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The Galton Laboratory, University College London

The value of the Human Genome Project and the prospects for the Human Variome project, illustrated by Tuberous sclerosis. Sue Povey. The Galton Laboratory, University College London. Learning objectives The goals of the Human Genome Project The achievements of the Human Genome project

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The Galton Laboratory, University College London

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  1. The value of the Human Genome Project and the prospects for the Human Variome project, illustrated by Tuberous sclerosis. Sue Povey The Galton Laboratory, University College London

  2. Learning objectives • The goals of the Human Genome Project • The achievements of the Human Genome project • provide some examples of practical value in Medicine of the Human genome project. • Be able to discuss one example is some detail (tuberous sclerosis considered here) • Appreciate the biological value of the ‘Human variome project’

  3. Human Gene mapping 1951-1990 • Many groups trying to find the cause of Mendelian diseases by studying inheritance of the disease in families • and mapping the disease to a chromosome. • Major problems: • No good genetic map of human chromosomes • No physical map at all • Great achievement to find which chromosome involved • (i.e. which of 22 haystacks in which to look for the needle) • and this was the easy bit. Cystic fibrosis was localised to chromosome 7q in 1985 , and the defective gene was identified in 1989 following intensive effort from many scientists.

  4. Human Genome project Goals 1991 A. Mapping and Sequencing the Human Genome B. Model Organisms C. Informatics: Data Collection and Analysis D. Ethical, Legal, and Social Considerations E. Research Training F. Technology Development G. Technology Transfer Main goals expected to take at least till 2005

  5. Human Genome Project Progress A. Mapping and Sequencing the Human Genome Genetic map High resolution genetic map(5000 markers) 1994 Physical Map 52,000 STSs (points of reference with sequence tags)1998 Human DNA Sequence draft 2001 Feb15 publicly funded Nature 2001 Feb16th Celera Genomics Science ‘99% finished’ ‘(excluding heterochromatin) 2004 Nature publications of each human chromosome separatelycompleted May2006

  6. Human Genome project progress • B. Model organisms • Saccharomyces cerevisiae (baker's yeast) 12MB 1996 • Caenorhabditis elegans(worm) 100MB 1998 • Drosophila melanogaster (fruit fly) 118MB 2000/02 • Mouse C57BL/6Japprox 3000MB 2003 • All finished ahead of schedule

  7. Why did it go so fast? • Competition! • Publicly funded international collaboration (USA, UK, and some from France Germany and Japan) • Wellcome Trust funded 30% of human genome sequence at Sanger Centre • Used mixed DNA from different individuals • Worked from individual chromosome maps • All data visible on world wide web within 24 hours Funding provided only if accuracy better than 1 mistake in 10,000 bases

  8. Why did it go so fast? • Competition! • Commercial firm Celera Genomics • Sequenced one individual • Did not use (many) maps but sequenced large numbers of random clones and enormous computing power to fit the pieces together • Did not release data freely till May 2006

  9. Human Genome project progress C Informatics: Data Collection and Analysis Three browsers: http://www.ensembl.org/index.html http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?txid=9606 http://genome.cse.ucsc.edu/ Human Gene names associated with correct sequence http://www.gene.ucl.ac.uk

  10. Will the Human Genome Project… • Help in understanding human disease? • Help in predicting/preventing disease? • Help in treating disease?

  11. Causes of Death *=complextrait • Duchenne muscular dystrophy • Cystic fibrosis • Other Mendelian disorders • Type 2 diabetes* • Heart disease* • Bi-polar depression* • Cancer* • Adverse effects of medicines • Starvation • High altitude climbing accident • Air crash (scheduled flight) • Struck by lightning when in bed Environment Genetics

  12. Has the Human Genome Project contributed to: Understanding? ,Prevention?Treatment? • Duchenne muscular dystrophy N N N • Cystic fibrosis N N N • Other Mendelian Disorders (hundreds) YY some • Type 2 diabetes Y ? ? • Heart disease Y ? ? • Bi-polar depression ? ? ? • Cancer YY some • Adverse effects of medicines Y?Y?Y • Starvation N N N • High altitude climbing accident ?Y N N • Air crash (scheduled flight) N N N • Struck by lightning when in bed N N N

  13. Tuberous Sclerosis: clinical features • epilepsy • learning disabled • characteristic skin rash • White patches on skin • ‘tubers’ in brain • Brain tumours(SEGAs) • Kidney tumours • Heart tumours -rhabdomyomas

  14. Tuberous Sclerosis: Genetics • Autosomal Dominant disorder • Hamartomas in many tissues • Prevalence about 1 in 10,000 at birth • 70% of cases are new mutations

