Incidental germline findings during molecular profiling of tumor tissues for precision oncology: molecular survey and methodological obstacles

Background A fraction of patients referred for complex molecular profiling of biopsied tumors may harbor germline variants in genes associated with the development of hereditary cancer syndromes (HCS). Neither the bioinformatic analysis nor the reporting of such incidental germline findings are standardized. Methods Data from Next-Generation Sequencing (NGS) of biopsied tumor samples referred for complex molecular profiling were analyzed for germline variants in HCS-associated genes. Analysis of variant origin was performed employing bioinformatic algorithms followed by manual curation. When possible, the origin of the variant was validated by Sanger sequencing of the sample of normal tissue. The variants’ pathogenicity was assessed according to ACMG/AMP. Results Tumors were sampled from 183 patients (Males: 75 [41.0%]; Females: 108 [59.0%]; mean [SD] age, 57.7 [13.3] years) and analysed by targeted NGS. The most common tumor types were colorectal (19%), pancreatic (13%), and lung cancer (10%). A total of 56 sequence variants in genes associated with HCS were detected in 40 patients. Of them, 17 variants found in 14 patients were predicted to be of germline origin, with 6 variants interpreted as pathogenic (PV) or likely pathogenic (LPV), and 9 as variants of uncertain significance (VUS). For the 41 out of 42 (97%) missense variants in HCS-associated genes, the results of computational prediction of variant origin were concordant with that of experimental examination. We estimate that Sanger sequencing of a sample of normal tissue would be required for ~ 1–7% of the total assessed cases with PV or LPV, when necessity to follow with genetic counselling referral in ~ 2–15% of total assessed cases (PV, LPV or VUS found in HCS genes). Conclusion Incidental findings of pathogenic germline variants are common in data from cancer patients referred for complex molecular profiling. We propose an algorithm for the management of patients with newly detected variants in genes associated with HCS.

SAG3 Toxoplasma gondii cloning reveals unexpected fivefold infection in the blood of feral cats in the Mexican Caribbean

Background Currently, more than 300 genotypes of Toxoplasma gondii (T. gondii) have been described throughout the world, demonstrating its wide genetic diversity. The SAG3 locus is one of the genes included in the genotyping panel of this parasite. It is associated with its virulence since it participates during the invasion process of the host cells. Therefore, cloning, sequencing, and bioinformatic analysis were used to deepen the understanding of the SAG3 locus genetic diversity of T. gondii in blood samples from feral cats. Results Six different SAG3 sequences were detected, five of which were detected in one feline. Three sequences were first reported here; one of them was an intragenic recombinant. In the cladogram, four out of ten SAG3 sequences did not share nodes with others reported worldwide. Conclusions Cloning and sequencing of samples with more than one restriction pattern by PCR-RFLP were very helpful tools to demonstrate the presence of more than three genotypes of T. gondii in the blood of feral cats from southeastern Mexico. This suggests a potential mixed infection of multiple T. gondii strains and high genetic diversity of the parasites in felines in this tropical region of Mexico.

Relationship Between the Pyroptosis Pathway and Epilepsy: A Bioinformatic Analysis

PurposeThis study aimed to analyse the correlation between the pyroptosis pathway and epilepsy using bioinformatics analysis technology. We analyzed the expression of gasdermin D (GSDMD) and gasdermin E (GSDME), the key molecules of pyroptosis, in kainic acid-induced epileptic mice.MethodsWeighted gene co-expression network analysis (WGCNA) was used to construct a signed co-expression network from expression data to screen gene sets closely related to epilepsy. The correlation between the module and epilepsy was verified through module conservative analysis, gene ontology (GO) annotation analysis, and correlation analysis with known epilepsy genes. We obtained currently recognized pyroptosis-related molecules through literature review, and correlation analysis was used to evaluate their correlation with epilepsy. Differentially expressed gene (DEG) analysis was used to analyse expression changes of pyroptosis-related molecules at the transcriptome level, compared to the sham group. We subsequently established a kainic acid-induced status epilepticus (SE) model in mice and validated the mRNA and protein expression of GSDMD and GSDME, the key molecules of pyroptosis, by quantitative reverse transcription PCR (qRT-PCR) and western blotting (WB).ResultsUsing WGCNA, module conservative analysis, and correlation analysis with known epilepsy genes, we screened out a module (a gene set of interest) closely related to epilepsy that was prominently enriched in immune and inflammatory-related biological processes. Correlation analysis results suggest that pyroptosis-related molecules are closely related to this module, but have no obvious correlation with others. DEG analysis of molecules associated with pyroptosis suggests that most of the pyroptosis-related molecules had significantly increased expression after SE, such as IL1b, Casp1, Casp4, Pycard, Gsdmd, Nlrp3, Aim2, Mefv, Tlr2, Tlr3, and Tlr4. qRT-PCR and WB analysis confirmed that the mRNA and protein levels of GSDMD in the mouse hippocampus were significantly upregulated after SE. The mRNA expression of GSDME was not different between the epilepsy group and sham group. However, the WB results showed that the expression of full-length GSDME was decreased and GSDME-N-terminus were significantly increased after SE.ConclusionsOur study highlights that the pyroptosis pathway may be closely related to epilepsy. GSDMD and GSDME, the key executive molecules of pyroptosis, will help to understand the pathogenesis of epilepsy and aid in discovering new targets for anti-epileptic drug treatments.

