Genetics
Abstract
Congenital long QT syndrome (LQTS) promotes risk for life-threatening cardiac arrhythmia and sudden death in children and young adults. Pathogenic variants in the voltage-gated potassium channel KCNQ1 are the most frequently discovered genetic cause. Most LQTS-associated KCNQ1 variants cause loss-of-function secondary to impaired trafficking of the channel to the plasma membrane. There are currently no therapeutic approaches that address this underlying molecular defect. Using a high-throughput screening paradigm, we identified VU0494372, a small molecule that increases total and cell surface levels and trafficking efficiency of WT KCNQ1 as well as three LQTS-associated variants. Additionally, 16-hour treatment of cells with VU0494372 increased IKs (KCNQ1-KCNE1 current) for WT KCNQ1 and the LQTS-associated variant V207M in cells co-expressing KCNE1. VU0494372 had no impact on KCNQ1 transcription, degradation, or thermal stability, and increased the rate of KCNQ1 reaching the cell surface. We identified a potential direct interaction site with KCNQ1 at or near the binding site of the KCNQ1 potentiator ML277. Together, these findings demonstrate that small molecules can increase the expression levels and cell surface trafficking efficiency of KCNQ1 and introduce a potential new pharmacological approach for treating LQTS.
Authors
Katherine R. Clowes Moster, Carlos G. Vanoye, Ana C. Chang-Gonzalez, Ian M. Romaine, Katherine M. Stefanski, Mason C. Wilkinson, Joshua A. Bauer, Thomas P. Hasaka, Emily L. Days, Reshma R. Desai, Kathryn R. Butcher, Gary A. Sulikowski, Alex G. Waterson, Jens Meiler, Kaitlyn V. Ledwitch, Alfred L. George, Jr., Charles R. Sanders
Abstract
Background: Sleep is increasingly recognized as essential to human health, yet the adverse health consequences of acute sleep deprivation are unknown. We hypothesized that acute sleep deprivation is associated with health outcomes and modulated by sleep-associated genotypes. Methods: LOESS smoothing was performed on sleep estimates from Fitbit users (N = 14,681) between June 1, 2016 and July 1, 2022. Dates when population minutes slept were less than the 90% confidence interval of the LOESS regression were named acute sleep deprivation events (ASDEs). Phenome-wide disease incidence among the AoU population (N = 287,012) in the 10 days post-ASDE was compared to a preceding reference period by McNemar test. Circadian rhythm and sleep duration-associated SNPs were screened to identify genotypes associated with shorter ASDE sleep duration. Influences of sleep and circadian genotype on post-ASDE influenza risk were modeled using binomial family generalized estimating equations. Results: We identified 32 ASDEs spanning major national events. A phenome-wide screen found increased risk of influenza (OR = 1.54 [1.40, 1.70], P-value = 1.00 x 10-18) following ASDEs. 56 SNPs were associated with decreased sleep duration on ASDEs. Higher quantiles of ASDE-related SNP genotype burden were associated with less ASDE sleep duration and a greater risk of influenza-associated healthcare visits. Conclusion: Major national events are associated with acute sleep deprivation and greater influenza risk which is amplified by sleep genotypes. These findings should inform public health vigilance surrounding major national events.
Authors
Neil J. Kelly, Rahul Chaudhary, Wadih El Khoury, Nishita Kalepalli, Jesse Wang, Priya Patel, Irene Chan, Haris Rahman, Aisha Saiyed, Anisha N. Shah, Colleen A. McClung, Satoshi Okawa, Mehdi Nouraie, Stephen Y. Chan
Abstract
The transcription factor IKAROS, encoded by IKZF1, is crucial for lymphocyte development and differentiation. Germline heterozygous IKZF1 mutations cause B cell immunodeficiency, but also affect T cells. Patients with IKZF1 haploinsufficiency (HI) or dimerization-defective (DD) variants show reduced naive and increased memory T cells, while dominant-negative (DN) mutations result in the opposite phenotype. Gain-of-function patients display variable patterns. To investigate IKAROS’s role in shaping the human naive/memory T cell phenotype, we performed IKAROS immunomodulation and knockdown experiments and analyzed early T cell development in an artificial thymic organoid (ATO) system using CD34+ cells from patients with representative IKZF1 variants. IKAROS inhibition by lenalidomide or silencing by small hairpin RNA directly altered expression of HNRNPLL, the master regulator of CD45 isoform splicing that defines CD45RA+/naive and CD45RO+/memory phenotypes. In the ATO system, IKAROS-DN precursor cells were blocked at the CD4–CD8–/double-negative stage and retained a CD45RA+ phenotype, whereas IKAROS-HI cells inefficiently reached the CD4+CD8+/double-positive stage and partially transitioned from CD45RA to CD45RO. Analysis of public gene expression data showed high HNRNPLL expression in double-positive thymic cells, beyond the stages affected by IKZF1 DN and HI mutations. Collectively, these findings indicate that IKAROS regulates early and late T cell development by mechanisms, including HNRNPLL modulation.
