Recurrent ATP1A1 variant Gly903Arg causes developmental delay, intellectual disability, and autism.

ATP1A1 encodes a sodium-potassium ATPase that has been linked to several neurological diseases. Using exome and genome sequencing, we identified the heterozygous ATP1A1 variant NM_000701.8: c.2707G>A;p.(Gly903Arg) in two unrelated children presenting with delayed motor and speech development and autism. While absent in controls, the variant occurred de novo in one proband and co-segregated in two affected half-siblings, with mosaicism in the healthy mother. Using a specific ouabain resistance assay in mutant transfected HEK cells, we found significantly reduced cell viability. Demonstrating loss of ATPase function, we conclude that this novel variant is pathogenic, expanding the phenotype spectrum of ATP1A1.

[Neuropathic pain as a symptom in autonomic neuropathies and other rare diseases : Small fiber neuropathy: its recognition, diagnosis, and treatment]. / Neuropathische Schmerzen als Symptom bei autonomen Neuropathien und anderen seltenen Erkrankungen : Kleinfaserneuropathie erkennen, diagnostizieren und behandeln.

BACKGROUND: Neuropathic pain is difficult to diagnose and treat. Small fiber neuropathy (SFN) flies under the radar of nerve conduction studies. OBJECTIVES: The importance of a structured patient history and physical examination in the context of neuropathic pain is emphasized. Describing SFN as an important cause, the authors consider rare but partially treatable differential diagnoses. They conclude that autonomic symptoms are frequently associated, often presenting with diverse symptoms. METHODS: A selective literature research to present SFN symptoms as well as differential diagnostic and therapeutic steps in the context of SFN and rare diseases focusing on the autonomic nervous system. RESULTS: Neuropathic pain significantly reduces quality of life. To shorten the time until diagnosis and to initiate therapy, the authors recommend a structured patient history including sensory plus and minus symptoms and non-specific autonomic signs. If the initial search for the cause is not successful, rare causes such as treatable transthyretin (ATTR) amyloidosis and Fabry's disease or autoimmune causes should be considered, particularly in the case of progressive and/or autonomic symptoms. CONCLUSION: The diagnosis and therapy of rare SFN requires interdisciplinary collaboration and, in many cases, a referral to specialized centers to achieve the best patient care.

A modelling study to dissect the potential role of voltage-gated ion channels in activity-dependent conduction velocity changes as identified in small fiber neuropathy patients.

Objective: Patients with small fiber neuropathy (SFN) suffer from neuropathic pain, which is still a therapeutic problem. Changed activation patterns of mechano-insensitive peripheral nerve fibers (CMi) could cause neuropathic pain. However, there is sparse knowledge about mechanisms leading to CMi dysfunction since it is difficult to dissect specific molecular mechanisms in humans. We used an in-silico model to elucidate molecular causes of CMi dysfunction as observed in single nerve fiber recordings (microneurography) of SFN patients. Approach: We analyzed microneurography data from 97 CMi-fibers from healthy individuals and 34 of SFN patients to identify activity-dependent changes in conduction velocity. Using the NEURON environment, we adapted a biophysical realistic preexisting CMi-fiber model with ion channels described by Hodgkin-Huxley dynamics for identifying molecular mechanisms leading to those changes. Via a grid search optimization, we assessed the interplay between different ion channels, Na-K-pump, and resting membrane potential. Main results: Changing a single ion channel conductance, Na-K-pump or membrane potential individually is not sufficient to reproduce in-silico CMi-fiber dysfunction of unchanged activity-dependent conduction velocity slowing and quicker normalization of conduction velocity after stimulation as observed in microneurography. We identified the best combination of mechanisms: increased conductance of potassium delayed-rectifier and decreased conductance of Na-K-pump and depolarized membrane potential. When the membrane potential is unchanged, opposite changes in Na-K-pump and ion channels generate the same effect. Significance: Our study suggests that not one single mechanism accounts for pain-relevant changes in CMi-fibers, but a combination of mechanisms. A depolarized membrane potential, as previously observed in patients with neuropathic pain, leads to changes in the contribution of ion channels and the Na-K-pump. Thus, when searching for targets for the treatment of neuropathic pain, combinations of several molecules in interplay with the membrane potential should be regarded.

