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Non-insulin-dependent diabetes mellitus, or type 2 diabetes (T2DM), has become a major public health problem in India. The high prevalence, relatively young age of onset, and strong familial aggregation of T2DM in some Indian communities remains to be explained. Many of the traditional risk factors established for European populations do not appear to be present in Asian Indians. Phase I of the Sikh Diabetes Study (SDS) was launched to build the population resources required to initiate a large-scale genetic epidemiological study of diabetes in an Asian Indian population. The SDS is focused on the Khatri Sikh population of North India. In all, 1,892 subjects were enrolled to participate in the family-based study; 1,623 of these subjects belong to 324 families, each of which has at least 2 siblings affected with T2DM. The sample included 1,288 individuals affected with T2DM (siblings, parents, or relatives) and 335 unaffected siblings, parents, or relatives. The remaining 269 subjects were unrelated nondiabetic control subjects, including unaffected spouses of probands or siblings. This primarily nonvegetarian, nonsmoking endogamous caste group has presented an unusual clinical picture of uneven distribution of adiposity and a high rate of T2DM with secondary complications. Such well-characterized population isolates may offer unique advantages in mapping genes for common complex diseases.

Elevated plasma lipoprotein(a) [Lp(a)] level has been established as an independent risk factor for atherosclerosis and coronary heart disease. Considerable ethnic group differences in the distribution of plasma Lp(a) levels have raised public health concerns. Recently, we have reported that Samoans have the lowest plasma Lp(a) levels of any population group. In the present investigation, we report the contribution of two apolipoprotein(a) (APOA) polymorphisms, the kringle 4 type 2 (K4) repeat and the pentanucleotide repeat (PNR), in affecting plasma Lp(a) levels in an American Samoan sample (n = 309). The K4 repeats ranged in size from 15 to 40. The common alleles contained repeats ranging from 26 to 36 with allele frequencies between 5.5% to 9.7%, and these accounted for 82% of all alleles. An inverse relationship between K4 repeat number and plasma Lp(a) level was observed for single-banded (r = -0.59, p = 0.0001) and double-banded phenotypes (r = -0.50, p = 0.0001). This polymorphism explained 60% of the variation in plasma Lp(a) level in American Samoans. For the PNR polymorphism, five different repeat alleles and eight different genotypes were identified; the most common allele was eight repeats. The *8 PNR allele was associated with a wide range of K4 repeats, the *9 PNR allele with larger K4 repeats (25-40), and the *10 PNR with smaller K4 repeats (15-24). Analysis of variance (ANOVA) revealed that the PNR polymorphism accounts for 2.1% of the variability in plasma Lp(a) levels in this sample, when the K4 repeat polymorphism was taken into account. Our data show that common polymorphisms in the APOA gene are major determinants of plasma Lp(a) variation in American Samoans.

Elevated levels of plasma lipoprotein(a) [Lp(a)] are thought to be a risk factor for atherosclerosis and coronary heart disease. Plasma levels of Lp(a) are highly variable between individuals, ranging from 0.1 mg/dL to > 100 mg/dL These levels are under strict genetic control, and genetic variation in the apolipoprotein A (APOA) gene accounts for almost all variation in Lp(a) levels. In this study, we investigated the relationship between two APOA polymorphisms (kringle 4 and 5' pentanucleotide repeat) and plasma Lp(a) levels in normoglycemic non-Hispanic Whites (NHWs) (n = 390) and Hispanics (n = 214) from the San Luis Valley, Colorado. Mean (+/- SD) and median Lp(a) levels were 9.6 +/- 12.5 mg/dL and 3.8 mg/dL, respectively, in NHWs and 12.1 +/- 15.6 mg/dL and 4.9 mg/dL, respectively, in Hispanics. The number of observed kringle 4 repeats ranged from 11 to 38 in NHWs and from 10 to 41 in Hispanics. Spearman rank correlation revealed an inverse relationship between the size of the kringle 4 repeat and plasma Lp(a) levels in both populations (r = -0.38; p < 0.0001 in NHWs and r = -0.64; p < 0.0001 in Hispanics). About 30% and 48% of the variation in plasma Lp(a) was explained by this polymorphism in NHWs and Hispanics, respectively. This study confirms that the kringle 4 polymorphism in the APOA gene is a significant determinant of Lp(a) levels in both study groups. A pentanucleotide repeat polymorphism in the 5' promoter region of the APOA gene did not show significant impact on plasma Lp(a) levels in either NHWs or Hispanics.

