The involvement of upstream stimulatory factor 1 in Dutch patients with familial combined hyperlipidemia
2006; Elsevier BV; Volume: 48; Issue: 1 Linguagem: Inglês
10.1194/jlr.m600184-jlr200
ISSN1539-7262
AutoresGerly M. van der Vleuten, Aaron Isaacs, Anneke Hijmans, Cornelia M. van Duijn, Anton F. H. Stalenhoef, Jacqueline de Graaf,
Tópico(s)Lipid metabolism and disorders
ResumoRecently, the upstream stimulatory factor 1 gene (USF1) was proposed as a candidate gene for familial combined hyperlipidemia (FCH). In this study, we examined the previously identified risk haplotype of USF1 with respect to FCH and its related phenotypes in 36 Dutch FCH families. The diagnosis of FCH was based on both the traditional diagnostic criteria and a nomogram. The two polymorphisms, USF1s1 and USF1s2, were in complete linkage disequilibrium. No association was found for the individual single nucleotide polymorphisms (SNPs) with FCH defined by the nomogram (USF1s1, P = 0.53; USF1s2, P = 0.53), whereas suggestive associations were found when using the traditional diagnostic criteria for FCH (USF1s1, P = 0.08; USF1s2, P = 0.07). USF1 was associated with total cholesterol (USF1s1, P = 0.05; USF1s2, P = 0.04) and apolipoprotein B (USF1s1, P = 0.06; USF1s2, P = 0.04). Small dense LDL showed a suggestive association (USF1s1, P = 0.10; USF1s2, P = 0.09). The results from the haplotype analyses supported the results obtained for the individual SNPs. In conclusion, the previously identified risk haplotype of USF1 showed a suggestive association with FCH and contributed to the related lipid traits in our Dutch FCH families. Recently, the upstream stimulatory factor 1 gene (USF1) was proposed as a candidate gene for familial combined hyperlipidemia (FCH). In this study, we examined the previously identified risk haplotype of USF1 with respect to FCH and its related phenotypes in 36 Dutch FCH families. The diagnosis of FCH was based on both the traditional diagnostic criteria and a nomogram. The two polymorphisms, USF1s1 and USF1s2, were in complete linkage disequilibrium. No association was found for the individual single nucleotide polymorphisms (SNPs) with FCH defined by the nomogram (USF1s1, P = 0.53; USF1s2, P = 0.53), whereas suggestive associations were found when using the traditional diagnostic criteria for FCH (USF1s1, P = 0.08; USF1s2, P = 0.07). USF1 was associated with total cholesterol (USF1s1, P = 0.05; USF1s2, P = 0.04) and apolipoprotein B (USF1s1, P = 0.06; USF1s2, P = 0.04). Small dense LDL showed a suggestive association (USF1s1, P = 0.10; USF1s2, P = 0.09). The results from the haplotype analyses supported the results obtained for the individual SNPs. In conclusion, the previously identified risk haplotype of USF1 showed a suggestive association with FCH and contributed to the related lipid traits in our Dutch FCH families. Familial combined hyperlipidemia (FCH; Online Mendelian Inheritance in Man 144250) is the most common genetic lipid disorder of unknown cause in humans, affecting up to 5% of the general population (1Hopkins P.N. Heiss G. Ellison R.C. Province M.A. Pankow J.S. Eckfeldt J.H. Hunt S.C. Coronary artery disease risk in familial combined hyperlipidemia and familial hypertriglyceridemia: a case-control comparison from the National Heart, Lung, and Blood Institute Family Heart Study..Circulation. 2003; 108: 519-523Crossref PubMed Scopus (174) Google Scholar). Major characteristics of FCH include increased levels of apolipoprotein B (apoB), triglycerides (TGs), and/or plasma total cholesterol (TC). Other FCH phenotypes are decreased levels of high density lipoprotein cholesterol and the presence of small, dense low density lipoprotein (sdLDL). In addition, patients with FCH have a 2-fold increased risk of cardiovascular disease (CVD) and are often obese and insulin-resistant (2de Graaf J. van der Vleuten G. Stalenhoef A.F. Diagnostic criteria in relation to the pathogenesis of familial combined hyperlipidemia..Semin. Vasc. Med. 2004; 4: 229-240Crossref PubMed Scopus (28) Google Scholar).During the past few years, several groups have performed linkage analyses in an attempt to determine the genetic defects causing FCH (3Aouizerat B.E. Allayee H. Cantor R.M. Davis R.C. Lanning C.D. Wen P.Z. Linga-Thie G.M. de Bruin T.W. Rotter J.I. Lusis A.J. A genome scan for familial combined hyperlipidemia reveals evidence of linkage with a locus on chromosome 11..Am. J. Hum. Genet. 