Celiac Disease: Methods and Protocols

249 Pages·2015·6.429 MB·English

by  Anthony W. Ryan

Methods in
Molecular Biology 1326
Anthony W. Ryan Editor
Celiac
Disease
Methods and Protocols
M M B
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hat fi eld, Hertfordshire, AL10 9AB, UK
For further volumes:
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Celiac Disease
Methods and Protocols
Edited by
Anthony W. Ryan
Department of Clinical Medicine, Trinity College Dublin; Institute of Molecular Medicine,
Trinity Centre for Health Sciences, St James’s Hospital, Dublin, Ireland.
Editor
Anthony W . Ryan
Department of Clinical Medicine
Trinity College Dublin
Dublin, Ireland
Institute of Molecular Medicine
Trinity Centre for Health Sciences
St James’s Hospital
Dublin, Ireland
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-2838-5 ISBN 978-1-4939-2839-2 (eBook)
DOI 10.1007/978-1-4939-2839-2
Library of Congress Control Number: 2015944312
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Prefa ce
Recent decades have seen considerable advances in our understanding of celiac disease. The
condition, once thought to be limited to individuals of European ancestry, has been discov-
ered at varying prevalence in North Africa, the Middle East, India, and China. The precipi-
tating auto-antigen has been characterized. The genetic association of the HLA region was
discovered early and refi ned in the years that followed. However, conclusive identifi cation
of non-HLA genetic risk proved elusive until the advent of genome-wide association stud-
ies, which have extended our understanding of the genetic component far beyond what
could have been envisaged a short time ago. Current estimates suggest that more than 50%
of the population variability associated with celiac disease risk can be explained by known
genetic loci. Despite these advances, there is still a great deal to be learned, both about the
nature of genetic risk and the functional genomic consequences of the established risk
factors.
Building on this knowledge will require detailed molecular analysis of the associated
pathways and many cell types involved in the disease, as well as embracing new technologies
such as next-generation sequencing. At the same time, long established molecular and
immunology methods will continue to have a place for some time to come. This book
brings together novel and more traditional methods in molecular biology and immunology,
in order to provide a tool-kit for all stages of celiac disease research, from the practicalities
of obtaining high-quality samples, to molecular analysis and bioinformatics.
Part I of this book sets the background with a number of reviews to describe the history
and nature of the disease, its diagnosis, the role of animal models, and study designs for
investigating genetic susceptibility. Part II describes the molecular techniques, including
tissue culture, isolation and cloning of relevant cell types, high content analysis of biopsies,
and HLA genotyping. Subsequent chapters describe analyses of gene expression and func-
tional analysis of genetic variants: detecting allelic expression imbalance, reporter assays,
and siRNA knockdown. The fi nal chapter in this part describes a number of protocols for
epigenetic analysis, which has attracted little attention until recently.
A great deal of modern molecular biology relies on high-throughput automated analy-
ses, which produce vast quantities of data. Coming to grips with this fl ow of information
represents a new set of challenges, dealt with in Part III. The fi nal three chapters describe
analysis pipelines for bioinformatic prediction of antigenicity, quality control and analysis of
GWAS data, and transcriptome analysis by next-generation sequencing. These techniques
require a certain level of scripting skills, not commonly held by laboratory based research-
ers. For this reason, Part III begins with an outline of scripting for data management, focus-
ing on tools which are freely available to researchers who wish to explore them.
To conclude, I would like to thank John Walker and Humana Press for their guidance and
assistance throughout this project, and I extend my gratitude to all contributors to the book,
who took time out from their busy research and clinical schedules to write their chapters.
Dublin, Ireland A nthony W. R yan
v

Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i x
PART I BACKGROUND
1 Celiac Disease: Background and Historical Context. . . . . . . . . . . . . . . . . . . . . 3
Graham D. Turner , Margaret R . D unne , and Anthony W. Ryan
2 Celiac Disease: Diagnosis.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Greg Byrne and C onleth F . F eighery
3 G enerating Transgenic Mouse Models for Studying Celiac Disease.. . . . . . . . . 2 3
Josephine M. J u , E ric V . M arietta , and Joseph A . Murray
4 S tudy Designs for Exploring the Non-HLA Genetics in Celiac Disease.. . . . . . 3 5
Åsa Torinsson Naluai
PART II LABORATORY PROTOCOLS
5 Twenty-Four Hour Ex Vivo Culture of Celiac Duodenal Biopsies. . . . . . . . . . 4 7
Sarah E. J . Cooper , Sharon W ilson , and Conleth F . F eighery
6 Isolation and Cloning of Gluten-Specific T Cells in Celiac Disease. . . . . . . . . . 5 3
Yvonne Kooy-Winkelaar and Frits K oning
7 F low Cytometric Analysis of Human Small Intestinal Lymphoid Cells. . . . . . . 6 1
Margaret R. D unne
8 A daptation of a Cell-Based High Content Screening System
for the In-Depth Analysis of Celiac Biopsy Tissue . . . . . . . . . . . . . . . . . . . . . . 6 7
Sarah E. J . Cooper , Bashir M . Mohamed , Louise Elliott ,
Anthony Mitchell Davies , Conleth F . F eighery , J acinta K elly ,
and Jean D unne
9 H LA Genotyping: Methods for the Identification of the HLA-DQ2,-DQ8
Heterodimers Implicated in Celiac Disease (CD) Susceptibility . . . . . . . . . . . . 7 9
Maria Edvige Fasano , E nnia D ametto , and Sandra D ’Alfonso
10 Detecting Allelic Expression Imbalance at Candidate Genes
Using 5′ Exonuclease Genotyping Technology . . . . . . . . . . . . . . . . . . . . . . . . 9 3
Jillian M. G ahan , M ikaela M . Byrne , Matthew Hill , E mma M. Quinn ,
Ross T . Murphy , Richard J . L. Anney , and A nthony W. Ryan
11 Gene Expression Profiling of Celiac Biopsies and Peripheral
Blood Monocytes Using Taqman Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 05
Martina G alatola , R enata Auricchio , and Luigi G reco
12 C loning Gene Variants and Reporter Assays . . . . . . . . . . . . . . . . . . . . . . . . . . 1 17
Ben Molloy and R oss M cManus
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viii Contents
13 Epigenetic Methodologies for the Study of Celiac Disease. . . . . . . . . . . . . . . . 1 31
Antoinette S. P erry , Anne-Marie Baird , and Steven G . G ray
14 C andidate Gene Knockdown in Celiac Disease . . . . . . . . . . . . . . . . . . . . . . . . 1 59
Ben Molloy , M ichael F reeley , Aideen Long , and Ross McManus
PART III BIOINFORMATICS
15 Perl One-Liners: Bridging the Gap Between Large Data Sets
and Analysis Tools. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Karsten H okamp
16 B ioinformatic Analysis of Antigenic Proteins in Celiac Disease. . . . . . . . . . . . . 1 93
Cathal P. O’Brien
17 Q uality Control Procedures for High-Throughput Genetic
Association Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 03
Ciara Coleman , E mma M . Q uinn , and Ross McManus
18 Quality Control and Analysis of NGS RNA Sequencing Data . . . . . . . . . . . . . 2 17
Emma M. Quinn and Ross M cManus
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Contributors
RICHARD J. L . A NNEY • Institute of Molecular Medicine, Trinity Centre for Health Sciences,
St James’s Hospital , Dublin, I reland ; D epartment of Psychiatry , T rinity College Dublin,
Dublin, Ireland
RENATA AURICCHIO • Department of Translational Medicine, Section of Pediatrics,
University of Naples Federico II, Via S. Pansini 5, Naples , I taly ; European Laboratory for
the Investigation of Food Induced Diseases (ELFID), U niversity of Naples Federico II,
Via S. Pansini 5 , N aples , I taly
ANNE-MARIE B AIRD • Department of Clinical Medicine, Trinity College Dublin , D ublin,
Ireland ; T horacic Oncology Research Group, Institute of Molecular Medicine,
Trinity Centre for Health Science, St. James’s Hospital , D ublin, I reland ; C ancer
and Aging Research Program, Q ueensland University of Technology , B risbane, Australia
GREG BYRNE • School of Biological Sciences, D ublin Institute of Technology , D ublin, I reland
MIKAELA M. B YRNE • Department of Clinical Medicine, Trinity College Dublin , D ublin,
Ireland ; I nstitute of Molecular Medicine, T rinity College Dublin , D ublin, I reland
CIARA COLEMAN • Department of Medicine, I nstitute of Molecular Medicine, Trinity College
Dublin, College Green , D ublin, Ireland ; St. James’s Hospital , D ublin, I reland
SARAH E. J . COOPER • Immunology Department , T rinity College Dublin , D ublin, Ireland
SANDRA D’ ALFONSO • Department of Health Sciences, U niversity of Eastern Piedmont ,
Novara, I taly ; I nterdisciplinary Research Centre of Autoimmune Disease (IRCAD),
University of Eastern Piedmont , Novara, I taly
ENNIA DAMETTO • Immunogenetica e Biologia dei Trapianti, AOU Città della salute e della
scienza di torino, via Santena , Torino , I taly
ANTHONY MITCHELL DAVIES • INCHSA, Institute for Molecular Medicine, Trinity College
Dublin , D ublin, Ireland
JEAN DUNNE • Immunology Department , T rinity College Dublin , D ublin, I reland ;
Immunology Department, S t James’s Hospital , D ublin, I reland
MARGARET R. DUNNE • Department of Surgery, Trinity Centre for Health Sciences,
St James’s Hospital, Dublin, Ireland; National Children’s Research Centre, Our Lady’s
Children’s Hospital, Dublin, Ireland; Department of Immunology, Institute of Molecular
Medicine, St. James’s Hospital, Trinity College Dublin, Dublin, Ireland
LOUISE ELLIOT • Immunology Department , Trinity College Dublin , D ublin, I reland
MARIA E DVIGE F ASANO • Immunogenetica e Biologia dei Trapianti, A OU Città della salute e
della scienza di torino , via Santena, Torino, Italy
CONLETH F. F EIGHERY • Immunology Department , Trinity College Dublin , D ublin,
Ireland ; I mmunology Department, St. James’s Hospital , D ublin, I reland
MICHAEL FREELEY • Department of Medicine, I nstitute of Molecular Medicine, Trinity
College Dublin, St. James’s Hospital , Dublin, I reland
JILLIAN M. GAHAN • Department of Clinical Medicine, Trinity College Dublin , D ublin,
Ireland ; I nstitute of Molecular Medicine, Trinity Centre for Health Sciences,
St James’s Hospital , D ublin, Ireland
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