Characterization of HNRNPA1 mutations defines diversity in pathogenic mechanisms and clinical presentation.

Mutations in HNRNPA1 encoding heterogeneous nuclear ribonucleoprotein (hnRNP) A1 are a rare cause of amyotrophic lateral sclerosis (ALS) and multisystem proteinopathy (MSP). hnRNPA1 is part of the group of RNA-binding proteins (RBPs) that assemble with RNA to form RNPs. hnRNPs are concentrated in the nucleus and function in pre-mRNA splicing, mRNA stability, and the regulation of transcription and translation. During stress, hnRNPs, mRNA, and other RBPs condense in the cytoplasm to form stress granules (SGs). SGs are implicated in the pathogenesis of (neuro-)degenerative diseases, including ALS and inclusion body myopathy (IBM). Mutations in RBPs that affect SG biology, including FUS, TDP-43, hnRNPA1, hnRNPA2B1, and TIA1, underlie ALS, IBM, and other neurodegenerative diseases. Here, we characterize 4 potentially novel HNRNPA1 mutations (yielding 3 protein variants: *321Eext*6, *321Qext*6, and G304Nfs*3) and 2 known HNRNPA1 mutations (P288A and D262V), previously connected to ALS and MSP, in a broad spectrum of patients with hereditary motor neuropathy, ALS, and myopathy. We establish that the mutations can have different effects on hnRNPA1 fibrillization, liquid-liquid phase separation, and SG dynamics. P288A accelerated fibrillization and decelerated SG disassembly, whereas *321Eext*6 had no effect on fibrillization but decelerated SG disassembly. By contrast, G304Nfs*3 decelerated fibrillization and impaired liquid phase separation. Our findings suggest different underlying pathomechanisms for HNRNPA1 mutations with a possible link to clinical phenotypes.

Truncating SLC5A7 mutations underlie a spectrum of dominant hereditary motor neuropathies.

OBJECTIVE: To identify the genetic cause of disease in 2 previously unreported families with forms of distal hereditary motor neuropathies (dHMNs). METHODS: The first family comprises individuals affected by dHMN type V, which lacks the cardinal clinical feature of vocal cord paralysis characteristic of dHMN-VII observed in the second family. Next-generation sequencing was performed on the proband of each family. Variants were annotated and filtered, initially focusing on genes associated with neuropathy. Candidate variants were further investigated and confirmed by dideoxy sequence analysis and cosegregation studies. Thorough patient phenotyping was completed, comprising clinical history, examination, and neurologic investigation. RESULTS: dHMNs are a heterogeneous group of peripheral motor neuron disorders characterized by length-dependent neuropathy and progressive distal limb muscle weakness and wasting. We previously reported a dominant-negative frameshift mutation located in the concluding exon of the SLC5A7 gene encoding the choline transporter (CHT), leading to protein truncation, as the likely cause of dominantly-inherited dHMN-VII in an extended UK family. In this study, our genetic studies identified distinct heterozygous frameshift mutations located in the last coding exon of SLC5A7, predicted to result in the truncation of the CHT C-terminus, as the likely cause of the condition in each family. CONCLUSIONS: This study corroborates C-terminal CHT truncation as a cause of autosomal dominant dHMN, confirming upper limb predominating over lower limb involvement, and broadening the clinical spectrum arising from CHT malfunction.

Substrate interaction defects in histidyl-tRNA synthetase linked to dominant axonal peripheral neuropathy.

Histidyl-tRNA synthetase (HARS) ligates histidine to cognate tRNA molecules, which is required for protein translation. Mutations in HARS cause the dominant axonal peripheral neuropathy Charcot-Marie-Tooth disease type 2W (CMT2W); however, the precise molecular mechanism remains undefined. Here, we investigated three HARS missense mutations associated with CMT2W (p.Tyr330Cys, p.Ser356Asn, and p.Val155Gly). The three mutations localize to the HARS catalytic domain and failed to complement deletion of the yeast ortholog (HTS1). Enzyme kinetics, differential scanning fluorimetry (DSF), and analytical ultracentrifugation (AUC) were employed to assess the effect of these substitutions on primary aminoacylation function and overall dimeric structure. Notably, the p.Tyr330Cys, p.Ser356Asn, and p.Val155Gly HARS substitutions all led to reduced aminoacylation, providing a direct connection between CMT2W-linked HARS mutations and loss of canonical ARS function. While DSF assays revealed that only one of the variants (p.Val155Gly) was less thermally stable relative to wild-type, all three HARS mutants formed stable dimers, as measured by AUC. Our work represents the first biochemical analysis of CMT-associated HARS mutations and underscores how loss of the primary aminoacylation function can contribute to disease pathology.

