Mitochondrial fusion and function in Charcot-Marie-Tooth type 2A patient fibroblasts with mitofusin 2 mutations

Charcot-Marie-Tooth Type 2A is a dominantly inherited peripheral neuropathy characterized by axonal degeneration of sensory and motor nerves. The disease is caused by mutations in the mitochondrial fusion gene MFN2. Mfn2 is an integral outer mitochondrial membrane protein composed of a large GTPase domain and two heptad repeat (HR) domains that face the cytoplasm. Mitochondrial membrane fusion and division are balanced processes that are necessary to maintain tubular mitochondrial morphology, respiratory function, and uniform distribution of the organelle throughout the cell. We have utilized primary fibroblasts from CMT2A patients to survey mitochondrial phenotypes associated with heterozygous MFN2 alleles expressed at physiological levels. Our results indicate that, in fibroblasts, mitofusin expression, mitochondrial morphology, ultrastructure, mtDNA content, and respiratory capacity are not affected by the presence of mutant Mfn2 protein. Consistent with a lack of mitochondrial dysfunction, we also show that mitochondrial fusion occurs efficiently in CMT2A patient-derived fibroblasts. Our observations are in agreement with the neuronal specificity of the disease and are consistent with a recent finding that mitochondrial fusion can be maintained in cells that express mutant Mfn2 protein due to complementation by a second mitofusin, Mfn1. We discuss our results and those of others in terms of a comprehensive model for the mechanism(s) by which mutations in MFN2 may lead to CMT2A disease.     

Role of mitofusin 2 mutations in the physiopathology of Charcot–Marie–Tooth disease type 2A

Charcot–Marie–Tooth disease (CMT) is the most common form of hereditary peripheral neuropathy. The main axonal form of CMT, CMT2A, preferentially affects peripheral neurons with the longest neurites. CMT2A has been recently linked to mutations in the mitofusin 2 (Mfn2) gene. Mfn2 participates in mitochondrial fusion a process that together with mitochondrial fission, contributes to mitochondrial morphology. Many hypotheses have been postulated to understand how mutations in Mfn2 lead to CMT2A. In this review, we will describe the physiological role of Mfn2, the pathophysiology of CMT2A and current hypotheses about the deleterious role of mutant Mfn2 in neuronal function.     

Mutational mechanisms in MFN2‐related neuropathy: compound heterozygosity for recessive and semidominant mutations

Mitofusin‐2 (MFN2) mutations are the most common cause of autosomal dominant axonal Charcot‐Marie‐Tooth disease (CMT, type 2A), sometimes complicated by additional features such as optic atrophy (CMT6) and upper motor neuron involvement (CMT5). Several pathogenic mutations are reported, mainly acting in a dominant fashion, although few sequence variants behaved as recessive or semidominant in rare homozygous or compound heterozygous patients. We describe a 49‐year‐old woman with CMT5 associated with compound heterozygosity for two MFN2 variants, one already reported missense mutation (c.748C>T, p.R250W) and a novel nonsense sequence change (c.1426C>T, p.R476*). Her mother, carrying the p.R250W variant, had very late‐onset minimal axonal neuropathy, whilst the father harboring the nonsense sequence change had neither clinical nor electrophysiological neuropathy. The missense mutation is likely pathogenic according to in silico analyses and a previous report, while the nonsense variant is predicted to behave as a null allele. The p.R250W variant behaves as semidominant by causing only a mild, almost subclinical, neuropathy when heterozygous; the nonsense mutation in the father was phenotypically silent, suggesting that haploinsufficiency for MFN2 is not disease causative, but was deleterious in the daughter who had only one active mutated MFN2 allele.     

