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Kate Garrard 4th April 2008

Setting up a Diagnostic Service to Investigate the Production of Type I Collagen in Patients with Osteogenesis Imperfecta . Kate Garrard 4th April 2008. Summary. Introduction to Osteogenesis Imperfecta (OI) Current Service Collagen Production Protocol for investigating collagen production

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Kate Garrard 4th April 2008

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  1. Setting up a Diagnostic Service to Investigate the Production of Type I Collagen in Patients with Osteogenesis Imperfecta Kate Garrard 4th April 2008 Kate Garrard BSc Pre-Registration Scientist

  2. Summary • Introduction to Osteogenesis Imperfecta (OI) • Current Service • Collagen Production • Protocol for investigating collagen production • Results and Discussion • Conclusions Kate Garrard BSc Pre-Registration Scientist

  3. Introduction • Features include: • Bone deformity - Wormian bones, reduced bone density, barrel shaped ribs • Dentogenesis imperfecta • Blue sclera • Short stature • Joint laxity • Hearing loss (caused by breakage of the ear bones) • Characteristic faces – frontal bossing Kate Garrard BSc Pre-Registration Scientist

  4. Clinical Features • Dentiogenesis imperfecta Kate Garrard BSc Pre-Registration Scientist

  5. Clinical Features • Wormian bones Kate Garrard BSc Pre-Registration Scientist

  6. Clinical Features • Beaded ribs Kate Garrard BSc Pre-Registration Scientist

  7. Clinical Features • Blue Sclera Kate Garrard BSc Pre-Registration Scientist

  8. Classification Severe Mild • Type II OI • Fractures occur in the womb • Severe bone deformities • Often lethal at birth • May have blue sclerae • Dentiogenesis imperfecta • Type III OI • Fractures occur during or just after birth • Progressive bone deformities • Mobility constrained to wheelchair • May have blue sclerae • Dentiogenesis imperfecta common • Type IV OI • Fractures occur mostly during childhood and adolescence • Progressive bone deformities • Mobility may be impaired • Normal coloured sclerae • May have Dentogenesis imperfecta • Type I OI • Fractures occur during childhood and adolescence • Few fractures • Low bone density • Mobility normal • May have blue sclerae • May have Dentogenesis imperfecta Kate Garrard BSc Pre-Registration Scientist

  9. Classification • OI is genetically heterogeneous • Most cases are due to mutations in the genes which form collagen1 • As would be expected the classes of OI correspond with the type of defect in collagen 1 • Types of defect correlate with the mutation present in the COL1A1 or COL1A2 gene. Severe Mild • Type II OI • Normal Levels of Collagen 1 • Aberrant Collagen 1 • Type III OI • Normal Levels of Collagen 1 • Aberrant Collagen 1 • Type IV OI • Normal Levels of Collagen 1 • Aberrant Collagen 1 • Type I OI • Low Levels of Collagen 1 • Normal Collagen 1 Kate Garrard BSc Pre-Registration Scientist

  10. Current Screening Strategy Sample received in the lab for OI testing. COL1A1 Sequencing MLPA Analysis of collagen production Classical Mutation Detected Classical Mutation Not Detected COL1A2 Sequencing Mutation Not Detected Report issued Kate Garrard BSc Pre-Registration Scientist

  11. Structure of Collagen 1 Gly Gly Short triple helical domain C-telopeptide N-propeptide N-telopeptide Main triple helical domain C-propeptide Gly Y X Y X Kate Garrard BSc Pre-Registration Scientist

  12. Collagen Production Precursor mRNA Spliceosomes Transcription factor Ribosome Mature mRNA Preproα chain Post translational modification enzymes Procollagen Proα chain Mature Collagen ProcollagenN DNA Cell Membrane Kate Garrard BSc Pre-Registration Scientist

  13. Collagen Fibrils Kate Garrard BSc Pre-Registration Scientist

  14. Collagen Analysis • So if we want to look for aberrant collagen how do we do this? Kate Garrard BSc Pre-Registration Scientist

  15. Cohort Kate Garrard BSc Pre-Registration Scientist

  16. Technique Culture fibroblasts with ascorbic acid. Incubate radioactively labelled proline for 24hrs Harvest secreted collagens (media) Harvest intra cellular collagens Isolate whole cells and retrieve contents Solubilise intracellular collagens in specific acetic acid concentration Ethanol precipitate standardise radioactivity in samples Pepsin digest (mature collagens) Leave undigested (procollagens) Lyophilise Electrophorese Autoradiograph Kate Garrard BSc Pre-Registration Scientist

  17. Distribution of Collagen • All intracellular proteins will be procollagens These are then digested to form mature collagens for analysis purposes. • Secreted collagens will be a mixture of procollagens and mature collagens. Mature Collagen 1 Procollagen 1 Disassociated Procollagen1A1 and Procollagen1A2 Chains Kate Garrard BSc Pre-Registration Scientist

