Search

Abstract SPOUT1/CENP-32 encodes a putative SPOUT RNA methyltransferase previously identified as a mitotic chromosome associated protein. SPOUT1/CENP-32 depletion leads to centrosome detachment from the spindle poles and chromosome misalignment. Aided by gene matching platforms, here we identify 28 individuals with neurodevelopmental delays from 21 families with bi-allelic variants in SPOUT1/CENP-32 detected by exome/genome sequencing. Zebrafish spout1/cenp-32 mutants show reduction in larval head size with concomitant apoptosis likely associated with altered cell cycle progression. In vivo complementation assays in zebrafish indicate that SPOUT1/CENP-32 missense variants identified in humans are pathogenic. Crystal structure analysis of SPOUT1/CENP-32 reveals that most disease-associated missense variants are located within the catalytic domain. Additionally, SPOUT1/CENP-32 recurrent missense variants show reduced methyltransferase activity in vitro and compromised centrosome tethering to the spindle poles in human cells. Thus, SPOUT1/CENP-32 pathogenic variants cause an autosomal recessive neurodevelopmental disorder: SpADMiSS (SPOUT1 Associated Development delay Microcephaly Seizures Short stature) underpinned by mitotic spindle organization defects and consequent chromosome segregation errors.

Abstract HPV-positive and HPV-negative head and neck squamous cell carcinoma (HNSCC) are recognized as distinct entities. There remains uncertainty surrounding the causal effects of smoking and alcohol on the development of these two cancer types. Here we perform multivariable Mendelian randomization (MR) to evaluate the causal effects of smoking and alcohol on the risk of HPV-positive and HPV-negative HNSCC in 3431 cases and 3469 controls. Lifetime smoking exposure, as measured by the Comprehensive Smoking Index (CSI), is associated with increased risk of both HPV-negative HNSCC (OR = 3.03, 95%CI:1.75-5.24, P = 7.00E-05) and HPV-positive HNSCC (OR = 2.73, 95%CI:1.39-5.36, P = 0.003). Drinks Per Week is also linked with increased risk of both HPV-negative HNSCC (OR = 7.72, 95%CI:3.63-16.4, P = 1.00E-07) and HPV-positive HNSCC (OR = 2.66, 95%CI:1.06-6.68, P = 0.038). Smoking and alcohol independently increase the risk of both HPV-positive and HPV-negative HNSCC. These findings have important implications for understanding the modifying risk factors between HNSCC subtypes.

ABSTRACT Background The main technique for identification of free flap perforator vessels is Doppler sonography, which is not always accurate, user dependent and affected by the patient's body habitus. Methods Adult patients undergoing head and neck resection and free flap reconstruction at two academic institutions were enrolled. Doppler sonography was used to identify perforators, and were marked using a skin marker. The donor site was cooled down for 3 min using a sterile iced saline bag. FLIR‐ONE (FLIR Systems Inc., Wilsonville, OR) camera was used to assess for “hot spots” during a 3–5 min period of re‐warming as a surrogate for cutaneous blood flow. The distance between the Doppler signal location, and the “hot spot” was recorded. The position of the perforator was then identified intraoperatively and the distances between the surgical position, the Doppler and “hot spot” were recorded. Results A total of 28 patients were included. For all flap types, FLIR thermal imaging measurements consistently tended to be closer to the surgical site compared to Doppler ultrasound. In anterolateral thigh flaps (n = 20), thoracodorsal artery perforator flaps (n = 5), and fibula osteocutaneous flaps (n = 3), absolute mean differences ranged from 0.62 to 1.33 cm, with trends favoring FLIR. While paired t‐tests did not reach statistical significance, both methods correlated with intraoperatively identified skin perforators, and distances generally ranged between 0 and 2 cm. Conclusion We demonstrate that a smartphone‐based thermal imaging system has the potential to serve as an adjunct for identifying flap perforators, with the possibility of reducing operative times and minimizing patient morbidity. Level of Evidence Level 3.

Abstract Background Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting over 300,000 people worldwide. It is characterized by the progressive decline of the nervous system that leads to the weakening of muscles which impacts physical function. Approximately, 15% of individuals diagnosed with ALS have a known genetic variant that contributes to their disease. As therapies that slow or prevent symptoms continue to develop, such as antisense oligonucleotides, it is important to discover novel genes that could be targets for treatment. Additionally, as cohorts continue to grow, performing analyses in ALS subtypes, such as primary lateral sclerosis (PLS), becomes possible due to an increase in power. These analyses could highlight novel pathways in disease manifestation. Methods Building on our previous discoveries using rare variant association analyses, we conducted rare variant burden testing on a substantially larger multi-ethnic cohort of 6,970 ALS patients, 166 PLS patients, and 22,524 controls. We used intolerant domain percentiles based on sub-region Residual Variation Intolerance Score (subRVIS) that have been described previously in conjunction with gene based collapsing approaches to conduct burden testing to identify genes that associate with ALS and PLS. Results A gene based collapsing model showed significant associations with SOD1, TARDBP, and TBK1 (OR = 19.18, p = 3.67 × 10–39; OR = 4.73, p = 2 × 10–10; OR = 2.3, p = 7.49 × 10–9, respectively). These genes have been previously associated with ALS. Additionally, a significant novel control enriched gene, ALKBH3 (p = 4.88 × 10–7), was protective for ALS in this model. An intolerant domain-based collapsing model showed a significant improvement in identifying regions in TARDBP that associated with ALS (OR = 10.08, p = 3.62 × 10–16). Our PLS protein truncating variant collapsing analysis demonstrated significant case enrichment in ANTXR2 (p = 8.38 × 10–6). Conclusions In a large multi-ethnic cohort of 6,970 ALS patients, collapsing analyses validated known ALS genes and identified a novel potentially protective gene, ALKBH3. A first-ever analysis in 166 patients with PLS found a candidate association with loss-of-function mutations in ANTXR2.

Abstract Post-infectious myalgic encephalomyelitis/chronic fatigue syndrome (PI-ME/CFS) is a disabling disorder, yet the clinical phenotype is poorly defined, the pathophysiology is unknown, and no disease-modifying treatments are available. We used rigorous criteria to recruit PI-ME/CFS participants with matched controls to conduct deep phenotyping. Among the many physical and cognitive complaints, one defining feature of PI-ME/CFS was an alteration of effort preference, rather than physical or central fatigue, due to dysfunction of integrative brain regions potentially associated with central catechol pathway dysregulation, with consequences on autonomic functioning and physical conditioning. Immune profiling suggested chronic antigenic stimulation with increase in naïve and decrease in switched memory B-cells. Alterations in gene expression profiles of peripheral blood mononuclear cells and metabolic pathways were consistent with cellular phenotypic studies and demonstrated differences according to sex. Together these clinical abnormalities and biomarker differences provide unique insight into the underlying pathophysiology of PI-ME/CFS, which may guide future intervention.

