The Gene Scanning In A Family Affected With Choroideremia

Backgrounds and AimsChoroideremia(CHM)is a progressive disorder of chorioretinal dystrophy with an incidence between 1 in 50000 and 1 in 100000 people,which is a rare X-linked recessive inheritance.Affected male patients initially appear night blindness in adolescence and peripheral visual field loss gradually.Progressive degeneration of the retinal pigment epithelium,photoreceptors,and choroid develops with age.Blindness has been reported in the late years.Female carriers generally have normal vision and may have patchy pigmentation or retinal pigment epithelial degeneration.Choroideremia is caused by CHM gene mutation which leads to the absence of REP1 function.So far there is no effective drug therapy,while the subsequent preclinical data and initial findings from a phase 1/2 clinical trial have been described,which have good prospects and challenge.Due to the difficulty in differentiating CHM from retinitis pigmentosa,gene sequencing technology is used to definite clinical diagnosis.The aim of this study is to screen pathogenic mutation sites in a pedigree affected with choroideremia by using whole exon sequencing technology(WES)and analyze the relationship between the mutation and phenotype.Material and Methods1.The proband of this pedigree was diagnosed with retinitis pigmentosa in other hospital and gene test did not detect the relevant pathogenic mutations.According to these clinical manifestations,this pedigree was diagnosed with choroideremia in our department.To identify this disease,detailed systemic and ophthalmological examinations of nine members in three generations were collected.A total of 100 volunteers without ocular and systemic diseases were randomly selected as normal controls.2.DNA was extracted from peripheral venous blood and whole exon gene sequencing was used to screen the candidate gene mutation after comparison with the database.The protein coding region of the putative mutations selected by the next generation sequencing(NGS)was amplified by polymerase chain reaction(PCR).All putative mutations found by NGS were validated by Sanger sequencing.The pathogenicity of the mutation site was predicted by CADD and other methods.Results1.In this pedigree,affected male proband(III2)manifested night blindness,scattered osteocytoid pigmentation and posterior choroid atrophy followed by progressive peripheral visual field loss with normal central vision preserved.A female carrier(II2)is asymptomatic,while the fundus occurred patchy pigmentation and chorioretinal degeneration followed by the decreased vision of left eye.No similar clinical manifestations and eye diseases were found in other family members.2.The proband harbored a nonsense mutation site with CHM gene on chromosome X by whole exon sequencing.The nucleotide 1441 of exon 12 in CHM gene was detected from guanine to thymine(c.1441 G > T),and its coding amino acid changed to p.E481 X,which generate the premature termination of peptide chain synthesis.CADD predicted this mutation site was pathogenic which was highly correlated with CHM.Sanger sequencing demonstrated the presence of this mutation on II2,while none of the normal members and 100 controls had this mutation.According to the manifestations of pedigree,X-linked recessive inheritance was proved.Gene bank data demonstrated that this mutation site has not been reported which was ascertained to be a novel mutation.Conclusions1.The study definited the diagnosis of CHM pedigree clinically and discovered a novel gene mutation site(c.1441 G > T,p.E481X)in exon 12 of CHM gene on X chromosome by using whole exon sequencing.The mutation was a nonsense mutation,which resulted in the loss of the function of the protein encoded by CHM gene.2.Sanger sequencing confirmed that there was no such mutation in the other family members and normal controls.Software such as CADD and Mutation Taster predicted that the mutation was a pathogenic mutation,and the inheritance mode of this family was recessive inheritance of X chromosome.