Indians, Twins and Scleroderma:
Genetic and Genomic Studies
Prof. Frank C. Arnett
The screen versions of these slides have full details of copyright and acknowledgements 1
INDIANS, TWINS AND SCLERODERMA:
GENETIC AND GENOMIC
STUDIESFrank C. Arnett, MD
UTHHSC
2
SCLERODERMA
equals
SYSTEMIC SCLEROSIS
equals
SSc
3
SYSTEMIC SCLEROSIS
�A complex disorder (genetic + non-germline / ? environmental)
�Affects 3/10,000 Americans
� 3:1 Female : Male
�Mortality from diffuse form 30%-50% at 5 years
Indians, Twins and Scleroderma:
Genetic and Genomic Studies
Prof. Frank C. Arnett
The screen versions of these slides have full details of copyright and acknowledgements 2
4
Autoimmunity Vasculopathy
Endothelium,
Smooth muscle
Fibroblast activation
T and B-cellsAutoantibodies
Raynaud’sPulm HTNRenal crisis
Skin thickeningPulm & GI Fibrosis
ECM abnormalities
SYSTEMIC SCLEROSIS
Anti-Centromere (ACA)Anti-Topoisomerase 1 (Scl70)
5
CLINICAL MANIFESTATIONS
OF SCLERODERMA
6
SCLERODERMA -PATHOLOGY
Indians, Twins and Scleroderma:
Genetic and Genomic Studies
Prof. Frank C. Arnett
The screen versions of these slides have full details of copyright and acknowledgements 3
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SYSTEMIC SCLEROSISVASCULOPATHY AND FIBROSIS
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ARTERIAL FIBROSIS -PATHOLOGY
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SYSTEMIC SCLEROSIS NAILFOLD CAPILLARY
ABNORMALITIES
Indians, Twins and Scleroderma:
Genetic and Genomic Studies
Prof. Frank C. Arnett
The screen versions of these slides have full details of copyright and acknowledgements 4
10
SYSTEMIC SCLEROSIS
� Population frequency in US is 0.026%
�Recurs in families 1.6%
�Relative risk in family members = 27-50
� Twin studies (to follow)
Evidence for Genetic Effects
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SYSTEMIC SCLEROSIS
Twin Concordance
Monozygotic Pairs Dizygotic Pairs
SSc concordant
ANA positive
1/24 (4.2%) 1/18 (5.6%)
(90%) (40%)
Feghali-Bostwick et al , Arthritis Rheum 2003 Jul;48(7):1956-63
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SYSTEMIC SCLEROSIS
Prevalences in American Indians
FrequenciesActual cases/
population
Estimated cases/
100,000
Full-blooded
Non-full blooded
Oklahoma Choctaw
8/1704 469 1/213
6/19,551 31 1/3259
Mississippi Choctaw
Full-blooded 0/4500 0 --------
Other-
Oklahoma indians 20/210,811 10 1/10,541
US Caucasians ------------ 30 1/3300
Indians, Twins and Scleroderma:
Genetic and Genomic Studies
Prof. Frank C. Arnett
The screen versions of these slides have full details of copyright and acknowledgements 5
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Talahina Service Unit AreaAtoka, Choctaw, Haskell, Latimer, LeFlore, McCurtain,
Pittsburg, Pushmataha
Red river
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GENEAOLOGY OF SSc IN THE CHOCTAW
Genealogic origins of modern Choctaw subjects with scleroderma
and their Fibrillin-1 haplotype status
15
PUSHMATAHA
Indians, Twins and Scleroderma:
Genetic and Genomic Studies