Abstract Exome sequencing (ES) has been used in a variety of clinical settings but there are limited data on its utility for diagnosis and/or prediction of monogenic liver diseases. We developed a curated list of 502 genes for monogenic disorders associated with liver phenotypes and analyzed ES data for these genes in 758 patients with chronic liver diseases (CLD). For comparison, we examined ES data in 7856 self-declared healthy controls (HC), and 2187 patients with chronic kidney disease (CKD). Candidate pathogenic (P) or likely pathogenic (LP) variants were initially identified in 19.9% of participants, most of which were attributable to previously reported pathogenic variants with implausibly high allele frequencies. After variant annotation and filtering based on population minor allele frequency (MAF ≤ 10–4 for dominant disorders and MAF ≤ 10–3 for recessive disorders), we detected a significant enrichment of P/LP variants in the CLD cohort compared to the HC cohort (X 2 test OR 5.00, 95% CI 3.06–8.18, p value = 4.5e−12). A second-level manual annotation was necessary to capture true pathogenic variants that were removed by stringent allele frequency and quality filters. After these sequential steps, the diagnostic rate of monogenic disorders was 5.7% in the CLD cohort, attributable to P/LP variants in 25 genes. We also identified concordant liver disease phenotypes for 15/22 kidney disease patients with P/LP variants in liver genes, mostly associated with cystic liver disease phenotypes. Sequencing results had many implications for clinical management, including familial testing for early diagnosis and management, preventative screening for associated comorbidities, and in some cases for therapy. Exome sequencing provided a 5.7% diagnostic rate in CLD patients and required multiple rounds of review to reduce both false positive and false negative findings. The identification of concordant phenotypes in many patients with P/LP variants and no known liver disease also indicates a potential for predictive testing for selected monogenic liver disorders.

Abstract African Americans have a significantly higher risk of developing chronic kidney disease, especially focal segmental glomerulosclerosis -, than European Americans. Two coding variants (G1 and G2) in the APOL1 gene play a major role in this disparity. While 13% of African Americans carry the high-risk recessive genotypes, only a fraction of these individuals develops FSGS or kidney failure, indicating the involvement of additional disease modifiers. Here, we show that the presence of the APOL1 p.N264K missense variant, when co-inherited with the G2 APOL1 risk allele, substantially reduces the penetrance of the G1G2 and G2G2 high-risk genotypes by rendering these genotypes low-risk. These results align with prior functional evidence showing that the p.N264K variant reduces the toxicity of the APOL1 high-risk alleles. These findings have important implications for our understanding of the mechanisms of APOL1-associated nephropathy, as well as for the clinical management of individuals with high-risk genotypes that include the G2 allele.

Abstract The mechanisms that prevent regeneration of irradiated (IR) salivary glands remain elusive. Bulk RNAseq of IR versus non-IR human salivary glands showed that neurotrophin signaling is highly disrupted post-radiation. Neurotrophin receptors (NTRs) were significantly upregulated in myoepithelial cells (MECs) post-IR, and single cell RNAseq revealed that MECs pericytes, and duct cells are the main sources of neurotrophin ligands. Using two ex vivo models, we show that nerve growth factor (NGF) induces expression of MEC genes during development, and upregulation of NTRs in adult MECs is associated with stress-induced plasticity and morphological abnormalities in IR human glands. As MECs are epithelial progenitors after gland damage and are required for proper acinar cell contraction and secretion, we propose that MEC-specific upregulation of NTRs post-IR disrupts MEC differentiation and potentially impedes the ability of the gland to regenerate.

Heterozygous de novo loss-of-function mutations in the gene expression regulator HNRNPU cause an early-onset developmental and epileptic encephalopathy. To gain insight into pathological mechanisms and lay the potential groundwork for developing targeted therapies, we characterized the neurophysiologic and cell-type-specific transcriptomic consequences of a mouse model of HNRNPU haploinsufficiency. Heterozygous mutants demonstrated global developmental delay, impaired ultrasonic vocalizations, cognitive dysfunction and increased seizure susceptibility, thus modeling aspects of the human disease. Single-cell RNA-sequencing of hippocampal and neocortical cells revealed widespread, yet modest, dysregulation of gene expression across mutant neuronal subtypes. We observed an increased burden of differentially-expressed genes in mutant excitatory neurons of the subiculum-a region of the hippocampus implicated in temporal lobe epilepsy. Evaluation of transcriptomic signature reversal as a therapeutic strategy highlights the potential importance of generating cell-type-specific signatures. Overall, this work provides insight into HNRNPU-mediated disease mechanisms and provides a framework for using single-cell RNA-sequencing to study transcriptional regulators implicated in disease.

HNRNPU is an RNA splicing protein associated with brain disorders such as early onset seizures. Here they show that HNRNPU functions to maintain neural progenitors and their progeny by regulating splicing of key neuronal genes.

Abstract Background Parkinson’s disease (PD) is characterized by intra-neuronal deposition of the protein α-synuclein (α-syn) and by deficiencies of the catecholamines dopamine and norepinephrine (NE) in the brain and heart. Accumulation of α-syn in sympathetic noradrenergic nerves may provide a useful PD biomarker; however, whether α-syn buildup is pathophysiological has been unclear. If it were, one would expect associations of intra-neuronal α-syn deposition with catecholaminergic denervation and with decreased NE contents in the same samples. Methods We assayed immunoreactive α-syn and tyrosine hydroxylase (TH, a marker of catecholaminergic innervation) concurrently with catecholamines in coded post-mortem scalp skin, submandibular gland (SMG), and apical left ventricular myocardial tissue samples from 14 patients with autopsy-proven PD and 12 age-matched control subjects who did not have a neurodegenerative disease. Results The PD group had increased α-syn in sympathetic noradrenergically innervated arrector pili muscles (5.7 times control, P

De novo mutations in GNB1, encoding the Gβ1 subunit of G proteins, cause a neurodevelopmental disorder with global developmental delay and epilepsy, GNB1 encephalopathy. Here, we show that mice carrying a pathogenic mutation, K78R, recapitulate aspects of the disorder, including developmental delay and generalized seizures. Cultured mutant cortical neurons also display aberrant bursting activity on multi-electrode arrays. Strikingly, the antiepileptic drug ethosuximide (ETX) restores normal neuronal network behavior in vitro and suppresses spike-and-wave discharges (SWD) in vivo. ETX is a known blocker of T-type voltage-gated Ca2+ channels and G protein-coupled potassium (GIRK) channels. Accordingly, we present evidence that K78R results in a gain-of-function (GoF) effect by increasing the activation of GIRK channels in cultured neurons and a heterologous model (Xenopus oocytes)—an effect we show can be potently inhibited by ETX. This work implicates a GoF mechanism for GIRK channels in epilepsy, identifies a new mechanism of action for ETX in preventing seizures, and establishes this mouse model as a pre-clinical tool for translational research with predicative value for GNB1 encephalopathy.

