Acta Neuropathologica Communications

Repeat expansion diseases, particularly those involving GC-rich motifs, have been increasingly recognized as contributors to neurological and neuromuscular disorders. Amyotrophic lateral sclerosis (ALS) has been linked to several such expansions, including intermediate-length repeats in genes implicated in oculopharyngodistal myopathy (OPDM). To investigate the possible involvement of CGG repeat expansions in ALS, 424 ALS patients and 312 controls of Japanese descent were screened for expansions in five genes associated with repeat expansion disorders, namely GIPC1, RILPL1, FMR1, AFF2, and NUTM2BAS1. Repeat-primed PCR and fragment analysis revealed that four ALS patients exhibited abnormal CGG expansions in GIPC1 (33-55 repeats), whereas two control individuals harbored expanded alleles (67 and 83 repeats). No expansions in the other genes were detected. Long-read sequencing confirmed repeat sizes and showed sequence instability. Histopathological analysis of ALS patients with GIPC1 expansion demonstrated classical ALS pathology, including phosphorylated TDP-43-positive inclusions. RNA fluorescence in situ hybridization revealed nuclear foci containing GIPC1 repeat RNA exclusively in ALS patients with GIPC1 expansions, suggesting RNA-mediated toxicity. These findings indicate that a subset of ALS patients present with intermediate CGG expansions in GIPC1, which may represent a novel pathogenic mechanism analogous to other noncoding repeat disorders. Given that GIPC1 full expansions are associated with OPDM, these results support the hypothesis of a pathological continuum between neurodegeneration and myopathy driven by repeat length and sequence context. Nonetheless, further investigations into the potential of GIPC1 CGG expansions as genetic risk factors or modifiers in ALS are warranted.

BackgroundChitotriosidase (Chit-1) and chitinase-3-like protein 1 (CHI3L1) protein levels are increased in the cerebrospinal fluid (CSF) of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimers disease (AD). Few studies have examined the spatial expression of chitinase expressing cells with respect to neuropathologic hallmarks of disease. MethodsRNA-sequencing was used to examine Chit-1 and CHI3L1 gene expression in the spinal cord and motor cortex. Immunohistochemistry was used to characterize the distribution of Chit-1 and CHI3L1 expressing cells in ALS, C9-ALS, FTLD, AD, and non-neurologic disease controls. Immunofluorescence confocal microscopy was used to correlate distribution of Chit-1 and CHI3L1 expressing cells to TDP pathology. ResultsChit-1 gene expression was increased in the spinal cord, and CHI3L1 expression was increased in both the spinal cord and motor cortex of sALS and C9-ALS patients when compared to controls. Highest levels of Chit-1+ glia were in cortical regions that contain hallmark neuropathology for each neurodegenerative disease. CHI3L1+ glia were only significantly increased in sALS. Neither Chit-1+ nor CHI3L1+ glia were in close proximity to pTDP containing neurons in the motor cortex gray matter; however, there was a significant co-localization of glial pTDP with Chit-1 and CHI3L1 in the motor cortex white matter. ConclusionsChit-1 and CHI3L1 expressing cells were most abundant in the white matter of cortical regions affected by each neurodegenerative disease and the spinal cord. Chit-1 or CHI3L1 expressing cells in the white matter also contained phosphorylated TDP-43. We also observed correlations between levels of Chit-1 or CHI3L1 expressing cells in the white matter to disease duration. KEY MESSAGESO_ST_ABSWhat is already known on this topicC_ST_ABSPrior studies identified elevated levels of Chit-1 and CHI3L1 proteins in the CSF of various neurodegenerative conditions, though few studies examined levels of Chit-1 and CHI3L1 expressing cells both spatially and in relation to disease pathology. What this study addsWe performed an extensive spatial characterization of Chit-1 and CHI3L1 protein levels across multiple regions and neurodegenerative conditions. This study also correlates Chit-1 and CHI3L1 expression to TDP pathology and other clinical parameters of disease duration. How this study might affect research, practice or policyOur findings indicate that the majority of Chit-1 and CHI3L1 expressing glia are located in the cortical subpial layer and the white matter, suggesting a role for chitinases in modulating neuroinflammatory mechanisms or reparative/regenerative responses in the white matter of ALS and other neurodegenerative diseases. This study suggests new therapeutic opportunities for targeting chitinase expressing cells in neurodegenerative diseases.

