identification of novel anatomical risk factors and susceptibility loci/genes for primary angle closure

345 Pages·2015·15.47 MB·English

by  Nongpiur Monisha Esther

IDENTIFICATION OF NOVEL ANATOMICAL RISK FACTORS
AND SUSCEPTIBILITY LOCI/GENES FOR PRIMARY ANGLE
CLOSURE GLAUCOMA (PACG)

NONGPIUR MONISHA ESTHER
(M.D)

A THESIS SUBMITTED

FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
DEPARTMENT OF OPHTHALMOLOGY
NATIONAL UNIVERSITY OF SINGAPORE

2015
DECLARATION

I hereby declare that the thesis is my original work and it has been written
by me in its entirety. I have duly acknowledged all the sources of
information which have been used in the thesis.

This thesis has also not been submitted for any degree in any university
previously.

_______________________________

Nongpiur Monisha Esther
15 January 2015

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THESIS SUPERVISORS AND COMMITTEE
Thesis Supervisors:
Tin Aung, Ph.D., Professor, Dept. of Ophthalmology, Yong Loo Lin School of
Medicine, National University of Singapore. (Main)

Eranga Vithana, Ph.D., Adjunct Associate Professor, Dept. of Ophthalmology,
National University of Singapore. (Co-supervisor)

Thesis Advisory Committee (TAC):
Tai E Shyong, PhD., Associate Professor, Dept. of Medicine, Yong Loo Lin School of
Medicine, National University of Singapore.

Ching-Yu Cheng, PhD., A/Professor, Dept. of Ophthalmology, Yong Loo Lin School
of Medicine, National University of Singapore

Tin Aung, Ph.D., Professor, Dept. of Ophthalmology, Yong Loo Lin School of
Medicine, National University of Singapore.
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ACKNOWLEDGEMENTS
First and foremost, I would like to thank my supervisors, Prof Tin Aung and
Assoc Prof Eranga Vithana for their unwavering support, encouragement and advice
throughout the course of this PhD. I am truly indebted to Tin for providing me with
the opportunity to undertake this journey and to steer me through my academic
development. Eranga is a mentor and friend from whom I have learnt the valuable
skills of critical thinking and technical writing. She has always found the time for
insightful and detailed discussions, and advice. Thank you Tin and Eranga. I am also
grateful to Prof Wong, Asst Prof CC Khor, and to the members of my Thesis
Committee A/Prof Ching-Yu Cheng and A/Prof E Shyong Tai for their invaluable
advice, guidance and assistance on analyses, methodology and inputs for publications.
Thank you Baskar, Arun, and Dr Htoon for the numerous exciting discussions
and it has been a pleasure working together. To Belinda, Wan Ting, Tun and JiaYu,
thank you for your help. I extend my gratitude to all the members of the Glaucoma
team at SERI, specially Serena Ting, Victor, and Xiao Yu. You guys are awesome!!
The ever-expanding genetics database and the smooth coordination with GIS would
not have been possible without you guys. I would also like to thank all our
collaborators, both within Singapore and overseas who have helped recruit glaucoma
patients and collect their DNA samples. Without their passionate participation, the
genetic aspect of this project could not have been successfully conducted. I am
thankful to Dr Mingguang He for providing the image analysis software (Zhongshan
Angle Assessment Program) to process the ASOCT images.
I would like to thank my family for all their love, continual encouragement,
support and prayers. I thank all my friends and colleagues who have supported me
over the last few years, especially Mhalo, James, Neha, Anand, Smitha and Purnima.
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TABLE OF CONTENTS
Declaration Page ............................................................................................................ ii
Thesis Supervisors and Committee..…………………….……….…………..……… iii
Acknowledgements ....................................................................................................... iv
Table of Contents ........................................................................................................... v
Summary ...................................................................................................................... xii
List of Tables .............................................................................................................. xiv
List of Figures ............................................................................................................ xvii
List of Abbreviations ………………………………………………………………. xix
List of Publications ……...………………………………………..……………….. xxi