1999; 65: 397-412Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 4Coon H. Myers R.H. Borecki I.B. Arnett D.K. Hunt S.C. Province M.A. Djousse L. Leppert M.F. Replication of linkage of familial combined hyperlipidemia to chromosome 1q with additional heterogeneous effect of apolipoprotein A-I/C-III/A-IV locus. The NHLBI Family Heart Study..Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2275-2280Crossref PubMed Scopus (83) Google Scholar, 5Huertas-Vazquez A. Guilar-Salinas C. Lusis A.J. Cantor R.M. Canizales-Quinteros S. 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Al Yahyaee S.A. Hui R. Wu X. Liu L. Busjahn A. Luft F.C. et al.Support for linkage of familial combined hyperlipidemia to chromosome 1q21-q23 in Chinese and German families..Clin. Genet. 2000; 57: 29-34Crossref PubMed Scopus (73) Google Scholar). By doing this, a locus on chromosome 1q21-23 was identified (4Coon H. Myers R.H. Borecki I.B. Arnett D.K. Hunt S.C. Province M.A. Djousse L. Leppert M.F. Replication of linkage of familial combined hyperlipidemia to chromosome 1q with additional heterogeneous effect of apolipoprotein A-I/C-III/A-IV locus. The NHLBI Family Heart Study..Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2275-2280Crossref PubMed Scopus (83) Google Scholar, 7Pajukanta P. Nuotio I. Terwilliger J.D. Porkka K.V. Ylitalo K. Pihlajamaki J. Suomalainen A.J. Syvanen A.C. Lehtimaki T. Viikari J.S. et al.Linkage of familial combined hyperlipidaemia to chromosome 1q21-q23..Nat. Genet. 1998; 18: 369-373Crossref PubMed Scopus (235) Google Scholar, 8Pei W. Baron H. Muller-Myhsok B. Knoblauch H. Al Yahyaee S.A. Hui R. Wu X. Liu L. Busjahn A. Luft F.C. et al.Support for linkage of familial combined hyperlipidemia to chromosome 1q21-q23 in Chinese and German families..Clin. Genet. 2000; 57: 29-34Crossref PubMed Scopus (73) Google Scholar, 9Huertas-Vazquez A. del Rincon J.P. Canizales-Quinteros S. Riba L. Vega-Hernandez G. Ramirez-Jimenez S. Uron-Gomez M. Gomez-Perez F.J. Guilar-Salinas C.A. Tusie-Luna M.T. Contribution of chromosome 1q21-q23 to familial combined hyperlipidemia in Mexican families..Ann. Hum. Genet. 2004; 68: 419-427Crossref PubMed Scopus (24) Google Scholar). The first candidate gene proposed in this region was the thioredoxin-interacting protein gene (TXNIP) (10Bodnar J.S. Chatterjee A. Castellani L.W. Ross D.A. Ohmen J. Cavalcoli J. Wu C. Dains K.M. Catanese J. Chu M. et al.Positional cloning of the combined hyperlipidemia gene Hyplip1..Nat. Genet. 2002; 30: 110-116Crossref PubMed Scopus (178) Google Scholar). It was demonstrated, however, that TXNIP was not a major contributor to the FCH phenotype (11Coon H. Singh N. Dunn D. Eckfeldt J.H. Province M.A. Hopkins P.N. Weiss R. Hunt S.C. Leppert M.F. TXNIP gene not associated with familial combined hyperlipidemia in the NHLBI Family Heart Study..Atherosclerosis. 2004; 174: 357-362Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 12Pajukanta P. Lilja H.E. Sinsheimer J.S. Cantor R.M. Lusis A.J. Gentile M. Duan X.J. Soro-Paavonen A. Naukkarinen J. Saarela J. et al.Familial combined hyperlipidemia is associated with upstream transcription factor 1 (USF1)..Nat. Genet. 2004; 36: 371-376Crossref PubMed Scopus (259) Google Scholar, 13van der Vleuten G.M. Hijmans A. Kluijtmans L.A. Blom H.J. Stalenhoef A.F. de Graaf J. Thioredoxin interacting protein in Dutch families with familial combined hyperlipidemia..Am. J. Med. Genet. A. 2004; 130: 73-75Crossref Scopus (16) Google Scholar). More recently, a second candidate gene, the upstream stimulatory factor 1 gene (USF1), was suggested as the prime candidate gene for FCH in this linkage region in 60 Finnish FCH families (12Pajukanta P. Lilja H.E. Sinsheimer J.S. Cantor R.M. Lusis A.J. Gentile M. Duan X.J. Soro-Paavonen A. Naukkarinen J. Saarela J. et al.Familial combined hyperlipidemia is associated with upstream transcription factor 1 (USF1)..Nat. Genet. 