Quantitative Trait Loci for Apolipoprotein B, Cholesterol, and Triglycerides in Familial Combined Hyperlipidemia Pedigrees

Quantitative Trait Loci for Apolipoprotein B, Cholesterol, and Triglycerides in Familial Combined Hyperlipidemia Pedigrees Rita M. Cantor, Tjerk de Bruin, Naoko Kono, Susan Napier, Atila van Nas, Hooman Allayee, Aldons J. Lusis Objective Familial combined hyperlipidemia (FCHL) is a genetically complex lipid disorder that is diagnosed in families by combinations of increased cholesterol, triglycerides, and/or apolipoprotein B (apob) levels in patients and their first-degree relatives. Identifying the predisposing genes promises to reveal the primary risk factors and susceptibility pathways and suggest methods of prevention and treatment. As with most genetically complex disorders, a clinical definition of disease may not be the most useful phenotype for finding the complement of predisposing genes, and the quantitative traits used to define the disorder can provide important information. This is a report of a quantitative trait loci (QTL) analysis of FCHL. Methods and Results A full genome scan of 377 multi-allelic markers genotyped at 10 centimorgan (cm) intervals was conducted in 150 sibling pairs from 22 nuclear families in FCHL pedigrees. These data were analyzed by 2 multipoint QTL linkage methods using the nonparametric and Haseman Elston procedures of the Genehunter software. Using a criterion of P 0.001 by the nonparametric analysis, we found evidence of 2 apob QTL at 1p21-31 (P 0.000009) and 17p11-q21 (P 0.000009), a total serum cholesterol QTL at 12p13 (P 0.0001), and a serum triglycerides QTL at 4p15-16 (P 0.0002). Using the criterion of P 0.03 for at least 2 traits at the same locus, additional evidence for cholesterol (P 0.01) and a triglycerides P 0.02) was observed at 17p11-21, as well as suggestive evidence for apob (P 0.02) and triglycerides (P 0.01) at 4q34-35, and cholesterol (P 0.01) and triglycerides (P 0.02) and a binary FCHL trait (lod 1.5) at 16p12-13. Conclusions QTL analyses of the traits that define FCHL are effective for localizing disease-predisposing genes. (Arterioscler Thromb Vasc Biol. 2004;24:1935-1941.) Key Words: FCHL QTL apolipoprotein B cholesterol triglycerides Familial combined hyperlipidemia (FCHL) is a genetically complex lipid disorder that predisposes to coronary artery disease. Since its recognition in 1973, 1 3 it has been clinically defined by multiple lipid abnormalities that are exhibited by first-degree relatives within families. A prevalence of 1% among white populations makes it an important contributor to the burden of coronary artery disease. 4 Finding the genes that predispose to FCHL promises to reveal its primary risk factors and susceptibility pathways, and to suggest possible methods of prevention and treatment. Increased levels of serum triglycerides, total cholesterol, and/or apolipoprotein B (apob) in a patient indicate the possibility of FCHL. On the examination of family members, FCHL is diagnosed if at least 2 of the lipid abnormalities seen in the patient (usually increases in cholesterol and triglycerides) also segregate among the patient s first-degree relatives. As with most genetically complex disorders, the binary classification of family members into affected and unaffected may not be the best phenotype for finding disease-predisposing genes, because such classifications do not follow a classical Mendelian pattern of inheritance. 5 7 These increased lipid levels are likely to result from the interaction of multiple genes and environmental factors, such as adiposity and the degree of exercise, and the individual quantitative lipid profiles may be closer to the genetic defects than the binary definition of FCHL. In addition, it is anticipated that the wide variance of quantitative traits will provide more statistical power to detect linked loci than the binary categorization of FCHL. We report, herein, the results of a full genome scan in FCHL families when the individual quantitative lipid traits used to diagnose FCHL have been the focus of the linkage analyses. Searches for lipid disorder genes have been conducted using multiple genetic epidemiologic approaches, including Original received April 15, 2004; final version accepted July 30, 2004. From the Departments of Human Genetics (R.M.C., N.K., S.N., A.v.N., H.A., A.J.L.) and Medicine (A.J.L.), David Geffen School of Medicine at UCLA, Los Angeles, Calif; and the Department of Medicine and Cardiovascular Research Institute (T.B.), Academic Hospital, Maastricht, the Netherlands. Correspondence to Dr Rita M. Cantor, Department of Human Genetics, David Geffen School of Medicine at UCLA, 695 Charles E. Young Dr. South, Los Angeles, CA 90095-7088. E-mail rcantor@mednet.ucla.edu 2004 American Heart Association, Inc. Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000142358.46276.a7 1935

