Pathogen dynamics and discovery of novel viruses and enzymes by deep nucleic acid sequencing of wastewater.

Wastewater contains an extensive reservoir of genetic information, yet largely unexplored. Here, we analyzed by high-throughput sequencing total nucleic acids extracted from wastewater samples collected during a 17 month-period in Berlin, Germany. By integrating global wastewater datasets and applying a novel computational approach to accurately identify viral strains within sewage RNA-sequencing data, we demonstrated the emergence and global dissemination of a specific astrovirus strain. Astrovirus abundance and sequence variation mirrored temporal and spatial patterns of infection, potentially serving as footprints of specific timeframes and geographical locations. Additionally, we revealed more than 100,000 sequence contigs likely originating from novel viral species, exhibiting distinct profiles in total RNA and DNA datasets and including undescribed bunyaviruses and parvoviruses. Finally, we identified thousands of new CRISPR-associated protein sequences, including Transposase B (TnpB), a class of compact, RNA-guided DNA editing enzymes. Collectively, our findings underscore the potential of high-throughput sequencing of total nucleic acids derived from wastewater for a broad range of applications.

Mutation-induced LZTR1 polymerization provokes cardiac pathology in recessive Noonan syndrome.

Noonan syndrome patients harboring causative variants in LZTR1 are particularly at risk to develop severe and early-onset hypertrophic cardiomyopathy. In this study, we investigate the mechanistic consequences of a homozygous variant LZTR1L580P by using patient-specific and CRISPR-Cas9-corrected induced pluripotent stem cell (iPSC) cardiomyocytes. Molecular, cellular, and functional phenotyping in combination with in silico prediction identify an LZTR1L580P-specific disease mechanism provoking cardiac hypertrophy. The variant is predicted to alter the binding affinity of the dimerization domains facilitating the formation of linear LZTR1 polymers. LZTR1 complex dysfunction results in the accumulation of RAS GTPases, thereby provoking global pathological changes of the proteomic landscape ultimately leading to cellular hypertrophy. Furthermore, our data show that cardiomyocyte-specific MRAS degradation is mediated by LZTR1 via non-proteasomal pathways, whereas RIT1 degradation is mediated by both LZTR1-dependent and LZTR1-independent pathways. Uni- or biallelic genetic correction of the LZTR1L580P missense variant rescues the molecular and cellular disease phenotype, providing proof of concept for CRISPR-based therapies.

The role of microRNAs in defining LSECs cellular identity and in regulating F8 gene expression.

Introduction: Coagulation Factor VIII (FVIII) plays a pivotal role in the coagulation cascade, and deficiencies in its levels, as seen in Hemophilia A, can lead to significant health implications. Liver sinusoidal endothelial cells (LSECs) are the main producers and contributors of FVIII in blood, a fact we have previously elucidated through mRNA expression profiling when comparing these cells to other endothelial cell types. Methods: Our current investigation focuses on small microRNAs, analyzing their distinct expression patterns across various endothelial cells and hepatocytes. Results: The outcome of this exploration underscores the discernible microRNAs expression differences that set LSECs apart from both hepatocytes (193 microRNAs at p < 0.05) and other endothelial cells (72 microRNAs at p < 0.05). Notably, the 134 and 35 overexpressed microRNAs in LSECs compared to hepatocytes and other endothelial cells, respectively, shed light on the unique functions of LSECs in the liver. Discussion: Our investigation identified a panel of 10 microRNAs (miR-429, miR-200b-3p, miR-200a-3p, miR-216b-5p, miR-1185-5p, miR-19b-3p, miR-192-5p, miR-122-5p, miR-30c-2-3p, and miR-30a-5p) that distinctly define LSEC identity. Furthermore, our scrutiny extended to microRNAs implicated in F8 regulation, revealing a subset (miR-122-5p, miR-214-3p, miR-204-3p, and miR-2682-5p) whose expression intricately correlates with F8 expression within LSECs. This microRNA cohort emerges as a crucial modulator of F8, both directly through suppression and indirect effects on established F8-related transcription factors. The above microRNAs emerged as potential targets for innovative therapies in Hemophilia A patients.

Bi-allelic PRRT2 variants may predispose to Self-limited Familial Infantile Epilepsy.

Heterozygous PRRT2 variants are frequently implicated in Self-limited Infantile Epilepsy, whereas homozygous variants are so far linked to severe presentations including developmental and epileptic encephalopathy, movement disorders, and intellectual disability. In a study aiming to explore the genetics of epilepsy in the Sudanese population, we investigated several families including a consanguineous family with three siblings diagnosed with self-limited infantile epilepsy. We evaluated both dominant and recessive inheritance using whole exome sequencing and genomic arrays. We identified a pathogenic homozygous splice-site variant in the first intron of PRRT2 [NC_000016.10(NM_145239.3):c.-65-1G > A] that segregated with the phenotype in this family. This work taps into the genetics of epilepsy in an underrepresented African population and suggests that the phenotypes of homozygous PRRT2 variants may include milder epilepsy presentations without movement disorders.