Supplementary Table 2: Summary of gene expression profiling studies conducted on human biosamples from patients with PD
Study
Samples
Tissue
preparation
Cell sample
RNA quality
control
Platform
Zheng et al.1*
Middleton-1: 10 PD;
8 controls
Middleton-2: 10 PD;
10 controls
Middleton-3: 8 PD, 8
controls
Subclinical PD: 16
with subclinical Lewy
bodies; 17 controls
15 PD; 7 normal
controls ; 1 MS
Middleton-1/2:
Frozen
Middleton-3:
EBVtransformed
peripheral
lymphocytes
Middleton-1/2: LCM of dopaminergic
neurons from SNpc and neurons from
globus pallidus interna
Middleton-3: Lymphoblastoid cells
Subclinical PD: LCM of dopaminergic
neurons from SNpc
Electrophoresis
Frozen
Mixed-cell sample from lateral and
medial SN and the SFG
Electrophoresis
Middleton-1/2/3:
Affymetrix HG
U133 plus 2.0
(~55,000
transcripts)
Subclinical PD:
Illumina
HumanHT-12v3
(>25,000 genes)
Affymetrix HG
U133AB (~33,000
genes)
Vogt et al4
8 PD; 8 MSA; 8
normal controls
Frozen
Mixed-cell sample from putamen,
cerebellum and occipital cortex.
Electrophoresis
Affymetrix HG
U133A (~14,500
genes)
Hauser et al5
6 PD; 2 PSP; 1 FTD
with parkinsonism; 5
normal controls
15 PD with
dyskinesia; 16 PD
without dyskinesia,
32 normal controls
Frozen
Mixed-cell sample from SN and
surrounding midbrain
Affymetrix HG
U133A
Frozen
Mixed cell sample from putamen
3’:5’ ratio of <5
from the actual
microarray
Electrophoresis
15 PD; 15 normal
controls
Frozen
Mixed-cell sample from SN, putamen
and Brodmann area 9
Electrophoresis;
ribosomal ratio
≥1.5
Affymetrix HG
U133A
Moran et al3
Naydenov et al6
Zhang et al7
1
Affymetrix HG
U133A 2.0
(~38,500 genes)
Measure of
differential
expression (no. of
differentially
expressed genes in
disease versus
control)
Genes rank ordered
by their association
with PD
Enrichment
analysis
Validation
Gene set
enrichment
analysis2
QRT-PCR in
independent
samples;
overexpression of
PGC1α tested in
cell models of PD
T-test, P <0.001; FC
≥100%; BH multiple
test correction
(489↓ and 81↑)
Multiway ANOVA;
FWER controlling
multiple test correction
(40 in putamen, 88 in
cerebellum, 38 in
occipital cortex)
T-test, P <0.05
(142, majority
downregulated)
ANOVA and ANCOVA
with age and
postmortem interval as
covariates; BH
multiple test
correction; post-hoc ttest; P ≤0.05
One-way ANOVA; BH
multiple test correction
(329)
Percentage of
genes within
functional
categories
None
QRT-PCR
None
None
Fisher’s exact
test
None
Pathstat8
RT-PCR and semiquantitative RTPCR; ISH
QRT-PCR; IHC
CantutiCastelvetri et al9
8 PD; 8 normal
controls
Frozen
LCM of dopaminergic neurons from
SNpc
Electrophoresis
Affymetrix Human
X3P (~47,000
genes)
Simunovic et
al10
10 PD and 9
controls
Frozen
LCM of dopaminergic neurons of SNpc
Electrophoresis
Affymetrix HG
U133A
Stamper et al11
13 PD with
dementia; 15 PD
without dementia; 14
normal controls
50 PD; 55 normal
and disease controls
Frozen
LCM of layer V–VI pyramidal neurons
from posterior cingulate
Affymetrix HG
U133 plus 2.0
PAXgene tubes
Venous blood
5 idiopathic PD, 3
LRRK2 PD, 1
asymptomatic
LRRK2 mutation
carrier, 5 normal
controls
10 LRRK2 PD, 1
asymptomatic
LRRK2 mutation
carrier, 7 normal
controls, additional
sample from 40
pooled controls
12 idiopathic PD, 7
controls
Frozen
Mixed-cell sample from putamen
1% Tris-acetate
EDTA gel to check
range of transcript
sizes
Electrophoresis
and RNA 6000
Nanochip
Electrophoresis
Sodium citrate
tube
Monocytes removed from whole blood
Frozen
Elstner et al19
11 PD, 11 agematched controls, 8
young controls
Matigian et al 20
Mar et al21
Scherzer et al12
Botta-Orfila et al
13
Mutez et al17
Durrenberger et
al18
SAM 2.0 with FDR
<11% (102↓ and 186↑
in females; 232↓ and
60↑ in males)
Multiway ANOVA,
FDR 10% (465↓ and
580↑)
