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Dissecting plant responses to the environment is key to understanding whether and how plants adapt to anthropogenic climate change. Stomata, plants' pores for gas exchange, are expected to decrease in density following increased CO2 concentrations, a trend already observed in multiple plant species. However, it is unclear whether such responses are based on genetic changes and evolutionary adaptation. Here we make use of extensive knowledge of 43 genes in the stomatal development pathway and newly generated genome information of 191 Arabidopsis thaliana historical herbarium specimens collected over 193 years to directly link genetic variation with climate change. While we find that the essential transcription factors SPCH, MUTE and FAMA, central to stomatal development, are under strong evolutionary constraints, several regulators of stomatal development show signs of local adaptation in contemporary samples from different geographic regions. We then develop a functional score based on known effects of gene knock-out on stomatal development that recovers a classic pattern of stomatal density decrease over the past centuries, suggesting a genetic component contributing to this change. This approach combining historical genomics with functional experimental knowledge could allow further investigations of how different, even in historical samples unmeasurable, cellular plant phenotypes may have already responded to climate change through adaptive evolution.

Genome-wide association studies (GWAS) have become well-powered to detect loci associated with telomere length. However, no prior work has validated genes nominated by GWAS to examine their role in telomere length regulation. We conducted a multi-ancestry meta-analysis of 211,369 individuals and identified five novel association signals. Enrichment analyses of chromatin state and cell-type heritability suggested that blood/immune cells are the most relevant cell type to examine telomere length association signals. We validated specific GWAS associations by overexpressing KBTBD6 or POP5 and demonstrated that both lengthened telomeres. CRISPR/Cas9 deletion of the predicted causal regions in K562 blood cells reduced expression of these genes, demonstrating that these loci are related to transcriptional regulation of KBTBD6 and POP5. Our results demonstrate the utility of telomere length GWAS in the identification of telomere length regulation mechanisms and validate KBTBD6 and POP5 as genes affecting telomere length regulation.

We will attempt a synthesis from various research perspectives that have analyzed the alterations that knowledge inevitably goes through while percolating into classroom activities. We will try to identify some of their causes and will illustrate them with examples in genetics. First, we will discuss some research on knowledge transformation when genetics knowledge is popularized (Staerklé and Clémence 2002), to show how knowledge is transformed in specific predictable manners as it moves from experts to general public. Then, we will draw from a large body of (French) research in didactic transposition (Chevallard 1991) in order to highlight what knowledge characteristically thrives or is lost as it percolates into school practice and learner's knowledge. We will then draw from Huberman's ("Science Communicatio," "4"(4), 478-510, 1983) analysis of knowledge use in schools the specificities of knowledge that teachers effectively use. All three perspectives reveal that cognitive and social environment are crucial determinants of what knowledge will be found in schools. An ecological metaphor explains how different cognitive environments from research into education favor knowledge "adapted" to specificities of this ecosystem. This transposition of knowledge is therefore not decay but inescapable and necessary. Ignoring this transposition has considerably reduced the effectiveness of many educational reforms. We will combine these three to propose an evolutionary perspective that could inform ways of expressing research into educational recommendations fed into the system to optimize the didactic transposition process.

An introduction to digital stochastic simulations for modeling a variety of physical and chemical processes is presented. Despite the importance of stochastic simulations in chemistry, the prevalence of turn-key software solutions can impose a layer of abstraction between the user and the underlying approach obscuring the methodology being employed. This article introduces the use of random variable generators in simulating physical and chemical processes suitable for use in undergraduate chemistry courses through detailed illustrative examples which include DNA sequences, Brownian motion, diffusion, chemical kinetics, and chain-growth polymerization. The results of the stochastic simulations are also compared to theoretical models. Jupyter notebooks containing Python code for running these simulations are provided in the Supporting Information section.

BackgroundRecently shown to regulate cardiac development, the secreted axon guidance molecule SLIT3 maintains its expression in the postnatal heart. Despite its known expression in the cardiovascular system after birth, SLIT3's relevance to cardiovascular function in the postnatal state remains unknown. As such, the objectives of this study were to determine the postnatal myocardial sources of SLIT3 and to evaluate its functional role in regulating the cardiac response to pressure overload stress.MethodsWe performed in vitro studies on cardiomyocytes and myocardial tissue samples from patients and performed in vivo investigation with SLIT3 and ROBO1 (roundabout homolog 1) mutant mice undergoing transverse aortic constriction to establish the role of SLIT3-ROBO1 in adverse cardiac remodeling.ResultsWe first found that SLIT3 transcription was increased in myocardial tissue obtained from patients with congenital heart defects that caused ventricular pressure overload. Immunostaining of hearts from WT (wild-type) and reporter mice revealed that SLIT3 is secreted by cardiac stromal cells, namely fibroblasts and vascular mural cells, within the heart. Conditioned media from cardiac fibroblasts and vascular mural cells both stimulated cardiomyocyte hypertrophy in vitro, an effect that was partially inhibited by an anti-SLIT3 antibody. Also, the N-terminal, but not the C-terminal, fragment of SLIT3 and the forced overexpression of SLIT3 stimulated cardiomyocyte hypertrophy and the transcription of hypertrophy-related genes. We next determined that ROBO1 was the most highly expressed roundabout receptor in cardiomyocytes and that ROBO1 mediated SLIT3's hypertrophic effects in vitro. In vivo, Tcf21+ fibroblast and Tbx18+ vascular mural cell-specific knockout of SLIT3 in mice resulted in decreased left ventricular hypertrophy and cardiac fibrosis after transverse aortic constriction. Furthermore, α-MHC+ cardiomyocyte-specific deletion of ROBO1 also preserved left ventricular function and abrogated hypertrophy, but not fibrosis, after transverse aortic constriction.ConclusionsCollectively, these results indicate a novel role for the SLIT3-ROBO1-signaling axis in regulating postnatal cardiomyocyte hypertrophy induced by pressure overload.

Neuroblastoma is the most common extracranial solid tumor of childhood. While MYCN and mutant anaplastic lymphoma kinase (ALKF1174L) cooperate in tumorigenesis, how ALK contributes to tumor formation remains unclear. Here, we used a human stem cell-based model of neuroblastoma. Mis-expression of ALKF1174L and MYCN resulted in shorter latency compared to MYCN alone. MYCN tumors resembled adrenergic, while ALK/MYCN tumors resembled mesenchymal, neuroblastoma. Transcriptomic analysis revealed enrichment in focal adhesion signaling, particularly the extracellular matrix genes POSTN and FN1 in ALK/MYCN tumors. Patients with ALK-mutant tumors similarly demonstrated elevated levels of POSTN and FN1. Knockdown of POSTN, but not FN1, delayed adhesion and suppressed proliferation of ALK/MYCN tumors. Furthermore, loss of POSTN reduced ALK-dependent activation of WNT signaling. Reciprocally, inhibition of the WNT pathway reduced expression of POSTN and growth of ALK/MYCN tumor cells. Thus, ALK drives neuroblastoma in part through a feedforward loop between POSTN and WNT signaling.

