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1 A rare variant in MYH6 is associated with high risk of sick sinus syndrome Hilma Holm 1,8, Daniel F. Gudbjartsson 1,8, Patrick Sulem 1, Gisli Masson 1, Hafdis Th. Helgadottir 1, Carlo Zanon 1, Olafur Th. Magnusson 1, Agnar Helgason 1, Jona Saemundsdottir 1, Arnaldur Gylfason 1, Hrafnhildur Stefansdottir 2, Solveig Gretarsdottir 1, Stefan E. Matthiasson 3, Guðmundur Thorgeirsson 2,4, Aslaug Jonasdottir 1, Asgeir Sigurdsson 1, Hreinn Stefansson 1, Thomas Werge 5, Thorunn Rafnar 1, Lambertus A. Kiemeney 6, Babar Parvez 7, Raafia Muhammad 7, Dan M. Roden 7, Dawood Darbar 7, Gudmar Thorleifsson 1, G. Bragi Walters 1, Augustine Kong 1, Unnur Thorsteinsdottir 1,4, David O. Arnar 2,4, Kari Stefansson 1,4. 1 decode genetics, Sturlugata 8, 101 Reykjavik, Iceland, 2 Department of Medicine, Landspitali University Hospital, 101 Reykjavik, Iceland, 3 Laekning, Medical Clinics, 108 Reykjavik, Iceland, 4 University of Iceland, Faculty of Medicine, 101 Reykjavik, Iceland, 5 Research Institute of Biological Psychiatry, Mental Health Centre Sct. Hans Copenhagen University Hospital, 4000 Roskilde, Denmark, 6 Department of Epidemiology, Biostatistics and Health Technology Assessment and Department of Urology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands, 7 Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA. 8 These authors contributed equally to this work. Correspondence and request for materials should be addressed to H.H. (hilma.holm@decode.is) or K.S. (kstefans@decode.is). Supplementary Information 1

2 Contents: Supplementary Table 1: Supplementary Table 2: The Icelandic sick sinus syndrome study population. Strongest association results at 14q11 in the Icelandic sick sinus syndrome discovery population. Supplementary Table 3: Number of SNPs discovered by whole-genome sequencing and overlap with SNPs from the 1000 Genome project and dbsnp. Supplementary Table 4: Supplementary Table 5: Rediscovery rates of SNPs on the 300K Illumina chips. Whole-genome sequencing depth and chip genotype comparison per sample. Supplementary Table 6: Supplementary Table 7: Refinement of the sick sinus syndrome association with R721W. Replication of the association between R721W and sick sinus syndrome. Supplementary Table 8: Supplementary Figure 1: The Icelandic electrocardiogram study population. The sequencing depth of the 87 whole-genome sequenced individuals. Supplementary Figure 2: Supplementary Figure 3: Supplementary Figure 4: Total sequencing depth at the 14q11 region. Imputed allele frequency of R721W as a function of year of birth. Gene, transcript and protein structures of MYH6. 2

3 Supplementary Table 1 The Icelandic sick sinus syndrome study population. SSS Controls Men Women Men Women Discovery sample Count ,162 21,423 Year of birth (SD) (10.8) (13.1) (22.1) (21.0) Replication sample Count Year of birth (SD) (12.2) (15.6) (14.7) (15.4) SSS = sick sinus syndrome, SD = standard deviation. 3

4 Supplementary Table 2 Strongest association results at 14q11 in the Icelandic sick sinus syndrome discovery population. Source SNP P value OR MAF Directly genotyped rs Imputed from HapMap2 rs Imputed from the 1000 Genomes project Imputed from the Human1M-Duo chip rs Imputed from the HumanOmni1-Quad chip rs Imputed from the HumanOmni1-Quad chip rs Results from the sick sinus syndrome genome-wide association study. OR = odds ratio. MAF = minor allele frequency. 4

