Abstract
Background/Aim: Thrombophilia is a multifactorial predisposition for thromboembolism affecting about a tenth of any population. We investigated whether genetic counseling combined with molecular testing for two common dominant mutations (coagulation factor V Leiden and prothrombin G20210A) may increase prevention of venous thromboembolic incidents in individuals with a positive family history compared to the general population. Patients and Methods: Mutation detection was carried out by Restriction Fragment Length Polymorphism analysis in DNA samples of 96 unrelated healthy Greeks (group A) who asked for genetic counseling for various reasons and had at least two relatives with thromboembolic incidents and 100 unrelated healthy Greeks (group B). Results: In group A, both mutations were detected at five-fold higher frequencies (33.33% for Leiden and 19.79% for G20210A) compared to group B, which had frequencies typically found in the Greek population (6% and 4%, respectively). Conclusion: In populations with a high prevalence for these two common mutations, genetic counseling and molecular testing of at-risk individuals may significantly increase prevention of thromboembolic disease.
Thrombophilia (OMIM 188050) is a multifactorial predisposition for thrombosis, caused by inborn hypercoagulation of the blood and affecting 10-15% of individuals in any given population (1-3). Thromboembolic incidents include brain stroke, myocardial infarction, deep vein thrombosis and obstetrical complications, including about 60% of spontaneous abortions (3-10). The disorder may occur on a familial basis as a result of one or more mutant genes encoding any one of a considerable number of clotting, anticoagulant or thrombolytic factors functioning alone, or in association with other genetic and environmental factors (2-9). Genetic causes are present in approximately one third of unselected thrombosis cases and up to two thirds of familial cases (3).
One of the most common inherited defects causing thrombophilia in Europeans is the coagulation factor V Leiden mutation (G1691A) (11-13). The factor V gene encodes a plasma glycoprotein which is activated by thrombin/factor Xa and converted into factor Va. The mutation destroys a cleavage site of the anticoagulant-activated protein C in factor Va. Several studies have reported a high frequency of factor V Leiden mutation in Caucasians (1.5-8.8%), while in other populations, the mutation is rare (3, 11, 14-18). This mutation is responsible for 20-25% of isolated thrombotic events and for 40-45% of cases of familial thrombophilia and fetal loss (1, 4-6, 9, 10, 17, 19-23).
Another common defect, with an allelic frequency of 1.3-4.5% in Caucasian populations, is a guanine to adenine transition in the 3′ untranslated region of the prothrombin gene (G20210A) (3, 16, 24). Also known as coagulation factor II, prothrombin is a plasma glycoprotein which is activated to thrombin by factors Xa and Va. The G20210A mutation is related to elevated plasma prothrombin levels, and increased risk of venous thrombosis (20, 24, 25).
Accumulating evidence suggests that these two genetic alterations display a dominant predisposition effect (1, 3, 16). Heterozygotes with the factor V Leiden mutation have a 5 to 10-fold risk, and heterozygotes with the prothrombin G20210A mutation have a 2 to 4-fold risk of venous thrombotic incidents compared with individuals without these mutations (1, 3, 16). Homozygotes for one mutation or double heterozygotes have a 50 to 100-fold risk compared to normal population (1, 3, 16).
Genetic counseling may play an important role in the prevention of thrombophilia (25, 26). Evaluation of family data may reveal parameters helpful for risk calculation and the ability to routinely detect the inherited gene defects may significantly contribute to early diagnosis. Thromboembolic events are associated with significant risk of morbidity and mortality, therefore, when a person's genetic predisposition is known, preventive anticoagulant therapy may safeguard that individual from life-threatening incidents and improve their health status (3, 27).
Leiden and G20210A mutations are important susceptibility factors for thromboembolic incidents in certain populations, such as in Greeks, in whom high frequencies of 5% and 4.5%, respectively, have been detected (15, 17, 18, 28). Here, we report the molecular testing results for these two common thrombophilia mutations in two cohorts of healthy Greeks: a) index cases with a family history of idiopathic thrombosis, and b) individuals of the general population. Analysis of the presented data illustrates the significant impact of genetic counseling on prevention of thromboembolic incidents.
Patients and Methods
Participants. Blood samples were collected from two different groups of unrelated and apparently healthy individuals of Greek origin (N=196). All studied individuals were fully informed about the potential meaning of test results and willingly participated in the study.
