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Textbook of
Autoinflammation
Philip J. Hashkes
Ronald M. Laxer
Anna Simon
Editors
123
Textbook of Autoinflammation
Philip J. Hashkes • Ronald M. Laxer
Anna Simon
Editors
Textbook of
Autoinflammation
Editors
Philip J. Hashkes Ronald M. Laxer
Pediatric Rheumatology Unit Department Paediatrics
Shaare Zedek Medical Center and Division of Rheumatology
Hebrew University The Hospital for Sick Children and
Jerusalem University of Toronto
Israel Toronto, ON
Canada
Anna Simon
Department of Internal Medicine
Radboudumc Expertisecenter for
Immunodeficiency and Autoinflammation,
Radboud University Medical Center
Nijmegen
The Netherlands
ISBN 978-3-319-98604-3 ISBN 978-3-319-98605-0 (eBook)

https://doi.org/10.1007/978-3-319-98605-0
Library of Congress Control Number: 2018963832
© Springer Nature Switzerland AG 2019

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or
part of the material is concerned, specifically the rights of translation, reprinting, reuse of
illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way,
and transmission or information storage and retrieval, electronic adaptation, computer software,
or by similar or dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this
publication does not imply, even in the absence of a specific statement, that such names are
exempt from the relevant protective laws and regulations and therefore free for general use.
The publisher, the authors, and the editors are safe to assume that the advice and information in
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This Springer imprint is published by the registered company Springer Nature Switzerland AG
The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
Preface
The innate immune system pervades the whole of human life, in all its aspects,
and defects in it may have consequences for all organ systems. This makes
the autoinflammatory disorders very complex and challenging, with variable
clinical presentations and sometimes devastating consequences.
In the two decades since the term ‘autoinflammation’ was first coined,
tremendous strides have been made in both the clinical field and the basic
science of this fascinating new area that involves many disciplines including
immunology, rheumatology, dermatology and genetics. With the coming of
age of the field comes the need for a textbook as a resource to pull together
the current knowledge and insights. This is a rapidly changing and develop-
ing area of research, with new disorders and pathogenic mechanisms
described every year. We hope this book provides a much-needed background
to encourage such developments.
This textbook will provide the clinician with detailed clinical information
on the monogenic as well as some of the more complex or polygenic autoin-
flammatory disorders. In addition it provides background information on the
cellular, immunologic and genetic mechanisms underlying many of these dis-
orders. The textbook also contains chapters that are meant to give the clini-
cian tools on how to approach patients with a suspected autoinflammatory
disorder and how to monitor their course. We have included chapters on
genetics, diagnosis, therapeutics and management in general. For basic scien-
tists interested in the field, this book aims to provide a resource which high-
lights connections between different areas of autoinflammation and gives
insight into the consequences of perturbations of the innate immune system
in patients, and the relationship with other disorders of the immune system.
We thank the international experts, many of whom are the pioneers and
leaders in the field of autoinflammation, who have contributed to this first
edition by providing ‘state-of-the-art’ chapters in their field of expertise.
Jerusalem, Israel Philip J. Hashkes

Toronto, ON, Canada Ronald M. Laxer
Nijmegen, The Netherlands Anna Simon
December 2018
v
Contents
Part I Introduction
1 Autoinflammation: Past, Present, and Future�� 3

Daniel L. Kastner
Part II Basic Science and Biology of Autoinflammation
2 Genetic Aspects of Investigating and Understanding

Autoinflammation�� 19
Isabella Ceccherini, Marta Rusmini,
and Juan Ignacio Arostegui
3 Epigenetics in Autoinflammation�� 49
Clara Lorente-Sorolla, Mihai G. Netea, and Esteban Ballestar
4 Pattern Recognition Receptors in Autoinflammation�� 61
Victor Saavedra, Fiona Moghaddas, Eicke Latz,
and Seth L. Masters
5 Inflammasomes and Autoinflammation�� 89
Lori Broderick
6 Cytokines in Autoinflammation�� 111
Angela Rösen-Wolff and Anna Rubartelli
7 Proteasomes in Autoinflammation�� 123
Anja Brehm, Frédéric Ebstein, and Elke Krüger
8 Disruption of Protein Homeostasis and Activation
of Cellular Stress Pathways in Autoinflammation�� 137
Cornelia D. Cudrici and Richard M. Siegel
9 S100 Proteins in Autoinflammation�� 149
Dirk Holzinger, Christoph Kessel, and Dirk Foell
Part III General Approach to Autoinflammatory Diseases
10 Classification of Genetically Defined

Autoinflammatory Diseases�� 167
Raphaela Goldbach-Mansky and Adriana A. de Jesus
vii
viii Contents
11 Clinical Approach to the Diagnosis of

Philip J. Hashkes, Karyl S. Barron, and Ronald M. Laxer
12 Genetic Approach to the Diagnosis of
Isabelle Touitou and Ivona Aksentijevich
13 Monitoring Disease Activity, Damage and Quality of Life�� 239
Nienke ter Haar, Maryam Piram, and Isabelle Koné-Paut
14 The Role of International Registries for Rare
Martina Finetti and Marco Gattorno
15 Systemic Amyloidosis�� 267
Tamer Rezk and Philip N. Hawkins
Part IV Monogenic Autoinflammatory Diseases
16 Familial Mediterranean Fever�� 293

Shai Padeh, Yelda Bilginer, and Seza Ozen
17 Mevalonate Kinase Deficiency�� 315
Joost Frenkel and Anna Simon
18 Tumor Necrosis Factor (TNF) Receptor-Associated
Periodic Syndrome (TRAPS)�� 329
Sinisa Savic and Michael F. McDermott
19 Cryopyrin-Associated Periodic Syndromes (CAPS)�� 347
Hal M. Hoffman, Jasmin B. Kuemmerle-Deschner,
and Raphaela Goldbach-Mansky
20 Autoinflammatory Granulomatous Disease:
Blau Syndrome �� 367
Carlos D. Rose and Carine H. Wouters
21 Very Early Onset Inflammatory Bowel Disease (VEOIBD)�� 383
Aleixo M. Muise
22 Pyogenic Arthritis Pyoderma Gangrenosum
and Acne (PAPA) Syndrome �� 405
Marilynn G. Punaro and Carol A. Wise
23 Deficiency of Adenosine Deaminase 2 (DADA2) �� 417
Amanda Ombrello and Reeval Segel
24 Genetic Interferonopathies�� 433
Despina Eleftheriou, Antonio Torrelo, and Paul A. Brogan
25 Genetic Causes of Inflammatory Bone Disease�� 455
James Verbsky and Polly J. Ferguson
Contents ix
26 Pustular Forms of Psoriasis Related to Autoinflammation�� 471

Satveer K. Mahil, Jonathan N. Barker, and Francesca Capon
27 Hydatidiform Moles�� 485
Ngoc Minh Phuong Nguyen, Pierre-Adrien Bolze,
and Rima Slim
28 Monogenic Autoinflammatory Diseases Associated
with Immunodeficiency �� 499
Michael J. Ombrello
29 Other Rare Monogenic Autoinflammatory Diseases�� 515
Isabelle Jéru, Scott W. Canna, and Eric P. Hanson
Part V Complex Autoinflammatory Diseases
30 Periodic Fever, Aphthous Stomatitis, Pharyngitis

and Cervical Adenitis (PFAPA) Syndrome�� 541
Kathryn M. Edwards and Michael Hofer
31 Chronic Non-Bacterial Osteomyelitis�� 563
Christian M. Hedrich and Hermann J. Girschick
32 Systemic Juvenile Idiopathic Arthritis and Adult
Onset Still Disease�� 587
Peter A. Nigrovic and Rayfel Schneider
33 Macrophage Activation Syndrome in Rheumatic Diseases�� 617
Alexei A. Grom and Edward M. Behrens
34 Gouty Inflammation�� 635
Naomi Schlesinger and Johnson C. Kay
35 Behçet Disease�� 647
Ahmet Gül
36 Idiopathic Recurrent Pericarditis�� 667
Massimo Imazio, Anna Valenti, Antonio Brucato,
and Alberto Martini
37 Schnitzler Syndrome�� 679
Heleen D. de Koning and Karoline Krause
Part VI The Relationship Between Autoinflammation

and Other Inflammatory and Common Diseases
38 Autoinflammation and Autoimmunity �� 693

Dennis McGonagle and Abdulla Watad
39 Interleukin-1 Mediated Autoinflammation
from Heart Disease to Cancer�� 711
Charles A. Dinarello
x Contents
Part VII Pharmaceutical Agents for Treatment

of the Autoinflammatory Diseases
40 Colchicine�� 729
Eldad Ben-Chetrit
41 Interleukin (IL)-1 Blocking Compounds
and Their Use in Autoinflammatory Diseases �� 751
Tilmann Kallinich and Fabrizio de Benedetti
42 Corticosteroid, Other Biologic and Small Molecule
Therapies in Systemic Autoinflammatory Disorders�� 775
Helen J. Lachmann
Index�� 793
Part I
Introduction
Autoinflammation: Past, Present,
and Future 1
Daniel L. Kastner
Abstract tory diseases, such as Behçet disease. During

The concept of autoinflammation arose from the next decade, the universe of autoinflam-
the recognition of monogenic disorders with matory diseases will continue to expand, but it
seemingly unprovoked inflammation without is likely that distinctions between clinical dis-
the high-titer autoantibodies or antigen- ease and normal variation will blur, and that
specific T cells seen in classic autoimmune treatments developed for autoinflammation
diseases. During the first decade of the ‘auto- will be applied to a much broader range of
inflammatory era’, a clear connection was human illnesses.
established between autoinflammatory dis-
ease and the innate immune system, with tar- Keywords
geted therapies providing a powerful Autoinflammation · Innate immunity ·
affirmation of mechanistic hypotheses. Inflammasome · Interleukin (IL)-1β · Type I
Although the ‘inflammasomopathies’, which interferon · Next-generation sequencing ·
are associated with marked interleukin (IL)-1β Genome-wide association study (GWAS) ·
production, were some of the earliest recog- Mosaicism · Nomenclature · Targeted
nized autoinflammatory diseases, it soon therapy · Aphthous ulcers
became clear that autoinflammation can be
caused by a variety of genetic lesions affecting
a range of innate immune pathways, including
nuclear factor kappa B (NF-κB) activation and
type I interferon production. The advent of
next-generation sequencing has resulted in the Abbreviations
discovery of multiple new diseases, genes, and
pathways, while genome-wide association CAPS Cryopyrin-associated periodic
studies (GWAS) have shed light on the patho- syndromes
genesis of genetically complex autoinflamma- CINCA Chronic infantile neurologic cutane-
ous and articular syndrome
CNO Chronic non-bacterial osteomyelitis
D. L. Kastner (*)
National Human Genome Research Institute CRMO Chronic recurrent multifocal
(NHGRI), National Institutes of Health (NIH), osteomyelitis
Bethesda, MD, USA DIRA Deficiency of interleukin-1 receptor
e-mail: dan.kastner@nih.gov; antagonist
Kastnerd@mail.nih.gov
© Springer Nature Switzerland AG 2019 3

P. J. Hashkes et al. (eds.), Textbook of Autoinflammation,
https://doi.org/10.1007/978-3-319-98605-0_1
4 D. L. Kastner
FMF Familial Mediterranean fever decade promises to draw connections

