Drosophila Arc1 is not required for male fertility or sperm competition success.

Activity-regulated cytoskeleton associated protein (Arc1), which is required for synaptic plasticity and metabolism in Drosophila , self-assembles into capsid-like structures that transport mRNAs in extracellular vesicles. In addition to expression in the brain and nervous system, Arc1 is expressed in the male accessory glands, an endothelial tissue that produces male seminal proteins and exosomes that impact male fertility. We thus hypothesized that Arc1 might impact male fertility. We measured the fertility, mating latency, mating duration, and sperm competition performance of Arc1 males relative to controls and found no evidence that Arc1 is required for any of these measures of male fertility.

Morphologic, immunophenotypic, molecular genetic, and clinical characterization in patients with SRSF2-mutated acute myeloid leukemia.

OBJECTIVES: SRSF2 mutations are known to be associated with poor outcomes in myelodysplastic neoplasm, but studies on their prognostic impact on acute myeloid leukemia (AML) remain limited. In this retrospective study, we analyzed clinical and pathologic characteristics of patients with AML and correlated the outcomes with SRSF2 mutations. METHODS: We characterized the morphologic, immunophenotypic, molecular, and clinical findings in AML with mutated SRSF2 and compared them with SRSF2 wild-type (WT) myeloid neoplasms (MNs). RESULTS: Using next-generation sequencing, we identified 134 patients with MNs and SRSF2 mutations (85 with AML and 49 with MNs) in addition to 342 SRSF2-WT AMLs. Fifty-two (62%) patients with altered SRSF2 demonstrated a variable degree of morphologic dysplasia. The most frequent immunophenotypic aberrancies in SRSF2-mutant AML included diminished CD33 expression and overexpression of CD7, CD56, or CD123, similar to WT AML. More IDH1/2 (P = .015) and NPM1 (P = .002) mutations were seen in SRSF2-mutant AML than in SRSF2-mutant non-AML. Further, more IDH1/2, ASXL1, RUNX1, and STAG2 mutations were observed in SRSF2-mutant AML than in SRSF2-WT AML (P < .0001 to P = .001). Finally, patients with SRSF2-mutant AML showed a significantly worse overall survival (OS) than patients with SRSF2-WT AML (P < .0001), but this worse OS appeared to be rescued by allogeneic stem cell transplant (allo-SCT). CONCLUSIONS: Acute myeloid leukemia with altered SRSF2 shows a variable degree of morphologic dysplasia without uniform immunophenotypic aberrancies. SRSF2 mutations appear to be independent poor prognostic factors, but allo-SCT has improved the clinical outcomes in patients with SRSF2-mutant AML.

Interpretation and reporting of large regions of homozygosity and suspected consanguinity/uniparental disomy, 2021 revision: A technical standard of the American College of Medical Genetics and Genomics (ACMG).

Genomic testing, including single-nucleotide variation (formerly single-nucleotide polymorphism)-based chromosomal microarray and exome and genome sequencing, can detect long regions of homozygosity (ROH) within the genome. Genomic testing can also detect possible uniparental disomy (UPD). Platforms that can detect ROH and possible UPD have matured since the initial American College of Medical Genetics and Genomics (ACMG) standard was published in 2013, and the detection of ROH and UPD by these platforms has shown utility in diagnosis of patients with genetic/genomic disorders. The presence of these segments, when distributed across multiple chromosomes, may indicate a familial relationship between the proband's parents. This technical standard describes the detection of possible consanguinity and UPD by genomic testing, as well as the factors confounding the inference of a specific parental relationship or UPD. Current bioethical and legal issues regarding detection and reporting of consanguinity are also discussed.

High-resolution within-sewer SARS-CoV-2 surveillance facilitates informed intervention.

To assist in the COVID-19 public health guidance on a college campus, daily composite wastewater samples were withdrawn at 20 manhole locations across the University of Colorado Boulder campus. Low-cost autosamplers were fabricated in-house to enable an economical approach to this distributed study. These sample stations operated from August 25th until November 23rd during the fall 2020 semester, with 1512 samples collected. The concentration of SARS-CoV-2 in each sample was quantified through two comparative reverse transcription quantitative polymerase chain reactions (RT-qPCRs). These methods were distinct in the utilization of technical replicates and normalization to an endogenous control. (1) Higher temporal resolution compensates for supply chain or other constraints that prevent technical or biological replicates. (2) The data normalized by an endogenous control agreed with the raw concentration data, minimizing the utility of normalization. The raw wastewater concentration values reflected SARS-CoV-2 prevalence on campus as detected by clinical services. Overall, combining the low-cost composite sampler with a method that quantifies the SARS-CoV-2 signal within six hours enabled actionable and time-responsive data delivered to key stakeholders. With daily reporting of the findings, wastewater surveillance assisted in decision making during critical phases of the pandemic on campus, from detecting individual cases within populations ranging from 109 to 2048 individuals to monitoring the success of on-campus interventions.

