3. Mutation Detection in Familial Aneurysm Syndrome Using High-Resolution Melting Analysis

• Published in: Journal of Nippon Medical School Vol. 76; no. 2; p. 114
• Main Authors: Banyar Than Naing, Atsushi Watanabe, Takashi Shimada
• Format: Journal Article
• Language: Japanese
• Published: The Medical Association of Nippon Medical School 2009
• ISSN: 1345-4676

Familial Aneurysm Syndrome (FAS) is caused by mutations in 3 genes, mainly in the triple helix region of Col3A1 (total 44 exons), but also in TGFBR 1 & 2 genes (total 16 exons). In Col3A1, the mutations may be single-base mutations in glycine (Gly) or splicing mutations in the exon-intron junction. I have already set up a mutation detection system using a reverse transcriptase PCR (RT-PCR) direct sequencing method for total RNA and a PCR direct sequencing method for genomic DNA, and am now developing a mutation detection system using high-resolution melting analysis (HRM). High-resolution melting analysis of PCR products for mutation scanning is based on monitoring of the fluorescence released during the melting of double-stranded DNA labeled with a specifically developed saturation dye such as LCGreen. A sensitivity of 100% and specificity of 99.4% can be expected if the PCR product is less than 300 bp, allowing us to predict mutation in specific exons before sequencing by comparing the melting curves in normal subjects and FAS patients using genomic DNA and PCR with LCGreen dye. Only the suspected exons would need to be confirmed by sequencing. I am developing a mutation detection system for the Col3A1 gene and TGFBR 1 & 2 genes(except Exon 1) using HRM with normal genomic DNA. The next step is to compare the melting curves of genomic DNA from 3 FAS patients and normal genomic DNA to detect mutations. To date I have not obtained good melting curves for some exons and am trying to resolve this problem. For Exon 1 of TGFBR 1 & 2, I am using a PCR direct sequencing method. I hope that my system will allow quicker and easier detection of mutations than previous methods such as PCR direct sequencing.