Alternative methods to a TaqMan assay to detect a tri-allelic single nucleotide polymorphism rs757210 in the HNF1β gene.
BACKGROUND
Several studies report difficulties in genotyping HNF1β rs757210 using TaqMan probes. This is possibly due to the tri-allelic nature of this single nucleotide polymorphism (SNP). The aim of the present research was to develop alternative methods for genotyping rs757210.


METHODS
Pyrosequencing and high resolution melting analysis of small amplicons (HRM) were developed and tested in panels of type 2 diabetes mellitus patients (n=258) and healthy blood donors (n=183). Results were confirmed by Sanger sequencing.


RESULTS
With pyrosequencing, allele frequencies for the A, G and C allele of 0.42, 0.56, 0.02 and 0.37, 0.62, 0.01 were established in the panel of type 2 diabetes mellitus patients and healthy blood donors, respectively. Similar results were found using the more routinely available HRM method. Results for pyrosequencing and HRM were in 99.6% concordance.


CONCLUSIONS
Pyrosequencing and HRM can be used to genotype the tri-allelic SNP rs757210 in the HNF1β gene and have the advantage over the commercially available TaqMan analysis that they can determine the rare C-allele variant.
Department of Clinical Pharmacy and Toxicology, Leiden, University Medical Center, Leiden, The Netherlands.
Jesse J
JJ
Swen
Renee F
RF
Baak-Pablo
Henk-Jan
HJ
Guchelaar
Tahar
T
van der Straaten
D000483:Alleles; D005260:Female; D060005:Genotyping Techniques; D051539:Hepatocyte Nuclear Factor 1-beta / Q000235:genetics*; D006358:Hot Temperature; D006801:Humans; D008297:Male; D020641:Polymorphism, Single Nucleotide
C496495:HNF1B protein, human; D051539:Hepatocyte Nuclear Factor 1-beta