Proteomic studies in VWA1-related neuromyopathy allowed new pathophysiological insights and the definition of blood biomarkers.

Bi-allelic variants in VWA1, encoding Von Willebrand Factor A domain containing 1 protein localized to the extracellular matrix (ECM), were linked to a neuromuscular disorder with manifestation in child- or adulthood. Clinical findings indicate a neuromyopathy presenting with muscle weakness. Given that pathophysiological processes are still incompletely understood, and biomarkers are still missing, we aimed to identify blood biomarkers of pathophysiological relevance: white blood cells (WBC) and plasma derived from six VWA1-patients were investigated by proteomics. Four proteins, BET1, HNRNPDL, NEFM and PHGDH, known to be involved in neurological diseases and dysregulated in WBC were further validated by muscle-immunostainings unravelling HNRNPDL as a protein showing differences between VWA1-patients, healthy controls and patients suffering from neurogenic muscular atrophy and BICD2-related neuromyopathy. Immunostaining studies of PHGDH indicate its involvement in apoptotic processes via co-localisation with caspase-3. NEFM showed an increase in cells within the ECM in biopsies of all patients studied. Plasma proteomics unravelled dysregulation of 15 proteins serving as biomarker candidates among which a profound proportion of increased ones (6/11) are mostly related to antioxidative processes and have even partially been described as blood biomarkers for other entities of neuromuscular disorders before. CRP elevated in plasma also showed an increase in the extracellular space of VWA1-mutant muscle. Results of our combined studies for the first time describe pathophysiologically relevant biomarkers for VWA1-related neuromyopathy and suggest that VWA1-patient derived blood might hold the potential to study disease processes of clinical relevance, an important aspect for further preclinical studies.

Alteration of LARGE1 abundance in patients and a mouse model of 5q-associated spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by recessive pathogenic variants affecting the survival of motor neuron (SMN1) gene (localized on 5q). In consequence, cells lack expression of the corresponding protein. This pathophysiological condition is clinically associated with motor neuron (MN) degeneration leading to severe muscular atrophy. Additionally, vulnerability of other cellular populations and tissues including skeletal muscle has been demonstrated. Although the therapeutic options for SMA have considerably changed, treatment responses may differ thus underlining the persistent need for validated biomarkers. To address this need and to identify novel marker proteins for SMA, we performed unbiased proteomic profiling on cerebrospinal fluid derived (CSF) from genetically proven SMA type 1-3 cases and afterwards performed ELISA studies on CSF and serum samples to validate the potential of a novel biomarker candidates in both body fluids. To further decipher the pathophysiological impact of this biomarker, immunofluorescence studies were carried out on spinal cord and skeletal muscle derived from a 5q-SMA mouse model. Proteomics revealed increase of LARGE1 in CSF derived from adult patients showing a clinical response upon treatment with nusinersen. Moreover, LARGE1 levels were validated in CSF samples of further SMA patients (type 1-3) by ELISA. These studies also unveiled a distinguishment between groups in improvement of motor skills: adult patients do present with lowered level per se at baseline visit while no elevation upon treatment in the pediatric cohort can be observed. ELISA-based studies of serum samples showed no changes in the pediatric cohort but unraveled elevated level in adult patients responding to future intervention with nusinersen, while non-responders did not show a significant increase. Additional immunofluorescence studies of LARGE1 in MN and skeletal muscle of a SMA type 3 mouse model revealed an increase of LARGE1 during disease progression. Our combined data unraveled LARGE1 as a protein dysregulated in serum and CSF of SMA-patients (and in MN and skeletal muscle of SMA mice) holding the potential to serve as a disease marker for SMA and enabling to differentiate between patients responding and non-responding to therapy with nusinersen.

A Homozygous NDUFS6 Variant Associated with Neuropathy and Optic Atrophy.

Background: The NADH dehydrogenase [ubiquinone] iron-sulfur protein 6 (NDUFS6) gene encodes for an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (complex I). Bi-allelic NDUFS6 variants have been linked with a severe disorder mostly reported as a lethal infantile mitochondrial disease (LMID) or Leigh syndrome (LS). Objective: Here, we identified a homozygous variant (c.309 + 5 G > A) in NDUFS6 in one male patient with axonal neuropathy accompanied by loss of small fibers in skin biopsy and further complicated by optic atrophy and borderline intellectual disability. Methods: To address the pathogenicity of the variant, biochemical studies (mtDNA copy number quantification, ELISA, Proteomic profiling) of patient-derived leukocytes were performed. Results: The analyses revealed loss of NDUFS6 protein associated with a decrease of three further mitochondrial NADH dehydrogenase subunit/assembly proteins (NDUFA12, NDUFS4 and NDUFV1). Mitochondrial copy number is not altered in leukocytes and the mitochondrial biomarker GDF15 is not significantly changed in serum. Conclusions: Hence, our combined clinical and biochemical data strengthen the concept of NDUFS6 being causative for a very rare form of axonal neuropathy associated with optic atrophy and borderline intellectual disability, and thus expand (i) the molecular genetic landscape of neuropathies and (ii) the clinical spectrum of NDUFS6-associated phenotypes.