2004; 36: 371-376Crossref PubMed Scopus (259) Google Scholar). Twenty-three single nucleotide polymorphisms (SNPs) were reported in the USF1 gene. Two of these SNPs, USF1s1 (3′ untranslated region; rs3737787) and USF1s2 (intron 7; rs2073658), individually and combined into a haplotype, showed linkage and association with FCH and multiple phenotypes of FCH, including TG, apoB, TC, and sdLDL, implying that USF1 contributes to the complex pathophysiology of FCH in these Finnish FCH families (12Pajukanta P. Lilja H.E. Sinsheimer J.S. Cantor R.M. Lusis A.J. Gentile M. Duan X.J. Soro-Paavonen A. Naukkarinen J. Saarela J. et al.Familial combined hyperlipidemia is associated with upstream transcription factor 1 (USF1)..Nat. Genet. 2004; 36: 371-376Crossref PubMed Scopus (259) Google Scholar). The association of USF1 with FCH, however, was strongest in males with increased levels of TGs (12Pajukanta P. Lilja H.E. Sinsheimer J.S. Cantor R.M. Lusis A.J. Gentile M. Duan X.J. Soro-Paavonen A. Naukkarinen J. Saarela J. et al.Familial combined hyperlipidemia is associated with upstream transcription factor 1 (USF1)..Nat. Genet. 2004; 36: 371-376Crossref PubMed Scopus (259) Google Scholar).Huertas-Vazquez et al. (5Huertas-Vazquez A. Guilar-Salinas C. Lusis A.J. Cantor R.M. Canizales-Quinteros S. Lee J.C. Mariana-Nunez L. Riba-Ramirez R. M.L. Jokiaho A. Tusie-Luna T. et al.Familial combined hyperlipidemia in Mexicans: association with upstream transcription factor 1 and linkage on chromosome 16q24.1..Arterioscler. Thromb. Vasc. Biol. 2005; 25: 1985-1991Crossref PubMed Scopus (64) Google Scholar) reported that USF1 was associated with FCH and increased TG levels in 24 Mexican FCH families, whereas other phenotypes were not investigated. No sex-specific effect of USF1 was found in these families. Coon et al. (14Coon H. Xin Y. Hopkins P.N. Cawthon R.M. Hasstedt S.J. Hunt S.C. Upstream stimulatory factor 1 associated with familial combined hyperlipidemia, LDL cholesterol, and triglycerides..Hum. Genet. 2005; 117: 444-451Crossref PubMed Scopus (58) Google Scholar) investigated the association of USF1 with FCH in Utah pedigrees ascertained for early death attributable to coronary heart disease (CHD), early stroke, or early-onset hypertension. They reported suggestive associations for FCH, TG, and low density lipoprotein cholesterol (LDL-c) levels in the pedigrees ascertained for early stroke and early onset of hypertension. However, in the families ascertained for early death attributable to CHD, in which 60% of the FCH patients were present, no association of USF1 with FCH was found (14Coon H. Xin Y. Hopkins P.N. Cawthon R.M. Hasstedt S.J. Hunt S.C. Upstream stimulatory factor 1 associated with familial combined hyperlipidemia, LDL cholesterol, and triglycerides..Hum. Genet. 2005; 117: 444-451Crossref PubMed Scopus (58) Google Scholar).The USF1 protein regulates the transcriptional activation of a variety of genes involved in glucose and lipid metabolism and in the development of atherosclerosis (15Casado M. Vallet V.S. Kahn A. Vaulont S. Essential role in vivo of upstream stimulatory factors for a normal dietary response of the fatty acid synthase gene in the liver..J. Biol. Chem. 1999; 274: 2009-2013Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 16Ribeiro A. Pastier D. Kardassis D. Chambaz J. Cardot P. Cooperative binding of upstream stimulatory factor and hepatic nuclear factor 4 drives the transcription of the human apolipoprotein A-II gene..J. Biol. Chem. 1999; 274: 1216-1225Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). USF1 plays a pivotal role in adipose tissue, where it influences de novo lipogenesis by mediating the glucose-regulated expression of hormone-sensitive lipase (HSL), a key enzyme in the regulation of lipid storage in adipose tissue (17Smih F. Rouet P. Lucas S. Mairal A. Sengenes C. Lafontan M. Vaulont S. Casado M. Langin D. Transcriptional regulation of adipocyte hormone-sensitive lipase by glucose..Diabetes. 