Multiomic ALS signatures highlight subclusters and sex differences suggesting the MAPK pathway as therapeutic target.

Amyotrophic lateral sclerosis (ALS) is a debilitating motor neuron disease and lacks effective disease-modifying treatments. This study utilizes a comprehensive multiomic approach to investigate the early and sex-specific molecular mechanisms underlying ALS. By analyzing the prefrontal cortex of 51 patients with sporadic ALS and 50 control subjects, alongside four transgenic mouse models (C9orf72-, SOD1-, TDP-43-, and FUS-ALS), we have uncovered significant molecular alterations associated with the disease. Here, we show that males exhibit more pronounced changes in molecular pathways compared to females. Our integrated analysis of transcriptomes, (phospho)proteomes, and miRNAomes also identified distinct ALS subclusters in humans, characterized by variations in immune response, extracellular matrix composition, mitochondrial function, and RNA processing. The molecular signatures of human subclusters were reflected in specific mouse models. Our study highlighted the mitogen-activated protein kinase (MAPK) pathway as an early disease mechanism. We further demonstrate that trametinib, a MAPK inhibitor, has potential therapeutic benefits in vitro and in vivo, particularly in females, suggesting a direction for developing targeted ALS treatments.

Exome-based gene panel analysis in a cohort of acute juvenile ischemic stroke patients:relevance of NOTCH3 and GLA variants.

BACKGROUND: Genetic variants are considered to have a crucial impact on the occurrence of ischemic stroke. In clinical routine, the diagnostic value of next-generation sequencing (NGS) in the medical clarification of acute juvenile stroke has not been investigated so far. MATERIAL AND METHODS: We analyzed an exome-based gene panel of 349 genes in 172 clinically well-characterized patients with magnetic resonance imaging (MRI)-proven, juvenile (age ≤ 55 years), ischemic stroke admitted to a single comprehensive stroke center. RESULTS: Monogenetic diseases causing ischemic stroke were observed in five patients (2.9%): In three patients with lacunar stroke (1.7%), we identified pathogenic variants in NOTCH3 causing cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Hence, CADASIL was identified at a frequency of 12.5% in the lacunar stroke subgroup. Further, in two male patients (1.2%) suffering from lacunar and cardioembolic stroke, pathogenic variants in GLA causing Fabry's disease were present. Additionally, genetic variants in monogenetic diseases lacking impact on stroke occurrence, variants of unclear significance (VUS) in monogenetic diseases, and (cardiovascular-) risk genes in ischemic stroke were observed in a total of 15 patients (15.7%). CONCLUSION: Genetic screening for Fabry's disease in cardioembolic and lacunar stroke as well as CADASIL in lacunar stroke might be beneficial in routine medical work-up of acute juvenile ischemic stroke.

Adult-Onset Neurodegeneration in Nucleotide Excision Repair Disorders (NERDND ): Time to Move Beyond the Skin.

BACKGROUND: Variants in genes of the nucleotide excision repair (NER) pathway have been associated with heterogeneous clinical presentations ranging from xeroderma pigmentosum to Cockayne syndrome and trichothiodystrophy. NER deficiencies manifest with photosensitivity and skin cancer, but also developmental delay and early-onset neurological degeneration. Adult-onset neurological features have been reported in only a few xeroderma pigmentosum cases, all showing at least mild skin manifestations. OBJECTIVE: The aim of this multicenter study was to investigate the frequency and clinical features of patients with biallelic variants in NER genes who are predominantly presenting with neurological signs. METHODS: In-house exome and genome datasets of 14,303 patients, including 3543 neurological cases, were screened for deleterious variants in NER-related genes. Clinical workup included in-depth neurological and dermatological assessments. RESULTS: We identified 13 patients with variants in ERCC4 (n = 8), ERCC2 (n = 4), or XPA (n = 1), mostly proven biallelic, including five different recurrent and six novel variants. All individuals had adult-onset progressive neurological deterioration with ataxia, dementia, and frequently chorea, neuropathy, and spasticity. Brain magnetic resonance imaging showed profound global brain atrophy in all patients. Dermatological examination did not show any skin cancer or pronounced ultraviolet damage. CONCLUSIONS: We introduce NERDND as adult-onset neurodegeneration (ND ) within the spectrum of autosomal recessive NER disorders (NERD). Our study demonstrates that NERDND is probably an underdiagnosed cause of neurodegeneration in adulthood and should be considered in patients with overlapping cognitive and movement abnormalities. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

GFPT1-Associated Congenital Myasthenic Syndrome Mimicking a Glycogen Storage Disease - Diagnostic Pitfalls in Myopathology Solved by Next-Generation-Sequencing.

