Journal of Human Genetics (2009) 54, 531–537
& 2009 The Japan Society of Human Genetics All rights reserved 1434-5161/09 $32.00
www.nature.com/jhg
ORIGINAL ARTICLE
Identification of novel candidate loci for anorexia
nervosa at 1q41 and 11q22 in Japanese by a genomewide association analysis with microsatellite markers
Kazuhiko Nakabayashi1,2,7, Gen Komaki3, Atsushi Tajima4, Tetsuya Ando3, Mayuko Ishikawa1, Junko Nomoto1,
Kenichiro Hata5, Akira Oka4, Hidetoshi Inoko4, Takehiko Sasazuki6, Japanese Genetic Research Group for
Eating Disorders (JGRED) and Senji Shirasawa2
The Japanese Genetic Research Group for Eating Disorders (JGRED) is a multisite collaborative study group that was organized
for the systematic recruitment of patients with an eating disorder for the purpose of genetic study in Japan. We conducted a
genome-wide case–control association study using 23 465 highly polymorphic microsatellite (MS) markers to identify genomic
loci related to anorexia nervosa (AN). Pooled DNA typing in two screening stages, followed by individual typing of 320 AN cases
and 341 controls, allowed us to identify 10 MS markers to be associated with AN. To narrow down genomic regions responsible
for the association of these MS markers, we further conducted a single-nucleotide polymorphism (SNP) association analysis for
7 of the 10 loci in 331 AN cases and 872 controls, which include the 320 AN cases and the 341 controls genotyped in the MS
screening, respectively. Two loci, namely 1q41 and 11q22, remained significantly associated with AN in the SNP-based fine
mapping, indicating the success in narrowing down susceptibility regions for AN. Neither of these loci showed a positive
evidence of association with bulimia nervosa. The most significant association was observed at SNP rs2048332 (allelic
P-value¼0.00023) located at 3¢-downstream of the SPATA17 gene on the 1q41 locus. The association analysis for MS-SNP
haplotypes detected a statistically significant association (permutation P-value¼0.00003) of the A-4-G-T haplotype that
comprised four SNP/MS markers (rs6590474–D11S0268i–rs737582–rs7947224) on the 11q22 locus with AN. This linkage
disequilibrium block spanning a 20.2-kb interval contains exon 9 of the CNTN5 gene encoding contactin 5.
Journal of Human Genetics (2009) 54, 531–537; doi:10.1038/jhg.2009.74; published online 14 August 2009
Keywords: anorexia nervosa; bulimia nervosa; eating disorders; genome-wide association study; microsatellite markers; SNPs
INTRODUCTION
Eating disorders (EDs) are psychiatric conditions characterized by
severe disturbances in eating behavior, and can be classified into three
major types, namely anorexia nervosa (AN), bulimia nervosa (BN)
and ED not otherwise specified. AN and BN are not mutually exclusive
conditions as some individuals cross over between both conditions.1,2
It is generally believed that ED has become more frequent over recent
decades. Fairburn and Harrison3 summarized the prevalence of AN
and BN to be 0.7% in teenage girls and 1–2% in 16–35-year-old
females, and the incidence (per 100 000 per year) of AN and BN to be
19 in females and 2 in males, and 29 in females and 1 in males,
respectively.3 AN has the highest mortality rate (5–6%) of any
psychiatric disease,4 whereas the mortality rate of BN is reported to
be 0.3%.5
The cause of ED is complex and poorly understood. However, the
involvement of genetic factors in the etiology of ED has been demonstrated in family and twin studies.6–9 Twin studies have estimated the
contribution of genetic factors in AN to be between 33 and 84%9 and
that in BN to be between 28 and 83%.8 To search for the genetic
etiology of ED, two types of molecular genetic approaches, namely
linkage studies and association studies, have been carried out (reviewed
in Bulik et al.10 and Pinheiro et al.11). Linkage studies for AN have
detected significant linkage at two regions on chromosome 112,13 and
an additional suggestive linkage at a number of loci.13,14 From the
1p33-p36 region, one of the regions showing significant linkage to
AN,12 serotonin 1D receptor (HTR1D) and opioid delta receptor
(OPRD1) genes were further evaluated and found to exhibit significant
association with AN.15 Linkage analysis of a BN cohort detected
1Department of Pathology, Research Institute, International Medical Center of Japan, Shinjuku, Tokyo, Japan; 2Department of Cell Biology, Faculty of Medicine, Fukuoka
University, Jonan, Fukuoka, Japan; 3Department of Psychosomatic Research, National Institute of Mental Health, National Center of Neurology and Psychiatry, Ogawahigashicho,
Kodaira, Japan; 4Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Japan, 5Department of Maternal-Fetal Biology, National Research Institute
for Child Health and Development, Setagaya, Tokyo, Japan and 6International Medical Center of Japan, Shinjuku, Tokyo, Japan
7Current address: Department of Maternal-Fetal Biology, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo 157-8535, Japan
Correspondence: Dr K Nakabayashi, Department of Maternal-Fetal Biology, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo
157-8535, Japan.
