Loss of δ-catenin function in severe autism.

Autism is a multifactorial neurodevelopmental disorder affecting more males than females; consequently, under a multifactorial genetic hypothesis, females are affected only when they cross a higher biological threshold. We hypothesize that deleterious variants at conserved residues are enriched in severely affected patients arising from female-enriched multiplex families with severe disease, enhancing the detection of key autism genes in modest numbers of cases. Here we show the use of this strategy by identifying missense and dosage sequence variants in the gene encoding the adhesive junction-associated δ-catenin protein (CTNND2) in female-enriched multiplex families and demonstrating their loss-of-function effect by functional analyses in zebrafish embryos and cultured hippocampal neurons from wild-type and Ctnnd2 null mouse embryos. Finally, through gene expression and network analyses, we highlight a critical role for CTNND2 in neuronal development and an intimate connection to chromatin biology. Our data contribute to the understanding of the genetic architecture of autism and suggest that genetic analyses of phenotypic extremes, such as female-enriched multiplex families, are of innate value in multifactorial disorders.

A Potential Contributory Role for Ciliary Dysfunction in the 16p11.2 600 kb BP4-BP5 Pathology.

The 16p11.2 600 kb copy-number variants (CNVs) are associated with mirror phenotypes on BMI, head circumference, and brain volume and represent frequent genetic lesions in autism spectrum disorders (ASDs) and schizophrenia. Here we interrogated the transcriptome of individuals carrying reciprocal 16p11.2 CNVs. Transcript perturbations correlated with clinical endophenotypes and were enriched for genes associated with ASDs, abnormalities of head size, and ciliopathies. Ciliary gene expression was also perturbed in orthologous mouse models, raising the possibility that ciliary dysfunction contributes to 16p11.2 pathologies. In support of this hypothesis, we found structural ciliary defects in the CA1 hippocampal region of 16p11.2 duplication mice. Moreover, by using an established zebrafish model, we show genetic interaction between KCTD13, a key driver of the mirrored neuroanatomical phenotypes of the 16p11.2 CNV, and ciliopathy-associated genes. Overexpression of BBS7 rescues head size and neuroanatomical defects of kctd13 morphants, whereas suppression or overexpression of CEP290 rescues phenotypes induced by KCTD13 under- or overexpression, respectively. Our data suggest that dysregulation of ciliopathy genes contributes to the clinical phenotypes of these CNVs.

KCTD13 is a major driver of mirrored neuroanatomical phenotypes of the 16p11.2 copy number variant.

Copy number variants (CNVs) are major contributors to genetic disorders. We have dissected a region of the 16p11.2 chromosome--which encompasses 29 genes--that confers susceptibility to neurocognitive defects when deleted or duplicated. Overexpression of each human transcript in zebrafish embryos identified KCTD13 as the sole message capable of inducing the microcephaly phenotype associated with the 16p11.2 duplication, whereas suppression of the same locus yielded the macrocephalic phenotype associated with the 16p11.2 deletion, capturing the mirror phenotypes of humans. Analyses of zebrafish and mouse embryos suggest that microcephaly is caused by decreased proliferation of neuronal progenitors with concomitant increase in apoptosis in the developing brain, whereas macrocephaly arises by increased proliferation and no changes in apoptosis. A role for KCTD13 dosage changes is consistent with autism in both a recently reported family with a reduced 16p11.2 deletion and a subject reported here with a complex 16p11.2 rearrangement involving de novo structural alteration of KCTD13. Our data suggest that KCTD13 is a major driver for the neurodevelopmental phenotypes associated with the 16p11.2 CNV, reinforce the idea that one or a small number of transcripts within a CNV can underpin clinical phenotypes, and offer an efficient route to identifying dosage-sensitive loci.

DISC1-dependent switch from progenitor proliferation to migration in the developing cortex.