GWAS Genome-wide association studies between autoinflammatory diseases and the
HIDS Hyperimmunoglobulinemia D with ‘range of normal’ phenotypes, and to apply
periodic fever syndrome the treatments developed for autoinflam-
IL Interleukin matory diseases to a broad spectrum of
ISSAID International Society for Systemic illnesses
Autoinflammatory Diseases
MKD Mevalonate kinase deficiency
MWS Muckle-Wells syndrome 1.1 ‘Ancient’ History
NF-κB Nuclear factor kappa B
NLR Nucleotide-binding domain, leucine- For over a century, medical science has been fas-
rich repeat cinated with the questions of if, when, and how
NLRP3 NLR family, pyrin domain contain- the immune system might turn against its host. At
ing 3 the beginning of the twentieth century, the Nobel
NOMID Neonatal-onset multisystem inflam- Prize-winning immunologist Paul Ehrlich pro-
matory disorder posed the concept of horror autotoxicus to argue
PAAND Pyrin-associated autoinflammation that the consequences of autoimmunity would be
with neutrophilic dermatosis so dire that an organism would have multiple
PAPA Pyogenic arthritis, pyoderma gan- mechanisms in place to prevent self-reactivity
grenosum and acne from ever happening [1]. However, the subse-
PFAPA Periodic fever, aphthous stomatitis, quent decades provided ample evidence that
pharyngitis, cervical adenitis there are in fact numerous human illnesses in
SAVI STING-associated vasculopathy which such safeguards break down, giving rise to
with onset in infancy either systemic or organ-specific autoimmunity.
SIFD Sideroblastic anemia with immuno- Self-reactive antibodies and T lymphocytes have
deficiency, fevers, and developmen- been implicated in the pathogenesis of many of
tal delay these disorders.
STING Stimulator of interferon genes By the latter half of the twentieth century
TNF Tumor necrosis factor there remained a group of illnesses characterized
TRAPS TNF receptor-associated periodic by episodes of seemingly unprovoked systemic
syndrome or localized inflammation, without the apparent
involvement of high-titer autoantibodies or
antigen-specific T lymphocytes. Astute clinicians
Key Points recognized that for several of these illnesses,
• The autoinflammatory diseases were ini- recurrent fevers were a prominent feature, and
tially recognized for seemingly unprovoked that they appeared to be hereditary. These
inflammation, but were soon discovered to included familial Mediterranean fever (FMF),
be disorders of innate immunity familial Hibernian fever, hyperimmunoglobu-
• Next-generation sequencing has led to an linemia D with periodic fever syndrome (HIDS),
explosion of discovery of monogenic auto- Muckle-Wells syndrome (MWS), and familial
inflammatory diseases and newly recog- cold urticaria. The advent of the Human Genome
nized innate immune pathways Project provided the tools to search for the under-
• Genome-wide association studies (GWAS) lying genes in a hypothesis-neutral, comprehen-
provide insight into the etiology of geneti- sive fashion known as positional cloning,
cally complex autoinflammatory diseases enabling the discovery of previously unknown
• In addition to continued discovery of new regulators of immunity gone awry in these ill-
diseases, genes, and pathways, the next nesses (see Chap. 2).
1 Autoinflammation: Past, Present, and Future 5
1.1.1 First Discoveries: The Birth cold urticaria, MWS—see Chap. 19), granuloma-
of Autoinflammation tous disorders (Blau syndrome—see Chap. 20),
metabolic disorders (crystalline arthropathies—
Owing both to its relatively well-defined pheno- see Chap. 34), storage diseases (Gaucher disease,
type and to the availability of the large numbers of Hermansky-Pudlak syndrome), fibrosing disor-
families needed for high-resolution genetic map- ders, and Behçet disease ([7]; see Chap. 35).
ping, FMF was the first of the recurrent fever syn- Recognizing the heterogeneity of human disease,
dromes to be analyzed in this way. In the summer this analysis included both monogenic and genet-
of 1997 two independent consortia discovered ically complex illnesses. However, at this early
recessive mutations in the causative gene, MEFV, stage the schema was based solely on the whim-
which encodes what was then a novel protein sical notion of a family of diseases manifesting
denoted pyrin (or marenostrin) ([2, 3]; see Chap. unprovoked inflammation without high-titer
16). Although not known at the time, pyrin forms autoantibodies or antigen-specific T cells, in the
the nucleus of a macromolecular complex absence of more detailed genetic or functional
(denoted the pyrin inflammasome) that activates insight.
interleukin-(IL)1β, IL-18, and the executioner
protein gasdermin D in response to certain bacte-
rial toxins ([4, 5]; see Chap. 5). FMF-associated 1.2 The ‘Eureka’ Decade
mutations in pyrin lower the threshold for activa-
tion. The ~90 N-terminal residues of pyrin consti- During the next decade, two independent lines of
tute a motif that is the prototype for a cognate investigation converged to corroborate the concept
interaction domain (the PYRIN domain) found in of autoinflammation. On the one hand, the field of
some 20 immune-related human proteins. The human genetics accelerated the discovery of genes
discovery of MEFV not only fulfilled the promise underlying the newly recognized autoinflamma-
of positional cloning, but also allowed the tory diseases. On the other hand, advances in basic
unequivocal determination that certain other peri- immunology firmly established the role of the
odic fever syndromes were not FMF, thus opening innate immune system in host defense [8]. Whereas
up a new area of clinical investigation. In 1999, the adaptive immune system is mediated by lym-
mutations in TNFRSF1A, encoding the 55 kDa phocytes with membrane receptors encoded by
tumor necrosis factor receptor, were shown to genes that somatically rearrange and mutate, the
define a recurrent fever syndrome now called the evolutionarily more ancient innate immune system
tumor necrosis factor (TNF) receptor-associated utilizes myeloid effector cells with both extracel-
periodic syndrome (TRAPS), which subsumed lular and intracellular receptors that are ‘hard-
familial Hibernian fever and several other domi- wired’ in the genome to recognize
nantly-inherited fever syndromes seen in multiple ‘pathogen-associated molecular patterns’ (see
ethnicities ([6]; see Chap. 18). Chap. 4). Genetics and immunobiology advanced
The authors of the paper describing TRAPS hand-in-hand, with the growing realization that
proposed the term ‘autoinflammatory’ to denote many of the disorders defined clinically as ‘autoin-
what appeared to be an emerging family of ill- flammatory’ are caused by genetic mutations that
nesses characterized by seemingly unprovoked perturb the innate immune system. Disease-gene
systemic or localized inflammation, but without discoveries provided clinical relevance for innate
the cardinal features of autoimmunity. A year immunity, and advances in immunology explained
later the concept was refined and extended, with newly recognized autoinflammatory illnesses.
the proposal of a classification scheme that Highly successful trials of therapies predicted to
included the recurrent fever syndromes, certain target the relevant pathways were the heady affir-
complement disorders (such as hereditary angio- mation of an emerging understanding of a new
edema), familial urticarial syndromes (familial field of medicine ([9–11]; see Chaps. 41 and 42).
6 D. L. Kastner
Nowhere was this paradigm more evident than technologies of the time, the new disease gene
in the elucidation of the cryopyrin-associated discoveries were the result of either positional
periodic syndromes (CAPS). In 2001 Hal cloning or candidate gene approaches, sometimes
Hoffman and his colleagues discovered suggesting extensions of known innate immune
dominantly- inherited mutations in the gene pathways (see Chap. 2). For example, the discov-
encoding a PYRIN domain-containing protein ery of loss-of-function mutations in IL1RN,
(denoted cryopyrin) as the cause of both familial encoding the endogenous IL-1 receptor antago-
cold autoinflammatory syndrome (formerly nist (a recombinant form of which is anakinra, a
familial cold urticaria) and MWS ([12]; see Chap. biologic used in the treatment of CAPS), causing
19). Within a year, two other groups discovered the disease deficiency of IL-1 receptor antagonist
mutations in the same gene as the cause of (DIRA), highlighted the need for tight IL-1 regu-
neonatal-onset multisystem inflammatory disor- lation in normal homeostasis ([18, 19]; see Chap.
der (NOMID; also called (mainly in Europe) 25). The discovery of dominantly inherited muta-
chronic infantile neurologic cutaneous and artic- tions in PSTPIP1, which encodes a pyrin-binding
ular [CINCA] syndrome), a devastating disorder protein also involved in regulating the cytoskele-
manifesting chronic aseptic meningitis [13, 14]. ton, in pyogenic arthritis, pyoderma gangreno-
All of these diseases are collectively denoted sum and acne (PAPA) syndrome [20, 21],
CAPS. Independently and nearly simultaneously, suggested a connection between innate immunity
other groups discovered a role for cryopyrin and the cytoskeleton that is still under active
(alternatively termed ‘PYPAF1,’ ‘NALP3,’ and investigation (see Chap. 22). The discovery of
now ‘NLRP3’) in the activation of IL-1β [15, 16]. autoinflammatory phenotypes associated with
The late Jürg Tschopp and his colleagues pro- CARD15/NOD2 (see Chap. 20) and NLRP12 (see
posed a macromolecular complex they termed Chap. 29) expanded the spectrum of disorders
the inflammasome, one variant of which includes associated with this large family of NACHT-
nucleotide-binding domain, leucine-rich repeat domain-containing proteins, raising the possibil-
(NLR) family, pyrin domain containing 3 ity of even more [22–24]. The discovery of
(NLRP3), that leads to the autocatalysis of cas- mevalonate kinase (MVK) mutations in HIDS
pase-1 and the release of biologically active [25, 26], now called mevalonate kinase defi-
IL-1β from leukocytes ([17]; see Chap. 5). ciency (MKD) due to this discovery, established
CAPS-associated mutations were soon found to a link between metabolism and autoinflammation
cause constitutive activation of the NLRP3 that has only recently been explained.
inflammasome, thus suggesting a possible role
for IL-1 inhibition in the treatment of CAPS. The
life-altering effects of IL-1 inhibition in CAPS 1.2.2 Early Thoughts
have been a triumph of molecular medicine and a on Pathophysiologic
true vindication of the importance of IL-1 in Mechanisms
human immunobiology [9–11].
During this first decade, many of the advances in
disease mechanism and treatment centered on
1.2.1 Expanding the Discovery IL-1β and related proteins, leading some to sug-
of Diseases Caused by Genetic gest an equivalence of autoinflammation with
Mutations IL-1-mediated disease (see Chaps. 5 and 10).
Evidence emerged that the prototypic autoin-
The early years of the ‘autoinflammatory era’ flammatory disease, FMF, is driven by IL-1β
witnessed the discovery of several new disease- [27], and that uric acid crystals activate the
causing genes (Table 1.1), the deepening of our NLRP3 inflammasome, thus supporting the
understanding of innate immune pathways, and hypothesis that gout, a genetically complex dis-
further therapeutic advances. Given the genomic order, is also autoinflammatory and driven by
Table 1.1 Timeline of monogenic autoinflammatory disease gene discoveries

Disorder Gene Protein Year Chapter
FMF MEFV Pyrin/Marenostrin 1997 16
TRAPS TNFRSF1A TNFR1 1999 18
HIDS/MKD MVK Mevalonate kinase 1999 17
CAPS NLRP3 Cryopyrin/NLRP3 2001 19
Blau NOD2 NOD2 2001 20
Cherubism SH3BP2 SH3BP2 2001 25
PAPA PSTPIP1 PSTPIP1 2002 22
Majeed LPIN2 LPIN2 2005 25
Hydatidiform mole NLRP7 NLRP7 2006 27
FCAS2 NLRP12 NLRP12 2008 29
Histiocytosis- SLC29A3 hENT3 2008 NIB
lymphadenopathy plus
DIRA IL1RN IL-1 receptor 2009 25
antagonist
VEOIBD IL10RA, IL10RB IL10 IL-10 receptor 2009 21
IL-10 2010
DITRA IL36RN IL-36 receptor 2011 26
antagonist
JMP/NNS/CANDLE PSMB8 β5i 2010– 24
Immunoproteasome 2012
CAMPS/PSORS2 CARD14 CARD14 2012 26
APLAID PLCG2 PLCγ2 2012 28
HOIL-1 deficiency RBCK1 HOIL-1 2012 28
DADA2 ADA2 (formerly CECR1) ADA2 2014 23
SAVI TMEM173 STING 2014 24
NLRC4-MAS NLRC4 NLRC4 2014 29
SIFD TRNT1 TRNT1 2014 28
TRAPS11 TNFRSF11A TNFRSF11A 2014 29
HOIP deficiency HOIP HOIP 2015 28
sJIA LACC1 FAMIN 2015 32
PRAAS PSMA3, PSMB4, PSMB9; digenic Proteasome 2015 24
inheritance components
Adult-onset CAPS NLRP3 NLRP3 2015 19, 37
HA20 TNFAIP3 A20 2016 29
PAAND MEFV Pyrin/Marenostrin 2016 29
Vibratory urticaria ADGRE2 ADGRE2 2016 NIB
MSPC/FKLC NLRP1 NLRP1 2016 29
Otulipenia, ORAS OTULIN OTULIN 2016 29
NAIAD NLRP1 NLRP1 2017 29
PFIT WDR1 WDR1 2017 28
PRAID POMP POMP 2018 NIB
Diseases: FMF Familial Mediterranean fever, TRAPS Tumor necrosis factor receptor associated periodic syn-
drome, HIDS Hyperimmunoglobulinemia D with periodic fever syndrome, MKD Mevalonate kinase deficiency,
CAPS Cryopyrin-associated periodic syndromes, PAPA Pyogenic arthritis, pyoderma gangrenosum and acne,
FCAS2 Familial cold autoinflammatory syndrome 2, DIRA Deficiency of IL-1 receptor antagonist, VEOIBD Very-
early onset inflammatory bowel disease, DITRA Deficiency of IL-36 receptor antagonist, JMP Joint contractures,
muscle atrophy, microcytic anemia and panniculitis-induced lipodystrophy syndrome, NNS Nakajo-Nishimura
syndrome, CANDLE Chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature,
CAMPS Caspase activation and recruitment domain (CARD) 14 mediated psoriasis, PSORS2 Psoriasis suscepti-
bility locus 2, APLAID Autoinflammatory PLCγ2-associated antibody deficiency and immune dysregulation,
HOIL-1 Heme-oxidized IRP2 ubiquitin ligase 1, DADA2 Deficiency of adenosine deaminase 2, SAVI Stimulator of
(continued)
8 D. L. Kastner
Table 1.1 (continued)

interferon genes (STING)-associated vasculopathy with onset in infancy, NLRC4-MAS NLRC: Nucleotide oligo-
merization domain (NOD)-like receptor family CARD domain-containing protein 4-macrophage activation syn-
drome, SIFD Sideroblastic anemia with immunodeficiency, fevers, and developmental delay, TRAPS 11 TRAPS
due to mutations in TNFRSF11A, HOIP HOIL-1 interacting protein, sJIA systemic juvenile idiopathic arthritis,
PRAAS Proteasome-associated autoinflammatory syndromes, HA20 A20 haploinsufficiency, PAAND Pyrin-
associated autoinflammation with neutrophilic dermatosis, MSPC Multiple self-healing palmoplantar carcinoma,
FKLC Familial keratosis lichenoides chronica, ORAS Otulin-related autoinflammatory syndrome, NAIAD NLRP1-
associated autoinflammation with arthritis and dyskeratosis, PFIT Periodic fever, immunodeficiency and thrombo-
cytopenia, PRAID Proteasome maturation protein (POMP)-related autoinflammation and immune dysregulation
disease
Proteins: TNFR1 Tumor necrosis factor receptor 1, NLRP Nucleotide oligomerization domain (NOD)-like receptor
family, leucine rich repeat, pyrin domain, SH3BP2 SH3 binding protein 2, PSTPIP Proline-serine-threonine phospha-
tase interacting protein, LPIN2 Lipin 2 gene symbol, hENT3 Human equilibrative nucleoside transporter-3, IL
Interleukin, CARD Caspase activation and recruitment domain, PLCγ2 Phospholipase Cγ2, HOIL-1 Heme-oxidized
IRP2 ubiquitin ligase 1, ADA2 Adenosine deaminase 2, STING Stimulator of interferon genes, NLRC Nucleotide oligo-
merization domain (NOD)-like receptor family CARD domain-containing protein, TRNT tRNA nucleotidyltransferase,
TNFRSF11A TNF receptor superfamily 11a, HOIP HOIL-1 interacting protein, FAMIN Fatty acid metabolic immune
nexus, ADGRE2 Adhesion G protein-coupled receptor E2, WDR1 WD domain repeat containing protein 1, POMP
Proteasome maturation protein
NIB Not in book
IL-1β ([28]; see Chap. 34). Nevertheless, even revolution in next-generation sequencing tech-
during this early era there was mounting evidence nology that has led to drastic reductions in costs
for other molecular mechanisms, such as nuclear and a concomitant boom in the availability of
factor kappa B (NF-κB) activation in Blau syn- whole-exome and now whole-genome sequenc-
drome ([29]; see Chap. 20). This is not surprising (see Chap. 2). The number of monogenic
ing, given the broad scope of innate immune autoinflammatory diseases has gone up dramati-
sensing and signaling. As was noted a decade cally, shedding light on new innate immune path-
ago, the autoinflammatory diseases are a sam- ways and disease mechanisms. While the cases
pling from the universe of natural variation in the have become ever rarer, they are ‘experiments of
innate immune system that is severe enough to nature’ by which, as Sir William Harvey noted
cause illness, but not so severe to be embryonic four centuries ago, “Nature is nowhere [more]
lethal [30]. The ensuing decade has given us a accustomed to display her secret mysteries than
glimpse of just how diverse a universe this is. in cases where she shows traces of her workings
apart from the beaten path” [31].
1.3 orror Autoinflammaticus:

H
The Golden Age 1.3.1 ew Discoveries of Rare
N
of Autoinflammation Mongenic Autoinflammatory
Diseases
The second decade of the autoinflammatory era
began in 2009 with the publication of ‘Horror Some of the newly recognized disease-causing
Autoinflammaticus: The Molecular genes encode known innate immune sensors for
Pathophysiology of Autoinflammatory Disease,’ which a monogenic human disease had not already
a comprehensive review of the field that proposed been discovered. NLRC4 encodes the lynchpin of
a classification scheme based on molecular an inflammasome that senses bacterial flagellin;
insights garnered to that point ([30]; see Chap. gain-of-function mutations have now been shown
10). Autoinflammation had come of age. Building to cause colitis, a CAPS-like spectrum, and an
on this foundation, the last decade has witnessed increased risk of macrophage activation syndrome
a genomic explosion, catalyzed in large part by a (MAS) ([32, 33]; see Chap. 29). NLRP1 encodes a
protein that nucleates the main inflammasome in domain cause vibratory urticaria [50]. LACC1
the skin; activating mutations were shown to cause encodes a key regulator of metabolism in macro-
dyskeratosis with or without arthritis ([34, 35]; see phages; biallelic loss-of-function mutations
Chap. 29). TMEM173 encodes the stimulator of cause a monogenic form of systemic juvenile
interferon genes (STING), a major sensor of intra- idiopathic arthritis ([51]; see Chap. 32). Perhaps
cellular double-stranded DNA; de novo gain-of- most surprising of all, TRNT1 encodes a ubiqui-
function mutations are now known to cause tously expressed enzyme that adds the 3-nt CCA
vasculopathy, peripheral gangrene, and interstitial sequence to the 3′ ends of all tRNA molecules.
fibrosis (STING-associated vasculopathy with Biallelic hypomorphic mutations cause an auto-
onset in infancy, SAVI) ([36]; see Chap. 24). inflammatory syndrome denoted sideroblastic
MEFV encodes pyrin, the protein mutated in FMF; anemia with immunodeficiency, fevers, and
mutations in a critical phosphorylation site have developmental delay (SIFD) ([52]; see Chap. 28).
been shown to cause a dominantly-inherited
chronic neutrophilic dermatosis termed pyrin-
associated autoinflammation with neutrophilic 1.3.2 Expanded Understanding
dermatosis (PAAND) ([37]; see Chap. 29). of Disease Pathophysiology
In other cases, next-generation sequencing has Related to the Innate Immune
led to the identification of genes defining entirely System and Novel Genetic
new mechanisms of innate immune regulation. Mechanisms
WDR1 encodes a protein that regulates the actin
cytoskeleton; loss-of-function mutations lead to Over the last decade there have also been sub-
activation of the pyrin inflammasome and stantial advances in our understanding of the
increased IL-18 production ([38]; see Chap. 28). biology of innate immunity and in targeted ther-
ADA2 (formerly CECR1) encodes what is thought apies, although, not surprisingly, these have not
to be a growth factor expressed in myeloid cells; kept pace with new disease gene discoveries. It
loss-of-function mutations cause recurrent fevers, is no secret that the timeline for functional and
early-onset strokes, vasculopathy, and sometimes mechanistic analysis is much slower than for
bone marrow failure and immunodeficiency ([39, monogenic disease gene discovery, especially in
40]; see Chap. 23). PSMB8 encodes a component the world of next-generation sequencing and
of the immunoproteasome that degrades K48- large clinics dedicated to undiagnosed autoin-
ubiquitinated proteins; biallelic loss-of-function flammatory patients. As a case in point, it took
mutations cause a syndrome of fevers, panniculi- almost 20 years to understand the role of pyrin
tis, and lipodystrophy ([41–44]; see Chap. 24). in the sensing of bacterial toxins that inactivate
TNFAIP3, OTULIN, HOIL-1, and HOIP encode RhoA and the pathway by which the pyrin
proteins that regulate ubiquitination, a major inflammasome is activated [4, 53]. It took an
form of post-translational protein modification. even longer time to discover gasdermin D and
Haploinsufficiency of TNFAIP3 or biallelic loss- its role in IL-1β release from leukocytes [54–
of-function mutations at the other three loci cause 56]. Nevertheless, the advances of the last
a spectrum of autoinflammatory phenotypes decade have made it abundantly clear that, not-
([45–48]; see Chap. 29). PLCG2 encodes a sig- withstanding the great importance of IL-1 in
naling molecule expressed in hematopoietic human biology, there is much more to autoin-
cells; heterozygous gain-of-function missense flammation than this cytokine. For example, the
mutations cause an autoinflammatory syndrome type I interferons play a central role in the
of rash, ocular inflammation, mild immunodefi- pathogenesis of several autoinflammatory dis-
ciency, and interstitial lung disease ([49]; see eases, such as SAVI and PRAAS [36, 57], and
Chap. 28). ADGRE2 encodes a membrane mech- targeted therapies with JAK inhibitors show
anosensor expressed on mast cells; heterozygous great promise in a number of these disorders
loss-of-function mutations in an autoinhibitory ([58]; see Chap. 24).
10 D. L. Kastner
The last decade has witnessed not only a dizzy- bacterial osteomyelitis (CNO), previously called
ing expansion in the quantity of monogenic dis- chronic recurrent multifocal osteomyelitis
eases and innate immune pathways, but new (CRMO) (see Chap. 31), the syndrome of peri-
qualitative insights into broader mechanisms of odic fever with aphthous stomatitis, pharyngitis,
human disease, driven by the study of autoinflam- and cervical adenitis (PFAPA) (see Chap. 30), the
mation. Of extraordinary potential impact is the crystalline arthropathies (see Chap. 34), sarcoid-
careful documentation of somatic mosaicism (see osis, fibrosing diseases, and, by some definitions,
Chaps. 2 and 12) not only in infantile-onset forms atherosclerosis, type 2 diabetes, cancer, and neu-
of NOMID/CINCA [59] but also in adult-onset rodegenerative diseases (see Chap. 39). Probably
CAPS and Schnitzler syndrome ([60, 61]; see the best-studied is Behçet disease, which presents
Chap. 37). We simply do not know how many with the classic triad of painful oral ulcers, ocular
adult-onset cases of (nonmalignant) unexplained inflammation, and genital ulcers. Advances in
recurrent fever and/or autoinflammation are due to genotyping chips have begun to shape our under-
somatic mutations, but the precedent of cancer standing of genetically complex autoinflamma-
teaches us that such events are not rare. Of similar tory diseases. Through the careful collection of
general import is the recent documentation of well-phenotyped patients and ethnically-matched
digenic inheritance (see Chap. 12) in the controls, combined with genome-wide associa-
proteasome- associated autoinflammatory syn- tion studies (GWAS) and targeted deep-
dromes (PRAAS) [57]. Consideration of the multi- resequencing, a total of 17 susceptibility loci for
molecular proteasome complex gave rise to the Behçet disease have been identified: HLA-B*51,
hypothesis of digenic inheritance in unexplained ERAP1, IL10, IL23R, STAT4, CCR1-CCR3,
cases of PRAAS, but it is eminently possible that KLRC4, CEBPB-PTPN1, ADO-EGR2, IRF8,
similar gene-gene interactions are operative in RIPK2, LACC1, FUT2, IL12A, MEFV-p.
other multistep pathways, offering potential expla- Met694Val, IL1A-IL1B, and TNFAIP3 [62–66].
nations for unsolved cases (see Chap. 24). Finally, Although it often has been observed that most
and not surprisingly, with the discovery of ever GWAS ‘hits’ confer relatively little risk to dis-
more genes underlying monogenic autoinflamma- ease susceptibility in any given individual, there
tion, there are now an increasing number of cases nevertheless is a remarkable convergence among
in which there is an overlap among autoinflamma- GWAS studies in immune diseases, suggesting
tion, autoimmunity, and immunodeficiency ([47, commonalities in pathogenesis among disorders,
48]; see Chaps. 28 and 38). In the case of the ubiq- and the possibility of targeted therapies. GWAS
uitination disorders, this has been shown to be due studies of Behçet disease indicate a role for adap-
to the differential effects of regulatory events in tive immunity (given the remarkable epistasis
multiple cell types. It would be absurd to believe between HLA-B*51 and ERAP1), shared patho-
that such overlaps would not be found. genesis with spondyloarthropathies and certain
infectious diseases, and the possibility of thera-
pies targeting the IL-23 axis (see Chap. 38). As
1.3.3 Expansion noted below, GWAS also draws a shocking but
of Autoinflammation to Non- totally logical connection between Behçet dis-
monogenic and Common ease and everyday life.
Diseases
Nearly since the outset, it has been clear that not 1.4 Nomenclature
all of the illnesses that fit under the autoinflam- of the Autoinflammatory
matory rubric are monogenic. As noted above, Diseases
some are now known to exhibit a digenic mode of
inheritance, but still others are genetically com- As a consequence of the burgeoning list of auto-
plex. The latter include Behçet disease (see Chap. inflammatory diseases, there are now vigorous
35), systemic juvenile idiopathic arthritis, adult- discussions about nomenclature and nosology.
onset Still disease (see Chap. 32), chronic non- Since language is very much a matter of
convention, it would be presumptuous for one tions, especially about the future.” Nevertheless,
individual to impose any specific naming scheme. the developments of the last 10 years likely fore-
In any area of discourse, history matters, and thus shadow the next ten, and so it would be reason-
it would be difficult to advocate against terms ably safe to predict more disease genes, more
like ‘familial Mediterranean fever,’ regardless of pathways, more biology, and more targeted thera-
whether all cases are familial, or Mediterranean, pies. There has been no evidence that we are
or exhibit fever, simply because FMF is thor- approaching an asymptote in new discoveries in
oughly entrenched in our lexicon. For similar this arena, and it is likely that as we peel the
reasons, it is sometimes difficult to dislodge onion we will be greeted with successive layers
firmly established eponyms. Nevertheless, going of regulatory complexity. There is nothing wrong
forward I do subscribe to the view that eponyms in prognosticating ‘more of the same’ for the next
should be avoided, so as not to torment our junior decade. And it would be grand.
colleagues with a litany of people who didn’t However, two recent advances augur addi-
actually have the diseases attached to their names. tional more profound tectonic shifts. The first is
Instead, I favor disease names and classification an abstract presented by the direct-to-consumer
schemes that reflect the underlying biology, genomic testing company 23andMe at the 2017
whether that is best reflected in a gene name or annual meeting of the American Society of
the name of its encoded protein – or even a path- Human Genetics [67]. This abstract presented a
way (‘inflammasomopathy,’ ‘interferonopa- GWAS of canker sores/aphthous ulcers in 178,409
thy’)—rather than a string of clinical affected individuals and 66,609 controls.
manifestations that spell out a memorable acro- Individuals were scored as affected through their
nym. Just as we classify and name infectious dis- response to a questionnaire (“Have you ever had a
eases by their causative microorganisms, so too canker sore [an open sore on the soft tissue inside
should we classify and name autoinflammatory the mouth]? Yes/No/Not sure”). There was no
diseases according to their underlying etiology. medical or dental examination, no review of med-
Such a schema shapes our thinking, stimulates ical records. Remarkably, 47 loci reached
hypotheses, and suggests targeted therapies. As genome-wide significance, including 8 loci
noted above, as we learn more there will be an known to be associated with Behçet disease (IL10,
inevitable blurring of the boundaries between STAT4, CCR3, IL12A, RIPK2, NOD2, IRF8,
autoinflammatory and autoimmune or immuno- CEBPB). Whereas the 23andMe study had very
deficiency (see Chaps. 28 and 38). That is simply large numbers of subjects but little opportunity for
the nature of nature, and any useful schema will clinical observation, the studies of Behçet disease
need to deal with it. The responsibility for estab- were roughly 100 times smaller, but relied on
lishing naming conventions should rest with the meticulous phenotyping. The fact that there was
community that uses them most. In this particular significant overlap between the two studies sug-
case, that is probably the International Society gests that, at least for some phenotypes, a yes-no
for Systemic Autoinflammatory Diseases questionnaire applied to many subjects may reach
(ISSAID) or its designees. the same conclusions as a careful clinical study of
a much smaller number of subjects. The overlap
between the two GWAS studies also suggests that
1.5 uō vādis? Autoinflammation
Q some of the same loci that confer susceptibility to
and the Human Condition severe diseases may also confer susceptibility to
more common, ‘every day’ problems like canker
The third decade of the autoinflammatory era sores. It is tempting to speculate that the loci that
will begin auspiciously with the publication of were not in common between the two studies
this, the first medical text on autoinflammation. (such as HLA-B*51, ERAP1, and IL23R), deter-
Anticipating what is in store for this next decade, mine who gets Behçet disease rather than simple
it is fitting to recall the observation of the twenti- canker sores. It is also possible that other disor-
eth century American ‘philosopher’, the baseball ders manifesting with oral ulcers may share some
player Yogi Berra: “It’s tough to make predic- of these susceptibility loci, and that the k nowledge
12 D. L. Kastner
of these loci will eventually lead to targeted thera- 1. What accounts for the intermittent nature of
pies for aphthae. many of the autoinflammatory diseases?
With the increasing dissemination of genomic 2. What is the molecular basis of phenotypic
sequencing and genotyping across the population, heterogeneity among individuals with the
and the advent of large cohort studies such as the same or similar genotypes?
All of Us Research Program, it will be increasingly 3. What is the penetrance of monogenic autoin-
possible to connect genes and loci associated with flammatory variants in the general
autoinflammatory diseases with phenotypes that population?
we would consider in the range of normal experi- 4. To what extent does somatic mutation
ence. While the experience to date with targeted explain late-onset autoinflammatory disease
therapies for rare autoinflammatory diseases could (see Chaps. 2 and 12)?
certainly be considered to be personalized or pre- 5. What is the role of the microbiome in autoin-
cision medicine, the more universal approach will flammatory disease?
take the field to an entirely new level. 6. To what extent do epigenetic factors (see
A second advance was the publication, in Chap. 3) influence the course of monogenic
August 2017, of two papers summarizing the ini- and genetically complex autoinflammatory
tial results of a randomized, double-blind, disorders?
placebo-controlled trial of canakinumab (a human 7. How do the various inflammasomes differ in
monoclonal anti-IL-1β antibody) in 10,061 sub- their processing of IL-1β, IL-18, and gasder-
jects with a previous myocardial infarction and an min D, and how do these differences corre-
elevated C-reactive protein level of 2 mg or more late with disease phenotype (see Chaps. 5
per liter. In a paper published in the New England and 6)?
Journal of Medicine, canakinumab at a dose of 8. To what extent do monogenic diseases
150 mg given every 3 months significantly low- inform our understanding of genetically
ered the rate of recurrent cardiovascular events, complex autoinflammatory diseases (see
relative to placebo, regardless of lipid-level low- Chap. 38)?
ering ([68]; see Chap. 39). The same research 9. How will disease discovery evolve with new
group simultaneously published a paper in The technologies, such as whole genome
Lancet demonstrating reductions in lung cancer sequencing (see Chap. 2) and
and total cancer mortality among subjects treated metabolomics?
with canakinumab in the same clinical protocol 10. What will be the relative roles of biologics,
([69]; see Chap. 39). Together, these papers sug- small molecules, and bone marrow trans-
gest an important role for inflammation in both plantation in the therapy of these illnesses
cardiovascular disease and cancer, and the possi- (see Chap. 42)?
bility of therapies targeting innate immunity in
preventing or treating these common illnesses. It is an exciting time to be working in the field
These two advances promise a much greater of autoinflammation. This textbook offers a mul-
role for autoinflammation in the general human tidisciplinary approach to a maturing discipline
condition. Not only will the boundaries blur that truly transcends the arenas of internal medi-
between autoinflammation and autoimmunity or cine, pediatrics, genetics and genomics, clinical
immunodeficiency, but the boundaries between and basic immunology, and cell biology, and I
health and disease will also blur. expect that practitioners and trainees from all of
these fields will derive great benefit from its com-
prehensive and systematic approach. I hope that
1.6 Questions for the Next you, too, will find yourself as captivated as I am,
Decade and that this text will be your passport to an
exhilarating journey in autoinflammation.
There also remain a number of questions for the Dan Kastner, MD, PhD
field to address in the next decade. Ten of those I Bethesda, Maryland
consider of primary importance are listed below: July 1, 2018
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Part II
Basic Science and Biology of
Autoinflammation
Genetic Aspects of Investigating
and Understanding 2
Autoinflammation
Isabella Ceccherini, Marta Rusmini,

and Juan Ignacio Arostegui
Abstract NGS methods in the clinics allows detection

At present, more than 30 different autoinflam- of (new) monogenic diseases in a growing
matory diseases have been described at molec- number of previously undiagnosed patients
ular and genetic level. The importance of with no familial history. This has resulted
genetic tests to reach a definitive diagnosis has in the increased awareness of the clinical
become evident during the past few years. In diversity of these diseases, best therapeutic
parallel to the description of these diseases, approaches and follow-up schemes for the
several technical changes have occurred that patients and appropriate genetic counseling
have revolutionized the field of human genet- for families.
ics. Ten years ago, the gold-standard method
for genetic studies was the Sanger method of Keywords
DNA sequencing. Currently, studies based on Next generation sequencing (NGS) · Gene
next generation sequencing (NGS) methods discovery · Mutation screening · NGS-based
are the standard methods in most genetic labo- gene panel · Comparative genomic hybridiza-
ratories around the world. NGS makes it pos- tion (CGH) · Gene expression · Real-time
sible to achieve a diagnosis both by analysis of polymerase chain reaction (rtPCR) · Gene
single families with extremely rare conditions, mosaicism · Post-zygotic mutations
thus identifying new genes, or simultaneous Amplicon-based deep sequencing
genotyping of multiple genes in groups of
patients. Moreover, in the past few years, dif-
ferent insights demonstrated an unexpected
role of post-zygotic mutations and gene mosa-
icism in the pathogenesis of some monogenic
autoinflammatory diseases. The availability of Abbreviations
ACMG America College of Medical Genetics

I. Ceccherini (*) · M. Rusmini
U.O.C. Genetica Medica, Istituto Giannina Gaslini, and Genomics
Genoa, Italy CAPS Cryopyrin-associated periodic syndrome
e-mail: isa.c@unige.it CGH Comparative genomic hybridization
J. I. Arostegui CINCA Chronic infantile neurological, cutane-
Department of Immunology, IDIBAPS, Hospital ous and articular
Clínic, Barcelona, Spain CNV Copy number variations
e-mail: JIAROSTE@clinic.ub.es
© Springer Nature Switzerland AG 2019 19