Higher Viral Load Drives Infrequent Severe Acute Respiratory Syndrome Coronavirus 2 Transmission Between Asymptomatic Residence Hall Roommates.

BACKGROUND: The coronavirus disease 2019 pandemic spread to >200 countries in <6 months. To understand coronavirus spread, determining transmission rate and defining factors that increase transmission risk are essential. Most cases are asymptomatic, but people with asymptomatic infection have viral loads indistinguishable from those in symptomatic people, and they do transmit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, asymptomatic cases are often undetected. METHODS: Given high residence hall student density, the University of Colorado Boulder established a mandatory weekly screening test program. We analyzed longitudinal data from 6408 students and identified 116 likely transmission events in which a second roommate tested positive within 14 days of the index roommate. RESULTS: Although the infection rate was lower in single-occupancy rooms (10%) than in multiple-occupancy rooms (19%), interroommate transmission occurred only about 20% of the time. Cases were usually asymptomatic at the time of detection. Notably, individuals who likely transmitted had an average viral load approximately 6.5-fold higher than individuals who did not (mean quantification cycle [Cq], 26.2 vs 28.9). Although students with diagnosed SARS-CoV-2 infection moved to isolation rooms, there was no difference in time to isolation between cases with or without interroommate transmission. CONCLUSIONS: This analysis argues that interroommate transmission occurs infrequently in residence halls and provides strong correlative evidence that viral load is proportional to transmission probability.

Just 2% of SARS-CoV-2-positive individuals carry 90% of the virus circulating in communities.

We analyze data from the fall 2020 pandemic response efforts at the University of Colorado Boulder, where more than 72,500 saliva samples were tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using qRT-PCR. All samples were collected from individuals who reported no symptoms associated with COVID-19 on the day of collection. From these, 1,405 positive cases were identified. The distribution of viral loads within these asymptomatic individuals was indistinguishable from what has been previously observed in symptomatic individuals. Regardless of symptomatic status, ∼50% of individuals who test positive for SARS-CoV-2 seem to be in noninfectious phases of the disease, based on having low viral loads in a range from which live virus has rarely been isolated. We find that, at any given time, just 2% of individuals carry 90% of the virions circulating within communities, serving as viral "supercarriers" and possibly also superspreaders.

Application of a bioinformatic pipeline to RNA-seq data identifies novel virus-like sequence in human blood.

Numerous reports have suggested that infectious agents could play a role in neurodegenerative diseases, but specific etiological agents have not been convincingly demonstrated. To search for candidate agents in an unbiased fashion, we have developed a bioinformatic pipeline that identifies microbial sequences in mammalian RNA-seq data, including sequences with no significant nucleotide similarity hits in GenBank. Effectiveness of the pipeline was tested using publicly available RNA-seq data and in a reconstruction experiment using synthetic data. We then applied this pipeline to a novel RNA-seq dataset generated from a cohort of 120 samples from amyotrophic lateral sclerosis patients and controls, and identified sequences corresponding to known bacteria and viruses, as well as novel virus-like sequences. The presence of these novel virus-like sequences, which were identified in subsets of both patients and controls, were confirmed by quantitative RT-PCR. We believe this pipeline will be a useful tool for the identification of potential etiological agents in the many RNA-seq datasets currently being generated.

Saliva TwoStep for rapid detection of asymptomatic SARS-CoV-2 carriers.

Here, we develop a simple molecular test for SARS-CoV-2 in saliva based on reverse transcription loop-mediated isothermal amplification. The test has two steps: (1) heat saliva with a stabilization solution and (2) detect virus by incubating with a primer/enzyme mix. After incubation, saliva samples containing the SARS-CoV-2 genome turn bright yellow. Because this test is pH dependent, it can react falsely to some naturally acidic saliva samples. We report unique saliva stabilization protocols that rendered 295 healthy saliva samples compatible with the test, producing zero false positives. We also evaluated the test on 278 saliva samples from individuals who were infected with SARS-CoV-2 but had no symptoms at the time of saliva collection, and from 54 matched pairs of saliva and anterior nasal samples from infected individuals. The Saliva TwoStep test described herein identified infections with 94% sensitivity and >99% specificity in individuals with sub-clinical (asymptomatic or pre-symptomatic) infections.