The phenotypic spectrum of pathogenic ATP1A1 variants expands: the novel p.P600R substitution causes demyelinating Charcot-Marie-Tooth disease.

BACKGROUND: Charcot-Marie-Tooth disease (CMT) is a genetically and clinically heterogeneous group of inherited neuropathies. Monoallelic pathogenic variants in ATP1A1 were associated with axonal and intermediate CMT. ATP1A1 encodes for the catalytic α1 subunit of the Na+/ K+ ATPase. Besides neuropathy, other associated phenotypes are spastic paraplegia, intellectual disability, and renal hypomagnesemia. We hereby report the first demyelinating CMT case due to a novel ATP1A1 variant. METHODS: Whole-exome sequencing on the patient's genomic DNA and Sanger sequencing to validate and confirm the segregation of the identified p.P600R ATP1A1 variation were performed. To evaluate functional effects, blood-derived mRNA and protein levels of ATP1A1 and the auxiliary ß1 subunit encoded by ATP1B1 were investigated. The ouabain-survival assay was performed in transfected HEK cells to assess cell viability, and two-electrode voltage clamp studies were performed in Xenopus oocytes. RESULTS: The variant was absent in the local and global control datasets, falls within a highly conserved protein position, and is in a missense-constrained region. The expression levels of ATP1A1 and ATP1B1 were significantly reduced in the patient compared to healthy controls. Electrophysiology indicated that ATP1A1p.P600R injected Xenopus oocytes have reduced Na+/ K+ ATPase function. Moreover, HEK cells transfected with a construct encoding ATP1A1p.P600R harbouring variants that confers ouabain insensitivity displayed a significant decrease in cell viability after ouabain treatment compared to the wild type, further supporting the pathogenicity of this variant. CONCLUSION: Our results further confirm the causative role of ATP1A1 in peripheral neuropathy and broaden the mutational and phenotypic spectrum of ATP1A1-associated CMT.

BiP inactivation due to loss of the deAMPylation function of FICD causes a motor neuron disease.

PURPOSE: The chaperone protein BiP is the master regulator of the unfolded protein response in the endoplasmic reticulum. BiP chaperone activity is regulated by the post-translational modification AMPylation, exclusively provided by FICD. We investigated whether FICD variants identified in patients with motor neuron disease could interfere with BiP activity regulation. METHODS: Exome sequencing was performed to identify causative pathogenic variants associated with motor neuron diseases. Functional studies were conducted on fibroblasts from patients to explore the molecular mechanism of the disease. RESULTS: We identified biallelic variants in FICD causing a neurodegenerative disease of upper and lower motor neurons. Affected individuals harbor a specific missense variant, Arg374His, positioned in the catalytic motif of the enzyme and important for adenosine triphosphate binding. The mutated residue abolishes intramolecular interaction with the regulatory residue Glu234, essential to inhibit AMPylation and to promote de-AMPylation by FICD. Consequently, fibroblasts from patients with FICD variants have abnormally increased levels of AMPylated and thus inactivated BiP. CONCLUSION: Loss of BiP chaperone activity in patients likely results in a chronic impairment of the protein quality control system in the endoplasmic reticulum. These findings will guide the development of therapeutic strategies for motoneuron and related diseases linked to proteotoxic stress.

Deoxy-sphingolipids, oxidative stress, and vitamin C correlate with qualitative and quantitative patterns of small fiber dysfunction and degeneration.

