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Murugan Sudhakar, Rashmi Rikhi, Sathish Kumar Loganathan, Deepti Suri, Surjit Singh Department of Pediatrics, Advanced Pediatrics Center, Postgraduate Institute of Medical Education and Research, Chandigarh, IndiaCorrespondence: Deepti SuriDepartment of Pediatrics, Advanced Pediatrics Centre, Postgraduate Institute of Medical Education and Research, Chandigarh, 160012, IndiaEmail surideepti@gmail.comAbstract: Wiskott–Aldrich syndrome (WAS) is an uncommon X-linked combined-immunodeficiency disorder characterized by a triad of thrombocytopenia, eczema, and immunodeficiency. Patients with WAS are also predisposed to autoimmunity and malignancy. Autoimmune manifestations have been reported in 26%– 72% of patients with WAS. Autoimmunity is an independent predictor of poor prognosis and predisposes to malignancy. Development of autoimmunity is also an early pointer of the need for hematopoietic stem–cell transplantation. In this manuscript, we have collated the published data and present a narrative review on autoimmune manifestations in WAS. A summary of currently proposed immunopathogenic mechanisms and genetic variants associated with development of autoimmunity in WAS is also included.Keywords: thrombocytopenia, vasculitis, genetics, hematopoietic stem–cell transplant, bleeding, malignancy

Kimura's disease (KD) is a chronic inflammatory disorder characterized by nontender lymphadenopathy involving the head and neck region. Renal involvement in KD is rare, especially in children. We report a 12-year-old boy who had been previously treated for classical KD and had presented with anasarca and oliguria after 4 years. There were no swellings or lymphadenopathy. The kidney biopsy revealed membranous nephropathy. Remission was achieved with oral prednisolone and tacrolimus therapy. This patient highlights the need to regularly monitor patients with KD for the evolution of renal diseases, even if lymphadenopathy regresses. Serial monitoring for eosinophilia, inflammatory markers, and urine examination is needed to help identify subclinical disease early and prompt initiation of specific therapy., (© 2024 Asia Pacific League of Associations for Rheumatology and John Wiley & Sons Australia, Ltd.)

Background: Dedicator of cytokinesis protein 8 (DOCK8) deficiency is an autosomal recessive form of combined immunodeficiency. This rare disorder is characterized by an increased predisposition to allergy, autoimmunity and malignancies., Objectives: To analyse clinical, immunological and molecular profiles of patients with DOCK8 deficiency., Methods: Clinic records of all patients attending the primary immunodeficiency clinic from 2018 to 2021 were reviewed. Six patients from five families were found to have DOCK8 deficiency., Results: Median age at diagnosis was 7.5 years (range 2-13), with a male/female ratio of 5 : 1. Among the six patients, recurrent eczematous skin lesions were the predominant cutaneous manifestation, present in five patients (83%). Warts and molluscum contagiosum were evident in two patients (33%) and one patient (16%), respectively. Two patients had recalcitrant prurigo nodularis lesions and two had epidermodysplasia verruciformis-like lesions. Food allergies and asthma were reported by one patient each. Of the six patients, recurrent sinopulmonary infections were detected in five (83%). Epstein-Barr virus-driven non-Hodgkin lymphoma with liver metastases was the only case of malignancy, in a 4-year-old boy. IgE was elevated in all patients. Lymphopenia and eosinophilia were observed in three patients (50%) and five patients (83.3%), respectively. Genetic analysis showed DOCK8 pathogenic variants in all patients: homozygous deletion mutations in two patients, compound heterozygous deletion mutations in one, and homozygous nonsense mutations in two. A novel pathogenic homozygous missense variant in the DOCK8 gene was identified in one patient., Conclusions: DOCK8 deficiency should be considered as a possibility in any patient with early onset eczema, cutaneous viral infections and increased predisposition to allergy, autoimmunity and malignancy., Competing Interests: Conflicts of interest All authors declare no conflicts of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Association of Dermatologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Rett syndrome (RTT) is a neurological disorder that mostly affects females, with a frequency of 1 in 10,000 to 20,000 live birth cases. Symptoms include stereotyped hand movements; impaired learning, language, and communication skills; sudden loss of speech; reduced lifespan; retarded growth; disturbance of sleep and breathing; seizures; autism; and gait apraxia. Pneumonia is the most common cause of death for patients with Rett syndrome, with a survival rate of 77.8% at 25 years of age. Survival into the fifth decade is typical in Rett syndrome, and the leading cause of death is cardiorespiratory compromise. Rett syndrome progression has multiple stages; however, most phenotypes are associated with the nervous system and brain. In total, 95% of Rett syndrome cases are due to mutations in the MECP2 gene, an X-linked gene that encodes for the methyl CpG binding protein, a regulator of gene expression. In this review, we summarize the recent developments in the field of Rett syndrome and therapeutics targeting MECP2.

