Homozygous Mutations In MITF May Be Associated With Waardenburg Syndrome 4

Objective Waardenburg syndrome(WS)is a rare polygenic auditory pigmented disease,typically characterized by moderate to severe sensorineural hearing loss and a variety of pigmented abnormalities,including iris heterochromia,patchy skin discoloration or excessive brown freckles,white forelock or premature graying,and other manifestations include dystopia canthorum,skeletal muscle development abnormalities and gastrointestinal abnormalities.The purpose of this study is to study the genetic etiology of a WS family with complex phenotype,to find the hereditary pathogenic factors of the family,and to provide genetic counseling and fertility guidance for offspring.Methods All subjects in this study were recruited by the Otorhinolaryngology-head and neck surgery Clinic of Taizhou people’s Hospital in Jiangsu Province.In order to clarify the clinical phenotype and draw the genetic map,all family members were subjected to detailed medical history collection and complete physical examinations such as otology,ophthalmology,dermatology,orthopedics,and digestive system.Peripheral venous blood samples of family members were collected after signing the informed consent form.All coding exons and flanking sequences in three common deafness genes(GJB2,SLC26A4,MT-RNR1)and WS candidate genes(PAX3,MITF,SNAI2,EDN3,EDNRB,SOX10)were amplified by polymerase chain reaction(PCR).The PCR products were sequenced by Sanger sequencing and analyzed by Sequencher 5.4.6.The suspected pathogenic mutations were identified by computational tools,verified by family member samples,and screened in 400 Chinese Han population with normal hearing.The structure differences between wild-type and mutant-type complexed were analyzed by homology modeling with the software Modeller 9.17 and Py MOL 1.8.x.Results 1)Phenotype: Typical WS2 phenotypes such as congenital severe sensorineural deafness,premature graying,iris heterochromia,facial freckles and abnormal pigmentation of trunk were identified in the proband Ⅲ-1 and his younger brother Ⅲ-2.Additional congenital intestinal symptoms,such as severe constipation,abdominal distension and abdominal pain,indicated the possibility of WS4 type.Mild phenotypes of pigment abnormalities such as premature graying and local white forelock were found in their parents.2)Genotype: No pathogenic mutations were found in three common deafness genes in all members.Novel mutation c.668 G > A(p.R223H)in MITF gene identified in this family was very likely the pathogenic one associated with WS phenotypes.Homozygous p.R223 H was identified in the proband Ⅲ-1 and his younger brother Ⅲ-2,and heterozygous one found in parents II-1 and II-2 with mild phenotype.3)The novel mutation was not found in 400 Han nationality with normal hearing.4)p.R223 H in MITF gene were not found in Exome Variant Server,gnom AD,and 1000 G databases.Disease causing variant was predicted by computational tools such as Mutation Taster,Polyphen-2,PROVEAN and SIFT.5)Homology modeling revealed structural changes and decreased dimer affinity.Conclusion The novel mutation p.R223 H in MITF gene might be the hereditary pathogenic factor in this WS family.It is the first time to reminding the association between homozygous mutation in MITF gene and WS4.Haplo-insufficiency result from decreased affinity of dimer might be its pathogenic mechanism.This study further enriched the genotypic and phenotypic spectrum of Waardenburg syndrome,and provided a further theoretical basis for clinical genetic counseling.