E-mail: knakabayashi@nch.go.jp
Received 15 April 2009; revised 2 July 2009; accepted 15 July 2009; published online 14 August 2009
Novel susceptibility loci for AN
K Nakabayashi et al
532
significant linkage at 10p13 and a suggestive linkage at 14q22.2-23.1.16
Fine mapping of these regions with significant/suggestive linkage
signals will clarify whether the regions contain gene(s) relevant to
AN or BN. Association studies for ED have so far been limited to
candidate gene approaches that focused on the genes, such as those
involved in the regulation of feeding and body composition and those
implicated in neurotransmitter pathways regulating behavior.10,11,17
Although many association studies performed for ED are considered
to be statistically underpowered because of their small sample sizes
and/or suffer from multiple testing,10 positive findings on HTR1D,
OPRD1 and BDNF genes seem promising, as their association with
AN/BN has been replicated in more than one study in which relatively
large numbers of samples were enrolled (reviewed in Bulik et al.10).
Completion of the Human Genome Project18 and the rapid progress of the International HapMap Project19 dramatically increased the
amount of information on genetic markers, such as microsatellite
(MS) markers and single-nucleotide polymorphisms (SNPs). Consequently, statistical strategies and genotyping platforms for genomewide association studies (GWASs) have been established and prevailed
as a means of identifying disease susceptibility genes. Disease association studies using MS markers distributed across the human genome
have advantages over linkage analysis and the candidate gene
approach. MS markers are highly polymorphic and show a high
degree of heterozygosity (B70% on average), and their linkage
disequilibrium (LD) lengths are in the 100 kb range.20–22
In this study, we adopted a practical and efficient GWAS strategy for
AN using a set of 23 465 MS markers22,23 and the DNA pooling
method, which has been adopted to identify novel susceptibility genes
of rheumatoid arthritis22 and candidate loci for hypertension24 and
adult height.23 We identified 10 novel loci related to AN by the MS
marker-based GWAS strategy, and subsequently conducted an SNPbased association analysis for 7 of the 10 loci to further narrow down
candidate genomic intervals responsible for AN susceptibility.
MATERIALS AND METHODS
Subjects
The patients enrolled in this study were recruited through the efforts of the
Japanese Genetic Research Group for Eating Disorders (JGRED), which
comprises 67 nation-wide hospitals/institutions (the full list is available in
Supplementary Table 1). A total of 456 unrelated Japanese female patients with
ED (331 cases with AN and 125 cases with BN) participated in this study.
According to the Diagnostic and Statistical Manual of Mental Disorders,25
218 and 113 cases were diagnosed as AN-restricting type (AN-R) and as AN
with binge eating/purging type (AN-BP), respectively. Among the
125 BN cases, 46 had histories of AN and 79 cases did not. The average age
at assessment was 23.2±7.91 (s.d.) years for AN-R, 25.7±7.22 years for AN-BP
and 26.3±6.77 years for BN. The lifetime minimum body mass index was
12.4±1.90 (s.d.) kg m2 for AN-R, 12.8±2.67 kg m2 for AN-BP and
16.2±3.47 kg m2 for BN. A total of 872 Japanese healthy individuals participated in this study: 180 female individuals whose average age was 34.5 years
(control group 1) and 692 male and female volunteers recruited among
university students (control group 2). The average age and gender ratio of
control group 2 were unavailable. The ethics committees of all facilities
approved the investigation. All subjects gave their written informed consent
before participation in the study.
Pooled DNA construction and MS Genotyping
Among the 27 037 MS markers developed by Tamiya et al.,22 23 465 (with
average spacing 118.0-kb) were used in this study. Detailed information on the
27 037 MS markers is also available at the Japan Biological Information
Research Center website (http://jbirc.jbic.or.jp/gdbs/database/viewer/download/list.jsp). The pooled DNA method for MS typing was performed according to the protocol of Collins et al.26 with a slight modification.21 Genomic
Journal of Human Genetics
DNA was extracted from peripheral blood using the Qiagen blood mini kit
(Qiagen, Hilden, Germany). DNA concentration was determined using PicoGreen fluorescence assay (Molecular Probes, Eugene, OR, USA) as described
previously.22,26 The detailed conditions for PCR amplification and peak
detection for pooled DNA and individual genotyping were described previously.22,24 A total of 320 AN cases and 341 controls were subjected to MS
genotyping. The number of subjects for pooled DNA typing was 90 AN-R cases
and 90 female controls from control group 1 in the first-stage screening, and
another 90 AN-R cases and another 90 female controls from control group 1 in
the second-stage screening. The number of subjects for individual genotyping
in the third screening was 140 AN cases (composed of 32 AN-R and 108 ANBP cases) and 161 controls from control group 2. As the final step of MS
screening, positive markers were subjected to individual genotyping of all 320
AN cases and 341 controls used in the three screening stages. MS markers
showing statistical significance (Po0.05) in Fisher’s exact test for the 22
contingency table in the first screening were subjected to the second screening.
