Genetic Variation in Nitric Oxide Synthase 2A (NOS2A) and Risk for Multiple Sclerosis

Supplementary Tables for:

Genetic variation in nitric oxide synthase 2A (NOS2A) and risk for multiple sclerosis

Lisa F. Barcellos1,2,3,*,Patricia P. Ramsay1, StacyJ. Caillier2,Stephen Sawcer4, Jonathan Haines5, Silke Schmidt6, Margaret Pericak-Vance7,D. Alastair S. Compston4, Pablo Gabatto2, Stephen L. Hauser2, Jorge R. Oksenberg

1

Table S1. Primers and PCR conditions for NOS2A gene re-sequencing

NOS2A region / Forward primer (5'-3') / Reverse primer (5'-3') / MgCl2 concentration/annealing temperature
promoter 1 / CTCCTGCATTGCTCAGTCCTT / TCTTGGTGGGTGAAGGAAGAA / 3.5mM/61°C
promoter 2 / TCTCCAATGCCCACAGATGAA / GACGTGTGGCTGTGCCAGTAA / 2.5mM/61°C
promoter 3 / ATGCTCAGAAAACCGGTGTAA / TGGTAGCTCATGCCTGTAAAT / 1.5mM/57°C
promoter 4 / GTTGGCAAGGCTGGTCTCAAA / GCTGCTCCCAGGGAGGTTAAA / 1.5mM/61°C
promoter 5 / CCTCCATTTGACCCAATTCTAAA / GAGCCCTATGTCATTTGTCAA / 1.5mM/61°C
promoter 6 / CACGCCCTCCAGTGGTCACTT / TGCTCTGGTGCTTGCTCCATAAA / 2.5mM/61°C
promoter 7 / CTCGGTTTCCTTATTGGTGAA / CGGGACAAACAGTGAGCTT / 2.5mM/59°C
promoter 8 / CTGCTGCATCCATCATCAGAA / AATGCCTTCAGCTGGAGATAA / 2.5mM/59°C
promoter 9 / CCTGGGACCTTTCAGGCTAAA / TGCCTGGAATCCCAGCAATTT / 1.5mM/59°C
promoter 10 / TGCCCAGGCTGGTCTCGAA / TGCCGGTTCAGTTCAGGTAAA / 1.5mM/61°C
promoter 11 / CTGGTGTCCTCTCCCTTGTAAA / GCACCACCACACCCAGCTAA / 1.5mM/59°C
promoter 12 / GTGGGCAGATCACTTGAGCTT / GCCTGTGGGTGACCTGATCTT / 1.5mM/59°C
Exon 1 / GCTTCCTGGACTCCTGTCACAA / GGATTTGTGGGGAGGTTACATGTT / 1.5mM/59°C
Exon 2 / GCCTGTGGCCAGTGATGAA / GGCCAAGCTCCTTCCCATATT / 2.5mM/57°C
Exon 3 / CTGCTGGGCAGTGCTGCTA / TCCTTCCCTGGGAGCATGTAA / 2.5mM/59°C
Exon 4 / GCCCAGGCACCTGGAGCTCTT / GTCCAGCTCTGAGCAGTTCTGTT / 2.5mM/59°C
Exon 5 / CAGCCAGTGGAAGAGAGGAGAT / GATGCCAGGACACTGTTCTGAA / 1.5mM/61°C
Exon 6 / CTTGCCCTTCTGATGTCCTATTT / AGCCCCAGGCTAGGTCCTTT / 2.5mM/61°C
Exon 7 / GCTGGTGTGGGAAAGGACCTA / AGCCCCAACCCTGAATAGTGAA / 1.5mM/59°C
Exon 8 / GAGGCTGGCTCAGTCAGCTT / CTGCTTCCCCTCAGCATCAA / 2.5mM/61°C
Exon 9 / GAGGCTGGCTCAGTCAGCTT / CTGCTTCCCCTCAGCATCAA / 2.5mM/61°C
Exon 10 / ATAGCTCTCTGCAGCCTCTAA / ACCTGCAGCAAACACATGTATT / 2.5mM/61°C
Exon 11 / AGGCCAAGTAGATGGGATTCTT / TCTTTCCCAAGTTAAATTCAGCAT / 2.5mM/61°C
Exon 12 / AGCCGCAGCATTGAGCTGAA / GGTGGAATGGCAGGTAGGATTAAA / 2.5mM/61°C
Exon 13 / CCTTCCAGGAGGACACTAATGAGAA / TGCCGTGGCTCACATCTGTAA / 3.5mM/57°C
Exon 14 / TGCCCAGGTTGGTCTTGAA / GAGGCCTGGATTCATTGTGAAA / 1.5mM/61°C
Exon 15 / TGGGTGGGCCTGGATAGAA / TAGGAAAGGTGGCACTCCCATAT / 1.5mM/61°C
Exon 16 / GCCAGGTAGCCCCATATGTAAA / GAGGGAGAGCAGCAGTGACCTAA / 2.5mM/61°C
Exon 17 / GCCAGGTAGCCCCATATGTAAA / GAGGGAGAGCAGCAGTGACCTAA / 2.5mM/61°C
Exon 18 / CCAAGCCCAGACACCTCTTT / GAATCGCCTCTGTGACACCTT / 1.5mM/59°C
Exon 19 / ATGCCCCAAGGGTGGTGTT / TAGGCCACCACCCTGAGCTT / 1.5mM/59°C
Exon 20 / TGGTGGCCTAGCTTGTGGAA / AGTGGCTGGGATTGGGATAA / 1.5mM/61°C
Exon 21 / TTCAGGTCTGGGCCAGAGAA / CATCCATCCCCCAATACATCTTAA / 3.5mM/61°C
Exon 22 / CCTGGCACATGGACTTGTTTGTT / TGCGTTCAGGCTGCATGATT / 2.5mM/57°C
Exon 23 / GTGCCAGAGCAGGCTGAAA / GTCCTGCAGGCTGCTTCACTT / 2.5mM/59°C
Exon 24 / GCAGGACGTGGGAAATCAAA / ATTTTCGAGCTCAGGAGGTGAA / 2.5mM/61°C
Exon 25 / GCAGGACGTGGGAAATCAAA / ATTTTCGAGCTCAGGAGGTGAA / 2.5mM/61°C
Exon 26 / GCAGGACTGTGTCCAGCATTT / TTGCTTTGGCCACACAGATACTTT / 3.5mM/61°C
Exon 27 / GCAGGAAATGGGTATGCTAAGTAAA / CCTGCCCTACCACAGGTCTTT / 3.5mM/61°C

