Flexibility of Melting Temperature Assay for Rapid Detection of Insertions, Deletions, and Single-Point Mutations of the AGXT Gene Responsible for Type 1 Primary Hyperoxaluria

Primary hyperoxaluria type 1 (PH1; OMIM 259900) is a rare autosomal recessive disorder characterized by impaired hepatic detoxification of glyoxylate. PH1 is caused by a deficiency of alanine:glyoxylate aminotransferase (AGT; EC 2.6.1.44), which catalyzes the transamination of glyoxylate to glycine. This defect leads to endogenous overproduction of oxalate and glycolate, producing oxalic and glycolic hyperacidurias, which are the hallmarks of the disease (1). The AGT enzyme is encoded by a single-copy gene (AGXT), which consists of 11 exons ranging from 65 to 407 bp and spanning a 10-kb DNA segment in the 2q37.3 human region. AGT is a 392-amino acid protein with a molecular mass of 43 kDa (2). Several technical approaches have been used to identify 7 polymorphisms and 26 mutations in the AGXT gene (3)(4)(5)(6). Here we describe a rapid, flexible, and inexpensive method for detection of the different types of mutations (insertions, deletions, point mutations) of the AGXT gene. Our method is based on the ability to distinguish between PCR amplification products by their melting temperatures (Tm) (7)(8)(9).