In the First Screening, 1,818 MS Markers Showed Significant Association with CAD and They

Supplementary Text S1. Multistep genome-wide screening of CAD loci using a total of 18,880 MS markers

Materials & Methods

Preparation of screeningsamples

Constitutions of three screening samples were as follows: the first set(100 MI cases with 3 VD versus 100 controls), the second set (100 MI cases with 2 VDversus100 controls), and the third set (192MI cases consisting of two 3VD, forty six 2VD and one hundred forty four 1 VD with onset age younger than 50 years-old cases versus 192 controls) from the Japanese group A. To preparethe pooled DNA samples, genomic DNA concentration was measured in triplicate as described by Collins et al(Collins et al. 2000).

Statistical Analysis

Allele frequencies in the pooled-DNA were estimated from the height of peaks: each allele frequency was determined by dividing the height of each allele by the summed height of all alleles as described previously (Tamiya et al. 2005). Significance of differences in allele frequencies between two pooled DNAs was evaluated by chi-square test, with the use of 2×2 and 2×m (where m is the number of alleles) contingency tables (Tamiya et al. 2005; Pritchard and Rosenberg 1999). Alleles of six MS markers as the representatives of candidate loci for CAD were evaluated by fisher’s exact test. P values less than 0.05 were supposed to be significant throughout this study.

Results

We performed amultistep genome-wide screening using 18,880 MS markers by pooled DNA method in a Japanese population (Japanese group A, Table 1) to identify the candidate loci for CAD. In the first screening, 1,818MS markersshowed significant association with CAD and they were further analyzed in the second screening to obtain 190MS markers with statistical significance.To confirm the observed associations, these markers were subjected to individual typing in192(or 96) cases and 192 (or 96) controls randomly chosen from the samples usedin the first and second screening preparation of pooled DNAs and 42 markers were confirmed for the significant associations. These MS markers were next tested for the association in the third screening by individual typing, resulting in the identification of six MS as the representatives of candidate loci for CAD (Table S1 and Table S2).

Linkage disequilibrium (LD) structures of these six candidate loci wereanalyzed based on HapMap JPT+CHB data. It was found that three MS markers in the candidate loci CAD3, CAD4 and CAD5 were located in the LD blocks containing no known genes, while MS markers representative of CAD1, CAD2, and CAD6 were within the LD-blocks carrying MKL1, PCSK9, and AK125001, respectively (Table S1).

Reference

Collins HE, Li H, Inda SE, Anderson J, Laiho K, et al.(2000) A simple and accurate method for determination of microsatellite total allele content differences between DNA pools. Hum Genet106: 218-226.

Pritchard JK, Rosenberg NA (1999) Use of unlinked genetic markers to detect population stratification in association studies.Am J Hum Genet65: 220-228.

Tamiya G, Shinya M, Imanishi T, Ikuta T, Makino S, et al.(2005) Whole genome association study of rheumatoid arthritis using 27 039 microsatellites. Hum Mol Genet 14: 2305-2321.