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Background Aims: Metabolic dysfunction-associated steatotic liver disease (MASLD) affects about a third of adults worldwide and is projected soon to be the leading cause of cirrhosis. It occurs when fat accumulates in hepatocytes and can progress to metabolic dysfunction-associated steatohepatitis (MASH), liver cirrhosis, and hepatocellular carcinoma. MASLD pathogenesis is believed to involve a combination of genetic and environmental risk factors. Single nucleotide polymorphisms have been implicated but non-syndromic monogenic causes are lacking., Methods: We identified a novel genetic variant in a familial case of MASH and performed deep variant functional analysis including protein modeling, dynamics, and cell-based assays to assess molecular mechanisms of dysfunction and altered cellular signaling. We analyzed exome sequencing data of 3,904 individuals with steatotic liver disease (SLD) to identify additional cases and establish the link between specific gene variants and SLD diagnosis., Results: We discovered and functionally validated the NM_000245.4:c.3505A>T; p.(Ile1169Phe) variant in the MET (mesenchymal epithelial transition) kinase domain as a monogenic cause of SLD. Subsequently, we detected additional ultra-rare, previously un-interpreted, and likely deleterious variants in MET from screening sequencing data. Among individuals with confirmed SLD based on electronic record review, 1.1% (45/3,904) had rare predicted deleterious MET variants. Eight of 45 (17.7%) individuals had predicted deleterious variants in the MET kinase domain confirmed to be functionally like the familial case variant., Conclusions: We report the first germline non-malignant rare MET-driven disease, a monogenic form of SLD., (Copyright © 2025 American Association for the Study of Liver Diseases.)

Reduced function or hypomorphic variants in recombination-activating genes (RAG) 1 or 2 result in a broad clinical phenotype including common variable immunodeficiency (CVID) and even adult-onset disease. Milder RAG variants are less characterized. Here we describe the longitudinal course of a milder combined RAG deficiency in 3 of 7 siblings sharing the same RAG2 mutations over a 50-year study. Whole-genome and repertoire sequencing, bacteriophage immunizations, and deep immunophenotyping were used to compare affected and unaffected family members. The clinical phenotype of three affected siblings with hypomorphic RAG deficiency ranged from combined immunodeficiency and early mortality to a late-onset CID with hyper-IgM phenotype. T cells were remarkably similar across affected siblings, yet CDR3 skewing and regulatory T cell defects were not observed. B cell analysis showed elevated unswitched CD27+ and CD21 low cells as well as features of an autoreactive antibody repertoire and presence of secreted autoantibodies, yet no clinical autoimmunity was present. Most striking was an expanded polyclonal marginal zone-like B cell population (IgM+IgD+CD27+) utilizing the self-reactive unmutated VH4-34 receptor demonstrating that hypomorphic RAG deficiency can promote expansion of self-reactive B cells. This process, however, was not sufficient to trigger clinical autoimmunity. Utilizing multiple approaches, we functionally measured the specific RAG2 variant effects and assessed how selection and secondary triggers altered the BCR repertoire and immunophenotype overtime. Overall, we demonstrate a broad disease spectrum in siblings with identical hypomorphic RAG deficiency, highlighting that phenotypic divergence can result from expansion of IgM + memory B cells., Competing Interests: Declarations. Conflicts of Interest: The authors declare no competing interests., (© 2025. The Author(s).)

Aims: Epigenomics has significantly advanced through the incorporation of Systems Biology approaches. This study aims to investigate the human lysine methylome as a system, using a data-science approach to reveal its emergent properties, particularly focusing on histone mimicry and the broader implications of lysine methylation across the proteome., Methods: We employed a data-science-driven OMICS approach, leveraging high-dimensional proteomic data to study the lysine methylome. The analysis focused on identifying sequence-based recognition motifs of lysine methyltransferases and evaluating the prevalence and distribution of lysine methylation across the human proteome., Results: Our analysis revealed that lysine methylation impacts 15% of the known proteome, with a notable bias toward mono-methylation. We identified sequence-based recognition motifs of 13 lysine methyltransferases, highlighting candidates for histone mimicry. These findings suggest that the selective inhibition of individual lysine methyltransferases could have systemic effects rather than merely targeting histone methylation., Conclusions: The lysine methylome has significant mechanistic value and should be considered in the design and testing of therapeutic strategies, particularly in precision oncology. The study underscores the importance of considering non-histone proteins involved in DNA damage and repair, cell signaling, metabolism, and cell cycle pathways when targeting lysine methyltransferases.

Background: Expanded analysis of tumor genomics data enables current and future patients to gain more benefits, such as improving diagnosis, prognosis, and therapeutics. Methods: Here, we report tumor genomic data from 1146 cases accompanied by simultaneous expert analysis from patients visiting our oncological clinic. We developed an analytical approach that leverages combined germline and cancer genetics knowledge to evaluate opportunities, challenges, and yield of potentially medically relevant data. Results: We identified 499 cases (44%) with variants of interest, defined as either potentially actionable or pathogenic in a germline setting, and that were reported in the original analysis as variants of uncertain significance (VUS). Of the 7405 total unique tumor variants reported, 462 (6.2%) were reported as VUS at the time of diagnosis, yet information from germline analyses identified them as (likely) pathogenic. Notably, we find that a sizable number of these variants (36%-79%) had been reported in heritable disorders and deposited in public databases before the year of tumor testing. Conclusions: This finding indicates the need to develop data systems to bridge current gaps in variant annotation and interpretation and to develop more complete digital representations of actionable pathways. We outline our process for achieving such methodologic integration. Sharing genomics data across medical specialties can enable more robust, equitable, and thorough use of patient's genomics data. This comprehensive analytical approach and the new knowledge derived from its results highlight its multi-specialty value in precision oncology settings.

Artificial Intelligence (AI)-based deep learning methods for predicting protein structures are reshaping knowledge development and scientific discovery. Recent large-scale application of AI models for protein structure prediction has changed perceptions about complicated biological problems and empowered a new generation of structure-based hypothesis testing. It is well-recognized that proteins have a modular organization according to archetypal folds. However, it is yet to be determined if predicted structures are tuned to one conformation of flexible proteins or if they represent average conformations. Further, whether or not the answer is protein fold-dependent. Therefore, in this study, we analyzed 2878 proteins with at least ten distinct experimental structures available, from which we can estimate protein topological rigidity verses heterogeneity from experimental measurements. We found that AlphaFold v2 (AF2) predictions consistently return one specific form to high accuracy, with 99.68% of distinct folds (n = 623 out of 628) having an experimental structure within 2.5Å RMSD from a predicted structure. Yet, 27.70% and 10.82% of folds (174 and 68 out of 628 folds) have at least one experimental structure over 2.5Å and 5Å RMSD, respectively, from their AI-predicted structure. This information is important for how researchers apply and interpret the output of AF2 and similar tools. Additionally, it enabled us to score fold types according to how homogeneous versus heterogeneous their conformations are. Importantly, folds with high heterogeneity are enriched among proteins which regulate vital biological processes including immune cell differentiation, immune activation, and metabolism. This result demonstrates that a large amount of protein fold flexibility has already been experimentally measured, is vital for critical cellular processes, and is currently unaccounted for in structure prediction databases. Therefore, the structure-prediction revolution begets the protein dynamics revolution!, Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Haque et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Clinical genomics sequencing is rapidly expanding the number of variants that need to be functionally elucidated. Interpreting genetic variants (i.e., mutations) usually begins by identifying how they affect protein-coding sequences. Still, the three-dimensional (3D) protein molecule is rarely considered for large-scale variant analysis, nor in analyses of how proteins interact with each other and their environment. We propose a standardized approach to scoring protein surface property changes as a new dimension for functionally and mechanistically interpreting genomic variants. Further, it directs hypothesis generation for functional genomics research to learn more about the encoded protein's function. We developed a novel method leveraging 3D structures and time-dependent simulations to score and statistically evaluate protein surface property changes. We evaluated positive controls composed of eight thermophilic versus mesophilic orthologs and variants that experimentally change the protein's solubility, which all showed large and statistically significant differences in charge distribution ( p < 0.01). We scored static 3D structures and dynamic ensembles for 43 independent variants (23 pathogenic and 20 uninterpreted) across four proteins. Focusing on the potassium ion channel, KCNK9, the average local surface potential shifts were 0.41 k B T/ec with an average p -value of 1 × 10 -2 . In contrast, dynamic ensemble shifts averaged 1.15 k B T/ec with an average p -value of 1 × 10 -5 , enabling the identification of changes far from mutated sites. This study demonstrates that an objective assessment of how mutations affect electrostatic distributions of protein surfaces can aid in interpreting genomic variants discovered through clinical genomic sequencing.

