DEVELOPING THERAPIES FOR C3G: REPORT OF THE KIDNEY HEALTH INITIATIVE C3G TRIAL ENDPOINTS WORK GROUP.

Randomized clinical trials are underway to evaluate the efficacy of novel agents targeting the alternative complement pathway in patients with C3G, a rare glomerular disease. The Kidney Health Initiative (KHI) convened a panel of experts in C3G to: (1) assess the data supporting the use of the prespecified trial endpoints as measures of clinical benefit; and (2) opine on efficacy findings they would consider compelling as treatment(s) for C3G in native kidneys. Two subpanels of the C3G Trial Endpoints Work group reviewed the available evidence and uncertainties for the association between the three prespecified endpoints -- (1) proteinuria; (2) estimated glomerular filtration rate (eGFR); and (3) histopathology -- and anticipated outcomes. The full work group provided feedback on the summaries provided by the subpanels and on what potential treatment effects on the proposed endpoints they would consider compelling to support evidence of an investigational product's effectiveness for treating C3G. Members of the full work group agreed with the characterization of the data, the evidence, and uncertainties, supporting the endpoints. Given the limitations of the available data, the workgroup was unable to define a minimum threshold for change in any of the endpoints that might be considered clinically meaningful. The workgroup concluded that a favorable treatment effect on all three endpoints would provide convincing evidence of efficacy in the setting of a therapy that targeted the complement pathway. A therapy might be considered effective in the absence of complete alignment in all three endpoints if there was meaningful lowering of proteinuria and stabilization or improvement in eGFR. The panel unanimously supported efforts to foster data sharing between academic and industry partners to address the gaps in the current knowledge identified by the review of the endpoints in the aforementioned trials.

The Role of Complement in Kidney Disease: Conclusions From a Kidney Disease: Improving Global Outcomes (KDIGO) Controversies Conference.

Uncontrolled complement activation can cause or contribute to glomerular injury in multiple kidney diseases. While complement activation plays a causal role in atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G), over the past decade a rapidly accumulating body of evidence has shown a role for complement activation in multiple other kidney diseases, including diabetic nephropathy and several glomerulonephritides. The number of available complement inhibitor therapies has also increased during the same period. In 2022 KDIGO convened a Controversies Conference, The Role of Complement in Kidney Disease, to address the expanding role of complement dysregulation in the pathophysiology, diagnosis, and management of various glomerular diseases, diabetic nephropathy, and other forms of HUS. Conference participants reviewed the evidence for complement playing a primary causal or secondary role in progression for several disease states and considered how evidence of complement involvement might inform management. Participating patients with various complement-mediated diseases and caregivers described concerns related to life planning, implications surrounding genetic testing, and the need for inclusive implementation of effective novel therapies into clinical practice. The value of biomarkers in monitoring disease course and the role of the glomerular microenvironment in complement response were examined, and key gaps in knowledge and research priorities were identified.

Prevalence of kidney health genetic variants in adults with sickle cell nephropathy.

The pathophysiology and genetic risk for sickle cell disease (SCD)-related chronic kidney disease (CKD) are not well understood. In 70 adults with SCD-related CKD and without APOL1 inherited in a high-risk pattern, 24 (34%) had pathogenic variants in candidate genes using KidneySeq™. A moderate impact INF2 variant was observed in 20 (29%) patients and those with 3 versus 0-2 pathogenic or moderate impact glomerular genetic variants had higher albuminuria and lower estimated glomerular filtration rate (adjusted p ≤ 0.015). Using a panel of preselected genes implicated in kidney health, we observed several variants in people with sickle cell nephropathy.

The natural history and genotype-phenotype correlations of TMPRSS3 hearing loss: an international, multi-center, cohort analysis.