Analysis of LINE1 Retrotransposons in Huntington’s Disease

Transposable elements (TEs) are mobile genetic elements that made up about half the human genome. Among them, the autonomous non-LTR retrotransposon long interspersed nuclear element-1 (L1) is the only currently active TE in mammals and covers about 17% of the mammalian genome. L1s exert their function as structural elements in the genome, as transcribed RNAs to influence chromatin structure and as retrotransposed elements to shape genomic variation in somatic cells. L1s activity has been shown altered in several diseases of the nervous system. Huntington disease (HD) is a dominantly inherited neurodegenerative disorder caused by an expansion of a CAG repeat in the HTT gene which leads to a gradual loss of neurons most prominently in the striatum and, to a lesser extent, in cortical brain regions. The length of the expanded CAG tract is related to age at disease onset, with longer repeats leading to earlier onset. Here we carried out bioinformatic analysis of public RNA-seq data of a panel of HD mouse models showing that a decrease of L1 RNA expression recapitulates two hallmarks of the disease: it correlates to CAG repeat length and it occurs in the striatum, the site of neurodegeneration. Results were then experimentally validated in HttQ111 knock-in mice. The expression of L1-encoded proteins was independent from L1 RNA levels and differentially regulated in time and tissues. The pattern of expression L1 RNAs in human HD post-mortem brains showed similarity to mouse models of the disease. This work suggests the need for further study of L1s in HD and adds support to the current hypothesis that dysregulation of TEs may be involved in neurodegenerative diseases.

Identification and Expression Profile of Chemosensory Receptor Genes in Aromia bungii (Faldermann) Antennal Transcriptome

The red-necked longicorn beetle, Aromia bungii (Faldermann) (Coleoptera: Cerambycidae), is a major destructive, wood-boring pest, which is widespread throughout the world. The sex pheromone of A. bungii was reported earlier; however, the chemosensory mechanism of the beetle remains almost unknown. In this study, 45 AbunORs, 6 AbunGRs and 2 AbunIRs were identified among 42,197 unigenes derived from the antennal transcriptome bioinformatic analysis of A. bungii adults. The sequence of putative Orco (AbunOR25) found in this study is highly conserved with the known Orcos from other Coleoptera species, and these Orco genes might be potentially used as target genes for the future development of novel and effective control strategies. Tissue expression analysis showed that 29 AbunOR genes were highly expressed in antennae, especially in the antennae of females, which was consistent with the idea that females might express more pheromone receptors for sensing pheromones, especially the sex pheromones produced by males. AbunOR5, 29, 31 and 37 were clustered with the pheromone receptors of the cerambycid Megacyllene caryae, suggesting that they might be putative pheromone receptors of A. bungii. All six AbunGRs were highly expressed in the mouthparts, indicating that these GRs may be involved in the taste perception process. Both AbunIRs were shown to be female-mouthparts-biased, suggesting that they might also be related to the tasting processes. Our study provides some basic information towards a deeper understanding of the chemosensing mechanism of A. bungii at a molecular level.

Mutational screening of the TPO and DUOX2 genes in Argentinian children with congenital hypothyroidism due to thyroid dyshormonogenesis.

Purpose Primary congenital hypothyroidism (CH) is the most common endocrine disease in children and one of the preventable causes of both cognitive and motor deficits. We present a genetic and bioinformatics investigation of rational clinical design in 16 Argentine patients suspected of CH due to thyroid dyshormonogenesis (TDH). Methods Next-Generation Sequencing approach was used to identify variants in Thyroid Peroxidase (TPO) and Dual Oxidase 2 (DUOX2) genes. A custom panel targeting 7 genes associated with TDH [(TPO, Iodothyrosine Deiodinase I (IYD), Solute Carrier Family 26 Member 4 (SLC26A4), Thyroglobulin (TG), (DUOX2), Dual Oxidase Maturation Factor 2 (DUOXA2), Solute Carrier Family 5 Member 5 (SLC5A5)] and 4 associated with thyroid dysembryogenesis [PAX8, FOXE1, NKX2-1, Thyroid Stimulating Hormone Receptor (TSHR)] has been designed. Additionally, bioinformatic analysis and structural modeling were carried out to predict the disease-causing potential variants. Results Five novel variants have been identified, two in TPO: c.2749-2A>C and c.2752_2753delAG, [p.Ser918Cysfs*62] and three variants in DUOX2 gene: c.425C>G [p.Pro142Arg]; c.790delC [p.Leu264Cysfs*57] and c.2695delC [p.Gln899Serfs*21]. Seventeen identified TPO, DUOX2 and IYD variants were previously described. We identified potentially pahogenic bi-allelic variants in TPO and DUOX2 in 8 and 2 patients, respectively. We also detected a potentially pathogenic mono-allelic variant in TPO and DUOX2 in 4 and 1 patients respectively. Only two patients were heterozygous for digenic variants in TPO/IYD and in TPO/DUOX2 genes. Conclusions 22 variants have been identified associated with TDH. All described novel mutations occur in domains important for protein structure and function, predicting the TDH phenotype.