Clinical and genetic diversities of Charcot‐Marie‐Tooth disease with MFN2 mutations in a large case study

Charcot‐Marie‐Tooth disease (CMT) constitutes a heterogeneous group affecting motor and sensory neurons in the peripheral nervous system. MFN2 mutations are the most common cause of axonal CMT. We describe the clinical and mutational spectra of CMT patients harboring MFN2 mutations in Japan. We analyzed 1,334 unrelated patients with clinically suspected CMT referred by neurological and neuropediatric departments throughout Japan. We conducted mutation screening using a DNA microarray, targeted resequencing, and whole‐exome sequencing. We identified pathogenic or likely pathogenic MFN2 variants from 79 CMT patients, comprising 44 heterozygous and 1 compound heterozygous variants. A total of 15 novel variants were detected. An autosomal dominant family history was determined in 43 cases, and the remaining 36 cases were reported as sporadic with no family history. The mean onset age of CMT in these patients was 12 ± 14 (range 0–59) years. We observed neuropathic symptoms in all patients. Some had optic atrophy, vocal cord paralysis, or spasticity. We detected a compound heterozygous MFN2 mutation in a patient with a severe phenotype and the co‐occurrence of MFN2 and PMP22 mutations in a patient with an uncommon phenotype. MFN2 is the most frequent causative gene of CMT2 in Japan. We present 15 novel variants and broad clinical and mutational spectra of Japanese MFN2‐related CMT patients. Regardless of the onset age and inheritance pattern, MFN2 gene analysis should be performed. Combinations of causative genes should be considered to explain the phenotypic diversity.     

A mutation in the heptad repeat 2 domain of MFN2 in a large CMT2A family

Dominant mutations in MFN2 cause a range of phenotypes, including severe, early‐onset axonal neuropathy, “classical CMT2,” and late‐onset axonal neuropathies. We report a large family with an axonal polyneuropathy, with clinical onset in the 20s, followed by slow progression.     

Early onset Charcot‐Marie‐Tooth neuropathy type 2A and severe developmental delay: expanding the clinical phenotype of MFN2‐related neuropathy

Charcot‐Marie‐Tooth (CMT) syndromes are a group of clinically heterogeneous disorders of the peripheral nervous system. Mutations of mitofusin 2 (MFN2) have been recognized to be associated with CMT type 2A (CMT2A). CMT2A is primarily an axonal disorder resulting in motor and sensory neuropathy. We report a male child with psychomotor delay, dysmorphic features, and weakness of lower limbs associated with electrophysiological features of severe, sensory‐motor, axonal neuropathy. The patient was diagnosed with early onset CMT2A and severe psychomotor retardation associated with c.310C>T mutation (p.R104W) in MFN2 gene. CMT2A should be considered in patients with both axonal sensory‐motor neuropathy and developmental delay.     

MFN2‐related genetic and clinical features in a cohort of Chinese CMT2 patients

Charcot‐Marie‐Tooth disease 2A (CMT2A), caused by mutations in the mitofusin 2 gene (MFN2), is the most common CMT2 subtype. The aim of our study is to assess the frequency and summarize the genetic and clinical characteristics of Chinese CMT2A patients. A total of 17 coding exons of MFN2 were detected by direct sequencing in 82 unrelated Chinese families diagnosed as CMT2. Clinical evaluations were analyzed among CMT2A patients. We identified 14 missense variants in 9 sporadic and 6 familial cases, including four novel mutations (T129A, S249F, Q367P, and Q674L), 4 known mutations (R94W, R94Q, T105M, C132Y, M376V and Q751X), and 4 rare missense variants (K120E, C217F, K307E and T356S). A total of 23 patients had early‐onset phenotype. Two patients had a CMTNS score of 0 to 10; 16 had a score of 11 to 20; and 7 had a score greater than 20. Five patients were confirmed a de novo origin. Six of 14 variants were located or closed to the GTPase domain. We report four novel mutations and four rare missense variants. MFN2 mutations account for 18% of CMT2 families in mainland China. The common characteristics of Chinese pedigree are early disease onset and moderate phenotypes.     

Mechanisms of disease and clinical features of mutations of the gene for mitofusin 2: an important cause of hereditary peripheral neuropathy with striking clinical variability in children and adults

Mitofusin 2, a large transmembrane GTPase located in the outer mitochondrial membrane, promotes membrane fusion and is involved in the maintenance of the morphology of axonal mitochondria. Mutations of the gene encoding mitofusin 2 (MFN2) have recently been identified as the cause of approximately one‐third of dominantly inherited cases of the axonal degenerative forms of Charcot–Marie–Tooth disease (CMT type 2A) and of rarer variants. The latter include a severe, early‐onset axonal neuropathy, which may occur in autosomal dominant or recessive forms, as well as some instances associated with pyramidal tract involvement (CMT type 5), with optic atrophy (CMT type 6), and, occasionally, with alterations of cerebral white matter. All individuals with a dominantly or recessively inherited or otherwise unexplained, chronic progressive axonal degenerative polyneuropathy should be tested for mutations of MFN2.     