5 Supplementary Table 3 Number of SNPs discovered by whole-genome sequencing and overlap with SNPs from the 1000 Genome project and dbsnp. Source Number of SNPs Number of SNPs overlapping with decode whole-genome sequencing Fraction of SNPs that are transitions decode wholegenome sequencing 10,944, dbsnp version ,876,586 6,714, dbsnp version ,564,596 8,321, Genomes imputation set 6,858,242 6,133, Number of SNPs discovered by the decode whole-genome sequencing project and the 1000 Genomes project and archived in versions 130 and 132 of dbsnp. Also shown are the overlap between the decode whole-genome sequencing project and external sources and the fraction of SNPs from each source that are transitions (purine to purine or pyrimidine to pyrimidine mutations). 5

6 Supplementary Table 4 Rediscovery rates of SNPs on the 300K Illumina chips. Number of minor alleles observed Number of SNPs Number of SNPs not rediscovered Percentage of SNPs not rediscovered , , , , , , , , , , , , , , , , , > , The fraction of SNPs not rediscovered through sequencing as a function of how many times the minor allele of the SNP was observed in the chip genotypes. 6

7 Supplementary Table 5 Whole-genome sequencing depth and chip genotype comparison per sample. Sequencing depth Fraction of chip SNPs called Percentage of mismatching chip SNPs

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10 Supplementary Table 6 Refinement of the sick sinus syndrome association with R721W. Phenotype status R721W carrier Not R721W carrier Sick sinus syndrome 40 4 Controls Among carriers of rs [t]: 91% of sick sinus syndrome carriers also carry R721W while only 38% of controls also carry R721W (P = ). 10

11 Supplementary Table 7 Replication of the association between R721W and sick sinus syndrome. N cases N ctrls Freq cases Freq ctrls OR (95% CI) P value 469 1, (3.83, 43.80) This table shows the association between R721W and sick sinus syndrome in Icelandic individuals that were genotyped for the R721W mutation with the R721W Centaurus single SNP assay but were not chip genotyped and did not overlap with the original discovery set. Frequencies are allelic frequencies. The odds ratio (OR) and P value are obtained from a logistic regression model using sex, year of birth and year of birth squared as covariates. N = number, ctrls = controls, freq = frequency. 11

12 Supplementary Table 8 The Icelandic electrocardiogram study population. Chip typed Single assay typed Men Women Men Women Count (%) 5,081 (46.6%) 5,823 (53.4%) 3,949 (48.2%) 4,250 (51.8%) Year of birth (SD) (14.9) (16.1) (15.1) (15.8) CAD (%) 2,298 (45.2%) 1,256 (21.6%) 956 (24.2%) 528 (12.4%) SD = standard deviation, CAD = coronary artery disease. 12

13 Supplementary Figure 1 The sequencing depth of the 87 whole-genome sequenced individuals. 13

14 Supplementary Figure 2 Total sequencing depth at the 14q11 region. The grey line represents the sequencing depth at each base. The red line represents the mean sequencing depth over one kilobase. The blue rectangles show the position of MYH6 and MYH7. 14

15 Supplementary Figure 3 Imputed allele frequency of R721W as a function of year of birth. The ~38,000 individuals with imputed data were grouped into ten groups of roughly equal size. 15

16 Supplementary Figure 4 Gene, transcript and protein structures of MYH6. Bottom panel: Gene structure with chromosomal coordinates at the bottom and intron numbering at the top. The gene is coded by the minus-strand of chromosome 14, hence its orientation is represented right to left. Full hight colored bars depict coding exons, half-hight bars depict non-coding exons. Middle panel: Transcript structure with basepairs 16

17 positions at the bottom and exon numbering at the top. Full hight colored bars depict coding exons, half-hight bars depict 5 and 3 UTR s. Top panel: Domain composition of the protein with amino-acid positions at the bottom and domain descriptions at the top. Grey thin lines connect the panels according to exon boundaries. The position of the SNP in exon 18 is highlighted by a red line in the trascript and a red dot in the protein. 17

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