Cryptic Amyloidogenic Elements in the 3' UTRs of Neurofilament Genes Trigger Axonal Neuropathy.

Abnormal protein aggregation is observed in an expanding number of neurodegenerative diseases. Here, we describe a mechanism for intracellular toxic protein aggregation induced by an unusual mutation event in families affected by axonal neuropathy. These families carry distinct frameshift variants in NEFH (neurofilament heavy), leading to a loss of the terminating codon and translation of the 3' UTR into an extra 40 amino acids. In silico aggregation prediction suggested the terminal 20 residues of the altered NEFH to be amyloidogenic, which we confirmed experimentally by serial deletion analysis. The presence of this amyloidogenic motif fused to NEFH caused prominent and toxic protein aggregates in transfected cells and disrupted motor neurons in zebrafish. We identified a similar aggregation-inducing mechanism in NEFL (neurofilament light) and FUS (fused in sarcoma), in which mutations are known to cause aggregation in Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis, respectively. In summary, we present a protein-aggregation-triggering mechanism that should be taken into consideration during the evaluation of stop-loss variants.

Absence of HINT1 mutations in a UK and Spanish cohort of patients with inherited neuropathies.

Biallelic mutations in the HINT1 gene were recently identified as the cause of axonal neuropathy with neuromyotonia. It has been suggested that HINT1 mutations may indeed account for 11% of all inherited neuropathies with autosomal recessive inheritance. However, 81% of patients HINT1-related neuropathies reported to date are originally from five European countries and the global prevalence of the disorder is still unknown. In our study, we aimed to determine the frequency of HINT1 mutations by direct sequencing in a cohort of 152 patients with inherited neuropathies from the UK and Spain, where no cases have been described to date. We failed to identify patients with clinical myotonia, neuromyotonia or pathogenic mutations in HINT1. Our results support that HINT1-related neuropathies are not homogeneously distributed among European populations, which may be explained by founder effects. This geographical variability also underlines the importance of considering the ethnic background when screening for mutations in neuropathy-related genes.

Truncating and missense mutations in IGHMBP2 cause Charcot-Marie Tooth disease type 2.

Using a combination of exome sequencing and linkage analysis, we investigated an English family with two affected siblings in their 40s with recessive Charcot-Marie Tooth disease type 2 (CMT2). Compound heterozygous mutations in the immunoglobulin-helicase-µ-binding protein 2 (IGHMBP2) gene were identified. Further sequencing revealed a total of 11 CMT2 families with recessively inherited IGHMBP2 gene mutations. IGHMBP2 mutations usually lead to spinal muscular atrophy with respiratory distress type 1 (SMARD1), where most infants die before 1 year of age. The individuals with CMT2 described here, have slowly progressive weakness, wasting and sensory loss, with an axonal neuropathy typical of CMT2, but no significant respiratory compromise. Segregating IGHMBP2 mutations in CMT2 were mainly loss-of-function nonsense in the 5' region of the gene in combination with a truncating frameshift, missense, or homozygous frameshift mutations in the last exon. Mutations in CMT2 were predicted to be less aggressive as compared to those in SMARD1, and fibroblast and lymphoblast studies indicate that the IGHMBP2 protein levels are significantly higher in CMT2 than SMARD1, but lower than controls, suggesting that the clinical phenotype differences are related to the IGHMBP2 protein levels.

Mutations in γ adducin are associated with inherited cerebral palsy.

OBJECTIVE: Cerebral palsy is estimated to affect nearly 1 in 500 children, and although prenatal and perinatal contributors have been well characterized, at least 20% of cases are believed to be inherited. Previous studies have identified mutations in the actin-capping protein KANK1 and the adaptor protein-4 complex in forms of inherited cerebral palsy, suggesting a role for components of the dynamic cytoskeleton in the genesis of the disease. METHODS: We studied a multiplex consanguineous Jordanian family by homozygosity mapping and exome sequencing, then used patient-derived fibroblasts to examine functional consequences of the mutation we identified in vitro. We subsequently studied the effects of adducin loss of function in Drosophila. RESULTS: We identified a homozygous c.1100G>A (p.G367D) mutation in ADD3, encoding gamma adducin in all affected members of the index family. Follow-up experiments in patient fibroblasts found that the p.G367D mutation, which occurs within the putative oligomerization critical region, impairs the ability of gamma adducin to associate with the alpha subunit. This mutation impairs the normal actin-capping function of adducin, leading to both abnormal proliferation and migration in cultured patient fibroblasts. Loss of function studies of the Drosophila adducin ortholog hts confirmed a critical role for adducin in locomotion. INTERPRETATION: Although likely a rare cause of cerebral palsy, our findings indicate a critical role for adducins in regulating the activity of the actin cytoskeleton, suggesting that impaired adducin function may lead to neuromotor impairment and further implicating abnormalities of the dynamic cytoskeleton as a pathogenic mechanism contributing to cerebral palsy.