Authors
Jennifer Stoddard, Hye Sun Kuehn, Ravichandra Tagirasa, Marita Bosticardo, Francesca Pala, Julie E. Niemela, Agustin A. Gil Silva, Kayla Amini, Eduardo Anaya, Mario Framil Seoane, Carolina Bouso, Dimana Dimitrova, Jennifer A. Kanakry, Laia Alsina, Matias Oleastro, Steven M. Holland, Thomas A. Fleisher, Richard L. Wasserman, Luigi D. Notarangelo, Sergio D. Rosenzweig
Abstract
Pathogenic variants in kinesin KIF11 underlie microcephaly-lymphedema-chorioretinopathy (MLC) syndrome. Although well known for regulating spindle dynamics ensuring successful cell division, the association of KIF11 (encoding EG5) with development of the lymphatic system, and how KIF11 pathogenic variants lead to lymphatic dysfunction and lymphedema remain unknown. Using patient-derived lymphoblastoid cells, we demonstrated that MLC patients carrying pathogenic stop-gain variants in KIF11 have reduced mRNA and protein levels. Lymphoscintigraphy showed reduced tracer absorption, and intestinal lymphangiectasia was detected in one patient, pointing to impairment of lymphatic function caused by KIF11 haploinsufficiency. We revealed that KIF11 is expressed in early human and mouse development with the lymphatic markers VEGFR3, Podoplanin and PROX1. In zebrafish, scRNA-seq identified kif11 specifically expressed in endothelial precursors. In human lymphatic endothelial cells (LECs), EG5 inhibition with Ispinesib, reduced VEGFC-driven AKT phosphorylation, migration and spheroid sprouting. KIF11 knockdown reduced PROX1 and VEGFR3 expression, providing for the first time a link between KIF11 and drivers of lymphangiogenesis and lymphatic identity.
Authors
Kazim Ogmen, Sara E. Dobbins, Rose Yinghan Behncke, Ines Martinez-Corral, Ryan C.S. Brown, Michelle Meier, Sascha Ulferts, Nils Rouven Hansmeier, Ege Sackey, Ahlam Alqahtani, Christina Karapouliou, Dionysios Grigoriadis, Juan C. Del Rey Jimenez, Michael Oberlin, Denise Williams, Arzu Ekici, Kadri Karaer, Steve Jeffery, Peter Mortimer, Kristiana Gordon, Kazuhide S. Okuda, Benjamin M. Hogan, Taija Mäkinen, René Hägerling, Sahar Mansour, Silvia Martin-Almedina, Pia Ostergaard
Abstract
Kidney organoids are an emerging tool for disease modeling, especially genetic diseases. Among these diseases, X-linked Alport syndrome (XLAS) is a hematuric nephropathy affecting the glomerular basement membrane (GBM) secondary to pathogenic variations in the COL4A5 gene encoding the α5 subunit of type IV collagen [α5(IV)]. In patients carrying pathogenic variations affecting splicing, the use of antisense oligonucleotides (ASOs) offers immense therapeutic hope. In this study, we develop a framework combining the use of patient-derived cells and kidney organoids to provide evidence of the therapeutic efficacy of ASOs in XLAS patients. Using multiomics analysis, we describe the development of GBM in wild-type and mutated human kidney organoids. We show that GBM maturation is a dynamic process, which requires long organoid culture. Then, using semi-automated quantification of α5(IV) at basement membranes in organoids carrying the splicing variants identified in patients, we demonstrate the efficacy of ASO treatment for α5(IV) restoration. These data contribute to our understanding of the development of GBM in kidney organoids and pave the way for a therapeutic screening platform for patients.
