Transcriptome changes in the temporal cortex of subjects with

autism

Károly MirnicsDept Psychiatry and Kennedy Center for Human DevelopmentVanderbilt University School of Medicine

April 1, 2009

DEVELOPMENT

INFECTIONS NUTRITION CHEMICALS

ENVIRONMENTAL INSULTS

GENETIC SUSCEPTIBILITY

GENE EXPRESSIONIMPAIRMENTS

NEUROCHEMICAL &METABOLICCHANGES

ALTEREDCONNECTIVITY

IMPAIREDINFORMATIONPROCESSSING

BEHAVIORCOGNITIONEMOTION

MET, GABRB3, NRXN, NLGN,RELN, CNVs…

Courchesne Lab

Watson-Crick Complementarity• Four nucleotide types: A,C,G,T

• A’s paired with T’s (2 hydrogen bonds)

• C’s paired with G’s (3 hydrogen bonds)

The Central Dogma of Molecular Biology

Different methods, different applications

Number of samples

Number of genes queried

RT qPCR

NorthernISH

10

1

1,000

1 10 100 1,000

100

10,000

Expression microarrays

SAGEMPSSTOGA

Mirnics Lab

THE BASIC PRINCIPLE OF DNA MICROARRAYS IS COMPLEMETARY HYBRIDIZATION

Solid support (glass, membrane, metal)

Hybridization Wash

http://www.affymetrix.com

http://www.affymetrix.com

Affymetrix GeneChips

HIGH-TROUGHPUT TRANSCRIPTOME PROFILING METHODS ALLOW, BUT

DO NOT REQUIRE AN A PRIORY HYPOTHESIS

“HYPOTHESIS-FREE APPROACH”“DATA-DRIVEN APPROACH”“IGNORANCE-BASED APPROACH” (Dr. Eric Lander)

“FISHING EXPEDITION” (malicious reviewers)

… and what is wrong with fishing?! If you don’t go fishing, you won’t catch a fish.

To catch fish, you must wisely choose your equipment, bait and a fishing spot.

You are never certain what will you catch.

Once you start catching fish, everyone will want to fish at your fishing spot.

Poor fisherman greatly outnumber skilled fisherman, but they usually catch only Pomoxis annularis (crappies).

Once you start fishing around a “taken spot”, the other fishermen will try anything and everything to chase you away.

If you want to impress the tribe elders, you will have to catch, clean, cook and properly serve the fish.

When you talk to non-fishermen about your adventures, they will find them boring and they will rarely believe your stories.

Not everyone likes fish, fishing or fishermen.http://www.gofisherie.com/

a.k.a. Lina’s Rules

EXPERIMENTAL DESIGN

6 matched pairs of ASD-control subjects

(from >25 pairs surveyed)

HG_U133plusV2 Affymetrix DNA microarrays

(RMA normalization, pairwise analysis)

qPCR verification (SybrGreen method, ddCt method)

RESULTS

• MET pathway

• PKC

• OAS family

• Immune changes

I. Expression of MET signaling pathway

genes is impaired in ASD

Campbell et al, Ann Neurol. Vol.62, 3 Pages: 243-250

Notice the decreased expression of MET and significantly increased expression of messenger RNA for proteins involved in the activation of MET signaling.

MET pathway: DNA microarray – qPCR data

Campbell et al, Ann Neurol. Vol.62, 3 Pages: 243-250

HEPATOCYTE GROWTH FACTOR (HGF)

• neurotrophic factor in the developing brain

• both hepatocyte growth factor and its receptor (MET) are present in third trimester placentas, suggesting that HGF serves as a paracrine mediator to control placental development and growth

Notice that MET and HGF transcript levels are positively correlated in controls, and negatively correlated in ASD.

Campbell et al, Ann Neurol. Vol.62, 3 Pages: 243-250

Dysregulation of MET/HGF ratio in autism

II. In addition to genetic susceptibility, PKCB1 expression is

reduced in the temporal cortex of subjects with ASD

Lintas, C., Sacco, R., Gaita, L., D’Oronzio, R., Garbett, K., Mirnics, K., Militerni, R., Bravaccio, C., Curatolo, P., Manzi, P., Schneider, C., Melmed, R., Elia, M. & Persico, A.M. (2007) Involvement of the PRKCB1 gene in autistic disorder: significant genetic association and reduced neocortical gene expression. Mol Psychiatry, in press.

