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Genome-wide association study implicates immune activation of
multiple integrin genes in inflammatory bowel disease
Citation for published version:
de Lange, KM, Moutsianas, L, Lee, JC, Lamb, CA, Luo, Y, Kennedy, NA, Jostins, L, Rice, DL, Gutierrez-
Achury, J, Ji, S-G, Heap, G, Nimmo, ER, Edwards, C, Henderson, P, Mowatt, C, Sanderson, J, Satsangi, J,
Simmons, A, Wilson, DC, Tremelling, M, Hart, A, Matthew, CG, Newman, WG, Parkes, M, Lees, CW, Uhlig,
H, Hawkey, C, Prescott, NJ, Ahmad, T, Mansfield, JC, Anderson, CA & Barrett, JC 2017, 'Genome-wide
association study implicates immune activation of multiple integrin genes in inflammatory bowel disease',
Nature Genetics, vol. 49, no. 2, pp. 256–261. https://doi.org/10.1038/ng.3760
Digital Object Identifier (DOI):
10.1038/ng.3760
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Nature Genetics
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Download date: 26. Aug. 2022
1
Genome-wide association study implicates immune activation of
1
multiple integrin genes in inflammatory bowel disease
2
Katrina M. de Lange*
1
, Loukas Moutsianas*
1
, James C. Lee*
2
, Christopher A. Lamb
3
, Yang Luo
1,4,5
,
3
Nicholas A. Kennedy
6,7
, Luke Jostins
8,9
, Daniel L. Rice
1
, Javier Gutierrez-Achury
1
, Sun-Gou Ji
1
,
4
Graham Heap
6,7
, Elaine R. Nimmo
10
, Cathryn Edwards
11
, Paul Henderson
12,13
, Craig Mowat
14
,
5
Jeremy Sanderson
15
, Jack Satsangi
10
, Alison Simmons
16,17
, David C. Wilson
18,19
, Mark Tremelling
20
,
6
Ailsa Hart
21
, Christopher G. Mathew
22,23
, William G. Newman
24,25
, Miles Parkes
2
, Charlie W. Lees
10
,
7
Holm Uhlig
26
, Chris Hawkey
27
, Natalie J. Prescott
22
, Tariq Ahmad
6,7
, John C. Mansfield
28
, Carl A.
8
Anderson
✝1
, Jeffrey C. Barrett
✝1
9
[1] Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, UK
10
[2] Inflammatory Bowel Disease Research Group, Addenbrooke's Hospital, Cambridge, UK
11
[3] Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne
12
[4] Division of Genetics and Rheumatology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA
13
[5] Program in Medical and Population Genetics, Broad Institute of Harvard and MIT, Cambridge, MA, USA
14
[6] Precision Medicine Exeter, University of Exeter, Exeter, UK
15
[7] IBD Pharmacogenetics, Royal Devon and Exeter Foundation Trust, Exeter, UK
16
[8] Wellcome Trust Centre for Human Genetics, University of Oxford, Headington, UK
17
[9] Christ Church, University of Oxford, St Aldates, UK
18
[10] Gastrointestinal Unit, Wester General Hospital University of Edinburgh, Edinburgh, UK
19
[11] Department of Gastroenterology, Torbay Hospital, Torbay, Devon, UK
20
[12] Department of Child Life and Health, University of Edinburgh, Edinburgh, UK
21
[13] Department of Paediatric Gastroenterology and Nutrition, Royal Hospital for Sick Children,Edinburgh, UK
22
[14] Department of Medicine, Ninewells Hospital and Medical School, Dundee, UK
23
[15] Guy’s & St Thomas’ NHS Foundation Trust, St Thomas’ Hospital, Department of Gastroenterology, London, UK
24
[16] Translational Gastroenterology Unit, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, UK
25
[17] Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK
26
[18] Paediatric Gastroenterology and Nutrition, Royal Hospital for Sick Children, Edinburgh, UK
27
[19] Child Life and Health, University of Edinburgh, Edinburgh, Scotland, UK
28
[20] Gastroenterology & General Medicine, Norfolk and Norwich University Hospital, Norwich, UK
29
[21] Department of Medicine, St Mark's Hospital, Harrow, Middlesex, UK
30
[22] Department of Medical and Molecular Genetics, Faculty of Life Science and Medicine, King's College London, Guy's
31
Hospital, London, UK
32
[23] Sydney Brenner Institute for Molecular Bioscience, Faculty of Health Sciences, University of Witwatersrand, South Africa.