Introduction: In North America and in most European countries, Human Papillomavirus (HPV) is responsible for over 70% of oropharyngeal squamous cell carcinomas. The burden of OPSCC, in high-income countries, has been steadily increasing over the past 20 years. As a result, in the USA and in the UK, the burden of HPV-related oropharyngeal squamous cell carcinoma in men has now surpassed that of cervical cancer in women. However, the oncogenic impact of high-risk HPV integration in oropharyngeal squamous cell carcinomas hasn't been extensively studied. The present study aimed to explore the patterns of HPV integration in oropharyngeal squamous cell carcinomas and to assess the feasibility and reliability of long-read sequencing technology in detecting viral integration events in oropharyngeal head and neck cancers., Methods: A cohort of eight HPV-positive OPSCC pre-treatment patient tumors (four males and four females), were selected. All patients received a p16 INK4A positive OPSCC diagnosis and were treated at the McGill University Health Centre, a quaternary center in Montreal. A minimum of 20mg of tumor tissue was used for DNA extraction. Extracted DNA was subjected to Nanopore long-read sequencing to detect and analyze for the presence of high-risk HPV sequences. PCR and Sanger sequencing experiments were performed to confirm Nanopore long-read sequencing readings., Results: Nanopore long-read sequencing showed that seven out of eight patient samples displayed either integrated or episomal high-risk HPV sequences. Out of these seven samples, four displayed verifiable integration events upon bioinformatic analysis. Integration confirmation experiments were designed for all four samples using PCR-based methods. Sanger sequencing was also performed. Four distinct HPV integration patterns were identified: concatemer chromosomal integration in a single chromosome, bi-chromosomal concatemer integration, single chromosome complete integration and bi-chromosomal complete integration. HPV concatemer integration also proved more common than full HPV integration events., Conclusion and Relevance: Long-read sequencing technologies can be effectively used to assess HPV integration patterns in OPSCC tumors. Clinically, more research should be conducted on the prognostication value of high-risk HPV integration in OPSCC tumors using long-read sequencing technology., 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 Gauthier, Kadam, Leveque, Golabi, Zeitouni, Richardson, Mascarella, Sadeghi and Loganathan.)

How the genetic landscape governs a tumor's response to immunotherapy remains poorly understood. To assess the immune-modulatory capabilities of 573 genes associated with altered cytotoxicity in human cancers, here we perform CRISPR/Cas9 screens directly in mouse lung cancer models. We recover the known immune evasion factors Stat1 and Serpinb9 and identify the cancer testis antigen Adam2 as an immune modulator, whose expression is induced by Kras G12D and further elevated by immunotherapy. Using loss- and gain-of-function experiments, we show that ADAM2 functions as an oncogene by restraining interferon and TNF cytokine signaling causing reduced presentation of tumor-associated antigens. ADAM2 also restricts expression of the immune checkpoint inhibitors PDL1, LAG3, TIGIT and TIM3 in the tumor microenvironment, which might explain why ex vivo expanded and adoptively transferred cytotoxic T-cells show enhanced cytotoxic efficacy in ADAM2 overexpressing tumors. Together, direct in vivo CRISPR/Cas9 screens can uncover genetic alterations that control responses to immunotherapies., (© 2023. The Author(s).)

Acute myeloid leukemia (AML) is fueled by leukemic stem cells (LSC) whose determinants are challenging to discern from hematopoietic stem cells (HSC) or uncover by approaches focused on general cell properties. We have identified a set of RNA-binding proteins (RBP) selectively enriched in human AML LSCs. Using an in vivo two-step CRISPR-Cas9 screen to assay stem cell functionality, we found 32 RBPs essential for LSCs in MLL-AF9;NrasG12D AML. Loss-of-function approaches targeting key hit RBP ELAVL1 compromised LSC-driven in vivo leukemic reconstitution, and selectively depleted primitive malignant versus healthy cells. Integrative multiomics revealed differentiation, splicing, and mitochondrial metabolism as key features defining the leukemic ELAVL1-mRNA interactome with mitochondrial import protein, TOMM34, being a direct ELAVL1-stabilized target whose repression impairs AML propagation. Altogether, using a stem cell-adapted in vivo CRISPR screen, this work demonstrates pervasive reliance on RBPs as regulators of LSCs and highlights their potential as therapeutic targets in AML., Significance: LSC-targeted therapies remain a significant unmet need in AML. We developed a stem-cell-adapted in vivo CRISPR screen to identify key LSC drivers. We uncover widespread RNA-binding protein dependencies in LSCs, including ELAVL1, which we identify as a novel therapeutic vulnerability through its regulation of mitochondrial metabolism. This article is highlighted in the In This Issue feature, p. 171., (©2023 The Authors; Published by the American Association for Cancer Research.)

Background: Juvenile localized scleroderma (JLS) or morphoea, a rare chronic autoimmune disease predominantly affects skin, subcutaneous tissue and occasionally the adjacent muscle, fascia and bone. We report the largest single-centre cohort of patients with JLS from India., Methods: Patients who were diagnosed to have JLS were enrolled from the Paediatric Dermatology Clinic and the Paediatric Rheumatology Clinic of a tertiary care referral hospital in India. Collected data included details of the clinical profile, laboratory investigations and management., Results: We analysed 84 patients with Juvenile localized scleroderma. Median age of disease onset was 5 years, and median age at diagnosis was 8 years. Commonest subtype was linear scleroderma (57 patients, 67.7%) followed by plaque morphoea and generalized morphoea. Fourteen patients (16.6%) were noted to have extracutaneous manifestations (ECMs). These included arthritis in eight (33.3%), brain parenchymal abnormalities in four (4.7%) and pulmonary involvement in two (8.3%) patients. Antinuclear antibody (ANA) was positive in eight/25 patients (32%; diffuse and speckled pattern in four patients each). One amongst these also had elevated anti-dsDNA titres. Positive ANA was found to have no association with ECMs (p 1.000). Patients were treated using methotrexate (61 patients; 72.6%), dexamethasone oral mini-pulse (OMP; 35 patients; 41.6%), calcipotriol (39 patients; 46.4%), topical corticosteroids (32 patients; 38%) and topical tacrolimus (three patients; 3.7%). Using linear regression analysis, administration of dexamethasone OMP and calcipotriol was found to be a predictor of good treatment response (p 0.034 and 0.019, respectively)., Conclusion: Early use of systemic corticosteroids along with methotrexate may be more beneficial than methotrexate therapy alone., (© 2022 European Academy of Dermatology and Venereology.)