Microtubule affinity-regulating kinase 2 (MARK2) contributes to establishing neuronal polarity and developing dendritic spines. Although large-scale sequencing studies have associated MARK2 variants with autism spectrum disorder (ASD), the clinical features and variant spectrum in affected individuals with MARK2 variants, early developmental phenotypes in mutant human neurons, and the pathogenic mechanism underlying effects on neuronal development have remained unclear. Here, we report 31 individuals with MARK2 variants and presenting with ASD, other neurodevelopmental disorders, and distinctive facial features. Loss-of-function (LoF) variants predominate (81%) in affected individuals, while computational analysis and in vitro expression assay of missense variants supported the effect of MARK2 loss. Using proband-derived and CRISPR-engineered isogenic induced pluripotent stem cells (iPSCs), we show that MARK2 loss leads to early neuronal developmental and functional deficits, including anomalous polarity and dis-organization in neural rosettes, as well as imbalanced proliferation and differentiation in neural progenitor cells (NPCs). Mark2 +/- mice showed abnormal cortical formation and partition and ASD-like behavior. Through the use of RNA sequencing (RNA-seq) and lithium treatment, we link MARK2 loss to downregulation of the WNT/β-catenin signaling pathway and identify lithium as a potential drug for treating MARK2-associated ASD., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: Changes in quantitative magnetic resonance imaging (qMRI) are frequently observed during chemotherapy or radiation therapy (RT). It is hypothesized that qMRI features are reflective of underlying tissue responses. It's unknown what underlying genomic characteristics underly qMRI changes. We hypothesized that qMRI changes may correlate with DNA damage response (DDR) capacity within human tumors. Therefore, we designed the current study to correlate qMRI changes from daily RT treatment with underlying tumor transcriptomic profiles., Methods and Materials: Study participants were prospectively enrolled (National Clinical Trial 03500081). RNA expression levels for 757 genes from pretreatment biopsies were obtained using a custom panel that included signatures of radiation sensitivity and DDR. Daily qMRI data were obtained from a 1.5 Tesla MR linear accelerator. Using these images, d-slow, d-star, perfusion, and apparent diffusion coefficient-mean values in tumors were plotted per-fraction, over time, and associated with genomic pathways., Results: A total of 1022 qMRIs were obtained from 39 patients and both genomic data and qMRI data from 27 total patients. For 20 of those patients, we also generated normal tissue transcriptomic data. Radio sensitivity index values most closely associated with tissue of origin. Multiple genomic pathways including DNA repair, peroxisome, late estrogen receptor responses, KRAS signaling, and UV response were significantly associated with qMRI feature changes (P < .001)., Conclusions: Genomic pathway associations across metabolic, RT sensitivity, and DDR pathways indicate common tumor biology that may correlate with qMRI changes during a course of treatment. Such data provide hypothesis-generating novel mechanistic insight into the biologic meaning of qMRI changes during treatment and enable optimal selection of imaging biomarkers for biologically MR-guided RT., (Published by Elsevier Inc.)

Introduction: The Euchromatic Histone Methyl Transferase Protein 2 (EHMT2), also known as G9a, deposits transcriptionally repressive chromatin marks that play pivotal roles in the maturation and homeostasis of multiple organs. Recently, we have shown that Ehmt2 inactivation in the mouse pancreas alters growth and immune gene expression networks, antagonizing Kras-mediated pancreatic cancer initiation and promotion. Here, we elucidate the essential role of Ehmt2 in maintaining a transcriptional landscape that protects organs from inflammation. Methods: Comparative RNA-seq studies between normal postnatal and young adult pancreatic tissue from Ehmt2 conditional knockout animals ( Ehmt2 fl/fl ) targeted to the exocrine pancreatic epithelial cells ( Pdx1-Cre and P48 Cre/+ ), reveal alterations in gene expression networks in the whole organ related to injury-inflammation-repair, suggesting an increased predisposition to damage. Thus, we induced an inflammation repair response in the Ehmt2 fl/fl pancreas and used a data science-based approach to integrate RNA-seq-derived pathways and networks, deconvolution digital cytology, and spatial transcriptomics. We also analyzed the tissue response to damage at the morphological, biochemical, and molecular pathology levels. Results and discussion: The Ehmt2 fl/fl pancreas displays an enhanced injury-inflammation-repair response, offering insights into fundamental molecular and cellular mechanisms involved in this process. More importantly, these data show that conditional Ehmt2 inactivation in exocrine cells reprograms the local environment to recruit mesenchymal and immunological cells needed to mount an increased inflammatory response. Mechanistically, this response is an enhanced injury-inflammation-repair reaction with a small contribution of specific Ehmt2-regulated transcripts. Thus, this new knowledge extends the mechanisms underlying the role of the Ehmt2-mediated pathway in suppressing pancreatic cancer initiation and modulating inflammatory pancreatic diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Pollin, Mathison, de Assuncao, Thomas, Zeighami, Salmonson, Liu, Urrutia, Vankayala, Pandol, Hong, Zimmermann, Iovanna, Jin, Urrutia and Lomberk.)

Despite the promise of concurrent radiotherapy (RT) and immunotherapy in head and neck cancer (HNC), multiple randomized trials of this combination have had disappointing results. To evaluate potential immunologic mechanisms of RT resistance, we compared pre-treatment HNCs that developed RT resistance to a matched cohort that achieved curative status. Gene set enrichment analysis demonstrated that a pre-treatment pro-immunogenic tumor microenvironment (TME), including type II interferon [interferon gamma (IFNγ)] and tumor necrosis factor alpha (TNFα) signaling, predicted cure while type I interferon [interferon alpha (IFNα)] enrichment was associated with an immunosuppressive TME found in tumors that went on to recur. We then used immune deconvolution of RNA sequencing datasets to evaluate immunologic cell subset enrichment. This identified M2 macrophage signaling associated with type I IFN pathway expression in RT-recurrent disease. To further dissect mechanism, we then evaluated differential gene expression between pre-treatment and RT-resistant HNCs from sampled from the same patients at the same anatomical location in the oral cavity. Here, recurrent samples exhibited upregulation of type I IFN-stimulated genes (ISGs) including members of the IFN-induced protein with tetratricopeptide repeats (IFIT) and IFN-induced transmembrane (IFITM) gene families. While several ISGs were upregulated in each recurrent cancer, IFIT2 was significantly upregulated in all recurrent tumors when compared with the matched pre-RT specimens. Based on these observations, we hypothesized sustained type I IFN signaling through ISGs, such as IFIT2, may suppress the intra-tumoral immune response thereby promoting radiation resistance., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-23-2104/coif). The authors have no conflicts of interest to declare., (2024 Translational Cancer Research. All rights reserved.)

