Darryl D’Lima, M.D. The Donald P. Shiley Visiting Lectureship Wednesday, Feb. 11, 2009 Scripps Green Hospital Cartilage Disease & Regeneration Genomics in Darryl D. D’Lima, MD, PhD Osteoarthritis Clifford W. Colwell Jr, MD Shiley Center for Shiley Center for Orthopaedic Research & Education Orthopaedic Research & Education Scripps Clinic Scripps Clinic Donald P Shiley Visiting Lectureship Donald P Shiley Visiting Lectureship February 11, 2009 February 11, 2009 Genetics Single gene disorders GENOMICS – Cystic fibrosis – Huntington’s Study of genes and how genetic variations affect function and disease Multifactorial – Heart disease – Diabetes –OA Human Genome Project Human Genome Project A GENOME the complete set of DNA HUMAN GENOME ~ 3 billion pairs of nucleotides for an organism 23 pairs of chromosomes Location of each gene on the chromosome How many genes are there? (normal volunteers) 20,000 - 75,000 C. elegans GeneSweep Winner: 24,947 1 Darryl D’Lima, M.D. The Donald P. Shiley Visiting Lectureship Wednesday, Feb. 11, 2009 Scripps Green Hospital Genome Trivia Human Genome Project Chromosome 1 has the most genes = 2968 Less than 2% of the genome codes for proteins Y (Male) chromosome has the fewest = 231 The functions are unknown for over 50% of discovered genes X (Female) chromosome = 2000 Junk DNA = repeated sequences that do not code for proteins make up at least 50% of the human genome Is This JUNK? Human Genome Project O lny srmat poelpe can raed tihs. I cdnuolt blveiee taht I cluod aulaclty uesdnatnrd waht I was rdanieg. 99.9% nucleotide bases are exactly the The phaonmneal pweor of the hmuan mnid, aoccdrnig to a same in all people rscheearch at Cmabrigde Uinervtisy, it deosn't mttaer in waht oredr the ltteers in a wrod are, the olny iprmoatnt tihng is taht the frist and lsat ltteer be in the rghit pclae. The rset can be a taotl mses and you can sitll raed it wouthit a porbelm. Tihs is bcuseae the huamn mnid deos not raed ervey lteter by istlef, but the wrod as a wlohe. Amzanig huh? yaeh adn I awlyas tghuhot slpeling was ipmorantt! Single Nucleotide Polymorphism Success in Genomics SNP Macular degeneration Humans differ in genetic makeup by only 0.1% Rheumatoid arthritis Crohn’s disease Osteoarthritis? 1.4 million locations where single-base DNA Bipolar disorder differences (SNPs) occur in humans Breast cancer Hypertension Diabetes 2 Darryl D’Lima, M.D. The Donald P. Shiley Visiting Lectureship Wednesday, Feb. 11, 2009 Scripps Green Hospital MRI Femoral Condyles 72 yr 52 yr Current Status Current Status Linkage Analysis • Linkage Analysis Rare Mendelian Families •COL2A1 • Affected Sibling Pairs – Type II procollagen/Chr 12 • Candidate Gene • Single base mutation Knowlton RG, N Engl J Med 1990 Palotie A, Lancet 1989 Current Status Current Status Linkage Analysis First Large Twin OA Study • Chr 2, 4, 16 • 250 twins • No known genes • Monozygotic • Uncommon OA – 64% concordance • Dizygotic – 39% concordance Review Loughlin J, Curr Opin Rheum 2001 Spector TD, BMJ 1996 3 Darryl D’Lima, M.D. The Donald P. Shiley Visiting Lectureship Wednesday, Feb. 11, 2009 Scripps Green Hospital Current Status Current Status Candidate Genes • Linkage Analysis Rare Mendelian Families • Matrix proteins • Affected Sibling Pairs •VitD • Growth factors • Candidate Gene • Whole Genome Approach Single Nucleotide Polymorphism Whole Genome Approach SNP • LRCH1 – N = 335 (Females) Humans differ in genetic makeup by only 0.1% – Knee OA 1.4 million locations where single-base DNA – DNA pooling differences (SNPs) occur in humans – ~25,000 SNPs – 13q14 – Odds ratio = 1.4 Spector TD, Arth Rheum 2006 Study Design Study Design • Primary Knee OA • N = 500 • Total Knee Arthroplasty • 10 cc blood •Exclusion • Saliva – Secondary OA • DNA extraction • SNP detection • Statistical Treatment 4 Darryl D’Lima, M.D. The Donald P. Shiley Visiting Lectureship Wednesday, Feb. 11, 2009 Scripps Green Hospital Scripps Genomic Medicine Scripps Translational Science Institute Validation Study Design • Control • NIH OAI • 80 years • Mayo Clinic • No Major Disease • Hospital for Special Surgery • “Wellderly” • Eric Topol, MD • Scripps Clinic Trained Fellows • Nick Schork, PhD • Kelly Frazer, PhD • Sarah Murray, PhD SCRDA Grant Award, CRS, Brian Issell, MD Relevance Stem Cells in • Identify “at-risk” candidates Osteoarthritis • Identify “high-risk” candidates • Discover new treatments Shiley Center for • Tailor treatment specific to each patient Orthopaedic Research & Education Scripps Clinic Bovine Chondrocytes in Agarose Gel Tissue Engineering • Obtain cells • Physiologic environment Day 5 Day 10 Day 20 • Cell proliferation • Matrix production • Phenotype 5 Darryl D’Lima, M.D. The Donald P. Shiley Visiting Lectureship Wednesday, Feb. 11, 2009 Scripps Green Hospital Tissue Engineering Tissue Engineering Status in laboratory Clinical Status Obtain cells Obtain cells Physiologic environment Physiologic environment Cell proliferation Cell proliferation Matrix production Matrix production Phenotype Phenotype Human Embryonic Stem Cells Stem Cells • Rapid “unlimited” proliferation • Differentiate into several types of cells Cell Pellets • Ready source Pellet Culture of Stem-Cell-Derived Cells Stem Cell Pellet in Human Cartilage 3 weeks Control TGFβ3 Transforming Growth Factor Cartilage Bone Empty Defect Cell Pellets 6 Darryl D’Lima, M.D. The Donald P. Shiley Visiting Lectureship Wednesday, Feb. 11, 2009 Scripps Green Hospital Stem Cell Pellet in Human Cartilage Stem Cell Pellet in Human Cartilage 10 X 40 X Cartilage Slice Stem Cell Pellet in Human Cartilage 6 mm 300 micron Cartilage Slice Embryonic Stem Cells •Ethics • Allogeneic 7 Darryl D’Lima, M.D. The Donald P. Shiley Visiting Lectureship Wednesday, Feb. 11, 2009 Scripps Green Hospital Induced Pluripotent Stem Cells Nuclear Transfer Cell Fusion Lowry WE & Plath K, Nature Biotech 2008 Induced Pluripotent Stem Cells • Repair • Regenerate •Replace iPSC from Human Chondrocytes Donald P. and Darlene V. Shiley TISSUE Shiley Center for Orthopaedic Research & Education ENGINEERING STEM CELLS Scripps Clinic GENOMICS OSTEOARTHRITIS PREVENTION DRUG THERAPY SURGICAL PROCEDURES 8 Darryl D’Lima, M.D. The Donald P. Shiley Visiting Lectureship Wednesday, Feb. 11, 2009 Scripps Green Hospital Cartilage Disease & Regeneration Darryl D. D’Lima, MD, PhD Clifford W. Colwell Jr, MD Shiley Center for Orthopaedic Research & Education Scripps Clinic Donald P Shiley Visiting Lectureship February 11, 2009 9.