Receptor-interacting serine/threonine protein kinase 4 (RIPK4) and its kinase substrate the transcription factor interferon regulatory factor 6 (IRF6) play critical roles in the development and maintenance of the epidermis. In addition, ourselves and others have previously shown that RIPK4 is a NOTCH target gene that suppresses the development of cutaneous and head and neck squamous cell carcinomas (HNSCCs). In this study, we used autochthonous mouse models, where the expression of Pik3ca H1047R oncogene predisposes the skin and oral cavity to tumor development, and show that not only loss of Ripk4 , but also loss of its kinase substrate Irf6 , triggers rapid SCC development. In vivo rescue experiments using Ripk4 or a kinase-dead Ripk4 mutant showed that the tumor suppressive function of Ripk4 is dependent on its kinase activity. To elucidate critical mediators of this tumor suppressive pathway, we performed transcriptional profiling of Ripk4 -deficient epidermal cells followed by multiplexed in vivo CRISPR screening to identify genes with tumor suppressive capabilities. We show that Elovl4 is a critical Notch-Ripk4-Irf6 downstream target gene, and that Elovl4 loss itself triggers SCC development. Importantly, overexpression of Elovl4 suppressed tumor growth of Ripk4 -deficient keratinocytes. Altogether, our work identifies a potent Notch1-Ripk4-Irf6-Elovl4 tumor suppressor axis.

Systematically investigating the scores of genes mutated in cancer and discerning disease drivers from inconsequential bystanders is a prerequisite for precision medicine but remains challenging. Here, we developed a somatic CRISPR/Cas9 mutagenesis screen to study 215 recurrent "long-tail" breast cancer genes, which revealed epigenetic regulation as a major tumor-suppressive mechanism. We report that components of the BAP1 and COMPASS-like complexes, including KMT2C/D, KDM6A, BAP1, and ASXL1/2 ("EpiDrivers"), cooperate with PIK3CAH1047R to transform mouse and human breast epithelial cells. Mechanistically, we find that activation of PIK3CAH1047R and concomitant EpiDriver loss triggered an alveolar-like lineage conversion of basal mammary epithelial cells and accelerated formation of luminal-like tumors, suggesting a basal origin for luminal tumors. EpiDriver mutations are found in ∼39% of human breast cancers, and ∼50% of ductal carcinoma in situ express casein, suggesting that lineage infidelity and alveogenic mimicry may significantly contribute to early steps of breast cancer etiology., Significance: Infrequently mutated genes comprise most of the mutational burden in breast tumors but are poorly understood. In vivo CRISPR screening identified functional tumor suppressors that converged on epigenetic regulation. Loss of epigenetic regulators accelerated tumorigenesis and revealed lineage infidelity and aberrant expression of alveogenesis genes as potential early events in tumorigenesis. This article is highlighted in the In This Issue feature, p. 2711., (©2022 American Association for Cancer Research.)

Establishing causal links between inherited polymorphisms and cancer risk is challenging. Here, we focus on the single-nucleotide polymorphism rs55705857, which confers a sixfold greater risk of isocitrate dehydrogenase ( IDH) -mutant low-grade glioma (LGG). We reveal that rs55705857 itself is the causal variant and is associated with molecular pathways that drive LGG. Mechanistically, we show that rs55705857 resides within a brain-specific enhancer, where the risk allele disrupts OCT2/4 binding, allowing increased interaction with the Myc promoter and increased Myc expression. Mutating the orthologous mouse rs55705857 locus accelerated tumor development in an Idh1 R132H -driven LGG mouse model from 472 to 172 days and increased penetrance from 30% to 75%. Our work reveals mechanisms of the heritable predisposition to lethal glioma in ~40% of LGG patients.

Coronaviruses have led to three major outbreaks to date-Severe Acute Respiratory Syndrome (SARS; 2002), Middle East Respiratory Syndrome (MERS; 2012) and the ongoing pandemic, Coronavirus Disease (COVID-19; 2019). Coronavirus infections are usually mild in children. However, a few children with MERS had presented with a severe phenotype in the acute phase resulting in progressive pneumonic changes with increasing oxygen dependency and acute respiratory distress requiring ventilatory support. A subset of children with a history of SARS-CoV-2 infection develops a multisystem hyper-inflammatory phenotype known as Multisystem Inflammatory Syndrome in Children (MIS-C). This syndrome occurs 4-6 weeks after infection with SARS-CoV-2 and has been reported more often from areas with high community transmission. Children with MIS-C present with high fever and often have involvement of cardiovascular, gastrointestinal and hematologic systems leading to multiorgan failure. This is accompanied by elevation of pro-inflammatory cytokines such as IL-6 and IL-10. MIS-C has several similarities with Kawasaki disease (KD) considering children with both conditions present with fever, rash, conjunctival injection, mucosal symptoms and swelling of hands and feet. For reasons that are still not clear, both KD and MIS-C were not reported during the SARS-CoV and MERS-CoV outbreaks. As SARS-CoV-2 differs from SARS-CoV by 19.5% and MERS by 50% in terms of sequence identity, differences in genomic and proteomic profiles may explain the varied disease immunopathology and host responses. Left untreated, MIS-C may lead to severe abdominal pain, ventricular dysfunction and shock. Immunological investigations reveal reduced numbers of follicular B cells, increased numbers of terminally differentiated CD4 + T lymphocytes, and decreased IL-17A. There is still ambiguity about the clinical and immunologic risk factors that predispose some children to development of MIS-C while sparing others. Host-pathogen interactions in SARS, MERS and COVID-19 are likely to play a crucial role in the clinical phenotypes that manifest. This narrative review focuses on the immunological basis for development of MIS-C syndrome in the ongoing SARS-CoV-2 pandemic. To the best of our knowledge, these aspects have not been reviewed before., 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 © 2022 Dhaliwal, Tyagi, Malhotra, Barman, Loganathan, Sharma, Sharma, Mondal, Rawat and Singh.)

