Genome-Wide Mapping of Susceptibility to Coronary Artery Disease Identifies a Novel Replicated Locus on Chromosome 17

Coronary artery disease (CAD) is a leading cause of death world-wide, and most cases have a complex, multifactorial aetiology that includes a substantial heritable component. Identification of new genes involved in CAD may inform pathogenesis and provide new therapeutic targets. The PROCARDIS study recruited 2,658 affected sibling pairs (ASPs) with onset of CAD before age 66 y from four European countries to map susceptibility loci for CAD. ASPs were defined as having CAD phenotype if both had CAD, or myocardial infarction (MI) phenotype if both had a MI. In a first study, involving a genome-wide linkage screen, tentative loci were mapped to Chromosomes 3 and 11 with the CAD phenotype (1,464 ASPs), and to Chromosome 17 with the MI phenotype (739 ASPs). In a second study, these loci were examined with a dense panel of grid-tightening markers in an independent set of families (1,194 CAD and 344 MI ASPs). This replication study showed a significant result on Chromosome 17 (MI phenotype; p = 0.009 after adjustment for three independent replication tests). An exclusion analysis suggests that further genes of effect size λ(sib) > 1.24 are unlikely to exist in these populations of European ancestry. To our knowledge, this is the first genome-wide linkage analysis to map, and replicate, a CAD locus. The region on Chromosome 17 provides a compelling target within which to identify novel genes underlying CAD. Understanding the genetic aetiology of CAD may lead to novel preventative and/or therapeutic strategies

A Common Polymorphism in the Promoter Region of the TNFSF4 Gene Is Associated with Lower Allele-Specific Expression and Risk of Myocardial Infarction

BACKGROUND: The TNFSF4/TNFRSF4 system, along with several other receptor-ligand pairs, is involved in the recruitment and activation of T-cells and is therefore tentatively implicated in atherosclerosis and acute coronary syndromes. We have previously shown that genetic variants in TNFSF4 are associated with myocardial infarction (MI) in women. This prompted functional studies of TNFSF4 expression. METHODS AND RESULTS: Based on a screening of the TNFSF4 genomic region, a promoter polymorphism (rs45454293) and a haplotype were identified, conceivably involved in gene regulation. The rs45454293T-allele, in agreement with the linked rs3850641G-allele, proved to be associated with increased risk of MI in women. Haplotype-specific chromatin immunoprecipitation of activated polymerase II, as a measure of transcriptional activity in vivo, suggested that the haplotype including the rs45454293 and rs3850641 polymorphisms is functionally important, the rs45454293T- and rs3850641G-alleles being associated with lower transcriptional activity in cells heterozygous for both polymorphisms. The functional role of rs45454293 on transcriptional levels of TNFSF4 was clarified by luciferase reporter assays, where the rs45454293T-allele decreased gene expression when compared with the rs45454293C-allele, while the rs3850641 SNP did not have any effect on TNFSF4 promoter activity. Electromobility shift assay showed that the rs45454293 polymorphism, but not rs3850641, affects the binding of nuclear factors, thus suggesting that the lower transcriptional activity is attributed to binding of one or more transcriptional repressor(s) to the T-allele. CONCLUSIONS: Our data indicate that the TNFSF4 rs45454293T-allele is associated with lower TNFSF4 expression and increased risk of MI

Isolation and characterization of genes within the region for multiple endocrine neoplasia type 1 on chromosome 11q13

