Reference materials (RMs) for analysis of the human factor II (prothrombin) gene G20210A mutation

Abstract

The Scientific Committee of Molecular Biology Techniques (C-MBT) in Clinical Chemistry of the IFCC has initiated a joint project in co-operation with the European Commission, Joint Research Centre, Institute of Reference Materials and Measurements to develop and produce plasmid-type reference materials (RMs) for the analysis of the human prothrombin gene G20210A mutation. Although DNA tests have a high impact on clinical decision-making and the number of tests performed in diagnostic laboratories is high, issues of quality and quality assurance exist, and currently only a few RMs for clinical genetic testing are available. A gene fragment chosen was produced that spans all primer annealing sites published to date. Both the wild-type and mutant alleles of this gene fragment were cloned into a pUC18 plasmid and two plasmid RMs were produced. In addition, a mixture of both plasmids was produced to mimic the heterozygous genotype. The present study describes the performance of these reference materials in a commutability study, in which they were tested by nine different methods in 13 expert laboratories. This series of plasmid RMs are, to the best of our knowledge, the first plasmid-type clinical genetic RMs introduced worldwide.

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

“…Recently, in a collaborative effort, the Scientific Committee of Molecular Biology Techniques in Clinical Chemistry of the IFCC and the IRMM have developed a set of 3 plasmid-type certified reference materials (CRMs) for the analysis of the 20210GϾA (G20210A) mutation in F2, the coagulation factor II (prothrombin) gene (15)(16)(17)(18)(19). These plasmids contain a wild-type or G20210A mutant prothrombin gene fragment that spans all primer annealing sites published to date.…”

“…Development and processing of the wild-type and G20210A mutant plasmid CRMs has been reported (15 ). These plasmids include a well-characterized fragment of the human prothrombin gene (GenBank M17262; nucleotide position 26 302-26 910) comprising the SNP of interest and spanning all published primer annealing sites.…”

Preparing compound heterozygous reference material using gene synthesis technology: a model of thrombophilic mutations

“…In genotyping assays for rare allelic variants, heterozygous control materials are formed by mixing equal numbers of cells or DNA clones containing wild-type and mutant sequences. This approach enabled the development of certified RMs for G20210A mutation in the Factor II (FII) gene 6 and G1691A Leiden mutation in the Factor V (FV) gene 7 . Both these mutations are associated with a higher risk of venous thrombosis and appear in healthy people of European ancestry at frequencies of 1-2% (G20210A) and 5-6% (G1691A), respectively 8,9 .…”