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Genetics and Rheumatology Lessons from Extreme Phenotypes & Subtle Genotypes Paul Wordsworth NIHR Oxford Musculoskeletal BRU Heberden Oration BSR AGM 2012 Paul Wordsworth Genetics and Rheumatology Lessons from Extreme Phenotypes & Subtle Genotypes Educational grants, speakers fees and consultation fees from Abbott, Pfizer and Merck. I am an investigator in the Novartis AIN 457 study in AS and a NASS trustee William Heberden (the elder) Heberden Society Heberden Round Heberden Oration MENTORS Matt Brown in the Gambia 1994 I DO TRY BUT…… “One of the advantages of being disorderly is that one is constantly making exciting discoveries.” AA Milne “Probably the only advantage!” CIW April 11th 2012 William Heberden (1710-1801) • 1710 born in London, son of an inn-keeper • 1724 St John’s College Cambridge, aged 14 years • 1730 Fellow St John’s • 1739 MD • 1746 FRCP • 1749 FRS • Extensive medical practice • “Commentaries on the History & Cure of Diseases” Heberden’s nodes • Best known to rheumatologists for Heberden’s nodes • Distinguished RA from gout • How he would have defined OA today? • What would he have made of these? OMIM – McKusick’s legacy • All you ever wanted to know about “Juvenile Hyaline Fibromatosis” • On-Line Mendelian Inheritance in Man • Compendium of human genetic disease • Hours of fun on the net. Just Google “OMIM” • William Heberden would have enjoyed it it! Principles of genetic mapping JUST LIKE SHUFFLING CARDS The closer the marker is to the gene of interest the better We are just one big happy family 150,000 yrs ago 80,000 yrs ago 75,000 yrs ago 70,000 yrs ago 74,000 yrs ago Mount Toba 74,000 yrs ago 40,000 yrs ago 30-25,000 yrs ago 22-19,000 yrs ago 22-19,000 yrs ago 12,500 yrs ago 25% 8% 50% 3% 0% 0% 0% Early Human Migration Routes 74k 80-75,000 yrs ago 73,000 yrs ago 45-25,000 yrs ago Stephen Oppenheimer and Bryan Sykes Sir Archibald Garrod 1896 100 yrs ago ALKAPTONURIA “a rather harmless disease” Homogentisic acid oxidase “Inborn factors in Disease” Garrod 1931 1999 CPPD arthritis and ANKH Lesson 1 - an extreme phenotype may give clues to common disorders 6 10 11 1 2 7 12 13 14 8 4 15 5 16 3 18 19 9 22 23 17 24 31 32 28 34 25 40 26 36 37 45 33 38 39 35 47 46 48 49 50 51 30 41 42 20 27 29 21 43 44 G→A in 5’UTR (-4bp) associated with CPPD in general population (p<0.0006) Andrew et al Am J Hum Genet 1999; 64: 136-45 Zhang et al Am J Hum Genet 2002; 71: 985-91 Hydroxyapatite Chondrocalcinosis Hydroxyapatite Crystal Formation (–) iPPi ANKH ePPi (+) ATP Alkaline phosphatase (AP) Pi (inorganic phosphate) Ca2+ NPPS AMP+PPi Nucleotide pyrophosphatase CPPD Crystal Formation CPPD Chondrocalcinosis Gain of function mutations in ANKH increase extracellular pyrophosphate → chondrocalcinosis Fibrodysplasia ossificans progressiva Bone in the wrong places Activating mutations in ACVR1 Not many families with FOP about! Narrowed to a Chr 2 region with 120 genes (including ACVR1) Increased signal in the absence of BMP Cell Signalling Through TGFβ Receptors Miyazono, K., et al.17:17 J Biochem (2010) Page 31 Receptor/SMAD interaction Huse, M., et al. Mol Cell (2001), Miyazono, K., et al. J Biochem (2010) 17:17 Page 32 TGFβ Signalling through ACVR1 Inactive Form • FKPB12 inhibitor FKBP12 inhibitor –Binds to GS Domain unphosphorylated Gly/Ser domain Kinase –Stabilises the Domain inactive receptor –Binding to GS ensures kinase is Chaikuad, A., et al. PDB (2009), Huse, M., et al. Mol Cell (2001) inactive 17:17 Page 33 Structure-activity Relationships GS Enhanced Inhibitors LDN-193189 ALK1 in complex with compound LDN-193189 A structural analog of Dorsomorphin Might these be useful treatments? Kinase Domain 3MTF - Chaikuad, A., et al. PDB (2010) 17:17 Page 34 COMPLEX DISORDERS? Classic forms of AS CT Reconstruction Bridging syndesmophytes SOIL & SEED Nature or Nurture? Heberden Orations