Schizophrenia-associated HapICE haplotype is associated with increased NRG1 Type III expression and high nucleotide diversity
Shannon Weickert C, Tiwari Y, Schofield PR, Mowry BJ and Fullerton JM
Supplementary material
Supplementary methods
Human postmortem cohort and tissue extraction
Dissection of the DLPFC from the coronal slab rostral to the corpus collosum along the middle frontal gyrus was isolated as previously described1-3. Unaffected controls were screened by telephone interviews of family members and/or police records for a history of medical and/or psychiatric problems, including alcohol abuse and elicit drug use.Any positive history of a psychiatric problem or excessive alcohol or drug use led to the exclusion of that case from the normal control group. Cases with an unclear psychiatric diagnosis, evidence of cocaine or phencyclidine (PCP) abuse by history and/or toxicology, cerebrovascular disease, autolysis, subdural hematoma, neuritic pathology or other pathological features were excluded from the cohort.
Detection of nucleotide variation in NRG1 putative regulatory regions
Sequence traces were visualized using PhredPhrap software4-5, and SNPs identified by manual inspection of each chromatogram. Base pair positions reported herein refer to the March 2006 human genomereference assembly (NCBI36/hg18). We report all SNPs with frequency as low as 0.7% (detected on one out of 148 chromosomes), which were compared to NCBI dbSNP (build 130) to determine their novelty. Bioinformatic predictions of the functionality each SNP allele was assessed with TFSEARCH6, using the vertebrate transcription factor binding matrixwith a minimum score of 85 constituting a hit. Nucleotide diversity was assessed using methods previously described7-8, using the following equation:
In brief, nucleotide diversity (θ) takes into account the number of SNPs (K) identified in a genomic length (L)in a sample of (n) alleles. Haplotype reconstruction was performed using the --hap-phase function in PLINK9, andLD structure was assessed using HAPLOVIEW10.
Australian control cohort
All participants provided written informed consent prior to participation in this study, which was approved by the Wolston Park Hospital Institutional Ethics Committee, Wacol, Brisbane. Participants were drawn from a12,745 square kilometre area of south-east Queensland via advertisements placed in local newspapers and community venues. Inclusion criteria were: (i) screen negative on the Diagnostic Interview of Psychosis 11; (ii) Caucasian ethnicity; (iii) adequate English proficiency in order to complete the interview; and (iv) DNA availability at the time of testing. The sample comprised 128 normal individuals: 85 men and 43 women (mean age=37.82±10.86 and 45.91±12.69 respectively).
Quantification of NRG1 isoform expression
Three reverse transcriptase reactions (a total of 9µg RNA from the DLPFC) using random hexamers and the SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) were generated from each individual, and pooled before transcript quantification.
Transcripts were quantified on an ABI Prism 7900 sequence detection system with Sequence Detector Software version 2.0 (Applied Biosystems, Foster City, CA)by a relative standard curve method, using serial dilutions of pooled cDNA derived from RNA obtained from brain tissue of 6 subjects. In each experiment, the r2 value of the standard curve was more than 0.99 and no-template control assays resulted in no detectable signal. Each 20μl PCR reaction contained 6μl of cDNA, 1l of 20X primer/probe mixture and 10μl of RT-PCR Mastermix Plus (Eurogentec, Seraing, Belgium) containing Hot Goldstar DNA Polymerase, dNTPs with dUTP, uracil-N-glycosylase, passive reference, and optimized buffer components with standard PCR cycling conditions. All samples were measured in triplicate, and replicates were excluded if the coefficient of variation of the three measures was greater than 40%. The replicate to be excluded was determined by Grubbs’ outlier test. Individual samples were excluded as population outliers if the normalized quantity was greater than two standard deviations above or below their diagnostic group means.
Supplementary results
Type IVNRG1 mRNA expression
Type IV isoform expression in the DLPFC was at the limit of detection and the data unreliable, so caution must be used in any interpretation. Type IV mRNA failed to amplify in 19 individuals, and most others required exclusion of at least one replicate due to measurement error.With data from 55 individuals, we found that females had higher Type IV mRNA by t-test (t value= -3.654(1,53), p=0.0006). On performing a 2-way ANCOVA with diagnosis and genderfor NRG1 TypeIV mRNA (age, RIN and brain pH as covariates), we did not detect a main effect of diagnosis (F=0.459(1,49), p= 0.50), but did find a main effect of gender (F=11.159(1,51), p=0.0016), suggesting that females have more Type IV mRNA expression compared to males, both in case of schizophrenia (p=0.017) and controls (p=0.007).