2002; 51: 293-300Crossref PubMed Scopus (63) Google Scholar). An interaction between SNPs in the HSL and USF1 genes in postprandial TG levels has been reported previously (18Putt W. Palmen J. Nicaud V. Tregouet D.A. Tahri-Daizadeh N. Flavell D.M. Humphries S.E. Talmud P.J. Variation in USF1 shows haplotype effects, gene:gene and gene:environment associations with glucose and lipid parameters in the European Atherosclerosis Research Study II..Hum. Mol. Genet. 2004; 13: 1587-1597Crossref PubMed Scopus (67) Google Scholar). Despite this, HSL activity in adipose tissue was unchanged in Finnish patients with FCH (19Ylitalo K. Large V. Pajukanta P. Reynisdottir S. Porkka K.V. Vakkilainen J. Nuotio I. Taskinen M.R. Arner P. Reduced hormone-sensitive lipase activity is not a major metabolic defect in Finnish FCHL families..Atherosclerosis. 2000; 153: 373-381Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). USF1 also influences the transcription of fatty acid synthase (FAS), which is involved in the synthesis of fatty acids (15Casado M. Vallet V.S. Kahn A. Vaulont S. Essential role in vivo of upstream stimulatory factors for a normal dietary response of the fatty acid synthase gene in the liver..J. Biol. Chem. 1999; 274: 2009-2013Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 20Wang D. Sul H.S. Upstream stimulatory factor binding to the E-box at −65 is required for insulin regulation of the fatty acid synthase promoter..J. Biol. Chem. 1997; 272: 26367-26374Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). FAS might play a role in FCH, as patients with FCH have decreased circulating levels of FAS ligand (21Blanco-Colio L.M. Martin-Ventura J.L. Sol J.M. Diaz C. Hernandez G. Egido J. Decreased circulating Fas ligand in patients with familial combined hyperlipidemia or carotid atherosclerosis: normalization by atorvastatin..J. Am. Coll. Cardiol. 2004; 43: 1188-1194Crossref PubMed Scopus (37) Google Scholar). USF1 also plays a role in the transcription of several apolipoproteins (APOCIII, APOAII, and APOE) (16Ribeiro A. Pastier D. Kardassis D. Chambaz J. Cardot P. Cooperative binding of upstream stimulatory factor and hepatic nuclear factor 4 drives the transcription of the human apolipoprotein A-II gene..J. Biol. Chem. 1999; 274: 1216-1225Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 22Pastier D. Lacorte J.M. Chambaz J. Cardot P. Ribeiro A. Two initiator-like elements are required for the combined activation of the human apolipoprotein C-III promoter by upstream stimulatory factor and hepatic nuclear factor-4..J. Biol. Chem. 2002; 277: 15199-15206Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), glucokinase (23Iynedjian P.B. Identification of upstream stimulatory factor as transcriptional activator of the liver promoter of the glucokinase gene..Biochem. J. 1998; 333: 705-712Crossref PubMed Scopus (55) Google Scholar), hepatic lipase (24Botma G.J. Verhoeven A.J. Jansen H. Hepatic lipase promoter activity is reduced by the C-480T and G-216A substitutions present in the common LIPC gene variant, and is increased by upstream stimulatory factor..Atherosclerosis. 2001; 154: 625-632Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), angiotensinogen (25Yanai K. Saito T. Hirota K. Kobayashi H. Murakami K. Fukamizu A. Molecular variation of the human angiotensinogen core promoter element located between the TATA box and transcription initiation site affects its transcriptional activity..J. Biol. Chem. 1997; 272: 30558-30562Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), and ABCA1 (26Yang X.P. Freeman L.A. Knapper C.L. Amar M.J. Remaley A. Brewer Jr., H.B. Santamarina-Fojo S. The E-box motif in the proximal ABCA1 promoter mediates transcriptional repression of the ABCA1 gene..J. Lipid Res. 2002; 43: 297-306Abstract Full Text Full Text PDF PubMed Google Scholar). Genetic variations in APOCIII, APOAII, and hepatic lipase have been associated with FCH (27Martin-Campos J.M. Escola-Gil J.C. Ribas V. Blanco-Vaca F. Apolipoprotein A-II, genetic variation on chromosome 1q21-q24, and disease susceptibility..Curr. Opin. Lipidol. 