1936 Arterioscler Thromb Vasc Biol. October 2004 full genome scans, 8 14 extensive candidate gene analyses, 15 and analyses of the FCHL diagnosis in large pedigrees 10 and in nuclear families. 11 Recently, very promising findings were generated by analyzing the FCHL diagnosis or a related binary trait derived from increases over age/sex-specific cutoff values. These include linkage to 1q in large Finnish pedigrees, followed by successful association analyses of a positional candidate gene, upstream transcription factor 1, 16 replication of a linked region on 11p, 11,12 and a combined analysis of Finnish and Dutch families, which showed linkage to 16q. 17 Although some analyses of the quantitative lipid traits associated with FCHL have been included in these gene finding investigations, they have rarely been the main focus of the studies. For example, when analyzing FCHLassociated quantitative traits, Allayee et al 18 found linkage of apob to chromosome region 1p31 in a cohort of Dutch FCHL pedigrees when the trait was treated as binary. That is, if the individual exceeded the 90th percentile for their age and sex category, they were considered as affected. Using a similar analytic approach, Pajukanta et al 17 treated high-density lipoprotein (HDL) cholesterol as a binary trait, and mapped a locus for HDL cholesterol to chromosome 16q. Almasy et al 19 used the full quantitative distribution of HDL cholesterol and localized 2 putative genes influencing this trait in large pedigrees that were not ascertained for a specific lipid disorder, such as FCHL. Although the quantitative traits have been the focus of these analyses, we can hypothesize that against different genetic backgrounds (morbid or healthy), a different genetic cause in the same or different pathways may be operating to define one s profile of lipid traits. The linkage analyses we report herein were undertaken to identify the QTL for cholesterol, triglycerides, and apob, which are the component traits contributing to a diagnosis of FCHL. We conducted these analyses in a sample of FCHL pedigrees containing affected and unaffected individuals, and in which the disease-defining quantitative traits show extensive variation that is not normally distributed. By searching for lipid QTL within FCHL families, we hypothesized genes that normally make small contributions to individual lipid profiles may have larger effects and act as major genes that are easier to detect in these FCHL families. Materials and Methods Study Sample Pedigrees were ascertained in Maastricht, the Netherlands via probands with multiple lipid abnormalities associated with FCHL. The ascertainment criteria were: (1) a proband with a primary hyperlipidemia, including fasting total plasma cholesterol 6.5mmol/L (250 mg/dl) and/or fasting plasma triglyceride concentrations 2.0 mmol/l (180 mg/dl); (2) multiple lipoprotein phenotypes within the family (Fredrickson classification IIa, IIb, or IV); and (3) a positive family history of premature cardiovascular disease, defined as the occurrence of a myocardial infarction or cerebrovascular accident before the age of 60 reported in medical records. Exclusion criteria were secondary causes of hyperlipidemia (renal or hepatic insufficiency, hypothyroidism, and medication), the apo E2/E2 genotype, and subjects with tendon xanthomas or a diagnosis matching familial hypercholesterolemia. To further reduce potential genetic heterogeneity, pedigrees from among those satisfying the aforementioned criteria were included in the current analyses only if the proband had levels of serum triglycerides and serum cholesterol Figure 1. Frequency distributions of apob, cholesterol, and triglycerides in FCHL sibships. exceeding age/sex-specific cutoff values and at least 1 first-degree relative had an increased age-specific and sex-specific serum triglyceride or serum cholesterol level Specifically, the proband in each family included in these analyses exceeded the 90th percentiles for total serum cholesterol and triglyceride levels within the subject s age and sex category according to the distributions from a large-scale Finnish population-based study. 20 These percentiles may be more stringent than those for the Dutch, which are not available. ApoB, cholesterol, and triglycerides levels are not normally distributed in these pedigrees, as illustrated in Figure 1. The means