Oculopharyngodistal myopathy is an adult-onset degenerative muscle disorder characterized by ptosis, ophthalmoplegia and weakness of the facial, pharyngeal and limb muscles. Trinucleotide repeat expansions in non-coding regions of LRP12, G1PC1and NOTCH2NLC were recently reported to be the etiologies for OPDM. However, a significant portion of OPDM patients still have unknown genetic causes. In this study, we performed long-read whole-genome sequencing in a large five-generation family of 156 individuals, including 22 patients diagnosed with typical OPDM and identified CGG repeat expansions in RILPL1 gene in all patients we tested while not in unaffected family members. Methylation analysis indicated that methylation levels of the RILPL1 gene were unaltered in OPDM patients, which was in consistent with previous reports. Our findings first provided evidences that RILPL1 were associated OPDM which we suggested as OPDM type 4.

The pathologies of oculopharyngeal muscular dystrophy (OPMD) and oculopharyngodistal myopathy (OPDM) are indistinguishable. We found that p62-positive intra-nuclear inclusions (INIs) in myonuclei (myo-INIs) were significantly more frequent in OPMD (11.4 {+/-} 4.1%, range 5.0- 17.5%) than in OPDM and other rimmed vacuolar myopathies (RVMs) (1-2% on average, range 0.0-3.5%, p<0.0001). In contrast, INIs in nonmuscle cells (nonmuscle-INIs) were present in OPDM, but absent in other RVMs, including OPMD. These results indicate that OPMD can be differentiated from OPDM and other RVMs by the frequent presence of myo-INIs ([&ge;]5%) and the absence of nonmuscle-INIs in muscle pathology.

ObjectiveTo define the diagnostic utility and clinicopathologic features correlating with anti-cytosolic 5'-nucleotidase 1A (NT5C1A) antibody positivity in idiopathic inflammatory myopathies (IIMs). Methods4987 patients had anti-NT5C1A status clinically tested between 2014 and 2019 in the Washington University neuromuscular clinical laboratory. Using clinicopathologic information available for 630 of these patients, we classified them as inclusion body myositis (IBM), dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy (IMNM), non-specific myositis, or non-inflammatory muscle diseases. ResultsAnti-NT5C1A was found in 182/287 patients with IBM (63%), 11/53 patients with dermatomyositis (21%), 7/27 patients with antisynthetase syndrome (26%), 9/76 patients with IMNM (12%), 20/84 patients with non-specific myositis (24%), and 6/103 patients with non-inflammatory muscle diseases (6%). The sensitivity and specificity of anti-NT5C1A seropositivity for the diagnosis of IBM was 63% and 85%, respectively. Anti-NT5C1A positive IBM patients had a higher frequency of finger flexion weakness (p<0.01) and their biopsies harbored more COX negative fibers (p=0.04). 18% of anti-NT5C1A negative IBM patients seroconverted to anti-NT5C1A positive on repeat testing. ConclusionsThis is the largest description of patients tested by a clinical diagnostic lab for anti-NT5C1A. We confirm the sensitivity and specificity of anti-NT5C1A for IBM and identified clinicopathologic features in IBM which correlate with anti-NT5C1A status. Notably, anti-NT5C1A testing increased both the diagnostic sensitivity and specificity of IBM when combined with patient age, gender and creatine kinase (CK) level. We propose that future IBM diagnostic criteria include anti-NT5C1A testing.