With respect to clinical factors, duration of illness was positively correlated with NRG1 Type IV mRNA (r=0.507, p=0.008), suggesting an up-regulation of Type IV mRNA may occur with disease progression.
Supplementary Figures
Supplementary Figure 1: Position of novel variants with respect to putative functional regions in promoter and intronic regions. For each region interrogated, the position of each novel variation is indicated by the red arrow, with SNPs which were detected only in individuals with schizophrenia enclosed in a red box. The numbers of heterozygous individuals (hets) detected with each variant is shown, and any change in transcription binding prediction is indicated. The predicted regulatory potential track (light blue) and 28-way sequence conservation track (dark blue) from the UCSC database is shown, along with the known SNPs described in dbSNP130.
Supplementary Figure 2:An increased novel variant load in schizophrenia cases (dark grey bars) compared to controls (light grey bars) (Fishers exact test: χ2=7.815; p=0.05; df=3). One control individual with 3 novel variants is represented in the ≥3 category, compared to 7 schizophrenic patients with 3, 5 and 6 novel variants (n=3, 3 and 1 respectively).
Supplementary Figure 3:Relationship betweenthe five HapICE SNPs and rs7014762 in the promoter IV/II region of NRG1. A) SNP rs7014762 is in highLD (D’=1.0, r2=0.913-0.241) with four of the five HapICE SNPs. The alleles represented in the original schizophrenia-associated HapICE haplotype are indicated after the underscore at the end of each SNP name. The block represented was determined via the solid spine of LD method in HAPLOVIEW10. B)Seven haplotypes were identified using the four HapICE SNPs and rs7014762 as per the haplotype block shown in panel A. The most common haplotype, indicated with the red box, contains each of the risk alleles from the four HapICE SNPs, plus the major allele of rs7014762 (allele frequencies for rs7014762 were: A=0.2838; T=0.7162). Note that the designation of either the A or T for the minor/major alleles for rs7014762 is dependent on the strand on which the genotyping assay was designed, so caution must be used when comparing risk alleles across studies.
Supplementary Figure 4:Loss of hemispheric asymmetry of EGFisoform mRNA expression in schizophrenia.Mean (±standard errors) EGFβ expression is shown for controls (light grey bars) compared to schizophrenia patients (dark grey bars) for DLPFC from the left and right hemispheres. For controls, there were 14 samples from the left and 23 from the right hemisphere. For schizophrenia cases, there were 19 samples from the left and 17 from the right hemisphere. Post-hoc LSD comparisons for mean group differences were made between control left and control right (mean: 63.47±5.36 vs 51.56±4.12 respectively) revealing a significant hemispheric asymmetry (p=0.019). This asymmetry is absent in schizophrenia cases (left: 52.69±3.06 vs right: 50.20±4.11; p=0.61). Expression in the left hemisphere was significantly lower in brains of schizophrenia patients compared to controls (mean 52.69±3.06 vs 63.47±5.36 respectively; p=0.040).
Supplementary Figure 5: Protein binding assay for novel SNP 1395_1 comparing binding efficiencies of the novel variant to wild-type allele. For each lane, a 32P-labelled complementary primer heteroduplex (novel A allele probe: agctTAAAAGTTAAGTTTCATTAT; wild-type T allele probe: agctTAAAAGTTATGTTTCATTAT) was incubated with nuclear extracts from cultured HEK293 cells, or from dorsolateral prefrontal cortex tissue which was pooled from individual human brains from the Stanley Medical Research Institute cohort.A probe with an NFkB binding site (agctGGGTCTGTGAATTCCCGGGGGT) was used as a positive control with HEK293 nuclear protein. For each SNP variant, a hot reaction (H) containing only 32P-labeled probe is loaded adjacent to a cold competitor reaction (C) which contains an approximate 10 fold excess of unlabelled “cold” probe. Arrows indicate specific protein binding. With both protein extracts, the novel allele demonstrated reduced protein binding efficiency compared to the wild-type allele.
