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Kate Garrard BSc

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Setting up a Diagnostic Service to Investigate the Production of Type I Collagen in Patients with Osteogenesis Imperfecta Kate Garrard 4th April 2008 – PowerPoint PPT presentation

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Title: Kate Garrard BSc


1
Setting up a Diagnostic Service to Investigate
the Production of Type I Collagen in Patients
with Osteogenesis Imperfecta
Kate Garrard 4th April 2008
2
Summary
  • Introduction to Osteogenesis Imperfecta (OI)
  • Current Service
  • Collagen Production
  • Protocol for investigating collagen production
  • Results and Discussion
  • Conclusions

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

4
Clinical Features
  • Dentiogenesis imperfecta


5
Clinical Features
  • Wormian bones

6
Clinical Features
  • Beaded ribs

7
Clinical Features
  • Blue Sclera


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

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

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
11
Structure of Collagen 1
Gly
Y
X
Y
X
12
Collagen Production
DNA
Cell Membrane
13
Collagen Fibrils
14
Collagen Analysis
  • So if we want to look for aberrant collagen how
    do we do this?

15
Cohort
Identifier 1A1 sequenced 1A2 sequenced Gene Mutation (cDNA No.) Mutation (Protein No.) Type OI
1AR yes no COL1A1 c.2643CgtT p.Arg882X termination IV
2AB yes no COL1A1 c.3581delG p.Gly1195fs frameshift I
3HE yes yes - NN none atypical
4PP yes yes COL1A2 c.604GgtC p.Gly202Arg glycine sub IV
5MG yes no COL1A1 c.589GgtC p.Gly187Arg glycine sub IV
7DD yes no COL1A1 c.994GgtA p.Gly332Arg glycine sub III
8FH yes no COL1A1 c.2436GgtA p.Gly773Ser glycine sub III
9SA yes no COL1A1 c.814GgtT p.Gly272Cys glycine sub IV
11MW yes no COL1A1 c.1378_1379insC p.Gly461fs frameshift IV
12GF yes no COL1A1 c.3806GgtA p.Trp1269X termination IV
13LH no no - - - - IV
14BB yes (in father) yes (in father) - NN - - I
15DI no no - - - - IV
17CM yes no COL1A1 c.1939GgtA p.Gly647Ser glycine sub atypical
18MO yes no COL1A1 c.1012GgtT p.Gly338Cys glycine sub IV
19MF yes no COL1A1 c.3580_3581delGC p.Ala1194fs frameshift III
20LI no no - - - - NK
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
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
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
19
Secreted Procollagen Gels
1 2 3 4 5 6 7
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
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
21
Results and Discussion
Identifier 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
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
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
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
23
Results and Discussion
Identifier 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
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
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
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
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.

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.

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.

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.

28
Clinical Features
  • Wormian Bones

29
Secreted Procollagen Gels
1 2 3 4 5 6 7
8 9 10 11 12 13 14
pro3A1
pro1A1
proN1A1
pro1A2
a1(III) and a1(I)
Procollagena2(I)
ProNa2(I)
a2(I)
15 16 17 18 19 20 21 NC1 NC2
NC3 NC4 NC5 NC6
pro3A1
pro1A1
proN1A1
pro1A2
a1(III) and a1(I)
Procollagena2(I)
ProNa2(I)
a2(I)
30
Structure of Collagen 1
  • The three procollagens spiral together to form a
    heterotrimeric triple helix.
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