Cantor et al ApoB, Cholesterol, and Triglycerides QTL in FCHL 1937 (m) and standard deviations (s) of these traits taken respectively in the siblings in this sample are: apob (m 121.2; s 33.1), cholesterol (m 5.9; s 1.7), and triglycerides (m 2.4; s 2.8). The pedigrees selected for the analyses also vary markedly in their sizes, with some nuclear families and other quite large multigenerational families. These factors discouraged us from undertaking a variance component linkage analysis, which assumes normality of the quantitative traits and is most effective in larger pedigrees. We chose to use a nonparametric statistical linkage method that correlates allele sharing identical-by-descent (ibd) in sibling pairs with the differences in their trait values, instead. Table 1 gives the sizes of the sibships in this sample, the numbers of sibships of each of those sizes, and the numbers of sibling pairs provided to the analyses. This study was approved by Human Investigation Research Committee of the Academic Hospital of Maastricht and the UCLA institutional review board. Lipid Measurements The lipid measurements were performed in the morning (8:00 AM to 11:00 AM) after an overnight fast (12 to 14 hours). Subjects refrained from smoking and did not drink coffee or tea in the morning and had abstained from alcohol for at least 72 hours. Any lipid-lowering medication had been withdrawn for 2 weeks before all measurements were made. Total cholesterol and fasting triglyceride concentrations were measured in duplicate by a commercially available colorimetric assay (Monotest Cholesterol kit, Boehringer Mannheim 1442350 and GPO-PAP, Boehringer Mannheim, 701912, respectively). Plasma apob measurements were obtained using a commercially available immunophelometric assay using calibrated standards according to the International Federation for Clinical Chemistry (Behringwerke, Marburg, Germany). Plasma cholesterol and triglycerides are reported in mmol/l. Each sibling had values for the 3 quantitative traits undergoing analysis. Genotyping Three hundred seventy-seven informative microsatellite markers with an average intermarker distance of 9.4 cm were genotyped by the Marshfield Genotyping service (http://www.marshfieldclinic. org/research/genetics/) using the markers of Marshfield Panel 10. Gaps between markers 15 cm were located at 5p1314 (23 cm), 6p24-25 (17 cm), 9q34 (17 cm), 11q12-3 (17 cm), and 11q24-25 (19 cm). All siblings and 76% of the parents in these nuclear families were genotyped. Mendelian errors were detected in 4.2% of the sibling genotypes, and the inconsistent marker was then coded with zeros for that sibling or family. QTL Linkage Analyses A multipoint nonparametric or model-free statistical method that correlates the differences in trait levels within sibling pairs with their degree of allele sharing ibd along the chromosomes was used. When there is linkage to a region, similar trait values are expected to occur in sibling pairs with increased marker allele sharing, whereas those pairs that have different trait values will exhibit reduced marker allele sharing. With this test, for each sibling pair, the trait difference is calculated and ranked over the entire sample. Siblings in a pair who have a similar lipid value will have a low rank, such as 10 of 100, whereas siblings in a pair who differ by a relatively large amount will have a larger rank, such as 90 of 100. The statistical procedure relates the ranks of the sibling trait differences to their estimated degrees of allele sharing, using the nonparametric Kruskal Wallis test, 21 which is similar to an analysis of variance on the ranks of the trait differences. This test is programmed in the nonparametric option in the Genehunter software, 22 and the test statistic is a z-score in which the probability value is calculated from the 1-sided standard normal distribution. The multipoint nature of the analysis results from estimating the probabilities of sharing 0, 1, and 2 alleles ibd across the chromosome at 2-cM intervals using the alleles of the siblings at the genotyped markers and the genetic map across the region. A Haldane map function was used. Significant QTL have been identified by P 0.001 with this analysis. We also identified those regions having 2 or more traits each with P 0.03 at the same locus. Additional QTL Analyses A second analytic approach, the Haseman Elston (HE) test, 23 which correlates allele-sharing ibd with the squares of raw trait differences in