Mutation Analysis Of Infectious And Hereditary Diseases Using Next-generation Sequencing

Genetic diseases and infectious diseases are serious threats to human health. Genome analysis of pathogenic micro-organisms and identification of causal or susceptible genes of herediatary diseases contribute to the elucidation of the pathogenesis and disease prevention. In this study we characterized the profile of genome-wide hepatitis B virus (HBV) quasispecies, and located the causl genes of a Berker muscular dystrophy (BMD) pedigree and a hereditary spastic paraplegia (HSP) pedigree with the help of high-throughput next-generation sequencing.The distinct but closely related HBV genetic variants in affected patients constitute quasispecies, and its composition determines clinical liver disease progression and the outcome of antiviral therapy. But there is no study foucing on the whole picture of HBV quasispecies composition. We characterized population of viral strains by massively parallel pyrosequencing at the whole HBV genome level in17patients with advanced liver diseases (ALD) and30chronic carriers (CC). Internal reference sequence analysis showed high accuracy and sensitivity of our system. An average sequencing coverage of1952X and634X in ALD and CC groups were achieved respectively, and resolved a more complicated quasispecies composition than previously observed. The values of substitution frequencies in quasispecies were clustered as either more than80%or lower than20%, forming a unique U-shaped distribution pattern in both clinical groups. Furthermore, quantitative comparison of mutation frequencies of each site between two groups resulted in nine mutations associated with liver disease progression, and among which, C2288A/T, C2304A, and A/G2525C/T, were novel candidates. Moreover, distinct deletion patterns in preS, X, and C regions were illustrated between the two groups. In conclusion, we resolved the panorama of the viral mutations not only at the early onset of chronic infection but also at the end stage of liver disease progression. Comparison of mutation profiles between different clinical groups helped us determine the quasispecies evolution after long-term infection and viral factors associated with the development of ALD.BMD is the most common X-linked recessive childhood neuromuscular disease. All BMD patients and a young individual in our herediatary family carry a deletion spanning exons45to53and an unreported missense mutation on exon11of the DMD gene. Determination of deletion breakpoints demonstrated a330-kb deletion and there was a9-bp insertion between the breakpoints. This9-bp could match a reference sequence located within the deleted region which may caused by short-nucleotide sequence capture. The point mutation was screened in412healthy individuals and heterozygous genotype was found in two females. The pathogenic potential of this point mutation requires further investigation. HSP is a group of neurodegenerative disorders with highly clinical and genetic heterogeneity and are usually caused by damage to the corticospinal tract. Screening of the known causal genes failed to detect causal mutaions in our family and linkage analysis using the genome-wide SNP genotyping data showed no significant linkage signal. Combining exome sequencing of two affected patients and the genetic structure with three patients in one generation, a candidate causal gene SERPINA7was identified in the idential genomic region of the three patients. Candidate pathogenic locus was quite conservative in multi-species comparison and genotyping of430normal samples showed no mutaion. TBG protein encoded by SERPINA7is the main carrier of thyroid hormone in the blood. TBG interact with MCT8which mediates transmembrane transport of thyroid hormone and MCT8was encoded by a HSP known causal genes SLC16A2. Candidate pathogenic mutations lead to changes in polarity and hydrophilicity of amino acid413which located in the TBG surface, suggesting that it may hinder interaction of TBG and MCT8, which will in turn affect thyroid hormone absorption of cells and lead to HSP. We intend to use zebrafish model to verify the pathogenicity of SERPINA7.