Background and objectivesTropomyosin receptor kinase A (TrkA), a high-affinity receptor for nerve growth factor (NGF), is highly expressed in nociceptive neurons and plays a key role in nociception. Increased NGF levels have been related to local angiogenesis. This study investigates skin biopsies of multiple myeloma patients who developed peripheral neuropathy during bortezomib treatment for localization and abundance of TrkA. MethodsMultiple myeloma patients with bortezomib-induced peripheral neuropathy (BIPN) were recruited at the University Hospital Wurzburg between 2021 and 2024. Age-and sex-matched controls without neuropathy were enrolled for comparison. Skin biopsies from the distal leg were analyzed to determine intraepidermal nerve fiber density (IENFD) and area, TrkA mean fluorescence intensity (MFI) and gene expression levels of TrkA and NGF. Additionally, the proximity between nerve fibers and blood vessels was measured. ResultsWe enrolled 50 patients with BIPN (age 64.1 {+/-} 9.2 years; 76% male) and 27 controls (age 60.7 {+/-} 7.1 years; 81.5% male). Sensory abnormalities included reduced sural nerve action potential amplitude (63.3%), elevated cold (36%) and heat pain detection thresholds (44%) compared to normative population values (95% CI). IENFD was reduced in patients (median 2.6 [1.6-4.2] fibers/mm) compared to controls (4.7 [3.1-6.8] fibers/mm; p < 0.05). TrkA protein abundance in epidermal nerve fibers (normalized MFI) was higher in patients (0.93 [0.56-1.25]) than controls (0.13 [0.08-0.17]; p < 0.0001) and moderately correlated with the number of bortezomib treatment cycles (r = 0.36, p < 0.05). TrkA mRNA levels did not differ between groups, while NGF mRNA levels were lower in BIPN patients (p = 0.01). Among BIPN patients, those reporting pain had higher NGF mRNA levels than those without pain (p = 0.04). Dermal vascular area was increased in BIPN patients (926.0 [714.8-1710] {micro}m2) compared to controls (705.1 [496.5-1190] {micro}m2; p < 0.05). DiscussionThis study identifies reduced IENFD, increased TrkA abundance in remaining fibers, and enhanced dermal vascularization in multiple myeloma patients with BIPN. These findings raise the possibility that dysregulated NGF/TrkA signaling may contribute to small fiber pathology and the associated alterations in sensory function observed in BIPN.

TDP-43 proteinopathy is a neuropathological hallmark of nearly all amyotrophic lateral sclerosis (ALS) and approximately half of frontotemporal dementia (FTD) cases. Nuclear loss of TDP-43 leads to widespread RNA misprocessing, such as the inclusion of cryptic exons that are no longer repressed by TDP-43. Notably, in-frame cryptic exons encode novel cryptic peptides that can be detected in biofluids, including that found in the HDGFL2 transcript. Here, we quantified HDGFL2 cryptic peptide and neurofilament light chain (NfL) in paired cerebrospinal fluid (CSF) and plasma samples from ALS and FTD patients. Cryptic HDGFL2 peptide was detected in the CSF of ALS patients, whereas no significant differences were observed between genetic and behavioral FTD subgroups. In contrast, NfL levels were elevated in both ALS and FTD, although this biomarker does not reflect TDP-43 pathology. Notably, NfL:HDGFL2 cryptic peptide ratio outperformed either marker alone in discriminating ALS and FTD cases from controls, achieving high specificity. Moreover, this ratio correlated with disease progression in ALS, suggesting added prognostic value. Collectively, our findings support the NfL:HDGFL2 cryptic peptide ratio as a promising fluid biomarker that integrates neurodegeneration with TDP-43 dysfunction, potentially improving diagnostic accuracy, disease stratification, and longitudinal monitoring in TDP-43-associated neurodegenerative disorders.

Variants of the oxygen free radical scavenging enzyme superoxide dismutase-1 (SOD1) are associated with the neurodegenerative disease amyotrophic lateral sclerosis (ALS). These variants occur in roughly 20% of familial ALS cases, and 1% of sporadic ALS cases. Here, we identified a novel SOD1 variant in a patient in their 50s who presented with movement deficiencies and neuropsychiatric features. The variant was heterozygous and resulted in the isoleucine at position 149 being substituted with a serine (I149S). In silico analysis predicted the variant to be destabilising to the SOD1 protein structure. Expression of the SOD1I149S variant with a C-terminal EGFP tag in neuronal-like NSC-34 cells resulted in extensive inclusion formation and reduced cell viability. Immunoblotting revealed that the intramolecular disulphide between Cys57 and Cys146 was fully reduced for SOD1I149S. Furthermore, SOD1I149S was highly susceptible to proteolytic digestion, suggesting a large degree of instability to the protein fold. Finally, fluorescence correlation spectroscopy and native-PAGE of cell lysates showed that SOD1I149S was monomeric in solution in comparison to the dimeric SOD1WT. This experimental data was obtained within 3 months and resulted in the rapid re-classification of the variant from a variant of unknown significance to a clinically actionable likely pathogenic variant.