CHAPTER 1
GENERAL INTRODUCTION
1.1 PRIMARY ANGLE CLOSURE GLAUCOMA …………........................… 01
1.1.1 Classification of Angle Closure ……................….…………………………. 03
1.1.2 Global Prevalence of PACG and Impact on Vision …................................... 05
1.1.3 Magnitude of the Problem in Singapore ….................................................... 13
1.1.4 Mechanisms Responsible for Angle Closure ….............................................. 14
1.1.5 Known Risk Factors for Angle Closure ……………………………............. 18
1.2 ANGLE CLOSURE IS NOT FULLY EXPLAINED BY VARIATIONS
IN THE KNOWN BIOMETRIC FEATURES .............................................. 23
1.3 GENETIC ETIOLOGY OF ANGLE CLOSURE …..................................... 24
1.3.1 Evidence for the Genetic Basis of Angle Closure. …………...............….….. 24
1.3.2 Potential Candidate Genes for Angle Closure …........................................... 27
1.3.3 Recently Identified Novel PACG Loci ………………………....................… 29
1.4 UNANSWERED RESEARCH QUESTIONS IN ANGLE CLOSURE ....... 30
1.5 TECHNOLOGICAL ADVANCES RELEVANT TO ANGLE
CLOSURE...................................................................................................... 31
1.6 STUDY AIMS …………………………………..................................….… 33
1.7 OVERALL STUDY PLAN …....................................................................... 35

CHAPTER 2
SUBJECTS AND METHODS
2.1 STUDY POPULATIONS ………………………………………………….. 36
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2.1.1 Hospital-based Subjects…………………………………………….....……. 36
2.1.2 Population-based Studies ……………………………….……………..…… 36
2.2 CLINICAL EXAMINATIONS ………………………………..........….….. 40
2.3 ASOCT IMAGING ……………………………………………….……...… 43
2.3.1 Principle of ASOCT ……………………………….……………………..… 44
2.3.2 Processing of ASOCT Images ……………………………………………… 45
2.3.3 Scleral Spur Identification in ASOCT Images …………………….......…… 45
2.3.4 Anterior Segment Parameters Measured by ZAAP ………………………… 47
2.3.5 Features of ‘Good software Delineation …………………………………… 49
2.3.6 Limitations of the ZAAP ……………………………………………………. 49
2.3.7 Reproducibility of the ZAAP Software ………………………...…………… 50
2.4 GENOME-WIDE ASSOCIATION STUDIES: PRINCIPLE AND
REQUISITES OF A ROBUST STUDY …...………………………………. 52
2.4.1 Design and Conduct of GWA Studies ………………………………………. 53
2.4.1.1 GWA Study Designs ……………………………………………………...… 53
2.4.1.2 Selection of Study Subjects ………………………………………......…….. 55
2.4.1.3 Genotyping …………………………………………………………………. 56
2.4.1.4 Quality Control …………………………………………………………….. 56
2.4.1.5 Association Analyses and Correction for Multiple Testing ……...……....… 58
2.4.1.6 Visualization and Interpretation of Results ………………………………… 59
2.4.1.7 Replication and Meta-analysis ……………………………………………... 60
2.4.1.8 Refining Genome-wide Association Signals ………………………...……… 61
2.4.2 Limitations of Genome-wide Association Studies ………………………...….. 62
2.5 GENOTYPING AND STATISTICAL ANALYSIS …....................................... 62