Objective: The adverse effects of ischemia-reperfusion injury (IRI) remain a principal barrier to a successful outcome after lifesaving orthotopic liver transplantation (OLT). Gene expression during different phases of IRI is dynamic and modified by individual exposures, making it attractive for identifying potential therapeutic targets for improving the number of suitable organs for transplantation and patient outcomes. However, data remain limited on the functional landscape of gene expression during liver graft IRI, spanning procurement to reperfusion and recovery. Therefore, we sought to characterize transcriptomic profiles of IRI during multiple phases in human OLT., Methods: We conducted clinical data analyses, histologic evaluation, and RNA sequencing of 17 consecutive human primary OLT. We performed liver allograft biopsies at 4 time points: baseline (B, before donor cross-clamp), at the end of cold ischemia (CI), during early reperfusion (ER, after revascularization), and during late reperfusion (LR). Data were generated and then recipients grouped by post-OLT outcomes categories: immediate allograft function (IAF; n = 11) versus early allograft dysfunction (EAD; n = 6) groups., Results: We observed that CI (vs B) modified a transcriptomic landscape enriched for a metabolic and immune process. Expression levels of hallmark inflammatory response genes were higher transitioning from CI to ER and decreased from ER to LR. IAF group predominantly showed higher bile and fatty acid metabolism activity during LR compared with EAD group, while EAD group maintained more immunomodulatory activities. Throughout all time points, EAD specimens exhibited decreased metabolic activity in both bile and fatty acid pathways., Conclusions: We report transcriptomic profiles of human liver allograft IRI from prepreservation in the donor to posttransplantation in the recipient. Immunomodulatory and metabolic landscapes across ER and LR phases were different between IAF and EAD allografts. Our study also highlights marker genes for these biological processes that we plan to explore as novel therapeutic targets or surrogate markers for severe allograft injury in clinical OLT., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

The polybromo, brahma-related gene 1-associated factors (PBAF) chromatin remodeling complex subunit polybromo-1 (PBRM1) contains six bromodomains that recognize and bind acetylated lysine residues on histone tails and other nuclear proteins. PBRM1 bromodomains thus provide a link between epigenetic posttranslational modifications and PBAF modulation of chromatin accessibility and transcription. As a putative tumor suppressor in several cancers, PBRM1 protein expression is often abrogated by truncations and deletions. However, ∼33% of PBRM1 mutations in cancer are missense and cluster within its bromodomains. Such mutations may generate full-length PBRM1 variant proteins with undetermined structural and functional characteristics. Here, we employed computational, biophysical, and cellular assays to interrogate the effects of PBRM1 bromodomain missense variants on bromodomain stability and function. Since mutations in the fourth bromodomain of PBRM1 (PBRM1-BD4) comprise nearly 20% of all cancer-associated PBRM1 missense mutations, we focused our analysis on PBRM1-BD4 missense protein variants. Selecting 16 potentially deleterious PBRM1-BD4 missense protein variants for further study based on high residue mutational frequency and/or conservation, we show that cancer-associated PBRM1-BD4 missense variants exhibit varied bromodomain stability and ability to bind acetylated histones. Our results demonstrate the effectiveness of identifying the unique impacts of individual PBRM1-BD4 missense variants on protein structure and function, based on affected residue location within the bromodomain. This knowledge provides a foundation for drawing correlations between specific cancer-associated PBRM1 missense variants and distinct alterations in PBRM1 function, informing future cancer personalized medicine approaches., Competing Interests: Conflict of interest The authors declare they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The PI3K enzymes modify phospholipids to regulate cell growth and differentiation. Somatic variants in PI3K are recurrent in cancer and drive a proliferative phenotype. Somatic mosaicism of PIK3R1 and PIK3CA are associated with vascular anomalies and overgrowth syndromes. Germline PIK3R1 variants are associated with varying phenotypes, including immunodeficiency or facial dysmorphism with growth delay, lipoatrophy, and insulin resistance associated with SHORT syndrome. There has been limited study of the molecular mechanism to unify our understanding of how variants in PIK3R1 drive both undergrowth and overgrowth phenotypes. Thus, we compiled genomic variants from cancer and rare vascular anomalies and sought to interpret their effects using an unbiased physics-based simulation approach for the protein complex. We applied molecular dynamics simulations to mechanistically understand how genetic variants affect PIK3R1 and its interactions with PIK3CA. Notably, iSH2 genetic variants associated with undergrowth destabilize molecular interactions with the PIK3CA receptor binding domain in simulations, which is expected to decrease activity. On the other hand, overgrowth and cancer variants lead to loss of inhibitory interactions in simulations, which is expected to increase activity. We find that all disease variants display dysfunctions on either structural characteristics or intermolecular interaction energy. Thus, this comprehensive characterization of novel mosaic somatic variants associated with two opposing phenotypes has mechanistic importance and biomedical relevance and may aid in future therapeutic developments.

Multiple Myeloma (MM) is a plasma cell neoplasm originating in the bone marrow and is the second most common blood cancer in the United States. One challenge in understanding the pathogenesis of MM and improving treatment is a lack of immunocompetent mouse models. We previously developed the IL6Myc mouse that generates plasmacytomas at 100% penetrance that phenotypically resemble aggressive MM. Using comprehensive genomic analysis, we found that the IL6Myc tumors resemble aggressive MM by RNA and protein expression. We also found that IL6Myc tumors accumulated fusions and missense mutations in genes that overlap significantly with human myeloma, indicating that the mouse is good model for studying disease etiology. Lastly, we derived cell lines from IL6Myc tumors that express cell surface markers typical of MM and readily engraft into mice, home to the bone marrow, and induce osteolytic disease. The cell lines may be useful in developing immunotherapies directed against BAFF-R and TACI, though not BCMA, and may also be a good model for studying dexamethasone resistance. These data indicate that the IL6Myc model is useful for studying development of aggressive MM and for developing new treatments against such forms of the disease.

Introduction: Kleefstra Syndrome type 2 (KLEFS-2) is a genetic, neurodevelopmental disorder characterized by intellectual disability, infantile hypotonia, severe expressive language delay, and characteristic facial appearance, with a spectrum of other distinct clinical manifestations. Pathogenic mutations in the epigenetic modifier type 2 lysine methyltransferase KMT2C have been identified to be causative in KLEFS-2 individuals. Methods: This work reports a translational genomic study that applies a multidimensional computational approach for deep variant phenotyping, combining conventional genomic analyses, advanced protein bioinformatics, computational biophysics, biochemistry, and biostatistics-based modeling. We use standard variant annotation, paralog annotation analyses, molecular mechanics, and molecular dynamics simulations to evaluate damaging scores and provide potential mechanisms underlying KMT2C variant dysfunction. Results: We integrated data derived from the structure and dynamics of KMT2C to classify variants into SV (Structural Variant), DV (Dynamic Variant), SDV (Structural and Dynamic Variant), and VUS (Variant of Uncertain Significance). When compared with controls, these variants show values reflecting alterations in molecular fitness in both structure and dynamics. Discussion: We demonstrate that our 3D models for KMT2C variants suggest distinct mechanisms that lead to their imbalance and are not predictable from sequence alone. Thus, the missense variants studied here cause destabilizing effects on KMT2C function by different biophysical and biochemical mechanisms which we adeptly describe. This new knowledge extends our understanding of how variations in the KMT2C gene cause the dysfunction of its methyltransferase enzyme product, thereby bearing significant biomedical relevance for carriers of KLEFS2-associated genomic mutations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Jorge, Chi, Mazaba, Haque, Wagenknecht, Smith, Volkman, Mathison, Lomberk, Zimmermann and Urrutia.)