An undergraduate biochemistry laboratory experiment is described that utilizes free online bioinformatics tools along with readily available exonucleases to study the effects of base stacking and hydrogen bonding on the UV absorbance of DNA samples. UV absorbance of double-stranded DNA at the ?[subscript max] is decreased when the DNA bases are involved in hydrogen bonding and formation of secondary structure. When an exonuclease is added to the solution containing the DNA, the strand is digested and the interactions disappear, leading to an increase in the absorbance called hyperchromicity. This experiment utilizes exonuclease digestion of DNA to show students how base interactions and secondary structure can alter the spectroscopic properties of a sample and, by extension, how apparent concentration as calculated with Beer-Lambert's law is not necessarily representative of the true concentration of DNA in solution. Teaching applications of this laboratory experiment include enzyme kinetics and activity, secondary structure of single-stranded DNA (and its parallel to RNA structure), and an introduction to bioinformatics.

SNCAIP duplication may promote Group 4 medulloblastoma via induction of PRDM6, a poorly characterized member of the PRDF1 and RIZ1 homology domain-containing (PRDM) family of transcription factors. Here, we investigated the function of PRDM6 in human hindbrain neuroepithelial stem cells and tested PRDM6 as a driver of Group 4 medulloblastoma. We report that human PRDM6 localizes predominantly to the nucleus, where it causes widespread repression of chromatin accessibility and complex alterations of gene expression patterns. Genome-wide mapping of PRDM6 binding reveals that PRDM6 binds to chromatin regions marked by histone H3 lysine 27 trimethylation that are located within, or proximal to, genes. Moreover, we show that PRDM6 expression in neuroepithelial stem cells promotes medulloblastoma. Surprisingly, medulloblastomas derived from PRDM6-expressing neuroepithelial stem cells match human Group 3, but not Group 4, medulloblastoma. We conclude that PRDM6 expression has oncogenic potential but is insufficient to drive Group 4 medulloblastoma from neuroepithelial stem cells. We propose that both PRDM6 and additional factors, such as specific cell-of-origin features, are required for Group 4 medulloblastoma. Given the lack of PRDM6 expression in normal tissues and its oncogenic potential shown here, we suggest that PRDM6 inhibition may have therapeutic value in PRDM6-expressing medulloblastomas.

BackgroundNeuroblastoma is the most common extra-cranial pediatric solid tumor. 131I-metaiodobenzylguanidine (MIBG) is a targeted radiopharmaceutical highly specific for neuroblastoma tumors, providing potent radiotherapy to widely metastatic disease. Aurora kinase A (AURKA) plays a role in mitosis and stabilization of the MYCN protein in neuroblastoma. We aimed to study the impact of AURKA inhibitors on DNA damage and tumor cell death in combination with 131I-MIBG therapy in a pre-clinical model of high-risk neuroblastoma.ResultsUsing an in vivo model of high-risk neuroblastoma, we demonstrated a marked combinatorial effect of 131I-MIBG and alisertib on tumor growth. In MYCN amplified cell lines, the combination of radiation and an AURKA A inhibitor increased DNA damage and apoptosis and decreased MYCN protein levels.ConclusionThe combination of AURKA inhibition with 131I-MIBG treatment is active in resistant neuroblastoma models.

The Environmental Influences on Child Health Outcomes (ECHO)-Wide Cohort Study (EWC), a collaborative research design comprising 69 cohorts in 31 consortia, was funded by the National Institutes of Health (NIH) in 2016 to improve children's health in the United States. The EWC harmonizes extant data and collects new data using a standardized protocol, the ECHO-Wide Cohort Data Collection Protocol (EWCP). EWCP visits occur at least once per life stage, but the frequency and timing of the visits vary across cohorts. As of March 4, 2022, the EWC cohorts contributed data from 60,553 children and consented 29,622 children for new EWCP data and biospecimen collection. The median (interquartile range) age of EWCP-enrolled children was 7.5 years (3.7-11.1). Surveys, interviews, standardized examinations, laboratory analyses, and medical record abstraction are used to obtain information in 5 main outcome areas: pre-, peri-, and postnatal outcomes; neurodevelopment; obesity; airways; and positive health. Exposures include factors at the level of place (e.g., air pollution, neighborhood socioeconomic status), family (e.g., parental mental health), and individuals (e.g., diet, genomics).

Abstract Genomic data holds huge potential for medical progress but requires strict safety measures due to its sensitive nature to comply with data protection laws. This conflict is especially pronounced in genome-wide association studies (GWAS) which rely on vast amounts of genomic data to improve medical diagnoses. To ensure both their benefits and sufficient data security, we propose a federated approach in combination with privacy-enhancing technologies utilising the findings from a systematic review on federated learning and legal regulations in general and applying these to GWAS.

The basic helix-loop-helix (bHLH) family of transcription factors recognizes DNA motifs known as E-boxes (CANNTG) and includes 108 members1. Here we investigate how chromatinized E-boxes are engaged by two structurally diverse bHLH proteins: the proto-oncogene MYC-MAX and the circadian transcription factor CLOCK-BMAL1 (refs. 2,3). Both transcription factors bind to E-boxes preferentially near the nucleosomal entry-exit sites. Structural studies with engineered or native nucleosome sequences show that MYC-MAX or CLOCK-BMAL1 triggers the release of DNA from histones to gain access. Atop the H2A-H2B acidic patch4, the CLOCK-BMAL1 Per-Arnt-Sim (PAS) dimerization domains engage the histone octamer disc. Binding of tandem E-boxes5-7 at endogenous DNA sequences occurs through direct interactions between two CLOCK-BMAL1 protomers and histones and is important for circadian cycling. At internal E-boxes, the MYC-MAX leucine zipper can also interact with histones H2B and H3, and its binding is indirectly enhanced by OCT4 elsewhere on the nucleosome. The nucleosomal E-box position and the type of bHLH dimerization domain jointly determine the histone contact, the affinity and the degree of competition and cooperativity with other nucleosome-bound factors.