TMPRSS3-related hearing loss presents challenges in correlating genotypic variants with clinical phenotypes due to the small sample sizes of previous studies. We conducted a cross-sectional genomics study coupled with retrospective clinical phenotype analysis on 127 individuals. These individuals were from 16 academic medical centers across 6 countries. Key findings revealed 47 unique TMPRSS3 variants with significant differences in hearing thresholds between those with missense variants versus those with loss-of-function genotypes. The hearing loss progression rate for the DFNB8 subtype was 0.3 dB/year. Post-cochlear implantation, an average word recognition score of 76% was observed. Of the 51 individuals with two missense variants, 10 had DFNB10 with profound hearing loss. These 10 all had at least one of 4 TMPRSS3 variants predicted by computational modeling to be damaging to TMPRSS3 structure and function. To our knowledge, this is the largest study of TMPRSS3 genotype-phenotype correlations. We find significant differences in hearing thresholds, hearing loss progression, and age of presentation, by TMPRSS3 genotype and protein domain affected. Most individuals with TMPRSS3 variants perform well on speech recognition tests after cochlear implant, however increased age at implant is associated with worse outcomes. These findings provide insight for genetic counseling and the on-going design of novel therapeutic approaches.

Label-free Brillouin endo-microscopy for the quantitative 3D imaging of sub-micrometre biology.

This report presents an optical fibre-based endo-microscopic imaging tool that simultaneously measures the topographic profile and 3D viscoelastic properties of biological specimens through the phenomenon of time-resolved Brillouin scattering. This uses the intrinsic viscoelasticity of the specimen as a contrast mechanism without fluorescent tags or photoacoustic contrast mechanisms. We demonstrate 2 µm lateral resolution and 320 nm axial resolution for the 3D imaging of biological cells and Caenorhabditis elegans larvae. This has enabled the first ever 3D stiffness imaging and characterisation of the C. elegans larva cuticle in-situ. A label-free, subcellular resolution, and endoscopic compatible technique that reveals structural biologically-relevant material properties of tissue could pave the way toward in-vivo elasticity-based diagnostics down to the single cell level.

A case-based narrative review of pregnancy-associated atypical hemolytic uremic syndrome/complement-mediated thrombotic microangiopathy.

Atypical hemolytic uremic syndrome is a complement-mediated thrombotic microangiopathy caused by uncontrolled activation of the alternative complement pathway in the setting of autoantibodies to or rare pathogenic genetic variants in complement proteins. Pregnancy may serve as a trigger and unmask atypical hemolytic uremic syndrome/complement-mediated thrombotic microangiopathy (aHUS/CM-TMA), which has severe, life-threatening consequences. It can be difficult to diagnose aHUS/CM-TMA in pregnancy due to overlapping clinical features with other thrombotic microangiopathy syndromes including hypertensive disorders of pregnancy. However, the distinction among thrombotic microangiopathy etiologies in pregnancy is important because each syndrome has specific disease management and treatment. In this narrative review, we discuss 2 cases to illustrate the diagnostic challenges and evolving approach in the management of pregnancy-associated aHUS/CM-TMA. The first case involves a 30-year-old woman presenting in the first trimester who was diagnosed with aHUS/CM-TMA and treated with eculizumab from 19 weeks' gestation. Genetic testing revealed a likely pathogenic variant in CFI. She successfully delivered a healthy infant at 30 weeks' gestation. In the second case, a 22-year-old woman developed severe postpartum HELLP syndrome, requiring hemodialysis. Her condition improved with supportive management, yet investigations assessing for aHUS/CM-TMA remained abnormal 6 months postpartum consistent with persistent complement activation but negative genetic testing. Through detailed case discussion describing tests assessing for placental health, fetal anatomy, complement activation, autoantibodies to complement regulatory proteins, and genetic testing for aHUS/CM-TMA, we describe how these results aided in the clinical diagnosis of pregnancy-associated aHUS/CM-TMA and assisted in guiding patient management, including the use of anticomplement therapy.

Iptacopan in Idiopathic Immune Complex-Mediated Membranoproliferative Glomerulonephritis: Protocol of the APPARENT Multicenter, Randomized Phase 3 Study.