Histone H3 lysine 9 methylation (H3K9me), which is written by the Euchromatic Histone Lysine Methyltransferases EHMT1 and EHMT2 and read by the heterochromatin protein 1 (HP1) chromobox (CBX) protein family, is dysregulated in many types of cancers. Approaches to inhibit regulators of this pathway are currently being evaluated for therapeutic purposes. Thus, knowledge of the complexes supporting the function of these writers and readers during the process of cell proliferation is critical for our understanding of their role in carcinogenesis. Here, we immunopurified each of these proteins and used mass spectrometry to define their associated non-histone proteins, individually and at two different phases of the cell cycle, namely G1/S and G2/M. Our findings identify novel binding proteins for these writers and readers, as well as corroborate known interactors, to show the formation of distinct protein complex networks in a cell cycle phase-specific manner. Furthermore, there is an organizational switch between cell cycle phases for interactions among specific writer-reader pairs. Through a multi-tiered bioinformatics-based approach, we reveal that many interacting proteins exhibit histone mimicry, based on an H3K9-like linear motif. Gene ontology analyses, pathway enrichment, and network reconstruction inferred that these comprehensive EHMT and CBX-associated interacting protein networks participate in various functions, including transcription, DNA repair, splicing, and membrane disassembly. Combined, our data reveals novel complexes that provide insight into key functions of cell cycle-associated epigenomic processes that are highly relevant for better understanding these chromatin-modifying proteins during cell cycle and carcinogenesis., (© 2023 The Author(s).)

This study investigates the functional significance of assorted variants of uncertain significance (VUS) in euchromatic histone lysine methyltransferase 1 (EHMT1), which is critical for early development and normal physiology. EHMT1 mutations cause Kleefstra syndrome and are linked to various human cancers. However, accurate functional interpretations of these variants are yet to be made, limiting diagnoses and future research. To overcome this, we integrate conventional tools for variant calling with computational biophysics and biochemistry to conduct multi-layered mechanistic analyses of the SET catalytic domain of EHMT1, which is critical for this protein function. We use molecular mechanics and molecular dynamics (MD)-based metrics to analyze the SET domain structure and functional motions resulting from 97 Kleefstra syndrome missense variants within the domain. Our approach allows us to classify the variants in a mechanistic manner into SV (Structural Variant), DV (Dynamic Variant), SDV (Structural and Dynamic Variant), and VUS (Variant of Uncertain Significance). Our findings reveal that the damaging variants are mostly mapped around the active site, substrate binding site, and pre-SET regions. Overall, we report an improvement for this method over conventional tools for variant interpretation and simultaneously provide a molecular mechanism for variant dysfunction., Competing Interests: The authors declare that they have no competing interests., (© 2023 The Authors.)

Current capabilities in genomic sequencing outpace functional interpretations. Our previous work showed that 3D protein structure calculations enhance mechanistic understanding of genetic variation in sequenced tumors and patients with rare diseases. The KRAS GTPase is among the critical genetic factors driving cancer and germline conditions. Because KRAS-altered tumors frequently harbor one of three classic hotspot mutations, nearly all studies have focused on these mutations, leaving significant functional ambiguity across the broader KRAS genomic landscape observed in cancer and non-cancer diseases. Herein, we extend structural bioinformatics with molecular simulations to study an expanded landscape of 86 KRAS mutations. We identify multiple coordinated changes strongly associated with experimentally established KRAS biophysical and biochemical properties. The patterns we observe span hotspot and non-hotspot alterations, which can all dysregulate Switch regions, producing mutation-restricted conformations with different effector binding propensities. We experimentally measured mutation thermostability and identified shared and distinct patterns with simulations. Our results indicate mutation-specific conformations, which show potential for future research into how these alterations reverberate into different molecular and cellular functions. The data we present is not predictable using current genomic tools, demonstrating the added functional information derived from molecular simulations for interpreting human genetic variation., Competing Interests: BV declares ownership interests in Protein Foundry and XLock Biosciences. All other authors declare no conflicts of interest., (© 2023 The Authors.)

Interpreting genetic changes observed in individual patients is a critical challenge. The array of immune deficiency syndromes is typically caused by genetic variation unique to individuals. Therefore, new approaches are needed to interpret functional variation and accelerate genomics interpretation. We constructed the first full-length structural model of human RAG recombinase across four functional states of the recombination process. We functionally tested 182 clinically observed RAG missense mutations. These experiments revealed dysfunction due to recombinase dysfunction and altered chromatin interactions. Structural modeling identified mechanical and energetic roles for each mutation. We built regression models for RAG1 (R 2  = 0.91) and RAG2 (R 2  = 0.97) to predict RAG activity changes. We applied our model to 711 additional RAG variants observed in population studies and identified a subset that may impair RAG function. Thus, we demonstrated a fundamental advance in the mechanistic interpretation of human genetic variations spanning from rare and undiagnosed diseases to population health., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

X-Linked Hyper-IgM Syndrome is caused by pathogenic variants in CD40LG. Three patients with atypical clinical and immunological features were identified with variants in CD40LG requiring further characterization. Flow cytometry was used to evaluate CD40L protein expression and binding capacity to a surrogate receptor, CD40-muIg. Though functional anomalies were observed, there was still a lack of clarity regarding the underlying mechanism. We developed structural models for wild-type and the three variants of CD40L protein observed in these patients (p. Lys143Asn, Leu225Ser and Met36Arg) to evaluate structural alterations by molecular mechanic calculations, and assess protein movement by molecular dynamic simulations. These studies demonstrate that functional analysis of variants of unknown significance in CD40LG can be supplemented by advanced computational analysis in atypical clinical contexts. These studies in combination identify the deleterious effects of these variants and potential mechanisms for protein dysfunction., Competing Interests: Declaration of Competing Interest The authors declare no relevant conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Statins are a mainstay intervention for cardiovascular disease prevention, yet their use can cause rare severe myopathy. HMG-CoA reductase, an essential enzyme in the mevalonate pathway, is the target of statins. We identified nine individuals from five unrelated families with unexplained limb-girdle like muscular dystrophy and bi-allelic variants in HMGCR via clinical and research exome sequencing. The clinical features resembled other genetic causes of muscular dystrophy with incidental high CPK levels (>1,000 U/L), proximal muscle weakness, variable age of onset, and progression leading to impaired ambulation. Muscle biopsies in most affected individuals showed non-specific dystrophic changes with non-diagnostic immunohistochemistry. Molecular modeling analyses revealed variants to be destabilizing and affecting protein oligomerization. Protein activity studies using three variants (p.Asp623Asn, p.Tyr792Cys, and p.Arg443Gln) identified in affected individuals confirmed decreased enzymatic activity and reduced protein stability. In summary, we showed that individuals with bi-allelic amorphic (i.e., null and/or hypomorphic) variants in HMGCR display phenotypes that resemble non-genetic causes of myopathy involving this reductase. This study expands our knowledge regarding the mechanisms leading to muscular dystrophy through dysregulation of the mevalonate pathway, autoimmune myopathy, and statin-induced myopathy., Competing Interests: Declaration of interests The Department of Molecular and Human Genetics at Baylor College of Medicine receives revenue from clinical genetic testing from Baylor Genetics Laboratories. J.R.L. has stock ownership in 23andMe, is a paid consultant for Regeneron Pharmaceuticals, and is a co-inventor on multiple United States and European patents related to molecular diagnostics for inherited neuropathies, eye diseases, genomic disorders, and bacterial genomic fingerprinting., (Copyright © 2023. Published by Elsevier Inc.)

