Routine mutation screening of HNF-1 alpha and GCK genes in MODY diagnosis:How effective are the techniques of DHPLC and direct sequencing used in combination?

Aims/hypothesis. Mutations in the hepatocyte nuclear factor (HNF)-1 alpha a nd glucokinase (GCK) genes are the major causes of monogenic forms of Type II (non-insulin-dependent) diabetes mellitus (Maturity-Onset Diabetes of th e Young subtypes, MODY). We evaluated the effectiveness of fluorescent sing le-strand conformation polymorphism (F-SSCP), denaturing high-performance l iquid chromatography (DHPLC) and sequencing based mutation detection in the molecular diagnosis of MODY. Our goal is to identify a rapid, efficient an d cost effective mutation detection method for the molecular diagnosis of M ODY and other human genetic disorders. Methods. We evaluated the accuracy of DHPLC in screening for MODY 2 and 3 m utations. In addition, we compared the sensitivity, specificity, cost, hand ling time and analysis time of fluorescent single-strand conformation polym orphism, denaturing high-performance liquid chromatography and direct seque ncing screening methods. Results. Denaturing high-performance liquid chromatography is a recently de veloped method for mutation detection. It is cost effective, powerful and r eliable and quite suitable for 22 out of the 24 fragments required for MODY 2 and 3 testing. However, exons 1 and 7 of the HNF-1 alpha gene are very p olymorphic and so direct sequencing is faster as well as more efficient and reliable. Conclusion/interpretation. Our results suggest that combining denaturing hi gh-performance liquid chromatography and direct sequencing is a good approa ch for the routine detection of HNF-1 alpha and GCK mutations in MODY famil ies. Denaturing high-performance liquid chromatography appears to be a powe rful tool in genetic testing and the method could be applied to the molecul ar diagnosis of other human genetic diseases.