ABSTRACT: Defined by dysfunction or degeneration of Aδ and C fibers, small fiber neuropathies (SFNs) entail a relevant health burden. In 50% of cases, the underlying cause cannot be identified or treated. In 100 individuals (70% female individuals; mean age: 44.8 years) with an idiopathic, skin biopsy-confirmed SFN, we characterized the symptomatic spectrum and measured markers of oxidative stress (vitamin C, selenium, and glutathione) and inflammation (transforming growth factor beta, tumor necrosis factor alpha), as well as neurotoxic 1-deoxy-sphingolipids. Neuropathic pain was the most abundant symptom (95%) and cause of daily life impairment (72%). Despite the common use of pain killers (64%), the painDETECT questionnaire revealed scores above 13 points in 80% of patients. In the quantitative sensory testing (QST), a dysfunction of Aδ fibers was observed in 70% and of C fibers in 44%, affecting the face, hands, or feet. Despite normal nerve conduction studies, QST revealed Aß fiber involvement in 46% of patients' test areas. Despite absence of diabetes mellitus or mutations in SPTLC1 or SPTLC2 , plasma 1-deoxy-sphingolipids were significantly higher in the sensory loss patient cluster when compared with those in patients with thermal hyperalgesia ( P < 0.01) or those in the healthy category ( P < 0.1), correlating inversely with the intraepidermal nerve fiber density (1-deoxy-SA: P < 0.05, 1-deoxy-SO: P < 0.01). Patients with arterial hypertension, overweight (body mass index > 25 kg/m 2 ), or hyperlipidemia showed significantly lower L-serine (arterial hypertension: P < 0.01) and higher 1-deoxy-sphingolipid levels (arterial hypertension: P < 0.001, overweight: P < 0.001, hyperlipidemia: P < 0.01). Lower vitamin C levels correlated with functional Aß involvement ( P < 0.05). Reduced glutathione was lower in patients with Aδ dysfunction ( P < 0.05). Idiopathic SFNs are heterogeneous. As a new pathomechanism, plasma 1-deoxy-sphingolipids might link the metabolic syndrome with small fiber degeneration.

De Novo ATP1A1 Variants in an Early-Onset Complex Neurodevelopmental Syndrome.

ATP1A1 encodes the α1 subunit of the sodium-potassium ATPase, an electrogenic cation pump highly expressed in the nervous system. Pathogenic variants in other subunits of the same ATPase, encoded by ATP1A2 or ATP1A3, are associated with syndromes such as hemiplegic migraine, dystonia, or cerebellar ataxia. Worldwide, only 16 families have been reported carrying pathogenic ATP1A1 variants to date. Associated phenotypes are axonal neuropathies, spastic paraplegia, and hypomagnesemia with seizures and intellectual disability. By whole exome or genome sequencing, we identified 5 heterozygous ATP1A1 variants, c.674A>G;p.Gln225Arg, c.1003G>T;p.Gly335Cys, c.1526G>A;p.Gly509Asp, c.2152G>A;p.Gly718Ser, and c.2768T>A;p.Phe923Tyr, in 5 unrelated children with intellectual disability, spasticity, and peripheral, motor predominant neuropathy. Additional features were sensory loss, sleep disturbances, and seizures. All variants occurred de novo and are absent from control populations (MAF GnomAD = 0). Affecting conserved amino acid residues and constrained regions, all variants have high pathogenicity in silico prediction scores. In HEK cells transfected with ouabain-insensitive ATP1A1 constructs, cell viability was significantly decreased in mutants after 72h treatment with the ATPase inhibitor ouabain, demonstrating loss of ATPase function. Replicating the haploinsufficiency mechanism of disease with a gene-specific assay provides pathogenicity information and increases certainty in variant interpretation. This study further expands the genotype-phenotype spectrum of ATP1A1.

Are we creating a new phenotype? Physiological barriers and ethical considerations in the treatment of hereditary transthyretin-amyloidosis.