Background: Systems biology leveraging multi-OMICs technologies, is rapidly advancing development of precision therapies and matching patients to targeted therapies, leading to improved responses. A new pillar of precision oncology lies in the power of chemogenomics to discover drugs that sensitizes malignant cells to other therapies. Here, we test a chemogenomic approach using epigenomic inhibitors (epidrugs) to reset patterns of gene expression driving the malignant behavior of pancreatic tumors., Methods: We tested a targeted library of ten epidrugs targeting regulators of enhancers and super-enhancers on reprogramming gene expression networks in seventeen patient-derived primary pancreatic cancer cell cultures (PDPCCs), of both basal and classical subtypes. We subsequently evaluated the ability of these epidrugs to sensitize pancreatic cancer cells to five chemotherapeutic drugs that are clinically used for this malignancy., Findings: To comprehend the impact of epidrug priming at the molecular level, we evaluated the effect of each epidrugs at the transcriptomic level of PDPCCs. The activating epidrugs showed a higher number of upregulated genes than the repressive epidrugs (χ 2 test p-value <0.01). Furthermore, we developed a classifier using the baseline transcriptome of epidrug-primed-chemosensitized PDPCCs to predict the best epidrug-priming regime to a given chemotherapy. Six signatures with a significant association with the chemosensitization centroid (R ≤ -0.80; p-value < 0.01) were identified and validated in a subset of PDPCCs., Interpretation: We conclude that targeting enhancer-initiated pathways in patient-derived primary cells, represents a promising approach for developing new therapies for human pancreatic cancer., Funding: This work was supported by INCa (Grants number 2018-078 to ND and 2018- 079 to JI), Canceropole PACA (ND), Amidex Foundation (ND), and INSERM (JI)., Competing Interests: Declaration of interests All the authors declare no competing financial or non-financial interests., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Competing Interests: Conflict of Interest The authors declare no conflicts of interest.

Pancreatic cancer is characterized by abundant desmoplasia, a dense stroma composed of extra-cellular and cellular components, with cancer associated fibroblasts (CAFs) being the major cellular component. However, the tissue(s) of origin for CAFs remains controversial. Here we determine the tissue origin of pancreatic CAFs through comprehensive lineage tracing studies in mice. We find that the splanchnic mesenchyme, the fetal cell layer surrounding the endoderm from which the pancreatic epithelium originates, gives rise to the majority of resident fibroblasts in the normal pancreas. In a genetic mouse model of pancreatic cancer, resident fibroblasts expand and constitute the bulk of CAFs. Single cell RNA profiling identifies gene expression signatures that are shared among the fetal splanchnic mesenchyme, adult fibroblasts and CAFs, suggesting a persistent transcriptional program underlies splanchnic lineage differentiation. Together, this study defines the phylogeny of the mesenchymal component of the pancreas and provides insights into pancreatic morphogenesis and tumorigenesis., (© 2023. The Author(s).)

Background: High-impact pathogenic variants in more than a thousand genes are involved in Mendelian forms of neurodevelopmental disorders (NDD)., Methods: This study describes the molecular and clinical characterisation of 28 probands with NDD harbouring heterozygous AGO1 coding variants, occurring de novo for all those whose transmission could have been verified (26/28)., Results: A total of 15 unique variants leading to amino acid changes or deletions were identified: 12 missense variants, two in-frame deletions of one codon, and one canonical splice variant leading to a deletion of two amino acid residues. Recurrently identified variants were present in several unrelated individuals: p.(Phe180del), p.(Leu190Pro), p.(Leu190Arg), p.(Gly199Ser), p.(Val254Ile) and p.(Glu376del). AGO1 encodes the Argonaute 1 protein, which functions in gene-silencing pathways mediated by small non-coding RNAs. Three-dimensional protein structure predictions suggest that these variants might alter the flexibility of the AGO1 linker domains, which likely would impair its function in mRNA processing. Affected individuals present with intellectual disability of varying severity, as well as speech and motor delay, autistic behaviour and additional behavioural manifestations., Conclusion: Our study establishes that de novo coding variants in AGO1 are involved in a novel monogenic form of NDD, highly similar to the recently reported AGO2 -related NDD., Competing Interests: Competing interests: KMW, ET, FM, AD and MJT are employees of GeneDx. ZP and KM are employees of Ambry Genetics., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Genomics enables individualized diagnosis and treatment, but large challenges remain to functionally interpret rare variants. To date, only one causative variant has been described for KCNK9 imprinting syndrome (KIS). The genotypic and phenotypic spectrum of KIS has yet to be described and the precise mechanism of disease fully understood., Methods: This study discovers mechanisms underlying KCNK9 imprinting syndrome (KIS) by describing 15 novel KCNK9 alterations from 47 KIS-affected individuals. We use clinical genetics and computer-assisted facial phenotyping to describe the phenotypic spectrum of KIS. We then interrogate the functional effects of the variants in the encoded TASK3 channel using sequence-based analysis, 3D molecular mechanic and dynamic protein modeling, and in vitro electrophysiological and functional methodologies., Results: We describe the broader genetic and phenotypic variability for KIS in a cohort of individuals identifying an additional mutational hotspot at p.Arg131 and demonstrating the common features of this neurodevelopmental disorder to include motor and speech delay, intellectual disability, early feeding difficulties, muscular hypotonia, behavioral abnormalities, and dysmorphic features. The computational protein modeling and in vitro electrophysiological studies discover variability of the impact of KCNK9 variants on TASK3 channel function identifying variants causing gain and others causing loss of conductance. The most consistent functional impact of KCNK9 genetic variants, however, was altered channel regulation., Conclusions: This study extends our understanding of KIS mechanisms demonstrating its complex etiology including gain and loss of channel function and consistent loss of channel regulation. These data are rapidly applicable to diagnostic strategies, as KIS is not identifiable from clinical features alone and thus should be molecularly diagnosed. Furthermore, our data suggests unique therapeutic strategies may be needed to address the specific functional consequences of KCNK9 variation on channel function and regulation., (© 2022. The Author(s).)

The histone demethylase KDM6A has recently elicited significant attention because its mutations are associated with a rare congenital disorder (Kabuki syndrome) and various types of human cancers. However, distinguishing KDM6A mutations that are deleterious to the enzyme and their underlying mechanisms of dysfunction remain to be fully understood. Here, we report the results from a multi-tiered approach evaluating the impact of 197 KDM6A somatic mutations using information derived from combining conventional genomics data with computational biophysics. This comprehensive approach incorporates multiple scores derived from alterations in protein sequence, structure, and molecular dynamics. Using this method, we classify the KDM6A mutations into 136 damaging variants (69.0%), 32 tolerated variants (16.2%), and 29 variants of uncertain significance (VUS, 14.7%), which is a significant improvement from the previous classification based on the conventional tools (over 40% VUS). We further classify the damaging variants into 15 structural variants (SV), 88 dynamic variants (DV), and 33 structural and dynamic variants (SDV). Comparison with variant scoring methods used in current clinical diagnosis guidelines demonstrates that our approach provides a more comprehensive evaluation of damaging potential and reveals mechanisms of dysfunction. Thus, these results should be taken into consideration for clinical assessment of the damaging potential of each mutation, as they provide hypotheses for experimental validation and critical information for the development of mutant-specific drugs to fight diseases caused by KDM6A dysfunctions., (© 2022 The Authors.)