Introduction: Immune complex-mediated membranoproliferative glomerulonephritis (IC-MPGN) is an ultra-rare, fast-progressing kidney disease that may be idiopathic (primary) or secondary to chronic infection, autoimmune disorders, or monoclonal gammopathies. Dysregulation of the alternative complement pathway is implicated in the pathophysiology of IC-MPGN; and currently, there are no approved targeted treatments. Iptacopan is an oral, highly potent proximal complement inhibitor that specifically binds to factor B and inhibits the alternative pathway (AP). Methods: This randomized, double-blind, placebo-controlled phase 3 study (APPARENT; NCT05755386) will evaluate the efficacy and safety of iptacopan in patients with idiopathic (primary) IC-MPGN, enrolling up to 68 patients (minimum of 10 adolescents) aged 12 to 60 years with biopsy-confirmed IC-MPGN, proteinuria ≥1 g/g, and estimated glomerular filtration rate (eGFR) ≥30 ml/min per 1.73 m2. All patients will receive maximally tolerated angiotensin-converting enzyme inhibitor/angiotensin receptor blocker and vaccination against encapsulated bacteria. Patients with any organ transplant, progressive crescentic glomerulonephritis, or kidney biopsy with >50% interstitial fibrosis/tubular atrophy, will be excluded. Patients will be randomized 1:1 to receive either iptacopan 200 mg twice daily (bid) or placebo for 6 months, followed by open-label treatment with iptacopan 200 mg bid for all patients for 6 months. The primary objective of the study is to evaluate the efficacy of iptacopan versus placebo in proteinuria reduction measured as urine protein-to-creatinine ratio (UPCR) (24-h urine) at 6 months. Key secondary end points will assess kidney function measured by eGFR, patients who achieve a proteinuria-eGFR composite end point, and patient-reported fatigue. Conclusion: This study will provide evidence toward the efficacy and safety of iptacopan in idiopathic (primary) IC-MPGN.

Congenital Cytomegalovirus Testing Outcomes From the ValEAR Trial.

OBJECTIVE: To determine the positivity rate of congenital cytomegalovirus (cCMV) testing among universal, hearing-targeted CMV testing (HT-cCMV) and delayed targeted dried blood spot (DBS) testing newborn screening programs, and to examine the characteristics of successful HT-cCMV testing programs. STUDY DESIGN: Prospective survey of birth hospitals performing early CMV testing. SETTING: Multiple institutions. METHODS: Birth hospitals participating in the National Institutes of Health ValEAR clinical trial were surveyed to determine the rates of cCMV positivity associated with 3 different testing approaches: universal testing, HT-cCMV, and DBS testing. A mixed methods model was created to determine associations between successful HT-cCMV screening and specific screening protocols. RESULTS: Eighty-two birth hospitals were surveyed from February 2019 to December 2021. Seven thousand six hundred seventy infants underwent universal screening, 9017 infants HT-cCMV and 535 infants delayed DBS testing. The rates of cCMV positivity were 0.5%, 1.5%, and 7.3%, respectively. The positivity rate for universal CMV screening was less during the COVID-19 pandemic than that reported prior to the pandemic. There were no statistically significant drops in positivity for any approach during the pandemic. For HT-cCMV testing, unique order sets and rigorous posttesting protocols were associated with successful screening programs. CONCLUSION: Rates of cCMV positivity differed among the 3 approaches. The rates are comparable to cohort studies reported in the literature. Universal CMV prevalence decreased during the pandemic but not significantly. Institutions with specific order set for CMV testing where the primary care physician orders the test and the nurse facilitates the testing process exhibited higher rates of HT-cCMV testing.

Defining Nephritic Factors as Diverse Drivers of Systemic Complement Dysregulation in C3 Glomerulopathy.

