New in-house molecular diagnostic tests for thrombosis

Published on : 28 September 2016

As part of our continuous efforts to improve the quality of our services and provide our clients results with quicker turnaround time, we have recently expanded our growing in-house tests menu with several tests and panels for thrombosis, as one of the most common causes of morbidity and mortality in developed societies. The tests are performed at our flagship Molecular Diagnostics Laboratory in the frames of our main testing site in ICAD, Abu Dhabi. 

Please see more information below.

 

Order Code

Order Code Name

CPT Code

Specimen type

Volume

Specimen container

Storage

TAT days

512103

Thrombosis SNP Panel Assay

Test Includes: Factor V Leiden Mutation, MTHFR, Prothrombin II Mutation

81240 81241 81291

Whole blood

Specimens should be tested within 72 hours of collection

 

3.0 ml

Minimum: 1.0  ml

 

Lavender-top (EDTA) tube

Refrigerate

5-7 days

 

511154

 

Factor V Leiden Mutation

81241

Whole blood

Specimens should be tested within 72 hours of collection

3.0 ml

Minimum: 1.0  ml

Lavender-top (EDTA) tube EDTA tube

 

 

 

 

Refrigerate

5-7 days

511238

Methylenetetrahydrofolate Reductase (MTHFR) 2 Variants

81291

Whole blood

Specimens should be tested within 72 hours of collection

3.0 ml

Minimum: 1.0  ml

Lavender-top (EDTA) tube EDTA tube

Refrigerate

5-7 days

511162

Prothrombin (Factor II) Mutation

81240

Whole blood

Specimens should be tested within 72 hours of collection

3.0 ml

Minimum: 1.0  ml

Lavender-top (EDTA) tube EDTA tube

Refrigerate

5-7 days

Testing Frequency: Sunday and Tuesday

Analyzer: Bio Rad CFX96TM REAL-TIME PCR

Methodology: Multiplex Real Time PCR

 

Clinical Significance

 

1. Thrombosis

Thrombosis represents one of the most common causes of morbidity and mortality in western societies. Haemostatic disequilibrium is the key mechanism at the origin of all types of thrombosis. The pathogenesis of venous thromboembolism, a complex and multifactorial process, involves the interaction of acquired factors such as ageing, obesity, prolonged immobilization (bed rest, long travel, and fracture), surgery, pregnancy and post-partum, and oral contraceptives or cancer, with a series of genetic predisposing conditions. During recent decades, several alterations in the genetics of haemostatic factors increasing the risk of thrombosis have been described.

 

2. Factor V Leiden (R506Q)

Since its discovery in 1994, the FV Leiden (FVL) mutation has been demonstrated to represent the most frequent cause of activated protein C (APC) resistance. In healthy individuals of Caucasian origin, the prevalence of FVL is between 2 and 10%, making the FVL mutation at least ten times more common than other genetic defects that cause thrombosis. The highest prevalence of the mutation is found in Europe, most notably in Cyprus, Southern Sweden and Germany, but the mutation is also very common in Saudi Arabia and in Arab and Jewish populations of Israel. Although widely present in Caucasians, the FVL mutation is not found in other ethnic groups such as in individuals of African, Chinese or Japanese origin. Haplotyping of FVL homozygotes suggested a founder effect and that a single mutational event occurred approximately 21,000 years ago. Among patients with venous thromboembolism, FVL occurs in 20% of all cases and in up to 50% of selected patients with thrombophilia. The FVL mutation seems to be a risk factor of much the same strength as the deficiencies of coagulation inhibitors, increasing the risk about five-fold in heterozygous carriers. Homozygous carriers of the mutation are considered to have a 50- to 100-fold increased risk of thrombosis. The most common clinical manifestations of APC resistance caused by the FVL mutation are venous thrombosis and pulmonary embolism. It is generally accepted that there is a much less clear link between the mutation and arterial thrombosis.

 

2. Factor II G20210A

Prothrombin (Factor II, FII) is an important component of the coagulation cascade. It is a vitamin K-dependent protein that participates in coagulation and its regulation. Prothrombin participates in the final stages of the blood coagulation cascade where it is converted to thrombin in the presence of factor Xa, factor Va, calcium ions, and phospholipids. A few years after identification of APC resistance and the causative FVL mutation, the second most frequent, and important inherited risk factor for thrombosis, was identified. The sequence alteration c.20210G>A is located in the 39-untranslated region of the FII and is associated with slightly increased plasma prothrombin concentrations. The c.20210G>A mutation in FII represents a gain-of-function mutation, causing increased recognition of the cleavage site, increased 39 end processing, and increased accumulation of mRNA and protein synthesis. These changes can result in a hypercoagulable state. The relative risk for venous thrombosis associated with the mutation is two to four fold for heterozygotes.

Homozygosity for the c.20210G>A mutation is rare, but it increases the risk of thrombosis above that which has been observed for heterozygotes. The prevalence of the mutation varies and is dependent on geographic location and ethnic origin. It is found in 0-4% of the general population. The prevalence of the mutation in Southern Europe is twice as high as in northern Europe. It is rare in Asian and African descendants, and in native Australians and Americans. The mutation is also found in 6-8% of patients with venous thromboembolism (VTE). It was found that 6-12% of patients with VTE who were heterozygous for FVL also had the FII mutation c.20210G>A (compound heterozygotes). The relative risk for venous thrombosis in the individuals carrying both mutations are higher than in individuals without either mutation.

 

3. MTHFR C677T

Methylenetetrahydrofolate reductase (MTHFR) is the enzyme that catalyses the transformation of homocysteine to methionine via the remethylation pathway (gene located in 1p36). Hyperhomocysteinemia (HHC), a known prothrombotic condition, is the consequence of decreased activity of MTHFR. Interestingly, both genetic and acquired (vitamins B-6, B-12 or folic acid deficiencies) factors may lower the activity of MTHFR. The C677T mutation, causing an amino acid change from alanine to valine and rendering the enzyme thermolabile, and about half as efficient, is the most common genetic cause of HHC. This polymorphism is only associated with elevated homocysteine levels in cases of low folate intake. Conflicting results have been reported regarding the prothrombotic role of the 677T variant, which in fact seems far less pronounced than that of FVL or FII G20210A. The prevalence varies widely according to populations (allelic frequency: 0.06-0.59; homozygosity frequency: 0-0.35) and seems closely related to folic acid food content. In Europe (and also in Asia), there is a North to South gradient with a very high prevalence among Mediterranean countries. For instance, in Spain, the 677T allele frequency is higher than its wild counterpart. In addition, a certain level of microheterogeneity is seen in some populations, e.g., between northern and southern parts of Italy (allele frequencies being 0.448 and 0.556, respectively). On the other hand, in Africans from the Sub-Saharan zone, this frequency is among the lowest in the world, due perhaps to undernutrition and infectious diseases that diminish the intestinal absorption of folic acid.

 

4. MTHFR A1298C

A second variant of the MTHFR enzyme, with a substitution of A to C at nucleotide 1298, has also been identified. Unlike the MTHFR C677T polymorphism, the enzyme activities of the variants of MTHFR A1298C polymorphism are not thermolabile but the enzyme activity is reduced by approximately 40% of the wild type (AA genotype) in the variant genotype. Altered homocysteine levels have not been found in individuals with these variants. The prevalence of the 1298CC genotype varies, with the homozygous genotype found in 7-12% of Caucasians, in Europeans, 4-12%, while in China, Japan, and Hawaiian studies the prevalence ranged between 1 and 4%