Prof. Frank C. Arnett
The screen versions of these slides have full details of copyright and acknowledgements 6
16
FIBRILLIN-1 GENE
(FBN1)
�Associated with SSc
in the Choctaw and Japanese
�fbn-1 duplication causes tight skin (tsk)
murine phenotype
17
�Case-control study of SSc in Choctaw Native
Americans - 20 SSc patients and 76 matched controls
� 400 microsatellite markers with average 10 cM
resolution across the genome (Applied Biosystems)
�Monte Carlo simulation to test for differences
in microsatellite allelic distribution
GENOME-WIDE SCAN FOR
DISEASE LOCI ASSOCIATED
WITH SSc
Zhou X, et al: A&R 2003;48(9):2585 - 2592
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COMPARISON OF ALLELIC DISTRIBUTIONS IN CHOCTAW CASES
AND CONTROLS
Region on chromosome Marker p
1p32-31 D1S255 0.00681p21.2 D1S206 <0.00011q42.3 D1S2800 0.0012
5q33.2 D5S410 <0.00016p22.3 D6S422 0.03686q23-27 D6S264 0.0176
7p12-11 D7S510 0.00177q35 D7S661 0.005
8q24.12 D8S514 0.0015
14q21 D14S63 0.017315q21.1 D15S978 0.018919p13.2 D19S221 0.0016
19q13.2 D19S220 0.017520q12 D20S107 0.009522q13.1 D22S423 0.0005
Xp11.4 DXS1068 0.0026Xq21-23 DXS8055 0.0082
Indians, Twins and Scleroderma:
Genetic and Genomic Studies
Prof. Frank C. Arnett
The screen versions of these slides have full details of copyright and acknowledgements 7
19Wandstrat and Wakeland, Nat Immunol, 2001 Sep;2(9):802-9.
AUTOIMMUNITY REGIONS
FROM GENOME SCANS
SSc
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AUTOIMMUNITY GENES� Major Histocompatibility Complex (MHC) class II
� RUNX family - transcription regulation
� PDCD1 (2q37.3)
� SLC9A3R1 (?)
� SLC22A4 (5q31)
� PTPN22 – protein tyrosine phosphatase (1p13)
� TYK2 – tyrosine kinase 2 (19p13.2)
� IRF5 – interferon regulator (7q32)
� MHC2TA (16p13)
� Others – major gene at 1q41- 42
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GENE MAPPING IN SCLERODERMA
Case-control approach
ofCandidate loci (SNP genotyping)
fromChoctaw genome scan and genes found
in other autoimmune diseases
Indians, Twins and Scleroderma:
Genetic and Genomic Studies
Prof. Frank C. Arnett
The screen versions of these slides have full details of copyright and acknowledgements 8
22
MAJOR HISTOCOMPATIBILITY
COMPLEX CLASS II HAPLOTYPES
With SSc Autoantibodies:
�DRB1*11 – Anti-topo I
�DQB1*0501 – Anti-centromere
�DRB1*1302 – Anti-fibrillarin
�DRB1*0301 – Anti-PM-Scl
With Scleroderma (over 1100 cases):�DRB1*11, DQB1*0301
23
PROTEIN TYROSINE PHOSPHATASE (PTPN22)
� Lymphoid-specific regulator of T- cell signal
transduction (maps at 1p13)
� Position 1858 C to T single nucleotide
polymorphism (SNP)
� T allele associated with susceptibility to SLE,
RA, IDDM, Grave’s disease and Hashimoto’s
thyroiditis but not multiple sclerosis
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PTPN22 IN SCLERODERMA
SSc ControlsN=1006 N=826
p value*
T allele 16.2% 5.9% 0.002*
*OR = 1.49, C.I. = 1.15-1.92 (combined whites,blacks and hispanics)
Associated with Anti-Topo I Autoantibodies
SSc topo I + SSc topo I - Controls
