Chapter 8. Evidence for a Major Quantitative Trait Locus on Chromosome 17q21 Affecting LDL Peak Particle Diameter

Yohan Bossé, Louis Pérusse, Jean-Pierre Després, Benoît Lamarche, Yvon C Chagnon, Treva Rice, D.C. Rao, Claude Bouchard, and Marie-Claude Vohl

Des études d’héritabilité et de ségrégation ont démontré que la taille des particules LDL est caractérisée par une grande contribution génétique et la présence d’un gène à effet majeur. L’objectif de cette étude était d’identifier les régions chromosomiques influençant le diamètre principal des particules LDL (DP-LDL). Un criblage génomique a donc été effectué chez 681 sujets participant à l’Étude des familles de Québec. La plus forte évidence de liaison a été retrouvée sur le chromosome 17q21.33 avec un rapport de cote logarithmique (LOD) à 6.76 pour le DP-LDL ajusté pour l’âge, l’indice de masse corporelle et les niveaux de triglycérides. Des évidences de liaison suggestive (LOD > 1.75) ont aussi été retrouvées sur les régions 1q31, 2q33.2, 4p15.2, 5q12.3 et 14q31. Ces résultats suggèrent fortement la présence d’un locus majeur sur le chromosome 17q ainsi que de plusieurs autres loci prometteurs influençant le DP-LDL.

Evidence for a Major Quantitative Trait Locus on Chromosome 17q21 Affecting LDL Peak Particle Diameter

Yohan Bossé1,2, Louis Pérusse3, Jean-Pierre Després1,2,4, Benoît Lamarche1,5, Yvon C. Chagnon6, Treva Rice7, D.C. Rao7, Claude Bouchard8, and Marie-Claude Vohl1,2,5

1- Lipid Research Center, CHUL Research Center; 2- Department of Food Sciences and Nutrition; 3- Division of Kinesiology, Department of Social and Preventive Medicine, Laval University, Ste-Foy, Québec; 4- The Quebec Heart Institute; 5- Institute on Nutraceutical and Functional Food; 6- Genetic and Molecular Psychiatric Unit, Laval University Robert-Giffard Research Center, Beauport, Québec, Canada; 7- Division of Biostatistics, Washington University School of Medicine, St. Louis, Missouri; and 8- Pennington Biomedical Research Center, Baton Rouge, Louisiana, USA.

Short title: Genome Scan on LDL Size.

Address all correspondence to:

Marie-Claude Vohl, Ph.D. Lipid Research Center, CHUL Research Center, TR-93, 2705, Boul. Laurier, Sainte-Foy, Quebec, G1V 4G2, Canada.

Fax: (418) 654-2145; Tel: (418) 656-4141 extension 8280; E-mail: marie-claude.vohl@crchul.ulaval.ca

Total word count: title page = 181; abstracts = 348; text = 2354; references = 1443; tables = 859; figure legends = 124.

Journal Subject Heads: [89] Genetics of cardiovascular disease.

Abstract

Background ―Several lines of evidence suggest that small dense low-density lipoprotein (LDL) particles are associated with the risk of coronary heart disease. Heritability and segregation studies suggest that LDL particle size is characterized by a large genetic contribution and the presence of a putative major genetic locus. However, association and linkage analyses have been thus far inconclusive in identifying the underlying gene(s).

Methods and Results ―An autosomal genome-wide scan for LDL peak particle diameter (LDL-PPD) was performed in the Québec Family Study. A total of 442 markers were genotyped with an average intermarker distance of 7.2 centimorgans. LDL-PPD was measured by gradient gel electrophoresis in 681 subjects from 236 nuclear families. Linkage was tested using both sibpair- and variance components-based linkage methods. The strongest evidence of linkage was found on chromosome 17q21.33 at marker D17S1301 with a LOD score of 6.76 using variance components method for the phenotype adjusted for age, BMI and triglyceride levels. Similar results were obtained with the sibpair method (p < 0.0001). Other chromosomal regions harboring markers with highly suggestive evidence of linkage (p ≤ 0.0023; LOD ≥ 1.75) includes 1p31, 2q33.2, 4p15.2, 5q12.3 and 14q31. Several candidate genes are localized under the peak linkages, including apolipoprotein H on chromosome 17q, the apolipoprotein E receptor 2 and members of the phospholipase A2 family on chromosome 1p as well as the HMG-CoA reductase on chromosome 5q.

