An in-house multilocus SNP genotyping assay for evaluation of complex genetic diseases

<p><b>Background:</b> With an increase in the discovery of newer genetic loci/polymorphisms in complex multifactorial diseases, there is also an increased need for methods that can simultaneously genotype multiple loci in a cost-effective manner. Using coronary artery disease (CAD) as a model, the study aimed to develop an in-house multilocus assay for simultaneous detection of 17 genetic variants in 11 genes implicated in CAD.</p> <p><b>Methods:</b> A multiplex polymerase chain reaction (PCR)-based reverse line blot hybridization (MPCR-RLBH) approach was used, where each DNA sample was amplified using two separate MPCRs, and the alleles were genotyped using covalently immobilized, amino-linked sequence-specific oligonucleotide probes using an enhanced chemiluminescence system. The assay performance was tested on 75 healthy controls and 75 angiographically proven CAD cases. Validation was done by automated Sanger sequencing.</p> <p><b>Results:</b> The assay could successfully discriminate both the alleles at <i>CETP</i> (I405V), <i>LPL</i> (D9N), <i>NOS3</i> (T-786G and E298D), <i>LIPC</i> (C-514T), <i>FGB</i> (G-455A), <i>ITGB3</i> (L33P), <i>AGT</i> (M235T), and <i>MTR</i> (A2756G) loci. Certain mutations included in this assay such as ins242G, ins397G, E387K, L393K in the <i>LDLR</i>; N291S in the <i>LPL</i>; D442G in the <i>CETP</i>; and T833C in the <i>CBS</i> genes were found to be absent. The genotype results obtained using this assay showed 100% concordance with sequencing.</p> <p><b>Conclusion:</b> The study demonstrated development and validation of a multiplex SNP genotyping assay that can be used to assess genetic risk factors in CAD. The assay provides a cost-effective alternative to expensive high throughput genotyping systems in common molecular research laboratories.</p