Identification of 16 novel Alzheimer's disease susceptibility loci using multi-ancestry meta-analyses of clinical Alzheimer's disease and AD-by-proxy cases from four whole genome sequencing datasets.

Alzheimer's disease (AD) is the most prevalent form of dementia. While many AD-associated genetic determinants have been previously identified, few studies have analyzed individuals of non-European ancestry. Here, we describe a multi-ancestry genome-wide association study of clinically-diagnosed AD and AD-by-proxy using whole genome sequencing data from NIAGADS, NIMH, UKB, and All of Us (AoU) consisting of 49,149 cases (12,074 clinically-diagnosed and 37,075 AD-by-proxy) and 383,225 controls. Nearly half of NIAGADS and AoU participants are of non-European ancestry. For clinically-diagnosed AD, we identified 14 new loci - five common (FBN2,/SCL27A6, AC090115.1, DYM, KCNG1/AL121785.1, TIAM1) and nine rare (VWA5B1, RNU6-755P/LMX1A, MOB1A, MORC1-AS1, LINC00989, PDE4D, RNU2-49P/CDO1, NEO1, and SLC35G3/AC022916.1). Meta-analysis of UKB and AoU AD-by-proxy cases yielded two new rare loci (RPL23/LASP1 and CEBPA/ AC008738.6) which were also nominally significant in NIAGADS. In summary, we provide evidence for 16 novel AD loci and advocate for more studies using WGS-based GWAS of diverse cohorts.

Joint, multifaceted genomic analysis enables diagnosis of diverse, ultra-rare monogenic presentations.

Genomics for rare disease diagnosis has advanced at a rapid pace due to our ability to perform "N-of-1" analyses on individual patients with ultra-rare diseases. The increasing sizes of ultra-rare disease cohorts internationally newly enables cohort-wide analyses for new discoveries, but well-calibrated statistical genetics approaches for jointly analyzing these patients are still under development.1,2 The Undiagnosed Diseases Network (UDN) brings multiple clinical, research and experimental centers under the same umbrella across the United States to facilitate and scale N-of-1 analyses. Here, we present the first joint analysis of whole genome sequencing data of UDN patients across the network. We introduce new, well-calibrated statistical methods for prioritizing disease genes with de novo recurrence and compound heterozygosity. We also detect pathways enriched with candidate and known diagnostic genes. Our computational analysis, coupled with a systematic clinical review, recapitulated known diagnoses and revealed new disease associations. We further release a software package, RaMeDiES, enabling automated cross-analysis of deidentified sequenced cohorts for new diagnostic and research discoveries. Gene-level findings and variant-level information across the cohort are available in a public-facing browser (https://dbmi-bgm.github.io/udn-browser/). These results show that N-of-1 efforts should be supplemented by a joint genomic analysis across cohorts.

SARS-CoV-2 rapidly evolves lineage-specific phenotypic differences when passaged repeatedly in immune-naïve mice.

The persistence of SARS-CoV-2 despite the development of vaccines and a degree of herd immunity is partly due to viral evolution reducing vaccine and treatment efficacy. Serial infections of wild-type (WT) SARS-CoV-2 in Balb/c mice yield mouse-adapted strains with greater infectivity and mortality. We investigate if passaging unmodified B.1.351 (Beta) and B.1.617.2 (Delta) 20 times in K18-ACE2 mice, expressing the human ACE2 receptor, in a BSL-3 laboratory without selective pressures, drives human health-relevant evolution and if evolution is lineage-dependent. Late-passage virus causes more severe disease, at organism and lung tissue scales, with late-passage Delta demonstrating antibody resistance and interferon suppression. This resistance co-occurs with a de novo spike S371F mutation, linked with both traits. S371F, an Omicron-characteristic mutation, is co-inherited at times with spike E1182G per Nanopore sequencing, existing in different within-sample viral variants at others. Both S371F and E1182G are linked to mammalian GOLGA7 and ZDHHC5 interactions, which mediate viral-cell entry and antiviral response. This study demonstrates SARS-CoV-2's tendency to evolve with phenotypic consequences, its evolution varying by lineage, and suggests non-dominant quasi-species contribution.

Alternative splicing in lung influences COVID-19 severity and respiratory diseases.

