Päivi Pajukanta

Familial combined hyperlipidemia is associated with upstream transcription factor 1 ( USF1 )

Familial combined hyperlipidemia (FCHL), characterized by elevated levels of serum total cholesterol, triglycerides or both, is observed in about 20% of individuals with premature coronary heart disease. We previously identified a locus linked to FCHL on 1q21–q23 in Finnish families with the disease. This region has also been linked to FCHL in families from other populations as well as to type 2 diabetes mellitus. These clinical entities have several overlapping phenotypic features, raising the possibility that the same gene may underlie the obtained linkage results. Here, we show that the human gene encoding thioredoxin interacting protein (TXNIP) on 1q, which underlies combined hyperlipidemia in mice, is not associated with FCHL. We show that FCHL is linked and associated with the gene encoding upstream transcription factor 1 (USF1) in 60 extended families with FCHL, including 721 genotyped individuals (P = 0.00002), especially in males with high triglycerides (P = 0.0000009). Expression profiles in fat biopsy samples from individuals with FCHL seemed to differ depending on their carrier status for the associated USF1 haplotype. USF1 encodes a transcription factor known to regulate several genes of glucose and lipid metabolism.

Two Loci on Chromosomes 2 and X for Premature Coronary Heart Disease Identified in Early- and Late-Settlement Populations of Finland

Coronary heart disease (CHD) is a complex disorder constituting a major health problem in Western societies. To assess the genetic background of CHD, we performed a genomewide linkage scan in two study samples from the genetically isolated population of Finland. An initial study sample consisted of family material from the northeastern part of Finland, settled by a small number of founders approximately 300 years ago. A second study sample originated from the southwestern region of Finland, settled approximately 2,000 years ago. Families were ascertained through probands exhibiting premature CHD, defined as >50% stenosis of at least two coronary arteries at a young age, as verified by coronary angiography. Both study samples and the pooled data set provided evidence for linkage in two chromosomal regions. A region on chromosome 2q21.1-22 yielded two-point LOD scores of 3.2, 1.9, and 3.7, in the affected sib-pair (ASP) analyses of the northeastern, southwestern, and pooled study samples. The corresponding multipoint maximum-likelihood scores (MLSs) for these three study samples were 2.4, 1.3, and 3.0. In addition, a region on chromosome Xq23-26 resulted in two-point LOD scores of 1.9, 3.5, and 2.9 and in multipoint MLSs of 3.4, 3.1, and 2.5, respectively. In conclusion, this study identifies two loci likely to contribute to premature CHD: one on chromosome 2q21.1-22 and another on chromosome Xq23-26.

Genomewide Scan for Familial Combined Hyperlipidemia Genes in Finnish Families, Suggesting Multiple Susceptibility Loci Influencing Triglyceride, Cholesterol, and Apolipoprotein B Levels

Familial combined hyperlipidemia (FCHL) is a common dyslipidemia predisposing to premature coronary heart disease (CHD). The disease is characterized by increased levels of serum total cholesterol (TC), triglycerides (TGs), or both. We recently localized the first locus for FCHL, on chromosome 1q21-q23. In the present study, a genomewide screen for additional FCHL loci was performed. In stage 1, we genotyped 368 polymorphic markers in 35 carefully characterized Finnish FCHL families. We identified six chromosomal regions with markers showing LOD score (Z) values >1.0, by using a dominant mode of inheritance for the FCHL trait. In addition, two more regions emerged showing Z>2.0 with a TG trait. In stage 2, we genotyped 26 more markers and seven additional FCHL families for these interesting regions. Two chromosomal regions revealed Z>2.0 in the linkage analysis: 10p11.2, Z=3.20 (theta=.00), with the TG trait; and 21q21, Z=2.24 (theta=.10), with the apoB trait. Furthermore, two more chromosomal regions produced Z>2.0 in the affected-sib-pair analysis: 10q11.2-10qter produced Z=2.59 with the TC trait and Z=2.29 with FCHL, and 2q31 produced Z=2.25 with the TG trait. Our results suggest additional putative loci influencing FCHL in Finnish families, some potentially affecting TG levels and some potentially affecting TC or apoB levels.