Regulatory mechanisms governing the sequence from progenitor cell proliferation to neuronal migration during corticogenesis are poorly understood. Here we report that phosphorylation of DISC1, a major susceptibility factor for several mental disorders, acts as a molecular switch from maintaining proliferation of mitotic progenitor cells to activating migration of postmitotic neurons in mice. Unphosphorylated DISC1 regulates canonical Wnt signalling via an interaction with GSK3ß, whereas specific phosphorylation at serine 710 (S710) triggers the recruitment of Bardet-Biedl syndrome (BBS) proteins to the centrosome. In support of this model, loss of BBS1 leads to defects in migration, but not proliferation, whereas DISC1 knockdown leads to deficits in both. A phospho-dead mutant can only rescue proliferation, whereas a phospho-mimic mutant rescues exclusively migration defects. These data highlight a dual role for DISC1 in corticogenesis and indicate that phosphorylation of this protein at S710 activates a key developmental switch.

Discovery and functional annotation of SIX6 variants in primary open-angle glaucoma.

Glaucoma is a leading cause of blindness worldwide. Primary open-angle glaucoma (POAG) is the most common subtype and is a complex trait with multigenic inheritance. Genome-wide association studies have previously identified a significant association between POAG and the SIX6 locus (rs10483727, odds ratio (OR) = 1.32, p = 3.87×10(-11)). SIX6 plays a role in ocular development and has been associated with the morphology of the optic nerve. We sequenced the SIX6 coding and regulatory regions in 262 POAG cases and 256 controls and identified six nonsynonymous coding variants, including five rare and one common variant, Asn141His (rs33912345), which was associated significantly with POAG (OR = 1.27, p = 4.2×10(-10)) in the NEIGHBOR/GLAUGEN datasets. These variants were tested in an in vivo Danio rerio (zebrafish) complementation assay to evaluate ocular metrics such as eye size and optic nerve structure. Five variants, found primarily in POAG cases, were hypomorphic or null, while the sixth variant, found only in controls, was benign. One variant in the SIX6 enhancer increased expression of SIX6 and disrupted its regulation. Finally, to our knowledge for the first time, we have identified a clinical feature in POAG patients that appears to be dependent upon SIX6 genotype: patients who are homozygous for the SIX6 risk allele (His141) have a statistically thinner retinal nerve fiber layer than patients homozygous for the SIX6 non-risk allele (Asn141). Our results, in combination with previous SIX6 work, lead us to hypothesize that SIX6 risk variants disrupt the development of the neural retina, leading to a reduced number of retinal ganglion cells, thereby increasing the risk of glaucoma-associated vision loss.

Whole genome sequencing in patients with retinitis pigmentosa reveals pathogenic DNA structural changes and NEK2 as a new disease gene.

We performed whole genome sequencing in 16 unrelated patients with autosomal recessive retinitis pigmentosa (ARRP), a disease characterized by progressive retinal degeneration and caused by mutations in over 50 genes, in search of pathogenic DNA variants. Eight patients were from North America, whereas eight were Japanese, a population for which ARRP seems to have different genetic drivers. Using a specific workflow, we assessed both the coding and noncoding regions of the human genome, including the evaluation of highly polymorphic SNPs, structural and copy number variations, as well as 69 control genomes sequenced by the same procedures. We detected homozygous or compound heterozygous mutations in 7 genes associated with ARRP (USH2A, RDH12, CNGB1, EYS, PDE6B, DFNB31, and CERKL) in eight patients, three Japanese and five Americans. Fourteen of the 16 mutant alleles identified were previously unknown. Among these, there was a 2.3-kb deletion in USH2A and an inverted duplication of ~446 kb in EYS, which would have likely escaped conventional screening techniques or exome sequencing. Moreover, in another Japanese patient, we identified a homozygous frameshift (p.L206fs), absent in more than 2,500 chromosomes from ethnically matched controls, in the ciliary gene NEK2, encoding a serine/threonine-protein kinase. Inactivation of this gene in zebrafish induced retinal photoreceptor defects that were rescued by human NEK2 mRNA. In addition to identifying a previously undescribed ARRP gene, our study highlights the importance of rare structural DNA variations in Mendelian diseases and advocates the need for screening approaches that transcend the analysis of the coding sequences of the human genome.