Hereditary transthyretin (TTR) amyloidosis (ATTRv) is an autosomal dominant, systemic disease transmitted by amyloidogenic mutations in the TTR gene. To prevent the otherwise fatal disease course, TTR stabilizers and mRNA silencing antisense drugs are currently approved treatment options. With 90% of the amyloidogenic protein produced by the liver, disease progression including polyneuropathy and cardiomyopathy, the two most prominent manifestations, can successfully be halted by hepatic drug targeting or-formerly-liver transplantation. Certain TTR variants, however, favor disease manifestations in the central nervous system (CNS) or eyes, which is mostly associated with TTR production in the choroid plexus and retina. These compartments cannot be sufficiently reached by any of the approved medications. From liver-transplanted patients, we have learned that with longer lifespans, such CNS manifestations become more relevant over time, even if the underlying TTR mutation is not primarily associated with such. Are we therefore creating a new phenotype? Prolonging life will most likely lead to a shift in the phenotypic spectrum, enabling manifestations like blindness, dementia, and cerebral hemorrhage to come out of the disease background. To overcome the first therapeutic limitation, the blood-brain barrier, we might be able to learn from other antisense drugs currently being used in research or even being approved for primary neurodegenerative CNS diseases like spinal muscular atrophy or Alzheimer's disease. But what effects will unselective CNS TTR knock-down have considering its role in neuroprotection? A potential approach to overcome this second limitiation might be allele-specific targeting, which is, however, still far from clinical trials. Ethical standpoints underline the need for seamless data collection to enable more evidence-based decisions and for thoughtful consenting in research and clinical practice. We conclude that the current advances in treating ATTRv amyloidosis have become a meaningful example for mechanism-based treatment. With its great success in improving patient life spans, we will still have to face new challenges including shifts in the phenotype spectrum and the ongoing need for improved treatment precision. Further investigation is needed to address these closed barriers and open questions.

New Keys to Early Diagnosis: Muscle Echogenicity, Nerve Ultrasound Patterns, Electrodiagnostic, and Clinical Parameters in 150 Patients with Hereditary Polyneuropathies.

Hereditary neuropathies are of variable genotype and phenotype. With upcoming therapies, there is urgent need for early disease recognition and outcome measures. High-resolution nerve and muscle ultrasound is a dynamic, non-invasive, well-established tool in the field of inflammatory and traumatic neuropathies. In this study, we defined nerve and muscle ultrasound parameters as recognition and progression markers in 150 patients with genetically confirmed hereditary neuropathies, including Charcot-Marie-Tooth (CMT) disease (CMT1A, n = 55; other CMT1/4, n = 28; axonal CMT, n = 15; CMTX, n = 15), hereditary neuropathy with liability to pressure palsies (HNPP, n = 16), hereditary transthyretin-amyloidosis (ATTRv, n = 14), and Fabry's disease (n = 7). The CMT1A, followed by the CMT1/4 group, had the most homogeneous enlargement of the nerve cross-sectional areas (CSA) in the ultrasound pattern sum (UPSS) and homogeneity score. Entrapment scores were highest in HNPP, ATTRv amyloidosis, and Fabry's disease patients. In demyelinating neuropathies, the CSA correlated inversely with nerve conduction studies. The muscle echo intensity was significantly highest in the clinically most affected muscles, which was independent from the underlying disease cause and correlated with muscle strength and disease duration. Further correlations were seen with combined clinical (CMTES-2) and electrophysiological (CMTNS-2) scores of disease severity. We conclude that nerve ultrasound is a helpful tool to distinguish different types of hereditary neuropathies by pattern recognition, whereas muscle ultrasound is an objective parameter for disease severity. The implementation of neuromuscular ultrasound might enrich diagnostic procedures both in clinical routines and research.

Progressive multifocal leukoencephalopathy and immune reconstitution inflammatory syndrome in seven patients with sarcoidosis: a critical discussion of treatment and prognosis.

Progressive multifocal leukoencephalopathy (PML) is a subacute brain infection by the opportunistic John Cunningham (JC) virus. Herein, we describe seven patients with PML, lymphopenia, and sarcoidosis, in three of whom PML was the first manifestation of sarcoidosis. At onset, the clinical picture comprised rapidly progressive spastic hemi- or limb pareses as well as disturbances of vision, speech, and orientation. Cerebral magnetic resonance imaging showed T2-hyperintense, confluent, mainly supratentorial lesions. Four patients developed punctate contrast enhancement as a radiological sign of an immune reconstitution inflammatory syndrome (IRIS), three of them having a fatal course. In the cerebrospinal fluid, the initial JC virus load (8-25,787 copies/ml) did not correlate with interindividual severity; however, virus load corresponded to clinical dynamics. Brain biopsies (n = 2), performed 2 months after symptom onset, showed spotted demyelination and microglial activation. All patients had lymphopenia in the range of 270-1150/µl. To control JC virus, three patients received a combination of mirtazapine and mefloquine, another two patients additionally took cidofovir. One patient was treated with cidofovir only, and one patient had a combined regimen with mirtazapine, mefloquine, cidofovir, intravenous interleukin 2, and JC capsid vaccination. To treat sarcoidosis, the four previously untreated patients received prednisolone. Three patients had taken immunosuppressants prior to PML onset, which were subsequently stopped as a potential accelerator of opportunistic infections. After 6-54 months of follow up, three patients reached an incomplete recovery, one patient progressed, but survived so far, and two patients died. One further patient was additionally diagnosed with lung cancer, which he died from after 24 months. We conclude that the combination of PML and sarcoidosis is a diagnostic and therapeutic challenge. PML can occur as the first sign of sarcoidosis without preceding immunosuppressive treatment. The development of IRIS might be an indicator of poor outcome.