33
[24] Genetic Medicine, Manchester Academic Health Science Centre, Manchester, UK
34
[25] The Manchester Centre for Genomic Medicine, University of Manchester, Manchester, UK
35
[26] Translational Gastroenterology Unit and the Department of Paediatrics, University of Oxford, Oxford, United Kingdom
36
[27] Nottingham Digestive Diseases Centre, Queens Medical Centre, Nottingham, UK
37
[28] Institute of Human Genetics, Newcastle University, Newcastle upon Tyne, UK
38
* These authors contributed equally to this work
39
✝ These authors jointly supervised this work
40
Correspondence should be addressed to Jeffrey C. Barrett (jb26@sanger.ac.uk) and Carl A. Anderson (ca3@sanger.ac.uk)
41
2
Genetic association studies have identified 215 risk loci for inflammatory bowel disease
1–8
,
42
which have revealed fundamental aspects of its molecular biology. We performed a genome-
43
wide association study of 25,305 individuals, and meta-analyzed with published summary
44
statistics, yielding a total sample size of 59,957 subjects. We identified 25 new loci, three of
45
which contain integrin genes that encode proteins in pathways identified as important
46
therapeutic targets in inflammatory bowel disease. The associated variants are correlated
47
with expression changes in response to immune stimulus at two of these genes (ITGA4,
48
ITGB8) and at previously implicated loci (ITGAL, ICAM1). In all four cases, the expression
49
increasing allele also increases disease risk. We also identified likely causal missense
50
variants in the primary immune deficiency gene PLCG2 and a negative regulator of
51
inflammation, SLAMF8. Our results demonstrate that new common variant associations
52
continue to identify genes relevant to therapeutic target identification and prioritization.
53
Inflammatory bowel disease (IBD) is a chronic, debilitating, disorder of the gastrointestinal tract that
54
includes two common disease subtypes, Crohn’s disease and ulcerative colitis. Disease
55
pathogenesis is poorly understood but is likely driven by a dysregulated immune response to
56
unknown environmental triggers in genetically susceptible individuals. Treatment regimes often use
57
potent immunomodulators to achieve and maintain remission of symptoms. However, patients
58
commonly experience side effects, lose response to treatment, or develop complications of IBD, with
59
many ultimately requiring major abdominal surgery. Previous genome-wide association studies
60
(GWAS) and targeted follow-up using the Immunochip have been very successful at identifying
61
genetic risk loci for IBD, but increased biological understanding has not yet had a significant impact
62
on therapy for these disorders.
63
In order to further expand our understanding of the biology of these disorders we carried out a
64
GWAS of 12,160 IBD cases and 13,145 population controls of European ancestry that had not been
65
included in any genome-wide meta-analysis of IBD to date (Supplementary Table 1, Online
66
Methods). We imputed genotypes using a reference panel comprising whole genome sequences
67
from 4,686 IBD cases
9
and 6,285 publically available population controls
10,11
. Following quality
68
control (Online Methods) we tested 9.7 million sites for association. At the 232 IBD associated SNPs
69
in the latest meta-analysis by the International IBD Genetics Consortium
1
, 228 had effects in the
70
3
same direction in our data, 188 showed at least nominal evidence of replication (P<0.05) and none
71
showed significant evidence of heterogeneity of effect by Cochrane’s Q test. Among these replicated
72
loci was a genome-wide significant association on chromosome 10q25 that was only previously
73
significantly associated with Crohn’s disease in individuals of East Asian ancestry
3,7
, further
74
supporting near complete sharing of genetic risk loci across populations
1
. We meta-analyzed our
75
new GWAS data with previously published summary statistics from 12,882 IBD cases and 21,770
76
population controls imputed using the 1000 Genomes Project reference panel
1
(Supplementary
77
Figures 1-3, Supplementary Table 2). We observed inflation of the summary statistics (λ
GC
= 1.23
78
and 1.29 for Crohn’s and ulcerative colitis, respectively), but LD score regression demonstrated that
79
this was due to broad polygenic signal, rather than confounding population substructure (both
80
intercepts = 1.09, Online Methods).
