Genetic analysis of eight candidate genes in South Asian and Caucasian rheumatoid arthritis ...

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Genetic analysis of eight
candidate genes in South
Asian and Caucasian
rheumatoid arthritis patients
of the East Midlands
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• A Doctoral Thesis. Submitted in partial fulfilment of the requirements
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Genetic Analysis of Eight Candidate Genes in South Asian and Caucasian
Rheumatoid Arthritis Patients of the East Midlands
ANANT GHELANI
B.Sc., M.Sc.
Supervisor: Dr S S Mastana
Director of Research: Professor N Cameron
Doctoral Thesis
Submitted in partial fulfilment of the requirements for the award of
PhD in Human Genetics of Loughborough University
Human Sciences Department
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ABSTRACT
Rheumatoid Arthritis (RA) is a chronic musculoskeletal disease of unknown
aetiology. RA is a complex polygenic and multifactorial disease and has not been
analysed comprehensively among South Asians, specifically in the East Midlands.
Two genetic approaches were used; case-control and sib-TDT analyses. Ten
polymorphisms of eight genes (ACE, VDR, A2M, GSTTt and GSTMt, TNFRII,
FcyRIIIA and CRH) were analysed in South Asians (134 patients, 66 unaffected sibs,
149 random controls) and Caucasians (137 pateints, 83 unaffected sibs, 150 random
controls).
The epidemiological results suggest that the South Asians were progressing to the
disease significantly earlier (X2 = 21.01, 5 df, P < 0.005). The gender distribution
(male:female) was 1:4 in South Asians and 1:3 in Caucasians.
Significant genetic associations were observed with VDR Bsrn I B-B genotype (OR=
2.08, Cl 1.23- 3.52, P < 0.05), A2M 2-2 genotype (OR= 3.99, Cl 1.19- 17.18, P <
0.05), and GST Ttnull genotype (OR= 2.81, Cl 1.40- 5.77, P < 0.002) among South
Asian RAs. In Caucasians, TNFRII R-R (OR= 3.16, Cl 1.20-9.26, P < 0.05), A2M 1-
1 (OR= 2.09, Cl 1.21-3.64, P < 0.05) and GST Ttnull (OR= 1.97, Cl 1.07- 3.68, P <
0.05) genotypes were associated with RA. In the majority of cases, recessive and
multiplicative modes of inheritance explained the observed associations. There were
no confounding interactions between the genotypes showing significant associations
in both groups.
Overall this study demonstrates that ethnic and genetic variation plays a significant
role in RA susceptibility and progression. The observed genetic associations may
have pharmacogenetic and diagnostic implications. Mechanisms to some of the
possible pathogenic changes observed in RA are suggested by the study.
I
ACKNOWLEDGMENTS
I am greatly indebted to Dr. Ash Samanta, Senior Consultant Rheumatologist,
Rheumatology Department, Leicester Royal Infirmary NHS Trust, Leicester, UK.,
LEl 5WW, for permitting access to the Rheumatoid Arthritis patients at his clinics in
Leicestershire Hospitals, and for requesting co-operation from Dr. Leslie Goh, Senior
Registrar, other Rheumatology Consultants, Specialist Nurses, Nursing Staff in
Leicestershire Hospitals and a few General Practitioners in Leicestershire. For an
additional cohort of participants, I am thankful to Dr Adrian Jones, Consultant
Rheumatologist based at City Hospital, Nottingham, NG5 lPB, and Queens Medical
Centre, Nottingham, NG7 2UH, Nottinghamshire, UK. Without any exception, I am
most grateful to all the participants who kindly volunteered in this study.
I am extremely grateful to Dr. Sarabjit Singh Mastana, Senior Lecturer in Human
Genetics, Human Sciences Department, Loughborough University, Loughborough,
for accepting me as Research Student for the higher degree of Doctor of Philosophy
in Human Genetics. Dr. Mastana, whilst supervising me, constantly gave me valuable
advice, assistance and encouragement throughout the period of my study and allowed
me full access to departmental research and other facilities. I am indebted to Dr.
Mastana for providing access to his DNA collection of Random Controls of either
ethnicity.