P. J. Hashkes et al. (eds.), Textbook of Autoinflammation,
https://doi.org/10.1007/978-3-319-98605-0_2
20 I. Ceccherini et al.
DADA2 Deficiency of adenosine deaminase 2 notypically overlapping autoinflammatory

ddNTP Dideoxynucleotide disorders, though the success rate is still low
DIRA Deficiency of IL-1 receptor antagonist • Whole exome sequencing and whole
DSAP Disseminated superficial actinic genome sequencing, along with array-com-
porokeratosis parative genomic hybridization (aCGH),
FCAS Familial cold autoinflammatory real-time polymerase chain reaction (PCR)
syndrome and other gene expression studies, repre-
FMF Familial Mediterranean fever sent effective means to identify new genes
IL Interleukin and new pathogenic mechanisms in autoin-
InDels Insertions or deletions flammatory disorders
JMP Joint contractures, muscle atrophy, • In the last decade, post-zygotic mutations
microcytic anemia, and panniculitis- and gene mosaicism have been described in
induced lipodystrophy syndrome a growing number of patients with several
LPS Lipopolysaccharide monogenic autoinflammatory diseases,
MKD Mevalonate kinase deficiency mainly as a result of using NGS-based meth-
MWS Muckle Wells syndrome ods during the routine genetic screening
NGS Next generation sequencing
NOMID Neonatal-onset multisystem inflamma-
tory disease 2.1 Introduction
PCR Polymerase chain reaction
PID Primary immunodeficiency diseases Systemic autoinflammatory diseases are a large
POADS Postaxial acrofacial dysostosis and heterogeneous group of disorders of the
SAVI STING-associated vasculopathy with innate immune system. The search for their
onset in infancy underlying genetic causative components has
SNP Single nucleotide polymorphism been pursued for decades as a means to discover
SNV Single nucleotide variant pathogenic mechanisms, a first step to assess pos-
STING Stimulator of interferon genes sible medication targets and to develop the most
TGF Transforming growth factor effective pharmacological treatments.
TNF Tumor necrosis factor The emerging complexity of the different clin-
TRAPS TNF receptor-associated periodic ical phenotypes within the autoinflammatory
syndrome spectrum, ranging from isolated periodic fevers
VUS Variant of uncertain significance to involvement of the gastrointestinal tract, bone,
WES Whole exome sequencing skin, etc, is not fully accounted for by the many
WGS Whole genome sequencing genes identified to date. For this reason, the rate
of undiagnosed patients is still remarkably high.
The technological developments during the
last decades in the methods of genetic investiga-
Key Points tions have largely contributed to the evolution of
• Next generation sequencing (NGS)-based the molecular genetic approaches used to under-
approaches have replaced previous strate- stand autoinflammatory disorders. In this chapter
gies for discovery of new genes, and muta- we will review the methods currently used in an
tion detection in already known genes attempt to analyze the genetic components of the
responsible for monogenic autoinflamma- simple Mendelian autoinflammatory disorders.
tory diseases We will first describe the Sanger sequencing
• NGS-based gene panels, allowing parallel technique, which was critical in detecting gene
sequencing of multiple small fragments of mutations both in the first autoinflammatory dis-
any given DNA target, are suitable to diag- eases in which disease-causing genes were i dentified
nose the many genetically diverse but phe- and even today in performing molecular genetic
2 Genetic Aspects of Investigating and Understanding Autoinflammation 21
diagnosis of single genes or among few candidate 2.2.1 xperimental Methods Used
E
genes. The next generation sequencing (NGS)- in the Pre-NGS Era
based approaches have become crucial to speed up
the identification of new genes and also for simul- The “positional cloning” strategy has driven the
taneous multiple gene testing, especially in those discovery of genes responsible for monogenic
patients whose phenotype cannot easily be linked diseases for years, before the advent of the NGS-
to an already recognized autoinflammatory disor- based technologies. To this end, genome-wide
der. The role of additional genetic techniques in the genetic linkage analysis was used to identify
diagnostic process is also going to be reported. chromosomal regions involved in the diseases
Last, somatic mosaicism has emerged as an under study. After narrowing the intervals and
important pathogenic mechanism, which is able cloning the chromosomal segments of interest,
to account for the development of a full clinical the disease genes were eventually identified, fol-
picture despite limited proportions of cells bear- lowed by detection of specific mutations. Despite
ing the mutation. The latter part of the chapter the great importance and significance that this
will be devoted to understanding this important methodologic approach has had in the discovery
mechanism of inheritance. of genes causing familial Mediterranean fever
A glossary of genetic terms for readers with- (FMF) [1, 2], tumor necrosis factor (TNF)
out a genetics background can be found at https:// receptor-associated periodic syndrome (TRAPS)
www.genome.gov/glossary. [3], mevalonate kinase deficiency (MKD) [4, 5],
and cryopyrin-associated periodic syndromes
(CAPS) [6], such a labor intensive and time con-
suming strategy has slowly been abandoned, to
2.2 Autoinflammatory Diseases: be replaced by more accurate and powerful tech-
Approaches to Gene niques [7].
Identification The “homozygosity mapping” approach con-
tinues to be successfully applied to the present to
• Linkage analysis and homozygosity map- complement the search for genes responsible for
ping have allowed the positional cloning recessively inherited diseases. Homozygosity
strategy to drive the discovery of genes mapping relates to the identification of disease
responsible for monogenic autoinflamma- causing gene regions in consanguineous families,
tory diseases in the past decades or in populations subjected to founder effects,
• Mutation search in candidate genes has where affected individuals are likely to have two
been carried out through Sanger sequenc- “replication” copies of the disease allele, as well
ing and more recently by the next genera- as additional identical alleles located near the
tion sequencing (NGS) technology, allowing disease locus from a common ancestor. Rare
parallel sequencing of multiple small frag- recessive traits can therefore be identified through
ments of any given DNA target regions of homozygosity that are shared by dif-
• NGS-based gene panels are appropriate to ferent affected individuals. Informative polymor-
diagnose the many genetically diverse but phic markers have been used to perform
phenotypically overlapping autoinflamma- homozygosity mapping in the case of FMF [1, 2]
tory disorders. However, the success rate is and Majeed syndrome [8]. More recently, the
still low genotyping methods have evolved and single
• Whole exome sequencing data can be lim- nucleotide polymorphism (SNP) arrays have led
ited to in silico panels, thus avoiding many to the discovery of the deficiency of interleukin
variants of unknown significance and (IL)-1 receptor antagonist (DIRA) [9, 10] and the
unwanted detection of secondary findings, joint contractures, muscle atrophy, microcytic
and, if no causative variant is found, the anemia, and panniculitis-induced lipodystrophy
entire exome can still be analyzed syndrome (JMP) [11].
When a linkage based approach cannot be sequencing”, the method developed by the British
applied to identify a disease-causing gene, for biochemist Dr. Frederick Sanger. This method
instance in the case of a limited number of avail- makes use of dideoxynucleotides (ddNTPs),
able families, the analysis of candidate genes can chemically modified bases that terminate the
serve as a possible alternative. Candidate genes chain when incorporated into the new strands
can be selected based on different criteria, such while these are synthetized by DNA polymerase
as genes whose functions is related to the pheno- during a PCR [48]. In particular, single strand
type, within an already identified chromosomal DNA is used as the template in four PCR reac-
interval, as in the case of TRAPS [3]; the absence tions, each including one different ddNTP labeled
or reduction of enzymatic activity, or any other with one of four different colored fluorescent
measurable disease marker, as in the case of tags, besides unmodified nucleotides (dNTPs).
MKD [5]; the response to a known treatment, as By the time the cycling is complete, a ddNTP
in the case of DIRA [9] and the homology with will have been incorporated at every single posi-
genes belonging to the same family and involved tion of the target DNA in each tube reaction,
in similar autoinflammatory disorders, as in the namely, the tube will contain fragments of differ-
case of NLRP12 mutations in familial cold auto- ent lengths, ending at each of the nucleotide posi-
inflammatory syndrome 2 (FCAS2) [12], which tions in the original DNA, which will be labeled
was discovered due to the known involvement of with final nucleotide specific dyes. In the end, the
NLRP3 gene in FCAS1. four reaction mixtures can be combined and
Finally, the presence of chromosomal rear- applied to a single lane of a capillary electropho-
rangements or structural variations occurring in resis. The color of each fragment is detected
patients, the availability of animal models with using a laser beam and the information is col-
human disease phenotypes, and other possible lected by a computer that generates chromato-
meaningful clues have in many instances driven grams showing peaks for each color, from which
the identification of the disease-causing gene. the template DNA sequence can be determined.
Table 2.1 reports the up to date list of genes This sequencing method is accurate for sequences
responsible for autoinflammatory disorders. In up to a maximum of about 700–800 base-pairs in
addition, Table 2.2 focuses on a more recent sub- length (Fig. 2.1).
set of autoinflammatory disorders, termed “type
1 interferonopathy”, characterized by mutations
in genes encoding proteins involved in nucleotide 2.2.3 DNA Sequencing: The NGS
metabolism and resulting in the upregulation of Method
interferon stimulated genes. The advent of whole
exome sequencing has greatly facilitated the NGS is a revolutionary diagnostic tool for
identification of all these genes (see also Sects. genetic investigations, allowing the simultane-
2.2.4 and 2.3.2.3). ous analysis of multiple genes and the effective
detection of gene mosaicism (see below). There
are a variety of different NGS technologic plat-
2.2.2 DNA Sequencing: The Sanger forms making use of different sequencing chem-
Method istries [49, 50]. However, most share a common
set of features concerning sequencing reactions
DNA sequencing, the process of reading the such as: (1) taking place in parallel, at the same
sequence of nucleotides present in a DNA mole- time, (2) micro scaled so that a very high num-
cule that verifies the presence of variants in genes ber of genes can be accommodated on the same
of interest, is a crucial final step common to all chip, (3) requiring a very tiny amount of DNA
the genetic approaches. per test, (4) cheaper than Sanger sequencing, (5)
The gold standard for DNA sequencing has producing shorter reads (typically 50–700 nt in
been for many years the so-called “Sanger length).
2
Table 2.1 List of genes causing systemic autoinflammatory diseases, with experimental approaches used for their first descriptions and initial referencesa
Trait Experimental approach used for gene
Gene name OMIM Disease OMIM trans-mission identification References
AP1S3 615781 PSORS15 616106 Dominant Whole exome sequencing Setta-Kaffetzi et al., 2014 [13]
CARD14 607211 PSORS2 602723 Dominant Linkage analysis coupled with targeted Jordan et al., 2012 [14]
whole-exome sequencing and candidate-gene
screening
CARD14 607211 PRP 173200 Dominant Linkage analysis followed by targeted Fuchs-Telem et al., 2012 [15]
whole-exome sequencing and candidate-gene
screening
ADA2 607575 PAN/DADA2 615688 Recessive Whole exome sequencing Zhou et al., 2014 [16]; Navon-Elkan
et al., 2014 [17]
IL10 124092 IL-10D Recessive Candidate gene Glocker et al., 2010 [18]
IL10RA 146933 IBD28/IL-10R1D 613148 Recessive Linkage analysis and candidate-gene sequencing Glocker et al., 2009 [19]
IL10RB 123889 IBD25/IL-10R2D 612567 Recessive Linkage analysis and candidate-gene sequencing
IL1RN 147679 OMPP/DIRA 612852 Recessive Candidate gene Aksentijevich et al., 2009 [9]; Reddy
et al., 2009 [10]
IL36RN 605507 DITRA 614204 Recessive Homozygosity mapping and direct Marrakchi et al., 2011 [20]; Onoufriadis
sequencing + whole exome sequencing et al., 2011 [21]
LPIN2 605519 Majeed syndrome 609628 Recessive Homozygosity mapping and direct sequencing Ferguson et al., 2005 [8]
of genes in the region
MEFV 608107 FMF 249100 Recessive Positional cloning International FMF Consortium, 1997
[1]; French FMF Consortium, 1997 [2]
MVK 251170 HIDS 260920 Recessive Positional cloning + candidate gene Drenth et al., 1999 [4]; Houten et al.,
Genetic Aspects of Investigating and Understanding Autoinflammation
1999 [5]
MVK 251170 MEVA/MA 610377 Recessive Candidate gene Schafer et al., 1992 [22]
MVK 251170 POROK3/DSAP 175900 Dominant Linkage analysis and targeted whole exome Zhang et al., 2012 [23]
sequencing
NLRC4 606831 AIFEC 616050 Dominant Whole exome sequencing Romberg et al., 2014 [24]; Canna SW,
et al. 2014 [25]
NLRP12 609648 FCAS2/NAPS12 611762 Dominant Candidate gene Jeru et al., 2008 [12]
NLRP3 606416 FCAS1/FCU/ CAPS1 120100 Dominant Positional cloning (linkage analysis followed by Hoffman et al., 2001 [6]
NLRP3 606416 MWS/CAPS2 191900 Dominant direct sequencing in the candidate region) Dode et al., 2002 [26]
(continued)
23
24
Trait Experimental approach used for gene
OMIM
Gene name OMIM Disease trans-mission identification References
NLRP3 606416 NOMID/CINCA/ 607115 Dominant Candidate gene Aksentijevich et al., 2002 [27]; Feldman
CAPS3 et al., 2002 [28]
NLRP7 609661 HYDM1/RHM 231090 Recessive Positional cloning Murdoch et al., 2006 [29]
NOD2 605956 Blau syndrome 186580 Dominant Linkage analysis and candidate-gene sequencing Miceli-Richard et al., 2001 [30]
NOD2 605956 Early-onset 609464 Dominant Candidate gene Kanazawa et al., 2004 [31]
sarcoidosis
NOD2 605956 IBD1 266600 Dominant Positional cloning based on linkage analysis Ogura et al., 2001 [32]; Hugot et al.,
followed by linkage disequilibrium mapping 2001 [33]
OTULIN 615712 AIPDS 617099 Recessive Homozygosity mapping followed by candidate Damgaard et al., 2016 [34]; Zhou et al.,
gene and exome sequencing 2016 [35]
PLCG2 600220 APLAID 614878 Dominant Whole exome sequencing Zhou et al., 2012 [36]
PLCG2 600220 FCAS3 614468 Dominant Linkage analysis, targeted Sanger sequencing, Ombrello et al., 2012 [37]
and next generation/whole genome sequencing
POMP 613386 CANDLE/PRAAS 2 618048 Dominant Candidate gene Brehm et al., 2015 [38]
PSMA3 176843 CANDLE/PRAAS Candidate gene
PSMB4 176846 CANDLE/PRAAS Candidate gene
PSMB8 177046 ALDD/JMP/ NNS/ 256040 Recessive Homozygosity mapping followed by sequencing Agarwal et al., 2010 [11]; Arima et al.,
CANDLE of candidate genes in the region 2011 [39]
PSMB9 177045 CANDLE/PRAAS Candidate gene Brehm et al., 2015 [38]
PSTPIP1 606347 PAPA 604416 Dominant Positional cloning Wise et al., 2002 [40]
PSTPIP1 606347 Hyperzyncemia and Dominant Candidate gene Holzinger D et al., 2015 [41]
hypercalprotectinemia
RBCK1 610924 PBMEI/ HOIL-1D 615895 Recessive Whole exome sequencing coupled with single Boisson et al., 2012 [42]
nucleotide polymorphism array
SH3BP2 602104 Cherubism 118400 Dominant Positional cloning Ueki et al., 2001 [43]
SLC29A3 612373 Histiocytosis- 602782 Recessive Genome-wide linkage analysis followed by Morgan et al., 2010 [44]
lymphadeno-pathy sequencing of candidate genes in the region
plus syndrome
TMEM173 612374 SAVI 615934 Dominant Whole exome sequencing Liu et al., 2014 [103]
I. Ceccherini et al.
2
TNFAIP3 191163 AISBL/ A20 616744 Dominant Whole exome sequencing Zhou et al., 2016 [46]
Haploinsufficiency
TNFRSF11A 603499 TRAPS11 Dominant Identification by means of a structural variant Jeru et al., 2014 [47]
(aCGH) followed by gene sequencing
TNFRSF1A 191190 TRAPS 142680 Dominant Positional cloning based on linkage analysis McDermott et al., 1999 [3]
followed by linkage disequilibrium mapping
a
Empty cells refer to information not available yet
PSORS15 Psoriasis 15, pustular, susceptibility to, HAE1/HANE Hereditary angioEdema type I/Hereditary angioneurotic edema, PSORS2/PRP Psoriasis 2/Pityriasis rubra pilaris,
PAN/DADA2 Polyarteritis nodosa, childhood-onset/Deficiency of adenosine deaminase 2, IL-10D IL-10 deficiency, IBD28/IL10R1D Inflammatory bowel disease 28, early onset/
IL-10 receptor 1 deficiency, IBD25/IL-10R2D Inflammatory bowel disease 25, early onset/IL-10 receptor 2 deficiency, OMPP/DIRA Osteomyelitis, sterile multifocal, with peri-
ostitis and pustulosis/Deficiency of interleukin 1 receptor antagonist, PSORP/DITRA Generalized pustular psoriasis/deficiency of interleukin 36 receptor antagonist, FMF
Familial Mediterranean fever, HIDS Hyperimmunoglobulinemia D syndrome (periodic fever, Dutch type), MEVA/MA Mevalonic aciduria, POROK3/DSAP Porokeratosis 3,
disseminated superficial actinic type/Disseminated superficial actinic porokeratosis, AIFEC autoinflammation with infantile enterocolitis, FCAS2/NAPS12 Familial cold autoin-
flammatory syndrome 2/NLRP12-associated periodic syndrome, FCAS1/FCU/CAPS1 Familial cold autoinflammatory syndrome 1/Familial cold urticaria/CIAS1-associated
periodic syndrome 1, MWS/CAPS2 Muckle-Wells syndrome/CIAS1 associated periodic syndrome 2, CINCA/NOMID/CAPS3 Chronic infantile neurological cutaneous and
articular syndrome/neonatal-onset multisystem inflammatory disease/CIAS1 associated periodic syndrome 3, HYDM1/RHM Hydatidiform mole, recurrent, 1/Recurrent hydati-
form moles, BLAU-JABS/EOS/IBD1 Blau and Jabss Syndromes/Early onset sarcoidosis/Inflammatory bowel disease 1(Crohn disease), AIPDS Autoinflammation, panniculitis,
and dermatosis syndrome, APLAID Autoinflammation, antibody deficiency, and immune dysregulation, PLCG2-associated syndrome, FCAS3 Familial cold autoinflammatory
syndrome 3, CANDLE/PRAAS Chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature syndrome/Proteasome-associated autoinflammatory syn-
dromes, ALDD/JMP/NNS/CANDLE Autoinflammation, lipodystrophy, and dermatosis syndrome/Joint contractures, muscle atrophy, microcytic anemia, and panniculitis-induced
lipodystrophy syndrome/Nakajo-Nishimura syndrome/Chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature syndrome, PAPA Pyogenic sterile
arthritis, pyoderma gangrenosum, and acne, PBMEI/HOIL-1D Polyglucosan body myopathy, early-onset, with or without immunodeficiency/HOIL1 deficiency, SAVI STING-
associated vasculopathy, infantile-onset, AISBL Autoinflammatory syndrome, familial, Behçet-like
25
Table 2.2 List of interferonopathies and corresponding causative genes

