Citations

BACKGROUND: Genetic testing for cardiac channelopathies is the standard of care. However, many rare genetic variants remain classified as variants of uncertain significance (VUS) due to lack of epidemiological and functional data. Whether deep protein language models may aid in VUS resolution remains unknown. Here, we set out to compare how 2 deep protein language models perform at VUS resolution in the 3 most common long-QT syndrome–causative genes compared with the gold-standard patch clamp. METHODS: A total of 72 rare nonsynonymous VUS (9 KCNQ1, 19 KCNH2 , and 50 SCN5A ) were engineered by site-directed mutagenesis and expressed in either HEK293 cells or TSA201 cells. Whole-cell patch-clamp technique was used to functionally characterize these variants. The protein language models, evolutionary scale modeling, version 1b and AlphaMissense, were used to predict the variant effect of missense variants and compared with patch clamp. RESULTS: Considering variants in all 3 genes, the evolutionary scale modeling, version 1b model had a receiver operating characteristic curve–area under the curve of 0.75 ( P =0.0003). It had a sensitivity of 88% and a specificity of 50%. AlphaMissense performed well compared with patch-clamp with an receiver operating characteristic curve-area under the curve of 0.85 ( P <0.0001), sensitivity of 80%, and specificity of 76%. CONCLUSIONS: Deep protein language models aid in VUS resolution with high sensitivity but lower specificity. Thus, these tools cannot fully replace functional characterization but can aid in reducing the number of variants that may require functional analysis.

Key Points Common kidney stones are unlikely to be an independent and direct cause of CKD in the general population. CKD may protect against kidney stones because of changes in key urinary factors critical for stone formation. Background Kidney stones and CKD are common disorders with a substantial interaction. Although observational studies have suggested a potential for enhanced CKD risk after prior kidney stones, the exact relationship remains ambiguous. Methods Shared comorbidities between two diseases were identified using unbiased screening. Genome-wide association study summary statistics were obtained from the UK Biobank (UKBB), FinnGen, and CKDGen, followed by genetic association analyses across various traits. Bidirectional Mendelian randomization (MR) analyses were performed to define causal links, complemented by multivariable MR that included the shared comorbidities including hypertension, diabetes, and obesity. Observational analyses were undertaken using cohorts from the Mayo Clinic and a UKBB subset. Results Despite identifying a total of 123 conditions as shared comorbidities, there was no significant genetic correlation between kidney stones and CKD. Unadjusted MR analysis revealed no significant association between kidney stones and CKD risk (UKBB [exposure]/FinnGen [outcome]: odds ratio [OR]=0.97, 95% confidence interval [CI], 0.88 to 1.06; FinnGen/UKBB: OR=1.17, 95% CI, 0.98 to 1.39). Kidney stones did significantly associate with a higher urinary albumin-creatinine ratio ( β =0.014, 95% CI, 0.002 to –0.025), but this association disappeared in the multivariable MR model ( β =0.009, 95% CI, −0.003 to 0.020). Furthermore, in a cross-sectional analysis limited to the UKBB cohort, a robust regression model did not detect an independent association between kidney stones and urinary albumin-creatinine ratio ( β =0.16, 95% CI, −0.04 to 0.35) or eGFR ( β =0.10, 95% CI, −0.07 to 0.28). Conversely, CKD associated with a diminished risk of kidney stones in multivariable MR models (UKBB/FinnGen: OR=0.77, 95% CI, 0.69 to 0.87; FinnGen/UKBB: OR=0.73, 95% CI, 0.66 to 0.81). Furthermore, in the Mayo Clinic cohort with available urinary biochemistries, lower eGFR was associated with lower urinary calcium excretion and urinary calcium oxalate/phosphate supersaturation. Conclusions In this study, kidney stones were not independently associated with CKD. Conversely, CKD was associated with a lower risk of calcium kidney stones likely via changes in key urinary traits, including lower calcium excretion.

Abstract Although the functions of tyrosine phosphatases in T‐cell biology have been extensively studied, our knowledge on the contribution of serine/threonine phosphatases in T cells remains poor. Protein phosphatase 2A (PP2A) is one of the most abundantly expressed serine/threonine phosphatases. It is important in thymocyte development and CD4 + T‐cell differentiation. Utilizing a genetic model in which its catalytic subunit alpha isoform (PP2A Cα) is deleted in T cells, we investigated its contribution to CD8 + T‐cell homeostasis and effector functions. Our results demonstrate that T‐cell intrinsic PP2A Cα is critically required for CD8 + T‐cell homeostasis in secondary lymphoid organs and intestinal mucosal site. Importantly, PP2A Cα–deficient CD8 + T cells exhibit reduced proliferation and survival. CD8 + T‐cell antibacterial response is strictly dependent on PP2A Cα. Expression of Bcl2 transgene rescues CD8 + T‐cell homeostasis in spleens, but not in intestinal mucosal site, nor does it restore defective antibacterial responses. Finally, proteomics and phosphoproteomics analyses reveal potential targets dependent on PP2A Cα, including mTORC1 and AKT. Thus, PP2A Cα is a key modulator of CD8 + T‐cell homeostasis and effector functions.