1

Table S2. The 41 NOS2A SNPs identified from re-sequencing the promoter region, exons,

intron/exon boundaries and chosen for further genotyping

NOS2A SNP / type / nt position
rs4795080 / promoter / -8087
rs12709502 / promoter / -7793
rs12709501 / promoter / -7741
rs12709500 / promoter / -7427
rs2734299 / promoter / -7411
rs8073782 / promoter / -7164
rs2531860 / promoter / -7122
rs9902311 / promoter / -6689
rs2779252 / promoter / -3824
rs2779251 / promoter / -3780
rs11080358 / promoter / -3051
rs2301369 / promoter / -2447
rs2779249 / promoter / -1026
rs2779248 / promoter / -277
rs10459953 / 3'UTR / 38
rs3730013 / intron 1/2 / 1638
rs3730014 / exon 2 / 1813
rs2072324 / intron 2/3 / 10660
rs8072199 / intron 2/3 / 10708
rs16966563 / exon 4 / 11607
rs28942370 / intron 5/6 / 13168
rs944725 / intron 6/7 / 17985
rs944724 / intron 6/7 / 18139
rs3729508 / intron 7/8 / 18526
rs2297520 / intron 8/9 / 19516
rs1137933 / exon 10 / 21624
rs3729966 / intron 12/13 / 26319
rs2248814 / intron 12/13 / 27235
rs2274894 / intron 14/15 / 28385
rs2297518 / exon 16 / 30959
rs2297515 / intron 19/20 / 34223
rs2297514 / intron 19/20 / 34241
NOT REPORTED / intron 19/20 / 34434
rs2297513 / intron 19/20 / 34572
rs1060822 / exon 20 / 34925
rs2297511 / intron 20/21 / 36329
rs2297510 / intron 21/22 / 36802
rs1060826 / exon 22 / 37689
rs28944190 / intron 22/23 / 37954
rs28944192 / intron 22/23 / 38146
rs3729661 / intron 24/25 / 40111

