A novel LRSAM1 mutation is associated with autosomal dominant axonal Charcot-Marie-Tooth disease.
ABSTRACT: Charcot-Marie-Tooth (CMT) disease is the most common hereditary neuropathy resulting from mutations in >30 genes expressed in either the Schwann cells or the axon of peripheral nerves. The disease is classified into demyelinating (CMT1), axonal (CMT2) or intermediate (CMTI) based on electrophysiological and pathological findings. Our study focused on the identification of a novel disease mutation in a large Sardinian family with CMT2 of autosomal dominant (AD) inheritance. All available family members were clinically evaluated and samples were collected from consenting individuals. Initially, we excluded known CMT2 genes/loci in this family. We then conducted a genome-wide linkage analysis and mapped the gene to chromosome 9q33-q34. Refined linkage and haplotype analyses defined an 11.6-Mb candidate region with a maximum LOD score of 8.06. Following exclusion of several candidate genes from the region, we targeted the LRSAM1 (leucine-rich repeat and sterile alpha motif-containing 1) gene, very recently found to be associated with autosomal recessive CMT2 in one family. For a more efficient investigation of this large gene, already available proband RNA (cDNA) was initially analyzed. Targeted DNA analysis then confirmed a novel LRSAM1 splice-site (c.2047-1G>A) mutation, causing a frameshift that introduces a stop codon three amino acids further down the new reading frame (p.Ala683ProfsX3). This mutation is located in the C-terminal RING finger motif of the encoded protein and leads to premature truncation of the protein. In the course of our work, a second LRSAM1 mutation dominantly transmitted was identified by another group. Our data further confirms that LRSAM1 mutations are associated with CMT2 of AD inheritance.
Project description:Despite the high number of genes identified in hereditary polyneuropathies/Charcot-Marie-Tooth (CMT) disease, the genetic defect in many families is still unknown. Here we report the identification of a new gene for autosomal dominant axonal neuropathy in a large three-generation family. Linkage analysis identified a 5 Mb region on 9q33-34 with a LOD score of 5.12. Sequence capture and next-generation sequencing of the region of interest identified five previously unreported non-synonymous heterozygous single nucleotide changes or indels, four of which were confirmed by Sanger sequencing. Two sequence variants co-segregated with the disease, and one, a 2 bp insertion in the last exon of LRSAM1, was also absent in 676 ethnicity-matched control chromosomes. This frameshift mutation (p.Leu708Argfx28) is located in the C-terminal RING finger motif of the encoded protein. Ubiquitin ligase activity in transfected cells with constructs carrying the patient mutation was affected as measured by a higher level of abundance of TSG101, the only reported target of LRSAM1. Injections of morpholino oligonucleotides in zebrafish embryos directed against the ATG or last splice site of zebrafish Lrsam1 disturbed neurodevelopment, showing a less organized neural structure and, in addition, affected tail formation and movement. LRSAM1 is highly expressed in adult spinal cord motoneurons as well as in fetal spinal cord and muscle tissue. Recently, a homozygous mutation in LRSAM1 was proposed as a strong candidate for the disease in a family with recessive axonal polyneuropathy. Our data strongly support the hypothesis that LRSAM1 mutations can cause both dominant and recessive forms of CMT.
Project description:Charcot-Marie-Tooth disease type 2 (CMT2) is an autosomal dominant axonal neuropathy caused by mutations in various genes. The subtype CMT2B results from missense mutations in RAB7A, member RAS oncogene family gene, whereas missense mutations in the Leucine-rich repeat and sterile alpha motif-containing protein 1 (LRSAM1) gene cause CMT2P. We describe the genotype/phenotype analysis of a family in which a previously described mutation in the RAB7A gene and a novel mutation in the LRSAM1 gene were identified. In this family, none of the individuals had ulceromutilating features, and there was a marked variability in the age of onset. We discuss the possible etiology of the observed phenotypic variability including the role of gender and possible RAB7A/LRSAM1 gene interactions.
Project description:Charcot-Marie-Tooth disease (CMT) represents a family of related sensorimotor neuropathies. We studied a large family from a rural eastern Canadian community, with multiple individuals suffering from a condition clinically most similar to autosomal recessive axonal CMT, or AR-CMT2. Homozygosity mapping with high-density SNP genotyping of six affected individuals from the family excluded 23 known genes for various subtypes of CMT and instead identified a single homozygous region on chromosome 9, at 122,423,730-129,841,977 Mbp, shared identical by state in all six affected individuals. A homozygous pathogenic variant was identified in the gene encoding leucine rich repeat and sterile alpha motif 1 (LRSAM1) by direct DNA sequencing of genes within the region in affected DNA samples. The single nucleotide change mutates an intronic consensus acceptor splicing site from AG to AA. Direct analysis of RNA from patient blood demonstrated aberrant splicing of the affected exon, causing an obligatory frameshift and premature truncation of the protein. Western blotting of immortalized cells from a homozygous patient showed complete absence of detectable protein, consistent with the splice site defect. LRSAM1 plays a role in membrane vesicle fusion during viral maturation and for proper adhesion of neuronal cells in culture. Other ubiquitin ligases play documented roles in neurodegenerative diseases. LRSAM1 is a strong candidate for the causal gene for the genetic disorder in our kindred.