Disease OMIM Inheritance Gene (s) OMIM Protein function/pathway
CANDLE (Chronic Atypical 256040 AR (digenic PSMA3, 176,843, Proteasome pathway:
Neutrophilic Dermatosis with transmission PSMB4, 602177, responsible for
Lipodystrophy and Elevated has also been PSMB8, 177046, regulating proteolysis
temperature) syndrome; also described) PSMB9, 177045, in eukaryotic cells
referred to as Proteasome POMP 613386
associated autoinflammatory
syndromes (PRAAS)
STING associated vasculitis 615934 AR TMEM173 612374 Adapter molecule
with onset in infancy (SAVI) involved in IFN
production
Aicardi-Goutières syndromes 225750, AD/AR TREX1, 606609, Regulation of
(types 1–7) 610329, AR RNASEH2C, 610330, cytoplasmic DNA/RNA
610181, AR RNASEH2B, 610326,
610333, AR RNASEH2A, 606034,
612952, AR SAMHD1, 606754,
615010, AR ADAR, IFIH1 146920,
615846 AD 606951
Retinal vasculopathy with 192315 AD TREX1 606609 Regulation of
cerebral leukodystrophy cytoplasmic DNA/RNA
(RVCL)
Spondyloenchondrodysplasia 607944 AR ACP5 171640 Lysosomal acid
(SPENCD) phosphatase activity/
osteoclastic dysfunction
Singleton-Merten Syndrome 182250, AD IFIH1, 606951, Cytosolic sensor of
(types 1–2) 616298 AD DDX58 609631 ds-RNA
ISG15 deficiency 616126 AR ISG15 147571 Negative regulator of
(immunodeficiency 38) type I IFN by
stabilisation of USP18
USP18 deficiency (pseudo- 617397 AR USP18 607057 Negative feedback
TORCH syndrome) regulator of type I IFN
signalling
Trichohepatoenteric 614602 AR SKIV2L 600478 RNA helicase
syndrome 2
The NGS technology is therefore based on the ogy” where the effective costs are significantly
parallel sequencing of multiple small fragments reduced and the quantity of information/sequences
of a given DNA target [49, 51], which are ligated produced markedly increased [50, 52]. Due to
to proper adaptors and pooled in so-called “librar- these great improvements, NGS can be used to
ies” for the successive sequencing, rather than on sequence very large DNA targets, such as specific
the sequencing of single fragments like in the regions of interest that may span even hundreds of
Sanger sequencing technology. Next generation thousands of base pairs, the whole “exome”, repre-
methods of DNA sequencing have therefore three senting the coding portion of the genome (around
main steps: (1) creation of DNA libraries includ- 30–40 Megabases), up to the whole genome, cor-
ing the whole target DNA, first captured in the responding to ≈3 × 10E9 base pairs.
form of DNA segments that are then ligated to
custom linkers, (2) amplification of the libraries
using clonal methods to separate each fragment, 2.2.4 NGS-Based Techniques
and (3) sequencing of each fragment of the for New Gene Discovery
library using one of several different approaches.
Due to high speed and remarkably rich outputs, Whole exome sequencing (WES) is the most
NGS can be defined as a “high-throughput technol- widely used application of NGS when searching
obtain information regarding allele frequencies,

impact at protein level, pathogenicity prediction,
degree of conservation of the protein domain(s),
and possible associations with disease pheno-
types, etc (Table 2.3). Moreover, the most likely
pattern of disease inheritance should be consid-
ered in order to filter for de novo, heterozygous,
homozygous, compound heterozygous or hemi-
zygous variants. To this end, the analysis of unaf-
fected members of the patient’s family may
become indispensable for the segregation study
of selected variants. Finally, a thorough investi-
gation of the gene function, if already known,
and how it is affected by the presence of the vari-
ant will help to correlate the genotype thus
selected with the clinical phenotype of the patient
Fig. 2.1 Diagram schematically showing the Sanger under study. For this reason, patients’ history and
sequencing procedure. Molecular steps start with the
establishment of four PCR reactions, each including one clinical phenotypes should be also carefully
different ddNTP labeled with one of four different colored assessed [55].
fluorescent tags. Single strand DNA is used as the tem- Despite the tangible progress made in the
plate (not shown) (Step 1). Fragments of different lengths, technology underlying massive sequencing, the
ending at each of the nucleotide positions in the original
DNA template, will be labeled with nucleotide specific genetic interpretation of the results still remains a
dyes (Step 2). In the end, the four reaction mixtures are critical aspect.
combined and applied to a single lane of a capillary elec- An early application of WES has been the
trophoresis coupled with a laser beam so that the DNA rapid discovery of new genes in patients affected
fragments can be detected according to their length fluo-
rescent tag, thus determining the template DNA sequence by simple Mendelian disorders [56]. Among the
(Step 3) first publications of a genetic diagnosis achieved
by WES is the report of congenital chloride diar-
rhea, based on the finding of a homozygous mis-
for the gene responsible for a rare disease [53]. sense variant at the SLC26A3 gene in a patient
Patients with rare diseases benefit from WES referred with a different diagnosis [57], as well as
application that can facilitate gene discovery, the discovery of the gene responsible for Miller
thus attaining a correct and timely clinical diag- syndrome. This syndrome also known as postax-
nosis, providing insights into biological mecha- ial acrofacial dysostosis (POADS) is character-
nisms, and increasing therapeutic ized by mandibulofacial dysostosis with postaxial
opportunities. limb anomalies. The discovery started from four
Despite the great versatility of the NGS patients in three independent kindreds, finding
method and its applications, the bio-informatics that all shared homozygous or compound hetero-
data analysis, critically needed to interpret the zygous mutations of the DHODH gene [58].
huge amount of sequences obtained, can be com- Since then, many other reports have demon-
plicated. WES is typically used to detect single strated the great utility of WES [56]. WES has
nucleotide variants (SNVs) and small insertions been particularly successful in the case of rare
or deletions (InDels). But the identification of diseases where identifying a gene or a potentially
causative variants responsible for phenotypes altered pathway may be extremely difficult and
under study requires careful filtering and ranking often otherwise impossible.
(prioritizing) candidate genes, according to sev- Nearly one third of the genes responsible for
eral criteria [54]. In particular, public databases hereditary autoinflammatory disorders have been
should be searched for each variant in order to identified using NGS technology (Table 2.1). In
Table 2.3 Public databases that can be used to annotate variants found
Information that can be
retrieved Database URL
Allele frequencies The Exome Aggregation Consortium exac.broadinstitute.org/
(ExAC)
The Genome Aggregation Database http://gnomad.broadinstitute.org/
(gnomAD)
The Single Nucleotide Polymorphism https://www.ncbi.nlm.nih.gov/SNP/
database (dbSNP)
NHLBI Exome Sequencing Project http://evs.gs.washington.edu/EVS/
(ESP): Exome Variant Server
The 1000 Genomes Project http://www.internationalgenome.org/
data
Impact of variants at ENSEMBL https://www.ensembl.org/
protein level The Protein Variation Effect Analyzer http://provean.jcvi.org
(PROVEAN)
PANTHER http://www.pantherdb.org/
SNPs&GO http://snps.biofold.org/snps-and-go
Prediction of variant Combined Annotation Dependent http://cadd.gs.washington.edu/
pathogenicity Depletion (CADD)
Sorting Intolerant From Tolerant (SIFT) http://sift.jcvi.org/ift
Polymorphism Phenotyping v2 (Polyphen http://genetics.bwh.harvard.edu/pph2/
2)
SNPeff http://snpeff.sourceforge.net/SnpEff_
manual.html
MutationTaster http://www.mutationtaster.org/
Degree of conservation of The Genomic Evolutionary Rate Profiling http://mendel.stanford.edu/SidowLab/
the protein domain(s) (GERP) downloads/gerp/
Single nucleotide variants Variant Effect Predictor (VEP) http://www.ensembl.org/
(SNV) annotation tools VarSome: The Human Genomic Variant https://varsome.com/
Search Engine
ANNOVAR http://annovar.openbioinformatics.org/
Jannovar https://github.com/charite/jannovar
Overlaps with regulatory AnnTools http://anntools.sourceforge.net/
elements, known segmental SCAN http://www.scandb.org/newinterface/
duplications, etc about.html
Phenotypic abnormalities The Human Phenotype Ontology (HPO) http://human-phenotype-ontology.
encountered in human github.io/
disease
Collections of disease DisGeNET http://www.disgenet.org/web/
genes and human genetic DisGeNET/menu
diseases Online Mendelian Inheritance in Man https://www.omim.org/
(OMIM)
many cases, an unbiased WES protocol was ble for three recessive diseases, have been dis-
applied and the gene variants causative of the covered by targeted massive sequencing after
corresponding patients’ phenotype were assessed identifying the chromosomal segment containing
upon the application of the filters mentioned the disease-causing gene through genome-wide
above. As reported in Table 2.1, this was the case linkage analysis. Similarly, in two diseases, link-
for the AP1S3, ADA2, NLRC4, PLCG2, age analysis followed by targeted NGS approach
TMEM173, and TNFAIP3 genes and correspond- has expanded the phenotypic spectrum associ-
ing, mostly dominant, disorders. On the other ated with mutations of the MVK and PLCγ2
hand, IL36RN, OTULIN, and RBCK1, responsi- genes, after detecting new damaging variants of
these genes in two unexpected clinical entities, tions [62–66], the application of Sanger sequenc-
disseminated superficial actinic porokeratosis ing in the diagnosis of hereditary autoinflammatory
(DSAP) and FCAS3 syndrome, respectively disorders should nowadays be limited to those
(Table 2.1). patients showing unequivocal clinical pheno-
One of the great powers of WES is its versatil- types and whose diagnosis can be predicted with
ity. Creation of in silico panels, including the a reasonably high confidence. This has recently
genes already known to be responsible for most been confirmed in more than 2000 patients who
cases of the disease under study (i.e. autoinflam- were screened by Sanger sequencing for three
matory disease), allows for a wide number of autoinflammatory genes, namely NLRP3, MVK
genes to be examined simultaneously. If no and TNFRSF1A in addition to some portions of
pathogenic variants are found, the entire exome other genes, without finding any mutations in
can then potentially be analyzed. In silico panels 86% of samples [67]. In general clinical genetic
are particularly suitable when the involvement of settings, it has been reported that the diagnostic
a specific pathway can be postulated or when the rate of Sanger sequencing is not more than 50%,
disease under study belongs to a very large phe- and the rate becomes much lower for patients
notypic spectrum known for a wide genetic het- who have already been through one unsuccessful
erogeneity. Moreover, use of in silico panels can genetic evaluation [68, 69]. Possible explanations
avoid the unwanted detection of secondary or may be, among others, the limited number of
incidental findings from the whole exome data. genes tested but also possible clinical misdiagno-
sis, a wider than expected genetic heterogeneity,
complex modes of inheritance, gene mosaicism,
2.3 Autoinflammatory Diseases: poor yield of the Sanger sequencing approach for
Approaches to Molecular specific gene portions and missed mutations.
Genetic Diagnosis For all these reasons, other methodologies,
enabling the testing of multiple genes or detect-
The accuracy of the NGS approach [59–61] is ing genetic defects other than SNVs or small
high, though dependent on the platform and insertions/deletions (indels), should be preferred
chemistry used, thus making this the method of to Sanger sequencing for most patients with auto-
choice for the detection of causative mutations in inflammatory disorders.
already known genes, no matter whether typical,
atypical or new variants. A diagnostic application
of NGS is very much feasible, though limited by 2.3.1 NGS-Based Gene Panels
a few circumstances which must be kept in mind:
(1) patients should be well characterized in term Allowing the sequencing of several genes simul-
of clinical phenotype, a condition not always sat- taneously, the use of NGS-based gene panels can
isfied because of the strong heterogeneity of facilitate the diagnosis in patients with autoin-
autoinflammatory disorders and the difficulties to flammatory disorders, often hampered by the
correctly classify patients suffering from differ- wide heterogeneity of the many genetically
ent disorders with overlapping symptoms, and diverse but phenotypically overlapping diseases
(2) genetic heterogeneity may exceed our expec- belonging to the autoinflammatory spectrum.
tations thus preventing the identification of the The development of NGS-based gene panels
causative variant. Indeed, in many instances, an represents a perfect application of scientific
expensive and time consuming NGS search for knowledge gained about autoinflammatory disor-
mutations in the candidate gene(s) might lead to ders and genes involved in their pathogenesis for
an inconclusive result. diagnostic purposes. Indeed, knowing genes asso-
Based on the considerations previously dis- ciated with pathogenesis allows for the ad hoc
cussed, and despite having been used for years as creation of panels, including all genes or gene
the only means to screen patients for gene muta- portions of interest. Early commercial panels,
limited to ≤10 genes, were developed and applied and genetic heterogeneity of the included disor-
to patients with autoinflammatory disorders tak- ders, as well as, for recessive diseases, on the
ing advantage of new NGS technologies ([70], level of consanguinity of parents (correlated to
https://www.genedx.com/). More recently, these the degree of inbreeding of the populations which
panels have been revised with updated gene sets, patients belong to). For instance, different tar-
up to 166 genes, to study both systemic autoin- geted gene panels for primary immunodeficiency
flammatory disorders and vasculitis [71]. These diseases (PID) including between 162 and 170
panels uncovered a number of unexpected tech- genes allowed a definitive diagnosis in 15–25%
nological drawbacks, that potentially limit the in a total of 165 patients [72], while the diagnos-
diagnostic performance of the new tool. This is tic yield was much more heterogeneous in
the case, for instance, for the degree of represen- patients with epilepsy ranging from 10 to 48.5%
tation of the submitted target (in other words, the using panels including from 35 to 265 genes [73].
effectiveness of target capture), for the sequenc- In autoinflammatory disorders, the gene panels
ing depth (i.e. the mean coverage of samples), for published to date report a satisfactory validation
the ability of the panel to recognize variants pres- of positive controls which in all the cases revealed
ent in the sample under analysis (sensitivity) and high sensitivity and specificity of the panels
not to find variants that in fact are not present in under development [70, 71]. Omoyinmi and col-
the sample (specificity). Therefore, several tech- leagues tested 50 patients with undefined autoin-
nologic aspects can affect the performance of a flammatory disorders using their “vasculitis and
NGS panel, such as (1) the sequencing chemis- inflammation panel” that contains 166 genes,
tries adopted by different available commercial finding either pathogenic or likely pathogenic
platforms, (2) the selection methods applied to mutations in 16 samples, corresponding to a yield
capture of the desired targets, and (3) unlike in of 32% [71]. A further panel with a set of 41
Sanger sequencing, the bioinformatics step can genes specific for autoinflammatory disorders
make a difference in data analysis needed to was applied to 50 undifferentiated patients, with-
reach a genetic diagnosis [70]. This highlights out any improvement in the mutation detection
the need to first validate panels with DNA sam- rate compared to Sanger sequencing. This again
ples from patients already diagnosed. suggests that patients with undifferentiated phe-
This new experimental approach for the diag- notypes may have either a complex multifacto-
nosis of heterogeneous autoinflammatory disor- rial/multigenic etiology or the involvement of
ders has given a great impulse to the study not still unknown genes (IC, personal observation).
only of simple cases, with a clear clinical phe- Finally, among 246 children with a suspected pri-
notype, but also and especially of the so-called mary immunodeficiency, a NGS-based panel
“undifferentiated” patients, namely those clini- containing 302 genes, including 23 genes for
cally undiagnosed patients, with non-autoinflammatory disorders, revealed 15 subjects
confirmatory genetic test and/or atypical with a likely genetic diagnosis of NLRP12-
presentations, complicated by unexpected associated autoinflammatory disorder and pri-
symptoms. mary immunodeficiency, thus highlighting still
The demand for NGS-based testing has grown undisclosed associations and confirming the
rapidly without a corresponding increase in the powerfulness of the NGS tools to investigate
rate of detection of causative mutations, a cir- complex and/or heterogeneous disorders like
cumstance that has had a strong impact on the autoinflammatory disorders [74].
yield of NGS panels, in terms of the proportion of Therefore, an NGS-based gene panel for auto-
patients that have been diagnosed through the inflammatory disorders may often be unsatisfac-
molecular approach. Indeed, in the literature, the tory in routine confirmation of a genetic diagnosis
yield of the most focused disease panels varies in clinical practice or to solve complicated phe-
from a higher yield (40–50%) to a lower yield notypic pictures. Nevertheless, NGS-based auto-
(15–25%), likely depending on the phenotype inflammatory gene panels are still widely used,
as there are many patients suspected to have an The use of NGS-panels has lead to the detec-
autoinflammatory disorder and the turnover time tion of an enormous number of sequence variants
of the test is quite fast. Gene panels might be whose significance is often uncertain and whose
regarded as a first screening test before deciding correlation with the phenotype is anything but
about further investigations. Only a limited num- straightforward. Indeed, as the number of gene
ber of unsolved patients can ultimately undergo variants detected is continuously growing, we
further analysis, like WES, as this is still quite have realized the need of a consensus or agree-
demanding in terms of both costs and time. ment for scoring variant pathogenicity. A role,
According to present data, there does not seem either pathogenic or benign, can be assigned to
to be a strict correlation between the number of each variant based on a number of observations
genes in NGS-panels specific for autoinflamma- within the framework of the American College of
tory disorders and the diagnostic success rate, as Medical Genetics and Genomics (ACMG) rec-
stringency of inclusion criteria and tested genes ommendations [75]. These include (1) the allele
also affect the final performance of the panel. In frequency of the variant in large control popula-
addition, both small and large panels have advan- tions, (2) the type of gene variant (splice, stop
tages and also drawbacks: if small panels have an codon, missense, etc), (3) whether the variant is
undersized representation of disease-related already reported in relevant patients or registered
genes, the number of variants of unknown signifi- in databases (publications, Infevers, ClinVar,
cance thus detected is limited and the panel etc), (4) whether it affects a mutational hotspot,
results are more “manageable”, the opposite hap- namely a codon in which a mutation has already
pens with large panels. To overcome such an been detected previously, (5) in silico predic-
inconvenience, the use of sub-panels has been tions, including evolutionary conservation of the
introduced in many labs, each covering a differ- codon, location in putative functional sites of the
ent class of autoinflammatory disorders (recur- protein, type of amino acid substitution, (6)
rent fevers, skin related diseases, chronic familial co-segregation of the variant with the
urticaria, syndromes with intestinal involvement, phenotype, and finally (7) results from available
etc) with highly specific genes, some of which functional in vivo and in vitro studies (Fig. 2.2).
can be redundant, overlapping different Some studies have reported variant classifica-
sub-panels. tions for selected genes causing autoinflamma-
tory disorders [76, 77]. One of these studies
Fig. 2.2 List of criteria

