A novel variant on chromosome 7q22.3 associated with mean platelet volume, counts, and function
Blood Soranzo et al.
113: 3831
Supplemental materials for: Soranzo et al
Production Samples
UK Blood Services Donor Panel 1 and 2 (UKBS-CC1 and UKBS-CC2) Sample
The UKBS collection of Common Controls is an anonymised collection of DNA samples from 3,000 healthy blood donors. The collection has been established by the three British Blood Services of England, Scotland and Wales as part of the Wellcome Trust Case Control Consortium study and 1,500 of the samples (panel 1) served as shared controls in this study.1 The remaining 1,500 samples were selected for replication (panel 2). The study has been approved by the Cambridgeshire 3 Research Ethics Committee (Peterborough & Fenland REC).
Blood count measurements
Venous blood was taken from the dry pouch (attached to whole blood donation set) and placed in EDTA containing tube which was used to perform full blood counts (FBCs) on a Beckman-Coulter GenS automated blood count analyser. All samples were tested within 48 hours from collection.
Genotyping and imputation
1,500 samples from the (UKBS panel 1) were genotyped using the Affymetrix 500K Gene Chip as part of the Wellcome Trust Case Control consortium.1 The following QC criteria were applied to the data: HWE test P-value ≥ 1 × 10−6 (11,407 markers excluded), per-SNP missingness ≥ 0.05 (19,272 markers excluded), MAF ≥ 0.05 (116,025 SNPs excluded) leaving 361,352 SNPs in the analysis. The genomic inflation factor in the remaining dataset was 1.006. Imputation of SNPs in the 1-MB interval surrounding rs342293 was performed using HapMap2 phased data (www.hapmap.org) and the software IMPUTE.2 An additional set of 1,500 individuals from the same collection (UKBS Panel 2) were genotyped as part of the replication study using Sequenom iPLEX.
KORA F3 500K Sample
The study population for the GWAS (KORA F3 500K, http://epi.gsf.de/kora-gen/) was recruited from the KORA S3 survey. It is an independent population-based sample from the general population living in the region of Augsburg, Southern Germany, examined in the years 1994/95 (KORA S3). The standardized examinations have been described in detail elsewhere.3 Ten years age-sex strata have been sampled from the 25 to 74 years old population with a stratum size of 640 subjects in the MONICA S3 study (4,856 subjects, response 75%). A total of 3,006 subjects participated in a follow-up examination of S3 in 2004/05 (KORA F3). For KORA F3 500K we selected 1,644 subjects of these participants then aged 35 to 79 years. Informed consent has been given. The study has been approved by the local ethical committee.
Blood count measurements
DNA was extracted from fresh blood, and was stored at −80°C. FBCs were performed on fresh venous EDTA-anticoagulated blood using an automatic blood counter (Beckman Coulter STKS).
Genotyping and imputation
Genotyping of KORA F3 samples was performed using Affymetrix 500K Array Set as described in 4. For fine scale analysis genotypes were imputed for all polymorphic HapMap SNPs using a Hidden Markov Model as programmed in MACH (Y Li and GR Abecasis, unpublished). For association testing of imputed genotypes, we used the program MACH2QTL, which uses dosage value (0.0–2.0) as predictor in a linear regression framework.
TwinsUK Sample
The TwinsUK cohort (KCL) is an adult twin British registry shown to be representative of singleton populations and the United Kingdom population.5 A total of 1,763 twins (100 % females) were available with FBCs, of which 1,050 had data for MPV. The mean (SD) age of the TwinsUK cohort with FBC was 50.86 (13.36). Ethics approval was obtained from the Guy’s and St. Thomas’ Hospital Ethics Committee. Written informed consent was obtained from every participant to the study.
Blood count measurements
Venous blood was anticoagulated with EDTA and FBCs were performed using either an ADVIA 2120 Haematology System (Siemens Healthcare Diagnostics, Deerfield, IL, US) or a XE2100 automated hematology analyzer (Sysmex, Kobe, Japan) 6 on average within 24 hours from venesection (range 20–30 hrs). The two instruments displayed differences in measurements range, with means (SD) of 9.69 (0.96) and 11.17 (1.03) respectively. Hence association analyses were adjusted for instrument type.
Genotyping and imputation
All samples were typed with Infinium assay (Illumina, San Diego, USA) as described in 5. Imputation of genotypes was carried out using the software IMPUTE.2 The following QC filters were applied prior to imputation: per sample call rate > 95% (130 SNPs excluded), heterozygosity <33% and >37% (46), MAF ≥ 1% (9489 SNPs excluded), HWE P-value ≥ 10−6 (1555 SNPs excluded) and per SNP call rate ≥ 95% (777 SNPs excluded). Genotypes with imputation posterior probability on individual genotype calls ≤ 0.9 were discarded. The number of SNPs that passed quality control was 2,434,545.
Cambridge Bioresource (CBR) Sample
The Cambridge BioResource (CBR) is a collection of pseudo-anonymised DNA samples from 4,000 healthy blood donors that has been established by the Cambridge Biomedical Research Centre in collaboration with NHS Blood and Transplant for use in genotype-phenotype association studies. The study has been approved by the Cambridgeshire 1 Research Ethics Committee.
Blood count measurements
Blood was taken from the dry pouch (attached to whole blood donation set) and placed into an EDTA tube. FBCs were performed on a ABX Pentra 60 automated hematology analyzer (Horiba ABX). All samples were analysed within 24 hours from collection.
Genotyping of SNP rs342293 in non-GWAS samples
Genotyping of SNP rs342293 in replication cohorts for haematological traits (CBR, UKBS-CC2) was carried out at the Wellcome Trust Sanger Institute using Sequenom iPLEX using standard conditions. Primer details are available upon request from the principle author.
Acknowledgments
This study was funded by the Wellcome Trust, the European Union funded FP-5 project (QLG2-CT-2002¬01254) and FP-6 integrated project Bloodomics (LSHM-CT-2004-503485) and by a National Institute of Heath Research (England) grant to NHSBT and the Cambridge BioResource. We thank the study participants and staff from the TwinsUK, the DNA Collections and Genotyping Facilities at the Wellcome Trust Sanger Institute, Le Centre National de Génotypage, France (led by Mark Lathrop) for genotyping; Duke University, North Carolina, USA (led by David Goldstein) for genotyping; and the Finnish Institute of Molecular Medicine, Finnish Genome Center, University of Helsinki (led by Aarno Palotie). The MONICA/KORA Augsburg studies were financed by the Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany and supported by grants from the German Federal Ministry of Education and Research (BMBF). Part of this work was financed by the German National Genome Research Network (NGFN) and supported within the Munich Center of Health Sciences (MC Health) as part of LMUinnovativ. We gratefully acknowledge the contribution of P. Lichtner, G. Eckstein, T. Strom and all other members of the Helmholtz Zentrum München genotyping staff in generating and analyzing the SNP dataset and G. Fischer for data management. We thank all members of field staffs who were involved in the planning and conduct of the MONICA/KORA Augsburg studies. NJS holds a Chair funded by the British Heart Foundation. Part of this work, as well as the work of Heribert Schunkert and Jeanette Erdmann, was financed by the German National Genome Network (NGFN) and the EU-funded integrated project Cardiogenics.
REFERENCES
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3. Lowel H, Doring A, Schneider A, Heier M, Thorand B, Meisinger C. The MONICA Augsburg surveys—basis for prospective cohort studies. Gesundheitswesen. 2005;67 Suppl 1:S13–S18.
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