Chance or challenge, spoilt for choice? New recommendations on diagnostic and therapeutic considerations in hereditary transthyretin amyloidosis with polyneuropathy: the German/Austrian position and review of the literature.

Hereditary transthyretin amyloidosis is caused by pathogenic variants (ATTRv) in the TTR gene. Alongside cardiac dysfunction, the disease typically manifests with a severely progressive sensorimotor and autonomic polyneuropathy. Three different drugs, tafamidis, patisiran, and inotersen, are approved in several countries, including the European Union and the United States of America. By stabilizing the TTR protein or degrading its mRNA, all types of treatment aim at preventing amyloid deposition and stopping the otherwise fatal course. Therefore, it is of utmost importance to recognize both onset and progression of neuropathy as early as possible. To establish recommendations for diagnostic and therapeutic procedures in the follow-up of both pre-symptomatic mutation carriers and patients with manifest ATTRv amyloidosis with polyneuropathy, German and Austrian experts elaborated a harmonized position. This paper is further based on a systematic review of the literature. Potential challenges in the early recognition of disease onset and progression are the clinical heterogeneity and the subjectivity of sensory and autonomic symptoms. Progression cannot be defined by a single test or score alone but has to be evaluated considering various disease aspects and their dynamics over time. The first-line therapy should be chosen based on individual symptom constellations and contra-indications. If symptoms worsen, this should promptly implicate to consider optimizing treatment. Due to the rareness and variability of ATTRv amyloidosis, the clinical course is most importantly directive in doubtful cases. Therefore, a systematic follow-up at an experienced center is crucial to identify progression and reassure patients and carriers.

Targeting transthyretin - Mechanism-based treatment approaches and future perspectives in hereditary amyloidosis.

The liver-derived, circulating transport protein transthyretin (TTR) is the cause of systemic hereditary (ATTRv) and wild-type (ATTRwt) amyloidosis. TTR stabilization and knockdown are approved therapies to mitigate the otherwise lethal disease course. To date, the variety in phenotypic penetrance is not fully understood. This systematic review summarizes the current literature on TTR pathophysiology with its therapeutic implications. Tetramer dissociation is the rate-limiting step of amyloidogenesis. Besides destabilizing TTR mutations, other genetic (RBP4, APCS, AR, ATX2, C1q, C3) and external (extracellular matrix, Schwann cell interaction) factors influence the type of onset and organ tropism. The approved small molecule tafamidis stabilizes the tetramer and significantly decelerates the clinical course. By sequence-specific mRNA knockdown, the approved small interfering RNA (siRNA) patisiran and antisense oligonucleotide (ASO) inotersen both significantly reduce plasma TTR levels and improve neuropathy and quality of life compared to placebo. With enhanced hepatic targeting capabilities, GalNac-conjugated siRNA and ASOs have recently entered phase III clinical trials. Bivalent TTR stabilizers occupy both binding groves in vitro, but have not been tested in trials so far. Tolcapone is another stabilizer with the potential to cross the blood-brain barrier, but its half-life is short and liver failure a potential side effect. Amyloid-directed antibodies and substances like doxycycline aim at reducing the amyloid load, however, none of the yet developed antibodies has successfully passed clinical trials. ATTR-amyloidosis has become a model disease for pathophysiology-based treatment. Further understanding of disease mechanisms will help to overcome the remaining limitations, including application burden, side effects, and blood-brain barrier permeability.