Supplementary tables
Diagnostic group / Age at death (years) / PMI (hours) / pH / RIN / Gender / Hemisphere / Age of onset (years) / Illness Duration (years) / daily CPZ (grams)SCZ (n=37) / 51.32 ± 14.13 / 28.45 ± 13.77 / 6.61 ± 0.30 / 7.27 ± 0.58 / 24M, 13F / 23R, 14L / 23.70 ± 13.82 / 27.62 ± 13.82 / 691.63 ± 502.20
CON (n=37) / 51.13 ± 14.62 / 24.79 ± 10.97 / 6.65 ± 0.30 / 7.30 ± 0.57 / 30M, 7F / 17R, 20L / N/A / N/A / N/A
Supplementary Table 1: Summary of demographic variables for post-mortem brain cohort. Average values for schizophrenia patients (SCZ) and controls (CON) are given ± standard deviations. In the SCZ group, seven individuals were diagnosed with schizoaffective disorder. Post mortem interval (PMI), acidity of DLPFC brain tissue (pH) and RNA integrity number (RIN). The numbers of individuals in the two groups are given for gender (M=male, F=female), and hemisphere (R=right, L=left). Average daily chlorpromazine equivalent neuroleptic dose (CPZ) ± standard deviations are given. Age of onset, illness duration and daily CPZ values are not applicable (N/A) in the control group.
Gene / probe name / isoforms detected / inventoried assay / forward / reverse / probe(FAM-MGB)
NRG1 / Type I(Ig2) / Ig2 and s1 domains (excluding GGF-2, HRG-β1d, HRG-β3b, HRG-γ3, SMDF) / - / GCCAATATCACCATCGTGGAA / CCTTCAGTTGAGGCTGGCATA / CAAACGAGATCATCACTG
NRG1 / Type II / GGF-2, HRGβ-1d, HRG-β3b, HRG-γ3 / - / GAATCAAACGCTACATCTACATCCA / CCTTCTCCGCACATTTTACAAGA / CACTGGGACAAGCC
NRG1 / Type IV / HRG-β1b, HRG-β1c & HRG-β1d / - / GCTCCGGCAGCAGCAT / GAACCTGCAGCCGATTCCT / ACCACAGCCTTGCCT
NRG1 / Type III / SMDF / Hs01103792_m1 / - / - / -
NRG1 / pan-NRG1 / all isoforms (excluding SMDF, ndf43c) / Hs00247620_m1 / - / - / -
NRG1 / Type I / HRG-β3b and HRG-γ3 (excluding HRG-β1b, HRG-β1c) / Hs01108479_m1 / - / - / -
NRG1 / EGFα / HRG-α, ndf43 and ndf43c / Hs01103794_m1 / - / - / -
NRG1 / EGFβ / SMDF, GGF, GGF2, all HRG-β isoforms / Hs00247624_m1 / - / - / -
ACTB / - / beta-actin / Hs99999903_m1 / - / - / -
GAPDH / - / glyceraldehyde-3-phosphate dehydrogenase / Hs99999905_m1 / - / - / -
UBC / - / ubiquitin C / Hs00824723_m1 / - / - / -
TBP / - / TATA box binding protein / Hs00427620_m1 / - / - / -
Supplementary Table 2: Taqman probes for quantification and normalization of NRG1 isoform expression. Custom designed primer and probe combinations were used to specifically targetparticular NRG1 isoforms previously investigated 12-13, while inventoried assays (Applied Biosystems, Foster City, CA)were used for all other isoforms. The isoforms detected by each probe as described 14-15 in are indicated. The genometric mean of four endogenous control genes was used for transcript normalization.
region name / region location, bp (NCBI Build 36) / DNA length screened, bp / # dbSNPs annotated / observed SNPs (ALL) / observed SNPs (SZ) / observed SNPs (CON) / nucleotide diversity (θ×10-4)dbSNPs / novel SNPs / dbSNPs / novel SNPs / dbSNPs / novel SNPs
upstream HapICE / 31592860-31593868 / 1,008 / 5 / 2 / 3 / 3 / 3 (1) / 3 / 2 (0) / 17.5
promoter IV/II / 31613334-31616575 / 3,241 / 11 / 9 / 5 / 9 / 5 (2) / 7 / 3 (0) / 11.1
478B14-848 / 31708115-31708936 / 821 / 4 / 3 / 4 / 3 / 3 (3) / 3 / 1 (1) / 26.8
420M9-1395 / 31784622-31785559 / 937 / 7 / 7 / 3 / 3 / 3 (2) / 3 / 1 (0) / 30.1
promoter I / 32522326-32525340 / 3,014 / 33 / 15 / 1 / 13 / 1 (1) / 13 / 0 (0) / 13.2
promoter III / 32620730-32624137 / 3,407 / 11 / 6 / 10 / 6 / 6 (2) / 6 / 7 (4) / 11.7
total screened / 12,428 / 71 / 42 / 26 / 37 / 21 (11) / 35 / 14 (5) / 10.0
Supplementary Table 3: Summary of the nucleotide diversity in upstream regulatory and intronic regions of NRG1. The average minor allele frequency (MAF) of novel SNP variations was 0.044±0.038. The total number of novel SNPs observed in each group is shown, with the number of SNPs unique to that group shown in parentheses. Nucleotide diversity (θ)was assessed using the methods previously described 7-8.