Plasma lipoprotein(a) [Lp(a)]-consisting of a disulfide-linked complex of apolipoprotein B and apolipoprotein (a)--levels are considered to be an independent risk factor for coronary heart disease. There are considerable ethnic group differences in the distribution of plasma Lp(a) levels that raise public health concerns. Although plasma Lp(a) distribution has been determined in various ethnic groups, no such information is available in Pacific Islanders. In this study we have determined the distribution and correlates of plasma Lp(a) in population-based samples of 361 American Samoans (145 men, 216 women) and 560 Western Samoans (265 men, 295 women), aged 20-70 years. Plasma Lp(a) levels were measured using a commercial enzyme-linked immunosorbent assay. The distribution of plasma Lp(a) levels in both groups was highly skewed with 73% and 65% of values in the 0-5 mg/dl range in American Samoans and Western Samoans, respectively. The mean (6.4 mg/dl) and median (2.2 mg/dl) Lp(a) levels in pooled Samoans were significantly lower when compared with other ethnic groups using the same measurement kit. Plasma Lp(a) correlated significantly with total and LDL cholesterol in both genders after correcting for the contribution of Lp(a) cholesterol, and with apolipoprotein B in women after the correction for Lp(a)-apoB, but not with age, smoking, alcohol intake, or body mass index. Our data show that Samoans, Polynesians of Pacific Islands, have strikingly lower Lp(a) levels than all other reported population groups. These data are consistent with the hypothesis that genetic factors account for interethnic group variation in plasma Lp(a) levels.

Apolipoprotein J (apoJ, protein; APOJ, gene) is found in serum associated with high-density lipoprotein (HDL) subfractions, which also contain apolipoprotein A-I (apoA1) and cholesteryl ester transfer protein. ApoJ has been shown to be involved in a variety of physiological functions, including lipid transport. In earlier studies we reported the existence of a common genetic polymorphism (APOJ*1 and APOJ*2 alleles) using isoelectric focusing (IEF) and immunoblotting. In this study we determined the molecular basis of this polymorphism and together with another polymorphism at codon 328 (G-->A) evaluated its relationship with serum HDL cholesterol and apoA1 levels in 767 African blacks stratified by staff level: junior (less affluent, n = 450) and senior (more affluent, n = 317). The molecular analysis of the cathodally shifted APOJ*2 allele on IEF gels revealed an amino acid substitution of asparagine by histidine resulting from a missense mutation (A-->C) at codon 317 in exon 7. The frequency of the APOJ*2 (C) allele of codon 317 in the total sample was 0.267, whereas that of the less common allele A of codon 328 was 0.04. Despite their close proximity, no linkage disequilibrium was observed between the 2 polymorphisms. The impact of the codon 317 polymorphic variation was significant on serum HDL cholesterol (p = 0.003) and HDL3 cholesterol (p = 0.001) in junior staff. The adjusted mean values of these traits were higher in the codon 317 APOJ*2/*2 genotype than in the *1/*1 and *1/*2 genotypes. Overall, the APOJ codon 317 polymorphism explained 10.2% and 8.3% of the phenotypic variation in HDL cholesterol and HDL3 cholesterol, respectively, in junior staff. The codon 328 polymorphism showed a significant effect on HDL2 cholesterol (p = 0.039) and apoA1 (p = 0.007) only in junior women and accounted for 2.5% and 4.2% of the phenotypic variation in HDL2 cholesterol and apoA1, respectively. We also analyzed the combined effects of these genotypes at the 2 polymorphic sites. Significant effects on HDL cholesterol (p = 0.004) and HDL3 cholesterol (p = 0.008) in junior men and on HDL2 cholesterol (p = 0.003) in junior women were observed in the combined genotype data. The 2-locus genotypes explained 6.0% and 5.3% of the residual phenotypic variation of HDL cholesterol and HDL3 cholesterol in junior men and 10.4% of HDL2 cholesterol in junior women. These data indicate that the effect of the APOJ polymorphism on HDL cholesterol levels is modulated by socioeconomic status, as measured by staff level. Given the association of HDL and its subfractions with cardiovascular disease, these polymorphisms may lead to a better understanding of interracial differences in the risk of cardiovascular disease.