Conclusions ―This genome-wide scan for LDL-PPD indicates the presence of a major QTL located on chromosome 17q and others interesting loci influencing the phenotype.

Condensed abstract

In order to identify genetic loci involved in LDL peak particle diameter (LDL-PPD), an autosomal genome-wide scan was performed in 681 subjects enrolled in the Québec Family Study. A variance-component linkage analysis revealed a strong evidence of linkage on chromosome 17q21.33 at marker D17S1301 for LDL-PPD adjusted for age, BMI and triglyceride levels (LOD = 6.76). Other chromosomal regions harboring markers with highly suggestive evidence of linkage for LDL-PPD includes 1p31, 2q33.2, 4p15.2, 5q12.3 and 14q31 (LOD > 1.75). Several candidate genes were located in the vicinity of the genomic regions showing evidence of linkage.

Key words: genome scan, LDL size, genetics, lipoproteins, candidate genes.

A number of case-control as well as prospective studies reveal an increased risk of coronary heart disease (CHD) in patients with small, dense low density lipoprotein (LDL) compared with those having larger, more buoyant LDL particles1. Heritability studies, based on twins, suggested that approximately one third to one half of the variation in the LDL peak particle size can be attributed to genetic influences2,3. Complex segregation analyses of small dense LDL phenotypes have been performed with data from different types of family structures, different criteria for proband ascertainment and the use of different techniques to characterize LDL heterogeneity4-9. Indeed, the model providing the best fit to the data included either a dominant, a recessive or an undetermined mode of inheritance for the trait. Furthermore, the allele frequency determining the small dense LDL phenotype ranges from 19 to 42%, with reduced penetrances in young males and premenopausal women. However, these studies unanimously provided evidence in favor of a gene with a major effect on LDL particle phenotypes.

Association studies with candidate genes have been inconsistent in finding genes associated with small dense LDL. The –250G→A polymorphism within the hepatic lipase promoter was associated with buoyant LDL particles10. However, the –514C→T polymorphism, which is in complete linkage disequilibrium with the –250G→A polymorphism11, showed no effect on LDL particle size12,13. The apolipoprotein (apo) E genotype was also associated with the small dense LDL phenotype. However, some have reported smaller particles for subjects carrying the E4 allele14-16, while others did for subjects carrying the E2 allele17,18. In contrast, others have show that LDL particle size did not differ among the apo E genotypes19. Additional candidate genes, including cholesteryl ester transfer protein (CETP)20, microsomal triglyceride transfer protein21, cholesterol 7alpha hydroxylase22, apo B-10023, apo C-III24 and angiotensin-converting enzyme16 were investigated for potential effects on small dense LDL phenotypes. These studies revealed either absence of an association or presence of an association only in particular subgroups.

Results from linkage studies are equivocal. After excluding linkage of small dense LDL with the apo B (the protein moiety of LDL) gene locus on chromosome 225,26, suggestive linkage to the LDL receptor locus on chromosome 19 has been reported27,28. However, subsequent sequencing of the entire coding regions of the LDL-receptor gene did not reveal any sequence variants, thus weakening the hypothesis that a mutant LDL-receptor allele is responsible for the dense LDL phenotype29. Other candidate loci, including hepatic lipase12, lipoprotein lipase30, CETP28,31,32, apo A1-CIII-AIV complex28,32, and the manganese superoxide dismutase28,32, have been shown to be linked with the small dense LDL phenotype. Unfortunately, most of these linkages have not been replicated33,34. Based on these results, Austin et al.34 emphasized the necessity of finding new genetic loci, other than those harboring known candidate genes, in order to identify the genes potentially involved in determining the small dense LDL phenotype. Genome-wide scans are particularly suited for this purpose. Prior genome wide scan have focus on variation in cholesterol concentrations of LDL size fractions. Rainwater et al.35 found two QTLs on chromosome 3 and 4 with LOD scores > 3 for LDL size fraction 3 (LDL-3), a fraction that contains small LDL particles. This study demonstrates the existence of QTLs affecting the concentration of cholesterol within a particular sub-population of LDL, but do not provide evidence of QTLs responsible for the size of the LDL particle by itself. To the best of our knowledge, the only whole genome scan on LDL particle size have been performed on 240 individuals ascertain through 18 unrelated familial combined hyperlipidemic probands12. Results suggest a locus, over the hepatic lipase gene on chromosome 15, with a LOD score of 2.2. Here we report the results of an autosomal genomic scan for LDL peak particle diameter (LDL-PPD) measured by gradient gel electrophoresis.

