High-risk genotype for type 1 diabetes: a new simple microtiter plate-based ELOSA assay

Abstract

The main contribution to genetic susceptibility for type 1 diabetes (T1D) is conferred by the HLA class II genes, with a major involvement of the DQB1*02 and 0302 alleles. The aim of our study was to develop a simple and rapid method suitable for identifying individuals with an HLA-associated T1D risk using whole blood as a source of DNA and reverse hybridization on microtiter plates (ELOSA). DNA was extracted from whole blood using various extraction methods. The PCR-amplified second exon of the DQB1 gene was hybridized at 37 degrees C for 1 hr to a set of 11 capture probes immobilized on a microtiter plate (eight-well strip per test) and corresponding to T1D susceptibility (S), protection (P), or neutral (N) alleles. Colorimetric analysis was then performed using specific oligonucleotides coupled to horseradish peroxidase and OrthoPhenyl Peroxidase (OPD) substrate. DNA samples corresponding to French (Rhône-Alpes area) T1D patients (n = 128) have been genotyped with the HLA-T1D prototype. A strong correlation is observed between susceptible genotypes and the disease, because 92.2% of the T1D individuals screened have at least one susceptible allele (DQB1*02 or *0302), thereby strengthening interest in analyzing DQB1 alleles as HLA-linked T1D markers in our Rhône-Alpes area population. Interestingly, clear T1D-associated genotyping results have been observed when using DNA samples extracted from dried blood spots, making it possible to envisage such genotyping in geographically dispersed affected families, for large-scale newborn screening, and for the inclusion of high-risk patients in clinical trials aimed at preventing the disease.