81
We identified 25 new loci at genome-wide significance (Table 1). In order to identify causal variants,
82
genes and mechanisms, we performed a summary-statistic fine-mapping analysis on these loci, as
83
well as 40 previously discovered loci that were genome-wide significant in our data but where fine-
84
mapping had not yet been attempted
12
(Online Methods, Supplementary Table 3). In order to be
85
confident about fine-mapping inferences, we restricted subsequent analyses to 12 signals where we
86
had high quality imputed data for all relevant variants (Online Methods). At 6 of these 12 loci we
87
identified a single variant with >50% probability of being causal (Table 2, Supplementary Figures 4-
88
6). Among these were two loci where a single variant had >99% probability of being causal: a
89
missense variant predicted to affect protein function in SLAMF8, (rs34687326, p.Gly99Ser, Figure
90
1a), and an intronic variant in the key regulator of Th17 cell differentiation, RORC
13
. SLAMF8 is a
91
cell surface receptor that is expressed on activated myeloid cells and has been reported to
92
negatively regulate inflammatory responses by inhibiting their migration to sites of inflammation
14
93
and repressing their production of reactive oxygen species (ROS)
15
. This, together with the
94
observation that the risk-decreasing allele (MAF=0.1) is predicted to affect protein function
95
(CADD=32.0, 92
nd
percentile of missense variants)
16
, suggests further experiments evaluating a
96
possible gain-of-function mechanism may be worthwhile. RORC encodes RORγt, the master
97
transcriptional regulator of Th17 cells
13
and group 3 innate lymphoid cells
17
. Both of these cell types
98
play important roles in defence at mucosal surfaces, especially in the intestine, and have been
99
shown to contribute to the homeostasis between the intestinal immune system and gut
100
4
microbiota
18,19
, an equilibrium that is known to be lost in inflammatory bowel disease
20
.
101
Pharmacologic inhibition of RORγt has been shown to offer therapeutic benefit in mouse models of
102
intestinal inflammation, and reduces the frequency of Th17 cells isolated from primary intestinal
103
samples of IBD patients
21
.
104
In loci where fine-mapping was less clearly resolved, we searched for likely functional variants,
105
observing a missense variant (CADD=16.5, 50.2% probability of causality) in PLCG2. Furthermore,
106
after conditioning on this variant, we discovered a second, independent, likely functional
107
(CADD=34.0, 74.6% probability of causality) missense variant in the same gene (P=2x10
-8
). PLCG2
108
encodes a phospholipase enzyme that plays a critical role in regulating immune pathway
109
signalling
22
, and has previously been implicated in two autosomal dominant immune disorders.
110
Intragenic deletions in its autoinhibitory domain cause antibody deficiency and immune dysregulation
111
(familial cold autoinflammatory syndrome 3, MIM 614468)
23
and heterozygous missense variants
112
(e.g. p.Ser707Tyr) lead to a phenotype that includes intestinal inflammation
24
(Figure 1b).
113
A more general overlap between candidate IBD GWAS genes and Mendelian disorders of
114
inflammation and immunity has been previously observed in 163 loci discovered at that time
25
. We
115
replicated this finding in our list of 241 loci (p < 10
-6
, Supplementary Table 4), and observed that this
116
enrichment is even stronger when considering just the 26 loci where a gene can be confidently
117
implicated by fine-mapping to a coding variant or colocalisation with an eQTL (27% vs 3%, p=2x10
-
118
5
). In addition to PLCG2 we identified an association between Crohn’s disease and an intronic
119
variant in NCF4 (P=1.76x10
-8
). This gene encodes p40phox, a component of the NADPH-oxidase
120
system that is responsible for the oxidative burst in innate immune cells and which is a key
121
mechanism of killing phagocytosed bacteria. Rare pathogenic variants in NCF4 cause autosomal
122
recessive chronic granulomatous disease, characterized by Crohn’s disease-like intestinal
123
inflammation and defective ROS production in neutrophils
26
. Our associated variant, rs4821544, had
124
previously been suggestively associated with small bowel Crohn’s disease
27,28
, and when we
125
stratified patients by disease location we found that the effect was consistently stronger for small
126
bowel compared to large bowel disease (Supplementary Figure 7).
127
Among the remaining 21 novel loci we noted three that were within 150kb of integrin genes (ITGA4,
128
ITGAV and ITGB8), while a previously associated locus overlaps with a fourth integrin, ITGAL.
129