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 genome reference 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 matrix with a minimum score of 85
constituting a hit. Nucleotide diversity was assessed using methods previously described78
, 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, and LD 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 a 12,745 square kilometre area of southeast 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, 1l 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 IV NRG1 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 gender for NRG1 Type IV 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 between the five HapICE SNPs and rs7014762
in the promoter IV/II region of NRG1. A) SNP rs7014762 is in high LD (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 HAPLOVIEW 10. 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 EGFisoform 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
NRG1
Type
I(Ig2)
NRG1
Type II
NRG1
NRG1
NRG1
Type IV
Type III
panNRG1
NRG1
NRG1
Type I
EGFα
NRG1
ACTB
EGFβ
-
isoforms detected
Ig2 and s1 domains
(excluding GGF-2, HRG-β1d,
HRG-β3b, HRG-γ3, SMDF)
GGF-2, HRGβ-1d, HRG-β3b,
HRG-γ3
HRG-β1b, HRG-β1c & HRGβ1d
SMDF
all isoforms (excluding
SMDF, ndf43c)
HRG-β3b and HRG-γ3
(excluding HRG-β1b, HRGβ1c)
HRG-α, ndf43 and ndf43c
SMDF, GGF, GGF2, all
HRG-β isoforms
beta-actin
inventoried assay
forward
reverse
probe
(FAM-MGB)
-
GCCAATATCACC
ATCGTGGAA
CCTTCAGTTGAG
GCTGGCATA
CAAACGAGATC
ATCACTG
-
GAATCAAACGCT
ACATCTACATCCA
CCTTCTCCGCAC
ATTTTACAAGA
CACTGGGACAA
GCC
GCTCCGGCAGCA
GAACCTGCAGCC
GATTCCT
ACCACAGCCTTG
CCT
-
-
Hs00247620_m1 -
-
-
Hs01108479_m1 Hs01103794_m1 -
-
-
Hs00247624_m1 Hs99999903_m1 -
-
-
GCAT
Hs01103792_m1 -
glyceraldehyde-3-phosphate
GAPDH 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 target particular 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
upstream
HapICE
promoter IV/II
478B14-848
420M9-1395
promoter I
promoter III
region
location,
bp (NCBI
Build 36)
3159286031593868
3161333431616575
3170811531708936
3178462231785559
3252232632525340
3262073032624137
total screened
observed SNPs
(ALL)
novel
dbSNPs
SNPs
observed SNPs
(SZ)
novel
dbSNPs
SNPs
observed SNPs
(CON)
novel
dbSNPs
SNPs
DNA
length
screened,
bp
# dbSNPs
annotated
1,008
5
2
3
3
3 (1)
3
2 (0)
17.5
3,241
11
9
5
9
5 (2)
7
3 (0)
11.1
821
4
3
4
3
3 (3)
3
1 (1)
26.8
937
7
7
3
3
3 (2)
3
1 (0)
30.1
3,014
33
15
1
13
1 (1)
13
0 (0)
13.2
3,407
11
6
10
6
6 (2)
6
7 (4)
11.7
12,428
71
42
26
37
21 (11)
35
14 (5)
10.0
nucleotide
diversity
(θ×10-4)
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
n
74
pan-NRG1
age
-0.23
<0.05
pH
0.43
<0.001
RIN
0.29
<0.05
PMI
0.41
<0.001
73
EGFβ
age
-0.21
ns
pH
0.55
<0.001
RIN
0.37
<0.01
PMI
0.06
ns
73
Type I
age
0.08
ns
pH
0.21
ns