GFPT1-related congenital myasthenic syndrome (CMS) is characterized by progressive limb girdle weakness, and less prominent involvement of facial, bulbar, or respiratory muscles. While tubular aggregates in muscle biopsy are considered highly indicative in GFPT1-associated CMS, excessive glycogen storage has not been described. Here, we report on three affected siblings with limb-girdle myasthenia due to biallelic pathogenic variants in GFPT1: the previously reported missense variant c.41G > A (p.Arg14Gln) and the novel truncating variant c.1265_1268del (p.Phe422TrpfsTer26). Patients showed progressive proximal atrophic muscular weakness with respiratory involvement, and a lethal disease course in adulthood. In the diagnostic workup at that time, muscle biopsy suggested a glycogen storage disease. Initially, Pompe disease was suspected. However, enzymatic activity of acid alpha-glucosidase was normal, and gene panel analysis including 38 genes associated with limb-girdle weakness (GAA included) remained unevocative. Hence, a non-specified glycogen storage myopathy was diagnosed. A decade later, the diagnosis of GFPT1-related CMS was established by genome sequencing. Myopathological reexamination showed pronounced glycogen accumulations, that were exclusively found in denervated muscle fibers. Only single fibers showed very small tubular aggregates, identified in evaluation of serial sections. This family demonstrates how diagnostic pitfalls can be addressed by an integrative approach including broad genetic analysis and re-evaluation of clinical as well as myopathological findings.

Detection of mobile elements insertions for routine clinical diagnostics in targeted sequencing data.

BACKGROUND: Targeted sequencing approaches such as gene panel or exome sequencing have become standard of care for the diagnosis of rare and common genetic disease. The detection and interpretation of point mutations, small insertions and deletions, and even exon-level copy number variants are well established in clinical genetic testing. Other types of genetic variation such as mobile elements insertions (MEIs) are technically difficult to detect. In addition, their downstream clinical interpretation is more complex compared to point mutations due to a larger genomic footprint that can not only predict a clear loss of protein function but might disturb gene regulation and splicing even when located within the non-coding regions. As a consequence, the contribution of MEIs to disease and tumor development remains largely unexplored in routine diagnostics. METHODS: In this study, we investigated the occurrence of MEIs in 7,693 exome datasets from individuals with rare diseases and healthy relatives as well as 788 cancer patients analyzed by panel sequencing. RESULTS: We present several exemplary cases highlighting the diagnostic value of MEIs and propose a strategy for the detection, prioritization, and clinical interpretation of MEIs in routine clinical diagnostics. CONCLUSION: In this paper, we state that detection and interpretation of MEIs in clinical practice in targeted NGS data can be performed relatively easy despite the fact that MEIs very rarely occur in coding parts of the human genome. Large scale reanalysis of MEIs in existing cohorts may solve otherwise unsolvable cases.

Nusinersen in adults with 5q spinal muscular atrophy: a non-interventional, multicentre, observational cohort study.