Mutant SPART causes defects in mitochondrial protein import and bioenergetics reversed by Coenzyme Q.

Pathogenic variants in SPART cause Troyer syndrome, characterized by lower extremity spasticity and weakness, short stature and cognitive impairment, and a severe mitochondrial impairment. Herein, we report the identification of a role of Spartin in nuclear-encoded mitochondrial proteins. SPART biallelic missense variants were detected in a 5-year-old boy with short stature, developmental delay and muscle weakness with impaired walking distance. Patient-derived fibroblasts showed an altered mitochondrial network, decreased mitochondrial respiration, increased mitochondrial reactive oxygen species and altered Ca2+ versus control cells. We investigated the mitochondrial import of nuclear-encoded proteins in these fibroblasts and in another cell model carrying a SPART loss-of-function mutation. In both cell models the mitochondrial import was impaired, leading to a significant decrease in different proteins, including two key enzymes involved in CoQ10 (CoQ) synthesis, COQ7 and COQ9, with a severe reduction in CoQ content, versus control cells. CoQ supplementation restored cellular ATP levels to the same extent shown by the re-expression of wild-type SPART, suggesting CoQ treatment as a promising therapeutic approach for patients carrying mutations in SPART.

The emerging spectrum of fetal acetylcholine receptor antibody-related disorders (FARAD).

In utero exposure to maternal antibodies targeting the fetal acetylcholine receptor isoform (fAChR) can impair fetal movement, leading to arthrogryposis multiplex congenita (AMC). Fetal AChR antibodies have also been implicated in apparently rare, milder myopathic presentations termed fetal acetylcholine receptor inactivation syndrome (FARIS). The full spectrum associated with fAChR antibodies is still poorly understood. Moreover, since some mothers have no myasthenic symptoms, the condition is likely underreported, resulting in failure to implement effective preventive strategies. Here we report clinical and immunological data from a multicentre cohort (n = 46 cases) associated with maternal fAChR antibodies, including 29 novel and 17 previously reported with novel follow-up data. Remarkably, in 50% of mothers there was no previously established myasthenia gravis (MG) diagnosis. All mothers (n = 30) had AChR antibodies and, when tested, binding to fAChR was often much greater than that to the adult AChR isoform. Offspring death occurred in 11/46 (23.9%) cases, mainly antenatally due to termination of pregnancy prompted by severe AMC (7/46, 15.2%), or during early infancy, mainly from respiratory failure (4/46, 8.7%). Weakness, contractures, bulbar and respiratory involvement were prominent early in life, but improved gradually over time. Facial (25/34; 73.5%) and variable peripheral weakness (14/32; 43.8%), velopharyngeal insufficiency (18/24; 75%) and feeding difficulties (16/36; 44.4%) were the most common sequelae in long-term survivors. Other unexpected features included hearing loss (12/32; 37.5%), diaphragmatic paresis (5/35; 14.3%), CNS involvement (7/40; 17.5%) and pyloric stenosis (3/37; 8.1%). Oral salbutamol used empirically in 16/37 (43.2%) offspring resulted in symptom improvement in 13/16 (81.3%). Combining our series with all previously published cases, we identified 21/85 mothers treated with variable combinations of immunotherapies (corticosteroids/intravenous immunoglobulin/plasmapheresis) during pregnancy either for maternal MG symptom control (12/21 cases) or for fetal protection (9/21 cases). Compared to untreated pregnancies (64/85), maternal treatment resulted in a significant reduction in offspring deaths (P < 0.05) and other complications, with treatment approaches involving intravenous immunoglobulin/ plasmapheresis administered early in pregnancy most effective. We conclude that presentations due to in utero exposure to maternal (fetal) AChR antibodies are more common than currently recognized and may mimic a wide range of neuromuscular disorders. Considering the wide clinical spectrum and likely diversity of underlying mechanisms, we propose 'fetal acetylcholine receptor antibody-related disorders' (FARAD) as the most accurate term for these presentations. FARAD is vitally important to recognize, to institute appropriate management strategies for affected offspring and to improve outcomes in future pregnancies. Oral salbutamol is a symptomatic treatment option in survivors.

