Extracellular matrix gene sequence variant analysis and Achilles tendinopathy

262 Pages·2013·5.72 MB·English

by  Colleen Jayne Saunders

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The copyright of this thesis vests in othe author. No
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quotation from it or information derived from it is to be

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published without full acknowledgement of the source.
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The thesis is to be used fora private study or non-
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commercial research purposes only.

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Published by the Universyity of Cape Town (UCT) in terms
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of the non-exclusive liciense granted to UCT by the author.
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Extracellular matrix gene sequence
variant analyses and Achilles
tendinopathy
By
Colleen Jayne Saunders

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Submitted to the University of Cape Town
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in fulfilment of the requirements for the degree of
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DOCTOR OF PHILOaSOPHY
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in the Department of Hum an Biology, Faculty of Health Sciences
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UsNIVERSITY OF CAPE TOWN
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v April 2013
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Supervisors:
Prof. Malcolm Collins2,1
Dr. Alison September1
Prof. Martin Schwellnus1

1MRC/UCT Research Unit for Exercise Science and Sports Medicine, Department of Human
Biology, Faculty of Health Sciences, University of Cape Town, Newlands, South Africa
2South African Medical Research Council, Cape Town, South Africa
DECLARATION
Doctoral Degrees Board
University of Cape Town
Private Bag X3, Rondebosch
7701 South Africa
Tel: [phone]
Fax: [phone]
[email protected]

PhD THESIS TITLE: EXTRACELLULAR MATRIX GENE SEQUENCE VARIANT ANALYSES AND ACHILLES
TENDINOPATHY
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1. DECLARATIONS: w
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I, Colleen Jayne Saunders, hereby: T

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(a) grant the University of Cape Town free licencpe to reproduce the above thesis in
whole or in part, for the purpose of research;a
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(b) declare that:
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(i) the above thesis yis my own unaided work, both in concept and execution, and
that apart frotm the normal guidance from my supervisor, I have received no
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assistance except as stated below:
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I gratefully received guidance and assistance with the more complex statistical analyses
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included in this thesis from Dr. Lize van der Merwe, as included in the
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acknowledgments.________________________________________________________
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(ii) neither the substance nor any part of the above thesis has been submitted in the
past, or is being, or is to be submitted for a degree at this University or at any
other university, except as stated below:
___________________N/A_________________________________________________
_______________________________________________________________________
I am now presenting the thesis for examination for the degree of PhD.
Candidate’s Signature Date

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DEDICATION
This thesis is dedicated to my father, Butch, who will never see the completion of this
dream, and my mother, Teena, for their love and uncompromising support throughout my
student career. Words do not adequately convey my appreciation for them allowing me
the freedom to follow my dreams.

TEACH YOUR CHILDREN n
Crosby, Stills, Nash and Young w
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You who are on the road, must have a code that you can live by.
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And so, become yourself, because the past is just a good bye.

Teach your children well, their father's hell edid slowly go by,
And feed them on your dreams, the one they pickepd is the one you're known by.
Don't you ever ask them why, if they atold you you would cry,
So just look at them and sigh, aCnd know they love you.

And you of the tender years, can't kfnow the fears that your elders grew by.
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And so please help them with your years, they seek the truth before they can die.

Teach your parents weyll, their children's hell will slowly go by.
And feed them on your dreamts, the one they picked is the one you're known by.
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Don't you ever assk them why, if they told you you would cry,
So just lookr at them and sigh, and know they love you.
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ACKNOWLEDGEMENTS
Thank-you to my extremely patient supervisors, Dr. Alison September and Prof. Malcolm
Collins, for sharing this experience with me. Your knowledge, guidance and support have
been invaluable to me and I will be forever grateful for the solid foundation you have given
my academic career. Alison – your technical expertise in the laboratory and pep talks
helped me push through many obstacles. Malcolm – your open-door policy and engaging
teaching practice has been invaluable and helped me build confidence in my own abilities.
Thank-you to my “stats guru”, Dr. Lize van der Merwe, for your guidnance and assistance
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with the more complex statistical analyses undertaken in this thesis.
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The love and mountains of support I received from my partner, (soon-to-be Dr.) Benoit
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Capostagno, has been invaluable. I hope I can return thpe favour!
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Thank-you to my sister, Janice, who has been continuously supportive and kept me supplied

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with home-cooked meals and cookies whenever the going got tough.