  18. Technique Culture fibroblasts with ascorbic acid. Incubate radioactively labelled proline for 24hrs Harvest secreted collagens (media) Harvest intra cellular collagens Isolate whole cells and retrieve contents Solubilise intracellular collagens in specific acetic acid concentration Ethanol precipitate Ethanol precipitate standardise radioactivity in samples Leave undigested (procollagens) Pepsin digest (mature collagens) Pepsin digest (mature collagens) Lyophilise Electrophorese Electrophorese Autoradiograph Kate Garrard BSc Pre-Registration Scientist

  19. Secreted Procollagen Gels 1 2 3 4 5 67 8 NC1 NC2 NC3 NC4 NC5 NC6 pro3A1 pro1A1 proN1A1 pro1A2 3A1 and 1A1 procollagen1A2 proN1A2 COL1A2 9 10 11 12 13 14 15 16 17 18 19 20 21 pro3A1 pro1A1 proN1A1 pro1A2 Kate Garrard BSc Pre-Registration Scientist

  20. Intracellular Collagen Gels 3 2 4 6 7 8 9 10 NC1 NC2 NC3 NC4 NC5 NC6 COL3A1 COL5A1 COL5A3 COL5A2 COL1A1 COL1A2 14 15 16 17 18 19 20 COL3A1 COL5A1 COL5A3 COL5A2 COL1A1 COL1A2 Kate Garrard BSc Pre-Registration Scientist

  21. Results and Discussion Kate Garrard BSc Pre-Registration Scientist

  22. Intracellular Collagen Gels 3 2 4 6 7 8 9 10 NC1 NC2 NC3 NC4 NC5 NC6 COL1A1 COL1A2 14 15 16 17 18 19 20 COL1A1 COL1A2 Kate Garrard BSc Pre-Registration Scientist

  23. Results and Discussion Kate Garrard BSc Pre-Registration Scientist

  24. Summary • Correlation can be seen between gel banding and mutation types. • Correlation can be seen between gel banding and severity of OI. However: • Gels are difficult to interpret. • Processing of samples is technically challenging. • Processing of samples is lengthy. Kate Garrard BSc Pre-Registration Scientist

  25. Conclusion • Although this technique can be useful in providing extra information for patients with OI, especially in complex cases, it would not be useful in the majority of cases and therefore throughput of samples is unlikely to justify the cost of offering this service. Kate Garrard BSc Pre-Registration Scientist

  26. Further Work • Screening of further samples to define the associations between the weak bands in the procollagen gels and the severity of OI. • More gels with same samples to determine the degree of reproducibility. • pHing of secreted collagen samples to allow pepsin digestion and optimise electrophoresis conditions. • Sequencing of COL1A1 and COL1A2 in individuals with abnormal patterns in whom it had not already been completed. • Determination of the mutations/polymorphisms causes the pepsin digest band in normal control 6 and individuals 7 and 17. Kate Garrard BSc Pre-Registration Scientist

  27. Acknowledgements • This project has been a hugely collaborative body of work and would not have been possible without the extensive help and support of a number of people. Thank you to: • Mandy Nesbit, Rebecca Pollitt and Amal Affifi for their extensive knowledge of the genetics behind OI. • Simon Olpin, Shirley Clark and Helen Franks of Clinical Chemistry for use of their facilities and all of their help and support with cell culturing and radio-labelling techniques. • The Centre for Medical Genetics in Ghent, Belgium for all their help, particularly to Sofie Symoens for her help in troubleshooting and interpretation of gels. • Professor Nick Bishop for providing a cohort. • Nicola Jakins for additional work sequencing the COL1A1 and COL1A2 genes. • All my colleagues in molecular genetics for allowing me the free time to complete my project work. Kate Garrard BSc Pre-Registration Scientist

  28. Clinical Features • Wormian Bones Kate Garrard BSc Pre-Registration Scientist

  29. Secreted Procollagen Gels 1 2 3 4 5 6 7 8 91011 12 13 14 pro3A1 pro1A1 proN1A1 pro1A2 α1(III) and α1(I) Procollagenα2(I) ProNα2(I) α2(I) 15 16 17 18 19 20 21 NC1 NC2 NC3 NC4 NC5 NC6 pro3A1 pro1A1 proN1A1 pro1A2 α1(III) and α1(I) Procollagenα2(I) ProNα2(I) α2(I) Kate Garrard BSc Pre-Registration Scientist

  30. Structure of Collagen 1 • The three procollagens spiral together to form a heterotrimeric triple helix. Kate Garrard BSc Pre-Registration Scientist

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