used mostly for variant
Mendelian segregation in families
prioritization
Variant Allele frequency in populations
distribution
Consequence of the nucleotide change

Does the mutation affect a mutational hotspot
Variant
effects In silico prediction of variant effect
Association with a disease phenotype

Data from in vivo and in vitro studies
Observations
described a consensus-driven process by experts lacking a high diagnostic yield in the very hetero-
for the pathogenicity assessment, which resulted geneous field of the autoinflammatory disorders,
in the classification of almost all variants reported target resequencing, namely the (re)sequencing
so far in the four main genes causing hereditary of a small subset of the genome such as with a
recurrent fever syndromes (MEFV, TNFRSF1A, gene panel, results in a higher diagnostic power
NLRP3 and MVK genes). According to the and a reduction of costs, compared to the classi-
ACMG recommendations, these variants have cal method of investigation [78].
been classified as (1) benign, (2) likely benign, The still low diagnostic yield might be due to
(3) variants of uncertain significance (VUS), (4) mutations in regions not included in the NGS
likely pathogenic, and (5) pathogenic. The results panel used (either non-coding regions or alterna-
of this classification have been made available on tive transcripts) or in other genes. When these
the INFEVERS database at https://fmf.igh.cnrs. circumstances are suspected, either whole exome
fr/ISSAID/infevers/ [77]. or whole genome sequencing should be consid-
Discussion between laboratories involved in ered. Indeed, additional genes causing autoin-
the study of patients affected with autoinflamma- flammatory disorders and still undisclosed
tory disorders about variants detected in genetic mechanisms need to be identified to
autoinflammatory- related genes is crucial to explain the full genetic heterogeneity of autoin-
increase the quality and speed of the interpreta- flammatory disorders and NGS is already con-
tion of NGS data, to minimize discordant variant tributing to this.
classifications between laboratories, to limit mis-
interpretation of DNA variants, and, finally, to
recognize variants detected sporadically in diag- 2.3.2 ther Approaches Employed
O
nostic labs that are not yet contained in public in Patients
databases. with Autoinflammatory
As discussed above, NGS is an approach that, Disorders
for technical reasons, may be prone to yield both
false positive and false negative results. Therefore, 2.3.2.1 Array-Comparative Genomic
despite the high reliability of the method, it is of Hybridization (aCGH)
utmost importance to validate the detected vari- Microarray-based comparative genomic hybrid-
ants by the Sanger sequencing method, still the ization (Array-CGH, also known as aCGH) is a
gold standard for the DNA sequencing, to avoid molecular cytogenetic technique that allows for
wrong conclusions, especially in a diagnostic the detection of chromosomal imbalances involv-
setting. ing either loss or gain of genomic regions.
Last, the costs associated with the new NGS Balanced structural variations, such as balanced
technological approach represent another advan- chromosomal translocations, cannot be revealed
tage. Indeed, it has been estimated that a NGS by aCGH. The principle of the method is based on
panel of more than 150 genes costs as much as the comparison (hybridization) of two DNA sam-
the screening of one single gene performed by ples, typically one patient and one control, labeled
the Sanger sequencing approach ([71], IC, per- with different fluorescent tags, to detect any dif-
sonal observation). However, the time and cost ference in the relative quantity of individual
spent on bioinformatics analysis should also be regions of the genome. For this reason, aCGH is
taken into account when comparing Sanger the test of choice to investigate so-called “copy
sequencing and NGS-based tests. number variations” (CNVs), with a resolution
In conclusion, NGS is a reliable diagnostic ranging from about 20 to 200 Kb [79]. After dena-
tool for autoinflammatory disorders, which can turation, the two DNA samples are mixed and
be applied when the Sanger sequencing approach loaded onto a so-called array containing thou-
is not appropriate, testing progressively larger sands of synthetic short single-stranded immobi-
targets according to needs (Fig. 2.3). Though lized DNA fragments representing the whole
Fig. 2.3 A suggested

diagnostic workflow for Is the clinical
the genetic analyses of a phenotype clearly
candidate patient. In distinguishable?
case Sanger sequencing
and next generation
sequencing (NGS)-based
gene panels do not
identify a consistent
Sanger sequencing for NGS panel
variant, whole exome or
the suspected gene
whole-genome
sequencing should be
considered Are the variants thus
Patient’s identified related to the
diagnosis disease?
confirmed
Sanger Whole Exome

Sequencing Sequencing
validation
Do we have any clue

Patient’s for variant selection?
diagnosis
confirmed
In silico panel Analyses of the

with candidate whole dataset
genes
NO
YES
genome. Because the fluorescently labeled DNA identified in an infant of Puerto Rican origin
of the patient and the control compete with these affected with DIRA [9, 10]. Another, is the reces-
oligonucleotides, conclusions can be drawn from sive HOIL1 deficiency, reported in Table 2.1 as a
the ratio of the color signals of the patient and clear example of the combined use of WES and
control DNAs regarding their relative gene dos- aCGH, found a patient with a compound hetero-
age. For instance, when the color signal of the zygosity for a stop mutation of the RBCK1 gene
patient DNA prevails on the control DNA in a and a genomic 30 Mb deletion that includes this
given genomic region, a gain of DNA is sus- gene [42]. In this case the HOIL1 deficiency
pected, and vice versa for DNA loss. There are a derives from the failure of either allele to produce
few examples of patients with autoinflammatory a correct and functioning RBCK1 protein as one
disorders that were identified through this method. allele carries a nonsense mutation and the other a
One is the case of the already mentioned 175 kb null allele, namely no gene is present in the cor-
large homozygous deletion of the interleukin-1 responding chromosomal region due to an inter-
family gene cluster, including the IL1RN gene, stitial deletion. While the first mutation was
detected by WES, the second genetic defect was guish patients with cryopyrin-associated periodic
assessed by aCGH [42]. Finally, very recently, a syndromes (CAPS) from controls. Interestingly,
13.13 Mb deletion on chromosome 6, encompass- several differentially expressed genes turned out
ing 53 genes including the TNFAIP3 gene, has to be shared among other systemic inflammatory
been identified by using aCGH in a patient with a diseases [83]. A similar study increased knowl-
complex phenotype consistent with the domi- edge of pathogenic mechanisms in TRAPS. Gene
nantly inherited A20 haploinsufficiency [80]. expression profiles in resting monocytes from
Therefore, it has been recommended to include TRAPS patients confirmed the patients’ chronic
CGH arrays in the routine diagnostic methods for inflammatory condition, while additional path-
comprehensive analysis of patients with syn- ways, not yet associated with the disease, were
dromic features and immune dysregulation. discovered, such as interferon types I and II
response to lipopolysaccharide (LPS) stimulation
2.3.2.2 R eal-Time Polymerase Chain and a downregulation of the transforming growth
Reaction (PCR) factor (TGF)-β pathway in the basal condition
Quantitative polymerase chain reaction (Q-PCR), [84].
sometimes referred to as real-time PCR, is a Analysis of gene expression has become cru-
method by which the amount of the PCR product cial in suspected interferonopathies, a group of
can be determined, in real-time, by the use of fluo- Mendelian diseases associated with an upregula-
rescent or DNA intercalating dyes, typically used tion of interferon and consequently with a specific
to measure gene expression. A further application “signature” given by the simultaneous upregula-
of Q-PCR is to estimate the copy number of a gene tion of genes whose expression is stimulated by
or a genomic region, a quantification that allows to interferon (see Table 2.2 and Chap. 24) [85]. In
detect CNVs, either deletions or duplications [81]. particular, the expression of interferon-stimulated
This technique was successful in the case of two genes is measured by quantitative PCR, and the
sisters with a phenotype consistent with DADA2, median fold change is used to create an interferon
who were initially found to be only heterozygous score. This interferon score is higher in patients
for a missense pathogenic variant at the ADA2 than among controls. Type 1 interferonopathies
(previously known as CECR1) gene. Q-PCR have emerged during the latest few years and
revealed an additional heterozygous deletion of their number is still growing as interferon signa-
exon 7 predicted to lead to a frameshift and trun- tures are often recognized in patients with novel
cated protein in the opposite ADA2 allele [82]. autoinflammatory and/or autoimmune pheno-
types [86–88].
2.3.2.3 Gene Expression
in Autoinflammatory Disease
Analysis of gene expression in autoinflammatory 2.4 Gene Mosaicism
diseases has often provided a powerful means to
obtain hints about pathogenic mechanisms of dis- • According to the tissue distribution, gene
ease. A microarray (also chip) is used for such mosaicism can be divided into gonadal
studies. Such a microarray contains a large set mosaicism, somatic mosaicism and gono-
(up to millions) of DNA probes attached to a somal mosaicism. In this latter case the
solid surface that can be hybridized with tran- post-zygotic mutation affects both gonadal
scripts (targets), thus assessing, simultaneously, and somatic cells
through fluorescence or chemiluminescence sig- • The allele frequency of post-zygotic
nals, the expression levels of large numbers of (somatic) mutations ranges from 1 to 40%,
genes. and often less than 20%. These mutations
Taking advantage of such technology, gene can be missed when using conventional
expression patterns were analyzed to define a methods of genetic analyses (i.e Sanger
specific gene expression signature able to distin- method of DNA sequencing), and their
detection usually requires NGS-based clearly different from germline mutations.

methods with great depth Germline mutations are already present in the
• During the last few years various patients first fertilized egg, and consequently are present
with somatic NLRP3 mosaicism restricted in all cells of the body from conception.
to cells from myeloid lineage (neutrophils Moreover, these germline mutations can easily be
and monocytes) have been described. detected by conventional methods of genetic
• Somatic gene mosaicism has also been analysis (i.e. Sanger sequencing) in any analyzed
described as a disease-causing mechanism tissue, and their expected allele frequency in het-
in monogenic autoinflammatory diseases erozygosity is around 50%. On the contrary, post-
other than cryopyrin-associated periodic zygotic mutations are strictly de novo mutations,
syndromes (CAPS), but in smaller numbers which are absent in the individual’s parents. Their
of patients body distribution may differ among individuals
• The presence of post-zygotic mutations in carrying mosaicism, the main factor that deter-
gonadal tissue may cause an unexpected mines this distribution being the precise time
recurrence of a dominantly-inherited dis- when the post-zygotic mutational event occurred.
ease in a subsequent child of a healthy cou- The allele frequency of post-zygotic mutations is
ple with no mutations detected in previous less than 50%, ranging from 1 to 40%, and often
standard genetic analyses, thus emphasiz- less than 20%. This low or extremely low fre-
ing the importance of considering the pos- quency of the mutant allele means that these
sibility of parental gene mosaicism in gene mutations can be missed when using conven-
counseling of families tional methods of genetic analyses, and their
detection usually requires the use of novel tech-
In genetics, the term mosaicism describes an nologies such as NGS-based methods with great
individual who has developed from a single depth.
zygote, but carries two, or more than two, cell
types with distinct genotypes [89]. In a strict
sense, gene mosaicism should be clearly distin- 2.4.2 issue Distribution of Gene
T
guished from the related phenomenon of chime- Mosaicism
rism, which describes an individual who carries
cell types with distinct genotypes, but these cells As mentioned, the precise time when the post-
derived from distinct fertilized eggs (i.e. a recipi- zygotical mutational event occurs will determine
ent of an allogeneic transplant or cell fusion from the body distribution of gene mosaicism in a
an aborted dizygote twin early in given individual. When the mutational event
embryogenesis). occurs early during embryonic development, the
post-zygotic mutation will probably be present in
tissues derived from all three embryonic layers
2.4.1 Germline and Post-zygotic and provoke a type of mosaicism called extended
Mutations gene mosaicism. By contrast, the mosaicism
could be tissue-restricted when the post-zygotic
In an individual with gene mosaicism, the differ- mutation occurs later, during post-natal life.
ences observed among genetically different cells According to the tissue distribution of post-
are a consequence of mutational events that occur zygotic mutations, three main types of gene mosa-
post-zygotically, either during the embryonic icism can be distinguished, each having different
development in the ≈1016 mitotic cell divisions clinical consequences. When the post- zygotic
required to generate an adult organism, or later, mutation is restricted to the gonadal tissue, the
after birth, in a similar manner as in the case of gene mosaicism is named gonadal mosaicism. In
somatic gene variants involved in carcinogenesis. this case, the individual carrying the mosaicism is
The post-zygotic (or somatic) mutations are healthy, but at moderate-to-high risk to transmit
the mutant allele to his/her offspring, who will 2.4.3 tate of the Art in Monogenic
S
receive it as a germline mutation because it will be Autoinflammatory Diseases
already present at the first zygote.
When the post-zygotic mutation is restricted to The history of gene mosaicism in the field of
body (somatic) cells, the gene mosaicism is called monogenic autoinflammatory diseases started
somatic mosaicism. In this case, the individual in 2005, when a Japanese group identified for
carrying the post-zygotic mutation could be the first time a somatic NLRP3 mosaicism as
healthy or affected, depending on different vari- the underlying disease-causing mechanism in
ables such as the frequency of the mutant allele, a patient diagnosed with CAPS [90]. Since
the precise type of mutation and its consequences then, at least 68 individuals, most with clinical
on the function of the normal protein, and the pre- illness, have been identified carrying post-
cise relationship among tissues carrying the post- zygotic mutations in different genes associ-
zygotic mutation and the tissues where the specific ated with monogenic autoinflammatory
mutated gene is expressed. However, unlike indi- diseases. Most of reported gene mosaicisms
viduals with gonadal mosaicism, individuals with belong to the group of somatic mosaicism,
pure somatic mosaicism are not at risk of trans- with only seven cases belonging to the group
mitting the mutant allele to their offspring. of gonosomal mosaicism. Interestingly, no
Finally, when the post-zygotic mutation pure gonadal gene mosaicism has been
affects both gonadal and somatic cells, the gene described to date. With regard to the genes
mosaicism is termed gonosomal mosaicism, causing autoinflammatory diseases, post-
representing the most complex type among gene zygotic mutations have been detected in six
mosaicism. In this case, the individual carrying different genes, with most of the cases (87%)
gonosomal mosaicism is at moderate-to-high risk identified in the NLRP3 gene (Fig. 2.4).
to transmit the mutant allele to his/her offspring, Table 2.4 contains a summary of the cases of
and also could develop clinical symptoms gene mosaicism in the NLRP3 gene reported
depending on the variables mentioned previously to date, whereas Table 2.5 contains those
in the definition of somatic mosaicism. reported in genes other than NLRP3.
Fig. 2.4 Summary of n: 1 n: 2 n: 1 NLRP3

