Variants in tropomyosins TPM2 and TPM3 causing muscle hypertonia.

Patients with myopathies caused by pathogenic variants in tropomyosin genes TPM2 and TPM3 usually have muscle hypotonia and weakness, their muscle biopsies often showing fibre size disproportion and nemaline bodies. Here, we describe a series of patients with hypercontractile molecular phenotypes, high muscle tone, and mostly non-specific myopathic biopsy findings without nemaline bodies. Three of the patients had trismus, whilst in one patient, the distal joints of her fingers flexed on extension of the wrists. In one biopsy from a patient with a rare TPM3 pathogenic variant, cores and minicores were observed, an unusual finding in TPM3-caused myopathy. The variants alter conserved contact sites between tropomyosin and actin.

Effectiveness of clinical exome sequencing in adult patients with difficult-to-diagnose neurological disorders.

OBJECTIVES: Clinical diagnostics in adults with hereditary neurological diseases is complicated by clinical and genetic heterogeneity, as well as lifestyle effects. Here, we evaluate the effectiveness of exome sequencing and clinical costs in our difficult-to-diagnose adult patient cohort. Additionally, we expand the phenotypic and genetic spectrum of hereditary neurological disorders in Finland. METHODS: We performed clinical exome sequencing (CES) to 100 adult patients from Finland with neurological symptoms of suspected genetic cause. The patients were classified as myopathy (n = 57), peripheral neuropathy (n = 16), ataxia (n = 15), spastic paraplegia (n = 4), Parkinsonism (n = 3), and mixed (n = 5). In addition, we gathered the costs of prior diagnostic work-up to retrospectively assess the cost-effectiveness of CES as a first-line diagnostic tool. RESULTS: The overall diagnostic yield of CES was 27%. Pathogenic variants were found for 14 patients (in genes ANO5, CHCHD10, CLCN1, DES, DOK7, FKBP14, POLG, PYROXD1, SCN4A, TUBB3, and TTN) and likely pathogenic previously undescribed variants for 13 patients (in genes ABCD1, AFG3L2, ATL1, CACNA1A, COL6A1, DYSF, IRF2BPL, KCNA1, MT-ATP6, SAMD9L, SGCB, and TPM2). Age of onset below 40 years increased the probability of finding a genetic cause. Our cost evaluation of prior diagnostic work-up suggested that early CES would be cost-effective in this patient group, in which diagnostic costs increase linearly with prolonged investigations. CONCLUSIONS: Based on our results, CES is a cost-effective, powerful first-line diagnostic tool in establishing the molecular diagnosis in adult neurological patients with variable symptoms. Importantly, CES can markedly shorten the diagnostic odysseys of about one third of patients.

Threshold of heteroplasmic truncating MT-ATP6 mutation in reprogramming, Notch hyperactivation and motor neuron metabolism.

Mutations in mitochondrial DNA encoded subunit of ATP synthase, MT-ATP6, are frequent causes of neurological mitochondrial diseases with a range of phenotypes from Leigh syndrome and NARP to ataxias and neuropathies. Here we investigated the functional consequences of an unusual heteroplasmic truncating mutation m.9154C>T in MT-ATP6, which caused peripheral neuropathy, ataxia and IgA nephropathy. ATP synthase not only generates cellular ATP, but its dimerization is required for mitochondrial cristae formation. Accordingly, the MT-ATP6 truncating mutation impaired the assembly of ATP synthase and disrupted cristae morphology, supporting our molecular dynamics simulations that predicted destabilized a/c subunit subcomplex. Next, we modeled the effects of the truncating mutation using patient-specific induced pluripotent stem cells. Unexpectedly, depending on mutation heteroplasmy level, the truncation showed multiple threshold effects in cellular reprogramming, neurogenesis and in metabolism of mature motor neurons (MN). Interestingly, MN differentiation beyond progenitor stage was impaired by Notch hyperactivation in the MT-ATP6 mutant, but not by rotenone-induced inhibition of mitochondrial respiration, suggesting that altered mitochondrial morphology contributed to Notch hyperactivation. Finally, we also identified a lower mutation threshold for a metabolic shift in mature MN, affecting lactate utilization, which may be relevant for understanding the mechanisms of mitochondrial involvement in peripheral motor neuropathies. These results establish a critical and disease-relevant role for ATP synthase in human cell fate decisions and neuronal metabolism.