Several epidemiological studies have established that elevated serum lipoprotein (a) [Lp(a)] levels are independent risk factors for coronary heart disease, stroke, and restenosis of coronary lesions in white and Asian populations. Serum Lp(a) levels vary over a 1000-fold range among individuals and are under strict genetic control. Serum Lp(a) levels are significantly higher in populations with African ancestry than in populations of European ancestry. The APOA gene exhibits hypervariable length polymorphism resulting from a variable number of expressed kringle 4 repeats. An inverse relationship exists between the size of kringle 4 repeats and serum Lp(a) levels. However, most studies have been conducted in whites, and the data are scanty in African populations. To explore this relationship among Africans, we determined serum Lp(a) levels and APOA Kringle 4 size polymorphisms in 781 unrelated Africans (490 men, 291 women) from Benin City, Nigeria. Mean and median serum Lp(a) values were 25.6 +/- 0.6 mg/dl and 20.9 mg/dl, respectively. Although there was no difference in mean Lp(a) values between men and women, median Lp(a) values were higher in women than in men (p = 0.02). Using SDS-agarose gel electrophoresis, we detected 38 APOA isoforms, the highest number recorded to data. There were 10 consecutive medium-size alleles whose frequencies ranged between 4.2% and 10.9%, and together they accounted for 72.8% of the alleles observed in this population. Spearman's correlation coefficients showed an inverse relationship between the size of the APOA isoform and Lp(a) levels using either single-banded (r = 0.46; p < 0.0001) or double-banded (r = 0.42; p < 0.0001) phenotypes. Using random effects analysis of variance on the entire sample, the APOA size polymorphism explained about 15% of the phenotypic variation in Lp(a) levels. These data suggest that despite significant correlation between the APOA kringle 4 size polymorphism and Lp(a) levels, other sequence variations either in the APOA gene or closely linked genes may account for relatively higher Lp(a) levels found in Africans.

Two DNA polymorphisms, insertion/deletion (ins/del) and XbaI, of the apolipoprotein B (APOB) gene and their relationships with serum lipoprotein and lipid levels were examined in two Nigerian populations from Benin (n = 828) and Sokoto (n = 394). Allele frequencies for both polymorphisms were comparable between the two populations, and genotype frequencies conformed to Hardy-Weinberg equilibrium expectations. Because lipid profiles are affected by many known sex differences, males and females were analyzed separately. Analysis of variance was performed to test equality of means of quantitative traits across genotypes; the dependent variables were first adjusted by stepwise linear regression for environmental covariates in both populations. Plasma apoB and HDL2 cholesterol levels varied significantly between ins/del genotypes in Benin males (p = 0.05) and Sokoto males (p = 0.02), respectively. No significant variation between XbaI genotypes was observed for any lipid trait in either population. Combining the ins/del and XbaI polymorphisms to form haplotypes revealed significant linkage disequilibrium in both the Benin (p < 0.0001) and Sokoto (p < 0.001) populations. Analysis of the two-site haplotype showed a significant effect on serum lipoprotein (a) levels in Benin females (p < 0.04) and on HDL2 cholesterol among Sokoto males (p = 0.01). The haplotype explained 4% of the adjusted phenotypic variation in lipoprotein (a) levels in Benin females and 8.5% of the adjusted phenotypic variation in HDL2 cholesterol levels in Sokoto males. These data indicate that the ins/del and XbaI polymorphisms in the APOB gene affect interindividual variation in serum lipoprotein and lipid levels in African populations.

