Complex Mutations Research Articles

Persistent Molecular Disease in Adult Patients With AML Evaluated With Whole-Exome and Targeted Error-Corrected DNA Sequencing.

Persistent molecular disease (PMD) after induction chemotherapy predicts relapse in AML. In this study, we used whole-exome sequencing (WES) and targeted error-corrected sequencing to assess the frequency and mutational patterns of PMD in 30 patients with AML. The study cohort included 30 patients with adult AML younger than 65 years who were uniformly treated with standard induction chemotherapy. Tumor/normal WES was performed for all patients at presentation. PMD analysis was evaluated in bone marrow samples obtained during clinicopathologic remission using repeat WES and analysis of patient-specific mutations and error-corrected sequencing of 40 recurrently mutated AML genes (MyeloSeq). WES for patient-specific mutations detected PMD in 63% of patients (19/30) using a minimum variant allele fraction (VAF) of 2.5%. In comparison, MyeloSeq identified persistent mutations above 0.1% VAF in 77% of patients (23/30). PMD was usually present at relatively high levels (>2.5% VAFs), such that WES and MyeloSeq agreed for 73% of patients despite differences in detection limits. Mutations in DNMT3A, ASXL1, and TET2 (ie, DTA mutations) were persistent in 16 of 17 patients, but WES also detected non-DTA mutations in 14 of these patients, which for some patients distinguished residual AML cells from clonal hematopoiesis. Surprisingly, MyeloSeq detected additional variants not identified at presentation in 73% of patients that were consistent with new clonal cell populations after chemotherapy. PMD and clonal hematopoiesis are both common in patients with AML in first remission. These findings demonstrate the importance of baseline testing for accurate interpretation of mutation-based tumor monitoring assays for patients with AML and highlight the need for clinical trials to determine whether these complex mutation patterns correlate with clinical outcomes in AML.

The DREAM complex functions as conserved master regulator of somatic DNA-repair capacities

The DNA-repair capacity in somatic cells is limited compared with that in germ cells. It has remained unknown whether not only lesion-type-specific, but overall repair capacities could be improved. Here we show that the DREAM repressor complex curbs the DNA-repair capacities in somatic tissues of Caenorhabditis elegans. Mutations in the DREAM complex induce germline-like expression patterns of multiple mechanisms of DNA repair in the soma. Consequently, DREAM mutants confer resistance to a wide range of DNA-damage types during development and aging. Similarly, inhibition of the DREAM complex in human cells boosts DNA-repair gene expression and resistance to distinct DNA-damage types. DREAM inhibition leads to decreased DNA damage and prevents photoreceptor loss in progeroid Ercc1−/− mice. We show that the DREAM complex transcriptionally represses essentially all DNA-repair systems and thus operates as a highly conserved master regulator of the somatic limitation of DNA-repair capacities.

Determinants of subtype-selectivity of the anthelmintic paraherquamide A on Caenorhabditis elegans nicotinic acetylcholine receptors.

The anthelmintic paraherquamide A acts selectively on the nematode L-type nicotinic acetylcholine receptors (nAChRs) but the mechanism of its selectivity is unknown. This study targeted the basis of paraherquamide A selectivity by determining an X-ray crystal structure of the acetylcholine binding protein (AChBP), a surrogate nAChR ligand-binding domain, complexed with the compound and by measuring its actions on wild-type and mutant Caenorhabditis elegans nematodes and functionally expressed C. elegans nAChRs. Paraherquamide A showed a higher efficacy for the levamisole-sensitive (L-type (UNC-38/UNC-29/UNC-63/LEV8/LEV-1)) nAChR than the nicotine-sensitive (N-type (ACR-16)) nAChR, a result consistent with in vivo studies on wild type worms and worms with mutations in subunits of these two classes of receptors. The X-ray crystal structure of the Ls-AChBP-paraherquamide A complex and site-directed amino acid mutation studies showed for the first time that loop C, loop E and loop F of the orthosteric receptor binding site play critical roles in the observed L-type nAChR selective actions of paraherquamide A. Significance Statement Paraherquamide A, an oxindole alkaloid, has been shown to act selectively on the L-type over N-type nAChRs in nematodes, but the mechanism of selectivity is unknown. We have co-crystallized paraherquamide A with the acetylcholine binding protein, a surrogate of nAChRs, and found that structural features of loop C, loop E and loop F contribute to the L-type nAChR selectivity of the alkaloid. The results create a new platform for the design of anthelmintic drugs targeting cholinergic neurotransmission in parasitic nematodes.