ObjectiveReliable, circulating biomarkers for Duchenne, Becker and facioscapulohumeral muscular dystrophies (DBMD and FSHD) remain unvalidated. Here, we investigated the plasma extracellular vesicle (EV) proteome to identify disease-specific biomarkers that could accelerate therapy approvals. MethodsWe extracted EVs from the plasma of DBMD and FSHD patients and healthy controls using size-exclusion chromatography, conducted mass spectrometry on the extracted EV proteins, and performed comparative analysis to identify disease-specific biomarkers. We correlated the levels of these biomarkers with clinical outcome measures and confounding factors. ResultsThe muscle-associated proteins PYGM, MYOM3, FLNC, MYH2 and TTN were exclusively present in DBMD EVs. PYGM, MYOM3, and TTN negatively correlated with age. PYGM and MYOM3 levels were elevated in patients without cardiomyopathy, and PYGM levels were specifically elevated in ambulatory DMD patients. On the other hand, female FSHD patients displayed significantly higher MBL2 and lower GPLD1 levels. However, male FSHD patients exhibited higher C9 and lower C4BPB levels. Additionally, desmosome proteins JUP and DSP were uniquely found in FSHD males. MBL2 positively correlated with age and C4BPB negatively correlated with FSHD severity in male patients. InterpretationOur findings underscore the sensitivity of analyzing circulating EV content to identify disease-specific protein biomarkers for DBMD and FSHD. Our results also emphasize the potential of EV-based biomarker discovery as a promising approach to monitor disease progression as well as effectiveness of therapies in muscular dystrophy, potentially contributing to their approval. Further research with larger cohorts is needed to validate these biomarkers and explore their clinical implications.

Amyotrophic lateral sclerosis (ALS) is a neurogenerative disease resulting from progressive degeneration of motor neurons leading to systemic consequences. Despite being the most common motor neuron disease, with increasing global prevalence, limited treatment options exist. Emerging evidence from genetic studies and pathology analyses implicates RNA dysregulation in ALS pathogenesis, however, deep, comprehensive RNA sequencing studies have not been carried out. Here, we analysed >240 ALS and control whole blood transcriptome samples. Cross-sectional (Ncases=121, Ncontrols=53) and longitudinal (Nobservations=103) cohorts supported complementary expression analyses of disease mechanisms across disease stages. Both short (N=241) and long-read (N=16) technologies were utilised to discover splicing changes. Total RNA was extracted from PAXgene whole blood RNA tubes before libraries (Illumina Stranded Total RNA RiboZero Plus) were prepared and sequenced ([~]50M PE reads per sample). Long-read sequencing was performed using the Mas-Seq protocol with Kinnex full-length RNA prep kit and sequenced (PacBio Revio platform, 10M reads per sample) for full-length transcripts. Case-control cohort analyses identified 50 significantly differentially expressed genes, with pathway analyses implicating RNA processing and immune system regulation. Findings were corroborated using existing ALS RNAseq datasets from blood (correlation >0.4), iPSC-MN and post-mortem tissues. Alternative splicing (AS) analyses (LeafCutter) identified 62 clusters. Within-case analyses involved ALS cases with multiple (2-4) visits, detected 144 genes associated with disability progression over time. The long-read sequencing (Ncases=8, Ncontrols=8) provided novel discovery insights, in particular in the HLA region. This comprehensive blood-based transcriptomic dataset reveals both known and novel disease mechanisms in ALS, offering valuable insights that could inform future research and therapeutic development. The results of this study may inform and refine the prioritization of candidate genes and loci in future ALS research.

BackgroundAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by abnormal protein aggregates in motor neurons. Present and earlier proteomic studies to characterize peptides in cerebrospinal fluid (CSF) associated with motoneuron pathology did not target the low molecular weight proteins and peptides. We generated the hypothesis that specific changes in CSF peptides or low molecular weight proteins are significantly changed in ALS, and that these changes may support deciphering molecular pathophysiology and even guide approaches towards therapeutic interventions. MethodsCerebrospinal fluid (CSF) from 50 ALS patients and 50 non-ALS controls was collected, centrifuged immediately after collection, aliquoted into polypropylene test tubes, frozen within 30-40 min after the puncture and stored at -80{degrees}C until use. Peptides were sequenced using capillary electrophoresis or liquid chromatography / mass spectrometry (CE-MS/MS or LC-MS/MS) analyses. FindingsIn cerebrospinal CSF from 50 patients and 50 non-ALS controls 33 peptides were found, of which 14 could be sequenced using a non-lytic single pot proteomic detection method, CE/MS. ALS deregulated peptides vs. controls included Integral membrane protein 2B, Neurosecretory protein VGF, Osteopontin, Neuroendocrine protein 7B2 (Secretogranin-V), EGF-containing fibulin-like extracellular matrix protein 1, Xylosyltransferase 1 XT-1, Chromogranin-A, Superoxide dismutase SOD-1, Secretogranin-1 (Chromogranin B), NR2F2 Nuclear Receptor Subfamily 2 Group F Member 2 and Collagen alpha-1(VII) chain. InterpretationMost striking deregulations in CSF from ALS patients were found in VGF, Osteopontin, SOD-1 and EFEMP1 peptides. No associations of disease severity, duration and region of onset with sequenced peptides were found. Declarations of InterestS. Petri received honoraria as speaker/consultant from Biogen GmbH, Roche, Novartis, Teva, Cytokinetics Inc., Desitin, Italfarmaco, Ferrer, Amylyx, and Zambon; and grants from DGM e.V, Federal Ministry of Education and Research, German Israeli Foundation for Scientific Research and Development, EU Joint Program for Neurodegenerative Disease Research. J. Beige received funding from GSK and German Federal Ministries of Research and Health. FundingThere was no funding to the presented investigation Ethical ApprovalThis study was approved by the ethics committee of Hannover Medical School. Written informed consent was obtained from all participants in accordance with the Declaration of Helsinki. Key words: ALS, CSF, proteomics, biomarker, peptidomics, peptide deregulation

A subset of gliomas has DNA repair defects that lead to hypermutated genomes. While such tumors are resistant to alkylating chemotherapies, they may also express more mutant neoantigens on their cell surfaces, and thus be more responsive to immunotherapies. A fast, inexpensive method of screening for hypermutated gliomas would therefore be of great clinical value. Since immunohistochemistry (IHC) for the DNA mismatch repair (MMR) proteins Msh2, Msh6, Mlh1, and Pms2 is already used to screen for hypermutated colorectal cancers, we sought to determine whether that panel might have similar utility in gliomas. MMR IHC was scored in 100 WHO grade I-IV gliomas with known tumor mutation burdens (TMB), while blinded to TMB data. Eight of 100 cases showed loss of one or more MMR proteins by IHC, and all 8 were hypermutated. Among the remaining 94 gliomas with intact MMR IHC, only one was hypermutated; that tumor had an inactivating mutation in another DNA repair gene, ATM. Overall accuracy, sensitivity, and specificity were 99%, 89%, and 100%, respectively. The strongest correlates with hypermutation were prior TMZ treatment, MGMT promoter methylation, and IDH1 mutation. Among MMR-deficient hypermutated gliomas, 50% contained both MMR-lost and MMR-retained tumor cells. Together, these data suggest that MMR IHC could be a viable front-line screening test for gliomas in which immunotherapy is being considered. They also suggest that not all cells in a hypermutated glioma may actually be MMR-deficient, a finding that might need to be considered when treating such tumors with immunotherapies.

Sporadic amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disease with unknown causes and few treatment options. Here we report the first multi-dimensional single-cell landscape of the peripheral blood mononuclear cell samples from seven ALS patients representing three distinct disease stages: Early, Mid and Late. Our multipronged analyses revealed the over-activation of immune cells especially in the Mid and Late ALS stages, which is characterized by the increased cytotoxic CD8+ effector cells, expanded TCR clonotypes, upregulated oxidative phosphorylation and ribosomal genes across many cell types. More importantly, the current study discovered novel genes strongly correlated with ALS conditions, such as FOXO1 and TGFBR2 responsible for the aberrant T cell activation, and XBP1 and SPIB accountable for excessive oxidative stress. We further identified 60 genes that distinguished ALS patients from healthy donors with an unprecedented accuracy of 93% against an independent cohort of 792 specimens. Our findings provide new molecular and cellular insights into the mechanisms of ALS onset and progression, and offer novel strategies for accurate diagnosis and potential therapeutic development.