In the second and third screening stages, besides statistical significance
(Po0.1), consistency in the directions of effect of the associated MS allele
on AN susceptibility was considered; when the allele holding statistical
significance showed the opposite direction of effect compared with that in
the previous screening stage, such a marker was excluded from further analysis.
SNP genotyping
Single-nucleotide polymorphisms in candidate regions were selected from the
SNP database (http://www2.appliedbiosystems.com/) using SNPbrowser software 3.5 (Applied Biosystems, Foster City, CA, USA). SNPs were genotyped
using TaqMan assays, which were carried out using standard protocols for ABI
PRISM 7900HT Sequence Detection Systems (Applied Biosystems). A total of
331 AN cases, 125 BN cases and 872 control individuals were subjected to SNP
genotyping. The 331 AN cases consisted of the 320 cases genotyped in the MS
screening and 11 additional cases. The 872 controls consisted of 180 female
controls from control group 1 and 692 controls from control group 2.
Statistical analyses
Allelic frequencies in pooled DNA genotyping were estimated from the height
of peaks measured using the PickPeak and MultiPeaks programs (Applied
Biosystems). To calculate allelic P-values from MS genotyping data, we used
Fisher’s exact test for the 22 contingency tables for each individual allele and
for the 2m contingency tables for each locus, where m refers to the number of
marker alleles observed in the genotyped population.
The power of each of the three-stage MS screenings was calculated using a
‘Genetic Power Calculator27 (http://pngu.mgh.harvard.edu/~purcell/gpc/)’ in
the AN prevalence rate of 1%. In the three successive screening, the overall
power of the study was 72 and B50% for detecting an AN-susceptible allele
with a genotype relative risk of 2.0 and 1.8, respectively, under an additive
model in log-odds scale when the susceptible allele (with a frequency of 0.2)
and an AN-associated MS marker were in complete LD.
For SNP genotyping, disease associations were assessed by the w2 test mainly
using Haploview 4.0 software (http://www.broad.mit.edu/mpg/haploview/).28
As a multistep analysis was used, nominal P-values were corrected with 10 000
iterated permutations for a series of SNPs selected for each candidate genomic
interval. The significance level for SNP association was set at 0.05 throughout
the study. Haplotype association analysis (100 000 iterated permutations) was
also performed using Haploview 4.0. LD blocks in each candidate locus were
defined using the default algorithm, namely confidence intervals,29 of Haploview 4.0. SNPAlyze v7.0 software (Dynacom, Mobara, Japan) was used to
perform LD calculation, haplotype inference, identification of haplotypetagging SNP/MS and case–control haplotype analysis (100 000 iterated permutations) for combined data of MS and SNP genotypes.
RESULTS
Phased genomic screens using DNA pools
In the first-stage screening, among the 23 465 MS markers subjected to
genotyping, 1414 (6.0%) were considered to be statistically significant
(Po0.05) and were subjected to the next screening stage. In the
second-stage screening, among the 1414 markers tested, 158 satisfied
Novel susceptibility loci for AN
K Nakabayashi et al
533
our selection criteria: statistical significance (Po0.1) and the same
direction of allelic effect on AN susceptibility between the first and the
second screening results. In the third screening, among the 158
markers tested, 16 satisfied our selection criteria: statistical significance
(Po0.1) and the same direction of allelic effect throughout the MS
screening stages. We determined a significance threshold to control
false-positive rates (nominal a¼0.05) in the first stage of MS screening. In the second and third stages, considering that our sample sizes
of cases and controls are not large, we set the significance thresholds
(nominal a¼0.1) to maintain the overall statistical power of the
screening.
To determine the definite allele frequencies of the selected 16 MS
markers, we performed individual genotyping on all the AN cases
(n¼320) and controls (n¼341) used in the first to third screenings.
Of the 16 markers, 10 showed a statistically significant difference by
Fisher’s exact test in the comparison between controls and the AN
cohort (Table 1). After correction of multiple tests with the number of
alleles, 7 of 10 markers remained statistically significant (Pco0.05).