mRNA / r (X,Y) / p / npan-NRG1
age
pH
RIN
PMI / -0.23
0.43
0.29
0.41 / <0.05
<0.001
<0.05
<0.001 / 74
EGFβ
age
pH
RIN
PMI / -0.21
0.55
0.37
0.06 / ns
<0.001
<0.01
ns / 73
Type I
age
pH
RIN
PMI / 0.08
0.21
0.37
-0.11 / ns
ns
<0.01
ns / 73
Type I(Ig2)
age
pH
RIN
PMI / -0.12
0.01
-0.04
-0.26 / ns
ns
ns
<0.05 / 72
Type II
age
pH
RIN
PMI / 0.10
-0.33
-0.25
0.05 / ns
<0.01
<0.05
ns / 70
Type III
age
pH
RIN
PMI / -0.10
0.32
0.39
0.03 / ns
<0.01
<0.001
ns / 74
Type IV
age
pH
RIN
PMI / 0.28
-0.25
-0.37
-0.01 / <0.05
ns
<0.01
ns / 55
Supplementary Table 4: Demographic factors and NRG1 mRNA expression. Significant correlations with brain pH and RIN values at the p<0.05 level were observed for all isoforms excluding Type I, Type I(Ig2) and Type IV, which were significant for RIN only, PMI only or age and RIN respectively.Pan-NRG1 showed significant correlations at the p<0.05 level for all continuous demographic factors. P values are reported as < 0.001, <0.01, < 0.05, nor not significant (ns).
NRG1 gene region / BP position (build 36) / SNP / dbSNP name / minor allele / major allele / MAF (ALL) / MAF (SCZ) / MAF (CON) / TRANSFAC binding changeupstream prom IV/II / 31593076 / JF221XXX_4 / ss472054944 / C / T / 0.007 / 0.014 / 0.000 / removes TATA
upstream prom IV/II / 31593103 / JF221XXX_1 / rs71523425* / C / T / 0.061 / 0.068 / 0.054 / removes Hb, creates cap
upstream prom IV/II / 31593216 / JF221XXX_2 / rs74506441* / C / T / 0.068 / 0.095 / 0.041 / creates CDP-CR
upstream prom IV/II / 31613752 / JF4.3_1 / ss472054949 / C / T / 0.007 / 0.014 / 0.000 / removes CdxA, reduces Oct-1
upstream prom IV/II / 31614472 / JF4.2_4 / rs77626248* / C / A / 0.108 / 0.108 / 0.041 / creates c-ETS, GATA
upstream prom IV/II / 31614502 / YT4.2_1 / rs73584584* / C / T / 0.034 / 0.054 / 0.014 / reduces Sox-5, removes SRY
upstream prom IV/II / 31615552 / YT4.2_2 / ss472054956 / C / T / 0.007 / 0.014 / 0.000 / increases TATA
upstream prom IV/II / 31615555 / YT4.2_3 / rs76063839* / A / G / 0.034 / 0.027 / 0.041 / creates SRY, improves HNF-3
intron 1 / 31708375 / JF848_2 / ss472054950 / C / T / 0.029 / 0.042 / 0.000 / removes CdxA
intron 1 / 31708615 / JF848_3 / ss472054951 / A / G / 0.028 / 0.056 / 0.000 / removes SRY, creates CdxA
intron 1 / 31708702 / YT848_1 / rs117129618* / G / A / 0.030 / 0.054 / 0.000 / no change
intron 1 / 31708937 / NOR_848_4 / ss472054952 / C / T / 0.007 / 0.000 / 0.027 / increases AP-1
intron 1 / 31784714 / JF1395_2 / ss472054942 / A / G / 0.008 / 0.014 / 0.000 / increases Sox-5
intron 1 / 31784774 / JF1395_3 / ss472054943 / A / G / 0.028 / 0.054 / 0.000 / reduces SRY, removes c-Myb
