Genetics of Vascular Diseases Jake Lusis 3-730 MRL Jlusis@mednet.ucla

1. Genetics of Vascular Diseases Jake Lusis 3-730 MRL Jlusis@mednet.ucla.edu

3. Carl Miller, ~1938, Oslo County Hospital, Norway Inheritance of: Cholesterol level (mg/dl): Tendon xanthomas: Early coronary artery disease (under 55 years of age): 330210 210 200 210 180 430 410 490 190 420 460

4. Cosegregation of triad of xanthomas, high cholesterol and early heart disease Tendon Xanthomas

5. Inheritance of: Cholesterol level (mg/dl): Tendon xanthomas: Early coronary artery disease (under 55 years of age): 210230 190 400 210 210 420 180 430 490 420 810 420 170 This individual died from severe CAD at age 7

6. Lecture delivered in Sweden Dec 9, 1985 when they received the Nobel Prize in Physiology. Published in Science, 232, 34-47, 1986

7. 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

8. Experiments with fibroblasts from normal and FH individuals Normal FH homozygote

9. Sequential steps in LDL receptor pathway: Discovery of receptor mediated endocytosis

10. Degradation of LDL by lymphocytes and following injection

11. 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- probe cDNA and genomic libraries. Confirm identity of clones by sequencing and expression studies based on the amino acid sequence, synthesize corresponding oligonucleotide probes ….CACTCGACAGCGAAC...

12. Domain structure of the LDL receptor

13. Five classes of LDL receptor mutations

14. Development of drugs to treat hypercholesterolemia based on concepts learned from FH studies

15. Combined heart-liver transplant in an individual with homozygous FH

16. 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).

17. Francis Collins (1995)

18. Schematic of the approach generally utilized for positional cloning BACs Normal Candidate cDNAs Physical Mapping and Cloning Families Finer Genetic Mapping ..A A C T C Linkage Analysis Transcript Identification Mutation Search + ..A A G T C Mutation Genetic Markers

20. 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

21. Fibrillin 1 Gene Mutated in Marfan Syndrome Patients • Single-strand conformational polymorphism screen • DNA sequence

22. Letter Nature Genetics33, 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

23. Lung histopathology and morphometry of mice deficient in fibrillin-1. Next

24. Figure 3 Active TGF-B expression and signaling in lung tissue of mice deficient in fibrillin-1.

25. Neutralizing antibody to TGF- rescues lung maturation in mice deficient in fibrillin-1.

26. Mice deficient in fibrillin-1 have preserved lung cell proliferation but greater apoptosis.

27. 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

28. Glazer, et al. (2002) Science

29. Ian

33. Article Nature Genetics34, 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 Al-Chalabi5, 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

34. Figure 1.Genetic association of VEGF with ALS. Figure 1.Genetic association of VEGF with ALS. VEGF Polymorphisms

35. Figure 1.Genetic association of VEGF with ALS.

39. Lesson Basic vascular biology studies (regulation of VEGF expression in response to hypoxia) can lead to an understanding of a complex disease