Regional Variation in Cardiovascular Genes Enables a Tractable Genome Editing Strategy.

BACKGROUND: To realize the potential of genome engineering therapeutics, tractable strategies must be identified that balance personalized therapy with the need for off-the-shelf availability. We hypothesized that regional clustering of pathogenic variants can inform the design of rational prime editing therapeutics to treat the majority of genetic cardiovascular diseases with a limited number of reagents. METHODS: We collated 2435 high-confidence pathogenic/likely pathogenic (P/LP) variants in 82 cardiovascular disease genes from ClinVar. We assessed the regional density of these variants by defining a regional clustering index. We then combined a highly active base editor with prime editing to demonstrate the feasibility of a P/LP hotspot-directed genome engineering therapeutic strategy in vitro. RESULTS: P/LP variants in cardiovascular disease genes display higher regional density than rare variants found in the general population. P/LP missense variants displayed higher average regional density than P/LP truncating variants. Following hypermutagenesis at a pathogenic hotspot, mean prime editing efficiency across introduced variants was 57±27%. CONCLUSIONS: Designing therapeutics that target pathogenic hotspots will not only address known missense P/LP variants but also novel P/LP variants identified in these hotspots as well. Moreover, the clustering of P/LP missense rather than truncating variants in these hotspots suggests that prime editing technology is particularly valuable for dominant negative disease. Although prime editing technology in relation to cardiac health continues to improve, this study presents an approach to targeting the most impactful regions of the genome for inherited cardiovascular disease.

Deconvoluting complex correlates of COVID-19 severity with a multi-omic pandemic tracking strategy.

The SARS-CoV-2 pandemic has differentially impacted populations across race and ethnicity. A multi-omic approach represents a powerful tool to examine risk across multi-ancestry genomes. We leverage a pandemic tracking strategy in which we sequence viral and host genomes and transcriptomes from nasopharyngeal swabs of 1049 individuals (736 SARS-CoV-2 positive and 313 SARS-CoV-2 negative) and integrate them with digital phenotypes from electronic health records from a diverse catchment area in Northern California. Genome-wide association disaggregated by admixture mapping reveals novel COVID-19-severity-associated regions containing previously reported markers of neurologic, pulmonary and viral disease susceptibility. Phylodynamic tracking of consensus viral genomes reveals no association with disease severity or inferred ancestry. Summary data from multiomic investigation reveals metagenomic and HLA associations with severe COVID-19. The wealth of data available from residual nasopharyngeal swabs in combination with clinical data abstracted automatically at scale highlights a powerful strategy for pandemic tracking, and reveals distinct epidemiologic, genetic, and biological associations for those at the highest risk.

Wnt Signaling Interactor WTIP (Wilms Tumor Interacting Protein) Underlies Novel Mechanism for Cardiac Hypertrophy.

BACKGROUND: The study of hypertrophic cardiomyopathy (HCM) can yield insight into the mechanisms underlying the complex trait of cardiac hypertrophy. To date, most genetic variants associated with HCM have been found in sarcomeric genes. Here, we describe a novel HCM-associated variant in the noncanonical Wnt signaling interactor WTIP (Wilms tumor interacting protein) and provide evidence of a role for WTIP in complex disease. METHODS: In a family affected by HCM, we used exome sequencing and identity-by-descent analysis to identify a novel variant in WTIP (p.Y233F). We knocked down WTIP in isolated neonatal rat ventricular myocytes with lentivirally delivered short hairpin ribonucleic acids and in Danio rerio via morpholino injection. We performed weighted gene coexpression network analysis for WTIP in human cardiac tissue, as well as association analysis for WTIP variation and left ventricular hypertrophy. Finally, we generated induced pluripotent stem cell-derived cardiomyocytes from patient tissue, characterized size and calcium cycling, and determined the effect of verapamil treatment on calcium dynamics. RESULTS: WTIP knockdown caused hypertrophy in neonatal rat ventricular myocytes and increased cardiac hypertrophy, peak calcium, and resting calcium in D rerio. Network analysis of human cardiac tissue indicated WTIP as a central coordinator of prohypertrophic networks, while common variation at the WTIP locus was associated with human left ventricular hypertrophy. Patient-derived WTIP p.Y233F-induced pluripotent stem cell-derived cardiomyocytes recapitulated cellular hypertrophy and increased resting calcium, which was ameliorated by verapamil. CONCLUSIONS: We demonstrate that a novel genetic variant found in a family with HCM disrupts binding to a known Wnt signaling protein, misregulating cardiomyocyte calcium dynamics. Further, in orthogonal model systems, we show that expression of the gene WTIP is important in complex cardiac hypertrophy phenotypes. These findings, derived from the observation of a rare Mendelian disease variant, uncover a novel disease mechanism with implications across diverse forms of cardiac hypertrophy.