This thesis summarizes the investigations of three genes isolated from the locus for multiple endocrine neoplasia type 1 (MEN1) within the chromosomal region 11q13. As a common strategy for all three genes, the cDNA was sequenced, the genomic structure characterized, and the expression pattern analyzed by Northern blotting on human tissues and by in situ RNA hybridization on slides from rodent embryos. The phospholipase C beta3 gene (PLCB3) encodes for a key enzyme in signal transduction. Due to its loss of expression in some endocrine tumors we had considered PLCB3 as a promising MEN1 candidate. In paper I the genomic organization and complete cDNA sequence is presented. The previously published sequence of PLCB3 was extended with 876 bp in the 5' direction giving a transcript of 4400 bp and a total open reading frame of 1234 amino acids, the same size as detected by Northern blot analysis. The complete transcription unit of PLCB3 is approximately 15 kb, contains 31 exons, and is preceded by a highly GC rich 5'flanking region without TATA and CAAT boxes. This analysis was a prerequisite for the investigation in paper III in which PLCB3 is excluded as a MEN1 candidate, by mutation analysis both on cDNA and on genomic level. Paper II describes the expression in rat. High transcript levels were mainly seen in the dorsal root ganglia (DRGs) and the trigeminal ganglion of the nervous system. Slightly lower levels were seen in the skin, the mucous lining of the airways, the gastrointestinal canal, the lung and the thymus. The pituitary, pancreas and thyroid gland did not give any positive signals. The analysis supported the idea that PLCB3 does not play a role in endocrine tissues. The PLCB3 neighboring gene (PNG) which was identified only a few kb upstream of PLCB3 had not been described before. In paper IV the cDNA sequence, Northern blot analysis, and genomic structure of PNG including a complete sequence of the region between the 5' ends of PLCB3 and PNG is presented. PNG is expressed in most tissues as a 1kb transcript, comparable to the size of the cDNA clone. On somatic cell hybrid panels cross-hybridization to chromosomes 2, 4, 7 and 22 was detected, possibly indicating a gene family. The highly GC-rich sequence did not show any significant similarities with other known genes. The predicted protein of 199 amino acids has a high content of glycine, alanine, and proline which may result in a collagenous structure. PNG spans over 2.5 kb and contains 4 exons. The intergenic region between PNG and PLCB3 revealed two separate CpG islands without TATA or CAAT boxes, one in front of each gene. In paper V the cDNA sequence, and expression during development, of the mouse homologue (png) is described. In the developing mouse embryo expression was seen in most organs, with higher levels over parts of the brain, spinal cord, heart, liver, intestines and tongue. In the adult mouse, thymus and testis showed the strongest expression while most tissues including those related to MEN1 exhibited low or no specific expression. The third cDNA corresponds to a novel gene highly similar to the gene for vascular endothelial growth factor (VEGF), a potent mitogen for endothelial cells and an inducer of blood vessel permeability. Paper VI describes the sequence, the genomic structure, the expression by Northern blot analysis, and the splice pattern of the novel gene which we named the VEGF Related Factor gene (VRF). Two alternate splicing forms were detected. The shorter of the two was created by splicing of the 6th of 8 exons. This version preserves similarity with VEGF for the whole predicted protein while maintenance of exon 6 results in a frame shift for the terminal third part of the gene. Northern blot analysis detected bands of 5.5 kb and 2.0 kb. Paper VII describes the cDNA sequence, genomic structure and expression of the mouse VRF gene (vrf). vrf is highly homologous to its human counterpart, in sequence and genomic organization, and also generates the two alternative splicing forms. Northern blots showed only one band of 1.3 kb in size. Paper VIII describes the expression of vrf in the prenatal mouse. Expression was primarily seen over heart and in the nervous system. Just prior to and after birth strong expression was seen in heart, brown fat and in some locations in the central nervous system. Key words: Multiple Endocrine Neoplasia type 1 (MEN1), Chromosome 11q13, Phospholipase C Beta 3 gene (PLCB3), Phospholipase C Beta 3 Neighbouring Gene (PNG), Vascular Endothelial Growth Factor (VEGF), VEGF Related Factor gene (VRF). ISBN 91-628-2157-1 Stockholm, 199

Genome-Wide Mapping of Susceptibility to Coronary Artery Disease Identifies a Novel Replicated Locus on Chromosome 17

Coronary artery disease (CAD) is a leading cause of death world-wide, and most cases have a complex, multifactorial aetiology that includes a substantial heritable component. Identification of new genes involved in CAD may inform pathogenesis and provide new therapeutic targets. The PROCARDIS study recruited 2,658 affected sibling pairs (ASPs) with onset of CAD before age 66 y from four European countries to map susceptibility loci for CAD. ASPs were defined as having CAD phenotype if both had CAD, or myocardial infarction (MI) phenotype if both had a MI. In a first study, involving a genome-wide linkage screen, tentative loci were mapped to Chromosomes 3 and 11 with the CAD phenotype (1,464 ASPs), and to Chromosome 17 with the MI phenotype (739 ASPs). In a second study, these loci were examined with a dense panel of grid-tightening markers in an independent set of families (1,194 CAD and 344 MI ASPs). This replication study showed a significant result on Chromosome 17 (MI phenotype; p¼0.009 after adjustment for three independent replication tests). An exclusion analysis suggests that further genes of effect size ksib . 1.24 are unlikely to exist in these populations of European ancestry. To our knowledge, this is the first genome-wide linkage analysis to map, and replicate, a CAD locus. The region on Chromosome 17 provides a compelling target within which to identify novel genes underlying CAD. Understanding the genetic aetiology of CAD may lead to novel preventative and/or therapeutic strategies