Screening And Identification Of Disease-Causing Gene In A Mental Retardation Family

Mental retardation(MR),also called as mental deficiency,is caused by various factors and is charactered by intelligence of patients lower than that of average of the same age.It is a human disease that exists defects to requirement of adaptive capacity.The genetic factors include monogenic disease,chromosome structural aberration,chromosome number aberration and so on.Morbidity of group is about 1-3%,and the morbidity of moderate and severe mental retardation is about 0.3-0.4%.Mental retardation causes severely harm to the children’s physical and psychological health,and is a big social issue around the world.Objective We investigated the nosogenesis in a Chinese MR family and found out the disease-causing gene.We hope our results can be advisory opinion and evidence for genetic counseling and prenatal diagnosis to MR family.Methods research object:we chose a mental retardation family genealogy.Proband is a male,aged 22 years old.His parents was not consanguineous marry.He started to walk at the 3 years of age and speak at the 5 years of age.Electroencephalogram(EEG)and magnetic resonance imaging(MRI)of brain showed normal.Examination of the nervous system revealed that pathological reflex was negative.According to International Classification of Diseases,10th edition(ICD-10),MR was judged as moderate mental retardation,IQ50.His parents,sister,and brother did not had obvious MR symptom.But several cases exist in his other family relation.After they signed the informed consent,we collected data of proband’ family and peripheral blood sample.Cytogenetics analysis:we tested the chromosome morphology and number by using G-banding karyotype method and fluorescence in situ hybridization.Molecular genetics analysis:whole genome exome was sequenced.(1)Genomic DNA of peripheral blood sample was extracted and randomly broke into 150-200 bp.Whole genome exome sequencing library was built and then sequenced in the Hiseq2000 platform.(2)Original image files were deal with to remove low quality sequencing data.SNP and Indel were anlysed,so we could obtain SNP and Indel list data.(3)We filtered variants to obtain candidate SNP and Indel list data by blasting in NCBI Build 37,NCBI dbSNP Build 132,1000 Genomes project,YH Database.(4)We screened out the candidate disease-causing gene of the MR family by bioanalytical methods,such as SIFT,KEGG pathway analysis,and site conservative analysis,coupling with clinical information of the family genealogy.Candidate disease-causing gene verification:we designed primer to amplify the gene.We used Sanger sequencing to verify distrubution of the candidate MR disease-causing gene in the patient’s family and 100 health control.Result The family pedigree analysis found that the family might be chromosome X-linked MR genealogy.G-banding karyotype analysis and FISH confirmed that proband karyotype was(46,XY).Chromosome morphology and number did not show obvious abnormalities.Using whole-exome sequencing,we identified 14678 mutations(Nonsense,Readthrough,Missens,Splice site,Frameshift,Cds-Indel)in exome gene of the patient,including 1392]SNPs and 757 Indels.Combining clinical information and bioinformatics anlysis,such as databases filtering,SIFT,GO,and KEGG anlysis,we found ARHGAP4 that had been reported was candidate MR disease-causing gene.The candidate mutation site ARHGAP4(chrX:153173202[g.153173202G>A;p.T941M])kept highly conservation in many species.PCR and Sanger sequencing results were consistent with whole exome sequencing.ARHGAP4(T491M)mutation was present in the genome of the proband and his mother is a carrier,while his father,sister,brother,and other healthy control do not carry this mutation.Conclusion whole genome exome sequencing is a effective tool to screen out MR disease-causing gene,which can screen all genes have been reported in the MR in a single experiment.According to clinical information,whole exome sequencing results and Sanger verification results,ARHGAP4(T491M)mutation may be disease-causing gene of the MR patient.However,whole genome exome sequencing is hard in clinical application due to its difficult operation.The relation between ARHGAP4 mutation and MR clinical characteristic is need to be illuminated with participation of more MR patients.