Increased mitochondrial fusion in a autosomal recessive CMT2A family with mitochondrial GTPase mitofusin 2 mutations

Charcot‐Marie‐Tooth type 2A disease (CMT2A) is an inherited peripheral neuropathy mainly caused by mutations in the MFN2 gene coding for the mitochondrial fusion protein mitofusin 2. Although the disease is mainly inherited in a dominant fashion, few cases of early‐onset autosomal recessive CMT2A (AR‐CMT2A) have been reported in recent years. In this study, we characterized the structure of the mitochondrial network in cultured primary fibroblasts obtained from AR‐CMT2A family members. The patient‐derived cells showed an increase of the mitochondrial fusion with large connected networks and an increase of the mitochondrial volume. Interestingly, fibroblasts derived from the two asymptomatic parents showed similar changes to a lesser extent. These results support the hypothesis that AR‐CMT2A‐related MFN2 mutations acts through a semi‐dominant negative mechanism and suggest that other biological parameters might show mild alterations in asymptomatic heterozygote AR‐CMT2A patients. Such alterations could be useful biomarkers helping to distinguish MFN2 mutations from variants, a growing challenge with the advent of next generation sequencing into routine clinical practice.     

Mutation screening of <Emphasis Type="Italic">mitofusin 2</Emphasis> in Charcot-Marie-Tooth disease type 2

Charcot-Marie-Tooth (CMT) disease is among the most common inherited neurological disorders. Mutations in the gene mitofusin 2 (MFN2) cause the axonal subtype CMT2A, which has also been shown to be associated with optic atrophy, clinical signs of first motor neuron involvement, and early onset stroke. Mutations in MFN2 account for up to 20–30% of all axonal CMT type 2 cases. To further investigate the prevalence of MFN2 mutations and to add to the genotypic spectrum, we sequenced all exons of MFN2 in a cohort of 39 CMT2 patients. We identified seven variants, four of which are novel. One previously described change was co-inherited with a PMP22 duplication, which itself causes the demyelinating form CMT1A. Another mutation was a novel in frame deletion, which is a rare occurrence in the genotypic spectrum of MFN2 characterized mainly by missense mutations. Our results confirm a MFN2 mutation rate of ~15–20% in CMT2.     

MFN2 deletion of exons 7 and 8: founder mutation in the UK population

Mitofusin 2 (MFN2) mutations are the most common cause of axonal Charcot‐Marie‐Tooth disease (CMT2). The majority are inherited in an autosomal dominant manner but recessive and semi‐dominant kindreds have also been described. We previously reported a deletion of exons 7 and 8 resulting in nonsense‐mediated decay, segregating with disease when present in trans with another pathogenic MFN2 mutation. Detailed clinical and electrophysiological data on a series of five affected patients from four kindreds and, when available, their parents and relatives were collected. MFN2 Sanger sequencing, multiplex ligation probe amplification, and haplotype analysis were performed. A severe early‐onset CMT phenotype was seen in all cases: progressive distal weakness, wasting, and sensory loss from infancy or early childhood. Optic atrophy (four of five) and wheelchair dependency in childhood were common (four of five). All were compound heterozygous for a deletion of exons 7 and 8 in MFN2 with another previously reported pathogenic mutation (Phe216Ser, Thr362Met, and Arg707Trp). Carrier parents and relatives were unaffected (age range: 24–82 years). Haplotype analysis confirmed that the deletion had a common founder in all families.     

