Prothrombin G20210A and Factor V Leiden Polymorphisms in Stroke
Thierry Paluku They-They&Omar Battas&
Ilham Slassi&Mohamed Abdou Rafai&
Desire Tshala Katumbay&Sellama Nadifi
Received: 26 December 2010 / Accepted: 12 June 2011 / Published online: 24 June 2011
#Springer Science+Business Media, LLC 2011
Abstract
The molecular epidemiology of stroke is critically lacking in the developing world. We explored the relation- ships between genetics polymorphism and risk for ischemic stroke among the residents of Casablanca, Morocco. Ninety- one stroke patients matched 1:2 for their age, gender, and ethnic background to 182 healthy controls who were genotyped for the prothrombin G20210A mutation and factor V ( FV ) Leiden and were assessed for conventional risk factors for stroke. No significant association was found between prothrombin gene mutation with stroke ( p =.054).
Regarding stroke subtypes, significant relationships between patients with a large artery disease subtype of stroke and this mutation was found compared to controls ( p =.046). As a
genetic risk factor to develop this event, a strong association was observed when adjusted for conventional vascular risk factors (adjusted OR, 4.3; p = .029). No FV Leiden was found. We suggest that prothrombin mutation but not FV Leiden should be considered as a modest genetic risk factor for large artery disease stroke subtype in the Moroccan population.
Keywords
Factor V Leiden . Genetic polymorphism . Prothrombin . Thrombosis . Stroke
Introduction
Stroke is an emerging public health problem not only in developing societies, but also in low- and middle-income countries. Its development involves the interaction of multiple genetic factors and environmental influences (e.g., hypertension, diabetes, and smoking). Numerous polymorphisms have been identified within genes encoding peptides directly and indirectly involved in pathways in relation to stroke etiology such as thrombosis.
Recent advances in molecular epidemiology suggested that the identification of genetic and modifiable lifestyle risk factors for stroke is needed to elucidate the pathogenesis of stroke, notably that of ischemic origin, and designed target- specific interventions. While the genetic risk associated with ischemic stroke still appears controversial. In relation to genetic susceptibility to stroke, several studies have focused on the prothrombin and factor V ( FV ) genes because of the presumed but still controversial roles of their mutations in thrombotic events, possibly associated with ischemic events.
One of these potential risk factors is a mutation within the 3′-untranslated region of the prothrombin gene. A GA transition of nucleotide 20210 has been associated with
T. P. They-They:
S. NadifiLaboratory of Genetic and Molecular Pathology, Medical School, Hassan II University,
19, rue Tarik-Ibn-Ziad, BP 9154, 10000 Casablanca, Morocco T. P. They-They
:
O. BattasUFR of Clinical Neuroscience and Biological Psychiatry, Medical School, Hassan II University,
19, rue Tarik-Ibn-Ziad, BP 9154, 10000 Casablanca, Morocco I. Slassi
:
M. A. RafaiNeurology Department, Ibn Rochd Hospital, District Hospitals, Casablanca, Morocco
D. T. Katumbay
Neurology Department, Oregon Health & Science University, Portland, OR, USA
T. P. They-They (*)
UFR Clinic of Neurosciences and Laboratory of Genetic and Molecular Pathology, Medical School, Hassan II University, Casablanca, Morocco
e-mail: thierrypal@yahoo.fr DOI 10.1007/s12031-011-9580-9
higher prothrombin clotting activity and a two to fourfold higher risk for venous thrombosis (Poort et al.
1996;Ridker et al.
1999). However, the role of the G20210Amutation in arterial vascular disease has remained contro- versial. An increased prevalence of G20210A mutation has been reported in patients with myocardial infarction and cerebral artery disease (Arruda et al.
1997, 1998;Rosendaal et al.
1997; Longstreth et al. 1998), findingsthat other studies have failed to confirm (Ferrerasi et al.
1997; Zenz 1998; Croft and Daly 1999; Voetsch et al.