Neurofilament Light Regulates Axon Caliber, Synaptic Activity, and Organelle Trafficking in Cultured Human Motor Neurons.

Neurofilament light (NFL) is one of the proteins forming multimeric neuron-specific intermediate filaments, neurofilaments, which fill the axonal cytoplasm, establish caliber growth, and provide structural support. Dominant missense mutations and recessive nonsense mutations in the neurofilament light gene (NEFL) are among the causes of Charcot-Marie-Tooth (CMT) neuropathy, which affects the peripheral nerves with the longest axons. We previously demonstrated that a neuropathy-causing homozygous nonsense mutation in NEFL led to the absence of NFL in patient-specific neurons. To understand the disease-causing mechanisms, we investigate here the functional effects of NFL loss in human motor neurons differentiated from induced pluripotent stem cells (iPSC). We used genome editing to generate NEFL knockouts and compared them to patient-specific nonsense mutants and isogenic controls. iPSC lacking NFL differentiated efficiently into motor neurons with normal axon growth and regrowth after mechanical axotomy and contained neurofilaments. Electrophysiological analysis revealed that motor neurons without NFL fired spontaneous and evoked action potentials with similar characteristics as controls. However, we found that, in the absence of NFL, human motor neurons 1) had reduced axonal caliber, 2) the amplitude of miniature excitatory postsynaptic currents (mEPSC) was decreased, 3) neurofilament heavy (NFH) levels were reduced and no compensatory increases in other filament subunits were observed, and 4) the movement of mitochondria and to a lesser extent lysosomes was increased. Our findings elaborate the functional roles of NFL in human motor neurons. NFL is not only a structural protein forming neurofilaments and filling the axonal cytoplasm, but our study supports the role of NFL in the regulation of synaptic transmission and organelle trafficking. To rescue the NFL deficiency in the patient-specific nonsense mutant motor neurons, we used three drugs, amlexanox, ataluren (PTC-124), and gentamicin to induce translational read-through or inhibit nonsense-mediated decay. However, the drugs failed to increase the amount of NFL protein to detectable levels and were toxic to iPSC-derived motor neurons.

ALS and Parkinson's disease genes CHCHD10 and CHCHD2 modify synaptic transcriptomes in human iPSC-derived motor neurons.

Mitochondrial intermembrane space proteins CHCHD2 and CHCHD10 have roles in motor neuron diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy and axonal neuropathy and in Parkinson's disease. They form a complex of unknown function. Here we address the importance of these two proteins in human motor neurons. We show that gene edited human induced pluripotent stem cells (iPSC) lacking either CHCHD2 or CHCHD10 are viable and can be differentiated into functional motor neurons that fire spontaneous and evoked action potentials. Mitochondria in knockout iPSC and motor neurons sustain ultrastructure but show increased proton leakage and respiration, and reciprocal compensatory increases in CHCHD2 or CHCHD10. Knockout motor neurons have largely overlapping transcriptome profiles compared to isogenic control line, in particular for synaptic gene expression. Our results show that the absence of either CHCHD2 or CHCHD10 alters mitochondrial respiration in human motor neurons, inducing similar compensatory responses. Thus, pathogenic mechanisms may involve loss of synaptic function resulting from defective energy metabolism.

Recessive PYROXD1 mutations cause adult-onset limb-girdle-type muscular dystrophy.