Alternative splicing generates functional diversity in isoforms, impacting immune response to infection. Here, we evaluate the causal role of alternative splicing in COVID-19 severity and susceptibility by applying two-sample Mendelian randomization to cis-splicing quantitative trait loci and the results from COVID-19 Host Genetics Initiative. We identify that alternative splicing in lung, rather than total expression of OAS1, ATP11A, DPP9 and NPNT, is associated with COVID-19 severity. MUC1 and PMF1 splicing is associated with COVID-19 susceptibility. Colocalization analyses support a shared genetic mechanism between COVID-19 severity with idiopathic pulmonary fibrosis at the ATP11A and DPP9 loci, and with chronic obstructive lung diseases at the NPNT locus. Last, we show that ATP11A, DPP9, NPNT, and MUC1 are highly expressed in lung alveolar epithelial cells, both in COVID-19 uninfected and infected samples. These findings clarify the importance of alternative splicing in lung for COVID-19 and respiratory diseases, providing isoform-based targets for drug discovery.

Colocalization of expression transcripts with COVID-19 outcomes is rare across cell states, cell types and organs.

Identifying causal genes at GWAS loci can help pinpoint targets for therapeutic interventions. Expression studies can disentangle such loci but signals from expression quantitative trait loci (eQTLs) often fail to colocalize-which means that the genetic control of measured expression is not shared with the genetic control of disease risk. This may be because gene expression is measured in the wrong cell type, physiological state, or organ. We tested whether Mendelian randomization (MR) could identify genes at loci influencing COVID-19 outcomes and whether the colocalization of genetic control of expression and COVID-19 outcomes was influenced by cell type, cell stimulation, and organ. We conducted MR of cis-eQTLs from single cell (scRNA-seq) and bulk RNA sequencing. We then tested variables that could influence colocalization, including cell type, cell stimulation, RNA sequencing modality, organ, symptoms of COVID-19, and SARS-CoV-2 status among individuals with symptoms of COVID-19. The outcomes used to test colocalization were COVID-19 severity and susceptibility as assessed in the Host Genetics Initiative release 7. Most transcripts identified using MR did not colocalize when tested across cell types, cell state and in different organs. Most that did colocalize likely represented false positives due to linkage disequilibrium. In general, colocalization was highly variable and at times inconsistent for the same transcript across cell type, cell stimulation and organ. While we identified factors that influenced colocalization for select transcripts, identifying 33 that mediate COVID-19 outcomes, our study suggests that colocalization of expression with COVID-19 outcomes is partially due to noisy signals even after following quality control and sensitivity testing. These findings illustrate the present difficulty of linking expression transcripts to disease outcomes and the need for skepticism when observing eQTL MR results, even accounting for cell types, stimulation state and different organs.

Proteome-wide Mendelian randomization implicates nephronectin as an actionable mediator of the effect of obesity on COVID-19 severity.

Obesity is a major risk factor for Coronavirus disease (COVID-19) severity; however, the mechanisms underlying this relationship are not fully understood. As obesity influences the plasma proteome, we sought to identify circulating proteins mediating the effects of obesity on COVID-19 severity in humans. Here, we screened 4,907 plasma proteins to identify proteins influenced by body mass index using Mendelian randomization. This yielded 1,216 proteins, whose effect on COVID-19 severity was assessed, again using Mendelian randomization. We found that an s.d. increase in nephronectin (NPNT) was associated with increased odds of critically ill COVID-19 (OR = 1.71, P = 1.63 × 10-10). The effect was driven by an NPNT splice isoform. Mediation analyses supported NPNT as a mediator. In single-cell RNA-sequencing, NPNT was expressed in alveolar cells and fibroblasts of the lung in individuals who died of COVID-19. Finally, decreasing body fat mass and increasing fat-free mass were found to lower NPNT levels. These findings provide actionable insights into how obesity influences COVID-19 severity.

Genomic atlas of the plasma metabolome prioritizes metabolites implicated in human diseases.

Metabolic processes can influence disease risk and provide therapeutic targets. By conducting genome-wide association studies of 1,091 blood metabolites and 309 metabolite ratios, we identified associations with 690 metabolites at 248 loci and associations with 143 metabolite ratios at 69 loci. Integrating metabolite-gene and gene expression information identified 94 effector genes for 109 metabolites and 48 metabolite ratios. Using Mendelian randomization (MR), we identified 22 metabolites and 20 metabolite ratios having estimated causal effect on 12 traits and diseases, including orotate for estimated bone mineral density, α-hydroxyisovalerate for body mass index and ergothioneine for inflammatory bowel disease and asthma. We further measured the orotate level in a separate cohort and demonstrated that, consistent with MR, orotate levels were positively associated with incident hip fractures. This study provides a valuable resource describing the genetic architecture of metabolites and delivers insights into their roles in common diseases, thereby offering opportunities for therapeutic targets.

