Indians, Twins and Scleroderma: Genetic and Genomic Studies … · 2015-12-25 · 19q13.2 D19S220 0.0175 20q12 D20S107 0.0095 22q13.1 D22S423 0.0005 Xp11.4 DXS1068 0.0026 Xq21-23

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





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





7

SYSTEMIC SCLEROSISVASCULOPATHY AND FIBROSIS

8

ARTERIAL FIBROSIS -PATHOLOGY

9

SYSTEMIC SCLEROSIS NAILFOLD CAPILLARY

ABNORMALITIES





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

11

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

12

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





13

Talahina Service Unit AreaAtoka, Choctaw, Haskell, Latimer, LeFlore, McCurtain,

Pittsburg, Pushmataha

Red river

14

GENEAOLOGY OF SSc IN THE CHOCTAW

Genealogic origins of modern Choctaw subjects with scleroderma

and their Fibrillin-1 haplotype status

15

PUSHMATAHA





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

18

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





19Wandstrat and Wakeland, Nat Immunol, 2001 Sep;2(9):802-9.

AUTOIMMUNITY REGIONS

FROM GENOME SCANS

SSc

20

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

21

GENE MAPPING IN SCLERODERMA

Case-control approach

ofCandidate loci (SNP genotyping)

fromChoctaw genome scan and genes found

in other autoimmune diseases





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

24

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





25

GENETIC ASSOCIATIONS IN SCLERODERMA

Anti-

centromere

AIF1, MHC2TA

HLA-DQB1

*05

Anti-topo-I

TYK2

PTPN22

HLA-DRB1

*11

Males

HLA-DQA1

*0501

26

SYSTEMIC SCLEROSIS

Uses for DNA Microarrays

�To determine genetic vs.non-genetic contributions

�Identify pathogenetic pathways

27

SYSTEMIC SCLEROSIS

Twin Concordance


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





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

29

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





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


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.

33

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?





34

*

*

*

*

*

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

35

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

36

SYSTEMIC SCLEROSIS

�Gene discovery

�Define potential therapeutic targets

Uses for DNA Microarrays





37

SSc fibroblast cells Control fibroblast cells

Expression of SPARC gene

cDNA MICROARRAY

38

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





40

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





43

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





46

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

47

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

48

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





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)





52

Documents

Indians, Twins and Scleroderma: Genetic and Genomic Studies … · 2015-12-25 · 19q13.2 D19S220 0.0175 20q12 D20S107 0.0095 22q13.1 D22S423 0.0005 Xp11.4 DXS1068 0.0026 Xq21-23