1

Table S3. Results from NOS2A single SNP association tests in MS families

All families from each dataset (p-value)5 / Families with DRB1*15+ cases4 (p-value) / Families with DRB1*15- cases4 (p-value)
SNP / location / MAF1 / HWE1 / 720 trios, 50 SNPs2 / 930 trios, 38 SNPs2 / 1648 families, 15 SNPs3 / 1372 trios, 15 SNPs2 / 422 trios, 50 SNPs2 / 527 trios, 38 SNPs2 / 528 trios, 15 SNPs2 / 298 trios, 52 SNPs2 / 396 trios, 40 SNPs2 / 389 trios, 15 SNPs2
rs12450575 / 23019858 / 0.26 / 0.99 / 0.28 / 0.33 / * / * / 0.16 / 0.33 / * / 1.00 / 0.95 / *
rs747959 / 23026451 / 0.26 / 0.95 / 0.21 / 0.40 / * / * / 0.14 / 0.31 / * / 0.84 / 0.91 / *
rs12452310 / 23045550 / 0.46 / 0.22 / 0.79 / 0.23 / * / * / 0.36 / 0.68 / * / 0.51 / 0.79 / *
rs12452385 / 23046031 / 0.45 / 0.39 / 0.56 / 0.44 / * / * / 0.31 / 0.46 / * / 0.77 / 0.96 / *
rs6505416 / 23046682 / 0.43 / 0.28 / 0.35 / 0.40 / * / * / 0.38 / 0.54 / * / 0.69 / 0.45 / *
rs4796017 / 23099118 / 0.42 / 0.40 / 0.46 / 0.86 / * / * / 0.30 / 0.67 / * / 0.91 / 0.88 / *
rs7215373 / 23099627 / 0.46 / 0.59 / 0.67 / 0.50 / * / * / 0.33 / 0.45 / * / 0.58 / 0.96 / *
rs7207044 / 23099651 / 0.42 / 0.40 / 0.44 / 0.30 / * / * / 0.23 / 0.51 / * / 0.82 / 0.92 / *
rs9906835 / 23113501 / 0.40 / 0.54 / 0.49 / 0.60 / * / * / 0.58 / 0.53 / * / 0.68 / 0.84 / *
rs2297513 / 23117111 / 0.20 / 0.73 / 0.96 / * / 0.58 / 0.78 / 0.71 / * / 0.91 / 0.72 / * / 0.71
rs2297514 / 23117442 / 0.42 / 0.27 / 0.68 / * / 0.23 / 0.89 / 0.88 / * / 0.26 / 0.65 / * / 0.48
rs2297515 / 23117460 / 0.16 / 0.20 / 0.88 / * / 0.19 / 0.14 / 0.83 / * / 0.21 / 1.00 / * / 0.68
rs2314809 / 23119505 / 0.41 / 0.96 / 0.43 / 0.51 / * / * / 0.85 / 0.73 / * / 0.32 / 0.13 / *
rs2872753 / 23119689 / 0.41 / 0.71 / 0.56 / 0.51 / * / * / 0.88 / 0.82 / * / 0.26 / 0.17 / *
rs2297516 / 23119857 / 0.39 / 0.77 / 0.36 / 0.62 / * / * / 0.72 / 0.86 / * / 0.32 / 0.42 / *
rs2297518 / 23120724 / 0.21 / 0.85 / 0.78 / 0.20 / * / * / 0.95 / 0.70 / * / 0.72 / 0.29 / *
rs9797244 / 23121258 / 0.21 / 0.74 / 0.93 / 0.27 / * / * / 1.00 / 0.70 / * / 0.89 / 0.49 / *
rs4796052 / 23127161 / 0.22 / 0.37 / 0.86 / 0.57 / * / * / 0.90 / 0.91 / * / 0.89 / 0.63 / *
rs1137933 / 23130059 / 0.23 / 0.93 / 0.72 / * / 0.91 / 0.82 / 0.56 / * / 0.15 / 0.89 / * / 0.27
rs4795067 / 23130802 / 0.35 / 0.51 / 0.75 / 0.25 / * / * / 0.76 / 0.41 / * / 0.38 / 0.08 / *
rs3729508 / 23133157 / 0.39 / 0.30 / 0.57 / 0.37 / 0.62 / 0.98 / 0.88 / 0.44 / 0.30 / 0.30 / 0.08 / 0.33
rs944724 / 23133544 / 0.23 / 0.02 / 0.38 / 0.57 / 0.14 / 0.92 / 0.73 / 0.88 / 0.38 / 0.39 / 0.61 / 0.68
rs944725 / 23133698 / 0.40 / 0.34 / 0.53 / * / 0.17 / 0.91 / 0.60 / * / 0.10 / 0.73 / * / 0.14
