while stoke incidence has declined in developed countries over the last decade , its incidence rose during the same period in low- and middle- income countries . although stroke is an ever - increasing problem in developing countries , little is known about its incidence and outcomes in sub - saharan africa ( ssa ) . the interstroke study showed the commonality of the main risk factors for stroke worldwide , including ssa : hypertension , obesity , diabetes mellitus ( dm ) , smoking , alcohol abuse , hypercholesterolemia , diet and physical activity . in addition , genetic factors may act either by predisposing to vascular risk factors , such as hypertension , heart disease , dm , hyperlipidemia or by a direct independent effect on stroke risk . these genes include apolipoproteine ( apoe ) , plasminogen activator inhibitor 1 ( serpine1 ) , platelet receptor gp1ba , angiotensin - converting enzyme ( ace ) , and methylenetetrahydrofolate reductase ( mthfr ) . case - control studies of stroke candidate genes in different ethnic groups have produced contrary results . a meta - analysis of chinese , japanese , and korean ancestry reported significant association of polymorphisms in the apoe , mthfr and ace genes with ischemic stroke . however , in other studies , only minor or non - significant risk associations were observed , and it was concluded that it is a combination of several genotypes interacting which increases the odds ratio for risk of ischemic stroke . studies on stroke conducted within african populations mainly focus on the prevalence , risk factors , subtypes and outcome of stroke . to date , there are only a handful of studies examining genetic association of apoe , mthfr , ace or other candidate genes with cardiovascular disease in african populations . these candidate genes provide some of the most likely genetic variants which are involved in stroke risk and have been among the most studied . common apoe alleles has been shown to affect risk of cardio- and cerebrovascular disease and is considered an established genetic risk factor , however common variation in the mthfr gene ( related to folate metabolism ) and the ace gene although implicated in disease risk are not confirmed . the aim of the present study therefore , was to investigate association of the apoe , mthfr and ace gene polymorphisms with stroke in zambian patients . forty one consecutive patients with mean age 5416 years admitted to the university teaching hospital ( uth ) lusaka , zambia , between june and december 2010 with a diagnosis of stroke , were enrolled . we included only patients who had stroke confirmed by neurological examination and brain ct scan . daily physical checks / reviews were made of emergency room , admission ward and in - patient wards for stroke patients . within 24 hours of admission , we obtained medical history , conducted general and neurological examination and brain ct scan was performed . the patient s demographic details and risk factors for stroke were documented on a data collection sheet by study physician . risk factors for stroke included : hypertension , dm , elevated cholesterol , heart disease , tobacco smoking / sniffing , alcohol consumption , history of transient ischemic attack , and a family history of stroke . as a risk factor of stroke hypertension was defined as blood pressure of  140/90 mmhg and current use of antihypertensive medication . elevated cholesterol was considered if serum fasting cholesterol was > 5.2 mmol / l or prestroke treatment with a cholesterol lowering agent . the severity of stroke was evaluated on admission using the national institutes of health stroke scale ( nihss ) . data on risk factors and stroke severity in our study population was published in our previous article . as a control population , 116 subjects ( 68 women , 48 men ) matched for age , sex , free of cerebrovascular diseases and other risk factors including hypertension , dm and hiv , were recruited from the department of internal medicine of uth . only indigenous zambian patients and control subjects were included in the study . all participants gave written informed consent and the study was approved by the biomedical research ethics committee of the university of zambia . whole blood was collected in 4 ml edta tubes and stored at 4 degrees celsius prior to dna extraction which was performed using silica - gel - based membrane with vacuum technology qiaamp dna mini kit ( 250 ) ( qiagen , london , uk ) . the common apoe epsilon alleles defined by the p.c112r ( rs429358 ) and p.r158c ( rs7412 ) substitutions were selected for analysis , as well