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

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

3. Kate Garrard BSc Pre-Registration Scientist 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

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

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

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

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

8. Kate Garrard BSc Pre-Registration Scientist Classification 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 SevereMild

9. Kate Garrard BSc Pre-Registration Scientist 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. 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 SevereMild

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

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

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

13. Kate Garrard BSc Pre-Registration Scientist Collagen Fibrils

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

15. Kate Garrard BSc Pre-Registration Scientist Cohort Identifier 1A1 sequenced 1A2 sequencedGene Mutation (cDNA No.) Mutation (Protein No.)TypeOI 1ARyesnoCOL1A1c.2643C>Tp.Arg882XterminationIV 2AByesnoCOL1A1c.3581delGp.Gly1195fsframeshiftI 3HEyes -N:Nnoneatypical 4PPyes COL1A2c.604G>Cp.Gly202Argglycine subIV 5MGyesnoCOL1A1c.589G>Cp.Gly187Argglycine subIV 7DDyesnoCOL1A1c.994G>Ap.Gly332Argglycine subIII 8FHyesnoCOL1A1c.2436G>Ap.Gly773Serglycine subIII 9SAyesnoCOL1A1c.814G>Tp.Gly272Cysglycine subIV 11MWyesnoCOL1A1c.1378_1379insCp.Gly461fsframeshiftIV 12GFyesnoCOL1A1c.3806G>Ap.Trp1269XterminationIV 13LHno ----IV 14BByes (in father) -N:N--I 15DIno ----IV 17CMyesnoCOL1A1c.1939G>Ap.Gly647Serglycine subatypical 18MOyesnoCOL1A1c.1012G>Tp.Gly338Cysglycine subIV 19MFyesnoCOL1A1c.3580_3581delGCp.Ala1194fsframeshiftIII 20LIno ----NK

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

17. Kate Garrard BSc Pre-Registration Scientist 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

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

19. Kate Garrard BSc Pre-Registration Scientist Secreted Procollagen Gels NC1 NC2 NC3 NC4 NC5 NC6 1 2 3 4 5 6 7 8 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

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

21. Kate Garrard BSc Pre-Registration Scientist Results and Discussion IdentifierSecreted ProcollagenIntracellular CollagenTypeOI 1ARN-terminationIV 2AB--frameshiftI 3HE-Nnoneatypical 4PPweak pro1A1-glycine subIV 5MGweak pro1A1 weak pro1A2-glycine subIV 7DDweak pro3A1 weak proN1A1pepsin variantglycine subIII 8FHweak pro3A1 weak proN1A1?overhydroxylationglycine subIII 9SANglycine-cysteine subglycine subIV 11MWN-frameshiftIV 12GFN-terminationIV 13LHN--IV 14BBNN-I 15DIweak proN1A1overhydroxylation-NK 17CMweak pro1A1 weak pro1A2overhydroxylation and pepsin variantglycine subAtypical 18MOweak pro1A1 weak pro1A2-glycine subIV 19MFweak proN1A1NframeshiftIII 20LIN--NK IdentifierSecreted ProcollagenIntracellular CollagenTypeOI 1ARN-terminationIV 2AB--frameshiftI 3HE-Nnoneatypical 4PPweak pro1A1-glycine subIV 5MGweak pro1A1 weak pro1A2-glycine subIV 7DDweak pro3A1 weak proN1A1pepsin variantglycine subIII 8FHweak pro3A1 weak proN1A1?overhydroxylationglycine subIII 9SANglycine-cysteine subglycine subIV 11MWN-frameshiftIV 12GFN-terminationIV 13LHN--IV 14BBNN-I 15DIweak proN1A1overhydroxylation-NK 17CMweak pro1A1 weak pro1A2overhydroxylation and pepsin variantglycine subAtypical 18MOweak pro1A1 weak pro1A2-glycine subIV 19MFweak proN1A1NframeshiftIII 20LIN--NK IdentifierSecreted ProcollagenIntracellular CollagenTypeOI 1ARN-terminationIV 2AB--frameshiftI 3HE-Nnoneatypical 4PPweak pro1A1-glycine subIV 5MGweak pro1A1 weak pro1A2-glycine subIV 7DDweak pro3A1 weak proN1A1pepsin variantglycine subIII 8FHweak pro3A1 weak proN1A1?overhydroxylationglycine subIII 9SANglycine-cysteine subglycine subIV 11MWN-frameshiftIV 12GFN-terminationIV 13LHN--IV 14BBNN-I 15DIweak proN1A1overhydroxylation-NK 17CMweak pro1A1 weak pro1A2overhydroxylation and pepsin variantglycine subAtypical 18MOweak pro1A1 weak pro1A2-glycine subIV 19MFweak proN1A1NframeshiftIII 20LIN--NK