Introduction: C3 glomerulopathy (C3G) is an ultrarare renal disease characterized by deposition of complement component C3 in the glomerular basement membrane (GBM). Rare and novel genetic variation in complement genes and autoantibodies to complement proteins are commonly identified in the C3G population and thought to drive the underlying complement dysregulation that results in renal damage. However, disease heterogeneity and rarity make accurately defining characteristics of the C3G population difficult. Methods: Here, we present a retrospective analysis of the Molecular Otolaryngology and Renal Research Laboratories C3G cohort. This study integrated complement biomarker testing and in vitro tests of autoantibody function to achieve the following 3 primary goals: (i) define disease profiles of C3G based on disease drivers, complement biomarkers, and age; (ii) determine the relationship between in vitro autoantibody tests and in vivo complement dysregulation; and (iii) evaluate the association between autoantibody function and disease progression. Results: The largest disease profiles of C3G included patients with autoantibodies to complement proteins (48%) and patients for whom no genetic and/or acquired drivers of disease could be identified (43%). The correlation between the stabilization of convertases by complement autoantibodies as measured by in vitro modified hemolytic assays and systemic biomarkers that reflect in vivo complement dysregulation was remarkably strong. In patients positive for autoantibodies, the degree of stabilization capacity predicted worse renal function. Conclusion: This study implicates complement autoantibodies as robust drivers of systemic complement dysregulation in approximately 50% of C3G but also highlights the need for continued discovery-based research to identify novel drivers of disease.

Surveillance of antimicrobial resistance in Escherichia coli, Salmonella, and Campylobacter recovered from laying hens, their environment and products in Canada indicated a stable level of resistance to critically important antimicrobials, in varying time periods between 2007 and 2021.

The study objective was to determine the occurrence and antimicrobial resistance (AMR) attributes of select foodborne bacteria recovered from egg-producing (layer) chickens between 2007 and 2021 using different sample matrices (Study 1: liquid whole eggs, Study 2: spent hen cecal samples, Study 3: environmental sponge swabs and fecal samples from layer chicken barns, and Study 4: fecal samples from layer chicken barns). Samples from each study were submitted for the culture of Escherichia coli and Salmonella. In addition, samples from layer chicken barns were submitted for the culture of Campylobacter. Isolates were tested by microbroth dilution and interpreted using both clinical breakpoints and epidemiological cut-offs (ECOFFs). The ECOFFs were applied to detect non-wild type (NWT) strains. The proportion of resistant, NWT, and distribution of minimum inhibitory concentrations (MIC) were assessed. Ceftriaxone resistance was detected at a low-level in E. coli (< 2 %, all studies) and Salmonella (4.3 %, Study 2). Very low-level ciprofloxacin resistance was detected in E. coli (<1 %, Study 1) but a slightly elevated ciprofloxacin NWT E. coli (1 % to 6 %) observed. Only the farm fecal samples in Studies 2 and 3 were tested for Campylobacter as part of its study design, and moderate level ciprofloxacin resistance (<15 %) was observed. The MIC distribution patterns were similar across the organisms tested/studies and no substantial shifts in the distributions were detected. This analysis demonstrated that resistance to very important antimicrobials in bacteria from layers in Canada remains low, however, the detection of ciprofloxacin-resistant Campylobacter and the implications of this observation to the safety of egg products, and the role of laying flocks (i.e., as reservoir for resistant organisms) needs to be investigated.

Renin and renin blockade have no role in complement activity.

