Exome Sequencing Analysis of
Candidate Genes
VEO-IBD
Judith Kelsen, MD
12/14/2013
The Children’s Hospital of Philadelphia
Objectives
• Introduction to very early onset IBD
• Pathogenesis of VEO-IBD
• Rationale for targeted genomic approach in
VEO-IBD
• WES: workflow and outcomes
• Future Plans
Very Early-Onset IBD: The Challenge
• Diagnosed ≤5 years of age
• Frequently different phenotype and more
severe disease presentation
• Often unresponsive to conventional therapy
• No standard guidelines on evaluation and
treatment
IBD Susceptibility
Adapted from Inflamm Bowel Dis 2010:16;152
Genomics and VEO-IBD
• GWAS approach
– Primarily included adults and children>10
– Frequently misses rare variants, MAF <5%
• Role of primary immunodeficiency
• Are we missing important pathways in this
cohort?
Whole Exome Sequencing
• Exons:
– Short functionally
important DNA sequences
– Regions of DNA translated
into protein and the
untranslated region
flanking them
• Protein coding regions85% of disease-causing
mutations
Hypothesis
• Rare or novel variants are enriched in patients
with severe VEO-IBD
– Includes genes associated with primary
immunodeficiencies
– Variants may contribute to the development of
disease
Methods
Subjects: Patients with IBD ≤5 and parents
Controls: 1000 Genomes Project, CHOP cohort >400
DNA extracted from whole blood
Exome capture: Agilent SureSelect V4
Sequencing: Illumina HiSeq platform at an average coverage
depth of 100X
Alignment to human genome GRCh37 followed by post processing
and variant calling
Variant filter: MAF <5%
Variants likely to alter protein function, (i.e missense and loss of
function mutations) were kept for subsequent analysis
Primary focus of analysis was on known IBD risk loci and genes
associated with primary immunodeficiences and related pathways
(>870 genes)
Sequencing Work Flow
Alignment
NovoAlign
(GRCh37)
BAM Postprocessing
GATK/Picard
Variant
Calling
GATK
Annotation
SnpEff/Ensembl
Custom
Analysis
De novo
Gene Burden
Compound
Het
Autosomal
Recessive
Autosomal
Dominant
Sample Filter
Cases
•1 – 175,414 var
•2 – 229,750 var
Annotation/Filter
MAF filter (1kg, EVS)
Cases
•1 – 2,125 var
•2 – 2,241 var
Filter 3
Gene list
Cases
•1 – 43,493 var
•2 – 60,962 var
Filter 2
Non-Synonomous or
High/Medium Impact
Cases
•1 – 130 var
•2 – 150 var
Results
• Cohort: 44 samples
• Probands: 28 (ages 8 weeks-4 yrs, 21 <2 years old)
• Parents: 16 (trios: 8)
• IBD risk loci and genes associated with
primary immunodeficiency
• 14,060 coding exons totaling more than 2.5 Mbp
• 87.7% of coding exons were fully covered at
more than 20x
Identified Rare Variants in Candidate Genes
Chr
Position
dbSNP ID
Ref
Alt
DNA Alteration
Gene
Immunodeficiency
1
6
16
4
16
1
114699
31727677
28950051
151793903
50745926
183532364
rs114666761
Novel
rs43763945
rs72719663
rs2066844
rs35012521
C
G
G
A
C
T
G
C
A
G
T
A
p.T43R
p.S554T
p.r514h
p. T1724V
p. R702W
p.N419I
16
11
3293880
117869853
rs11466045
rs143538561
A
C
G
T
p.1591T
p.R412W
TNFRSF18
MSH5
CD19
LRBA
NOD2
NCF2
CYBA
MEFV
IL10RA
19
17945695
17953949
rs5577834
rs3213409
C
G
T
C
p.v7221
JAK3
CVID
CVID
CVID
CVID
IBD risk loci
CGD
CGD
FMF
IL-10R (see IL10)
Primary
immunodeficiency;
Multiple
immunodeficiency
pathways
Variants of Immunodeficiency
Pathways: Case 1
• Male, presented with diarrhea, folliculitis and
FTT as infant
• Diagnosed at 6 months with ileocolonic CD,
refractory to medical therapy
• Immunology evaluation: unremarkable
• Non-synonymous variant: rare heterozygous
IL-10RA variant (rs143538561; c.1234C>T,
p.R412W)
Case 1: IL-10R Pathway
• IL-10 blunts excessive immune
response
• Secreted by wide variety of cells
and inhibits the secretion of proinflammatory cytokines
• Receptor IL10R has two subunits:
– Alpha (A / 1)- required for
IL10
– Beta (B / 2) -Required for
IL10, IL‐22, IL‐26
• Acts through JAK1, TYK2, and
STAT3 (all identified in IBD GWAS)
• Defects in IL10R A/B and IL10
have been identified
Variants of Immunodeficiency
Pathways: Case 2
• Female presented as infant: diarrhea,
anemia
• Required transfusion and ICU
• Diagnosed with gastric, colonic VEOIBD
•
NCF2: rare missense variant
(rs35012521; c.1256A>T, p.N419I)
was detected in heterozygosity
• DHR: Dihydrorhodamine flow
showed abnormal burst
• Mother, with same variant, newly
diagnosed with CD
Case 2: NADPH Oxidase pathway
• Chronic Granulomatous disease (CGD)
– Genetic immunodeficiency defects in NADPH oxidase
complex genes
– Phagocytes are unable to kill certain bacteria and fungi as
a result of reduced production of superoxide and hydrogen
peroxide reactive oxygen species (ROS)
• NCF2: Component of the NADPH complex
Future Studies
• Functional analysis is ongoing
• Further variants are being analyzed and these
involve other primary immunodeficiency
pathways
• Larger cohort project utilizing WES is
underway
Conclusions
• Candidate causative mutations in VEO-IBD can
be identified by exome sequencing
• Likely some patients harbor mutations in
several genes in the same or different
pathways
• Additional causative variants may occur in
genes not previously associated with IBD
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Thank You
Marcella Devoto
Robert Baldassano
Gary Wu
Jim Lewis
Mahdi Sarmady
Ariella Sasson
Petar Mamula
David Piccoli
Lindsey Albenberg
Christopher Moran
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Wei Liu
Hakon Hakonarson
Helen Pauly-Hubbard
Andrew Grossman
Kernika Gupta