Chromatin structure influences rate and spectrum of spontaneous mutations in Neurospora crassa.

Although mutation rates have been extensively studied, variation in mutation rates throughout the genome is poorly understood. To understand patterns of genetic variation, it is important to understand how mutation rates vary. Chromatin modifications may be an important factor in determining variation in mutation rates in eukaryotic genomes. To study variation in mutation rates, we performed a mutation accumulation (MA) experiment in the filamentous fungus Neurospora crassa and sequenced the genomes of the 40 MA lines that had been propagated asexually for approximately 1015 [Formula: see text] mitoses. We detected 1322 mutations in total and observed that the mutation rate was higher in regions of low GC, in domains of H3K9 trimethylation, in centromeric regions, and in domains of H3K27 trimethylation. The rate of single-nucleotide mutations in euchromatin was [Formula: see text] In contrast, the mutation rate in H3K9me3 domains was 10-fold higher: 2.43 [Formula: see text] We also observed that the spectrum of single-nucleotide mutations was different between H3K9me3 and euchromatic domains. Our statistical model of mutation rate variation predicted a moderate amount of extant genetic variation, suggesting that the mutation rate is an important factor in determining levels of natural genetic variation. Furthermore, we characterized mutation rates of structural variants, complex mutations, and the effect of local sequence context on the mutation rate. Our study highlights that chromatin modifications are associated with mutation rates, and accurate evolutionary inferences should take variation in mutation rates across the genome into account.

Molecular spectrum of α- and β-thalassemia among individuals of reproductive age in the Zhuhai region of southern China.

Thalassemia is the most common monogenic disease in South and Southeast Asia. An accurate assessment of the relative frequency and composition of thalassemia mutations is important for the design of appropriate strategies to prevent the disease. In this study, we aimed to decode the molecular characterization of thalassemia mutations in Zhuhai region of southern China. A total of 8048 individuals who were potential thalassemia carriers were enrolled. Gap-polymerase chain reaction (Gap-PCR) and reverse dot-blot (RDB) hybridization methods were employed to detect common deletional and non-deletional thalassemia mutations. Multiplex ligation dependent probe amplification (MLPA) and Sanger sequencing were used to analyze and verify rare and complex mutations. We diagnosed 3433 individuals as thalassemia carriers or patients. Of these, 2395 (69.76%) individuals with α-thalassemia harbored 13 α-globin gene mutations. The three most common α-thalassemia mutations were --SEA (60.08%), -α3.7 (20.62%) and -α4.2 (9.25%). We diagnosed 903 (26.30%) individuals with β-thalassemia and identified 20 β-globin gene mutations, of which the three most frequent were CD41/42 (-TCTT) (38.10%), IVS-II-654 (C>T) (23.69%) and TATAbox-28 (A>G) (15.18%). In addition, we identified 15 rare thalassemia variants. We also summarized the association between the thalassemia genotype and hematological parameters, which demonstrated the broad phenotypic heterogeneity caused by globin gene mutations. This is the first survey of thalassemia molecular epidemiology and hematological phenotype in Zhuhai region. It uncovered a high prevalence and complex molecular spectrum of thalassemia. These findings can be used as a basis for thalassemia diagnosis, counseling and prevention management.