Renin, an aspartate protease, regulates the renin-angiotensin system by cleaving its only known substrate angiotensinogen to angiotensin. Recent studies have suggested that renin may also cleave complement component C3 to activate complement or contribute to its dysregulation. Typically, C3 is cleaved by C3 convertase, a serine protease that uses the hydroxyl group of a serine residue as a nucleophile. Here, we provide seven lines of evidence to show that renin does not cleave C3. First, there is no association between renin plasma levels and C3 levels in patients with C3 Glomerulopathies (C3G) and atypical Hemolytic Uremic Syndrome (aHUS), implying that serum C3 consumption is not increased in the presence of high renin. Second, in vitro tests of C3 conversion to C3b do not detect differences when sera from patients with high renin levels are compared to sera from patients with normal/low renin levels. Third, aliskiren, a renin inhibitor, does not block abnormal complement activity introduced by nephritic factors in the fluid phase. Fourth, aliskiren does not block dysregulated complement activity on cell surfaces. Fifth, recombinant renin from different sources does not cleave C3 even after 24 hours of incubation at 37 °C. Sixth, direct spiking of recombinant renin into sera samples of patients with C3G and aHUS does not enhance complement activity in either the fluid phase or on cell surfaces. And seventh, molecular modeling and docking place C3 in the active site of renin in a position that is not consistent with a productive ground state complex for catalytic hydrolysis. Thus, our study does not support a role for renin in the activation of complement.

A bifunctional kinase-phosphatase module balances mitotic checkpoint strength and kinetochore-microtubule attachment stability.

Two major mechanisms safeguard genome stability during mitosis: the mitotic checkpoint delays mitosis until all chromosomes have attached to microtubules, and the kinetochore-microtubule error-correction pathway keeps this attachment process free from errors. We demonstrate here that the optimal strength and dynamics of these processes are set by a kinase-phosphatase pair (PLK1-PP2A) that engage in negative feedback from adjacent phospho-binding motifs on the BUB complex. Uncoupling this feedback to skew the balance towards PLK1 produces a strong checkpoint, hypostable microtubule attachments and mitotic delays. Conversely, skewing the balance towards PP2A causes a weak checkpoint, hyperstable microtubule attachments and chromosome segregation errors. These phenotypes are associated with altered BUB complex recruitment to KNL1-MELT motifs, implicating PLK1-PP2A in controlling auto-amplification of MELT phosphorylation. In support, KNL1-BUB disassembly becomes contingent on PLK1 inhibition when KNL1 is engineered to contain excess MELT motifs. This elevates BUB-PLK1/PP2A complex levels on metaphase kinetochores, stabilises kinetochore-microtubule attachments, induces chromosome segregation defects and prevents KNL1-BUB disassembly at anaphase. Together, these data demonstrate how a bifunctional PLK1/PP2A module has evolved together with the MELT motifs to optimise BUB complex dynamics and ensure accurate chromosome segregation.

Classification of cancer cells at the sub-cellular level by phonon microscopy using deep learning.

There is a consensus about the strong correlation between the elasticity of cells and tissue and their normal, dysplastic, and cancerous states. However, developments in cell mechanics have not seen significant progress in clinical applications. In this work, we explore the possibility of using phonon acoustics for this purpose. We used phonon microscopy to obtain a measure of the elastic properties between cancerous and normal breast cells. Utilising the raw time-resolved phonon-derived data (300 k individual inputs), we employed a deep learning technique to differentiate between MDA-MB-231 and MCF10a cell lines. We achieved a 93% accuracy using a single phonon measurement in a volume of approximately 2.5 µm3. We also investigated means for classification based on a physical model that suggest the presence of unidentified mechanical markers. We have successfully created a compact sensor design as a proof of principle, demonstrating its compatibility for use with needles and endoscopes, opening up exciting possibilities for future applications.

Advances in cochlear gene therapies.

PURPOSE OF REVIEW: Hearing loss is the most common sensory deficit and in young children sensorineural hearing loss is most frequently genetic in etiology. Hearing aids and cochlear implant do not restore normal hearing. There is significant research and commercial interest in directly addressing the root cause of hearing loss through gene therapies. This article provides an overview of major barriers to cochlear gene therapy and recent advances in preclinical development of precision treatments of genetic deafness. RECENT FINDINGS: Several investigators have recently described successful gene therapies in many common forms of genetic hearing loss in animal models. Elegant strategies that do not target a specific pathogenic variant, such as mini gene replacement and mutation-agnostic RNA interference (RNAi) with engineered replacement, facilitate translation of these findings to development of human therapeutics. Clinical trials for human gene therapies are in active recruitment. SUMMARY: Gene therapies for hearing loss are expected to enter clinical trials in the immediate future. To provide referral for appropriate trials and counseling regarding benefits of genetic hearing loss evaluation, specialists serving children with hearing loss such as pediatricians, geneticists, genetic counselors, and otolaryngologists should be acquainted with ongoing developments in precision therapies.