genes related to TMEM173
autoinflammatory NOD2
diseases in which gene n: 1 NLRC4
mosaicism has been
TNFAIP3
identified. The numbers n: 4
TNFRSF1A
for each gene indicate
the total number of
unrelated individuals
carrying a specific gene
mosaicism
n: 59
2
Table 2.4 Summary of the currently known individuals carrying NLRP3 mosaicism
Germline
Amino acid MAF (%) in counterpart
Gene Exon Nucleotide exchange exchange whole blood Phenotype Country Type of mosaicism Reference phenotype
NLRP3 3 c.779G>C p.Arg260Pro 10.9 MWS Italy Somatic [91] Yes—NOMID
NLRP3 3 c.790C>T p.Leu264Phe 4.3 NOMID Japan Somatic [92] Yes—NOMID
NLRP3 3 c.906C>A p.Phe302Leu 9.8 NOMID Japan Somatic [93] No
NLRP3 3 c.907G>A p.Asp303Asn 7.2 Asymptomatic Mexico Gonosomal Personal Yes—NOMID
unpublished data
NLRP3 3 c.907G>C p.Asp303His 19.1 NOMID Spain Somatic [94] Yes—NOMID
NLRP3 3 c.907G>C p.Asp303His 4.2 NOMID France Somatic [95] Yes—NOMID
NLRP3 3 c.907G>C p.Asp303His 11.9 NOMID Japan Somatic [95] Yes—NOMID
NLRP3 3 c.907G>C p.Asp303His 7.1 NOMID Japan Somatic [93] Yes—NOMID
NLRP3 3 c.907G>C p.Asp303His 13.8 NOMID Spain Somatic Personal Yes—NOMID
unpublished data
NLRP3 3 c.908A>C p.Asp303Ala 31.3 MWS Spain Somatic [96] No
NLRP3 3 c.918A>T p.Gln306His 5.1 Late-Onset Spain Somatic Personal No
MWS unpublished data
NLRP3 3 c.919G>A p.Gly307Ser 4.3 NOMID Japan Somatic [92] No
NLRP3 3 c.920G>A p.Gly307Ala 4.5 CAPS Turkey Somatic [97] No
NLRP3 3 c.920G>T p.Gly307Val 9.6 NOMID Spain Somatic [95] Yes—NOMID
NLRP3 3 c.1000A>G p.Ile334Val 34.9 MWS Japan Somatic [96] Yes—NOMID
NLRP3 3 c.1040C>T p.Thr347Ile 4.9 MWS USA Somatic Personal No
unpublished data
NLRP3 3 c.1043C>T p.Thr348Met 2.8 Asymptomatic Spain Gonosomal [98] Yes—MWS

NLRP3 3 c.1054G>A p.Ala352Thr 14.6 Late-Onset UK Somatic [99] Yes—NOMID
MWS
NLRP3 3 c.1054G>A p.Ala352Thr 21.3 Late-Onset Spain Somatic Personal Yes—NOMID
NLRP3 3 c.1064A>C p.Lys355Thr 20.2 MWS Japan Somatic [96] No
NLRP3 3 c.1065A>T p.Lys355Asn 18.8 NOMID USA Somatic [95] No
NLRP3 3 c.1216A>G p.Met406Val 9.2 NOMID France Somatic [95] No
NLRP3 3 c.[1231C>T;1233G>T] p.Leu411Phe 14.4 MWS Spain Somatic [96] No
NLRP3 3 c.1298C>T p.Thr433Ile 5.2 NOMID France Somatic [95] No
NLRP3 3 c.1298C>T p.Thr433Ile 3.2 NOMID Italy Somatic [100] No
(continued)
37
38
Germline
Amino acid MAF (%) in counterpart
Gene Exon Nucleotide exchange exchange whole blood Phenotype Country Type of mosaicism Reference phenotype
NLRP3 3 c.1298C>T p.Thr433Ile 5.5 NOMID Italy Somatic [91] No
NLRP3 3 c.1303A>G p.Lys435Glu 27.0 Variant-type Netherlands Myeloid-restricted, [101] No
SS somatic
NLRP3 3 c.1305G>T p.Lys435Asn 9.0 MWS Brazil Somatic Personal No
unpublished data
NLRP3 3 c.1315G>C p.Ala439Pro 21.9 NOMID France Somatic [95] Yes—NOMID
NLRP3 3 c.1564A>T p.Thr522Ser 28.9 NOMID Colombia Somatic Personal No
unpublished data
NLRP3 3 c.1569C>A p.Phe523Leu 8.7 MWS Spain Somatic [96] Yes—NOMID
NLRP3 3 c.1569C>G p.Phe523Leu 8 Variant-type Netherlands Myeloid-restricted, [101] Yes—NOMID
SS somatic
NLRP3 3 c.1688A>G p.Tyr563Cys 2.7 NOMID Italy Somatic [91] No
NLRP3 3 c.1688A>G p.Tyr563Cys 5.1 Late-Onset UK Somatic [99] No
MWS
MWS
MWS
NLRP3 3 c.1688A>G p.Tyr563Cys 8.0 Late-Onset Spain Somatic Personal No
NLRP3 3 c.1690G>A p.Gly564Ser 8.1 NOMID Italy Somatic [91] No
NLRP3 3 c.1691G>A p.Gly564Asp 5.0 Late-Onset UK Somatic [99] No
MWS
NLRP3 3 c.1691G>A p.Gly564Asp 8.5 MWS Belgium Somatic Personal No
unpublished data
NLRP3 3 c.1698C>A p.Phe566Leu 11.5 NOMID France Somatic [95] No
NLRP3 3 c.1698C>A p.Phe566Leu 14.6 NOMID USA Somatic [95] No
NLRP3 3 c.1698C>A p.Phe566Leu 14.5 NOMID UK Somatic [102] No
NLRP3 3 c.1699G>A p.Glu567Lys 6.5 MWS Japan Somatic [92] No
NLRP3 3 c.1699G>A p.Glu567Lys 6.3 NOMID Netherlands Somatic [95] No
NLRP3 3 c.1699G>A p.Glu567Lys 5.8 NOMID Japan Somatic [93] No
NLRP3 3 c.1699G>A p.Glu567Lys 18.3 NOMID Japan Somatic [93] No
NLRP3 3 c.1699G > A p.Glu567Lys 5.6 MWS Japan Somatic [96] No
I. Ceccherini et al.
2
NLRP3 3 c.1699G>A p.Glu567Lys 5.5 MWS Japan Somatic [96] No

NLRP3 3 c.1699G>A p.Glu567Lys 5.4 Late-Onset UK Somatic [99] No
MWS
NLRP3 3 c.1700G>C p.Glu567Gln 15.0 Late-Onset UK Somatic [99]
MWS
NLRP3 3 c.1704G>C p.Lys568Asn 9.4 NOMID USA Somatic [95] No
NLRP3 3 c.1706G>T p.Gly569Val 21.1 Late-Onset UK Somatic [99] No
MWS
NLRP3 3 c.1708T>C p.Tyr570His 11.9 Severe CAPS Turkey Somatic [97] No
NLRP3 3 c.1709A>G p.Tyr570Cys 16.7 NOMID Japan Somatic [90] Yes—NOMID
NLRP3 3 c.1709A>G p.Tyr570Cys 10.9 Late-Onset USA Myeloid-restricted, [103] Yes—NOMID
MWS somatic
NLRP3 3 c.1906C>G p.Gln636Glu 18.4 Late-Onset Spain Myeloid-restricted, [104] No
MWS somatic
NLRP3 4 c.2263G>A p.Gly755Arg 35.8 NOMID USA Somatic [95] Yes—NOMID
NLRP3 4 c.2263G>A p.Gly755Arg 6.3 NOMID Netherlands Somatic [95] Yes—NOMID
MAF Mutant allele frequency, MWS Muckle-Wells syndrome, NOMID Neonatal-onset multisystem inflammatory disease, CAPS Cryopyrin-associated periodic syndromes, SS
Schnitzler syndrome, USA United States of America, UK United Kingdom
39
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Charles softly answered, “Yes, sir.”
“Well, I believe he is ashamed of his greediness,” said Mr.
Mansfield; “I only advise him another time to be more upon his guard
for fear we should take him for a pig.”
As they were sauntering about, a sow with a fine litter of pigs at
her heels came across the yard.
“Pray, sir,” asked Arthur, “how many pigs may a sow have at
once?”
“From ten to twenty,” said Mr. Mansfield. “But as she has not milk
enough to suckle so many, she casts off some, and seldom brings up
more than twelve.”
Arthur. What food do they like best, sir?
Page 24.
The Pigs.
London. Published by W. Darton Junʳ. Oct. 5, 1815.
Grandpapa. They are not very difficult. They will eat almost any
kind of rubbish and offal: but vegetables of all sorts are best for
them. Cabbage stalks, potatoe parings, bean and pea shells, they
like very well; and it is a good way to turn them out into the forests,
where they meet with plenty of acorns, and mast nuts that grow upon
beach trees. With their long snouts they turn up the ground, that they
may get at the roots or plants: to prevent this, we are obliged to have
a ring thrust through their noses, otherwise they would do a great
deal of mischief.
Charles. Are they of much use, grandpapa?
Grandpapa. Not whilst they are alive. When dead, the flesh, you
know, is eaten, and is called pork, or bacon if salted in a particular
manner. The lard, or some of the fat, is used in making many sorts of
plasters, and the bristles are formed into brushes of various kinds;
and are used by shoemakers and others in sewing leather, instead of
needles.
Arthur. I like little pigs much better than I do great old ones.
Grandpapa. I cannot say the hog is a favourite animal with me. He
is not only ugly, but his habits of life are disagreeable. You may have
observed that he is very fond of grouting in the mire. Neither his
grunting nor his squeaking is pleasant music; and the whole race are
so greedy, that, if they have food enough, they will eat till they are
too heavy to stand on their legs; even then they will lie on their sides,
and eat still. Sometimes the sow will go so far as to devour her own
young.
Arthur. Indeed? The unnatural brute!
Grandpapa. I should have told you that their stomach is made very
large, and requires an unusual quantity of food. But if we are
disgusted with the manners of a hog, we should be careful not to
imitate them; as filth, gluttony, and want of natural affection, must
surely be ten times more shocking in the creature man, who is
blessed with reason.
CHAPTER IV.
Sheep-Shearing.
The following day being appointed for sheep-shearing, a number

of men and boys assembled at an early hour in the great barn.
Arthur and Charles went with their grandpapa to see the process,
and were greatly pleased with the hurry and bustle of the scene. The
sheep were penned in a fold close to the barn, and were fetched
away by the lads one by one, as fast as the shearers were ready for
them. A few days before, they had all been washed at a mill-pond,
so that their fleeces were beautifully white, and they were so thick as
to make the animals appear almost twice as large as they really
were.
Page 28.
Sheep Shearing.
London. Published by W. Darton Junʳ. Octʳ. 5, 1815.
Arthur observed with surprise, that the poor creatures were