2000; Szolnoki et al. 2002). There is also evidence that
activated protein C resistance or its underlying genetic defect, the FV G1691A mutation which results in an arg506-to-gln (R506Q) substitution (Bertina et al.
1994),is associated with an increased risk for venous thrombosis (Ridker et al.
1995; Caprini et al. 2004) and ischemicstroke (Zenz
1998; Szolnoki et al.2002). These findings,however, also remained controversial (Catto et al.
1995;Press et al.
1996; Madonna et al. 2002; Meseguer et al.2004). Such conflicting results suggest that the genetic
predisposition for ischemic stroke may vary according the ethnic and geographical distribution of these mutations (Ioannidis et al.
2004).The influence of the prothrombin G20210A mutation and FV Leiden in the Moroccan population is lacking. We hypothesized that the prothrombin G20210A mutation and FV Leiden may be a relevant factor in patients with ischemic stroke, but with a limited role.
We investigated whether the presence of both prothrombin G20210A mutation and FV Leiden G1691A were associated with increased ischemic stroke compared to control groups matched for age, sex, and same ethnic background population.
Materials and Methods
Study Population Patients and Controls
We conducted a case–control study involving subjects admitted for ischemic stroke between May 2008 and September 2009 at the
“IBN ROCHD”unit of the University Hospital Center, Casablanca, Morocco. Cases were defined as subjects who rapidly developed clinical signs of focal or global cerebral dysfunction of which the ischemic lesion was confirmed by at least one computed tomography (CT) scanner or magnetic resonance imaging (MRI) examination. Patients were enrolled during admission, and the other cases were referred from other local hospitals for detailed investigation after the acute stroke. We also included patients with a previous history of stroke.
Information on major risk factors for arterial disease was collected from clinical files. Hypertension was defined as systolic and diastolic blood pressure above 140 and 90 mmHg, respectively or previous use of antihypertensive drugs. Diabetes mellitus was defined by serum glucose levels above 1.20 mg/dl or previous treatment for diabetes mellitus. Current use or history of cigarette smoking or alcohol intake was assessed.
Stroke subtypes were classified according to the TOAST classification system (Adams et al.
1993): Cortical orcerebellar lesions and brain stem or subcortical hemi- spheric infarcts greater than 1.5 cm in diameter on CT or MRI were considered to be potentially of large artery atherosclerotic origin.
1. Large artery disease (atherothrombotic) included patients with clinical and brain imaging findings of either significant (>50%) stenosis or occlusion of a major brain artery or branch cortical artery. Clinical findings included those of cerebral cortical impairment (aphasia, neglect, restricted motor involvement, etc.) or brain stem or cerebellar dysfunction. Cortical or cerebellar lesions and brain stem or subcortical hemi- spheric infarcts greater than 1.5 cm in diameter on CT or MRI were considered to be of potential large artery atherosclerotic origin.
2. Small vessel occlusion (lacunar) included those with one of the traditional clinical lacunar syndromes and without evidence of cerebral cortical dysfunction. A history of diabetes mellitus or hypertension supported the clinical diagnosis. The patient also had a normal CT/MRI examination or a relevant brain stem or subcortical hemispheric lesion with a diameter of less than 1.5 cm.
3. Cardioembolism included patients with arterial occlu- sions presumably due to an embolus arising in the heart such as ischemic heart disease, moderate aortic insuf- ficiency, and mitral stenosis. At least one cardiac source for an embolus was identified for a possible or probable diagnosis of cardioembolic stroke. Clinical and brain imaging findings are similar to those described for large artery atherosclerosis.
4. Acute stroke of other determined etiology included patients with rare causes of stroke, such as carotid dissection, subarachnoid hemorrhage, polycythemia, middle cerebral artery vasculitis, cerebral thrombophle- bitis, eclampsia, and a history of cervical trauma.
5. Stroke of unknown etiology included patients who had no likely etiology determined despite an extensive evaluation. Patients with hemorrhagic stroke were excluded.