SNP association analysis to narrow down the regions responsible
for AN susceptibility
From the 10 MS markers that were found to be associated with AN,
we selected 7 (shown in bold in Table 1), on the basis of the gene
content around each marker, as targets for SNP association analysis to
narrow down disease susceptibility loci. We primarily selected a
collection of evenly spaced SNPs (11.1-kb interval on average) within
a several 100-kb region surrounding each candidate MS marker,
although it should be noted that intragenic SNPs were preferentially
selected from the loci of 1q41 (spermatogenesis associated 17
(SPATA17)), 5q15 (CDH18) and 18q22 (NETO1). The number of
SNPs subjected to association analysis and the size and nucleotide
positions of the corresponding genomic interval are listed for each
locus in Table 2.
In total, we performed genotyping for 333 SNPs on 331 AN cases
and 872 controls. Among the 251 SNPs that satisfied thresholds for the
Hardy–Weinberg equilibrium (exact test P40.01) and minor allele
frequency (45%), 24 showed a statistically significant association
(nominal Po0.05) with the AN cohort (Table 2). For each of the
seven loci analyzed, nominal P-values were corrected with 10 000
iterated permutations using Haploview 4.0. In all, 3 SNPs, all of which
are located on 1q41, out of the 24 SNPs remained statistically
significant (Pco0.05) (underlined in Table 2).
Subsequently, using Haploview 4.0, we inferred LD block structures
for each candidate chromosomal region, and performed a haplotype
association analysis (100 000 iterated permutations) for the constructed LD blocks. Significant association (Pco0.05) with the AN
cohort was detected in three of the six SNP haplotype blocks defined
in the 1q41 locus, and in one of the eight blocks defined in the 11q22
locus (Figure 1 and Table 3).
1q41
A total of 38 SNPs were selected for genotyping within a 337.3-kb
interval, including the AN-associated MS marker D1S0562i. Among
30 SNPs subjected to association analysis, 7 showed a statistically
significant association (Po0.05) with the AN cohort (Table 2).
All the seven SNPs were located at 3¢-downstream of the SPATA17
gene (Figure 1, left). SNP rs2048332 showed the most significant
association (allelic P¼0.00023) and was further analyzed under
different genetic models. Association analysis under a recessive
model for rs2048332 showed the lowest P-value of 0.00015 with the
CC genotype, indicating that the CC genotype of rs2048332 has a
susceptible effect on the AN phenotype in the Japanese (odds
ratio¼1.73, confidence interval, 1.30–2.31). Among the three ANassociated haplotype blocks (1q41-#4, #5 and #6 in Figure 1, left and
Table 3), 1q41-#5 that comprised two SNPs, namely rs1397178 and
rs2048332, spanning a 10.2-kb interval, was found to be most
significantly associated (Pc¼0.0039).
The AN-associated MS marker D1S0562i was located in block
1q41-#6, which comprised five SNPs spanning a 38.1-kb interval,
and was also associated with AN (Pc¼0.038). Four of the five SNPs
binned to haplotype block 1q41-#6, rs17691163, rs34418611,
rs1934216 and rs1538555, were in a relatively strong pairwise LD
(D¢¼0.72–0.75) with D1S0562i, whereas the most significant SNP,
rs2048332, in 1q41-#5 block was in modest LD (D¢¼0.46) with it.
The four SNPs and D1S0562i (rs17691163–D1S0562i–rs34418611–
rs1934216–rs1538555) were selected as tags captured through LD in
block 1q41-#6. These haplotype tags were subjected to an MS-SNP
haplotype-based association analysis: one haplotype (G-2-A-T-G),
tagged by an AN-associated risk allele of D1S0562i (Supplementary
Table 2), was significantly associated with AN (Pc¼0.0065) (Table 4).
Table 1 Ten microsatellite markers showing statistically significant differences in the individual genotyping
Allele frequencies
22
2m
No. of alleles
Positive allelesa
Control (N¼341)
AN (N¼320)
1p36
1q41
12
9
2
2
13.0%
12.4%
20.3%
18.1%
D5S0853i
5q15
17
1
12.5%
7.7%
D11S0389i
D11S0268i
11q13
11q22
16
16
1
2
6.5%
17.8%
10.8%
26.0%
D12S0245i
D12S0848i
12q14.1
12q23.2
3
11
2
1
50.0%
14.6%
G09961
D18S0019i
16q12
18q22
11
13
1
3
D19S0081i
19p13.3
13
1
MS Marker
Cytoband
D1S0016i
D1S0562i
P
Pc
P
Odds ratio
95% CI
0.00047
0.0043
0.0056
0.039
0.014
0.054
1.70
1.57
1.26–2.28
1.16–2.12
0.0047
0.080
0.29
0.59
0.41–0.85
0.038
0.00039
0.61
0.0062
0.85
0.0057
1.75
1.62
1.18–2.60
1.24–2.11
57.5%
21.1%
0.0067
0.0029
0.020
0.032
0.024
0.040
1.36
1.56
1.09–1.69
1.17–2.08
17.6%
36.8%
11.1%
26.5%
0.00075
0.000070
0.008
0.00091
0.0043
0.0071
0.58
0.61
0.43–0.80
0.48–0.77
14.2%
10.4%
0.044
0.57
0.017
0.70
0.50–0.98
Abbreviations: P, Fisher’s exact test P-value; Pc, P-value corrected for the number of alleles.