We examined the distributions of seven polymorphic sites in three apolipoprotein genes (APOE, APOA4, and APOH) and their relationships with quantitative lipid levels (total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides) among the Evenki reindeer herders of central Siberia. The polymorphism data reveal several distinctive features that differentiate the Evenki from white populations: the near absence of the APOE*2 allele, the highest ever recorded frequency of the APOH*3 allele, the complete absence of the APOA*2 allele at codon 360, and significantly different frequencies at three other APOA4 polymorphic sites. Our analyses of the relationships of common apolipoprotein polymorphism and plasma lipid levels also revealed interesting results. The well-established positive association between the APOE*4 allele and LDL cholesterol level reported in white populations was not seen in the Evenki despite a comparable frequency of the APOE*4 allele. Because the Evenki have significantly lower cholesterol levels than Westernized whites, this difference in allelic effect probably reflects gene-diet interaction, which modulates the effect of APOE polymorphism on LDL cholesterol. At the APOA4 locus the HincII polymorphism at codon 127 shows a significant impact on plasma triglyceride variation in the Evenki sample: The HincII - allele was associated with higher triglyceride levels than the HincII + allele. Our data indicate that both the genetic and the environmental factors conventionally associated with cardiovascular disease risk in Western societies are different in the Evenki.

Apolipoprotein E (apoE, protein; APOE, gene) plays an important role in lipoprotein metabolism by acting as a ligand for two specific cell receptors to mediate the uptake of apoE-containing lipoproteins by cells. The APOE gene, located on chromosome 19, exhibits a genetic polymorphism with three common alleles, APOE*2, APOE*3, and APOE*4, which show marked variation in their distribution among various ethnic groups. APOE polymorphism has a profound effect on interindividual variation in plasma cholesterol level in the general population, and this effect has made the APOE gene one of the most recognized and appreciated genes today. In addition to its pivotal involvement in lipoprotein metabolism, apoE is thought to participate in seemingly unrelated metabolic pathways, including the normal development of the nervous system and peripheral nerve regeneration after injury. The most dramatic and unexpected finding in this regard was made in early 1993, when it was reported that the presence of the APOE*4 allele is a significant risk factor for the development of late-onset familial Alzheimer's disease, a debilitating brain disorder. Since then, a number of studies have confirmed this provocative association and also have found that the APOE*4 allele is a major risk factor for Alzheimer's disease regardless of age at onset or family history. ApoE appears to contribute directly to the pathogenesis of Alzheimer's disease because it has been immunochemically localized in three defining lesions of the disease (extracellular amyloid plaques, intracellular neurofibrillary tangles, and vascular amyloid deposits). In this article I review current data about the association between APOE polymorphism and Alzheimer's disease and possible physiological mechanisms behind this association.

The Samoan islands were politically separated into American Samoa and Western Samoa in the early 1900s. Economic modernization is far more extensive in American Samoa. However, the Samoan archipelago has maintained a remarkable degree of sociocultural homogeneity, including intermarriage. The sociocultural exchanges presumably led to genetic homogeneity between the two Samoas. Detailed genetic comparisons and characterizations of Samoans are scanty, however. As part of a multidisciplinary study of modernization and cardiovascular risk factors in adults, we analyzed nine hypervariable nuclear DNA (HVR) and four serum protein polymorphisms in the two Samoan groups. The average heterozygosities at both DNA and serum protein loci are comparable in the two groups. As expected, the HVR loci reveal a high degree of variability (heterozygosity 30-87%) compared with the serum protein loci (heterozygosity 1-52%). A large proportion of alleles at the HVR loci, ranging from 50% to 100%, are shared between American and Western Samoa. With the exceptions of the D1S80 locus in American Samoa and the D13S118 locus in Western Samoa, the genotype distributions at all loci conform to their respective Hardy-Weinberg expectations. Sporadic occurrence of the F13B*2 allele at the F13B locus in Samoans indicates a low level of European admixture because this allele is unique to Europeans. The calculated zero values of kinship coefficients and standard genetic distances indicate minimal population differentiation between the two Samoan groups.

