Placental DNA methylation changes in detection of tetralogy of Fallot.

OBJECTIVES: To determine whether the methylation level of cytosine nucleotides in placental DNA can be used to predict tetralogy of Fallot (TOF) and provide insights into the developmental mechanism of this condition. METHODS: Tissue sections were obtained from formalin-fixed paraffin-embedded specimens of placental tissue obtained at birth from eight cases with non-chromosomal, non-syndromic TOF and 10 unaffected newborns. The Illumina Infinium HumanMethylation450 BeadChip assay was used to measure cytosine ('CpG' or 'cg') methylation levels at loci throughout the placental genome. Differential methylation was assessed by comparing the ß-values (a measure of the extent of cytosine methylation) for individual CpG loci in fetuses with TOF vs in controls. The most discriminating CpG sites were determined based on a preset cut-off of ≥ 2.0-fold change in the methylation level. The predictive accuracy of CpG loci with significant methylation changes for TOF was determined by the area under the receiver-operating-characteristics curve (AUC). A false-discovery-rate (FDR) P-value < 0.05 was used to define a statistically significant difference in the methylation level. Ingenuity Pathway Analysis (IPA) (Qiagen) was used to identify gene pathways that were significantly overexpressed, and thus altered, in TOF cases compared with controls. RESULTS: We found a total of 165 significantly differentially methylated CpG loci in TOF cases compared with controls, in 165 separate genes. These biomarkers demonstrated from fair to excellent individual predictive accuracy for TOF detection, with AUCs ≥ 0.75 (FDR P-value < 0.001 for all). The following CpG loci (gene) had the highest predictive accuracy: cg05273049 (ARHGAP22; AUC = 1.00; 95% CI, 1.00-1.00), cg02540011 (CDK5; AUC = 0.96; 95% CI, 0.87-1.00), cg08404201 (TRIM27; AUC = 0.95; 95% CI, 0.84-1.00) and cg00687252 (IER3; AUC = 0.95; 95% CI, 0.84-1.00). IPA revealed over-representation (dysregulation) of 14 gene pathways involved in normal cardiac development, including cardiomyocyte differentiation via bone morphogenetic protein receptors, cardiac hypertrophy signaling and role of nuclear factor of activated T cells in cardiac hypertrophy. Cardiac hypertrophy is an important feature of TOF. CONCLUSIONS: Analysis of placental DNA cytosine methylation changes yielded accurate markers for TOF detection and provided mechanistic information on TOF development. Our work appears to confirm the central role of epigenetic changes and of the placenta in the development of TOF. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

A new algorithm for the alignment of multiple protein structures using Monte Carlo optimization.

We have developed a new algorithm for the alignment of multiple protein structures based on a Monte Carlo optimization technique. The algorithm uses pair-wise structural alignments as a starting point. Four different types of moves were designed to generate random changes in the alignment. A distance-based score is calculated for each trial move and moves are accepted or rejected based on the improvement in the alignment score until the alignment is converged. Initial tests on 66 protein structural families show promising results, the score increases by 69% on average. The increase in score is accompanied by an increase (12%) in the number of residue positions incorporated into the alignment. Two specific families, protein kinases and aspartic proteinases were tested and compared against curated alignments from HOMSTRAD and manual alignments. This algorithm has improved the overall number of aligned residues while preserving key catalytic residues. Further refinement of the method and its application to generate multiple alignments for all protein families in the PDB, is currently in progress.

Stable expression of a biodegradable protein-based polymer in tobacco chloroplasts.

Bioelastic protein-based polymers (PBP) have several medical (prevention of post-surgical adhesions) and non-medical (biodegradable plastic) applications. This study compares expression levels of PBP genes (synthetic) integrated into the nuclear genome or the large single-copy (LSC) or inverted repeat (IR) region of the chloroplast genome in transgenic tobacco plants. Polymer transcripts accumulated up to 100-fold higher in the IR plants than in those of nuclear transgenic plants. Integration of foreign genes into all of the chloroplast genomes (homoplasmy) and higher levels of polymer transcripts were observed only in the IR and not in LSC transgenic plants. Expression of the polymer protein was further confirmed by Western blot analysis.