T-Cell Prolymphocytic Leukemia With t(X;14)(q28;q11.2): A Clinicopathologic Study of 15 Cases.

T-cell prolymphocytic leukemia (T-PLL) is a rare mature T-cell leukemia usually characterized by inv(14)(q11.2q32)/t(14;14)(q11.2;q32). In this study, we aimed to investigate the clinicopathologic features and molecular profile of T-PLL associated with t(X;14)(q28;q11.2). The study group included 10 women and 5 men with a median age of 64 years. All 15 patients had a diagnosis of T-PLL with t(X;14)(q28;q11.2). All 15 patients had lymphocytosis at initial diagnosis. Morphologically, the leukemic cells had features of prolymphocytes in 11 patients, small cell variant in 3, and cerebriform variant in 1. All 15 patients had hypercellular bone marrow with an interstitial infiltrate in 12 (80%) cases. By flow cytometry, the leukemic cells were surface CD3+/CD5+/CD7+/CD26+/CD52+/TCR α/β+ in 15 (100%) cases, CD2+ in 14 (93%) cases, CD4+/CD8+ in 8 (53%) cases, CD4+/CD8- in 6 (40%) cases, and CD4-/CD8 + in 1 (7%) case. At the cytogenetic level, complex karyotypes with t(X;14)(q28;q11.2) were seen in all 15 patients assessed. Mutational analysis showed mutations of JAK3 in 5 of 6 and STAT5B p.N642H in 2 of 6 patients. Patients received variable treatments, including 12 with alemtuzumab. After a median follow-up of 17.2 months, 8 of 15 (53%) patients died. T-PLL with t(X;14)(q28;q11.2) frequently shows a complex karyotype and mutations involving JAK/STAT pathway, and it is an aggressive disease with a poor outcome.

Comparison of the disease severity with infection of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) Delta and Omicron variants: A meta‐analysis

Comparison of the disease severity with infection of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) Delta and Omicron variants: A meta‐analysis

The Interplay Between Developmental Stage and Environment Underlies the Adaptive Effect of a Natural Transposable Element Insertion.

Establishing causal links between adaptive mutations and ecologically relevant phenotypes is key to understanding the process of adaptation, which is a central goal in evolutionary biology with applications for conservation, medicine, and agriculture. Yet despite recent progress, the number of identified causal adaptive mutations remains limited. Linking genetic variation to fitness-related effects is complicated by gene-by-gene and gene-by-environment interactions, among other processes. Transposable elements, which are often ignored in the quest for the genetic basis of adaptive evolution, are a genome-wide source of regulatory elements across organisms that can potentially result in adaptive phenotypes. In this work, we combine gene expression, in vivo reporter assays, CRISPR/Cas9 genome editing, and survival experiments to characterize in detail the molecular and phenotypic consequences of a natural Drosophila melanogaster transposable element insertion: the roo solo-LTR FBti0019985. This transposable element provides an alternative promoter to the transcription factor Lime, involved in cold- and immune-stress responses. We found that the effect of FBti0019985 on Lime expression depends on the interplay between the developmental stage and environmental condition. We further establish a causal link between the presence of FBti0019985 and increased survival to cold- and immune-stress. Our results exemplify how several developmental stages and environmental conditions need to be considered to characterize the molecular and functional effects of a genetic variant, and add to the growing body of evidence that transposable elements can induce complex mutations with ecologically relevant effects.

The Role of TRiC-enhanced Actin Folding in Leber Congenital Amaurosis.

