Charles F. Sing

DNA sequence diversity in a 9.7-kb region of the human lipoprotein lipase gene

Lipoprotein lipase plays a central role in lipid metabolism and the gene that encodes this enzyme (LPL) is a candidate susceptibility gene for cardiovascular disease. Here we report the complete sequence of a fraction of the LPL gene for 71 individuals (142 chromosomes) from three populations that may have different histories affecting the organization of the sequence variation. Eighty-eight sites in this 9.7 kb vary among individuals from these three populations. Of these, 79 were single nucleotide substitutions and 9 sites involved insertion-deletion variations. The average nucleotide diversity across the region was 0.2% (or on average 1 variable site every 500 bp). At 34 of these sites, the variation was found in only one of the populations, reflecting the differing population and mutational histories. If LPL is a typical human gene, the pattern of sequence variation that exists in introns as well as exons, even for the small number of samples considered here, will present challenges for the identification of sites, or combinations of sites, that influence variation in risk of disease in the population at large.

Haplotype Structure and Population Genetic Inferences from Nucleotide- Sequence Variation in Human Lipoprotein Lipase

Allelic variation in 9.7 kb of genomic DNA sequence from the human lipoprotein lipase gene (LPL) was scored in 71 healthy individuals (142 chromosomes) from three populations: African Americans (24) from Jackson, MS; Finns (24) from North Karelia, Finland; and non-Hispanic Whites (23) from Rochester, MN. The sequences had a total of 88 variable sites, with a nucleotide diversity (site-specific heterozygosity) of .002+/-.001 across this 9.7-kb region. The frequency spectrum of nucleotide variation exhibited a slight excess of heterozygosity, but, in general, the data fit expectations of the infinite-sites model of mutation and genetic drift. Allele-specific PCR helped resolve linkage phases, and a total of 88 distinct haplotypes were identified. For 1,410 (64%) of the 2,211 site pairs, all four possible gametes were present in these haplotypes, reflecting a rich history of past recombination. Despite the strong evidence for recombination, extensive linkage disequilibrium was observed. The number of haplotypes generally is much greater than the number expected under the infinite-sites model, but there was sufficient multisite linkage disequilibrium to reveal two major clades, which appear to be very old. Variation in this region of LPL may depart from the variation expected under a simple, neutral model, owing to complex historical patterns of population founding, drift, selection, and recombination. These data suggest that the design and interpretation of disease-association studies may not be as straightforward as often is assumed.

Apolipoprotein E Variation at the Sequence Haplotype Level: Implications for the Origin and Maintenance of a Major Human Polymorphism

Three common protein isoforms of apolipoprotein E (apoE), encoded by the epsilon2, epsilon3, and epsilon4 alleles of the APOE gene, differ in their association with cardiovascular and Alzheimers disease risk. To gain a better understanding of the genetic variation underlying this important polymorphism, we identified sequence haplotype variation in 5.5 kb of genomic DNA encompassing the whole of the APOE locus and adjoining flanking regions in 96 individuals from four populations: blacks from Jackson, MS (n=48 chromosomes), Mayans from Campeche, Mexico (n=48), Finns from North Karelia, Finland (n=48), and non-Hispanic whites from Rochester, MN (n=48). In the region sequenced, 23 sites varied (21 single nucleotide polymorphisms, or SNPs, 1 diallelic indel, and 1 multiallelic indel). The 22 diallelic sites defined 31 distinct haplotypes in the sample. The estimate of nucleotide diversity (site-specific heterozygosity) for the locus was 0.0005+/-0.0003. Sequence analysis of the chimpanzee APOE gene showed that it was most closely related to human epsilon4-type haplotypes, differing from the human consensus sequence at 67 synonymous (54 substitutions and 13 indels) and 9 nonsynonymous fixed positions. The evolutionary history of allelic divergence within humans was inferred from the pattern of haplotype relationships. This analysis suggests that haplotypes defining the epsilon3 and epsilon2 alleles are derived from the ancestral epsilon4s and that the epsilon3 group of haplotypes have increased in frequency, relative to epsilon4s, in the past 200,000 years. Substantial heterogeneity exists within all three classes of sequence haplotypes, and there are important interpopulation differences in the sequence variation underlying the protein isoforms that may be relevant to interpreting conflicting reports of phenotypic associations with variation in the common protein isoforms.

The use of measured genotype information in the analysis of quantitative phenotypes in man.

