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. |