Protein Kinase C-beta (PRKCB1)

• located on chromosome 16 p

• a family of serine- and threonine-specific protein kinases that can be activated by calcium and second messenger diacylglycerol

• plays an important role in signal transduction, regulation of gene expression and control of cell division and differentiation in the brain and epithelium

• Previous reports of association with autism (Philippi et al, 2005)

Both PRKCB1 probesets show a downregulation

(DNA microarrays and qPCR data)

III. The OAS gene expression is increased in autism(2’-5’- OLIGOADENYLATE SYNTHETASE)

• 3 genes, OAS1, OAS2, OAS3

• Bind to and activate RNase L, which degrades viral and cellular RNAs, leading to inhibition of cellular protein synthesis and impairment of viral replication

• mediates resistance to virus infection

• implicated in the control of cell growth, differentiation, and apoptosis.

Postmortem expression data lead to identification of the

OAS (2',5'-oligoadenylate synthetase) gene cluster as an

ASD susceptibility locus

Confidential information, collaboration between the Levitt, Sutcliffe, Persico and Mirnics laboratories, to be submitted.

12q24.2

629 pedigrees including 1,228 individuals with ASD plus 312 unrelated controls

Association of the OAS1 marker rs3741981 allele G (P = 0.032) and a four-marker haplotype spanning the OAS1, OAS3 and OAS2 genes (P = 0.008).

292 individuals with narrowly-defined autism revealed significant association of the OAS3 marker rs2285932 allele C by both family-based association test (P = 0.008) and case-control analyses (P = 0.019).

The relative risk of autism diagnosis was 2.745 (95% CI: 1.169-6.449) for the rs2285932 C/C genotype and 3.277 (95% CI: 1.386-7.747) for the C/T genotype compared to the T/T genotype.

OAS gene cluster is an ASD

susceptibility locus

IV. Profound dysregulation of immune

system genes in the temporal cortex with

ASD

Garbett et al, Neurobiol Dis, 30 (3):303-11, 2008

PROBESET GENE SYMBOL prPval grPval Fold

244699_at heat shock 70kDa protein 6 (HSP70B') HSPA6 0.03223 0.03902 12.9

242138_at serpin peptidase inhibitor, clade H (heat shock protein 47), mem 1 SERPINH1 0.02988 0.01320 11.1

241758_at BCL2-associated athanogene 3 BAG3 0.02634 0.01613 8.4

240282_at adrenomedullin ADM 0.01214 0.00691 7.7

239001_at chitinase 3-like 1 (cartilage glycoprotein-39) CHI3L1 0.04795 0.03390 7.3

238320_at GTP binding protein overexpressed in skeletal muscle GEM 0.03275 0.02341 6.8

237584_at growth arrest and DNA-damage-inducible, beta GADD45B 0.03507 0.02532 5.6

236106_at heat shock 27kDa protein 1 /// Meis1, homolog 3 (mouse) HSPB1 0.01708 0.01592 5.5

236034_at trophoblast-derived noncoding RNA TncRNA 0.01877 0.01900 5.4

235420_at growth arrest and DNA-damage-inducible, beta GADD45B 0.01990 0.02254 5.1

234989_at nucleoside phosphorylase NP 0.04096 0.02375 5.0

234024_at tubulin, beta 6 TUBB6 0.01193 0.01366 4.8

233471_at TIMP metallopeptidase inhibitor 1 TIMP1 0.04213 0.02646 4.8

233364_s_at TGFB-induced factor (TALE family homeobox) TGIF 0.00522 0.00125 4.5

232882_at cold shock domain protein A CSDA 0.00614 0.01571 4.5

232693_s_at C-type lectin domain family 2, member B CLEC2B 0.01113 0.00592 4.3

232668_at cysteine-rich, angiogenic inducer, 61 CYR61 0.01124 0.03898 4.2

230482_at growth arrest and DNA-damage-inducible, beta GADD45B 0.04512 0.03109 4.2

