Genetics of inherited peripheral neuropathies and the next frontier: looking backwards to progress forwards.

Inherited peripheral neuropathies (IPNs) encompass a clinically and genetically heterogeneous group of disorders causing length-dependent degeneration of peripheral autonomic, motor and/or sensory nerves. Despite gold-standard diagnostic testing for pathogenic variants in over 100 known associated genes, many patients with IPN remain genetically unsolved. Providing patients with a diagnosis is critical for reducing their 'diagnostic odyssey', improving clinical care, and for informed genetic counselling. The last decade of massively parallel sequencing technologies has seen a rapid increase in the number of newly described IPN-associated gene variants contributing to IPN pathogenesis. However, the scarcity of additional families and functional data supporting variants in potential novel genes is prolonging patient diagnostic uncertainty and contributing to the missing heritability of IPNs. We review the last decade of IPN disease gene discovery to highlight novel genes, structural variation and short tandem repeat expansions contributing to IPN pathogenesis. From the lessons learnt, we provide our vision for IPN research as we anticipate the future, providing examples of emerging technologies, resources and tools that we propose that will expedite the genetic diagnosis of unsolved IPN families.

An Update on the Adult-Onset Hereditary Cerebellar Ataxias: Novel Genetic Causes and New Diagnostic Approaches.

The hereditary cerebellar ataxias (HCAs) are rare, progressive neurologic disorders caused by variants in many different genes. Inheritance may follow autosomal dominant, autosomal recessive, X-linked or mitochondrial patterns. The list of genes associated with adult-onset cerebellar ataxia is continuously growing, with several new genes discovered in the last few years. This includes short-tandem repeat (STR) expansions in RFC1, causing cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS), FGF14-GAA causing spinocerebellar ataxia type 27B (SCA27B), and THAP11. In addition, the genetic basis for SCA4, has recently been identified as a STR expansion in ZFHX3. Given the large and growing number of genes, and different gene variant types, the approach to diagnostic testing for adult-onset HCA can be complex. Testing methods include targeted evaluation of STR expansions (e.g. SCAs, Friedreich ataxia, fragile X-associated tremor/ataxia syndrome, dentatorubral-pallidoluysian atrophy), next generation sequencing for conventional variants, which may include targeted gene panels, whole exome, or whole genome sequencing, followed by various potential additional tests. This review proposes a diagnostic approach for clinical testing, highlights the challenges with current testing technologies, and discusses future advances which may overcome these limitations. Implementing long-read sequencing has the potential to transform the diagnostic approach in HCA, with the overall aim to improve the diagnostic yield.

Two New Families and a Literature Review of ELOVL4-Associated Spinocerebellar Ataxia Type 34.

Autosomal dominant variants in ELOVL4 cause spinocerebellar ataxia type 34 (SCA34; ATX-ELOVL4), classically associated with a skin condition known as erythrokeratoderma. Here, we report a large Italian-Maltese-Australian family with spinocerebellar ataxia. Notably, while there were dermatological manifestations (eczema), erythrokeratoderma was not present. Using a next-generation sequencing panel, we identified a previously reported ELOVL4 variant, NM_022726.4: c.698C > T p.(Thr233Met). The variant was initially classified as a variant of uncertain significance; however, through segregation studies, we reclassified the variant as likely pathogenic. We next identified an individual from another family (Algerian-Maltese-Australian) with the same ELOVL4 variant with spinocerebellar ataxia but without dermatological manifestations. We subsequently performed the first dedicated literature review of ELOVL4-associated ataxia to gain further insights into genotype-phenotype relationships. We identified a total of 60 reported cases of SCA34 to date. The majority had gait ataxia (88.3%), limb ataxia (76.7%), dysarthria (63.3%), and nystagmus (58.3%). Of note, skin lesions related to erythrokeratoderma were seen in a minority of cases (33.3%). Other extracerebellar manifestations included pyramidal tract signs, autonomic disturbances, retinitis pigmentosa, and cognitive impairment. For brain MRI data, cerebellar atrophy was seen in all cases (100%), whereas the hot cross bun sign (typically associated with multiple system atrophy type C) was seen in 32.4% of cases. Our family study and literature review highlight the variable phenotypic spectrum of SCA34. Importantly, it shows that erythrokeratoderma is not found in most cases and that, while a dermatological assessment may be helpful in these patients, SCA34 diagnosis should be considered irrespective of dermatological manifestations.

A deep intronic variant in MME causes autosomal recessive Charcot-Marie-Tooth neuropathy through aberrant splicing.