  15. The search for Tuberous sclerosis • 1935 disease is autosomal dominant; many cases are new mutations • 1987 In families it is inherited with the ABO blood group! It must be on chromosome 9 . • 1988 some cases are NOT on chromosome 9.Must be at least two genes TSC1 and TSC2 • 1990 TSC1 between two genetic markers in 9q34.1-9q34.2 Let’s make a physical map 1996achieved 1.6 megabase contig across region

  16. Success in finding genes! • Caused by mutation in either of two genes • TSC1 9q34 - coding for hamartin (23 exons) found 1997(contig sequenced as part of Human Genome project) • TSC2 16p13 - coding for tuberin (41 exons) found 1994 (helped by chromosome tranlocation)

  17. Mutation Sites in TSC1 and TSC2 nonsense splice site deletion insertion missense in-frame deletion X TSC1 TSC2 Kwiatkowski DJ (2003) Ann Hum Genet 67: 87-96 (review)

  18. Tuberous Sclerosis: Genetics • Genetic counselling difficult because • The disease is so variable in severity • One established case of non-penetrance means that unaffected sibs worry that they may have affected children • Occasionally completely healthy parents have more than one child with tuberous sclerosis

  19. Two practical points learnt from mutation testing:

  20. ? ? A documented case of non-penetrance ? I:1 I:2 II:4 II:5 II:6 II:7 II:3 II:3 ? III:1 III:3 III:2 III:4 III:6 III:5 III:8 III:9 IV:1 IV:2 III.5 healthy in all respects with normal CT scan

  21. ? ? Haplotypes on chromosome 9q34 (TSC1) I:1 I:2 II:4 II:5 II:6 II:7 II:3 II:3 ? ? III:1 III:3 III:2 III:4 III:6 III:5 III:8 III:9 Most of family has mutation inTSC1 IV.2 has new mutation in TSC2 IV:1 IV:2

  22. Tuberous Sclerosis: Genetics • No proven example of non-penetrance. • An adult at risk of inheriting TSC who has no clinical findings and normal CT scan is extremely unlikely to have inherited the mutation

  23. ASS ABL D9S10 D9S66 ASS ABL D9S10 D9S66 -another problem No mut found I:1 I:2 So I:2 is a mosaic 2 1 3 4 4 4 2 4 If this is a mutation in TSC1II.1 should be affected If TSC2II.2 should be affected 2 2 1 2 2 2 1 2 D16S85 2 2 1 4 D16S291 2 2 3 5 D16S283 2 5 2 1 No mut found TSC1mut TSC1mut II:1 II:2 II:3 II:4 2 4 2 3 2 4 2 4 4 4 4 2 4 4 4 4 2 2 2 1 2 2 2 2 2 2 2 1 2 2 2 2 2 1 2 4 2 4 2 4 D16S85 2 3 2 5 2 5 2 5 D16S291 5 2 2 1 2 1 2 1 D16S283

  24. Tuberous Sclerosis: Genetics • But there IS Mosaicism…...both germline and somatic. • The clinically normal parent of an affected child who is regarded as a sporadic case is still at higher risk of having another affected child than the general population. • And the first case in a family may be a mosaic

  25. What did genetics or biochemistry tell us about function? • Loss of heterozygosity suggests a tumour suppressor mechanism • i.e. tumour caused by second mutational hit on normal allele (Possibly not in all lesions)

  26. What did genetics or biochemistry tell us about function? • Tuberin binds to rabaptin • Hamartin binds to ezrin (cytoskeleton) • Strong binding interaction between Tuberin and Hamartin • High level of expression of either protein in many cell systems gives growth arrest • A Bit Confusing……..

  27. What did rodent models tell us about gene function? • Spontaneous TSC2 mutation in Eker rat gives renal carcinomas and occasional brain lesions • Mouse models Tsc1+/- or Tsc2+/- get renal cystadenomas and hepatic hemangiomas • Interesting, but not very like human disease …..

  28. To The Rescue….. Drosophila Genetics

  29. Mutation of Tsc1 Increases Cell and Organ Size Tsc1+ Tsc1- Mutant cells in mosaic eye control Tsc1 mutant Potter CJ et al., (2001) Cell 105: 357-368.

  30. Co-overexpression of Tsc1 and Tsc2 Decreases Cell Size, Cell Number and Organ Size. Tsc1 overexpression Tsc2 overexpression Tsc1 & Tsc2 co-overexpression Potter CJ et al., (2001) Cell 105: 357-368.

  31. Co-overexpression of Tsc1 and Tsc2 Decreases Cell Size, Cell Number and Organ Size. Wild-type Tsc1 overexpression Tsc2 overexpression Tsc1 & Tsc2 co-overexpression Potter CJ et al., (2001) Cell 105: 357-368.