Abstract Background Report the incidence of contralateral nodal failure rates in well-lateralized oropharyngeal carcinoma treated with upfront surgery and unilateral neck management. Methods Lateralized oropharyngeal carcinomas treated with upfront surgery using transoral robotic surgery (TORS) and unilateral neck management (unilateral neck dissection ± unilateral radiation treatment) were identified. Primary endpoint was contralateral regional control (CRC). Secondary endpoints were local control (LC), and overall survival (OS). Results Thirty-two patients were included. Pathologic T categories included 66% pT1, 31% pT2 and 3% pT3. Nodal diseases comprised 41% N0 and 47% N1 (AJCC 8th). Twenty-three (72%) patients had HPV related tumors. 3-years CRC, LC and OS were 100%, 96% (89–100) and 96% (CI 89–100). One patient developed a second primary with contralateral nodal disease. Only one patient died from another primary cancer. Conclusion In selected patients with lateralized oropharyngeal cancer, treatment with TORS and ipsilateral management of the neck may be oncologically safe without significant risk of contralateral failure. Level of evidence: Level 2. Graphical abstract

Abstract Purpose Drug development strategies for genetic diseases depend critically on accurate knowledge of how pathogenic variants cause disease. For some well‐studied genes, the direct effects of pathogenic variants are well documented as loss‐of‐function, gain‐of‐function or hypermorphic, or a combination of the two. For many genes, however, even the direction of effect of variants remains unclear. Classification of Mendelian disease genes in terms of whether pathogenic variants are loss‐ or gain‐of‐function would directly inform drug development strategies. Methods We leveraged the recent dramatic increase in reported pathogenic variants to provide a novel approach to inferring the direction of effect of pathogenic variants. Specifically, we quantify the ratio of reported pathogenic variants that are missense compared to loss‐of‐function. Results We first show that for many genes that cause dominant Mendelian disease, the ratio of reported pathogenic missense variants is diagnostic of whether the gene causes disease through loss‐ or gain‐of‐function, or a combination. Second, we identify a set of genes that appear to cause disease largely or entirely through gain‐of‐function or hypermorphic pathogenic variants. Conclusions We suggest a set of 16 genes suitable for drug developmental efforts utilizing direct inhibition.

Summary: Generating effective therapies for neurodevelopmental disorders has remained elusive. An emerging drug discovery approach for neurodevelopmental disorders is to characterize transcriptome-wide dysregulation in an appropriate model system and screen therapeutics based on their capacity to restore functionally relevant expression patterns. We characterized transcriptomic dysregulation in a human model of HNRNPU-related disorder to explore the potential of such a paradigm. We identified widespread dysregulation in functionally relevant pathways and then compared dysregulation in a human model to transcriptomic differences in embryonic and perinatal mice to determine whether dysregulation in an in vitro human model is partially replicated in an in vivo model of HNRNPU-related disorder. Strikingly, we find enrichment of co-dysregulation between 45-day-old human organoids and embryonic, but not perinatal, mice from distinct models of HNRNPU-related disorder. Thus, hnRNPU deficient human organoids may only be suitable to model transcriptional dysregulation in certain cell types within a specific developmental time window.

Abstract Purpose Patients undergoing head and neck surgery are often elderly and frail with significant comorbidities. Discussion and documentation of what patients would desire for end‐of‐life care and decision‐making is, therefore, essential for delivering patient‐centered care. Materials and Methods This was a retrospective, cross‐sectional study of patients undergoing surgery for head and neck cancer at two large, academic, tertiary care centers in Canada. Advance care planning was defined as any documentation of advance directives, resuscitation orders, or end‐of‐life care preferences. Results Among 301 patients, advance care planning was documented for 31 (10.3%). Patients with locally advanced disease (T3+) were twice as likely to have advance care planning documentation compared to those with early disease (RR 1.97, 95%CI [0.98, 3.97]). Conclusions In this multi‐institutional cross‐sectional study of two large academic centers, we have demonstrated that advance care planning and documentation is overall poor in patients undergoing surgery for oral cancer. These findings may have health policy implications, as advance care planning is associated with increased patient and provider satisfaction and improved alignment of patient goals and care delivered. Future work will investigate barriers and facilitators to advance care‐planning documentation in this setting.

Abstract Active surveillance (AS) of small, low-risk papillary thyroid cancers (PTCs) is increasingly studied in prospective observational studies. Ultrasound is the primary imaging modality for case selection. While researchers have put forward selection criteria for PTCs based on size, absence of suspicious lymph nodes and tumor location, there are limited reported data highlighting inherent ultrasound limitations and guidelines for case selection and follow-up. We report our experience including imaging limitations encountered in the ongoing AS prospective observational study for PTCs measuring 3 mm in the largest dimension of nodule or evidence of metastatic disease or extrathyroidal extension. Accurate, consistent and reproducible measurements of PTCs are essential in risk stratifying patients for the option of AS or disease progression. Interobserver discrepancy, shadowing from coarse calcification and background parenchyma heterogeneity or thyroiditis can limit accurate PTC size assessment and therefore hinder patient eligibility evaluation or AS follow-up. Following the ACR Thyroid Imaging, Reporting and Data System (TI-RADS) protocol of three-axes technique to measure a thyroid nodule enables reproducibility of measurements. In patients with multi-nodular goiter, accurate identification and labeling of the PTC is important to avoid mistaking with adjacent benign nodules at follow-up. Ultrasound assessment for extrathyroid extension of PTC, and relationship of PTC to trachea and the anatomic course of the recurrent laryngeal nerve are important considerations in evaluation for AS eligibility.

Host genetics is a key determinant of COVID-19 outcomes. Previously, the COVID-19 Host Genetics Initiative genome-wide association study used common variants to identify multiple loci associated with COVID-19 outcomes. However, variants with the largest impact on COVID-19 outcomes are expected to be rare in the population. Hence, studying rare variants may provide additional insights into disease susceptibility and pathogenesis, thereby informing therapeutics development. Here, we combined whole-exome and whole-genome sequencing from 21 cohorts across 12 countries and performed rare variant exome-wide burden analyses for COVID-19 outcomes. In an analysis of 5,085 severe disease cases and 571,737 controls, we observed that carrying a rare deleterious variant in the SARS-CoV-2 sensor toll-like receptor TLR7 (on chromosome X) was associated with a 5.3-fold increase in severe disease (95% CI: 2.75-10.05, p = 5.41x10-7). This association was consistent across sexes. These results further support TLR7 as a genetic determinant of severe disease and suggest that larger studies on rare variants influencing COVID-19 outcomes could provide additional insights.

Abstract Rare monogenic disorders often share molecular etiologies involved in the pathogenesis of common diseases. Congenital disorders of glycosylation (CDG) and deglycosylation (CDDG) are rare pediatric disorders with symptoms that range from mild to life threatening. A biological mechanism shared among CDG and CDDG as well as more common neurodegenerative diseases such as Alzheimer’s disease and amyotrophic lateral sclerosis, is endoplasmic reticulum (ER) stress. We developed isogenic human cellular models of two types of CDG and the only known CDDG to discover drugs that can alleviate ER stress. Systematic phenotyping confirmed ER stress and identified elevated autophagy among other phenotypes in each model. We screened 1049 compounds and scored their ability to correct aberrant morphology in each model using an agnostic cell-painting assay based on >300 cellular features. This primary screen identified multiple compounds able to correct morphological phenotypes. Independent validation shows they also correct cellular phenotypes and alleviate each of the ER stress markers identified in each model. Many of the active compounds are associated with microtubule dynamics, which points to new therapeutic opportunities for both rare and more common disorders presenting with ER stress, such as Alzheimer’s disease and amyotrophic lateral sclerosis.