A novel missense mutation of Wilms' Tumor 1 causes autosomal dominant FSGS.

FSGS is a clinical disorder characterized by focal scarring of the glomerular capillary tuft, podocyte injury, and nephrotic syndrome. Although idiopathic forms of FSGS predominate, recent insights into the molecular and genetic causes of FSGS have enhanced our understanding of disease pathogenesis. Here, we report a novel missense mutation of the transcriptional regulator Wilms' Tumor 1 (WT1) as the cause of nonsyndromic, autosomal dominant FSGS in two Northern European kindreds from the United States. We performed sequential genome-wide linkage analysis and whole-exome sequencing to evaluate participants from family DUK6524. Subsequently, whole-exome sequencing and direct sequencing were performed on proband DNA from family DUK6975. We identified multiple suggestive loci on chromosomes 6, 11, and 13 in family DUK6524 and identified a segregating missense mutation (R458Q) in WT1 isoform D as the cause of FSGS in this family. The identical mutation was found in family DUK6975. The R458Q mutation was not found in 1600 control chromosomes and was predicted as damaging by in silico simulation. We depleted wt1a in zebrafish embryos and observed glomerular injury and filtration defects, both of which were rescued with wild-type but not mutant human WT1D mRNA. Finally, we explored the subcellular mechanism of the mutation in vitro. WT1(R458Q) overexpression significantly downregulated nephrin and synaptopodin expression, promoted apoptosis in HEK293 cells and impaired focal contact formation in podocytes. Taken together, these data suggest that the WT1(R458Q) mutation alters the regulation of podocyte homeostasis and causes nonsyndromic FSGS.

Mutations in LOXHD1, a recessive-deafness locus, cause dominant late-onset Fuchs corneal dystrophy.

Fuchs corneal dystrophy (FCD) is a genetic disorder of the corneal endothelium and is the most common cause of corneal transplantation in the United States. Previously, we mapped a late-onset FCD locus, FCD2, on chromosome 18q. Here, we present next-generation sequencing of all coding exons in the FCD2 critical interval in a multigenerational pedigree in which FCD segregates as an autosomal-dominant trait. We identified a missense change in LOXHD1, a gene causing progressive hearing loss in humans, as the sole variant capable of explaining the phenotype in this pedigree. We observed LOXHD1 mRNA in cultured human corneal endothelial cells, whereas antibody staining of both human and mouse corneas showed staining in the corneal epithelium and endothelium. Corneal sections of the original proband were stained for LOXHD1 and demonstrated a distinct increase in antibody punctate staining in the endothelium and Descemet membrane; punctate staining was absent from both normal corneas and FCD corneas negative for causal LOXHD1 mutations. Subsequent interrogation of a cohort of >200 sporadic affected individuals identified another 15 heterozygous missense mutations that were absent from >800 control chromosomes. Furthermore, in silico analyses predicted that these mutations reside on the surface of the protein and are likely to affect the protein's interface and protein-protein interactions. Finally, expression of the familial LOXHD1 mutant allele as well as two sporadic mutations in cells revealed prominent cytoplasmic aggregates reminiscent of the corneal phenotype. All together, our data implicate rare alleles in LOXHD1 in the pathogenesis of FCD and highlight how different mutations in the same locus can potentially produce diverse phenotypes.

Recurrent de novo mutations in PACS1 cause defective cranial-neural-crest migration and define a recognizable intellectual-disability syndrome.