BACKGROUND: Nusinersen is approved for the treatment of 5q spinal muscular atrophy of all types and stages in patients of all ages. Although clinical trials have shown improvements in motor function in infants and children treated with the drug, data for adults are scarce. We aimed to assess the safety and efficacy of nusinersen in adults with 5q spinal muscular atrophy. METHODS: We did an observational cohort study at ten academic clinical sites in Germany. Patients with genetically confirmed 5q spinal muscular atrophy (age 16-65 years) with a homozygous deletion of exons 7, 8, or both, or with compound heterozygous mutations were eligible for inclusion and received nusinersen treatment in accordance with the label for a minimum treatment time of 6 months to a follow-up of up to 14 months. The primary outcome was the change in the total Hammersmith Functional Motor Scale Expanded (HFMSE) score, assessed at months 6, 10, and 14, and based on pre-post comparisons. This study is registered with the German Clinical Trials Register (number DRKS00015702). FINDINGS: Between July 13, 2017, and May 1, 2019, 173 patients were screened, of whom 139 (80%) were eligible for data analysis. Of these, 124 (89%) were included in the 6-month analysis, 92 (66%) in the 10-month analysis, and 57 (41%) in the 14-month analysis; patients with missing baseline HFMSE scores were excluded from these analyses. Mean HFMSE scores were significantly increased compared with baseline at 6 months (mean difference 1·73 [95% CI 1·05-2·41], p<0·0001), 10 months (2·58 [1·76-3·39], p<0·0001), and 14 months (3·12 [2·06-4·19], p<0·0001). Clinically meaningful improvements (≥3 points increase) in HFMSE scores were seen in 35 (28%) of 124 patients at 6 months, 33 (35%) of 92 at 10 months, and 23 (40%) of 57 at 14 months. To 14-month follow-up, the most frequent adverse effects among 173 patients were headache (61 [35%] patients), back pain (38 [22%]), and nausea (19 [11%]). No serious adverse events were reported. INTERPRETATION: Despite the limitations of the observational study design and a slow functional decline throughout the natural disease course, our data provide evidence for the safety and efficacy of nusinersen in the treatment of adults with 5q spinal muscular atrophy, with clinically meaningful improvements in motor function in a real-world cohort. FUNDING: None.

Radiation dose reduction for CT-guided intrathecal nusinersen administration in adult patients with spinal muscular atrophy.

Intrathecal administration of nusinersen in adult spinal muscular atrophy (SMA) patients with scoliosis and spondylodesis requires image guidance, which is preferably achieved with multi-detector computed tomography (MDCT). As long-term treatment is necessary and patients are young, radiation doses should be reduced to a minimum whilst a sufficient image quality for precise interventional performance should be kept. We compared 44 MDCT standard-dose scans (133.0-200.0 mA) with a hybrid iterative reconstruction (iDose4) to 20 low-dose scans (20.0-67.0 mA) with iterative model reconstruction (IMR), which were performed for procedure planning of intrathecal nusinersen administration in 13 adult patients with SMA and complex spinal conditions. Qualitative image evaluation, including confidence for intervention planning, was performed by two neuroradiologists for standard- and low-dose scans. All 64 MDCT-guided intrathecal administrations of nusinersen were successful. The dose length product (DLP) was significantly lower when using low-dose scanning with IMR (median DLP of standard-dose scans: 92.0 mGy•cm vs. low-dose scans: 34.5 mGy•cm; p < 0.0001). Image quality was significantly reduced for low-dose compared to standard-dose scanning. However, bone/soft tissue contrast and confidence for intervention planning were not significantly impaired in low-dose MDCT according to both readers, showing good inter-reader agreement. Thus, we hereby demonstrate a low-dose MDCT protocol combined with advanced image reconstruction for scanning during procedure planning as a viable option for image guidance in intrathecal nusinersen treatment of adult SMA patients with complex spinal conditions.

Intrathecal nusinersen administration in adult spinal muscular atrophy patients with complex spinal anatomy.

BACKGROUND: Intrathecal administration of nusinersen in adult spinal muscular atrophy (SMA) patients presents challenges owing to severe scoliosis and previous spinal surgery with metal implantation. In patients with a complex spinal situation, the potential risks of the intrathecal administration may lead to delayed treatment initiation. METHODS: In this study, we analyzed 53 CT-guided lumbar punctures of 11 adult nonambulatory SMA type 2 and 3 patients. All patients had scoliosis and six patients had previously undergone metal implantation. RESULTS: Drug administration was successful in 100% of the patients and none of the patients opted for treatment discontinuation. Complete osseous fusion precluded conventional posterior interlaminar access in eight lumbar punctures in four patients, which required alternative routes including transforaminal punctures and translaminar drilling. Median duration of all lumbar punctures was 9 min and median radiation exposure was 100 mGy* cm. The most common adverse event was post-lumbar puncture syndrome that occurred in five lumbar punctures (9.4%). CONCLUSIONS: Our data demonstrate that nusinersen can be successfully, safely, and rapidly administered in adult SMA patients with complex spinal conditions and suggest the translaminar drilling technique as an alternative delivery route. Therefore, intrathecal nusinersen treatment should not be withheld from patients because of severe spine deformities, however, drug efficacy in adult SMA patients needs to be investigated in further studies.