intron 1 / 31785235 / YT1395_1 / rs117469567* / A / T / 0.027 / 0.054 / 0.000 / reduces SRY, removes CdxA
upstream prom I / 32524132 / JF1.2_1 / ss472054941 / T / G / 0.014 / 0.027 / 0.000 / creates SRY
upstream prom III / 32620979 / NOR3.3_1 / ss472054954 / A / G / 0.007 / 0.000 / 0.014 / introduces GATA-1, removes AML-1a
upstream prom III / 32621144 / JF3.3_5 / ss472054948 / del / AA / 0.014 / 0.014 / 0.014 / removes HFH-2
upstream prom III / 32621357 / JF3.3_6 / rs111526496* / A / G / 0.020 / 0.014 / 0.027 / removes AML-1a
upstream prom III / 32621907 / JF3.3_7 / rs113060920* / T / C / 0.016 / 0.029 / 0.000 / creates GATA, reduces deltaE
upstream prom III / 32622150 / NOR3.2_4 / rs117532293* / T / C / 0.007 / 0.000 / 0.014 / creates S8
upstream prom III / 32622768 / NOR3.2_2 / rs117347889* / G / A / 0.007 / 0.000 / 0.014 / no change
upstream prom III / 32622867 / NOR3.2_3 / ss472054953 / A / G / 0.007 / 0.000 / 0.014 / no change
upstream prom III / 32622943 / JF3.2_2 / ss472054947 / del / CA / 0.020 / 0.027 / 0.014 / removes SRY, HFH-2
upstream prom III / 32623136 / JF3.1_3 / ss472054946 / del / TGA / 0.014 / 0.014 / 0.000 / introduces C-/EBP
upstream prom III / 32624045 / JF3.1_2 / ss472054945 / C / T / 0.131 / 0.014 / 0.000 / no change
Supplementary Table 5: Summary of novel variants (dbSNP130) identified in NRG1 re-sequenced regions. The gene region and base pair location (NCBI build 36) of each SNP identified is given, along with the minor and major alleles of the variant. The NCBI dbSNP submission numbers (ss) for each variant is shown, as are the SNPs names subsequently identified through the 1000 Genomes project (dbSNP132; August 2011 release), are indicated with an asterisk. The minor allele frequency (MAF) in all 74 individuals (ALL), the 37 cases with schizophrenia (SCZ) and the 37 controls (CON) are given. The predicted transcription factor binding changes for each SNP (TRANSFAC) are listed.
SNPname / SNPsequence / TRANSFAC predicted change (minor allele)420M9-1395 / ATTTCCTTCTTTTTTAAGGCTCAAGAGTATTCGC[GTn]ATCACATTTTCTTTATTCATCTGTTGATG / no change
478B14-848 / AAGTTTTAAAAGTAGGATACAAAATTATGTCATA[CAn]TTTTACAAAAACCAAAATATATGTATG / no change
JF1.2_1 (ss472054941) / TTTACAGTGAAATACTCTTGTKTTGTGGTCGGGAAGTGGTGAGTT / creates SRY
JF1395_2 (ss472054942) / TTTAAGTCTGCAATACAGTATTGTTGACCATARGAACAATATTGTATAGTGGATTTCTAGCATTT / increases Sox-5
JF1395_3 (ss472054943) / TTCTTAACTGTAATTTTGTGCCCRTTGTTTAGTAACTCTAAATTTTCCTC / reduces SRY, removes c-Myb
rs71523425 / ataagctactcaatttaacttttYatttttgaattcaagctttttt / increases CdxA
JF221XXX_1
(rs71523425*) / ATAAGCTACTCAATTTAACTTTTYATTTTTGAATTCAAGCTTTTTT / removes Hb, creates cap