We studied two unrelated boys with intellectual disability (ID) and a striking facial resemblance suggestive of a hitherto unappreciated syndrome. Exome sequencing in both families identified identical de novo mutations in PACS1, suggestive of causality. To support these genetic findings and to understand the pathomechanism of the mutation, we studied the protein in vitro and in vivo. Altered PACS1 forms cytoplasmic aggregates in vitro with concomitant increased protein stability and shows impaired binding to an isoform-specific variant of TRPV4, but not the full-length protein. Furthermore, consistent with the human pathology, expression of mutant PACS1 mRNA in zebrafish embryos induces craniofacial defects most likely in a dominant-negative fashion. This phenotype is driven by aberrant specification and migration of SOX10-positive cranial, but not enteric, neural-crest cells. Our findings suggest that PACS1 is necessary for the formation of craniofacial structures and that perturbation of its functions results in a specific syndromic ID phenotype.

Cilia in vertebrate development and disease.

Through the combined study of model organisms, cell biology, cell signaling and medical genetics we have significantly increased our understanding of the structure and functions of the vertebrate cilium. This ancient organelle has now emerged as a crucial component of certain signaling and sensory perception pathways in both developmental and homeostatic contexts. Here, we provide a snapshot of the structure, function and distribution of the vertebrate cilium and of the pathologies that are associated with its dysfunction.

Development and plasticity of outer retinal circuitry following genetic removal of horizontal cells.

The present study examined the consequences of eliminating horizontal cells from the outer retina during embryogenesis upon the organization and assembly of the outer plexiform layer (OPL). Retinal horizontal cells exhibit a migration defect in Lim1-conditional knock-out (Lim1-CKO) mice and become mispositioned in the inner retina before birth, redirecting their dendrites into the inner plexiform layer. The resultant (mature) OPL, developing in the absence of horizontal cells, shows a retraction of rod spherules into the outer nuclear layer and a sprouting of rod bipolar cell dendrites to reach ectopic ribbon-protein puncta. Cone pedicles and the dendrites of type 7 cone bipolar cells retain their characteristic stratification and colocalization within the collapsed OPL, although both are atrophic and the spatial distribution of the pedicles is disrupted. Developmental analysis of Lim1-CKO retina reveals that components of the rod and cone pathways initially co-assemble within their normal strata in the OPL, indicating that horizontal cells are not required for the correct targeting of photoreceptor terminals or bipolar cell dendrites. As the rod spherules begin to retract during the second postnatal week, rod bipolar cells initially show no signs of ectopic growth, sprouting only subsequently and continuing to do so well after the eighth postnatal week. These results demonstrate the critical yet distinctive roles for horizontal cells on the rod and cone pathways and highlight a unique and as-yet-unrecognized maintenance function of an inhibitory interneuron that is not required for the initial targeting and co-stratification of other components in the circuit.

Context-dependent regulation of Wnt signaling through the primary cilium.

The primary cilium is a highly conserved environmental sensor and modulator of fluid movement in tubular structures. The growing recognition of mutations among its many components has led to the discovery of new disorders collectively called ciliopathies. Ciliary dysfunction disturbs a variety of signaling pathways along its basal body and axoneme that are critical for embryonic development and cell and organ homeostasis. Among the many pathways, here we discuss the emerging role of Wnt proteins in morphogenic signaling and ciliary biology during health and disease.

Detecting time-varying genetic effects in Alzheimer's disease using a longitudinal genome-wide association studies model.