N=176 N=826 N=826
11.9% 8.7% 5.9%
p value 0.036 0.0002
Odds ratio 2.04 1.48
Indians, Twins and Scleroderma:
Genetic and Genomic Studies
Prof. Frank C. Arnett
The screen versions of these slides have full details of copyright and acknowledgements 9
25
GENETIC ASSOCIATIONS IN SCLERODERMA
Anti-
centromere
AIF1, MHC2TA
HLA-DQB1
*05
Anti-topo-I
TYK2
PTPN22
HLA-DRB1
*11
Males
HLA-DQA1
*0501
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SYSTEMIC SCLEROSIS
Uses for DNA Microarrays
�To determine genetic vs.non-genetic contributions
�Identify pathogenetic pathways
27
SYSTEMIC SCLEROSIS
Twin Concordance
Monozygotic Pairs Dizygotic Pairs
SSc concordant
ANA positive
1/24 (4.2%) 1/18 (5.6%)
(90%) (40%)
Feghali-Bostwick et al , Arthritis Rheum 2003; 48(7):1956-63
Indians, Twins and Scleroderma:
Genetic and Genomic Studies
Prof. Frank C. Arnett
The screen versions of these slides have full details of copyright and acknowledgements 10
28
� Affected tw ins (MZ and DZ) showed significantly
different gene expression profiles than unaffected
DZ tw ins and normal controls BUT NOT
unaffected MZ tw ins*
* student t tests
SYSTEMIC SCLEROSIS
FIBROBLAST DNA
MICROARRAYS
� 10 discordant MZ pairs
� 5 discordant DZ pairs
� 5 normal controls
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MICROARRAY ANALYSES
�Classification analysis: select genes significantly different (for instance p< 0.01) between classes (disease vs controls)
�Computer software clusters genes based on their similarities of expression
Gene Clusters
DENDROGRAM FOR CLUSTERING
EXPERIMENTS, USING CENTERED CORRELATION AND COMPLETE LINKAGE
Log-ratios
6
-6
Indians, Twins and Scleroderma:
Genetic and Genomic Studies
Prof. Frank C. Arnett
The screen versions of these slides have full details of copyright and acknowledgements 11
31
�Connective tissue growth factor
�Tropomyosin 1 (alpha)
�Collagen, type I, alpha 2
�Secreted protein, acidic, cysteine-rich
or SPARC (osteonectin)
�Phosphatidylinositol 4-kinase type II
�NY-REN-24 antigen
�Ras homolog gene family, member B
�APG4 autophagy 4 homolog B
�Sperm protein SSP411
�Apical protein-like (Xenopus laevis)
�Yip1 interacting factor homolog
�UDP glycosyltransferase 2 family,
polypeptide B7
�Activin A receptor, type IB
�Thy-1 cell surface antigen
�Angiotensin receptor-like 2
�Dopamine responsive protein
�MAM domain containing
glycosylphosphatidylinositol anchor 1
32
SYSTEMIC SCLEROSIS
Twin Concordance
Monozygotic Pairs Dizygotic Pairs
SSc concordant
ANA positive
1/24 (4.2%) 1/18 (5.6%)
(90%) (40%)
Feghali-Bostwick et al , Arthritis Rheum 2003 Jul; 48(7):1956-63
Fibroblast gene
profiles concordant5/10 (50%) 0/5 (0%)
Zhou et al., Arthritis Rheum. 2005 Oct; 52(10):3305-14.
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SYSTEMIC SCLEROSIS
FIBROBLAST MICROARRAYS
� 40%-50% of unaffected MZ twins but none of the unaffected dizygotic twins have “activated fibroblasts”
� Implies strong genetic effects (40-50% heritability)� Inherent to the fibroblast?
� Induced by exogenous stimulus?