BACKGROUND: Loss-of-function variants in MME (membrane metalloendopeptidase) are a known cause of recessive Charcot-Marie-Tooth Neuropathy (CMT). A deep intronic variant, MME c.1188+428A>G (NM_000902.5), was identified through whole genome sequencing (WGS) of two Australian families with recessive inheritance of axonal CMT using the seqr platform. MME c.1188+428A>G was detected in a homozygous state in Family 1, and in a compound heterozygous state with a known pathogenic MME variant (c.467del; p.Pro156Leufs*14) in Family 2. AIMS: We aimed to determine the pathogenicity of the MME c.1188+428A>G variant through segregation and splicing analysis. METHODS: The splicing impact of the deep intronic MME variant c.1188+428A>G was assessed using an in vitro exon-trapping assay. RESULTS: The exon-trapping assay demonstrated that the MME c.1188+428A>G variant created a novel splice donor site resulting in the inclusion of an 83 bp pseudoexon between MME exons 12 and 13. The incorporation of the pseudoexon into MME transcript is predicted to lead to a coding frameshift and premature termination codon (PTC) in MME exon 14 (p.Ala397ProfsTer47). This PTC is likely to result in nonsense mediated decay (NMD) of MME transcript leading to a pathogenic loss-of-function. INTERPRETATION: To our knowledge, this is the first report of a pathogenic deep intronic MME variant causing CMT. This is of significance as deep intronic variants are missed using whole exome sequencing screening methods. Individuals with CMT should be reassessed for deep intronic variants, with splicing impacts being considered in relation to the potential pathogenicity of variants.

Novel gene-intergenic fusion involving ubiquitin E3 ligase UBE3C causes distal hereditary motor neuropathy.

Distal hereditary motor neuropathies (dHMNs) are a group of inherited diseases involving the progressive, length-dependent axonal degeneration of the lower motor neurons. There are currently 29 reported causative genes and four disease loci implicated in dHMN. Despite the high genetic heterogeneity, mutations in the known genes account for less than 20% of dHMN cases, with the mutations identified predominantly being point mutations or indels. We have expanded the spectrum of dHMN mutations with the identification of a 1.35 Mb complex structural variation (SV) causing a form of autosomal dominant dHMN (DHMN1 OMIM %182906). Given the complex nature of SV mutations and the importance of studying pathogenic mechanisms in a neuronal setting, we generated a patient-derived DHMN1 motor neuron model harbouring the 1.35 Mb complex insertion. The DHMN1 complex insertion creates a duplicated copy of the first 10 exons of the ubiquitin-protein E3 ligase gene (UBE3C) and forms a novel gene-intergenic fusion sense transcript by incorporating a terminal pseudo-exon from intergenic sequence within the DHMN1 locus. The UBE3C intergenic fusion (UBE3C-IF) transcript does not undergo nonsense-mediated decay and results in a significant reduction of wild-type full-length UBE3C (UBE3C-WT) protein levels in DHMN1 iPSC-derived motor neurons. An engineered transgenic Caenorhabditis elegans model expressing the UBE3C-IF transcript in GABA-ergic motor neurons shows neuronal synaptic transmission deficits. Furthermore, the transgenic animals are susceptible to heat stress, which may implicate defective protein homeostasis underlying DHMN1 pathogenesis. Identification of the novel UBE3C-IF gene-intergenic fusion transcript in motor neurons highlights a potential new disease mechanism underlying axonal and motor neuron degeneration. These complementary models serve as a powerful paradigm for studying the DHMN1 complex SV and an invaluable tool for defining therapeutic targets for DHMN1.

Charcot-Marie-tooth disease causing mutation (p.R158H) in pyruvate dehydrogenase kinase 3 (PDK3) affects synaptic transmission, ATP production and causes neurodegeneration in a CMTX6 C. elegans model.