Project description:Currently only 25-30% of patients with axonal forms of Charcot-Marie-Tooth disease (CMT) receive a genetic diagnosis. We aimed to identify the causative gene of CMT type 2 in 8 non-related French families with a distinct clinical phenotype. We collected clinical, electrophysiological, and laboratory findings and performed genetic analyses in four different French laboratories. Seventy-two patients with autosomal dominant inheritance were identified. The disease usually started in the fourth decade and the clinical picture was dominated by sensory ataxia (80%), neuropathic pain (38%), and length-dependent sensory loss to all modalities. Electrophysiological studies showed a primarily axonal neuropathy, with possible isolated sensory involvement in milder phenotypes. Disease severity varied greatly but the clinical course was generally mild. We identified 2 novel variants in LRSAM1 gene: a deletion of 4 amino acids, p.(Gln698_Gln701del), was found in 7 families and a duplication of a neighboring region of 10 amino acids, p.(Pro702_Gln711dup), in the remaining family. A common haplotype of ~450?kb suggesting a founder effect was noted around LRSAM1 in 4 families carrying the first variant. LRSAM1 gene encodes for an E3 ubiquitin ligase important for neural functioning. Our results confirm the localization of variants in its catalytic C-terminal RING domain and broaden the phenotypic spectrum of LRSAM1-related neuropathies, including painful and predominantly sensory ataxic forms.
Project description:In the past decade, mutations in LRSAM1 were identified as the genetic cause of both dominant and recessive forms of axonal CMT type 2P (CMT2P). Despite demonstrating different inheritance patterns, dominant CMT2P is usually characterized by relatively mild, slowly progressive axonal neuropathy, mainly involving lower limbs, with age of onset between the second and fifth decades of life. Asymptomatic individuals were identified in several pedigrees exemplifying the strong phenotypic variability of these patients requiring serial clinical evaluation to establish correct diagnosis; in this respect, magnetic resonance imaging of lower-limb musculature showing fatty atrophy might be helpful in detecting subclinical gene mutation carriers. LRSAM1 is a universally expressed RING-type E3 ubiquitin protein ligase catalysing the final step in the ubiquitination cascade. Strikingly, TSG101 remains the only known ubiquitination target hampering our mechanistic understanding of the role of LRSAM1 in the cell. The recessive CMT mutations lead to complete loss of LRSAM1, contrary to the heterozygous dominant variants. These tightly cluster in the C-terminal RING domain highlighting its importance in governing the CMT disease. The domain is crucial for the ubiquitination function of LRSAM1 and CMT mutations disrupt its function, however it remains unknown how this leads to the peripheral neuropathy. Additionally, recent studies have linked LRSAM1 with other neurodegenerative diseases of peripheral and central nervous systems. In this review we share our experience with the challenging clinical diagnosis of CMT2P and summarize the mechanistic insights about the LRSAM1 dysfunction that might be helpful for the neurodegenerative field at large.
Project description:LRSAM1 mutations have been found in recessive and dominant forms of Charcot-Marie-Tooth disease. Within one generation of the original Dutch family in which the dominant LRSAM1 mutation was identified, three of the five affected family members have developed Parkinson's disease between ages 50 and 65 years, many years after neuropathy onset. We speculate that this late-onset parkinsonism is part of the LRSAM1 phenotype, thus associating a hitherto peripheral nerve disease with a central nervous system phenotype. How the mutated Lrsam1 protein, which normally has E3 ubiquitin ligase activity and is expressed in the nervous system, impacts on substantia nigra neurons is unclear.