perfectly quiet during the time of their being shorn; although they
struggled with terror when they were first brought out, and bleated
piteously as soon as they were set at liberty.
He wondered at the ease with which the men laid them on the
ground, and afterwards turned them over from side to side, as was
necessary in the course of the shearing. After watching one of the
shearers for some time, he began the following conversation with
him:—
Arthur. Good man, does not it hurt the sheep to be pulled about in
that way?
The Man. They do not like it; but I try to hurt them as little as I can.
Arthur. Are you not afraid of cutting them with the shears, when
you put them down into the middle of the wool?
The Man. We take care to feel our way, but now and then they get
an unlucky snip. That man there, that stands by the door, has some
tar that he puts to them if they chance to be hurt.
Arthur. Poor things! how cold they must feel when they lose such a
quantity of wool!
The Man. It is time they should be shorn now, master. This is their
winter coat, as one may say; and if it was left much longer, by little
and little it would fall off of itself.
Arthur. Then why don’t you let it come off of itself, instead of taking
all this trouble, and teasing the sheep?
The Man. My service to you, sir! What, are we to lose the wool, or
to follow the sheep from place to place wherever they choose to
stray, in order to gather it after them? No, no; they may suffer a little
at first, but if the weather is warm they soon get over it.
Arthur. How many can you shear in a day, good man?
The Man. Why, fifty, more or less. The quickest hands can finish
one in ten minutes.
Charles during this time was helping a little girl to pick up the loose
locks of wool that were scattered over the floor. His brother turned
round, and saw how he was employed. What should he do? Every
one was busy besides himself, and he could not bear to be the only
idle person. A message came to fetch away one of the women,
whose task it was to roll up the fleeces and pile them together on a
heap. Arthur offered to take her place; and, after a few trials, he
learned to tie them up very dexterously. He continued at this
employment for some time, and rejoiced to find himself of some use.
Mr. Mansfield at length called the two boys to go away. They
immediately obeyed; and Charles, taking hold of his grandpapa’s
hand, asked him if he did not think a sheep-shearing was a most
charming thing.
Grandpapa. It does very well in its season, my dear boy. Wool is
so useful, that the shearing-time always gives me pleasure.
Arthur. What shall you do with it, grandpapa?
Grandpapa. I shall sell it to the wool-stapler; and, after it has
passed through the hands of different manufacturers, you may
perhaps meet with it again in some shop, though so altered as not to
be known for the same. It will then be in the shape of flannel,
worsted, cloth, or perhaps some kind of stuff.
“That is all very droll,” said Charles. “But when will there be
another sheep-shearing, grandpapa?”
“Not till this day twelvemonth, my dear,” returned Mr. Mansfield.
“Wool does not grow very fast. In two or three weeks you will see the
sheep covered with a little short wool; and the traces of the shears
will then be worn away. As winter comes on, it grows thicker and
longer; but that is not a time to rob them of their fleece. At last the
year will come round, and then they will be again ready for the
shearer.”
“I am fond of sheep,” said Arthur; “and I like little lambs, they look
so innocent.”
Grandpapa. They are gentle, timid creatures, and require the care
of man more than almost any other animal; as they have neither
strength to defend themselves when attacked by their enemies, nor
swiftness to run from danger.
Arthur. And they pay us for the care we take of them, by letting us
have their wool?
Grandpapa. Indeed they do, Arthur; but not by their wool alone, for
they are useful in more ways than one. Mutton, which you know is
the flesh of the sheep, is one of the most wholesome meats we
have; some parts of the fat are melted down to make tallow. The skin
is sometimes made into parchment, and sometimes into leather, for
gloves, shoes, and other things: and parts of the guts are twisted into
strings for musical instruments.
Charles. What enemies have sheep, grandpapa? You have said
they can’t defend themselves against their enemies.
Grandpapa. Wherever there are wild beasts, Charles, they have
many enemies, as they all prey upon the sheep. Eagles will attack
young lambs; so will foxes; and even dogs, if they are fierce, and not
properly trained.
Arthur. But I have often seen a dog along with a flock of sheep.
Grandpapa. Yes; the breed that is called the shepherd’s dog is
very useful in managing them. They seldom bite, but will fetch those
back that have gone astray; and by barking at them alone, guide the
whole flock much more easily than a man can do. When they have
done their business, you may see them come back to the shepherd,
and follow him as quietly as possible.
In the evening a supper was provided to refresh the shearers after
their hard day’s work, consisting of legs of mutton, and plum-
puddings, with plenty of good ale. All was jollity and mirth. During the
day a constant buz of many voices might have been heard even at
some distance from the barn; but the business they were engaged in
did not allow time for much talk. At night, on the contrary, they had
nothing to do but to divert themselves, and every tongue was heard.
They told merry stories without end, sang songs, and drank to the
health of their kind master. Mr. Mansfield himself staid with them for
some time, encouraging them to be cheerful, and walked about to
see that every body was helped. At length, he left the party, followed
by his grand-children, who immediately retired to rest, highly
satisfied with the pleasures of the day.
CHAPTER V.
A Walk through the Fields.
The next morning, Mr. Mansfield asked the little boys if they were
disposed for a walk. Arthur replied that he should like it very much;
but Charles said he would rather stay at home with his
grandmamma; accordingly they set off without him.
“What pretty purple flowers grow in that field!” observed Arthur,
when they had proceeded a little way. “Pray, grandpapa, what are
they?”
“That is a field of clover,” replied Mr. Mansfield; “and it will soon be
cut for hay.”
Arthur. I never saw such pretty hay as that.
Grandpapa. Oh, there will be no beauty in it. On the contrary, it
looks much coarser and browner than what is made of common
grass, which is called meadow hay.
Arthur. What becomes of the flowers then?
Grandpapa. They dry and wither away. You do not suppose they
would live when cut down. Did you ever see how hay is made?
Arthur. Yes, a great many times. A number of men and women go
into a field and turn the grass, and then they put it into cocks, and
afterwards make a stack of it.
Grandpapa. Why do they do all that?
Arthur. To make it into hay.
Grandpapa. Yes. But why does turning it about make grass into
hay?
Arthur said he did not know.
Grandpapa. Then I will tell you. The grass when cut down is full of
moisture. If you squeeze a blade in your fingers, it will be damp; and
that dampness is called sap. Now, while the sap is in it the grass will
not keep. If you were to make it into a stack, it would soon rot, and
smell so putrid you would not like to go near it. But when it is turned
about to the sun and the wind, till the sap is dried away, there is no
more danger, and you may stack it, and keep it for a long time.
Arthur. But if I had a field, grandpapa, I would never make hay. My
horses should go in and eat the grass when they wanted it; and I
would save myself the trouble of working for them.
Grandpapa. I am afraid, Arthur, you would make a lazy farmer. Do
not you know that nothing in this world is to be had without trouble?
and if you are so very sparing of your pains, I fear you will not
succeed very well.
Arthur. Why not, pray, sir?
Grandpapa. Did you ever take notice of the grass in the winter?
Arthur. Yes; I believe it is then short and black.
Grandpapa. The blackness is nothing but the earth among it; it is
very thin at that time of the year. Did you ever observe a field just
before it was cut for hay?
Arthur. Oh, yes. Do you know, grandpapa, we all took a walk in a
field a little while ago; and the grass was so very long that it came up
to the top of my legs; and little Kate cried, and could not get on at all.
Grandpapa. You see then, that as there is much grass in summer
and but little in winter, your horses at one time would have more than
they could eat, and at another would starve. Yet this would be owing
to your own fault: for God gives enough for the whole year; and all
he requires of us is, that we should in the season of plenty lay up for
the time of need.
CHAPTER VI.
The Walk continued.
The next field they came to was sown with rye, which Mr.
Mansfield said was a species of corn; and, although much coarser
than wheat, was frequently made into bread, and in many places
formed the chief food of the poor. He desired his grandson to gather
an ear or two, that he might learn to distinguish between that and
barley, which grew in the field through which they were next to pass.
Arthur pulled up a root of rye, and then ran to overtake his
grandfather, who by this time had got over the stile, and was slowly
crossing the barley field.
“Well, Arthur, what difference do you find in the growth of these
two kinds of corn?” asked Mr. Mansfield.
Arthur. Indeed, grandpapa, I don’t see any, except that the rye
grows very high, as high as the top of your hat, and that the barley
only comes to my elbow.
Mr. Mansfield. That is one difference, to be sure. Examine them
well, and perhaps you may discover some other.
Arthur. Oh, yes, I see, sir. The spikes of the rye are neither so fine
nor so long as in the barley.
Mr. Mansfield. Very true again. So you see you need never
mistake between them. The straw of the rye is the longest, but the
beard (you should not call it the spikes) is shorter and coarser.
Arthur. I think the long beard of the barley gives it rather a silky
look, as it waves about with the wind. Pray, grandpapa, is barley
sown to make bread too?
Mr. Mansfield. Sometimes it is used for that purpose; but the
greatest part of what we grow in England is for making beer.
Arthur. Beer! Is it possible that barley can make beer? Do you
know, sir, how it is done?
Mr. Mansfield. Yes; and you shall hear, if you wish to know. All
grain is the seed of the plant; and before it can be put to any use it
must be taken out of the ear. Now, to do that, it is thrashed with an
instrument called a flail. I suppose you have seen one, have you
not?
Arthur. I remember once passing at some distance from a barn,
where a man was swinging something about, that looked like a bent
stick; and he beat the ground with it, and somebody said he was
thrashing.
Mr. Mansfield. That he certainly was. The corn was spread upon
the barn-floor, and he was beating out the grain with a flail. The next
business is to separate it from the chaff, or outside skin. This is
sometimes done by turning a machine very quickly so as to cause a
wind, which blows away the chaff, for it is as light as a feather. A
more simple method is, to throw the corn across from one side of the
barn to the other, against the wind. The chaff, being so light, is soon
blown back, whilst the corn goes on a little further, and falls in a heap
by itself.
Arthur. But, dear grandpapa, what has this to do with making
beer?
Mr. Mansfield. All in good time, my dear boy. You must get at the
barley before you can use it, must you not? The method of
winnowing I have described, relates principally to wheat (for barley is
without chaff); but the barley must be thrashed, and separated from
the ear; after which it is put for some days into a cistern of water. It is
then taken out and laid in heaps; when it ferments, and is ready to
shoot out in the same manner as if sown in the ground. Afterwards it
is spread thinly over a floor, and frequently turned; and when partly
dry is carried to a kiln, a kind of oven, where it is dried. Having
passed through all this process it is called malt, and the man whose
business it is, is termed a maltster.
Arthur. I thought brewers made beer?
Mr. Mansfield. You were right. Brewers buy malt. They grind it, and
then pour hot water upon it, to get out its strength and goodness.
The liquor thus obtained, which is sweet-wort, becomes the most
valuable part of the commodity; for the malt has lost its virtue, and is
called grains, and is only used to feed pigs and cattle. The wort is
afterwards boiled with hops, which give it a bitterish taste instead of
a sickly sweet, and keep it wholesome and good. Then it takes the
name of beer; and after fermenting for a little while may be put into
casks and kept for use. And now, Arthur, do you think that you
understand brewing? Shall you recollect that malt is barley prepared
in a particular way? and that beer is made by pouring warm water on
the malt, and afterwards boiling it with hops?
Arthur. I think I shall, grandpapa.
CHAPTER VII.
The Pony.
As Mr. Mansfield and Arthur were returning from their walk, in a

lane at a little distance from the house they were met by Charles,
who had mounted a pony belonging to his grandfather: it had taken
fright, and was running away at full speed. Mr. Mansfield stopped it
by catching hold of the bridle; and as soon as he was satisfied that
no mischief had happened, and Charles was sufficiently recovered to
be able to talk, he inquired what had led him to try his skill in
horsemanship.
Page 52.
Charles on the Poney.
London. Published by W. Darton Junʳ. Oct. 5, 1815.
“Why, grandpapa,” replied Charles, “Robert had just come home

with the pony, and left him at the gate; and I wanted to ride; so I got
upon him, and he ran away with me.”
Mr. Mansfield. As you have never been used to ride, my dear
Charles, you had better not get upon strange horses when you are
alone. I wonder too that Plover should run away; he is in general
very gentle.
Charles. At first he would keep his head over the gate, and I could
not get him to move. So I hit him with a stick I had in my hand, and
that set him off in a gallop.
Mr. Mansfield. I fancy all was owing to your want of skill; for Plover
is a very quiet creature, and easily managed; but he will not bear ill
usage; therefore, if you beat him much, I am not surprised at the
accident.
Arthur. I am sure, grandpapa, Charles did not mean to be cruel,
and use the horse ill.
Mr. Mansfield. He is so good a boy that I do not suspect him of it;
and I only meant to give him a caution against another time. No, my
dear children, I hope you will never take pleasure in wanton cruelty.
My heart has often ached at the barbarities I have seen practised on
poor dumb creatures.
Arthur. Once when I was walking with papa, we saw a man
beating a horse about the head with the butt end of his whip, and my
papa advised him not to do so; but he said it was his own horse, and
he had a right to do as he liked.
Mr. Mansfield. Nothing can give a man a right to be cruel. We may,
it is true, make what use we please of our beasts, as long as we
treat them well, for they were made for our convenience; but God
Almighty has given to them life and feeling the same as he has to us;
and we make him angry with us whenever we use them ill.
“I often think, grandpapa, that it is very strange such large
creatures as these,” said Arthur, patting Plover, who now walked
quietly by the side of his master, “should suffer us to get upon their
backs, and manage them as we please. They are much stronger
than we are; and I wonder they do not drive us away, and not carry
us, and refuse to draw our coaches and do every thing we like.”
Mr. Mansfield. It would be astonishing, Arthur, if we did not
consider that our reason gives us a great advantage over all brutes.
Some of them, it is true, are much larger, some much stronger, and
others much swifter than we; but by means of our understanding we
can conquer the strongest, and tame the fiercest of them.
Charles. How can we tame them, pray, sir?
Mr. Mansfield. By methods which they cannot resist. Plover is
stronger than you, but a boy of your size who understands riding
would be able to manage him. He would pull the bridle on this side,
or on that, according as he wished him to turn; and as he pulled, the
bit would hurt the horse’s mouth just enough to make him willing to
go where he was wanted: therefore, by our knowing how to manage
a bit and a bridle, we are more than a match for a horse in spite of
his great strength.
Arthur. I understand you now, grandpapa. And I have something to
tell you. As we were taking a walk a little while ago, a dog came
barking and snapping, and I thought he was going to bite me; but my
mamma called out, “Don’t be frightened, Arthur; pick up a stone to
throw at the dog, and it will send him away.” So I did, and to be sure
he slunk off at once. Now was not it my reason that made me
conquer the dog, though the dog could bite harder than myself?
Mr. Mansfield. Exactly so. You see, then, that although our bodies
are naturally weak and helpless, yet by our reason we are furnished
with the means of strength and defence. So God has ordained; and
therefore, though he will not suffer us to be cruel to any of his
creatures, yet, as our Bible tells us, he said at the beginning of the
world, that the fear and dread of man should be for ever upon all
animals.
As Mr. Mansfield finished these words, they reached the stable
yard, and Ralph came forward to unharness the pony.
“Plover must be shod to-morrow, sir,” said he, as he looked at one
of his hinder feet.
“Is not it cruel, grandpapa,” asked Charles, “to drive nails into the
horse’s feet?”
Mr. Mansfield. No, my dear, it is not. The nails only go into the
hoofs, which are very hard, and have not any feeling; but if we did
not put on these iron shoes, the hoofs, hard as they are, would soon
be battered to pieces when they travel over rough and gravelled
ground.
Arthur. Dead horses are of no use; are they, sir?
Mr. Mansfield. Their flesh is given to dogs; but the skin, when
converted into leather, is used for making harness and some other
things.
CHAPTER VIII.
A Visit to the Windmill.
“Do you know, my dear,” said Mrs. Mansfield to her husband,

when they were sitting at tea, “the miller has forgotten to send home
the flour he promised to let us have last week; and Sarah has just
told me we have not enough in the house to bake to-morrow! So
what must we do? Can you spare one of the men to go over and
inquire about it?”
“I am afraid, my dear,” said Mr. Mansfield, “they are all busy at
present; but when Ralph comes in he may go of the errand.”
“It has just occurred to me,” rejoined Mrs. Mansfield, “that if you
are disposed for another walk this fine evening, you might go
yourself and take the children with you; and it will be a nice treat to
them, for I know they have never seen a mill.”
“Ah, do go, grandpapa; will you, grandpapa? it will be so very
delightful!” said both the boys at the same instant.
“Well, bring me my hat then,” said their indulgent grandfather. “I
did not intend to stir again to-night; but if it will give you pleasure, my
dear boys——”
“Thank you, sir! thank you, sir!” cried Charles, running for the hat.
“I hope you won’t be tired though,” said Arthur. “You shall rest
upon my shoulder all the way; and do not be afraid of leaning all your
weight, for I shall be able to bear it very well.”
“You shall have my shoulder to rest upon too,” exclaimed Charles:
“So I dare say, grandpapa, you will not be tired.”
“Indeed,” replied Mr. Mansfield, putting one hand upon the
shoulder of each, as he rose from the chair, “with two such kind little
supporters, I shall not be easily fatigued.”

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Textbook of autoinflammation 1st

Edition Philip J. Hashkes

Illustrated Textbook of Clinical Diagnosis in Farm

Textbook of Interventional Cardiology 8th Edition Eric

Neuromuscular Assessments of Form and Function

The Cambridge handbook of personality psychology 2nd ed

Handbook of Radiotherapy Physics 2nd Edition Philip

Principles of Investigative Documentation 2nd Edition

Creole Societies in the Portuguese Colonial Empire 1st

World Civilizations: Volume II: Since 1500, 8th Edition

ISBN 978-3-319-98604-3 ISBN 978-3-319-98605-0 (eBook)

Library of Congress Control Number: 2018963832

© Springer Nature Switzerland AG 2019

Jerusalem, Israel Philip J. Hashkes

1 Autoinflammation: Past, Present, and Future������������������������������ 3

Part II Basic Science and Biology of Autoinflammation

2 Genetic Aspects of Investigating and Understanding

Part III General Approach to Autoinflammatory Diseases

10 Classification of Genetically Defined

11 Clinical Approach to the Diagnosis of

Part IV Monogenic Autoinflammatory Diseases

16 Familial Mediterranean Fever�������������������������������������������������������� 293

26 Pustular Forms of Psoriasis Related to Autoinflammation���������� 471

Part V Complex Autoinflammatory Diseases

30 Periodic Fever, Aphthous Stomatitis, Pharyngitis

Part VI The Relationship Between Autoinflammation

38 Autoinflammation and Autoimmunity ������������������������������������������ 693

Part VII Pharmaceutical Agents for Treatment

Abstract tory diseases, such as Behçet disease. During

© Springer Nature Switzerland AG 2019 3

FMF Familial Mediterranean fever decade promises to draw connections

Table 1.1 Timeline of monogenic autoinflammatory disease gene discoveries

Table 1.1 (continued)

1.3  orror Autoinflammaticus:

References 1-­dependent cytokine processing. J Biol Chem.

Isabella Ceccherini, Marta Rusmini,

Abstract NGS methods in the clinics allows detection

ACMG America College of Medical Genetics

© Springer Nature Switzerland AG 2019 19

DADA2 Deficiency of adenosine deaminase 2 notypically overlapping autoinflammatory

Table 2.2 List of interferonopathies and corresponding causative genes

obtain information regarding allele frequencies,

Fig. 2.2 List of criteria

Consequence of the nucleotide change

Association with a disease phenotype

Fig. 2.3 A suggested

Sanger Whole Exome

Do we have any clue

In silico panel Analyses of the

detection usually requires NGS-based clearly different from germline mutations.

Fig. 2.4 Summary of n: 1 n: 2 n: 1 NLRP3

NLRP3 3 c.1043C>T p.Thr348Met 2.8 Asymptomatic Spain Gonosomal [98] Yes—MWS

NLRP3 3 c.1699G>A p.Glu567Lys 5.5 MWS Japan Somatic [96] No

The following day being appointed for sheep-shearing, a number

Arthur observed with surprise, that the poor creatures were

As Mr. Mansfield and Arthur were returning from their walk, in a

“Why, grandpapa,” replied Charles, “Robert had just come home

“Do you know, my dear,” said Mrs. Mansfield to her husband,

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Jerusalem, Israel Philip J. Hashkes

1 Autoinflammation: Past, Present, and Future�� 3

Part II Basic Science and Biology of Autoinflammation

2 Genetic Aspects of Investigating and Understanding

Part III General Approach to Autoinflammatory Diseases

10 Classification of Genetically Defined

11 Clinical Approach to the Diagnosis of

Part IV Monogenic Autoinflammatory Diseases

16 Familial Mediterranean Fever�� 293

26 Pustular Forms of Psoriasis Related to Autoinflammation�� 471

Part V Complex Autoinflammatory Diseases

30 Periodic Fever, Aphthous Stomatitis, Pharyngitis

Part VI The Relationship Between Autoinflammation

38 Autoinflammation and Autoimmunity �� 693

Part VII Pharmaceutical Agents for Treatment

1.3 orror Autoinflammaticus:

References 1-dependent cytokine processing. J Biol Chem.

ACMG America College of Medical Genetics