rs2072324 / 23141023 / 0.21 / 0.07 / 0.25 / * / 0.21 / 0.11 / 0.28 / * / 0.60 / 0.63 / * / 0.34
rs16949 / 23148826 / 0.25 / 0.85 / 0.83 / 0.06 / * / * / 0.69 / 0.88 / * / 0.89 / 0.45 / *
rs3730013 / 23150045 / 0.33 / 0.51 / 0.04 / * / 0.21 / 0.17 / 0.21 / * / 0.43 / 0.08 / * / 0.24
rs10459953 / 23151645 / 0.35 / 0.58 / 0.73 / * / 0.70 / 0.93 / 0.96 / * / 0.23 / 0.54 / * / 0.37
rs2301369 / 23154123 / 0.38 / 0.02 / 0.48 / * / 0.17 / 0.14 / 0.58 / * / 0.06 / 0.66 / * / 0.75
rs2779251 / 23155453 / 0.17 / 1.00 / 0.77 / * / 0.19 / 0.74 / 0.60 / * / 0.17 / 0.88 / * / 0.64
rs2531860 / 23158796 / 0.15 / 0.90 / 0.84 / * / 0.75 / 0.51 / 0.75 / * / 0.17 / 0.94 / * / 0.60
rs2734299 / 23159085 / 0.17 / 0.85 / 0.69 / 0.73 / 0.90 / 0.72 / 1.00 / 0.91 / 0.29 / 0.59 / 0.39 / 0.69
rs4795080 / 23159761 / 0.29 / 0.46 / 0.11 / * / 0.31 / 0.08 / 0.31 / * / 0.14 / 0.21 / * / 0.61
rs16966598 / 23162249 / 0.15 / 0.49 / 0.63 / 0.46 / * / * / 0.16 / 0.45 / * / 0.37 / 0.83 / *
rs2779259 / 23172411 / 0.38 / 0.51 / 0.85 / 0.63 / * / * / 0.12 / 0.31 / * / 0.14 / 0.48 / *
rs12600739 / 23173454 / 0.38 / 0.92 / 0.84 / 0.63 / * / * / 0.42 / 0.70 / * / 0.23 / 0.76 / *
rs2779267 / 23188621 / 0.38 / 0.49 / 0.91 / 0.71 / * / * / 0.15 / 0.33 / * / 0.14 / 0.51 / *
rs7218952 / 23220299 / 0.34 / 0.80 / 0.67 / 0.81 / * / * / 0.76 / 0.75 / * / 0.76 / 0.96 / *
rs6505524 / 23221896 / 0.45 / 0.29 / 0.42 / 0.97 / * / * / 0.17 / 0.42 / * / 0.72 / 0.61 / *
rs9901262 / 23225345 / 0.20 / 0.25 / 0.34 / 0.81 / * / * / 0.47 / 0.42 / * / 0.53 / 0.34 / *
rs2079714 / 23246647 / 0.25 / 0.70 / 0.86 / 0.68 / * / * / 0.35 / 0.56 / * / 0.42 / 0.53 / *
rs4796228 / 23246937 / 0.32 / 0.30 / 0.72 / 0.33 / * / * / 0.79 / 0.89 / * / 0.81 / 0.75 / *
rs8078361 / 23254607 / 0.25 / 1.00 / 0.65 / 0.19 / * / * / 0.19 / 0.34 / * / 0.42 / 0.45 / *
rs11653617 / 23272015 / 0.07 / 0.44 / 0.40 / 1.00 / * / * / 0.40 / 0.52 / * / 0.03 / 0.05 / *
rs8079838 / 23290559 / 0.14 / 0.86 / 0.71 / 0.75 / * / * / 0.21 / 0.46 / * / 0.35 / 0.13 / *
rs7222245 / 23293540 / 0.13 / 0.72 / 0.78 / 0.84 / * / * / 0.21 / 0.44 / * / 0.29 / 0.09 / *
rs4483903 / 23329994 / 0.26 / 0.81 / 0.63 / 0.38 / * / * / 0.06 / 0.20 / * / 0.14 / 0.05 / *
rs4239211 / 23333545 / 0.27 / 1.00 / 0.80 / 0.71 / * / * / 0.30 / 0.66 / * / 0.10 / 0.04 / *
rs9909148 / 23334619 / 0.28 / 0.47 / 0.93 / 0.62 / * / * / 0.74 / 0.30 / * / 0.79 / 0.87 / *
rs4416064 / 23338353 / 0.48 / 0.23 / 0.82 / 0.82 / * / * / 0.39 / 0.69 / * / 0.17 / 0.36 / *
rs4404123 / 23355401 / 0.09 / 0.57 / 0.15 / 0.36 / * / * / 0.12 / 0.13 / * / 0.69 / 0.60 / *

