Targeted Double-Stranded cDNA Sequencing-Based Phase Analysis to Identify Compound Heterozygous Mutations and Differential Allelic Expression.

Hiroki Ura,Yo Niida,Sumihito Togi

doi:10.3390/biology10040256

Hiroki Ura, Yo Niida + Show 1 more

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https://doi.org/10.3390/biology10040256

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Abstract

Simple SummaryPhase analysis to distinguish between in cis and in trans heterozygous mutations is important for clinical diagnosis because in trans compound heterozygous mutations cause autosomal recessive diseases. However, conventional phase analysis is limited because of the large target size of genomic DNA. Here, we performed a targeted double-stranded cDNA sequencing-based phase analysis to resolve the limitation of distance using direct adapter ligation library preparation and paired-end sequencing; we elucidated that two heterozygous mutations on a patient with Wilson disease are in trans compound heterozygous mutations. Furthermore, we detected the differential allelic expression. Our results indicate that a targeted double-stranded cDNA sequencing-based phase analysis is useful for determining compound heterozygous mutations and confers information on allelic expression.There are two combinations of heterozygous mutation, i.e., in trans, which carries mutations on different alleles, and in cis, which carries mutations on the same allele. Because only in trans compound heterozygous mutations have been implicated in autosomal recessive diseases, it is important to distinguish them for clinical diagnosis. However, conventional phase analysis is limited because of the large target size of genomic DNA. Here, we performed a genetic analysis on a patient with Wilson disease, and we detected two heterozygous mutations chr13:51958362;G>GG (NM_000053.4:c.2304dup r.2304dup p.Met769HisfsTer26) and chr13:51964900;C>T (NM_000053.4:c.1841G>A r.1841g>a p.Gly614Asp) in the causative gene ATP7B. The distance between the two mutations was 6.5 kb in genomic DNA but 464 bp in mRNA. Targeted double-stranded cDNA sequencing-based phase analysis was performed using direct adapter ligation library preparation and paired-end sequencing, and we elucidated they are in trans compound heterozygous mutations. Trio analysis showed that the mutation (chr13:51964900;C>T) derived from the father and the other mutation from the mother, validating that the mutations are in trans composition. Furthermore, targeted double-stranded cDNA sequencing-based phase analysis detected the differential allelic expression, suggesting that the mutation (chr13:51958362;G>GG) caused downregulation of expression by nonsense-mediated mRNA decay. Our results indicate that targeted double-stranded cDNA sequencing-based phase analysis is useful for determining compound heterozygous mutations and confers information on allelic expression.

Highlights

Next-generation sequencing (NGS) is a powerful technology used in the clinical field for genetic diagnosis [1,2,3]
The distance between our identified compound heterozygous mutations in the ATP7B gene tested in this study is 464 bp in mRNA and 6.5 kb in genomic DNA
Analyzing differential allelic expression by conventional phase analysis using sanger sequencing is difficult because sanger sequencing of many clones is required

Summary

Introduction

Next-generation sequencing (NGS) is a powerful technology used in the clinical field for genetic diagnosis [1,2,3]. The use of NGS in clinical diagnosis is largely for comprehensive analysis, such as whole-genome sequencing, whole-exome sequencing, and gene targeting panel sequencing. Phase analysis, which detects specific compound heterozygous mutations, is not commonly performed using NGS technology. Multiple heterozygous mutations could be detected at specific loci. In cis heterozygous loss-of-function mutation still retains one functionally active allele as both mutations are located at the same allele (Figure 1A), whereas in trans compound heterozygous mutation does not retain any functionally active alleles as each mutation is found at a different allele. The clinical diagnoses must distinguish between in trans and in cis heterozygous mutations when there are more than two heterozygous mutations at a particular gene locus

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Conclusion