Emotional Regulation in Children With ND: the Role of Genomic Variation, Proteomic Patterns, and Early Experience

Genomic DNA will be extracted from 0.4 ml aliquots of each saliva sample using the kit manufacturer's suggested protocol, quantified with Qubit 2.0 (Invitrogen), and stored at -20°C. Aliquots of 250 ng of each DNA will be edited for methylation analysis with the EZ DNA Methylation Lightning kit (Zymo Research). Amplification of samples and their preparation for NGS sequencing will be performed as described. Samples will be sequenced on NextSeq 500 (Illumina). Individual processed sequences (PE reads) will be independently aligned to reference sequences using a parallel Smith-Waterman algorithm. Only reads that consistently align to the same reference sequence will be retained. At each CpG site in each analyzed sequence, the frequencies of the four bases will be evaluated and tabulated.

Urine samples are collected using non-invasive methods and are prepared according to a procedure preparatory to quantitative recovery of exosomes: once thawed and centrifuged at 17,000 x g for 10 min at 4°C, the recovered supernatants are separated and centrifuged at 200,000 x g for 1 hr at 4°C. Exosome pellets are separated, washed repeatedly and resuspended in buffer (NH4HCO3, 0.1 mM ph=7.8). Protein concentration is estimated with the SPNTM Protein Assay kit and each sample (50 ± 0.5 μg protein) is digested with trypsin using a 1:50 (w/w) enzyme/substrate ratio at 37 °C over night (o/w). A second tryptic digestion is performed with an enzyme:substrate ratio of 1:100 (w/w) for 4h. Digested samples, centrifuged at 13,000 × g for 10 min, are purified and concentrated using PepClean C-18 columns. The samples obtained are analyzed by reversed-phase liquid chromatography coupled to high-resolution mass spectrometry.

During an observational session, a short video recording of the mother-child interaction of approximately 10 minutes will be made in a semi-structured setting to assess the child's emotional regulation and social behavior. The interaction will be structured in 5 different phases according to the Still Face paradigm (Tronick et al., 1978): Play, Still#1, Reunion#1, Still#2, Reunion#2. Play: Mothers will be invited to interact with their babies for 10 minutes; Still: mothers will be asked to remain still while maintaining an unresponsive expressionless face and not to smile, touch, or talk to the child for 2 minutes (Still#1: 2 minutes; Still#2: 2 minutes); Reunion: mothers will be asked to resume the play activity with their own for an additional 2 minutes (Reunion#1: 2 minutes; Reunion#2: 2 minutes).