title: An in-house multilocus SNP genotyping assay for evaluation of complex genetic diseases.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  The assay could successfully discriminate both the alleles at CETP (I405V), LPL (D9N), NOS3 (T-786G and E298D), LIPC (C-514T), FGB (G-455A), ITGB3 (L33P), AGT (M235T), and MTR (A2756G) loci. Certain mutations included in this assay such as ins242G, ins397G, E387K, L393K in the LDLR; N291S in the LPL; D442G in the CETP; and T833C in the CBS genes were found to be absent. The genotype results obtained using this assay showed 100% concordance with sequencing.
  
  
  CONCLUSIONS
  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.
authors: a Research Laboratories , P. D. Hinduja National Hospital and Medical Research Centre , Mumbai , India.
authors: Swarup A V
authors: SA
authors: Shah
authors: a Research Laboratories , P. D. Hinduja National Hospital and Medical Research Centre , Mumbai , India.
authors: Altaf A
authors: AA
authors: Kondkar
authors: a Research Laboratories , P. D. Hinduja National Hospital and Medical Research Centre , Mumbai , India.|b Department of Biochemistry , P. D. Hinduja National Hospital and Medical Research Centre , Mumbai , India.
authors: Tester F
authors: TF
authors: Ashavaid
mesh_terms: D000483:Alleles; D001798:Blood Proteins / Q000235:genetics* / Q000378:metabolism; D016022:Case-Control Studies; D003324:Coronary Artery Disease / Q000097:blood / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology; D056426:Genetic Loci; D020022:Genetic Predisposition to Disease; D060005:Genotyping Techniques; D006801:Humans; D006867:Hydrolases / Q000097:blood / Q000235:genetics*; D058885:Multilocus Sequence Typing / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D015345:Oligonucleotide Probes / Q000138:chemical synthesis / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D012307:Risk Factors; D017422:Sequence Analysis, DNA
chemical_list: D001798:Blood Proteins; D015345:Oligonucleotide Probes; D006867:Hydrolases
keywords: Genetic predisposition; genetic testing; multifactorial inheritance; multiplex PCR; reverse line blot hybridization