Impaired endothelium-dependent vasodilation has been suggested to be a key component of coronary microvascular dysfunction (CMD). A better understanding of endothelial pathways involved in vasodilation in human arterioles may provide new insight into the mechanisms of CMD. The goal of this study is to investigate the role of TRPV4, NOX4, and their interaction in human arterioles and examine the underlying mechanisms. Arterioles were freshly isolated from adipose and heart tissues obtained from 71 patients without coronary artery disease, and vascular reactivity was studied by videomicroscopy. In human adipose arterioles (HAA), ACh-induced dilation was significantly reduced by TRPV4 inhibitor HC067047 and by NOX 1/4 inhibitor GKT137831, but GKT137831 did not further affect the dilation in the presence of TRPV4 inhibitors. GKT137831 also inhibited TRPV4 agonist GSK1016790A-induced dilation in HAA and human coronary arterioles (HCA). NOX4 transcripts and proteins were detected in endothelial cells of HAA and HCA. Using fura-2 imaging, GKT137831 significantly reduced GSK1016790A-induced Ca 2+ influx in the primary culture of endothelial cells and TRPV4-WT-overexpressing human coronary artery endothelial cells (HCAEC). However, GKT137831 did not affect TRPV4-mediated Ca 2+ influx in non-phosphorylatable TRPV4-S823A/S824A-overexpressing HCAEC. In addition, treatment of HCAEC with GKT137831 decreased the phosphorylation level of Ser824 in TRPV4. Finally, proximity ligation assay (PLA) revealed co-localization of NOX4 and TRPV4 proteins. In conclusion, both TRPV4 and NOX4 contribute to ACh-induced dilation in human arterioles from patients without coronary artery disease. NOX4 increases TRPV4 phosphorylation in endothelial cells, which in turn enhances TRPV4-mediated Ca 2+ entry and subsequent endothelium-dependent dilation in human arterioles., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.)

Hundreds of human membrane proteins form channels that transport necessary ions and compounds, including drugs and metabolites, yet details of their normal function or how function is altered by genetic variants to cause diseases are often unknown. Without this knowledge, researchers are less equipped to develop approaches to diagnose and treat channelopathies. High-resolution computational approaches such as molecular modeling enable researchers to investigate channelopathy protein function, facilitate detailed hypothesis generation, and produce data that is difficult to gather experimentally. Molecular modeling can be tailored to each physiologic context that a protein may act within, some of which may currently be difficult or impossible to assay experimentally. Because many genomic variants are observed in channelopathy proteins from high-throughput sequencing studies, methods with mechanistic value are needed to interpret their effects. The eminent field of structural bioinformatics integrates techniques from multiple disciplines including molecular modeling, computational chemistry, biophysics, and biochemistry, to develop mechanistic hypotheses and enhance the information available for understanding function. Molecular modeling and simulation access 3D and time-dependent information, not currently predictable from sequence. Thus, molecular modeling is valuable for increasing the resolution with which the natural function of protein channels can be investigated, and for interpreting how genomic variants alter them to produce physiologic changes that manifest as channelopathies. © 2022 American Physiological Society. Compr Physiol 12:3141-3166, 2022., (Copyright © 2022 American Physiological Society. All rights reserved.)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which upper and lower motor neuron loss is the primary phenotype, leading to muscle weakness and wasting, respiratory failure, and death. Although a portion of ALS cases are linked to one of over 50 unique genes, the vast majority of cases are sporadic in nature. However, the mechanisms underlying the motor neuron loss in either familial or sporadic ALS are not entirely clear. Here, we used induced pluripotent stem cells derived from a set of identical twin brothers discordant for ALS to assess the role of astrocytes and microglia on the expression and accumulation of neurofilament proteins in motor neurons. We found that motor neurons derived from the affected twin which exhibited increased transcript levels of all three neurofilament isoforms and increased expression of phosphorylated neurofilament puncta. We further found that treatment of the motor neurons with astrocyte-conditioned medium and microglial-conditioned medium significantly impacted neurofilament deposition. Together, these data suggest that glial-secreted factors can alter neurofilament pathology in ALS iPSC-derived motor neurons.

Disruptor of telomeric silencing 1-like (DOT1L) is the only non-SET domain histone lysine methyltransferase (KMT) and writer of H3K79 methylation on nucleosomes marked by H2B ubiquitination. DOT1L has elicited significant attention because of its interaction or fusion with members of the AF protein family in blood cell biology and leukemogenic transformation. Here, our goal was to extend previous structural information by performing a robust molecular dynamic study of DOT1L and its leukemogenic partners combined with mutational analysis. We show that statically and dynamically, D161, G163, E186, and F223 make frequent time-dependent interactions with SAM, while additional residues T139, K187, and N241 interact with SAM only under dynamics. Dynamics models reveal DOT1L, SAM, and H4 moving as one and show that more than twice the number of DOT1L residues interacts with these partners, relative to the static structure. Mutational analyses indicate that six of these residues are intolerant to substitution. We describe the dynamic behavior of DOT1L interacting with AF10 and AF9. Studies on the dynamics of a heterotrimeric complex of DOT1L1-AF10 illuminated describe coordinated motions that impact the relative position of the DOT1L HMT domain to the nucleosome. The molecular motions of the DOT1L-AF9 complex are less extensive and highly dynamic, resembling a swivel-like mechanics. Through molecular dynamics and mutational analysis, we extend the knowledge previous provided by static measurements. These results are important to consider when describing the biochemical properties of DOT1L, under normal and in disease conditions, as well as for the development of novel therapeutic agents., (© 2021 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

In the current study, we report computational scores for advancing genomic interpretation of disease-associated genomic variation in members of the RAS family of genes. For this purpose, we applied 31 sequence- and 3D structure-based computational scores, chosen by their breadth of biophysical properties. We parametrized our data by assembling a numerically homogenized experimentally-derived dataset, which when use in our calculations reveal that computational scores using 3D structure highly correlate with experimental measures (e.g., GAP-mediated hydrolysis R Spearman  = 0.80 and RAF affinity R spearman  = 0.82), while sequence-based scores are discordant with this data. Performing all-against-all comparisons, we applied this parametrized modeling approach to the study of 935 RAS variants from 7 RAS genes, which led us to identify 4 groups of mutations according to distinct biochemical scores within each group. Each group was comprised of hotspot and non-hotspot KRAS variants, indicating that poorly characterized variants could functionally behave like pathogenic mutations. Combining computational scores using dimensionality reduction indicated that changes to local unfolding propensity associate with changes in enzyme activity by genomic variants. Hence, our systematic approach, combining methodologies from both clinical genomics and 3D structural bioinformatics, represents an expansion for interpreting genomic data, provides information of mechanistic value, and that is transferable to other proteins., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Objectives: Otitis media (OM) is the most common pediatric diagnosis in the United States. However, our understanding of the molecular pathogenesis of OM remains relatively poor. Investigation of molecular pathways involved in OM may improve the understanding of this disease process and elucidate novel therapeutic targets. In this study, RNA sequencing (RNA-Seq) was used to discern cellular changes associated with OME compared to healthy middle ear epithelium (MEE)., Study Design: Ex vivo case-control translational., Methods: Middle ear epithelia was collected from five pediatric patients diagnosed with OME undergoing tympanostomy tube placement and five otherwise healthy pediatric patients undergoing cochlear implantation. Specimens underwent RNA-Seq and pathways analyses., Results: A total of 1,292 genes exhibited differential expression in MEE from OME patients compared to controls including genes involved in inflammation, immune response to bacterial OM pathogens, mucociliary clearance, regulation of proliferation and transformation, and auditory cell differentiation. Top networks identified in OME were organismal injury and abnormalities, cell morphology, and auditory disease. Top Ingenuity canonical pathways identified were axonal guidance signaling, which contains genes associated with auditory development and disease and nicotine degradation II and III pathways. Associated upstream regulators included β-estradiol, dexamethasone, and G-protein-coupled estrogen receptor-1 (GPER1), which are associated with otoprotection or inflammation during insult., Conclusions: RNA-Seq demonstrates differential gene expression in MEE from patients with OME compared to healthy controls with important implications for infection susceptibility, hearing loss, and a role for tobacco exposure in the development and/or severity of OME in pediatric patients., Level of Evidence: 4 Laryngoscope, 131:2590-2597, 2021., (© 2021 The American Laryngological, Rhinological and Otological Society, Inc.)

