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Related Experiment Videos

Denaturing temperature selection may underestimate keratin mutation detection by DHPLC.

Pavel Strnad1, Tim Christian Lienau, Guo-Zhong Tao

  • 1Department of Medicine, Palo Alto Veterus Affairs Medical Center, Palo Alto, California 94304, USA.

Human Mutation
|April 1, 2006
PubMed
Summary

Keratin variants (KRT8/KRT18) are linked to liver disease. Optimizing denaturing temperatures in DHPLC analysis improved detection of these critical KRT8 and KRT18 gene variants.

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Area of Science:

  • Genetics
  • Molecular Biology
  • Hepatology

Background:

  • Keratin 8 and 18 (KRT8/KRT18) gene variants are associated with end-stage liver disease, inflammatory bowel disease, and chronic pancreatitis.
  • Population-specific frequencies of KRT8/KRT18 variants have been reported, with discrepancies between studies using denaturing gradient gel electrophoresis (DHPLC).

Purpose of the Study:

  • To investigate if experimental conditions, specifically denaturing temperatures in DHPLC, contribute to the differing frequencies of KRT8/KRT18 variants observed in previous studies.
  • To optimize DHPLC conditions for reliable detection of KRT8 and KRT18 variants.

Main Methods:

  • Utilized previously described and a novel KRT18 variant (K18 p.R353G) to test DHPLC conditions across a range of denaturing temperatures for 10 exons.
  • Compared variant detection rates using standard predicted temperatures versus optimized higher temperatures.

Related Experiment Videos

  • Screened genomic DNA from German liver disease patients for KRT8 variants in exons 1 and 6 using optimized DHPLC conditions.

    • Main Results:

    • Six of 16 tested KRT8/KRT18 variants were undetectable at predicted DHPLC temperatures but detectable at 2°C higher.
    • Optimized DHPLC identified 12 exonic and 2 intronic KRT8 variants in 151 German liver disease patients, including previously reported and novel mutations.
    • Identified frequent KRT8 variants (K8 p.G62C, K8 p.I63V, K8 p.R341H) and a novel KRT8 variant (K8 p.V380I).

    Conclusions:

    • DHPLC is a robust, high-throughput method for KRT8/KRT18 mutation analysis.
    • Careful assessment of denaturing temperature ranges and inclusion of control mutants are crucial for reliable DHPLC variant detection.
    • Optimized DHPLC conditions can resolve discrepancies in KRT8/KRT18 variant frequency studies.