as the common mthfr c677 t ( p.a222v ; rs1801133 ) variant and the ace exonic synonymous variant p.t202 t ( rs4343 ; a / g ) ; which acts as a surrogate marker for the common intronic alui insertion / deletion ( i / d ; rs4646994 ) polymorphism . genotyping was performed with abi predesigned taqman probes and chemistry ( applied biosystems , foster city , ca ) with analysis performed using sds 2.2.2 software on an abi 7900 for taqman . positive and negative controls where included on all taqman assay plates . positive or ambiguous results in the taqman assay were also resolved with direct sequencing . the fishers test was used to calculate for p - value and the two tailed probability levels for statistical significance with p<0.05 being considered significant . an odds ratio ( or ) was calculated as a measure the strength of association forty one consecutive patients with mean age 5416 years admitted to the university teaching hospital ( uth ) lusaka , zambia , between june and december 2010 with a diagnosis of stroke , were enrolled . we included only patients who had stroke confirmed by neurological examination and brain ct scan . daily physical checks / reviews were made of emergency room , admission ward and in - patient wards for stroke patients . within 24 hours of admission , we obtained medical history , conducted general and neurological examination and brain ct scan was performed . the patient s demographic details and risk factors for stroke risk factors for stroke included : hypertension , dm , elevated cholesterol , heart disease , tobacco smoking / sniffing , alcohol consumption , history of transient ischemic attack , and a family history of stroke . as a risk factor of stroke hypertension was defined as blood pressure of  140/90 mmhg and current use of antihypertensive medication . elevated cholesterol was considered if serum fasting cholesterol was > 5.2 mmol / l or prestroke treatment with a cholesterol lowering agent . the severity of stroke was evaluated on admission using the national institutes of health stroke scale ( nihss ) . data on risk factors and stroke severity in our study population was published in our previous article . as a control population , 116 subjects ( 68 women , 48 men ) matched for age , sex , free of cerebrovascular diseases and other risk factors including hypertension , dm and hiv , were recruited from the department of internal medicine of uth . only indigenous zambian patients and control subjects were included in the study . all participants gave written informed consent and the study was approved by the biomedical research ethics committee of the university of zambia . whole blood was collected in 4 ml edta tubes and stored at 4 degrees celsius prior to dna extraction which was performed using silica - gel - based membrane with vacuum technology qiaamp dna mini kit ( 250 ) ( qiagen , london , uk ) . the common apoe epsilon alleles defined by the p.c112r ( rs429358 ) and p.r158c ( rs7412 ) substitutions were selected for analysis , as well as the common mthfr c677 t ( p.a222v ; rs1801133 ) variant and the ace exonic synonymous variant p.t202 t ( rs4343 ; a / g ) ; which acts as a surrogate marker for the common intronic alui insertion / deletion ( i / d ; rs4646994 ) polymorphism . genotyping was performed with abi predesigned taqman probes and chemistry ( applied biosystems , foster city , ca ) with analysis performed using sds 2.2.2 software on an abi 7900 for taqman . positive and negative controls where included on all taqman assay plates . positive or ambiguous results in the taqman assay were also resolved with direct sequencing . the fishers test was used to calculate for p - value and the two tailed probability levels for statistical significance with p<0.05 being considered significant . an odds ratio ( or ) was calculated as a measure the strength of association the genotype and allele frequencies in the study and control groups are shown in table 1 . there was no significant difference between the two groups in the frequencies of apoe , mthfr and ace genotypes and alleles . the apoe , mthfr and ace genotype frequencies for the case group were compatible with the hardy - weinberg equilibrium . the apoe 22 and the mthfr tt genotypes were absent in the zambian population . however , in the control group , the apoe , mthfr genotype frequencies were compatible with the hardy - weinberg equilibrium ( hwe ) , while the ace genotype was not in agreement with hwe . the frequency of control subjects homozygous for apoe 4 was 7.7% compared to 2.4% for stroke patients . the mthfr t allele frequency was 8.6% in