Methods

Population

The Québec Family Study (QFS) is an ongoing project composed of French-Canadian families that has been described previously36. In the present study, a total of 681 subjects from 236 nuclear families had available data on LDL-PPD. Table 1 presents the characteristics of subjects in each of the sex and generation groups. The study was approved by the Laval University Medical Ethics Commitee, and all subjects provided written informed consent. All the procedures followed were in accordance with institutional guidelines.

Phenotypes

LDL peak particle diameter (LDL-PPD) was measured by gradient gel electrophoresis from plasma obtained after a 12-hour fast. Details on the technique have been provided previously37.

Genotypes

Genomic DNA was prepared by the proteinase K and phenol/chloroform technique. DNA preparation, polymerase chain reaction conditions, and genotyping are described in details elsewhere38. Genotypes for each marker were typed using automatic DNA sequencers and the computer software SAGA from LICOR (Lincoln, NE). The results were stored in a local dBase IV database, GENEMARK, which inspects results for Mendelian inheritance incompatibilities within nuclear families and extended pedigrees. A total of 335 microsatellite markers (dinucleotide, trinucleotide, and tetranucleotide repeats) selected from different sources, but mainly from the Marshfield panel version 8a, were available for this genome scan. The location of markers on the chromosomes in centimorgan (cM) were taken from version 9.0 of the Marshfield Institute map (http://research.marshfieldclinic.org/genetics/) and the Location Database map (http://cedar.genetics.soton.ac.uk/public_html). In addition, 107 polymorphisms in 63 candidate genes were included. The average intermarker distance for the whole set of 442 markers was 7.2 cM. The Genome Database (http://gdbwww.gdb.org/) and the OMIM gene map (http://www.ncbi.nlm.nih.gov/htbin-post/Omim/getmap) were used to identify candidate genes.

Statistical analyses

LDL-PPD was adjusted for covariates using a stepwise multiple regression procedure retaining only terms that were significant at the 5% level. Regression parameters were estimated within six age- (<30, 30-50, and ≥50 years) by-sex (male vs. female) groups after exclusion of outliers (± 4SD) and residuals were computed for all subjects. Residual scores were then standardized to a mean of 0 and a standard deviation of 1. LDL-PPD were adjusted for three different sets of covariates: 1) age up to the cubic polynomial, 2) age and BMI, 3) age, BMI and triglyceride levels. These adjustments gave three phenotypes arbitrary called LDL-PPD1, LDL-PPD2 and LDL-PPD3, respectively. Adjustment of the phenotypes were performed using SAS (version 8.02).

The search for linkage between the phenotypes and the genetic markers was performed using two different approaches. First, linkage was tested using the new Haseman-Elston regression-based method which models the trait covariance between sibpairs, instead of the squared sibpair trait difference used in the original method. It regresses the mean-corrected trait cross-product on the number of alleles shared identical by descent (IBD). Singlepoint and multi-point estimates of alleles shared IBD were generated using the GENIBD software and linkage was tested using the SIBPAL2 software from the S.A.G.E. 4.0 statistical package (S.A.G.E., 2001)39. The maximum number of sibpairs was 352. Linkage was also investigated using the variance components-based approach implemented in the quantitative transmission disequilibrium test (QTDT) computer software40. Under this approach, the phenotypic covariance among members of a family is assumed to result from the additive effects of linkage due to a major locus (a), a residual familial component due to polygenes (g) and a residual non-shared environmental component (e) that represents environmental effects unique to each family member. Linkage is tested by contrasting the null hypothesis of no linkage (σa = 0) to the alternative hypothesis (σa ≠ 0) using a likelihood ratio test as described previously41. The LOD score was computed as χ2/(2 loge 10). The interpretation of linkage evidence was considered as suggestive (p ≤ 0.01; LOD ≥ 1.18), highly suggestive (p ≤ 0.0023; LOD ≥ 1.75) or evidence of linkage (p ≤ 0.0001; LOD ≥ 3.0)42.

