Supplementary Information. Werner Koch, Petra Hoppmann, Jakob C. Mueller, Albert Schömig & Adnan Kastrati

Transcription

1 Supplementary Information Werner Koch, Petra Hoppmann, Jakob C. Mueller, Albert Schömig & Adnan Kastrati The Supplementary Information has the following sections in order: 1. Supplementary Methods 2. Supplementary Table 1 3. Supplementary Table 2 4. Supplementary Table 3 5. Supplementary Figure 1 6. Supplementary Figure 2

2 2 SUPPLEMENTARY METHODS Patients and Controls. Participants were recruited from Southern Germany and examined at Deutsches Herzzentrum München or 1. Medizinische Klinik rechts der Isar der Technischen Universität München from 1993 to After catheterization, 5,264 individuals were deemed eligible for inclusion into the MI or control group. Written informed consent for genetic analysis was obtained from 97.1% (n = 5,111) of these individuals. In no case, consent was withdrawn. Blood samples assigned for DNA preparation had been collected from 95.2% (n = 4,868) of the individuals who agreed to participate in the study. These individuals, 3,657 patients with MI and 1,211 controls, constituted the study population. Mean age of the patients was higher than that of the control individuals (64.0 ± 12.0 y vs ± 11.9 y), the proportion of women was lower in the patient group than in the control group (24.2% vs. 49.4%), and history of arterial hypertension (61.4% vs. 48.6%) and hypercholesterolemia (56.5% vs. 49.7%), current cigarrette smoking (50.6% vs. 15.2%), and diabetes mellitus (20.6% vs. 5.4%) were more prevalent in the patient group than in the control group. The study protocol was approved by the institutional ethics committee (Ethikkommission der Fakultät für Medizin der Technischen Universität München, Munich, Germany) and the reported investigations were in accordance with the principles of the current version of the Declaration of Helsinki. Obviously, as with other similar investigations, the study does not provide data on those patients with acute MI who died before reaching the hospital. Their genetic and clinical risk profile may have been different from that of patients who survived the initial attack. Population stratification is a potential problem for case-control studies and may lead to falsepositive results or mask an existing relationship 1. In this study, the probability of a falsenegative association result due to population stratification is relatively low because the study

3 3 participants were recruited from a defined geographic area of southern Germany with limited recent immigration. Definitions. The diagnosis of MI was established in the presence of chest pain lasting > 20 min combined with ST-segment elevation or pathologic Q waves on a surface electrocardiogram. Patients with MI had to show either an angiographically occluded infarctrelated artery or regional wall motion abnormalities corresponding to the electrocardiographic infarct localization, or both. Individuals were considered disease-free and, therefore, eligible as controls when their coronary arteries were angiographically normal and when they had no history of MI, no symptoms suggestive of MI, no electrocardiographic signs of MI, and no regional wall motion abnormalities. Systemic arterial hypertension was defined as a systolic blood pressure of 140 mm Hg, and/or a diastolic blood pressure of 90 mm Hg (ref. 2), at least on two separate occasions, or antihypertensive treatment. Hypercholesterolemia was defined as a documented total cholesterol value 240 mg dl 1 ( 6.2 mmol l 1 ) or current treatment with cholesterol-lowering medication. Persons reporting regular smoking in the previous six months were considered as current smokers. Diabetes mellitus was defined as the presence of an active treatment with insulin or an oral antidiabetic agent; for patients on dietary treatment, documentation of an abnormal fasting blood glucose or glucose tolerance test based on the World Health Organisation criteria 3 was required for establishing this diagnosis. SNP selection. Tag SNPs of the TNFSF4 gene region were inferred from HapMap data with the use of the software package Haploview (refs. 4,5). On the basis of the CEU population sample and with cut offs for pairwise r 2 values set at 0.8 and minimum minor allele frequency at 0.10, 15 tag SNPs were captured between positions 171,411,000 and 171,451,000 of

4 4 chromosome 1 (HapMap data release 22/phase II Apr07, on National Center for Biotechnology Information B36 assembly, SNP database build 126). Genotyping of at least 200 different DNA samples showed no base variations in 2 instances (rs and rs ), which reduced the number of informative SNPs to 13. Among the informative SNPs were 4 of the 5 SNPs that were examined in a prior case-control study of MI (ref. 6). One SNP (rs ) analyzed in the prior study 6 was also included in the present investigation, though it was not retrieved as a tag SNP in the analysis with Haploview. Supplementary Fig. 1 indicates the positions of the 14 SNPs in the TNFSF4 gene region selected for examination in this study. Genotype analysis. TaqMan allelic discrimination assays 7,8 were designed and used for SNP genotyping. The sequences of oligonucleotide primers and TaqMan probes and reaction protocols are available upon request. About 100 different PCR products obtained with each TaqMan system were sequenced to test whether one or more additional polymorphisms were present in the probe-binding section of the amplicons, because they may interfere with TaqMan reactions and result in wrong genotype assignments. The selected SNPs were identified as the only sequence variabilities in the probe-binding regions, which implicated that the probability of genotyping errors because of possible further sequence variations was relatively low. In addition, the genotype results obtained with sequence analyses and the corresponding TaqMan reactions were in full agreement. Genotype distributions in the control and case groups were consistent with those expected for samples in Hardy-Weinberg equilibrium. In addition, genotype and allele frequencies of the SNPs in the control group and MI group were not substantially different from those observed in reference populations including white individuals. With each SNP, re-typing of 20% of the DNA samples was done

