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 1␣, 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 Leu 32 Pro; Ile49Thr; 88 del G; Gln50Arg; Arg24Pro; Met53Ile; Met53Thr; Arg58stop; Pro81Leu; Asp84Ala; Arg80stop; Gly101Trp; Val106Val; Ala148Thr; and in positions (؊2) in intron 1 (C 3 T); and in the 3 UTR, nucleotide 500 (C 3 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 Cutaneous melanomas are being detected at an increasing rate worldwide. Even though many patients are diagnosed at an early stage, the death rate continues to rise due to the increasing incidence of more advanced lesions. 1,2 Genetic and environmental factors such as family history, skin type, previous tumors, and sun exposure have been identified as important risk factors. [3][4][5][6] In addition, germline mutations or variants of certain genes have been proposed as risk factors for the development of melanomas. One of these genes, the CDKN2A or INK4A, encodes for p16, an important cell cycle regulator capable of arresting cells in G1-phase by inhibiting the phosphorylation of the retinoblastoma protein by cyclinD1/Cdk4 complexes. 7 The INK4A gene has been found silenced by point mutation, deletion, and methylation of the promoter region in several sporadic tumor types. 8 -16 Analyses of INK4A in sporadic melanomas revealed a frequency of mutations and deletions that ranges from approximately 75% in cell lines 8 to 15% in primary multiple melanoma tumors. 17 In addition, INK4A germline mutations have been found in melanoma kindreds, ranging in prevalence from 10.3 to 72.2%, 18 -19 although in overall approximately 20% of the families that have been studied show mutations in this gene. 20 In an attempt to better define the gene-environment interactions in sporadic melanoma, our group expects to enroll 4000 newly diagnosed subjects to determine the relationship between germline INK4A mutations and environmental factors such as sun exposure. Typically, INK4A gene mutations have been analyzed by polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) and sequencing. 16,18,21 D...

Mutation and expression analyses of the MET and CDKN2A genes in rhabdomyosarcoma with emphasis on MET overexpression

“…For CDKN2A, one nonsense mutation (CGA > TGA) (2.6%; 95% CI, 0.5-13.2%), resulting in a truncated protein at codon 80 of p16 INK4A , was observed in an ARMS cell line, SJRH-18. The same mutation was reported previously in melanoma (Orlow et al, 2001). This C > T transition also results in a missense mutation (Pro135Leu) Using RQ-PCR, the expression of MET was detected in all of the RMS samples and normal skeletal muscles.…”

Prevalence of CDKN2A mutations in pancreatic cancer patients: implications for genetic counseling

“…The former has not been previously reported, while the latter has been reported at least once in a melanoma patient. 28 It is difficult to know the significance of the variants apparently affecting p14ARF alone except that both patients had young-onset disease (ages 58 and 45), while neither carried additional family histories of pancreatic cancer or melanoma. However, their presence lends further evidence to the question of the significance of p14ARF in inherited risk for pancreatic cancer.…”

The Prevalence of CDKN2A Germ-Line Mutations and Relative Risk for Cutaneous Malignant Melanoma: An International Population-Based Study

“…Our methods for identifying sequence variation have been described in detail in a previous article (14). Briefly, PCR products spanning exons 1a, 2, and 3 plus the adjacent intronic regions of all samples were initially screened by denaturing high-performance liquid chromatography analysis (17). All samples showing an altered denaturing high-performance liquid chromatography chromatographic profile were reamplified from genomic DNA in an independent PCR reaction for sequencing.…”