sibling pairs by regressing the squared differences against the allele sharing, is more vulnerable to the influences of non-normally distributed data. Multipoint HE analyses were also conducted with the Genehunter software, but the results were not used to identify the linked regions and were included for informational purposes only in those the regions first identified by the nonparametric analyses. One large sibship in the sample consisted of 13 siblings as well as additional sibships that contained their offspring. We conducted all of the analyses described, on this subsample alone, to infer which of the linkage signals derived primarily from this family. Linkage Analysis of ApoB and Triglycerides as a Discrete Trait To also investigate the binary phenotype of FCHL, we used the quantitative traits of apob and triglycerides as suggested by the most recent workshop on FCHL 24 to define a binary FCHL phenotype. Their consensus was that increased apob and triglyceride levels, rather than the classical phenotype of combined elevated cholesterol and triglyceride levels, be applied in the diagnosis of FCHL. This was based on previous studies of the FCHL trait that showed impaired clearance of postprandial lipoproteins and increased verylow-density lipoprotein (VLDL) secretion lead to increased plasma levels of apob and triglycerides, 25,26 implying that apob may be a more precise measure of increased VLDL. Thus, all pedigree members were assessed as to whether their apob or triglyceride levels exceeded the 90th percentile for their age and sex categories, again using the Finnish distributions. 20 Among the pedigrees, 20 contained at least 2 individuals who exceeded either of these criteria and they were included, resulting in 125 individuals who were classified as affected. A single-point parametric linkage analysis of the genome scan data was conducted using a dominant fully penetrant model for the inheritance of the putative disease gene. Results Table 2 summarizes the positive QTL results from the nonparametric linkage analyses as defined by P 0.001 for 1 trait, or P 0.03 for each of 2 or more traits. The table is organized vertically by the chromosome, and within that the trait names in alphabetic order. The first column gives the name of the trait, the second column gives the name of the marker closest to the linkage peak, and the third column gives the chromosome band(s) at which the QTL occurs. The fourth column gives the distance of the peak marker from the p-terminal end of the chromosome, the fifth column gives the z-score for the nonparametric analysis and the corresponding probability value, the sixth column gives the HE lod score, and the last column gives the nonparametric z-score and the corresponding probability value for the analysis of the largest family of 13 siblings and 3 nuclear families containing their offspring. Four QTL were identified by the criterion of P 0.001. They are at 1p21-31 (P 0.000009) and 17p11-21 (P 0.000009) for apob, 12p13 (P 0.0001) for cholesterol, and 4p15-16 (P 0002) for triglycerides. Detailed plots of the significant QTL as defined by P 0.001 by the multipoint nonparametric analysis of the full genome scan of apob, total cholesterol, and triglycerides are presented in Figure 2. The QTL are displayed in both directions from the peak to the point where the linkage signal is zero. The nonparametric and HE linkage statistics are

1938 Arterioscler Thromb Vasc Biol. October 2004 TABLE 1. Sibships in the FCHL Quantitative Trait Locus Analyses Sibship Size 2 3 4 5 8 13 Total N of sibships 13 5 1 1 1 1 22 N of individuals 26 15 4 5 8 13 71 N of sibpairs 13 15 6 10 28 78 150 presented on the same axes, with the nonparametric z-score in solid black lines and the HE lod scores in dashed gray lines. The results for regions showing 1 QTL, as defined by P 0.03 for each trait, are 4q34-35 for apob (P 0.02) and triglycerides (P 0.01), 16p12-13 for cholesterol (P 0.01) and triglycerides (P 0.02), and 17p11-q21 shows linkage to cholesterol (P 0.008) and triglycerides (P 0.02) in addition to apob. The traits are highly correlated in the probands of these sibships; the Spearman correlations are: 0.71 (P 0.0002) for apob and cholesterol, 0.42 (P 0.05) for apob and triglycerides, and 0.62 (P 0.002) for cholesterol and triglycerides. Nonparametric analysis z-scores and probability values indicate that the largest sibship of 13 siblings and 3 nuclear families with their offspring can explain the linkage of triglycerides to 4p15-16. However, although linkage of apob to 1p21-31 is also observed in the large