Novel Mutations In The USH2A Gene In A Family Affected With Usher Syndrome Type2

Backgrounds and AimsUsher syndrome(USH)is an autosomal recessive disorder characterized by sensorineural hearing loss,retinitis pigmentosa(RP)and varying degrees of vestibular dysfunction.The prevalence of USH was estimated at 3.2-6.2 per 100,000 persons.Usher syndrome is both genetically and clinically heterogeneous.It is the most common form of deaf-blindness and frequent case of recessive retinitis pigmentosa.The major clinical manifestations are different degrees of sensorineural hearing loss,retinitis pigmentosa with or without vestibular dysfunction.Three clinical subtypes of Usher syndrome have been identified according to the extent and onset of deafness and whether vestibular dysfunctionaccompany.The most common type of these three type is USH2 whose penetrance is 100%.So far,14 pethogenitic gene have been identified and USH2 A is the most common one,which occupying 74-90% among all patients with USH.Therefore,USH2 A is the first choice to conduct genetic screening to make genetic diagnosis of USH.Material and MethodsAn autosomal recessive Chinese family with three generationswas diagnosed USH in our hospital in may 2016 was investigated.There are nine members in the family,which includes 2 cases of male patients,and 6 normal family members.Blood samples of all these nine family members and 100 unrelated normal healthy persons were collected.All participants underwent the general physical examination to exclude systemic diseases and all family members underwent comprehensive bilateral ophthalmological and examinations,including best-corrected visual acuity(BCVA),intra ocular pressure,anterior segment imaging,fundus examination using a slit-lamp biomicroscope and colour fundus photography,and electroretinogram(ERG),optical coherence tomography,visual field test,vestibular function assessment and audiometry.This study was conducted in accordance with the World Medical Association’s Declaration of Helsinki and ethical approval was obtained from Ethics Committee of the First Affiliated Hospital of Zhengzhou University,Zhengzhou University.Informed consent was obtained from all participants or from the guardians of patients younger than 18 years old.Peripheral blood was collected from three members include two patients and five unaffected family members after informed consent was obtained.DNA was isolated with a whole blood DNA Extraction kit(TIANGEN,Beijing)according to the manufacturer’s instructions and quantified with NanoVue 2000(GE Healthcare Life Sciences,SWE).We used a gene capture panel including 136 hereditary retinal disease target genesand covering 14 USH related genes.The capture probes were designed and produced by Mygenostics(Beijing,China).A minimum of 3 μg DNA was used for the indexed Illumina libraries preparation.DNA was sheared into 150 bp by sonication.The resultant DNA was blunt-end–repaired followed by the addition of over-hanging A,ligation of single-index adaptors,polymerase chain reaction(PCR)enrichment,and then size-selected and were quantified as mentioned previously.The last step of the protocol was to sequence on Illumina HiSeq 2500 after hybridization,elution,amplified and purified.The sequencing results was detected in patients were annotated by the following data sources: Human Gene Mutation Database(HGMD),NCBI SNP,database of SNP(dbSNP),1000 Genomes,Human genome database(HG19),and our internal data containing 1000 controls.Then,the linkage analysis was conducted.ResultsWe diagnosed this family with USH according to the clinical examination of opthalmology and otology,detailed family history and investigation about the pedigree of this family.All affected individuals manifest ophthalmic abnormality and hearing disorders not associated with any other systemic diseases.Affected individuals were dignosed to USH2 accroding to USH diagnosis and typing standard,and the genetic pattern correspond to autosomal recessive manner.The phenotype of these two affected individuals are alike,including onset of night blindness during puberty,progressive constricted visual fields(tunnel vision),typical fund performances: atrophy of the retinal pigment epithelium in peripheral area,and moderate to severe bilateral hearing impairments.Target sequence capture was used to identify mutations in the USH2 A gene: c.5459 T>C(p.M1820T),c.802 G>A(p.G268R)and c.1190 T>A(p.I397K)and muations in the GPR98 gene: c.4703 G>A(p.S1568N)and c.15701 A>G(p.K5234R).These mutations were not detected in the normal subjects and not found in gene databases.Sanger sequence analysis was done to demonstrate co-segregation of the USH2 A and GPR98 mutations.The analysis result indicates that the complex heterozygous mutation in the USH2 A co-segregated with the disease in the family,whereas mutations in the GPR98 gene cannot co-segregated with the disease,indicating that these mutations in the GPR98 gene may not be the pathogenetic mutations.Conclusions1.In this study,we identified a novel complex heterozygous mutation in the USH2A(c.5459T>C(p.M1820T)、 c.1190T>A(p.I397K)and c.802G>A(p.G268R))2.This discovery not only expands the USH2 A mutation spectrum,but also provides references to the molecular biology research ofusher syndrome,genetic counseling and prenatal diagnosis.