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. 2006 May 2:5:11.
doi: 10.1186/1476-511X-5-11.

Allelic drop-out may occur with a primer binding site polymorphism for the commonly used RFLP assay for the -1131T>C polymorphism of the Apolipoprotein AV gene

Kirsten J Ward  1 , Sian Ellard, Chittaranjan S Yajnik, Timothy M Frayling, Andrew T Hattersley, Prathyusha N S Venigalla, Giriraj R Chandak
Affiliations

Allelic drop-out may occur with a primer binding site polymorphism for the commonly used RFLP assay for the -1131T>C polymorphism of the Apolipoprotein AV gene

Kirsten J Ward et al. Lipids Health Dis. .

Abstract

Apolipoprotein AV (ApoAV) gene variant, -1131T>C, is associated with increased triglyceride concentrations in all ethnic groups studied. An MseI based RFLP analysis is the most commonly used method for genotyping this SNP. We genotyped a large cohort comprising 1185 Asian Indians and 173 UK Caucasians for -1131T>C using an ARMS-PCR based tetra-primer method. For quality control, we re-genotyped approximately 10% random samples from this cohort utilizing the MseI RFLP, which showed a 2.9% (3/102) genotyping error rate between the two methods. To investigate further, we sequenced the 900 bp region around the -1131T>C polymorphism in 25 Asian Indians and 15 UK Caucasians and found a number of polymorphisms including the -987C>T polymorphism. Further analysis of the -987C>T SNP showed a higher rare allele frequency of 0.23 in Asian Indians (n = 158) compared to 0.09 in the UK Caucasians (n = 157). This SNP is located 4 bp from the 3' end of the RFLP forward primer and is in weak linkage disequilibrium with -1131T>C variant (r2 = 0.084 and D' = 1). Repeated RFLP analysis of seven subjects heterozygous for -987C>T (seven times), showed discordant results with the sequence at -1131T>C SNP nearly one third (15/49) of the time. We conclude that presence of -987C>T polymorphism in the forward primer of the MseI RFLP assay may lead to allelic drop-out and generate unforeseen errors in genotyping the -1131T>C polymorphism. Our results also emphasise the need for careful quality control in all molecular genetic studies, particularly while transferring genotyping methods between various ethnic groups.

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Figures

Figure 1
Figure 1
Schematic of the ApoAV gene, its variants and primer positions.
Figure 2
Figure 2
This shows the -987C>T polymorphism which potentially alters primer binding for the RFLP method of genotyping (primer sequence is placed within the boxed area, with the site of the polymorphism in capital letter).
See this image and copyright information in PMC

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