control subjects and was observed at a frequency of 11.0% in patients . the frequency for ace rs4343 g - allele corresponding to the d allele was 13.8% for subjects in the control group compared to 12.2% for patients . there was significant genotypic variations between cases and controls for apoe 24 and 23 genotypes , statistical analysis demonstrated a higher risk for hemorrhagic stroke in apoe 24 carriers [ or of 4.45 ( p<0.05 ) ] while the presence of apoe 23 genotype seemed to significantly lower the risk for hemorrhagic stroke ( or 0.097 ; 0.006 - 1.67 , p<0.05 ) ( table 2 ) . apoe 22 and mthfr 677tt polymorphisms were absent in both ischemic and hemorrhagic stroke . though not significant , the frequency of the mthfr 677 t allele was higher ( double ) in ischemic stroke ( 17.4% ) compared to hemorrhagic stroke ( 8.3% ) . studies conducted over the last decade has shown that among blacks , stroke is more common , more severe , and carries higher mortality when compared with other races ; blacks also have increased frequency of risk factors such as dm , hypertension , and obesity . in this study we have analyzed for the first time common polymorphisms of the apoe , mthfr and ace genes in zambian stroke patients and in controls . this constitutes an approach to start characterization of the genetic risk architecture of zambian patients with cerebrovascular disease . the study also describes the prevalence of apoe , mthfr and ace polymorphisms corresponding to stroke in the zambian population . we observed a relatively high frequency of the apoe 4 allele and a complete absence of the apoe 22 genotype in both patients and controls . we did not find any association between the apoe 4 allele and stroke in our patients . however , there was an increased risk to hemorrhagic stroke by the apoe 24 genotype with or at 4.45 p<0.05 . this may suggest that zambian carriers of apoe 2 and 4 have an increased risk of hemorrhagic stroke . of note , in our previous study , the prevalence of hemorrhagic stroke was observed to be higher than in reported western cohorts . a similar high frequency for hemorrhagic stroke has been reported for other african countries with estimates up to 52% in democratic republic of congo , up to 60% in ghana , and tanzania . according to the literature , carriers of apoe 2 and 4 have an increased risk of intracerebral hemorrhage perhaps because of the effects of these gene variants on risk of cerebral amyloid angiopathy . a recent metaanalysis in caucasian populations found apoe 2 genotypes to be associated with increased risk of hemorrhagic stroke . we found the effect of apoe 2 on carriers to be inconclusive as it increased hemorrhagic stroke risk in apoe 24 subjects while this risk seemed to be reversed in the apoe 23 subjects . these conflicting results may be due to a possible synergistic interaction effect of the 2 allele towards the 4 or simply the low sample size and low frequency of these alleles . found the frequency of 2 allele to be generally higher among sub - saharans ( 0.116 ) compared to europeans with the range of 0.044 - 0.108 ( with the exception of swedish with frequency 0.119 ) . hence , it would be plausible to hypothesize that an increase in the apoe 2 allele frequency ( 0.122 , table 1 ) increases the risk for development of hemorrhagic stroke in a population with a generally high 4 allele frequency ( 0.256 in the zambian population ) . we observed low minor allele frequencies for the mthfr c677 t and ace i / d polymorphisms in the zambian population . no significant association were found between the mthfr and ace genotypes and stroke risk in the zambian population . association between these polymorphisms and stroke have , however , been demonstrated in other populations including chinese population , hungarian population , and the northern irish population . this may reflect population heterogeneity or specificity in disease risk . to clarify the role of these variants in stroke risk in sub - saharan populations it is important to note that the major limitation of this study is the sample size which does not yet allow exclusion of apoe , mthfr and ace polymorphisms as a risk factor in zambian patients with stroke , and therefore , large scale prospective studies are needed to confirm these findings . however , as genetic technologies become more available to african countries and in particular those between the sahara and south africa we will gain a better understanding of how genetics will determine health outcomes . these data will help ease health - related social and economic burdens within these developing nations .