Frequency of Triple Mutations Involving Factor V,Prothrombin,and Methylenetetrahydrofolate Reductase Genes Among Patients Referred for Molecular Thrombophilia Workup in a Tertiary Care Center in Lebanon

Abstract

Aim: Molecular diagnostics has markedly improved the diagnosis and workup of different clinical conditions including hypercoagulable state or thrombophilia where different genes are involved. In this report, which is the largest report in the medical literature and the first in Lebanon, we describe the prevalence of simultaneous mutations in the three major thrombophilia genes Factor V, Factor II, and methylenetetrahydrofolate reductase. Materials and Methods: Using a polymerase chain reaction and reverse hybridization assay for the corresponding mutations identification, 2248 referred cases were analyzed. Results: Only 25 cases were found to be simultaneously positive for the three mutations at a prevalence rate of 1.1%. Conclusion: Compared with other populations, this prevalence rate is considered high, possibly the highest, and warrants future clinical studies and follow-up.

Introduction

Factor V (F5) or proaccelerin labile factor, Factor II (F2) or Prothrombin, and methylenetetrahydrofolate reductase (MTHFR) are essential components of the blood coagulation system. The genes encoding for F5, F2, and MTHFR are located on chromosomes 1q23, 11p11-q12, and 1p36.3, respectively (Botto and Yang, 2000; Duga et al., 2004; Jayandharan et al., 2005), and mutations affecting these genes, including the F5 G1691A mutation (known as Factor V Leiden: FVL), the F2 G20210A mutation, and the MTHFR C677T mutation, increase the incidence rate of thrombophilias (Sabbagh et al., 2008; Caramaschi et al., 2010; Severinsen et al., 2010). Patients with one or more of these mutations may suffer from venous thromboembolism including deep vein thrombosis (DVT) and pulmonary embolism (PE) (Otrock et al., 2008) or from adverse pregnancy outcomes including recurrent abortions, severe preeclampsia, premature delivery, and intrauterine growth restriction (Zahed et al., 2006).

The frequency of FVL, F2 G20210A, and MTHFR C677T mutations differs between various racial and ethnic groups (Mozafari et al., 2009). FVL mutation is detected in about 5% of Caucasians and 1% of Africans and Asians, and it is higher among Eastern Mediterraneans. F2 G20210A mutation prevalence ranges between 1% and 3% among European Caucasians but is rare in other ethnicities. Finally, the prevalence of MTHFR C667T mutation ranges between 5% and 15% among different populations (Kfoury et al., 2009). In Lebanon, the prevalence of FVL was found to be 14% among healthy individuals and 40% among patients with DVT, and prothrombin G20210A was found in 3% of healthy individuals and 12.5% in DVT patients (Taher et al., 2001). The carrier frequency of the C677T mutation of the MTHFR gene has been reported to be in the range of 10%-34% (Taher et al., 2001). Heterozygosis for FVL mutation increases the risk of venous thrombosis 5-10 times, whereas homozygosis for the same mutation increases the risk 80- to 100-fold (Arslan et al., 2011). Individuals with these combined genetic defects are at higher risk of thrombosis than those with a single gene mutation (Zoller et al., 1998). Double heterozygosity for FVL and G20210A is the most common combination (Makris et al., 1997; Ehrenforth et al., 1998) and has been reported to confer an increased risk of thrombosis, presumably because of a synergistic effect between these two mutations (Margaglione et al., 1999). Unlike double heterozygosity, triple mutations involving these three genes are not frequently seen. A study performed in Algeria among 172 unrelated individuals (Bourouba et al., 2009) and another performed among 147 healthy participants in Ukraine (Tatarskyy et al., 2010) revealed a 0% frequency of triple mutation.

F5, F2, and MTHFR gene single-nucleotide polymorphisms can be detected using several methods including the FV-PTH-MTHFR StripAssay (ViennaLab) that uses reverse hybridization. It is an easy, reliable, and affordable technique that involves three relatively simple steps: DNA isolation, PCR amplification using biotinylated primers, and hybridization of amplification products to a test strip containing allele-specific oligonucleotide probes (Spiroski et al., 2008; Sabbagh et al., 2009).

In this study, we assessed the frequency of triple mutations among 2248 Lebanese patients referred for molecular thrombophilia workup at a major tertiary care center.

Materials and Methods

Samples and DNA extraction

The results of 2248 cases referred to the American University of Beirut Medical Center, a major tertiary care center, were analyzed. The patients, and based on the specialty of the referring physicians, were mainly referred for workup of recurrent abortions, DVT, stroke, PE, and catheter thrombosis between January 2003 and February 2011. DNA extraction was done using the PEL-FREEZ extraction kit (PEL-FREEZ; DYNAL) and genomic material stored at −80°C for later use.

