Exome analysis in an Estonian multiplex family with neural tube defects-a case report.

INTRODUCTION: Neural tube defects (NTDs) are a group of common and severe congenital birth defects that occur during early embryonic development due to incomplete closure of the neural tube. The genetic architecture of human NTDs, including spina bifida and hydrocephalus, is highly heterogeneous, with multiple genes/loci and both gene-gene and gene-environment interactions involved. Hence, the variation in outcomes also most likely relates to a combination of the severity of different variants in multiple genes and genetic modifiers affecting the biochemical traits. METHODS: Here, we present a multiple-spouse family with one pedigree lineage where three brothers are affected with NTDs-two lumbar spina bifidas without hydrocephalus and one obstructive hydrocephalus. We sequenced the exomes of three NTD patients and their parents. RESULTS: The analysis revealed a heterozygous c.844ins68 variant in CBS, which was carried by all affected individuals and inherited from their mother. All affected individuals had a variable set of additional low frequency deleterious variants in PTK7, PLCD4, IL4I1 or RASSF4 as likely causal loci contributing to the disease development. CONCLUSION: This report extends the current knowledge of the genetic background of NTDs and proposes that common and low frequency variants in genes involved mostly in one-carbon metabolism or planar cell polarity (PCP) pathways can act in an additive manner to increase the genetic risk of the disease.

Sequence variation in nuclear ribosomal small subunit, internal transcribed spacer and large subunit regions of Rhizophagus irregularis and Gigaspora margarita is high and isolate-dependent.

Arbuscular mycorrhizal (AM) fungi are known to exhibit high intra-organism genetic variation. However, information about intra- vs. interspecific variation among the genes commonly used in diversity surveys is limited. Here, the nuclear small subunit (SSU) rRNA gene, internal transcribed spacer (ITS) region and large subunit (LSU) rRNA gene portions were sequenced from 3 to 5 individual spores from each of two isolates of Rhizophagus irregularis and Gigaspora margarita. A total of 1482 Sanger sequences (0.5 Mb) from 239 clones were obtained, spanning ~4370 bp of the ribosomal operon when concatenated. Intrasporal and intra-isolate sequence variation was high for all three regions even though variant numbers were not exhausted by sequencing 12-40 clones per isolate. Intra-isolate nucleotide variation levels followed the expected order of ITS > LSU > SSU, but the values were strongly dependent on isolate identity. Single nucleotide polymorphism (SNP) densities over 4 SNP/kb in the ribosomal operon were detected in all four isolates. Automated operational taxonomic unit picking within the sequence set of known identity overestimated species richness with almost all cut-off levels, markers and isolates. Average intraspecific sequence similarity values were 99%, 96% and 94% for amplicons in SSU, LSU and ITS, respectively. The suitability of the central part of the SSU as a marker for AM fungal community surveys was further supported by its level of nucleotide variation, which is similar to that of the ITS region; its alignability across the entire phylum; its appropriate length for next-generation sequencing; and its ease of amplification in single-step PCR.

Role of autophagy in cell-penetrating peptide transfection model.

Cell-penetrating peptides (CPPs) uptake mechanism is still in need of more clarification to have a better understanding of their action in the mediation of oligonucleotide transfection. In this study, the effect on early events (1 h treatment) in transfection by PepFect14 (PF14), with or without oligonucleotide cargo on gene expression, in HeLa cells, have been investigated. The RNA expression profile was characterized by RNA sequencing and confirmed by qPCR analysis. The gene regulations were then related to the biological processes by the study of signaling pathways that showed the induction of autophagy-related genes in early transfection. A ligand library interfering with the detected intracellular pathways showed concentration-dependent effects on the transfection efficiency of splice correction oligonucleotide complexed with PepFect14, proving that the autophagy process is induced upon the uptake of complexes. Finally, the autophagy induction and colocalization with autophagosomes have been confirmed by confocal microscopy and transmission electron microscopy. We conclude that autophagy, an inherent cellular response process, is triggered by the cellular uptake of CPP-based transfection system. This finding opens novel possibilities to use autophagy modifiers in future gene therapy.