We report a rare case of agranulocytosis and lymphopenia complicated with hemophagocytic lymphohistiocytosis. Diagnosis of reticular dysgenesis was made by detection of a pathogenic stop gain variant in the AK2 gene on targeted next generation sequencing and confirmed by Sanger sequencing. Parents were found to be carriers for this variant. Bone marrow aspirate and biopsy was also performed with a clinical diagnosis of severe combined immunodeficiency with HLH. However, no hemophagocytosis was noted in the bone marrow aspirate or trephine biopsy. Instead, it showed aggregates of large histiocyte-like cells, scattered erythroid precursors and megakaryocytes. These cells were confused to be some form of storage cells, but did not resemble storage cells seen in Gaucher's disease or Niemann Pick disease. Myeloid precursors were very few in number. Reticular dysgenesis was not suspected during admission due to a lack of awareness of this entity. Testing for sensorineural deafness in neonates with severe agranulocytosis and lymphopenia would facilitate an early diagnosis of reticular dysgenesis. To the best of our knowledge, hemophagocytic lymphohistiocytosis has not been previously reported in association with reticular dysgenesis., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Acute respiratory distress syndrome is a life-threatening complication of scrub typhus; we sought to describe its mortality and associated factors, namely eschar, hepatomegaly and severe thrombocytopenia, in a retrospective study in India of 65 children.

Background: Chronic granulomatous disease (CGD) is an inherited defect in phagocytic respiratory burst that results in severe and life-threatening infections in affected children. Single center studies from India have shown that proportion of autosomal recessive (AR) CGD is more than that reported from the West. Further, affected patients have high mortality rates due to late referrals and difficulties in accessing appropriate treatment. However, there is lack of multicentric collaborative data on CGD from India., Objective: To describe infection patterns, immunological, and molecular features of CGD from multiple centers in India., Methods: A detailed proforma that included clinical and laboratory details was prepared and sent to multiple centers in India that are involved in the care and management of patients with inborn errors of immunity. Twelve centers have provided data which were later pooled together and analyzed., Results: Of the 236 patients analyzed in our study, X-linked and AR-CGD was seen in 77 and 97, respectively. Male female ratio was 172:64. Median age at onset of symptoms and diagnosis was 8 and 24 months, respectively. Common infections documented include pneumonia (71.6%), lymphadenitis (31.6%), skin and subcutaneous abscess (23.7%), blood-stream infection (13.6%), osteomyelitis (8.6%), liver abscess (7.2%), lung abscess (2.9%), meningoencephalitis (2.5%), splenic abscess (1.7%), and brain abscess (0.9%). Forty-four patients (18.6%) had evidence of mycobacterial infection. Results of molecular assay were available for 141 patients (59.7%)- CYBB (44.7%) gene defect was most common, followed by NCF1 (31.9%), NCF2 (14.9%), and CYBA (8.5%). While CYBA variants were documented only in Southern and Western parts of India, a common dinucleotide deletion in NCF2 (c.835_836delAC) was noted only in North Indian population. Of the 174 patients with available outcome data, 67 (38.5%) had expired. Hematopoietic stem cell transplantation was carried out in 23 patients, and 12 are doing well on follow-up., Conclusions: In India, proportion of patients with AR-CGD is higher as compared to Western cohorts, though regional differences in types of AR-CGD exist. Clinical profile and mortality rates are similar in both X-linked and AR-CGD. However, this may be a reflection of the fact that milder forms of AR-CGD are probably being missed., 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 Rawat, Vignesh, Sudhakar, Sharma, Suri, Jindal, Gupta, Shandilya, Loganathan, Kaur, Chawla, Patra, Khadwal, Saikia, Minz, Aggarwal, Taur, Pandrowala, Gowri, Desai, Kulkarni, Hule, Bargir, Kambli, Madkaikar, Bhattad, Ginigeri, Kumar, Jayaram, Munirathnam, Sivasankaran, Raj, Uppuluri, Na, George, Lashkari, Kalra, Sachdeva, Seth, Sabui, Gupta, van Leeuwen, de Boer, Chan, Imai, Ohara, Nonoyama, Lau and Singh.)