1Derived using founders from 720 trios (n=1,440 individuals).

2 P-value from allelic test of association using HAPLOVIEW (v.4.0).

3 P-value from allelic test of association using PDT (v.5.1); results based on the PDT-sum statistic. No significant results were observed for PDT-genotype tests of NOS2A SNPs (data not shown).

4 Families were considered DRB1*15 positive if the MS case carried at least one copy of DRB1*15. Families were considered DRB1*15 negative if the MS case carried no copies of DRB1*15. DRB1*15 carrier status was available for 1,114 of total 2,159 MS cases (51.9 %) included in this study. Medium-resolution typing of HLA-DRB1 (two to fourdigits) was performed (see Methods). A total of six of 1,045 MS cases withoutHLA-DRB1 genotype data were genotypedfor the rs3135388 (A/G) SNP to identify the MS-associated DRB1*15 allele, since the presence of thisallele and the rs3135388A SNP are highly correlated (see Methods).

5A total of 427 US families from the original study were included 20.

Table S4. Results from NOS2A haplotype analyses in MS families2, 3

720 trios, 50 SNPs / 930 trios, 38 SNPs / 1372 trios, 15 SNPs
rs# / location / All trios / Trios with cases who carry at least one DRB1*15 allele / Trios with cases who carry no DRB1*15 allele / All trios / Trios with cases who carry at least one DRB1*15 allele / Trios with cases who carry no DRB1*15 allele / All trios / Trios with cases who carry at least one DRB1*15 allele / Trios with cases who carry no DRB1*15 allele
rs12450575 / 23019858 / Block 11 / 0.355 / 0.246 / 0.496 / Block 1 / 0.46 / 0.41 / 0.84 / *
rs747959 / 23026451 / *
rs12452310 / 23045550 / Block 2 / 0.489 / 0.442 / 0.237 / Block 2 / 0.22 / 0.47 / 0.31 / *
rs12452385 / 23046031 / *
rs6505416 / 23046682 / *
rs4796017 / 23099118 / *
rs7215373 / 23099627 / *
rs7207044 / 23099651 / *
rs9906835 / 23113501 / *
rs2297513 / 23117111 / Block 3 / 0.948 / 0.969 / 0.881 / * / Block 1 / 0.35 / 0.43 / 0.59
rs2297514 / 23117442 / *
rs2297515 / 23117460 / *
rs2314809 / 23119505 / Block 4 / 0.759 / 0.990 / 0.472 / Block 3 / 0.35 / 0.64 / 0.39 / *
rs2872753 / 23119689 / *
rs2297516 / 23119857 / *
rs2297518 / 23120724 / *
rs9797244 / 23121258 / Block 5 / 0.570 / 0.803 / 0.265 / Block 4 / 0.27 / 0.60 / 0.27 / *
rs4796052 / 23127161 / *
rs1137933 / 23130059 / * / Block 2 / 0.98 / 0.33 / 0.59
rs4795067 / 23130802 / Block 6 / 0.847 / 0.544 / 0.325 / Block 5 / 0.15 / 0.67 / 0.21 / *
rs3729508 / 23133157 / Block 2
rs944724 / 23133544
rs944725 / 23133698 / Block 7 / 0.002 / 0.009 / 0.232 / *
rs2072324 / 23141023 / *
rs16949 / 23148826 / *
rs3730013 / 23150045 / *
rs10459953 / 23151645 / Block 8 / 0.727 / 0.399 / 0.926 / * / Block 3 / 0.15 / 0.40 / 0.30
rs2301369 / 23154123 / *
rs2779251 / 23155453 / *
rs2531860 / 23158796 / * / Block 4 / 0.01 / 0.42 / 0.73
rs2734299 / 23159085 / Block 6 / 0.89 / 0.84 / 0.59
rs4795080 / 23159761 / *
rs16966598 / 23162249 / Block 9 / 0.741 / 0.654 / 0.113 / Block 6 / *
rs2779259 / 23172411 / *
rs12600739 / 23173454 / *
rs2779267 / 23188621 / *
rs7218952 / 23220299 / Block 10 / 0.347 / 0.147 / 0.569 / Block 7 / 0.31 / 0.32 / 0.92 / *
rs6505524 / 23221896 / *
rs9901262 / 23225345 / *
rs2079714 / 23246647 / Block 11 / 0.405 / 0.269 / 0.183 / Block 8 / 0.44 / 0.49 / 0.12 / *
rs4796228 / 23246937 / *
rs8078361 / 23254607 / *
rs11653617 / 23272015 / *
rs8079838 / 23290559 / Block 12 / 0.177 / 0.146 / 0.473 / Block 9 / 0.17 / 0.22 / 0.22 / *
rs7222245 / 23293540 / *
rs4483903 / 23329994 / Block 13 / 0.420 / 0.122 / 0.428 / Block 10 / 0.31 / 0.23 / 0.37 / *
rs4239211 / 23333545 / *
rs9909148 / 23334619 / *
rs4416064 / 23338353 / *
rs4404123 / 23355401 / *

