Detecting rare thrombophilia variants by high-resolution melting.

Genotyping by amplicon melting is a minimalistic approach to geno-typing, requiring only PCR, a fluorescent DNA dye, and single-color acquisition of a melting curve in a closed tube. Multiplex genotyping for the F5 1 [coagulation factor V (proaccelerin, labile factor)] Le-iden (1691GϾA), F2 [coagulation factor II (thrombin)] 20210GϾA, and MTHFR [methylenetetrahy-drofolate reductase (NAD(P)H)] 1298AϾC and 677CϾT thrombo-philia mutations distinguishes up to 81 (3 4) combinations in solution without physical processing (1). Furthermore, we previously suggested that it might be possible to detect additional rare variants within the sequences of the quadruplex am-plicons by a combination of melting temperature (T m) and shape discrimination enabled by high-resolution melting analysis. Amplified heterozygotes have a complex melting curve shape consisting of 2 homoduplexes (wild type and mutant) and 2 heteroduplexes that are influenced by the class of single-nucleotide polymorphism and other variants within the amplicon. These qualities yield unique melting curves for most heterozygotes (2). Because we perform thousands of single-target genotyping assays annually on these thrombo-philia mutations, some unexpected rare variants have been identified by abnormal dual hybridization probe melting (3). Archived, de-identified DNA samples (ARUP Laboratories protocol IRB #7275) with rare heterozygous variants confirmed by sequencing were analyzed by the quadruplex melting assay with a new instrument (LS-32; Idaho Technology) that both amplifies and melts 32 samples with a resolution similar to that of the original LightCycler/HR-1 method (4). For melting analysis, we used custom software to perform temperature correction and T m assignment (1, 4, 5). The rare thrombophilia variants included those between the primers of each amplicon (MTHFR variants 685AϾG, 679GϾA, and 1317CϾT; F5 variant 1690CϾT; and F2 variant 20209CϾT), those under the primers (MTHFR variant 1293TϾC and F5 variant 1689GϾA), and 1 sample with 2 rare variants [one between the primers and the other under a primer (MTHFR 1317CϾT/1331CϾT)]. Melting data, including T m s, ⌬T m s, the peak width at one-half the peak height, and the position of the variant within the amplicon, were collected for all of the variants for each of the 4 loci (Table 1). The variants that were located between primers (1690CϾT, 1298AϾC/1317TϾC, 679GϾA, 685AϾG, and 20209CϾT) were easily detectable by either unique T m shifts and/or melting curve shapes that differed from the controls for each of the 4 loci. Variants located under the primers (1689GϾA and 1293TϾC) had melting profiles that matched the profile of the corresponding wild-type control. One …