title: Risk of Misdiagnosis Due to Allele Dropout and False-Positive PCR Artifacts in Molecular Diagnostics: Analysis of 30,769 Genotypes.
abstract: Quality control is a complex issue for clinical molecular diagnostic applications. In the case of genotyping assays, artifacts such as allele dropout represent a risk of misdiagnosis for amplification-based methods. However, its frequency of occurrence in PCR-based diagnostic assays remains unknown. To maximize the likelihood of detecting allele dropout, our clinical genotyping PCR-based assays are designed with two independent assays for each allele (nonoverlapping primers on each DNA strand). To estimate the incidence of allelic dropout, we took advantage of the capacity of our clinical assays to detect such events. We retrospectively studied their occurrence in the initial PCR assay for 30,769 patient reports for mutations involved in four diseases produced over 8 years. Ninety-three allele dropout events were detected and all were solved before reporting. In addition, 42 cases of artifacts caused by amplification of an allele ultimately confirmed to not be part of the genotype (drop-in events) were detected and solved. These artifacts affected 1:227 genotypes, 94% of which were due to nonreproducible PCR failures rather than sequence variants interfering with the assay, suggesting that careful primer design cannot prevent most of these errors. This provides a quantitative estimate for clinical laboratories to take this phenomenon into account in quality management and to favor assay designs that can detect (and minimize) occurrence of these artifacts in routine clinical use.
authors.affiliation: Service of Medical Biochemistry, Department of Medical Biology, CHU de Québec, Quebec City, Quebec, Canada; Human and Molecular Genetics Research Unit, Research Center, CHU de Québec, Quebec City, Quebec, Canada; Department of Molecular Biology, Medical Biochemistry, and Pathology, Faculty of Medicine, Université Laval, Quebec City, Quebec, Canada.
authors.forename: Jonatan
authors.initials: J
authors.lastname: Blais
authors.affiliation: Service of Medical Biochemistry, Department of Medical Biology, CHU de Québec, Quebec City, Quebec, Canada.
authors.forename: Sébastien B
authors.initials: SB
authors.lastname: Lavoie
authors.affiliation: Human and Molecular Genetics Research Unit, Research Center, CHU de Québec, Quebec City, Quebec, Canada.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Giroux
authors.affiliation: Human and Molecular Genetics Research Unit, Research Center, CHU de Québec, Quebec City, Quebec, Canada.
authors.forename: Johanne
authors.initials: J
authors.lastname: Bussières
authors.affiliation: Human and Molecular Genetics Research Unit, Research Center, CHU de Québec, Quebec City, Quebec, Canada.
authors.forename: Carmen
authors.initials: C
authors.lastname: Lindsay
authors.affiliation: Service of Medical Biochemistry, Department of Medical Biology, CHU de Québec, Quebec City, Quebec, Canada.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Dionne
authors.affiliation: Service of Medical Biochemistry, Department of Medical Biology, CHU de Québec, Quebec City, Quebec, Canada.
authors.forename: Mélissa
authors.initials: M
authors.lastname: Laroche
authors.affiliation: Service of Medical Biochemistry, Department of Medical Biology, CHU de Québec, Quebec City, Quebec, Canada; Department of Molecular Biology, Medical Biochemistry, and Pathology, Faculty of Medicine, Université Laval, Quebec City, Quebec, Canada; APOGEE-Net/CanGèneTest Research and Knowledge Network on Genetic Health Services and Policy, Quebec City, Quebec, Canada.
authors.forename: Yves
authors.initials: Y
authors.lastname: Giguère
authors.affiliation: Service of Medical Biochemistry, Department of Medical Biology, CHU de Québec, Quebec City, Quebec, Canada; Human and Molecular Genetics Research Unit, Research Center, CHU de Québec, Quebec City, Quebec, Canada; Department of Molecular Biology, Medical Biochemistry, and Pathology, Faculty of Medicine, Université Laval, Quebec City, Quebec, Canada; APOGEE-Net/CanGèneTest Research and Knowledge Network on Genetic Health Services and Policy, Quebec City, Quebec, Canada. Electronic address: francois.rousseau@mac.com.
authors.forename: François
authors.initials: F
authors.lastname: Rousseau
mesh_terms: D000483:Alleles; D016477:Artifacts; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D003951:Diagnostic Errors / Q000706:statistics & numerical data; D005189:False Positive Reactions; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods; D006801:Humans; D019656:Loss of Heterozygosity; D025202:Molecular Diagnostic Techniques / Q000379:methods* / Q000592:standards; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D020641:Polymorphism, Single Nucleotide; D012307:Risk Factors
chemical_list: C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator