Recognition of the E1kE1f cholinesterase genotype in a family segregating three rare genes, E1k, E1f and E1a

The first identification of the cholinesterase variant E1kE1f is reported from a family study. The evidence is based on the determination of enzymic activity, dibucaine, fluoride and RO2 numbers. Three individuals appear to have this genotype, and family evidence is not at variance with our conclusions. All three individuals will be sensitive to suxamethonium.     

Suxamethonium sensitivity and segregation of human serum cholinesterase variants at locus, Ch1, among twelve British families.

Sera from 39 individuals with suxamethonium apnoea have been examined. The likelihood of the ChU1ChD1 heterozygotes developing suxamethonium apnoea has been calculated. The results indicate that 1 in 4,000 of normal homozygotes and 1 in 400 of ChU1ChD1 heterozygotes develop suxamethonium apnoea. The period of suxamethonium apnoea in these individuals has been found shorter than that usually seen in ChD1ChD1 homozygotes. An approximate estimate of the frequency of the ChF1 and ChS1 genes has also been calculated. Twelve British families of these suxamethonium apnoea propositi have been examined. The inheritance pattern in all these families was found to be of the Mendelian type. Three of these families have been found to be segregating for the rarer ChF1 gene and two for ChS1 gene, respectively. This present study provides an additional piece of support to the hypothesis that the ChF1 and ChS1 are alleles determining the synthesis of usual and atypical cholinesterase together with the likelihood of ChU1ChD1 heterozygotes having occasional suxamethonium apnoea. In addition, the present report indicates that there may be cholinesterase variants besides dibucaine and fluoride-resistant, 'silent' and C5.     

Recognition of the Ek1Ek1 homozygote for plasma cholinesterase

A family is reported in which the propositus was found to be sensitive to suxamethonium. Both parents were heterozygote each having genotype Ea1Ek1. The sibling of the propositus had the most common phenotype as defined by dibucaine, fluoride and RO2 numbers. Genetic analysis, however, indicated that this sibling must be an Ek1Ek1 homozygote.     

The activation of pigeon cytochrome c-specific T cell hybridomas by antigenic peptides is influenced by non-native sequences at the amino terminus of the determinant

We recently demonstrated that the sequence 95-104 contains all the residues necessary for direct recognition of the I-Ek restricted pigeon cytochrome c determinant but that residues located in sequences to the amino-terminal side of residue 95 improve the ability of peptides containing the sequence 95-104 to stimulate Ag-specific T cell clones. In this study we use synthetic peptides with amino-terminal leader sequences containing residues that differ with respect to their conformational stabilizing effects, charge, and hydrophilicity to examine the mechanism by which they modulate T cell recognition. Our findings indicate that the role of these residues in T cell stimulation is not related to their ability to stabilize alpha-helical secondary structure, nor do they appear to be processed differently. The leader sequences do not differentially influence the ability of the peptides to be presented by APC displaying Ia molecules of related haplotype, i.e., E alpha kE beta k, E alpha kE beta b, and E alpha kE beta s, to T cells which recognize the pigeon cytochrome c determinant on such presenting cells. Because antigenic potency correlates with the inclusion of hydrophobic residues and positively charged residues in the leader sequences, we discuss our findings with reference to the possibility that they non-specifically enhance the interaction of the antigenic peptides with the APC membrane.     

Global phylogeography and genetic diversity of the zoonotic tapeworm Echinococcus granulosus sensu stricto genotype G1

Echinococcus granulosus sensu stricto (s.s.) is the major cause of human cystic echinococcosis worldwide and is listed among the most severe parasitic diseases of humans. To date, numerous studies have investigated the genetic diversity and population structure of E. granulosus s.s. in various geographic regions. However, there has been no global study. Recently, using mitochondrial DNA, it was shown that E. granulosus s.s. G1 and G3 are distinct genotypes, but a larger dataset is required to confirm the distinction of these genotypes. The objectives of this study were to: (i) investigate the distinction of genotypes G1 and G3 using a large global dataset; and (ii) analyse the genetic diversity and phylogeography of genotype G1 on a global scale using near-complete mitogenome sequences. For this study, 222 globally distributed E. granulosus s.s. samples were used, of which 212 belonged to genotype G1 and 10 to G3. Using a total sequence length of 11,682?bp, we inferred phylogenetic networks for three datasets: E. granulosus s.s. (n?=?222), G1 (n?=?212) and human G1 samples (n?=?41). In addition, the Bayesian phylogenetic and phylogeographic analyses were performed. The latter yielded several strongly supported diffusion routes of genotype G1 originating from Turkey, Tunisia and Argentina. We conclude that: (i) using a considerably larger dataset than employed previously, E. granulosus s.s. G1 and G3 are indeed distinct mitochondrial genotypes; (ii) the genetic diversity of E. granulosus s.s. G1 is high globally, with lower values in South America; and (iii) the complex phylogeographic patterns emerging from the phylogenetic and geographic analyses suggest that the current distribution of genotype G1 has been shaped by intensive animal trade.     