Authors
Hassan Saei, Bruno Estebe, Nicolas Goudin, Mahsa Esmailpour, Julie Haure, Olivier Gribouval, Christelle Arrondel, Vincent Moriniere, Pinyuan Tian, Rachel Lennon, Corinne Antignac, Geraldine Mollet, Guillaume Dorval
Abstract
Urinary tract infections (UTIs) are the most common severe bacterial infections in young children, often associated with vesicoureteral reflux (VUR). To explore host genetic-microbiota interactions and their clinical implications, we analyzed the urinary microbiota (urobiota) and conducted genome-wide association studies (GWAS) for bacterial abundance traits in pediatric UTI and VUR patients from the RIVUR and CUTIE cohorts. We identified four urobiota community types based on relative abundance, characterized by the genera Enterococcus, Prevotella, Pseudomonas, and Escherichia/Shigella, and their associations with VUR, age, and toilet training. Children with VUR exhibited decreased microbial diversity and increased abundance of genera that included opportunistic pathogens, suggesting a disrupted urobiota. We detected genome-wide significant genetic associations with urinary bacterial relative abundances, in or near candidate genes including CXCL12, ABCC1, and ROBO1, which are implicated in urinary tract development and response to infection. We showed that Cxcl12 is induced 12 hours after uropathogenic bacterial infection in mouse bladder. The association with CXCL12 suggests a genetic link between UTI, VUR and cardiovascular phenotypes later in life. These findings provide the first characterization of host genetic influences on the pediatric urobiota in UTI and VUR, offering insights into the interplay between disease, host genetics and the urobiota composition.
Authors
Miguel Verbitsky, Pavan Khosla, Daniel Bivona, Atlas Khan, Yask Gupta, Heekuk Park, Tian H. Shen, Aryan Ghotra, Katherine Xu, Iman A. Ghavami, Priya Krithivasan, Jeremiah Martino, Tanya Sezin, Tze Y. Lim, Victoria Kolupaeva, Nita A. Limdi, Yuan Luo, Hakon Hakonarson, Simone Sanna-Cherchi, Krzysztof Kiryluk, Cathy L. Mendelsohn, Anne-Catrin Uhlemann, Jonathan Barasch, Ali G. Gharavi
Abstract
Autoimmune diabetes encompasses rapidly progressive type 1 diabetes mellitus (T1D) and indolent latent autoimmune diabetes in adults (LADA), representing distinct inflammatory set points along a shared autoimmune spectrum. Yet the immunological mechanisms that determine these divergent inflammatory states remain unresolved. We performed single-cell RNA sequencing with paired T and B cell receptor profiling on over 400,000 peripheral blood mononuclear cells (PBMCs) from patients with LADA, newly diagnosed T1D, and healthy controls. PBMC composition was comparable across cohorts, indicating that qualitative rather than quantitative immune differences underlie disease heterogeneity. In T1D, pan-lineage activation of NF-κB, EGFR, MAPK, and hypoxia pathways, coupled with a TNF-centered communication hub, enhanced MHC signaling, and disrupted adhesion, promoted systemic inflammation. LADA, by contrast, exhibited global suppression of NF-κB/EGFR activity, retention of moderate JAK/STAT tone, reinforced natural killer cell inhibitory checkpoints via HLA-C–KIR2DL3/3DL1 interaction, and stabilized CD8⁺ T cell synapses through HLA-C–CD8 binding, collectively restraining effector activation. Single-cell V(D)J analysis revealed multiclonal, patient-unique adaptive repertoires, emphasizing the primacy of signaling context over receptor convergence. These findings define autoimmune diabetes as an inflammatory–inhibitory set-point continuum, positioning the NF-κB/EGFR–JAK/STAT gradient and HLA-C–KIR axis as potential therapeutic targets to preserve residual β-cell function.
