A Pilot Study on the Development of a PCR-Sanger Sequencing Method for Detecting the NOTCH1 c.17_18delinsTT Variant in Individuals with Aortopathy History
Keywords:
Aortopathy, NOTCH1 gene, Polymerase chain reaction, Sanger sequencingAbstract
Aortopathy encompasses various structural and degenerative disorders of the aorta, posing significant clinical challenges. Among the genetic variants reported in individuals with cardiovascular disorders, the c.17_18delinsTT variant in the NOTCH1 gene has attracted interest due to its potential biological relevance. This pilot study aimed to develop and evaluate a PCR-Sanger sequencing workflow for detecting the NOTCH1 c.17_18delinsTT variant in members of a Malaysian family with a history of aortopathy. DNA samples from six family members were analysed using polymerase chain reaction (PCR) and Sanger sequencing for mutation detection. Optimized dimethyl sulfoxide (DMSO) concentration significantly improved amplification, yielding a DNA fragment size of 893 bp. The mutation exhibited both a homozygous normal genotype (CG/CG) and a heterozygous genotype (CG/TT). Further studies are required to determine whether this variant affects NOTCH1 protein function. The developed workflow successfully amplified and identified the target NOTCH1 variant in selected samples, demonstrating its potential utility for future genetic screening and research applications. However, due to the limited sample size and the variant's classification as a variant of uncertain significance (VUS), no conclusions regarding its causal role in aortopathy can be drawn from this pilot study.
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