Single-cell RNA-sequencing of stria vascularis cells in the adult Slc26a4-/- mouse.

BACKGROUND: The primary pathological alterations of Pendred syndrome are endolymphatic pH acidification and luminal enlargement of the inner ear. However, the molecular contributions of specific cell types remain poorly characterized. Therefore, we aimed to identify pH regulators in pendrin-expressing cells that may contribute to the homeostasis of endolymph pH and define the cellular pathogenic mechanisms that contribute to the dysregulation of cochlear endolymph pH in Slc26a4-/- mice. METHODS: We used single-cell RNA sequencing to identify both Slc26a4-expressing cells and Kcnj10-expressing cells in wild-type (WT, Slc26a4+/+) and Slc26a4-/- mice. Bioinformatic analysis of expression data confirmed marker genes defining the different cell types of the stria vascularis. In addition, specific findings were confirmed at the protein level by immunofluorescence. RESULTS: We found that spindle cells, which express pendrin, contain extrinsic cellular components, a factor that enables cell-to-cell communication. In addition, the gene expression profile informed the pH of the spindle cells. Compared to WT, the transcriptional profiles in Slc26a4-/- mice showed downregulation of extracellular exosome-related genes in spindle cells. Immunofluorescence studies in spindle cells of Slc26a4-/- mice validated the increased expression of the exosome-related protein, annexin A1, and the clathrin-mediated endocytosis-related protein, adaptor protein 2. CONCLUSION: Overall, cell isolation of stria vascularis from WT and Slc26a4-/- samples combined with cell type-specific transcriptomic analyses revealed pH-dependent alternations in spindle cells and intermediate cells, inspiring further studies into the dysfunctional role of stria vascularis cells in SLC26A4-related hearing loss.

A scoping review of factors associated with antimicrobial-resistant Campylobacter species infections in humans.

Human infection with antimicrobial-resistant Campylobacter species is an important public health concern due to the potentially increased severity of illness and risk of death. Our objective was to synthesise the knowledge of factors associated with human infections with antimicrobial-resistant strains of Campylobacter. This scoping review followed systematic methods, including a protocol developed a priori. Comprehensive literature searches were developed in consultation with a research librarian and performed in five primary and three grey literature databases. Criteria for inclusion were analytical and English-language publications investigating human infections with an antimicrobial-resistant (macrolides, tetracyclines, fluoroquinolones, and/or quinolones) Campylobacter that reported factors potentially linked with the infection. The primary and secondary screening were completed by two independent reviewers using Distiller SR®. The search identified 8,527 unique articles and included 27 articles in the review. Factors were broadly categorised into animal contact, prior antimicrobial use, participant characteristics, food consumption and handling, travel, underlying health conditions, and water consumption/exposure. Important factors linked to an increased risk of infection with a fluoroquinolone-resistant strain included foreign travel and prior antimicrobial use. Identifying consistent risk factors was challenging due to the heterogeneity of results, inconsistent analysis, and the lack of data in low- and middle-income countries, highlighting the need for future research.

Factors associated with antimicrobial resistant enterococci in Canadian beef cattle: A scoping review.