RIN
0.37
<0.01
PMI
-0.11
ns
72
Type I(Ig2)
age
-0.12
ns
pH
0.01
ns
RIN
-0.04
ns
PMI
-0.26
<0.05
70
Type II
age
0.10
ns
pH
-0.33
<0.01
RIN
-0.25
<0.05
PMI
0.05
ns
74
Type III
age
-0.10
ns
pH
0.32
<0.01
RIN
0.39
<0.001
PMI
0.03
ns
55
Type IV
age
0.28
<0.05
pH
-0.25
ns
RIN
-0.37
<0.01
PMI
-0.01
ns
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
upstream prom IV/II
upstream prom IV/II
upstream prom IV/II
upstream prom IV/II
upstream prom IV/II
upstream prom IV/II
upstream prom IV/II
upstream prom IV/II
intron 1
intron 1
intron 1
intron 1
intron 1
intron 1
intron 1
upstream prom I
upstream prom III
upstream prom III
upstream prom III
upstream prom III
upstream prom III
upstream prom III
upstream prom III
upstream prom III
BP position
(build 36)
31593076
31593103
31593216
31613752
31614472
31614502
31615552
31615555
31708375
31708615
31708702
31708937
31784714
31784774
31785235
32524132
32620979
32621144
32621357
32621907
32622150
32622768
32622867
32622943
SNP
JF221XXX_4
JF221XXX_1
JF221XXX_2
JF4.3_1
JF4.2_4
YT4.2_1
YT4.2_2
YT4.2_3
JF848_2
JF848_3
YT848_1
NOR_848_4
JF1395_2
JF1395_3
YT1395_1
JF1.2_1
NOR3.3_1
JF3.3_5
JF3.3_6
JF3.3_7
NOR3.2_4
NOR3.2_2
NOR3.2_3
JF3.2_2
dbSNP name
ss472054944
rs71523425*
rs74506441*
ss472054949
rs77626248*
rs73584584*
ss472054956
rs76063839*
ss472054950
ss472054951
rs117129618*
ss472054952
ss472054942
ss472054943
rs117469567*
ss472054941
ss472054954
ss472054948
rs111526496*
rs113060920*
rs117532293*
rs117347889*
ss472054953
ss472054947
minor major MAF
MAF
MAF
allele allele (ALL) (SCZ) (CON) TRANSFAC binding change
C
T
0.007 0.014 0.000 removes TATA
C
T
0.061 0.068 0.054 removes Hb, creates cap
C
T
0.068 0.095 0.041 creates CDP-CR
C
T
0.007 0.014 0.000 removes CdxA, reduces Oct-1
C
A
0.108 0.108 0.041 creates c-ETS, GATA
C
T
0.034 0.054 0.014 reduces Sox-5, removes SRY
C
T
0.007 0.014 0.000 increases TATA
A
G
0.034 0.027 0.041 creates SRY, improves HNF-3
C
T
0.029 0.042 0.000 removes CdxA
A
G
0.028 0.056 0.000 removes SRY, creates CdxA
G
A
0.030 0.054 0.000 no change
C
T
0.007 0.000 0.027 increases AP-1
A
G
0.008 0.014 0.000 increases Sox-5
A
G
0.028 0.054 0.000 reduces SRY, removes c-Myb
A
T
0.027 0.054 0.000 reduces SRY, removes CdxA
T
G
0.014 0.027 0.000 creates SRY
A
G
0.007 0.000 0.014 introduces GATA-1, removes AML-1a
del
AA
0.014 0.014 0.014 removes HFH-2
A
G
0.020 0.014 0.027 removes AML-1a
T
C
0.016 0.029 0.000 creates GATA, reduces deltaE
T
C
0.007 0.000 0.014 creates S8
G
A
0.007 0.000 0.014 no change
A
G
0.007 0.000 0.014 no change
del
CA
0.020 0.027 0.014 removes SRY, HFH-2
upstream prom III
upstream prom III
32623136
32624045
JF3.1_3
JF3.1_2
ss472054946
ss472054945
del
C
TGA
T
0.014
0.131
0.014
0.014
0.000
0.000
introduces C-/EBP
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.