Background: Pancreatic ductal adenocarcinoma initiation is most frequently caused by Kras mutations., Results: Here, we apply biological, biochemical, and network biology methods to validate GEMM-derived cell models using inducible Kras G12D expression. We describe the time-dependent, chromatin remodeling program that impacts function during early oncogenic signaling. We find that the Kras G12D -induced transcriptional response is dominated by downregulated expression concordant with layers of epigenetic events. More open chromatin characterizes the ATAC-seq profile associated with a smaller group of upregulated genes and epigenetic marks. RRBS demonstrates that promoter hypermethylation does not account for the silencing of the extensive gene promoter network. Moreover, ChIP-Seq reveals that heterochromatin reorganization plays little role in this early transcriptional program. Notably, both gene activation and silencing primarily depend on the marking of genes with a combination of H3K27ac, H3K4me3, and H3K36me3. Indeed, integrated modeling of all these datasets shows that Kras G12D regulates its transcriptional program primarily through unique super-enhancers and enhancers, and marking specific gene promoters and bodies. We also report chromatin remodeling across genomic areas that, although not contributing directly to cis-gene transcription, are likely important for Kras G12D functions., Conclusions: In summary, we report a comprehensive, time-dependent, and coordinated early epigenomic program for Kras G12D in pancreatic cells, which is mechanistically relevant to understanding chromatin remodeling events underlying transcriptional outcomes needed for the function of this oncogene., (© 2021. The Author(s).)

Purpose: Somatic activating variants in the PI3K-AKT pathway cause vascular malformations with and without overgrowth. We previously reported an individual with capillary and lymphatic malformation harboring a pathogenic somatic variant in PIK3R1, which encodes three PI3K complex regulatory subunits. Here, we investigate PIK3R1 in a large cohort with vascular anomalies and identify an additional 16 individuals with somatic mosaic variants in PIK3R1., Methods: Affected tissue from individuals with vascular lesions and overgrowth recruited from a multisite collaborative network was studied. Next-generation sequencing targeting coding regions of cell-signaling and cancer-associated genes was performed followed by assessment of variant pathogenicity., Results: The phenotypic and variant spectrum associated with somatic variation in PIK3R1 is reported herein. Variants occurred in the inter-SH2 or N-terminal SH2 domains of all three PIK3R1 protein products. Phenotypic features overlapped those of the PIK3CA-related overgrowth spectrum (PROS). These overlapping features included mixed vascular malformations, sandal toe gap deformity with macrodactyly, lymphatic malformations, venous ectasias, and overgrowth of soft tissue or bone., Conclusion: Somatic PIK3R1 variants sharing attributes with cancer-associated variants cause complex vascular malformations and overgrowth. The PIK3R1-associated phenotypic spectrum overlaps with PROS. These data extend understanding of the diverse phenotypic spectrum attributable to genetic variation in the PI3K-AKT pathway., (© 2021. The Author(s).)

Sterile alpha motif (SAM) and Src homology-3 (SH3) domain-containing 3 (SASH3), also called SH3-containing lymphocyte protein (SLY1), is a putative adaptor protein that is postulated to play an important role in the organization of signaling complexes and propagation of signal transduction cascades in lymphocytes. The SASH3 gene is located on the X-chromosome. Here, we identified 3 novel SASH3 deleterious variants in 4 unrelated male patients with a history of combined immunodeficiency and immune dysregulation that manifested as recurrent sinopulmonary, cutaneous, and mucosal infections and refractory autoimmune cytopenias. Patients exhibited CD4+ T-cell lymphopenia, decreased T-cell proliferation, cell cycle progression, and increased T-cell apoptosis in response to mitogens. In vitro T-cell differentiation of CD34+ cells and molecular signatures of rearrangements at the T-cell receptor α (TRA) locus were indicative of impaired thymocyte survival. These patients also manifested neutropenia and B-cell and natural killer (NK)-cell lymphopenia. Lentivirus-mediated transfer of the SASH3 complementary DNA-corrected protein expression, in vitro proliferation, and signaling in SASH3-deficient Jurkat and patient-derived T cells. These findings define a new type of X-linked combined immunodeficiency in humans that recapitulates many of the abnormalities reported in mice with Sly1-/- and Sly1Δ/Δ mutations, highlighting an important role of SASH3 in human lymphocyte function and survival., (© 2021 by The American Society of Hematology.)

Motivation: Genomic data are prevalent, leading to frequent encounters with uninterpreted variants or mutations with unknown mechanisms of effect. Researchers must manually aggregate data from multiple sources and across related proteins, mentally translating effects between the genome and proteome, to attempt to understand mechanisms., Materials and Methods: P 2 T 2 presents diverse data and annotation types in a unified protein-centric view, facilitating the interpretation of coding variants and hypothesis generation. Information from primary sequence, domain, motif, and structural levels are presented and also organized into the first Paralog Annotation Analysis across the human proteome., Results: Our tool assists research efforts to interpret genomic variation by aggregating diverse, relevant, and proteome-wide information into a unified interactive web-based interface. Additionally, we provide a REST API enabling automated data queries, or repurposing data for other studies., Conclusion: The unified protein-centric interface presented in P 2 T 2 will help researchers interpret novel variants identified through next-generation sequencing. Code and server link available at github.com/GenomicInterpretation/p2t2., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Medical Informatics Association.)

SPTBN1 encodes βII-spectrin, the ubiquitously expressed β-spectrin that forms micrometer-scale networks associated with plasma membranes. Mice deficient in neuronal βII-spectrin have defects in cortical organization, developmental delay and behavioral deficiencies. These phenotypes, while less severe, are observed in haploinsufficient animals, suggesting that individuals carrying heterozygous SPTBN1 variants may also show measurable compromise of neural development and function. Here we identify heterozygous SPTBN1 variants in 29 individuals with developmental, language and motor delays; mild to severe intellectual disability; autistic features; seizures; behavioral and movement abnormalities; hypotonia; and variable dysmorphic facial features. We show that these SPTBN1 variants lead to effects that affect βII-spectrin stability, disrupt binding to key molecular partners, and disturb cytoskeleton organization and dynamics. Our studies define SPTBN1 variants as the genetic basis of a neurodevelopmental syndrome, expand the set of spectrinopathies affecting the brain and underscore the critical role of βII-spectrin in the central nervous system., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, painful disease with a 5-year survival rate of only 9%. Recent evidence indicates that distinct epigenomic landscapes underlie PDAC progression, identifying the H3K9me pathway as important to its pathobiology. Here, we delineate the role of Euchromatic Histone-lysine N -Methyltransferase 2 (EHMT2), the enzyme that generates H3K9me, as a downstream effector of oncogenic KRAS during PDAC initiation and pancreatitis-associated promotion. EHMT2 inactivation in pancreatic cells reduces H3K9me2 and antagonizes Kras G 12 D -mediated acinar-to-ductal metaplasia (ADM) and Pancreatic Intraepithelial Neoplasia (PanIN) formation in both the Pdx1-Cre and P48 Cre/+ Kras G 12 D mouse models. Ex vivo acinar explants also show impaired EGFR-KRAS-MAPK pathway-mediated ADM upon EHMT2 deletion. Notably, Kras G 12 D increases EHMT2 protein levels and EHMT2-EHMT1-WIZ complex formation. Transcriptome analysis reveals that EHMT2 inactivation upregulates a cell cycle inhibitory gene expression network that converges on the Cdkn1a/p21-Chek2 pathway. Congruently, pancreas tissue from Kras G 12 D animals with EHMT2 inactivation have increased P21 protein levels and enhanced senescence. Furthermore, loss of EHMT2 reduces inflammatory cell infiltration typically induced during Kras G 12 D -mediated initiation. The inhibitory effect on Kras G 12 D -induced growth is maintained in the pancreatitis-accelerated model, while simultaneously modifying immunoregulatory gene networks that also contribute to carcinogenesis. This study outlines the existence of a novel KRAS-EHMT2 pathway that is critical for mediating the growth-promoting and immunoregulatory effects of this oncogene in vivo , extending human observations to support a pathophysiological role for the H3K9me pathway in PDAC., Competing Interests: MBD is a co-founder and has ownership interests in Protein Foundry, LLC and Xlock Biosciences, LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Urrutia, de Assuncao, Mathison, Salmonson, Kerketta, Zeighami, Stodola, Adsay, Pehlivanoglu, Dwinell, Zimmermann, Iovanna, Urrutia and Lomberk.)