2004; 15: 247-253Crossref PubMed Scopus (42) Google Scholar, 28Hoffer M.J. Snieder H. Bredie S.J. Demacker P.N. Kastelein J.J. Frants R.R. Stalenhoef A.F. The V73M mutation in the hepatic lipase gene is associated with elevated cholesterol levels in four Dutch pedigrees with familial combined hyperlipidemia..Atherosclerosis. 2000; 151: 443-450Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 29McNeely M.J. Edwards K.L. Marcovina S.M. Brunzell J.D. Motulsky A.G. Austin M.A. Lipoprotein and apolipoprotein abnormalities in familial combined hyperlipidemia: a 20-year prospective study..Atherosclerosis. 2001; 159: 471-481Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 30Groenendijk M. Cantor R.M. Funke H. Linga-Thie G.M. Two newly identified SNPs in the APO AI-CIII intergenic region are strongly associated with familial combined hyperlipidaemia..Eur. J. Clin. Invest. 2001; 31: 852-859Crossref PubMed Scopus (8) Google Scholar); however, the role of glucokinase, angiotensinogen, and ABCA1 in FCH has not been explored yet.In this study, we evaluated the putative role of the USF1 gene in Dutch FCH families. We investigated the effect of two individual SNPs, and the haplotypes formed by these two SNPs, on FCH and its associated phenotypes, including not only lipids and lipoproteins but also parameters of obesity and insulin resistance.METHODSStudy populationThe FCH population was ascertained in 1994 and followed up and expanded in 1999. The families were ascertained through probands recruited from the outpatient lipid clinic of the Radboud University Nijmegen Medical Center. In 1994 and 1999, we ascertained families in which the proband exhibited a combined hyperlipidemia, with both plasma TC and TG levels above the age- and gender-related 90th percentile (31Assmann G. Schulte H. Relation of high-density lipoprotein cholesterol and triglycerides to incidence of atherosclerotic coronary artery disease (the PROCAM experience). Prospective Cardiovascular Munster study..Am. J. Cardiol. 1992; 70: 733-737Abstract Full Text PDF PubMed Scopus (827) Google Scholar), during several periods in which they were not treated with lipid-lowering drugs and despite dietary advice. Additionally, a first-degree relative had increased levels of TC and/or TG above the 90th percentile, and the proband, or a first-degree relative, suffered from premature (before the age of 60 years) CVD. Families were excluded when probands were diagnosed with underlying diseases causing hyperlipidemia (i.e., diabetes mellitus types 1 and 2, hypothyroidism, and hepatic or renal impairment), a first-degree relative had tendon xanthomata, or probands were homozygous for the APOE2 allele.In this study, we used the data of all participating subjects in 1999, including the spouses. The total population consists of 36 families from multiple (two to four) generations and comprises 611 subjects with known genealogic and phenotypic data. The diagnosis of FCH was based on 1) the traditional diagnostic criteria, including TC and/or TG levels above the 90th percentile (32Goldstein J.L. Schrott H.G. Hazzard W.R. Bierman E.L. Motulsky A.G. 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: 1544-1568Crossref PubMed Scopus (1089) Google Scholar), and 2) the nomogram, as recently described by our group (33Veerkamp M.J. de Graaf J. Hendriks J.C. Demacker P.N. Stalenhoef A.F. Nomogram to diagnose familial combined hyperlipidemia on the basis of results of a 5-year follow-up study..Circulation. 2004; 109: 2980-2985Crossref PubMed Scopus (110) Google Scholar). Plasma TG and TC levels, adjusted for age and gender, and absolute apoB levels were applied to the nomogram to calculate the probability of being affected with FCH. When this probability is >60%, the diagnostic phenotype is present in at least one first-degree relative, and premature CVD (before the age of 60 years) is present in at least one individual in the family, the individual is defined as affected by FCH. In 26 of the 36 FCH families, ascertained through the traditional criteria (32Goldstein J.L. Schrott H.G. Hazzard W.R. Bierman E.L. Motulsky A.G. 