Impaired processing of FLP and NLP peptides in carboxypeptidase E (EGL-21)-deficient Caenorhabditis elegans as analyzed by mass spectrometry

Biologically active peptides are synthesized from inactive pre-proproteins or peptide precursors by the sequential actions of processing enzymes. Proprotein convertases cleave the precursor at pairs of basic amino acids, which are then removed from the carboxyl terminus of the generated fragments by a specific carboxypeptidase. Caenorhabditis elegans strains lacking proprotein convertase EGL-3 display a severely impaired neuropeptide profile (Husson et al. 2006, J. Neurochem.98, 1999-2012). In the present study, we examined the role of the C. elegans carboxypeptidase E orthologue EGL-21 in the processing of peptide precursors. More than 100 carboxy-terminally extended neuropeptides were detected in egl-21 mutant strains. These findings suggest that EGL-21 is a major carboxypeptidase involved in the processing of FMRFamide-like peptide (FLP) precursors and neuropeptide-like protein (NLP) precursors. The impaired peptide profile of egl-3 and egl-21 mutants is reflected in some similar phenotypes. They both share a severe widening of the intestinal lumen, locomotion defects, and retention of embryos. In addition, egl-3 animals have decreased intestinal fat content. Taken together, these results suggest that EGL-3 and EGL-21 are key enzymes for the proper processing of neuropeptides that control egg-laying, locomotion, fat storage and the nutritional status.status: publishe

Using a chat-based informed consent tool in large-scale genomic research

OBJECTIVE: We implemented a chatbot consent tool to shift the time burden from study staff in support of a national genomics research study. MATERIALS AND METHODS: We created an Institutional Review Board-approved script for automated chat-based consent. We compared data from prospective participants who used the tool or had traditional consent conversations with study staff. RESULTS: Chat-based consent, completed on a user\u27s schedule, was shorter than the traditional conversation. This did not lead to a significant change in affirmative consents. Within affirmative consents and declines, more prospective participants completed the chat-based process. A quiz to assess chat-based consent user understanding had a high pass rate with no reported negative experiences. CONCLUSION: Our report shows that a structured script can convey important information while realizing the benefits of automation and burden shifting. Analysis suggests that it may be advantageous to use chatbots to scale this rate-limiting step in large research projects

Increased diagnostic yield from negative whole genome-slice panels using automated reanalysis

We evaluated the diagnostic yield using genome-slice panel reanalysis in the clinical setting using an automated phenotype/gene ranking system. We analyzed whole genome sequencing (WGS) data produced from clinically ordered panels built as bioinformatic slices for 16 clinically diverse, undiagnosed cases referred to the Pediatric Mendelian Genomics Research Center, an NHGRI-funded GREGoR Consortium site. Genome-wide reanalysis was performed using Moon™, a machine-learning-based tool for variant prioritization. In five out of 16 cases, we discovered a potentially clinically significant variant. In four of these cases, the variant was found in a gene not included in the original panel due to phenotypic expansion of a disorder or incomplete initial phenotyping of the patient. In the fifth case, the gene containing the variant was included in the original panel, but being a complex structural rearrangement with intronic breakpoints outside the clinically analyzed regions, it was not initially identified. Automated genome-wide reanalysis of clinical WGS data generated during targeted panels testing yielded a 25% increase in diagnostic findings and a possibly clinically relevant finding in one additional case, underscoring the added value of analyses versus those routinely performed in the clinical setting

Targeting ferroptosis: A novel therapeutic strategy for the treatment of mitochondrial disease-related epilepsy.

BACKGROUND:Mitochondrial disease is a family of genetic disorders characterized by defects in the generation and regulation of energy. Epilepsy is a common symptom of mitochondrial disease, and in the vast majority of cases, refractory to commonly used antiepileptic drugs. Ferroptosis is a recently-described form of iron- and lipid-dependent regulated cell death associated with glutathione depletion and production of lipid peroxides by lipoxygenase enzymes. Activation of the ferroptosis pathway has been implicated in a growing number of disorders, including epilepsy. Given that ferroptosis is regulated by balancing the activities of glutathione peroxidase-4 (GPX4) and 15-lipoxygenase (15-LO), targeting these enzymes may provide a rational therapeutic strategy to modulate seizure. The clinical-stage therapeutic vatiquinone (EPI-743, α-tocotrienol quinone) was reported to reduce seizure frequency and associated morbidity in children with the mitochondrial disorder pontocerebellar hypoplasia type 6. We sought to elucidate the molecular mechanism of EPI-743 and explore the potential of targeting 15-LO to treat additional mitochondrial disease-associated epilepsies. METHODS:Primary fibroblasts and B-lymphocytes derived from patients with mitochondrial disease-associated epilepsy were cultured under standardized conditions. Ferroptosis was induced by treatment with the irreversible GPX4 inhibitor RSL3 or a combination of pharmacological glutathione depletion and excess iron. EPI-743 was co-administered and endpoints, including cell viability and 15-LO-dependent lipid oxidation, were measured. RESULTS:EPI-743 potently prevented ferroptosis in patient cells representing five distinct pediatric disease syndromes with associated epilepsy. Cytoprotection was preceded by a dose-dependent decrease in general lipid oxidation and the specific 15-LO product 15-hydroxyeicosatetraenoic acid (15-HETE). CONCLUSIONS:These findings support the continued clinical evaluation of EPI-743 as a therapeutic agent for PCH6 and other mitochondrial diseases with associated epilepsy

Rare predicted loss-of-function variants of type I IFN immunity genes are associated with life-threatening COVID-19.

BACKGROUND: We previously reported that impaired type I IFN activity, due to inborn errors of TLR3- and TLR7-dependent type I interferon (IFN) immunity or to autoantibodies against type I IFN, account for 15-20% of cases of life-threatening COVID-19 in unvaccinated patients. Therefore, the determinants of life-threatening COVID-19 remain to be identified in ~ 80% of cases. METHODS: We report here a genome-wide rare variant burden association analysis in 3269 unvaccinated patients with life-threatening COVID-19, and 1373 unvaccinated SARS-CoV-2-infected individuals without pneumonia. Among the 928 patients tested for autoantibodies against type I IFN, a quarter (234) were positive and were excluded. RESULTS: No gene reached genome-wide significance. Under a recessive model, the most significant gene with at-risk variants was TLR7, with an OR of 27.68 (95%CI 1.5-528.7, P = 1.1 × 10 CONCLUSIONS: Rare variants of TLR3- and TLR7-dependent type I IFN immunity genes can underlie life-threatening COVID-19, particularly with recessive inheritance, in patients under 60 years old