1Blocks were assigned using HAPLOVIEW (v.4.0) and standard parameters as previously described50.

2 P-value from global haplotype test of heterogeneity using UNPHASED (v.3.0.10)

3 Families were considered DRB1*15 positive if the MS case carried at least one copy of DRB1*15. Families were considered DRB1*15 negative if the MS case carried no copies of DRB1*15. DRB1*15 carrier status was available for 1,114 of total 2,159 MS cases (51.9 %) included in this study. Medium-resolution typing of HLA-DRB1 (two to fourdigits) was performed (see Methods). A total of six of 1,045 MS cases withoutHLA-DRB1 genotype data were genotypedfor the rs3135388 (A/G) SNP to identify the MS-associated DRB1*15 allele, since the presence of thisallele and the rs3135388A SNP are highly correlated (see Methods). A total of 427 US families from the original study were included 20.

Table S5. NOS2Asingle SNP association and global haplotype tests in mild and severe MS cases1

NOS2A SNP / Map position / MAF / P-value2 / Haplotype Block3 / P-value4
rs2297513 / 23117111 / 0.2 / 0.79 / 1 / 0.64
rs2297514 / 23117442 / 0.42 / 0.45
rs2297515 / 23117460 / 0.16 / 0.59
rs1137933 / 23130059 / 0.23 / 0.26 / 2 / 0.52
rs3729508 / 23133157 / 0.39 / 0.45
rs944724 / 23133544 / 0.23 / 0.58
rs944725 / 23133698 / 0.4 / 0.9
rs2072324 / 23141023 / 0.21 / 0.28
rs3730013 / 23150045 / 0.33 / 0.92
rs10459953 / 23151645 / 0.35 / 0.5 / 3 / 0.47
rs2301369 / 23154123 / 0.38 / 0.21
rs2779251 / 23155453 / 0.17 / 0.59
rs2531860 / 23158796 / 0.15 / 0.36 / 4 / 0.04
rs2734299 / 23159085 / 0.17 / 0.6
rs4795080 / 23159761 / 0.29 / 0.02

1Mild MS (N=104 MS cases) is defined as maintaining EDSS 3 after 15 years or more. Severe MS (N=101)

is defined as reaching EDSS >6 within 10 years.

2P-value from allelic test of association using HAPLOVIEW (v.4.0).

3Blocks were assigned using HAPLOVIEW (v.4.0) and standard parameters as previously described50.

4P-value from global haplotype test of association using UNPHASED (v.3.0.10).

1