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Deep RNA-seq of male and female murine sensory neuron subtypes after nerve injury
Abstract Dorsal root ganglia (DRG) neurons have been well described for their role in driving both acute and chronic pain. Although nerve injury is known to cause transcriptional dysregulation, how this differs across neuronal subtypes and the impact of sex is unclear. Here, we study the deep transcriptional profiles of multiple murine DRG populations in early and late pain states while considering sex. We have exploited currently available transgenics to label numerous subpopulations for fluorescent-activated cell sorting and subsequent transcriptomic analysis. Using bulk tissue samples, we are able to circumvent the issues of low transcript coverage and drop-outs seen with single-cell data sets. This increases our power to detect novel and even subtle changes in gene expression within neuronal subtypes and discuss sexual dimorphism at the neuronal subtype level. We have curated this resource into an accessible database for other researchers (https://livedataoxford.shinyapps.io/drg-directory/). We see both stereotyped and unique subtype signatures in injured states after nerve injury at both an early and late timepoint. Although all populations contribute to a general injury signature, subtype enrichment changes can also be seen. Within populations, there is not a strong intersection of sex and injury, but previously unknown sex differences in naïve states—particularly in Aβ-RA + Aδ-low threshold mechanoreceptors—still contribute to differences in injured neurons.
Poststroke Executive Function in Relation to White Matter Damage on Clinically Acquired CT Brain Imaging.
BACKGROUND: Executive function (EF) impairments are prevalent post stroke and are associated with white matter (WM) damage on MRI. However, less is known about the relationship between poststroke EF and WM damage on CT imaging. OBJECTIVE: To investigate the relationship between poststroke EF and WM damage associated with stroke lesions and WM hypointensities (WMHs) on clinically acquired CT imaging. METHOD: This study analyzed data from the Oxford Cognitive Screening Program, which recruited individuals aged ≥18 years with a confirmed stroke from an acute stroke unit. The individuals completed a follow-up assessment 6 months post stroke. We included individuals with a CT scan showing a visible stroke who completed follow-up EF assessment using the Oxford Cognitive Screen-Plus rule-finding task. We manually delineated stroke lesions and quantified then dichotomized WM damage caused by the stroke using the HCP-842 atlas. We visually rated then dichotomized WMHs using the Age-Related White Matter Changes Scale. RESULTS: Among 87 stroke survivors (Mage = 73.60 ± 11.75; 41 female; 61 ischemic stroke), multivariable linear regression showed that stroke damage to the medial lemniscus (B = -8.86, P < 0.001) and the presence of WMHs (B = -5.42, P = 0.005) were associated with poorer EF 6 months post stroke after adjusting for covariates including age and education. CONCLUSION: Poorer EF was associated with WM damage caused by stroke lesions and WMHs on CT. These results confirm the importance of WM integrity for EF post stroke and demonstrate the prognostic utility of CT-derived imaging markers for poststroke cognitive outcomes.
A multicentre, prospective, double-blind study comparing the accuracy of autoantibody diagnostic assays in myasthenia gravis: the SCREAM study.
BACKGROUND: Laboratory determination of autoantibodies against acetylcholine receptor (AChR), muscle-specific kinase (MuSK) and other autoantigens have been integrated into the diagnosis of myasthenia gravis (MG). However, evidence supporting the selection of methodologies is lacking. METHODS: In this prospective, multicentre cohort study, we recruited patients with suspected MG to evaluate the diagnostic accuracy of cell-based assay (CBA), radioimmunoprecipitation assay (RIPA) and enzyme-linked immunosorbent assay (ELISA) in detecting AChR and MuSK autoantibodies. This study is registered with www.clinicaltrials.gov, number NCT05219097. FINDINGS: 2272 eligible participants were recruited, including 2043 MG, 229 non-MG subjects. AChR antibodies were detected in 1478, 1310, and 1280 out of a total of 2043 MG patients by CBA, RIPA, and ELISA, respectively; sensitivity, 72.3% (95% CI, 70.3-74.3), 64.1% (95% CI, 62.0-66.2), 62.7% (95% CI, 60.5-64.8); specificity, 97.8% (95% CI, 95.0-99.3), 97.8% (95% CI, 95.0-99.3), 94.8% (95% CI, 91.9-97.7). MuSK antibodies were found in 59, 50, and 54 from 2043 MG patients by CBA, RIPA and ELISA, respectively; sensitivity, 2.9% (95% CI, 2.2-3.7), 2.4% (95% CI, 1.8-3.2), 2.6% (95% CI, 2.0-3.4); specificity, 100% (95% CI, 98.4-100), 100% (95% CI, 98.4-100), and 99.1% (95% CI, 96.9-99.9). The area under the curve of AChR antibodies tested by CBA was 0.858, and there were statistical differences with RIPA (0.843; p = 0.03) and ELISA (0.809; p
Choroideremia: Toward Regulatory Approval of Retinal Gene Therapy.