Motivation: Protein-coding genetic alterations are frequently observed in Clinical Genetics, but the high yield of variants of uncertain significance remains a limitation in decision making. RAS-family GTPases are cancer drivers, but only 54 variants, across all family members, fall within well-known hotspots. However, extensive sequencing has identified 881 non-hotspot variants for which significance remains to be investigated., Results: Here, we evaluate 935 missense variants from seven RAS genes, observed in cancer, RASopathies and the healthy adult population. We characterized hotspot variants, previously studied experimentally, using 63 sequence- and 3D structure-based scores, chosen by their breadth of biophysical properties. Applying scores that display best correlation with experimental measures, we report new valuable mechanistic inferences for both hot-spot and non-hotspot variants. Moreover, we demonstrate that 3D scores have little-to-no correlation with those based on DNA sequence, which are commonly used in Clinical Genetics. Thus, combined, these new knowledge bear significant relevance., Availability and Implementation: All genomic and 3D scores, and markdown for generating figures, are provided in our supplemental data., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2020. Published by Oxford University Press.)

Tumor genomic profiling (TGP) has the potential to identify germline variants in addition to its primary use of informing cancer treatment based on genetic alterations within the tumor. However, there are no formal consensus guidelines to identify patients who would be eligible for genetic counseling (GC) and germline testing (GT) testing in patients undergoing TGP. The purpose of this study is to describe an institutionally developed Germline Review Protocol (GRP) to evaluate adult cancer patient cases already undergoing TGP to determine GC referral eligibility. We report on our retrospective experience implementing this protocol into practice wherein 172 patients out of 638 patients reviewed (27%) were recommended for a GC referral over a 17-month time period. Of those 172 patients recommended for a GC referral, only 34 patients (20%) completed GC and GT. Among patients who received GT, 15 (44%) were positive for at least one pathogenic or likely pathogenic (P/LP) variant, seven patients (21%) were negative and 12 patients (35%) had at least 1 variant of uncertain significance (VUS). The primary reason GC and GT was not completed was because the patient moved to hospice care or was deceased. This is one of the first studies outlining the process and results of a formalized institutional protocol to facilitate patient referrals for GC and GT based on TGP results., (© 2021 National Society of Genetic Counselors.)

Two inherent challenges in the mechanistic interpretation of protease-deficient phenotypes are defining the specific substrate cleavages whose reduction generates the phenotypes and determining whether the phenotypes result from loss of substrate function, substrate accumulation, or loss of a function(s) embodied in the substrate fragments. Hence, recapitulation of a protease-deficient phenotype by a cleavage-resistant substrate would stringently validate the importance of a proteolytic event and clarify the underlying mechanisms. Versican is a large proteoglycan required for development of the circulatory system and proper limb development, and is cleaved by ADAMTS proteases at the Glu 441 -Ala 442 peptide bond located in its alternatively spliced GAGβ domain. Specific ADAMTS protease mutants have impaired interdigit web regression leading to soft tissue syndactyly that is associated with reduced versican proteolysis. Versikine, the N-terminal proteolytic fragment generated by this cleavage, restores interdigit apoptosis in ADAMTS mutant webs. Here, we report a new mouse transgene, Vcan AA , with validated mutations in the GAGβ domain that specifically abolish this proteolytic event. Vcan AA/AA mice have partially penetrant hindlimb soft tissue syndactyly. However, Adamts20 inactivation in Vcan AA/AA mice leads to fully penetrant, more severe syndactyly affecting all limbs, suggesting that ADAMTS20 cleavage of versican at other sites or of other substrates is an additional requirement for web regression. Indeed, immunostaining with a neoepitope antibody against a cleavage site in the versican GAGα domain demonstrated reduced staining in the absence of ADAMTS20. Significantly, mice with deletion of Vcan exon 8, encoding the GAGβ domain, consistently developed soft tissue syndactyly, whereas mice unable to include exon 7, encoding the GAGα domain in Vcan transcripts, consistently had fully separated digits. These findings suggest that versican is cleaved within each GAG-bearing domain during web regression, and affirms that proteolysis in the GAGβ domain, via generation of versikine, has an essential role in interdigital web regression., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors. Published by Elsevier B.V.)

We investigated germline variation in pancreatic ductal adenocarcinoma (PDAC) predisposition genes in 535 patients, using a custom-built panel and a new complementary bioinformatic approach. Our panel assessed genes belonging to DNA repair, cell cycle checkpoints, migration, and preneoplastic pancreatic conditions. Our bioinformatics approach integrated annotations of variants by using data derived from both germline and somatic references. This integrated approach with expanded evidence enabled us to consider patterns even among private mutations, supporting a functional role for certain alleles, which we believe enhances individualized medicine beyond classic gene-centric approaches. Concurrent evaluation of three levels of evidence, at the gene, sample, and cohort level, has not been previously done. Overall, we identified in PDAC patient germline samples, 12% with mutations previously observed in pancreatic cancers, 23% with mutations previously discovered by sequencing other human tumors, and 46% with mutations with germline associations to cancer. Non-polymorphic protein-coding pathogenic variants were found in 18.4% of patient samples. Moreover, among patients with metastatic PDAC, 16% carried at least one pathogenic variant, and this subgroup was found to have an improved overall survival (22.0 months versus 9.8; p=0.008) despite a higher pre-treatment CA19-9 level (p=0.02). Genetic alterations in DNA damage repair genes were associated with longer overall survival among patients who underwent resection surgery (92 months vs. 46; p=0.06). ATM alterations were associated with more frequent metastatic stage (p = 0.04) while patients with BRCA1 or BRCA2 alterations had improved overall survival (79 months vs. 39; p=0.05). We found that mutations in genes associated with chronic pancreatitis were more common in non-white patients (p<0.001) and associated with longer overall survival (52 months vs. 26; p=0.004), indicating the need for greater study of the relationship among these factors. More than 90% of patients were found to have variants of uncertain significance, which is higher than previously reported. Furthermore, we generated 3D models for selected mutant proteins, which suggested distinct mechanisms underlying their dysfunction, likely caused by genetic alterations. Notably, this type of information is not predictable from sequence alone, underscoring the value of structural bioinformatics to improve genomic interpretation. In conclusion, the variation in PDAC predisposition genes appears to be more extensive than anticipated. This information adds to the growing body of literature on the genomic landscape of PDAC and brings us closer to a more widespread use of precision medicine for this challenging disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest, (Copyright © 2021 Zimmermann, Mathison, Stodola, Evans, Abrudan, Demos, Tschannen, Aldakkak, Geurts, Lomberk, Tsai and Urrutia.)