James E. Hixson

Genetic and environmental contributions to cardiovascular risk factors in Mexican Americans: The San Antonio Family Heart Study

BACKGROUND The familial aggregation of coronary heart disease can be in large part accounted for by a clustering of cardiovascular disease risk factors. To elucidate the determinants of cardiovascular disease, many epidemiological studies have focused on the behavioral and lifestyle determinants of these risk factors, whereas others have examined whether specific candidate genes influence quantitative variation in these phenotypes. METHODS AND RESULTS Among Mexican Americans from San Antonio (Tex), we quantified the relative contributions of both genetic and environmental influences to a large panel of cardiovascular risk factors, including serum levels of lipids, lipoproteins, glucose, hormones, adiposity, and blood pressure. Members of 42 extended families were studied, including 1236 first-, second-, and third-degree relatives of randomly ascertained probands and their spouses. In addition to the phenotypic assessments, information was obtained regarding usual dietary and physical activity patterns, medication use, smoking habits, alcohol consumption, and other lifestyle behaviors and medical factors. Maximum likelihood methods were used to partition the variance of each phenotype into components attributable to the measured covariates, additive genetic effects (heritability), household effects, and an unmeasured environmental residual. For the lipid and lipoprotein phenotypes, age, gender, and other environmental covariates accounted in general for < 15% of the total phenotypic variance, whereas genes accounted for 30% to 45% of the phenotypic variation. Similarly, genes accounted for 15% to 30% of the phenotypic variation in measures of glucose, hormones, adiposity, and blood pressure. CONCLUSIONS These results highlight the importance of considering genetic factors in studies of risk factors for cardiovascular disease.

Apolipoprotein E polymorphisms affect atherosclerosis in young males. Pathobiological Determinants of Atherosclerosis in Youth (PDAY) Research Group.

Investigators in eight communities collected aortas, right coronary arteries, blood and liver samples, and associated information from 720 young males, aged 15-34 years, who died of external causes. Genotypes for apolipoprotein (apo) E isoforms (E2, E3, and E4) were determined from hepatic DNA by restriction isotyping (restriction enzyme isoform genotyping) of amplified apo E sequences. Pathologists graded the arteries for atherosclerotic lesions, and a central laboratory measured lipoprotein cholesterol concentrations. Allele frequencies were different between blacks and whites (p less than 0.0001). E2 and E4 frequencies were higher and E3 frequency was lower in blacks than in whites. Among the common genotypes in both whites and blacks, E2E3 heterozygotes had the lowest levels of total serum cholesterol and low density lipoproteins, E3E4 had the highest levels, and E3E3 had intermediate levels. Apo E genotypes differed in mean percent surface area involvement with lesions in the thoracic aorta and the abdominal aorta of both whites and blacks (p less than or equal to 0.0002). Among the common genotypes, E2E3 heterozygotes had the least involvement of both thoracic and abdominal aortas with lesions, E3E4 had the greatest involvement (with the exception of the thoracic aorta in whites), and E3E3 had intermediate involvement. Apo E genotype accounted for 5.7% in whites and 5.9% in blacks of the observed variation in lesions for the thoracic aorta, and for 5.9% in whites and 7.0% in blacks for the abdominal aorta. Adjusting for cholesterol levels did not change apo E genotypic effects appreciably, an observation suggesting that genotypic effects on arterial lesions may not be mediated entirely by changes in serum cholesterol concentrations.

Human Pedigree-Based Quantitative-Trait–Locus Mapping: Localization of Two Genes Influencing HDL-Cholesterol Metabolism

Common disorders with genetic susceptibilities involve the action of multiple genes interacting with each other and with environmental factors, making it difficult to localize the specific genetic loci responsible. An important route to the disentangling of this complex inheritance is through the study of normal physiological variation in quantitative risk factors that may underlie liability to disease. We present an analysis of HDL-cholesterol (HDL-C), which is inversely correlated with risk of heart disease. A variety of HDL subphenotypes were analyzed, including HDL particle-size classes and the concentrations and proportions of esterified and unesterified HDL-C. Results of a complete genomic screen in large, randomly ascertained pedigrees implicated two loci, one on chromosome 8 and the other on chromosome 15, that influence a component of HDL-C-namely, unesterified HDL2a-C. Multivariate analyses of multiple HDL phenotypes and simultaneous multilocus analysis of the quantitative-trait loci identified permit further characterization of the genetic effects on HDL-C. These analyses suggest that the action of the chromosome 8 locus is specific to unesterified cholesterol levels, whereas the chromosome 15 locus appears to influence both HDL-C concentration and distribution of cholesterol among HDL particle sizes.