Group A consisted of 96 unrelated healthy individuals (26-55 years old), who were referred for genetic counseling for various reasons (Table I), and their family pedigree analysis revealed that, besides their reason of reference, they had at least one first-degree and one second-degree relative with a thromboembolic incident (in brain, heart, lungs, deep veins etc.). Thrombophilia-related details of family history of these individuals are presented in Table I, while three characteristic pedigrees are presented in Figure 1. Regarding the etiology of reference for genetic counseling, there were seven defined categories: a) neurogenetic disorders (N=19), including cases of neurofibromatosis I, Charcot-Marie-Tooth I, Becker muscular dystrophy, Duchenne muscular dystrophy, myotonic dystrophy, and X-linked spastic paraplegia; b) syndromes (N=11), including cases of Alagille, CADASIL (cerebellar autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy), cerebro-oculo-facio-skeletal, Cowden, cri-du-chat, de Lange, Dubowitz, frontonasal dysplasia, Opitz-G, Saethre-Chotzen and Waardenburg syndromes; c) familial cancer (N=6); d) endocrinological problems (N=6); e) fertility problems (N=8); f) recurrent spontaneous abortion (N=8); g) familial idiopathic thrombosis (N=38).
Group B consisted of 100 unrelated healthy individuals of comparative sex and age (23-61 years old), who were tested in the frame-work of a routine biochemical, hematological and molecular check-up, without obtaining any information on their family history. There were no exclusion criteria for these individuals.This group served as a control group representing the general Greek population.
Molecular testing. DNA was extracted from blood with the use of NucleoSpin™ kit (Macherey-Nagel GmbH & Co, Dfiren, Germany). Molecular analysis was performed as previously described, with a combination of PCR and endonuclease Taq I digestion analysis for thrombophilia-causing mutations Leiden (G1691A) and G20210A in the genes of coagulation factors V and II (prothrombin), respectively (13, 18, 24). In both cases, a Taq I recognition site found in normal alleles is lost in PCR products containing a mutant allele. All molecular analyses were blindly performed twice.
Statistical analysis. Carrier frequencies for both mutations found in group A were compared to the respective frequencies of the control group B. Statistical analysis for comparisons between categorical variables was performed using the chi-square test and Fisher's exact test. All statistical differences were two-sided, and the significance level was set at p<0.05.
Results
In group A, mutant alleles were detected in 47 out of 96 individuals (48.96%), while they were found in 10 out of 100 individuals of group B (10%). More specifically, factor V Leiden was detected in 32 out of 96 individuals of group A (33.33% carrier frequency), while it was detected in 6 out of 100 individuals of the group B (6% carrier frequency). The prothrombin G20210A mutation was detected in 19 out of 96 individuals in group A (19.79% carrier frequency) and in 4 out of 100 individuals in group B (4% carrier frequency). Both mutations were detected in only four individuals of group A (Table I). A significant difference between the two groups was found for both mutations (χ2=9.36, p<0.005 for Leiden and χ2=6.03, p<0.025 for G20210A).
Further analysis was performed in two subgroups of group A, examining the frequency of both mutations compared to the general population. The first subgroup included 46 individuals whose referral for genetic counseling was possibly related to thrombophilia: 38 with a family history of thromboembolic episodes and 8 with recurrent spontaneous abortions. As one would expect, the prevalence of both mutations in this subgroup was much higher in comparison to those of the control group: 41.3% for factor V Leiden (χ2=30.17, p<0.001) and 32.6% for the prothrombin mutation (χ2=25.09, p<0.001).
In the remaining 50 cases of group A, with a referral reason irrelevant to thrombophilia, only the observed frequency for Leiden mutation was also statistically different compared to the control group: 26% (χ2=15.39, p<0.001). On the contrary, in this subgroup the detected frequency for G20210A (8%) was not significantly different from that observed for the control group (0.25<p<0.10).
Characteristic pedigrees of index cases of group A. An arrow marks studied index cases with their age besides them. An unknown predisposition for thrombosis at the time of family history taking is depicted by a question mark. a: Forty-year-old woman who was referred for genetic counseling because of three spontaneous abortions at first trimester (family 51); b: 35-year-old father of a child with a syndrome which was diagnosed as cri-du-chat (family 23); c: 33-year-old man with oligospermia (family 49). The latter two index cases were found to have the factor V Leiden mutation.
Discussion
Thrombophilia is a common multifactorial predisposition for thromboembolism, that may cause life-threatening cardiovascular disease, as well as serious complications after surgery (3, 8, 29-31). Several susceptibility genes for thrombophilia are known but very few of them have been shown to be singularly significant for thrombosis, such as the common mutated ones coding for coagulation factor V and prothrombin. It is clear that a combination of genetic variations along with other factors increases the risk for thromboembolic events (1-3, 8, 21). A characteristic and rather common example of such an effect of interaction is homocysteinemia due to homozygosity for 677C→T polymorphism in the methylenetrahydrofolate reductase gene in combination with limited dietary uptake of folic acid (3, 32, 33).