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Thank-you to all my colleagues aind friends at ESSM who help foster a supportive and
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productive environment. In eparticular Dr. Kristina Plattner, Dr. Caroline D’Alton, Nicholas
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Tam, Dr. Liesl de Milander, Dr. Yumna Albertus-Kajee, Dr. Michael Posthumus, Dr. Robert
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Lamberts and Mark Kirkman have travelled this path with me and made it an enjoyable
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journey. I could not have wished for more supportive and inspiring office companions than
the hard-working Dr. Sharief Hendricks, James Brown and Benoit Capostagno – keep the
coffee and laughs rolling! My close friends, particularly Sarah Sandmann and Kim
Germishuys, have loved and supported me for many years – I hope to spend more time on
the beach with you now!
Thank-you to the wonderful administrative staff at ESSM who have assisted me with a smile
so often, as well as Adri Winkler and her team at the Faculty of Heath Sciences postgraduate
office who are always helpful, efficient and supportive. Thank you also to Neezaam Kariem
and Trevino Larry for their willing assistance in the laboratory.
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Thank you to the South African and Australian participants who donated their time and DNA
samples to these studies. Thank you to Dr. George Mokone, Prof. Martin Schwellnus, Prof.
Jill Cook and Prof. Christopher Handley who were involved in the initial recruitment and
diagnosis of the South African and Australian participants. I would also like to acknowledge
the contribution of Abrahams et al.,6 El Khoury et al.,76 Nell et al.,205 Posthumus et al.224 and
September et al.255,257 who completed the previously published genotyping for the
polymorphisms included in chapter 5 of this thesis.
Thank you to the examiners who kindly agreed to review and examine this thesis. Your time
and valuable input is greatly appreciated, and your constructive comments greatly helped to
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improve the quality of this thesis. w
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Finally, my studies would not have been possible without financial assistance gratefully

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received from the University of Cape Town postgraduate funding office (Benfarra
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scholarship, Harry Crossley Foundation scholarshipa, Marion Beatrice Waddell scholarship,
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University Research Committee scholarship and UCT conference travel grant) and the

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National Research Foundation (NRF Granthoolder bursary).

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TABLE OF CONTENTS
DECLARATION ............................................................................................................................ 1
DEDICATION ............................................................................................................................... 3
ACKNOWLEDGEMENTS .............................................................................................................. 5
TABLE OF CONTENTS.................................................................................................................. 7
SCIENTIFIC OUTPUTS ASSOCIATED WITH THIS THESIS ............................................................ 13
Publications in peer-reviewed journals ................................................................................ 13
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Presentations at international conferences ......................................................................... 13
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Presentations at local conferences ...................................................................................... 14

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ABBREVIATIONS ...............................................................p........................................................ 15
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LIST OF TABLES ........................................................C................................................................. 19

LIST OF FIGURES .............................................f.......................................................................... 23
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ETHICAL APPROVAL & FUNDING ..........y................................................................................... 29
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THESIS ABSTRACT ..................................................................................................................... 31
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CHAPTER 1: ACHILLES TvENDINOPATHY: A REVIEW ............................................................ 35
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1.1. Introduction and scope of thesis ............................................................................. 35
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1.2. Tendon anatomy ...................................................................................................... 36
1.2.1. Macro-structure of tendons .............................................................................. 36
1.2.2. Hierarchical micro-structure of tendons ........................................................... 38
1.3. Molecular structure of tendons ............................................................................... 40
1.3.1. Collagen .............................................................................................................. 41
1.3.2. Non-collagenous proteins of the extracellular matrix ....................................... 45
1.4. Achilles Tendon Pathology ....................................................................................... 47
1.4.1. Prevalence of Achilles tendon rupture and tendinopathy ................................ 47
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1.4.2. Aetiology ............................................................................................................ 48
1.4.3. Gene expression in tendinopathy ...................................................................... 59
1.5. Risk factors for Achilles tendon pathology .............................................................. 61
1.5.1. Extrinsic risk factors ........................................................................................... 62
1.5.2. Intrinsic risk factors ............................................................................................ 71
1.6. Summary and conclusions of the literature review ................................................. 85
1.7. Aims and objectives of the thesis ............................................................................ 86
CHAPTER 2: VARIANTS WITHIN THE COMP AND THBS2 GENES ARE NOT ASSOCIATED
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WITH ACHILLES TENDINOPATHY .............................................................................................. 89
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2.1. Introduction .................................................................T............................................ 89

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2.2. Materials and methods ............................................................................................ 92
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2.2.1. Participants ........................................................................................................ 92
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2.2.2. DNA extraction ............................ ....................................................................... 93
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2.2.3. Variant selection ................ ................................................................................ 94
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2.2.4. Genotyping ..............s........................................................................................... 95
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2.2.5. Statistical analyses ............................................................................................. 99
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2.3. Results ..........n.......................................................................................................... 100
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2.3.1. Descriptive characteristics ............................................................................... 100
2.3.2. Genotype and allele frequencies ..................................................................... 101
2.4. Discussion............................................................................................................... 106
2.5. Conclusion .............................................................................................................. 109
CHAPTER 3: HAPLOTYPE ANALYSIS OF COL27A1 & TNC CANDIDATE GENES AS RISK
FACTORS FOR ACHILLES TENDINOPATHY .............................................................................. 111
3.1. Introduction ........................................................................................................... 111
3.2. Materials and methods .......................................................................................... 117
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