T-tests, P value <0.01;
FC >2 (21 cognitively
normal, 73 in PD with
dementia)
SAM with FDR 3%
(22)
Fisher’s exact
test
QRT-PCR
Modified Fisher’s
exact test
QRT-PCR
None
QRT-PCR from
different cells
isolated from the
same tissue
QRT-PCR
Affymetrix 1.0
Exon
Limma; FC >2; BH
multiple test correction
(232 in idiopathic PD)
DAVID
bioinformatics
resources14,15,
Fisher’s exact
test, Onto-tools16
QRT-PCR
Electrophoresis
Agilent Human
44K (~44,000
genes)
Welch t-test, P <0.05;
FC >2 (132)
Fisher’s exact
test
QRT-PCR
Mixed cell sample from SNpc
Electrophoresis
Student t-test, P
<0.01; Multiple test
correction
Gene set
enrichment
analysis2
QRT-PCR
Frozen
LCM of dopaminergic neurons of SNpc
Electrophoresis
Illumina
HumanRef-8 v2
BeadChip
(>22,000 probes)
Illumina WG6v1
BeadChip
(>20,000 genes)
Fisher’s exact
test; BH multiple
test correction
FDR 5%
None
19 PD, 9
schizophrenia and
14 controls
Neurosphere
preparation
from nasal
biopsy
Olfactory-neurosphere-derived cells
Electrophoresis
Illumina
HumanRef-8 v2
BeadChip
(>22,000 probes)
ANOVA, P <0.05; BH
multiple test correction
(750↓ and 435↑ in
PD;147↓ and
109↑with age)
Two-way ANOVA, P
<0.05; BH multiple test
correction (514)
QRT-PCR
13 PD, 9
schizophrenia and
Neurosphere
preparation
Olfactory neurosphere derived cells
Electrophoresis
Illumina
HumanRef-8 v2
Fisher’s exact
test,
DAVID
bioinformatics
resources14,15
GOstats (version
2.14.0), P <0.05
2
Affymetrix HG
U133A
Compared numbers of
genes with given
None
None
11 controls
from nasal
biopsy
Miller et al22
6 PD (6 SN,4
striatum), 8 controls
(8 SN, 4 striatum)
Frozen
Mixed-cell sample from SN and/or
striatum
Electrophoresis
Papapetrpoulos
et al23
22 PD, 23 controls
Frozen
28S:18S ratio
Shehadeh et
al24
17 PD, 11 controls
PAXgene tubes
Mixed-cell sample from SN, ventral
tegmental area, Brodmann area 35,
insular, amygdala, nucleus basalis,
caudate, putamen, nucleus
accumbens, globus pallidus,
mediodorsal thalamus, pulvinar,
subthalamic nucleus, trigeminal
nucleus, cerebellar hemisphere,
cerebellar vermis, dorsal raphe, locus
coeruleus, hypothalamus, reticular
formation, hippocampus
Venous blood
Bossers et al25
4 PD, 4 controls
Frozen
Mixed-cell sample from spared areas
of SN
Electrophoresis
Electrophoresis
BeadChip
(>22,000 targets)
and v3 (>25,000
targets)
CodeLink Human
UniSet 20 K
bioarray (20,469
probes)
Affymetrix HG
U133 plus 2.0
variability in disease
and control groups by
Chi-squared test, P
<0.001
Student t-test, P
<0.01; FC >1.5 (211↓
and 112↑ in SN; 1,036
in striatum)
One-way ANOVA, P
<0.05; FC >1.3
Affymetrix 1.0
Exon
Gene level: one-way
ANOVA, P <0.05; FC
>2 (58)
Exon level: splicing
ANOVA, P <0.05, BH
multiple testing
correction, filtered
splicing index 0.87
(218)
Limma, P <0.05.
Bonferroni multiple
test correction
None
QRT-PCR, IHC
None
QRT-PCR
GeneSpring GX
10
QRT-PCR
Agilent 22k 60mer
Functional Class
QRT-PCR
oligonucleotide
Scoring Method26
array (~22,5000
probe sets)
*Only studies not published elsewhere are described. Abbreviations: ANCOVA, analysis of covariance; ANOVA, analysis of variance; BH, Benjamini–Hochberg; DAVID, Database for Annotation, Visualization and
Integrated Discovery; EBV, Epstein–Barr virus; FC, fold change; FDR, false discovery rate; FTD, frontotemporal dementia; FWER, familywise error rate; IHC, immunohistochemistry; ISH, in situ hybridization; LCM, laser
capture microdissection; LRRK2, leucine-rich repeat kinase 2; MS, multiple sclerosis; MSA, multiple system atrophy; PD, Parkinson disease; PSP, progressive supranuclear palsy; QRT, quantitative reverse transcription;
RT, reverse transcription; SAM, significance analysis of microarrays; SFG, superior fusiform gyrus; SN, substantia nigra; SNpc, substantia nigra pars compacta.