MYO9A deficiency in motor neurons is associated with reduced neuromuscular agrin secretion.

Congenital myasthenic syndromes (CMS) are a group of rare, inherited disorders characterized by compromised function of the neuromuscular junction, manifesting with fatigable muscle weakness. Mutations in MYO9A were previously identified as causative for CMS but the precise pathomechanism remained to be characterized. On the basis of the role of MYO9A as an actin-based molecular motor and as a negative regulator of RhoA, we hypothesized that loss of MYO9A may affect the neuronal cytoskeleton, leading to impaired intracellular transport. To investigate this, we used MYO9A-depleted NSC-34 cells (mouse motor neuron-derived cells), revealing altered expression of a number of cytoskeletal proteins important for neuron structure and intracellular transport. On the basis of these findings, the effect on protein transport was determined using a vesicular recycling assay which revealed impaired recycling of a neuronal growth factor receptor. In addition, an unbiased approach utilizing proteomic profiling of the secretome revealed a key role for defective intracellular transport affecting proper protein secretion in the pathophysiology of MYO9A-related CMS. This also led to the identification of agrin as being affected by the defective transport. Zebrafish with reduced MYO9A orthologue expression were treated with an artificial agrin compound, ameliorating defects in neurite extension and improving motility. In summary, loss of MYO9A affects the neuronal cytoskeleton and leads to impaired transport of proteins, including agrin, which may provide a new and unexpected treatment option.

Screening for lipoprotein receptor-related protein 4-, agrin-, and titin-antibodies and exploring the autoimmune spectrum in myasthenia gravis.

In autoimmune myasthenia gravis (MG), the identification of antibodies and characterization of serological subgroups is of great importance for diagnosis and management of the disease. Our aims were to study the frequency of antibodies against lipoprotein-related protein 4 (LRP4), agrin, and titin using the most recent techniques, and to characterize corresponding clinical features and autoimmune diseases (AID) in 100 MG-patients. The antibody frequencies in the 55 AChR-antibody positive patients were 7% LRP4, 5% agrin, 53% titin, and in the 45 AChR-antibody negative patients 2% MuSK, 2% LRP4, 2% agrin, and 27% titin. LRP4-MG presented late-onset age, mild symptoms, good therapeutic response, and no thymic changes. Agrin-MG showed early onset age, mild-to-severe symptoms, and moderate treatment response. The phenotype of titin-MG depended on AChR-antibodies: AChR-antibody negative patients presented with mostly mild limb muscle weakness, whereas AChR-antibody positive patients showed more frequently severe symptoms, including myasthenic crisis, bulbar predominance, and thymoma. Additional AID were detected in 32% of MG-patients, most frequently Hashimoto's thyroiditis (21%). Based on our data, we recommend the detection of LRP4-antibodies for at least AChR-antibody negative MG-patients and titin-antibodies for all MG-patients. We propose taking an accurate medical history for typical symptoms of Hashimoto's thyroiditis in MG-patients.

Tubular aggregates in autoimmune Lambert-Eaton myasthenic syndrome.

Tubular aggregates are accumulations of densely packed tubules in muscle fibers, occurring in distinct hereditary and acquired disorders. We present a patient with tubular aggregates and autoimmune Lambert-Eaton myasthenic syndrome. Initially, he showed mild proximal weakness, borderline decrement on 3 Hz stimulation, and slightly elevated creatine kinase. Muscle biopsy revealed tubular aggregates in type II fibers. Due to a good response to pyridostigmine, a limb-girdle myasthenia with tubular aggregates was suspected, but genetic analyses of GFPT1, DPGAT1, and ALG2 were normal. Two years later, the patient presented with progressive weakness and autonomic dysfunction. 17% decrement on 3 Hz stimulation and 100% increment after brief exercise were revealed. Autoantibodies to voltage-gated calcium-channels confirmed the diagnosis of Lambert-Eaton myasthenic syndrome. Steroids, azathioprine, and 3,4-diaminopyridine significantly improved symptoms. No tumor was found during follow-up. This is the first report about tubular aggregates associated with an acquired myasthenic syndrome. Our findings are important because of the therapeutic implications.