Abstract Background Therapeutic targeting of host-cell factors required for SARS-CoV-2 entry is an alternative strategy to ameliorate COVID-19 severity. SARS-CoV-2 entry into lung epithelium requires the TMPRSS2 cell surface protease. Pre-clinical and correlative data in humans suggest that anti-androgenic therapies can reduce the expression of TMPRSS2 on lung epithelium. Accordingly, we hypothesize that therapeutic targeting of androgen receptor signaling via degarelix, a luteinizing hormone-releasing hormone (LHRH) antagonist, will suppress COVID-19 infection and ameliorate symptom severity. Methods This is a randomized phase 2, placebo-controlled, double-blind clinical trial in 198 patients to compare efficacy of degarelix plus best supportive care versus placebo plus best supportive care on improving the clinical outcomes of male Veterans who have been hospitalized due to COVID-19. Enrolled patients must have documented infection with SARS-CoV-2 based on a positive reverse transcriptase polymerase chain reaction result performed on a nasopharyngeal swab and have a severity of illness of level 3–5 (hospitalized but not requiring invasive mechanical ventilation). Patients stratified by age, history of hypertension, and severity are centrally randomized 2:1 (degarelix: placebo). The composite primary endpoint is mortality, ongoing need for hospitalization, or requirement for mechanical ventilation at 15 after randomization. Important secondary endpoints include time to clinical improvement, inpatient mortality, length of hospitalization, duration of mechanical ventilation, time to achieve a normal temperature, and the maximum severity of COVID-19 illness. Exploratory analyses aim to assess the association of cytokines, viral load, and various comorbidities with outcome. In addition, TMPRSS2 expression in target tissue and development of anti-viral antibodies will also be investigated. Discussion In this trial, we repurpose the FDA approved LHRH antagonist degarelix, commonly used for prostate cancer, to suppress TMPRSS2, a host cell surface protease required for SARS-CoV-2 cell entry. The objective is to determine if temporary androgen suppression with a single dose of degarelix improves the clinical outcomes of patients hospitalized due to COVID-19. Trial registration ClinicalTrials.gov NCT04397718. Registered on May 21, 2020

Abstract Background A common approach for sequencing studies is to do joint-calling and store variants of all samples in a single file. If new samples are continually added or controls are re-used for several studies, the cost and time required to perform joint-calling for each analysis can become prohibitive. Results We present ATAV, an analysis platform for large-scale whole-exome and whole-genome sequencing projects. ATAV stores variant and per site coverage data for all samples in a centralized database, which is efficiently queried by ATAV to support diagnostic analyses for trios and singletons, as well as rare-variant collapsing analyses for finding disease associations in complex diseases. Runtime logs ensure full reproducibility and the modularized ATAV framework makes it extensible to continuous development. Besides helping with the identification of disease-causing variants for a range of diseases, ATAV has also enabled the discovery of disease-genes by rare-variant collapsing on datasets containing more than 20,000 samples. Analyses to date have been performed on data of more than 110,000 individuals demonstrating the scalability of the framework. To allow users to easily access variant-level data directly from the database, we provide a web-based interface, the ATAV data browser ( http://atavdb.org/ ). Through this browser, summary-level data for more than 40,000 samples can be queried by the general public representing a mix of cases and controls of diverse ancestries. Users have access to phenotype categories of variant carriers, as well as predicted ancestry, gender, and quality metrics. In contrast to many other platforms, the data browser is able to show data of newly-added samples in real-time and therefore evolves rapidly as more and more samples are sequenced. Conclusions Through ATAV, users have public access to one of the largest variant databases for patients sequenced at a tertiary care center and can look up any genes or variants of interest. Additionally, since the entire code is freely available on GitHub, ATAV can easily be deployed by other groups that wish to build their own platform, database, and user interface.

Abstract Background Out‐of‐pocket costs (OOPC) associated with treatment have significant implications on quality of life and survival in cancer patients. Head and neck cancer patients face unique treatment‐related challenges, but to date OOPC have been understudied in this population. Aims This study aims to identify and measure OOPC for patients with head and neck cancer (HNC) in Ontario. Methods HNC patients between 2015 and 2018 at Princess Margaret Cancer Centre in Toronto were recruited. Participants completed OOPC questionnaires and lost income questions during radiation, post‐surgery, and 3, 6, 12, and 24 months after completion of treatment. Associations between OOPC and treatment modality and disease site were tested with multivariable hurdle regression. Results A total of 1545 questionnaires were completed by 657 patients. Median estimated OOPC for the total duration of treatment for participants undergoing chemoradiation was $1452 [$0–14 616], for surgery with adjuvant radiation or chemoradiation (C/RT) was $1626, for radiation therapy alone was $635, and for surgery alone was $360. The major expenses for participants at the mid‐treatment time‐point was travel (mean $424, standard error of the mean [SEM] $34) and meals, parking, and accommodations (mean $617, SEM $67). In multivariable analysis, chemoradiation, surgery with C/RT, and radiation were associated with significantly higher OOPC than surgery alone during treatment (791% higher, p

Background Lewy body diseases (LBDs) feature deficiency of the sympathetic neurotransmitter norepinephrine in the left ventricular myocardium and sympathetic intra‐neuronal deposition of the protein alpha‐synuclein (αS). LBDs therefore are autonomic synucleinopathies. Computational modeling has revealed multiple functional abnormalities in residual myocardial sympathetic noradrenergic nerves in LBDs, including decreased norepinephrine synthesis, vesicular storage, and recycling. We report an extended model that enables predictions about the progression of LBDs and effects of genetic predispositions and treatments on that progression. Methods and Results The model combines cardiac sympathetic activation with autotoxicity mediated by the dopamine metabolite 3,4‐dihydroxyphenylacetaldehyde. We tested the model by its ability to predict longitudinal empirical data based on cardiac sympathetic neuroimaging, effects of genetic variations related to particular intra‐neuronal reactions, treatment by monoamine oxidase inhibition to decrease 3,4‐dihydroxyphenylacetaldehyde production, and post‐mortem myocardial tissue contents of catecholamines and αS. The new model generated a triphasic decline in myocardial norepinephrine content. This pattern was confirmed by empirical data from serial cardiac 18F‐dopamine positron emission tomographic scanning in patients with LBDs. The model also correctly predicted empirical data about effects of genetic variants and monoamine oxidase inhibition and about myocardial levels of catecholamines and αS. Conclusions The present computational model predicts a triphasic decline in myocardial norepinephrine content as LBDs progress. According to the model, disease‐modifying interventions begun at the transition from the first to the second phase delay the onset of symptomatic disease. Computational modeling coupled with biomarkers of preclinical autonomic synucleinopathy may enable early detection and more effective treatment of LBDs.

