Dharambir K. Sanghera

Genetic Polymorphism of Paraoxonase and the Risk of Coronary Heart Disease

Recent studies have implicated paraoxonase, an HDL-associated enzyme, in providing protection against LDL oxidation, thus affecting the risk of coronary heart disease (CHD) in the general population. In this study, we evaluated the distribution of a biallelic PON polymorphism at codon 192 (A and B alleles) and its relationship with plasma lipids and CHD in two racial groups comprising Asian Indians and Chinese from Singapore. The frequency of the B allele was significantly higher in Chinese control subjects than in Indian control subjects (0.58 versus 0.33; P < .0001). With the exception of a marginal effect on apolipoprotein A-I levels in Indians, no other significant association was observed between the PON polymorphism and quantitative lipid traits in either racial group. However, there was a race-specific association of the B allele with CHD in Indians but not in Chinese. The Indian CHD patients had a significantly higher frequency of the B allele than control subjects (.43 versus .33; P = .014). The age- and sex-adjusted odds ratio for developing CHD with the B allele (BB+AB genotypes) was 2.01 (95% CI, 1.17 to 3.45; P = .011) compared with the A allele (AA genotype). When the Indian patients were stratified into subgroups, the association remained significant in nondiabetic patients (odds ratio, 2.29; P = .008), and it became stronger in patients with myocardial infarction (odds ratio, 2.94; P = .004) than in patients without myocardial infarction (odds ratio, 1.11; P = .76). These data indicate that a common polymorphism in the PON gene is an independent risk factor for CHD in populations with white ancestry.

The codon 55 polymorphism in the paraoxonase 1 gene is not associated with the risk of coronary heart disease in Asian Indians and Chinese

Recently several but not all studies have implicated the codon 192 polymorphism in the paraoxonase 1 (PON1) gene with the risk of coronary heart disease (CHD). These findings suggest that this polymorphism is not functional but rather may be in linkage disequilibrium with a functional mutation in the PON1 or a nearby gene. In this investigation, we have evaluated the role of another common polymorphism in the PON1 gene at codon 55 with the risk of CHD in a biracial sample of Asian Indians and Chinese. We observed a significant inter-racial variability in the allelic distribution as the frequency of the less common allele, codon 55/L, was significantly higher in Indians than Chinese (0.202 versus 0.036; P < 0.0001). However, despite this inter-racial difference the codon 55 polymorphism was neither associated with CHD risk nor with plasma lipoprotein-lipids variation in both racial groups. We also used two site haplotype data (codons 55 and 192) to assess the combined contribution of the two polymorphisms to the risk of CHD. There was a strong linkage disequilibrium between the two polymorphic sites in both racial groups (P < 0.0001). While the haplotype data revealed no association with CHD in Chinese, the frequency of the BL haplotype was significantly higher (0.430 versus 0.311; P = 0.004) and the frequency of the AL haplotype was significantly lower (0.368 versus 0.483; P = 0.006) in Indian patients than controls. Since the B allele of the codon 192 polymorphism was shown to be an independent risk factor for CHD in Indians in our previous study, the positive association of the BL haplotype with CHD appears to be mediated by the B allele with no independent contribution from the codon 55 polymorphism.

Genetic variation in apolipoprotein H (β2-glycoprotein I) affects the occurrence of antiphospholipid antibodies and apolipoprotein H concentrations in systemic lupus erythematosus

Apolipoprotein H (apoH, protein; APOH, gene) is a required cofactor for the production of antiphospholipid antibodies (APA). In this study we have examined whether genetic variation in the APOH gene affects variation in risk for systemic lupus erythematosus (SLE), occurrence of antiphospholipid antibodies (APA), anti-apoH, and plasma apoH concentrations. A total of 222 white SLE women were screened for four APOHpolymorphisms (codons 88, 247, 306, and 316) by polymerase chain reaction, and for plasma apoH concentrations by ELISA. Of these, 29.3% were positive for APA (APA-positive group) and 31.1% for anti-apoH. None of the four APOH polymorphisms were significantly associated with variation in risk for SLE. The codons 306 and 316 polymorphisms showed significant, gene-dosage effects on plasma apoH concentrations (P<0.0001) and explained 30% and 13%, respectively, of the residual variation in apoH concentrations. No significant association was observed between anti-apoH status and APOH polymorphisms or plasma apoH levels. However, plasma apoH concentrations were significantly higher in patients positive for APA than in patients negative for APA (18.5±4.0 mg/dl vs 17.1+3.8 mg/dl; P=0.02). The distribution of the Trp3l6Ser polymorphism was significantly different between the APA-positive and APA-negative groups. The frequency of the mutant allele (Ser316) was significantly lower in the APA-positive group than the APA-negative group (3.1% vs 12.1% P < 0.04), indicating that the Ser316 mutation is protective against the production of phospholipid-apoH dependent APA. Our data indicate that common genetic variation in the APOH gene is a significant determinant of plasma apoH variation in SLE patients, and the Trp3l6Ser polymorphism appears to provide protection against the production of APA in SLE patients.

