| Background and objective:Cerebral hemorrhage(CH) is a major cause of death in the world and is characterized by high rates of cases fatality and disability.CH is a multifactorial disease and the prevalence of CH varies widely among different population.Epidemiology study showed that in Europe and North America,up to 6.5-19.6%of strokes are attributed to CH,but there is a tendency for a higher proportion of CH in China.Especially in the recent Changsha city study,the author concluded that Changsha was one of the areas with high incidence of CH reported and also had a high proportion of CH among all subtypes of stroke (accounted for 55.4%of all strokes).The prevalence risk factors of stroke and CH cannot give a perfect explanation for the reason of the high incidence and high proportion of CH in this area,so we carried out a series of etiology researches about CH.We find the phenomenon of familial aggregation of CH,which strongly indicate that genetic influence in the pathogenesis of CH.Genetic study may play an important role for uncovering the etiology of CH.Results from pathologic studies of CH showed that most CHs originate from the rupture of small,deep arteries with diameters between 50 and 200μm and remodeling(including hyalinosis and aneurysm formation) of these arterioles is the underlying pathologic change resulting in CH.It is well known that vascular remodeling and aneurysm formation are partially caused by gene defect.The kallikrein-kinin system(KKS) is an endogenous metabolic cascade,triggering of which results in the release of vasoactive kinins.The pharmacologically active kinins are implicated in many physiological and pathological processes such as hypertension,vascular remodeling and aneurysm formation, which are involved in the pathogenesis of CH.In this study,we observed the relationship between CH and SNPs in the genes coding for the key enzymes(KLK1 and ACE) and precursors(KNG1) involved in the kinins metabolism pathway.The aim is to determine whether genetic variation in KKS system is associated with increased risk of CH in Changsha Han Chinese.Methods:Collecting the CH patients who were in the department of Neurology,Xiangya Hospital of Central South University from Jan 2006 to Oct 2008 and part of their family members.All cases were diagnosed by CT and/or MRI and fulfilled the diagnose criteria of the fourth national annual meeting of cerebrovascular disease.The subjects of the control group were eliminated cerebrovascular disease by clinical examination,CT and/or MRI,and were made sure without family history of cerebrovascular disease.All subjects were Han Chinese in Changsha area and given informed consent,which were composed of three groups:①38 Chinese Han pedigree of CH including 200 subjects which consisted of 61 CH patients,95 first degree relatives and 23 second degree relatives and 21 non-blood relationships spouse of the family members.②273 sporadic cerebral hemorrhage(SCH) patients③140 healthy control subjects.The age and sex between SCH and control group were not significant.We examined single-nucleotide polymorphisms (SNPs) of genes involved in the kinins metabolism pathway in attempting to examine whether SNPs or haplotype molecular markers would confer a significant susceptibility to CH.The SNPs present in the genes coding for KLK1(rs3212855,rs5515,rs5516 and rs5517),ACE(rs4291 and rs4343) and KNG1(rs1656922 and rs2304456) were analyzed by Multiplex Snapshot method;The ACE I/D polymorphism(rs4646994) was analyzed by PCR and agarose gel electrophoresis.For the genotyping data,we performed a two-step analysis.The first step was to carry out the polymorphism screening for a genetic association between SNPs and phenotypes in 273 SCH patients and in 140 healthy subjects.The second step was to validate the initial findings resulting from the above case-control study by using the family-based association tests(FBAT) in 38 pedigree of CH,aiming to determine weather the SNPs or haplotype is associated with CH in Changsha Han Chinese.Result:1.We completed an association analysis with four SNPs(rs3212855, rs5515,rs5516 and rs5517) located within the KLK1 gene using the case control association tests,the results showed that the rs5515 is not a polymorphic site in Changsha Han Chinese.As for other three SNPs,the results showed that the frequecy of A allele in rs5517 were significantly higher in SCH patients than control subjects(P＜0.05);while genotye and allele frequecy in rs3212855 and rs5516 were found not different between SCH patients and control subjects(P＞0.05).Furthermore,we completed a transmission disequilibrium test with three SNPs(rs3212855,rs5516 and rs5517) located within the KLK1 region using the family-based association tests(FBAT) in 38 CH pedigree.We found significant transmission desequilibrium between CH and two of the SNPs markers (rs5516 G:Z=2.422,P=0.015424 and rs5517 A:Z=2.963,P=0.003049, repectively).Three SNPs(rs3212855,rs5516 and rs5517) were in strong linkage disequilibrium by linkage disequilibrium analysis.They were in the same haplotype block.The global chi-square test for the haplotype transmission also revealed a strong association(x~2=12.664,df=4,P= 0.013042).Haplotype CGA showed the excess transmission to affected individuals(Z=2.881,P=0.003969). 2.We completed an association analysis with two SNP(rs1656922 and rs2304456) located within the KNG1 gene using the case control association tests,the results showed that genotye and allele frequecy in the two SNPs were foud not different between SCH patients and control subjects(P＞0.05).Furthermore,we completed a transmission disequilibrium test with two SNPs(rs1656922 and rs2304456) located within the KNG1 gene using the family-based association tests(FBAT) in 38 CH pedigree.We found no evidence for transmission disequilibrium with CH(P＞0.05).Two SNPs(rs1656922 and rs2304456) were in strong linkage disequilibrium by linkage disequilibrium analysis.They were in the same haplotype block.The haplotype analysis also revealed no evidence for transmission disequilibrium with CH(P＞0.05).3.We completed an association analysis with three SNP(rs4291, rs4646994 and rs4343) located within the ACE gene using the case control association tests,the results showed that genotye and allele frequecy in the three SNPs were found not different between SCH patients and control subjects(P＞0.05).Furthermore,we completed an transmission disequilibrium test with three SNPs(rs4291,rs4646994 and rs4343) located within the ACE gene using the family-based association tests(FBAT) in 38 CH pedigree.We found no evidence for transmission disequilibrium with the SNP markers(P＞0.05).Three SNPs(rs4291, rs4646994 and rs4343) were in strong linkage disequilibrium by linkage disequilibrium analysis.They were in the same haplotype block.The haplotype analysis also revealed no evidence for transmission disequilibrium with CH(P＞0.05). Conclusion:1.Our preliminary results provide evidence that the rs5517 A alelle of KLK1 gene may be important risk factors for the development of CH in the population.The haplotype CGA which composed of the rs3212855 C,rs5516 G and rs5517 A alleles significantly increased the susceptibility of CH.2.Our results do not support the association between these SNPs (rs1656922 and rs2304456) of KNG1 gene and CH susceptibility in the population.The results suggest that haploype composed of alleles of these SNPs of KNG1 gene do not contribute significantly to the predisposition to develop CH in our sample.3.Our results do not support the association between these SNPs (rs4291,rs4646994 and rs4343) of ACE gene and CH susceptibility in the population.The results suggest that haploype composed of alleles of these SNPs of ACE gene do not contribute significantly to the predisposition to develop CH in our sample.
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