Charcot-Marie-Tooth (CMT) is a commonly inherited, non-fatal neurodegenerative disorder that affects sensory and motor neurons in patients. More than 90 genes are known to cause axonal and demyelinating forms of CMT. The p.R158H mutation in the pyruvate dehydrogenase kinase 3 (PDK3) gene is the genetic cause for an X linked form of axonal CMT (CMTX6). In vitro studies using patient fibroblasts and iPSC-derived motor neurons have shown that this mutation causes deficits in energy metabolism and mitochondrial function. Animal models that recapitulate pathogenic in vivo events in patients are crucial for investigating mechanisms of axonal degeneration and developing therapies for CMT. We have developed a C. elegans model of CMTX6 by knocking-in the p.R158H mutation in pdhk-2, the ortholog of PDK3. In addition, we have developed animal models overexpressing the wild type and mutant form of human PDK3 specifically in the GABAergic motor neurons of C. elegans. CMTX6 mutants generated in this study exhibit synaptic transmission deficits, locomotion defects and show signs of progressive neurodegeneration. Furthermore, the CMTX6 in vivo models display energy deficits that recapitulate the phenotype observed in patient fibroblasts and iPSC-derived motor neurons. Our CMTX6 animals represent the first in vivo model for this form of CMT and have provided novel insights into the cellular function and metabolic pathways perturbed by the p.R158H mutation, all the while closely replicating the clinical presentation observed in CMTX6 patients.

Long read sequencing overcomes challenges in the diagnosis of SORD neuropathy.

Biallelic mutations in sorbitol dehydrogenase (SORD) have been recently identified as a common cause of recessive axonal Charcot-Marie-Tooth neuropathy (CMT2). We aimed to assess a novel long-read sequencing approach to overcome current limitations in SORD neuropathy diagnostics due to the SORD2P pseudogene and the phasing of biallelic mutations in recessive disease. We conducted a screen of our Australian whole exome sequencing (WES) CMT cohort to identify individuals with homozygous or compound heterozygous SORD variants. Individuals detected with SORD mutations then underwent long-read sequencing, clinical assessment, and serum sorbitol analysis. An individual was detected with compound heterozygous truncating mutations in SORD exon 7, NM_003104.5:c.625C>T (p.Arg209Ter) and NM_003104.5:c.757del (p.Ala253GlnfsTer27). Subsequent Oxford Nanopore Tech (ONT) long-read sequencing was used to successfully differentiate SORD from the highly homologous non-functional SORD2P pseudogene and confirmed that the mutations were biallelic through haplotype-resolved analysis. The patient presented with axonal sensorimotor polyneuropathy (CMT2) and ulnar neuropathy without compression at the elbow. Burning neuropathic pain in the forearms and feet was also reported and was exacerbated by alcohol consumption and improved with alcohol cessation. UPLC-tandem mass spectrometry confirmed that the patient had elevated serum sorbitol levels (12.0 mg/L) consistent with levels previously observed in patients with biallelic SORD mutations. This represents a novel clinical presentation and expands the phenotype associated with biallelic SORD mutations causing CMT2. Our study is the first report of long-read sequencing for an individual with CMT and demonstrates the utility of this approach for clinical genomics.

Revisiting the pathogenic mechanism of the GJB1 5' UTR c.-103C > T mutation causing CMTX1.

The second most common form of Charcot-Marie-Tooth neuropathy (CMT), X-linked CMT type X1 (CMTX1), is caused by coding and non-coding mutations in the gap junction beta 1 (GJB1) gene. The non-coding GJB1 c.-103C > T mutation (NM_000166.5) has been reported to cause CMTX1 in multiple families. This study assessed the internal ribosomal entry site (IRES) activity previously reported for the rat Gjb1 P2 5' untranslated region (UTR). Using a bicistronic assay and transfecting RT4 Schwann cells, IRES activity of the human GJB1 P2 5' UTR was compared to the GJB1 P2 5' UTR containing either the c.-103C > T mutation or the non-pathogenic c.-102G > A variant. No differences in GJB1 P2 5' UTR IRES activity were observed between the negative control, the wild-type P2 5' UTR, the c.-103C > T 5' UTR or the c.-102G > A 5' UTR, irrespective of the GJB1 intron being present (p = .429 with intron, and p = .865 without). A theoretical c.-131A > G variant was predicted to result in the same RNA secondary structure as the GJB1 c.-103C > T P2 5' UTR. However, no significant difference was observed between expression from the wild-type GJB1 P2 5' UTR and the GJB1 c.-131A > G variant (p = .688). Deletion of the conserved region surrounding the c.-103C > T mutation (c.-108_-103del) resulted in significantly higher expression than the c.-103C > T mutation alone (p = .019), suggesting that the conserved c.-108_-103 region was not essential for translation. The reporter assays in this study do not recapitulate the previously reported GJB1 IRES activity and suggest an alternate pathogenic mechanism for the c.-103C > T CMTX1 non-coding mutation.

Linkage analysis and whole exome sequencing reveals AHNAK2 as a novel genetic cause for autosomal recessive CMT in a Malaysian family.