Background: Chronic granulomatous disease (CGD) is an inherited defect in components of the nicotinamide adenine dinucleotide phosphate oxidase complex that results in potential life-threatening infective and noninfective complications. Hemophagocytic lymphohistiocytosis (HLH) is an unusual but important inflammatory complication of CGD. Optimal management strategies have not yet been identified in children with CGD who develop HLH., Objective: To analyze clinical and laboratory features of HLH in CGD from a tertiary-care center in North India., Methods: A retrospective review of medical records of children with CGD diagnosed in the last 20 years was performed. Clinical and laboratory features of children with CGD who developed HLH were analyzed., Results: Of 80 patients diagnosed with CGD, 5 (6.25%) had evidence of HLH. All 5 were males; 4 had X-linked CGD and 1 had autosomal recessive CGD (NCF2 defect). Two children with CGD had HLH as the predominant presenting manifestation mimicking the clinical presentation of congenital HLH. Infectious triggers identified were bloodstream infections (n = 3) (Candida albicans, Burkholderia cenocepacia, Francisella noatuensis), pneumonia (n = 4), and splenic abscess (n = 1). We document the first human infection with a fish pathogen, F. noatuensis, in a child with X-linked CGD. Although mortality was seen in 3 children who received only intravenous (IV) immunoglobulin therapy, the other 2 who received IV methylprednisolone pulse therapy survived., Conclusion: HLH can be a presenting manifestation of CGD, and workup for CGD must be considered in children with HLH. Early recognition with optimal management of both infectious trigger and HLH is very important to prevent mortality., (Copyright © 2020 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Genetically modified mouse models (GEMM) have been instrumental in assessing gene function, modeling human diseases, and serving as preclinical model to assess therapeutic avenues. However, their time-, labor- and cost-intensive nature limits their utility for systematic analysis of gene function. Recent advances in genome-editing technologies overcome those limitations and allow for the rapid generation of specific gene perturbations directly within specific mouse organs in a multiplexed and rapid manner. Here, we describe a CRISPR/Cas9-based method (Clustered Regularly Interspaced Short Palindromic Repeats) to generate thousands of gene knock-out clones within the epithelium of the skin and oral cavity of mice, and provide a protocol detailing the steps necessary to perform a direct in vivo CRISPR screen for tumor suppressor genes. This approach can be applied to other organs or other CRISPR/Cas9 technologies such as CRISPR-activation or CRISPR-inactivation to study the biological function of genes during tissue homeostasis or in various disease settings.

We have recently tested the transforming potential of 484 'long-tail' genes, which are recurrently albeit infrequently mutated in head and neck cancers (HNSCC). We identified 15 novel tumor suppressors and our top hits converge on regulating the NOTCH signaling pathway. Therapeutic approaches activating NOTCH signaling could be a promising strategy to treat two-thirds of human HNSCC patients., (© 2020 Taylor & Francis Group, LLC.)

In most human cancers, only a few genes are mutated at high frequencies; most are mutated at low frequencies. The functional consequences of these recurrent but infrequent "long tail" mutations are often unknown. We focused on 484 long tail genes in head and neck squamous cell carcinoma (HNSCC) and used in vivo CRISPR to screen for genes that, upon mutation, trigger tumor development in mice. Of the 15 tumor-suppressor genes identified, ADAM10 and AJUBA suppressed HNSCC in a haploinsufficient manner by promoting NOTCH receptor signaling. ADAM10 and AJUBA mutations or monoallelic loss occur in 28% of human HNSCC cases and are mutually exclusive with NOTCH receptor mutations. Our results show that oncogenic mutations in 67% of human HNSCC cases converge onto the NOTCH signaling pathway, making NOTCH inactivation a hallmark of HNSCC., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Two blinding corneal dystrophies, pediatric-onset congenital hereditary endothelial dystrophy (CHED) and some cases of late-onset Fuchs endothelial corneal dystrophy (FECD), are caused by SLC4A11 mutations. Three N-terminal SLC4A11 variants: v1, v2 and v3 are expressed in humans. We set out to determine which of these transcripts and what translated products, are present in corneal endothelium as these would be most relevant for CHED and FECD studies. Reverse transcription PCR (RT-PCR) and quantitative RT-PCR revealed only v2 and v3 mRNA in human cornea, but v2 was most abundant. Immunoblots probed with variant-specific antibodies revealed that v2 protein is about four times more abundant than v3 in human corneal endothelium. Bioinformatics and protein analysis using variant-specific antibodies revealed that second methionine in the open reading frame (M36) acts as translation initiation site on SLC4A11 v2 in human cornea. The v2 variants starting at M1 (v2-M1) and M36 (v2-M36) were indistinguishable in their cell surface trafficking and transport function (water flux). Structural homology models of v2-M36 and v3 suggest structural differences but their significance remains unclear. A combination of bioinformatics, RNA quantification and isoform-specific antibodies allows us to conclude that SLC4A11 variant 2 with start site M36 is predominant in corneal endothelium.

Rabies, a zoonotic viral encephalitis, continues to be a serious public health problem in India and several other countries in Asia and Africa. Survival is rarely reported in rabies, which is considered to be almost universally fatal. We report the clinical and radiological findings of eight patients with laboratory-confirmed rabies who survived the illness. With the exception of one patient who recovered with mild sequelae, all survivors had poor functional outcomes. The reported survival from rabies in recent years may reflect an increased awareness of the disease and greater access to better critical care facilities in rabies-endemic countries. Nonetheless, there is an urgent need to focus on preventive strategies to reduce the burden of this dreadful disease in rabies-endemic countries.