Revisiting the Non-Coding Nature of Pospiviroids

Viroids are small, circular, highly structured pathogens that infect a broad range of plants, causing economic losses. Since their discovery in the 1970s, they have been considered as non-coding pathogens. In the last few years, the discovery of other RNA entities, similar in terms of size and structure, that were shown to be translated (e.g., cirRNAs, precursors of miRNA, RNA satellites) as well as studies showing that some viroids are located in ribosomes, have reignited the idea that viroids may be translated. In this study, we used advanced bioinformatic analysis, in vitro experiments and LC-MS/MS to search for small viroid peptides of the PSTVd. Our results suggest that in our experimental conditions, even though the circular form of PSTVd is found in ribosomes, no produced peptides were identified. This indicates that the presence of PSTVd in ribosomes is most probably not related to peptide production but rather to another unknown function that requires further study.

A Novel Metastasis-related Genes Based Signature for Predicting the Progression-free Interval of Patients With Papillary Thyroid Carcinoma

Background: We aimed to build a novel model with metastasis-related genes (MTGs) signature and relevant clinical parameters for predicting progression-free interval (PFI) after surgery for papillary thyroid carcinoma (PTC).Methods: We performed a bioinformatic analysis of integrated PTC datasets with the MTGs to identify differentially expressed MTGs (DE-MTGs). Then we generated PFI-related DE-MTGs and established a novel MTGs based signature. After that, we validated the signature on multiple datasets and PTC cell lines. Further, we carried out uni- and multivariate analysis to identify independent prognostic characters. Finally, we established a signature and clinical parameters-based nomogram for predicting the PFI of PTC. Results: We identified 155 DE-MTGs related to PFI in PTC. The functional enrichment analysis showed that the DE-MTGs were associated with an essential oncogenic process. Consequently, we found a novel 10-gene signature and could distinguish patients with poorer prognoses and predicted PFI accurately. The novel signature had a C-index of 0.76 and the relevant nomogram had a C-index of 0.80. Also, it was closely related to pivotal clinical characters of datasets and invasiveness of cell lines. And the signature was confirmed a significant independent prognostic factor in PTC. Finally, we built a nomogram by including the signature and relevant clinical factors. Validation analysis showed that the nomogram's efficacy was satisfying in predicting PTC’s PFI. Conclusions: The MTG signature and nomogram were closely associated with PTC prognosis and may help clinicians improve the individualized prediction of PFI, especially for high-risk patients after surgery.

MiR-423-5p prevents MALAT1-mediated proliferation and metastasis in prostate cancer

Background The long non-coding RNA (lncRNA), MALAT1, plays a key role in the development of different cancers, and its expression is associated with worse prognosis in patients. However, its mechanism of action and its regulation are not well known in prostate cancer (PCa). A general mechanism of action of lncRNAs is their interaction with other epigenetic regulators including microRNAs (miRNAs). Methods Using lentiviral stable miRNA transfection together with cell biology functional assays and gene expression/target analysis, we investigated the interaction between MALAT1 and miR-423-5p, defined as a target with in silico prediction analysis, in PCa. Results Through bioinformatic analysis of data available from TCGA, we have found that MALAT1 expression correlates with high Gleason grade, metastasis occurrence, and reduced survival in PCa patients. These findings were validated on a TMA of PCa showing a significant correlation between MALAT1 expression with both stage and grading. We report that, in PCa cells, MALAT1 expression and activity is regulated by miR-423-5p that binds MALAT1, downregulates its expression and inhibits its activity in promoting proliferation, migration, and invasion. Using NanoString analysis, we unraveled downstream cell pathways that were affected by miR-423-5p expression and MALAT1 downregulation and identified several alterations in genes that are involved in metastatic response and angiogenic pathways. In addition, we showed that the overexpression of miR-423-5p increases survival and decreases metastases formation in a xenograft mouse model. Conclusions We provide evidence on the role of MALAT1 in PCa tumorigenesis and progression. Also, we identify a direct interaction between miR-423-5p and MALAT1, which results in the suppression of MALAT1 action in PCa.