Mutations in TCP-1 ring complex (TRiC) have been associated with Leber Congenital Amaurosis (LCA). TRiC is involved in protein folding and has 8 essential subunits including CCT5. Herein, we studied the retina of TRiC mutant zebrafish to evaluate the possible role of impaired actin and tubulin folding in LCA. The cct5 retina was histologically studied using Toluidine Blue staining as well as TUNEL, BrdU-labeling, and Phalloidin assays. Retinal organisation was assessed by quantification of the cellularity utilising DAPI. Laminar organization of cct5 retinas was intact. Enhanced apoptosis throughout the cct5 retina was not compensated by higher proliferation rates, leaving the cct5 retina smaller in size. Quantification of retinal layer cellularity demonstrated that specifically the numbers of the amacrine and the retinal ganglion cells were depleted, suggesting that the cct5 retina was not uniformly affected by the reduced actin folding. Whereas the current literature suggests that LCA is predominantly affecting retinal photoreceptor cells and the retinal pigment epithelium, cct5 analyses demonstrated the important role of folding of actin by TRiC, suggesting that cct5 is a useful tool to specifically analyze the role of F-actin filaments in the context of LCA.

S100a9 deficiency accelerates MDS-associated tumor escape via PD-1/PD-L1 overexpression.

In recent studies, the tolerable safety profile and positive bone marrow (BM) response suggest a beneficial use of anti-PD-1 agents in the treatment of Myelodysplastic Syndromes (MDS), but the underlying mechanism is still unknown. MDS is mainly characterized by ineffective hematopoiesis, which may contribute to inflammatory signaling or immune dysfunction. Our previous studies focused on inflammatory signaling, and the results showed that S100a9 expression was higher in low-risk MDS and lower in high-risk MDS. In this study, we combine the inflammatory signaling and immune dysfunction. SKM-1 cells and K562 cells co-cultured with S100a9 acquire apoptotic features. Moreover, we confirm the inhibitory effect of S100a9 on PD-1/PD-L1. Importantly, PD-1/PD-L1 blockade and S100a9 can both activate the PI3K/AKT/mTOR signaling pathway. The cytotoxicity is higher in lower-risk MDS-lymphocytes than in high-risk MDS-lymphocytes, and S100a9 partially rescues the exhausted cytotoxicity in lymphocytes. Our study demonstrates that S100a9 may inhibit MDS-associated tumor escape via PD-1/PD-L1 blockade through PI3K/AKT/mTOR signaling pathway activation. Our findings indicate the possible mechanisms by which anti-PD-1 agents may contribute to the treatment of MDS. These insights may provide mutation-specific treatment as a supplementary therapy for MDS patients with high-risk mutations, such as TP53, N-RAS or other complex mutations.

Dyskeratosis Congenita Links Telomere Attrition to Age-Related Systemic Energetics.

The underlying mechanisms of plasma metabolite signatures of human aging and age-related diseases are not clear but telomere attrition and dysfunction are central to both. Dyskeratosis congenita (DC) is associated with mutations in the telomerase enzyme complex (TERT, TERC, and DKC1) and progressive telomere attrition. We analyzed the effect of telomere attrition on senescence-associated metabolites in fibroblast-conditioned media and DC patient plasma. Samples were analyzed by gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. We showed extracellular citrate was repressed by canonical telomerase function in vitro and associated with DC leukocyte telomere attrition in vivo, leading to the hypothesis that altered citrate metabolism detects telomere dysfunction. However, elevated citrate and senescence factors only weakly distinguished DC patients from controls, whereas elevated levels of other tricarboxylic acid cycle (TCA) metabolites, lactate, and especially pyruvate distinguished them with high significance. The DC plasma signature most resembled that of patients with loss of function pyruvate dehydrogenase complex mutations and that of older subjects but significantly not those of type 2 diabetes, lactic acidosis, or elevated mitochondrial reactive oxygen species. Additionally, our data are consistent with further metabolism of citrate and lactate in the liver and kidneys. Citrate uptake in certain organs modulates age-related disease in mice and our data have similarities with age-related disease signatures in humans. Our results have implications for the role of telomere dysfunction in human aging in addition to its early diagnosis and the monitoring of anti-senescence therapeutics, especially those designed to improve telomere function.

Mutations in Complex I of the Mitochondrial Electron-Transport Chain Sensitize the Fruit Fly (Drosophila melanogaster) to Ether and Non-Ether Volatile Anesthetics.

The mitochondrial electron transport chain (mETC) contains molecular targets of volatile general anesthetics (VGAs), which places carriers of mutations at risk for anesthetic complications. The ND-2360114 and mt:ND2del1 lines of fruit flies (Drosophila melanogaster) that carry mutations in core subunits of Complex I of the mETC replicate numerous characteristics of Leigh syndrome (LS) caused by orthologous mutations in mammals and serve as models of LS. ND-2360114 flies are behaviorally hypersensitive to volatile anesthetic ethers and develop an age- and oxygen-dependent anesthetic-induced neurotoxicity (AiN) phenotype after exposure to isoflurane but not to the related anesthetic sevoflurane. The goal of this paper was to investigate whether the alkane volatile anesthetic halothane and other mutations in Complex I and in Complexes II-V of the mETC cause AiN. We found that (i) ND-2360114 and mt:ND2del1 were susceptible to toxicity from halothane; (ii) in wild-type flies, halothane was toxic under anoxic conditions; (iii) alleles of accessory subunits of Complex I predisposed to AiN; and (iv) mutations in Complexes II-V did not result in an AiN phenotype. We conclude that AiN is neither limited to ether anesthetics nor exclusive to mutations in core subunits of Complex I.

A Mutation in Mouse MT-ATP6 Gene Induces Respiration Defects and Opposed Effects on the Cell Tumorigenic Phenotype.

As the last step of the OXPHOS system, mitochondrial ATP synthase (or complex V) is responsible for ATP production by using the generated proton gradient, but also has an impact on other important functions linked to this system. Mutations either in complex V structural subunits, especially in mtDNA-encoded ATP6 gene, or in its assembly factors, are the molecular cause of a wide variety of human diseases, most of them classified as neurodegenerative disorders. The role of ATP synthase alterations in cancer development or metastasis has also been postulated. In this work, we reported the generation and characterization of the first mt-Atp6 pathological mutation in mouse cells, an m.8414A>G transition that promotes an amino acid change from Asn to Ser at a highly conserved residue of the protein (p.N163S), located near the path followed by protons from the intermembrane space to the mitochondrial matrix. The phenotypic consequences of the p.N163S change reproduce the effects of MT-ATP6 mutations in human diseases, such as dependence on glycolysis, defective OXPHOS activity, ATP synthesis impairment, increased ROS generation or mitochondrial membrane potential alteration. These observations demonstrate that this mutant cell line could be of great interest for the generation of mouse models with the aim of studying human diseases caused by alterations in ATP synthase. On the other hand, mutant cells showed lower migration capacity, higher expression of MHC-I and slightly lower levels of HIF-1α, indicating a possible reduction of their tumorigenic potential. These results could suggest a protective role of ATP synthase inhibition against tumor transformation that could open the door to new therapeutic strategies in those cancer types relying on OXPHOS metabolism.

DeteX: A highly accurate software for detecting SNV and InDel in single and paired NGS data in cancer research.

Background: Genetic testing is becoming more and more accepted in the auxiliary diagnosis and treatment of tumors. Due to the different performance of the existing bioinformatics software and the different analysis results, the needs of clinical diagnosis and treatment cannot be met. To this end, we combined Bayesian classification model (BC) and fisher exact test (FET), and develop an efficient software DeteX to detect SNV and InDel mutations. It can detect the somatic mutations in tumor-normal paired samples as well as mutations in a single sample. Methods: Combination of Bayesian classification model (BC) and fisher exact test (FET). Results: We detected SNVs and InDels in 11 TCGA glioma samples, 28 clinically targeted capture samples and 2 NCCL-EQA standard samples with DeteX, VarDict, Mutect, VarScan and GatkSNV. The results show that, among the three groups of samples, DeteX has higher sensitivity and precision whether it detects SNVs or InDels than other callers and the F1 value of DeteX is the highest. Especially in the detection of substitution and complex mutations, only DeteX can accurately detect these two kinds of mutations. In terms of single-sample mutation detection, DeteX is much more sensitive than the HaplotypeCaller program in Gatk. In addition, although DeteX has higher mutation detection capabilities, its running time is only .609 of VarDict, which is .704 and .343 longer than VarScan and MuTect, respectively. Conclusion: In this study, we developed DeteX to detect SNV and InDel mutations in single and paired samples. DeteX has high sensitivity and precision especially in the detection of substitution and complex mutations. In summary, DeteX from NGS data is a good SNV and InDel caller.