family, other sibships in the sample also appear to contribute to this QTL. This same pattern is evident at 17p11-q21 for apob. The QTL for cholesterol at 12p13 appears to derive very little of its linkage signal from this large family. The parametric 2-point analyses of FCHL, when it was defined as binary trait by either increased age/sex-specific apob or triglyceride levels, yielded all but 2 lod scores 1.5. Both were in one 20-cM region of 16p12-13. Because only one of the markers yielded a high lod score of 4.0, we also conducted a multipoint analysis to include the linkage information from the surrounding markers. Those results were, as expected, more modulated, with a multipoint lod score of 1.5. In a parametric analysis of a binary trait, lack of evidence for linkage can occur either because there is no linkage to a region or because an incorrect model for the inheritance TABLE 2. pattern of the FCHL genes was posed. We report these results, however, because the alternative definition of the FCHL trait is linked to a chromosome region that overlaps with the cholesterol and triglycerides QTL on 16p12-13 as defined by P 0.03 for both traits. Discussion The overwhelming success of gene-finding efforts for Mendelian diseases encourages a focus on genetically complex disorders such as FCHL. Recent reports indicate that a number of predisposing FCHL genes are on their way toward being identified and confirmed, but much remains to be performed before the genetics of this disorder is fully understood, and thoughts can turn to risk prediction and specific treatment targets. A successful example is the report of linkage 10 and association of USF-1 in Finnish FCHL pedigrees, 16 with support for linkage of triglycerides to this region found in Dutch FCHL families, 18 as well as diabetes-related traits found in other independent cohorts. 27 29 FCHL is a genetically complex disease, and the consensus is that innovative and more in-depth trait definitions and alternative analytic strategies are required to obtain insight. Toward that end, we examined individual FCHL-related quantitative traits in pedigrees to map their QTL. A model-free nonparametric statistical linkage procedure that makes no assumptions regarding trait normality and correlates the degree of allele sharing with the degree of trait sharing in the sibling pairs was used. The analyses provided evidence for apob QTL on 1p21-31 and 17p11-q23, a cholesterol QTL on 12p13, and a triglycerides QTL on 4p15-16. Using a less stringent criterion, we found evidence of apob and triglycerides QTL on 4q34-35, cholesterol and triglycerides QTL as well as linkage of a binary FCHL trait defined by increased apob or serum triglycerides to 16p13-12, and all 3 quantitative traits to 17p11-21. Although the criterion of P 0.001 was used for reporting linkage, 3 correlated quantitative traits and 1 bivariate binary trait have been analyzed separately, and multiple testing must be considered in the interpretation of these results. The QTL Linkage Peaks for ApoB, Cholesterol, and Triglycerides QTL in FCHL Sibships Quantitative Trait Peak Marker QTL Chromosome Band(s) Marker Distance Nonparametric z-score (P ) HE Lod Nonparametric z-score (P ), Large Sibship apob D1S1627 1p21-31 139 4.3 (0.000009) 2.2 3.7 (0.0001) Triglycerides GATA70E01 4p15-16 35 3.6 (0.0002) 1.2 4.0 (0.00003) apob D4S2417 4q34 182 2.1 (0.02) 0.5 2.5 (0.006) Triglycerides 2.3 (0.01) 1.0 2.8 (0.003) Cholesterol D12S372 12p13 6 3.7 (0.0001) 3.7 1.1 (NS) Cholesterol D16S2616 16p13 12 2.2 (0.01) 1.9 1.8 (0.04) Triglycerides 2.0 (0.02) 0.4 2.1 (0.02) apob D17S1294 17p11-q21 51 4.3 (0.000009) 3.7 3.6 (0.0002) Cholesterol 2.4 (0.008) 0.7 1.7 (0.04) Triglycerides 2.0 (0.02) 0.5 1.3 (NS) A binary trait defined by an age-specific and sex-specific increase in apob or triglycerides only showed linkage to 16p12-13 with a multipoint lod of 1.5.

Cantor et al ApoB, Cholesterol, and Triglycerides QTL in FCHL 1939 Figure 2. Plots in bold black lines of nonparametric z-scores QTL defined by P.001 for apob, cholesterol, and triglycerides in FCHL sibships. The HE lod scores for these data are plotted with dashed gray lines on the same axes. The distance along the horizontal axis is in centimorgans (cm) and the genotyped markers are indicated. The distance along the vertical axis is either nonparametric z-score or HE lod score. reported in Table 2 and Figure 2 are significant at the P 0.001 level, even if a stringent Bonferroni correction is applied. This is not true for the QTL identified by P 0.03 for 2 or more traits, so we report them but, without