Apolipoprotein A-IV (apoA-IV protein; APOA4 gene) is structurally polymorphic in various mammalian species, including human, baboon, dog, horse, and mouse. To analyze the extent of genetic variation in the chimpanzee APOA4 gene, we screened 115 common chimpanzees (Pan troglodytes) (86 unrelated wild captured parents and 29 captive-born offspring) using isoelectric focusing followed by immunoblotting for protein polymorphism and using polymerase chain reaction (PCR) assay for DNA polymorphism. At the protein level the unrelated sample of chimpanzees is highly variable, having four alleles, APOA4*1, APOA4*2, APOA4*3, and APOA4*4, with frequencies of 0.192, 0.430, 0.331, and 0.047, respectively. The chimpanzee APOA4 locus, with four common alleles and a gene diversity of 67%, is more variable than previously reported variations in baboons (five alleles with 52% gene diversity) and humans (two alleles with 15% gene diversity). PCR amplification of chimpanzee DNAs, using a pair of human oligonucleotide primers covering a region of 300 nucleotides in the third exon, revealed a common 12-nucleotide deletion (allele frequency = 0.192) that correlates exactly with the APOA4*1 allele detected by isoelectric focusing and immunoblotting. DNA sequencing of the 300-nucleotide PCR amplified product revealed the deletion of 12 nucleotides near the carboxyl terminal region of the mature apoA-IV protein. This in-frame deletion, which codes for and eliminates four amino acids [glutamic acid (GAG), glutamine (CAG), glutamine (CAG), and glutamine (CAG)], occurs in a region that is evolutionarily conserved among rats, mice, chimpanzees, and humans. The partial DNA sequencing of the 3' end of the chimpanzee APOA4 gene revealed 99% identity with the human APOA4 gene.

Using isoelectric focusing and immunoblotting techniques, we have screened 937 plasma or serum samples from Nigerian blacks (N = 380), Papua New Guineans (N = 110), Aleuts (N = 62), Mayans (N = 139), Dogrib Indians (N = 45), and Eskimos from Kodiak and St. Lawrence islands (N = 201) for the hemopexin (HPX) polymorphism. We compared these data with our previously published data for US whites (N = 267) and US blacks (N = 194). Except for Nigerian blacks and US blacks, HPX was found to be monomorphic for the common HPX*1 allele in all populations tested. In addition to the commonly occurring HPX*1 allele, two other less common alleles, HPX*2 and HPX*3, were observed with respective frequencies of 1.8% and 4.6% in US blacks and 1.7% and 9.0% in Nigerian blacks. These data strongly suggest that the HPX*2 and HPX*3 alleles are unique alleles restricted to the black gene pool and are of potential significance in microevolutionary studies and in defining African admixture in hybrid populations. In addition to their importance in anthropogenetic studies, these unique HPX mutations also have potential biological significance in hemolytic disorders.