Mutational signature of mtDNA confers mechanistic insight into oxidative metabolism remodeling in colorectal cancer.

Rationale: Mitochondrial dysfunction caused by mitochondrial DNA (mtDNA) mutations and subsequent metabolic defects are closely involved in tumorigenesis and progression in a cancer-type specific manner. To date, the mutational pattern of mtDNA somatic mutations in colorectal cancer (CRC) tissues and its clinical implication are still not completely clear. Methods: In the present study, we generated a large mtDNA somatic mutation dataset from three CRC cohorts (432, 1,015, and 845 patients, respectively) and then most comprehensively characterized the CRC-specific evolutionary pattern and its clinical implication. Results: Our results showed that the mtDNA control region (mtCTR) with a high mutation density exhibited a distinct mutation spectrum characterizing a high enrichment of L-strand C > T mutations, which was contrary to the H-strand C > T mutational bias observed in the mtDNA coding region (mtCDR) (P < 0.001). Further analysis clearly confirmed the relaxed evolutionary selection of mtCTR mutations, which was mainly characterized by the similar distribution of hypervariable region (HVS) and non-HVS mutation density. Moreover, significant negative selection was identified in mutations of mtDNA complex V (ATP6/ATP8) and tRNA loop regions. Although our data showed that oxidative metabolism was commonly increased in CRC cells, mtDNA somatic mutations in CRC tissues were not closely associated with mitochondrial biogenesis, oxidative metabolism, and clinical progression, suggesting a cancer-type specific relationship between mtDNA mutations and mitochondrial metabolic functions in CRC cells. Conclusion: Our study identified the CRC-specific evolutionary mode of mtDNA mutations, which is possibly matched to specific mitochondrial metabolic remodeling and confers new mechanic insight into CRC tumorigenesis.

Dual guide RNA-mediated concurrent C&G-to-T&A and A&T-to-G&C conversions using CRISPR base editors

Base editing tools enable precise genome modifications, disease modeling, and promising gene therapy. However, many human genetic diseases are elicited by multi-nucleotide variants (MNVs) with heterogeneous substitutions at the same genomic locus. Based on the adenine and cytosine base editors, dual base editors that can catalyze concurrent C-to-T and A-to-G editing have been developed, while simultaneous C&G-to-T&A and A&T-to-G&C conversions on the same allele have not been achieved at the desirable site. Here we propose a strategy of combining base editors with dual guide RNAs (gRNAs) that target two overlapped neighboring loci on the opposite strands, which can induce simultaneous C&G-to-T&A and A&T-to-G&C conversions within their overlapping targeting windows. Moreover, one of the paired gRNAs is mutated to perfectly match another gRNA-edited sequence, efficiently facilitating concurrent base conversions on the same allele. To further expand the targeting scopes, PAMless SpRY Cas9-mediated base editors are combined with our optimized dual gRNAs system to induce expected concurrent base editing and to install neighboring pathogenic MNVs in TP53 in cancer cells. In addition, more complex mutation types can be achieved by integrating dual base editors and our dual gRNAs strategy. Thus, we establish a general strategy to efficiently induce MNVs in human genome, helping to dissect the functions of pathogenic MNVs with multifarious types.