Purpose: Patients who have multiple sources of care are at risk for fragmented and uncoordinated care, which can lead to poorer outcomes. Veteran Medicare beneficiaries who use the Veterans Health Administration (VHA) system (VA users), particularly racial/ethnic minorities, often have complex medical conditions that may require care from multiple sources, leaving them especially vulnerable to the effects of fragmented care. We examined racial/ethnic differences in the level of satisfaction with care coordination among Medicare beneficiaries, comparing those who do and do not use the VHA healthcare system. Methods: We conducted a retrospective, pooled, cross-sectional study of Medicare beneficiaries using the 2009-2011 Medicare Current Beneficiary Survey. The outcomes are self-reported satisfaction with care items related to three dimensions of care coordination: (1) integrated care, (2) care continuity, and (3) follow-up care. We present descriptive statistics and use generalized linear models to examine racial/ethnic differences across VA and non-VA users, after accounting for other demographic characteristics, health status, functional limitations, insurance coverage, and geographic variation. Results: VA users are more likely to be very satisfied with receiving both integrated and follow-up care compared with non-VA users. Despite the existence of significant racial/ethnic disparities in the likelihood of being very satisfied with receiving well-coordinated care in the larger Medicare population, racial/ethnic minority VA users are just as likely as White non-Hispanics to be very satisfied with receiving well-coordinated care. Conclusions: Future research should continue to study care coordination among VA users and reasons for preferring the VA over other healthcare systems, especially among racial/ethnic minority groups., Competing Interests: No competing financial interests exist.

SLC4A11, a member of the SLC4 family of bicarbonate transporters, is a widely expressed integral membrane protein, abundant in kidney and cornea. Mutations of SLC4A11 cause some cases of the blinding corneal dystrophies, congenital hereditary endothelial dystrophy, and Fuchs endothelial corneal dystrophy. These diseases are marked by fluid accumulation in the corneal stroma, secondary to defective fluid reabsorption by the corneal endothelium. The role of SLC4A11 in these corneal dystrophies is not firmly established, as SLC4A11 function remains unclear. To clarify the normal function(s) of SLC4A11, we characterized the protein following expression in the simple, low-background expression system Xenopus laevis oocytes. Since plant and fungal SLC4A11 orthologs transport borate, we measured cell swelling associated with accumulation of solute borate. The plant water/borate transporter NIP5;1 manifested borate transport, whereas human SLC4A11 did not. SLC4A11 supported osmotically driven water accumulation that was electroneutral and Na + independent. Studies in oocytes and HEK293 cells could not detect Na + -coupled HCO 3 - transport or Cl - /HCO 3 - exchange by SLC4A11. SLC4A11 mediated electroneutral NH 3 transport in oocytes. Voltage-dependent OH - or H + movement was not measurable in SLC4A11-expressing oocytes, but SLC4A11-expressing HEK293 cells manifested low-level cytosolic acidification at baseline. In mammalian cells, but not oocytes, OH - /H + conductance may arise when SLC4A11 activates another protein or itself is activated by another protein. These data argue against a role of human SLC4A11 in bicarbonate or borate transport. This work provides additional support for water and ammonia transport by SLC4A11. When expressed in oocytes, SLC4A11 transported NH 3 , not NH 3 /H ., (Copyright © 2016 the American Physiological Society.)

Large cytoplasmic domains (CD) are a common feature among integral membrane proteins. In virtually all cases, these CD have a function (e.g., binding cytoskeleton or regulatory factors) separate from that of the membrane domain (MD). Strong associations between CD and MD are rare. Here we studied SLC4A11, a membrane transport protein of corneal endothelial cells, the mutations of which cause genetic corneal blindness. SLC4A11 has a 41-kDa CD and a 57-kDa integral MD. One disease-causing mutation in the CD, R125H, manifests a catalytic defect, suggesting a role of the CD in transport function. Expressed in HEK-293 cells without the CD, MD-SLC4A11 is retained in the endoplasmic reticulum, indicating a folding defect. Replacement of CD-SLC4A11 with green fluorescent protein did not rescue MD-SLC4A11, suggesting some specific role of CD-SLC4A11. Homology modeling revealed that the structure of CD-SLC4A11 is similar to that of the Cl(-)/HCO3(-) exchange protein AE1 (SLC4A1) CD. Fusion to CD-AE1 partially rescued MD-SLC4A11 to the cell surface, suggesting that the structure of CD-AE1 is similar to that of CD-SLC4A11. The CD-AE1-MD-SLC4a11 chimera, however, had no functional activity. We conclude that CD-SLC4A11 has an indispensable role in the transport function of SLC4A11. CD-SLC4A11 forms insoluble precipitates when expressed in bacteria, suggesting that the domain cannot fold properly when expressed alone. Consistent with a strong association between CD-SLC4A11 and MD-SLC4A11, these domains specifically associate when coexpressed in HEK-293 cells. We conclude that SLC4A11 is a rare integral membrane protein in which the CD has strong associations with the integral MD, which contributes to membrane transport function., (Copyright © 2016 the American Physiological Society.)