Just 2% of SARS-CoV-2-positive individuals carry 90% of the virus circulating in communities.

We analyze data from the Fall 2020 pandemic response efforts at the University of Colorado Boulder (USA), where more than 72,500 saliva samples were tested for SARS-CoV-2 using quantitative RT-PCR. All samples were collected from individuals who reported no symptoms associated with COVID-19 on the day of collection. From these, 1,405 positive cases were identified. The distribution of viral loads within these asymptomatic individuals was indistinguishable from what has been previously reported in symptomatic individuals. Regardless of symptomatic status, approximately 50% of individuals who test positive for SARS-CoV-2 seem to be in non-infectious phases of the disease, based on having low viral loads in a range from which live virus has rarely been isolated. We find that, at any given time, just 2% of individuals carry 90% of the virions circulating within communities, serving as viral "super-carriers" and possibly also super-spreaders.

Saliva TwoStep for rapid detection of asymptomatic SARS-CoV-2 carriers.

Here, we develop a simple molecular test for SARS-CoV-2 in saliva based on reverse transcription loop-mediated isothermal amplification (RT-LAMP). The test has two steps: 1) heat saliva with a stabilization solution, and 2) detect virus by incubating with a primer/enzyme mix. After incubation, saliva samples containing the SARS-CoV-2 genome turn bright yellow. Because this test is pH dependent, it can react falsely to some naturally acidic saliva samples. We report unique saliva stabilization protocols that rendered 295 healthy saliva samples compatible with the test, producing zero false positives. We also evaluated the test on 278 saliva samples from individuals who were infected with SARS-CoV-2 but had no symptoms at the time of saliva collection, and from 54 matched pairs of saliva and anterior nasal samples from infected individuals. The Saliva TwoStep test described herein identified infections with 94% sensitivity and >99% specificity in individuals with sub-clinical (asymptomatic or pre-symptomatic) infections.

De Novo Acute Myeloid Leukemia with Combined CBFB-MYH11 and BCR-ABL1 Gene Rearrangements: A Case Report and Review of Literature.

Acute myeloid leukemia (AML) with inv(16)(p13.1q22) resulting in CBFB-MYH11 fusion is associated with a favorable prognosis. The presence of a KIT mutation modifies it to an intermediate prognosis. Additionally, inv(16) can cooperate with other genetic aberrations to further increase cell proliferation. Coexistence of inv(16) and t(9;22) is extremely rare (20 cases). We present a case of a 55-year-old male with elevated white blood cell count. Bone marrow evaluation and flow cytometry analysis were compatible with AML with monocytic features. Cytogenetic studies revealed two-related clones, a minor clone with inv(16) and a major clone with concurrent inv(16) and t(9;22) rearrangements. Fluorescent in situ hybridization studies confirmed these rearrangements. Molecular analysis detected a p190 BCR-ABL1 transcript protein. KIT mutations were negative. The patient was initially treated with standard induction regimen; 7 daily doses of cytarabine from day 1-day 7, 3 daily doses of daunorubicin from day 1-day 3, and 1 dose of Mylotarg (gemtuzumab ozogamicin) on day 1. The detection of t(9;22) led to the addition of daily doses of dasatinib (tyrosine kinase inhibitor) from day 7 onwards. The patient achieved complete remission on day 45. During his treatment course, he acquired disseminated Fusarium infection. Day 180 bone marrow evaluation revealed florid relapse with 64% blasts. Cytogenetic study showed clonal evolution of the inv(16) clone with no evidence of the t(9;22) subclone. Eventually, bone marrow transplantation was contraindicated, and the patient was transferred to palliative care. Literature review revealed that AML with co-occurrence of CBFB-MYH11 and BCR-ABL1 gene rearrangements was involved by only a small number of cases with de novo and therapy-related AML. Most cases were in myeloid blast crisis of chronic myeloid leukemia (CML). Treatment and prognosis among the de novo AML cases varied and majority of them achieved clinical remission. In contrast, these cytogenetic abnormalities in the blast phase of CML had a poor prognosis. As the prognosis and management of AML is dependent upon the underlying genetic characteristics of the neoplasm, it is imperative to include clinical outcome with such rare combinations of genetic alterations.

Heat shock in C. elegans induces downstream of gene transcription and accumulation of double-stranded RNA.

