ATP7B Gene Mutation Analysis In The Patients With Wilson Disease

ObjectiveWilson disease (WD) is an autosomal recessive genetic disease with copper metabolic barrier.The worldwide prevalence of Wilson disease is estimated to be between 1 in 30, 000 and 1 in100,000. Clinical symptoms caused by the toxic accumulation of overload copper in the body,mainly for liver dysfunction, neuropsychiatric system dysfunction, Kayser–Fleischer ring andabnormal renal function. Wilson disease is caused by mutations of ATP7B gene, located in13q14.3. To date, almost 400 variations reported as causative mutations of WD, almostthroughout the coding region. The deep research for WD gene mutations may help the earlydiagnosis and treatment of WD. In this study, we will use the DNA extraction, PCRamplification and DNA sequence analysis techniques on WD gene ATP7B all 21 exons andadjacent intron regions of mutation scanning to obtain the exact gene mutation. we preliminaryanalysis the relationship between genotype and phenotype of WD patients, provide data to themolecular etiology of the WD to help WD patients in Shanxi province with the diagnosis andgenetic counseling.MethodsTotal genomic DNA was extracted by the Miller classic proteinase K-sodium chloridesalting, from the peripheral blood leukocytes from 12 patients with WD and 50 randomly testedsubjects. Polymerase chain reaction (PCR) and direct DNA sequencing were used to detectATP7B mutations of 12 WD patients. Restriction enzyme analysis was used to confirm thepathogenicity of a mutation. The genotype and phenotype of WD patients were gathered in orderto analyse the genotype-phenotype correlations with statistical.Results1. Four mutations were detected in the ATP7B gene in this study: R778L, A874V, T935Mand P992L, all of them have been reported.2. The newly identified mutation R778L was not found in PCR amplified products from 50randomly tested subjects by restriction enzyme analysis.3. Eight single nucleotide polymorphisms (SNPS): A406S, L456V, L770L, K832R and2866-13 g>c were detected in this study. All of them have been found in SNP database of NCBI.4. All gene mutations in this study were point mutation.5. The mutation rate of R778L is 41.7% in this study.6. R778L and T935M mutation were not related to gender, the onset age and clinicalphenotype. ConclusionConclusions1. R778L is the hot point mutation of ATP7B gene in ShanXi Han Patients with Wilson'disease;2. In clinic, HpaII restriction enzyme analysis can be used as an initial screening method inpatients with WD.3. R778L and T935M mutation were not related to gender, the onset age and clinicalphenotype.4. The results of this study consistent of previous study results only detectied of hot spotmutation. Whether genetic diagnosis of WD only detected the hot spot mutation, would bedetermined by the sample size increased.