A paired sibling analysis of the beta-3 adrenergic receptor and obesity in Mexican Americans.

The beta3 adrenergic receptor, located on chromosome 8, is a regulator of energy expenditure and lipolysis. A missense mutation in this gene, characterized by the replacement of tryptophan by arginine at codon 64 (Trp64Arg), is associated with obesity in some studies. We examined the effect of this variant on obesity in Mexican Americans, using a paired sibling design to minimize variability due to genetic background and a previously identified major susceptibility locus for obesity. We identified 45 sib-pairs that were concordant (identical by descent) for a locus on chromosome 2 which we have shown previously to be tightly linked to obesity in this population. The Trp64Arg variant, detected by PCR-restriction fragment length polymorphism analysis, was present in one sibling within each of the 45 sib-pairs. Presence of the variant was associated with significantly higher values in body mass index (P = 0.04), fat mass (P = 0.04), and waist circumference (P = 0.05). We conclude that the Trp64Arg variant is associated with obesity in this Mexican American population. The paired sibling design probably enhanced our ability to detect the effects of this variant by allowing us to account for variation attributable to another obesity susceptibility locus and to background genes.

A Genome Search Identifies Major Quantitative Trait Loci on Human Chromosomes 3 and 4 That Influence Cholesterol Concentrations in Small LDL Particles

Small, dense LDL particles are associated with increased risk of cardiovascular disease. To identify the genes that influence LDL size variation, we performed a genome-wide screen for cholesterol concentrations in 4 LDL size fractions. Samples from 470 members of randomly ascertained families were typed for 331 microsatellite markers spaced at approximately 15 cM intervals. Plasma LDLs were resolved by using nondenaturing gradient gel electrophoresis into 4 fraction sizes (LDL-1, 26.4 to 29.0 nm; LDL-2, 25.5 to 26.4 nm; LDL-3, 24.2 to 25.5 nm; and LDL-4, 21.0 to 24.2 nm) and cholesterol concentrations were estimated by staining with Sudan Black B. Linkage analyses used variance component methods that exploited all of the genotypic and phenotypic information in the large extended pedigrees. In multipoint linkage analyses with quantitative trait loci for the 4 fraction sizes, only LDL-3, a fraction containing small LDL particles, gave peak multipoint log10 odds in favor of linkage (LOD) scores that exceeded 3.0, a nominal criterion for evidence of significant linkage. The highest LOD scores for LDL-3 were found on chromosomes 3 (LOD=4.1), 4 (LOD=4.1), and 6 (LOD=2.9). In oligogenic analyses, the 2-locus LOD score (for chromosomes 3 and 4) increased significantly (P=0.0012) to 6.1, but including the third locus on chromosome 6 did not significantly improve the LOD score (P=0.064). Thus, we have localized 2 major quantitative trait loci that influence variation in cholesterol concentrations of small LDL particles. The 2 quantitative trait loci on chromosomes 3 and 4 are located in regions that contain the genes for apoD and the large subunit of the microsomal triglyceride transfer protein, respectively.