follow-up studies, interpret them as interesting results. In addition, we recognize that this 10-cM genome scan is only a first step in the gene identification process for FCHL. It can only localize the predisposing genes to wide chromosome regions of 35 to 40 cm. However, we envision that reports, such as this one, will provide the basis for future collaborations to fine-map specific regions in combined study samples to localize the disease-predisposing genes with greater precision. As SNP technologies become more cost-effective, the individual risk genes will likely be discovered. This is a long process, and these initial investigations are necessary for future success. With this in mind, we examined the literature for other positive linkage signals in the QTL regions revealed by these analyses. The apob QTL at 1p21-31 with a peak at 139 cm near marker D1S1627 in this sample is 35 cm from a linkage peak for apob at marker D1S1665 found in an independent Dutch FCHL cohort from Utrecht. In that sample, apob was treated as a binary trait, and those with an apob level exceeding the 90th percentile for their age and sex were coded as affected in an affecteds only linkage analysis. 18 Although it is tempting, the distance of 35 cm between the peaks may be too large to allow us to infer that these samples are mapping the same apob gene. We are most encouraged by the coincident location of QTL for apob, cholesterol, and triglycerides within the 17p11-21 chromosomal region. Although the traits are correlated in this sample, the fact that all 3 have QTL in the same region may reflect a gene contributing to the increase in multiple lipid profiles associated with FCHL. A 2-point HE analysis of triglyceride levels in an independent sample of 75 obese families resulted in a peak z-score of 2.58 at 65 cm, although the linked region ranged from 40 to 70 cm, and is consistent with our findings. 13 Although it was not among the strongest results, linkage to this region with P 0.014 has also been reported for cholesterol in an independent cohort of British FCHL families. 12 Because cholesterol-carrying lipoproteins have apob as their structural apoprotein, and FCHL is characterized by elevated plasma levels of apob containing VLDL and low-density lipoproteins (LDL), this is not unexpected. The putative gene in this region may have a pleiotropic effect on increased total serum cholesterol and

1940 Arterioscler Thromb Vasc Biol. October 2004 apob within FCHL pedigrees. In addition, VLDL subclasses have direct impacts on the plasma concentrations of LDL subclasses in FCHL, specifically VLDL1 and small dense LDL, 30 suggesting a VLDL triglyceride pathway in FCHL. What is known about its biochemistry corresponds well with linkage and association results for apoa-ciii-aiv, apoaii, and apob in FCHL. 30 The USF-1 gene may also act in the VLDL triglyceride pathway by stimulating lipogenesis, by fatty acid synthase, for export in VLDL, apob, cholesterol, and triglycerides. 31 17q11 contains the gene for NOS2A, the inducible nitric oxide synthase, which we found to be downregulated in adipose tissue biopsy samples from FCHL subjects in comparison with age-, sex-, and body mass index-matched controls (data not shown). NOS2A acts in the liver and may have other less well-characterized functions than regulation of vascular tone. 32 The linkage found with apob in the region of marker D1S1627 is of interest, because it was also found to be linked with plasma leptin concentrations that reflect adiposity in an independent cohort of Dutch FCHL subjects from Utrecht. 33 It was suggested that the linkage may be caused by DNA variation in the leptin receptor (LEPR) gene, close to D1S1627. We also find that as adiposity, assessed by the waist-to-hip ratio, increases, FCHL subjects express higher plasma apob concentrations than matched controls (data not shown). Thus, the linkage between plasma apob concentrations and D1S1627 is consistent with the influence of the LEPR gene on apob in FCHL. Extensive single nucleotide association studies would have to be conducted to pursue this possibility. These findings illustrate that a QTL approach in combination with refined phenotypes will yield insight into novel genes and pathways responsible for FCHL and its associated risk of heart disease. Acknowledgments This work was supported by National Institutes of Health grant HL-28481 and the Netherlands Organization of Scientific Research (900-95-299). Genotyping was performed by the Mammalian Genotyping Service of the Marshfield Medical Research Foundation. We thank Eric Keulen, MD and Josefine van Lin, research nurse, for invaluable help with the collection of the families, and Margee Robertus-Teunissen for technical assistance. References 1. Goldstein JL, Schrott HG, Hazzard WR, Bierman EL, Motulsky AG. Hyperlipidemia in coronary heart disease. II. Genetic analysis of lipid levels in 176 families and delineation of a new inherited disorder, combined hyperlipidemia. J Clin Invest. 