by Lawrence and Weatherall 1973 A good year for the Westminster Hospital Nail patella syndrome Linked to ABO blood groups Ankylosing spondylitis in 1973 • Familial nature well recognised • More penetrant with female line? • HLA genes were a great source of interest • Tissue/bone marrow transplantation Brewerton et al Lancet 1973 • 72/75 patients with AS HL-A27 positive • 3/75 controls HL-A27 positive • Odds ratio for HLA-B27~120, still about the strongest for a polygenic disorder The Seventies – when everyone was hairy Look who’s come to play! Professor Brewerton • Taught me at Westminster Hospital • Investigated genetics of AS • Played full back • Is a published geologist Sir James Hutton 1726-1797 Father of British Geology Siccar Point 345M 425M “Concerning the system of the Earth” Royal Society of Edinburgh 1767 Alpine orogeny PreCambrian pillow lava Destruction of Ephesus Diastrophism in rocks The original union of UK 400M years ago “Concerning the system of the Earth” Royal Society of Edinburgh 1767 Alpine orogeny PreCambrian pillow lava Destruction of Ephesus Diastrophism in rocks What was the effect of the Act of Union? Diastrophism in bones! Diastrophic Dysplasia – a rare chondrodysplasia (sometimes called catastrophic dysplasia by RS) Diastrophism in Osteogenesis Imperfecta Problems with structural collagen genes “Star wars skull” “Popcorn bones” OI - a classic collagen disorder Lesson 2 – it’s never as straightforward as you think OI Type 1A OI Type 4B An international collaboration in osteogenesis imperfecta Collagen winds up from the C-terminus N-terminal mutations less severe? Than C-terminal mutations? Over-modification Lethal OI maps to matrix interaction domains in Type 1 collagen (integrins, fibronectin, COMP etc) MID2 MID3 Di Lullo et al J Biol Chem 2002; 277:4223-31 Marini et al Hum Mut 2007; 28:209-21 Role for processing enzymes and molecular chaperones in rare forms of severe OI Families of matrix disorders • Type 1 collagenopathies OI, EDS • Type 2 collagenopathies achondrogenesis, SED (c), Kniest, Stickler • Type 9 collagen Multiple epiphyseal dysplasia • Type 10 collagen Metaphyseal chondrodysplasia (Schmid) • Other cartilage components (COMP/matrilin/proteoglycans) Pseudoacondroplasia, MED • Type 1, 3 and 5 collagen Classic, vascular and OI/EDS overlap • Fibrillinopathies Marfan, congenital contractural arachnodactyly Type 2 collagenopathies Achondrogenesis/hypochondrogenesis Kniest dysplasia SED (congenita) Stickler syndrome Multiple Epiphyseal Dysplasia – various types (eg. Collagen 9, matrilin, COMP, sulfate transporter) Type 9 Collagen Pseudoachondroplasia and COMP Metaphyseal chondrodysplasia (type Schmid) Type 10 collagen. Transiently expressed by hypertrophic chondrocytes in the growth plate (cf rickets) Diastrophic dwarfism & MED (Type 5) “Hitch-hiker thumb” Abnormal sulfation of proteoglycans Double-layered patella 1st Century A.D. Founder population with striking linkage disequilibrium and a high mutant gene frequency Hastbacka et al Nat Genet 1992; 2: 204 FIBRILLINOPATHIES Lessons from Marfan Syndrome TGFβ signalling is important Lesson 2 AGAIN - never as simple as it seems! Marfan Syndrome and FBN1 not just a structural gene problem FBN1 - Cysteine mutations alter folding, cause abnormal microfibrils and lead to ocular lens dislocation Intrachain disulfide bonding Features of MFS unexplained by FBN1 • Tall stature • Fat/muscle hypoplasia • Excessive collagen • Lung abnormalities • Craniofacial features FBN1 microfibril FBN1 -/- Mice • Neptune Nat Gen 2003; 33:407-411 • Abnormalities in prealveolar saccules • Correlates with TGFβ • Lung phenotype blocked by neutralising TGFβ abs Dysregulated TGFβ signaling in Marfan syndrome Unexpected therapeutic opportunities with ACE receptor blockers Bone formation and remodelling Potential treatments from extreme phenotypes Activity of osteoblasts and