Validation of a Large Custom-Designed Pharmacogenomics Panel on an Array Genotyping Platform.

BACKGROUND: Pharmacogenomics has the potential to improve patient outcomes through predicting drug response. We designed and evaluated the analytical performance of a custom OpenArray® pharmacogenomics panel targeting 478 single-nucleotide variants (SNVs). METHODS: Forty Coriell Institute cell line (CCL) DNA samples and DNA isolated from 28 whole-blood samples were used for accuracy evaluation. Genotyping calls were compared to at least 1 reference method: next-generation sequencing, Sequenom MassARRAY®, or Sanger sequencing. For precision evaluation, 23 CCL samples were analyzed 3 times and reproducibility of the assays was assessed. For sensitivity evaluation, 6 CCL samples and 5 whole-blood DNA samples were analyzed at DNA concentrations of 10 ng/µL and 50 ng/µL, and their reproducibility and genotyping call rates were compared. RESULTS: For 443 variants, all samples assayed had concordant calls with at least 1 reference genotype and also demonstrated reproducibility. However, 6 of these 443 variants showed an unsatisfactory performance, such as low PCR amplification or insufficient separation of genotypes in scatter plots. Call rates were comparable between 50 ng/µL DNA (99.6%) and 10 ng/µL (99.2%). Use of 10 ng/µL DNA resulted in an incorrect call for a single sample for a single variant. Thus, as recommended by the manufacturer, 50 ng/µL is the preferred concentration for patient genotyping. CONCLUSIONS: We evaluated a custom-designed pharmacogenomics panel and found that it reliably interrogated 437 variants. Clinically actionable results from selected variants on this panel are currently used in clinical studies employing pharmacogenomics for clinical decision-making.

Enhanced Carrier Screening for Spinal Muscular Atrophy: Detection of Silent (SMN1: 2 + 0) Carriers Utilizing a Novel TaqMan Genotyping Method.

BACKGROUND: Individuals whose copies of the survival motor neuron 1 (SMN1) gene exist on the same chromosome are considered silent carriers for spinal muscular atrophy (SMA). Conventional screening for SMA only determines SMN1 copy number without any information regarding how those copies are arranged. A single nucleotide variant (SNV) rs143838139 is highly linked with the silent carrier genotype, so testing for this SNV can more accurately assess risk to a patient of having an affected child. METHODS: Using a custom-designed SNV-specific Taqman genotyping assay, we determined and validated a model for silent-carrier detection in the laboratory. RESULTS: An initial cohort of 21 pilot specimens demonstrated results that were 100% concordant with a reference laboratory method; this cohort was utilized to define the reportable range. An additional 177 specimens were utilized for a broader evaluation of clinical validity and reproducibility. Allelic-discrimination analysis demonstrated tight clustering of genotype groupings and excellent reproducibility, with a coefficient of variation for all genotypes ranging from 1% to 4%. CONCLUSION: The custom-developed Taqman SNV genotyping assay we tested provides a rapid, accurate, and cost-effective method for routine SMA silent-carrier screening and considerably improves detection rates of residual risk for SMA carriers.