Download - Vawter Functional Genomics Lab

PBMCs and LCLs in Complex
Neuropsychiatric Disorders
Marquis P. Vawter, Ph.D.
Functional Genomics Laboratory
Psychiatry and Human Behavior
University of California, Irvine

Objectives -- Biomarkers
Transformation of PBMCs (Peripheral Blood
Mononuclear Cells) Into LCLs (Lymphoblastic
Cell Lines) , Compare To Whole Blood
Correlation of PBMCs And Brain Expression
Klinefelter’s Syndrome, Cognition, and Gene
Expression In LCLs.
Trans-Regulatory Loci Stress Response in
LCLs

Psychiatric Illnesses And Microarray Gene
Expression
Psychiatric Disorder N PBMC/LCL/Whole Author Year
Panic Disorder 60 LCL Philibert et al. 2007
Nicotine Dependence 94 LCL Philibert et al. 2007
Bipolar Disorder 40
54
PBMC
PBMC
Konradi et al.
Glatt et al.
2007
2005
Klinefelter’s Syndrome 17 LCL Vawter et al. 2007
Alzheimer’s Disease 14
32
Schizophrenia And
Bipolar Disorder
74
76
118
PBMC
PBMC
Whole Blood
PBMC
LCL
Maes et al.
Kálmán et al.
Tsuang et al.
Middleton et al.
Iwamoto et al.
2006
2005
2005
2005
2005
Huntington’s Disease 83 Whole Blood Borovecki et al. 2005
Neurofibromatosis Type 1 /
Epilepsy/Tourette’s Syndrome
129 Whole Blood Tang et al. 2005
Down’s Syndrome 4 LCL Giannone et al. 2004
Schizophrenia 10
14
LCL
LCL
Vawter et al.
Vawter et al.
2006
2004

Advantage to cells and cell lines
• Less agonal artifacts
• Medication(s) might wash out in repeated passage, although epigenetic
effects may persist.
• Easier and more abundant access than brain
• Can be used in experimental research for repeated measures, such as doseresponse
titration.
• Variance in gene expression trait in cells may be linked to genetic loci
• Can be obtained repeatedly
• Expression might be more stable than Ficoll isolated PBMCs?
Some disadvantages to cell lines
• Not related to brain tissue
• Immortalized which might alter gene transcription machinery

Intrinsic interindividual differences in
gene expression patterns (PBMC).
PBMC: Whole Blood Comparison (Whitney et al., 2003)

Expression Whole Blood, PBMCs, LCLs?
Subject
1
2
3
4
5
6
7
8
Whole Blood
(Tempus,
ABI)
T
Pre Post
PBMC
(Ficoll)
F
Transformed
P0
Passage 1
P1
Passage 2
P2

QC-Affymetrix Exon Array
Positive vs. Negative ROC AUC : positive-negative controls
Gender T F P0 P1 P2
F 0.862 0.9 0.9 0.9 0.9
F 0.871 0.9 0.9 0.9 0.9
F 0.867 0.9 0.9 0.9 0.9
F 0.859 0.9 0.9 0.9 0.9
Mean 0.865 0.902 0.902 0.905 0.903
SD 0.006 0.007 0.004 0.004 0.006
M 0.873 0.9 0.9 0.9 0.9
M 0.869 0.9 0.9 NA NA
M 0.881 0.9 0.9 0.9 0.9
M 0.866 0.9 0.9 0.9 0.9
Mean 0.872 0.891 0.897 0.879 0.895
SD 0.007 0.010 0.002 0.007 0.004
p-value 0.140 0.099 0.083 0.002 0.119
Grand mean 0.869 0.896 0.899 0.894 0.900
SD 0.007 0.010 0.004 0.014 0.006