  32. InR Insulinreceptor Pi3K92E Phosphoinositol-3-kinase Pten Phosphatase & tensinhomolog Akt1 Protein kinase B Tsc1 / Tsc2 Tor Target of rapamycin Tor S6k Ribosomal protein S6 kinase S6k Genetic Model for the Function of Tsc1 and Tsc2 in Drosophila Kwiatkowski DJ (2003) Ann Hum Genet 67: 87-96 (review)

  33. Signalling Pathway Model for TSC1 and TSC2 in Mammalian Cells Kwiatkowski DJ (2003) Ann Hum Genet 67: 87-96 (review)

  34. So is this the main/sole function of TSC1/TSC2 ??? • It is probably not as simple as that! • Some recent evidence that TSC1/TSC2 can interact directly with S6K. • Various other apparently unrelated observations • But some encouraging studies in Eker rat, treated with rapamycin

  35. Effects of Rapamycin on Eker Rat Renal Tumours: Western blot analysis of tumour lysates showing biochemical responses to i.p. rapamycin. Kenerson HL et al., (2002) Cancer Res 62: 5645-5650.

  36. Tsc1 and Tsc2 Null Mice WT Null • Null Embryos Die between E10 and E12 • Cause of death is cardiac enlargement and liver hypoplasia

  37. Mouse models difficult;signs in mouse quite different fron TSC in humans The mouse model is not entirely satisfactory because it dies of complications not seen in human (eg liver haemangiomas) before it has a chance to develop brain disease.So perhaps the answer will be a conditional mouse model ? This has flox sites around the gene of interest (Tsc1,)and a mouse carrying this construct is mated to a mouse carrying a cre-recombinase driven from a brain- specific -promoter.The Tsc1 should only be knocked out in those brain cells in which the cre recombinase has acted on the flox sites. So with luck the Tsc1 will only be knocked out in brain.

  38. Expression of Cre Recombinase causes the LoxP sites to recombine thus producing a null (knock out) allele Spatial and temporal Control of Cre expression can be achieved by using different promoters to drive expression. Using the Synapsin I promoter the group of Kwiatkowski knocked out Tsc1 in differentiating neurons of the mouse brain starting at E13.5. Mm Tsc1 16 17 18 19 20 Conditional allele 16 17 18 19 20 cre recombinase 16 19 20 null allele Tsc1 Conditional Model

  39. Tsc1c-synI-cre+ mice: survival, weight gain, phenotype, and loss of Tsc1 in the brain. C D Affected mice live longer when treated with rapamycin

  40. A recent report of successful treatment! • Franz DN, Leonard J, Tudor C, Chuck G, Care M, Sethuraman G, Dinopoulos A, Thomas G, Crone KR. Ann Neurol. 2006 Mar;59(3):490-8. • ‘Rapamycin causes regression of astrocytomas in Tuberous sclerosis complex.’

  41. Case 2. Reduction in size of subependymal giant cell astrocytoma after 5 months of rapamycin therapy. (Left) Pretreatment; lesion volume is 6cc. (Right) After treatment; lesion volume is 2.4cc. Axial T1 contrast-enhanced magnetic resonance imaging

  42. Current controlled trials Two small but carefully designed clinical trials of rapamycin in adult patients with kidney and lung lesions (one in Cincinnati ,one in the UK) are about to report at mid-point of trial. Benefit found in about half the treated patients. Why treat the kidney and lung tumours? Why not treat the babies with fits or brain tumours? Because of need for safety and consent.

  43. references Kwiatkowski DJ (2003) Tuberous Sclerosis: from Tubers to mTOR Ann Hum Genet 67: 87-96 (review) Potter, C. J., Huang, H. & Xu, T. (2001). Drosophila tsc1 functions with tsc2 to antagonize insulin signaling in regulating cell growth, cell proliferation, and organ size. Cell 105, 357-68. Kwiatkowski DJ, Manning BD. Tuberous sclerosis: a GAP at the crossroads of multiple signaling pathways. Hum Mol Genet. 2005 Oct 15;14 Spec No. 2:R251-8. Review.

  44. The Human Variome Project • An attempt to characterise all the clinically significant variation in the human population, usually ascertained through disease • So far more than 500 different locus-specific mutation databases linked from the Human Genome Variation Society website

  45. The Human Variome Project • Such databases have great practical value for diagnostic laboratories. • They can also tell us a lot about basic biology They allow us to determine what DNA changes actually affect transcription, splicing, RNA stability, protein folding and functioning , in an enormous and very well phenotyped collection of mammals which cost the scientist nothing to keep. Eg http://www.LOVD.nl/TSC2

  46. The Tuberous sclerosis association UK (TSA) has supported much of the research described in this talk, from 1985. The TS Alliance (USA) is supporting the new database . Many scientists, students,clinicians, patients and their families have contributed to the work. Special thanks to David Kwiatkowski and Lynsey Meikle for unpublished data and to John Osborne,John Yates, Julian Sampson, Dickey Halley and David Kwiatkowski for their long-term collaboration

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