kate garrard bsc pre-registration scientist setting up a diagnostic service to investigate the...

Kate Garrard BScPre-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


Summary

• Introduction to Osteogenesis Imperfecta (OI)

• Current Service

• Collagen Production

• Protocol for investigating collagen production

• Results and Discussion

• Conclusions


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


Clinical Features

• Dentiogenesis imperfecta


Clinical Features

• Wormian bones


Clinical Features

• Beaded ribs


Clinical Features

• Blue Sclera


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

Severe Mild


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

Severe Mild


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


Structure of Collagen 1

C-telopeptideShort triple helical domain

N-propeptide N-telopeptide Main triple helical domain

C-propeptide

GlyX

YY

XGly

Gly


Collagen ProductionDNA

Cell Membrane

Post translational modification enzymes

Ribosome

Transcription factor

Precursor mRNA Spliceosomes

Mature mRNA

Preproα chain

Proα chain

Procollagen

Mature CollagenProcollagenN


Collagen Fibrils


Collagen Analysis

• So if we want to look for aberrant collagen how do we do this?


CohortIdentifier

1A1sequenced

1A2 sequenced Gene

Mutation(cDNA No.)

Mutation(Protein No.) Type OI

1AR yes no COL1A1 c.2643C>T p.Arg882X termination IV

2AB yes no COL1A1 c.3581delG p.Gly1195fs frameshift I

3HE yes yes - N:N none atypical

4PP yes yes COL1A2 c.604G>C p.Gly202Arg glycine sub IV

5MG yes no COL1A1 c.589G>C p.Gly187Arg glycine sub IV

7DD yes no COL1A1 c.994G>A p.Gly332Arg glycine sub III

8FH yes no COL1A1 c.2436G>A p.Gly773Ser glycine sub III

9SA yes no COL1A1 c.814G>T p.Gly272Cys glycine sub IV

11MW yes no COL1A1 c.1378_1379insC p.Gly461fs frameshift IV

12GF yes no COL1A1 c.3806G>A p.Trp1269X termination IV

13LH no no - - - - IV

14BB yes (in father) yes (in father) - N:N - - I

15DI no no - - - - IV

17CM yes no COL1A1 c.1939G>A p.Gly647Ser glycine sub atypical

18MO yes no COL1A1 c.1012G>T p.Gly338Cys glycine sub IV

19MF yes no COL1A1 c.3580_3581delGC p.Ala1194fs frameshift III

20LI no no - - - - NK


TechniqueCulture fibroblasts with ascorbic acid.

Incubate radioactively labelled proline for 24hrs

Harvest secreted collagens (media) Harvest intra cellular collagens

Ethanol precipitateSolubilise 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)


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


TechniqueCulture fibroblasts with ascorbic acid.

Incubate radioactively labelled proline for 24hrs

Harvest secreted collagens (media) Harvest intra cellular collagens

Ethanol precipitateSolubilise intracellular collagens in specific acetic acid concentration

Isolate whole cells and retrieve contents


standardise radioactivity in samples

Lyophilise

Electrophorese

Autoradiograph

Leave undigested (procollagens)

Ethanol precipitate

Electrophorese



Secreted Procollagen GelsNC1 NC2 NC3 NC4 NC5 NC6 1 2 3 4 5 6 7 8

pro3A1

pro1A1

proN1A1pro1A23A1 and 1A1procollagen1A2

proN1A2

COL1A2

9 10 11 12 13 14 15 16 17 18 19 20 21

pro3A1

pro1A1

proN1A1pro1A2


Intracellular Collagen GelsNC1 NC2 NC3 NC4 NC5 NC6 3 2 4 6 7 8 9 10

COL5A1

COL3A1

COL5A2COL1A1

COL1A2

COL5A3

COL1A1

COL1A2

14 15 16 17 18 19 20

COL5A1

COL3A1

COL5A2COL5A3


Results and DiscussionIdentifier Secreted Procollagen Intracellular Collagen Type OI

1AR N - termination IV

2AB - - frameshift I

3HE - N none atypical

4PP weak pro1A1 - glycine sub IV

5MG weak pro1A1 weak pro1A2 - glycine sub IV

7DD weak pro3A1 weak proN1A1 pepsin variant glycine sub III

8FH weak pro3A1 weak proN1A1 ?overhydroxylation glycine sub III

9SA N glycine-cysteine sub glycine sub IV

11MW N - frameshift IV

12GF N - termination IV

13LH N - - IV

14BB N N - I

15DI weak proN1A1 overhydroxylation - NK

17CM weak pro1A1 weak pro1A2 overhydroxylation and pepsin variant glycine sub Atypical

18MO weak pro1A1 weak pro1A2 - glycine sub IV

19MF weak proN1A1 N frameshift III

20LI N - - NK

Identifier Secreted Procollagen Intracellular Collagen Type OI











13LH N - - IV

14BB N N - I





20LI N - - NK












13LH N - - IV

14BB N N - I





20LI N - - NK


Intracellular Collagen GelsNC1 NC2 NC3 NC4 NC5 NC6 3 2 4 6 7 8 9 10

COL1A1

COL1A2

COL1A1

COL1A2

14 15 16 17 18 19 20


Results and DiscussionIdentifier Secreted Procollagen Intracellular Collagen Type OI











13LH N - - IV

14BB N N - I





20LI N - - NK












13LH N - - IV

14BB N N - I





20LI N - - NK












13LH N - - IV

14BB N N - I





20LI N - - NK












13LH N - - IV

14BB N N - I





20LI N - - NK


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.


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.


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.


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.


Clinical Features

• Wormian Bones


Secreted Procollagen Gels1 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)


Structure of Collagen 1• The three procollagens spiral together to form a

heterotrimeric triple helix.

kate garrard bsc pre-registration scientist setting up a diagnostic service to investigate the...

Documents

normal collagen

aberrant collagen

normal levels of collagen

collagen production

low levels of collagen

production of type

dentogenesis imperfecta

type of defect