Heterotopic ossification (HO), defined as the formation of extraskeletal bone in muscle and soft tissues, is a diverse pathological process caused by either genetic mutations or inciting trauma. Fibrodysplasia ossificans progressiva (FOP) is a genetic form of HO caused by mutations in the bone morphogenetic protein (BMP) type I receptor gene activin A receptor type 1 ( ACVR1 ). These mutations make ACVR1 hypersensitive to BMP and responsive to activin A. Hedgehog (Hh) signaling also contributes to HO development. However, the exact pathophysiology of how skeletogenic cells contribute to endochondral ossification in FOP remains unknown. Here, we showed that the wild-type or FOP-mutant ACVR1 localized in the cilia of stem cells from human exfoliated deciduous teeth with key FOP signaling components, including activin A receptor type 2A/2B, SMAD family member 1/5, and FK506-binding protein 12kD. Cilia suppression by deletion of intraflagellar transport 88 or ADP ribosylation factor like GTPase 3 effectively inhibited pathological BMP and Hh signaling, subdued aberrant chondro-osteogenic differentiation in primary mouse or human FOP cells, and diminished in vivo extraskeletal ossification in Acvr1 Q207D , Sox2-Cre ; Acvr1 R206H/+ FOP mice and in burn tenotomy–treated wild-type mice. Our results provide a rationale for early and localized suppression of cilia in affected tissues after injury as a therapeutic strategy against either genetic or acquired HO.

We developed a generally applicable method, CRISPR/Cas9-targeted long-read sequencing (CTLR-Seq), to resolve, haplotype-specifically, the large and complex regions in the human genome that had been previously impenetrable to sequencing analysis, such as large segmental duplications (SegDups) and their associated genome rearrangements. CTLR-Seq combines in vitro Cas9-mediated cutting of the genome and pulse-field gel electrophoresis to isolate intact large (i.e., up to 2,000 kb) genomic regions that encompass previously unresolvable genomic sequences. These targets are then sequenced (amplification-free) at high on-target coverage using long-read sequencing, allowing for their complete sequence assembly. We applied CTLR-Seq to the SegDup-mediated rearrangements that constitute the boundaries of, and give rise to, the 22q11.2 Deletion Syndrome (22q11DS), the most common human microdeletion disorder. We then performed de novo assembly to resolve, at base-pair resolution, the full sequence rearrangements and exact chromosomal breakpoints of 22q11.2DS (including all common subtypes). Across multiple patients, we found a high degree of variability for both the rearranged SegDup sequences and the exact chromosomal breakpoint locations, which coincide with various transposons within the 22q11.2 SegDups, suggesting that 22q11DS can be driven by transposon-mediated genome recombination. Guided by CTLR-Seq results from two 22q11DS patients, we performed three-dimensional chromosomal folding analysis for the 22q11.2 SegDups from patient-derived neurons and astrocytes and found chromosome interactions anchored within the SegDups to be both cell type-specific and patient-specific. Lastly, we demonstrated that CTLR-Seq enables cell-type specific analysis of DNA methylation patterns within the deletion haplotype of 22q11DS.

The effectiveness of coronavirus disease 2019 (COVID-19) vaccines against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain rapidly wanes over time. Growing evidence from epidemiological studies suggests that influenza vaccination is associated with a reduction in the risk of SARS-CoV-2 infection and COVID-19 severity. However, the underlying mechanisms remain elusive. Here, we investigate the cross-reactive immune responses of influenza vaccination to SARS-CoV-2 spike protein peptides based on in vitro study. Our data indicate enhanced activation-induced-marker (AIM) expression on CD4+ T cells in influenza-vaccination (IV)-treated peripheral blood mononuclear cells (PBMCs) upon stimulation with spike-protein-peptide pools. The fractions of other immune cell subtypes, including CD8+ T cells, monocytes, NK cells, and antigen-presenting cells, were not changed between IV-treated and control PBMCs following ex vivo spike-protein-peptide stimulation. However, the classical antiviral (IFN-γ) and anti-inflammatory (IL-1RA) cytokine responses to spike-protein-peptide stimulation were still enhanced in PBMCs from both IV-immunized adult and aged mice. Decreased expression of proinflammatory IL-1β, IL-12p40, and TNF-α is associated with inhibited levels of histone acetylation in PBMCs from IV-treated mice. Remarkably, prior immunity to SARS-CoV-2 does not result in modification of histone acetylation or hemagglutinin-protein-induced cytokine responses. This response is antibody-independent but can be mediated by manipulating the histone acetylation of PBMCs. These data experimentally support that influenza vaccination could induce modification of histone acetylation in immune cells and reveal the existence of potential cross-reactive immunity to SARS-CoV-2 antigens, which may provide insights for the adjuvant of influenza vaccine to limit COVID-19-related inflammatory responses.

