Denaturing temperature selection may underestimate keratin mutation detection by DHPLC

Keratins 8 and 18 (KRT8 and KRT18 genes; 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 novel KRT18 c. 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 frequent KRT8 c. 184G > T (K8 p.G62C), KRT8 c. 187A > G (K8 p.I63V), and KRT8 c. 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: one KRT8 c. 184G > T (K8 p.G62C), two KRT8 c. 187A > G (K8 p.I63V), seven KRT8 c. 1022G > A (K8 p.R341H), one KRT8 c. 1128G > A (K8 p.E376E), two intronic KRT8 c. 1202+46 A > T, and one hitherto undescribed KRT8 c. 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.