Indians, Twins and Scleroderma:
Genetic and Genomic Studies
Prof. Frank C. Arnett
The screen versions of these slides have full details of copyright and acknowledgements 12
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*
*
*
*
*
0
1
2
3
4
5
6
control normal sera unaffected
twin sera
affected twin
sera
comparison of transcript levels
COL1A2
COL3A1
MMP1
TIMP3
SPARC
CTGF
IL6
SSc TWIN SERA STIMULATE
NORMAL FIBROBLASTS
* = p<0.05
RT-PCR
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SCLERODERMAOccurrence and Risk in Families
Populations
First-degree relatives
Siblings
Identical (MZ) twins
MZ fibroblast gene profiles
0.03% --
1.6% 50
0.7% 27
5% 192
50% 1923
Frequency Risk
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SYSTEMIC SCLEROSIS
�Gene discovery
�Define potential therapeutic targets
Uses for DNA Microarrays
Indians, Twins and Scleroderma:
Genetic and Genomic Studies
Prof. Frank C. Arnett
The screen versions of these slides have full details of copyright and acknowledgements 13
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SSc fibroblast cells Control fibroblast cells
Expression of SPARC gene
cDNA MICROARRAY
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INCREASED SPARC
EXPRESSION IN SYSTEMIC
SCLEROSIS FIBROBLASTS
Normal control fibroblast
A. W estern Blot
B. Immunostaining
SSc fibroblast
FLUORESCENT STAINS FOR
SPARC IN FIBROBLASTS
AFTER SPARC siRNA
SPARC silencing siRNAtransfected fibroblasts
Non-silencing siRNAtransfected fibroblasts
Untransfectedcontrol fibroblasts
Indians, Twins and Scleroderma:
Genetic and Genomic Studies
Prof. Frank C. Arnett
The screen versions of these slides have full details of copyright and acknowledgements 14
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WESTERN BLOTS OF COLLAGEN
TYPE I AND SPARC IN FIBROBLASTS
AFTER SPARC siRNA
1: control fibroblasts;
2: control fibroblasts in response to TGFB1;
3: SPARC siR%A transfected fibroblasts; 4: SPARC siR%A transfected fibroblasts in response to TGFB1;
5: non-silencing siR%A transfected fibroblasts;
6: non-silencing siR%A transfected fibroblasts in response to TGFB1.
41
SYSTEMIC SCLEROSIS
�Prognosis or disease outcome
�Select patients at highest risk for more aggressive therapy
Uses for DNA microarrays
42
+ ++
+ + +
+ + + +
Death
Variable
MRSS>20
ILD
Race H C C H A H C H C C A
+
+ +
+ + + + + + + + ++
+ + + + +
A A A H H C
+
A C C H
A B
• = p<0.01• Fibroblast array
Indians, Twins and Scleroderma:
Genetic and Genomic Studies
Prof. Frank C. Arnett
The screen versions of these slides have full details of copyright and acknowledgements 15
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MICROARRAYS OF
PERIPHERAL BLOOD CELLS
�More practical in clinical setting
�RNA stabilized as it enters the PAXgene tube (Stratagene): less artifacts; long storage time
� Serial samples (? predict disease prognosis, activity, progression)
44
45
�Ser/Thr-like kinase �Beta-transducin repeat containing�Recombining binding protein suppressor of hairless-like
�chloride channel 4�Plexin B2�TYRO protein tyrosine kinase binding protein�Selectin L (lymphocyte adhesion molecule 1)�Vanin 2�Interferon, gamma-inducible protein 30�Fc fragment of IgE, high affinity I, receptor�Lipid phosphate phosphatase-related protein type 2
�Tumor protein p53 inducible protein 11�Dysferlin , limb girdle muscular dystrophy 2B�ATPase, H+ transporting, lysosomal 9kD�Myeloid cell nuclear differentiation antigen�Formin-like�Apoptosis-associated speck-like protein containing a CARD
