Study On The Association Of Single Nucleotide Polymorphisms In Apolipoprotein APOA1/C3/A4/A5 Gene Cluster With Coronary Heart Disease And Type Ⅱ Diabetes

Coronary heart disease (CHD) and Type â…¡ diabetes mellitus (non-insulin-independent diabetes mellitus, NIDDM) are two complex disorders caused by multiple genetic and environmental factors. Some risk factors, such as diet, heredity, age and obesity likely contributed to the generation and development of these two diseases. The genetic factor is generally considered as one of risk factors, and the hypertriglyceridaemia is also the prominent feature of the dyslipidemia commonly observed in CHD and NIDDM, as an independent risk factor.Apolipoprotein A5 gene (APOA5) identified newly is a fourth member ofthe chromosome 11 apolipoprotein gene cluster with apolipoprotein A1/C3/A4 together. The previous researches on AP0A5 indicate that AP0A5 is an important determinant of plasma tyiglyceride levels in human and mice. The models of transgenetic mice overexpressing human AP0A5 and knock-out mice lacking Apoa5 observed that the triglyceride levels of the former were about one-third of these of control group, but the latter were about four times as much plasma triglyceride as their controls. Meanwhile, the function influencing triglyceride levels is independent on others apolipoprotein genes in this cluster. Therefore, AP0A5 gene is an important gene regulating plasma triglyceride levels.To investigate the impossible association and roles of APOA1/C3/A4/A5 gene cluster in CHD and NIDDM, firstly, we examined the distribution of 12 known SNPs (-75bp G>A, +83bp C>A(+84bp G>A) in APOA1; 3238bp C>G, -455bp T>C,-482bp C>T,-2854bp T>C in APOC3; T347S, Q360H in AP0A4; -12238bp T>C, -1131T>C, S19W, C185G in AP0A5) in 483 Chinese CHD patients,465 NIDDM patients and 502 controls by PCR-RFLP. Secondly, we screened primarily the unknown SNPs of the APOA5 gene by high-throughput denaturing high-performance liquid chromatography (DHPLC) and DNA sequencing techniques. Finally, we analysed the association of these SNPs with lipid metabolism in patients with CHD and NIDDM.The results were as follows:1. The frequency of APOAl -75bp A allele increased significantly in CHD patients (33.7% vs. 27.8%, X2= 8.21, p=0.004) while which was similar to that in NIDDM patients (30.5% vs. 27.8%, X2= 1.768, P > 0.05).2. No significant difference of allelic or genotypic frequencies either of +83bp OT polymorphism in APOAl gene or of 3238bp OG polymorphism in APOC3 gene between the CHD, NIDDM patients and control subjects were observed.3. The frequencies of -455bp C allele in the APOC3 gene were found to be significantly higher in CHD and NIDDM patients ( for CHD: X2= 7.341, p=0.007 OR=1.277, 95%CI=1.070-1.524; for NIDDM: X2= 4.524, P=0.033 OR=1.214,95%CI=1.015-1.451.) There is no significant difference of allelic or genotypic frequencies either in -482bp C>T polymorphism or in -2854bp T>G polymorphism in APOC3 gene between the CHD,NIDDM patients and control subjects.4. Two known SNPs in the APOA4 gene, T347S and Q360H, have not been found in the 1450 subjects, indicating that they are rare in Chinese Han population.5. The -12238bp C allele frequency in APOA5gene was significantly lower in the CHD patients (X2=7.68, p=0.006, OR=0.775 , 95%CI=0.647-0.928), but not in NIDDM patients.6. The C allele as well as C/C genotype frequency in the -1131bp of APOA5 gene was found to be significantly higher in CHD patients (by allele X2= 18.662, P=0.000; by genotype X2= 11.178, P=0.001); the frequencies of C allele and C/C genotype were also significantly higher in NIDDM patients( for allele, X2= 17.948, P=0.000; for genotype, X2= 8.629, P=0.003).7. The frequencies of APOA5 19W allele and W carrier genotype increased significantly in CHD patients (X2= 47.864, P=0.000; X2= 47.869, P=0.000, respectively ) while they were also higher significantly in NIDDMpatients (X2= 45.221, p=0.000; X2= 45.046, P=0.000, respectively).8. The T allele as well as T/T genotype frequency in the code 185 of APOA5 gene was found to be significantly higher in CHD patients (by allele X2= 116.784, p=0.000; by genotype X2= 18.947, P=0.000); the frequencies of T allele and T/T genotype were also significantly higher in NIDDM patients(X2= 62.879, p=0.000; X2= 11.173, P=0.000, respectively).9. The TG, TC and LDL-C levels of patients with APOC3 3238bp C/G genotype were higher significantly than those with C/C or G/G in CHD (F = 4.54, p = 0.0111; F= 13.84, P = 0.0000; F = 7.63, P = 0.0005, respectively); the CHD patients with C/C homozygote of APOA5- 1131bp had the highest TG levels (F=4.59, p = 0.0106); T/T genotype CHD patients in APOA5 185 code exhibited a significant increase in plasma TG and TC concentrations (F= 13.13, P = 0.0000; F=3.57, P = 0.0288;) , while G/G homozygote CHD patients had the highest HDL-C levels (F = 6.48, P = 0.0017).10. In NIDDM patients, the homozygotes of T/T of APOC3 -482bp, C/C of APOA5 -1131bp, T/T of APOA5 code 185 all had the highest TG concentrations(F =4.13, P = 0.0167; F= 4.50, P = 0.0117; F= 3.81, P = 0.0229 , respectively).11. In control groups, the homozygote of G/G of APOC3 3238bp, heterozygote C/G of APOA5 -1131bp and the homozygote of G/G of APOC3 -482bp all had the highest TG concentrations(F = 5.68, P = 0.0036; F = 6.46, P = 0.0017; F= 3.81, P = 0.0229 , respectively).12. Haplotype analyses with PHASE 2.0 program showed that the frequencies of haplotype T-C-G-C-G (2₂₁₁₁) was highest in 32haplotype consisting 5 SNPs (-482bp C>T and -455bp T>C in APOC3, -75bp G>T and + 83bp OT in APOA1 , C185G in AP0A5) . The frequencies of haplotype C-T-G-C-G (1₁₁₁₁) and haplotype C-T-A-C-G (1₁₂₁₁) were the second and third highest.13. Haplotype analyses with EH program demonstrated that the haplotypes frequencies containing 5 SNPs were significantly different between NIDDM and control groups (P =0.000000).14. Following three haplotypes showed the highest TQ TC, HDLC-C levels: for TG, 1₂₂₁₂, 2₁₁₁₂, 2₂₂₁₂; for TC, 1₁₁₂₂, 1₂₂₂₁, 2₂₂₁₁; for HDL-C, 1₁₁₂₁ , 2₂₂₁₁, 2₂₁₂₁.15. Screening of the APOA5 genetic polymorphisms revealed 1 novel SNP: -1029G>A.