ELF5 is a potential respiratory epithelial cell-specific risk gene for severe COVID-19.

Despite two years of intense global research activity, host genetic factors that predispose to a poorer prognosis of COVID-19 infection remain poorly understood. Here, we prioritise eight robust (e.g., ELF5) or suggestive but unreported (e.g., RAB2A) candidate protein mediators of COVID-19 outcomes by integrating results from the COVID-19 Host Genetics Initiative with population-based plasma proteomics using statistical colocalisation. The transcription factor ELF5 (ELF5) shows robust and directionally consistent associations across different outcome definitions, including a >4-fold higher risk (odds ratio: 4.88; 95%-CI: 2.47-9.63; p-value < 5.0 × 10-6) for severe COVID-19 per 1 s.d. higher genetically predicted plasma ELF5. We show that ELF5 is specifically expressed in epithelial cells of the respiratory system, such as secretory and alveolar type 2 cells, using single-cell RNA sequencing and immunohistochemistry. These cells are also likely targets of SARS-CoV-2 by colocalisation with key host factors, including ACE2 and TMPRSS2. In summary, large-scale human genetic studies together with gene expression at single-cell resolution highlight ELF5 as a risk gene for severe COVID-19, supporting a role of epithelial cells of the respiratory system in the adverse host response to SARS-CoV-2.

A tetraethylene glycol coat gives gold nanoparticles long in vivo half-lives with minimal increase in size.

In this study, we describe the experiments determining whether coating gold nanoparticles with tetraethylene glycol (TEG) provides pharmacologically relevant advantages, such as increased serum half-life and resistance to protein adsorption. Monodisperse TEG-coated, NaBH4-reduced gold nanoparticles with a hydrodynamic size comparable to albumin were synthesized by reducing gold chloride with NaBH4 under alkaline conditions in the presence of TEG-SH. The particles were characterized by gel electrophoresis, column chromatography, and transmission electron microscopy. The nanoparticles were subsequently injected intravenously into mice, and their half-lives and final destinations were determined via photometric analysis, light microscopy (LM), and transmission electron microscopy. The TEG particles had a long half-life (~400 minutes) that was not influenced by splenectomy. After 500 minutes of injection, TEG particles were found in kidney proximal tubule cell vesicles and in spleen red and white pulp. The particles induced apoptosis in the spleen red pulp but not in white pulp or the kidney. Some of the TEG particles appeared to have undergone ligand exchange reactions that increased their charge. The TEG particles were shown to be resistant to nonspecific protein adsorption, as judged by gel electrophoresis and column chromatography. These results demonstrate that naturally monodisperse, small-sized gold nanoparticles coated with TEG have long in vivo plasma half-lives, are minimally toxic, and are resistant to protein adsorption. This suggests that a TEG coating should be considered as an alternative to a polyethylene glycol coating, which is polydisperse and of much larger size.

Permeation of macromolecules into the renal glomerular basement membrane and capture by the tubules.

How the kidney prevents urinary excretion of plasma proteins continues to be debated. Here, using unfixed whole-mount mouse kidneys, we show that fluorescent-tagged proteins and neutral dextrans permeate into the glomerular basement membrane (GBM), in general agreement with Ogston's 1958 equation describing how permeation into gels is related to molecular size. Electron-microscopic analyses of kidneys fixed seconds to hours after injecting gold-tagged albumin, negatively charged gold nanoparticles, and stable oligoclusters of gold nanoparticles show that permeation into the lamina densa of the GBM is size-sensitive. Nanoparticles comparable in size with IgG dimers do not permeate into it. IgG monomer-sized particles permeate to some extent. Albumin-sized particles permeate extensively into the lamina densa. Particles traversing the lamina densa tend to accumulate upstream of the podocyte glycocalyx that spans the slit, but none are observed upstream of the slit diaphragm. At low concentrations, ovalbumin-sized nanoparticles reach the primary filtrate, are captured by proximal tubule cells, and are endocytosed. At higher concentrations, tubular capture is saturated, and they reach the urine. In mouse models of Pierson's or Alport's proteinuric syndromes resulting from defects in GBM structural proteins (laminin ß2 or collagen α3 IV), the GBM is irregularly swollen, the lamina densa is absent, and permeation is increased. Our observations indicate that size-dependent permeation into the lamina densa of the GBM and the podocyte glycocalyx, together with saturable tubular capture, determines which macromolecules reach the urine without the need to invoke direct size selection by the slit diaphragm.