Supplement S1: Material and methods
1. Subjects
The BD patient group consisted of 41 % males and 59 % females. The SZ patient group
consisted of 64.9 % males and 35.1 % females and a control group of 51.4 % female and
48.6% male participants. Clinical characterization of the patients included the MiniInternational Neuropsychiatric Interview (MINI1), the Diagnostic Interview for Genetic
Studies (DIGS2), the Family Interview for Genetic Studies (FIGS3) and the Schedules for
Clinical Assessment in Neuropsychiatry (SCAN4), a highly reliable and valid method for
DSM-IV diagnoses5. The final diagnoses were made according to the DSMIV-TR
criteria6 and determined by consensus of 2 research psychiatrists. Only the patients where
consensus was reached were included in the study. The unrelated Swedish control
individuals, consisting of a large population-based sample representative of the general
population of the region, were randomly selected from the ‘Betula study’7, 8.
The population stratification of the association sample was analyzed with free software
package STRUCTURE (http://pritch.bsd.uchicago.edu/structure.html) using 37 short
tandem repeat markers. No population substructure was observed.
2. Gene selection
References for the ‘in silico’ miRNA selection:
Linkage/association evidence: 9, 10, 11, 12, 13, 14
Expression in mammalian brain evidence: 15, 16, 17, 18,19, 20, 21, 22, 23, 24
3. Sanger sequencing based variant discovery
10 ng of DNA, 1.5 µl 10X PCR buffer (Titatinum taq buffer, Clonetech, California,
USA), 0.15 µl of 25 mM dNTPs (Invitrogen, Life Technology, NY, USA), 0.075 µl of
100µM forward and reverse primer (IDT, Integrated DNA Technologies Inc, CA, USA)
and 0.075 µl of Titanium Taq (Clonetech, CA, USA) were used in each 15 µl PCR
amplification. For some of the reactions 1 M betaine (Thermo Scientific, DE, USA) was
used (when used, it is listed next to the melting temperature Tm of the amplicons). The
PCR conditions were as follows: initial denaturation step for 3 min at 95 °C, followed by
35 cycles of [30sec at 95 °C, 30 sec at Tm and 45 sec at 72 °C] and the final elongation
step for 5 min at 72 °C on Veriti thermal cycles (Applied Biosystems, Life technologies,
CA, USA).
Primers for sequencing miRNA (in brackets are melting temperatures Tm for each primer
pair and if used, concentration of betaine):
MIR34A-F: CAAACTTCTCCCAGCCAAAA
MIR34A-R: CTTTCCTCCCCACATTTCCT (Tm-63 °C, 1 M betaine)
MIR99B-F: TTCTATCAGGCCATGCCTTC
MIR99B-R: TGATCTCCTTGGGTGTCCTC (Tm-62 °C, 1 M betaine)
MIR103A1 and MIR103B1-F: AAAGCGTACTTCCCAATCCA
MIR103A1 and MIR103B1-R: CACAACCTAAATCCCTTGAGGA (Tm-58 °C)
MIR128-1-F: ATACTGTGAAGTACACTGCATATAAGG
MIR128-1-R: GCCAAAGATGTCACTTAAATTCT (Tm- 58 °C)
MIR132-F: CACGTGGGATCTTGACTCG
MIR132-R: GGCACCTTGGCTCTAGACTG (Tm-62 °C, 1 M betaine)
MIR135B-F: CCGATCCCAGGTTACCAGAT
MIR135B -R: CAAGAACTGGAAACTCATTACTGG (Tm-63 °C, 1 M betaine)
MIR137-F: CTTTCCGGTGGAACCAGTG
MIR137-R: GCACAGCTTTGGATCCTTCT (Tm-63 °C)
MIR301A-F: TCCAGACGTGTTTCATAATGC
MIR301A -R: TCATCAATAAGCAACATCACTTTG (Tm-63 °C, 1 M betaine)
MIR448-F: AGGCCAGAAGAGGCTTTCAT
MIR448-R: CAGATCTACTGGCCCTGAGC (Tm-62 °C)
MIR764-F: GGAGGAACTTGGTTTTTAAAGGA
MIR764-R: CAGTCCCTTTACCGCTGTTT (Tm-62 °C)
MIRLET7A2-F: AACCCGAGGAAACAGAATATGA
MIRLET7A2-R: TTGCTCCCTTCATGTTTTCA (Tm-58 °C)
MIRLET7E-F: ACCCGTAGAACCGACCTTG
MIRLET7E-R: GGAAAACAGATTTCAGGGGAAG (Tm-63 °C, 1M betaine)
PCR products were treated with ExoSAP-IT (GE Healthcare, Chalfont St. Giles, UK) and
sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit, according to the
instructions of the manufacturers (Applied Biosystems, Life technologies, CA, USA).