There is an unmet need for reliable biomarkers for amyotrophic lateral sclerosis (ALS). Recent studies demonstrated that the levels of the microtubule-associated protein tau are altered in plasma and cerebrospinal fluid (CSF) from people living with ALS. Our previous findings revealed that while the ratio between tau and phosphorylated tau at T181 (pTau-T181) is lower in ALS CSF compared to healthy controls (HC), higher CSF tau levels correlated with faster disease progression. Here, we measured total tau and pTau-T181 in plasma samples from two cohorts of participants with ALS and HC using two platforms: Quanterix Simoa and Meso Scale Discovery (MSD). Both assays demonstrated significantly higher pTau-T181 and pTau-T181:tau ratio levels in ALS compared to HC. Longitudinal analysis revealed that higher pTau-T181 levels at baseline correlated with faster decline on the revised ALS functional rating scale (ALSFRS-R). Although the MSD analysis revealed higher plasma tau levels in ALS, the Quanterix Simoa assay demonstrated lower total tau levels in ALS plasma than in HC. To investigate the source of this discrepancy, we measured brain-derived tau (BD-tau) using a new Quanterix assay that specifically detects tau originating from the central nervous system (CNS). This analysis demonstrated significantly elevated plasma BD-tau levels in ALS, mirroring the results obtained with the MSD assay, suggesting that the elevated plasma tau observed in ALS is primarily CNS-derived. Collectively, our data indicate that plasma BD-tau and pTau-T181 levels are elevated in ALS, and future studies will aim to define their potential utility as diagnostic or prognostic biomarkers.

Tofersen is the first effective and approved therapy for familial ALS caused by pathogenic variants in the SOD1 gene. Following treatment with tofersen, neurofilaments in patients CSF and serum display a faster response than clinical parameters, underlining their importance as a biomarker for treatment response in clinical trials. This evidence led us to hypothesize that this novel treatment might represent an opportunity to identify additional therapy-responsive biomarkers for ALS. We chose the commercial NUcleic acid Linked Immuno-Sandwich Assay (NULISA), to investigate a predefined panel of 120 neural, glial and inflammatory markers in CSF and serum samples longitudinally collected from SOD1-ALS patients at baseline and three months after tofersen treatment. We identified a set of proteins (beyond pNfH and NfL) whose levels differed between SOD1-ALS and the matched control group and that were responsive to treatment with tofersen, including A{beta}42, NPY and UCHL1. Even though our results warrant validation in larger cohorts and longer follow-up time, they may pave the way for a panel of responsive proteins solidifying biomarker endpoints in clinical trials.

Skin biopsies of patients with parkinsonism, mild cognitive impairment or dementia might offer a simple and relatively non-invasive means to determine whether -synuclein aggregates might be the underlying pathology. Accurate biomarkers are critically needed for Lewy body disease (LBD) clinical trials but currently there are none that have undergone full regulatory scrutiny. We sought to simulate the rigor of a diagnostic study done with regulatory oversight in this study of the accuracy of skin biopsies processed by -synuclein seeding amplification assays (SAA) in predicting the clinical diagnoses of Parkinson disease (PD), PD with dementia (PDD) and dementia with Lewy bodies (DLB). Our study design utilized parallel blinded performances of assays in two independent, experienced SAA assay laboratories. For control subjects without clinical LBD, we used a clinically heterogeneous set, simulating a cross-section of elderly, in comparison with previous studies that have mainly used cases and controls that have been prescreened to accentuate group differences. Subjects clinically diagnosed with PD, PDD and DLB as well as those without a suspected LBD were recruited from three sites, including the Mayo Clinic Arizona, Barrow Neurological Institute and Banner Sun Health Research Institute (BSHRI), all located in metropolitan Phoenix, Arizona. The LBD group was designated as Group 1 while the non-LBD groups were divided between a Group 2 and a Group 3, based on the absence (Group 2) or presence (Group 3) of potential clinical risk factors for LBD, including mild cognitive impairment, dementia, REM sleep behavior disorder and hyposmia. Skin punch biopsies were collected from the posterior cervical area and three biopsies were analyzed by SAA between the two labs. Sensitivity across assays ranged between 50.0% and 60.0% while specificity ranged between 69.6% and 100%. Comparisons of Group 1, with the highest clinical diagnostic confidence for the presence of an LBD, versus Group 2, with no clinical indicators of relevant abnormalities, produced the greatest specificities, between 77.3% and 100%, while sensitivities were less variable, ranging between 50% and 60% on group comparisons. Specificities were lower when Group 3 subjects, with potential LBD risk factors, were included in the calculations. Pairwise agreement between biopsy assays ranged from excellent, with a kappa of 0.816, to moderate, between 0.47 and 0.67. Agreement may have been affected by differing protocols, as substrate and testing strategies were different between the two laboratories. Also, it might be possible that variability in -synuclein seed concentrations within different skin biopsies might have contributed to differences in the outcome of the testing by the two labs. The greater agreement for Biopsy 3 was perhaps to be expected given that a single biopsy was analyzed by both labs and the homogenates were prepared by a single lab (Lab 2) and then shared between labs; homogenates for Biopsies 1 and 2 were separately prepared and separately assayed in Labs 1 and 2. A separate analysis of subjects diagnosed with PDD or DLB indicated that skin SAA may have a greater sensitivity for clinically advanced LBD. Nine of these eleven subjects had at least one positive assay, for a sensitivity of 81.82%, substantially better than the 57.1% sensitivity for probable PD subjects without dementia. This suggests that skin SAA may have greater utility as a diagnostic and progression biomarker of Lewy body dementias, as compared with PD subjects without dementia.

