| Background and objective:Cerebrovascular disease is the second commonest cause of death and leading cause of adult disability worldwide, of which cerebral hemorrhage (CH) represents a main subtype with high morbidity and disability. The etiology of CH is increasingly becoming an important concern in prevention and cure of cerebrovascular disease. Current studies on the clinical and molecular genetics stress this heterogeneous disease caused by the interactions among many minor genes and environmental factors. The study on candidate genes SNPs(single nucleotide polymorphisms) about CH will help us to understand the molecular nature of this disease and suggest a theory for prevention and treatment of CH.Hypertension is an independent risk factor of CH and atherosclerosis is an important risk factor of CH, too. Our clinical epidemiology study on CH in the past dozen years has verified the conclusion that hypertension and atherosclerosis are risk factors related to CH, in which hypertension is the most important one. The human endothelin family gene is thought to play an important role in the development of atherosclerosis, hypertension and may increase the risk of CH. Up to now, there are few studies on the relationship between the polymorphism of Endothelin family gene and CH. Identification of gene polymorphisms of endothelins that confer susceptibility to CH and the development of genetic risk diagnosis systems may contribute to the personalized prevention of these conditions. Our aim is to investigate the correlations of the polymorphism of Endothelin family gene with CH in the Changsha Han ethnic group.Methods:All subjects came from Changsha Han population which were composed of 3 groups:(1) 281 members of 65 family with CH aggregation, including:①65 CH patients with family history:46 men and 19 women, averagely aged 52.42±11.50 years;②133 first-degree relatives:69 men and 64 women, averagely aged 39.52±14.17 years;③36 second-degree relatives:26 men and 10 women, averagely aged 31.93±12.21 years;④10 third-degree relatives:7 men and 3 women, averagely aged 32.65±12.95 years;⑤37 relatives with no kinship with proband:30 men and 7 women, averagely aged 47.68±7.64 years, who are spouses of the proband or the first-degree and second-degree relatives. (2)Sporadic cerebral hemorrhage (SCH) group:195 patients with sporadic cerebral hemorrhage,133 men and 62 women, averagely aged 58.40±11.21 years. (3) Control group:116 healthy controls,41 men and 75 women, averagely aged 54.97±10.41 years.The measures of blood pressure:The blood pressure of every subject was measured and the hypertension was diagnosed according 2005 guidelines for The prevention and treatment of hypertension in China.Serum lipid levels were tested:Total cholesterol (TC), High density lipoprotein (HDL) and triglycerides (TG) were measured by standard enzymatic methods. LDL cholesterol levels were calculated with the Friedewald formula (LDL=TC-HDL-TG/5).Multiplex SNaPshot genotyping analysis and DNA sequencing were used to detect 10 SNPs in 4 genes associated with endogenous endothelins synthesis of some subjects, including:rs 1800541,rs2070699,rs5370 of endthelin-1 gene, rs1801708,rs5333,rs5335 of endothelin receptor A gene, rs3818416,rs5351 of endothelin receptor B gene and rs2212528,rs213045 of Endothelin-converting enzyme 1 gene.The frequencies of the alleles and genotypes between the SCH group and control group were analyzed byχ2 analysis, and blood pressure levels were compared among genotypes by ANOVA using SPSS. The SNP and haplotype transmissions of the CH pedigrees were analyzed by transmission disequilibrium test (TDT) using FBAT computer program. Results:1. The distribution of Endothelin family gene polymorphism in Changsha Hans population:There are EDN-1gene rs 1800541,rs2070699,rs5370 polymorphisms, EDNRA gene rs 1801708,rs5333,rs5335 polymorphisms, EDNRB gene rs3818416,rs5351 polymorphisms and ECE-1 gene rs3818416,rs5351 polymorphisms in Changsha Han population. These gene polymorphisms in Changsha Han population revealed obvious racial difference comparing with those of other regions in the world in different extents.2. The results of the case-control study in SCH and normal controls were as follows:①There is significant difference of genotype and allele frequencies of EDNRA gene rs180178,rs5333 between SCH group and control group(p<0.05).②The frequencies of G allele (MAF) of EDNRB gene rs5351 was significantly lower in SCH group than those in control group (p< 0.05).③There is no significant difference of genotype and alleles frequencies of EDN-lgene rs1800541,rs2070699,rs5370 between SCH group and control group (P>0.05).④There is no significant difference of genotype and alleles frequencies of ECE-1 gene rs3818416,rs5351 between SCH group and control group (P>0.05).3. The results of FBAT in those member of CIFH pedigrees showed the following characteristics:①TDT analysis revealed that the frequency of transmission (from parent to the offspring with CH) of allele C of rs5335 polymorphism in CH increased significantly (P<0.05).②Haplotype analysis of EDNRA gene rs 1801708,rs5333,rs5335 revealed there were significant evidence for transmission disequilibrium with CH (P>0.05) in these haplotype:EDNRA rs1801708 rs5335 G-C, EDNRA rs5333 rs5335 T-C.③EDN1 gene rs1800541,rs2070699,rs5370 revealed no evidence for transmission disequilibrium with CH (P >0.05). Haplotyping and Haplotype Frequency Estimation of the 3 sites hasn't revealed significant results too(P>0.05).④Application of TDT to genotype data from EDNRB gene rs3818416,rs5351 polymorphisms and their Haplotype Frequency Estimation haven't got any positive results too.4. Analysis of Endothelin family gene polymorphism and blood pressure level in Changsha Hans CH and control population:①In SCH group, the levels of diastolic pressure and mean arterial blood pressure of the GG gene type of rs5335 were significantly higher than those of the GC/CC gene type (p<0.05)。②In SCH group, the levels of systolic blood pressure of the AA gene type of ECE-1 gene rs5335 was significantly higher than that of the AG/GG gene type (p<0.05)。③There was no significant difference of levels of systolic blood pressure, diastolic pressure and mean arterial blood pressure between TT and GT/GG gene type of EDN1 gene rs1800541, between GG and GT/TT gene type of EDN1 gene rs2070699 and between GG and GT/TT gene type of EDN1 gene rs5370 (P>0.05)。④There was no significant difference of levels of systolic blood pressure, diastolic pressure and mean arterial blood pressure between AA and AG/GG gene type of EDNRB gene (P>0.05)。Conclusions:1. The EDNRA gene rs1801708,rs5333,rs5335 and EDNRB gene polymorphisms are associated with risks of CH in Changsha Han population.2. The haplotypes of EDNRA rs1801708 rs5333 rs5335 G-T-C, EDNRA rs1801708 rs5335 G-C and rs5333 rs5335T-C may be related to with CH in Changsha Han population.3. The polymorphism of EDNRA gene rs5335, ECE-1 gene rs212528 may have relation with the higher levels of blood pressure in the CH susceptible population in Changsha Han population, in which EDNRA gene rs5335 polymorphism may increase CH risk by influencing hypertension.4. There may be no correlation between the polymorphism EDN-1 gene rs 1800541,rs2070699,rs5370,EDNRB gene rs3818416 and ECE-1 gene rs213045 and CH in Changsha Han population; The polymorphism of ECE-1 gene rs212528 may be related to the levels of blood pressure but not related to the susceptibility for CH.
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