Introduction: Antimicrobial resistance (AMR) is a global health concern, occurring when bacteria evolve to render antimicrobials no longer effective. Antimicrobials have important roles in beef production; however, the potential to introduce AMR to people through beef products is a concern. This scoping review identifies factors associated with changes in the prevalence of antimicrobial-resistant Enterococcus spp. applicable to the Canadian farm-to-fork beef continuum. Methods: Five databases (MEDLINE, BIOSIS, Web of Science, Embase, and CAB Abstracts) were searched for articles published from January 1984 to March 2022, using a priori inclusion criteria. Peer-reviewed articles were included if they met all the following criteria: written in English, applicable to the Canadian beef production context, primary research, in vivo research, describing an intervention or exposure, and specific to Enterococcus spp. Results: Out of 804 screened articles, 26 were selected for inclusion. The included articles discussed 37 factors potentially associated with AMR in enterococci, with multiple articles discussing at least two of the same factors. Factors discussed included antimicrobial administration (n = 16), raised without antimicrobials (n = 6), metal supplementation (n = 4), probiotics supplementation (n = 3), pen environment (n = 2), essential oil supplementation (n = 1), grass feeding (n = 1), therapeutic versus subtherapeutic antimicrobial use (n = 1), feeding wet distiller grains with solubles (n = 1), nutritional supplementation (n = 1) and processing plant type (n = 1). Results were included irrespective of their quality of evidence. Discussion: Comparability issues arising throughout the review process were related to data aggregation, hierarchical structures, study design, and inconsistent data reporting. Findings from articles were often temporally specific in that resistance was associated with AMR outcomes at sampling times closer to exposure compared to studies that sampled at longer intervals after exposure. Resistance was often nuanced to unique gene and phenotypic resistance patterns that varied with species of enterococci. Intrinsic resistance and interpretation of minimum inhibitory concentration varied greatly among enterococcal species, highlighting the importance of caution when comparing articles and generalizing findings. Systematic Review Registration: [http://hdl.handle.net/1880/113592].

Parallel imaging with phonon microscopy using a multi-core fibre bundle detection.

In this paper, we show a proof-of-concept method to parallelise phonon microscopy measurements for cell elasticity imaging by demonstrating a 3-fold increase in acquisition speed which is limited by current acquisition hardware. Phonon microscopy is based on time-resolved Brillouin scattering, which uses a pump-probe method with asynchronous optical sampling (ASOPS) to generate and detect coherent phonons. This enables access to the cell elasticity via the Brillouin frequency with sub-optical axial resolution. Although systems based on ASOPS are typically faster compared to the ones built with a mechanical delay line, they are still very slow to study real time changes at the cellular level. Additionally, the biocompatibility is reduced due to long light exposure and scanning time. Using a multi-core fibre bundle rather than a single channel for detection, we acquire 6 channels simultaneously allowing us to speed-up measurements, and open a way to scale-up this method.

Assessing variants of uncertain significance implicated in hearing loss using a comprehensive deafness proteome.

Hearing loss is the leading sensory deficit, affecting ~ 5% of the population. It exhibits remarkable heterogeneity across 223 genes with 6328 pathogenic missense variants, making deafness-specific expertise a prerequisite for ascribing phenotypic consequences to genetic variants. Deafness-implicated variants are curated in the Deafness Variation Database (DVD) after classification by a genetic hearing loss expert panel and thorough informatics pipeline. However, seventy percent of the 128,167 missense variants in the DVD are "variants of uncertain significance" (VUS) due to insufficient evidence for classification. Here, we use the deep learning protein prediction algorithm, AlphaFold2, to curate structures for all DVD genes. We refine these structures with global optimization and the AMOEBA force field and use DDGun3D to predict folding free energy differences (∆∆GFold) for all DVD missense variants. We find that 5772 VUSs have a large, destabilizing ∆∆GFold that is consistent with pathogenic variants. When also filtered for CADD scores (> 25.7), we determine 3456 VUSs are likely pathogenic at a probability of 99.0%. Of the 224 genes in the DVD, 166 genes (74%) exhibit one or more missense variants predicted to cause a pathogenic change in protein folding stability. The VUSs prioritized here affect 119 patients (~ 3% of cases) sequenced by the OtoSCOPE targeted panel. Approximately half of these patients previously received an inconclusive report, and reclassification of these VUSs as pathogenic provides a new genetic diagnosis for six patients.