Charcot-Marie-Tooth (CMT) disease is a form of inherited peripheral neuropathy that affects motor and sensory neurons. To identify the causative gene in a consanguineous family with autosomal recessive CMT (AR-CMT), we employed a combination of linkage analysis and whole exome sequencing. After excluding known AR-CMT genes, genome-wide linkage analysis mapped the disease locus to a 7.48-Mb interval on chromosome 14q32.11-q32.33, flanked by the markers rs2124843 and rs4983409. Whole exome sequencing identified two non-synonymous variants (p.T40P and p.H915Y) in the AHNAK2 gene that segregated with the disease in the family. Pathogenic predictions indicated that p.T40P is the likely causative allele. Analysis of AHNAK2 expression in the AR-CMT patient fibroblasts showed significantly reduced mRNA and protein levels. AHNAK2 binds directly to periaxin which is encoded by the PRX gene, and PRX mutations are associated with another form of AR-CMT (CMT4F). The altered expression of mutant AHNAK2 may disrupt the AHNAK2-PRX interaction in which one of its known functions is to regulate myelination.

Whole Genome Sequencing Identifies a 78 kb Insertion from Chromosome 8 as the Cause of Charcot-Marie-Tooth Neuropathy CMTX3.

With the advent of whole exome sequencing, cases where no pathogenic coding mutations can be found are increasingly being observed in many diseases. In two large, distantly-related families that mapped to the Charcot-Marie-Tooth neuropathy CMTX3 locus at chromosome Xq26.3-q27.3, all coding mutations were excluded. Using whole genome sequencing we found a large DNA interchromosomal insertion within the CMTX3 locus. The 78 kb insertion originates from chromosome 8q24.3, segregates fully with the disease in the two families, and is absent from the general population as well as 627 neurologically normal chromosomes from in-house controls. Large insertions into chromosome Xq27.1 are known to cause a range of diseases and this is the first neuropathy phenotype caused by an interchromosomal insertion at this locus. The CMTX3 insertion represents an understudied pathogenic structural variation mechanism for inherited peripheral neuropathies. Our finding highlights the importance of considering all structural variation types when studying unsolved inherited peripheral neuropathy cases with no pathogenic coding mutations.

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.

MORC2 mutations cause axonal Charcot-Marie-Tooth disease with pyramidal signs.

OBJECTIVE: To use linkage analysis and whole exome sequencing to identify the genetic mutation in a multigenerational Australian family with Charcot-Marie-Tooth disease type 2 (CMT2) and pyramidal signs. METHODS: Genome-wide linkage analysis was performed to map the locus. Whole exome sequencing was undertaken on selected individuals (3 affected, 1 normal), and segregation analysis and mutation screening were carried out using high-resolution melt analysis. The GEM.app database was queried to identify additional families with mutations. RESULTS: Significant linkage (2-point LOD score ≥ +3) and haplotype analysis mapped a new locus for CMT2 and pyramidal signs to a 6.6Mb interval on chromosome 22q12.1-q12.3. Whole exome sequencing identified a novel mutation (p.R252W) in the microrchidia CW-type zinc finger 2 (MORC2) gene mapping within the linkage region. The mutation fully segregated with the disease phenotype in the family. Screening additional families and querying unsolved CMT2 exomes, we identified the p.R252W mutation in 2 unrelated early onset CMT2 families and a second mutation p.E236G in 2 unrelated CMT2 families. Both the mutations occurred at highly conserved amino acid residues and were absent in the normal population. INTERPRETATION: We have identified a new locus in which MORC2 mutations are the likely pathogenic cause of CMT2 and pyramidal signs in these families. MORC2 encodes the human CW-type zinc finger 2 protein, which is a chromatin modifier involved in the regulation of DNA repair as well as gene transcription.

Pathogenic mechanisms underlying X-linked Charcot-Marie-Tooth neuropathy (CMTX6) in patients with a pyruvate dehydrogenase kinase 3 mutation.

