Identification of NFT-associated genes, Miyashita A., et al. / 04:33 / February 12, 16
SUPPLEMENTARY INFORMATION
SUPPLEMENTARY METHODS
RT-qPCR
The first strand cDNA was synthesized from 200 ng of total RNA with High-Capacity
RNA-to-cDNA Master Mix (Applied Biosystems®) or a Superscript® VILOTM cDNA
Synthesis Kit (Life TechnologiesTM) in a total volume of 20 µl. After dilution of the
synthesized cDNA solution with distilled water, samples were subjected to RT-qPCR
amplification with a TaqMan® Gene Expression Assay (Life TechnologiesTM) on an ABI
PRISM 7900 HT instrument (Applied Biosystems®). In a total reaction volume of 10 µl
in the wells of a 384-well optical PCR plate, 2.5 µl of a diluted cDNA solution was
mixed with 5.0 µl of 2 x TaqMan® Universal PCR Master Mix II (Life TechnologiesTM),
0.5 µl of 20 x a TaqMan® Gene Expression Assay and 2.0 µl of distilled water. The
TaqMan® assays used were as follows: Hs03006670_m1 for DCHS2, Hs00610307_m1
for
GRB14,
Hs00218921_m1
for
MYO5C,
Hs00698866_m1
for
NPAS4,
Hs00288878_m1 for PHYHD1, Hs00153133_m1 for PTGS2, Hs01022646_m1 for
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Identification of NFT-associated genes, Miyashita A., et al. / 04:33 / February 12, 16
RELN, and Hs04188203_s1 for TRIL as target genes, and Hs00201226_m1 for CASC3,
Hs00153277_m1 for CDKN1B, Hs99999908_m1 for GUSB, Hs00183533_m1 for IPO8,
and Hs00734303_g1 for RPS17 as endogenous control genes. Each sample was
analyzed in duplicate, the values being used for statistical analysis after being averaged.
Relative gene expression levels were calculated as the cycle difference by means of the
delta-delta Ct (ddCt) method.1,2
For RT-qPCR analysis, five endogenous control genes, GUSB, IPO8,
CDKN1B, RPS17 and CASC3, were selected from among 33 housekeeping genes.
Thirty-two of these genes were contained in a TaqMan® Express Human Endogenous
Control Plate (Applied Biosystems®), and the other one, ANKFY1 (TaqMan® assay ID,
Hs00212598_m1), was discovered in our previous studies as a ubiquitously expressed
gene in mouse3 and man4. To evaluate the 33 genes by means of RT-qPCR analysis,
total RNA from the entorhinal (EC), temporal (TC), and frontal (FC) cortices of six
brain-donor subjects included in the ROW set (Supplementary Tables S1 and S8) was
utilized. For every brain region, we chose the gene exhibiting the lowest variation, as
judged from the standard deviation (SD), within the six subjects: IPO8 in EC (SD,
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Identification of NFT-associated genes, Miyashita A., et al. / 04:33 / February 12, 16
0.1676), CDKN1B in TC (SD, 0.1667), and RPS17 in FC (SD, 0.0978). Concerning FC,
CASC3 (SD, 0.1055) ranked second was additionally selected and used. GUSB was
commonly used across the three brain regions, EC, TC and FC, since this gene had
previously been shown to be stably expressed in postmortem human brain tissues.5
SUPPLEMENTARY REFERENCES
1.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using
real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;
25: 402-408.
2.
Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T)
method. Nat Protoc 2008; 3: 1101-1108.
3.
Ito K, Ishii N, Miyashita A, Tominaga K, Kuriyama H, Maruyama H et al.
Molecular cloning of a novel 130-kDa cytoplasmic protein, Ankhzn, containing
Ankyrin repeats hooked to a zinc finger motif. Biochem Biophys Res Commun
3
Identification of NFT-associated genes, Miyashita A., et al. / 04:33 / February 12, 16
1999; 257 : 206-213.
4.
Kuriyama H, Asakawa S, Minoshima S, Maruyama H, Ishii N, Ito K et al.
Characterization and chromosomal mapping of a novel human gene, ANKHZN.
Gene 2000; 253 : 151-160.
5.
Kuwano R, Miyashita A, Arai H, Asada T, Imagawa M, Shoji M et al.
Dynamin-binding protein gene on chromosome 10q is associated with late-onset
Alzheimer’s disease. Hum Mol Genet 2006; 15: 2170-2182.
SUPPLEMENTARY FIGURE LEGENDS
Supplementary Figure 1. Analytical flowchart. We initially surveyed transcripts at the
genome-wide level using exon arrays with the ROW set comprising 71 brain-donor
subjects (Supplementary Tables S1 and S8). Three brain regions, the entorhinal (EC),
temporal (TC) and frontal (FC) cortices, were utilized per subject in this sample set.
Two-type comparisons, Com-NFT(BR) and Com-BR(NFT), were performed, and genes
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Identification of NFT-associated genes, Miyashita A., et al. / 04:33 / February 12, 16
showing both alt-splicing ANOVA PFDR < 5.00E-02 and fold change > |1.5| were
preferentially chosen for subsequent analysis. Then, we examinded whether or not the
expression levels of genes identified through Com-NFT(BR) reproducibly exhibit
associations with Braak NFT stages on real-time quantitative PCR (RT-qPCR) analysis
with TaqMan expression assays in the same sample set, ROW. Using the NP set (brain
region, FC) comprising control subjects (N=30) and patients with AD (N=37), DLB
(N=17), and PD (N=36) (Supplementary Table S7), we further assessed the
disease-specificity of the genes verified by means of the RT-qPCR analysis. BR, brain
region.