Microbial population dynamics in response to Pectobacterium atrosepticum infection in potato tubers.

Endophytes are microbes and fungi that live inside plant tissues without damaging the host. Herein we examine the dynamic changes in the endophytic bacterial community in potato (Solanum tuberosum) tuber in response to pathogenic infection by Pectobacterium atrosepticum, which causes soft rot in numerous economically important crops. We quantified community changes using both cultivation and next-generation sequencing of the 16S rRNA gene and found that, despite observing significant variability in both the mass of macerated tissue and structure of the endophytic community between individual potato tubers, P. atrosepticum is always taken over by the endophytes during maceration. 16S rDNA sequencing revealed bacteria from the phyla Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes, Fusobacteria, Verrucomicrobia, Acidobacteria, TM7, and Deinococcus-Thermus. Prior to infection, Propionibacterium acnes is frequently among the dominant taxa, yet is out competed by relatively few dominant taxa as the infection proceeds. Two days post-infection, the most abundant sequences in macerated potato tissue are Gammaproteobacteria. The most dominant genera are Enterobacter and Pseudomonas. Eight days post-infection, the number of anaerobic pectolytic Clostridia increases, probably due to oxygen depletion. These results demonstrate that the pathogenesis is strictly initiated by the pathogen (sensu stricto) and proceeds with a major contribution from the endophytic community.

Coffin-Siris Syndrome with obesity, macrocephaly, hepatomegaly and hyperinsulinism caused by a mutation in the ARID1B gene.

Coffin-Siris Syndrome (CSS, MIM 135900) is a rare genetic disorder, and mutations in ARID1B were recently shown to cause CSS. In this study, we report a novel ARID1B mutation identified by whole-exome sequencing in a patient with clinical features of CSS. We identified a novel heterozygous frameshift mutation c.1584delG in exon 2 of ARID1B (NM_020732.3) predicting a premature stop codon p.(Leu528Phefs*65). Sanger sequencing confirmed the c.1584delG mutation as a de novo in the proband and that it was not present either in her parents, half-sister or half-brother. Clinically, the patient presented with extreme obesity, macrocephaly, hepatomegaly, hyperinsulinism and polycystic ovarian syndrome (PCOS), which have previously not been described in CSS patients. We suggest that obesity, macrocephaly, hepatomegaly and/or PCOS may be added to the list of clinical features of ARID1B mutations, but further clinical reports are required to make a definite conclusion.

Target site selection of Pseudomonas putida transposon Tn4652.

We analyzed the target preferences of a Tn3 family transposon Tn4652. Alignment of 93 different insertion sites revealed a consensus sequence which resembles that of Tn3, indicating that despite a low similarity between Tn4652 and Tn3 transposases, their target site recognition is conserved.

The ColRS two-component system regulates membrane functions and protects Pseudomonas putida against phenol.

As reported, the two-component system ColRS is involved in two completely different processes. It facilitates the root colonization ability of Pseudomonas fluorescens and is necessary for the Tn4652 transposition-dependent accumulation of phenol-utilizing mutants in Pseudomonas putida. To determine the role of the ColRS system in P. putida, we searched for target genes of response regulator ColR by use of a promoter library. Promoter screening was performed on phenol plates to mimic the conditions under which the effect of ColR on transposition was detected. The library screen revealed the porin-encoding gene oprQ and the alginate biosynthesis gene algD occurring under negative control of ColR. Binding of ColR to the promoter regions of oprQ and algD in vitro confirmed its direct involvement in regulation of these genes. Additionally, the porin-encoding gene ompA(PP0773) and the type I pilus gene csuB were also identified in the promoter screen. However, it turned out that ompA(PP0773) and csuB were actually affected by phenol and that the influence of ColR on these promoters was indirect. Namely, our results show that ColR is involved in phenol tolerance of P. putida. Phenol MIC measurement demonstrated that a colR mutant strain did not tolerate elevated phenol concentrations. Our data suggest that increased phenol susceptibility is also the reason for inhibition of transposition of Tn4652 in phenol-starving colR mutant bacteria. Thus, the current study revealed the role of the ColRS two-component system in regulation of membrane functionality, particularly in phenol tolerance of P. putida.