The fact remains that the factor V Leiden and the prothrombin G20210A dominant mutations are strong contributors to thrombosis since their combined frequencies are about 3.5-11% in Caucasian populations (16). This contribution seems to be most important in populations of South and East European decent such as Greeks, Italians, Argentineans and Polish, in which the distribution of the two mutations shows a nearly two-fold higher prevalence in comparison to Northern Europeans (15-18, 34). In such a population, in which the mutant allele frequencies of strong contributors to thrombophilia are high, genetic counseling may be very important for prevention of life-threatening thromboembolic incidents in individuals with an inherited predisposition for thrombosis (25-27). Molecular findings of thrombophilia susceptibility in individuals at risk may offer the possibility of early preventive anticoagulant therapy and prevention of serious thromboembolic complications.
In the present study, we investigated whether genetic counseling combined with molecular testing for two common thrombophilia-causing mutations could potentially contribute to increase prevention of disease in healthy individuals with a family history of idiopathic thrombosis, compared to random check-up of the general population. Molecular testing revealed that one third of the studied at risk individuals of group A had the factor V Leiden mutation and one fifth of them the G20210A mutation. The overall observed frequency of both mutations was 48.96%, corresponding surprisingly well to the expected respective risk of individuals who have a parent with an autosomal dominant disorder (50%). This overall frequency is five-fold greater than that of group B, which was found to be 10% (6% for factor V Leiden, and 4% for G20210A), corresponding roughly to the combined frequencies of both mutations (9.5-11%) previously observed in the Greek population (15, 17, 18, 23, 28).
Analytical presentation of individuals tested for thrombophilia-causing mutations (Group A): family history regarding thromboembolic events and molecular screening results. 1st, 2nd, 3rd, 4th: First, second, third and fourth degree of kinship relation. The numbers in these columns indicate the number of family members, of the respective relative degree, that presented with the observed problem. F: Female, M: male; FV: factor V Leiden mutation, PT: prothrombin G20210A mutation; m: presence of the mutation, −: absence of the mutation; THR-E: thromboembolic event; NF1: neurofibromatosis type 1; MD: muscular dystrophy; BS: brain stroke, MI: myocardial infarction, SA: spontaneous abortion, OTE: other thromboembolic events (deep venous thrombosis, pulmonary embolism); +: indicates death of that person due to the thromboembolic event.
All individuals from both groups who were found to be carriers of either mutation were referred to hematologists who advised some of them to receive preventive anticoagulant therapy. They were also advised to inform certain at-risk relatives about testing. Women in particular were counseled to a) avoid taking contraceptive pills without the advice of a hematologist, and b) consult a hematologist in addition to an obstetrician in case of pregnancy.
The optimal management of asymptomatic mutation carriers remains unclear, but it is generally agreed that thromboprophylaxis, at least during risk periods, should be provided (35-46). Asymptomatic close relatives of thrombotic patients who also carry a mutated gene may benefit from prophylactic treatment with anticoagulants, especially when their risk of thrombosis is increased by temporal factors, such as pregnancy or surgery (5, 39, 40, 43, 45, 46).
Prevention of idiopathic thrombosis is imperative, since it is very common and life-threatening. Genetic counseling is very important for people with a family history of thrombophilia (26). Geneticists may play a very significant role in prevention of this complex disorder if, during their routine collection of family history data in a counseling session, they recognize individuals at risk for thrombosis and inform them about preventive measures, including the available molecular tests (47). Obviously, during genetic counseling, it is important to address psychological issues in regard to the impact of predictive testing on the wellbeing of testees and their family, as in other late-onset hereditary diseases (48, 49). A number of studies have indicated that pre-test genetic counselling would be helpful in reducing confusion about thrombophilia facts and anxiety (26, 50, 51).
It seems imperative that both clinicians and patients obtain accurate information concerning appropriate use of genetic testing in order to achieve an acceptable cost/benefit ratio safeguarding the life and health status of individuals at risk. As genetic testing becomes a routine approach, it is expected that it will be extensively used both in hospital and community preventive medicine.
Acknowledgements
The Authors wish to thank everyone who participated in this study by donating blood, as well as referring clinicians Dr. George Antonopoulos and Dr. Helena Skouteli. This article is dedicated to the memory of friend and colleague Dr. Nikos Koufaliotis. This work was supported in part by a Bioerevna Research grant to C.Y.
- Received July 18, 2011.
- Revision received October 3, 2011.
- Accepted October 5, 2011.
- Copyright © 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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