Diagnosis Of Variants Causing Wilson’s Disease Based On DNA Sequencing And MRNA Sequencing

BackgroundsWilson’s disease(WD)is a rare autosomal recessive inherited disorder that is induced by defects of the ATP7B gene and characterized by damage to the liver and nervous system caused by aberrant copper metabolism.The identification of pathogenic mutations on two homologous chromosomes has become the gold standard for the diagnosis of WD.Aims1.ATP7B gene diagnosis was performed on patients with WD to determine the genetic cause of the disease at the DNA level.2.For WD patients who failed to establish genetic diagnosis through exon sequencing,we tried to explore at the RNA level to find clues to abnormal structure of mRNA.Methods1.Sanger sequencing and multiplex ligation-dependent probe amplification(MLPA)were combined to detect germline ATP7B mutations for probands and other members from 30 unrelated Chinese WD families.2.For three WD patients whose exon sequencing only detected a single heterozygous variation,next-generation and third-generation sequencing techniques were combined to find the clues of abnormal splicing of mRNA in peripheral blood.RT-PCR and Sanger sequencing was used to verify the suspicious splicing abnormalities and analyze the causes.Results1.The results of direct sequencing showed that biallelic mutations were detected by Sanger sequencing in 26 of the probands,while single heterozygous mutations were detected in the remaining four probands.A total of 30 diverse mutations that could be evaluated as "pathogenic" or "likely pathogenic" according to the ACMG guidelines were detected,including 19 missense mutations,2 splicing mutations,1 nonsense mutations,1 in-frame deletion,6 frameshift mutations and 1 synonymous mutation.Among them,four mutations,including three missense mutations(c.3215G>T,c.3265G>A,c.3542T>A)and one frameshift mutations(c.2887 del C),have not been reported before.MLPA was performed in these probands(No.27,28,29 and 30)in whom only one mutation was detected,but no large intragenic deletions/duplications of ATP7B gene were found.In addition,2 asymptomatic patients were screened from 6 siblings of probands from 5 non-one-child families participating in the study.2.ATP7B gene targeted RNA sequencing analysis showed that the proband of family 29 had abnormal skipping of exon 14.RT-PCR experiments confirmed that this phenomenon occurred both in the samples of the proband and his mother.A heterozygous deletion mutation(c.3060+434del G,g.65645 del G)in the deep part of intron 13 was found in the samples of this patient and his mother by Sanger sequencing,which may be the cause of abnormal splicing.In addition,the results of sequencing analysis suggests that there may be a novel transcript variant with exon 2-5 skipping in human peripheral blood,which needs to be further verified in subsequent experiments.Conclusions1.In this study,we established a clear genetic diagnosis for 26 WD probands at the DNA level and found 4 novel mutations.This study contributes to the enlargement of the mutational spectrum of the ATP7B gene among the population of China.2.Through targeted sequencing of ATP7B gene mRNA and RT-PCR,a splicing mutation was found at the RNA level,which confirmed the gene diagnosis for one of the three patients who had only found a single mutation before,and found the suspected corresponding germline mutation at the DNA level.3.A suspected new transcript of ATP7B gene was found by targeted sequencing of ATP7B gene mRNA,which needs further research in follow-up experiments.