Different levels of variability in subtypes 1b and 4a of hepatitis C viruses

We performed genetic and phenic analyses to evaluate nucleotide and amino-acid sequences of the amino-terminus of the E1 protein of HCV genotype 1b (extracted from databank) and 4a (characterised in this study). The non-synonymous (ka) mutation analysis demonstrated that the genome of genotype 1b was not saturated by variations, with a rate of transition/transversion (s/v) of 1.5, which is similar to the expected ratio (i.e., 2.0). The s/v ratio in genotype 4a isolates was lower (0.98), indicating saturation due long-term variability. Moreover, the genotype 1b sequences showed a higher number of ka mutations (s+v) (mean of 2.8 per sequence) than genotype 4a (mean of 1.5). The introduction of ka mutations resulted in a higher degree of amino acid variability in genotype 4a. In the genome of genotype 1b, each nucleotide mutation introduced new amino acids, with a Granthan distance of 3.35-42.5, whereas for genotype 4a the distances ranged from 48.8 to 102.1. The phenic analysis also indicated different and complex patterns of amino-acid substitution. Finally, diverse isoelectric points and hydrophobicity were predicted for the two genotypes, with a higher acidity for genotype 4a E1 proteins.     

Influence of GSTT1, mEH, CYP2E1 and RAD51 polymorphisms on diepoxybutane-induced SCE frequency in cultured human lymphocytes

1,3-Butadiene (BD) is a common chemical in the human environment. Diepoxybutane (DEB) is the most reactive epoxide metabolite of BD. The aim of the present study was to evaluate the influence of polymorphisms in enzymes operating in DEB-metabolism (epoxide hydrolase mEH, CYP2E1 and GSTT1), as well as in the DNA-repair enzyme RAD51, on the frequency of sister chromatid exchange (SCE) induced by DEB in lymphocyte cultures from 63 healthy donors. Their genotypes were determined using PCR and restriction fragment length polymorphism (RFLP)-PCR techniques. The analysis of xenobiotic-metabolizing genes revealed that GSTT1 and CYP2E1 polymorphisms have an influence on DEB-induced SCE frequency. Individuals with the GSTT1 null genotype and CYP2E1 c2 variant allele heterozygotes were observed to have significantly higher SCE frequency than individuals with more common genotypes. A correlation between sensitivity to DEB and GSTT1 null genotype indicates that this pathway is a major detoxification step in DEB metabolism in whole-blood lymphocyte cultures, which has been shown in many studies. The analysis of combined polymorphisms indicated that, in the presence of GSTT1, a significantly higher DEB-induced SCE frequency is observed in the CYP2E1 c2 variant allele heterozygotes than in individuals with the most common CYP2E1 genotype. In the absence of GSTT1, however, the CYP2E1 polymorphism has no influence on DEB-induced SCEs. A significant difference was also observed between individuals characterized by low and high mEH activity, but only in subjects with the GSTT1 null genotype. Lack of GSTT1 resulted in higher SCE frequency in individuals with mEH high-activity genotypes than in individuals with mEH low-activity genotype. In the present study no statistically significant difference in DEB-induced SCEs was observed for the RAD51 polymorphism. The influence of GSTT1 genotype on SCE-frequency in RAD51 variant allele carriers was not analysed as all individuals in this group (except one person) had the GSTT1 gene present. Our study shows that the combined analysis of polymorphisms in metabolizing enzymes may lead to a better understanding of their contribution to an individual's susceptibility to DEB.     

Mechanisms of interaction between human skin fibroblasts and elastin: differences between elastin fibres and derived peptides.