P- and Pc-values smaller than 0.05 are underlined.
aNumber of the alleles showing significant differences (Po0.05). When more than one positive allele were detected, the allele frequencies and the P and Pc-values of the positive allele showing
the most significant difference are listed.
Journal of Human Genetics
Journal of Human Genetics
rs3753499
rs106968
1p36
54 SNPs, 332.8 kb
chr.11 66568914
rs1477490
rs1477491
chr.18 68910075
chr.18 68910515
chr.18 68647991
chr.16 50958219
NETO1
TOX3
TOX3
TOX3
TOX3
CNTN5
CNTN5
CNTN5
SYT12
SYT12
UTS2
Gene
symbol
Intergenic
Intergenic
Intron
Intron
Intron
Intron
Intron
Intergenic
Intergenic
Intergenic
Intron
Intron
Intron
UTR 3
Intron
Intergenic
Intergenic
Intergenic
Intergenic
Intergenic
Intergenic
Intergenic
Intron
Intergenic
SNP Type
T
A
G
G
T
A
G
T
A
G
G
A
G
T
A
G
C
T
C
T
T
C
T
G
Allele
0.385
0.383
0.554
0.756
0.880
0.863
0.755
0.858
0.858
0.086
0.395
0.164
0.207
0.207
0.214
0.311
0.446
0.311
0.453
0.283
0.424
0.354
0.868
0.397
Control
(n¼872)
0.447
0.447
0.599
0.800
0.913
0.903
0.801
0.902
0.902
0.115
0.443
0.214
0.264
0.248
0.258
0.388
0.530
0.382
0.530
0.326
0.500
0.400
0.903
0.447
Case
(n¼331)
Frequency
0.92
0.84
0.04
0.72
0.016
0.30
0.73
0.23
0.49
0.29
0.49
0.68
0.74
0.82
0.39
0.71
0.61
0.75
0.60
0.16
0.41
0.29
0.59
0.017
HWE
P-value
0.0061
0.0044
0.047
0.022
0.020
0.0077
0.019
0.0040
0.0045
0.031
0.034
0.004
0.0027
0.029
0.019
0.00036
0.00023
0.00092
0.00074
0.039
0.00088
0.039
0.019
0.027
P-value
Abbreviations: P, chi-square test P-value; Pc, 10000 iterated permutation P-value.
Pc-values smaller than 0.05 are underlined.
For the 5q15 locus, 30 SNPs within a 521.8 kb interval (chr5: 19501188-20341733 ) were subjected to association analysis, and none of them showed a stastitically significant association (Po0.05) with AN.