Mutations in the mitofusin 2 gene are the most common cause of Charcot-Marie-Tooth type 2 disease

In contrast to Charcot-Marie-Tooth type 1 disease (CMT1), which is most commonly caused by 17p11.2-p12 duplication (in 70% of CMT1 cases), the axonal form of hereditary motor and sensory neuropathy (CMT2) seemed to be a genetically heterogeneous disease group, with no single gene playing a major pathogenetic role. In 2004, 10 mutations were identified in CMT2A families in the MFN2 gene coding for the mitochondrial protein mitofusin-2, previously mapped to the 1p35-36 locus. In the last two years, MFN2 gene mutations were shown to be the most common cause of autosomal dominant hereditary axonopathy. In addition, MFN2 gene mutations were also identified in CMT type 6 (axonal neuropathy with optic nerve atrophy). Recent reports indicate that some MFN2 gene mutations may by inherited as autosomal recessive traits. As MFN2 gene mutations are the most common cause of autosomal dominant CMT2 disease (33% of cases), MFN2 gene testing may be considered a diagnostic test for CMT2.     

Mitofusin 1 overexpression rescues the abnormal mitochondrial dynamics caused by the Mitofusin 2 K357T mutation in vitro

Background and aims

Mitofusin 1 (MFN1) and MFN2 are outer mitochondrial membrane fusogenic proteins regulating mitochondrial network morphology. MFN2 mutations cause Charcot-Marie-Tooth type 2A (CMT2A), an axonal neuropathy characterized by mitochondrial fusion defects, which in the case of a GTPase domain mutant, were rescued following wild-type MFN1/2 (MFN1/2^WT) overexpression. In this study, we compared the therapeutic efficiency between MFN1^WT and MFN2^WT overexpression in correcting mitochondrial defects induced by the novel MFN2^K357T mutation located in the highly conserved R3 region.

Methods

Constructs expressing either MFN2^K357T, MFN2^WT, or MFN1^WT under the ubiquitous chicken β-actin hybrid (CBh) promoter were generated. Flag or myc tag was used for their detection. Differentiated SH-SY5Y cells were single transfected with MFN1^WT, MFN2^WT, or MFN2^K357T, as well as double transfected with MFN2^K357T/MFN2^WT or MFN2^K357T/MFN1^WT.

Results

SH-SY5Y cells transfected with MFN2^K357T exhibited severe perinuclear mitochondrial clustering with axon-like processes devoid of mitochondria. Single transfection with MFN1^WT resulted in a more interconnected mitochondrial network than transfection with MFN2^WT, accompanied by mitochondrial clusters. Double transfection of MFN2^K357T with either MFN1^WT or MFN2^WT resolved the mutant-induced mitochondrial clusters and led to detectable mitochondria throughout the axon-like processes. MFN1^WT showed higher efficacy than MFN2^WT in rescuing these defects.

Interpretation

These results further demonstrate the higher potential of MFN1^WT over MFN2^WT overexpression to rescue CMT2A-induced mitochondrial network abnormalities due to mutations outside the GTPase domain. This higher phenotypic rescue conferred by MFN1^WT, possibly due to its higher mitochondrial fusogenic ability, may be applied to different CMT2A cases regardless of the MFN2 mutation type.     

Late‐onset hereditary sensory and autonomic neuropathy expands the phenotypic spectrum of MFN2‐related diseases

Mutations in the Mitofusin 2 (MFN2) gene have been identified in patients with autosomal dominant axonal motor and sensory neuropathy or Charcot–Marie‐Tooth 2A (CMT2A). Here we describe clinical and pathological changes in an adult patient with sporadic hereditary sensory and autonomic neuropathy (HSAN) due to an MFN2 mutation. The patient was a 53‐year‐old man who had sensory involvement and anhidrosis in all limbs without motor features. The electrophysiological assessment documented severe axonal sensory neuropathy. The sural nerve biopsy confirmed the electrophysiological findings, revealing severe loss of myelinated and unmyelinated fibers with regeneration clusters. Genetic analysis revealed the previously identified mutation c.776 G > A in MFN2. Our report expands the phenotypic spectrum of MFN2‐related diseases. Sequencing of MFN2 should be considered in all patients presenting with late‐onset HSAN.     

Dominant GDAP1 founder mutation is a common cause of axonal Charcot-Marie-Tooth disease in Finland

We describe a founder mutation in the gene encoding ganglioside-induced differentiation associated-protein 1 (GDAP1), leading to amino acid change p.H123R, as a common cause of autosomal dominant axonal Charcot-Marie-Tooth (CMT2) neuropathy in Finland. The mutation explains up to 14 % of CMT2 in Finland, where most patients with axonal neuropathy have remained without molecular diagnosis. Only three families out of 28 were found to carry putative disease mutations in the MFN2 gene encoding mitofusin 2. In addition, the MFN2 variant p.V705I was commonly found in our patients, but we provide evidence that this previously described mutation is a common polymorphism and not pathogenic. GDAP1-associated polyneuropathy caused predominantly a mild and slowly progressive phenotype. Besides distal leg muscle weakness, most patients showed mild proximal weakness, often with asymmetry and pes cavus. Our findings broaden the understanding of GDAP1 mutations in CMT2 phenotypes and provide support for the use of whole-exome sequencing in CMT gene diagnostics.     

Characterizing the phenotypic manifestations of MFN2 R104W mutation in Charcot-Marie-Tooth type 2

Mutations of the mitofusin 2 (MFN2) gene have been reported to be the most common cause of the axonal form of Charcot-Marie-Tooth disease (CMT). The aim of this study was to describe a de novo MFN2 p.R104W mutation and characterize the associated phenotype. We screened the entire coding region of MFN2 gene and characterized its clinical phenotype, nerve conduction studies and sural nerve biopsy. Neuropsychological tests and brain MRI were also performed. A de novo mutation was found in exon 4 (c.310C > T; p.R104W). In addition to a severe and early onset axonal neuropathy, the patient presented learning problems, obesity, glucose intolerance, leukoencephalopathy, brain atrophy and evidence of myelin involvement and mitochondrial structural changes on sural nerve biopsy. These results suggest that MFN2 p.R104W mutation is as a hot-spot for MFN2 gene associated to a large and complex range of phenotypes.     

Sonography of the median nerve in CMT1A,CMT2A,CMTX, and HNPP

Introduction: In this study we compare the ultrasound features in the median nerve in patients with different types of Charcot–Marie–Tooth (CMT) disease and hereditary neuropathies with liability to pressure palsies (HNPP) as a typical entrapment neuropathy. Methods: Median nerve ultrasound and conduction studies were performed in patients with CMT1A (n = 12), MFN2‐associated CMT2A (n = 7), CMTX (n = 5), and HNPP (n = 5), and in controls (n = 28). Results: Median nerve cross‐sectional area (CSA) was significantly increased in CMT1A, whereas, in axonal CMT2A, fascicle diameter (FD) was enlarged. CSA correlated with nerve conduction slowing in CMT1A and with axonal loss, as shown by motor and sensory nerve amplitudes in both CMT1A and CMT2A. A relatively low wrist‐to‐forearm‐ratio (WFR <0.8) or a relatively high WFR (>1.8) appeared to be unlikely in MFN2 and Cx32 mutations of CMT2A and CMTX, respectively. Conclusion: Differences in CSA, FD, and WFR of the median nerve can be helpful in defining subtypes of hereditary neuropathies. Muscle Nerve 47:385‐395, 2013     

Phenotypical features of the p.R120W mutation in the GDAP1 gene causing autosomal dominant Charcot-Marie-Tooth disease

Mutations in the ganglioside-induced-differentiation-associated protein 1 gene (GDAP1) can cause Charcot-Marie-Tooth (CMT) disease with demyelinating (CMT4A) or axonal forms (CMT2K and ARCMT2K). Most of these mutations present a recessive inheritance, but few autosomal dominant GDAP1 mutations have also been reported. We performed a GDAP1 gene screening in a clinically well-characterized series of 81 index cases with axonal CMT neuropathy, identifying 17 patients belonging to 4 unrelated families in whom the heterozygous p.R120W was found to be the only disease-causing mutation. The main objective was to fully characterize the neuropathy caused by this mutation. The clinical picture included a mild-moderate phenotype with onset around adolescence, but great variability. Consistently, ankle dorsiflexion and plantar flexion were impaired to a similar degree. Nerve conduction studies revealed an axonal neuropathy. Muscle magnetic resonance imaging studies demonstrated selective involvement of intrinsic foot muscles in all patients and a uniform pattern of fatty infiltration in the calf, with distal and superficial posterior predominance. Pathological abnormalities included depletion of myelinated fibers, regenerative clusters and features of axonal degeneration with mitochondrial aggregates. Our findings highlight the relevance of dominantly transmitted p.R120W GDAP1 gene mutations which can cause an axonal CMT with a wide clinical profile.