3H-Labelled kappa-elastin peptides (kE:75 kDa molecular weight) were shown to bind to confluent human skin fibroblast (HSF) cultures in a time-dependent and saturable manner. Scatchard analysis indicated the presence of high affinity binding sites with kD = 2.7 x 10(-10) M and 19,000 sites per cell. Binding of kE to its receptor on HSF accelerates and intensifies the adhesion of insoluble elastin fibres (iE) to confluent HSF. Optimal effect was attained for a kE concentration of 0.3 x 10(-9) M close to kD. This stimulatory effect of kE on the binding of iE to HSF could be inhibited by neomycin, retinal and pertussis toxin, substances which act at different levels of the transduction mechanism following the activation of the receptor and the subsequent triggering of cell biological events (chemotaxis, modification of calcium fluxes). The stimulation of iE adhesion to HSF induced by kE as well as kE binding to the cells could be inhibited by lactose and laminin but not by Arg-Gly-Asp-Ser(RGDS) peptides. This indicates that the elastin peptide receptor on HSF possesses lectin-like properties and shares homology with the laminin receptor as also shown for other cell types. None of the substances tested, that is inhibitors of the transduction mechanism, lactose, laminin and Arg-Gly-Asp-Ser(RGDS) peptides were shown to interfere significantly with the binding of iE (in the absence of added kE) to confluent HSF. The proteins adhering strongly to elastin fibres were isolated by a sequential extraction procedure and the final hydrochloride guanidinium-DTT extract was analysed by SDS-PAGE under reducing conditions, Western blots using specific antibodies against several connective tissue proteins and affinity for [3H]-kE following nitrocellulose electro-transfer of proteins. Fibronectin, vitronectin, tropoelastin(s), and a 120 kDa cysteine rich glycoprotein previously designated as elastonectin were identified. Among these proteins, [3H]-kE was found to bind exclusively to a 65 kDa protein that could be eluted selectively from elastin fibres with a neutral buffer containing 100 mM lactose. Therefore the elastin peptide receptor on human skin fibroblasts shares properties with the elastin receptor characterized from other cell types. Conformational differences between elastin peptides and elastin fibres could explain the differences in the mechanisms of interactions between elastin fibres and elastin peptides with HSF in culture. The stimulatory effect of elastin-derived peptides on the adhesion of elastin fibres to HSF could have implications in the oriented biosynthesis of elastin fibres.     

Significance of the glutamate-139 residue of the V-type Na+-ATPase NtpK subunit in catalytic turnover linked with salt tolerance of Enterococcus hirae

A Glu139Asp mutant of the NtpK subunit (kE139D) of Enterococcus hirae vacuolar-type ATPase (V-ATPase) lost tolerance to sodium but not to lithium at pH 10. Purified kE139D V-ATPase retained relatively high specific activity and affinity for the lithium ion compared to the sodium ion. The kE139 residue of V-ATPase is indispensable for its enzymatic activity that is linked with the salt tolerance of enterococci.     

Molecular Characterization of Echinococcus granulosus Cysts in North Indian Patients: Identification of G1, G3, G5 and G6 Genotypes

Background

Cystic echinococcosis (CE) caused by the Echinococcus granulosus, is a major public health problem worldwide, including India. The different genotypes of E. granulosus responsible for human hydatidosis have been reported from endemic areas throughout the world. However, the genetic characterization of E. granulosus infecting the human population in India is lacking. The aim of study was to ascertain the genotype(s) of the parasite responsible for human hydatidosis in North India.

Methodology/Principal Findings

To study the transmission patterns of E. granulosus, genotypic analysis was performed on hydatid cysts obtained from 32 cystic echinococcosis (CE) patients residing in 7 different states of North India. Mitochondrial cytochrome c oxidase subunit1 (cox1) sequencing was done for molecular identification of the isolates. Most of the CE patients (30/32) were found to be infected with hydatid cyst of either G3 (53.1%) or G1 (40.62%) genotype and one each of G5 (cattle strain) and G6 (camel strain) genotype.

Conclusions/Significance

These findings demonstrate the zoonotic potential of G1 (sheep strain) and G3 (buffalo strain) genotypes of E. granulosus as these emerged as predominant genotypes infecting the humans in India. In addition to this, the present study reports the first human CE case infected with G5 genotype (cattle strain) in an Asian country and presence of G6 genotype (camel strain) in India. The results may have important implications in the planning of control strategies for human hydatidosis.     

