for the further pathological research of Waardenburg syndrome.