Title Variations in the interleukin 7 receptor α chain gene influence the risk of multiple sclerosis Authors Frida Lundmark, Kristina Duvefelt, Ellen Iacobaeus, Ingrid Kockum, Erik Wallström, Mohsen Khademi, Annette Oturai, Lars P Ryder, Janna Saarela, Hanne F Harbo, Elisabeth G Celius, Hugh Salter, Tomas Olsson, Jan Hillert
Supplementary Table 1. Numbers of patients and controls included in the confirmatory study and the following tag- SNP fine-mapping study. Sweden 1 refers to the patients included in the paper by Zhang et al. Country MS CTR Confirmation study tagsnp study Denmark 606 584 yes no Finland 224 240 yes no Norway 410 576 yes no Sweden Group 1 * 630 0 no yes Sweden Group 2 580 1234 yes yes
Supplementary Table 2. Estimation of haplotype frequencies was performed in Haploview. We chose to describe the haplotypes consistently by the allelic nucleotides carried by the + strand, whereas in the previous report 4 haplotypes were described by a mixture of + and strand nucleotides. Global P-values were obtained performing 10,000 permutations of haplotype-count distributions and haplotype P-values were calculated using a chi-square test, as implemented in Haploview. Permutation tests with 10,000 permutations showed significant P-values for both associated haplotypes. Haplotype MS patients Controls Global P Haplotype P Haplotype P permutation Haplotype 1 CAA 0.483 0.452 0.0004 0.003 Haplotype 2 TTG 0.266 0.250 0.001 Haplotype 3 CTA 0.247 0.296 0.003 0.01
Supplementary Table 3. Tagging properties of SNP markers used.. SNP Location Alleles Capture Location of captured SNPS 1 2 rs10058453 3' C T X X rs10461959 intron 3 C T rs1353251 intron 1 rs11567698 intron 1 G T rs11567697 intron 1 rs11567714 intron 2 rs11567699 intron 1 rs11567701 intron 1 G T rs11567751 intron 4 rs1494554 intron 5 rs3822733 intron 6 rs987107 intron 6 rs3194051 exon 8, nonsyn rs10491435 rs10491434 rs1053496 rs700179 rs1494571 X X rs11567773 intron 3 G T rs4869492 rs13169780 A C rs6891095 intron 2 rs11567754 intron 4 rs11567761 intron 4 rs3822731 intron 6 rs9292618 rs13167136 rs1389835 rs1389834 rs13172065 rs10059688 rs6451234 X X rs2303137 A T rs1353252 intron1, rs3777090 intron 3 A T rs1389832 intron 1 rs1494560 intron1, rs1494558 exon2, nonsyn rs6451226 intron 2 rs6893142 intron 2 rs1389830 intron 2 rs10044838 intron 2 rs7711202 intron 2 rs11567737 intron 3 rs10074127 intron 3 rs10074095 intron 3
rs1494556 intron 3 rs10941267 intron 3 rs9292616 intron 3 rs10063445 intron 3 rs1494555 exon 4, nonsyn rs9292617 rs7716064 rs6451231 rs1862634 rs10044883 rs11567762 intron 4 rs6870944 A G rs1423657 rs1423658 rs4141646 rs12055232 rs6870944 rs6890853 rs6871748 C T rs11567694 intron 1 rs10213865 intron 1 rs7717955 intron 2 rs6897932 exon 6 rs6881270 rs6881706 rs11742240 rs11742270 rs10214237 rs10214273 rs6871748 rs6897932 exon 6 C T rs1494555 exon 4 C T