The 16p11.2 and 22q11.2 copy number variants (CNVs) are associated with neurobehavioral traits including autism spectrum disorder (ASD), schizophrenia, bipolar disorder, obesity, and intellectual disability. Identifying specific genes contributing to each disorder and dissecting the architecture of CNV-trait association has been difficult, inspiring hypotheses of more complex models, such as multiple genes acting together. Using multi-tissue data from the GTEx consortium, we generated pairwise expression imputation models for CNV genes and then applied these elastic net models to GWAS for: ASD, bipolar disorder, schizophrenia, BMI (obesity), and IQ (intellectual disability). We compared the variance in these five traits explained by gene pairs with the variance explained by single genes and by traditional interaction models. We also modeled polygene region-wide effects using summed predicted expression ranks across many genes to create a regionwide score. We found that in all CNV-trait pairs except for bipolar disorder at 22q11.2, pairwise effects explain more variance than single genes. Pairwise model superiority was specific to the CNV region for all 16p11.2 traits and ASD at 22q11.2. We identified novel individual genes over-represented in top pairs that did not show single-gene signal. We also found that BMI and IQ have significant regionwide association with both CNV regions. Overall, we observe that genetic architecture differs by trait and region, but 9/10 CNV-trait combinations demonstrate evidence for multigene contribution, and for most of these, the importance of combinatorial models appears unique to CNV regions. Our results suggest that mechanistic insights for CNV pathology may require combinational models.

Summary: Human genetic studies of critical COVID-19 pneumonia have revealed the essential role of type I interferon-dependent innate immunity to SARS-CoV-2 infection. Conversely, an association between the HLA-B∗15:01 allele and asymptomatic SARS-CoV-2 infection in unvaccinated individuals was recently reported, suggesting a contribution of pre-existing T cell-dependent adaptive immunity. We report a lack of association of classical HLA alleles, including HLA-B∗15:01, with pre-omicron asymptomatic SARS-CoV-2 infection in unvaccinated participants in a prospective population-based study in the United States (191 asymptomatic vs. 945 symptomatic COVID-19 cases). Moreover, we found no such association in the international COVID Human Genetic Effort cohort (206 asymptomatic vs. 574 mild or moderate COVID-19 cases and 1,625 severe or critical COVID-19 cases). Finally, in the Human Challenge Characterisation study, the three HLA-B∗15:01 individuals infected with SARS-CoV-2 developed symptoms. As with other acute primary infections studied, no classical HLA alleles favoring an asymptomatic course of SARS-CoV-2 infection were identified.

Objective: As researchers make progress in understanding genetic aspects of mental illness and its treatment, psychiatrists will increasingly need to understand and interpret genetic information specific to psychiatric disorders. Little is known about the extent to which residency programs are preparing psychiatrists for this new role. This study was designed to explore the current state of genetics education in psychiatric residency. Methods: U.S. psychiatric residency educators and residents were invited to participate in an Internet-based survey. Results: Out of 509 educators and at least 207 residents contacted, 100 educators and 135 residents from 55 geographically dispersed U.S. psychiatry training programs in diverse settings responded to the survey. Participants rated genetics as somewhat important as a curricular topic but less important than other subjects. Nearly half of the educators reported that their program had few or no faculty with adequate expertise in teaching this subject. Most residents routinely obtained family psychiatric histories when evaluating patients, but most did not feel competent to order and interpret genetic tests or to interpret papers on psychiatric genetics. Conclusion: This study presents a sobering picture of the state of genetics education in psychiatric residency training. Potential obstacles to improving genetics education include perceptions of the relative importance of genetics in comparison with other topics and a lack of faculty with appropriate expertise.

We have been teaching applied molecular genetics to engineers and adapted the teaching methodology to the European Credit Transfer System. We teach core principles of genetics that are universal and form the conceptual basis of most molecular technologies. The course then teaches widely used techniques and finally shows how different techniques can be put together to solve specific technological problems from the environmental and food industry. Students from Spain, France, Turkey, and Poland have been able to understand and use simple scientific literature and propose experimental strategies as essays to tackle simple problems of technical interest. (Contains 4 tables and 2 figures.)

Nononcogenic somatic mutations are thought to be uncommon and inconsequential. To test this, we analyzed 43,693 National Heart, Lung and Blood Institute Trans-Omics for Precision Medicine blood whole genomes from 37 cohorts and identified 7131 non-missense somatic mutations that are recurrently mutated in at least 50 individuals. These recurrent non-missense somatic mutations (RNMSMs) are not clearly explained by other clonal phenomena such as clonal hematopoiesis. RNMSM prevalence increased with age, with an average 50-year-old having 27 RNMSMs. Inherited germline variation associated with RNMSM acquisition. These variants were found in genes involved in adaptive immune function, proinflammatory cytokine production, and lymphoid lineage commitment. In addition, the presence of eight specific RNMSMs associated with blood cell traits at effect sizes comparable to Mendelian genetic mutations. Overall, we found that somatic mutations in blood are an unexpectedly common phenomenon with ancestry-specific determinants and human health consequences.

Summary: The maturation of genomic surveillance in the past decade has enabled tracking of the emergence and spread of epidemics at an unprecedented level. During the COVID-19 pandemic, for example, genomic data revealed that local epidemics varied considerably in the frequency of SARS-CoV-2 lineage importation and persistence, likely due to a combination of COVID-19 restrictions and changing connectivity. Here, we show that local COVID-19 epidemics are driven by regional transmission, including across international boundaries, but can become increasingly connected to distant locations following the relaxation of public health interventions. By integrating genomic, mobility, and epidemiological data, we find abundant transmission occurring between both adjacent and distant locations, supported by dynamic mobility patterns. We find that changing connectivity significantly influences local COVID-19 incidence. Our findings demonstrate a complex meaning of ‘local’ when investigating connected epidemics and emphasize the importance of collaborative interventions for pandemic prevention and mitigation.

MYC-driven medulloblastomas are highly aggressive childhood brain tumors, however, the molecular and genetic events triggering MYC amplification and malignant transformation remain elusive. Here we report that mutations in CTDNEP1, a CTD nuclear-envelope-phosphatase, are the most significantly enriched recurrent alterations in MYC-driven medulloblastomas, and define high-risk subsets with poorer prognosis. Ctdnep1 ablation promotes the transformation of murine cerebellar progenitors into Myc-amplified medulloblastomas, resembling their human counterparts. CTDNEP1 deficiency stabilizes and activates MYC activity by elevating MYC serine-62 phosphorylation, and triggers chromosomal instability to induce p53 loss and Myc amplifications. Further, phosphoproteomics reveals that CTDNEP1 post-translationally modulates the activities of key regulators for chromosome segregation and mitotic checkpoint regulators including topoisomerase TOP2A and checkpoint kinase CHEK1. Co-targeting MYC and CHEK1 activities synergistically inhibits CTDNEP1-deficient MYC-amplified tumor growth and prolongs animal survival. Together, our studies demonstrate that CTDNEP1 is a tumor suppressor in highly aggressive MYC-driven medulloblastomas by controlling MYC activity and mitotic fidelity, pointing to a CTDNEP1-dependent targetable therapeutic vulnerability.