Authors
Ivan I. Golodnikov, Elizaveta S. Podshivalova, Vadim I. Chechekhin, Anatoliy V. Zubritskiy, Alina A. Matrosova, Nikita A. Sergeev, Margarita D. Samsonova, Yaroslav V. Dvoryanchikov, Tatiana V. Nikonova, Ekaterina V. Bondarenko, Marina Yu. Loguinova, Yulia A. Medvedeva, Dmitry N. Laptev, Rita I. Khusainova, Ildar R. Minniakhmetov, Marina V. Shestakova, Natalia G. Mokrysheva, Ivan I. Dedov
Abstract
Duchenne muscular dystrophy (DMD) is a fatal genetic muscle-wasting disease characterized by loss of dystrophin protein. Therapeutic attempts to restore a functional copy of dystrophin to striated muscle are under active development, and many utilize adeno-associated viral (AAV) vectors. However, the limited cargo capacity of AAVs precludes delivery of full-length dystrophin, a 427 kDa protein, to target tissues. Recently, we developed a novel method to express large dystrophin constructs using the protein trans-splicing (PTS) mechanism mediated by split inteins and myotropic AAV vectors. The efficacy of this approach to restore muscle function in mdx4cv mice was previously assessed using histology, dystrophin immunolabeling, and western blotting. Here, we expand our molecular characterization of dystrophin constructs with variable lengths using a mass spectrometry-based proteomics approach, providing insight into unique protein expression profiles in skeletal muscles of wild-type, dystrophic mdx4cv, and AAV-treated mdx4cv. Our data reveal several affected cellular processes in mdx4cv skeletal muscles with changes in the expression profiles of key proteins to muscle homeostasis, whereas successful expression of dystrophin constructs results in an intermediate to complete restoration. This study highlights several biomarkers that could be used in future preclinical or clinical studies to evaluate the effectiveness of therapeutic strategies.
Authors
Erynn E. Johnson, Theodore R. Reyes, Jeffrey S. Chamberlain, James M. Ervasti, Hichem Tasfaout
Abstract
Over 95% of head and neck cancers are squamous cell carcinoma (HNSCC). HNSCC is mostly diagnosed late, causing a poor prognosis despite the application of invasive treatment protocols. Tumor-educated platelets (TEPs) have been shown to hold promise as a molecular tool for early cancer diagnosis. We sequenced platelet mRNA isolated from blood of 101 HNSCC patients and 101 propensity-score matched non-cancer controls. Two independent machine learning classification strategies were employed using a training and validation approach to identify a cancer predictor: a particle swarm optimized support vector machine (PSO-SVM) and a least absolute shrinkage and selection operator (LASSO) logistic regression model. The best performing PSO-SVM predictor consisted of 245 platelet transcripts and reached a maximum area under the curve (AUC) of 0.87. For the LASSO-based prediction model 1,198 mRNAs were selected, resulting in an median AUC of 0.84, independent of HPV status. Our data show that TEP RNA classification by different AI tools is promising in the diagnosis of HNSCC.
Authors
N.E. Wondergem, J.B. Poell, S.G.J.G In 't Veld, E. Post, S.W. Mes, M.G. Best, W.N. van Wieringen, T. Klausch, R.J. Baatenburg de Jong, C.H.J. Terhaard, R.P. Takes, J.A. Langendijk, I.M. Verdonck-de Leeuw, F. Lamers, C.R. Leemans, E. Bloemena, T. Würdinger, R.H Brakenhoff
Abstract
HOXB13 is a prostate-specific transcription factor best known for its role as an androgen receptor (AR) cofactor. Recent evidence suggests that HOXB13 plays critical AR-independent functions in repressing lipogenic programs and promoting prostate cancer (PCa) metastasis. However, the mechanisms linking HOXB13 loss to tumor metastasis remain unclear. Here, we show that p300 and CBP co-occupy lipogenic enhancers suppressed by HOXB13 and HDAC3 and are essential for enhancer activation and target gene expression following HOXB13 depletion. Loss of HOXB13 induces lipid-sensitive matrix metalloproteinases (MMPs), promoting increased cell motility. Importantly, pharmacological inhibition of p300 and CBP blocks HOXB13-loss-driven lipogenesis, reduces MMP expression, and decreases cell migration in vitro and tumor metastasis in vivo. Analysis of clinical samples revealed that HOXB13 expression is reduced in metastatic hormone-sensitive PCa compared with matched primary tumors, further supporting its role in tumor metastasis. These findings demonstrate that HOXB13 downregulation promotes PCa metastasis through p300- and CBP-dependent lipogenic and motility pathways, which may be targeted by p300 inhibition.
Authors
Xiaodong Lu, Liu Peng, Qi Chu, Samantha Ye, Mingyang Liu, Maha Hussain, Mehmet A. Bilen, Lara R. Harik, Jonathan Melamed, Jonathan C. Zhao, Jindan Yu
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