IntroductionSerum cardiac troponin T (cTnT) levels are elevated in the majority of amyotrophic lateral sclerosis (ALS) patients and increase over time. Neurofilament light chain (NfL) is an established therapy response biomarker in ALS as superoxide dismutase 1 (SOD1)-ALS patients treated with the antisense oligonucleotide tofersen show a decrease in NfL. In this study we assess the course of cTnT levels in SOD1-ALS at baseline and during tofersen treatment. MethodsSerum cTnT was analyzed at baseline and during tofersen treatment in 23 SOD1-ALS patients at two specialized ALS centers in Germany and compared to a control cohort of 74 ALS patients without SOD1-mutations and not treated with tofersen. ResultscTnT levels increased in the control ALS-cohort over time (p<0.0001) but not in the tofersen group (p=0.36). CK, and CK-MB levels did not show relevant changes over time. The median monthly increase of cTnT was 0.045 points (IQR 0.02-0.08) in the control ALS cohort and 0.01 points (IQR -0.01-0.03) in the tofersen group (p=0.0013). The fold change in cTnT levels of the tofersen-treated cohort (median 1.2; IQR 0.77-1.59) was significantly less and even showed a reduction in some patients compared to the control group (median 1.89; IQR 1.35-2.75) (p=0.0003). DiscussionIn this study, we find a response signal of cTnT to tofersen treatment, which supports the value of cTnT as an independent biomarker in ALS. These results contribute to the notion that cTnT may provide additional value as a progression and treatment response biomarker in ALS complementary to NfL and warrant further investigations. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/25322198v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1c4870aorg.highwire.dtl.DTLVardef@17ddef4org.highwire.dtl.DTLVardef@b86903org.highwire.dtl.DTLVardef@f67176_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background and ObjectivesSporadic inclusion body myositis (IBM) is the most common acquired myopathy in individuals over age 50. The disorder is slowly progressive and while many therapies have been investigated, response has generally been poor. Clinical heterogeneity may influence treatment responsiveness; however, data regarding heterogeneity in IBM is limited and often conflicting. We aim to identify clinically distinct subgroups within a large IBM cohort, as well as prognostic factors for disease progression. MethodsClinical, histologic, radiologic, and electrophysiologic data were analyzed for all patients with IBM and other forms of myositis enrolled in a longitudinal cohort from The Johns Hopkins Myositis Center from 2003-2018. Univariate, multivariate, and graphical analyses were used to identify prognostic factors in IBM patients. ResultsAmong the 335 IBM patients meeting inclusion criteria, 64% were male with an average age of disease onset of 58.7 years and a delay to diagnosis of 5.2 years. Initial misdiagnosis (52%) and immunosuppressant treatment (42%) were common. Less than half (43%) of muscle biopsies demonstrated all three pathologic hallmarks: endomysial inflammation, mononuclear cell invasion, and rimmed vacuoles. Black patients had significantly weaker arm abductors, hip flexors, and knee flexors compared to non-Black patients but were less likely to develop dysphagia. Female patients had stronger finger flexors and knee extensors compared to their male counterparts but were more likely to develop dysphagia. A significant number (20%) of patients had an age of onset less than 50 years. This group of younger patients was weaker at their first visit; however, this may be accounted for by a longer disease duration at first visit. DiscussionAlthough IBM has long been considered a disorder predominately of older, White men, female, and non-White patients comprise a significant proportion of the IBM population. Our study demonstrates that female and Black patients have distinct clinical phenotypes within the overarching IBM clinical phenotype.