Supplementary Figure 2. Expression levels of neural cell-type specific genes. We here
focused on three genes, AQP4, GFAP and S100B, for astrocytes (A, E, I), four, AIF1,
CD68, EMR1 and LGALS3, for microglia (B, F, J), four, MAG, MBP, MOG and SOX10,
for oligodendrocytes (C, G, K), and seven, CHGA, ENO2, NEFL, NEFM, NEFH,
SNAP25 and SYT1, for neurons (D, H, L) in exon array data obtained with the ROW set
(Supplementary Table S1). The mean expression levels of these genes are plotted for
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Identification of NFT-associated genes, Miyashita A., et al. / 04:33 / February 12, 16
every Braak NFT stage group. The numbers of subjects included in each Braak NFT
stage group are given in parentheses. A – D in Com-NFT(E), E – H in Com-NFT(T),
and I – L in Com-NFT(F). PFDR and fold changes are shown in Supplementary Table
S4.
Supplementary Figure 3. Transcript views of eight genes, RELN, PTGS2, MYO5C,
TRIL, DCHS2, GRB14, NPAS4 and PHYHD1, comparing four Braak NFT stage groups,
0, I-II, III-IV and V-VI, in three brain regions, EC, TC and FC: Com-NFT(BR). The
views of genes satisfying both PFDR < 5.00E-02 and fold change > |1.5| are boxed in red.
Significant PFDR values are depicted in boldface type. A, RELN in Com-NFT(E); B,
RELN in Com-NFT(T); C, RELN in Com-NFT(F); D, PTGS2 in Com-NFT(E); E,
PTGS2 in Com-NFT(T); F, PTGS2 in Com-NFT(F); G, MYO5C in Com-NFT(E); H,
MYO5C in Com-NFT(T); I, MYO5C in Com-NFT(F); J, TRIL in Com-NFT(E); K,
TRIL in Com-NFT(T); L, TRIL in Com-NFT(F); M, DCHS2 in Com-NFT(E); N,
DCHS2 in Com-NFT(T); O, DCHS2 in Com-NFT(F); P, GRB14 in Com-NFT(E); Q,
GRB14 in Com-NFT(T); R, GRB14 in Com-NFT(F); S, NPAS4 in Com-NFT(E); T,
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Identification of NFT-associated genes, Miyashita A., et al. / 04:33 / February 12, 16
NPAS4 in Com-NFT(T); U, NPAS4 in Com-NFT(F); V, PHYHD1 in Com-NFT(E); W,
PHYHD1 in Com-NFT(T); and X, PHYHD1 in Com-NFT(F).
Supplementary Figure 4. Correlation of expression levels between two genes included
in Clusters-1 (RELN and GRB14), 2 (NPAS4 and PTGS2), and 4 (MYO5C, PHYHD1
and TRIL) in three brain regions, EC, TC and FC. A-C, RELN (X-axis) vs. GRB14
(Y-axis) in Cluster-1; D-F, NPAS4 (X-axis) vs. PTGS2 (Y-axis) in Cluster-2; G-I,
MYO5C (X-axis) vs. PHYHD1 (Y-axis) in Cluster-4; J-L, MYO5C (X-axis) vs. TRIL
(Y-axis) in Cluster-4; and M-O, PHYHD1 (X-axis) vs. TRIL (Y-axis) in Cluster-4.
Pearson’s correlation coefficient, R, was calculated. Dotted lines indicate the 95%
confidence intervals for the correlation coefficients. P values exhibiting < 5.00E-02 are
depicted in boldface type.
Supplementary Figure 5. Overlapping of genes identified in Com-BR(NFT). The
sections of the Venn diagram are designated as Sec-A ~ Sec-O. The number of genes
included in each section is shown.
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Identification of NFT-associated genes, Miyashita A., et al. / 04:33 / February 12, 16
Supplementary Figure 6. Comparison of the expression levels of three genes, MYO5C,
GRB14, and PHYHD1, among four Braak NFT stage groups, 0, I-II, III-IV and V-VI.
For this analysis, we used TaqMan-based RT-qPCR data obtained with the ROW set
(Supplementary Tables S1 and S8). Box-and-whisker plots show the distributions of
the expression levels of these genes in every Braak NFT stage group: upper horizontal
line of box, 75th percentile; lower horizontal line of box, 25th percentile; horizontal bar
within box, median; upper horizontal bar outside box, 90th percentile; lower horizontal
bar outside box, 10th percentile; dot, outlier; plus, mean. For four-group comparison (0
vs. I-II vs. III-IV vs. V-VI) for each endogenous control gene, the Kruskal-Wallis test
was performed. When statistical significance (PKW < 5.00E-02, depicted in boldface
type) was observed, Dunn’s multiple comparison test was applied as a post hoc test.
Braak NFT stage 0 was used as a reference group for this test: multiplicity-adjusted
P-values were computed (*, P < 5.00E-02; and **, P < 1.00E-02). The numbers of
subjects included in each Braak NFT stage group are shown in parentheses. A, MYO5C
in EC; B, GRB14 in EC; and C, PHYHD1 in TC. NS, not significant; PKW, P-value
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Identification of NFT-associated genes, Miyashita A., et al. / 04:33 / February 12, 16
computed by means of the Kruskal-Wallis test.
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