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Mutation in the gene coding for coagulation factor V and resistance to activated protein C: detection of the genetic
R B Zotz1, B Maruhn-Debowski, R E Scharf
1Department of Hemostasis and Transfusion Medicine, Heinrich Heine University Medical Center, Düsseldorf, Germany.
Abstract:
Resistance of coagulation factor Va to inactivation by activated protein C (APCR) is associated with a point mutation in which adenine is substituted for guanine at nucleotide 1691 in the gene coding for factor V (FV Leiden). To date, this mutation of factor V is the most frequent genetic risk factor for venous thrombophilia. In this report, we describe the adaptation of an automatable oligonucleotide ligation assay (OLA) to detect the mutation in polymerase chain reaction-amplified DNA samples from 40 normal, 20 affected heterozygous, and 3 affected homozygous individuals. The genotypes determined by conventional allele-specific restriction enzyme site analysis were in complete concordance with the results obtained by ELISA-based oligonucleotide-ligation assay. The automated oligonucleotide ligation assay provides a rapid, reliable, nonisotopic method to detect the mutation responsible for APCR that can rapidly be applied to large population screening.
Insights
A common genetic mutation (FV Leiden) causes resistance to activated protein C (APCR), a major risk factor for venous thrombophilia. An automated oligonucleotide ligation assay reliably detects this mutation for large-scale screening.
Area of Science:
- Genetics
- Molecular Biology
- Hematology
Background:
- Activated protein C resistance (APCR) is a significant risk factor for venous thrombophilia.
- The most common genetic cause of APCR is the FV Leiden mutation, a specific point mutation in the factor V gene.
- Identifying this mutation is crucial for understanding thrombotic risk.
Purpose of the Study:
- To adapt and validate an automatable oligonucleotide ligation assay (OLA) for detecting the FV Leiden mutation.
- To assess the reliability and efficiency of the OLA method compared to conventional techniques.
- To establish a rapid, nonisotopic method for large population screening of the FV Leiden mutation.
Main Methods:
- Polymerase chain reaction (PCR) amplification of DNA samples.
- Adaptation of an oligonucleotide ligation assay (OLA) for mutation detection.
- Comparison of OLA results with allele-specific restriction enzyme analysis and ELISA-based OLA.
Main Results:
- The automated OLA successfully detected the FV Leiden mutation in all tested individuals (normal, heterozygous, and homozygous).
- Results from the OLA showed complete concordance with conventional allele-specific restriction enzyme site analysis.
- The ELISA-based OLA provided reliable genotype determination.
Conclusions:
- The automated OLA is a rapid, reliable, and nonisotopic method for detecting the FV Leiden mutation.
- This assay is suitable for large-scale population screening to identify individuals at risk for venous thrombophilia.
- The OLA offers a practical approach for genetic testing related to thrombotic disorders.