Control subjects originated from the same population
and were recruited to serve in population studies conducted
by the Genetic Laboratory of the Medical School of Casablanca, Morocco. Information on risk factors for stroke was assessed only as family history of hypertension or diabetes mellitus, and current use or history of alcohol drinking and smoking.
A total of 91 consecutively admitted unrelated patients with ischemic stroke (45 males, 46 females, mean±SD age, 49.01±15.28 years) were matched 1:2 (for their age, gender, and ethnic background) with 182 presumably healthy blood donors (94 males, 88 females; mean±SD age, 46.25 ± 11.24 years). Patients and their respective controls were enrolled after giving an informed consent. The study was approved by the institutional ethics committee.
Genotyping Analysis
Deoxyribonucleic acid was extracted from peripheral leukocyte with the use of salt method (Miller et al.,
1988).Prothrombin G20210A Mutation Detection
The G20210A mutation does not disrupt a natural recog- nition site for any restriction enzyme. Primers were therefore designed to introduce a Hin dIII cleavage site only if the mutant allele was present. This involves insertion of a single base into the wild-type sequence at the 3-end of the primer, so that the recognition site overlaps the primer 3-end and new strand 5-end, such was reported by Danneberg et al. (1998): primer 1, 5′-TCTAGAAA CAGTTGCCTGGC-3
′; primer 2, 5
′-ATAGCACTGGGA GCATTGAA*GC-3′ (Invitrogen, Carlsbad, New Mexico, USA) were used to amplify a 486-bp product from prothrombin gene. The nucleotide with the asterisk in mutagenic primer 2 is not present in the normal sequence, and introduces a Hin dIII site in the amplified fragment when the G to A transition is present.
Factor V Leiden Mutation Detection
Primers used in the reactions were based on the exon 10 sequence of FV , such as reported by Gandrille (1995).
Mismatched antisense primer was modified at the 3′-end to create a single Hin dIII site, and was used to amplify a 241- bp product from FV gene. FV primers included the sense strand primer 1, 5′-TCAGGCAGGAACAACACCAT-3′ and the antisense strand primer 2, 5
′-GGTTACTTCAAGGA CAAAATACCTGTAAAAGCT-3′; substituted with the three mismatched nucleotides were substitute, so that amplifica- tion of a mutant allele resulted in the generation of a new Hin dIII restriction endonuclease site. The PCR program on the GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, Inc.; Foster City, CA, USA) was as follows: a
7-min enzyme activation at 94°C, followed by 35 cycles of 1 min at 94°C, 1 min at 55°C, and 61°C for FV and prothrombin , respectively, 1 min at 72°C, and a final extension time for 5 min at 72°C. Ten microlitres of amplified products was then digested with 0.5
μl ofHin dIII (Promega Biosciences; San Luis Obispo, CA, USA) with 2
μl of ×10 enzyme buffer at 37°C for at least 3 h. Afterrestriction digested products were resolved by electropho- resis on a 3% agarose gel and stained with ethidium bromide for analysis. Hin dIII digestion of FV and prothrombin normal amplicon yielded a fragment of 241 bp and fragments of 413, 73 bp, respectively. Digestion of FV Leiden heterozygote resulted in fragments of 241, 209, and 32 bp, and digestion of prothrombin G20210A heterozygote resulted in fragments of 413, 384, 73, and 23 bp. FV Leiden homozygote digested with Hin dIII yielded fragments of 209 and 32 bp, and prothrombin 20210A homozygote digested yielded fragments of 384, 73, and 29 bp (figure not shown).
Statistical Analysis
The Statistical Package for Social Sciences (SPSS version 17; Chicago, IL, USA) was used for statistical analysis. The results are expressed as mean±SD. Continuous variables were analyzed by Student’s t test while the relationships between categorical variables were assessed by chi-square statistics. Odds ratios (ORs) and respective 95% confidence intervals (CIs) were computed for each variable. Logistic regression analysis was performed with an enter method.
P values < .05 were considered statistically significant.
