M. Wankerl et al.

Molecular correlates of early adversity and 5-HTTLPR

Effects of genetic and early environmental risk factors for depression on serotonin transporter expression and methylation profiles

Supplemental Information

SUPPLEMENT 1: Quantitative Real-Time PCRProtocol

Paxgene RNA blood tubes were thawn for 3 h at room temperature. RNA was isolated with simplyRNA blood isolation kit for Maxwell 16 (Promega) according to the manufacturers protocol for Paxgene blood RNA tubes. RNA was reverse transcribed with MMLV point mutant (Promega) for 90 minutes at 42°C. After diluting 1:10 in molecular grade water (sigma), gene expression was measured on the LightCycler 480 I (Roche) with the SensiFast Sybr green mix from Bioline. Cq were automatically called. ACTB and GAPDH were used as reference genes. After mean calculation and delta Cq generation (reference mean - target gene value) values were transformed by the2-2ΔΔCt method to form relative expression level on a linear scale.

Supplement Table 1: Primer sequences for SERT mRNA expression analysis.

Primer sequences
5HTT-F / GTGATTGGCTATGCTGTGGA
5HTT-R / ATGGTGTAGGGGAGGAGGAA
ACTB-F / GTGGGCATGGGTCAGAAG
ACTB-R / GATGGGGTACTTCAGGGTGAG
GAPDH-F / TGAGTACGTCGTGGAGTCCA
GAPDH-R / ATGTTCGTCATGGGTGTGAA

SUPPLEMENT 2: Bisulfite Pyrosequencing Protocol

Genomic DNA was bisulfite treated using the EZ DNA Methylation Gold Kit (Zymo Research, Range, CA, USA). 3 Amplicons were generated from bisulfite-treated DNA. PCR protocols were run as follow: fragments 5HTT_P1 and 5HTT_P2 : HotStarTaq polymerase (Qiagen, Hilden, Germany), 95°C 15', 49x (95°C 35'', 57°C 35'', 72°C 35''), 72°C 5'; fragment 5HTT_P3: HotStarTaq polymerase (Qiagen, Hilden, Germany) 95°C 15', 49x (95°C 35'', 52°C 35'', 72°C 35''); 72°C 5'.Given that pyrosequencing only reliably generates short reads, 10 sequencing primer were selected to cover all 83 CpG sites of interest within the SERT promoter associated island. Sample preparation was carried out using Vacuum Prep Tool according to standard procedures. 12-15μl PCR product was immobilized to 2μl Streptavidin Sepharose™ HP beads (GE Healthcare) followed by annealing to 0.8-1.0μl sequencing primer (5μM) for 2’ at 80°C. Amplicon and sequencing primers are depicted in Supplement Table 2.

Supplement Table 2: Amplicon and Sequencing Primers used for bisulfite pyrosequencing of the SERT promotor-associated CpG island. All primers refer to bisulfite treated DNA.

Amplicon Primer name / Amplicon Primer
Sequence 5´-3´ / Seqencing Primer Name / Seqencing Primer Sequence 5´-3´
Amplicon 1
(CpG sites 1-13) / 5HTT_P1-F / ggg ttt tta agt tga gtt tat at / 5HTT_P1-S1 (CpG 1-7) / ttt agt agg tta gtt aga taa a
5HTT_P1-R / Biotin-
cta act ttc cta ctc ttt aac tt / 5HTT_P1-S2 (CpG 8-13) / gag tag att ttt gtg tg
Amplicon 2
(CpG sites 14-42) / 5HTT_P2-F / aag agt agg aaa gtt agg a / 5HTT_P2-S1 (CpG 14-22) / gta gga aag tta gga ttt
5HTT_P2-R / Biotin-
ccc tca cat aat cta atc t / 5HTT_P2-S2 (CpG 23-32) / ttt tgg ttt tgg ggt
5HTT_P2-S3 (CpG 32-42) / ttg gag aga gta att tta
Amplicon 3
(CpG sites 43-83) / 5HTT_P3-F / ggg gaa gta tta agt tta t / 5HTT_P3-S1 (CpG 43-57) / att tag aga tta gat tat gtg
5HTT_P3-R / Biotin-
ccc cta caa caa taa aca / 5HTT_P3-S2 (CpG 58-64) / agg tta gtt agt ttg ttt ag
5HTT_P3-S3 (CpG 65-71) / att taa gtt ttt ttt tag at
5HTT_P3-S4 (CpG 72-77) / agg aga gga ggt gta t
5HTT_P3-S5 (CpG 78-83) / tta gta aga gtt aga gtt gaa