rs2000728
chr.16 51059819
chr.16 51068809
rs2287144
rs3095620
27 SNPs, 1026.4 kb
chr18: 68441726–69468090
chr.16 51033470
chr.16 51059669
rs8062936
rs2052287
34 SNPs, 324.9 kb
chr16: 50819155–51144082
18q22
chr.16 51021032
chr.16 51022612
rs2270355
rs1111482
rs11647880
16q12
chr.11 99333169
chr.11 99335431
chr.11 99358383
rs12574821
rs1349782
rs6590474
56 SNPs, 699.8 kb
chr11: 98931758–99631604
chr.11 66573083
11q22
rs11227668
rs1538555
rs3741190
chr.1 216224643
rs2048332
rs12125437
20 SNPs, 441.3 kb
chr11: 66365567–66806868
chr.1 216176389
chr.1 216186564
rs7538903
rs1397178
chr1: 215887318–216224643
11q13
chr.1 216135516
chr.1 216166200
rs11117962
rs907057
chr.1 216116886
chr.1 216125221
chr.1 7834024
chr.1 7897131
Location
(NCBI Build 36.1)
1q41
30 SNPs, 337.3 kb
chr1: 7695967–8028788
SNP ID
analysis, interval size, and nucleotide
positions (NCBI Build 36.1)
Cytoband,
# of SNPs subjected to association
Table 2 SNP allelic association with AN
0.137
0.103
0.667
0.397
0.376
0.171
0.347
0.094
0.102
0.512
0.678
0.137
0.100
0.330
0.243
0.039
0.009
0.062
0.022
0.643
0.106
0.710
0.375
0.480
Pc
1.29
1.30
1.20
1.29
1.44
1.48
1.30
1.52
1.52
1.38
1.22
1.39
1.38
1.26
1.28
1.40
1.40
1.37
1.36
1.23
1.36
1.21
1.42
1.23
Odds
ratio
1.07B1.55
1.09B1.56
1.00B1.44
1.04B1.61
1.06B1.96
1.11B1.99
1.04B1.62
1.14B2.04
1.14B2.03
1.03B1.84
1.01B1.46
1.11B1.75
1.12B1.69
1.02B1.56
1.04B1.58
1.16B1.69
1.17B1.68
1.14B1.65
1.14B1.63
1.01B1.49
1.13B1.62
1.01B1.46
1.06B1.90
1.02B1.47
95% CI
Novel susceptibility loci for AN
K Nakabayashi et al
534
Novel susceptibility loci for AN
K Nakabayashi et al
535
11q22
1q41
SPATA17
BC040896
#5
CNTN5
#5
#6
4
4
3
3
-log10P
-log10P
#4
2
1
2
1
0
0
100 kb
100 kb
rs6590633
rs7947224
rs737582
D11S0268i
rs1901860
rs7129985
rs6590474
rs1349782
rs12574821
rs2585885
rs1538555
rs1934216
rs34418611
D1S0562i
rs17691163
rs12125437
rs2048332
rs1397178
rs11585818
rs11576595
rs7538903
rs907057
rs11117962
rs958431
rs1930302
Figure 1 Single-nucleotide polymorphism (SNP) and haplotype association analyses for the 1q41 (left) and the 11q22 (right) loci. For each locus, the
linkage disequilibrium (LD) plot (top), resided gene(s) (middle) and the P-value plot (bottom) are shown. In LD plots, the extent of LD between two SNPs is
shown by the standard color scheme (D¢/LOD) of Haploview 4.0. In P-value plots, closed dots show the minus log P-value (y axis) and the physical location
(x axis) of SNPs. Minus log P-values were calculated by w2 tests for the genotyping data of anorexia nervosa (AN) cases (n¼331) and controls (n¼872). The
horizontal dashed line corresponds to the P-value of 0.05. Black and red horizontal bars above the P-value plots correspond to the LD blocks defined by the
confidence intervals method (Haploview 4.0). LD blocks showing statistical significance in the haplotype association analysis (Pco0.05 in Table 3) are
shown by red bars. The rs numbers of the SNPs showing statistical significance (Po0.05 in Table 1), the SNPs binned to the AN-associated LD blocks and
the SNPs at the ends of the genomic interval are shown underneath the P-value plot. The positions of AN-associated MS markers (D1S0562i and
D11S0268i) are shown by blue rectangles. The SPATA17 gene and an uncharacterized mRNA sequence, BC040896, which are transcribed from left to right
orientation, are mapped in the 337.3-kb interval between SNPs rs1930302 and rs1538555 on 1q41. For the 11q22 locus, the 474.6-kb region between
SNPs rs2585885 and rs6590633, which are located in intron 2 and intron 16 of the CNTN5 gene (NM_014361), respectively, is shown.
Table 3 LD blocks in 1q41 and 11q22 loci and haplotype association analysis
# of positive
Frequency Frequency
(controls)
(AN)
Pc (permutation
P-value)
End
1q41
#1
85.1
rs1930302
rs11578064 SPATA17
6
3
0
0.676
0.676
0.99
1
#2
#3
29.8
32.9
rs6604558
rs10495075 SPATA17
rs11578620 rs1510262
SPATA17
4
5
3
4
0
0
0.535
0.586
0.559
0.570
0.29
0.48
1
1
#4
#5
49.9
10.2
rs958431
rs1397178
6
2
7
3
2
2
0.520
0.548
0.447
0.461
0.0014
0.00010
0.031
0.0039
#6
38.1
rs12125437 rs1538555
5
4
2
0.139
0.191
0.0019
0.038
11q22
#1
48.0
rs2585885
rs1145408
CNTN5
5
5
0
0.487
0.508
0.37
1
#2
#3
8.6
22.7
rs4754649
rs3824932
rs11221713 CNTN5
rs10894179 CNTN5
2
3
3
3
1
0
0.037
0.654
0.020
0.623
0.032
0.16
0.56
0.99
#4
#5
6.5
20.2
rs11221996 rs1530997
rs6590474
rs7947224
CNTN5
CNTN5
2
5
3
3
0
2
0.445
0.730
0.431
0.655
0.55
0.00030
1
0.0078
#6
#7
32.0
45.9
rs770569
rs4754665
CNTN5
rs10750469 rs12806530 CNTN5
5
5
5
6
0
0
0.482
0.409
0.447
0.397
0.13
0.61
1
1
#8
7.3
rs7115626
2
3
0
0.798
0.793
0.81
1
Gene
rs11585818 SPATA17/BC040896
rs2048332
BC040896
rs6590633
CNTN5
# of
# of SNPs haplotypes
haplotypes
(Po0.05)
Locus &
block#
Size (kb) Start
P
Abbreviations: P, chi-square test P-value; Pc, 100 000 iterated permutation P-value.