osteoclasts is tightly linked in health Sclerostin Osteoporosis pseudoglioma EXTREME PHENOTYPES CAN REVEAL NEW TREATMENT OPTIONS “Sclerosteosis” • • • • Natural BMP inhibitor Deficiency of Sclerostin Sclersoteosis Sclerostin antibodies for osteoporosis? • Mouse studies successful (Li et al J Bone Min Res 2009; 24:578) Dutch founder populations and sclerosteosis Lesson 3 - Never take any for granted Different mutations – who would have believed it? Bone: the crucial balance between formation and resorption PAGET DISEASE RELATED PHENOTYPES Expansile Skeletal Hyperphosphatasia TNFRSF11A (RANK) Familial Expansile Osteolysis TNFRSF11A (RANK) Juvenile Paget (idiopathic hyperphosphatasia) TNFRSF11B (OPG) Familial Paget Disease SQSTMI (involved in NFκB pathways) RANKL activates osteoclast precursors Denosumab – an anti RANKL antibody We only have a skeleton crew working on dysplasias now! COMPLEX DISORDERS Classic forms of AS TWIN STUDIES IN ANKYLOSING SPONDYLITIS MZ Twins DZ Twins B27 + DZ UK (1997) 6/8 (75%) 4/32 (13%) 4/15 (27%) Others 11/19 (58%) 3/24 (13%) 3/15 (20%) Total 17/27 (63%) 7/56 (13%) 7/30 (23%) 92% of population variance due to genetic factors AS is a genetic disease that is not all due to B27 Brown et al, Arthritis Rheum 1997;40:1823 CHRO6 NASC+Oxford NASC Oxford All AS GFEGS: AS only GFEGS:SpA+AS All:SpA+AS 30.0 25.0 LOD 20.0 15.0 10.0 5.0 0.0 0 50 100 150 200 Distance (cM) Carter et al Rheumatol 2007;46:763 The Wellcome Trust Case-Control Consortium (WTCCC) 1 Second Study: 1,000 cases 1,500 controls 3,000 MHC SNPs 12,000 non-synonymous coding SNPs 4 diseases Ankylosing Spondylitis Autoimmune thyroid disease Breast Cancer Multiple sclerosis WTCCC & TASC Nat Genet 2007; 39: 1329-37 Wordsworth et al PNAS 1989 METHODS SLIDE Before and after Thermal Cyclers c.1987 UK RA Genetics Group SUMMARY OF RA GENES courtesy of Jane Worthington • 7 new RA loci confirmed at p<5x10-8 – SPRED2, ANKRD55, C5orf30, PXK, RBPJ, CCR6, IRF5 • 31RA loci confirmed at p<5x10-8 EIRE • Evidence for 9 further RA loci p 0.01-6.7x10-5 NARAC UKRAGG WTCCC SE hypothesis HLA-DRB1 1978 PTPN22 2004 CTLA4 2005 TNFAIP3 STAT4 TRAF1 IL2 / IL21 2007 REL BLK CD40 TAGAP CCL21 CD28 TNFRSF14 TRAF6 IL2RB PTPRC PRKCQ FCGR2A KIF5A PRDM1 IL2RA CD2-CD58 AFF3 2008 2009 IL6R CD247 ZEB1 SH2B3 BATF NFATC2IP IKZF3 UBASH3A UBE2L3 ANKRD55 SPRED2 C5orf30 RBPJ CCR6 IRF5 PXK 2010 GENETICS OF RHEUMATOID ARTHRITIS At least 30 genes involved Many are in the same pathways Most genetic effects are small Account for ~ 20% of variance Some are translatable Eg. CTLA4 (OR~1.05) Lesson 4 - CTLA4 (OR ~1.05) The biological importance of an association cannot be extrapolated from the strength of its genetic association with disease. CTLA4-Ig binds to B7 molecules and inhibits CD28-B7 interactions; suppresses T cell response ABATACEPT CD28 TCR T cell B7 MHC + peptide Antigen presenting cell Uncontrolled for DRB1/DQB1 association Type1 diabetes Controlled for DRB1/DQB1 association 89 Nejentsev, Nature, 2007. Effects from 5 amino acid residues in HLA-DR, HLA-DP and HLA-B Plenge et al 2012 A Retrospectoscope is all very well but…….. WHY STUDY GENETICS OF AS? • Curiosity • Diagnosis • Prognosis • New treatments • CURE/prevention IL23R US + UK vs UK ERAP1 ? New Peak US vs UK IL23R ERAP1 ? New Peak Revised T-helper-celldifferentiation model USTEKINUMAB TARGET STAT3/JAK2 STAT 3 Secukinumab TARGETS IL17 RORgT IL-6 TGFb and IL-6 promoteTH17 and Tregs. IL-23 does not act on naïve T cells It’S that retroSpectoScope agaIn…….. A BETTER INSTRUMENT (and a good team) ERAP1 resquencing Wild Type A/A Heterozygous A/G Homozygous G/G A→G = Lys→Arg 528 Oxford colleagues in AS Recombinant Protein Production • Site-directed mutagenesis • Cloned into baculovirus genome • Infection of insect cells with baculovirus DNA • Collect culture supernatant 72h PI • Purification by IMAC • Adjust to uniform protein concentrations Crystal