Purpose: Protein misfolding, causing retention of nascent protein in the endoplasmic reticulum (ER), is the most common molecular phenotype for disease alleles of membrane proteins. Strategies are needed to identify therapeutics able to correct such folding/trafficking defects. Mutations of SLC4A11, a plasma membrane transport protein of the human corneal endothelial cell layer, cause cases of congenital hereditary endothelial dystrophy, Harboyan syndrome, and Fuchs' endothelial corneal dystrophy. Most SLC4A11 mutations induce SLC4A11 misfolding and retention in the ER., Methods: An assay amenable to high-throughput screening was developed to quantify SLC4A11 at the plasma membrane, enabling a search for potential traffic-correcting small molecules. The assay was validated by comparing cell surface abundance of SLC4A11 mutants measured in the assay to observations from confocal immunofluorescence and values from cell surface biotinylation. Functionality of mutant proteins was assessed, using a confocal microscopic green fluorescent protein (GFP) water flux assay where relative rates of cell swelling are compared., Results: A small-scale screen revealed that the nonsteroidal anti-inflammatory drugs (NSAIDs), glafenine, ibuprofen, and acetylsalicylic acid dissolved in 0.2% dimethyl sulfoxide (DMSO), partially rescued the trafficking defect in some SLC4A11 mutants, expressed in HEK293 cells. These SLC4A11 mutants retained functional activity when rescued to the plasma membrane by glafenine treatment. Glafenine was effective with an EC50 of 1.5 ± 0.7 μM., Conclusions: These data suggest that glafenine, and perhaps other NSAIDs, hold potential as therapeutics for misfolded membrane proteins, like SLC4A11. The high throughput approach described here can be modified to identify correctors of other misfolded plasma membrane proteins that cause eye disease.

Aquaporin-1 (AQP1) enables greatly enhanced water flux across plasma membranes. The cytosolic carboxy terminus of AQP1 has two acidic motifs homologous to known carbonic anhydrase II (CAII) binding sequences. CAII colocalizes with AQP1 in the renal proximal tubule. Expression of AQP1 with CAII in Xenopus oocytes or mammalian cells increased water flux relative to AQP1 expression alone. This required the amino-terminal sequence of CAII, a region that binds other transport proteins. Expression of catalytically inactive CAII failed to increase water flux through AQP1. Proximity ligation assays revealed close association of CAII and AQP1, an effect requiring the second acidic cluster of AQP1. This motif was also necessary for CAII to increase AQP1-mediated water flux. Red blood cell ghosts resealed with CAII demonstrated increased osmotic water permeability compared with ghosts resealed with albumin. Water flux across renal cortical membrane vesicles, measured by stopped-flow light scattering, was reduced in CAII-deficient mice compared with wild-type mice. These data are consistent with CAII increasing water conductance through AQP1 by a physical interaction between the two proteins., (© 2015 Vilas, Krishnan, Loganathan, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

SLC4A11 mutations cause some cases of the corneal endothelial dystrophies, congenital hereditary endothelial corneal dystrophy type 2 (CHED2), Harboyan syndrome (HS), and Fuchs endothelial corneal dystrophy (FECD). SLC4A11 protein was recently identified as facilitating water flux across membranes. SLC4A11 point mutations usually cause SLC4A11 misfolding and retention in the endoplasmic reticulum (ER). We set about to test the feasibility of rescuing misfolded SLC4A11 protein to the plasma membrane as a therapeutic approach. Using a transfected HEK293 cell model, we measured functional activity present in cells expressing SLC4A11 variants in combinations representing the state found in CHED2 carriers, affected CHED2, FECD individuals, and unaffected individuals. These cells manifest respectively about 60%, 5%, and 25% of the water flux activity, relative to the unaffected (WT alone). ER-retained CHED2 mutant SLC4A11 protein could be rescued to the plasma membrane, where it conferred 25%-30% of WT water flux level. Further, some ER-retained CHED2 mutants expressed at 30°C supported increased water flux compared with 37°C cultures. Caspase activation and cell vitality assays revealed that expression of SLC4A11 mutants in HEK293 cells does not induce cell death. We conclude that therapeutics able to increase cell surface localization of ER-retained SLC4A11 mutants hold promise to treat CHED2 and FECD patients., (© 2014 WILEY PERIODICALS, INC.)

Late-onset Fuchs endothelial corneal dystrophy (FECD) shows genetic heterogeneity. Identification of SLC4A11 as a candidate gene for congenital hereditary endothelial dystrophy with similar corneal endothelial defects as FECD and reduced mRNA expression of SLC4A11 in the endothelium of FECD cases suggested that this gene may also be involved in pathogenesis of FECD. Mutations in SLC4A11 give rise to SLC4A11 protein marked by retention in the endoplasmic reticulum as a result of mis-folding. We screened 45 sporadic late-onset, 4 early-onset FECD patients and an early-onset autosomal dominant FECD family. We identified three previously unreported missense mutations: c.719G>C (p.W240S), c.1519G>A (p.V507I) and c.1304C>T (p.T434I) in unrelated individuals. These SLC4A11 mutants, expressed in HEK293 cells, had defects in either their cell surface expression or functional activity (rate of osmotically driven water flux). SLC4A11 mutations contribute to 11% (5/45) of sporadic late-onset FECD in the cohort studied. COL8A2, which causes some cases of early-onset FECD, was also screened in this cohort. No mutations were identified in COL8A2, in neither the late-onset cohort nor the early-onset family, suggesting genetic heterogeneity in this FECD family.