We have begun a measured genotype approach to the genetic analysis of lipid and lipoprotein variability. This approach enables one to simultaneously estimate the frequencies and effects of alleles at specific loci along with the residual polygenetic variance component. In this study we consider the contribution of three common alleles at the locus coding for apolipoprotein E to interindividual variation of total cholesterol, betalipoprotein, and triglyceride levels. A sample of 102 nuclear families consisting of 434 individuals was studied. The frequencies of the ε2, ε3, and ε4 alleles in this sample are 0·137,0·740, and 0·123, respectively. In separate analyses of cholesterol and betalipoprotein levels, a complete model that includes the effects of the six apo E genotypes, unmeasured polygenes, and individual specific environmental effects fits these data significantly better than a reduced model that does not include the effects of the apo E polymorphism or a reduced model that does not include the effects of polygenes. On the average the ε2 allele lowers total cholesterol and betalipoprotein levels by 0·425 mmol/l and 0·811 units, respectively. The ε4 allele is associated with an average increase of these phenotypes by 0·255 mmol/l and 0·628 units, respectively. Simultaneous estimates of the interindividual variability of total cholesterol levels attributable to the apo E polymorphism and to residual polygenic effects are 8% and 56%, respectively. For betalipoprotein levels, we simultaneously estimate these values to be 7% and 42%, respectively. A reduced model including the effects of polygenes but not the effects of the apo E polymorphism fitted the triglyceride data as well as the complete model. The estimate of the fraction of interindividual variability associated with polygenetic effects was 26.5%. We review our present understanding of the genetic architecture underlying variability of cholesterol levels in the population at large and infer that the majority of the genetic variability may be accounted for by polymorphic gene loci with moderate effects on cholesterol levels.

The use of measured genotype information in the analysis of quantitative phenotypes in man. I. Models and analytical methods.

Improved laboratory methods allow one to investigate the contribution of measured allelic variability at a locus physiologically involved in determining the expression of a quantitative trait. We present statistical methods that incorporate measured genotype information into the analysis of a quantitative phenotype that allows one simultaneously to detect and estimate the effects of a measured single locus and residual polygenic effects. Likelihoods are presented for the joint distribution of the quantitative phenotype and a measured genotype that are appropriate when the data are collected as a sample of unrelated individuals or as a sample of nuclear families. Application of this method to the analysis of serum cholesterol levels and the concentration of the group specific component (Gc) are presented. The analysis of the contribution of the common Gc polymorphism to the determination of quantitative variability in Gc using smaples of related and unrelated individuals presents, for the first time, the simultaneous estimation of the frequencies and the effects of the genotypes at a measured locus, and the contribution of residual unmeasured polygenes to phenotypic variability.

Genome-Wide Linkage Analyses of Systolic Blood Pressure Using Highly Discordant Siblings

BACKGROUND Elevated blood pressure is a risk factor for cardiovascular, cerebrovascular, and renal diseases. Complex mechanisms of blood pressure regulation pose a challenge to identifying genetic factors that influence interindividual blood pressure variation in the population at large. METHODS AND RESULTS We performed a genome-wide linkage analysis of systolic blood pressure in humans using an efficient, highly discordant, full-sibling design. We identified 4 regions of the human genome that show statistical significant linkage to genes that influence interindividual systolic blood pressure variation (2p22.1 to 2p21, 5q33.3 to 5q34, 6q23.1 to 6q24.1, and 15q25.1 to 15q26.1). These regions contain a number of candidate genes that are involved in physiological mechanisms of blood pressure regulation. CONCLUSIONS These results provide both novel information about genome regions in humans that influence interindividual blood pressure variation and a basis for identifying the contributing genes. Identification of the functional mutations in these genes may uncover novel mechanisms for blood pressure regulation and suggest new therapies and prevention strategies.