Baboons as an animal model for genetic studies of common human disease

In recent years, human genetic research has made draprospects for the use of baboons in the investigation of the genetics of common human diseases. matic advances in the identification of molecular defects that cause rare inherited disorders. However, today’s major challenge is the identification of genes that predispose General Characteristics of the Baboon Model for individuals to the common conditions that generate the Genetic Studies overwhelming majority of health problems and costs, inBaboons (genus Papio) belong to the larger taxonomic cluding heart disease, diabetes, osteoporosis, obesity, and grouping Old World monkeys (superfamily Cercopihypertension. Identifying the genetic basis of these disorthecoidea). The genetic similarity between baboons and ders is proving to be exceedingly difficult, because comhumans, evident at the level of overall DNA sequence mon diseases result from the complex actions and interac(Caccone and Powell 1989), the sequences of specific tions of many different genes and are strongly influenced genes (see below), and the arrangement of genetic loci by environmental factors such as nutrition and exercise. on chromosomes (Graves et al. 1995; Perelygin et al. Controlling, or even measuring, these environmental ef1996) reflect the close evolutionary relationship between fects in large studies of human subjects is daunting, althe two species. In addition, baboons and humans share though the influence of these variables may equal or exa broad range of physiological similarities that distinceed the effect that individual genes have on the guish us from other animal models and that make baphenotype of interest. Many common diseases are genetiboons particularly valuable for analyses of gene-gene cally heterogeneous, so that mutations in different genes and gene-environment interaction (Blangero 1993; Vanor combinations of genes may yield similar disease phenodeBerg and Williams-Blangero 1996). For example, types (phenocopies) in different families or populations. aging baboons exhibit a natural menopause that is not For these reasons, the research strategies that were succharacteristic of most other laboratory animals (Carey cessful for single-gene disorders may not suffice to idenand Rice 1996). Baboons, humans, and other anthrotify the genes involved in these complex diseases. poid primates also share important characteristics with Primate models offer a complementary approach for respect to neurophysiological function. There is a subgenetic studies of common human diseases. Baboons stantial literature describing behavior, temperament, (Papio hamadryas) provide an excellent model for genetic and various aspects of neurophysiological function in studies, because they exhibit the same physiological charprimates (e.g., see Kaplan et al. 1995; Higley et al. acteristics that are critical to common diseases in humans 1993), yet few studies have incorporated genetic variand because both breeding and environmental factors can ability into investigations of primate neurophysiology. be carefully controlled to suit experimental purposes. FurPedigreed primates will provide a unique opportunity thermore, recent advances toward a complete genetic linkfor such research in neurogenetics (see Palmour et al. age map of the baboon genome enable genomewide 1997 [in this issue]). searches to identify novel genes that represent risk factors Common human diseases result from the interaction for common disease. Here, we present a brief summary of inherited genetic constitution and environmental facof past applications, current research efforts, and future tors such as diet, exposure to pathogens, and exercise. Studies of the interaction of genes and environment are very difficult to conduct in human subjects because it is Received June 25, 1997; accepted for publication July 16, 1997. almost impossible to control or measure environmental Address for correspondence and reprints: Dr. Jeffrey Rogers, Department of Genetics, Southwest Foundation for Biomedical Research, conditions accurately over substantial periods of time. P.O. Box 760549, San Antonio, TX 78245-0549. In contrast, baboons can be maintained for generations This article represents the opinion of the authors and has not been in carefully controlled environments. For example, meapeer reviewed. suring serum cholesterol in hundreds of animals exposed 1997 by The American Society of Human Genetics. All rights reserved. 0002-9297/97/6103-0005

A Major Locus Influencing Plasma High-Density Lipoprotein Cholesterol Levels in the San Antonio Family Heart Study: Segregation and Linkage Analyses

02.00 long term to a low-fat, low-cholesterol diet and then

Apo B insertion/deletion polymorphisms are associated with atherosclerosis in young black but not young white males. Pathobiological Determinants of Atherosclerosis in Youth (PDAY) Research Group.