1973;52:1544 1568. 2. Nikkila EA, Aro A. Family study of serum lipids and lipoproteins in coronary heart-disease. Lancet. 1973;1:954 959. 3. Rose HG, Kranz P, Weinstock M, Juliano J, Haft JI. Inheritance of combined hyperlipoproteinemia: evidence for a new lipoprotein phenotype. Am J Med. 1973;54:148 160. 4. Grundy SM. Monounsaturated fatty acids, plasma cholesterol, and coronary heart disease. Am J Clin Nutr. 1987;45:1168 1175. 5. Jarvik GP, Beaty TH, Gallagher PR, Coates PM, Cortner JA. Genotype at a major locus with large effects on apolipoprotein B levels predicts familial combined hyperlipidemia. Genet Epidemiol. 1993;10:257 270. 6. Jarvik GP, Brunzell JD, Austin MA, Krauss RM, Motulsky AG, Wijsman E. Genetic predictors of FCHL in four large pedigrees. Influence of ApoB level major locus predicted genotype and LDL subclass phenotype. Arterioscler Thromb. 1994;14:1687 1694. 7. Bredie SJ, van Drongelen J, Kiemeney LA, Demacker PN, Beaty TH, Stalenhoef AF. Segregation analysis of plasma apolipoprotein B levels in familial combined hyperlipidemia. Arterioscler Thromb Vasc Biol. 1997; 17:834 840. 8. Austin MA, Edwards KL, Monks SA, Koprowicz KM, Brunzell JD, Motulsky AG, Mahaney MC, Hixson JE. Genome-wide scan for quantitative trait loci influencing LDL size and plasma triglyceride in familial hypertriglyceridemia. J Lipid Res. 2003;44:2161 2168. 9. Coon H, Leppert MF, Eckfeldt JH, Oberman A, Myers RH, Peacock JM, Province MA, Hopkins PN, Heiss G. Genome-wide linkage analysis of lipids in the Hypertension Genetic Epidemiology Network (HyperGEN) Blood Pressure Study. Arterioscler Thromb Vasc Biol. 2001;21: 1969 1976. 10. Pajukanta P, Terwilliger JD, Perola M, Hiekkalinna T, Nuotio I, Ellonen P, Parkkonen M, Hartiala J, Ylitalo K, Pihlajamaki J, Porkka K, Laakso M, Viikari J, Ehnholm C, Taskinen MR, Peltonen L. Genomewide scan for familial combined hyperlipidemia genes in Finnish families, suggesting multiple susceptibility loci influencing triglyceride, cholesterol, and apolipoprotein B levels. Am J Hum Genet. 1999;64:1453 1463. 11. Aouizerat BE, Allayee H, Cantor RM, Davis RC, Lanning CD, Wen PZ, Dallinga-Thie GM, de Bruin TW, Rotter JI, Lusis AJ. A genome scan for familial combined hyperlipidemia reveals evidence of linkage with a locus on chromosome 11. Am J Hum Genet. 1999;65:397 412. 12. Naoumova RP, Bonney SA, Eichenbaum-Voline S, Patel HN, Jones B, Jones EL, Amey J, Colilla S, Neuwirth CK, Allotey R, Seed M, Betteridge DJ, Galton DJ, Cox NJ, Bell GI, Scott J, Shoulders CC. Confirmed locus on chromosome 11p and candidate Loci on 6q and 8p for the triglyceride and cholesterol traits of combined hyperlipidemia. Arterioscler Thromb Vasc Biol. 2003;23:2070 2077. 13. Reed DR, Nanthakumar E, North M, Bell C, Price RA. A genome-wide scan suggests a locus on chromosome 1q21 q23 contributes to normal variation in plasma cholesterol concentration. J Mol Med. 2001;79: 262 269. 14. Francke S, Manraj M, Lacquemant C, Lecoeur C, Lepretre F, Passa P, Hebe A, Corset L, Yan SL, Lahmidi S, Jankee S, Gunness TK, Ramjuttun US, Balgobin V, Dina C, Froguel P. A genome-wide scan for coronary heart disease suggests in Indo-Mauritians a susceptibility locus on chromosome 16p13 and replicates linkage with the metabolic syndrome on 3q27. Hum Mol Genet. 2001;10:2751 2765. 15. Aouizerat BE, Allayee H, Cantor RM, Dallinga-Thie GM, Lanning CD, de Bruin TW, Lusis AJ, Rotter JI. Linkage of a candidate gene locus to familial combined hyperlipidemia: lecithin:cholesterol acyltransferase on 16q. Arterioscler Thromb Vasc Biol. 1999;19:2730 2736. 16. Pajukanta P, Lilja HE, Sinsheimer JS, Cantor RM, Lusis AJ, Gentile M, Duan XJ, Soro-Paavonen A, Naukkarinen J, Saarela J, Laakso M, Ehnholm C, Taskinen MR, Peltonen L. Familial combined hyperlipidemia is associated with upstream transcription factor 1 (USF1). Nat Genet. 2004;36:371 376. 17. Pajukanta P, Allayee H, Krass KL, Kuraishy A, Soro A, Lilja HE, Mar R, Taskinen MR, Nuotio I, Laakso M, Rotter JI, de Bruin TW, Cantor RM, Lusis AJ, Peltonen L. Combined analysis of genome scans of dutch and finnish families reveals a susceptibility locus for high-density lipoprotein cholesterol on chromosome 16q. Am J Hum Genet. 