Supplementary Table 4. Results from the logistic regression analysis, to determine the best logistic regression model. The analysis included one SNP in each model for six associated SNPs. Forward, stepwise and backward selection was used to determine the best fit. Model -2logL P-value Intercept only 3130.6 rs6897932 co-dominant 3119.0 0.0007 rs6897932 dominant 3119.5 0.0009 rs6897932 recessive 3126.9 0.06 rs2303137 co-dominant 3120.5 0.002 rs2303137 dominant 3123.1 0.007 rs2303137 recessive 3124.8 0.02 rs3194051 co-domiant 3130.0 ns rs3194051 domiant 3124.9 0.02 rs3194051 recessive 3130.5 ns rs987106 co-domiant 3124.8 0.02 rs987106 dominant 3125.2 0.03 rs987106 recessive 3128.1 ns rs6871748 co-domiant 3120.1 0.002 rs6871748 dominant 3119.7 0.001 rs6871748 recessive 3128.1 ns rs987107 co-domiant 3129.9 ns rs987107 dominant 3125.8 0.03 rs987107 recessive 3130.6 ns
Supplementary Table 5. Haplotype frequencies were estimated using Haploview, and haplotype P-values were calculated using a chi-square test. A permutation test based on 10,000 permutations was performed in order to correct for multiple testing. Haplotypes were constructed using SNPs 1-12, i.e. the markers within the LD block (Table 2). The 3-marker haplotype presented in Supplementary table 2 are indicated in bold. Number Haplotype MS patients Controls Haplotype P Haplotype P permutation 1 TGTAGTTCTATC 0.251 0.298 0.0004 0.0007 2 TTTAGTCTTGTT 0.262 0.251 0.4043 ns 3 TGCTGCCCAACT 0.195 0.189 0.6531 ns 4 TGTAGTCCAATT 0.140 0.128 0.2523 ns 5 GGTTGCCCAACT 0.131 0.112 0.0453 ns
Supplementary Table 6. Primer sequences of primers used in the SNP genotyping and mrna expression assays. Assay Amplification primer 1 Amplification primer 2 Extension primer rs10058453 ACGTTGGATGCTTCCTGATAGCTCTCACTC ACGTTGGATGCAAAAGCTTATGAAGAGGGTC GAAGAGGGTCATATGACA rs10461959 ACGTTGGATGCATCACGGAAGGCAATCTAC ACGTTGGATGGCGAATAGGGAATGCTAAAAG gaaaagtatttttaagacatggtt rs11567698 ACGTTGGATGAAAGCTGGGTTTTCACTCC ACGTTGGATGGACGTAATCATGTGAATAGGG AGGGTAATTATACATCTTCTAAAA rs11567701 ACGTTGGATGCAAGGCTTTACACTGGAGAC ACGTTGGATGGAAAGTGCTATTCGGACAG GGTCTCTGGGACAATCAACT rs11567773 ACGTTGGATGTGAAGAAGAGTGAGCCTCAG ACGTTGGATGTTCAGGCCCCTGTAAAATGG gagggtttaaagggatccaaat rs13169780 ACGTTGGATGTCCAGTTACCAAAGAACCTC ACGTTGGATGTTTTTGTTGTAGAGGCAGAC TAGAGGCAGACCTCTTC rs1494555 ACGTTGGATGCACCACAAAGTCATTGGCTC ACGTTGGATGTTTCCAGTTAAACCTGAGGC cctctccttttgacctgagtgtc rs2303137 ACGTTGGATGTTCCACATGTGCATAAACAG ACGTTGGATGAAGCCCAGCCTTCAACCAAG TTTCCTACCAGAGGCACTT rs3777090 ACGTTGGATGCATCTTCCAACCCAGTAAGG ACGTTGGATGACTCTGCTAAGAGAAGTGGG GAAGTGGGAAGGCAGGA rs6870944 ACGTTGGATGATGAGCTTACTCAACGTAGG ACGTTGGATGAGATTCGAATCTGTCAGCCC ccccctccaagagtagatcca rs6871748 ACGTTGGATGGGTTGTGTCCTCAAATTAAA ACGTTGGATGGTGACTATTCCTGAAGAGTG tacatcttcaacttgatacattac rs6897932 ACGTTGGATGAAGATGACCAACAGAGCGAC ACGTTGGATGCAGGGGAGATGGATCCTAT GGAGATGGATCCTATCTTACTAA rs987106 (hme) ACGTTGGATGGGCCATACGAAGGCTTAATC ACGTTGGATGAAGTGGGAAGACTCAGTGTG TGCCATTCACTTCATCT rs987106 (iplex) ACGTTGGATGGCCATACGATAGGCTTAATC ACGTTGGATGAAGTGGGAAGACTCAGTGTG TGCCATTCACTTCATCT rs3194051 (hme) ACGTTGGATGGGATGAATCTCTTCCAAAGC ACGTTGGATGATCTGAGAAGCAGAGGCTTG CCATCTGAGGATGTAGTC rs3194051 (iplex) ACGTTGGATGGGATGAATCTCTTCCAAAGC ACGTTGGATGATCTGAGAAGCAGAGGCTTG GGGCCATCTGAGGATGTAGTC rs987107 (iplex) ACGTTGGATGCCCCGTTGTTTACTCCCAAT ACGTTGGATGAATATTCCCCCACTTCCACC TAGCTGCCAGAGTTGCT rs987107 (hme) ACGTTGGATGATATTCCCCCACTTCCACCA ACGTTGGATGTAACCCCGTTGTTTACTCCC CTTTTTATTTCTTCTCTTTACTGA IL7 (mrna) CCAGTTGCGGTCATCATGAC GGATGGATCGAACTTTAATCTCATACA IL7R (mrna) GATGAAAACAAATGGACGATGT GGATGGATCGAACTTTAATCTCATACA GAPDH (mrna) AGGGCTGCTTTTAACTCTGGTAAA CATATTGGAACATGTAAACCATGTAGTTG