Expansion of newborn screening (NBS) has been driven primarily by a combination of advances in technology and medical treatment, and the sustained advocacy efforts of consumers and voluntary health organizations. The longstanding leadership of the March of Dimes has been credited by many as a critical factor in the expansion and improvement of state NBS programs. From the historic vantage point of four decades of March of Dimes involvement with newborn screening, this report reviews the unique origin of the first newborn screening test, and identifies from this point of origin several of the elements which still define the evolution of advocacy for NBS today. It also documents activities at the federal level and in seven states that have lead to expanded screening for newborns. Advances in NBS technology and medical treatment have informed policy development. Mobilization of volunteers and focused advocacy activities have brought about expansion of screening opportunities for newborns across the United States. But more work is needed. Continued application of the effective strategies identified in this report will help assure that all families have the best possible chance of assuring that their newborns do not have to suffer the complications of conditions that we know can be treated effectively. (Contains 2 figures.)

Abstract Cytokine-based therapeutics have been shown to mediate objective responses in certain tumor entities but suffer from insufficient selectivity, causing limiting toxicity which prevents dose escalation to therapeutically active regimens. The antibody-based delivery of cytokines significantly increases the therapeutic index of the corresponding payload but still suffers from side effects associated with peak concentrations of the product in blood upon intravenous administration. Here we devise a general strategy (named “Intra-Cork”) to mask systemic cytokine activity without impacting anti-cancer efficacy. Our technology features the use of antibody-cytokine fusions, capable of selective localization at the neoplastic site, in combination with pathway-selective inhibitors of the cytokine signaling, which rapidly clear from the body. This strategy, exemplified with a tumor-targeted IL12 in combination with a JAK2 inhibitor, allowed to abrogate cytokine-driven toxicity without affecting therapeutic activity in a preclinical model of cancer. This approach is readily applicable in clinical practice.

Abstract Huntington’s disease (HD) is an incurable inherited disorder caused by a repeated expansion of glutamines in the huntingtin gene (Htt). The mutant protein causes neuronal degeneration leading to severe motor and psychological symptoms. Selective downregulation of the mutant Htt gene expression is considered the most promising therapeutic approach for HD. We report the identification of small molecule inhibitors of Spt5-Pol II, SPI-24 and SPI-77, which selectively lower mutant Htt mRNA and protein levels in HD cells. In the BACHD mouse model, their direct delivery to the striatum diminished mutant Htt levels, ameliorated mitochondrial dysfunction, restored BDNF expression, and improved motor and anxiety-like phenotypes. Pharmacokinetic studies revealed that these SPIs pass the blood-brain-barrier. Prolonged subcutaneous injection or oral administration to early-stage mice significantly delayed disease deterioration. SPI-24 long-term treatment had no side effects or global changes in gene expression. Thus, lowering mutant Htt levels by small molecules can be an effective therapeutic strategy for HD.

Abstract Background To diagnose the full spectrum of hereditary and congenital diseases, genetic laboratories use many different workflows, ranging from karyotyping to exome sequencing. A single generic high-throughput workflow would greatly increase efficiency. We assessed whether genome sequencing (GS) can replace these existing workflows aimed at germline genetic diagnosis for rare disease. Methods We performed short-read GS (NovaSeq™6000; 150 bp paired-end reads, 37 × mean coverage) on 1000 cases with 1271 known clinically relevant variants, identified across different workflows, representative of our tertiary diagnostic centers. Variants were categorized into small variants (single nucleotide variants and indels

The S-Star Trial Bioinformatics on-line course (www.s-star.org) is a global experiment in bioinformatics distance education. Six universities from five continents have participated in this project. One hundred and fifty students participated in the first trial course of which 96 followed through the entire course and 70 fulfilled the overall course requirements. The course was built up of 12 lectures covering different basic and advanced topics within bioinformatics. The lectures consist of streaming video and synchronized slides with follow-up discussion forums and a final assessment. It draws on experts from diverse institutions to share their teaching expertise and resources via distance learning. A major goal of S-Star is to provide free introductory web-based education. Here we present the outline of the course, experiences from a global collaboration, and feedback from the first trial of the on-line S-Star course. (Contains 2 tables, 1 figure and 1 footnote.)

BackgroundLower-grade gliomas (LGG) are heterogeneous diseases by clinical, histological, and molecular criteria. We aimed to personalize the diagnosis and therapy of LGG patients by developing and validating robust cellular morphometric subtypes (CMS) and to uncover the molecular signatures underlying these subtypes.MethodsCellular morphometric biomarkers (CMBs) were identified with artificial intelligence technique from TCGA-LGG cohort. Consensus clustering was used to define CMS. Survival analysis was performed to assess the clinical impact of CMBs and CMS. A nomogram was constructed to predict 3- and 5-year overall survival (OS) of LGG patients. Tumor mutational burden (TMB) and immune cell infiltration between subtypes were analyzed using the Mann-Whitney U test. The double-blinded validation for important immunotherapy-related biomarkers was executed using immunohistochemistry (IHC).ResultsWe developed a machine learning (ML) pipeline to extract CMBs from whole-slide images of tissue histology; identifying and externally validating robust CMS of LGGs in multicenter cohorts. The subtypes had independent predicted OS across all three independent cohorts. In the TCGA-LGG cohort, patients within the poor-prognosis subtype responded poorly to primary and follow-up therapies. LGGs within the poor-prognosis subtype were characterized by high mutational burden, high frequencies of copy number alterations, and high levels of tumor-infiltrating lymphocytes and immune checkpoint genes. Higher levels of PD-1/PD-L1/CTLA-4 were confirmed by IHC staining. In addition, the subtypes learned from LGG demonstrate translational impact on glioblastoma (GBM).ConclusionsWe developed and validated a framework (CMS-ML) for CMS discovery in LGG associated with specific molecular alterations, immune microenvironment, prognosis, and treatment response.

