Download Lessons in rheumatology from extreme phenotypes and subtle

Genetics and Rheumatology
Lessons from Extreme Phenotypes
&
Subtle Genotypes
Paul Wordsworth
NIHR Oxford Musculoskeletal BRU
Heberden Oration BSR AGM 2012
Paul Wordsworth
Genetics and Rheumatology
Lessons from Extreme
Phenotypes
&
Subtle Genotypes
Educational grants, speakers fees and consultation fees from
Abbott, Pfizer and Merck. I am an investigator in the Novartis AIN
457 study in AS and a NASS trustee
William Heberden (the elder)
Heberden
Society
Heberden
Round
Heberden Oration
MENTORS
Matt Brown in the Gambia 1994
I DO TRY BUT……
“One of the advantages of
being disorderly is that
one is constantly making
exciting discoveries.”
AA Milne
“Probably the only
advantage!”
CIW
April 11th 2012
William Heberden (1710-1801)
• 1710 born in London, son of
an inn-keeper
• 1724 St John’s College
Cambridge, aged 14 years
• 1730 Fellow St John’s
• 1739 MD
• 1746 FRCP
• 1749 FRS
• Extensive medical practice
• “Commentaries on the
History & Cure of Diseases”
Heberden’s nodes
• Best known to
rheumatologists for
Heberden’s nodes
• Distinguished RA
from gout
• How he would have
defined OA today?
• What would he have
made of these?
OMIM – McKusick’s legacy
• All you ever wanted to know about “Juvenile
Hyaline Fibromatosis”
• On-Line Mendelian Inheritance in Man
• Compendium of human genetic disease
• Hours of fun on the net. Just Google “OMIM”
• William Heberden would have enjoyed it it!
Principles of genetic mapping
JUST LIKE SHUFFLING CARDS
The closer the marker is to the gene of interest the better
We are just one big happy family
150,000 yrs ago
80,000 yrs ago
75,000 yrs ago
70,000 yrs ago
74,000 yrs ago
Mount Toba
74,000 yrs ago
40,000 yrs ago
30-25,000 yrs ago
22-19,000 yrs ago
22-19,000 yrs ago
12,500 yrs ago
25%
8%
50%
3%
0%
0%
0%
Early Human Migration Routes
74k
80-75,000 yrs ago
73,000 yrs ago
45-25,000 yrs ago
Stephen Oppenheimer and Bryan Sykes
Sir Archibald Garrod 1896
100 yrs ago
ALKAPTONURIA
“a rather harmless disease”
Homogentisic acid oxidase
“Inborn factors in Disease” Garrod 1931
1999 CPPD arthritis and ANKH
Lesson 1 - an extreme phenotype
may give clues to common disorders
6
10 11
1
2
7
12 13 14
8
4 15
5
16
3
18 19
9
22
23 17
24
31 32
28 34 25 40
26
36 37
45
33
38 39
35
47
46
48 49 50 51
30
41 42
20
27 29
21
43
44
G→A in 5’UTR (-4bp) associated with
CPPD in general population (p<0.0006)
Andrew et al Am J Hum Genet 1999; 64: 136-45
Zhang et al Am J Hum Genet 2002; 71: 985-91
Hydroxyapatite Chondrocalcinosis
Hydroxyapatite Crystal Formation
(–)
iPPi
ANKH
ePPi
(+)
ATP
Alkaline phosphatase (AP)
Pi (inorganic phosphate)
Ca2+
NPPS
AMP+PPi
Nucleotide pyrophosphatase
CPPD Crystal Formation
CPPD Chondrocalcinosis
Gain of function mutations in ANKH increase
extracellular pyrophosphate → chondrocalcinosis
Fibrodysplasia ossificans progressiva
Bone in the wrong places
Activating mutations in ACVR1
Not many families with FOP about!
