Nonisotopic, PCR-based method for 5' insulin gene VNTR allele class assignment.

Abstract

The identification of genes that contribute to the susceptibility to polygenic human diseases is a laborious task that requires the analysis of multiple polymorphic markers in search of variants putatively associated with the disease phenotype. Besides, it is often necessary to study large cohorts of affected and unaffected individuals to achieve acceptable levels of significance. Once preliminary evidence of association has been obtained, a confirmation of results in independent samples from different populations is also required. If this type of research is to be cost effective, genotyping methods must be straightforward, easily scalable to large numbers of samples and results must be obtained in a short time. A clear example of a polygenic disease is insulin-dependent (type 1) diabetes mellitus, an autoimmune disorder in which T-cell-mediated destruction of the insulin-producing pancreatic beta cell occurs, and is currently the object of intense genetic investigation in the search for susceptibility genes. During the last few years, several groups have focused their attention towards the allelic variation at the polymorphic region situated 5' upstream of the human insulin gene (INS) on chromosome 11p15.5, which arises from a variable number of tandem repetitions (VNTR) of a 14–15 bp oligonucleotide with the consensus sequence 5' -ACAGGGGT(G/C)(T/C)GGGG-3'. Based on Southern blot analysis (2), these VNTR alleles fall into three distinctive size classes according to the number of repeats of the oligonucleotide (classes I, II and III average 570, 1200 and 2200 bp, respectively). It has been shown that in Caucasians, class I alleles are positively associated with type I diabetes, class II alleles are rare and class III alleles are dominantly protective. More recently, a PCR-based method has been used for class I allele subtyping, and allele-specific effects on disease susceptibility have been described (3,8). However, all reported methods for PCR amplification of the INS-VNTR polymorphism are limited to the analysis of class I alleles and fail to amplify either class II or class III alleles (3,5,6) probably because of (i) the increased length of these alleles, (ii) the strong secondary structure derived from the high GC-rich content of the template and (iii) the high frequency of class I/class III heterozygous in which the amplification of the short class I alleles is strongly favored over the long class III alleles. In fact, long alleles may only be detected by traditional Southern blot hybridization with an internal radioactive-specific probe, and this is a laborious, time-consuming technique unsuitable for large population studies requiring large amounts of purified, high molecular weight DNA, which is not always available. The analysis of diallelic markers in the 5' region of the insulin gene, especially the -23/HphI RFLP, has proven useful as an alternative to Southern blot typing of the INS-VNTR, at least in populations of European descent, in which the VNTR locus may be considered diallelic (class I/class III) and tight linkage disequilibrium between -23/ HphI and VNTR alleles has been demonstrated (4). However, it might be necessary to investigate both the frequency of class II alleles and the degree of linkage disequilibrium between the proposed marker and the INS-VNTR locus when a non-Caucasian population is investigated for the first time. We report a set of PCR conditions that allow efficient amplification of INSVNTR alleles of all classes, offering a nonradioactive and rapid alternative to the conventional, tedious Southern blot technique for reliable VNTR typing in large samples from any population. Genomic DNA was isolated from blood samples using NucleoSpin® Blood kit (Macherey-Nagel, Duren, Germany). DNA (150 ng) was subjected to PCR amplification with flanking primers (5) (5FP1: 5' -CACCTTGGCCCATCCATGGCGGCATC-3' and 5FP2: 5'-CTCCAGGAGAGCAAAGCCCTCACCTG-3', which anneal 21 and 64 bp beyond the 5' and 3' ends of the VNTR, respectively). The downstream primer is labeled with fluorochrome Cy5 on its 5' end, so the pair of primers is routinely used for INSVNTR class I subtyping on an ALFexpressTM automatic DNA sequencing apparatus (Amersham Pharmacia Biotech, Uppsala, Sweden). PCR amplifications were performed in 100-mL reactions containing NH4-based PCR buffer (16 mM ammonium sulfate, 67 mM TrisHCl, pH 8.8, and 0.01% Tween® 20), 2 mM MgCl2, 10% glycerol, 0.4 mM Benchmarks