BackgroundHereditary transthyretin-mediated (hATTR) amyloidosis is a rare, progressively debilitating, and fatal disease caused by deposition of aggregated transthyretin amyloid in multiple organs and tissues. Highly variable disease penetrance has made it difficult to predict disease onset and progression. Clinically validated, non-invasive plasma biomarkers may facilitate earlier diagnosis and aid monitoring of disease progression. MethodsPlasma levels of >1000 proteins were measured in patients with hATTR amyloidosis with polyneuropathy who received either placebo or patisiran in the phase 3 APOLLO study (NCT01960348) and in a cohort of healthy individuals. The impact of patisiran treatment on the time profile of each protein was determined by a linear mixed model at 0, 9, and 18 months. Neurofilament light chain (NfL) protein was further assessed using an orthogonal quantitative approach. FindingsA significant change in the levels of 66 proteins was observed with patisiran vs placebo, with change in NfL, a marker of neuronal damage, most significant (p<10-20). Analysis of the changes in protein levels demonstrated that the proteome of patients treated with patisiran trended towards healthy individuals at 18 months. Plasma NfL levels in healthy controls were four-fold lower than in patients with hATTR amyloidosis with polyneuropathy (16{middle dot}3 [SD 12{middle dot}0] pg/mL vs 69{middle dot}4 [SD 42{middle dot}1] pg/mL, p<10-16). Levels of NfL at 18 months increased with placebo (99{middle dot}5 [SD 60{middle dot}1] pg/mL) and decreased with patisiran treatment (48{middle dot}8 [SD 29{middle dot}9] pg/mL). At 18 months, improvement in modified Neuropathy Impairment Score+7 (mNIS+7) in patisiran-treated patients significantly correlated with a reduction in NfL levels (R=0{middle dot}43, p<10-7). InterpretationThe observed correlation of NfL reduction with patisiran treatment and improvement in mNIS+7 suggests it may serve as a biomarker of nerve damage and polyneuropathy in hATTR amyloidosis. This biomarker may enable earlier diagnosis of polyneuropathy in patients with hATTR amyloidosis and facilitate monitoring of disease progression.

BackgroundChitinases, including chitotriosidase (CHIT1) and chitinase-3-like protein 1 (CHI3L1), are markers of neuroinflammation, a key process in amyotrophic lateral sclerosis (ALS). Tear fluid (TF) can be collected non-invasively and may represent a promising alternative to CSF or blood to study chitinases. MethodsTF was collected from 50 ALS patients and 50 control subjects using Schirmer strips. CHIT1 and CHI3L1 levels in TF, serum, and CSF were quantified using ELISA. Serum NfL was measured using SIMOA. The frequency of a 24 bp-duplication polymorphism in the CHIT1 gene influencing CHIT1 expression was assessed by PCR. ResultsNo group differences in the distribution of the CHIT1 polymorphism were detected. Carriers of the polymorphism in both ALS and controls showed lower CHIT1 levels in serum and TF. CHI3L1 levels in TF were higher in ALS patients compared to controls (p = 0.007), consistent with changes in CSF but not serum. In ALS, males showed higher TF CHIT1-values compared to females (p = 0.009). Combining TF chitinase values with serum NfL values improved discrimination between ALS and controls. ConclusionsChitinases are detectable in TF, and CHI3L1 levels recapitulate changes observed in CSF, highlighting its potential for non-invasive longitudinal assessment. Furthermore, chitinase values in TF, together with serum NfL, may act complementary by capturing distinct aspects of the disease, neuroinflammation and axonal damage. These results suggest TF chitinases and serum NfL could complementarily contribute to the diagnosis and monitoring of the disease, and call for further evaluation of TF as a biomarker source in ALS.