Covariance PCA

Expression Differences RefSeq
Transcripts Between Preparations
Variable Name Cutoff Value # of Significant p-values
PREPARATION 8.10E-06 7358
PBMC- P0 4.63E-06 3973
PBMC – P1 4.52E-06 3798
PBMC – P2 4.72E-06 4105
PBMC - WB 3.71E-06 2341
P0 - P1 2.80E-06 14
P0 – P2 2.81E-06 43
P1 – P2 2.80E-06 5
P0, P1, P2 -- Passage 0, 1, 2
PBMC -- Peripheral Blood Mononuclear Cells
WB -- Whole Blood processed in Tempus Tubes
Whitney et al (2003)
reported “~2,000 transcripts
for this PBMC:WB
comparison with at least a
2-fold average difference. “

Transformation (Effect Across Transcripts)

Expected Gender Difference Across 5
RPS4Y1
Preparations

Genome Wide View Of Subject
(Gender effect), < 10-6

Highly Significant Expression
Difference Across Preparations
CPVL carboxypeptidase, vitellogenic-like
“Using differential display PCR,
we cloned a novel
carboxypeptidase expressed in
human macrophages but not in
other leukocytes.” 2001 Mar
15;72(3):243-51. Cloning and
characterization of CPVL, a novel
serine carboxypeptidase, from
human macrophages. Mahoney
JA, et al.

PBMC expression compared to LCLs
from same subjects.
Transcript differences across genome (pvalue
< 10 -6 ).

PBMC expression compared to
LCLs from same subjects.
Test chromosomal over-rep of
results that pass 10^6.

Over-representation by cytogenetic
band
Cytoband Count % Corrected P-value Differentially Expressed Genes In Cytoband
19q13.4 11 1.34% 1.31E-04 FPR1, LILRA5, LILRA1, LILRB2, LILRA2,
LILRA3, LILRA4, LILRB3, LILRB1, LAIR1,
LILRA6
2p12 6 0.73% 3.82E-04 IGKC, CD8A, CD8B, IGKV1-5, EIF2AK3,
IGKV1D-13
7q34 6 0.73% 2.20E-02 TRBV5-4, TRBV21-1, TRBC1, TRBV19,
TRBV4-1, TRBV3-1
1p31 4 0.49% 3.32E-02 MSH4, ACADM, RABGGTB, AK5
1q23 4 0.49% 3.90E-02 FCGR2A, NDUFS2, FCER1G, FCGR3A
1p31.1 4 0.49% 3.90E-02 IFI44, DNAJB4, CTH, IFI44L
2q35 5 0.61% 5.46E-02 CTDSP1, DSU, SLC11A1, IL8RB, IL8RA

EBI3 (Epstein Barr Virus Induced Gene 3)
Transformed
(P0,P1,P2)
Non Transformed
(Whole Blood, Ficoll WBC)

EBV Induced Gene 6

SH2D1A Expression Repressed By
EBV

Q-PCR Validation Relative to PBMC
Whole Blood
Tempus
Transformed
PBMCs
Passage 1 Passage 2
CCL22
Fold Change 9.8 3,774,507 2,404,824 1,504,355
(p)# 0.00064215 0.00000003 0.00000007 0.00000510
HCK
Fold Change 1.48 0.07 0.06 0.05
(p) 0.2209 0.0019 0.0019 0.0026
SH2D1A
Fold Change 0.90 0.31 0.37 0.15
(p) 0.1088 0.0178 0.0907 0.0157
TRAF1
Fold Change 1.75 45.19 44.63 47.53
(p) 0.0799 0.0001 0.0005 0.0007

Transformation Induces Consistent
Expression Changes Across Subjects

Which Preparations To Study
• PBMCs were isolated within 2 hr of blood draw
• Majority of PBMCs viable (Trypan blue )
• WB drawn into Tempus tubes was immediately „stopped‟
These findings show that a large difference in transcription
profiling occurs due to the isolation of PBMCs.
Somewhat surprising was the observation that once cells are
transformed, further passages of transformed cell lines at
confluency did not induce significant differential gene expression.
PBMC -- Peripheral Blood Mononuclear Cells
WB -- Whole Blood processed in Tempus Tubes