RNA Interference Approach Is a Good Strategy against SARS-CoV-2

COVID-19, caused by SARS-CoV-2, created a devastating outbreak worldwide and consequently became a global health concern. However, no verifiable, specifically targeted treatment has been devised for COVID-19. Several emerging vaccines have been used, but protection has not been satisfactory. The complex genetic composition and high mutation frequency of SARS-CoV-2 have caused an uncertain vaccine response. Small interfering RNA (siRNA)-based therapy is an efficient strategy to control various infectious diseases employing post-transcriptional gene silencing through the silencing of target complementary mRNA. Here, we designed two highly effective shRNAs targeting the conserved region of RNA-dependent RNA polymerase (RdRP) and spike proteins capable of significant SARS-CoV-2 replication suppression. The efficacy of this approach suggested that the rapid development of an shRNA-based therapeutic strategy might prove to be highly effective in treating COVID-19. However, it needs further clinical trials.

Knockout of TSC2 in Nav1.8+ neurons predisposes to the onset of normal weight obesity

ObjectiveObesity and nutrient oversupply increase mammalian target of rapamycin (mTOR) signaling in multiple cell types and organs, contributing to the onset of insulin resistance and complications of metabolic disease. However, it remains unclear when and where mTOR activation mediates these effects, limiting options for therapeutic intervention. The objective of this study was to isolate the role of constitutive mTOR activation in Nav1.8-expressing peripheral neurons in the onset of diet-induced obesity, bone loss, and metabolic disease. MethodsIn humans, loss of function mutations in tuberous sclerosis complex 2 (TSC2) lead to maximal constitutive activation of mTOR. To mirror this in mice, we bred Nav1.8-Cre with TSC2fl/fl animals to conditionally delete TSC2 in Nav1.8-expressing neurons. Male and female mice were studied from 4- to 34-weeks of age and a subset of animals were fed a high-fat diet (HFD) for 24-weeks. Assays of metabolism, body composition, bone morphology, and behavior were performed. ResultsBy lineage tracing, Nav1.8-Cre targeted peripheral sensory neurons, a subpopulation of postganglionic sympathetics, and several regions of the brain. Conditional knockout of TSC2 in Nav1.8-expressing neurons (Nav1.8-TSC2KO) selectively upregulated neuronal mTORC1 signaling. Male, but not female, Nav1.8-TSC2KO mice had a 4–10% decrease in body size at baseline. When challenged with HFD, both male and female Nav1.8-TSC2KO mice resisted diet-induced gains in body mass. However, this did not protect against HFD-induced metabolic dysfunction and bone loss. In addition, despite not gaining weight, Nav1.8-TSC2KO mice fed HFD still developed high body fat, a unique phenotype previously referred to as ‘normal weight obesity’. Nav1.8-TSC2KO mice also had signs of chronic itch, mild increases in anxiety-like behavior, and sex-specific alterations in HFD-induced fat distribution that led to enhanced visceral obesity in males and preferential deposition of subcutaneous fat in females. ConclusionsKnockout of TSC2 in Nav1.8+ neurons increases itch- and anxiety-like behaviors and substantially modifies fat storage and metabolic responses to HFD. Though this prevents HFD-induced weight gain, it masks depot-specific fat expansion and persistent detrimental effects on metabolic health and peripheral organs such as bone, mimicking the ‘normal weight obesity’ phenotype that is of growing concern. This supports a mechanism by which increased neuronal mTOR signaling can predispose to altered adipose tissue distribution, adipose tissue expansion, impaired peripheral metabolism, and detrimental changes to skeletal health with HFD – despite resistance to weight gain.

Interpretable and Predictive Deep Neural Network Modeling of the SARS-CoV-2 Spike Protein Sequence to Predict COVID-19 Disease Severity.

