
 stroke is the second highest cause of death in the world and the main cause of disability in people in productive ages . in indonesia ,
the average age of stroke patients is 58.8 years old and stroke is the leading cause of high mortality rate in people above 50 years old .
statistics of heart disease and stroke2013 update reveals that number of stroke patients aged between 2045 years has increased significantly in recent years . according to world health organization , stroke is rapidly developing clinical signs of focal ( or global ) disturbance of cerebral function .
the symptoms last in 24 hours or longer and may lead to death , with no apparent cause other than of vascular origin .
more than 87% of stroke cases are caused by ischemia due to thrombosis or cardio embolisms .
ischemic stroke is atherosclerosis - associated complication [ 35 ] which is progressive in process .
it requires 3 to 4 decades or more since the period of endothelial dysfunction and carotid intimamedia thickness ( cimt ) to develop until clinical manifestations appear .
a study on american children aged 1014 years old who died from motorcycle accidents showed that 50% of them have early atherosclerosis evidence .
similarly , bogalusa heart study revealed that 50% of its 204 subjects aged 215 years old had fatty streak in their coronary arteries .
thus , detecting atherosclerotic vascular changes in childhood helps identifying high - risk groups which is very useful for the early stroke management .
cimt is a surrogate marker that reveals atherosclerosis in general and constitutes a predictor of blood vessels condition in the future .
the increase of cimt has been proven to be related with the raise of ischemia stroke risk [ 1113 ] . in indonesia
, cimt measurement has not yet become a routine screening procedure for atherosclerosis prevention . to date , there is no normal value data of cimt for population in indonesia .
, this sign becomes very useful to be intermediate phenotype in genetic studies [ 14 , 15 ] .
epidemiological data show that the increase of cimt is influenced by genetic factors , with the prediction that 30% to 40% is heritable . in 2006 ,
genetique de l'infarctus cerebral [ genic ] study conducted a research on genetic factors related with cimt of cerebral infraction patients .
the study examined polymorphism association with vascular pathologies which incorporate inflammation , hypertension , coagulation , and lipid metabolism .
the findings revealed that there was a significant relationship between cimt with monocyte chemoattractant protein-1 [ mcp-1 ] and osteopontin ( opn ) polymorphisms .
mcp-1 is synthesized by various cells associated with atherosclerosis , including endothelial cells , muscle cells , fibroblasts , and macrophages . in early stages of atherogenesis , mcp-1 is primary chemokine which recruits monocytes into arterial subendothelium .
mcp-1 protein and rna are highly evidenced in atherosclerotic vessels , but not in normal vessels .
the mcp-1 polymorphism in the forms of snp-957 , snp-2518 , and snp-2578 causes earlier occurrence of cimt faster progression [ 12 , 1619 ] in chinese , japanese , french , and slovakian people .
however , the mechanism of earlier and faster cimt development could not be identified yet .
some researchers suggest that ethnic and geographic factors give different effects on genetic polymorphisms taking in mcp-1 polymorphism . thus far
, there has never been a study on mcp-1 polymorphism and its contribution toward cimt conducted for indonesian population .
it functions in atherosclerosis , in cell - mediated immunity , and in macrophage recruitment and activation . in early stages of atherosclerosis ,
opn attracts inflammatory cells , promotes the releases of proteinolytic enzymes , and stimulates smooth muscle cell proliferation .
the opn promoter polymorphism is studied in several areas , such as in snp-443 and snp-66 [ 12 , 21 ]
. this research attempts at explaining the effect of mcp-1 and opn promoter polymorphism on cimt changes in javanese indonesian children .
subjects were 54 javanese indonesian children who were classified into two groups : case and controlled .
the case group consisted of 27 children aged 1021 years whose parents had ischemic stroke historical background .
the controlled group consisted of 27 javanese children aged 1021 years whose parents had no ischemic stroke history .