JF221XXX_2 (rs74506441*) / ctctgtataacattggccattaatcYacatcaatatatggtgaagatatgtaa / creates CDP-CR
JF221XXX_4 (ss472054944) / tcttttaaaattctatggacYatataagctactcaattttaagttttca / removes TATA
JF3.1_2 (ss472054945) / AGACTGAAGCAGAGAAGAGCYGCAGAGGAAGAAAGTGAATGAGC / no change
JF3.1_3 (ss472054946) / CTGCAGTGTGGAGTCACCA[TGA/del]AAGGCTAACTCAAAAATGAAGTGGTA / introduces C-/EBP
JF3.2_2 (ss472054947) / AATGCTGACTGTTTTTTTCTTTTAAAA[CA/del]AACAGTCATTAAAACACTAGAAGAAATGCAC / removes SRY, HFH-2
JF3.3_5 (ss472054948) / TGGGCCAATGAAATAAAAAAAA[AA/del]TTTAAGATAAATATGGACTGTATGGGATTAGTGAA / removes HFH-2
JF3.3_6 (rs111526496*) / GGGGTGTTCCTGGGCTTTACTGGRGTGGAGCTTAAAGGGTTAAAATGATATATCCTT / removes AML-1a
JF3.3_7 (rs113060920*) / gagcaattattcaccttaYCTCAACCATTAAGAGCAAACATATTCAGCAG / creates GATA, reduces deltaE
JF4.2_4 (rs77626248*) / TTCATGGGGCAGACGGATCTCAMAGGATGCCTAAGTTCAGCAGTGGATTGTTTGC / creates c-ETS, GATA
JF4.3_1 (ss472054949) / ATTCATACCTTTTCTTAAGCATATGTTAATCAYATTAGAAATGCCATTCCCTTCTCGTGCAAAAG / removes CdxA, reduces Oct-1
JF848_2 (ss472054950) / ATGTCCAACTGAAGAGGAATTAGGGTTTAACGATTYAAGAAGATATCATGAAACTATTAAA / removes CdxA
JF848_3 (ss472054951) / AAATTAATACTTTTGGTCATACAGGATGTCTCTRTTTTTTTGAAATACATTTTTCAA / removes SRY, creates CdxA
NOR_3.2_4 (rs117532293*) / caactgggtgctttttgaagaaatYataaattaccctagtttagcataaacacc / creates S8
NOR_848_4 (ss472054952) / CAAGACATCTAATATGAGTCATYTTGACCCAATATTTTCCTTGT / increases AP-1
NOR3.2_2 (rs117347889*) / ATAGATTTAGAGAGAGTTTTACAGACTCCTRTTGACATAAGTGAACAAAATGGTTCCTTGGAA / no change
NOR3.2_3 (ss472054953) / TACAGGTTCAGATGCATATTGTGTGCAGTGATRTGCAGCACAGTGCTTGGGGAAGCCTGTGGTC / no change
NOR3.3_1 (ss472054954) / ATATTCTGGTTCTCATAGTCTCTCCTTGAAGTGRTATGTAATCAATAATATAATCAAATGCACCC / introduces GATA-1, removes AML-1a
rs10090954 / GTTGCAGTTTTTAAAAAAGTAATCTTTGTTTAMATTATCTTAAGTTACTTGATTTAAAAAAGTTA / introduces GATAm XFD, reduces CdxA
rs10096965 / TTACTTTATTTCATTGTAGTAAGAAAACTGAACRTGCTACTACCTTCAATAAATTTTTAAGTCTG / no change
rs11785744 / CACATGTCCAACTGAAGAGGAATTAGGGTTTAAYGATTTAAGAAGATATCATGAAACTATTAA / introduces CdxA, Oct-1
rs11989919 / CCCAATTTTGACCTAAACCAAACTATATACTCARTACACCAGCATTTCATC / no change
rs11998176 / AATATCCCGGGAGAGGATGGATTCTTGTTTTAGWCATAGCTCTTTAAATTTGGCAGGACATGTG / no change
rs12707707 / TCCTGACCACAGAGATGAATAATTTAAGGACAAYATCAAATTCTTGATAAATCTCATAAATGTT / reduces Oct-1
rs13253310 / GAAGTCCACATGTCCAACTGAAGAGGAATTAGGSTTTAATGATTTAAGAAGATATCATGAAACT / increases Oct-1
rs13256117 / TCTGTCTTCATGAAAGAGAYGGAGAGTTCCCATTTCTACTTA / removes GATA-1 & -3
rs13256229 / ATTTGTATCCACCCCCATCCCCAATCTACTGAAYCAGAAAATCTGGCGGCAGGGCCAGCAATCT / removes AP-1
rs13263989 / AATATTATTCTTATGTCAAGTGTGGAAAATACYAACCGAAGTCCACATGTCCAACTGAAGAGGA / introduces CdxA
