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ObjectiveWe 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.MethodsMotor 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.ResultsWe 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.ConclusionsThe 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.

Background: Tibial muscular dystrophy (TMD), a late-onset dominant distal myopathy, is caused by yet unknown mutations on chromosome 2q, whereas MD with myositis (MDM) is a muscular dystrophy of the mouse, also progressing with age and linked to mouse chromosome 2. For both disorders, linkage studies have implicated titin as a potential candidate gene.Methods: The authors analyzed major candidate regions in the titin gene by sequencing and Southern blot hybridization, and performed titin immunohistochemistry on TMD patient material to identify the underlying mutation. Western blot studies were performed on the known titin ligands in muscle samples of both disorders and controls, and analysis of apoptosis was also performed.Results: The authors identified almost complete loss of calpain3, a ligand of titin, in the patient with limb-girdle MD (LGMD) with a homozygous state of TMD haplotype when primary calpain3 gene defect was excluded. Apoptotic myonuclei with altered distribution of transcription factor NF-kB and its inhibitor IkBα were encountered in muscle samples of patients with either heterozygous or homozygous TMD haplotype. Similar findings were confirmed in the MDM mouse.Conclusions: These results imply that titin mutations may be responsible for TMD, and that the pathophysiologic pathway following calpain3 deficiency may overlap with LGMD2A. The loss of calpain3 could be a downstream effect of the deficient TMD gene product. The significance of the secondary calpain3 defect for the pathogenesis of TMD was emphasized by similar calpain3 deficiency in the MDM mouse, which is suggested to be a mouse model for TMD. Homozygous mutation at the 2q locus may thus be capable of producing yet another LGMD.

Objective: To elaborate the diagnostic methods used as “gold standard” in one of the most common glycogen storage diseases (GSDs), Tarui disease (GSDVII). Methods: Two siblings with disease suggestive of GSD underwent thorough clinical analysis, including muscle biopsy, muscle MRI, exercise tests, laboratory examinations, and wholeexome sequencing (WES). Results: Both siblings had juvenile-onset exercise intolerance with cramping and infrequent myoglobinuria. Muscle biopsy showed extralysosomal glycogen accumulation, but because of normal phosphofructokinase histochemistry, GSDVII was thought to be excluded. However, WES revealed a causative homozygous PFKM gene defect, R39Q, in both siblings, establishing the diagnosis of GSDVII, which was confirmed by very low residual phosphofructo-1-kinase (PFK) enzyme activity in biochemical studies. Conclusions: We suggest that in patients with suspicion of GSD and extralysosomal glycogen accumulation, biochemical activity assay of PFK followed by molecular genetics should be performed even when enzyme histochemistry is normal. Neurol Genet 2015;1:e7; doi: 10.1212/ NXG.0000000000000007

Objective: We describe the phenotype consistent with axonal Charcot-Marie-Tooth disease type 2 (CMT2) in 4 families with a c.197G>T (p.(Gly66Val)) variant in CHCHD10 . Methods: We sequenced the CHCHD10 gene in a cohort of 107 families with CMT2 of unknown etiology. The patients were characterized by clinical examination and electroneuromyography. Muscle MRI and biopsy of the muscle or nerve were performed in selected cases. Neuropathologic autopsy was performed in 1 case. Results: The c.197G>T variant in CHCHD10 was found in 6 families, 4 of which included multiple individuals available for detailed clinical study. Variants in this gene have recently been associated with amyotrophic lateral sclerosis-frontotemporal dementia, mitochondrial myopathy, or spinal muscular atrophy Jokela type (SMAJ), but not with CMT2. Our patients had a late-onset distal axonal neuropathy with motor predominance, progressing to involve sensory nerves. Neurophysiologic and neuropathologic studies confirmed the diagnosis of sensorimotor axonal neuropathy with no loss of anterior horn neurons. Muscle biopsies showed occasional cytochrome c oxidase–negative fibers, combined with small amounts of mitochondrial DNA deletions. Conclusions: CHCHD10 c.197G>T (p.(Gly66Val)) is a cause of sensorimotor axonal neuropathy. This gene should be considered in patients presenting with a pure CMT2 phenotype, particularly when motor symptoms predominate.