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: 1544-1568Crossref PubMed Scopus (1089) Google Scholar), the proband also fulfilled the criterion of the nomogram for the FCH diagnosis; for the other 10 probands, the nomogram could not be applied because of missing apoB data. The normolipidemic relatives (n = 390), unaffected spouses of both the FCH patients and the normolipidemic relatives (n = 64), and subjects without known phenotypic and/or genotypic data (n = 230) were included in the family-based analyses. After withdrawal of lipid-lowering medication for 4 weeks and an overnight fast, blood was drawn by venipuncture. The maximum waist circumference (cm) at the umbilical level was measured in the late exhalation phase while standing. All included subjects were Caucasian and older than 10 years. The ethical committee of the Radboud University Nijmegen Medical Center approved the study protocol, and the procedures followed were in accordance with institutional guidelines. All subjects gave informed consent.Biochemical analysesPlasma TC and TG were determined by commercially available enzymatic reagents (Boehringer-Mannheim, Mannheim, Germany, catalog No. 237574; and Sera Pak, Miles, Belgium, catalog No. 6639, respectively). Total plasma apoB concentrations were measured by immunonephelometry (34Demacker P.N. Veerkamp M.J. Bredie S.J. Marcovina S.M. de Graaf J. Stalenhoef A.F. Comparison of the measurement of lipids and lipoproteins versus assay of apolipoprotein B for estimation of coronary heart disease risk: a study in familial combined hyperlipidemia..Atherosclerosis. 2000; 153: 483-490Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). LDL subfractions were separated by single-spin density gradient ultracentrifugation, according to a previously described method (35Swinkels D.W. Hak-Lemmers H.L. Demacker P.N. Single spin density gradient ultracentrifugation method for the detection and isolation of light and heavy low density lipoprotein subfractions..J. Lipid Res. 1987; 28: 1233-1239Abstract Full Text PDF PubMed Google Scholar). A continuous variable, K, represents the LDL subfraction profile of each individual. A negative K value (K ⩽ −0.1) reflects a more dense LDL subfraction profile, and a positive K value (K > −0.1) reflects a more buoyant profile. Glucose concentrations were analyzed in duplicate using the oxidation technique (Beckman® Glucose Analyser2; Beckman Instruments, Inc.). Plasma insulin concentrations were ascertained using a double antibody method (36de Galan B.E. Tack C.J. Lenders J.W. Pasman J.W. Elving L.D. Russel F.G. Lutterman J.A. Smits P. Theophylline improves hypoglycemia unawareness in type 1 diabetes..Diabetes. 2002; 51: 790-796Crossref PubMed Scopus (50) Google Scholar). Insulin resistance was assessed by the homeostasis model assessment (HOMA) method (37Matthews D.R. Hosker J.P. Rudenski A.S. Naylor B.A. Treacher D.F. Turner R.C. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man..Diabetologia. 1985; 28: 412-419Crossref PubMed Scopus (25088) Google Scholar).DNA was obtained from peripheral blood lymphocytes using a standard technique. Microsatellite markers, D1s104 and D1s1677, were amplified by PCR and analyzed on polyacrylamide gels, as described by Hughes (38Hughes A.E. Optimization of microsatellite analysis for genetic mapping..Genomics. 1993; 15: 433-434Crossref PubMed Scopus (45) Google Scholar). D1s104 and D1s1677 were successfully genotyped in 611 and 545 individuals, respectively. The genotyping of D1s1677 failed in 38 FCH patients. For the USF1 variants, PCRs were performed in a final volume of 50 μl at an annealing temperature of 53.7°C for both polymorphisms. Primer sequences for USF1s1 were 5′-GGTGTGTCCTTGAACTGAG-3′ (forward) and 5′-CAAGCCAGAGCATCACCTG-3′ (reverse), and those for USF1s2 were 5′-CTTTAGTAGAGACAGGGTTTCAC-3′ (forward) and 5′-GATTTAGCAGGTATTAGGAGCA-3′ (reverse). The mismatch (underlined) for USF1s2 was introduced to create a restriction site for BsiHKA I. The PCR products were digested with either 10 units of BstF5 I (USF1s1) or 10 units of BsiHKA I (USF1s2) at 