References
1.
Zheng, B. et al. PGC-1α, a potential therapeutic target for early intervention in Parkinson’s disease. Sci. Transl. Med. 2, 52ra73 (2010).
2.
Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102,15545–15550 (2005).
3.
Moran, L. et al. Whole genome expression profiling of the medial and lateral substantia nigra in Parkinson’s disease. Neurogenetics 7, 1–11 (2006).
4.
Vogt, I. R. et al. Transcriptional changes in multiple system atrophy and Parkinson's disease putamen. Exp. Neurol. 199, 465–478 (2006).
3
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
Hauser, M. A. et al. Expression profiling of substantia nigra in Parkinson disease, progressive supranuclear palsy, and frontotemporal dementia with parkinsonism. Arch. Neurol. 62, 917–
921 (2005).
Naydenov, A., Vassoler, F., Luksik, A., Kaczmarska, J. & Konradi, C. Mitochondrial abnormalities in the putamen in Parkinson’s disease dyskinesia. Acta Neuropathol. 120, 623–631
(2010).
Zhang, Y., James, M., Middleton, F. A. & Davis, R. L. Transcriptional analysis of multiple brain regions in Parkinson's disease supports the involvement of specific protein processing,
energy metabolism, and signaling pathways, and suggests novel disease mechanisms. Am. J. Med. Genetics B Neuropsychiatr. Genet. 137B, 5–16 (2005).
Middleton, F. A. et al. Application of genomic technologies: DNA microarrays and metabolic profiling of obesity in the hypothalamus and in subcutaneous fat. Nutrition 20, 14–25 (2004).
Cantuti-Castelvetri, I. et al. Effects of gender on nigral gene expression and parkinson disease. Neurobiol. Dis. 26, 606–614 (2007).
Simunovic, F. et al. Gene expression profiling of substantia nigra dopamine neurons: further insights into Parkinson's disease pathology. Brain 132, 1795–1809 (2009).
Stamper, C. et al. Neuronal gene expression correlates of Parkinson's disease with dementia. Mov. Disord. 23, 1588–1595 (2008).
Scherzer C. R. et al. Molecular markers of early Parkinson's disease based on gene expression in blood. Proc. Natl Acad. Sci. USA 104, 955–960 (2007).
Botta-Orfila, T. et al. Microarray expression analysis in idiopathic and LRRK2-associated Parkinson's disease. Neurobiol. Dis. 45, 462–468 (2012).
Huang, D. W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2008).
Huang, D. W. et al. DAVID Bioinformatics Resources: expanded annotation database and novel algorithms to better extract biology from large gene lists. Nucleic Acids Res. 35, W169–
W175 (2007).
Khatri, P., Bhavsar, P., Bawa, G. & Draghici, S. Onto-Tools: an ensemble of web-accessible, ontology-based tools for the functional design and interpretation of high-throughput gene
expression experiments. Nucleic Acids Res. 32, W449–W456 (2004).
Mutez, E. et al. Transcriptional profile of Parkinson blood mononuclear cells with LRRK2 mutation. Neurobiol. Aging 32, 1839–1848 (2011).
Durrenberger, P. et al. Inflammatory pathways in Parkinson’s disease; a BNE microarray study. Parkinson’s Dis. 2012, 214714 (2012).
Elstner, M. et al. Expression analysis of dopaminergic neurons in Parkinson’s disease and aging links transcriptional dysregulation of energy metabolism to cell death. Acta Neuropathol.
122, 75–86 (2011).
Matigian, N. et al. Disease-specific, neurosphere-derived cells as models for brain disorders. Dis. Model. Mech. 3, 785–798 (2010).
Mar, J. C. et al. Variance of gene expression identifies altered network constraints in neurological disease. PLoS Genet. 7, e1002207 (2011).
Miller, R. M. et al. Robust dysregulation of gene expression in substantia nigra and striatum in Parkinson's disease. Neurobiol. Dis. 21, 305–313 (2006).
Papapetropoulos, S. et al. Multiregional gene expression profiling identifies MRPS6 as a possible candidate gene for Parkinson's disease. Gene Expr. 13, 205–215 (2006).
Shehadeh, L. A. et al. SRRM2, a potential blood biomarker revealing high alternative splicing in Parkinson’s disease. PLoS ONE 5, e9104 (2010).
Bossers, K. et al. Analysis of gene expression in Parkinson’s disease: possible involvement of neurotrophic support and axon guidance in dopaminergic cell death. Brain Pathol. 19, 91–
107 (2009).
Pavlidis, P., Qin, J., Arango, V., Mann, J. J. & Sibille, E. Using the Gene Ontology for microarray data mining: a comparison of methods and application to age effects in human prefrontal
cortex. Neurochem. Res. 29, 1213–1222 (2004).
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