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. An X-linked form of CMT (CMTX6) is caused by a missense mutation (R158H) in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene. PDK3 is one of 4 isoenzymes that negatively regulate the activity of the pyruvate dehydrogenase complex (PDC) by reversible phosphorylation of its first catalytic component pyruvate dehydrogenase (designated as E1). Mitochondrial PDC catalyses the oxidative decarboxylation of pyruvate to acetyl CoA and links glycolysis to the energy-producing Krebs cycle. We have previously shown the R158H mutation confers PDK3 enzyme hyperactivity. In this study we demonstrate that the increased PDK3 activity in patient fibroblasts (PDK3(R158H)) leads to the attenuation of PDC through hyper-phosphorylation of E1 at selected serine residues. This hyper-phosphorylation can be reversed by treating the PDK3(R158H) fibroblasts with the PDK inhibitor dichloroacetate (DCA). In the patient cells, down-regulation of PDC leads to increased lactate, decreased ATP and alteration of the mitochondrial network. Our findings highlight the potential to develop specific drug targeting of the mutant PDK3 as a therapeutic approach to treating CMTX6.

A 1.35 Mb DNA fragment is inserted into the DHMN1 locus on chromosome 7q34-q36.2.

Distal hereditary motor neuropathies predominantly affect the motor neurons of the peripheral nervous system leading to chronic disability. Using whole genome sequencing (WGS) we have identified a novel structural variation (SV) within the distal hereditary motor neuropathy locus on chromosome 7q34-q36.2 (DHMN1). The SV involves the insertion of a 1.35 Mb DNA fragment into the DHMN1 disease locus. The source of the inserted sequence is 2.3 Mb distal to the disease locus at chromosome 7q36.3. The insertion involves the duplication of five genes (LOC389602, RNF32, LMBR1, NOM1, MNX1) and partial duplication of UBE3C. The genomic structure of genes within the DHMN1 locus are not disrupted by the insertion and no disease causing point mutations within the locus were identified. This suggests the novel SV is the most likely DNA mutation disrupting the DHMN1 locus. Due to the size and position of the DNA insertion, the gene(s) directly affected by the genomic re-arrangement remains elusive. Our finding represents a new genetic cause for hereditary motor neuropathies and highlights the growing importance of interrogating the non-coding genome for SV mutations in families which have been excluded for genome wide coding mutations.

X-linked Charcot-Marie-Tooth disease type 6 (CMTX6) patients with a p.R158H mutation in the pyruvate dehydrogenase kinase isoenzyme 3 gene.

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. Mutations in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene have been found to cause X-linked dominant CMT type 6 (CMTX6). This study identified the p.R158H PDK3 mutation after screening 67 probable X-linked CMT families. The mutation fully segregated with the phenotype, and genotyping the family indicated the mutation arose on a different haplotype compared with the original Australian CMTX6 family. Results of bisulphite sequencing suggest that methylated deamination of a CpG dinucleotide may cause the recurrent p.R158H mutation. The frequency of the p.R158H PDK3 mutation in Koreans is very rare. Magnetic resonance imaging revealed fatty infiltration involving distal muscles in the lower extremities. In addition, fatty infiltrations were predominantly observed in the soleus muscles, with a lesser extent in tibialis anterior muscles. This differs from demyelinating CMT1A patients and is similar to axonal CMT2A patients. The clinical, neuroimaging, and electrophysiological findings from a second CMTX6 family with the p.R158H PDK3 mutation were similar to the axonal neuropathy reported in the Australian family.

A new locus for X-linked dominant Charcot-Marie-Tooth disease (CMTX6) is caused by mutations in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene.

Hereditary motor and sensory disorders of the peripheral nerve form one of the most common groups of human genetic diseases collectively called Charcot-Marie-Tooth (CMT) neuropathy. Using linkage analysis in a three generation kindred, we have mapped a new locus for X-linked dominant CMT to chromosome Xp22.11. A microsatellite scan of the X chromosome established significant linkage to several markers including DXS993 (Zmax = 3.16; θ = 0.05). Extended haplotype analysis refined the linkage region to a 1.43-Mb interval flanked by markers DXS7110 and DXS8027. Whole exome sequencing identified a missense mutation c.G473A (p.R158H) in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene. The change localized within the 1.43-Mb linkage interval, segregated with the affected phenotype and was excluded in ethnically matched control chromosomes. PDK3 is one of the four isoenzymes regulating the pyruvate dehydrogenase complex (PDC), by reversible phosphorylation, and is a nuclear-coded protein located in the mitochondrial matrix. PDC catalyzes the oxidative decarboxylation of pyruvate to acetyl CoA and is a key enzyme linking glycolysis to the energy-producing Krebs cycle and lipogenic pathways. We found that the R158H mutation confers enzyme hyperactivity and binds with stronger affinity than the wild-type to the inner-lipoyl (L2) domain of the E2p chain of PDC. Our findings suggest a reduced pyruvate flux due to R158H mutant PDK3-mediated hyper-phosphorylation of the PDC as the underlying pathogenic cause of peripheral neuropathy. The results highlight an important causative link between peripheral nerve degeneration and an essential bioenergetic or biosynthetic pathway required for the maintenance of peripheral nerves.

