Ploski, R. | bioRxiv Science

bioRxiv2022

Methylation genome wide profiling in lowly and highly efficient adipose somatic cell nuclear transfer in pigs

Swine is a common model organism for biomedical research. Epigenetic reprogramming in SCNT embryos does not fully recapitulate the natural DNA demethylation events at fertilisation. This study aimed to conduct a genome-wide methylation profiling to detect differentially methylated regions (DMRs) responsible for epigenetic differences in stem cells that displayed high and low efficiency of SCNT and to elucidate the low efficiency of cloning rate in pigs. Adipose tissue mesenchymal stem cells (AMSC)s lines were isolated from adipose tissue of adult male pigs (n=20; high-efficiency cells=10; low efficiency cells= 10). Reduced representation bisulfite sequencing (RRBS) was performed on an Illumina HiSeq1500. Paired-end reads were filtered to remove the adapter contamination, and low-quality reads using TrimGalore!. Filtered reads were mapped to the reference genome using Bismark. MethylKit was used to identify differentially methylated regions (DMRs) (bases and tiles), showing statistically significant differential methylation between two groups: high and low-efficiency AMSCs. Hierarchical cluster analysis according to methylation patterns clearly defined groups with low and high cloning efficiency. We report 3704 bases with statistically significant differences in methylation and 10062 tiles with statistically significant differences in methylation. Most differentially methylated sites are intergenic 62%, 31% are intronic, 4% are located in exons and 4% in promoters. 37% of differentially methylated sites are located in known CpG islands (CGIs) and 4% in CpG island shores (CGSs).

genetics↗

bioRxiv2022

A novel de novo FEM1C variant as a potential cause of neurodevelopmental disorder with absent speech, pyramidal signs, and limb ataxia.

The principal component of the protein homeostasis network is the ubiquitin-proteasome system. Ubiquitination is mediated by an enzymatic cascade involving, i.e., E3 ubiquitin ligases, many of which belong to the cullin-RING ligases family. Genetic defects in the ubiquitin-proteasome system components, including cullin-RING ligases, are known causes of neurodevelopmental disorders. Using exome sequencing to diagnose a pediatric patient with developmental delay, pyramidal signs, and limb ataxia, we identified a de novo missense variant c.376G>C; p.(Asp126His) in the FEM1C gene encoding a cullin-RING ligase substrate receptor. This variant alters a conserved amino acid located within a highly constrained coding region and is predicted as pathogenic by most in silico tools. In addition, a de novo FEM1C mutation of the same residue p.(Asp126Val) was associated with an undiagnosed developmental disorder, and the relevant variant (FEM1CAsp126Ala) was found to be functionally compromised in vitro. Our computational analysis showed that FEM1CAsp126His hampers protein substrate binding. To further assess its pathogenicity, we used the nematode Caenorhabditis elegans. We found that the FEM-1Asp133His animals (expressing variant homologous to the FEM1C p.(Asp126Val)) had normal muscle architecture yet impaired mobility. Mutant worms were sensitive to the acetylcholinesterase inhibitor aldicarb but not levamisole (acetylcholine receptor agonist), showing that their disabled locomotion is caused by synaptic abnormalities and not muscle dysfunction. In conclusion, we provide the first evidence from an animal model suggesting that a mutation in the evolutionarily conserved FEM1C Asp126 position causes a neurodevelopmental disorder in humans.

genetics↗