Deregulation of neuroblastoma-derived myc (N-myc) is a leading cause of malignant brain tumors in children. To target N-myc-driven medulloblastoma, most research has focused on identifying genomic alterations or on the analysis of the medulloblastoma transcriptome. Here, we have broadly characterized the translatome of medulloblastoma and shown that N-myc unexpectedly drives selective translation of transcripts that promote protein homeostasis. Cancer cells are constantly exposed to proteotoxic stress associated with alterations in protein production or folding. It remains poorly understood how cancers cope with proteotoxic stress to promote their growth. Here, our data revealed that N-myc regulates the expression of specific components (∼5%) of the protein folding machinery at the translational level through the major cap binding protein, eukaryotic initiation factor eIF4E. Reducing eIF4E levels in mouse models of medulloblastoma blocked tumorigenesis. Importantly, targeting Hsp70, a protein folding chaperone translationally regulated by N-myc, suppressed tumor growth in mouse and human medulloblastoma xenograft models. These findings reveal a previously hidden molecular program that promotes medulloblastoma formation and identify new therapies that may have impact in the clinic.SignificanceTranslatome analysis in medulloblastoma shows that N-myc drives selective translation of transcripts that promote protein homeostasis and that represent new therapeutic vulnerabilities.

A common pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the cytoplasmic mislocalization and aggregation of the DNA/RNA-binding protein TDP-43, but how loss of nuclear TDP-43 function contributes to ALS and FTD pathogenesis remains largely unknown. Here, using large-scale RNAi screening, we identify TARDBP, which encodes TDP-43, as a gene whose loss-of-function results in elevated DNA mutation rate and genomic instability. Consistent with this finding, we observe increased DNA damage in induced pluripotent stem cells (iPSCs) and iPSC-derived post-mitotic neurons generated from ALS patients harboring TARDBP mutations. We find that the increase in DNA damage in ALS iPSC-derived neurons is due to defects in two major pathways for DNA double-strand break repair: non-homologous end joining and homologous recombination. Cells with defects in DNA repair are sensitive to DNA damaging agents and, accordingly, we find that ALS iPSC-derived neurons show a marked reduction in survival following treatment with a DNA damaging agent. Importantly, we find that increased DNA damage is also observed in neurons with nuclear TDP-43 depletion from ALS/FTD patient brain tissues. Collectively, our results demonstrate that ALS neurons with loss of nuclear TDP-43 function have elevated levels of DNA damage and contribute to the idea that genomic instability is a defining pathological feature of ALS/FTD patients with TDP-43 pathology.

Metagenomic next-generation sequencing (mNGS) has enabled the high-throughput multiplexed identification of sequences from microbes of potential medical relevance. This approach has become indispensable for viral pathogen discovery and broad-based surveillance of emerging or re-emerging pathogens. From 2015 to 2019, plasma was collected from 9586 individuals in Cameroon and the Democratic Republic of the Congo enrolled in a combined hepatitis virus and retrovirus surveillance program. A subset (n = 726) of the patient specimens was analyzed by mNGS to identify viral co-infections. While co-infections from known blood-borne viruses were detected, divergent sequences from nine poorly characterized or previously uncharacterized viruses were also identified in two individuals. These were assigned to the following groups by genomic and phylogenetic analyses: densovirus, nodavirus, jingmenvirus, bastrovirus, dicistrovirus, picornavirus, and cyclovirus. Although of unclear pathogenicity, these viruses were found circulating at high enough concentrations in plasma for genomes to be assembled and were most closely related to those previously associated with bird or bat excrement. Phylogenetic analyses and in silico host predictions suggested that these are invertebrate viruses likely transmitted through feces containing consumed insects or through contaminated shellfish. This study highlights the power of metagenomics and in silico host prediction in characterizing novel viral infections in susceptible individuals, including those who are immunocompromised from hepatitis viruses and retroviruses, or potentially exposed to zoonotic viruses from animal reservoir species.

The diagnostic yield of exome sequencing (ES) has primarily been evaluated in individuals of European ancestry, with less focus on underrepresented minority (URM) and underserved (US) patients. We evaluated the diagnostic yield of ES in a cohort of predominantly US and URM pediatric and prenatal patients suspected to have a genetic disorder. Eligible pediatric patients had multiple congenital anomalies and/or neurocognitive disabilities and prenatal patients had one or more structural anomalies, disorders of fetal growth, or fetal effusions. URM and US patients were prioritized for enrollment and underwent ES at a single academic center. We identified definitive positive or probable positive results in 201/845 (23.8%) patients, with a significantly higher diagnostic rate in pediatric (26.7%) compared to prenatal patients (19.0%) (P = 0.01). For both pediatric and prenatal patients, the diagnostic yield and frequency of inconclusive findings did not differ significantly between URM and non-URM patients or between patients with US status and those without US status. Our results demonstrate a similar diagnostic yield of ES between prenatal and pediatric URM/US patients and non-URM/US patients for positive and inconclusive results. These data support the use of ES to identify clinically relevant variants in patients from diverse populations.

Anthropometric traits, measuring body size and shape, are highly heritable and significant clinical risk factors for cardiometabolic disorders. These traits have been extensively studied in genome-wide association studies (GWASs), with hundreds of genome-wide significant loci identified. We performed a whole-exome sequence analysis of the genetics of height, body mass index (BMI) and waist/hip ratio (WHR). We meta-analyzed single-variant and gene-based associations of whole-exome sequence variation with height, BMI, and WHR in up to 22,004 individuals, and we assessed replication of our findings in up to 16,418 individuals from 10 independent cohorts from Trans-Omics for Precision Medicine (TOPMed). We identified four trait associations with single-nucleotide variants (SNVs; two for height and two for BMI) and replicated the LECT2 gene association with height. Our expression quantitative trait locus (eQTL) analysis within previously reported GWAS loci implicated CEP63 and RFT1 as potential functional genes for known height loci. We further assessed enrichment of SNVs, which were monogenic or syndromic variants within loci associated with our three traits. This led to the significant enrichment results for height, whereas we observed no Bonferroni-corrected significance for all SNVs. With a sample size of ∼20,000 whole-exome sequences in our discovery dataset, our findings demonstrate the importance of genomic sequencing in genetic association studies, yet they also illustrate the challenges in identifying effects of rare genetic variants.

Valosin-containing protein (VCP) gene mutations have been associated with a rare autosomal dominant, adult-onset progressive disease known as multisystem proteinopathy 1 (MSP1), or inclusion body myopathy (IBM), Paget's disease of bone (PDB), frontotemporal dementia (FTD), (IBMPFD), and amyotrophic lateral sclerosis (ALS). We report the clinical and genetic analysis findings in five patients, three from the same family, with novel VCP gene variants: NM_007126.5 c.1106T>C (p.I369T), c.478G>A (p.A160T), and c.760A>T (p.I254F), associated with cardinal MSP1 manifestations including myopathy, PDB, and FTD. Our report adds to the spectrum of heterozygous pathogenic variants found in the VCP gene and the high degree of clinical heterogeneity. This case series prompts increased awareness and early consideration of MSP1 in the differential diagnosis of myopathies and/or PDB, dementia, or ALS to improve the diagnosis and early management of clinical symptoms.