Cowchock syndrome is associated with a mutation in apoptosis-inducing factor.

Cowchock syndrome (CMTX4) is a slowly progressive X-linked recessive disorder with axonal neuropathy, deafness, and cognitive impairment. The disease locus was previously mapped to an 11 cM region at chromosome X: q24-q26. Exome sequencing of an affected individual from the originally described family identified a missense change c.1478A>T (p.Glu493Val) in AIFM1, the gene encoding apoptosis-inducing factor (AIF) mitochondrion-associated 1. The change is at a highly conserved residue and cosegregated with the phenotype in the family. AIF is an FAD-dependent NADH oxidase that is imported into mitochondria. With apoptotic insults, a N-terminal transmembrane linker is cleaved off, producing a soluble fragment that is released into the cytosol and then transported into the nucleus, where it triggers caspase-independent apoptosis. Another AIFM1 mutation that predicts p.Arg201del has recently been associated with severe mitochondrial encephalomyopathy in two infants by impairing oxidative phosphorylation. The c.1478A>T (p.Glu493Val) mutation found in the family reported here alters the redox properties of the AIF protein and results in increased cell death via apoptosis, without affecting the activity of the respiratory chain complexes. Our findings expand the spectrum of AIF-related disease and provide insight into the effects of AIFM1 mutations.

Analysis of dynein intermediate chains, light intermediate chains and light chains in a cohort of hereditary peripheral neuropathies.

The cytoplasmic dynein heavy chain (DYNC1H1) gene has been increasingly associated with neurodegenerative disorders including axonal Charcot-Marie-Tooth disease (CMT2), intellectual disability and malformations of cortical development. In addition, evidence from mouse models (Loa, catabolite repressor-activator (Cra) and Sprawling (Swl)) has shown that mutations in Dync1h1 cause a range of neurodegenerative phenotypes with motor and sensory neuron involvement. In this current study, we examined the possible contribution of other cytoplasmic dynein subunits that bind to DYNC1H1 as a cause of inherited peripheral neuropathy. We focused on screening the cytoplasmic dynein intermediate, light intermediate and light chain genes in a cohort of families with inherited peripheral neuropathies. Nine genes were screened and ten variants were detected, but none was identified as pathogenic, indicating that cytoplasmic dynein intermediate, light intermediate and light chains are not a cause of neuropathy in our cohort.

Altered intracellular localization and valosin-containing protein (p97 VCP) interaction underlie ATP7A-related distal motor neuropathy.

ATP7A is a P-type ATPase that regulates cellular copper homeostasis by activity at the trans-Golgi network (TGN) and plasma membrane (PM), with the location normally governed by intracellular copper concentration. Defects in ATP7A lead to Menkes disease or its milder variant, occipital horn syndrome or to a newly discovered condition, ATP7A-related distal motor neuropathy (DMN), for which the precise pathophysiology has been obscure. We investigated two ATP7A motor neuropathy mutations (T994I, P1386S) previously associated with abnormal intracellular trafficking. In the patients' fibroblasts, total internal reflection fluorescence microscopy indicated a shift in steady-state equilibrium of ATP7A(T994I) and ATP7A(P1386S), with exaggerated PM localization. Transfection of Hek293T cells and NSC-34 motor neurons with the mutant alleles tagged with the Venus fluorescent protein also revealed excess PM localization. Endocytic retrieval of the mutant alleles from the PM to the TGN was impaired. Immunoprecipitation assays revealed an abnormal interaction between ATP7A(T994I) and p97/VCP, an ubiquitin-selective chaperone which is mutated in two autosomal dominant forms of motor neuron disease: amyotrophic lateral sclerosis and inclusion body myopathy with early-onset Paget disease and fronto-temporal dementia. Small-interfering RNA (SiRNA) knockdown of p97/VCP corrected ATP7A(T994I) mislocalization. Flow cytometry documented that non-permeabilized ATP7A(P1386S) fibroblasts bound a carboxyl-terminal ATP7A antibody, consistent with relocation of the ATP7A di-leucine endocytic retrieval signal to the extracellular surface and partially destabilized insertion of the eighth transmembrane helix. Our findings illuminate the mechanisms underlying ATP7A-related DMN and establish a link between p97/VCP and genetically distinct forms of motor neuron degeneration.