Risk of misdiagnosis due to allele dropout in molecular diagnostics: analysis of 30769 genotypes.
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Jonatan Blais1,2,3, Sébastien Lavoie1, Sylvie Giroux2, Johanne Bussières2, Carmen Lindsay2, Jacqueline Dionne1, Mélissa Laroche1, Yves Giguère1,3,4, François Rousseau1,2,3,41 Service de biochimie, Dépt. Biologie médicale, CHU de Québec, Québec, Canada 2 Unité de recherche en génétique humaine et moléculaire, Centre de recherche du CHU de Québec, Québec, Canada 3 Département de biologie moléculaire, biochimie médicale et pathologie, Faculté de Médecine, Université Laval, Québec, Canada 4 APOGEE-Net/CanGèneTest Research and Knowledge Network on Genetic Health Services and Policy, Canadian Institutes of Health Research
Allele-specific oligonucleotide (ASO) PCR assays
Two independent sets of primers hybridizing on opposite strands of target DNA for each mutation, maximizing the likelihood of allele dropout detection
Forward and reverse target ASO PCR genotyping assay
Incidence of allele dropout & drop-in estimated from number of discordant results between forward and reverse assays
Alleles initially detected in one assay but shown to be false positives in confirmatory testing were also detected and referred to as "allele drop-in"
Detection by SYBR Green 1 end point fluorescence, measured on a Fluoroskan Ascent (MTX Laboratory Syst.)
Raw fluorescence intensity for the forward and reverse assay
One patient with discordant genotype between the forward and reverse assay (heterozygous and mutant homozygous respectively)
Error rate (%) ± 95% CI for each of the 16 PCR assays
No significant difference between forward and reverse assays across loci (Wilcoxon matched pairs test: p=0.48)
Significant differences among assay targets (Fisher’s exact test: p < 0.0001, p-value estimated from Monte-Carlo simulation of 10000 replicates in R version 2.15.1.)