We observed that heat shock of Caenorhabditis elegans leads to the formation of nuclear double-stranded RNA (dsRNA) foci, detectable with a dsRNA-specific monoclonal antibody. These foci significantly overlap with nuclear HSF-1 granules. To investigate the molecular mechanism(s) underlying dsRNA foci formation, we used RNA-seq to globally characterize total RNA and immunoprecipitated dsRNA from control and heat shocked worms. We find a subset of both sense and antisense transcripts enriched in the dsRNA pool by heat shock overlap with dsRNA transcripts enriched by deletion of tdp-1, which encodes the C. elegans ortholog of TDP-43. Interestingly, transcripts involved in translation are over-represented in the dsRNAs induced by either heat shock or deletion of tdp-1. Also enriched in the dsRNA transcripts are sequences downstream of annotated genes (DoGs), which we globally quantified with a new algorithm. To validate these observations, we used fluorescence in situ hybridization (FISH) to confirm both antisense and downstream of gene transcription for eif-3.B, one of the affected loci we identified.

Neurodegeneration, Heterochromatin, and Double-Stranded RNA.

Changes in chromatin and epigenetic modifications have been associated with aging and aging-associated neurodegenerative diseases, although the causal relationship between these changes and disease-related pathology has been unclear. Recent studies have now made direct connections between neurodegeneration-associated proteins and derepression of repetitive element transcription due to changes in heterochromatin. We suggest that this derepression leads to an increased accumulation of intracellular double-stranded RNA (dsRNA), with an attendant induction of innate immune responses that contribute to the neuroinflammation found in essentially all age-associated neurodegenerative diseases.

Heterochromatin anomalies and double-stranded RNA accumulation underlie C9orf72 poly(PR) toxicity.

How hexanucleotide GGGGCC (G4C2) repeat expansions in C9orf72 cause frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is not understood. We developed a mouse model engineered to express poly(PR), a proline-arginine (PR) dipeptide repeat protein synthesized from expanded G4C2 repeats. The expression of green fluorescent protein-conjugated (PR)50 (a 50-repeat PR protein) throughout the mouse brain yielded progressive brain atrophy, neuron loss, loss of poly(PR)-positive cells, and gliosis, culminating in motor and memory impairments. We found that poly(PR) bound DNA, localized to heterochromatin, and caused heterochromatin protein 1α (HP1α) liquid-phase disruptions, decreases in HP1α expression, abnormal histone methylation, and nuclear lamina invaginations. These aberrations of histone methylation, lamins, and HP1α, which regulate heterochromatin structure and gene expression, were accompanied by repetitive element expression and double-stranded RNA accumulation. Thus, we uncovered mechanisms by which poly(PR) may contribute to the pathogenesis of C9orf72-associated FTD and ALS.

A previously unrecognized 22q13.2 microdeletion syndrome that encompasses TCF20 and TNFRSF13C.

Phelan-McDermid syndrome (PMS, OMIM 606232) is a heterozygous contiguous gene microdeletion syndrome occurring at the distal region of chromosome 22q13. This deletion encompasses the SHANK3 gene at 22q13.33, which is thought to be the critical gene for the neurodevelopmental features seen in this syndrome. PMS is typically characterized by intellectual disability, autism spectrum disorder, absent to severely delayed speech, neonatal hypotonia, and dysmorphic features. Two patients presenting with classic clinical features of PMS have been reported to have interstitial microdeletions in the 22q13.2 region that map proximal to the SHANK3 gene (0.54 and 0.72 Mb, respectively). Here, we describe a 13-month-old girl with a de novo 1.16 Mb interstitial deletion in the 22q13.2 region who presented with global developmental delay, subtle dysmorphic features, and immunodeficiency. This deletion overlaps with the two previously published cases and five cases from the DECIPHER database. All eight patients share features common to patients with PMS including developmental delay and language delay, which suggests that this represents a previously unrecognized microdeletion syndrome in the 22q13.2 region. Our patient's deletion encompasses the TCF20 and TNFRSF13C genes, which are thought to play causative roles in the patient's neurodevelopmental and immunological features, respectively.

Germline and Somatic NF1 Alterations Are Linked to Increased HER2 Expression in Breast Cancer.