Abstract Generative artificial intelligence (AI) holds immense potential for medical applications, but the lack of a comprehensive evaluation framework and methodological deficiencies in existing studies hinder its effective implementation. Standardized assessment guidelines are crucial for ensuring reliable and consistent evaluation of generative AI in healthcare. Our objective is to develop robust, standardized guidelines tailored for evaluating generative AI performance in medical contexts. Through a rigorous literature review utilizing the Web of Sciences, Cochrane Library, PubMed, and Google Scholar, we focused on research testing generative AI capabilities in medicine. Our multidisciplinary team of experts conducted discussion sessions to develop a comprehensive 32‐item checklist. This checklist encompasses critical evaluation aspects of generative AI in medical applications, addressing key dimensions such as question collection, querying methodologies, and assessment techniques. The checklist and its broader assessment framework provide a holistic evaluation of AI systems, delineating a clear pathway from question gathering to result assessment. It guides researchers through potential challenges and pitfalls, enhancing research quality and reporting and aiding the evolution of generative AI in medicine and life sciences. Our framework furnishes a standardized, systematic approach for testing generative AI's applicability in medicine. For a concise checklist, please refer to Table S or visit GenAIMed.org .

e19049 Background: Acala and zanu are 2nd generation Bruton tyrosine kinase inhibitors (BTKis) that have demonstrated improved safety profiles vs 1st generation BTKi ibrutinib in clinical trials. While head-to-head comparison of zanu vs acala is lacking, a recent meta-analysis of clinical trials provided a comprehensive, indirect comparison of the AE profiles in B-cell malignancies (Hwang, Hemasphere. 2023;7(S3):1134). This study aims to evaluate costs and impacts on quality of life (QoL) for zanu vs acala using their AE profiles. Methods: A health economic model was developed to determine cost and QoL associated with AE management for zanu and acala. The incidence rates ( IRs) of all grade and grade ≥3 AEs of interest (n=21, including bleeding events, hypertension, atrial fibrillation, cytopenias, infections, headache, arthralgia, diarrhea) were taken from the meta-analysis. Additional model inputs, such as disutility, average duration of AE, and unit cost of each AE were sourced from published literature and publicly available cost databases in consultation with clinical experts. The model was developed from the US perspective, considering Medicare costs, inflated to 2023 USD. Model outcomes were AE management cost (= IR * unit cost) and quality-adjusted life years (QALYs) lost due to AE (= IR * Disutility * Duration/365.25). Results: In the base-case scenario (considering all AEs), treating a hypothetical cohort of 1000 patients (pts) with zanu instead of acala yielded cost savings of $124K and 3.7 QALY gains (i.e., 3.7 years extra in full health). Subgroup analysis for severe (grade ≥3) and non-severe AEs (grade 1-2) demonstrated similar trends. A sensitivity analysis limited to AEs significantly different between zanu and acala (n=13) yielded consistent results. Additionally, a probabilistic sensitivity analysis, conducted with 1000 iterations to account for uncertainty in model parameters, confirmed robustness of the model, indicating stable conclusions across a wide range of parameter uncertainties. Conclusions: This analysis suggested that zanu is cost-saving and associated with added health benefits compared to acala in terms of AE management in pts with B-cell malignancies. Should these results derived from meta-analysis of clinical trial data be applicable to real-world pt populations across different indications, the potential cost savings could be considerable. These results could be further improved if the differences in efficacy of zanu vs acala are also incorporated. [Table: see text]

AbstractTargeting NLRP3 inflammasome has been recognized as a promising therapeutic strategy for the treatment of numerous common diseases. UK5099, a long‐established inhibitor of mitochondrial pyruvate carrier (MPC), is previously found to inhibit macrophage inflammatory responses independent of MPC expression. However, the mechanisms by which UK5099 inhibit inflammatory responses remain unclear. Here, it is shown that UK5099 is a potent inhibitor of the NLRP3 inflammasome in both mouse and human primary macrophages. UK5099 selectively suppresses the activation of the NLRP3 but not the NLRC4 or AIM2 inflammasomes. Of note, UK5099 retains activities on NLRP3 in macrophages devoid of MPC expression, indicating this inhibitory effect is MPC‐independent. Mechanistically, UK5099 abrogates mitochondria‐NLRP3 interaction and in turn inhibits the assembly of the NLRP3 inflammasome. Further, a single dose of UK5099 persistently reduces IL‐1β production in an endotoxemia mouse model. Importantly, structure modification reveals that the inhibitory activities of UK5099 on NLRP3 are unrelated to the existence of the activated double bond within the UK5099 molecule. Thus, this study uncovers a previously unknown molecular target for UK5099, which not only offers a new candidate for the treatment of NLRP3‐driven diseases but also confounds its use as an MPC inhibitor in immunometabolism studies.