Narrowed to a Chr 2 region with 120 genes (including ACVR1)
Increased signal in
the absence of BMP
Cell Signalling Through TGFβ
Receptors
Miyazono, K., et al.17:17
J Biochem (2010)
Page 31
Receptor/SMAD interaction
Huse, M., et al. Mol Cell (2001), Miyazono, K., et al. J Biochem (2010)
17:17
Page 32
TGFβ Signalling through ACVR1
Inactive Form
• FKPB12 inhibitor
FKBP12
inhibitor
–Binds to
GS Domain
unphosphorylated
Gly/Ser domain
Kinase
–Stabilises the
Domain
inactive receptor
–Binding to GS
ensures kinase is
Chaikuad, A., et al. PDB (2009), Huse, M., et al. Mol Cell (2001)
inactive
17:17
Page 33
Structure-activity Relationships GS
Enhanced Inhibitors
LDN-193189

ALK1 in complex with
compound LDN-193189
 A structural analog of
Dorsomorphin
Might these be useful
treatments?
Kinase
Domain
3MTF - Chaikuad, A., et al. PDB (2010)
17:17
Page 34
COMPLEX DISORDERS?
Classic forms of AS
CT Reconstruction
Bridging syndesmophytes
SOIL & SEED
Nature or Nurture?
Heberden Orations by Lawrence and Weatherall
1973 A good year for the
Westminster Hospital
Nail patella syndrome
Linked to ABO blood
groups
Ankylosing spondylitis in 1973
• Familial nature well recognised
• More penetrant with female line?
• HLA genes were a great source of interest
• Tissue/bone marrow transplantation
Brewerton et al Lancet 1973
• 72/75 patients with AS HL-A27 positive
• 3/75 controls HL-A27 positive
• Odds ratio for HLA-B27~120, still about
the strongest for a polygenic disorder
The Seventies – when everyone was hairy
Look who’s come to play!
Professor Brewerton
• Taught me at Westminster Hospital
• Investigated genetics of AS
• Played full back
• Is a published geologist
Sir James Hutton 1726-1797
Father of British Geology
Siccar Point
345M
425M
“Concerning the system of the Earth”
Royal Society of Edinburgh 1767
Alpine orogeny
PreCambrian
pillow lava
Destruction of Ephesus
Diastrophism in rocks
The original union of UK
400M years ago
“Concerning the system of the Earth”
Royal Society of Edinburgh 1767
Alpine orogeny
PreCambrian
pillow lava
Destruction of Ephesus
Diastrophism in rocks
What was the effect of the Act of Union?
Diastrophism in bones!
Diastrophic Dysplasia – a rare chondrodysplasia
(sometimes called catastrophic dysplasia by RS)
Diastrophism in Osteogenesis Imperfecta
Problems with structural collagen genes
“Star wars skull”
“Popcorn bones”
OI - a classic collagen disorder
Lesson 2 – it’s never as straightforward as you think
OI Type 1A
OI Type 4B
An international collaboration
in osteogenesis imperfecta
Collagen winds up from the C-terminus
N-terminal mutations less severe?
Than C-terminal mutations?
Over-modification
Lethal OI maps to matrix interaction domains in
Type 1 collagen (integrins, fibronectin, COMP etc)
MID2
MID3
Di Lullo et al J Biol Chem 2002; 277:4223-31
Marini et al Hum Mut 2007; 28:209-21
Role for processing enzymes and molecular
chaperones in rare forms of severe OI
Families of matrix disorders
• Type 1 collagenopathies
OI, EDS
• Type 2 collagenopathies
achondrogenesis, SED (c), Kniest, Stickler
• Type 9 collagen
Multiple epiphyseal dysplasia
• Type 10 collagen
Metaphyseal chondrodysplasia (Schmid)
• Other cartilage components (COMP/matrilin/proteoglycans)
Pseudoacondroplasia, MED
• Type 1, 3 and 5 collagen
Classic, vascular and OI/EDS overlap
• Fibrillinopathies
Marfan, congenital contractural arachnodactyly
Type 2 collagenopathies
Achondrogenesis/hypochondrogenesis
Kniest dysplasia
SED (congenita)
Stickler syndrome
Multiple Epiphyseal Dysplasia – various types
(eg. Collagen 9, matrilin, COMP, sulfate transporter)
Type 9
Collagen
Pseudoachondroplasia
and COMP
Metaphyseal chondrodysplasia (type Schmid)
Type 10 collagen.
