Denaturing temperature selection may underestimate keratin mutation detection by DHPLC

Pavel Strnad, Tim Christian Lienau, Guo Zhong Tao, Nam-on Ku, Thomas M. Magin, M. Bishr Omary

Research output: Contribution to journalArticle

14 Citations (Scopus)

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°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.

Original languageEnglish
Pages (from-to)444-452
Number of pages9
JournalHuman Mutation
Volume27
Issue number5
DOIs
Publication statusPublished - 2006 May 1

Fingerprint

Keratins
Mutation
Temperature
Exons
Liver Diseases
Keratin-8
Keratin-18
End Stage Liver Disease
Chronic Pancreatitis
Inflammatory Bowel Diseases
K-18 conjugate
Software
Amino Acids
Liver
DNA
Population
Genes
Proteins

All Science Journal Classification (ASJC) codes

  • Genetics
  • Genetics(clinical)

Cite this

Strnad, P., Lienau, T. C., Tao, G. Z., Ku, N., Magin, T. M., & Omary, M. B. (2006). Denaturing temperature selection may underestimate keratin mutation detection by DHPLC. Human Mutation, 27(5), 444-452. https://doi.org/10.1002/humu.20311
Strnad, Pavel ; Lienau, Tim Christian ; Tao, Guo Zhong ; Ku, Nam-on ; Magin, Thomas M. ; Omary, M. Bishr. / Denaturing temperature selection may underestimate keratin mutation detection by DHPLC. In: Human Mutation. 2006 ; Vol. 27, No. 5. pp. 444-452.
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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°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.",
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Strnad, P, Lienau, TC, Tao, GZ, Ku, N, Magin, TM & Omary, MB 2006, 'Denaturing temperature selection may underestimate keratin mutation detection by DHPLC', Human Mutation, vol. 27, no. 5, pp. 444-452. https://doi.org/10.1002/humu.20311

Denaturing temperature selection may underestimate keratin mutation detection by DHPLC. / Strnad, Pavel; Lienau, Tim Christian; Tao, Guo Zhong; Ku, Nam-on; Magin, Thomas M.; Omary, M. Bishr.

In: Human Mutation, Vol. 27, No. 5, 01.05.2006, p. 444-452.

Research output: Contribution to journalArticle

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T1 - Denaturing temperature selection may underestimate keratin mutation detection by DHPLC

AU - Strnad, Pavel

AU - Lienau, Tim Christian

AU - Tao, Guo Zhong

AU - Ku, Nam-on

AU - Magin, Thomas M.

AU - Omary, M. Bishr

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N2 - 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.

AB - 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.

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