230304_at collagen, type IV, alpha 1 COL4A1 0.02879 0.03452 4.1

230109_at phospholipase A1 member A PLA1A 0.03479 0.01116 4.0

229435_at Yes-associated protein 1, 65kDa YAP1 0.03955 0.03993 4.0

228758_at CDNA FLJ26120 fis, clone SYN00419 --- 0.00445 0.00372 4.0

~110 genes separate the ASD and CNT phenotypes

tncRNA

RFX4

F3

ADM

CHI3L1 SPP1

DTNA

TIMP1

CLEC2B

CYR61

COL4A1IL1R1

CDS2

IFITM3 IFITM2

S100A10 AQP4

ITGAV

ITGA5

ADAMTS9ALCAM

TJP2

TNPO1

EFNA1

CMKOR1

RAB13

DNMBP

INHBA

SERPINH1

TNFRSF10B

PTDSR BAG3

BAG2

HSPB1

DNAJB1

HSPB8

DNAJB6

HSPA6

TAP1

TGIF

NFKBIZ

FGF2

GEMGADD45B

CRYABBCL6

PLSCR1

IFI16

SCARA3FOXO1A

TNFRSF1A

TNFRSF1B

TNFAIP3RIT1

TP53BP2FAS

GADD45G

ZFP36L1

>3 ALR>2 ALR>1 ALR

qPCR confirmed<-1 ALR

FGF12

HSPA1A

DLX1

EMP1

EMP3

CSDA

HMG20B

ZAK

TPD52L1

MKNK2

ELAVL1

CHRNA7

Cell communication and motility Cell fate and differentiation

NEURONAL DEVELOPMENT IS DISRUPTED IN THE BRAINS OF SUBJECTS WITH ASD

Gene Set SIZE ES NES p-val q-val RANK

NFKBPATHWAY 22 0.83 2.10 0.0000 0.0000 2082

IL1RPATHWAY 30 0.77 2.05 0.0000 0.0000 2338

TOLLPATHWAY 32 0.75 2.05 0.0000 0.0000 2338

NKTPATHWAY 25 0.76 2.00 0.0000 0.0000 1019

INFLAMPATHWAY 28 0.73 1.96 0.0000 0.0000 2966

GSK3PATHWAY 25 0.73 1.91 0.0000 0.0020 2082

TOB1PATHWAY 16 0.79 1.87 0.0000 0.0040 2231

TNFR2PATHWAY 17 0.77 1.86 0.0000 0.0040 2207

EGFPATHWAY 16 0.79 1.86 0.0000 0.0040 2405

P38MAPKPATHWAY 37 0.66 1.84 0.0030 0.0050 2216

TIDPATHWAY 17 0.75 1.79 0.0000 0.0120 2600

G1PATHWAY 24 0.68 1.76 0.0000 0.0200 2697

HIVNEFPATHWAY 54 0.59 1.73 0.0010 0.0290 3323

41BBPATHWAY 16 0.73 1.73 0.0020 0.0270 2338

MCALPAINPATHWAY 23 0.66 1.70 0.0050 0.0340 1653

P53HYPOXIAPATHWAY 18 0.70 1.69 0.0050 0.0350 2740

METPATHWAY 36 0.61 1.68 0.0030 0.0360 1753

DEATHPATHWAY 32 0.60 1.67 0.0060 0.0370 3084

ATMPATHWAY 18 0.68 1.66 0.0160 0.0390 2338

IL6PATHWAY 20 0.67 1.66 0.0130 0.0400 2724

  GENE NAME SYMBOL AUT Sd CNT Sd Sd

1 serpin peptidase inhibitor A3 (alpha-1 antiproteinase) SERPINA3 4.21 0.88 3.33

2 hypothetical protein FLJ10847 FLJ10847 3.33 0.04 3.29

3 stratifin* SFN 3.25 0.06 3.19

4 secreted frizzled-related protein 2* SFRP2 3.44 0.29 3.15

5 secretory leukocyte peptidase inhibitor SLPI 3.13 0.07 3.06

6 stratifin* SFN 3.37 0.33 3.04

7 scavenger receptor class A, member 5* SCARA5 3.70 0.66 3.03

8 heat shock 70kDa protein 6* HSPA6 3.27 0.38 2.89

9 secreted frizzled-related protein 2* SFRP2 3.60 0.71 2.88

10 CD44 molecule* CD44 2.88 0.01 2.87

11 heat shock 70kDa protein 6* HSPA6 3.69 0.94 2.75

12 scavenger receptor class A, member 5* SCARA5 3.16 0.47 2.69

13 CD44 molecule* CD44 3.32 0.67 2.65

14 neuronal PAS domain protein 4 NPAS4 3.75 1.16 2.59

15 CD44 molecule* CD44 2.72 0.15 2.58

16 CD44 molecule* CD44 2.68 0.11 2.57

17 guanylate binding protein 2, interferon-inducible GBP2 2.64 0.07 2.57

18 chemokine (C-C motif) ligand 19 CCL19 3.07 0.51 2.56