CHAPTER 3
IMAGING-BASED RISK FACTORS FOR ANGLE CLOSURE
3.1 BACKGROUND …………………………………………………………... 65
3.2 SUBJECTS AND METHODS ….................................................................. 69
3.2.1 Study Populations …………………………………………………...….….. 69
3.2.2 Clinical Examinations and Biometry………………...........................………70
3.2.3 ASOCT imaging and Analysis ……………………………………....……… 71
3.2.4 Sample size estimation……………………………………………...….…… 72
3.2.5 Statistical Analyses ………………………………………………...….…… 72
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3.3 RESULTS ………………………………………………………………...... 74
3.3.1 LENS VAULT, THICKNESS AND POSITION IN ANGLE CLOSURE... 75
3.3.1.1 Distribution of lens parameters by decades ……………………….………. .75
3.3.1.2 Comparison between Angle closure and Normals …………………………. 76
3.3.1.3 Association of parameters with Angle closure ……………………….…….. 77
3.3.1.4 Correlation among the parameters ………………………………………… 79
3.3.1.5 Performance characteristics for detection of angle Closure ………………. 81
3.3.1.6 Effect of lens thickness on the other parameters …………………………… 82
3.3.1.7 Comparison between Angle closure subtypes ……………………………… 83
3.3.2 THE IMPACT OF LENS VAULT ON VISUAL ACUITY AND
REFRACTIVE ERROR ……………………………………….….….…….. 84
3.3.2.1 Patient demographics ………………………………………………………. 84
3.3.2.2 Visual acuity and spherical equivalent stratified by Lens vault ……………. 86
3.2.4.3 Linear regression for association with Lens vault …………………………. 88
3.3.3 INCREASED LENS VAULT AS A RISK FACTOR FOR ANGLE
CLOSURE: CONFIRMATION IN A JAPANESE POPULATION ………. 91
3.3.4 ANTERIOR CHAMBER WIDTH AND ANGLE CLOSURE …………..... 96
3.3.4.1 Patient demographics ………………………………………………………. 96
3.3.4.2 Distribution of Anterior chamber width by age and gender …..…………….97
3.3.4.3 Relationship of systemic and ocular factors with Anterior chamber width… 97
3.3.4.4 Association of Anterior chamber width with Angle closure …………...….. 102
3.3.4.5 Comparison with hospital-based subjects ………………………………… 103
3.3.5 LENS VAULT RELATED PARAMETERS AND ANGLE CLOSURE.... 105
3.3.5.1 Demographics and association of Lens vault related parameters with
Angle closure ……………………………………………………………... 107
3.3.5.2 Comparison between subtypes of Angle closure ………………………….. 114
3.4 DISCUSSION …………………………………………………………….. 116
3.4.1 Lens vault: A better marker for assessing role of the lens in Angle Closure 116
3.4.2 Concept and mechanism of Lens vault ……………………....……………. 118
3.4.3 Characteristics of Lens vault in normal …………………………………... 119
3.4.4 Characteristics of Lens vault in Angle closure …………........…………… 119
3.4.5 Lens vault is independent of Lens thickness ……………………………… 120
3.4.6 Confirmation of the importance of the LV in another ethnic population … 121
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3.4.7 Is the lens vault a good screening parameter for angle closure? ...……... 122
3.4.8 The magnitude of the Anterior Vault remains stable with age …………… 123
3.4.9 Lens extraction in Angle closure: Is there a role for the Lens vault
in the management of angle closure? …………………………………….. 123
3.4.10 Anterior chamber width: Effect on the anterior chamber drainage region. 126
3.4.11 Anterior chamber width: Associations with Age, BMI and Educational level127
3.4.12 Anterior chamber width and Angle closure ……………....………...…….. 128
3.4.13 Characteristics of ASOCT parameters in subtypes of angle closure ……... 129
3.5 SUMMARY AND CONCLUSIONS ……………………………………... 131
3.6 STRENGTHS AND OF THE IMAGING STUDIES …………...……….. 132
3.7 OVERALL STUDY LIMITATIONS ………....……………………….…. 134
3.8 CAN THE ESTABLISHED AND NOVEL ANATOMICAL RISK
FACTORS BE CONSIDERED AS INTERMEDIATE PHENOTYPE FOR
ANGLE CLOSURE? ……………………………………………….…….. 136