5 5 to control for correct sample handling and data acquisition. Clinicians responsible for diagnosis were not aware of the genetic data. All genetic analyses were blinded. Statistical analysis. The analysis consisted of comparing separately allele, genotype, and haplotype frequencies between the control group and the MI group. In addition, separate analyses of genotype and haplotype frequencies were performed in the groups of women and men because the possibility of sex-specific associations was suggested from results obtained in a prior study 6. Discrete variables were compared with the use of the χ 2 test. Continuous variables are expressed as mean ± SD and were compared by means of the unpaired 2-sided t- test. Hardy-Weinberg equilibrium was assessed with the use of the χ 2 test. Pairwise measures of linkage disequilibrium (D and r 2 ) between the SNPs were calculated from primary genotype data with the use of Haploview (ref. 5). Haplotypes were reconstructed with the use of the software package PHASE (ref. 9). Accession numbers

6 6 References 1. Lohmueller, K.E. et al. Nat. Genet. 33, (2003). 2. Chalmers, J. et al. Clin. Exp. Hypertens. 21, (1999). 3. World Health Organization. Tech. Rep. Ser. 727 (1985). 4. The International HapMap Consortium. Nature 437, (2005). 5. Barrett, J.C. et al. Bioinformatics 21, (2005). 6. Wang, X. et al. Nat. Genet. 37, (2005). 7. Livak, K.J. Genet. Anal. 14, (1999). 8. Kutyavin, I.V. et al. Nucleic Acids Res. 28, (2000). 9. Stephens, M. et al. Am. J. Hum. Genet. 68, (2001).

7 7 Supplementary Table 1 Major allele frequencies and genotype frequencies of TNFSF4 SNPs in the control and MI groups Genotype Major allele Control (n = 1,211) MI (n = 3,657) RefSNP ID a Position b Alleles Control MI P Maj Het Min Maj Het Min P rs C > T rs C > T rs C > G rs A > G rs T > C rs T > C rs G > A rs A > C rs G > A rs C > T rs T > C rs T > C rs C > T rs G > A The order of SNPs is according to the direction of TNFSF4 gene transcription. Maj (Min), frequency of subjects homozygous for the major (minor) allele. Het, frequency of heterozygous subjects. a National Center for Biotechnology Information SNP database ( b Positions of SNPs on chromosome 1 (genome build 36.2).

8 8 Supplementary Table 2 Genotype frequencies of the TNFSF4 SNPs in the women and men of the control and MI groups Women Men Control (n = 598) MI (n = 885) Control (n = 613) MI (n = 2,772) RefSNP ID a Maj Het Min Maj Het Min P Maj Het Min Maj Het Min P rs rs rs rs rs rs rs rs rs rs rs rs rs rs The order of SNPs is according to the direction of TNFSF4 gene transcription. Maj (Min), frequency of subjects homozygous for the major (minor) allele. Het, frequency of heterozygous subjects. a National Center for Biotechnology Information SNP database (

9 9 Supplementary Table 3 Haplotype frequencies, odds ratios (ORs) and 95% confidence intervals (CIs) for myocardial infarction in relation to the 9 most frequent 5-marker haplotypes of the TNFSF4 gene region Haplotype No. Allele combination a Frequencies b OR (95% CI) P 1 CAATC / (0.85 to 1.13) TAGTC / (0.88 to 1.20) CAGCC / (0.91 to 1.29) TGGCC / (0.81 to1.24) TGGTC / (0.75 to 1.36) CAGTC / (0.61 to 1.16) TAGCC / (0.61 to 1.28) TAGTT / (0.54 to 1.69) CAGTT / (0.49 to 1.55) 0.63 a The order of the alleles in the haplotypes is in accordance with the relative chromosomal positions of the SNPs (from left to right): rs , rs , rs , rs , rs Haplotype bases are depicted from the coding strand of the TNFSF4 gene; 0, major allele; 1, minor allele. b Haplotype frequencies (%) of controls/cases.

10 10 Supplementary Figure kb kb Exon 1 Exon 2 Exon 3 Positions of the SNPs in the TNFSF4 gene region on chromosome 1 (band q25.1) genotyped in the study participants. SNPs: 1 = rs , 2 = rs , 3 = rs , 4 = rs , 5 = rs , 6 = rs , 7 = s , 8 = rs , 9 = rs , 10 = rs , 11 = rs , 12 = rs , 13 = rs , 14 = rs

11 11 Supplementary Figure 2 Exon 1 Exon 2 Exon 3

12 12 Legend to Supplementary Figure 2 Pairwise linkage disequilibrium between SNPs in the TNFSF4 gene region. Linkage disequilibrium measures between SNPs are shown as r 2 values (numbers) and D' values (color intensities).