SNP name
SNP sequence
TRANSFAC predicted
change (minor allele)
420M9-1395
ATTTCCTTCTTTTTTAAGGCTCAAGAGTATTCGC[GT n]ATCACATTTTCTTTATTCATCTGTTGATG
no change
478B14-848
AAGTTTTAAAAGTAGGATACAAAATTATGTCATA[CAn]TTTTACAAAAACCAAAATATATGTATG
TTTACAGTGAAATACTCTTGTKTTGTGGTCGGGAAGTGGTGAGTT
no change
JF1.2_1
(ss472054941)
JF1395_2
(ss472054942)
JF1395_3
(ss472054943)
rs71523425
creates SRY
TTTAAGTCTGCAATACAGTATTGTTGACCATARGAACAATATTGTATAGTGGATTTCTAGCATTT
increases Sox-5
TTCTTAACTGTAATTTTGTGCCCRTTGTTTAGTAACTCTAAATTTTCCTC
reduces SRY, removes c-Myb
ATAAGCTACTCAATTTAACTTTTYATTTTTGAATTCAAGCTTTTTT
increases CdxA
JF221XXX_1
(rs71523425*)
ATAAGCTACTCAATTTAACTTTTYATTTTTGAATTCAAGCTTTTTT
removes Hb, creates cap
JF221XXX_2
(rs74506441*)
JF221XXX_4
(ss472054944)
JF3.1_2
(ss472054945)
JF3.1_3
(ss472054946)
JF3.2_2
(ss472054947)
JF3.3_5
(ss472054948)
JF3.3_6
(rs111526496*)
JF3.3_7
(rs113060920*)
JF4.2_4
CTCTGTATAACATTGGCCATTAATCYACATCAATATATGGTGAAGATATGTAA
creates CDP-CR
TCTTTTAAAATTCTATGGACYATATAAGCTACTCAATTTTAAGTTTTCA
removes TATA
AGACTGAAGCAGAGAAGAGCYGCAGAGGAAGAAAGTGAATGAGC
no change
CTGCAGTGTGGAGTCACCA[TGA/DEL]AAGGCTAACTCAAAAATGAAGTGGTA
introduces C-/EBP
AATGCTGACTGTTTTTTTCTTTTAAAA[CA/DEL]AACAGTCATTAAAACACTAGAAGAAATGCAC
removes SRY, HFH-2
TGGGCCAATGAAATAAAAAAAA[AA/DEL]TTTAAGATAAATATGGACTGTATGGGATTAGTGAA
removes HFH-2
GGGGTGTTCCTGGGCTTTACTGGRGTGGAGCTTAAAGGGTTAAAATGATATATCCTT
removes AML-1a
GAGCAATTATTCACCTTAYCTCAACCATTAAGAGCAAACATATTCAGCAG
creates GATA, reduces deltaE
TTCATGGGGCAGACGGATCTCAMAGGATGCCTAAGTTCAGCAGTGGATTGTTTGC
creates c-ETS, GATA
(rs77626248*)
JF4.3_1
(ss472054949)
JF848_2
(ss472054950)
JF848_3
(ss472054951)
NOR_3.2_4
(rs117532293*)
NOR_848_4
(ss472054952)
NOR3.2_2
(rs117347889*)
NOR3.2_3
(ss472054953)
NOR3.3_1
(ss472054954)
rs10090954
ATTCATACCTTTTCTTAAGCATATGTTAATCAYATTAGAAATGCCATTCCCTTCTCGTGCAAAAG
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
removes CdxA, reduces Oct-1
ATGTCCAACTGAAGAGGAATTAGGGTTTAACGATTYAAGAAGATATCATGAAACTATTAAA
removes CdxA
AAATTAATACTTTTGGTCATACAGGATGTCTCTRTTTTTTTGAAATACATTTTTCAA
removes SRY, creates CdxA
CAACTGGGTGCTTTTTGAAGAAATYATAAATTACCCTAGTTTAGCATAAACACC
creates S8
CAAGACATCTAATATGAGTCATYTTGACCCAATATTTTCCTTGT
increases AP-1
ATAGATTTAGAGAGAGTTTTACAGACTCCTRTTGACATAAGTGAACAAAATGGTTCCTTGGAA
no change
