Rapid Molecular Diagnosis of Genetic Diseases by High Resolution Melting Analysis: Fabry and Glycogen Storage 1A Diseases

Abstract

For inborn errors of metabolism, high resolution melting analysis (HRMA) is a rapid, efficient, simple, and inexpensive method for mutation/rare variant screening. HRMA is a recent molecular technique for genotyping single-nucleotide polymorphisms without using probes. Here we apply HRMA to the α-galactosidase a (GLA) and glucose-6-phosphatase-alpha (G6PC) genes for mutation detection of patients with Fabry disease (MIM 301500) and glycogen storage disease type 1A (GSD1A; MIM 232200), respectively. To evaluate the procedure, genomic DNAs were blindly tested for known GLA mutations (c.658C>T, c. 679C>T, c.772G>A, c.796G>A, or c.718-719delAA) in three affected males and two obligate heterozygotes with Fabry disease, a G6PC mutation (c.247C>T) in a patient homozygous for that lesion, and 10 healthy control Turkish individuals. HRMA clearly detected the mutant amplicons and discriminated them from all wild-type GLA or G6PC amplicons. HRMA proved to be a sensitive, specific, and cost-effective mutation screening method for the rapid molecular diagnosis of these inborn errors of metabolism, indicating that the technique can be readily adapted to other genetic diseases.

Mutation Detection by Analysis of DNA Heteroduplexes in TILLING Populations of Diploid Species

“…The melting process is fast, and an analysis can be made in a few minutes (Li et al 2011). These advantages have made HRM a powerful tool for many applications-in the diagnosis of human diseases (Ezgu et al 2014) and mutation scanning in humans (e.g. BRCA1 and BRCA2 genes, Hondow et al 2011).…”