INTRODUCTION: The development and progression of Alzheimer's disease (AD) is a complex process, during which genetic influences on phenotypes may also change. Incorporating longitudinal phenotypes in genome-wide association studies (GWAS) could unmask these genetic loci. METHODS: We conducted a longitudinal GWAS using a varying coefficient test to identify age-dependent single nucleotide polymorphisms (SNPs) in AD. Data from 1877 Alzheimer's Neuroimaging Data Initiative participants, including impairment status and amyloid positron emission tomography (PET) scan standardized uptake value ratio (SUVR) scores, were analyzed using a retrospective varying coefficient mixed model association test (RVMMAT). RESULTS: RVMMAT identified 244 SNPs with significant time-varying effects on AD impairment status, with 12 SNPs on chromosome 19 validated using National Alzheimer's Coordinating Center data. Age-stratified analyses showed these SNPs' effects peaked between 70 and 80 years. Additionally, 73 SNPs were linked to longitudinal amyloid accumulation changes. Pathway analyses implicated immune and neuroinflammation-related disruptions. DISCUSSION: Our findings demonstrate that longitudinal GWAS models can uncover time-varying genetic signals in AD. Highlights: Identify time-varying genetic effects using a longitudinal GWAS model in AD.Illustrate age-dependent genetic effects on both diagnoses and amyloid accumulation.Replicate time-varying effect of APOE in a second dataset.

RETROSPECTIVE VARYING COEFFICIENT ASSOCIATION ANALYSIS OF LONGITUDINAL BINARY TRAITS: APPLICATION TO THE IDENTIFICATION OF GENETIC LOCI ASSOCIATED WITH HYPERTENSION.

Many genetic studies contain rich information on longitudinal phenotypes that require powerful analytical tools for optimal analysis. Genetic analysis of longitudinal data that incorporates temporal variation is important for understanding the genetic architecture and biological variation of complex diseases. Most of the existing methods assume that the contribution of genetic variants is constant over time and fail to capture the dynamic pattern of disease progression. However, the relative influence of genetic variants on complex traits fluctuates over time. In this study, we propose a retrospective varying coefficient mixed model association test, RVMMAT, to detect time-varying genetic effect on longitudinal binary traits. We model dynamic genetic effect using smoothing splines, estimate model parameters by maximizing a double penalized quasi-likelihood function, design a joint test using a Cauchy combination method, and evaluate statistical significance via a retrospective approach to achieve robustness to model misspecification. Through simulations we illustrated that the retrospective varying-coefficient test was robust to model misspecification under different ascertainment schemes and gained power over the association methods assuming constant genetic effect. We applied RVMMAT to a genome-wide association analysis of longitudinal measure of hypertension in the Multi-Ethnic Study of Atherosclerosis. Pathway analysis identified two important pathways related to G-protein signaling and DNA damage. Our results demonstrated that RVMMAT could detect biologically relevant loci and pathways in a genome scan and provided insight into the genetic architecture of hypertension.

Classifying Alzheimer's Disease Neuropathology Using Clinical and MRI Measurements.

Background: Computer-aided machine learning models are being actively developed with clinically available biomarkers to diagnose Alzheimer's disease (AD) in living persons. Despite considerable work with cross-sectional in vivo data, many models lack validation against postmortem AD neuropathological data. Objective: Train machine learning models to classify the presence or absence of autopsy-confirmed severe AD neuropathology using clinically available features. Methods: AD neuropathological status are assessed at postmortem for participants from the National Alzheimer's Coordinating Center (NACC). Clinically available features are utilized, including demographics, Apolipoprotein E(APOE) genotype, and cortical thicknesses derived from ante-mortem MRI scans encompassing AD meta regions of interest (meta-ROI). Both logistic regression and random forest models are trained to identify linearly and nonlinearly separable features between participants with the presence (N = 91, age-at-MRI = 73.6±9.24, 38 women) or absence (N = 53, age-at-MRI = 68.93±19.69, 24 women) of severe AD neuropathology. The trained models are further validated in an external data set against in vivo amyloid biomarkers derived from PET imaging (amyloid-positive: N = 71, age-at-MRI = 74.17±6.37, 26 women; amyloid-negative: N = 73, age-at-MRI = 71.59±6.80, 41 women). Results: Our models achieve a cross-validation accuracy of 84.03% in classifying the presence or absence of severe AD neuropathology, and an external-validation accuracy of 70.14% in classifying in vivo amyloid positivity status. Conclusions: Our models show that clinically accessible features, including APOE genotype and cortical thinning encompassing AD meta-ROIs, are able to classify both postmortem confirmed AD neuropathological status and in vivo amyloid status with reasonable accuracies. These results suggest the potential utility of AD meta-ROIs in determining AD neuropathological status in living persons.