Molecular basis of the apolipoprotein H (β2-glycoprotein I) protein polymorphism

Abstract Apolipoprotein H (apoH, protein; APOH, gene) is considered to be an essential cofactor for the binding of certain antiphospholipid autoantibodies to anionic phospholipids. APOH exhibits a genetically determined structural polymorphism due to the presence of three common alleles (APOH*1, APOH*2 and APOH*3 ) detectable by isoelectric focusing (IEF) and immunoblotting. The APOH*3 allele can be further characterized into two subtypes, APOH*3W and APOH*3B, based upon its reactivity with monoclonal antibody 3D11. In this study we have determined the molecular basis of the APOH protein polymorphism and its distribution in three large U.S. population samples comprising 661 non-Hispanic whites, 444 Hispanics and 422 blacks. By direct DNA sequencing of PCR amplified fragments corresponding to the eight APOH exons, we identified two missense mutations that correspond to the APOH*1 and APOH*3W alleles. A missense mutation (G→A) in exon 3, which alters amino acid Ser to Asn at codon 88 and creates a restriction site for TSP509 I, was present in all APOH*1 allele carriers. A second missense mutation (G→C) at codon 316 in exon 8, which replaces amino acid Trp with Ser and creates a restriction site for BSTBI, was present in all APOH*3W carriers. The distribution of the Ser 88 Asn and Trp 316 Ser mutations was significantly different between the three racial groups. The frequency of the Asn-88 allele was 0.011, 0.043, and 0.056 in blacks, Hispanics and non-Hispanic whites, respectively. While the Ser-316 allele was observed sporadically in blacks (0.008), it was present at a polymorphic frequency in Hispanics (0.027) and non-Hispanic whites (0.059). The identification of the molecular basis of the APOH protein polymorphism will help to elucidate the structural – functional relationship of apoH in the production of antiphospholipid autoantibodies.

Quantitative effects of the apolipoprotein E polymorphism in a biracial sample of 9–10-year-old girls

Genetic polymorphism at the apolipoprotein E locus (APOE) has been shown to have a significant impact on quantitative risk factors for coronary artery disease (CAD) in diverse populations. However, despite the recognition that atherosclerosis begins in childhood and that genetic factors are related to the initial stages of atherosclerosis, prior studies were carried out mostly on adults and little attention has been paid to genetic risk factors for CAD in children. We have examined the impact of APOE polymorphism on quantitative risk factors for CAD (apoAI, apoB, TC, LDL-C, HDL-C and TG) in a sample of 647 African American and 573 White 9-10-year-old girls who were enrolled in the National Heart, Lung and Blood Institute Growth and Healthy Study. The frequencies of the APOE*2, APOE*3 and APOE*4 alleles were 0.09, 0.76 and 0.15 in Whites and 0.11, 0.70 and 0.19 in African Americans, respectively. The APOE*2 allele was significantly associated with lower mean levels of LDL-C and apoB and the APOE*4 allele with higher levels of LDL-C and apoB in both racial groups. Variation in maturation stage, body fat and fat patterning, as assessed by skin fold measures and waist/hip ratio, accounted for a significant proportion of the variation in quantitative CAD risk factors.