Hyperglycemia and a Common Variant of GCKR Are Associated With the Levels of Eight Amino Acids in 9,369 Finnish Men

We investigated the association of glycemia and 43 genetic risk variants for hyperglycemia/type 2 diabetes with amino acid levels in the population-based Metabolic Syndrome in Men (METSIM) Study, including 9,369 nondiabetic or newly diagnosed type 2 diabetic Finnish men. Plasma levels of eight amino acids were measured with proton nuclear magnetic resonance spectroscopy. Increasing fasting and 2-h plasma glucose levels were associated with increasing levels of several amino acids and decreasing levels of histidine and glutamine. Alanine, leucine, isoleucine, tyrosine, and glutamine predicted incident type 2 diabetes in a 4.7-year follow-up of the METSIM Study, and their effects were largely mediated by insulin resistance (except for glutamine). We also found significant correlations between insulin sensitivity (Matsuda insulin sensitivity index) and mRNA expression of genes regulating amino acid degradation in 200 subcutaneous adipose tissue samples. Only 1 of 43 risk single nucleotide polymorphisms for type 2 diabetes or hyperglycemia, the glucose-increasing major C allele of rs780094 of GCKR, was significantly associated with decreased levels of alanine and isoleucine and elevated levels of glutamine. In conclusion, the levels of branched-chain, aromatic amino acids and alanine increased and the levels of glutamine and histidine decreased with increasing glycemia, reflecting, at least in part, insulin resistance. Only one single nucleotide polymorphism regulating hyperglycemia was significantly associated with amino acid levels.

The SLC6A14 gene shows evidence of association with obesity

In our previous genome-wide scan of Finnish nuclear families, obesity was linked to chromosome Xq24. Here we analyzed this 15-Mb region by genotyping 9 microsatellite markers and 36 single nucleotide polymorphisms (SNPs) for 11 positional and functional candidate genes in an extended sample of 218 obese Finnish sibling pairs (sibpairs) (BMI > 30 kg/m2). Evidence of linkage emerged mainly from the obese male sibpairs, suggesting a gender-specific effect for the underlying gene. By constructing haplotypes among the obese male sibpairs, we restricted the region from 15 Mb to 4 Mb, between markers DXS8088 and DXS8067. Regional functional candidate genes were tested for association in an initial sample of 117 cases and 182 controls. Significant evidence was observed for association for an SNP in the 3-untranslated region of the solute carrier family 6 member 14 (SLC6A14) gene (P = 0.0002) and for SNP haplotypes of the SLC6A14 gene (P = 0.0007-0.006). Furthermore, an independent replication study sample of 837 cases and 968 controls from Finland and Sweden also showed significant differences in allele frequencies between obese and non-obese individuals (P = 0.003). The SLC6A14 gene is an interesting novel candidate for obesity because it encodes an amino acid transporter, which potentially regulates tryptophan availability for serotonin synthesis and thus possibly affects appetite control.

A Systems Genetics Approach Implicates USF1, FADS3, and Other Causal Candidate Genes for Familial Combined Hyperlipidemia

We hypothesized that a common SNP in the 3 untranslated region of the upstream transcription factor 1 (USF1), rs3737787, may affect lipid traits by influencing gene expression levels, and we investigated this possibility utilizing the Mexican population, which has a high predisposition to dyslipidemia. We first associated rs3737787 genotypes in Mexican Familial Combined Hyperlipidemia (FCHL) case/control fat biopsies, with global expression patterns. To identify sets of co-expressed genes co-regulated by similar factors such as transcription factors, genetic variants, or environmental effects, we utilized weighted gene co-expression network analysis (WGCNA). Through WGCNA in the Mexican FCHL fat biopsies we identified two significant Triglyceride (TG)-associated co-expression modules. One of these modules was also associated with FCHL, the other FCHL component traits, and rs3737787 genotypes. This USF1-regulated FCHL-associated (URFA) module was enriched for genes involved in lipid metabolic processes. Using systems genetics procedures we identified 18 causal candidate genes in the URFA module. The FCHL causal candidate gene fatty acid desaturase 3 (FADS3) was associated with TGs in a recent Caucasian genome-wide significant association study and we replicated this association in Mexican FCHL families. Based on a USF1-regulated FCHL-associated co-expression module and SNP rs3737787, we identify a set of causal candidate genes for FCHL-related traits. We then provide evidence from two independent datasets supporting FADS3 as a causal gene for FCHL and elevated TGs in Mexicans.

Genome Scans Provide Evidence for Low-HDL-C Loci on Chromosomes 8q23, 16q24.1-24.2, and 20q13.11 in Finnish Families

We performed a genomewide scan for genes that predispose to low serum HDL cholesterol (HDL-C) in 25 well-defined Finnish families that were ascertained for familial low HDL-C and premature coronary heart disease. The potential loci for low HDL-C that were identified initially were tested in an independent sample group of 29 Finnish families that were ascertained for familial combined hyperlipidemia (FCHL), expressing low HDL-C as one component trait. The data from the previous genome scan were also reanalyzed for this trait. We found evidence for linkage between the low-HDL-C trait and three loci, in a pooled data analysis of families with low HDL-C and FCHL. The strongest statistical evidence was obtained at a locus on chromosome 8q23, with a two-point LOD score of 4.7 under a recessive mode of inheritance and a multipoint LOD score of 3.3. Evidence for linkage also emerged for loci on chromosomes 16q24.1-24.2 and 20q13.11, the latter representing a recently characterized region for type 2 diabetes. Besides these three loci, loci on chromosomes 2p and 3p showed linkage in the families with low HDL-C and a locus on 2ptel in the families with FCHL.

