Evaluation of the genetic
impact on inflammatory bowel
disease
Natalie Bibb
Trainee Project
KGC
Presentation aims
• Overview of inflammatory bowel disease (IBD)
• Purpose of project
• Genome-wide association studies (GWAS) and
identification of IBD susceptibility genes
• Pathogenesis of IBD
• Assay design for detection of common variants
• Results
• Conclusions and further work
Overview of IBD
• Complex polygenic disorders influenced by genetic, environmental
and immunological factors
• Includes Crohn’s disease (CD) and ulcerative colitis (UC) both
characterised by chronic relapsing intestinal inflammation
• Peak age of onset 2nd – 4th decade with European prevalence of
100-150 per 100,000
Comparison of the clinical features of CD and UC
UC
CD
Extra-intestinal manifestations
Common
Common
Haematochesia
Common
Rare
Passage of mucus/pus
Common
Rare
Fistulas, skip lesions and
perianal disease
No
Yes
Transmural mucosal
inflammation
No
Yes
Clinical features
Comparison of the clinical features of CD and UC
Purpose of project
•
Grant proposal - Multidisciplinary study of 4000 IBD
patients and case controls
–
–
–
Immunological profiling - dendritic cell function
Microbiological profiling - quantify faecal and gut microbiota
Genetic profiling - Identify variants most significantly associated
with IBD
•
Current treatments follow non-targeted approach steroids and surgery can have adverse side effects
•
To assess if combined or individual factors indicate
requirement for specific treatments
•
To provide more effective and individualised treatments
for IBD
Genetic factors of IBD
IBD results from a defective immune response to intestinal
microbes in a genetically susceptible host
•
Evidence for genetic contribution
–
Familial aggregation - higher incidence of IBD in
individuals with a positive family history
–
Higher disease concordance in monozygotic twins
than dizygotic twins
–
These observations are more evident in CD than UC
–
Racial and ethnic differences - specific variants
observed more frequently in Ashkenazi Jews
–
Ethnic heterogeneity - different genetic variants
observed in different ethnic populations
Genome wide association studies
(GWAS)
• GWAS identified significant linkage disequilibrium in variant allele
frequencies between IBD cases and controls
• Associating 9 genomic regions known as IBD locus with susceptibility
to disease
• Implicated 30+ genes many with roles in intestinal immunity
– CD and UC share some genetic
susceptibilty loci; which is evident from
the co-occurrence in IBD families; but
differ at others
– IBD variants are present in the
normal healthy population so are not
sufficient or necessary to cause
disease
NOD2
• NOD2 (nucleotide-binding oligomerisation domain 2)
consists of 2 CARD domains, a central NBD and an LRR
domain
• LRR domain recognizes bacterial muramyl dipeptide
(MDP) which regulates NF activation and the
production of pro-inflammatory cytokines
• 3 variants in the LRR domain of NOD2 show significant
association with CD, but no association with UC
Role of NOD2 in IBD pathogenesis
• Exact
mechanism unclear
- proposed loss or gain of function
• Carriers of one NOD2 high risk
variants have a 2-4 fold increase
risk of developing CD
• Carriers of two NOD2 high risk
variants have a 20-40 fold
increased risk
• NOD2 high risk variant carriers
also show association with ileal
disease
IL23R
• IL23R (interleukin 23 receptor) encodes
a subunit of the receptor for the proinflammatory cytokine IL23
• IL23 binds to receptor (IL23R/IL-12RB1)
inducing a pro-inflammatory response
• A rare variant c.1442G>A (p.Arg381Gln)
confers a strong protective effect against
CD and UC
• The mechanism by which this variant
confers a protective effect is not yet
understood
ATG16L1
• ATG16L1 has a role in the autophagy pathway
• The variant c.898A>G, p.Thr300Ala is associated with CD and not UC
• Two recent studies have shown evidence of NOD2 and ATG16L1
functioning in a single pathway
Assay design
• Ethics approval for testing 4000 patients not yet received
• Genotype 25 healthy controls and 25 IBD patients for
variants with most significant association
• Multiplex PCR - detects 3 NOD2 variants and single
variant in IL23R
• Touchdown PCR method
• Allele specific fluorescent primer design using primer 3
program
• One fluorescent tagged common primer and two
untagged allele specific primers per variant
Results
• Identification of
IL23R variant in
healthy control
supports its reduced
frequency in IBD
IBD (/25)
No variants
identified
21/25 (84%)
22/25 (88%)
c.2104 het
1/25 (4%)
0
c.2722 het
0
1/25 (4%)
c.3020 het
1/25 (4%)
0
c.3020hom
1/25 (4%)
0
c.2104/c.3020
compound het
0
1/25 (4%)
c.2722/c.3020
compound het
0
1/25 (4%)
c.3020/c.1442
1/25 (4%)
0
Genotype
• NOD2 variants
only account for
~20% genetic
contribution to IBD
• Presence of
variants in healthy
controls is expected
NC (/25)
NOD2
variants
NOD2/
IL23R
variants
Electropherogram analysis
Electropherograms display a peak for each of the 3 NOD2 and single IL23R loci
NOD2 loci
c.2104C>T c.2722G>C c.3020insC
A tube NOD2 WT and
IL23R variant primers
B tube NOD2 variant
and IL23R WT primers
IL23R locus
c.1422G>A
NOD2 c.2104C>T
(p.Arg702Trp) heterozygote
NOD2 c.3020insC
(p.Leu1007fs) heterozygote
NOD2 c.3020insC (p.Leu1007fs)/IL23R c.1442G>A
(p.Arg381Gln) compound heterozygote
Conclusion
• A multiplex PCR assay has been designed to
detect IBD associated variants
• No significant associations can be concluded from
this small pilot cohort
• Multidisciplinary approach may provide better
understanding of disease type, severity and
requirement for treatment
• Pharmacogenomics - Genotyping could contribute
to providing targeted therapies and treatment
according to variant carrier status
Further work
• Development of high throughput variant
detection using pyrosequencing
• Assay expansion - incorporate further
known variants and include other genetic
associations as identified
• Testing of 4000 IBD patients once ethics
approval received
Acknowledgments
• Stewart Payne
• Lindsey Sutherland
• Marta Pereira
• Dr Alisa Hart (St Mark’s Hospital, London)
• David Walker (St Mary’s Hospital, London)
• Dr Andrew Milestone (Division of Medicine,
Imperial College London)