Sequencing ran for 25 cycles: 10 sec at 96°C, 5 sec at 50°C and 4 min at 60°C on Veriti
thermal cyclers (Applied Biosystems, Life technologies, CA, USA). Sequencing
reactions were run on an ABI 3730XL automated sequencer (Applied Biosystems, Life
Technologies, CA, USA), and the resulting trace files were analyzed for variants using
NovoSNP25.
4. Fragment analysis
Fragment analysis was carried out on the 3730XL DNA Analyzer (Applied Biosystems
Inc, Life Technologies, CA, USA) with a G5 filter. 2 µl of the PCR product was mixed
with 10 µl of Hi-Di formamide, and 0.3 µl Liz-500 GeneScanTM sizing standard (Applied
Biosystems, Life Technologies, CA, USA) in an optical 96-well plate. The mixture was
denatured at 95 °C for 5 minutes, immediately placed on ice for 15 minutes and subjected
to the analysis. Products were sized using an ABI 3730XL sequencer and the sizes 399,
414, 430, 445, 460, 475 and 489 corresponded to PCR products with 3, 4, 5, 6, 7, 8 and 9
(15 bp) VNTRs. The genotypes were assigned and scored using GeneScan® Analysis,
v3.5.2 (Applied Biosystems, Life Technologies, CA, USA).
Primers used for fragment analysis:
FAM labeled forward primer: 5’-CTTTCCGGTGGAACCAGTG-3’-FAM
reverse primer: 5’-GCACAGCTTTGGATCCTTCT-3’
5. Generation of SH-SY5Y stable cell lines expressing mature miR-137
SH-SY5Y cells were cultivated in a minimum essential medium (MEM) that included
10% (vol/vol) fetal calf serum, 1X non-essential amino acids, 2mM L-glutamine and
penicillin (100U/ml) /streptomycin (100µg/ml). The HEK293T cell line was purchased
from Invitrogen (Life Technologies, CA, USA) and cultivated in DMEM with 10 % fetal
calf serum, 2mM L-glutamine, and penicillin (100 U/ml), streptomycin (100 µg/ml), 1X
non-essential amino acids. Both cell lines were cultivated at 37 °C with 5 % CO2.
Gateway attB1 and attB2 sequences were added by PCR to the 5’ and 3’ of a miRNA
gene sequence in order to amplify specific constructs: MIR137 wt (3 VNTRs), MIR137
-4C>T, MIR137 4 VNTR and MIR137 8 VNTR. Constructs with or without specific
variants were ordered from IDT (Integrated DNA Technologies Inc, CA, USA) and
confirmed by sequencing.
Gateway primers:
mir137-F:
GGGGACAAGTTTGTACAAAAAAGCAGGCTGGCGGGCTCAGCGAGCAGCA
attB1
mir137-R:
GGGGACCACTTTGTACAAGAAAGCTGGGTAAACACCCGAGGAAATGAAAAGAAC
attB2
DNA constructs (Integrated DNA Technologies Inc, CA, USA)
MIR137 (wild type, 3 VNTR)
TCGAGGATCCAAACACCCGAGGAAATGAAAAGAACAAGAAAGTGCTACCTT
GGCAACCACGGGCGTTTAGTGGCCAGCTGGTGGGCTGGGGAGGGCGGCCGCT
GCCCCCCTGCCGCTGGTACTCTCCTCGACTACGCGTATTCTTAAGCAATAACA
ACGTAATCCGTATTATCCACCCAAGAATACCCGTCACCGAAGAGAGTCAGAG
GACCAAGCTGCCGCTGCCGCTGCTACCGCTGCCGCTGCTACCGCTGCCGCTGC
TACTGCCGCCGCCGCCGCCACCAGAACTCTTGCTGCTCGCTGAGCCCGCCCGC
TAGCTCGA
MIR137 (variant -4 T, 3 VNTR)
TCGAGGATCCAAACACCCGAGGAAATGAAAAGAACAAGAAAGTGCTACCTT