Using isoelectric focusing and immunoblotting techniques, we screened 96 serum samples from Yanomami Indians of northwestern Brazil to determine structural variation at three apolipoprotein loci: A4, E, and H. The APO-H locus, which is commonly polymorphic in white and black samples, was found to be monomorphic. At the APO-E locus only two alleles, APOE*3 and APOE*4, rather than the three-allele polymorphism commonly seen in Caucasians, was observed. At the APO-A4 locus no example of the APOA4*2 allele, found in Caucasians, was detected. However, the frequency of the less common APOA4*4 allele was above what has been observed in any other population. We investigated the impact of genetic variation at both polymorphic loci on quantitative differences in lipids, apolipoproteins, serum glucose, glycated hemoglobin, and uric acid. Contrary to the cholesterol-elevating effect of APOE*4 reported elsewhere, in both univariate analyses and after adjustments for age, sex, weight, and height, APOE*4 was associated with about a 4% lower mean serum cholesterol. Only after adjustment was this association statistically significant. The APOE*4 allele was significantly associated with unadjusted APO-A1 and APO-E levels but not with any other dependent variable; associations with adjusted APO-A1, APO-C2, and uric acid also approached standard levels of statistical significance (p < or = 0.05). In univariate analyses the APOA4*4 allele was significantly associated with APO-B, serum glucose, percent glycated hemoglobin, and uric acid, but no significant associations were observed after dependent variables were adjusted for age, sex, weight, and height. These results support the notion that apolipoprotein distributions and their associations with lipid and carbohydrate metabolism show ethnic variability.

Apolipoprotein A-IV exhibits a common two-allele polymorphism in several human populations studied to date. Using isoelectric focusing and immunoblotting, we have analyzed plasmas from 188 non-insulin-dependent diabetic and 238 normoglycemic Hispanic individuals from the San Luis Valley, Colorado, to determine APOA4 genotype frequencies and to estimate the impact of the genotypes on quantitative traits. The frequencies of the two common alleles, APOA4*1 and APOA4*2, were 0.929 and 0.069, respectively, in normal subjects and 0.901 and 0.096, respectively, in diabetics. The third rare allele, APOA4*3, was detected sporadically in both groups. We studied the impact of APOA4 polymorphism on the levels of total plasma cholesterol, HDL cholesterol and its subfractions (HDL3 and HDL2), LDL cholesterol, triglycerides, glucose, and insulin. We observed no significant effect of the APOA4 polymorphism on any trait in diabetics. However, we did note a significant sex-specific effect in normoglycemic females on the level of total HDL cholesterol (p = 0.001) and its subfractions HDL2 (p = 0.043) and HDL3 (p = 0.001). The effect of the APOA4*2 allele in normal Hispanic females was to lower the total HDL, HDL2, and HDL3 cholesterol by 8.75 mg/dl, 2.37 mg/dl, and 5.36 mg/dl, respectively, compared to the common APOA4*1 allele.

Apolipoprotein H (APOH) (beta-2-glycoprotein I) polymorphism has been studied in 1159 Asians. The sample included 872 Chinese, 179 Asiatic Indians (Dravidian), 91 Filipinos, and 17 Malays. APOH polymorphism was determined by isoelectric focusing of sera in thin-layer polyacrylamide gels containing 3 M urea followed by immunoblotting. The frequencies of the three alleles--APOH*1, APOH*2, and APOH*3--were found to be 0.031, 0.900, and 0.069 in the Chinese; 0.061, 0.866, and 0.073 in the Dravidian Indians; 0.055, 0.923, and 0.022 in the Filipinos; and 0.088, 0.882, and 0.029 in the Malays. The phenotypic distribution was at Hardy-Weinberg equilibrium in all the populations.

A tribal aboriginal community, the Mowanjum, from the Kimberley region in Western Australia has been screened to determine the extent of genetic variation in the products of genes coding for apolipoproteins, which are intimately involved in lipid metabolism. Of the seven systems tested, APOE and APOH revealed common structural variations, but their distribution patterns are significantly different from those found in European populations. Australian Aborigines were found to be unique because they have no APOE*2 and APOH*3 alleles and have strikingly high frequencies of the APOE*4 (26%) and APOH*1 (13%) alleles. The contrast in variation observed at these apolipoprotein loci between Australian Aborigines and Europeans not only makes these loci useful genetic markers in biologic anthropology studies but also provides a unique opportunity to investigate the role of genetic-environment interaction in determining interpopulation differences in cardiovascular disease risk factors.