NF1 germline mutation predisposes to breast cancer. NF1 mutations have also been proposed as oncogenic drivers in sporadic breast cancers. To understand the genomic and histologic characteristics of these breast cancers, we analyzed the tumors with NF1 germline mutations and also examined the genomic and proteomic profiles of unselected tumors. Among 14 breast cancer specimens from 13 women affected with neurofibromatosis type 1 (NF1), 9 samples (NF + BrCa) underwent genomic copy number (CN) and targeted sequencing analysis. Mutations of NF1 were identified in two samples and TP53 were in three. No mutation was detected in ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, NBN, PALB2, PTEN, RAD50, and STK11 HER2 (ErbB2) overexpression was detected by IHC in 69.2% (9/13) of the tumors. CN gain/amplification of ERBB2 was detected in 4 of 9 with DNA analysis. By evaluating HER2 expression and NF1 alterations in unselected invasive breast cancers in TCGA datasets, we discovered that among samples with ERBB2 CN gain/amplification, the HER2 mRNA and protein expression were much more pronounced in NF1-mutated/deleted samples in comparison with NF1-unaltered samples. This finding suggests a synergistic interplay between these two genes, potentially driving the development of breast cancer harboring NF1 mutation and ERBB2 CN gain/amplification. NF1 gene loss of heterozygosity was observed in 4 of 9 NF + BrCa samples. CDK4 appeared to have more CN gain in NF + BrCa and exhibited increased mRNA expression in TCGA NF1--altered samples. Cancer Prev Res; 11(10); 655-64. ©2018 AACR.

The Caenorhabditis elegans Ortholog of TDP-43 Regulates the Chromatin Localization of the Heterochromatin Protein 1 Homolog HPL-2.

TDP-1 is the Caenorhabditis elegans ortholog of mammalian TDP-43, which is strongly implicated in the etiology of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). We discovered that deletion of the tdp-1 gene results in enhanced nuclear RNA interference (RNAi). As nuclear RNAi in C. elegans involves chromatin changes moderated by HPL-2, a homolog of heterochromatin protein 1 (HP1), we investigated the interaction of TDP-1 and HPL-2. We found that TDP-1 and HPL-2 interact directly and that loss of TDP-1 dramatically alters the chromatin association of HPL-2. We showed previously that deletion of the tdp-1 gene results in transcriptional alterations and the accumulation of double-stranded RNA (dsRNA). These molecular changes are replicated in an hpl-2 deletion strain, consistent with HPL-2 acting in consort with TDP-1 to modulate these aspects of RNA metabolism. Our observations identify novel mechanisms by which HP1 homologs can be recruited to chromatin and by which nuclear depletion of human TDP-43 may lead to changes in RNA metabolism that are relevant to disease.

Novel de novo pathogenic variant in the NR2F2 gene in a boy with congenital heart defect and dysmorphic features.

The NR2F2 gene plays an important role in angiogenesis and heart development. Moreover, this gene is involved in organogenesis in many other organs in mouse models. Variants in this gene have been reported in a number of patients with nonsyndromic atrioventricular septal defect, and in one patient with congenital heart defect and dysmorphic features. Here we report an 11-month-old Caucasian male with global developmental delay, dysmorphic features, coarctation of the aorta, and ventricular septal defect. He was later found to have a pathogenic mutation in the NR2F2 gene by whole exome sequencing. This is the second instance in which an NR2F2 mutation has been identified in a child with a congenital heart defect and other anomalies. This case suggests that some variants in NR2F2 may cause syndromic forms of congenital heart defect.

Transcriptome analysis of genetically matched human induced pluripotent stem cells disomic or trisomic for chromosome 21.

Trisomy of chromosome 21, the genetic cause of Down syndrome, has the potential to alter expression of genes on chromosome 21, as well as other locations throughout the genome. These transcriptome changes are likely to underlie the Down syndrome clinical phenotypes. We have employed RNA-seq to undertake an in-depth analysis of transcriptome changes resulting from trisomy of chromosome 21, using induced pluripotent stem cells (iPSCs) derived from a single individual with Down syndrome. These cells were originally derived by Li et al, who genetically targeted chromosome 21 in trisomic iPSCs, allowing selection of disomic sibling iPSC clones. Analyses were conducted on trisomic/disomic cell pairs maintained as iPSCs or differentiated into cortical neuronal cultures. In addition to characterization of gene expression levels, we have also investigated patterns of RNA adenosine-to-inosine editing, alternative splicing, and repetitive element expression, aspects of the transcriptome that have not been significantly characterized in the context of Down syndrome. We identified significant changes in transcript accumulation associated with chromosome 21 trisomy, as well as changes in alternative splicing and repetitive element transcripts. Unexpectedly, the trisomic iPSCs we characterized expressed higher levels of neuronal transcripts than control disomic iPSCs, and readily differentiated into cortical neurons, in contrast to another reported study. Comparison of our transcriptome data with similar studies of trisomic iPSCs suggests that trisomy of chromosome 21 may not intrinsically limit neuronal differentiation, but instead may interfere with the maintenance of pluripotency.