Brain-Lymphocyte Potential
Biomarkers

Brain-Lymphocyte
Potential Biomarkers
A B

Tissue Diagnosis
Age
(yrs)
Gender
Brain-Lymphocyte
Potential Biomarkers
Agilent
Bioanalyser
28S/18S Ratio
OD 260/
280
Yield (microgram / mg) or
(microgram / 10^6 cells)
Array Type
Cerebellum Control 67 M 2.8 1.50 69.0 EXON
Lymphocyte Control 67 M 1.8 1.55 7.2 EXON
Cerebellum
Lymphocyte
Major
Depressive
Disorder
Major
Depressive
Disorder
77 M 2.8
77 M 1.9
1.72 31.9
1.61 17.5
Cerebellum Control 72 M 2.6 1.54 67.2 EXON
Lymphocyte Control 72 M 1.9 1.75 8.0 EXON
Cerebellum
Lymphocyte
Bipolar
Disorder
Bipolar
Disorder
51 F 2.5
51 F 1.6
1.74 31.7
1.61 4.4
Cerebellum Schizophrenia 52 M 2.3 1.76 56.9 U133P
Lymphocyte Schizophrenia 52 M 1.3 1.51 2.6 U133P
Cerebellum Schizophrenia 69 M 1.2 1.81 57.3 U133P
Lymphocyte Schizophrenia 69 M NA 1.74 18.9 U133P
Cerebellum Control 55 M 0.6 1.76 57.7 U133P
Lymphocyte Control 55 M NA 1.67 13.9 U133P
EXON
EXON
EXON
EXON

Brain-Lymphocyte
Potential Biomarkers
The same array type (Affymetrix Human 1.0 ST Exon Array) was used
to compare 4 matched brain and PBMC pairs of samples from the
same subjects.
For a separate comparison of brain and lymphocytes, 3 postmortem
donor subjects were additionally matched for brain and PBMC pairs
and analysed on the Affymetrix U133 Plus chips .
The overlap between both platforms for the two independent brain and
PBMC experiments was evaluated and reported.

Cerebellum-Lymphocyte Gene Summary For
18,560 RefSeq Transcripts

Filtered List Of 6,570 Refseq Transcripts Showing
Brain-Lymphocyte Coexpression
Filtering criteria were mean fold change within ± 1.4 , the mean tissue comparison
was not significant following a Bonferroni correction, and mean signal greater than
4 (log 2) in both tissues.

Biomarker— Working Definition
A. Expressed in WB, PBMC, LCL (not different among 5
preparations, 10 post-hoc comparisons, and above
background). 8,448
B. Expressed in PBMCs and brain from same matched
subjects , experiment repeated twice with different
platform and subjects. 4,103
The intersection of A+B is 2,124.

Hit Rate On Putative Biomarkers
Gene Brain Expression From Allen Brain Atlas
ACTL7B Very low expression in olfactory bulb.
ANKRD49 Widely expressed throughout brain, including cortex, hippocampal CA1-CA3, hypothalamus
and olfactory bulb.
ATF6 Extremely widely expressed throughout entire brain. Expressed in the cortex, hippocampus,
olfactory bulb, cerebellum, thalamus, hypothalamus, amygdale, basal ganglia.
HOXB1 Expressed in cortex, amygdala, basal ganglia, hippocampus, cerebellum.
KRT24 Expressed in cerebellum, cortex, basal ganglia, hippocampus, olfactory bulb.
RDH13 Not included in Allen Brain Atlas.
RPAP1 Expressed in cortex, cerebellum, hippocampus, olfactory bulb.
SERPINA6 Expressed widely throughout cortex, amygdala, caudate putamen, thalamus, cerebellum.
SRPX2 Expressed throughout cortex, amygdala, cerebellum, Moderate expression in CA1-3 and
dentate gyrus.
TNFAIP8L1 Very sparse, low expression in cortex and olfactory bulb (may be background). Appears to
outline cortex and olfactory bulb, possibly labeling meninges.
Random drawing of 10 genes from the biomarker list showed 7 / 9
transcripts widely expressed in brain, 2 sparse or low, 1 not
found.