�Fc fragment of IgG, receptor, transporter, alpha�Spleen focus forming virus (SFFV) proviral integration
� oncogene spi1�Fatty-acid-Coenzyme A ligase, long-chain 2�Hypothetical protein FLJ22662�S100 calcium-binding protein A12 (calgranulin C)�S100 calcium-binding protein A9 (calgranulin B)�Sirtuin 7�Rho GTPase activating protein 9�Flavin containing monooxygenase 5�Trinucleotide repeat containing 5�CASP8 and FADD-like apoptosis regulator�Protein tyrosine phosphatase, receptor type�Glia maturation factor, gamma�Carcinoembryonic antigen-related cell adhesion molecule 3
�Notch homolog 4 �Interleukin 10 receptor, beta�UDP-N-acetyl-alpha-D-galactosamine:polypeptide �N-acetylgalactosaminyltransferase 14
Indians, Twins and Scleroderma:
Genetic and Genomic Studies
Prof. Frank C. Arnett
The screen versions of these slides have full details of copyright and acknowledgements 16
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INTERFERON - RELATED
GENES�Interferon, alpha-inducible protein (clone IFI-6-16)
�Interferon-stimulated protein, 15 kDa
�Myeloid cell nuclear differentiation antigen
�Interferon regulatory factor 7
�Transporter, ATP-binding cassette, major histocompatibility complex, 1
�Interferon, gamma-inducible protein 30
�Interferon stimulated gene (20kD)
�Signal transducer and activ ator of transcription (STAT1)
�Interferon gamma receptor 2 (interferon gamma transducer 1)
�Major histocompatibility complex, class I, E
�Myxov irus (influenza) resistance 1, homolog of murine (interferon-inducible protein p78)
�Solute carrier family 11 (proton-coupled div alent metal ion transporters), member 1 (NRAMP1)
�Interferon induced with helicase C domain 1
�Suppressor of cytokine signaling 3
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VASCULOTROPHIC GENES
�Selectin L (lymphocyte adhesion molecule 1)
�Selectin P ligand (CD162)
�Integrin, alpha 2b (platelet glycoprotein IIb of IIb/IIIa
complex, antigen CD41B)
�Integrin, beta 2 (antigen CD18 (p95), lymphocyte
function-associated antigen 1
�Integrin, beta 5
�Integrin, alpha 6
�glycoprotein Ib (platelet), beta polypeptide
�Allograft inflammatory factor 1
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IMMUNE FUNCTION GENES
�CASP8 and FADD-like apoptosis regulator (FLIP)
�Cytotoxic T-lymphocyte-associated protein 4 (CTLA4)
�Interleukin 8
�Interleukin 23 p19 subunit
�Interleukin 1 receptor antagonist
�Interleukin 10 receptor, beta
�macrophage antigen 1
�Chemokine-like factor, alternatively spliced
�T-cell, immune regulator 1, ATPase, H+ transporting,
lysosomal V0 protein a isoform 33
�B-cell receptor-associated protein 29
Indians, Twins and Scleroderma:
Genetic and Genomic Studies
Prof. Frank C. Arnett
The screen versions of these slides have full details of copyright and acknowledgements 17
49
TO SEE MORE OF THE DATA
Go to:
www.uth.tmc.edu/scleroderma
50
ACKNOWLEDGEMENTS
Xiaodong Zhou, M.D.
Filemon K. Tan, M.D., Ph.D.
Maureen D. Mayes, M.D., MPH
Prav itt Gourh, MD
Dianna M.Milewicz, MD, Ph.D
Chul Ahn, Ph.D
Ranaj it Chakraborty, Ph.D
Carol Feghali-Bostwick, Ph.D
Momiao Xiong, Ph.D.
Li (Felix) Jin, Ph.D.
UT Rheumatology
UT Rheumatology
UT Rheumatology
UT Medical School
UT Medical Genetics
UT Medical SchoolUniversity of Cincinnati
University of Pittsburgh
UT Human Genetics Center
University of Cincinnati
51
SUPPORT
�NIAMS Specialized Center of Research (SCOR)
in Scleroderma (Dr Arnett)
�NIAMS Scleroderma Family Registry and DNA
Repository (Dr Mayes)
�UT NIH Clinical Research Center (CRC)
� Scleroderma Foundation and NIH R03 (Dr Zhou)