Racial differences in the distribution of a low density lipoprotein receptor-related protein (LRP) polymorphism and its association with serum lipoprotein, lipid and apolipoprotein levels

The low density lipoprotein (LDL) receptor-related protein (LRP) is a cell receptor that has close structural homology to the LDL and very low density lipoprotein receptors and thus is believed to play an important role in lipid metabolism. This study was carried out to evaluate the distribution of a known tetranucleotide repeat polymorphism in the LRP gene and its association with serum lipoprotein-lipid and apolipoprotein levels in four large samples comprising Hispanics (n=373) and non-Hispanic Whites (n=522) from the U.S. and Nigerian Blacks from Sokoto (n=390) and Benin (n=800). A total of four alleles, designated 83, 87, 91 and 95 bp, were observed. The 83 bp allele was observed at 0.4-1.1% in the two U.S. populations but was completely absent in African Blacks. Sokoto Blacks had significantly different frequencies of the 87 and 91 bp alleles compared to Hispanics (P=0.008) and non-Hispanic Whites (P=0.024). The frequency of the 91 bp allele was also significantly higher in Benin Blacks compared to Hispanics (P=0.026) and non-Hispanic Whites (P=0.054). The analysis of the relationship between the LRP polymorphism and serum lipid traits yielded some significant race and gender specific significant association for lipoprotein(a) in non-Hispanic White males (P=0.02); HDL2-cholesterol in Hispanic females (P=0.03) and apolipoprotein B in Benin males (P=0.04). We also observed an interaction between the LRP polymorphism and menopausal status for Lp(a) in Hispanic females (P=0.014). However, considering multiple comparisons were performed, these associations could be due to chance. Our data indicate that although the LRP tetranucleotide polymorphism exhibits inter-racial differences in its distribution, it does not appear to have a significant role in affecting serum lipid traits.

Gender-specific nonrandom association between the α1-antichymotrypsin and apolipoprotein E polymorphisms in the general population and its implication for the risk of Alzheimer's disease

A common polymorphism in the α1‐antichymotrypsin (ACT) gene has been found to modify the APOE*4‐associated risk of Alzheimers disease due to an apparent interaction between the two loci. This study was undertaken to determine the gender‐ and age‐related distributions of these two polymorphisms in two large population‐based samples of Caucasians (n = 803) and Nigerian Blacks (n = 730). Significantly higher frequencies of the ACT*A (78.6% vs. 48.4%; P < 0.001) and APOE*4 (25.6% vs. 15.6%; P < 0.001) alleles were observed in Nigerian Blacks than in Caucasians. In Caucasian women but not in men, the frequency of the APOE*4 allele was significantly lower in the ACT/AA genotype as compared to the ACT/AT and ACT/TT genotypes, while a reverse trend was seen for the APOE*3 allele frequency among the ACT genotypes. The distribution of the ACT*A allele between the APOE*4 carriers and non‐APOE*4 carriers was also different in Caucasian women but not in men. A similar gender‐specific nonrandom association between the two polymorphisms was observed in Black women but this was not as strong as observed in Caucasian women. When the two samples were stratified by age group, an association or trend of association was observed in all age groups in women only. These data indicate the existence of a nonrandom association between the APOE and ACT loci in women which may have an important implication for the higher prevalence of Alzheimers disease in women. Genet. Epidemiol. 14:169–180,1997.

Apolipoprotein H promoter polymorphisms in relation to lupus and lupus-related phenotypes

Objective. Sequence variation in gene promoters is often associated with disease risk. We tested the hypothesis that common promoter variation in the APOH gene (encoding for ß2-glycoprotein I) is associated with systemic lupus erythematosus (SLE) risk and SLE-related clinical phenotypes in a Caucasian cohort. Methods. We used a case-control design and genotyped 345 women with SLE and 454 healthy control women for 8 APOH promoter single-nucleotide polymorphisms (SNP; –1284C>G, –1219G>A, –1190G>C, –759A>G, –700C>A, –643T>C, –38G>A, and –32C>A).Association analyses were performed on single SNP and haplotypes. Haplotype analyses were performed using EH (Estimate Haplotype–frequencies) and Haploview programs. In vitro reporter gene assay was performed in COS-1 cells. Electrophoretic mobility shift assay (EMSA) was performed using HepG2 nuclear cells. Results. Overall haplotype distribution of the APOH promoter SNP was significantly different between cases and controls (p = 0.009). The –643C allele was found to be protective against carotid plaque formation (adjusted OR 0.37, p = 0.013) among patients with SLE. The –643C allele was associated with a ~2-fold decrease in promoter activity as compared to wild-type –643T allele (mean ± standard deviation: 3.94 ± 0.05 vs 6.99 ± 0.68, p = 0.016). EMSA showed that the –643T>C SNP harbors a binding site for a nuclear factor. The –1219G>A SNP showed a significant association with the risk of lupus nephritis (age-adjusted OR 0.36, p = 0.016). Conclusion. Our data indicate that APOH promoter variants may be involved in the etiology of SLE, especially the risk for autoimmune-mediated cardiovascular disease.