rs13282705 / TTTTTAAGCGATAAAGAATAAAGCTCTTTTCATYTTTTAACTGGAATTATTTTTTAGAAAATATT / introduces CdxA
rs13362886 / AATTGCCATTTCATCATCTTTCCTTAAAGTCCCYTCAATATTTATCATTGCAATTTTTGTCCTGTC / removes MZF1
rs17603786 / TTAAGATAAAACGAGTTTAACAGATAATTTAGYCCATTCACATTTGTTGTAATTCATGATATTTG / introduces S8
rs17722883 / CCCAGTTGAAATATACGGCACTGAATTCCCYAATTTTGACTAAACCAAACTATATACTCATACA / introduces MZF1, reduces Ik-2
rs2466044 / TAAAAAAAAAATTTAAGATAAATATGGACTGTRTGGGATTAGTGAAGATCAGAAATAATGTAT / increases Ik-2
rs28401439 / TGAGGTCACTGAATATATTTTTCACTATAAAAKAACATGAGAGAAAATATTTACCTTGAAATGC / introduces Cdxa, reduces TATA, SRY
rs28476555 / TAAAGCTTCTATGACATACTTTCAAGAAACTGYTAGAGGCAACACGTAGAATCCCAGAGTAAA / introduces v-Myb
rs33978908 / AGGAAGAATATTTTTGCTTTAAAAAAAAA[A/del]CCCAATTGTATAATTTAGAAATGATGACA / no change
rs34150028 / ttttttaaggctcaagagtattcg[CG/del]tgtgtgtgtgtgtgtgtgtgtgtgtgtatcacatt / no change
rs34178679 / AACCTAGCATCTTTAAGGTTCRCTTAGCCCTTCCTGTGCACCTG / introduces Nkx-2
rs34445647 / CTCTTTAGAATTCTCCACCAGAGGGARGACAAGGGAAGGAGTAGGTTTCACGCGCAG / no change
rs34595725 / GAAGTAGGTGTCAAGTTAC[C/del]TAAGATGTCCAAGAGACAGCTGAT / removes VBP, CRE-BP, C/EBP
rs36213229 / GGAGCGGGCAGCGAGAGCCTCGGGTCTCCKCCTGGGTTCCCGGGTCTCCGGGGCGCTGGCC / no change
rs36213230 / GGGGGTTCCCGGCAGCCGCGCCGCCACCCCYCGCCCGGCCAGCGCGGGAGGAAAAGGGGCT / no change
rs36213231 / GCGCCCGGGAGCGCCGAGCCCAGGCTCCTCCYGGTGGCGTGTCCGCGCCTCGGGGTGGGGGT / no change
rs3802158 / GATCTCCAGTTCTGTGTCTTTATTCTACYCCTCCCTGCCTGCTTTTCCACTCGGCAG / no change
rs3802159 / GTAGACAATTCTGGATCCTTCCGTGGTGCCCSTACCCTGGTCTTTAACTTTTGTCCTTTGCAGGG / no change
rs3802160 / GAGTATTTCACTGTAAATTAAGAGTCTAAGTTARCCACAGCTGTGGTATAACTCTGAGCAATGC / removes C/EBP
rs3802161 / AAAACCAAGAGCAAGTCACTTTTAAAGTGAMGCAATAGATTTTGAATATGGATTGTTCCAACTC / introduces AP-1, HLF, CrEB, reduces C/EBP
rs3808368 / TAGGGCACAATCTCCACTACTTTGAGGTATGTTWCAGCTTTAAACGGCAGGAGATAAGAATATT / removes SRY
rs4129812 / TTCAGAAACAATATCAGCAGGTGTTTATGCTRAACTAGGGTAATTTATGATTTCTTCAAAAAGC / introduces SRY
rs4281084 / TAAATGAACCAACAGGTCACCAAATGTTGAAGTRGTTTGTCATATAGTGACAGATAACTGATAC / introduces Nkx-2, AML-1a, removes SRY
rs4400337 / ATTTCTTCAAAAAGCACCCAGTTGAAATATAYGGCACTGAATTCCCCAATTTTGACCTAAACCA / reduces CdxA
rs4433107 / TTACAACATTTCCATAGAAATGGATTTTGAGCTTYTTTTTTTTAATTGGGGAAAATCTTATTT / no change
rs4531002 / GAAGGCAGAAAGGCAACTTCTGGGTCCTAGTCYCAAGGGTAGAACTAATGGAGAATTCTTTTAT / no change
rs4623366 / AAAATTCTATGGACTATAYAAGCTACTCAATTTTAAGT / increases TATA
rs55898258 / ACATTTATTGAGTACTTAATATTTGACCCAAAYTGGATCAAAACTGGGGAATATAGAAACTGTC / removes RORa1p
rs57147288 / TGTAAGCAGAATGTACARTGTATAAGACATGCATATAT / no change
rs57205530 / CCCTGCTTGTATCTCTGCTCTTTGGCATTGCAAYTTTTCAGGTCCTTATGTGAAGAGGTAGAGTC / no change
