Validation of denaturing high performance liquid chromatography as a rapid detection method for the identification of human INK4A gene mutations

Abstract

The incidence of melanoma is increasing rapidly in western countries. Genetic predisposition in familial and in some sporadic melanomas has been associated with the presence of INK4A gene mutations. To better define the risk for developing sporadic melanoma based on genetic and environmental interactions, large groups of cases need to be studied. Mutational analysis of genes lacking hot spots for sequence variations is time consuming and expensive. In this study we present the application of denaturing high performance liquid chromatography (DHPLC) for screening of mutations. Exons 1alpha, 2, and 3 were amplified from 129 samples and 13 known mutants, yielding 347 products that were examined at different temperatures. Forty-two of these amplicons showed a distinct non-wild-type profile on the chromatogram. Independent sequencing analysis confirmed 16 different nucleotide variations in Leu32Pro; Ile49Thr; 88 del G; Gln50Arg; Arg24Pro; Met53Ile; Met53Thr; Arg58stop; Pro81Leu; Asp84Ala; Arg80stop; Gly101Trp; Val106Val; Ala148Thr; and in positions (-2) in intron 1 (C --> T); and in the 3' UTR, nucleotide 500 (C --> G). No false negatives or false positives were obtained by DHPLC in samples with mutations or polymorphisms. We conclude that the DHPLC is a fast, sensitive, cost-efficient, and reliable method for the scanning of INK4A somatic or germline mutations and polymorphisms of large number of samples.

Figure 1.

Detection of INK4A mutations by DHPLC. Elution profiles obtained in melanoma cases or known mutants for INK4A exon 1α at 67°C (A, B, C); INK4A exon 2 at 65°C (D, E, F); and INK4A exon 2 at 65, 69, and 70°C (G, H, I). Representative normal profiles are depicted in panels A and D. Note the additional peaks obtained for the mutant control no. 1489F, case no. 2044, case no. 2049 and control BlTm50 (panels B, C, E, F; Table 2 ). The mutation present in cell line SK-Mel21 (c.58 Arg → stop) is evident at all of the temperatures analyzed for exon 2, as compared with a wild-type elution profile (dotted line) (G, H, I, note details of the mutant profile). The numbers on the x axis correspond to the column retention time in minutes. Only a portion of the chromatographic profile containing the homoduplex or heteroduplex peaks is shown.

Figure 2.

Detection of INK4A-exon 3 variants by DHPLC and direct sequencing. A: Homoduplex profile in normal human placental DNA obtained at 59°C (upper panel) and 60°C (middle panel). B: Heteroduplex profile corresponding to melanoma DNA no. 2046 mixed with wild-type DNA. C: Sequencing analysis of normal DNA showing the C/C variant in position 500. D: Sequencing analysis of case no. 2046 shows heterozygosity (C/G, arrow) at position 500.