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

23. Kate Garrard BSc Pre-Registration Scientist Results and Discussion IdentifierSecreted ProcollagenIntracellular CollagenTypeOI 1ARN-terminationIV 2AB--frameshiftI 3HE-Nnoneatypical 4PPweak pro1A1-glycine subIV 5MGweak pro1A1 weak pro1A2-glycine subIV 7DDweak pro3A1 weak proN1A1pepsin variantglycine subIII 8FHweak pro3A1 weak proN1A1?overhydroxylationglycine subIII 9SANglycine-cysteine subglycine subIV 11MWN-frameshiftIV 12GFN-terminationIV 13LHN--IV 14BBNN-I 15DIweak proN1A1overhydroxylation-NK 17CMweak pro1A1 weak pro1A2overhydroxylation and pepsin variantglycine subAtypical 18MOweak pro1A1 weak pro1A2-glycine subIV 19MFweak proN1A1NframeshiftIII 20LIN--NK IdentifierSecreted ProcollagenIntracellular CollagenTypeOI 1ARN-terminationIV 2AB--frameshiftI 3HE-Nnoneatypical 4PPweak pro1A1-glycine subIV 5MGweak pro1A1 weak pro1A2-glycine subIV 7DDweak pro3A1 weak proN1A1pepsin variantglycine subIII 8FHweak pro3A1 weak proN1A1?overhydroxylationglycine subIII 9SANglycine-cysteine subglycine subIV 11MWN-frameshiftIV 12GFN-terminationIV 13LHN--IV 14BBNN-I 15DIweak proN1A1overhydroxylation-NK 17CMweak pro1A1 weak pro1A2overhydroxylation and pepsin variantglycine subAtypical 18MOweak pro1A1 weak pro1A2-glycine subIV 19MFweak proN1A1NframeshiftIII 20LIN--NK IdentifierSecreted ProcollagenIntracellular CollagenTypeOI 1ARN-terminationIV 2AB--frameshiftI 3HE-Nnoneatypical 4PPweak pro1A1-glycine subIV 5MGweak pro1A1 weak pro1A2-glycine subIV 7DDweak pro3A1 weak proN1A1pepsin variantglycine subIII 8FHweak pro3A1 weak proN1A1?overhydroxylationglycine subIII 9SANglycine-cysteine subglycine subIV 11MWN-frameshiftIV 12GFN-terminationIV 13LHN--IV 14BBNN-I 15DIweak proN1A1overhydroxylation-NK 17CMweak pro1A1 weak pro1A2overhydroxylation and pepsin variantglycine subAtypical 18MOweak pro1A1 weak pro1A2-glycine subIV 19MFweak proN1A1NframeshiftIII 20LIN--NK IdentifierSecreted ProcollagenIntracellular CollagenTypeOI 1ARN-terminationIV 2AB--frameshiftI 3HE-Nnoneatypical 4PPweak pro1A1-glycine subIV 5MGweak pro1A1 weak pro1A2-glycine subIV 7DDweak pro3A1 weak proN1A1pepsin variantglycine subIII 8FHweak pro3A1 weak proN1A1?overhydroxylationglycine subIII 9SANglycine-cysteine subglycine subIV 11MWN-frameshiftIV 12GFN-terminationIV 13LHN--IV 14BBNN-I 15DIweak proN1A1overhydroxylation-NK 17CMweak pro1A1 weak pro1A2overhydroxylation and pepsin variantglycine subAtypical 18MOweak pro1A1 weak pro1A2-glycine subIV 19MFweak proN1A1NframeshiftIII 20LIN--NK

24. Kate Garrard BSc Pre-Registration Scientist 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.

25. Kate Garrard BSc Pre-Registration Scientist 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.

26. Kate Garrard BSc Pre-Registration Scientist 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.

27. Kate Garrard BSc Pre-Registration Scientist 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.

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

29. Kate Garrard BSc Pre-Registration Scientist Secreted Procollagen Gels 1 2 3 4 5 6 7 8 9 10 11 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)

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