all participants were required to complete a standardized questionnaire designed to obtain information about family history .
plasma concentrations of total cholesterol , high - density lipoprotein [ hdl ] , low - density lipoprotein [ ldl ] cholesterol concentrations , triglycerides , and fasting blood glucose were measured in accordance with standardized protocols in the physiology laboratory of medical faculty , university of brawijaya malang .
the research had been approved to be conducted by ethical committee , medical faculty , university of brawijaya , malang , indonesia . to measure cimt , all subjects went through carotid ultrasound process in the radiology department of saiful anwar general hospital in malang , indonesia .
high resolution carotid ultrasound measurements were performed using logiq s6 ultrasound ( ge healthcare ) , with 10  mhz linier transducer .
the measurement was done by scanning the far wall of common carotid arteries in 1.0  cm distal .
the crest at the origin of the bifurcation was used as an anatomical mark to identify segment to be visualized . in each examination ,
the sonographer used three different scanning angles , namely , transversal , anterolateral , and posterolateral angles , to record the greatest cimt ( figure 1 ) [ 2224 ] .
genomic dna was isolated from 1  ml blood using extraction kit ( wizard genomic dna purification kit / promega ) .
mcp and opn promoters were amplified by using specific primer mcp forward 5-ccgagatgttcccagcacag-3 and mcp reverse : 5-ctgctttgcttgtgcctctt-3 .
amplified dna was sequenced by employing automated sequencer ( macrogen , seoul , korea ) .
data characteristics , laboratory test results and ultrasound results were tested with data normality test ( kolmogorov - smirnov ) and data homogeneity test ( levence statistics ) prior to comparison test by using independent t - test .
the calculation of mutation influence on carotid artery toward cimt - increasing risks was done using the odd ratio [ or ] in a 2  2 table .
each sample received the same treatment in which right and left common carotid arteries were checked , and cimt and diameter of the carotid artery were measured and calculated .
result of correlation analysis on the average cimt with clinical characteristic variables is presented as in table 2 .
result of correlation test carried on using pearson product moment test showed that there was no correlation between average cimt with age , height , weight , body mass index , fasting glucose , total cholesterol , hdl , and ldl , as well as triglycerides . 
mutations took place in base number-2138 in which adenine base had substituted to thymine base .
the mutations occurred in two controlled samples ( 14.3% ) and one case sample ( 5% ) ( figure 2 ) .
further mutations took place in base number-2464 in which guanine base mutated into adenine base .
the calculation of mutation influence on cimt - increasing risks measured through odd ratio [ or ] using a 2  2 table appears as in table 3 . based on previous research
[ 11 , 24 , 26 ] , 0.5  mm was set as the normal value of cimt for odd ratio analysis .
this means that children with mutation have 1.471 times higher tendency of having cimt thickening than children without mutation .
all samples were analyzed for identifying t-66 g opn polymorphism promoter , but there was no mutation found in all samples .
from the research , two types of point mutation in mcp-1 gene promoters were found , that is , a-2138 t and g-2464a . either base-2138 or base-2464 was located in promoter site .
the promoter determines the efficiency of rna polymerase binding and , thus , it also determines the transcription efficiency .
the transcription factor is attached to the binding site in the promoter region and stimulates rna polymerase to bind with the promoter site .
in japan , mcp-1a-2138 t polymorphism has been reported to be identified in myocardial infarction patients above 65 years old .
it was found out that the mcp-1 a-2138 t polymorphism was significantly associated with mcp-1 serum level and incidence of myocardial infarction . in this research , mcp-1a-2138 t polymorphism
was found in children from parents with ischemic stroke history and children whose parents do not have ischemic stroke history as well .
the polymorphism has never been reported to be found in people with no ischemic stroke history previously .
children with a-2138 t polymorphism , either from the case or the controlled group , had a thicker cimt than the normal value .
the two children with a-2138 t polymorphisms had a family history of myocardial infarction disease with body mass index over 25 .