Transiently expressed
by hypertrophic
chondrocytes in the
growth plate (cf rickets)
Diastrophic dwarfism & MED (Type 5)
“Hitch-hiker thumb”
Abnormal sulfation
of proteoglycans
Double-layered
patella
1st Century A.D.
Founder population with striking
linkage disequilibrium and a high
mutant gene frequency
Hastbacka et al Nat Genet 1992; 2: 204
FIBRILLINOPATHIES
Lessons from Marfan Syndrome
TGFβ signalling is important
Lesson 2 AGAIN - never as simple as it seems!
Marfan Syndrome and FBN1
not just a structural gene problem
FBN1 - Cysteine mutations alter folding, cause abnormal
microfibrils and lead to ocular lens dislocation
Intrachain disulfide
bonding
Features of MFS unexplained by FBN1
• Tall stature
• Fat/muscle hypoplasia
• Excessive collagen
• Lung abnormalities
• Craniofacial features
FBN1 microfibril
FBN1 -/- Mice
• Neptune Nat Gen 2003;
33:407-411
• Abnormalities in prealveolar saccules
• Correlates with TGFβ
• Lung phenotype blocked
by neutralising TGFβ abs
Dysregulated TGFβ signaling in
Marfan syndrome
Unexpected therapeutic opportunities with ACE receptor blockers
Bone formation and remodelling
Potential treatments from extreme phenotypes
Activity of osteoblasts
and osteoclasts is
tightly linked in health
Sclerostin
Osteoporosis
pseudoglioma
EXTREME PHENOTYPES CAN REVEAL NEW
TREATMENT OPTIONS
“Sclerosteosis”
•
•
•
•
Natural BMP inhibitor
Deficiency of Sclerostin
Sclersoteosis
Sclerostin antibodies for
osteoporosis?
• Mouse studies successful
(Li et al J Bone Min Res 2009; 24:578)
Dutch founder populations
and sclerosteosis
Lesson 3 - Never take any for granted
Different mutations – who would have believed it?
Bone: the crucial balance between formation and
resorption
PAGET DISEASE RELATED PHENOTYPES
Expansile Skeletal
Hyperphosphatasia
TNFRSF11A (RANK)
Familial Expansile Osteolysis
TNFRSF11A (RANK)
Juvenile Paget
(idiopathic hyperphosphatasia)
TNFRSF11B (OPG)
Familial Paget Disease
SQSTMI (involved in NFκB
pathways)
RANKL activates osteoclast precursors
Denosumab – an anti RANKL antibody
We only have a skeleton crew working on dysplasias now!
COMPLEX DISORDERS
Classic forms of AS
TWIN STUDIES IN ANKYLOSING
SPONDYLITIS
MZ Twins
DZ Twins
B27 + DZ
UK (1997)
6/8 (75%)
4/32 (13%)
4/15 (27%)
Others
11/19 (58%)
3/24 (13%)
3/15 (20%)
Total
17/27 (63%)
7/56 (13%)
7/30 (23%)
 92% of population variance due to genetic factors
 AS is a genetic disease that is not all due to B27
Brown et al, Arthritis Rheum 1997;40:1823
CHRO6
NASC+Oxford
NASC
Oxford
All AS
GFEGS: AS only
GFEGS:SpA+AS
All:SpA+AS
30.0
25.0
LOD
20.0
15.0
10.0
5.0
0.0
0
50
100
150
200
Distance (cM)
Carter et al Rheumatol 2007;46:763
The Wellcome Trust Case-Control
Consortium (WTCCC) 1
Second Study:
1,000 cases
1,500 controls
3,000 MHC SNPs
12,000 non-synonymous coding SNPs
4 diseases
Ankylosing Spondylitis
Autoimmune thyroid disease
Breast Cancer
Multiple sclerosis
WTCCC & TASC Nat Genet 2007; 39: 1329-37
Wordsworth et al PNAS 1989
METHODS SLIDE
Before and after Thermal Cyclers
c.1987
UK RA Genetics Group