ASD SUBJECTS SHOW HIGH VARIABILITY IN GENE EXPRESSION

There are significant similarities between

immune system dysregulation in autism

and schizophrenia: a common gene

expression signature of developmental

origin?

Arion, D., Unger, T., Lewis, D.A., Levitt, P. & Mirnics, K. (2007) Molecular evidence for increased expression of genes related to immune and chaperone function in the prefrontal cortex in schizophrenia. Biol Psychiatry, 62, 711-721.

Schizophrenia is characterized by a systemic increase of immune system transcripts in the prefrontal cortex

Can we recapitulate the human gene

expression changes observed in ASD in a

mouse model?

Poly (I:C) treated mice

• polyinosinic-polycytidylic is an immunostimulant used to mimic viral infection

• binds to Toll-like receptors, elevates a wide range of cytokine, including IL1, IL6 and IFNgamma

• Pregnant mice exposed at E13 to poly(I:C) show sickness behavior

• Offspring shows behavioral deficits in social interactions.

Prediction: the offspring of poly(I:C)-treated mice will show the same gene expression deficit as the postmortem samples from ASD and/or schizophrenia

Result: not really. There are gene expression changes as a result of intrauterine poly(I:C) exposure, but they are not common with the postmortem findings of ASD or schizophrenia.

Question: is the immune activation seen in humans not an immune scar, but an active inflammatory process?

CONCLUSIONS• DNA microarray analysis of the postmortem tissue originating from autistic subjects is

feasible and yields high quality data.

• Subjects in autism show dysregulated gene expression of the MET pathway and activated immune system transcripts

• Some of the genes showing altered expression (PKCB, OAS) also confer genetic susceptibility to autism

• ASD shows significant inter-subject variability of gene expression profile, stressing the heterogeneity of disorder

• Immune activation in the brain may be characteristic of multiple brain disorders, including ASD and schizophrenia, and may be an ongoing, active process

• We all need to join efforts and mechanistically follow up the data generated in these transcriptome profiling studies.

COLLABORATORS on the AUTISM PROJECT Pat Levitt and Dan Campbell (Vanderbilt)

James Sutcliffe (Vanderbilt)

Tony Persico (Rome)

Paul Patterson (Caltech)

Eric Courchesne (UCSD)

MIRNICS LAB Phil Ebert

Krassimira Garbett

Dominique Arion

Annie Bedison

Kathie Douglass

Melissa Macioce

Travis Unger

Amanda Mitchell

Rodica Gal-Chis

We greatly appreciate the sacrifice that the families made by donating us the brain tissue of their loved ones. This amazing gift is the ultimate display of humanity and love that

triumphs over the tragedy of their loss, offering hope to those sill waiting for the cure.

THANK YOU !!!

THE AUTISM RESEARCH OF THE MIRNICS LAB HAVE BEEN FULLY FUNDED BY THE VANDERBILT

UNIVERSITY KENNEDY CENTER STARTUP FUNDS.

Thank you, ATP.