Results
Demographic and Clinical Characteristics of the Study Sample
The baseline characteristics of patients and control subjects are given in Table
1. Of the 91 stroke patients, 46 weremales (50.5%) versus 51.6% of controls (50.5%) while 45 (49.5%) were females, a distribution that did not significantly differ from that of the control group with 94 males (51.6%) and 88 females (48.4%) ( p > .05, t test).
The mean age ± SD of patients (49.01 ± 15.28 years) was statistically comparable to that of controls (46.25 ± 11.44 years) ( p= .095, t test).
Cases were characterized with most conventional vascular
risk factors more prevalent than controls group. In particular,
hypertension ( p <.001, chi-square test), habitual alcohol
intake ( p <.001, chi-square test), current smokers ( p =.001,
chi-square test), history of diabetes ( p =.029, chi-square test),
and age up to 50 years ( p =.016, chi-square test). Because of
the limited number of subjects, dyslipidemia, serum glucose level, and blood pressure were not taken into consideration in the analysis.
Prothrombin G20210A and FV Leiden Distribution among Patients and Controls
Cases have shown increasing prevalence for prothrombin GA genotype (12.1% versus 5.5%; unadjusted OR, 2.3;
95% CI, 0.97 to 5.7; p =.054, chi-square test; adjusted OR, 2.4; 95% CI, 0.82 to 7.1; p =.110, chi-square test) and A allele frequency (6% versus 2.7%; OR, 2.3; 95% CI, 0.58 to 8.9; p =.350, chi-square test) than controls but no signifi- cant association between prothrombin A allele carriers genetic predisposing factors and stroke was observed.
These results were unchanged when adjusted for age, sex, and conventional vascular risk factors. No prothrombin 20210AA, FV Leiden, or FV 1691AA, were found, neither in the cases nor in the controls (Table
1).Prothrombin G20210A and Allele in Relation to Stroke Subtypes
The proportion of cases with prothrombin GA genotype was marginally higher in cases relative to controls (12.1%
relative to 5.5%; p =.054, chi-square test; Table
1). Asimilar result was found for the frequency distribution of allele A (6% in cases versus 2.7% in controls; p =.350, chi- square test). However, our stratified analysis (per stroke subtypes) showed a statistically significant relationship between the prothrombin G20210A genotype and the large artery disease subtype event ( p =.046, chi-square test). No significant association was observed between nonathero- thrombotic events (e.g., cardioembolic stroke) and the aforementioned genotype; results are shown in Table
2.Prothrombin Mutation and Risk Factors in Large Artery Disease
Our analysis has suggested a significant relationship between the prothrombin G20210A genotype and the large
artery disease subtype event. Logistic regression analysis with enter method was performed to assess the risk for large artery disease event adjusted to age, gender, and conven- tional risk factors retained for this study. Adjusted ORs revealed a significant association between prothrombin GA phenotype and large artery disease (adjusted OR, 4.3;
95% CI, 1.16 to 16.4; p =.029). A significant risk was also found for subjects with prothrombin GA genotype and history of hypertension (adjusted OR, 15.6; 95% CI, 6 to 40.6; p <.001), alcohol drinking habits (adjusted OR, 11.2;
95% CI, 2.2 to 55.6; p =.003), smoking status (adjusted OR, 4.6; 95% CI, 1.27 to 16.7; p =.020), and female gender (adjusted OR, 3.3; 95% CI, 1.2 to 8.7; p =.016). No significant risk was found for diabetes or age>50, in prothrombin GA genotype carriers. Results are summarized in Table
3.Discussion
The present case–control study was, to our knowledge, the first to investigate the association of the prothrombin G20210A mutation and FV Leiden with ischemic stroke in Moroccan population.