2003;72:903 917. 18. Allayee H, Krass KL, Pajukanta P, Cantor RM, van der Kallen CJ, Mar R, Rotter JI, de Bruin TW, Peltonen L, Lusis AJ. Locus for elevated apolipoprotein B levels on chromosome 1p31 in families with familial combined hyperlipidemia. Circ Res. 2002;90:926 931. 19. Almasy L, Hixson JE, Rainwater DL, Cole S, Williams JT, Mahaney MC, VandeBerg JL, Stern MP, MacCluer JW, Blangero J. Human pedigree-based quantitative-trait-locus mapping: localization of two genes influencing HDL-cholesterol metabolism. Am J Hum Genet. 1999; 64:1686 1693. 20. Porkka KV, Viikari JS, Ronnemaa T, Marniemi J, Akerblom HK. Age and gender specific serum lipid and apolipoprotein fractiles of Finnish children and young adults. The Cardiovascular Risk in Young Finns Study. Acta Paediatr. 1994;83:838 848. 21. Kruskal WH, Wallis WA. Use of ranks in one-criterion analysis of variance. Journal of the Am Statistical Association. 1953;47:583 621. 22. Kruglyak L, Daly MJ, Reeve-Daly MP, Lander ES. Parametric and nonparametric linkage analysis: a unified multipoint approach. Am J Hum Genet. 1996;58:1347 1363. 23. Haseman JK, Elston RC. The investigation of linkage between a quantitative trait and a marker locus. Behav Genet. 1972;2:3 19. 24. Sniderman AD, Castro Cabezas M, Ribalta J, Carmena R, de Bruin TW, de Graaf J, Erkelens DW, Humphries SE, Masana L, Real JT, Talmud PJ,

Cantor et al ApoB, Cholesterol, and Triglycerides QTL in FCHL 1941 Taskinen MR. A proposal to redefine familial combined hyperlipidaemia third workshop on FCHL held in Barcelona from 3 to 5 May 2001, during the scientific sessions of the European Society for Clinical Investigation. Eur J Clin Invest. 2002;32:71 73. 25. Castro Cabezas M, de Bruin TW, de Valk HW, Shoulders CC, Jansen H, Willem Erkelens D. Impaired fatty acid metabolism in familial combined hyperlipidemia. A mechanism associating hepatic apolipoprotein B overproduction and insulin resistance. J Clin Invest. 1993;92:160 168. 26. de Graaf J, Stalenhoef AF. Defects of lipoprotein metabolism in familial combined hyperlipidaemia. Curr Opin Lipidol. 1998;9:189 196. 27. Hsueh WC, St Jean PL, Mitchell BD, Pollin TI, Knowler WC, Ehm MG, Bell CJ, Sakul H, Wagner MJ, Burns DK, Shuldiner AR. Genome-wide and fine-mapping linkage studies of type 2 diabetes and glucose traits in the Old Order Amish: evidence for a new diabetes locus on chromosome 14q11 and confirmation of a locus on chromosome 1q21 q24. Diabetes. 2003;52:550 557. 28. Vionnet N, Hani El H, Dupont S, Gallina S, Francke S, Dotte S, De Matos F, Durand E, Lepretre F, Lecoeur C, Gallina P, Zekiri L, Dina C, Froguel P. Genomewide search for type 2 diabetes-susceptibility genes in French whites: evidence for a novel susceptibility locus for early-onset diabetes on chromosome 3q27-qter and independent replication of a type 2-diabetes locus on chromosome 1q21 q24. Am J Hum Genet. 2000;67: 1470 1480. 29. Wiltshire S, Hattersley AT, Hitman GA, Walker M, Levy JC, Sampson M, O Rahilly S, Frayling TM, Bell JI, Lathrop GM, Bennett A, Dhillon R, Fletcher C, Groves CJ, Jones E, Prestwich P, Simecek N, Rao PV, Wishart M, Bottazzo GF, Foxon R, Howell S, Smedley D, Cardon LR, Menzel S, McCarthy MI. A genomewide scan for loci predisposing to type 2 diabetes in a U.K. population (the Diabetes UK Warren 2 Repository): analysis of 573 pedigrees provides independent replication of a susceptibility locus on chromosome 1q. Am J Hum Genet. 2001;69: 553 569. 30. Georgieva AM, Van Greevenbroek MM, Krauss RM, Brouwers MC, Vermeulen VM, Robertus-Teunissen MG, Van Der Kallen CJ, De Bruin TW. Subclasses of Low-Density Lipoprotein and Very Low-Density Lipoprotein in Familial Combined Hyperlipidemia: Relationship to Multiple Lipoprotein Phenotype. Arterioscler Thromb Vasc Biol. 2004; 24:744 749. 31. Chandler CE, Wilder DE, Pettini JL, Savoy YE, Petras SF, Chang G, Vincent J, Harwood HJ, Jr. CP-346086: an MTP inhibitor that lowers plasma cholesterol and triglycerides in experimental animals and in humans. J Lipid Res. 2003;44:1887 1901. 32. Rutherford S, Johnson MP, Curtain RP, Griffiths LR. Chromosome 17 and the inducible nitric oxide synthase gene in human essential hypertension. Hum Genet. 2001;109:408 415. 33. van der Kallen CJ, Cantor RM, van Greevenbroek MM, Geurts JM, Bouwman FG, Aouizerat BE, Allayee H, Buurman WA, Lusis AJ, Rotter JI, de Bruin TW. Genome scan for adiposity in Dutch dyslipidemic families reveals novel quantitative trait loci for leptin, body mass index and soluble tumor necrosis factor receptor superfamily 1A. Int J Obes Relat Metab Disord. 2000;24:1381 1391.