Abstract Background Being able to predict negative postoperative outcomes is important for helping select patients for treatment as well for informed decision‐making by patients. Frailty measures are often time and resource intensive to use as screening measures, whereas the Braden scale, a commonly used measure to assess patients at risk of developing pressure ulcers after surgery, may be a potential tool to predict postoperative complication rates and longer length of stay (LOS) in patients undergoing major head and neck cancer surgery. Methods A retrospective analysis of Braden scale scores was performed on a prospectively collected cohort of patients undergoing major head and neck surgery recruited between December 2011 and April 2014. The association of Braden scale score with the primary outcomes of complications and LOS was analyzed using logistic regression and linear regression models on univariate analysis (UVA), respectively. Multivariate analysis (MVA) was performed based on a backward stepwise selection algorithm. Results There were 232 patients with a mean (SD) Braden scale score of 14.9 (2.8) with a range from 9 to 23. The Braden scale (β = −.07 per point; 95% CI −0.09, −0.04, P

Abstract Objective Pure autonomic failure (PAF) is a rare disease characterized by neurogenic orthostatic hypotension (nOH), absence of signs of central neurodegeneration, and profound deficiency of the sympathetic neurotransmitter norepinephrine. Reports have disagreed about mechanisms of the noradrenergic lesion. Neuropathological studies have highlighted denervation, while functional studies have emphasized deficient vesicular sequestration of cytoplasmic catecholamines in extant neurons. We examined both aspects by a combined positron emission tomographic (PET) neuroimaging approach using 11C‐methylreboxetine (11C‐MRB), a selective ligand for the cell membrane norepinephrine transporter, to quantify interventricular septal myocardial noradrenergic innervation and using 18F‐dopamine (18F‐DA) to assess intraneuronal vesicular storage in the same subjects. Methods Seven comprehensively tested PAF patients and 11 controls underwent 11C‐MRB PET scanning for 45 minutes (dynamic 5X1’, 3X5’, 1X10’, static 15 minutes) and 18F‐DA scanning for 30 minutes (same dynamic imaging sequence) after 3‐minute infusions of the tracers on separate days. Results In the PAF group septal 11C‐MRB‐derived radioactivity in the static frame was decreased by 26.7% from control (p = 0.012). After adjustment for nonspecific binding of 11C‐MRB, the PAF group had a 41.1% mean decrease in myocardial 11C‐MRB‐derived radioactivity (p = 0.015). The PAF patients had five times faster postinfusion loss of 18F‐DA‐derived radioactivity (70 ± 3% vs. 14 ± 8% by 30 minutes, p

Abstract Background Advances in healthcare delivery have allowed for the increase in the number of ambulatory surgery procedures performed in Canada. Despite these advances, patients return to hospital following discharge. However, the reason for unplanned healthcare use after ambulatory surgery in Canada is not well understood. Aims To examine unplanned healthcare use, specifically emergency department visit and hospital admissions, in the 3 days after ambulatory surgery in Ontario, Canada. Methods This population-based retrospective cohort study was conducted using de-identified administrative databases. Participants were residents in the province of Ontario, Canada; 18 years and older; and underwent common ambulatory surgical procedures between 2014 and 2018. The outcomes included emergency department (ED) visit and hospital admission. Incidence rates were calculated for the total cohort, for each patient characteristic and for surgical category. The odds ratios and 95% confidence intervals were calculated for each outcome using bivariate and multivariate logistic regression. Results 484,670 adults underwent select common surgical procedures during the study period. Patients had healthcare use in the first 3 days after surgery, with 14,950 (3.1%) ED visits and 14,236 (2.9%) admissions. The incidence of ED use was highest after tonsillectomy (8.1%), cholecystectomy (4.2%) and appendectomy (4.0%). Incidence of admissions was highest after appendectomy (21%). Acute pain (19.7%) and haemorrhage (14.2%) were the most frequent reasons for an ED visit and “convalescence following surgery” (49.2%) followed by acute pain (6.2%) and haemorrhage (4.5%) were the main reasons for admission. Conclusions These findings can assist clinicians in identifying and intervening with patients at risk of healthcare use after ambulatory surgery. Pain management strategies that can be tailored to the patient, and earlier follow-up for some patients may be required. In addition, administrative decision-makers could use the results to estimate the impact of specific ambulatory procedures on hospital resources for planning and allocation of resources.

Abstract With the COVID-19 pandemic, there has been significant changes and challenges in the management of oncology patients. One of the major strategies to reduce transmission of the virus between patients and healthcare workers is deferral of follow-up visits. However, deferral may not be possible in total laryngectomy patients. Urgent procedures may be necessary to prevent complications related to ill-fitting tracheoesophageal puncture (TEP) voice prostheses, such as aspiration or loss of voicing. In this paper, we describe the Princess Margaret Cancer Center’s approach to managing this unique patient population.

Abstract Background Cope’s gray treefrog, Dryophytes chrysoscelis, withstands the physiological challenges of corporeal freezing, partly by accumulating cryoprotective compounds of hepatic origin, including glycerol, urea, and glucose. We hypothesized that expression of genes related to cryoprotectant mobilization and stress tolerance would be differentially regulated in response to cold. Using high-throughput RNA sequencing (RNA-Seq), a hepatic transcriptome was generated for D. chrysoscelis, and gene expression was compared among frogs that were warm-acclimated, cold-acclimated, and frozen. Results A total of 159,556 transcripts were generated; 39% showed homology with known transcripts, and 34% of all transcripts were annotated. Gene-level analyses identified 34,936 genes, 85% of which were annotated. Cold acclimation induced differential expression both of genes and non-coding transcripts; freezing induced few additional changes. Transcript-level analysis followed by gene-level aggregation revealed 3582 differentially expressed genes, whereas analysis at the gene level revealed 1324 differentially regulated genes. Approximately 3.6% of differentially expressed sequences were non-coding and of no identifiable homology. Expression of several genes associated with cryoprotectant accumulation was altered during cold acclimation. Of note, glycerol kinase expression decreased with cold exposure, possibly promoting accumulation of glycerol, whereas glucose export was transcriptionally promoted by upregulation of glucose-6-phosphatase and downregulation of genes of various glycolytic enzymes. Several genes related to heat shock protein response, DNA repair, and the ubiquitin proteasome pathway were upregulated in cold and frozen frogs, whereas genes involved in responses to oxidative stress and anoxia, both potential sources of cellular damage during freezing, were downregulated or unchanged. Conclusion Our study is the first to report transcriptomic responses to low temperature exposure in a freeze-tolerant vertebrate. The hepatic transcriptome of Dryophytes chrysoscelis is responsive to cold and freezing. Transcriptomic regulation of genes related to particular pathways, such as glycerol biosynthesis, were not all regulated in parallel. The physiological demands associated with cold and freezing, as well as the transcriptomic responses observed in this study, are shared with several organisms that face similar ecophysiological challenges, suggesting common regulatory mechanisms. The role of transcriptional regulation relative to other cellular processes, and of non-coding transcripts as elements of those responses, deserve further study.

