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 .
 the aim of the present study was to investigate the association of apoe , mthfr and ace polymorphisms with stroke in the zambian population . 
 we analyzed 41 stroke patients and 116 control subjects all of zambian origin for associations between the genotype of the apoe , mthfr and ace polymorphisms and stroke . 
 the apoe 24 genotype showed increased risk for hemorrhagic stroke ( p<0.05 ) and also a high risk for ischemic stroke ( p=0.05 ) . 
 there was complete absence of the apoe 22 and the mthfr tt genotypes in the zambian population . 
 the difference between cases and controls was not significant for the other genetic variants when analyzed for relationship between stroke , stroke subtype and genotype . 
 we show that genetic variation at the apoe locus affects susceptibility to stroke . 
 no detectable association were observed for the mthfr and ace genotypes and stroke in the zambian population . 
Introduction
Materials and Methods
Study population
Clinical procedure
Genetic analysis
Statistical analysis
Results
Discussion and Conclusions