Epigenetic reprogramming drives tumorigenesis in pediatric H3K27M diffuse midline glioma (DMG) by altering the canonical functions of chromatin remodeling complexes. These studies (i) identified BRG1 (encoded by SMARCA4), the catalytic subunit of the mammalian SWI/SNF (BAF) chromatin remodeling complex, as a novel dependency in pediatric H3K27M glioma; (ii) investigated the molecular mechanisms underlying the maintenance of the progenitor state; and (iii) demonstrated efficacy for BRG1 inhibitors.The authors identified the BRG1 ATPase as a dependency in pediatric H3K27M-mutant DMG. SOX10 recruits BRG1 to regulatory elements to drive progression. Pharmacologically targeting BRG1 reduced tumor volume and improved survival in vivo. Inhibiting BRG1 ATPase represents a potential therapeutic strategy for pediatric H3K27M DMG. See related article by Panditharatna et al., p. 2880 (5) See related article by Mo et al., p. 2906 (4) .

Background and objectivesWhile somatic mutations have been well-studied in cancer, their roles in other complex traits are much less understood. Our goal is to identify somatic variants that may contribute to the formation of saccular cerebral aneurysms.MethodsWe performed whole-exome sequencing on aneurysm tissues and paired peripheral blood. RNA sequencing and the CRISPR/Cas9 system were then used to perform functional validation of our results.ResultsSomatic variants involved in supervillin (SVIL) or its regulation were found in 17% of aneurysm tissues. In the presence of a mutation in the SVIL gene, the expression level of SVIL was downregulated in the aneurysm tissue compared with normal control vessels. Downstream signaling pathways that were induced by knockdown of SVIL via the CRISPR/Cas9 system in vascular smooth muscle cells (vSMCs) were determined by evaluating changes in gene expression and protein kinase phosphorylation. We found that SVIL regulated the phenotypic modulation of vSMCs to the synthetic phenotype via Krüppel-like factor 4 and platelet-derived growth factor and affected cell migration of vSMCs via the RhoA/ROCK pathway.DiscussionWe propose that somatic variants form a novel mechanism for the development of cerebral aneurysms. Specifically, somatic variants in SVIL result in the phenotypic modulation of vSMCs, which increases the susceptibility to aneurysm formation. This finding suggests a new avenue for the therapeutic intervention and prevention of cerebral aneurysms.

Nonhuman primates living in proximity to humans increase risks for sylvatic arbovirus transmission. We collected serum samples from nonhuman primates in Hlawga National Park near Yangon, Myanmar, and detected antibodies against chikungunya (33%) and Japanese encephalitis (4%) viruses. Buffer zones between primate and human communities might reduce cross-species arbovirus transmission.

Glioblastoma (GBM) is characterized by extensive cellular and genetic heterogeneity. Its initial presentation as primary disease (pGBM) has been subject to exhaustive molecular and cellular profiling. By contrast, our understanding of how GBM evolves to evade the selective pressure of therapy is starkly limited. The proteomic landscape of recurrent GBM (rGBM), which is refractory to most treatments used for pGBM, are poorly known. We, therefore, quantified the transcriptome and proteome of 134 patient-derived pGBM and rGBM samples, including 40 matched pGBM-rGBM pairs. GBM subtypes transition from pGBM to rGBM towards a preferentially mesenchymal state at recurrence, consistent with the increasingly invasive nature of rGBM. We identified immune regulatory/suppressive genes as important drivers of rGBM and in particular 2-5-oligoadenylate synthase 2 (OAS2) as an essential gene in recurrent disease. Our data identify a new class of therapeutic targets that emerge from the adaptive response of pGBM to therapy, emerging specifically in recurrent disease and may provide new therapeutic opportunities absent at pGBM diagnosis.

Building on an exercise that identified potential harms from simulated investigational releases of a population suppression gene drive for malaria vector control, a series of online workshops identified nine recommendations to advance future environmental risk assessment of gene drive applications.

Mutationally activated BRAF is detected in approximately 7% of human lung adenocarcinomas, with BRAFT1799A serving as a predictive biomarker for treatment of patients with FDA-approved inhibitors of BRAFV600E oncoprotein signaling. In genetically engineered mouse (GEM) models, expression of BRAFV600E in the lung epithelium initiates growth of benign lung tumors that, without additional genetic alterations, rarely progress to malignant lung adenocarcinoma. To identify genes that cooperate with BRAFV600E for malignant progression, we used Sleeping Beauty-mediated transposon mutagenesis, which dramatically accelerated the emergence of lethal lung cancers. Among the genes identified was Rbms3, which encodes an RNA-binding protein previously implicated as a putative tumor suppressor. Silencing of RBMS3 via CRISPR/Cas9 gene editing promoted growth of BRAFV600E lung organoids and promoted development of malignant lung cancers with a distinct micropapillary architecture in BRAFV600E and EGFRL858R GEM models. BRAFV600E/RBMS3Null lung tumors displayed elevated expression of Ctnnb1, Ccnd1, Axin2, Lgr5, and c-Myc mRNAs, suggesting that RBMS3 silencing elevates signaling through the WNT/β-catenin signaling axis. Although RBMS3 silencing rendered BRAFV600E-driven lung tumors resistant to the effects of dabrafenib plus trametinib, the tumors were sensitive to inhibition of porcupine, an acyltransferase of WNT ligands necessary for their secretion. Analysis of The Cancer Genome Atlas patient samples revealed that chromosome 3p24, which encompasses RBMS3, is frequently lost in non-small cell lung cancer and correlates with poor prognosis. Collectively, these data reveal the role of RBMS3 as a lung cancer suppressor and suggest that RBMS3 silencing may contribute to malignant NSCLC progression.SignificanceLoss of RBMS3 cooperates with BRAFV600E to induce lung tumorigenesis, providing a deeper understanding of the molecular mechanisms underlying mutant BRAF-driven lung cancer and potential strategies to more effectively target this disease.

UV radiation (UVR) has significant physiological effects on organisms living at or near the Earth's surface, yet the full suite of genes required for fitness of a photosynthetic organism in a UVR-rich environment remains unknown. This study reports a genome-wide fitness assessment of the genes that affect UVR tolerance under environmentally relevant UVR dosages in the model cyanobacterium Synechococcus elongatus PCC 7942. Our results highlight the importance of specific genes that encode proteins involved in DNA repair, glutathione synthesis, and the assembly and maintenance of photosystem II, as well as genes that encode hypothetical proteins and others without an obvious connection to canonical methods of UVR tolerance. Disruption of a gene that encodes a leucyl aminopeptidase (LAP) conferred the greatest UVR-specific decrease in fitness. Enzymatic assays demonstrated a strong pH-dependent affinity of the LAP for the dipeptide cysteinyl-glycine, suggesting an involvement in glutathione catabolism as a function of night-time cytosolic pH level. A low differential expression of the LAP gene under acute UVR exposure suggests that its relative importance would be overlooked in transcript-dependent screens. Subsequent experiments revealed a similar UVR-sensitivity phenotype in LAP knockouts of other organisms, indicating conservation of the functional role of LAPs in UVR tolerance.