Through the COVID-19 pandemic, SARS-CoV-2 has gained and lost multiple mutations in novel or unexpected combinations. Predicting how complex mutations affect COVID-19 disease severity is critical in planning public health responses as the virus continues to evolve. This paper presents a novel computational framework to complement conventional lineage classification and applies it to predict the severe disease potential of viral genetic variation. The transformer-based neural network model architecture has additional layers that provide sample embeddings and sequence-wide attention for interpretation and visualization. First, training a model to predict SARS-CoV-2 taxonomy validates the architecture's interpretability. Second, an interpretable predictive model of disease severity is trained on spike protein sequence and patient metadata from GISAID. Confounding effects of changing patient demographics, increasing vaccination rates, and improving treatment over time are addressed by including demographics and case date as independent input to the neural network model. The resulting model can be interpreted to identify potentially significant virus mutations and proves to be a robust predctive tool. Although trained on sequence data obtained entirely before the availability of empirical data for Omicron, the model can predict the Omicron's reduced risk of severe disease, in accord with epidemiological and experimental data.

Abstract B011: Single cell CRISPR screen of malignant peripheral nerve sheath tumors identifies SWI/SNF genes of regulatory importance

Abstract Background: Malignant peripheral nerve sheath tumors (MPNST) are rare and aggressive soft tissue sarcomas of the peripheral nervous system which are associated with a poor prognosis. Recurrent genetic mutations of the polycomb repressive complex 2 (PRC2) have been identified in up to 75% of MPNST cases. PRC2 is involved in an intricate epigenetic regulatory network in mammalian cells, where it is known to function antagonistically to the SWI/SNF protein complexes which are commonly associated with active transcription. The regulatory relationship between these complexes is critical to normal cell development and maintenance of cellular identity. However, little is known about how PRC2 aberrations affect this regulatory balance in MPNST. Therefore, this study aims to investigate the role of SWI/SNF in MPNST. Methods: A CRISPR knock-out (KO) screen combined with single cell RNA sequencing (RNAseq) was used to target 44 components of SWI/SNF complexes. The transcriptional regulatory effects of resulting genes of interest were further investigated via bulk CRISPR RNAseq experiments, while phenotypic effects were studied using loss-of-function assays coupled with colony formation assays. The dynamic complex formation was further deciphered using glycerol gradient sedimentation and co-immunoprecipitation (co-IP) assays. Results: Double PHD Fingers 1 gene, DPF1, was identified as a unique transcriptional regulator of MPNST via the single cell CRISPR KO screen. These findings were confirmed using a bulk RNAseq screen, where gene set enrichment analysis suggested DPF1 KO has an inhibitory effect on MPNST cell cycle and proliferation through enrichment of genes involved in the P53 pathway (FDR&amp;lt;0.0001) and the apoptotic pathway (FDR&amp;lt;0.0001). This hypothesis was validated using siRNA and CRISPR KO studies in MPNST cell lines, where DPF1 KO led to reduced proliferation and viability of these cells in both 2D and 3D cell culture assays. Soft agar assays were then used to highlight a role of DPF1 to in anchorage independent cell growth of MPNST cells. We then used glycerol gradient sedimentation assays to confirm DPF1 function as a member of the SWI/SNF complexes in MPNST, where DPF1 was observed to co-migrate with SMARCA4, a core ATPase commonly associated with the BAF complex of the SWI/SNF family. This assay was also used to eliminate DPF1 as a member of the GBAF and PBAF SWI/SNF complexes in MPNST, as we identified distinct sedimentation of DPF1 and proteins unique to these complexes. These findings were further validated using co-immunoprecipitation assays, where SMARCA4 had the ability to pull down DPF1, while components of the GBAF and PBAF complexes did not. Conclusions: DPF1 has been identified as a unique regulator of MPNST cells and acts as a member of the BAF SWI/SNF complex. Citation Format: Bega Murray, Xiyuan Zhang, Shahroze Abbas, Haiyan Lei, Hilda Jafarah, Michael C. Kelly, Jack F. Shern. Single cell CRISPR screen of malignant peripheral nerve sheath tumors identifies SWI/SNF genes of regulatory importance. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B011.