structure of ERAP1 crystal of ERAP1A SIRAS-phased electron density map Diamond Synchtrotron @ beamline I02 ERAP1 crystallized in 2 crystal forms Kochan et al PNAS 2011;108:7745-50 space group P622: closed space group P212121 open molecule 1 molecule 2 molecule 3 ERAP1A may be a “molecular ruler” ERAP1 SUBSTRATES: *Length 9-16aa *C-teminal aa – hydrophobic *X-P-X-X-X…. *X-X-P-X-X-X…. Shih-Chung Chang et al. PNAS 2005 ERAP1 (endoplasmic reticulum aminopeptidase) is synergistic with HLA-B27 A role for ERAP1 in peptide presentation? ERAP1 Activity of ERAP1 Variants Towards WRVYEKCALK ** ** ** p < 0.05 Lys528Arg 40% reduction in activity – p = 0.009 ** LocusZOOM, regional association plot for ERAP1 P=10-6 P-values lower mainly due to smaller control group… No association when comparing B27- cases & controls… Providing further evidence that ERAP1 plays an important role in disease mechanism by trimming peptides for presentation by B27.. ERAP1 (endoplasmic reticulum aminopeptidase) is synergistic with HLA-B27 A role for ERAP1 in peptide presentation? ERAP1 Small molecule ERAP1 inhibitors for AS? TASC Triple A (Australo-Anglo-American) Spondyloarthritis Consortium Paul Wordsworth Oxford John Revielle Houston Matt Brown Brisbane WTCCC2 TASC HLA-B27 HLA-B60 ERAP1 IL23R 1q32 2p15 21q22 CMG2 IL1R2 CARD9 STAT3 RUNX3 PTGER4 TBKBP1 TNFRSF1A IL12B ERAP1/B27 synergy IGAS Immunochip B27 structure ERAP1 structure ______________________________________________________________________ 1973 1989 2007 2010 2011 2012 Milestones in the genetics of AS Statistical genetic signals can disappear. Findings 1. SNPs in the MHC on chromosome 6p were strongly associated with AS (P = 5 × 10−304). 2. 25 SNPs from 6 independent loci significantly associated with AS Includes ERAP1 and IL23R Chr 2p15 Chr 21q22 IL1R2 ANTXR2 Findings P=5x10-304 25 SNPs from 6 loci associated with AS Includes ERAP1 IL23R Chr 2p15 Chr 21q22 IL1R2 ANTXR2 or CMG Manhattan plot of discovery genome-wide association study findings Overlap with Genetic Associations of Psoriasis IL12B HLA-C IL23R ERAP1 IL23A Overlap of IBD susceptibility loci and AS AS HLA IL23R ERAP IL12B STAT3 PTGER4 CARD9 IL1R2 IL7R KIF21B Lees C W et al. Gut doi:10.1136/gut.2009.199679 ©2011 by BMJ Publishing Group Ltd and British Society of Gastroenterology UK collaboration in AS genetics Lessons for AS from extreme phenotypes? X-linked Hypophosphataemia Looser zones and ossification of hip capsule & ligamentum flavum Excessive bone in pseudohypoparathyroidism ASIF Izmer 2011 This could not have happened without NASS March 15th 2012 What a night! To the planet Venus William Wordsworth 1838 Man now presides In power, where once he trembled in his weakness; Science advances with gigantic strides; But are we aught enriched in love and meekness? Musings on genetics Consider Queen Victoria, ‘Tis odd to contemplate How just one little chromosome Decreed the Royal fate. For had She been X and Y, Instead of double X, Victoria Regina Would have ruled as Victor Rex. Victor Wordsworth c1990 Acknowledgements OXFORD NIHR BRC/BU David Harvey Clare Farrar Alice Harin Jenny Pointon Tugce Karaderi Louise Appleton Tom Wordsworth Bristol University David Evans DIamentina Institute BRISBANE Matthew Brown Linda Bradbury TASC (John Reveille et al), IGAS Structural Genomics Consortium Grazyna Kochan Centre for Proteomics Tobias Kroja Benedikt Kessler Udo Oppermann FUNDING Wellcome Trust Human Immunology Unit (MRC) NIAMS Liye Chen Arthritis Research UK Simon Kollnberger National Institute for Health Research Paul Bowness NASS Henni Mester Acknowledgements FOP • • • • • • Wordsworth Group Dr Kirsten Petrie Prof Graham Russell Prof Jim Triffitt Dr Roger Smith Prof Matt Brown Dr Alex N. Bullock Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences Nuffield Department of Clinical Medicine 17:17 Page 128 “A New Hope” in Oxford “Twilight” 800M 2Billion