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Genetics of Vascular Diseases Jake Lusis 3-730 MRL [email protected] Carl Miller, ~1938, Oslo County Hospital, Norway Inheritance of: Cholesterol level (mg/dl): 330 210 Tendon xanthomas: Early coronary artery disease (under 55 years of age): 210 410 490 200 460 210 190 180 420 430 Cosegregation of triad of xanthomas, high cholesterol and early heart disease Tendon Xanthomas Inheritance of: Cholesterol level (mg/dl): 210 230 Tendon xanthomas: Early coronary artery disease (under 55 years of age): 190 400 210 210 430 420 170 420 810 490 180 420 This individual died from severe CAD at age 7 Lecture delivered in Sweden Dec 9, 1985 when they received the Nobel Prize in Physiology. Published in Science, 232, 34-47, 1986 Experiments with 125I-LDL and skin fibroblasts skin fibroblasts from normal and FH individuals add labeled 125I-LDL ( ) incubate to allow binding wash away unbound LDL and follow label - 125I-LDL on surface can be displaced from cells by anionic polymers - 125I-LDL that has been internalized cannot be displaced from cells - 125I-LDL that has been degraded to amino acids is soluble in tricholoroacetic acid Experiments with fibroblasts from normal and FH individuals Normal FH homozygote Sequential steps in LDL receptor pathway: Discovery of receptor mediated endocytosis Degradation of LDL by lymphocytes and following injection Cloning of the Human LDL Receptor isolate plasma membrane cultured skin fibroblasts purify such that a single protein remains upon electrophoresis extract proteins with detergent and subject to chromatography 125I-LDL bound digest the protein and sequence the resulting peptides -ASN-PHE-THR-SER- based on the amino acid sequence, synthesize corresponding oligonucleotide probes ….CACTCGACAGCGAAC... probe cDNA and genomic libraries. Confirm identity of clones by sequencing and expression studies Domain structure of the LDL receptor Five classes of LDL receptor mutations Development of drugs to treat hypercholesterolemia based on concepts learned from FH studies Combined heart-liver transplant in an individual with homozygous FH Lessons from familial hypercholesterolemia • Studies of an uncommon disease (FH) can have important implications for a common disease (coronary artery disease) • Genetics studies of clinical traits can lead to an understanding of basic biologic mechanisms (receptor mediated endocytosis, cholesterol homeostasis) as well as new therapies (statins). Francis Collins (1995) Schematic of the approach generally utilized for positional cloning BACs Families + Linkage Analysis Finer Genetic Mapping Physical Mapping and Cloning Normal Candidate cDNAs ..A A C T C Transcript Identification Mutation Search ..A A G T C Mutation Genetic Markers Marfan Syndrome -autosomal dominant disorder characterized by weakened connective tissue -most life-treatening is aortic dissection (an aneurysm of the aorta) -other symptoms include mitral valve prolapse, emphysema-like lung disease, and ocular and skin disorders -patients tend to be tall and lanky, with very long limbs, fingers and toes Fibrillin 1 Gene Mutated in Marfan Syndrome Patients (A) Single-strand conformational polymorphism screen (B) DNA sequence Letter Nature Genetics 33, 407 - 411 (2003) Published online: 24 February 2003; Dysregulation of TGF- activation contributes to pathogenesis in Marfan syndrome Enid R. Neptune1, 2, Pamela A. Frischmeyer2, Dan E. Arking2, Loretha Myers2, Tracie E. Bunton3, Barbara Gayraud4, Francesco Ramirez4, Lynn Y. Sakai5 & Harry C. Dietz2, 6 Lung histopathology and morphometry of mice deficient in fibrillin-1. Active TGF-B expression and signaling in lung tissue of mice deficient in fibrillin-1. Neutralizing antibody to TGF- rescues lung maturation in mice deficient in fibrillin-1. Mice deficient in fibrillin-1 have preserved lung cell proliferation but greater apoptosis. Model Fibrillin deficiency TGFB not sequestered TGFB activated Apoptosis in lung, bone overgrowth, etc. A lesson: Structural matrix elements serve crucial regulatory roles in cytokine activation and signalling Identification of Mendelian and complex trait genes 1980-2002 Number of Mendelian genes 1600 Mendelian traits 100 All complex traits 90 Human complex traits 80 1400 70 1200 60 1000 50 800 40 600 30 400 20 200 10 0 1980 Number of complex trait genes 1800 0 1985 1990 Year 1995 2000 Glazer, et al. (2002) Science Nature Genetics 34, 383 - 394 (2003) VEGF is a modifier of amyotrophic lateral sclerosis in mice and humans and protects motoneurons against ischemic death Diether Lambrechts1, Erik Storkebaum1, Masafumi Morimoto1, Jurgen Del-Favero2, Frederik Desmet1, Stefan L Marklund3, Sabine Wyns1, Vincent Thijs1, Jörgen Andersson3, Ingrid van Marion4, Ammar AlChalabi5, Stephanie Bornes6, Rhiannon Musson4, Valerie Hansen5, Lars Beckman3, Rolf Adolfsson7, Hardev Singh Pall4, Hervé Prats6, Severine Vermeire1, Paul Rutgeerts1, Shigehiro Katayama8, Takuya Awata8, Nigel Leigh5, Loïc Lang-Lazdunski9, Mieke Dewerchin1, Christopher Shaw5, Lieve Moons1, Robert Vlietinck1, 10, Karen E Morrison4, Wim Robberecht1, Christine Van Broeckhoven2, Désiré Collen1, Peter M Andersen3 & Peter Carmeliet1 VEGF Polymorphisms Lesson Basic vascular biology studies (regulation of VEGF expression in response to hypoxia) can lead to an understanding of a complex disease