Denaturing temperature selection may underestimate keratin mutation detection by DHPLC.

Abstract

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 degrees 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.

• Authors: Pavel Strnad, Tim Christian Lienau, Guo-Zhong Tao, Nam-On Ku, Thomas M Magin, M Bishr Omary
• Journal: Human mutation (Hum Mutat) Vol. 27 Issue 5 Pg. 444-52 (May 2006) ISSN: 1098-1004 [Electronic] United States
• PMID: 16575832 (Publication Type: Comparative Study, Evaluation Study, Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)
• Copyright: Published 2006 Wiley-Liss, Inc.

Chemical References

• KRT18 protein, human
• KRT8 protein, human
• Keratin-18
• Keratin-8
• Keratins

Topics

• Chromatography, High Pressure Liquid (methods)
• Cohort Studies
• DNA Mutational Analysis (methods)
• Humans
• Keratin-18
• Keratin-8
• Keratins (chemistry, genetics)
• Liver Diseases (diagnosis, genetics)
• Mutation
• Nucleic Acid Denaturation
• Protein Structure, Tertiary
• Temperature