I am grateful to Dr. Harriet Gross, Head of Postgraduate Research Studies for
accepting my preliminary thesis proposal in January 2000 and Professor Noel
Cameron for acting as Director of Research for the whole duration of my study at
Loughborough University. It gives me great pleasure to express my thanks to Mrs.
Alice Pacynko who patiently taught me molecular genetic techniques. Equally, I am
grateful to Miss Ashley Gilmour in assisting me with other techniques in the recently
refurbished Human Genetic Laboratories at the University and assisting in reviewing
the manuscript.
I am extremely grateful to Dr. John D. Isaacs, Professor of Clinical Rheumatology,
University ofNewcastle-Upon-Tyne, Framlington Place, Newcastle-Upon-Tyne, NE2
4HH, Dr. Ann W Morgan, ARC Clinical Scientist/Lecturer in Rheumatology,
11
and Jim Robinson, Research Assistant, both at the Molecular Medicine Unit, St
James's University Hospital, Beckett Street, Leeds, West Yorkshire, LS9 7TF for
assisting me with SSCP analysis for Fciiiy receptor gene. Equally, I am indebted to
Dr. Sophia Steer, ARC Clinical Lecturer and Miss Bhaneeta Lad, Research Assistant,
both at the Department of Rheumatology, GKT School of Medicine, Kings College,
London, SE1 9RT, for assisting with the use of ABI 377 Prism SNP analyser for the
detection of CRH RA1 and CRH RA short tandem repeat (STR} markers of CRH
2
gene and Sheila Fisher for performing statistical analysis of the microsatellite
markers. I am also indebted to Dr. Derek Mattey and Mrs Nicola B. Nixon, both of
Staffordshire Rheumatology Centre, Research Laboratory, Haywood Hospital, High
Lane, Burslem, Stoke-on-Trent, ST6 7AC for familiarising me with GST T1 and M1
genotyping techniques. Also, I am grateful to Dr Anne Barton, Clincal Research
Fellow, Dr Jane Worthington, Reader in Molecular Genetics and Professor Alan
Silman, ARC Director, Epidemiology and Health Sciences, University of Manchester,
Oxford Road, Manchester, UK M13 9PL for their kind suggestion with reference to
TNF genotyping.
I am greatly indebted to Nick Smith for familiarising me with the SPSS data analysis
package and Rosie Shier of Mathematics Learning Support Centre, Loughborough
University for assisting with binary logistic regression analysis of the data.
I am also thankful to the administrative and other technical staff at the University for
supporting me by suggesting appropriate modifications to my research project.
Lastly, but not least, I am grateful to my wife for the financial support and my son
who provided computer plus secretarial support. The research project was partly
funded, for a period of three years, by AstraZeneca, R&D Charnwood,
Pharmaceutical Research, Bakewell Road, Loughborough, Leicestershire, LE 11 5RH.
xxXXxx
iii
List of Abbreviations used in the text (Pages iv and v).
Abbreviation Terminology
A2M = Alpha-2-macroglobulin
ACE = Angiotensin converting enzyme
AD = Alzheimer's Disease
Anti-IL = Anti-interleukin
xz
= Chi-square
Cl = Confidence Interval
CRHRAl = Corticotropin Releasing Hormone RA!
CRHRA2 = Corticotropin Releasing Hormone RA2
COX! = Cyclooxygenase I (constitutive) enzyme
COXII = Cyclooxygenase II (inducible) enzyme
DMARDs = Disease modifying anti-rheumatic drugs
DHEA = Dehydroepiandrosterone
DNA = Deoxyribose nucleic acid
DZ = Dizygotic twins
FB = Complement factor B
FcyRIIIA = Fe receptor gamma receptor Ill A
GI = Gastrointestinal
GM-CSF = Granulocyte-macrophage colony stimulating factor
GMZL = Gastric mucosal zone lymphoma
GP = General practitioner
GSTM1 = Glutathione-S-Transferases Ml
GSTTI = Glutathione-S-Transferases Tl
HLA = Human leukocyte antigen
HNE = Human neutrophil elastase
HWE = Hardy-Weinberg Equilibrium
L-Osp = Lipidated outer surface protein
IL-l = Interleukin-1
iNO S = Inducible nitric oxide synthase
IV