Project description:BACKGROUND: Point mutations in the mitofusin 2 (MFN2) gene has been identified exclusively in Charcot-Marie-Tooth type 2 (CMT2), and in a single family with intermediate CMT. MFN2 point mutations are probably the most common cause of CMT2. METHODS: Two-hundred and thirty-two consecutive unselected and unrelated CMT families with available DNA from all regions in Norway were included. We screened for point mutations in the MFN2 gene. RESULTS: We identified four known and three novel point mutations in 8 unrelated CMT families. The novel point mutations were not found in 100 healthy controls. This corresponds to 3.4% (8/232) of CMT families have point mutations in the MFN2 gene. The phenotypes were compatible with CMT1 in two families, CMT2 in four families, intermediate CMT in one family and distal Hereditary Motor Neuropathy (dHMN) in one family. This corresponds to 2.3% of CMT1, 5.5% of CMT2, 12.5% of intermediate CMT and 6.7% of dHMN families have a point mutation in the MFN2 gene. Point mutations in the MFN2 gene is likely to be the fourth most common cause to CMT after duplication of the peripheral myelin protein 22 (PMP22) gene, and point mutations in the Connexin32 (Cx32) and myelin protein zero (MPZ) genes. CONCLUSIONS: The identified known and novel point mutations in the MFN2 gene expand the clinical spectrum from CMT2 and intermediate CMT to also include possibly CMT1 and the dHMN phenotypes. Thus, genetic analyses of the MFN2 gene should not be restricted to persons with CMT2.
Project description:The Charcot-Marie-Tooth (CMT) disorders comprise a group of clinically and genetically heterogeneous hereditary motor and sensory neuropathies, which are mainly characterized by muscle weakness and wasting, foot deformities, and electrophysiological, as well as histological, changes. A subtype, CMT2, is defined by a slight or absent reduction of nerve-conduction velocities together with the loss of large myelinated fibers and axonal degeneration. CMT2 phenotypes are also characterized by a large genetic heterogeneity, although only two genes---NF-L and KIF1Bbeta---have been identified to date. Homozygosity mapping in inbred Algerian families with autosomal recessive CMT2 (AR-CMT2) provided evidence of linkage to chromosome 1q21.2-q21.3 in two families (Zmax=4.14). All patients shared a common homozygous ancestral haplotype that was suggestive of a founder mutation as the cause of the phenotype. A unique homozygous mutation in LMNA (which encodes lamin A/C, a component of the nuclear envelope) was identified in all affected members and in additional patients with CMT2 from a third, unrelated family. Ultrastructural exploration of sciatic nerves of LMNA null (i.e., -/-) mice was performed and revealed a strong reduction of axon density, axonal enlargement, and the presence of nonmyelinated axons, all of which were highly similar to the phenotypes of human peripheral axonopathies. The finding of site-specific amino acid substitutions in limb-girdle muscular dystrophy type 1B, autosomal dominant Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy type 1A, autosomal dominant partial lipodystrophy, and, now, AR-CMT2 suggests the existence of distinct functional domains in lamin A/C that are essential for the maintenance and integrity of different cell lineages. To our knowledge, this report constitutes the first evidence of the recessive inheritance of a mutation that causes CMT2; additionally, we suggest that mutations in LMNA may also be the cause of the genetically overlapping disorder CMT2B1.
Project description:Charcot-Marie-Tooth disease type 1 (CMT1) or hereditary motor and sensory neuropathy type I (HMSNI) is an autosomal dominant peripheral neuropathy. In most families the disease segregates with a 1.5 Mb duplication on chromosome 17p11.2 (CMT1A). A few patients have been found with point mutations in the PMP-22 gene. In some families linkage has been found with markers located on chromosome 1q21-q25 (CMT1B) and more recently mutations have been identified in the P0 gene. We analysed an extended CMT1 pedigree (CMT-B) without the CMT1A duplication. Significant positive linkage with chromosome 1 indicated that this family is of the CMT1B subtype. Sequencing of the candidate gene P0 located in chromosome band 1q21-q23 showed a C to A point mutation at position 446 in exon 3 resulting in an Asp134Glu substitution. Since the P0 mutation cosegregated with CMT1 disease we suggest that this mutation is the primary genetic cause of CMT1B in family CMT-B.
Project description:Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. One form of CMT, CMT type 1A, is characterized by uniformly decreased nerve conduction velocities, usually shows autosomal dominant inheritance, and is associated with a large submicroscopic duplication of the p11.2-p12 region of chromosome 17. A cohort of 75 unrelated patients diagnosed clinically with CMT and evaluated by electrophysiological methods were analyzed molecularly for the presence of the CMT1A DNA duplication. Three methodologies were used to assess the duplication: measurement of dosage differences between RFLP alleles, analysis of polymorphic (GT)n repeats, and detection of a junction fragment by pulsed-field gel electrophoresis. The CMT1A duplication was found in 68% of the 63 unrelated CMT patients with electrophysiological studies consistent with CMT type 1 (CMT1). The CMT1A duplication was detected as a de novo event in two CMT1 families. Twelve CMT patients who did not have decreased nerve conduction velocities consistent with a diagnosis of CMT type 2 (CMT2) were found not to have the CMT1A duplication. The most informative molecular method was the detection of the CMT1A duplication-specific junction fragment. Given the high frequency of the CMT1A duplication in CMT patients and the high frequency of new mutations, we conclude that a molecular test for the CMT1A DNA duplication is very useful in the differential diagnosis of patients with peripheral neuropathies.