Transfer RNAs have been extensively explored as the molecules that translate the genetic code into proteins. At this interface of genetics and biochemistry, tRNAs direct the efficiency of every major step of translation by interacting with a multitude of binding partners. However, due to the variability of tRNA sequences and the abundance of diverse post-transcriptional modifications, a guidebook linking tRNA sequences to specific translational outcomes has yet to be elucidated. Here, we review substantial efforts that have collectively uncovered tRNA engineering principles that can be used as a guide for the tuning of translation fidelity. These principles have allowed for the development of basic research, expansion of the genetic code with non-canonical amino acids, and tRNA therapeutics.

Adaptations to infectious and dietary pressures shape mammalian physiology and disease risk. How such adaptations affect sex-biased diseases remains insufficiently studied. In this study, we show that sex-dependent hepatic gene programs confer a robust (~300%) survival advantage for male mice during lethal bacterial infection. The transcription factor B cell lymphoma 6 (BCL6), which masculinizes hepatic gene expression at puberty, is essential for this advantage. However, protection by BCL6 protein comes at a cost during conditions of dietary excess, which result in overt fatty liver and glucose intolerance in males. Deleting hepatic BCL6 reverses these phenotypes but markedly lowers male survival during infection, thus establishing a sex-dependent trade-off between host defense and metabolic systems. Our findings offer strong evidence that some current sex-biased diseases are rooted in ancient evolutionary trade-offs between immunity and metabolism.

Progressive dilation of the infrarenal aortic diameter is a consequence of the ageing process and is considered the main determinant of abdominal aortic aneurysm (AAA). We aimed to investigate the genetic and clinical determinants of abdominal aortic diameter (AAD). We conducted a meta-analysis of genome-wide association studies in 10 cohorts (n = 13 542) imputed to the 1000 Genome Project reference panel including 12 815 subjects in the discovery phase and 727 subjects [Partners Biobank cohort 1 (PBIO)] as replication. Maximum anterior-posterior diameter of the infrarenal aorta was used as AAD. We also included exome array data (n = 14 480) from seven epidemiologic studies. Single-variant and gene-based associations were done using SeqMeta package. A Mendelian randomization analysis was applied to investigate the causal effect of a number of clinical risk factors on AAD. In genome-wide association study (GWAS) on AAD, rs74448815 in the intronic region of LDLRAD4 reached genome-wide significance (beta = -0.02, SE = 0.004, P-value = 2.10 × 10-8). The association replicated in the PBIO1 cohort (P-value = 8.19 × 10-4). In exome-array single-variant analysis (P-value threshold = 9 × 10-7), the lowest P-value was found for rs239259 located in SLC22A20 (beta = 0.007, P-value = 1.2 × 10-5). In the gene-based analysis (P-value threshold = 1.85 × 10-6), PCSK5 showed an association with AAD (P-value = 8.03 × 10-7). Furthermore, in Mendelian randomization analyses, we found evidence for genetic association of pulse pressure (beta = -0.003, P-value = 0.02), triglycerides (beta = -0.16, P-value = 0.008) and height (beta = 0.03, P-value

Advances in genome and tissue engineering have spurred significant progress and opportunity for innovation in cancer modeling. Human induced pluripotent stem cells (iPSCs) are an established and powerful tool to study cellular processes in the context of disease-specific genetic backgrounds; however, their application to cancer has been limited by the resistance of many transformed cells to undergo successful reprogramming. Here, we review the status of human iPSC modeling of solid tumors in the context of genetic engineering, including how base and prime editing can be incorporated into "bottom-up" cancer modeling, a term we coined for iPSC-based cancer models using genetic engineering to induce transformation. This approach circumvents the need to reprogram cancer cells while allowing for dissection of the genetic mechanisms underlying transformation, progression, and metastasis with a high degree of precision and control. We also discuss the strengths and limitations of respective engineering approaches and outline experimental considerations for establishing future models.

Biallelic pathogenic variants in SZT2 result in a neurodevelopmental disorder with shared features, including early-onset epilepsy, developmental delay, macrocephaly, and corpus callosum abnormalities. SZT2 is as a critical scaffolding protein in the amino acid sensing arm of the mTORC1 signalling pathway. Due to its large size (3432 amino acids), lack of crystal structure, and absence of functional domains, it is difficult to determine the pathogenicity of SZT2 missense and in-frame deletions, but these variants are increasingly detected and reported by clinical genetic testing in individuals with epilepsy. To exemplify this latter point, here we describe a cohort of 12 individuals with biallelic SZT2 variants and phenotypic overlap with SZT2-related neurodevelopmental disorders. However, the majority of individuals carried one or more SZT2 variants of uncertain significance (VUS), highlighting the need for functional characterization to determine, which, if any, of these VUS were pathogenic. Thus, we developed a novel individualized platform to identify SZT2 loss-of-function variants in the context of mTORC1 signalling and reclassify VUS. Using this platform, we identified a recurrent in-frame deletion (SZT2 p.Val1984del) which was determined to be a loss-of-function variant and therefore likely pathogenic. Haplotype analysis revealed that this single in-frame deletion is a founder variant in those of Ashkenazi Jewish ancestry. Moreover, this approach allowed us to tentatively reclassify all of the VUS in our cohort of 12 individuals, identifying five individuals with biallelic pathogenic or likely pathogenic variants. Clinical features of these five individuals consisted of early-onset seizures (median 24 months), focal seizures, developmental delay and macrocephaly similar to previous reports. However, we also show a widening of the phenotypic spectrum, as none of the five individuals had corpus callosum abnormalities, in contrast to previous reports. Overall, we present a rapid assay to resolve VUS in SZT2, identify a founder variant in individuals of Ashkenazi Jewish ancestry, and demonstrate that corpus callosum abnormalities is not a hallmark feature of this condition. Our approach is widely applicable to other mTORopathies including the most common causes of the focal genetic epilepsies, DEPDC5, TSC1/2, MTOR and NPRL2/3.