Presenilin Expression In Brain and
Lymphocytes

Dysregulation Of X-linked Gene
Expression In Klinefelter’s Syndrome
And Association With Verbal Cognition
Klinefelter's syndrome --
• 47,XXY chromosomal pattern
• Associated with physical abnormalities, cognitive
impairments, and psychiatric pathology.
• May be a neuropsychiatric genetic model for
dysregulation involving X chromosome

Study Design XXY Gene Expression
Klinefelter's syndrome --
• The dysregulation involving X chromosome was the
target of our investigation of gene expression
• We studied 11 males (47, XXY confirmed
karyotypes) and compared to 7 XY males.
• LCLs (Lynn DeLisi, M.D.) and Trizol RNA extracts.
• U133Plus 2.0 chips.

Dysregulation Of X-linked Gene
Expression In Klinefelter’s Syndrome
Found gene expression differernces between XXY
and XY males (passed Benjamini Hochberg FDR,
Partek) for 128 ENSEMBL transcripts.
• The X-chromosome was over-represented.
• Correlated 128 transcripts with Verbal Cognitive
psychometric testing conducted by P. Harvey, Ph.D.
at Mt Sinai School of Medicine.

Genes Dysregulated X-linked
Fold change *Number of
Cytoband Symbol P-value (Klinefelter's/control) brain regions
Xq13.2 XIST 6.28E-15 136.53 3
Xq13.2 XIST 4.88E-15 79.06 3
Xp21.3 CXorf21 3.08E-07 2.62 17
Xq28 ZNF275 4.54E-08 2.02 13
Xp22.32 DXYS155E 2.49E-06 1.95 8
Xp11.22-p11.21 SMCX 1.85E-06 1.81 5
Xp21.3 ZFX 3.57E-09 1.81 5
Xp22.33/Yp11.32 GTPBP6 2.59E-06 1.65 8
Xq28 MECP2 3.07E-07 1.36 1
Xq13.1 RPS4X 6.83E-07 1.18 10
Xp11.23-p11.22 SLC35A2 2.66E-07 0.8 16
Xp11.23-p11.22 SLC35A2 1.50E-07 0.78 16
Xp22.2-p22.1 YY2 1.25E-07 0.72 6
Xp22.1-p22.2 MBTPS2 1.93E-08 0.54 7
Xq13 PGK1 2.11E-06 0.29 2
Xq13.1-q21.1 TAF9L 2.45 E-06 0.56 2
* Number of brain regions above median expression (Novartis SymAtlas)

XIST Expressed At High Levels In XXY
Previously reported XIST reference
Individuals

Gene XXY (ave
delta Ct)
QPCR Microarray
XY (ave
delta Ct)
Fold
change
t-test (Pvalue)
Gene XXY ave XY ave FC t-test (Pvalue)
XIST -2.29 11.03 10231.0 0.00006 XIST 4.77 -2.33 136.53 6.28E-15
ZNF275 -1.65 -0.23 2.68 0.00031 ZNF275 2.90 1.89 2.02 4.53E-08
CXorf21 -0.90 0.14 2.06 0.05957 CXorf21 1.07 -0.32 2.62 3.08E-07
SMCX 0.24 1.27 2.05 0.04849 SMCX 1.47 0.61 1.81 1.85E-06
GTBP6 2.06 3.03 1.96 0.00716 GTBP6 0.00 -0.38 1.65 2.59E-06
MECP2 2.05 2.06 1.00 0.99409 MECP2 0.70 0.26 1.36 3.07E-07
RECQL -0.62 -0.86 0.84 0.46164 RECQL -0.50 0.01 0.46 7.18E-07
SLC35A2 3.17 2.29 0.55 0.03687 SLC35A2 -1.18 -0.83 0.78 1.50E-07
TAF9L 3.53 2.66 0.55 0.09789 TAF9L -0.83 0.01 0.56 2.45E-06
MBTPS2 4.81 3.19 0.32 0.00040 MBTPS2 -0.89 0.01 0.54 1.93E-08
Q-PCR Study Results—Brandi Rollins