PCR and the Vienna Lab FV-PTH-MTHFR StripAssay

To test for the various genotypic profiles of the F5, prothrombin, and methylenetetrahydrofolate genes, the FV-PTH-MTHFR StripAssay (ViennaLab) was used and the manufacturer's protocol was followed as recommended. This assay screens for the G1691A, G20210A, and C677T mutations of the F5, prothrombin, and MTHFR genes, respectively, whereby in vitro, the different gene sequences are simultaneously amplified and biotin labeled in a single amplification reaction (Multiplexing). Briefly, 5 μL of DNA is added to 15 μL of already prepared PCR amplification mix in the presence of 5 μL of 0.2 U/μL Taq polymerase enzyme (AmpliTaq; Perkin Elmer). The thermocycler (Px2; ThermoHYBAID) program consists of an initial step of 94°C for 2 min, followed by 30 cycles of 94°C for 15 s, 58°C for 30 s, and 72°C for 30 s, and a final extension step of 72°C for 3 min. Finally, the amplification products are selectively hybridized to a test strip that contains allele-specific oligonucleotide probes immobilized as an array of parallel lines. Bound biotinylated sequences are detected using streptavidin-alkaline phosphatase and color substrates.

The results were interpreted as heterozygous, homozygous, or normal based on the probing pattern on the assays.

Results

Of the 2248 referred patients, 25 (1.11%) had F5, F2, and MTHFR gene mutations simultaneously. As per Table 1, and as deduced from the referral diagnosis stated by the physician, 13 patients out of the 25 had DVT, 5 had recurrent abortions, 4 had stroke, 2 patients experienced catheter thrombosis, and 1 patient had both DVT and PE.

Table 1.

Genotype of the 25 Referred Cases Showing Triple Mutations in the Factor V, Factor II, and MTHFR Genes

Referral Diagnosis	Number of patients	Factor V	Factor II	MTHFR
DVT	7	Heterozygous	Heterozygous	Heterozygous
	4	Heterozygous	Heterozygous	Homozygous
	1	Homozygous	Heterozygous	Heterozygous
	1	Homozygous	Heterozygous	Homozygous
RA	2	Heterozygous	Heterozygous	Heterozygous
	2	Heterozygous	Heterozygous	Homozygous
	1	Homozygous	Heterozygous	Homozygous
Stroke	2	Heterozygous	Heterozygous	Homozygous
	1	Homozygous	Heterozygous	Homozygous
	1	Heterozygous	Heterozygous	Heterozygous
CT	2	Heterozygous	Heterozygous	Heterozygous
DVT and PE	1	Heterozygous	Heterozygous	Heterozygous

DVT, deep vein thrombosis; RA, recurrent abortions; CT, catheter thrombosis; PE, pulmonary embolism.

Discussion

Genetic testing for thrombophilia is gaining more interest among different medical and surgical fields and has even been introduced into algorithms for management of DVT, stroke, recurrent abortions, and pulmonary embolization (Emmerich et al., 2001; Said et al., 2010). Even though the associated gene mutations only increase the risk of a thrombotic episode, most physicians and surgeons are currently resorting to genetic profiling of their referred patients in addition to serum or plasma coagulation studies.

The most common mutations studied for genetic thrombophilia workup are the G1691A, G20210A, and C677T for the F5, F2, and MTHFR genes, respectively (Ehrenforth et al., 1999). These mutations are associated with various conditions, for instance, in patients undergoing renal transplantation the risk of thrombosis and mortality is higher because of FVL mutation (Rahimi et al., 2011a). Moreover, a correlation exists between FVL mutation and sickle-cell anemia (Rahimi et al., 2011b). In addition, it is well known that the more the number of mutations in the previously mentioned genes the more the patient is at a higher risk for development of a thrombotic episode (Simioni et al., 2000).

In Lebanon, previous studies have shown high prevalence of mutations in this population; however, this is the first report that describes the prevalence of simultaneous mutations among referred cases in Lebanon. This is actually also the single report in the medical literature involving such a high number of referrals. Importantly, the Lebanese population (even though not referring here to the general healthy population) also seems to harbor a higher, probably the highest, prevalence of F5, F2, and MTHFR gene mutations occurring together in the same individual. It would be interesting to study the prevalence of triple mutations in the general healthy Lebanese population for completion of the comparison.

With this presumably high prevalence rate of triple mutations, the Lebanese community again shows a more peculiar and specific finding and may label the Lebanese people as “more prone” for thrombotic disorders. We highly advocate for clinical studies to be performed at a larger scale in Lebanon (as well as other countries) for better correlation with the genetic findings based on this and other national and international studies.

Footnotes

Disclosure Statement

No competing financial interests exist.

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