High resolution melting analysis for a rapid identification of heterozygous and homozygous sequence changes in the MUTYH gene

Abstract

Background: MUTYH-associated polyposis (MAP) is an autosomal recessive form of intestinal polyposis predisposing to colorectal carcinoma. High resolution melting analysis (HRMA) is a mutation scanning method that allows detection of heterozygous sequence changes with high sensitivity, whereas homozygosity for a nucleotide change may not lead to significant curve shape or melting temperature changes compared to homozygous wild-type samples. Therefore, HRMA has been mainly applied to the detection of mutations associated with autosomal dominant or X-linked disorders, while applications to autosomal recessive conditions are less common.

Methods: MUTYH coding sequence and UTRs were analyzed by both HRMA and sequencing on 88 leukocyte genomic DNA samples. Twenty-six samples were also examined by SSCP. Experiments were performed both with and without mixing the test samples with wild-type DNA.

Results: The results show that all MUTYH sequence variations, including G > C and A > T homozygous changes, can be reliably identified by HRMA when a condition of artificial heterozygosity is created by mixing test and reference DNA. HRMA had a sensitivity comparable to sequencing and higher than SSCP.

Conclusions: The availability of a rapid and inexpensive method for the identification of MUTYH sequence variants is relevant for the diagnosis of colorectal cancer susceptibility, since the MAP phenotype is highly variable.

Figure 1

HRMA of MUTYH exon 12. The homozygous c.1014G > C transversion is visible with HRMA only when samples are mixed with wild-type DNA. A) HRMA of native test samples: the normalized melting curves of c.1014G > C heterozygotes are distinct from those of GG wild-type homozygotes; the melting profiles of GG and CC homozygotes are clustered together. B) After mixing with a reference DNA, the CC sample is clearly distinguishable from both heterozygous GC and homozygous GG samples. Homo: homozygote; het: heterozygote; WT: wild-type; deg: degrees (°C).

Figure 2

HRMA of MUTYH exon 13. All heterozygous and homozygous variants in exon 13 are clearly distinguishable from each other and from wild-type samples without mixing with reference DNA. A) Normalized melting curves obtained for samples carrying variants c.1187G > A (both homozygous and heterozygous), c.1187-27C > T + c.1249C > A (double heterozygous), and wild-type samples; B) Normalized melting curves obtained for c.1187G > A (both homozygous and heterozygous) and homozygous c.1227_1228dupGG, and wild-type samples. Homo: homozygote; het: heterozygote; WT: wild-type; deg: degrees (°C).

Figure 3

HRMA of MUTYH exon 7. Two mutations (c.536A > G and c.544C > G) were identified in this exon. A) Normalized melting curves of c.536A > G heterozygotes and homozygotes are clearly distinct from wild-type homozygotes; on the other hand, wild-type and c.544C > G homozygous samples have identical melting profiles; B) After mixing with a reference DNA, the c.544C > G homozygous sample is clearly differentiated from wild-type, c.536A > G heterozygous and homozygus samples. Homo: homozygote; het: heterozygote; WT: wild-type; deg: degrees (°C).

Figure 4

HRMA of MUTYH exon 2. Four genotype combinations, in addition to wild-type, are distinguishable for this exon based on melting profile analysis, without prior mixing with a reference DNA. Homo: homozygote; het: heterozygote; WT: wild-type; deg: degrees (°C).