A recurrent WARS mutation is a novel cause of autosomal dominant distal hereditary motor neuropathy.

Distal hereditary motor neuropathy is a heterogeneous group of inherited neuropathies characterized by distal limb muscle weakness and atrophy. Although at least 15 genes have been implicated in distal hereditary motor neuropathy, the genetic causes remain elusive in many families. To identify an additional causal gene for distal hereditary motor neuropathy, we performed exome sequencing for two affected individuals and two unaffected members in a Taiwanese family with an autosomal dominant distal hereditary motor neuropathy in which mutations in common distal hereditary motor neuropathy-implicated genes had been excluded. The exome sequencing revealed a heterozygous mutation, c.770A > G (p.His257Arg), in the cytoplasmic tryptophanyl-tRNA synthetase (TrpRS) gene (WARS) that co-segregates with the neuropathy in the family. Further analyses of WARS in an additional 79 Taiwanese pedigrees with inherited neuropathies and 163 index cases from Australian, European, and Korean distal hereditary motor neuropathy families identified the same mutation in another Taiwanese distal hereditary motor neuropathy pedigree with different ancestries and one additional Belgian distal hereditary motor neuropathy family of Caucasian origin. Cell transfection studies demonstrated a dominant-negative effect of the p.His257Arg mutation on aminoacylation activity of TrpRS, which subsequently compromised protein synthesis and reduced cell viability. His257Arg TrpRS also inhibited neurite outgrowth and led to neurite degeneration in the neuronal cell lines and rat motor neurons. Further in vitro analyses showed that the WARS mutation could potentiate the angiostatic activities of TrpRS by enhancing its interaction with vascular endothelial-cadherin. Taken together, these findings establish WARS as a gene whose mutations may cause distal hereditary motor neuropathy and alter canonical and non-canonical functions of TrpRS.

A novel AARS mutation in a family with dominant myeloneuropathy.

OBJECTIVE: To determine the genetic cause of neurodegeneration in a family with myeloneuropathy. METHODS: We studied 5 siblings in a family with a mild, dominantly inherited neuropathy by clinical examination and electrophysiology. One patient had a sural nerve biopsy. After ruling out common genetic causes of axonal Charcot-Marie-Tooth disease, we sequenced 3 tRNA synthetase genes associated with neuropathy. RESULTS: All affected family members had a mild axonal neuropathy, and 3 of 4 had lower extremity hyperreflexia, evidence of a superimposed myelopathy. A nerve biopsy showed evidence of chronic axonal loss. All affected family members had a heterozygous missense mutation c.304G>C (p.Gly102Arg) in the alanyl-tRNA synthetase (AARS) gene; this allele was not identified in unaffected individuals or control samples. The equivalent change in the yeast ortholog failed to complement a strain of yeast lacking AARS function, suggesting that the mutation is damaging. CONCLUSION: A novel mutation in AARS causes a mild myeloneuropathy, a novel phenotype for patients with mutations in one of the tRNA synthetase genes.

Defects of mutant DNMT1 are linked to a spectrum of neurological disorders.