Mutations in BICD2 cause dominant congenital spinal muscular atrophy and hereditary spastic paraplegia.

Dominant congenital spinal muscular atrophy (DCSMA) is a disorder of developing anterior horn cells and shows lower-limb predominance and clinical overlap with hereditary spastic paraplegia (HSP), a lower-limb-predominant disorder of corticospinal motor neurons. We have identified four mutations in bicaudal D homolog 2 (Drosophila) (BICD2) in six kindreds affected by DCSMA, DCSMA with upper motor neuron features, or HSP. BICD2 encodes BICD2, a key adaptor protein that interacts with the dynein-dynactin motor complex, which facilitates trafficking of cellular cargos that are critical to motor neuron development and maintenance. We demonstrate that mutations resulting in amino acid substitutions in two binding regions of BICD2 increase its binding affinity for the cytoplasmic dynein-dynactin complex, which might result in the perturbation of BICD2-dynein-dynactin-mediated trafficking, and impair neurite outgrowth. These findings provide insight into the mechanism underlying both the static and the slowly progressive clinical features and the motor neuron pathology that characterize BICD2-associated diseases, and underscore the importance of the dynein-dynactin transport pathway in the development and survival of both lower and upper motor neurons.

Rapidly progressive asymmetrical weakness in Charcot-Marie-Tooth disease type 4J resembles chronic inflammatory demyelinating polyneuropathy.

Charcot-Marie-Tooth disease type 4J (CMT4J), a rare form of demyelinating CMT, caused by recessive mutations in the phosphoinositide phosphatase FIG4 gene, is characterised by progressive proximal and distal weakness and evidence of chronic denervation in both proximal and distal muscles. We describe a patient with a previous diagnosis of CMT1 who presented with a two year history of rapidly progressive weakness in a single limb, resembling an acquired inflammatory neuropathy. Nerve conduction studies showed an asymmetrical demyelinating neuropathy with conduction block and temporal dispersion. FIG4 sequencing identified a compound heterozygous I41T/K278YfsX5 genotype. CMT4J secondary to FIG4 mutations should be added to the list of inherited neuropathies that need to be considered in suspected cases of inflammatory demyelinating neuropathy, especially if there is a background history of a more slowly progressive neuropathy.

Genetic dysfunction of MT-ATP6 causes axonal Charcot-Marie-Tooth disease.

OBJECTIVE: Charcot-Marie-Tooth (CMT) disease is the most common inherited neuromuscular disorder, affecting 1 in 2,500 individuals. Mitochondrial DNA (mtDNA) mutations are not generally considered within the differential diagnosis of patients with uncomplicated inherited neuropathy, despite the essential requirement of ATP for axonal function. We identified the mtDNA mutation m.9185T>C in MT-ATP6, encoding the ATP6 subunit of the mitochondrial ATP synthase (OXPHOS complex V), at homoplasmic levels in a family with mitochondrial disease in whom a severe motor axonal neuropathy was a striking feature. This led us to hypothesize that mutations in the 2 mtDNA complex V subunit encoding genes, MT-ATP6 and MT-ATP8, might be an unrecognized cause of isolated axonal CMT and distal hereditary motor neuropathy (dHMN). METHODS: A total of 442 probands with CMT type 2 (CMT2) (270) and dHMN (172) were screened for MT-ATP6/8 mutations after exclusion of mutations in known CMT2/dHMN genes. Mutation load was quantified using restriction endonuclease analysis. Blue-native gel electrophoresis (BN-PAGE) was performed to analyze the effects of m.9185T>C on complex V structure and function. RESULTS: Three further probands with CMT2 harbored the m.9185T>C mutation. Some relatives had been classified as having dHMN. Patients could be separated into 4 groups according to their mutant m.9185T>C levels. BN-PAGE demonstrated both impaired assembly and reduced activity of the complex V holoenzyme. CONCLUSIONS: We have shown that m.9185T>C in MT-ATP6 causes CMT2 in 1.1% of genetically undefined cases. This has important implications for diagnosis and genetic counseling. Recognition that mutations in MT-ATP6 cause CMT2 enhances current understanding of the pathogenic basis of axonal neuropathy.