Choroideremia is an X-linked inherited retinal degeneration characterized by primary centripetal degeneration of the retinal pigment epithelium (RPE), with secondary degeneration of the choroid and retina. Affected individuals experience reduced night vision in early adulthood with blindness in late middle age. The underlying CHM gene encodes REP1, a protein involved in the prenylation of Rab GTPases essential for intracellular vesicle trafficking. Adeno-associated viral gene therapy has demonstrated some benefit in clinical trials for choroideremia. However, challenges remain in gaining regulatory approval. Choroideremia is slowly progressive, which presents difficulties in demonstrating benefit over short pivotal clinical trials that usually run for 1-2 years. Improvements in visual acuity are particularly challenging due to the initial negative effects of surgical detachment of the fovea. Despite these challenges, great progress toward a treatment has been made since choroideremia was first described in 1872.
Abnormal folate metabolism causes age-, sex- and parent-of-origin-specific haematological defects in mice.
KEY POINTS: Folate (folic acid) deficiency and mutations in folate-related genes in humans result in megaloblastic anaemia. Folate metabolism, which requires the enzyme methionine synthase reductase (MTRR), is necessary for DNA synthesis and the transmission of one-carbon methyl groups for cellular methylation. In this study, we show that the hypomorphic Mtrrgt/gt mutation in mice results in late-onset and sex-specific blood defects, including macrocytic anaemia, extramedullary haematopoiesis and lymphopenia. Notably, when either parent carries an Mtrrgt allele, blood phenotypes result in their genetically wildtype adult daughters, the effects of which are parent specific. Our data establish a new model for studying the mechanism of folate metabolism in macrocytic anaemia aetiology and suggest that assessing parental folate status might be important when diagnosing adult patients with unexplained anaemia. ABSTRACT: The importance of the vitamin folate (also known as folic acid) in erythrocyte formation, maturation and/or longevity is apparent since folate deficiency in humans causes megaloblastic anaemia. Megaloblastic anaemia is a type of macrocytic anaemia whereby erythrocytes are enlarged and fewer in number. Folate metabolism is required for thymidine synthesis and one-carbon metabolism, though its specific role in erythropoiesis is not well understood. Methionine synthase reductase (MTRR) is a key enzyme necessary for the progression of folate metabolism since knocking down the Mtrr gene in mice results in hyperhomocysteinaemia and global DNA hypomethylation. We demonstrate here that abnormal folate metabolism in mice caused by Mtrrgt/gt homozygosity leads to haematopoietic phenotypes that are sex and age dependent. Specifically, Mtrrgt/gt female mice displayed macrocytic anaemia, which might be due to defective erythroid differentiation at the exclusion of haemolysis. This was associated with increased renal Epo mRNA expression, hypercellular bone marrow, and splenic extramedullary haematopoiesis. In contrast, the male response differed since Mtrrgt/gt male mice were not anaemic but did display erythrocytic macrocytosis and lymphopenia. Regardless of sex, these phenotypes were late onset. Remarkably, we also show that when either parent carries an Mtrrgt allele, a haematological defect results in their adult wildtype daughters. However, the specific phenotype was dependent upon the sex of the parent. For instance, wildtype daughters of Mtrr+/gt females displayed normocytic anaemia. In contrast, wildtype daughters of Mtrr+/gt males exhibited erythrocytic microcytosis not associated with anaemia. Therefore, abnormal folate metabolism affects adult haematopoiesis in an age-, sex- and parent-specific manner.
The global prevalence of Wilson disease from next-generation sequencing data.
