Denaturing Temperature Selection May Underestimate Keratin Mutation Detection by DHPLC

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Keratins 8 and 18 (KRT8andKRT18genes; K8 and K18 proteins) variants are risk factors for developing end-stage liver disease and may be associated with inflammatory bowel disease and chronic pancreatitis. The frequency of K8/K18 variants in American, British, German, and Italian populations differs. For example, one study showed no amino acid-altering K8/K18 mutations in 256 German patients with liver disorders, while another found 58 out of 467 American liver disease patients with K8/K18 mutations. Both studies used the WaveSystem™, which utilizes DHPLC. We hypothesized that experimental conditions contribute to the discrepancy, and we tested this hypothesis using previously described K8/K18 variants and a novelKRT18c.1057C>G variant (K18 p.R353G) to optimize the DHPLC conditions in 10 examined exons under a range of denaturing temperatures. Six of 16 tested variants in three of the 10 exons, including the frequentKRT8c.184G>T (K8 p.G62C),KRT8c.187A>G (K8 p.I63V), andKRT8c.1022G>A (K8 p.R341H), could not be reliably detected when using temperatures suggested by the prediction software, but all these variants were readily detectable at 2°C higher denaturing temperatures. Using optimized temperatures, we then tested available genomic DNA from 151 out of the 256 German liver disease patients for the presence of K8 variants in exons 1 and 6, where most of the American cohort K8 variants occur. We identified 12 exonic and two intronic K8 variants: oneKRT8c.184G>T (K8 p.G62C), twoKRT8c.187A>G (K8 p.I63V), sevenKRT8c.1022G>A (K8 p.R341H), oneKRT8c.1128G>A (K8 p.E376E), two intronicKRT8c.1202+46 A>T, and one hitherto undescribedKRT8c.1138G>A (K8 p.V380I). Therefore, although DHPLC offers a robust and high throughput means for mutation analysis, assessment of denaturing temperature ranges, and possible inclusion of control mutants should be considered. Hum Mutat 27(5), 444-452, 2006. Published 2006 Wiley-Liss, Inc.†

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