COLD-HRM: A Combination of Methods to Infer the Nature of Somatic Mutations

High Resolution Melting (HRM) measures DNA melting patterns to detect mutations. Hitherto, HRM has not been used to report the nature of the mutation. We reasoned that in conditions with a minor allele (e.g. allelic loss and somatic mutation), CO-amplification at Lower Denaturation temperature (COLD) PCR could change HRM patterns allowing (i) detection of Loss of Heterozygosity (LOH) and (ii) discrimination of somatic mutations from germline variants. For LOH, we tested paired tumour/normal samples from 11 cases of colorectal cancer (CRC) which were heterozygous for the SMAD4 SNP rs12455792 and 1 case which was homozygous wild type. To discriminate somatic from germline mutation, we tested tumour from 4 CRCs with somatic mutation in codon 374 POLE, paired tumour/normal tissue from 10 CRCs heterozygous for POLESNP rs4077170 and matched normal samples wild type. All samples underwent both the standard and COLD PCR followed by HRM analysis. In 4/11 tumour samples which were heterozygous for the SMAD4 SNP, HRM patterns changed following COLD-PCR suggesting LOH. Changes in HRM patterns were also seen in 4/4 tumours with POLE codon 374 mutation suggesting somatic rather than germline mutation. No change was seen in the 20 tumour and matched normal samples heterozygous for POLESNP rs4077170. We conclude that combining COLD PCR with HRM (COLD-HRM) allows inference of LOH and somatic sequence mutation without the requirement for matched normal DNA.