Abstract Active surveillance (AS) in the management of small, low risk papillary thyroid cancer (PTC) as an alternative option to thyroidectomy, is an area of active research. A national Canadian study is proposed to evaluate the long-term outcomes of patients with small, low risk PTC who choose AS or surgery. This letter describes the proceedings of a national investigator meeting to plan the study. Graphical abstract

Certolizumab pegol (CZP) is a PEGylated Fc-free tumor necrosis factor (TNF) inhibitor antibody approved for use in the treatment of rheumatoid arthritis (RA), Crohn's disease, psoriatic arthritis, axial spondyloarthritis and psoriasis. In a clinical trial of patients with severe RA, CZP improved disease symptoms in approximately half of patients. However, variability in CZP efficacy remains a problem for clinicians, thus, the aim of this study was to identify genetic variants predictive of CZP response. We performed a genome-wide association study (GWAS) of 302 RA patients treated with CZP in the REALISTIC trial to identify common single nucleotide polymorphisms (SNPs) associated with treatment response. Whole-exome sequencing was also performed for 74 CZP extreme responders and non-responders within the same population, as well as 1546 population controls. No common SNPs or rare functional variants were significantly associated with CZP response, though a non-significant enrichment in the RA-implicated KCNK5 gene was observed. Two SNPs near spondin-1 and semaphorin-4G approached genome-wide significance. The results of the current study did not provide an unambiguous predictor of CZP response.

Recent reports indicate that Parkinson's disease (PD) involves specific functional abnormalities in residual neurons – decreased vesicular sequestration of cytoplasmic catecholamines via the vesicular monoamine transporter (VMAT) and decreased aldehyde dehydrogenase (ALDH) activity. This double hit builds up the autotoxic metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL), the focus of the catecholaldehyde hypothesis for the pathogenesis of PD. An animal model is needed that reproduces this abnormal catecholamine neurochemical pattern. Adult rats received subcutaneous vehicle or the mitochondrial complex 1 inhibitor rotenone (2 mg/kg/day via a minipump) for 10 days. Locomotor activity was recorded, and striatal tissue sampled for catechol contents and catechol ratios that indicate the above abnormalities. Compared to vehicle, rotenone reduced locomotor activity (P=0.002), decreased tissue dopamine concentrations (P=0.00001), reduced indices of vesicular sequestration (3,4-dihydroxyphenylacetic acid (DOPAC)/dopamine) and ALDH activity (DOPAC/DOPAL) (P=0.0025, P=0.036), and increased DOPAL levels (P=0.04). The rat rotenone model involves functional abnormalities in catecholaminergic neurons that replicate the pattern found in PD putamen. These include a vesicular storage defect, decreased ALDH activity and DOPAL build-up. The rat rotenone model provides a suitable in vivo platform for studying the catecholaldehyde hypothesis.

ObjectiveTo report the first use of a novel projected augmented reality (AR) system in open sinonasal tumor resections in preclinical models and to compare the AR approach with an advanced intraoperative navigation (IN) system.MethodsFour tumor models were created. Five head and neck surgeons participated in the study performing virtual osteotomies. Unguided, AR, IN, and AR + IN simulations were performed. Statistical comparisons between approaches were obtained. Intratumoral cut rate was the main outcome. The groups were also compared in terms of percentage of intratumoral, close, adequate, and excessive distances from the tumor. Information on a wearable gaze tracker headset and NASA Task Load Index questionnaire results were analyzed as well.ResultsA total of 335 cuts were simulated. Intratumoral cuts were observed in 20.7%, 9.4%, 1.2,% and 0% of the unguided, AR, IN, and AR + IN simulations, respectively (p

Sclerosing microcystic adenocarcinoma (SMA) of the salivary gland is an extremely rare entity described in the mucosal sites of the head and neck region. These tumours are noteworthy for their histological resemblance to cutaneous microcystic adnexal carcinoma (MAC). Here, we describe about an unusual parotid tumour which showed remarkable similarity to cutaneous MAC. In this report we will be discussing about the diagnostic dilemma created by this tumour as this entity was not previously described in the parotid gland, the differential diagnosis considered along with a brief review of literature.

Introduction Paediatric ambulatory surgery (same day surgery and planned same day discharge) is more frequently being performed more in Canada and around the world; however, after surgery children may return to hospital, either through the emergency department (ED) or through a hospital admission (HA). The aim of this study was to determine the patient characteristics associated with ED visits and HA in the 3 days following paediatric ambulatory surgery.Methods This population-based retrospective cohort study used de-identified health administrative database housed at ICES and included residents of Ontario, younger than 18 years of age, who underwent ambulatory surgery between 2014 and 2018. Patients were not involved in the design of this study. The proportion of ED visit and HA were calculated for the total cohort, and the type of surgery. The ORs and 95% CIs were calculated for each outcome using logistic regression.Results 83 468 children underwent select ambulatory surgeries. 2588 (3.1%) had an ED visit and 608 (0.7%) had a HA in the 3 days following surgery. The most common reasons for ED visits included pain (17.2%) and haemorrhage (10.5%). Reasons for HA included haemorrhage (24.8%), dehydration (21.9%), and pain (9.1%).Conclusions Our findings suggest that pain, bleeding and dehydration symptoms are associated with a return visit to the hospital. Implementing approaches to prevent, identify and manage these symptoms may be helpful in reducing ED visits or hospital admissions.

Summary: Mutations in the GNB1 gene, encoding the Gβ1 subunit of heterotrimeric G proteins, cause GNB1 Encephalopathy. Patients experience seizures, pointing to abnormal activity of ion channels or neurotransmitter receptors. We studied three Gβ1 mutations (K78R, I80N and I80T) using computational and functional approaches. In heterologous expression models, these mutations did not alter the coupling between G protein-coupled receptors to Gi/o, or the Gβγ regulation of the neuronal voltage-gated Ca2+ channel CaV2.2. However, the mutations profoundly affected the Gβγ regulation of the G protein-gated inwardly rectifying potassium channels (GIRK, or Kir3). Changes were observed in Gβ1 protein expression levels, Gβγ binding to cytosolic segments of GIRK subunits, and in Gβγ function, and included gain-of-function for K78R or loss-of-function for I80T/N, which were GIRK subunit-specific. Our findings offer new insights into subunit-dependent gating of GIRKs by Gβγ, and indicate diverse etiology of GNB1 Encephalopathy cases, bearing a potential for personalized treatment.