P- and Pc-values smaller than 0.05 are underlined.
When no haplotype shows statistically significant association among multiple haplotypes inferred, the frequencies, P value, and Pc value of the major haplotype are shown.
When more than one haplotype show statistically significant association, the frequencies, P value, and Pc value of the most significantly associated haplotype are shown.
Journal of Human Genetics
Novel susceptibility loci for AN
K Nakabayashi et al
536
Table 4 Case-control association analysis for MS-SNP haplotypes
Frequencies
Haplotype
control
case
Pc
rs17691163-D1S0562i-rs34418611-rs1934216-rs1538555 (1q41-#6)
G-6-A-T-A
G-7-A-T-A
0.279
0.141
0.218
0.127
0.025
0.51
G-2-A-T-G
G-4-G-A-G
0.098
0.111
0.153
0.105
0.0065
0.76
G-5-A-T-A
G-4-A-T-A
0.102
0.066
0.080
0.073
0.20
0.68
A-6-A-T-A
0.051
0.052
0.89
screening, an additional 692 control individuals enrolled in the later
stages (161 individuals in the third stage of MS screening and all of 692
individuals in the SNP association analysis) consisted of male and
female individuals. To assess whether the detected association of the
1q41 and 11q22 loci to AN was inflated by this partial mismatch in
gender between case and control populations, we conducted a stratified
analysis for 7 SNPs on 1q41 and for 3 SNPs on 11q22 (Table 2) that
showed a significant association with AN. When control group 1
(180 females) and the AN cohort were subjected to association analysis,
all 10 SNPs were detected to be associated (Po0.05) with AN
(Supplementary Table 4). These results assure that the association of
the 1q41 and 11q22 loci with AN detected in this study is not because of
inflation caused by the inclusion of male individuals in the control
population.
rs6590474-D11S0268i-rs737582-rs7947224 (11q22-#5)
C-2-G-T
C-3-G-T
0.479
0.288
0.408
0.242
0.023
0.105
A-4-G-T
C-5-A-C
0.124
0.050
0.212
0.062
0.00003
0.377
Abbreviation: Pc (permutation P-value, n¼100 000).
Haplotypes whose control frequency is 4¼0.05 are shown.
Pc-values smaller than 0.05 and the corresponding haplotypes are underlined.
This association was comparable with those observed in the SNPhaplotype analysis in two haplotype blocks 1q41-#5 (Pc¼0.0039) and
1q41-#6 (Pc¼0.038) in terms of statistical significance.
11q22
A total of 66 SNPs were selected from a 699.8-kb interval surrounding
the AN-associated MS marker D11S0268i. Among the 56 SNPs
subjected to association analysis, 3 (rs12574821, rs1349782 and
rs6590474) showed a statistically significant association (Po0.05)
with the AN cohort (Table 2). These associated SNPs were found to
be located in the eighth intron of the CNTN5 gene (GenBank
accession no. NM_014361). Although these three SNPs did not hold
statistical significance after multiple-testing correction by permutation
tests, a haplotype composed of five SNPs (rs6590474, rs7129985,
rs1901860, rs737582 and rs7947224) spanning a 20.2-kb interval
showed a statistically significant association with AN (Pc¼0.0082) in
the haplotype association analysis (11q22-#5 in Figure 1, right and
Table 3). Exon 9 of CNTN5 was included in the 20.2-kb interval.
The AN-associated MS marker, D11S0268i, was located in the ANassociated 11q22-#5 block. In this block, D11S0268i was in a strong
pairwise LD (D¢¼0.81–0.90) with each of the five SNPs binned to this
block. As three SNPs (rs6590474, rs737582 and rs7947224) and
D11S0268i were selected as tags captured through LD in the 11q22#5 block, we further conducted an MS-SNP haplotype-based association study within the block using these four markers (rs6590474,
D11S0268i, rs737582 and rs7947224). As shown in Table 4, the A-4-GT haplotype was overrepresented in AN cases with the greatest
statistical significance (Pc¼0.00003). This MS-SNP haplotype contained both of the significantly associated risk alleles (A allele and 4) at
SNP rs6590474 (Table 2) and D11S0268i (Supplementary Table 3).