Cerebrospinal fluid findings in patients with myelin oligodendrocyte glycoprotein (MOG) antibodies. Part 2: Results from 108 lumbar punctures in 80 pediatric patients.

BACKGROUND: New-generation, cell-based assays have demonstrated a robust association of serum autoantibodies to full-length human myelin oligodendrocyte glycoprotein (MOG-IgG) with (mostly recurrent) optic neuritis, myelitis, and brainstem encephalitis, as well as with neuromyelitis optica (NMO)-like or acute-disseminated encephalomyelitis (ADEM)-like presentations. However, only limited data are yet available on cerebrospinal fluid (CSF) findings in MOG-IgG-associated encephalomyelitis (MOG-EM; also termed MOG antibody-associated disease, MOGAD). OBJECTIVE: To describe systematically the CSF profile in children with MOG-EM. MATERIAL AND METHODS: Cytological and biochemical findings (including white cell counts [WCC] and differentiation; frequency and patterns of oligoclonal bands; IgG/IgM/IgA and albumin concentrations and CSF/serum ratios; intrathecal IgG/IgM/IgA fractions; locally produced IgG/IgM/IgA concentrations; immunoglobulin class patterns; IgG/IgA/IgM reibergrams; Link index; measles/rubella/zoster [MRZ] reaction; other anti-viral and anti-bacterial antibody indices; CSF total protein; CSF L-lactate) from 108 lumbar punctures in 80 pediatric patients of mainly Caucasian descent with MOG-EM were analyzed retrospectively. RESULTS: Most strikingly, CSF-restricted oligoclonal IgG bands, a hallmark of multiple sclerosis (MS), were absent in 89% of samples (N = 96), and the MRZ reaction, the most specific laboratory marker of MS known so far, in 100% (N = 29). If present at all, intrathecal IgG synthesis was low, often transient and mostly restricted to acute attacks. Intrathecal IgM synthesis was present in 21% and exclusively detectable during acute attacks. CSF WCC were elevated in 54% of samples (median 40 cells/µl; range 6-256; mostly lymphocytes and monocytes; > 100/µl in 11%). Neutrophils were present in 71% of samples; eosinophils, activated lymphocytes, and plasma cells were seen only rarely (all < 7%). Blood-CSF barrier dysfunction (as indicated by an elevated albumin CSF/serum ratio) was present in 46% of all samples (N = 79) and at least once in 48% of all patients (N = 67) tested. CSF alterations were significantly more frequent and/or more pronounced in patients with acute spinal cord or brain disease than in patients with acute ON and varied strongly depending on attack severity. CSF L-lactate levels correlated significantly with the spinal cord lesions load (measured in vertebral segments) in patients with acute myelitis (p = 0.0099). An analysis of pooled data from the pediatric and the adult cohort showed a significant relationship of QAlb (p < 0.0005), CST TP (p < 0.0001), and CSF L-lactate (p < 0.0003) during acute attacks with age. CONCLUSION: MOG-IgG-associated EM in children is characterized by CSF features that are distinct from those in MS. With regard to most parameters, no marked differences between the pediatric cohort and the adult cohort analyzed in Part 1 were noted. Our findings are important for the differential diagnosis of pediatric MS and MOG-EM and add to the understanding of the immunopathogenesis of this newly described autoimmune disease.

The Influence of BIS Monitoring on Sedative Dose in Pediatric Patients Undergoing Open Muscle Biopsies - a Randomized Controlled Trial.

BACKGROUND: The use of Propofol and Remifentanil for analgosedation in children is common and safe. For sedation monitoring clinical scores as Comfort Score (CS) as well as bispectral index (BIS) are frequently applied. The impact of BIS for sedation monitoring in pediatric patients is still under debate. This prospective study aims to investigate whether dual sedation monitoring of CS and BIS compared with monitoring of CS alone during muscle biopsies in children can reduce sedative doses, reduce awakening time and prevent complications. METHODS: 50 pediatric patients requiring sedation for open muscle biopsy were prospectively enrolled. Analgosedation was performed with remifentanil and propofol. Patients were randomly assigned to 2 groups: In 25 patients, sedation was monitored using CS alone, and in 25 patients CS and BIS monitoring were simultaneously applied. The primary outcome was the propofol dose applied during muscle biopsy. Secondary outcome parameters were recovery time and the frequency of complications. RESULTS: The median CS during the intervention in both groups was equal (11, P=1.000). The median BIS in group 2 was 59. No complications occurred in either group. There was no difference in propofol dose in either group (8.4 vs. 7.2 mg/kg/h; P=0.58) and no difference in the duration until eye opening (9 vs. 11 min; P=0.081). CONCLUSION: For children undergoing minor surgical procedures under analgosedation, BIS monitoring does not affect the sedative dose, the time until eye opening or the frequency of complications.