rs62500193 / GCAAAAATATTCTTCCTCTTTTCTCCATCCMTTGTTCTGGTCAGTTCCA / introduces GATA, removes SRY, Sox-5
rs62500194 / TTGTTCTGGTCAGTTCCARGGTTTTTTACAAATGCAAAAGAAATTCATTTGC / no change
rs7014762 / AAGCGCTCCATCAGGGTATGAGTAACAGGGAWCTCCCCTTGCCAAGACACACAGGGAGTGTGA / removes NF-kap, c-Rel
rs7350144 / TTTCCTACAAACATGCATGTTTTATCCAAARGAAATTCTGACCTCTAACCCCATTCACACTTTCC / introduces c-Ets, removes SRY
rs7812451 / AGACAGCTGATGGGTTATGARTTAAATTTTGGGTTCTGCTTATCATT / removes CdxA, Tst-1, Pbx-1
rs7817936 / AAAGTTAAGTCTAGCCAAGGAAAAATGTAGTGSCACACGATTGCTTTTCTCTTACGC / introduces AML-1a
rs7817942 / AGCCAAGGAAAAATGTAGTGGCACACGATTGCKTTTCTCTTACGCTGTCATTTAATGTGAGATC / reduces GATA
rs7823498 / TTCCTGTAGGAATCCTGCTTTTAYGTTTTATCTTAAAGCCACCATAGTATCTGTAAT / introduces CdxA, SRY, GATA-X
rs7825588 / CCAGCCTGCAGCTCTAGAGTGTGGGTAGAGAGCRGGGAGTGGGGGTTGGGAGAGGGGG / increases p300, removes MZF1
rs73235619# / tgtataagacatgcatatatcaatRtaaggtagtaatgtttattttaaa / no change
rs35753505# / GAGATATATGATATTTGGYAAAATAAAGATACATGGCTTCCA / increases C/EBPb & a, reduces C/EBP
rs62510682# / GAAATGAAATATGTGTGCAAACAGTTCTTAKTACTGAGCTGTTTAAAGAAGGCCTACCTTTGCA / removes CdxA, Oct-1
rs6994992# / GCTAGAAGCACCATGCAGGGTTCAAGTGAAYGTATACTGGAGGCCAGACCTGCCCAACTATGC / no change
SNP8NRG433E1006# / gggcggcggccggcaacgaggcggctcccgcgRgggcctcggtgtgctactcgtccccgccca / no change
YT1395_1 / ATTTTTTTGCTATTCTTTCATGATTAAAAGTTAWGTTTCATTATTATCAGGTTGTATATTTACATA / reduces SRY, removes CdxA
YT4.2_1 / CAAAGGATGCCTAAGTTCAGCAGTGGATTGTTYGCAGAAATGGCCTAATTCTTCCCCTGCTTGT / reduces Sox-5, removes SRY
YT4.2_2 / TCACTGAATATATTTTTCACTATAAAAGAACAYGAGAGAAAATATTTACCTTGAAATGCTAAAA / increases TATA
YT4.2_3 / CTGAATATATTTTTCACTATAAAAGAACATGARAGAAAATATTTACCTTGAAATGCTAAAAATG / creates SRY, improves HNF-3
YT848_1 / CAATAGTGTTATTTGAAAAATATTTAAACAGARCATAACTCAGTTAATATATATGTACTAAATA / no change
Supplementary Table 6: Flanking sequences of 68 nucleotide variants identified in resequencing study. For each SNP observed, bioinformatic predictions of the effect of the minor allele on transcription factor binding is presented. SNPs represented in the HapICE risk haplotype were SNP8NRG221132 (=rs73235619), SNP8NRG221533 (=rs35753505), SNP8NRG241930 (=rs62510682), SNP8NRG243177 (=rs6994992) and SNP8NRG433E1006 (not annotated in dbSNP130), and are annotated with a hash (#). Novel SNPs with respect to dbSNP130 release are given, with their NCBI submission numbers (ss) in parentheses. Thosenovel SNPs which have subsequently been discovered in the 1000 Genomes project(dbSNP132) and are indicated withan asterisk. Novel SNPswhich were exclusively observed in cases are in bold text.
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