For the first time, we have elucidated the role of prothrombin G20210A mutation and modifiable lifestyle risk factors in relation to the large artery disease subtype of ischemic stroke among the residents of Casablanca, in Morocco. While our analysis has shown no association between prothrombin G20210A and ischemic stroke at the aggregate level ( p= .054), the relationship appeared to be marginally significant when data were stratified according to stroke subtypes. Patients with large artery disease stroke subtype were more likely carriers of the prothrombin G20210A mutation ( p= .046) suggesting that genetic risk factors for ischemic stroke may vary according to the underlying pathogenetic mechanisms. This finding appears to be in agreement with results from a meta-analysis study that found a modest increased risk of large artery disease event in carriers of the prothrombin G20210A mutation (OR, 1.30; 95% CI, 0.91 to 1.87) (Kim and Becker
2003),Characteristics Cases,n=91 Controls,n=182 OR 95% CI pvalue
Age in yeas±SD 49.01±15.28 46.25±11.24 .095
Female gender,n(%) 45 (49.5) 88(48.4) 1.05 0.63–1.7 .864
Hypertension,n(%) 46 (50.5) 18 (9.9) 9.3 4.9–17.6 <.001*
Diabetes,n(%) 16 (17.6) 16 (8.8) 2.2 1.05–4.7 .037*
Smoking habits,n(%) 20 (22) 13 (7.1) 3.7 1.7–7.8 .001*
Alcohol intake,n(%) 14 (15.4) 4 (2.2) 8.1 2.6–25.4 <.001*
Prothrombin GA vs GG 11 (12.1) 10 (5.5) 2.3 0.97–5.8 .060
FVLeiden 91 (100) 182 (100) – – –
Table 1 Demographic and clinical characteristics of stroke patients and controls
pvalues were obtained using Fisher’s exact test. Continuous (age) and categorical variables were tested byttest and chi-square analysis, respectively SDstandard deviation,nnumber (percentage)
*pvalue<.05, significant
though not consistent with findings from previously published reports (Hankey et al.
2001; Szolnoki et al.2002; Meseguer et al.2004). Another interesting finding of
our study is that the risk for large artery disease events is modified by gender or select manageable conditions such as hypertension, cigarette smoking, and alcohol drinking habits, and vice versa. After adjustment, the risk of large artery disease event in patients with prothrombin GA genotype became statistically significant (OR, 4.3; 95%
CI, 1.16 to 16.4; p =.029). A similar trend, i.e., increased in risk for large artery disease event was noticed in patients with the abovementioned lifestyle risk factors for stroke.
As previously reported (Mathonnet et al.
2002), noFV Leiden was found in our subjects suggesting that this mutation may not be a risk factor for ischemic stroke in the Moroccan population. This proposal needs, however, to be tested by studies conducted across the two major ethnic groups, i.e., the Arabs and Berbers, which have different
Table 3 Distribution of prothrombin G20210A,FVLeiden, and conventional risk factors with unadjusted and adjusted odds ratio in large artery disease stroke patients
Risk factors,n(%) Patients,n=42 Controls,n=182 Unadjusted OR 95% CI pvalues Adjusted OR 95% CI pvalues Age
≤50 16 (38.1) 49 (26.9) 1
>50 26 (61.9) 133 (73.1) 1.7 0.83–3.4 .153 0.96 0.38–2.4 .922
Sex
Males 21 (50) 94 (51.6) 1
Females 21 (50) 88 (48.4) 0.9 0.47–1.8 .847 3.1 1.18–8.2 .022*
History of hypertension
No 21 (50) 164 (90.1) 1
Yes 21 (50) 18 (9.9) 9.1 4.1–19.8 <.001* 15.6 6–40.6 <.001*
History of diabetes
No 37 (88.1) 166 (81.3) 1
Yes 5 (11.9) 16 (8.8) 1.4 0.48–4.1 .534 0.86 0.21–3.4 .830
Tabac habits
No 31 (73.8) 169 (92.9) 1
Yes 11 (26.2) 13 (7.1) 4.6 1.9–11.2 .001* 3.5 1.3–16.7 .020*
Alcohol intake
No 35 (83.3) 178 (97.8) 1
Yes 7 (16.7) 4 (2.2) 8.9 2.5–32 .001* 13 2.3–55.6 .003*
Prothrombin risk factors
GA vs GG 6 (14.3) 10 (5.5) 2.8 0.98–8.3 .055 4.3 1.16–16.4 .029*
A 0.07 0.027 2.7 0.7–10.3 .220
FVLeiden GG 42 (100) 182 (100) – – –
