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Targeted Double-Stranded cDNA Sequencing-Based Phase Analysis to Identify Compound Heterozygous Mutations and
Hiroki Ura1,2, Sumihito Togi1,2, Yo Niida1,2
1Center for Clinical Genomics, Kanazawa Medical University Hospital, 1-1 Daigaku, Uchinada, Kahoku, Ishikawa 920-0923, Japan.
Biology
|April 3, 2021
Summary
Distinguishing between cis and trans heterozygous mutations is crucial for diagnosing autosomal recessive diseases. This study introduces a novel cDNA sequencing method to accurately identify trans compound heterozygous mutations and assess their impact on gene expression.
Area of Science:
- Genetics
- Molecular Biology
- Clinical Diagnostics
Background:
- Compound heterozygous mutations can occur in cis (same allele) or in trans (different alleles).
- Only in trans compound heterozygous mutations are linked to autosomal recessive diseases, necessitating accurate differentiation for clinical diagnosis.
- Conventional phase analysis methods face limitations due to the large size of genomic DNA targets.
Purpose of the Study:
- To develop and validate a targeted double-stranded cDNA sequencing-based phase analysis method.
- To accurately distinguish between in cis and in trans compound heterozygous mutations.
- To investigate the impact of identified mutations on gene expression, specifically in Wilson disease.
Main Methods:
- Genetic analysis of a Wilson disease patient with two heterozygous mutations in the ATP7B gene.
- Targeted double-stranded cDNA sequencing with direct adapter ligation and paired-end sequencing.
- Trio analysis (parental sequencing) to confirm mutation inheritance patterns.
Main Results:
- Two heterozygous mutations in ATP7B were identified: c.2304dup and c.1841G>A.
- The targeted cDNA sequencing method successfully elucidated that these mutations are in trans compound heterozygous.
- Trio analysis confirmed the maternal and paternal origin of the respective mutations.
- Differential allelic expression was detected, indicating the c.2304dup mutation likely causes reduced expression via nonsense-mediated mRNA decay.
Conclusions:
- Targeted double-stranded cDNA sequencing-based phase analysis is an effective method for determining the phase of compound heterozygous mutations.
- This technique provides valuable insights into allelic expression, aiding in the understanding of disease mechanisms.
- The findings support the clinical utility of this advanced sequencing approach for genetic diagnostics.