CHAPTER 4
GENETIC LOCI FOR ANGLE CLOSURE
4.1 ENDOPHENOTYPE APPROACH TOWARDS IDENTIFICATION OF
SUSCEPTIBILITY LOCI FOR ANGLE CLOSURE ………………....…. 137
4.1.1 Genome-wide approaches for the identification of genetic variants for
complex diseases ………………………………………………………….. 137
4.1.2 What is an endophenotype? ……………………….……………......…….. 138
4.1.3 Identification of disease genes through the quantitative traits/ endophenotype
approach ………………………………………………………………..... 139
4.1.4 Identification of PACG genes through the Endophenotype approach ….... 142
4.2 GENETIC DETERMINANTS OF ANTERIOR CHAMBER DEPTH ...... 143
4.2.1 Evidence for genetic basis for ACD ...………………….…………………. 144
4.2.2 Aims of the study ………………………………………………………….. 146
4.2.3 Material and methods ……………………………………………………...147
4.2.4 Statistical Analysis ………………………...……………………………… 149
4.2.5 Results …………………………………………………………………….. 151
4.2.5.1 Identification of a quantitative trait locus for anterior chamber depth ...… 151
4.2.5.2 Association between ABCC5 rs1401999 and PACG ……………………... 154
4.2.5.3 What is the effect of age and gender? …………………………………….. 159
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4.2.5.4 Conditional analysis with recently identified PACG loci ……………...…. 160
4.2.5.5 The effect of rs1401999 when using the left eye ACD measurement …...… 161
4.2.5.6 What is the Effect of ABCC5 rs1401999 on axial length? ………………... 161
4.2.5.7 Expression of ABCC5 in eye tissues …………………………………........ 162
4.2.5.8 Power Calculations …………………………………………………....….. 165
4.2.6 Discussion ………………………………………………………………....... 166
4.2.6.1 ABCC5 gene: Does it have a possible role in eye development? …….…… 166
4.2.6.2 Strength of association improves with the use of pre-selected
‘Super-controls’ …………………………………………………………... 169
4.2.6.3 Modest effect size of ABCC5 ……………...………………………………. 170
4.2.3.4 ABCC5 more strongly associated with Malays and Indians ………...…… 170
4.3 ASSOCIATION OF THE THREE PACG LOCI WITH ANTERIOR
CHAMBER DEPTH AND AXIAL LENGTH …………………………… 172
4.3.1 Purpose ……………………………………………………………...……. 172
4.3.2 Methods ……………………………...……………………………………. 172
4.3.3 Statistical Analysis ………………………………………………………... 173
4.3.4 Results …………………………………………………………………….. 174
4.3.5 Discussion …………………………………………………………...…..... 178
4.3.4.1 Lack of association with ACD and AL in Asian cohorts ………………….. 178
4.3.4.2 Are the loci associated with other anatomical traits? ………………...….. 179
4.3.4.3 Other likely reasons for the lack of significant associations between the PACG
loci with ACD and AL …………………..………………………………… 183
4.4 GENETIC DETERMINANTS OF ANTERIOR CHAMBER WIDTH ….. 185
4.4.1 Heritability, distribution and reproducibility of ACW …............................. 185
4.4.2 Methods ………...…………………………………………………………. 189
4.4.3 Statistical Analysis ………………………………………………...……… 190
4.4.4 Results …………………………………………..………………………… 190
4.4.4.1 Identification of the genetic determinants for anterior chamber width …... 190
4.4.4.2 Power of the currently available data and sample size required to achieve
adequate power …………………………………………………………… 194
4.4.4.3 The regions that shows promising SNPS ……………………...…….……. 195
4.4.4.4 Can MBNL2 be a potential gene for ACW? ………………….…………… 195
4.4.4.5 Are there genetic determinants for the other ASOCT-based risk factors for
angle closure? …………………………………………………………….. 196
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CHAPTER 5
GENERAL DISCUSSION AND IMPLICATIONS
5.1 MAIN FINDINGS OF THE IMAGING DATA ……….…………………. 198
5.1.1 Is there an appropriate and accurate screening test for Angle closure? …. 200
5.1.2 Can the novel parameters be useful as potential screening tools for angle
closure? ……………………………………………………………....…… 205
5.2 CLINICAL IMPLICATIONS AND PROGRESS MADE FROM THE
FINDINGS OF THIS THESIS ……………………………………………. 206
5.2.1 Determinants of Angle Width …................................................................... 207
5.2.2 Classification algorithms for detection of Angle Closure and the ‘Angle
closure Score’ ….......................................................................................... 210
5.3 MAIN FINDINGS OF THE DETERMINATION OF GENETIC LOCI FOR
ANGLE CLOSURE ……………………………………………........……. 213
5.3.1 What are the other endophenotypes for PACG? …...................................... 214
5.3.2 What is the role of the PACG associated genes identified to date? ………. 215
5.4 IMPLICATIONS OF THE NEW BIOMARKERS FOR ANGLE CLOSURE
GLAUCOMA ……………………………………………….…………….. 216
5.4.1 Improved screening and prevention of blindness …………………………. 216
5.4.2 Expand the spectrum of genetic loci associated with PACG disease …...… 217
5.4.3 Improved Risk Assessment for PACG…....................................................... 218
5.4.4 Economic Implications ……………………………………………………. 218

CHAPTER 6
FUTURE ENDEVOURS
6.1 TO IDENTIFY FURTHER SUSCEPTIBILITY LOCI FOR PACG …….. 221
6.1.1 Quantitative endophenotype approach using ASOCT-based quantitative
traits ……………...…………………………………………….……...….. 221
6.1.2 Other methodologies to identify PACG susceptibility loci ……….……..... 223
6.2 REFINING GWAS SIGNALS ………………………………………........ 224
6.3 DEVELOP/ CONSTRUCT A PREDICTIVE RISK MODEL FOR PACG 224

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