Combined analysis of genome scans of dutch and finnish families reveals a susceptibility locus for high-density lipoprotein cholesterol on chromosome 16q

Several genomewide screens have been performed to identify novel loci predisposing to unfavorable serum lipid levels and coronary heart disease (CHD). We hypothesized that the accumulating data of these screens in different study populations could be combined to verify which of the identified loci truly harbor susceptibility genes. The power of this strategy has recently been demonstrated with other complex diseases, such as inflammatory bowel disease and asthma. We assessed the largely unknown genetic background of CHD by investigating the most common dyslipidemia predisposing to CHD, familial combined hyperlipidemia (FCHL), affecting 1%-2% of Western populations and 10%-20% of families with premature CHD. To be able to perform a combined data analysis, we unified the diagnostic criteria for FCHL and its component traits and combined the data from two genomewide scans performed in two populations, the Finns and the Dutch. As a result of our pooled data analysis, we identified three chromosomal regions, on chromosomes 2p25.1, 9p23, and 16q24.1, exceeding the statistical significance level of a LOD score >2.0. The 2p25.1 region was detected for the FCHL trait, and the 9p23 and 16q24.1 regions were detected for the low high-density lipoprotein cholesterol (HDL-C) trait. In addition, the previously recognized 1q21 region also obtained additional support in the other study sample, when the triglyceride trait was used. Analysis of the 16q24.1 region resulted in a statistically significant LOD score of 3.6 when the data from Finnish families with low HDL-C were included in the analysis. To search for the underlying gene in the 16q24.1 region, we investigated a novel functional and positional candidate gene, helix/forkhead transcription factor (FOXC2), by sequencing and by genotyping of two single-nucleotide polymorphisms in the families.

Familial Combined Hyperlipidemia in Mexicans Association With Upstream Transcription Factor 1 and Linkage on Chromosome 16q24.1

Objective—To investigate the largely unknown genetic component of the common lipid disorder, familial combined hyperlipidemia (FCHL) in Mexicans, we analyzed the upstream transcription factor 1 (USF1) gene that was recently associated with FCHL and high triglycerides (TG) in Finns. We also analyzed the Mexican FCHL families for 26 microsatellite markers residing in the seven chromosomal regions on 2p25.1, 9p23, 10q11.23, 11q13, 16q24.1, 19q13, and 21q21, previously linked to FCHL in Whites. Methods and Results—We genotyped 314 individuals in 24 Mexican families for 13 SNPs spanning an 88-kb region, including USF1. The FCHL and TG traits showed significant evidence for association with 3 SNPs, hCV1459766, rs3737787, and rs2073658, and haplotype analyses further supported these findings (probability values of 0.05 to 0.0009 for SNPs and their haplotypes). Of these SNPs, hCV1459766 is located in the F11 receptor (F11R) gene, located next to USF1, making it difficult to exclude. Importantly, the association was restricted to a considerably smaller region than in the Finns (14 kb versus 46 kb), possibly because of a different underlying linkage disequilibrium structure. In addition, 1 of the 7 regions, 16q24.1, showed suggestive evidence for linkage (a lod score of 2.6) for total cholesterol in Mexicans. Conclusions—This study, the first to extensively investigate the genetic component of the common FCHL disorder in Mexicans, provides independent evidence for the role of USF1 in FCHL in an outbred population and links the 16q24.1 region to an FCHL-component trait in Mexicans.

USF1 Contributes to High Serum Lipid Levels in Dutch FCHL Families and U.S. Whites With Coronary Artery Disease

Objective— Familial combined hyperlipidemia (FCHL) characterized by high serum total cholesterol and/or triglycerides (TGs) is a common dyslipidemia predisposing to coronary artery disease (CAD). Recently, the upstream transcription factor 1 (USF1) was linked and associated with FCHL and TGs in Finnish FCHL families. Here we examined the previously associated rs3737787 SNP in extended Dutch FCHL families (n=532) and in a cohort of US subjects who underwent diagnostic coronary angiography (n=1533). Methods and Results— In males of the Dutch FCHL families, we observed significant sex-dependent associations between the common allele of rs3737787 and FCHL, TGs, and related metabolic traits (P=0.02 to 0.006). In the U.S. Whites, sex-dependent associations with TGs and related metabolic traits were observed for the common allele of rs3737787 in males (P=0.04 to 0.02) and rare allele in females (P=0.05 to 0.002). This intriguing relationship was further supported by the highly significant genotype x sex interactions observed for TGs in the Dutch and TGs and body mass index (BMI) in U.S. White subjects with CAD (P=0.0005 to 0.00004). Conclusion— These data show that USF1 influences several cardiovascular risk factors in a sex-dependent manner in Dutch FCHL families and U.S. Whites with CAD. A significant interaction between sex and genotype was shown to affect TGs and BMI.