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Detection of Factor V Leiden (G1691A), Factor II (G20210A) & MTHFR (C677T) using Pyrosequencing™

Background

Several genetic defects have been identified which have been shown to be associated with an increased risk of developing thrombosis. The most frequent inherited risk factor is the R506Q (G1691A) mutation in the factor V gene (Factor V Leiden). This mutation is found in the Caucasian population with an allele frequency ranging from 1 - 2% up to 15% in certain regions. The mutation conveys resistance to cleavage of factor V by activated protein C. Activated protein C resistance is found in approximately 20 - 60% of thrombophilia patients and, of these, more than 90% are caused by the Factor V Leiden mutation. Individuals who are heterozygous for the Factor V Leiden mutation are estimated to have a 6 - 8 fold greater risk of thromboembolic disease. Homozygotes have a 50 - 80 fold increased risk.

The second most common cause of familial thrombophilia is a mutation in the untranslated region of the prothrombin gene (G20210A). This has a prevalence of 1 - 2% in the population (and is found in 6 - 18% of patients with venous thrombosis). The mutation is associated with elevated levels of factor II activity and antigen and carries a 2 - 3 fold increased risk of deep vein thrombosis.

Mild hyperhomocysteinaemia has been identified as a risk factor for arterial disease and for venous thrombosis. Individuals homozygous for the methylene tetrahydrofolate reductase (MTHFR) gene C677T polymorphism have reduced enzyme activity leading to significantly elevated plasma homocysteine levels. Prevalence of the homozygous genotype ranges from 5 - 15% in the European and North American populations.

Methodology and Assay Design:

In this study Pyrosequencing was used to genotype samples for Factor V, Factor II and MTHFR mutations. 90 anonymised DNA samples were obtained from Dr L Lavender at the Molecular Pathology Unit, Southampton that had been previously genotyped using restriction enzyme digestion. These samples were re-tested using a multiplex assay developed by Pyrosequencing. Details of the Pyrosequencing assays can be found here.

Overall Conclusions:

SNP detection was 100% specific and sensitive compared to previously obtained genotypes.
5/90 samples had to be repeated to obtain all three genotypes using the triplex assay
The overall failure rate was 0 %.
The time taken to analyse 96 samples for the triplex assay was 3 hours and 30 minutes.
The cost per genotype for the triplex assay was £0.65/sample (excluding system costs and maintainance charges).
The PCR, PCR clean up and use of the Pyrosequencing system were undemanding
The software and data analysis were easy to use.

 


Last Updated: 7 August, 2008 by G. Watkins
 
© 2006 National Genetics Reference Laboratory (Wessex), Salisbury District Hospital, Salisbury SP2 8BJ; Tel: +44 (0)1722 429080; E-mail:ncpc@soton.ac.uk