SUPPLEMENT 3: Principal Component Analysis

In order to account for common variance in the degree of methylation across all 83 CpG cites investigatedwithin the 799-bp CpG island in SLC6A4, a principal component analysis was performed on their complete covariance structure. As we expected the existence of both a “general methylation factor” (e.g. due to global DNA methyltransferase activity) and “site-specific methylation clusters”, an oblique bi-factor rotation1was applied to these components. Parallel analyses were performed from 1.000 diagonal covariance matrices in order to limit all subsequent analyses to meaningful factors,2 which suggested a 6-component solution (see Figure S1). Supplement Table 3 lists these variance components and their intercorrelations. Multivariate analyses of variance revealed no significant associations between individual SERT methylation factors and prenatal, early or recent stress/trauma related variables (see Suppl. Table 4).

Supplement Table 3:Principal component analysis on DNA methylation within the 799-bp CpG island in SLC6A4.

Principal component / F1 / F2 / F3 / F4 / F5 / F6
Variance explained
Total amount / 103.13 / 66.87 / 61.94 / 42.00 / 35.55 / 30.53
Cumulative proportion / 14.5% / 23.9% / 32.5% / 38.4% / 43.4% / 47.7%
Correlations
F1 / 1
F2 / -0.053 / 1
F3 / 0.202 / 0.028 / 1
F4 / -0.281 / 0.160 / -0.241 / 1
F5 / 0.099 / 0.142 / 0.006 / 0.069 / 1
F6 / -0.089 / 0.013 / -0.099 / 0.272 / -0.137 / 1

Supplement Figure 1. Screeplot of the principal component analyses (PCA) of SERT methylation data, suggesting a solution of 6 variance components. Blue circles represent the empirically estimated components. Black dots indicate means and 95% confidence intervals of component estimations upon presence of no covariance in between the methylation of all 83 CpG sites.

Supplement Table 4:Main effects of stress/trauma-related variables on SERT methylation levels within subregions of the 799-bp CpG island in SLC6A4 as defined by principal component analysis(Mean  SD).

Prenatal trauma/stress / Early trauma/stress / Recent trauma/stress
Mean SERT methylation / Yes (n=39) / No (n= 46) / p / Yes (n=17) / No (N=116) / p / Yes (n=34) / No (N=99) / p
F 1 / -1.036 1.15 / 0.861 1.06 / 0.23 / -2.025 1.82 / 0.297 0.70 / 0.24 / 0.424 0.75 / -1.234 1.29 / 0.27
F 2 / 0.055 1.14 / -0.556 1.14 / 0.72 / 0.785 1.85 / -0.115 0.71 / 0.65 / -0.62  0.76 / 1.8081.30 / 0.11
F3 / -0.057 1.30 / 1.670 1.20 / 0.33 / 3.225 1.95 / -0.473 0.75 / 0.08 / 0.400 1.05 / -1.1561.39 / 0.34
F 4 / -0.705  0.92 / 1.086  0.85 / 0.16 / 0.418 1.49 / -0.061 0.57 / 0.76 / -0.123  0.62 / 0.3591.05 / 0.69
F 5 / -0.515 1.09 / 0.230 1.01 / 0.62 / -0.905 1.66 / 0.133 0.64 / 0.56 / 0.069 0.69 / -0.201.18 / 0.84
F 6 / -0.013  0.84 / 0.246 0.77 / 0.82 / 1.304 1.35 / -0.191 0.52 / 0.30 / -0.477  0.96 / 0.1640.56 / 0.56

SUPPLEMENTAL REFERENCES

1.Jennrich RI, Bentler, PM. Exploratory bi-factor analysis: The oblique case. Psychometrika 2012; 77: 442-454.

2.Buja A, Eyuboglu N. Remarks on parallel analysis. Multivariate Behavioral Research 1992;27: 509-540.

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