Received on July 8, 2008
Accepted on January 27, 2009

Molecular Diagnostics and Genetics

European External Quality Control Study on the Competence of Laboratories to Recognize Rare Sequence Variants Resulting in Unusual Genotyping Results

¹ European Commission, Joint Research Centre, Institute for Reference Materials and Measurements, Geel, Belgium, and Department of Biochemistry, Faculty of Medicine, University of Szeged, Szeged, Hungary
² European Commission, Joint Research Centre, Institute for Reference Materials and Measurements, Geel, Belgium, and European Commission, Joint Research Centre, Institute for Health and Consumer Protection, Ispra, Italy
³ European Commission, Joint Research Centre, Institute for Reference Materials and Measurements, Geel, Belgium, and European Commission, Directorate-General for Research, Health Biotechnology, Brussels, Belgium
⁴ European Commission, Joint Research Centre, Institute for Reference Materials and Measurements, Geel, Belgium
⁵ Department of Biochemistry, Faculty of Medicine, University of Szeged, Szeged, Hungary, and QualiCont Kht., Szeged, Hungary

^* To whom correspondence should be addressed. E-mail: Heinz.Schimmel{at}ec.europa.eu.

BACKGROUND: Depending on the method used, rare sequence variants adjacent to the single nucleotide polymorphism (SNP) of interest may cause unusual or erroneous genotyping results. Because such rare variants are known for many genes commonly tested in diagnostic laboratories, we organized a proficiency study to assess their influence on the accuracy of reported laboratory results.

METHODS: Four external quality control materials were processed and sent to 283 laboratories through 3 EQA organizers for analysis of the prothrombin 20210G>A mutation. Two of these quality control materials contained sequence variants introduced by site-directed mutagenesis.

RESULTS: One hundred eighty-nine laboratories participated in the study. When samples gave a usual result with the method applied, the error rate was 5.1%. Detailed analysis showed that more than 70% of the failures were reported from only 9 laboratories. Allele-specific amplification–based PCR had a much higher error rate than other methods (18.3% vs 2.9%). The variants 20209C>T and [20175T>G; 20179_20180delAC] resulted in unusual genotyping results in 67 and 85 laboratories, respectively. Eighty-three (54.6%) of these unusual results were not recognized, 32 (21.1%) were attributed to technical issues, and only 37 (24.3%) were recognized as another sequence variant.

CONCLUSIONS: Our findings revealed that some of the participating laboratories were not able to recognize and correctly interpret unusual genotyping results caused by rare SNPs. Our study indicates that the majority of the failures could be avoided by improved training and careful selection and validation of the methods applied.