PURPOSE: Wilson disease (WD) is an autosomal recessive disorder of copper metabolism, caused by pathogenic variants in ATP7B. We aimed to (1) perform a meta-analysis of previous WD prevalence estimates, (2) estimate the prevalence of WD from population sequencing data, and (3) generate an ATP7B gene variant database. METHODS: MEDLINE and EMBASE were systematically searched. Previous prevalence estimates were subjected to meta-analysis. All previously reported pathogenic ATP7B variants were compiled and annotated with gnomAD allele frequencies. Pooled global and ethnicity-specific genetic prevalences for WD were generated using the Hardy-Weinberg equation. RESULTS: Meta-analysis of genetic studies of WD prevalence gave an estimate 12.7 per 100,000 (95% confidence interval [CI]: 6.3-23.0). We developed a referenced, searchable ATP7B database comprising 11,520 variants including 782 previously reported disease variants, which can be found at http://www.wilsondisease.tk/ ; 216/782 of these were present in gnomAD, remained after filtering by allele frequency, and met American College of Medical Genetics and Genomics criteria. Based on these, the genetic prevalence of WD was 13.9 per 100,000 (95% CI: 12.9-14.9), or 1 per 7194. Combining this with 60 predicted pathogenic variants gave a birth prevalence of 15.4 per 100,000 (95% CI: 14.4-16.5). CONCLUSION: The genetic prevalence of Wilson disease may be greater than previous estimates.
The global prevalence and genetic spectrum of lysosomal acid lipase deficiency: A rare condition that mimics NAFLD.
BACKGROUND & AIMS: Lysosomal acid lipase deficiency (LAL-D) is an autosomal recessive condition that may present in a mild form (cholesteryl ester storage disease [CESD]), which mimics non-alcoholic fatty liver disease (NAFLD). It has been suggested that CESD may affect 1 in 40,000 and is under-diagnosed in NAFLD clinics. Therefore, we aimed to estimate the prevalence of LAL-D using analysis of genetic variation in LIPA. METHODS: MEDLINE and EMBASE were systematically searched for previously reported disease variants and prevalence estimates. Previous prevalence estimates were meta-analysed. Disease variants in LIPA were annotated with allele frequencies from gnomAD and combined with unreported major functional variants found in humans. Pooled ethnicity-specific prevalences for LAL-D and CESD were calculated using the Hardy-Weinberg equation. RESULTS: Meta-analysis of existing genetic studies estimated the prevalence of LAL-D as 1 per 160,000 (95% CI 1 per 65,025-761,652) using the allele frequency of c.894G>A in LIPA. A total of 98 previously reported disease variants in LIPA were identified, of which 32/98 were present in gnomAD, giving a prevalence of 1 per 307,482 (95% CI 257,672-366,865). Wolman disease was associated with more loss-of-function variants than CESD. When this was combined with 22 previously unreported major functional variants in LIPA identified in humans, the pooled prevalence of LAL-D was 1 per 177,452 (95% CI 149,467-210,683) with a carrier frequency of 1 per 421. The prevalence is lowest in those of East Asian, South Asian, and Finnish ancestry. CONCLUSION: Using 120 disease variants in LIPA, these data can reassure clinicians that LAL-D is an ultra-rare disorder. Given the therapeutic capability of sebelipase alpha, investigation for LAL-D might be included in second-line metabolic screening in NAFLD. LAY SUMMARY: Lysosomal Acid Lipase Deficiency (LAL-D) is a rare genetic condition that can cause severe liver disease, but it is difficult to diagnose and sometimes can look like simple fatty liver. It was not clear how common LAL-D was and whether many cases were being missed. To study this, we searched for all genetic mutations that could cause LAL-D, calculated how common those mutations were, and added them up. This let us estimate that LAL-D affects roughly 1 in 175,000 people. We conclude that LAL-D is a very rare condition, but it is treatable so may be included in a 'second-line' of tests for causes of fatty liver.
Development and validation of an expanded antibody toolset that captures alpha-synuclein pathological diversity in Lewy body diseases.
The abnormal aggregation and accumulation of alpha-synuclein (aSyn) in the brain is a defining hallmark of synucleinopathies. Various aSyn conformations and post-translationally modified forms accumulate in pathological inclusions and vary in abundance among these disorders. Relying on antibodies that have not been assessed for their ability to detect the diverse forms of aSyn may lead to inaccurate estimations of aSyn pathology in human brains or disease models. To address this challenge, we developed and characterized an expanded antibody panel that targets different sequences and post-translational modifications along the length of aSyn, and that recognizes all monomeric, oligomeric, and fibrillar aSyn conformations. Next, we profiled aSyn pathology across sporadic and familial Lewy body diseases (LBDs) and reveal heterogeneous forms of aSyn pathology, rich in Serine 129 phosphorylation, Tyrosine 39 nitration and N- and C-terminal tyrosine phosphorylations, scattered both to neurons and glia. In addition, we show that aSyn can become hyperphosphorylated during processes of aggregation and inclusion maturation in neuronal and animal models of aSyn seeding and spreading. The validation pipeline we describe for these antibodies paves the way for systematic investigations into aSyn pathological diversity in the human brain, peripheral tissues, as well as in cellular and animal models of synucleinopathies.