pvalues were obtained using logistic regression model adjusted for age, gender, smoking habits, alcohol intake, history of hypertension, and history of diabetes
*pvalue<.05, significant
Subjects studied n(%) Prothrombin Genotypes pvalues allele pvalues
GG,n(%) GA,n(%) G/A
Controls subjects 182 172 (94.5) 10 (5.5) 0.973/0.027
Ischemic stroke 91 80 (87.9) 11 (12.1) .054 0.94/0.06 .089
Large artery disease 42 (46.2) 36 (85.7) 6 (14.3) .046** 0.93/0.07 .157
Lacunar 24 (26.4) 21 (87.5) 3 (12.5) .185 0.937/0.063 .219
Cardioembolic 8 (8.8) 7 (87.5) 1 (12.5) .406 0.937/0.063 .219
Others etiology 11 (12.1) 10 (90.9) 1 (9.1) .617 0.955/0.045 .494
Unknown 6 (6.6) 6 (100) 0 (0) – – –
Table 2 Distribution of prothrombin genotypes in stroke subtypes patients and controls
pvalues were obtained using Pearson chi-square exact test
*pvalue<.05, significant
ethnographic origin and possibly, significant genetic varia- tions (Cavalli Sforza et al.
1988; Zhang et al. 2008). Inrelation to ischemic stroke in general, but not with stroke subtypes, our findings appear consistent with reports that suggest that the presence of the prothrombin G20210A mutation (Longstreth et al.
1998; Margaglione et al. 1999;Madonna et al.
2002) or that ofFV Leiden (Catto et al.
1995; Longstreth et al. 1998; Madonna et al. 2002) are not
associated with an increased risk for arterial thrombosis such as stroke.
However, they are not consistent with findings from studies which propose that prothrombin G20210A substi- tution (Ridker et al.
1999; Voetsch et al.2000; Casas et al.2004) or
FV Leiden (Margaglione et al.
1999) beconsidered as weak risk factors for ischemic stroke. While differences may be due to methodological and population factors, our study suggested that not taking into consider- ation the subtype of stroke may also account for such discrepancy.
Our study limitations include a convenient sampling technique and a relatively small sample size to assess the contribution of rare etiologies, e.g., a patent foramen ovale with or without atrial septal aneurysm in young subjects (Webster et al.
1988), frequent use of illicit drugs (Putaalaet al.
2009) or use of contraceptives in females, or increasethe precision of the study measurements and make robust conclusions. In addition, biological evidence linking the prothrombin G20210A mutation to changes in clotting mechanisms such as high plasma prothrombin levels (Poort et al.
1996) to validate our findings was lacking.Nevertheless, our case
–control design has shown that risk of large artery disease event is increased in females, or hypertensive subjects, or cigarette smokers, or alcohol drinkers who are carriers of the prothrombin G20210A mutation. These findings underscore the importance of assessing gene–gene and/or gene–environment interactions in relation to risk assessment and management of patients with or prone to ischemic stroke, as suggested in previous reports (Markus
2003).A shortcoming of our study is the relatively small size of the study group, which might in principle increase the possibility of spurious associations. In addition, the large confidence intervals calculated need to be confirmed in studies involving larger numbers of subjects.
Additional prospective, controlled studies of unselected ischemic stroke patients are needed to better assess the roles of these hereditary coagulation defects in the etiology of ischemic stroke among the Moroccan population.
Acknowledgments This study was supported by the Hassan II Academy of Science and Technology. We thank the population which readily wanted to lend itself to our study. We thank the Belgian Technical Cooperation (BTC) in the Democratic Republic of Congo for their support.
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