Results

An overview of the variance components-based linkage results for the three LDL-PPD phenotypes is given in Figure 1. Suggestive evidence of linkages (p ≤ 0.01 or LOD scores ≥ 1.18 for at least one of the phenotype) are summarized in Table 2. The strongest evidence of linkage, which was confirmed by both linkage methods, was found on chromosome 17q21.33. As shown in Figure 2, the peak linkages were found with marker D17S1301 for LDL-PPD1 (LOD = 4.72), LDL-PPD2 (LOD = 4.70) and LDL-PPD3 (LOD = 6.76). Marker D17S1290, located 1.6 cM from D17S1301, gave also fairly good evidence of linkage for the three phenotypes.

Other chromosomes exhibiting some evidence of linkage by the variance components-based method are displayed in Figure 1. Highly suggestive evidence of linkages were observed at 1p31 (leptin receptor locus), 2q33.2 (marker D2S1384), 4p15.2 (D4S2397), 5q12.3 (D5S1501) and 14q31.1 (D14S53). Markers at the leptin receptor locus and markers D5S1501 and D14S53 also provided evidence of linkage by the sibpair method (see Table 2).

Other markers gave highly suggestive evidence of linkage (p < 0.0023) with at least one of the linkage methods. For instance, marker D16S261 provided evidence of singlepoint linkage with the three phenotypes. The markers VWFP1 on chromosome 22q11.21 provided evidence of singlepoint and multipoint linkage for the three phenotypes. On the other hand, marker D4S1627 yielded highly suggestive evidence of linkage for LDL-PPD1 and LDL-PPD2 with the variance component method. D5S1457 at 5p12 shows highly suggestive evidence of linkage in multipoint analysis for the three phenotypes and in singlepoint for LDL-PPD3. Finally, several markers provided highly suggestive evidence of linkage with LDL-PPD3 only, including D1S198, D2S434, IRS1 (2q36.3), ADRB2 (5q31), TNFα (6p21.3), D8S1110, D9S1121, D16S410 and ACEDI (17q23).

Discussion

The primary objective of this study was to identify QTLs affecting LDL-PPD variation. The results provide evidence for a major locus affecting LDL-PPD located on chromosome 17q21. Interestingly, none of the candidate genes located in the area of this QTL were previously tested. The marker D17S1301 located on chromosome 17q21.33 was strongly linked with the LDL peak particle diameter, whether adjusted or not for covariates. However, the evidence for linkage was stronger when the phenotype was adjusted for plasma triglycerides, indicating that triglyceride levels may attenuate the penetrance of the locus. Marker D17S1290 located 1.6 cM from D17S1301 also provided good evidence of linkage (1.34 ≤ LOD ≤ 2.63). The apolipoprotein H (APOH) gene, also referred to as β2-glycoprotein I, is encoded under the peak linkage on 17q21. ApoH is a single chain glycoprotein that exists in plasma both in a free form and in combination with lipoprotein particles. It has been implicated in several physiologic pathways, including lipid metabolism, coagulation, and the production of antiphospholipid antibodies. This apolipoprotein activates lipoprotein lipase43 and genetic variations in this gene has been associated with variation in HDL-cholesterol and triglyceride levels44-46. The angiotensin-converting enzyme (ACE) is also located in this genomic region. This enzyme cleaves the final intravascular step resulting in the vasoactive peptide, angiotensin II. Angiotensin II has been shown to bind specifically to LDL47, which produces a modified form of LDL which is taken up by macrophages at an enhanced rate, leading to cellular cholesterol accumulation48. In the present study, the insertion/deletion polymorphism in intron 16 of the ACE gene provided evidence of linkage with LDL-PPD1 (LOD = 1.46), LDL-PPD2 (LOD = 1.57) and LDL-PPD3 (LOD = 2.35). Figure 2 shows the approximate location of candidate genes surrounding the major peak on chromosome 17.

Several other chromosomal regions provided highly suggestive (p < 0.0023) evidence of linkage. These regions include chromosomes 1p31, 5p12-p12.3 and 14q31.1, which show evidence of linkage with both linkage methods and for all LDL-PPD phenotypes. Some promising candidate genes are located within these regions. First, the strongest evidence of linkage on chromosome 1p comes from a marker located within the leptin receptor (LEPR) gene. By modulating the hypothalamic effects of leptin on food intake and energy expenditure, genetic variants in the LEPR may affect energy balance and the size of LDL particles as a consequence of body fatness alterations. However, adjusting the LDL-PPD for BMI did not affect the strength of the linkage. On 1p, three members of the phospholipase A2 (PLA2) gene family are present, namely PLA2 group IID (PLA2G2D), group V (PLA2G5) and group IIA (PLA2G2A). PLA2 is known to hydrolyze the phospholipid monolayers of LDL particles and change their physicochemical properties and size49. Apolipoprotein E receptor 2 (APOER2) is also located near the locus of interest. On chromosome 5, two markers (D5S1457 and D5S1501), located 20 cM apart, provided evidence of linkage with LDL-PPD. This region contains the 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMG CoA reductase, HMGCR), which is the rate limiting enzyme for cholesterol synthesis. A list of others potential candidate genes within the chromosomal regions linked to the LDL-PPD is provided in Table 3.