TACAGGTTCAGATGCATATTGTGTGCAGTGATRTGCAGCACAGTGCTTGGGGAAGCCTGTGGTC
ATATTCTGGTTCTCATAGTCTCTCCTTGAAGTGRTATGTAATCAATAATATAATCAAATGCACCC
GTTGCAGTTTTTAAAAAAGTAATCTTTGTTTAMATTATCTTAAGTTACTTGATTTAAAAAAGTTA
no change
introduces GATA-1, removes
AML-1a
introduces GATAm XFD,
reduces CdxA
rs17722883
CCCAGTTGAAATATACGGCACTGAATTCCCYAATTTTGACTAAACCAAACTATATACTCATACA
introduces MZF1, reduces Ik-2
rs2466044
TAAAAAAAAAATTTAAGATAAATATGGACTGTRTGGGATTAGTGAAGATCAGAAATAATGTAT
rs28401439
TGAGGTCACTGAATATATTTTTCACTATAAAAKAACATGAGAGAAAATATTTACCTTGAAATGC
increases Ik-2
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
rs34595725
GAAGTAGGTGTCAAGTTAC[C/DEL]TAAGATGTCCAAGAGACAGCTGAT
no change
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
rs3802161
AAAACCAAGAGCAAGTCACTTTTAAAGTGAMGCAATAGATTTTGAATATGGATTGTTCCAACTC
removes C/EBP
introduces AP-1, HLF, CrEB,
reduces C/EBP
rs3808368
TAGGGCACAATCTCCACTACTTTGAGGTATGTTWCAGCTTTAAACGGCAGGAGATAAGAATATT
removes SRY
rs4129812
TTCAGAAACAATATCAGCAGGTGTTTATGCTRAACTAGGGTAATTTATGATTTCTTCAAAAAGC
rs4281084
TAAATGAACCAACAGGTCACCAAATGTTGAAGTRGTTTGTCATATAGTGACAGATAACTGATAC
introduces SRY
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
rs62500193
GCAAAAATATTCTTCCTCTTTTCTCCATCCMTTGTTCTGGTCAGTTCCA
no change
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
rs7823498
TTCCTGTAGGAATCCTGCTTTTAYGTTTTATCTTAAAGCCACCATAGTATCTGTAAT
reduces GATA
introduces CdxA, SRY,
GATA-X
rs7825588
CCAGCCTGCAGCTCTAGAGTGTGGGTAGAGAGCRGGGAGTGGGGGTTGGGAGAGGGGG
increases p300, removes MZF1
rs73235619#
TGTATAAGACATGCATATATCAATRTAAGGTAGTAATGTTTATTTTAAA
rs35753505#
GAGATATATGATATTTGGYAAAATAAAGATACATGGCTTCCA
no change
increases C/EBPb & a, reduces
C/EBP
rs62510682#
GAAATGAAATATGTGTGCAAACAGTTCTTAKTACTGAGCTGTTTAAAGAAGGCCTACCTTTGCA
removes CdxA, Oct-1
rs6994992#
GCTAGAAGCACCATGCAGGGTTCAAGTGAAYGTATACTGGAGGCCAGACCTGCCCAACTATGC
no change
SNP8NRG433E
1006#
YT1395_1
GGGCGGCGGCCGGCAACGAGGCGGCTCCCGCGRGGGCCTCGGTGTGCTACTCGTCCCCGCCCA
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
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. Those novel SNPs which
have subsequently been discovered in the 1000 Genomes project (dbSNP132) and are indicated with an asterisk. Novel SNPs which
were exclusively observed in cases are in bold text.
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