Cerebral small vessel disease is a leading cause of stroke and a major contributor to cognitive decline and dementia, but our understanding of specific genes underlying the cause of sporadic cerebral small vessel disease is limited. We report a genome-wide association study and a whole-exome association study on a composite extreme phenotype of cerebral small vessel disease derived from its most common MRI features: white matter hyperintensities and lacunes. Seventeen population-based cohorts of older persons with MRI measurements and genome-wide genotyping (n = 41 326), whole-exome sequencing (n = 15 965), or exome chip (n = 5249) data contributed 13 776 and 7079 extreme small vessel disease samples for the genome-wide association study and whole-exome association study, respectively. The genome-wide association study identified significant association of common variants in 11 loci with extreme small vessel disease, of which the chr12q24.11 locus was not previously reported to be associated with any MRI marker of cerebral small vessel disease. The whole-exome association study identified significant associations of extreme small vessel disease with common variants in the 5' UTR region of EFEMP1 (chr2p16.1) and one probably damaging common missense variant in TRIM47 (chr17q25.1). Mendelian randomization supports the causal association of extensive small vessel disease severity with increased risk of stroke and Alzheimer's disease. Combined evidence from summary-based Mendelian randomization studies and profiling of human loss-of-function allele carriers showed an inverse relation between TRIM47 expression in the brain and blood vessels and extensive small vessel disease severity. We observed significant enrichment of Trim47 in isolated brain vessel preparations compared to total brain fraction in mice, in line with the literature showing Trim47 enrichment in brain endothelial cells at single cell level. Functional evaluation of TRIM47 by small interfering RNAs-mediated knockdown in human brain endothelial cells showed increased endothelial permeability, an important hallmark of cerebral small vessel disease pathology. Overall, our comprehensive gene-mapping study and preliminary functional evaluation suggests a putative role of TRIM47 in the pathophysiology of cerebral small vessel disease, making it an important candidate for extensive in vivo explorations and future translational work.

The factors driving therapy resistance in diffuse glioma remain poorly understood. To identify treatment-associated cellular and genetic changes, we analyzed RNA and/or DNA sequencing data from the temporally separated tumor pairs of 304 adult patients with isocitrate dehydrogenase (IDH)-wild-type and IDH-mutant glioma. Tumors recurred in distinct manners that were dependent on IDH mutation status and attributable to changes in histological feature composition, somatic alterations, and microenvironment interactions. Hypermutation and acquired CDKN2A deletions were associated with an increase in proliferating neoplastic cells at recurrence in both glioma subtypes, reflecting active tumor growth. IDH-wild-type tumors were more invasive at recurrence, and their neoplastic cells exhibited increased expression of neuronal signaling programs that reflected a possible role for neuronal interactions in promoting glioma progression. Mesenchymal transition was associated with the presence of a myeloid cell state defined by specific ligand-receptor interactions with neoplastic cells. Collectively, these recurrence-associated phenotypes represent potential targets to alter disease progression.

The oral microbiome is linked to oral and systemic health, but its fluctuation under frequent daily activities remains elusive. Here, we sampled saliva at 10- to 60-min intervals to track the high-resolution microbiome dynamics during the course of human activities. This dense time series data showed that eating activity markedly perturbed the salivary microbiota, with tongue-specific Campylobacter concisus and Oribacterium sinus and dental plaque-specific Lautropia mirabilis, Rothia aeria, and Neisseria oralis increased after every meal in a temporal order. The observation was reproducible in multiple subjects and across an 11-mo period. The microbiome composition showed significant diurnal oscillation patterns at different taxonomy levels with Prevotella/Alloprevotella increased at night and Bergeyella HMT 206/Haemophilus slowly increased during the daytime. We also identified microbial co-occurring patterns in saliva that are associated with the intricate biogeography of the oral microbiome. Microbial source tracking analysis showed that the contributions of distinct oral niches to the salivary microbiome were dynamically affected by daily activities, reflecting the role of saliva in exchanging microbes with other oral sites. Collectively, our study provides insights into the temporal microbiome variation in saliva and highlights the need to consider daily activities and diurnal factors in design of oral microbiome studies.

We have previously demonstrated that deletion of activin receptor-like kinase 1 (Alk1) or endoglin in a fraction of endothelial cells (ECs) induces brain arteriovenous malformations (bAVMs) in adult mice upon angiogenic stimulation. Here, we addressed three related questions: (1) could Alk1- mutant bone marrow (BM)-derived ECs (BMDECs) cause bAVMs? (2) is Alk1- ECs clonally expended during bAVM development? and (3) is the number of mutant ECs correlates to bAVM severity? For the first question, we transplanted BM from PdgfbiCreER;Alk12f/2f mice (EC-specific tamoxifen-inducible Cre with Alk1-floxed alleles) into wild-type mice, and then induced bAVMs by intra-brain injection of an adeno-associated viral vector expressing vascular endothelial growth factor and intra-peritoneal injection of tamoxifen. For the second question, clonal expansion was analyzed using PdgfbiCreER;Alk12f/2f;confetti+/- mice. For the third question, we titrated tamoxifen to limit Alk1 deletion and compared the severity of bAVM in mice treated with low and high tamoxifen doses. We found that wild-type mice with PdgfbiCreER;Alk12f/2f BM developed bAVMs upon VEGF stimulation and Alk1 gene deletion in BMDECs. We also observed clusters of ECs expressing the same confetti color within bAVMs and significant proliferation of Alk1- ECs at early stage of bAVM development, suggesting that Alk1- ECs clonally expanded by local proliferation. Tamoxifen dose titration revealed a direct correlation between the number of Alk1- ECs and the burden of dysplastic vessels in bAVMs. These results provide novel insights for the understanding of the mechanism by which a small fraction of Alk1 or endoglin mutant ECs contribute to development of bAVMs.

Human genetic studies support an inverse causal relationship between leukocyte telomere length (LTL) and coronary artery disease (CAD), but directionally mixed effects for LTL and diverse malignancies. Clonal hematopoiesis of indeterminate potential (CHIP), characterized by expansion of hematopoietic cells bearing leukemogenic mutations, predisposes both hematologic malignancy and CAD. TERT (which encodes telomerase reverse transcriptase) is the most significantly associated germline locus for CHIP in genome-wide association studies. Here, we investigated the relationship between CHIP, LTL, and CAD in the Trans-Omics for Precision Medicine (TOPMed) program (n = 63,302) and UK Biobank (n = 47,080). Bidirectional Mendelian randomization studies were consistent with longer genetically imputed LTL increasing propensity to develop CHIP, but CHIP then, in turn, hastens to shorten measured LTL (mLTL). We also demonstrated evidence of modest mediation between CHIP and CAD by mLTL. Our data promote an understanding of potential causal relationships across CHIP and LTL toward prevention of CAD.