Vawter et al., 2004
Human XIST DLPFC
ISH and PCR

Verbal Cognition Correlations
Gene symbol CHSY1
DKFZP564O2
43 GTPBP6 PKMYT1 ADPGK RECQL TAF9L DNAJA2 AMD1 TCF8
Klinefelter/control 1.84 1.77 1.65 1.31 1.27 0.46 0.56 0.64 0.66 0.79
FC #
Verbal IQ 0.14 0.38 -0.85 -0.64 -0.60 -0.52 0.02 -0.03 -0.26 0.04
Receptive
language
Token test 0.52 0.46 -0.29 -0.04 -0.02 -0.70 -0.45 -0.33 -0.51 -0.30
Peabody PVT 0.53 0.63 -0.76 -0.24 -0.28 -0.73 -0.44 -0.34 -0.55 -0.26
Expressive
language
Boston naming
test
0.53 0.63 -0.56 -0.08 0.08 -0.80 -0.71 -0.62 -.32 -0.23
COWA
Animal 0.03 0.05 -0.50 -0.08 0.14 -0.39 -0.36 -0.53 -0.16 -.0.10
Vegetable 0.63 0.45 -0.60 -0.16 -0.17 -0.48 -0.34 -0.11 -0.39 -0.12
Fruit 0.74 0.45 -0.43 -0.23 -0.09 -0.63 -0.49 -0.18 -0.44 -0.07
Similarities -0.19 0.25 -0.62 -0.64 -0.71 -0.22 0.36 0.17 -0.05 0.10
Phonological 0.24 0.47 -0.53 -0.48 -0.51 -0.61 -0.07 -0.07 0.09 0.17
LOGMEM1 0.07 0.63 -0.14 0.04 0.16 -0.63 -0.35 -0.42 -0.37 -0.63
LOGMEM2 0.07 0.50 -0.12 0.22 0.43 -0.59 -0.47 -0.57 -0.53 -0.78
Sentence
Repetition
0.68 0.48 -0.27 0.09 0.11 -0.79 -0.59 -0.40 -0.68 -0.54
Trails A -0.20 -0.32 0.58 0.04 0.22 0.42 0.36 0.43 0.26 0.09
Trails B
Academic skills
-0.11 0.04 0.27 -0.12 -0.38 0.44 0.52 0.45 0.64 0.47
WRAT 0.51 0.08 -0.60 -0.13 -0.19 -0.57 -0.36 -0.32 -0.23 0.02

GTPBP6 Expression
In Lymphoblast (cytes)

Trans-Regulatory Loci Implicated
with Gene Expression Stress
Response in Lymphoblastic Cell
Lines (LCLs)

Subjects
Diagnosis Pair Relationship
Schizophrenia 1 Siblings
Control 1 Siblings
Schizophrenia 2 Siblings (all four)
Control 2 Siblings (all four)
Schizophrenia 3 Siblings (all four)
Control 3 Siblings (all four)
Schizophrenia 4 Uncle
Control 4 Nephew
Schizophrenia 5 Cousins
Control 5 Cousins

Diagnosis and Glucose Effects
• Diagnosis 0
• Glucose 1
• Diagnosis * Exon 72
• Glucose * Exon 227
• Glucose * Diagnosis 1
•

E-QTL Trans Hotspots No Overlap
With Glucose Deprivation
Marker # of Transcripts
Normal D11S928 14
Glucose D15S158 10
D15S816 10
D15S1004 10
D9S1004 10
D1S2667 9
D3S1266 9
D3S3547 9
D12S2081 9
Glucose D3S1607 10
Deprivation