GGCAACCACGGGCGTTTAGTGGCCAGCTGGTGGGCTGGGGAGGGCGGCCGCT
GCCCCCCTGCCGCTGGTACTCTCCTCGACTACGCGTATTCTTAAGCAATAACA
ACGTAATCCGTATTATCCACCCAAGAATACCCGTCACCGAAGAGAGTCAGAG
GACCAAGTTGCCGCTGCCGCTGCTACCGCTGCCGCTGCTACCGCTGCCGCTGC
TACTGCCGCCGCCGCCGCCACCAGAACTCTTGCTGCTCGCTGAGCCCGCCCGC
TAGCTCGA
MIR137 (4 VNTR)
TCGAGGATCCAAACACCCGAGGAAATGAAAAGAACAAGAAAGTGCTACCTT
GGCAACCACGGGCGTTTAGTGGCCAGCTGGTGGGCTGGGGAGGGCGGCCGCT
GCCCCCCTGCCGCTGGTACTCTCCTCGACTACGCGTATTCTTAAGCAATAACA
ACGTAATCCGTATTATCCACCCAAGAATACCCGTCACCGAAGAGAGTCAGAG
GACCAAGCTGCCGCTGCCGCTGCTACCGCTGCCGCTGCTACCGCTGCCGCTGC
TACCGCTGCCGCTGCTACTGCCGCCGCCGCCGCCACCAGAACTCTTGCTGCTC
GCTGAGCCCGCCCGCTAGCTCGA
MIR137 (8 VNTR)
TCGAGGATCCAAACACCCGAGGAAATGAAAAGAACAAGAAAGTGCTACCTT
GGCAACCACGGGCGTTTAGTGGCCAGCTGGTGGGCTGGGGAGGGCGGCCGCT
GCCCCCCTGCCGCTGGTACTCTCCTCGACTACGCGTATTCTTAAGCAATAACA
ACGTAATCCGTATTATCCACCCAAGAATACCCGTCACCGAAGAGAGTCAGAG
GACCAAGCTGCCGCTGCCGCTGCTACCGCTGCCGCTGCTACCGCTGCCGCTGC
TACCGCTGCCGCTGCTACCGCTGCCGCTGCTACCGCTGCCGCTGCTACCGCTG
CCGCTGCTACCGCTGCCGCTGCTACTGCCGCCGCCGCCGCCACCAGAACTCTT
GCTGCTCGCTGAGCCCGCCCGCTAGCTCGA
Lentiviruses encoding MIR137-EGFP wild type and variant fusion constructs, where the
EGFP is fused at the 3’-terminal of the MIR137 sequence, were produced in HEK293T
cells by calcium phosphate co-transfection of 10 µg of pLenti-MIR137 expression
constructs and 3 µg of pMD2-VSV and 6.5 µg of pCMV-R8.91 viral packaging vectors.
The calcium transfection medium was removed 8 hours after transfection. Cells were
washed with PBS and replaced with medium containing 10 mM sodium butyrate to
enhance promoter activity. The following day the medium was replaced with complete
NB medium and incubated for 24 h. Lentiviruses were harvested by filtering the
supernatant of the HEK293T cells. SHY-SY5Y cells, seeded in the culture dishes were
transduced with lentiviral particles. After the infection, the cells were selected in MEM
media supplemented with 10% fetal calf serum, 1% non-essential amino acids, 2mM Lglutamine and 100 U/ml penicillin, 100 µg/ml streptomycin and 3 µg/ml blasticidin for
3–4 weeks.
Transduced SH-SY5Y cell lines were named as follows:
a) MIR137 wt: clones c1 (S51.2), c2(S52), c3(S53), c4, c5 and c6 were transduced
with MIR137 vector with 3 VNTRs and Chr1:98511731, -4 C (wt) in pri-miR137.
b) MIR137 4C>T: clones c1(S41), c2(S42), c3(S43.2), c4, c5, c6 were transduced
with MIR137 vector with 3 VNTRs and Chr1:98511731, -4 T.
c) MIR137 4VNTRs: clones c1(S61), c2(S62), c3(S63), c4, c5 and c6 were
transduced with MIR137 vector with 4 VNTRs and Chr1:98511731, -4 C.
d) MIR137 8VNTRs: clones c1(S71), c2(S72), c3(S73), c4, c5 and c6 were
transduced with MIR137 vector with 8 VNTRs and Chr1:98511731, -4 C.
For all transductions (a-d): clones c4, c5 and c6 were used for independent transduction
followed by replication of the RT-qPCR analysis.