High Prevalence of Hepatitis C Virus Genotype 1b Infection in a Small Town of Argentina. Phylogenetic and Bayesian Coalescent Analysis

Previous studies in Argentina have documented a general prevalence of Hepatitis C Virus (HCV) infection close to 2%. In addition, a high prevalence of HCV has been recently reported in different Argentinean small rural communities. In this work, we performed a study aimed at analyzing the origins and diversification patterns of an HCV outbreak in Wheelwright, a small rural town located in Santa Fe province (Argentina).A total of 89 out of 1814 blood samples collected from people living in Wheelwright, were positive for HCV infection. The highest prevalence (4.9%) was observed in people older than 50 years, with the highest level for the group aged between 70–79 years (22%). The RFLP analyses showed that 91% of the positive samples belonged to the HCV-1b genotype. The E1/E2 and NS5B genes were sequenced, and their phylogenetic analysis showed that the HCV-1b sequences from Wheelwright were monophyletic. Bayesian coalescent-based methods were used to estimate substitution rates and time of the most recent common ancestor (tMRCA). The mean estimated substitution rates and the tMRCA for E1/E2 with and without HVR1 and NS5B were 7.41E-03 s/s/y and 61 years, 5.05E-03 s/s/y and 58 years and 3.24E-03 s/s/y and 53 years, respectively. In summary, the tMRCA values, the demographic model with constant population size, and the fact that the highest prevalence of infection was observed in elder people support the hypothesis that the HCV-1b introduction in Wheelwright initially occurred at least five decades ago and that the early epidemic was characterized by a fast rate of virus transmission. The epidemic seems to have been controlled later on down to the standard transmission rates observed elsewhere.     

Incompatibility properties of Col E1 and pMB1 derivative plasmids: Random replication of multicopy replicons

The incompatibility properties of Col E1-like plasmids have been examined in Rec⁺ and RecA^? bacteria. Two Col E1- (or two pMB1-) derivative plasmids coreplicated in the same clone for many cell doublings, irrespective of the rec genotype of host bacteria. Their kinetics of segregation were found to be consistent with models that assume a random choice of template molecule for each plasmid replication event, but with models based on a single (master) template molecule per cell. In contrast, minimal coreplication of a Col E1- and a pMB1-derivative plasmid occurred, with the latter type rapidly excluding the former. We suggest here that the pMB1 derivatives, pMB9 and pBR322, are less sensitive than Col E1 derivatives to the putative inhibitor that regulates plasmid replication, due to base sequence differences in their target for the inhibitor, and consider one mechanism whereby the duplication of Col E1-like plasmids might be regulated.     

Expression of cytochrome P4502E1 in human liver: Relationship between genotype and phenotype in Chinese

Polymorphic cytochrome P4502E1 (CYP2E1) plays an important role in the metabolic activation of many carcinogens. We have previously shown that the c1/c1 genotype recognized byRsa I in the 5′-regulatory region of theCYP2E1 may be a susceptibility factor for developing esophageal cancer and lung cancer in Chinese. The present study was to investigate the relationship between theRsa I genotype and the expression of CYP2E1 in human livers. A total of 50 liver specimens were genotyped forCYP2E1 and assayed for CYP2E1 protein contents and functional activity by using specific antibody in immunoblot and a probe substrate,p-nitrophenol. A considerable interindividual variation in CYP2E1 protein (20-fold) and functional activity (56-fold) was observed among these liver samples. However, when they were categorized according to genotype, the mean content of CYP2E1 protein was significantly higher among individuals with the c1/c1 genotype than that among those having c1/c2 or c2/c2 genotype [124.0±83.9 pmol/mg (n = 28) versus 65.5 ±38.9 pmol/mg (n = 22),P<0.01]. The mean activity of CYP2E1 towardsp-nitrophenol for the c1/c1 genotype was also higher than that for the variant genotypes (198.4±27.8 pmol/min/mg versus 101.2 ±18.1 pmol^-1 · min^-1 · mg^-1,P<0.01). Also, the protein levels and functional activity showed a significant correlation (r = 0.68,P<0.01). These results demonstrate an association between theRsa I genotype and the phenotype of CYP2E1 in our samples, and the data are compatible with the assumption thatCYP2E1 c1/c1 genotype is a susceptibility factor for certain cancers in Chinese.     