To detect and measure the effects of a single locus on quantitative variation in plasma concentrations of HDL cholesterol (HDL-C), we conducted statistical genetic analyses on data from 526 Mexican American individuals in 25 randomly ascertained pedigrees. By using maximum-likelihood complex segregation analysis, we found evidence for a major locus with a codominant mixture model that included the phenotypic means, standard deviations, relative frequency of a low HDL-C allele, and heritability for plasma HDL-C levels, plus the effects of sex (genotype specific), age-by-sex, age2-by-sex, plasma concentrations of apolipoprotein (apo)AI and triglycerides (genotype specific), exogenous sex hormone use, and menopausal status under an unrestricted general model. Inclusion of the four covariates (in addition to the sex and age-by-sex effects) accounted for nearly 79% of the variance in total plasma HDL-C levels. Of the remaining 21% of the variance, the detected major locus accounted for approximately 55% in men and 21% in women; the total genetic contributions to the variance by genes were approximately 82% in men and 69% in women. Linkage analyses with penetrance parameter estimates from the segregation analysis excluded tight linkage between the detected major locus and markers for the following candidate loci: the apoAI/apoCIII genomic region (P < .05), apoB (P < .01), hepatic lipase (P < .001), lipoprotein lipase (P < .001), and the LDL receptor (P < .001). While not excluding the apoE locus (LOD = -0.348, P < .21), the analysis provided no support for tight linkage between it and the detected major locus.

Evidence for a Major Gene Affecting Postchallenge Insulin Levels in Mexican-Americans

Investigators in eight communities collected aortas, right coronary arteries, blood and liver samples, and associated information from 872 young males, aged 15-34 years, who died of external causes. Pathologists graded the arteries for atherosclerotic lesions, and a central laboratory measured lipoprotein cholesterol concentrations. Apolipoprotein (apo) B sequences were amplified in hepatic DNA samples to determine genotypes for length polymorphisms in the signal peptide of apo B. In addition to the insertion (ins) allele (27-amino acid signal peptide) and the deletion (del) allele (24 amino acids), we detected a rare allele (ins*) in whites with an in-frame insertion of two Leu codons in a region that normally contains six Leu codons. The frequency for the apo B del allele was lower in blacks than in whites (p less than 0.0001). In blacks, homozygotes for the ins allele had the lowest levels of serum cholesterol and very low plus low density lipoprotein cholesterol (VLDL + LDL-C), homozygotes for the del allele had the highest levels, and heterozygotes had intermediate levels (p = 0.0509 for cholesterol, p = 0.0530 for VLDL + LDL-C), but no differences were found in whites. In blacks, homozygotes for the ins allele had the least involvement of the thoracic and the abdominal aorta with lesions, homozygotes for the del allele had the greatest involvement, and heterozygotes had intermediate involvement (p = 0.0328 for thoracic aorta, p = 0.0104 for abdominal aorta), but no differences were found in whites.(ABSTRACT TRUNCATED AT 250 WORDS)

Two Major Loci Control Variation in β-Lipoprotein Cholesterol and Response to Dietary Fat and Cholesterol in Baboons

Hyperinsulinemia, which is considered a hallmark of insulin resistance, precedes the development of non-insulin-dependent diabetes mellitus (NIDDM). Results of family and twin studies have shown that heredity influences insulin resistance and insulin levels. In Caucasian families ascertained through two or more NIDDM siblings, it has been reported that single genes with large effects, i.e., major genes, influence both fasting and 1-h postchallenge insulin levels. To determine whether a major gene affects 2-h postchallenge insulin levels in Mexican-Americans, we conducted segregation analyses using data collected on 527 pedigreed individuals from 27 families in San Antonio, TX. Probands for the families were randomly ascertained and all first-, second-, and third-degree relatives aged 16 years and older were invited to participate. Subjects received a 2-h oral glucose tolerance test, and diabetes was diagnosed according to World Health Organization criteria. We found that an autosomal dominant major gene best described the inheritance of 2-h insulin levels (ln-transformed) in these 27 families. Of the individuals in the population, 17% were homozygous for the 2-h low-insulin allele (back-transformed mean = 125 pmol/l) and 83% were heterozygous or homozygous for the 2-h high-insulin allele (back-transformed mean = 406 pmol/l). This major gene accounted for 31% of the variance in ln(2-h insulin levels) in this population. Using quantitative trait linkage analyses, we excluded tight linkage between this gene affecting 2-h insulin levels and three candidate loci for insulin levels: the insulin receptor gene, the low-density lipoprotein receptor gene, and the glucokinase gene. In addition, it is likely that this major gene is not linked to the fatty acid-binding protein gene, a gene that has previously been associated with insulin resistance through sib-pair analysis.