We report a broader than previously appreciated clinical spectrum for hereditary sensory and autonomic neuropathy type 1E (HSAN1E) and a potential pathogenic mechanism for DNA methyltransferase (DNMT1) mutations. The clinical presentations and genetic characteristics of nine newly identified HSAN1E kinships (45 affected subjects) were investigated. Five novel mutations of DNMT1 were discovered; p.C353F, p.T481P, p.P491L, p.Y524D and p.I531N, all within the target-sequence domain, and two mutations (p.T481P, p.P491L) arising de novo. Recently, HSAN1E has been suggested as an allelic disorder of autosomal dominant cerebellar ataxia, deafness and narcolepsy. Our results indicate that all the mutations causal for HSAN1E are located in the middle part or N-terminus end of the TS domain, whereas all the mutations causal for autosomal dominant cerebellar ataxia, deafness and narcolepsy are located in the C-terminus end of the TS domain. The impact of the seven causal mutations in this cohort was studied by cellular localization experiments. The binding efficiency of the mutant DNMT proteins at the replication foci and heterochromatin were evaluated. Phenotypic characterizations included electromyography, brain magnetic resonance and nuclear imaging, electroencephalography, sural nerve biopsies, sleep evaluation and neuropsychometric testing. The average survival of HSAN1E was 53.6 years. [standard deviation = 7.7, range 43-75 years], and mean onset age was 37.7 years. (standard deviation = 8.6, range 18-51 years). Expanded phenotypes include myoclonic seizures, auditory or visual hallucinations, and renal failure. Hypersomnia, rapid eye movement sleep disorder and/or narcolepsy were identified in 11 subjects. Global brain atrophy was found in 12 of 14 who had brain MRI. EEGs showed low frequency (delta waves) frontal-predominant abnormality in five of six patients. Marked variability in cognitive deficits was observed, but the majority of patients (89%) developed significant cognitive deficit by the age of 45 years. Cognitive function decline often started with personality changes and psychiatric manifestations. A triad of hearing loss, sensory neuropathy and cognitive decline remains as the stereotypic presentation of HSAN1E. Moreover, we show that mutant DNMT1 proteins translocate to the cytoplasm and are prone to form aggresomes while losing their binding ability to heterochromatin during the G2 cell cycle. Our results suggest mutations in DNMT1 result in imbalanced protein homeostasis through aggresome-induced autophagy. This mechanism may explain why mutations in the sole DNA maintenance methyltransferase lead to selective central and peripheral neurodegeneration.

Sensory neuropathy with bone destruction due to a mutation in the membrane-shaping atlastin GTPase 3.

Many neurodegenerative disorders present with sensory loss. In the group of hereditary sensory and autonomic neuropathies loss of nociception is one of the disease hallmarks. To determine underlying factors of sensory neurodegeneration we performed whole-exome sequencing in affected individuals with the disorder. In a family with sensory neuropathy with loss of pain perception and destruction of the pedal skeleton we report a missense mutation in a highly conserved amino acid residue of atlastin GTPase 3 (ATL3), an endoplasmic reticulum-shaping GTPase. The same mutation (p.Tyr192Cys) was identified in a second family with similar clinical outcome by screening a large cohort of 115 patients with hereditary sensory and autonomic neuropathies. Both families show an autosomal dominant pattern of inheritance and the mutation segregates with complete penetrance. ATL3 is a paralogue of ATL1, a membrane curvature-generating molecule that is involved in spastic paraplegia and hereditary sensory neuropathy. ATL3 proteins are enriched in three-way junctions, branch points of the endoplasmic reticulum that connect membranous tubules to a continuous network. Mutant ATL3 p.Tyr192Cys fails to localize to branch points, but instead disrupts the structure of the tubular endoplasmic reticulum, suggesting that the mutation exerts a dominant-negative effect. Identification of ATL3 as novel disease-associated gene exemplifies that long-term sensory neuronal maintenance critically depends on the structural organisation of the endoplasmic reticulum. It emphasizes that alterations in membrane shaping-proteins are one of the major emerging pathways in axonal degeneration and suggests that this group of molecules should be considered in neuroprotective strategies.

De novo INF2 mutations expand the genetic spectrum of hereditary neuropathy with glomerulopathy.

OBJECTIVE: Identification of mutations in the inverted formin-2 (INF2) gene in patients with Charcot-Marie-Tooth (CMT) disease combined with focal segmental glomerulosclerosis (FSGS) in order to expand the genetic and phenotypic spectrum. METHODS: We sequenced INF2 in 5 patients with CMT disease and FSGS. Mutations were subsequently screened in family members of the index patient and 264 control individuals. RESULTS: In 3 patients, we detected 2 novel de novo INF2 mutations (p.Leu77Arg and p.Leu69_Ser72del) and a third, most likely de novo mutation (p.Gly114Asp). One of our patients displayed intellectual disability, a phenotypic characteristic not previously associated with INF2. The same patient also showed a more pronounced sensorineural hearing loss than described before. CONCLUSIONS: In exon 2 of INF2, we identified 3 novel mutations that likely affect the protein structure and function. Our findings expand the genetic spectrum of INF2-associated disorders and broaden the associated phenotype with the co-occurrence of intellectual disability and more severe hearing loss than previously reported. De novo INF2 mutations may be more common in patients with CMT disease and FSGS in comparison to FSGS alone. Furthermore, renal dysfunction is more severe and starts earlier in life when associated with CMT disease. Our study confirms that INF2 mutations are a major cause of disease in patients with CMT disease and early signs of nephropathy. Diagnostic screening of INF2 is strongly recommended in isolated patients presenting with CMT disease and FSGS.