SUPPLEMENTARY INFORMATION
METHODS
Study setting and financial support.
Data for the present investigation were obtained from an ongoing epidemiological and threeyear longitudinal intervention program of first-episode psychosis (PAFIP) conducted at the
outpatient clinic and the inpatient unit at the University Hospital Marques de Valdecilla,
Spain1. Conforming to international standards for research ethics, this program was approved
by the local institutional review board. Patients meeting inclusion criteria and their families
provided written informed consent to be included in the PAFIP. The Mental Health Services of
Cantabria and the Spanish Ministry of Science provided funding for implementing the program.
No pharmaceutical company supplied any financial support.
Study design.
Patients recruited into this study were drawn from a consecutive sample of non-affective
psychotic patients enrolled in our first episode psychosis program (PAFIP) from May 2010 to
May 2012. Patients had to meet the following criteria: 1) age 15-60 years, 2) living in the
catchment area, 3) experiencing their first episode of psychosis 4) never treated with
antipsychotic medication, 5) meeting DSM-IV criteria for brief psychotic disorder,
schizophreniform disorder, schizophrenia, or schizoaffective disorder, 6) they understood the
nature of the study and signed an informed consent document. Patients were excluded for any
of the following reasons: 1) meeting DSM-IV criteria for drug dependence, 2) meeting DSM-IV
criteria for mental retardation, 3) having a serious medical illness. The diagnoses were
confirmed according to the DSM-IV criteria, using the Structured Clinical Interview for DSM-IV
(SCID-I) by an expert psychiatrist after 6 months of the initial contact. Demographic and
clinical characteristics of patients are shown in Supplementary Table 2.
At the time of blood test patients were antipsychotic naïve although certain concomitant
medications (lormetazepam and clonazepam) were permitted for the management of
agitation, anxiety and/or insomnia.
The severity scale of the Clinical Global Impression (CGI) scale2, the Brief Psychiatric Rating
Scale (BPRS)3, the Scale for the Assessment of Positive symptoms (SAPS)4, the Scale for the
Assessment of Negative symptoms (SANS)5, the Calgary Depression Scale for Schizophrenia
(CDSS)6 and the Young Mania Rating Scale (YMRS)7 were used to evaluate clinical
symptomatology. The same trained psychiatrist (BC-F) completed all clinical assessments. The
severity of psychopathology at baseline is shown in Supplementary Table 3.
Laboratory Assessments.
Fasting venous blood samples were collected between 8:00 and 10:00 am at baseline.
RNA extraction.
Total RNA was extracted from blood using the Tempus™ Blood RNA Tube and Tempus™ Spin
RNA Isolation Kit (Applied Biosystems, Foster City, CA, USA) using the manufacturer protocols.
To define expression profiles, a key factor is that the RNA is intact. To select only RNA with
good quality, the RNA Integrity Number (RIN) was characterized with a Bioanalyzer (Agilent
Technologies, Santa Clara, CA, USA) and samples with a RIN of at least 7.2 were selected. The
selected samples have RINs that range from 7.2 to 9.8 with an average of 8.5.
RNA Next Generation Sequencing.
Sequences of 35 nucleotide single-end reads were produced using a Genome Analyzer IIx
(Illumina). Alignment of the reads was performed using Illumina CASAVA 1.8.1 software
specifically configured to process RNA-Seq data. Read length varied from 35bp to 38bp
between runs; thus, CASAVA was also configured to mask reads out to 35bp in order to avoid
mapping bias.
Differential Expression Statistical Analyses.
Bedtools 2.15.0 (bedtools coverage) was used to count the amount of reads mapped to each
gene. R Bioconductor DESeq 1.9.6 package was used to test for differential expression from
gene-count data setting up fit-only as fitting method. CASAVA 1.8.1 was used to calculate
RPKMs (Reads Per Kilobase per Million of mapped reads).
Validation of the expression data by qRT-PCR.
In order to validate the expression data obtained by RNA-Seq, we performed qRT-PCR
amplification of 10 selected genes on 30 randomly selected patient samples (15 cases and 15
controls). We used pre-designed Taqman probes (Life technologies®) using manufacturer
instructions for LAMP3 (Hs00180880_m1), RFX2 (Hs01100925_m1), CTAG2 (Hs00535628_m1),
GPR128 (Hs00262184_m1), IFI27 (Hs01086373_g1), ADAMTS2 (Hs01029111_m1), BRS3
(Hs00179951_m1), MSLN (Hs00245879_m1), RSAD2 (Hs00369813_m1) and IFI144L
(Hs00119115_m1) using GADPH (Hs02758991_g1) as control. The expression ratio obtained
from each gene compared to the housekeeping control gene (GAPDH) was compared, in each
sample, with the ratio of the same genes calculated from the RNA-Seq data. This comparison
was performed calculating the lineal Person product-moment correlation coefficient between
the data generated for both platforms using the Libre Office statistics package. On average we
obtained a correlation ratio of 87.15 % between the two technologies, with gene individual
correlation ratios varying from 75% (CTAG2) to 99.9 % (MSLN). We observed lower correlation
ratios on low expressed genes, probably due to a lower sensitivity of the qRT-PCR compared
with the RNA-Seq technology. Besides this, the high grade of correlation observed between
qRT-PCR and RNA-Seq supports the differential expression data observed in this study.
DIFFERENTIAL EXPRESSION GENES RELATED TO SCHIZOPHRENIA.
According to GeneRIF [http://www.ncbi.nlm.nih.gov/gene/about-generif; National Center for
Biotechnology Information (NCBI)], 8 out of the 200 differentially expressed genes have been
related to schizophrenia: GRIK3,8 LPL,9 S100B,10,11, SNCA,12 SYN2,13 TUBB2A,12 SELENBP1,14,15
CSMD1.16
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Supplementary Figure 1: Differential Expression Genes (Case/Control).
Graph of the log2 of the fold change (baseMean Cases/baseMean controls) versus the
baseMean of both conditions [(baseMean cases+basemean controls)/2]. BaseMean is defined
as the mean expression level of each gene. Red dots represent genes with significant
differential expression (Padj Value <0.05)
Supplementary Figure 2: Gene Ontology Processes significantly enriched with schizophrenia
differentially expressed genes.
Biological processes (colored) having significant over-representation of genes with differential
expression between schizophrenia patients and controls.