Among the panel of markers included in the genome scan, few candidate genes for LDL-PPD were present. First, an apo B marker gave no evidence of linkage with the phenotype. A significant linkage to apo B has been reported in a sibpair linkage analysis of dizygotic women twins33 but other linkage studies excluded the hypothesis of linkage for the apo B locus and LDL size25,26,28. Second, while no linkage was found with the lipoprotein lipase (LPL) locus in the current study or in two others28,34, a highly significant LOD score of 6.24 was obtained in another study of heterozygous LPL deficient families30. Third, the apo E gene gave no evidence of linkage as reported previously28,33,34. Finally, consistent with three other studies32-34, the LDL receptor also was not linked to LDL-PPD in the present study. In contrast, two previous evidence linked the LDL receptor locus to LDL subclass in families ascertained through probands with the atherogenic lipoprotein phenotype27 and in families with CHD28. However, no amino acid sequence changes in the LDL receptor were found in the former study27 making it unlikely that a mutant allele in the LDL receptor gene was responsible for the linkage29. In the present study, negative results were also obtained with other candidate genes including paraoxonase, hormone-sensitive lipase, CD36 and the intestinal fatty acid-binding protein.

In conclusion, the results of this study reveal the presence of a major locus located on chromosome 17q21.33 influencing LDL-PPD. This finding supports results from a handful of segregation analyses indicating the presence of a putative major locus for LDL particle size. Evidence of linkage was also found on chromosome 1p31, 2q33.2, 4p15.2, 5q12.3 and 14q31.1. These QTLs harbor a good number of candidate genes that have not been previously tested in association studies with LDL-PPD.

Acknowledgments

This study was supported by the Canadian Institutes of Health Research (MT-13960 and GR-15187). The authors would like to express their gratitude to the subjects for their excellent collaboration and to the staff of the Physical Activity Sciences Laboratory for their contribution to the study. Y. Bossé is the recipient of a studentship from the "Fonds pour la formation de chercheurs et l'aide à la recherche (FCAR) et le Fonds de la recherche en santé du Québec (FRSQ)". M.C. Vohl is a research scholars from the FRSQ. C. Bouchard was supported in part by the George A. Bray Chair in Nutrition. J.P. Després is chair professor of human nutrition, lipidology and prevention of cardiovascular disease supported by Provigo and Pfizer Canada. Benoît Lamarche is the recipient of the Canada Research Chair in Nutrition, Functional Foods and Cardiovascular Health. The results of this paper were obtained using the program package S.A.G.E., which is supported by a U.S. Public Health Service Resource Grant (1 P41 RR03655) from the National Center for Research Ressources.

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Table 1. Descriptive Statistics of LDL Peak Particle Diameter and Covariates in Each of the Sex and Generation Groups.

Table 2. Results from the Genome Scan: Markers showing evidence of linkage with the LDL-PPD phenotypes according to the linkage methods used.

Table 3. Candidate genes within chromosomic regions linked to LDL-PPD.

Figure 1. Quantitative transmission disequilibrium test linkage results for all autosomal chromosomes with LDL-PPD phenotypes. LOD scores are presented on the y-axis and genetic distance is presented on the x-axis in centimorgans. LDL-PPD1, LDL-PPD2 and LDL-PPD3 indicate LDL-PPD adjusted for: 1) age, 2) age and BMI, 3) age, BMI and triglyceride, respectively.

Figure 2. Quantitative transmission disequilibrium test linkage results for chromosome 17 with LDL-PPD phenotypes. Genetics markers used for linkage are indicated under the x-axis. The approximate location of candidate genes in the vicinity of the major peak are displayed on the graph. The dashed horizontal line represents a LOD score of 3.00. LDL-PPD1, LDL-PPD2 and LDL-PPD3 indicate LDL-PPD adjusted for: 1) age, 2) age and BMI, 3) age, BMI and triglyceride, respectively.