Spatial and Temporal Drug Usage Patterns in Wastewater Correlate with Socioeconomic and Demographic Indicators in Southern Nevada.

Evaluating drug use within populations in the United States poses significant challenges due to various social, ethical, and legal constraints, often impeding the collection of accurate and timely data. Here, we aimed to overcome these barriers by conducting a comprehensive analysis of drug consumption trends and measuring their association with socioeconomic and demographic factors. From May 2022 to April 2023, we analyzed 208 wastewater samples from eight sampling locations across six wastewater treatment plants in Southern Nevada, covering a population of 2.4 million residents with 50 million annual tourists. Using bi-weekly influent wastewater samples, we employed mass spectrometry to detect 39 analytes, including pharmaceuticals and personal care products (PPCPs) and high risk substances (HRS). Our results revealed a significant increase over time in the level of stimulants such as cocaine (pFDR=1.40×10-10) and opioids, particularly norfentanyl (pFDR =1.66×10-12), while PPCPs exhibited seasonal variation such as peak usage of DEET, an active ingredient in insect repellents, during the summer (pFDR =0.05). Wastewater from socioeconomically disadvantaged or rural areas, as determined by Area Deprivation Index (ADI) and Rural-Urban Commuting Area Codes (RUCA) scores, demonstrated distinct overall usage patterns, such as higher usage/concentration of HRS, including cocaine (p=0.05) and norfentanyl (p=1.64×10-5). Our approach offers a near real-time, comprehensive tool to assess drug consumption and personal care product usage at a community level, linking wastewater patterns to socioeconomic and demographic factors. This approach has the potential to significantly enhance public health monitoring strategies in the United States.

Environmental Surveillance of Flood Control Infrastructure Impacted by Unsheltered Individuals Leads to the Detection of SARS-CoV-2 and Novel Mutations in the Spike Gene.

In the United States, the growing number of people experiencing homelessness has become a socioeconomic crisis with public health ramifications, recently exacerbated by the COVID-19 pandemic. We hypothesized that the environmental surveillance of flood control infrastructure may be an effective approach to understand the prevalence of infectious disease. From December 2021 through July 2022, we tested for SARS-CoV-2 RNA from two flood control channels known to be impacted by unsheltered individuals residing in upstream tunnels. Using qPCR, we detected SARS-CoV-2 RNA in these environmental water samples when significant COVID-19 outbreaks were occurring in the surrounding community. We also performed whole genome sequencing to identify SARS-CoV-2 lineages. Variant compositions were consistent with those of geographically and temporally matched municipal wastewater samples and clinical specimens. However, we also detected 10 of 22 mutations specific to the Alpha variant in the environmental water samples collected during January 2022-one year after the Alpha infection peak. We also identified mutations in the spike gene that have never been identified in published reports. Our findings demonstrate that environmental surveillance of flood control infrastructure may be an effective tool to understand public health conditions among unsheltered individuals-a vulnerable population that is underrepresented in clinical surveillance data.

Science Education for the Youth (SEFTY): A Neuroscience Outreach Program for High School Students in Southern Nevada during the COVID-19 Pandemic.