SUMMARY OF RA GENES
courtesy of Jane Worthington
• 7 new RA loci confirmed at p<5x10-8
– SPRED2, ANKRD55, C5orf30, PXK, RBPJ, CCR6, IRF5
• 31RA loci confirmed at p<5x10-8
EIRE
• Evidence for 9 further RA loci p 0.01-6.7x10-5
NARAC
UKRAGG
WTCCC
SE hypothesis
HLA-DRB1
1978
PTPN22
2004
CTLA4
2005
TNFAIP3
STAT4
TRAF1
IL2 / IL21
2007
REL
BLK
CD40
TAGAP
CCL21
CD28
TNFRSF14
TRAF6
IL2RB
PTPRC
PRKCQ
FCGR2A
KIF5A
PRDM1
IL2RA
CD2-CD58
AFF3
2008
2009
IL6R
CD247
ZEB1
SH2B3
BATF
NFATC2IP
IKZF3
UBASH3A
UBE2L3
ANKRD55
SPRED2
C5orf30
RBPJ
CCR6
IRF5
PXK
2010
GENETICS OF RHEUMATOID ARTHRITIS
At least 30 genes involved
Many are in the same pathways
Most genetic effects are small
Account for ~ 20% of variance
Some are translatable
Eg. CTLA4 (OR~1.05)
Lesson 4 - CTLA4 (OR ~1.05)
The biological importance of an
association cannot be extrapolated
from the strength of its genetic
association with disease.
CTLA4-Ig binds to B7 molecules and inhibits
CD28-B7 interactions; suppresses T cell response
ABATACEPT
CD28
TCR
T cell
B7
MHC + peptide
Antigen presenting cell
Uncontrolled for DRB1/DQB1
association
Type1 diabetes
Controlled for DRB1/DQB1 association
89
Nejentsev, Nature, 2007.
Effects from 5 amino acid residues in HLA-DR, HLA-DP
and HLA-B
Plenge et al 2012
A Retrospectoscope is all
very well but……..
WHY STUDY GENETICS OF AS?
• Curiosity
• Diagnosis
• Prognosis
• New treatments
• CURE/prevention
IL23R
US + UK vs UK
ERAP1
? New Peak
US vs UK
IL23R
ERAP1
? New Peak
Revised T-helper-celldifferentiation model
USTEKINUMAB
TARGET
STAT3/JAK2
STAT 3
Secukinumab
TARGETS
IL17
RORgT
IL-6
TGFb and IL-6 promoteTH17 and Tregs. IL-23 does not act on naïve T cells
It’S that retroSpectoScope
agaIn……..
A BETTER INSTRUMENT
(and a good team)
ERAP1 resquencing
Wild
Type
A/A
Heterozygous
A/G
Homozygous
G/G
A→G = Lys→Arg 528
Oxford colleagues in AS
Recombinant Protein Production
• Site-directed mutagenesis
• Cloned into baculovirus genome
• Infection of insect cells with
baculovirus DNA
• Collect culture supernatant 72h PI
• Purification by IMAC
• Adjust to uniform protein
concentrations
Crystal structure of ERAP1
crystal of ERAP1A
SIRAS-phased electron density map
Diamond Synchtrotron @ beamline I02
ERAP1 crystallized in 2 crystal forms
Kochan et al PNAS 2011;108:7745-50
space group P622: closed
space group P212121
open
molecule 1
molecule 2
molecule 3
ERAP1A may be a “molecular ruler”
ERAP1 SUBSTRATES:
*Length 9-16aa
*C-teminal aa – hydrophobic
*X-P-X-X-X….
*X-X-P-X-X-X….
Shih-Chung Chang et al. PNAS 2005
ERAP1 (endoplasmic reticulum aminopeptidase)
is synergistic with HLA-B27
A role for ERAP1
in peptide presentation?
ERAP1
Activity of ERAP1 Variants Towards WRVYEKCALK
**
**
** p < 0.05
Lys528Arg
40% reduction in activity – p = 0.009
**
LocusZOOM, regional association plot for ERAP1
P=10-6
P-values lower mainly due to smaller control group…
No association when comparing B27- cases & controls…
Providing further evidence that ERAP1 plays an important role in disease mechanism
by trimming peptides for presentation by B27..
