Identification Of A Disease-causing Gene In STGD Family And Its Potential Role In Corneal Dystrophy

PurposeUse the Whole exome sequencing(WES)to identify the disease-associated mutations in a family of Stargardt's disease(stargardt macular dystrophy,STGD)in China,and extend the current pathogenic spectrum of mutation,and to further identify the genotype-phenotype correlation.MethodsA Chinese STGD family was collected along with 200 normal subjects as a control.The clinical data of the proband and his sister were recorded,and their medical history and family history were inquired and recorded.The general ophthalmologic examination and examination were performed on them,including fundus oculi color photography,yellow spot optical coherence tomography(OCT)and examination of visual field.Peripheral blood of patients and family members were sampled intravenously,and DNA was extracted by Blood DNA Kit.The Illumina HiSeq2500 sequencing platform was used to perform WES after the detection was done and qualified,and the biological process was analyzed and variation detection and annotation was performed.By the integration of data locus,filtration,functional prediction were combined with an autosomal dominant genetic model analysis to discover the pathogenic gene mutations.Sanger sequencing technique was used to carry out an analysis on mutation of family members in order to finish the confirmation of the result of next generation sequencing.Mutation screening was then performed through a high resolution melting(HRM)curve method aimed at 200 healthy controls to further exclude polymorphic variants.The ProtParam online software was used to calculate the physicochemical parameters of mutant and wild-type protein.SMART online software was used for online analysis of protein structural domains.HOPE online software was used to analyze the structural effect of mutation.Healthy C57BL/6 mice were used for immunofluorescence staining and Western blot(WB)to detect the protein expression level of the abovementioned genes.Real-time fluorescence quantitative polymerase chain reaction(PCR)technology was used to detect the mRNA expression.The content of ABCA4 in mouse's retina was analyzed.ResultsThe color photography of the proband and his sister's fundus oculi were visible.The fundus morphology was characterized by varying degrees of the macular region,shrunken range,and distribution of yellow spots on the fundus.The OCT of macular area showed that the neuroepithelial thickness was reduced in the macular area.The visual field examination showed that the central dark spot and the local dark spot were complicated with the absence of visual field.By using WES,a new homozygous mutation in the ABCA4 gene(NM 000350: c.G3190 C,p.G1064R)was identified.This mutation is co-segregated from the phenotype in the family system.This mutation was not detected among 200 unrelated healthy controls and was not recorded in any database,indicating that it was a new mutation.Five functional predictive software predicted that this mutation be "harmful" and highly conserved during the process of evolution.The AAA ATPase core of ABCA4 is found in amino acid 955 to 1145,while p.G1064 R is located in the AAA structural domain,which may force the local skeleton to form an incorrect conformation,disrupt local structures,and reduce the activity of ATPase,thus leading to the occurrence of disease.ABCA4 is highly expressed in ocular tissues of mice such as the retina.ConclusionWe have identified a new pathogenic mutation of STGD located at the ABCA4 gene(NM 000350: c.G3190 C,p.G1064R).This gene mutation is co-segregated from the phenotype of the family,and is predicted by the software function as "harmful",and it is highly conservative in evolution,both of which suggest its pathogenicity.The results of this study expand the existing pathogenic mutation spectrum and further enhance the understanding of genotype-phenotype correlation.