Abstract Background Patients with human papillomavirus-positive (HPV+) oropharyngeal squamous cell carcinoma (OPC) have substantially better treatment response and overall survival (OS) than patients with HPV-negative disease. Treatment options for HPV+ OPC can involve either a primary radiotherapy (RT) approach (± concomitant chemotherapy) or a primary surgical approach (± adjuvant radiation) with transoral surgery (TOS). These two treatment paradigms have different spectrums of toxicity. The goals of this study are to assess the OS of two de-escalation approaches (primary radiotherapy and primary TOS) compared to historical control, and to compare survival, toxicity and quality of life (QOL) profiles between the two approaches. Methods This is a multicenter phase II study randomizing one hundred and forty patients with T1–2 N0–2 HPV+ OPC in a 1:1 ratio between de-escalated primary radiotherapy (60 Gy) ± concomitant chemotherapy and TOS ± de-escalated adjuvant radiotherapy (50–60 Gy based on risk factors). Patients will be stratified based on smoking status (

Oral tongue squamous cell carcinoma (OTSCC) displays variable levels of immune cells within the tumor microenvironment. The quantity and localization of tumor infiltrating lymphocytes (TILs), specific functional TIL subsets (e.g., CD8+), and biomarker-expressing cells (e.g., PD-L1+) may have prognostic and predictive value. The purpose of this study was to evaluate the robustness and utility of computer-assisted image analysis tools to quantify and localize immunohistochemistry-based biomarkers within the tumor microenvironment on a tissue microarray (TMA). We stained a 91-patient OTSCC TMA with antibodies targeting CD3, CD4, CD8, FOXP3, IDO, and PD-L1. Cell populations were segmented into epithelial (tumor) or stromal compartments according to a mask derived from a pan-cytokeratin stain. Definiens Tissue Studio was used to enumerate marker-positive cells or to quantify the staining intensity. Automated methods were validated against manual tissue segmentation, cell count, and stain intensity quantification. Univariate associations of cell count and stain intensity with smoking status, stage, overall survival (OS), and disease-free survival (DFS) were determined. Our results revealed that the accuracy of automated tissue segmentation was dependent on the distance of the tissue section from the cytokeratin mask and the proportion of the tissue containing tumor vs. stroma. Automated and manual cell counts and stain intensities were highly correlated (Pearson coefficient range: 0.46–0.90; p

Abstract Objective A number of in vitro and clinical studies have suggested potential antineoplastic effects of metformin. The impact of this medication on outcomes in head and neck cancer is less clear. Our aim was to determine the effect of metformin on outcomes within our large cohort of head and neck cancer patients with Type II Diabetes (T2DM). Study design Retrospective cohort study. Setting Tertiary Cancer Centre. Subjects and methods A retrospective review of individuals with head and neck squamous carcinoma (HNSCC) and T2DM between January 2005 and December 2011 at Princess Margaret Cancer Centre was conducted. Medication history was obtained from surveys at initial presentation and electronic medical record review. Using Cox regression analyses, the association between metformin use and local, regional and distant failures was explored. Subgroup analyses were conducted for oral cavity, oropharynx and larynx. Results A total of 329 HNSCC patients with T2DM were identified, including 195 metformin users and 134 non-metformin users. Patients were well-matched in terms of clinical, pathologic, and treatment factors. No difference in local, regional, or distant failure was observed between diabetic metformin users and diabetic non-metformin users for the entire cohort or within subgroup analysis for subsite. No difference between the two groups was observed for overall survival, recurrence-free survival, and disease-specific survival at 5 years. Conclusion No association between metformin use and oncologic outcomes were observed in this large cohort of HNSCC patients. Multicenter, prospective studies may be needed to verify previous studies identifying a potential anti-neoplastic effect of this medication.

Abstract Objective The incidence of thyroid cancer (TC) is known to be very high in the Greater Toronto Area of Ontario, Canada. We performed a pilot survey study examining Toronto-area family physician (FP) perspectives on thyroid neoplasm evaluation (i.e. thyroid nodules [TNs] or thyroid cancer [TC]) in this region, to explore for potential factors leading to overdiagnosis. Methods We performed a cross-sectional mail-out written survey of a random sample of 300 FPs in active practice in the Greater Toronto Area (Markham and Brampton). Results The overall response rate was 22.3, 95% confidence interval (CI) 18.0, 27.4% (67/300); the effective response rate was 19.9, 95% CI 15.7, 24.9% (58/291), after excluding 6 FPs that reported TN evaluation was outside their scope of practice and three FPs with an invalid mailing address. There were no missing responses to questions. The demographic characteristics were as follows: 58.6% (34/58) from Markham, 55.2% (32/58) were female, 58.6% (34/58) were in practice > 10 years, and 32.8% (19/58) affiliated with a University. All FPs reported easy access to thyroid ultrasound (TUS). About half of FPs were concerned about overdiagnosis of TC and most did not believe that there was any TC survival advantage with routine screening TUS. Although appropriate indications for TUS were endorsed by most respondents (e.g. palpable TN, incidental TN on other imaging), inappropriate recommendations were observed in a third of FPs (19/57) who recommended TUS for abnormal thyroid blood tests about half of FPs (30/56) who endorsed biopsy of sub-centimeter nodules. About half of FPs (31/58) reported that their patients sometimes request medically unnecessary TUS. Conclusion There are likely multiple complex factors leading to potential overdiagnosis of TC in primary care, including some physicians’ knowledge gaps about appropriate indications for TN investigations as well as patients’ requests and expectations.

BackgroundThe traditional management of papillary thyroid cancer (PTC) is thyroidectomy (total or partial removal of the thyroid). Active surveillance (AS) may be considered as an alternative option for small, low risk PTC. AS involves close follow-up (including regularly scheduled clinical and radiological assessments), with the intention of intervening with surgery for disease progression or patient preference.MethodsThis is a protocol for a prospective, observational, long-term follow-up multi-centre Canadian cohort study. Consenting eligible adults with small, low risk PTC (< 2cm in maximal diameter, confined to the thyroid, and not immediately adjacent to critical structures in the neck) are offered the choice of AS or surgery for management of PTC. Patient participants are free to choose either option (AS or surgery) and the disease management course is thus not assigned by the investigators. Surgery is provided as usual care by a surgeon in an institution of the patient’s choice. Our primary objective is to determine the rate of ‘failure’ of disease management in respective AS and surgical arms as defined by: i) AS arm – surgery for progression of PTC, and ii) surgical arm - surgery or other treatment for disease persistence or progression after completing initial treatment. Secondary outcomes include long-term thyroid oncologic and treatment outcomes, as well as patient-reported outcomes.DiscussionThe results from this study will provide long-term clinical and patient reported outcome evidence regarding active surveillance or immediate surgery for management of small, low risk PTC. This will inform future clinical trials in disease management of small, low risk papillary thyroid cancer.Registration detailsThis prospective observational cohort study is registered on clinicaltrials.gov (NCT04624477), but it should not be considered a clinical trial as there is no assigned intervention and patients are free to choose either AS or surgery.