ERAP1 (endoplasmic reticulum aminopeptidase)
is synergistic with HLA-B27
A role for ERAP1
in peptide presentation?
ERAP1
Small molecule ERAP1 inhibitors for AS?
TASC
Triple A (Australo-Anglo-American) Spondyloarthritis
Consortium
Paul Wordsworth
Oxford
John Revielle
Houston
Matt Brown
Brisbane
WTCCC2
TASC
HLA-B27
HLA-B60
ERAP1
IL23R
1q32
2p15
21q22
CMG2
IL1R2
CARD9
STAT3
RUNX3
PTGER4
TBKBP1
TNFRSF1A
IL12B
ERAP1/B27
synergy
IGAS
Immunochip
B27 structure
ERAP1
structure
______________________________________________________________________
1973
1989
2007
2010
2011
2012
Milestones in the genetics of AS
Statistical genetic signals can
disappear.
Findings
1.
SNPs in the MHC on chromosome 6p were strongly associated
with AS (P = 5 × 10−304).
2. 25 SNPs from 6 independent loci significantly associated with AS
Includes ERAP1 and IL23R
Chr 2p15
Chr 21q22
IL1R2
ANTXR2
Findings
P=5x10-304
25 SNPs from 6 loci
associated with AS
Includes
ERAP1
IL23R
Chr 2p15
Chr 21q22
IL1R2
ANTXR2 or CMG
Manhattan plot of discovery genome-wide association study findings
Overlap with Genetic Associations
of Psoriasis
IL12B
HLA-C
IL23R ERAP1
IL23A
Overlap of IBD susceptibility loci and AS
AS
HLA
IL23R
ERAP
IL12B
STAT3
PTGER4
CARD9
IL1R2
IL7R
KIF21B
Lees C W et al. Gut doi:10.1136/gut.2009.199679
©2011 by BMJ Publishing Group Ltd and British Society of Gastroenterology
UK collaboration in AS genetics
Lessons for AS from extreme phenotypes?
X-linked Hypophosphataemia
Looser zones and ossification of hip capsule & ligamentum flavum
Excessive bone in
pseudohypoparathyroidism
ASIF Izmer 2011
This could not have happened without NASS
March 15th 2012
What a night!
To the planet Venus
William Wordsworth 1838
Man now presides
In power, where once he
trembled in his weakness;
Science advances with
gigantic strides;
But are we aught enriched in
love and meekness?
Musings on genetics
Consider Queen Victoria,
‘Tis odd to contemplate
How just one little chromosome
Decreed the Royal fate.
For had She been X and Y,
Instead of double X,
Victoria Regina
Would have ruled as Victor Rex.
Victor Wordsworth c1990
Acknowledgements
OXFORD
NIHR BRC/BU
David Harvey
Clare Farrar
Alice Harin
Jenny Pointon
Tugce Karaderi
Louise Appleton
Tom Wordsworth
Bristol University
David Evans
DIamentina Institute BRISBANE
Matthew Brown
Linda Bradbury
TASC (John Reveille et al), IGAS
Structural Genomics Consortium
Grazyna Kochan
Centre for Proteomics
Tobias Kroja
Benedikt Kessler
Udo Oppermann
FUNDING
Wellcome Trust
Human Immunology Unit (MRC)
NIAMS
Liye Chen
Arthritis Research UK
Simon Kollnberger
National Institute for Health Research
Paul Bowness
NASS
Henni Mester
Acknowledgements
FOP
•
•
•
•
•
•
Wordsworth Group
Dr Kirsten Petrie
Prof Graham Russell
Prof Jim Triffitt
Dr Roger Smith
Prof Matt Brown

Dr Alex N. Bullock
Nuffield Department of
Orthopaedics, Rheumatology and
Musculoskeletal Sciences
Nuffield Department of
Clinical Medicine
17:17
Page 128
“A New Hope” in Oxford
“Twilight”
800M
2Billion