Expression of cytochrome P4502E1 in human liver: Relationship between genotype and phenotype in Chinese

Polymorphic cytochrome P4502E1 (CYP2E1) plays an important role in the metabolic activation of many carcinogens. We have previously shown that the c1/c1 genotype recognized by Rsa I in the 5'-regulatory region of the CYP2E1 may be a susceptibility factor for developing eso-phageal cancer and lung cancer in Chinese. The present study was to investigate the relationship between the Rsa I genotype and the expression of CYP2E1 in human livers. A total of 50 liver specimens were genotyped for CYP2E1 and assayed for CYP2E1 protein contents and functional activity by using specific antibody in immunoblot and a probe substrate, p-nitrophenol. A considerable interindividual variation in CYP2E1 protein (20-fold) and functional activity (56-fold) was observed among these liver samples. However, when they were categorized according to genotype, the mean content of CYP2E1 protein was significantly higher among individuals with the c1/c1 genotype than that among those having c1/c2 or c2/c2 genotype [124.0 ±83.9 pmol/     

The Effect of Vaccination on the Evolution and Population Dynamics of Avian Paramyxovirus-1

Newcastle Disease Virus (NDV) is a pathogenic strain of avian paramyxovirus (aPMV-1) that is among the most serious of disease threats to the poultry industry worldwide. Viral diversity is high in aPMV-1; eight genotypes are recognized based on phylogenetic reconstruction of gene sequences. Modified live vaccines have been developed to decrease the economic losses caused by this virus. Vaccines derived from avirulent genotype II strains were developed in the 1950s and are in use globally, whereas Australian strains belonging to genotype I were developed as vaccines in the 1970s and are used mainly in Asia. In this study, we evaluated the consequences of attenuated live virus vaccination on the evolution of aPMV-1 genotypes. There was phylogenetic incongruence among trees based on individual genes and complete coding region of 54 full length aPMV-1 genomes, suggesting that recombinant sequences were present in the data set. Subsequently, five recombinant genomes were identified, four of which contained sequences from either genotype I or II. The population history of vaccine-related genotype II strains was distinct from other aPMV-1 genotypes; genotype II emerged in the late 19^th century and is evolving more slowly than other genotypes, which emerged in the 1960s. Despite vaccination efforts, genotype II viruses have experienced constant population growth to the present. In contrast, other contemporary genotypes showed population declines in the late 1990s. Additionally, genotype I and II viruses, which are circulating in the presence of homotypic vaccine pressure, have unique selection profiles compared to nonvaccine-related strains. Collectively, these data show that vaccination with live attenuated viruses has changed the evolution of aPMV-1 by maintaining a large effective population size of a vaccine-related genotype, allowing for coinfection and recombination of vaccine and wild type strains, and by applying unique selective pressures on viral glycoproteins.     

CYP1A1, GSTM1 and GSTT1 polymorphisms and lung cancer: a pooled analysis of gene-gene interactions

Gene-environment interactions have been extensively studied in lung cancer. It is likely that several genetic polymorphisms cooperate in increasing the individual risk. Therefore, the study of gene-gene interactions might be important to identify high-susceptibility subgroups. GSEC is an initiative aimed at collecting available data sets on metabolic polymorphisms and the risks of cancer at several sites and performing pooled analyses of the original data. Authors of published papers have provided original data sets. The present paper refers to gene-gene interactions in lung cancer and considers three polymorphisms in three metabolic genes: CYP1A1, GSTM1 and GSTT1. The present analyses compare the gene-gene interactions of the CYP1A1*2A, GSTM1 and GSTT1 polymorphisms from studies on lung cancer conducted in Europe and the USA between 1991 and 2000. Only Caucasians have been included. The data set includes 1466 cases and 1488 controls. The only clear-cut association was found with CYP1A1*2A. This association remained unchanged after stratification by polymorphisms in other genes (with an odds ratio [OR] of approximately 2.5), except when interaction with GSTM1 was considered. When the OR for CYP1A1*2A was stratified according to the GSTM1 genotype, the OR was increased only among the subjects who had the null (homozygous deletion) GSTM1 genotype (OR=2.8, 95% CI=0.9-8.4). The odds ratio for the interactive term (CYP1A1*2A by GSTM1) in logistic regression was 2.7 (95% CI=0.5-15.3). An association between lung cancer and the homozygous CYP1A1*2A genotype is confirmed. An apparent and biologically plausible interaction is suggested between this genotype and GSTM1.     

