Computational protocol: Maternal Sensitivity Buffers the Association between SLC6A4 Methylation and Socio-Emotional Stress Response in 3-Month-Old Full Term, but not very Preterm Infants

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[…] We analyzed a CpG-rich region of the SLC6A4 promoter (chr17:28562750.28562958, Human hg19 Assembly), between −69 and −213 relative to the transcriptional start site, which contains 20 CpG sites and is adjacent to exon 1A. Methylation levels were determined in DNA using bisulfite modification followed by PCR amplification and NGS. Genomic DNA was extracted from 0.2 ml of each sample using the GenElute Blood Genomic DNA kit (Sigma). Bisulfite conversion was performed on 500 ng of genomic DNA using the EZ DNA methylation kit (ZymoResearch, Inc., Irvine, CA, USA). Primers were designed using Bisulfite Primer Seeker. The gene-specific forward 5′-GYGGGTTTTTATATGGTTTGATTTTTAG-3′ and reverse 5′-CRAAAATCCCTCCCCTCCTAACTCTAAAATC-3′ primers were sequenced. A TruSeq amplicon-specific tail 5′-CCTACACGACGCTCTTCCGATCT-3′ was added to the forward primer, while the sequence 5′-TCAGACGTGTGCTCAACCGATCT-3′ was added to the reverse primer, in order to allow synthesis and sequencing of TruSeq libraries of methylated fragments. Primary PCR-amplification was performed on 20 ng of bisulfite-treated DNA using Taq Gold (Life Technologies, Inc.). Cycling comprised 5 min preactivation at 95°C, followed by 35 cycles of 94°C denaturation for 15 s, 58°C annealing for 20 s, 72°C elongation for 1.5 min. All PCR products were checked on 2% agarose gel and treated with Ilustra Exo Pro-STAR (GE Healthcare) to eliminate unincorporated primers. Secondary PCR was conducted on each sample using a TruSeq Custom Amplicon Index Kit (Illumina) containing eight forward (i5) and 12 reverse (i7) index primers. Optimal annealing temperature (68°C) and number of PCR cycles were experimentally determined. Cycling comprised 5 min preactivation at 95°C, followed by 16 cycles of 94°C denaturation for 15 s, 68°C annealing for 20 s, 72°C elongation for 1 min. All PCR products were checked on 2% agarose gel, and approximately equimolar aliquots of each product were pooled and purified on a 2% agarose gel. The purified library was quantified on a Bioanalyzer 2100 (Agilent) and sequenced on a MiSeq (Illumina) using a v2 Reagent kit, 300 cycles PE. Paired ends reads from each sample were independently aligned to all the reference sequences by a parallel striped Smith–Waterman algorithm. Only paired reads that aligned coherently to the same reference sequence were retained. At each CpG site in each sequence, the four bases frequencies were evaluated and reported along with the C-to-T percentage. As previous works on this topic documented that a significant change in SLC6A4 methylation from birth to discharge was detected at CpG2 and CpG5 (, ), these two CpG sites were selected for the present study purposes. [...] Very preterm and FT infants were compared for neonatal and sociodemographic variables by means of t-test and chi2 test for continuous and categorical variables, respectively. Socio-emotional stress response was compared between VPT and FT infants through mixed model analysis of variance, with negative emotionality as the output variable and FFSF episodes as the within-subject factor (5 levels: Play, Stil-Face#1, Reunion#1, Still-Face#2, Reunion#2) and Group as the between-subject factor (2 levels: VPT vs. FT). Maternal sensitivity during the Play episode was compared between the two groups. To assess the moderating role of maternal sensitivity, two sets of stepwise multiple linear regressions were performed separately for VPT and FT infants.Separate regression models are generally used when comparing preterm vs. FT infants’ outcomes [e.g., Ref. ()] because VPT and FT infants’ life experience is very different and different confounders need to be controlled for. To select predictors, preliminary bivariate correlations were run to test the association between (a) maternal sensitivity and infants’ negative emotionality across the FFSF episodes; (b) SLC6A4 CpG2 and CpG5 methylation and infants’ negative emotionality across the FFSF episodes. The final model was tested on infants’ negative emotionality considering only the FFSF episode(s) for which significant preliminary had emerged, and it included the following predictors: methylation of CpG sites significantly correlated with infants’ negative emotionality during the specific episode, maternal sensitivity during Play, and the interaction between CpG-specific SLC6A4 methylation and maternal sensitivity. Finally, the regression model was controlled for clinical confounders. For both VPT and FT infants, confounders were: gestational age (weeks), maternal age (years), maternal educational level (years of study), family SES, maternal depression, maternal anxiety. Moreover, for VPT infants only, pain-related stress index, days on ventilation, and length of stay in the NICU was added as confounders. All the analyses were carried out using IBM SPSS Statistics 21, at α < 5%. […]

Pipeline specifications

Software tools SSW, SPSS
Applications Miscellaneous, Nucleotide sequence alignment