SUCLA2 Deficiency: A Deafness-Dystonia Syndrome with Distinctive Metabolic Findings (Report of a New Patient and Review of the Literature).

SUCLA2 encodes for a subunit of succinyl-coenzyme A synthase, the enzyme that reversibly synthesises succinyl-coenzyme A and ATP from succinate, coenzyme A and ADP in the Krebs cycle. Disruption of SUCLA2 function can lead to mitochondrial DNA depletion. Patients with a SUCLA2 mutation present with a rare but distinctive deafness-dystonia syndrome. Additionally, they exhibit elevated levels of the characteristic biochemical markers: methylmalonate, C4-dicarboxylic carnitine and lactate are increased in both plasma and urine. Thus far, eight different disease-causing SUCLA2 mutations, of which six missense mutations and two splice site mutations, have been described in the literature. Here, we present the first patient with an intragenic deletion in SUCLA2 and review the patients described in literature.

NDUFS8-related Complex I Deficiency Extends Phenotype from "PEO Plus" to Leigh Syndrome.

With over 1,000 nuclear genes that could potentially cause a mitochondrial disorder, the current diagnostic approach requires targeted molecular analysis, guided by a combination of clinical and biochemical features. However, the expanding molecular and clinical spectrum means that this approach does not always yield a result. Here we report the unusual clinical presentation of "Progressive External Ophthalmoplegia (PEO) plus" Leigh syndrome in three children from a consanguineous family where exome sequencing identified mutations in NDUFS8. NDUFS8 is a nuclear-encoded structural core protein of complex I, and mutations are expected to cause infantile onset and severe disease. Our patients had a later onset, milder and a clinically distinct phenotype, and this gene would not normally be considered in this context. Being untargeted to specific genes, whole exome analysis has the potential to re-write the phenotype and reveal an unexpected molecular aetiology, as illustrated by this family.

Glucose transporter-1 deficiency syndrome: the expanding clinical and genetic spectrum of a treatable disorder.

Glucose transporter-1 deficiency syndrome is caused by mutations in the SLC2A1 gene in the majority of patients and results in impaired glucose transport into the brain. From 2004-2008, 132 requests for mutational analysis of the SLC2A1 gene were studied by automated Sanger sequencing and multiplex ligation-dependent probe amplification. Mutations in the SLC2A1 gene were detected in 54 patients (41%) and subsequently in three clinically affected family members. In these 57 patients we identified 49 different mutations, including six multiple exon deletions, six known mutations and 37 novel mutations (13 missense, five nonsense, 13 frame shift, four splice site and two translation initiation mutations). Clinical data were retrospectively collected from referring physicians by means of a questionnaire. Three different phenotypes were recognized: (i) the classical phenotype (84%), subdivided into early-onset (<2 years) (65%) and late-onset (18%); (ii) a non-classical phenotype, with mental retardation and movement disorder, without epilepsy (15%); and (iii) one adult case of glucose transporter-1 deficiency syndrome with minimal symptoms. Recognizing glucose transporter-1 deficiency syndrome is important, since a ketogenic diet was effective in most of the patients with epilepsy (86%) and also reduced movement disorders in 48% of the patients with a classical phenotype and 71% of the patients with a non-classical phenotype. The average delay in diagnosing classical glucose transporter-1 deficiency syndrome was 6.6 years (range 1 month-16 years). Cerebrospinal fluid glucose was below 2.5 mmol/l (range 0.9-2.4 mmol/l) in all patients and cerebrospinal fluid : blood glucose ratio was below 0.50 in all but one patient (range 0.19-0.52). Cerebrospinal fluid lactate was low to normal in all patients. Our relatively large series of 57 patients with glucose transporter-1 deficiency syndrome allowed us to identify correlations between genotype, phenotype and biochemical data. Type of mutation was related to the severity of mental retardation and the presence of complex movement disorders. Cerebrospinal fluid : blood glucose ratio was related to type of mutation and phenotype. In conclusion, a substantial number of the patients with glucose transporter-1 deficiency syndrome do not have epilepsy. Our study demonstrates that a lumbar puncture provides the diagnostic clue to glucose transporter-1 deficiency syndrome and can thereby dramatically reduce diagnostic delay to allow early start of the ketogenic diet.