( 2006 ) informed that in japan a-2138 t polymorphism was found in patients with body mass index of more than 25  kg / m with myocardial infarction .
, the polymorphism was found in the case sample of a 19-year - old girl who inherited it from the mother suffering from ischemic stroke at the age of 32 .
her grandfather also suffered from ischemic stroke with hypertension , while her grandmother suffered from hypertension .
her total cholesterol was 208  mg / dl and ldl cholesterol was 136  mg / dl , which indicated dyslipidemia .
her average cimt was 0.60  mm , or thicker than normal average cimt [ 11 , 26 ] .
results of this research reveal that children with polymorphism had thicker cimt than children without mutation .
risk measured with odd ratio shows that children with mcp-1 polymorphism promoter had 1.471 times possibility to have thicker cimt than children without polymorphism .
these results confirm the importance of genetic examination , especially the mcp-1 polymorphism promoter analysis in children with atherosclerosis risk factors .
result of this interaction is mcp-1/ccr2 messenger system increasing activity which leads to the local recruitment of monocytes at the site of injury in the arterial wall .
this , in turn , will lead to increasing atherosclerosis incidence [ 29 , 30 ] .
the relation of mcp-1 promoter polymorphism with increasing cimt has been reported in several studies , in which the mcp-1 g-927c and a-2578 g polymorphisms are significantly associated with the increasing cimt in patients with ischemic stroke .
the mcp-1 g-362c polymorphism in black population is associated with the increasing cimt and atherosclerosis risk .
the mcp-1 g-928c polymorphism is associated with the increased cimt and the atherosclerosis risk [ 29 , 30 ] .
in contrast to some other studies conducted on atherosclerosis - related conditions , this research did not result in any finding on mcp-1 a-2518 g polymorphism [ 16 , 31 ] .
the difference among the above genetic polymorphism findings is likely to be due to the ethnic and geographical differences .
high mcp-1 transcription rate contributes to the severity of stroke [ 32 , 33 ] .
the mcp-1 transcription rate is influenced by several risk factors such as hypertension , hypercholesterolemia , smoking habit , and diabetes .
in summary , two mcp-1 promoter polymorphisms were found in the research , namely , a-2138 t and g-2464a .
children with polymorphism demonstrate increasing cimt and have 1.471 times higher possibility to have thicker cimt .
cimt of javanese indonesian children from parents with ischemic stroke is thicker than cimt of children from healthy parents .
 carotid intima media thickness ( cimt ) is clearly associated with atherosclerosis . 
 studies in ischemic stroke patients reveal that there is a significant association between cimt with monocyte chemoattractant protein-1 ( mcp-1 ) and osteopontin ( opn ) promoter polymorphism . 
 this research aims to explain the effect of mcp-1 and opn promoter polymorphism toward cimt changes identified in javanese indonesian children . 
 subjects were 54 children : 27 were from parents with ischemic stroke ( cases ) , and 27 were from healthy parents ( controlled ) . 
 the cimt was examined by utilizing high resolution b - mode ultrasound . physical examination and genotyping analysis of mcp-1 promoter 
 were conducted by employing pcr method . 
 research results indicate that two polymorphisms were obtained , that is , a-2138 t and g-2464a , respectively . 
 a-2138 
 t polymorphism was found in 5% of case children and in 14.3% of controlled children . 
 g-2464a polymorphism was found in 5% of case children . 
 cimt of case children was significantly different from that of controlled children ( 0.61  0.012  mm versus , 0.52  0.015  mm , p = 0.021 ) . 
 subjects with mcp-1 promoter polymorphism have 1.471 times higher tendency to have thicker cimt than subjects with no polymorphism in mcp1 promoter . 
 opn promoter t-66 g was also studied but it did not indicate occurrence of polymorphism in samples . 
1. Introduction
2. Methods
3. Results
4. Discussion
5. Conclusion