Immunoglobulins to mitigate paraneoplastic Lambert Eaton Myasthenic Syndrome under checkpoint inhibition in Merkel cell carcinoma.

Lambert-Eaton myasthenic syndrome (LEMS) is a rare, autoimmune or paraneoplastic condition characterized by muscle weakness and fatigability. In cancer therapy, immune checkpoint inhibitors (ICI) sensitize the immune system for tumor antigens. We report a 62-year-old, female patient with paraneoplastic LEMS as first manifestation of Merkel cell carcinoma. Under avelumab, the LEMS exacerbated with worsening of limb weakness and a severely reduced vital capacity (< 1 l). To treat this immunological side effect, we added a regimen with intravenous immunoglobulins. Hereby, the LEMS improved significantly. As we were able to continue the cancer treatment, the Merkel cell carcinoma has been in remission so far. This is the first description of paraneoplastic LEMS, avelumab, and Merkel cell carcinoma. We conclude that immunoglobulins are an option to control an ICI-associated deterioration of paraneoplastic symptoms.

Role of high-resolution ultrasound in detection and monitoring of peripheral nerve tumor burden in neurofibromatosis in children.

PURPOSE: Peripheral nerve sheath tumors are hallmark findings in neurofibromatosis types 1 and 2. With increasing size, they typically lead to neurological symptoms, and NF1 patients have a lifetime risk of 8-13% for developing malignant peripheral nerve sheath tumors. Medical imaging is therefore highly needed for early detection and exact localization of symptomatic or potentially malignant tumors. This review will give an overview of the ultrasound characteristics of peripheral nerve sheath tumors and findings in patients with neurofibromatosis types 1 and 2. METHODS: A systematic search of electronic databases, reference lists, and unpublished literature was conducted including the keywords "schwannoma," "neurofibroma," "neurofibromatosis," "benign and malignant peripheral nerve sheath tumor." RESULTS: The high-resolution allows a clear analysis of tumor echotexture, definition of margins, and the relation to the parent nerve. The use of color duplex/Doppler and contrast agent adds valuable information for the differentiation of benign and malignant tumors. CONCLUSION: High-resolution ultrasound is a well-established, non-invasive, and easily repeatable first-line tool in diagnostic procedures of soft tissue tumors.

Diagnostic hallmarks and pitfalls in late-onset progressive transthyretin-related amyloid-neuropathy.

Familial amyloid polyneuropathy (FAP) is a progressive systemic autosomal dominant disease caused by pathogenic mutations in the transthyretin (TTR) gene. We studied clinical, electrophysiological, histopathological, and genetic characteristics in 15 (13 late-onset and two early-onset) patients belonging to 14 families with polyneuropathy and mutations in TTR. In comparison, we analysed the features of nine unrelated patients with an idiopathic polyneuropathy, in whom TTR mutations have been excluded. Disease occurrence was familial in 36 % of the patients with TTR-associated polyneuropathy and the late-onset type was observed in 86 % (mean age at onset 65.5 years). Clinically, all late-onset TTR-mutant patients presented with distal weakness, pansensory loss, absence of deep tendon reflexes, and sensorimotor hand involvement. Afferent-ataxic gait was present in 92 % leading to wheelchair dependence in 60 % after a mean duration of 4.6 years. Autonomic involvement was observed in 60 %, and ankle edema in 92 %. The sensorimotor polyneuropathy was from an axonal type in 82 %, demyelinating or mixed type in 9 % each. Compared to the TTR-unmutated idiopathic polyneuropathy patients, we identified rapid progression, early ambulatory loss, and autonomic disturbances, associated with a severe polyneuropathy as red flags for TTR-FAP. In 18 % of the late-onset TTR-FAP patients, no amyloid was found in nerve biopsies. Further diagnostic pitfalls were unspecific electrophysiology, and coincident diabetes mellitus (23 %) or monoclonal gammopathy (7 %). We conclude that a rapid disease course, severely ataxic gait, hand involvement, and autonomic dysfunction are diagnostic hallmarks of late-onset TTR-FAP. Genetic analysis should be performed even when amyloid deposits are lacking or when polyneuropathy-causing comorbidities are concomitant.