65°C (New England Biolabs); subsequently, the resulting fragments (242, 172, and 70 bp in heterozygotes for USF1s1 and 154, 136, and 18 bp in heterozygotes for USF1s2) were separated on agarose gels. USF1s1 and USF1s2 were genotyped in all 611 individuals, including 157 FCH patients. The genotyping of USF1s2, however, failed for six individuals, including three FCH patients.Quantitative real-time PCR analysis of USF1 expression in peripheral blood mononuclear cellsUSF1 mRNA expression levels were quantified in peripheral blood mononuclear cells (PBMCs) of 30 FCH patients and 30 sex-matched normolipidemic relatives randomly selected from our study population, as described previously (39van der Vleuten G.M. Hijmans A. Heil S. Blom H.J. Stalenhoef A.F. de Graaf J. Can we exclude the TXNIP gene as a candidate gene for familial combined hyperlipidemia?.Am. J. Med. Genet. A. 2006; 140: 1010-1012Crossref PubMed Scopus (3) Google Scholar). RNA was isolated from PBMCs, including both lymphocytes and monocytes, and reversed-transcribed to cDNA. Quantitative real-time PCR was carried out in a total of 25 μl containing 2 μl of cDNA, 0.25× SYBR® green solution (Invitrogen), 1× fluorescein (Bio-Rad), 2 mM MgCl2, 50 ng of forward (5′-ATGACCCAGGCGGTGATCCA-3′) and reverse (5′-GACGCTCCACTTCATTATGC-3′) primers, 100 μM deoxynucleoside triphosphates, 1× AmpliTaq Gold amplification buffer, and 1.5 unit of AmpliTaq Gold DNA polymerase (Applied Biosystems). PCR conditions were as follows: a hot start at 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. Samples were run in duplicate on the Icycler iQ real-time PCR detection system (Bio-Rad) to determine the threshold cycle (40Wilhelm J. Pingoud A. Real-time polymerase chain reaction..ChemBioChem. 2003; 4: 1120-1128Crossref PubMed Scopus (279) Google Scholar). Expression levels were normalized to beta-2-microglobulin by comparative quantification using the ΔΔCt method (41Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method..Methods. 2001; 25: 402-408Crossref PubMed Scopus (119422) Google Scholar).Statistical analysesThe characteristics of the study population are expressed as means ± SD. Before further statistical analyses, extended Mendelian error-checking was performed for 36 families, including 219 nuclear families (n = 611), with Pedcheck (42O'Connell J.R. Weeks D.E. PedCheck: a program for identification of genotype incompatibilities in linkage analysis..Am. J. Hum. Genet. 1998; 63: 259-266Abstract Full Text Full Text PDF PubMed Scopus (1820) Google Scholar). Data were available for only 25 of the 36 probands, because 11 probands were already recruited in 1994 and did not participate in 1999. For families with Mendelian inconsistencies, which can be attributable to paternity problems and/or misgenotyping, problematic genotypes were set to missing for the complete nuclear families or the isolated problematic individuals [n = 10 (1.6%)]. The parental data for each polymorphism were tested for Hardy-Weinberg equilibrium by use of an exact test. Variables with skewed distribution, including TGs and the HOMA index, were logarithmically transformed. Multipoint parametric linkage analysis of the two microsatellite markers on chromosome 1 and the two USF1 polymorphisms was performed for three traits, the FCH trait, defined by both the traditional diagnostic criteria and the nomogram, and the TG trait, defined by TG levels above the 90th percentile (31Assmann G. Schulte H. Relation of high-density lipoprotein cholesterol and triglycerides to incidence of atherosclerotic coronary artery disease (the PROCAM experience). Prospective Cardiovascular Munster study..Am. J. Cardiol. 1992; 70: 733-737Abstract Full Text PDF PubMed Scopus (827) Google Scholar), using SIMWALK2 (43Sobel E. Lange K. Descent graphs in pedigree analysis: applications to haplotyping, location scores, and marker-sharing statistics..Am. J. Hum. Genet. 1996; 58: 1323-1337PubMed Google Scholar). Data were prepared using MEGA2 (44Mukhopadhyay N. Almasy L. Schroeder M. Mulvihill W.P. Weeks D.E.
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