6. RNA isolation
Triplicates of approximately 1 x 106 cells originating from clone SH-SY5Y population
with the vector of interest were used for each extraction. The concentration and quality of
RNA was assessed by spectrophotometry on Nanodrop (Thermo Scientific, DE, USA)
and Agilent 2100 Bioanalyzer with Agilent Small RNA kit and Agilent RNA 6000 Pico
kit (all Agilent Technologies, CA, USA). Total RNA was treated with TURBO DNA-free
kit according to manufacturer’s protocol (Ambion, Life Technologies, CA, USA) prior to
RT-qPCR and microarray experiments.
7. Expression of miR-137 and EGFP
EGFP RT-qPCR primers
EGFP-F: GTGGTGCCCATCCTGGTC
EGFP-R:CCGTCGTCCTTGAAGAAGAT
Endogenous controls:
Following the manufacturer’s protocol, 4 µg aliquots of total RNA were transcribed with
Superscript III (Invitrogen, Life Technologies, CA, USA) using random hexamers prior
to RT-qPCR using SYBR Green I mix.
GAPDH-F: TGCACCACCAACTGCTTAGC
GAPDH-R: GGCATGGACTGTGGTCATGAG
HMBS-F: GAAACTCTGCTTCGCTGCATT
HMBS-R: TGCCCATCTTTCATCACTGTATG
SDHA-F: TGGGAACAAGAGGGCATCTG
SDHA-R: GGCATGGACTGTGGTCATGAG
TBP- F: CTACCGTGAATCTTGGCTGTAAA
TBP- R: TTCTCATGATGACTGCAGCAAA
8. Microarray analysis (SAM)
Statistical analysis of microarrays resulted in the identification of genes with significant
changes in expression by assimilating a set of gene specific scores. Each gene was
assigned a score based on the expression differences relative to the standard deviation of
repeated measurements of that gene. Genes with scores above the threshold are regarded
as significant. For statistical analysis of microarrays between miR-137 wild type (class
A) and miR-137 -4C>T (class B) datasets, the statistic design using SAM v1.0 was as
follows: two class unpaired analysis; delta 1.9660827, upper cutoff 4.572125 lower
cutoff –infinity. Data was permuted 500 times. FDR at median and at 90th percentile was
0,00000%.
For analysis using SAM on 3 datasets originating from the transcriptome of cells
transduced with MIR137 wild type (3VNTR), MIR137 4 VNTRs and 8 VNTRs) we used
a multiclass study design (3 classes); delta 1.075725, upper cutoff 2.5578384; lower
cutoff – infinity and 500 permutations. FDR at median and at 90th percentile was
0.00000%.
9. GSEA analysis
Each probe set in the expression dataset was collapsed into a single vector, identified by
the gene symbol and using the maximum probe expression value. Statistical significance
of the enrichment score was assessed by 1000 permutations of the gene sets (sets larger
than 500 genes and smaller than 15 genes were excluded from the analysis). For the
enrichment analysis we compared expression values between cells transduced with wild
type and variant MIR137 construct. For analyzing gene expression differences in groups
with different number of VNTRs in pri-miR-137 we compared the microarray expression
datasets originating from cells transduced with MIR137 wild type (3 VNTRs), 4VNTR
MIR137 and 8 VNTR MIR137 constructs.
All gene sets with a nominal p-value lower than 0.01 and FDR lower than 5% or lower
than 25% (if the 5% threshold was breached) were called as significant or enriched
between the analyzed groups.
10. Identification of predicted targets for miR-137
3’UTR sequences were extracted using Ensembl BioMart, v.0.7 from GRCh37.p10.
Possible target sites in the 3’UTR sequences were searched using the BLAST algorithm
with adjusted settings and with parameters: word size 7, E value of 50000 and number of
alignments of 100000. The final input query was mature miR-137 obtained from
miRBase version 1826. From the BLAST all possible transcripts of all genes with ‘seedhits’ (complementarity between 3’UTR and bases 2-8 of the mature miR-137) and ‘nonseed hits’ (complementarity of the 3’UTR with mature miR-137 outside the ‘seed’ but
not shorter than 7 consecutive bases) were extracted. We compared the output file with
all significantly differentially expressed genes derived from the microarray data by SAM
or GSEA. We further used PITA (August 2008 release)27, DIANAmT (v3.0)28,
MIRANDA (August 2010 release)29, MIRWALK (March 2011 release)30, PICTAR (5way; March 2007 release)31 and TARGETSCAN (v5.1)32 to detect other potential miR137 targets. The same analysis was done on the set of 21435 genes which were not
significantly differentially expressed and the results of the comparison and the statistical
evaluation is presented in Supplement S3.
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