Three genetic corneal dystrophies [congenital hereditary endothelial dystrophy type 2 (CHED2), Harboyan syndrome and Fuchs endothelial corneal dystrophy] arise from mutations of the SLC4a11 gene, which cause blindness from fluid accumulation in the corneal stroma. Selective transmembrane water conductance controls cell size, renal fluid reabsorption and cell division. All known water-channelling proteins belong to the major intrinsic protein family, exemplified by aquaporins (AQPs). Here we identified SLC4A11, a member of the solute carrier family 4 of bicarbonate transporters, as an unexpected addition to known transmembrane water movement facilitators. The rate of osmotic-gradient driven cell-swelling was monitored in Xenopus laevis oocytes and HEK293 cells, expressing human AQP1, NIP5;1 (a water channel protein from plant), hCNT3 (a human nucleoside transporter) and human SLC4A11. hCNT3-expressing cells swelled no faster than control cells, whereas SLC4A11-mediated water permeation at a rate about half that of some AQP proteins. SLC4A11-mediated water movement was: (i) similar to some AQPs in rate; (ii) uncoupled from solute-flux; (iii) inhibited by stilbene disulfonates (classical SLC4 inhibitors); (iv) inactivated in one CHED2 mutant (R125H). Localization of AQP1 and SLC4A11 in human and murine corneal (apical and basolateral, respectively) suggests a cooperative role in mediating trans-endothelial water reabsorption. Slc4a11(-/-) mice manifest corneal oedema and distorted endothelial cells, consistent with loss of a water-flux. Observed water-flux through SLC4A11 extends the repertoire of known water movement pathways and call for a re-examination of explanations for water movement in human tissues.

We recently reported a novel form of BMP2, designated nBMP2, which is translated from an alternative downstream start codon and is localized to the nucleus rather than secreted from the cell. To examine the function of nBMP2 in the nucleus, we engineered a gene-targeted mutant mouse model (nBmp2NLS(tm)) in which nBMP2 cannot be translocated to the nucleus. Immunohistochemistry demonstrated the presence of nBMP2 staining in the myonuclei of wild type but not mutant skeletal muscle. The nBmp2NLS(tm) mouse exhibits altered function of skeletal muscle as demonstrated by a significant increase in the time required for relaxation following a stimulated twitch contraction. Force frequency analysis showed elevated force production in mutant muscles compared to controls from 10 to 60 Hz stimulation frequency, consistent with the mutant muscle's reduced ability to relax between rapidly stimulated contractions. Muscle relaxation after contraction is mediated by the active transport of Ca(2+) from the cytoplasm to the sarcoplasmic reticulum by sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA), and enzyme activity assays revealed that SERCA activity in skeletal muscle from nBmp2NLS(tm) mice was reduced to approximately 80% of wild type. These results suggest that nBMP2 plays a role in the establishment or maintenance of intracellular Ca(2+) transport pathways in skeletal muscle.

Mutations in the SLC4A11 gene, which encodes a plasma membrane borate transporter, cause recessive congenital hereditary endothelial corneal dystrophy type 2 (CHED2), corneal dystrophy and perceptive deafness (Harboyan syndrome), and dominant late-onset Fuchs endothelial corneal dystrophy (FECD). We analyzed missense SLC4A11 mutations identified in FECD and CHED2 patients and expressed in transfected HEK 293 cells. Chemical cross-linking and migration in nondenaturing gels showed that SLC4A11 exists as a dimer. Furthermore, co-immunoprecipitation of epitope-tagged proteins revealed heteromeric interactions between wild-type (WT) and mutant SLC4A11 proteins. When expressed alone, FECD- and CHED2-causing mutant SLC4A11 proteins are primarily retained intracellularly. Co-expression with WT SLC4A11 partially rescued the cell surface trafficking of CHED2 mutants, but not FECD mutants. CHED2 alleles of SLC4A11 did not affect cell surface processing of WT SLC4A11. In contrast, FECD mutants reduced WT cell surface processing efficiency, consistent with dominant inheritance of FECD. The reduction in movement of WT protein to the cell surface caused by FECD SLC4A11 helps to explain the dominant inheritance of this disorder. Similarly, the failure of CHED2 mutant SLC4A11 to affect the processing of WT protein, explains the lack of symptoms found in CHED2 carriers and the recessive inheritance of the disorder., (© 2011 Wiley Periodicals, Inc.)

Mutations in the SLC4A11 protein, reported as a sodium-coup-led borate transporter of the human plasma membrane, are responsible for three corneal dystrophies (CD): congenital hereditary endothelial dystrophy type 2, Harboyan syndrome, and late-onset Fuch's CD. To develop a rational basis to understand these diseases, whose point mutations are found throughout the SLC4A11 sequence, we analyzed the protein biochemically. Hydropathy analysis and an existing topology model for SLC4A1 (AE1), a bicarbonate transporter with the lowest evolutionary sequence divergence from SLC4A11, formed the basis to propose an SLC4A11 topology model. Immunofluorescence studies revealed the cytosolic orientation of N- and C-termini of SLC4A11. Limited trypsinolysis of SLC4A11 partially mapped the folding of the membrane and cytoplasmic domains of the protein. The binding of SLC4A11 to a stilbenedisulfonate inhibitor resin (SITS-Affi-Gel) was prevented by preincubation with H(2)DIDS, with a significantly higher half-maximal effective concentration than AE1. We conclude that stilbenedisulfonates interact with SLC4A11 but with a lower affinity than other SLC4 proteins. Disease-causing mutants divided into two classes on the basis of the half-maximal [H(2)DIDS] required for resin displacement and the fraction of protein binding H(2)DIDS, likely representing mildly misfolded and grossly misfolded proteins. Disease-causing SLC4A11 mutants are retained in the endoplasmic reticulum of HEK 293 cells. This phenotype could be partially rescued in some cases by growing the cells at 30 °C.