Genetic Etiology Studies On Rare Cases With Chromosomal Structural Variation

Background Chromosome Structural variations refer to the abnormal reconnection of a broken chromosome,which causes the number,position or sequence of genes to change.In the past,researchers often believed that the two types of chromosome Structural variation,chromosome reciprocal translocation or inversion,mostly belong to balanced Structural variation,which generally has no serious impact on genome and individual development and does not produce phenotypic effects.However,studies in the past 30 years found that about 6.7% of chromosome reciprocal translocation or inversion carriers have mental retardation,congenital malformation,growth retardation,multiple organ damage and other serious causes of ID,disability even lethal phenotypes.Due to the rarity of such patients and the significant genetic heterogeneity among them,the majority of patients have unknown causes of disease,long-term misdiagnosis,missed diagnosis,and cannot receive timely and effective treatment.Therefore,precise positioning of chromosomal structural abnormalities and in-depth analysis of sequences and candidate gene functions are necessary,The analysis of genotype phenotype correlation and the study of genetic causes in patients not only contributes to the accurate diagnosis and consultation of rare diseases,but also promotes the precise treatment of Rare disease and the development of drugs for related diseases,which has extremely important scientific value and clinical significance.Objective(1)Research on rare cases with chromosome balanced Structural variation with abnormal phenotype(s),and establish a technical system of chromosome rearrangement and analysis derived from complex chromosome structural variation;(2)The NGS/WGS sequencing,Bionano,Nanopore sequencing and other technologies were used to precisely locate the breakpoints of chromosome Structural variation.The breakpoints were analyzed by bioinformatics,and the genetic etiology diagnosis of patients was clarified by combining the genotype phenotype correlation of patients and the gene function of breakpoints.Methods(1)Seven rare cases with chromosome balanced Structural variation with mental retardation,growth retardation,congenital malformation and other phenotypes collected earlier were sequenced by NGS/WGS sequencing,Bionano,Nanopore sequencing and other techniques.Through bioinformatics analysis,BWA-MEM was used to compare the sequencing data with the Human genome reference sequence(GRCh37/hg19),Generate BAM files,use Breakdancer-1.45 software to predict candidate breakpoints,and use the Integrated Genomic Viewer(IGV)software to conduct visual candidate breakpoints inspection,Sanger sequencing to verify breakpoints,determine the rearrangement order of derived chromosomes,and establish the rearrangement and analysis technology system of complex chromosome structural variation;(2)The established technical system was used to resuscitate biological immortality lymphocyte cell lines of rare cases,amplify and culture RNA,reverse transcribe RT-PCR to form c DNA,amplify candidate breakpoints fragments,use Exo Tap purification reaction system,use Big Dye Terminator to conduct Sanger sequencing,accurately locate chromosome breakpoints,detect and analyze breakpoints and upstream and downstream genes change through PCR,q PCR,Hi-C,compare the genotype phenotype correlation and breakpoint gene function of patients to clarify their genetic etiology diagnosis.Results(1)By studying 7 rare cases with chromosome balanced structural variation with abnormal phenotype(s),the technical system of chromosome rearrangement and analysis derived from complex chromosome structural variation was established;(2)The established technical system was used to carry out genetic etiology research on 7 rare cases.For one typical balanced translocation case of PWS,WGS sequencing method was used for the first time to accurately locate the translocation breakpoints.It was found that the breakpoint was located in the key area of PWS,and no coding genes or sno RNAs in the region were interrupted by the breakpoint but only the host gene SNHG14,causing the phenotypes.The breakpoint domain was analyzed by Hi-C,it is inferred that the cause of the disease may be chromosomal breakage causing gene domain breakage in the region where it is located;for a complex case of chromosome fragmentation initially diagnosed with Kabuki syndrome,WGS sequencing was used for detection,but the rearrangement of derived chromosome sequences was not clear.Nanopore sequencing was used for further detection.This study accurately rearranged the chromosomal recombination fragments(21 breakpoints,14 fragments)derived from this case with chromothripsis for the first time.By accurately querying the phenotypes of the case,thoroughly study the genes near the breakpoints and the missing region genes,and based on the genotype phenotype correlation analysis of the patient,it is clearly diagnosed that the patient is LGS combined with Cd LS4 syndrome,correcting the initial diagnosis results of the patient and clarifying the genetic etiology of the case;for a case of nail dysplasia,WGS sequencing method was used to precisely locate the inverted breakpoints,which were located in the KRT Gene family and KRTAP Gene family region.The structural domain near the breakpoints were analyzed,and it was inferred that the pathogenic reason might be the change of gene sequence caused by inverted breakpoints,which led to the phenotypes;for a case of azoospermia,Bionano mapping technology was used to accurately locate the breakpoints and interrupt the upstream of the AZFa region,affecting gene expression and causing the phenotypes.Conclusion(1)In this study,the technique system of chromosome rearrangement and analysis derived from structural variation of complex chromosomes was established for the first time;(2)The technical system was used to accurately locate the chromosome breakpoint information of7 cases with chromosomal structural abnormalities with rare phenotypes,and 4 cases of genetic etiology were identified;(3)The NGS/WGS sequencing and Bionano mapping technology can identify the breakpoints and rearranged sequence of some cases of chromosome structural abnormalities,but for cases of complex chromosome structural variation such as chromothripsis,genetic etiology diagnosis cannot be completed.Nanopore sequencing technology,due to the length advantage of its reading segments,it is superior to NGS in the diagnosis of complex chromosomal structural abnormalities such as chromothripsis.