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Exome Sequencing Analysis of Candidate Genes VEO-IBD

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

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Exome Sequencing Analysis of Candidate Genes VEO-IBD

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  1. Exome Sequencing Analysis of Candidate Genes VEO-IBD Judith Kelsen, MD 12/14/2013 The Children’s Hospital of Philadelphia

  2. 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

  3. 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

  4. IBD Susceptibility Adapted from Inflamm Bowel Dis 2010:16;152

  5. 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?

  6. Whole Exome Sequencing • Exons: • Short functionally important DNA sequences • Regions of DNA translated into protein and the untranslated region flanking them • Protein coding regions-85% of disease-causing mutations

  7. 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

  8. 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)

  9. Sequencing Work Flow BAM Post-processing GATK/Picard Variant Calling GATK AlignmentNovoAlign(GRCh37) Annotation SnpEff/Ensembl Custom Analysis Gene Burden De novo Compound Het Autosomal Recessive Autosomal Dominant

  10. Sample Filter Annotation/Filter MAF filter (1kg, EVS) Cases • 1 – 43,493 var • 2 – 60,962 var Filter 2 Non-Synonomous or High/Medium Impact Cases • 1 – 2,125 var • 2 – 2,241 var Filter 3 Gene list Cases • 1 – 130 var • 2 – 150 var Cases • 1 – 175,414 var • 2 – 229,750 var

  11. 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

  12. Identified Rare Variants in Candidate Genes

  13. 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)

  14. Case 1: IL-10R Pathway • IL-10 blunts excessive immune response • Secreted by wide variety of cells and inhibits the secretion of pro-inflammatory 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

  15. Variants of Immunodeficiency Pathways: Case 2 • Female presented as infant: diarrhea, anemia • Required transfusion and ICU • Diagnosed with gastric, colonic VEO-IBD • 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

  16. 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

  17. 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

  18. 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

  19. Thank You • Marcella Devoto • Robert Baldassano • Gary Wu • Jim Lewis • Mahdi Sarmady • Ariella Sasson • Petar Mamula • David Piccoli • Lindsey Albenberg • Christopher Moran • Wei Liu • Hakon Hakonarson • Helen Pauly-Hubbard • Andrew Grossman • Kernika Gupta

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