Free-Living, Psychrotrophic Bacteria of the Genus Psychrobacter Are Descendants of Pathobionts.

Host-adapted microorganisms are generally assumed to have evolved from free-living, environmental microorganisms, as examples of the reverse process are rare. In the phylum Gammaproteobacteria, family Moraxellaceae, the genus Psychrobacter includes strains from a broad ecological distribution including animal bodies as well as sea ice and other nonhost environments. To elucidate the relationship between these ecological niches and Psychrobacter's evolutionary history, we performed tandem genomic analyses with phenotyping of 85 Psychrobacter accessions. Phylogenomic analysis of the family Moraxellaceae reveals that basal members of the Psychrobacter clade are Moraxella spp., a group of often-pathogenic organisms. Psychrobacter exhibited two broad growth patterns in our phenotypic screen: one group that we called the "flexible ecotype" (FE) had the ability to grow between 4 and 37°C, and the other, which we called the "restricted ecotype" (RE), could grow between 4 and 25°C. The FE group includes phylogenetically basal strains, and FE strains exhibit increased transposon copy numbers, smaller genomes, and a higher likelihood to be bile salt resistant. The RE group contains only phylogenetically derived strains and has increased proportions of lipid metabolism and biofilm formation genes, functions that are adaptive to cold stress. In a 16S rRNA gene survey of polar bear fecal samples, we detect both FE and RE strains, but in in vivo colonizations of gnotobiotic mice, only FE strains persist. Our results indicate the ability to grow at 37°C, seemingly necessary for mammalian gut colonization, is an ancestral trait for Psychrobacter, which likely evolved from a pathobiont.IMPORTANCE Host-associated microbes are generally assumed to have evolved from free-living ones. The evolutionary transition of microbes in the opposite direction, from host associated toward free living, has been predicted based on phylogenetic data but not studied in depth. Here, we provide evidence that the genus Psychrobacter, particularly well known for inhabiting low-temperature, high-salt environments such as sea ice, permafrost soils, and frozen foodstuffs, has evolved from a mammalian-associated ancestor. We show that some Psychrobacter strains retain seemingly ancestral genomic and phenotypic traits that correspond with host association while others have diverged to psychrotrophic or psychrophilic lifestyles.

High-throughput SARS-CoV-2 and host genome sequencing from single nasopharyngeal swabs.

During COVID19 and other viral pandemics, rapid generation of host and pathogen genomic data is critical to tracking infection and informing therapies. There is an urgent need for efficient approaches to this data generation at scale. We have developed a scalable, high throughput approach to generate high fidelity low pass whole genome and HLA sequencing, viral genomes, and representation of human transcriptome from single nasopharyngeal swabs of COVID19 patients.

The NIa-Pro protein of Turnip mosaic virus improves growth and reproduction of the aphid vector, Myzus persicae (green peach aphid).

Many plant viruses depend on aphids and other phloem-feeding insects for transmission within and among host plants. Thus, viruses may promote their own transmission by manipulating plant physiology to attract aphids and increase aphid reproduction. Consistent with this hypothesis, Myzus persicae (green peach aphids) prefer to settle on Nicotiana benthamiana infected with Turnip mosaic virus (TuMV) and fecundity on virus-infected N. benthamiana and Arabidopsis thaliana (Arabidopsis) is higher than on uninfected controls. TuMV infection suppresses callose deposition, an important plant defense, and increases the amount of free amino acids, the major source of nitrogen for aphids. To investigate the underlying molecular mechanisms of this phenomenon, 10 TuMV genes were over-expressed in plants to determine their effects on aphid reproduction. Production of a single TuMV protein, nuclear inclusion a-protease domain (NIa-Pro), increased M. persicae reproduction on both N. benthamiana and Arabidopsis. Similar to the effects that are observed during TuMV infection, NIa-Pro expression alone increased aphid arrestment, suppressed callose deposition and increased the abundance of free amino acids. Together, these results suggest a function for the TuMV NIa-Pro protein in manipulating the physiology of host plants. By attracting aphid vectors and promoting their reproduction, TuMV may influence plant-aphid interactions to promote its own transmission.