Top 10 Exon * Diagnosis Interaction Effects
Affymetrix ID p-value RefSeq Symbol

DSC2 Expression Exon * Diagnosis

DPM2 Exon Expression in 10
Lymphocyte Samples
DPM2 Exon Expression
100 Fold Range Between Subjects
DPM2 (dolichyl-phosphate mannosyltransferase polypeptide 2, regulatory
subunit, # NM_003863.2)

Relative Expression
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Evidence for Allelic Specific
Hybridization of DPM2
Affy probe
7997- C
7997 - G
Exon 4
GG GG GG CG CG CC CC CC CC CC
Genotype

Summary Of LCL Schizophrenia Functional
Genomics Results
• Exon arrays were useful in a case – control design for
detection of expression differences in lymphoblastic cell
lines:
Detected exon expression differences in schizophrenia
for DSC2 that were validated by QPCR.
Expression changes were not related to hybridization
artifacts showing the reliability and accuracy of exon
arrays.
• Detected SNP genotypes on exon array.
• Detected allelic specific expression on exon array in
DPM2.
• Putative state-dependent eQTLs identified.

Summary
• Circulating levels of endogenous cell types and levels of EBV
affect the transcript profile.
• Other potential viral infections may be present in
PBMCs/Whole Blood.
• Appropriate controls for infections are needed to rule out
these causative factors.
• Other variables show large effects on profiles, e.g. circadian
and gender.

Conclusions
• Whole Blood and PBMCs And LCLs Present Biomarker
Opportunities in Psychiatric Disorders (and others).
• Large scale biomarker studies of normal controls are
required.
• Transcript profiling may distinguish healthy controls from
psychiatric patients.
• A subset of circulating mRNA transcripts may reflect
brain expression, although animal experiments are needed
to determine false positives.
• The expression levels of mRNA transcripts may be
related to brain functions.
• Further lymphocyte and brain work are needed.

Collaborative Studies For
Lymphocyte Biomarkers –
Functional Genomics Laboratory
Circadian rhythm – controls
Sleep deprivation – controls and bipolar disorder
Brain Imaging – controls and schizophrenia
Neuropsychological performance – controls
Genetics and functional genomics – bipolar disorder
Animal studies – blood and brain comparisons
Samples and / or data and collection completed.
Proposal awaiting regulatory approval or funding.

Acknolwedgements - XXY Study
Brandi Rollins, University of California, Department of
Psychiatry and Behavior for performing real-time PCR
experiments.
The Klinefelters project was funded by discretionary
funds allotted to Dr. DeLisi within the Mental Illness
Prevention Center of the Department of Psychiatry, New
York University School of Medicine.
Dr. Marquis P. Vawter research was supported by NIMH
RMH074307A award for Biomarkers in Neuropsychiatric
Disorders, and the William Lion Penzner Foundation.

Acknolwedgements – Other Studies
Jacque Berndt and the investigators and medical
examiners at the Orange County Coroner’s Office .
Dr. Preston Cartagena, Kathleen Burke and Claudia in
processing blood and brain samples.
F. Warren Lovell, M.D, performed a neuropathological
evaluation of the postmortem brains.
The procurement of brain samples supported by the
NIMH Conte Center Grant P50 MH60398 and the Pritzker
Family Philanthropic Fund.
Biomarker investigation was supported by Public Health
Service research grant M01 RR00827 from the National
Center for Research Resource, Biomarker research grant
MH 024370 from the National Institute of Mental Health,
and the William Lion Penzner Foundation.

Future Studies Based Upon
Klinefelter’s Results
Dan Rujescu, M.D. is currently testing for a genotype
association of GTPBP6 with Verbal Cognition in Normal
Controls at the Munich Genetics Research Centre.
GTPBP6 is proximal in the PAR to genes associated with
psychosis, and near a translocation site associated with
psychosis.
Lynn DeLisi, M.D. is currently seeking additional funding for
further study of XXY individuals, XX and XY controls, in whole
blood or PBMCs.