Variation in the Region of the Angiotensin-Converting Enzyme Gene Influences Interindividual Differences in Blood Pressure Levels in Young White Males

BACKGROUND The renin-angiotensin system regulates blood pressure through its effects on vascular tone, renal hemodynamics, and renal sodium and fluid balance. METHODS AND RESULTS Using data from a large population-based sample of 1488 siblings having a mean age of 14.8 years and belonging to the youngest generation of 583 randomly ascertained three-generation pedigrees from Rochester, Minn, we carried out variance components-based linkage analyses to evaluate the contribution of variation in four renin-angiotensin system gene regions (angiotensinogen, renin, angiotensin I-converting enzyme, and angiotensin II receptor type 1) to interindividual variation in systolic, diastolic, and mean arterial pressure. We rejected the null hypothesis that allelic variation in the region of the angiotensin-converting enzyme (ACE) gene does not contribute to interindividual blood pressure variability. After conditioning on measured covariates, variation in this region accounted for 0%, 13% (P=0.04), and 16% (P=0.04) of the interindividual variance in systolic, diastolic, and mean arterial pressures, respectively. These estimates were even greater in a subset of subjects with a positive family history of hypertension (0%, 29% [P=0.005], and 32% [P<0.005], respectively). In sex-specific analyses, genetic variation in the region of the ACE gene significantly influenced interindividual blood pressure variation in males (37% for SBP [P=0.03], 38% for DBP [P=0.04], and 53% for MAP [P<0.005]) but not in females. CONCLUSIONS Although it is possible that variation in a gene near the ACE gene may explain the observed results, knowledge about the physiological involvement of ACE in blood pressure regulation supports the proposition that the ACE gene itself influences blood pressure variability in a sex-specific manner.

Genes, Environment, and Cardiovascular Disease

Abstract—In this essay, we call to attention what every medical researcher knows about the etiology of cardiovascular disease but most deny, or choose to ignore, when designing, carrying out, and reporting genetic studies. Medical research is entering an era of synthesis that will take advantage of the successes of reductionism over the past decade in defining and describing human genome variations. Meaningful insights into the role of such variation requires a biological model of genome-phenotype relationships that incorporates interactions between subsets of possible genetic and environmental agents as causations in particular contexts indexed by time and space. We make recommendations for what needs to be done to cope with these complexities.

Deep resequencing reveals excess rare recent variants consistent with explosive population growth

Accurately determining the distribution of rare variants is an important goal of human genetics, but resequencing of a sample large enough for this purpose has been unfeasible until now. Here, we applied Sanger sequencing of genomic PCR amplicons to resequence the diabetes-associated genes KCNJ11 and HHEX in 13,715 people (10,422 European Americans and 3,293 African Americans) and validated amplicons potentially harbouring rare variants using 454 pyrosequencing. We observed far more variation (expected variant-site count ∼578) than would have been predicted on the basis of earlier surveys, which could only capture the distribution of common variants. By comparison with earlier estimates based on common variants, our model shows a clear genetic signal of accelerating population growth, suggesting that humanity harbours a myriad of rare, deleterious variants, and that disease risk and the burden of disease in contemporary populations may be heavily influenced by the distribution of rare variants.

Positional Genomic Analysis Identifies the β2-Adrenergic Receptor Gene as a Susceptibility Locus for Human Hypertension

BACKGROUND -After genome-wide linkage analyses of blood pressure levels, we resequenced 5 positional candidate genes in a linkage region on chromosome 5 and genotyped selected variants in several family samples from Rochester, Minn. METHODS AND RESULTS In a sample of 55 pedigrees containing >/=1 sibling-pair(s) discordant for systolic blood pressure, polymorphisms within the beta(2)-adrenergic receptor gene (Arg16Gly, P=0.009) and the glutathione peroxidase 3 gene (-302G-->A, P=0.037; -623A-->C, P=0.013) were significantly related to blood pressure levels. In a second sample of 298 nuclear families (n=1283 individuals), the Arg16Gly polymorphism was significantly associated with diastolic blood pressure in family-based analyses (P=0.016) and with both diastolic (P=0.009) and mean arterial blood pressure (P=0.038) in analyses of the parental generation only. Neither polymorphism in the glutathione peroxidase 3 gene was associated with blood pressure levels in this sample. An additional 291 families (n=1240 individuals) were added to the nuclear family sample, and the Gln27Glu polymorphism in the beta(2)-adrenergic receptor gene was significantly associated with both systolic (P=0.034) and mean arterial blood pressure (P=0.035) in the parental generation of the combined 589 families. The frequencies of both the Gly16 and Glu27 alleles were higher in hypertensives than in normotensives (0.649 versus 0.604 and 0.490 versus 0.429, respectively), and the odds ratio for the occurrence of hypertension was 1.80 (95% confidence interval, 1.08 to 3.00; P=0. 023) for the Glu27 allele. CONCLUSIONS The results of this study provide support for further detailed investigations of the mechanistic pathways by which variations in the beta(2)-adrenergic receptor gene may influence blood pressure levels.