Genetic diversity of Echinococcus granulosus sensu stricto in Sardinia (Italy)

Cystic echinococcosis (CE) is a severe parasitic zoonosis caused by the metacestode of the tapeworm Echinococcus granulosus sensu lato (s.l.). The disease has a global distribution representing a significant public health concern. Based on mitochondrial DNA analysis E. granulosus s.l. has been subdivided into five species: E. granulosus sensu stricto (s.s.) (G1, G3 genotype), E. equinus (G4 genotype), E. ortleppi (G5 genotype), E. canadensis (G6-G8, G10 genotype) and E. felidis. E. granulosus s.s., and in particular G1, is the most widespread genotype and the major responsible of human CE cases worldwide. In Italy G1 genotype is higly represented with larger percentages in some hyperendemic areas such as Sardinia. Molecular studies represent a valuable tool to improve our understanding of the E. granulosus epidemiology and CE control strategies. In the present study we investigated genetic variability of E. granulosus s.s. in Sardinia. To this purpose 83 hydatid cysts were collected from different animal species including humans and the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was partially sequenced (720 bp). Nucleotide sequences from Mediterranean basin were also analyzed for comparison. The phylogenetic network revealed 30 haplotypes grouped around a predominant isolate that had been already reported from other world regions. Haplotype diversity (0.8495 ± 0.0336) and nucleotide diversity (0.003305 ± 0.002014) were similar in Sardinia respect to other Mediterranean countries. Neutrality indices obtained by Tajima's D and Fu's Fs test were significantly negative (p ≤ .01) suggesting expansion of Sardinian population. Low Fixation indices (Fst), ranging from negative values (Algeria, Greece, Spain, other part of Italy) to 0.089 (Albania, France), indicated absence of genetic differentiation, and gene flow between Sardinia and other Mediterranean countries.     

Genetic polymorphism of cytochrome P450 2E1 in the Turkish population.

CYP 2E1 is involved in metabolic activation of carcinogenic N-nitrosamines, benzene, urethane and other low molecular weight compounds. CYP2E1 gene is present in the population in various polymorphic forms. We detected the RFLP of the human CYP2E1 gene with the restriction endonuclease PstI, RsaI and DraI in a group of 153 Turkish individuals. According to the results of the PstI/RsaI analysis, 96.07% of the subjects were of the c1/c1 genotype, and 3.93% were of the c1/c2 genotype. In the DraI RFLP analysis, 84.30% DD genotype, 15.03% CD genotype and 0.66% CC genotype were determined. The data obtained may be useful in epidemiological studies of the influence of CYP2E1 polymorphism on carcinogenesis.     

Synergistic effect between apolipoprotein E and apolipoprotein A1 gene polymorphisms in the risk for coronary artery disease

Alterations in lipid metabolism and genetic predisposition are major risk factors for coronary artery disease (CAD). Variations in genes involved in lipid metabolism may act synergistically to confer risk or protection against CAD. The objective of the present study was to determine such interactions in variants of apolipoprotein E and apolipoprotein A1 genes. One hundred and forty subjects with clinically confirmed CAD and 100 unrelated normal subjects participated in the study. Multiple regression analysis was used to relate lipid and apolipoprotein profiles with genotypes. Odd ratios were calculated for various combinations of ApoE and ApoA1 genotypes. Prevalence of ApoE 'E4' and ApoA1 'A' and 'T' alleles was significantly higher in patients than controls. Serum apolipoprotein E and apolipoprotein A1 levels were significantly lower in CAD patients than controls. When lipid parameters were related to genotypes, the polymorphisms associated to various markers were in agreement with previous reports. ApoE 2/4 genotype in combination with either ApoA1 heterozygous GA or CT genotype conferred higher risk of CAD. E3 allele in homozygous or heterozygous state in combination with ApoA1+83 CC genotype conferred highest protection (P < 0.05). Thus, it appears that ApoE and ApoA1 gene variants may act synergistically to associate with risk and protection against CAD.