Assessment of possible gender effects in the detected association of
1q41 and 11q22 with AN
Owing to the limited number of female individuals whose age matches
with the average age of the AN cases in our control samples, we adopted
a population-based control group to search for AN susceptibility loci.
Therefore, although 180 female controls (control group 1, average age:
34.5 years) were genotyped in the first and second stages of MS
Journal of Human Genetics
Association analysis for a BN cohort
To assess whether the genomic intervals identified to be associated with
AN in this study are also involved in the genetic etiology of BN, we
conducted an SNP association analysis for BN cases and controls.
The 7 SNPs from the 1q41 locus and the 3 SNPs from the 11q22 locus,
which showed a statistically significant association (Po0.05) with AN
before multiple-testing correction, were subjected to SNP genotyping
on the cohort of 125 BN cases. None of the 10 SNPs showed a
statistically significant association with BN (data not shown).
DISCUSSION
We have completed a genome-wide association analysis for AN
using 23 465 MS markers. To our knowledge, this is the first GWAS
performed for EDs. Among the 10 candidate loci we identified, 9 are
reported to be associated with AN for the first time in this study. Only
one locus, D1S0016i on 1p36, overlaps with the chromosomal region
of 1p33-p36 that has already been reported to show significant linkage
to AN.12
Through an SNP association analysis for the seven selected candidate regions to narrow down genomic intervals involved with AN
susceptibility, we tentatively identified a 10.2-kb genomic interval (the
haplotype block 1q41-#5) located at 3¢-downstream of the SPATA17
gene as a region associated with AN. The MS-SNP haplotype-based
association analysis also indicated the association of haplotype block
1q41-#6 with AN with a similar statistical significance. SPATA17
encodes a 361 amino-acid protein that contains three highly conserved
IQ motifs and is strongly expressed in the testis.30 It is unknown
whether the SPATA17 protein has any physiological roles in neuronal
tissues. It should be noted that the 10.2-kb critical interval coincides
with the exon–intron structure of the uncharacterized mRNA
sequence BC040896, which is derived from a cDNA library made
from brain (adult medulla) RNA.
Another genomic region identified to be associated with AN in this
study is a 20.2-kb interval (the haplotype block 11q22-#5), spanning
from the eighth to the ninth intron of the CNTN5 gene on 11q22.
Furthermore, we found that one MS-SNP haplotype (A-4-G-T),
which includes two AN-associated risk alleles at SNP rs6590474 and
MS D11S0268i, was significantly overrepresented in AN cases. CNTN5
encodes a member of the contactin family known to function during
the formation of neuronal interactions. It is reported that the mouse
line deficient of Cntn1, another member of the contactin family,
exhibits an ataxic and anorectic phenotype.31,32 In human adult
tissues examined using northern blot analysis, CNTN5 has been
shown to be predominantly expressed in the brain and thyroid.33
In various regions of an adult brain examined, the gene was found
to be expressed with highest levels in the occipital lobe and amygdala,
Novel susceptibility loci for AN
K Nakabayashi et al
537
followed by the cerebral cortex, frontal lobe, thalamus and temporal
lobe.33 Although neuronal activity in the auditory system is reported
to be impaired in the mouse line deficient for Cntn5, no anorexic
phenotype has been described.34
Although causative SNPs are not yet determined, we have successfully mapped genetic association with AN to at least two genomic
regions on 1q41 and 11q22 and narrowed down an AN-associated
genomic interval for each locus by haplotype association analysis.
Further replication analysis using independent patient/control populations for AN-associated SNPs and functional analyses for the genes
or for particular genomic regions in these loci will better clarify the
impact of these SNPs/genes in the genetic etiology of AN. It should
also be noted that additional common variants are likely to have roles
in the development of AN because this study was not well powered to
detect susceptible loci with relatively small genetic risks. Additional
gender/age-matched cohorts consisting of much larger numbers
of cases and controls need to be used to improve statistical power
in MS-based genome-wide association analysis.
ACKNOWLEDGEMENTS
This study was performed under the management of the Japan Biological
Informatics Consortium (JBIC) and was supported by grants from the New
Energy and Industrial Technology Development Organization (NEDO). This
study was also supported by Grant-in-Aid for Scientific Research on Priority
Areas from MEXT, and by Grant-in-Aid for Young Scientists (B) from JSPS.
We thank Karin Ohki for her technical assistance.
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Supplementary Information accompanies the paper on Journal of Human Genetics website (http://www.nature.com/jhg)
Journal of Human Genetics