Pathogenic variants in KCNQ2 and KCNQ3, paralogous genes encoding Kv7.2 and Kv7.3 voltage-gated K+ channel subunits, are responsible for early−onset developmental/epileptic disorders characterized by heterogeneous clinical phenotypes ranging from benign familial neonatal epilepsy (BFNE) to early−onset developmental and epileptic encephalopathy (DEE). KCNQ2 variants account for the majority of pedigrees with BFNE and KCNQ3 variants are responsible for a much smaller subgroup, but the reasons for this imbalance remain unclear. Analysis of additional pedigrees is needed to further clarify the nature of this genetic heterogeneity and to improve prediction of pathogenicity for novel variants. We identified a BFNE family with two siblings and a parent affected. Exome sequencing on samples from both parents and siblings revealed a novel KCNQ3 variant (c.719T>G; p.M240R), segregating in the three affected individuals. The M240 residue is conserved among human Kv7.2-5 and lies between the two arginines (R5 and R6) closest to the intracellular side of the voltage-sensing S4 transmembrane segment. Whole cell patch-clamp recordings in Chinese hamster ovary (CHO) cells revealed that homomeric Kv7.3 M240R channels were not functional, whereas heteromeric channels incorporating Kv7.3 M240R mutant subunits with Kv7.2 and Kv7.3 displayed a depolarizing shift of about 10 mV in activation gating. Molecular modeling results suggested that the M240R substitution preferentially stabilized the resting state and possibly destabilized the activated state of the Kv7.3 subunits, a result consistent with functional data. Exposure to β-hydroxybutyrate (BHB), a ketone body generated during the ketogenic diet (KD), reversed channel dysfunction induced by the M240R variant. In conclusion, we describe the first missense loss-of-function (LoF) pathogenic variant within the S4 segment of Kv7.3 identified in patients with BFNE. Studied under conditions mimicking heterozygosity, the M240R variant mainly affects the voltage sensitivity, in contrast to previously analyzed BFNE Kv7.3 variants that reduce current density. Our pharmacological results provide a rationale for the use of KD in patients carrying LoF variants in Kv7.2 or Kv7.3 subunits.

Parkinson's disease (PD) is a neurodegenerative disorder featuring progressive loss of midbrain dopaminergic (DA) neurons that leads to motor symptoms. The etiology and pathogenesis of PD are not clear. We found that expression of COUP-TFII, an orphan nuclear receptor, in DA neurons is upregulated in PD patients through the analysis of public datasets. We show here that through epigenetic regulation, COUP-TFII contributes to oxidative stress, suggesting that COUP-TFII may play a role in PD pathogenesis. Elevated COUP-TFII expression specifically in DA neurons evokes DA neuronal loss in mice and accelerates the progression of phenotypes in a PD mouse model, MitoPark. Compared to control mice, those with elevated COUP-TFII expression displayed reduced cristae in mitochondria and enhanced cellular electron-dense vacuoles in the substantia nigra pars compacta. Mechanistically, we found that overexpression of COUP-TFII disturbs mitochondrial pathways, resulting in mitochondrial dysfunction. In particular, there is repressed expression of genes encoding cytosolic aldehyde dehydrogenases, which could enhance oxidative stress and interfere with mitochondrial function via 3,4-dihydroxyphenylacetaldehyde (DOPAL) buildup in DA neurons. Importantly, under-expression of COUP-TFII in DA neurons slowed the deterioration in motor functions of MitoPark mice. Taken together, our results suggest that COUP-TFII may be an important contributor to PD development and a potential therapeutic target.

HLA-restricted T cell responses can induce antitumor effects in cancer patients. Previous human T cell research has largely focused on the few HLA alleles prevalent in a subset of ethnic groups. Here, using a panel of newly developed peptide-exchangeable peptide/HLA multimers and artificial antigen-presenting cells for 25 different class I alleles and greater than 800 peptides, we systematically and comprehensively mapped shared antigenic epitopes recognized by tumor-infiltrating T lymphocytes (TILs) from eight melanoma patients for all their class I alleles. We were able to determine the specificity, on average, of 12.2% of the TILs recognizing a mean of 3.1 shared antigen-derived epitopes across HLA-A, B, and C. Furthermore, we isolated a number of cognate T cell receptor genes with tumor reactivity. Our novel strategy allows for a more complete examination of the immune response and development of novel cancer immunotherapy not limited by HLA allele prevalence or tumor mutation burden.

Menkes disease is a lethal neurodegenerative disorder of copper metabolism caused by mutations in an evolutionarily conserved copper transporter, ATP7A. Based on our prior clinical and animal studies, we seek to develop a therapeutic approach suitable for application in affected human subjects, using the mottled-brindled (mo-br) mouse model that closely mimics the Menkes disease biochemical and clinical phenotypes. Here, we evaluate the efficacy of low-, intermediate-, and high-dose recombinant adeno-associated virus serotype 9 (rAAV9)-ATP7A delivered to the cerebrospinal fluid (CSF), in combination with subcutaneous administration of clinical-grade copper histidinate (sc CuHis, IND #34,166). Mutant mice that received high-dose (1.6 × 1010 vg) cerebrospinal fluid-directed rAAV9-rsATP7A plus sc copper histidinate showed 53.3% long-term (≥300-day) survival compared to 0% without treatment or with either treatment alone. The high-dose rAAV9-rsATP7A plus sc copper histidinate-treated mutant mice showed increased brain copper levels, normalized brain neurochemical levels, improvement of brain mitochondrial abnormalities, and normal growth and neurobehavioral outcomes. This synergistic treatment effect represents the most successful rescue to date of the mo-br mouse model. Based on these findings, and the absence of a large animal model, we propose cerebrospinal fluid-directed rAAV9-rsATP7A gene therapy plus subcutaneous copper histidinate as a potential therapeutic approach to cure or ameliorate Menkes disease. Keywords: adeno-associated virus, Menkes disease, ATP7A, copper, dopamine-beta-hydroxylase, choroid plexus epithelia

Abstract Nonalcoholic fatty liver disease (NAFLD) is a heterogeneous disease with highly variable outcomes. Patients with simple steatosis typically experience a benign course, whereas those with more advanced liver injury, nonalcoholic steatohepatitis (NASH), and advanced stage fibrosis suffer increased risk for complications such as cirrhosis, hepatic decompensation, and liver cancer. Genetic variants in patatin‐like phospholipase domain‐containing 3 (PNPLA3) and transmembrane 6 superfamily member 2 (TM6SF2) and clinical factors including diabetes, obesity, and older age increase a patient's risk for NASH, advanced fibrosis, and worse outcomes. Despite substantial investigation and identification of some common variants associated with NAFLD and advanced fibrosis, the genetics and functional mechanisms remain poorly understood. This study aimed to identify genetic variants by whole‐exome sequencing of NAFLD phenotypes to provide novel insights into mechanisms behind NAFLD pathogenesis and variability. We sequenced 82 patients with liver biopsy–confirmed NAFLD and 4455 population controls. NAFLD patients were divided into extreme phenotypes based on liver fibrosis stage and clinical risk factors to investigate rare variants that might predispose to or protect from advanced NAFLD fibrosis. We compared NAFLD extremes to each other and individually to population controls, exploring genetic variation at both the single‐variant and gene‐based level. We replicated known associations with PNPLA3 and TM6SF2 and advanced fibrosis, despite sample‐size limitations. We also observed enrichment of variation in distinct genes for progressor or protective NAFLD phenotypes, although these genes did not reach statistical significance. Conclusion: We report the first whole‐exome sequencing study of genetic variation in liver biopsy–confirmed NAFLD susceptibility and severity, using a small cohort of extreme NAFLD phenotypes and a large cohort of population controls.