OBJECTIVE: To describe adult-onset limb-girdle-type muscular dystrophy caused by biallelic variants in the PYROXD1 gene, which has been recently linked to early-onset congenital myofibrillar myopathy. METHODS: Whole exome sequencing was performed for adult-onset neuromuscular disease patients with no molecular diagnosis. Patients with PYROXD1 variants underwent clinical characterization, lower limb muscle MRI, muscle biopsy and spirometry. A yeast complementation assay was used to determine the biochemical consequences of the genetic variants. RESULTS: We identified four patients with biallelic PYROXD1 variants. Three patients, who had symptom onset in their 20s or 30s, were homozygous for the previously described p.Asn155Ser. The fourth patient, with symptom onset at age 49, was compound heterozygous for p.Asn155Ser variant and previously unknown p.Tyr354Cys. All patients presented with a LGMD-type phenotype of symmetric muscle weakness and wasting. Symptoms started in proximal muscles of the lower limbs, and progressed slowly to involve also upper limbs in a proximal-predominant fashion. All patients remained ambulant past the age of 60. They had restrictive lung disease but no cardiac impairment. Muscle MRI showed strong involvement of anterolateral thigh muscles. Muscle biopsy displayed chronic myopathic changes. Yeast complementation assay demonstrated the p.Tyr354Cys mutation to impair PYROXD1 oxidoreductase ability. CONCLUSION: PYROXD1 variants can cause an adult-onset slowly progressive LGMD-type phenotype.

Absence of NEFL in patient-specific neurons in early-onset Charcot-Marie-Tooth neuropathy.

OBJECTIVE: We used patient-specific neuronal cultures to characterize the molecular genetic mechanism of recessive nonsense mutations in neurofilament light (NEFL) underlying early-onset Charcot-Marie-Tooth (CMT) disease. METHODS: Motor neurons were differentiated from induced pluripotent stem cells of a patient with early-onset CMT carrying a novel homozygous nonsense mutation in NEFL. Quantitative PCR, protein analytics, immunocytochemistry, electron microscopy, and single-cell transcriptomics were used to investigate patient and control neurons. RESULTS: We show that the recessive nonsense mutation causes a nearly total loss of NEFL messenger RNA (mRNA), leading to the complete absence of NEFL protein in patient's cultured neurons. Yet the cultured neurons were able to differentiate and form neuronal networks and neurofilaments. Single-neuron gene expression fingerprinting pinpointed NEFL as the most downregulated gene in the patient neurons and provided data of intermediate filament transcript abundancy and dynamics in cultured neurons. Blocking of nonsense-mediated decay partially rescued the loss of NEFL mRNA. CONCLUSIONS: The strict neuronal specificity of neurofilament has hindered the mechanistic studies of recessive NEFL nonsense mutations. Here, we show that such mutation leads to the absence of NEFL, causing childhood-onset neuropathy through a loss-of-function mechanism. We propose that the neurofilament accumulation, a common feature of many neurodegenerative diseases, mimics the absence of NEFL seen in recessive CMT if aggregation prevents the proper localization of wild-type NEFL in neurons. Our results suggest that the removal of NEFL as a proposed treatment option is harmful in humans.

Increased circulating concentrations of mesencephalic astrocyte-derived neurotrophic factor in children with type 1 diabetes.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) was recently shown to be essential for the survival and proliferation of pancreatic ß-cells in mice, where deletion of MANF resulted in diabetes. The current study aimed at determining whether the concentration of circulating MANF is associated with the clinical manifestation of human type 1 diabetes (T1D). MANF expression in T1D or MANF levels in serum have not been previously studied. We developed an enzyme-linked immunosorbent assay (ELISA) for MANF and measured serum MANF concentrations from 186 newly diagnosed children and adolescents and 20 adults with longer-term T1D alongside with age-matched controls. In healthy controls the mean serum MANF concentration was 7.0 ng/ml. High MANF concentrations were found in children 1-9 years of age close to the diagnosis of T1D. The increased MANF concentrations were not associated with diabetes-predictive autoantibodies and autoantibodies against MANF were extremely rare. Patients with conspicuously high MANF serum concentrations had lower C-peptide levels compared to patients with moderate MANF concentrations. Our data indicate that increased MANF concentrations in serum are associated with the clinical manifestation of T1D in children, but the exact mechanism behind the increase remains elusive.