Laboratory outreach programs for K-12 students in the United States from 2020 to 2022 were suspended or delayed due to COVID-19 restrictions. While Southern Nevada also observed similar closures for onsite programs, we and others hypothesized that in-person laboratory activities could be prioritized after increasing vaccine doses were available to the public and masking was encouraged. Here, we describe how the Laboratory of Neurogenetics and Precision Medicine at the University of Nevada Las Vegas (UNLV) collaborated with administrators from a local school district to conduct training activities for high school students during the COVID-19 pandemic. The Science Education for the Youth (SEFTY) program's curriculum was constructed to incorporate experiential learning, fostering collaboration and peer-to-peer knowledge exchange. Leveraging neuroscience tools from our UNLV laboratory, we engaged with 117 high school applicants from 2021 to 2022. Our recruitment efforts yielded a diverse cohort, with >41% Pacific Islander and Asian students, >9% African American students, and >12% multiracial students. We assessed the impact of the SEFTY program through pre- and postassessment student evaluations, revealing a significant improvement of 20.3% in science proficiency (p < 0.001) after participating in the program. Collectively, our laboratory curriculum offers valuable insights into the capacity of an outreach program to actively foster diversity and cultivate opportunities for academic excellence, even in the challenging context of a global pandemic.

Science Education for the Youth (SEFTY): A Neuroscience Outreach Program for High School Students in Southern Nevada During the COVID-19 Pandemic.

Laboratory outreach programs for K-12 students in the United States from 2020-2022 were suspended or delayed due to COVID-19 restrictions. While Southern Nevada also observed similar closures for onsite programs, we and others hypothesized that in-person laboratory activities could be prioritized after increasing vaccine doses were available to the public and masking was encouraged. Here, we describe how the Laboratory of Neurogenetics and Precision Medicine at the University of Nevada Las Vegas (UNLV) collaborated with administrators from a local school district to conduct training activities for high school students during the COVID-19 pandemic. The Science Education for the Youth (SEFTY) program's curriculum was constructed to incorporate experiential learning, fostering collaboration and peer-to-peer knowledge exchange. Leveraging neuroscience tools from our UNLV laboratory, we engaged with 117 high school applicants from 2021-2022. Our recruitment efforts yielded a diverse cohort, with >41% Pacific Islander and Asian students, >9% African American students, and >12% multiracial students. We assessed the impact of the SEFTY program through pre- and post-assessment student evaluations, revealing a significant improvement of 20.3% in science proficiency ( p <0.001) after participating in the program. Collectively, our laboratory curriculum offers valuable insights into the capacity of an outreach program to actively foster diversity and cultivate opportunities for academic excellence, even in the challenging context of a global pandemic. Significance Statement: The Science Education for the Youth (SEFTY) program at UNLV successfully engaged 117 diverse high school students in neuroscience-based experiential learning, demonstrating the viability of in-person education during a pandemic. Significant improvements in science proficiency (20.3% increase) underscore the program's effectiveness in fostering academic excellence and diversity. This initiative potentially serves as a model for maintaining high-quality, inclusive science education in challenging times.

Early Detection of Novel SARS-CoV-2 Variants from Urban and Rural Wastewater through Genome Sequencing and Machine Learning.

Genome sequencing from wastewater has emerged as an accurate and cost-effective tool for identifying SARS-CoV-2 variants. However, existing methods for analyzing wastewater sequencing data are not designed to detect novel variants that have not been characterized in humans. Here, we present an unsupervised learning approach that clusters co-varying and time-evolving mutation patterns leading to the identification of SARS-CoV-2 variants. To build our model, we sequenced 3,659 wastewater samples collected over a span of more than two years from urban and rural locations in Southern Nevada. We then developed a multivariate independent component analysis (ICA)-based pipeline to transform mutation frequencies into independent sources with co-varying and time-evolving patterns and compared variant predictions to >5,000 SARS-CoV-2 clinical genomes isolated from Nevadans. Using the source patterns as data-driven reference "barcodes", we demonstrated the model's accuracy by successfully detecting the Delta variant in late 2021, Omicron variants in 2022, and emerging recombinant XBB variants in 2023. Our approach revealed the spatial and temporal dynamics of variants in both urban and rural regions; achieved earlier detection of most variants compared to other computational tools; and uncovered unique co-varying mutation patterns not associated with any known variant. The multivariate nature of our pipeline boosts statistical power and can support accurate and early detection of SARS-CoV-2 variants. This feature offers a unique opportunity for novel variant and pathogen detection, even in the absence of clinical testing.