Routine mutation screening of HNF-1alpha and GCK genes in MODY diagnosis: how effective are the techniques of DHPLC and direct sequencing used in combination?
OBJECTIVE
Mutations in the hepatocyte nuclear factor (HNF)-1alpha and glucokinase (GCK) genes are the major causes of monogenic forms of Type II (non-insulin-dependent) diabetes mellitus (Maturity-Onset Diabetes of the Young subtypes, MODY). We evaluated the effectiveness of fluorescent single-strand conformation polymorphism (F-SSCP), denaturing high-performance liquid chromatography (DHPLC) and sequencing based mutation detection in the molecular diagnosis of MODY. Our goal is to identify a rapid, efficient and cost effective mutation detection method for the molecular diagnosis of MODY and other human genetic disorders.


METHODS
We evaluated the accuracy of DHPLC in screening for MODY 2 and 3 mutations. In addition, we compared the sensitivity, specificity, cost, handling time and analysis time of fluorescent single-strand conformation polymorphism, denaturing high-performance liquid chromatography and direct sequencing screening methods.


RESULTS
Denaturing high-performance liquid chromatography is a recently developed method for mutation detection. It is cost effective, powerful and reliable 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-1alpha gene are very polymorphic and so direct sequencing is faster as well as more efficient and reliable.


CONCLUSIONS
Our results suggest that combining denaturing high-performance liquid chromatography and direct sequencing is a good approach for the routine detection of HNF-1alpha and GCK mutations in MODY families. Denaturing high-performance liquid chromatography appears to be a powerful tool in genetic testing and the method could be applied to the molecular diagnosis of other human genetic diseases.
Boutin
P
P
Department of Human Genetics-CNRS UPRES, CHRU, Pasteur Institute, Lille, France.
Vasseur
F
F
Samson
C
C
Wahl
C
C
Froguel
P
P
D001483:Base Sequence / Q000235:genetics; D002851:Chromatography, High Pressure Liquid / Q000191:economics / Q000379:methods / Q000592:standards; D003362:Cost-Benefit Analysis; D004268:DNA-Binding Proteins; D003924:Diabetes Mellitus, Type 2 / Q000145:classification / Q000175:diagnosis* / Q000235:genetics*; D005941:Glucokinase / Q000235:genetics*; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D006801:Humans; D009154:Mutation; D009687:Nuclear Proteins; D018807:Polymorphism, Single-Stranded Conformational; D012680:Sensitivity and Specificity; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics*
D004268:DNA-Binding Proteins; C496487:HNF1A protein, human; C496495:HNF1B protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; D009687:Nuclear Proteins; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D005941:Glucokinase