Figure 1. Identified new MFN2 mutation and affected kindred. A, DNA sequencing results showing the identified missense alteration within MFN2 at nucleotide base 436 C→T with resultant amino acid change of leucine (Leu) to phenylalanine (Phe) at codon position 146 CTC→TTC: Leu146Phe. B, The examined American kindred of Northern European and Cherokee Indian descent with genetic testing (indicated by asterisk) revealing the affected status associated with the demonstrated base alteration. In addition to the shown persons, 800 evaluable healthy controls underwent DNA sequencing without MFN2 436 C→T alteration identified and not reported at Web-based Human Gene Mutation Database Biobase and National Center for Biotechnology Information dbSNP. mut Indicates mutation; wt, wild type. The arrow signifies the proband; shaded squares (males) and circles (females), clinically affected by examination and history.
Klein CJ, Kimmel GW, Pittock SJ, Engelstad JE, Cunningham JM, Wu Y, Dyck PJ. Large Kindred Evaluation of Mitofusin 2 Novel Mutation, Extremes of Neurologic Presentations, and Preserved Nerve Mitochondria. Arch Neurol. 2011;68(10):1295-1302. doi:10.1001/archneurol.2011.225
Author Affiliations: Divisions of Neuroimmunology (Drs Klein and Pittock), Peripheral Nerve Diseases (Drs Klein and Dyck and Ms Engelstad), Department of Neurology, Laboratory Medicine and Pathology (Drs Cunningham and Wu), and Medical Genetics (Dr Klein), Mayo Clinic, Rochester, Minnesota; and Biological Sciences, University of Miami, Miami, Florida (Ms Kimmel).
Background Mitofusin 2 (MFN2) is a mitochondrial membrane protein mediating mitochondrial fusion and function. Mutated MFN2 is responsible for Charcot-Marie-Tooth type 2A2. In small kindreds, specific MFN2 mutations have been reported to associate with severity of axonal neuropathy, optic atrophy, and involvement of the central nervous system. The results of the nerve biopsy specimens suggested that the mitochondria are structurally abnormal in patients with MFN2 mutations.
Objective To study a newly identified MFN2 mutation, Leu146Phe, and the associated phenotypes in a large kindred.
Patients An American kindred of Northern European and Cherokee American Indian descent.
Results Genetic analysis revealed a novel GTPase domain MFN2 mutation Leu146Phe that associated with clinical status of 15 studied persons (10 affected and 5 unaffected) and not found in 800 control persons. Clinical manifestations were markedly different. In 1 affected person, optic atrophy and brain magnetic resonance imaging abnormalities led to multiple sclerosis diagnosis and interferon β-1a treatment when neuropathy was initially unrecognized. Age of onset ranged from 1 to 45 years. In some affected family members, severe and rapid-onset motor sensory neuropathy led to early loss of ambulation, whereas other family members experienced minimal neuropathic sensory symptoms. Despite histologically significant loss of nerve fibers, the mitochondria were not distinguishable from diseased sural nerve biopsy specimens and healthy controls.
Conclusions Novel MFN2 mutation Leu146Phe causes Charcot-Marie-Tooth type 2A2. Intrafamilial clinical phenotype variability is emphasized and has important implications in genetic counseling. The clinical phenotype may mimic multiple sclerosis when optic atrophy and the characteristic brain lesions of MFN2 on magnetic resonance imaging are present and neuropathy is mild or unrecognized. The predicted molecular pathogenesis may occur without evident histological abnormalities of mitochondria in nerve.
Mitofusin 2 (MFN2) mutations are responsible for the most common known axonal form of Charcot-Marie-Tooth (CMT) disease, CMT2A2.1- 5 Mutation in Kinesin 1B (KIF1B) was first identified and designated as CMT2A1. Kinesin 1b colocalizes with MFN2 at chromosome position 1p36.2, but KIF1B mutation has been identified in only 1 rare family.6 In contrast, more than 80 MFN2 mutations have been reported that cause CMT2A2, and in 1 study,4 the frequency of MFN2 mutations was as high as 33% in tested patients with inherited axonal CMT. Mitofusin 2 is a large transmembrane mitochondrial GTPase protein located on outer mitochondrial membranes. Axonal mitochondria morphology is thought to be maintained by MFN2, which promotes mitochondria membrane fusion.7 Mutations have been identified throughout the MFN2 protein, but the majority of them occur in the hydrophobic coiled-coil (HR1) and GTPase domains, both of which are essential for mitochondrial fusion.8 Mutations of MFN2 are theorized to have complex pathogenic effects. One predicted mechanism of disease is that energy production required for axonal mitochondrial transport may be reduced by defective MFN2.9 Mitochondrial tethering to the endoplasmic reticulum is also thought to be defective, with MFN2 mutations triggering neuronal apoptosis by calcium uptake dysregulation.10 These molecular pathogenic mechanisms possibly change mitochondria histologic appearance. A limited number of studies4,11,12 on nerves from persons with mutated MFN2 have suggested misshaped and aggregated mitochondria, but the specificity of those findings is not clear.
With expanding clinical experience, we increasingly recognize that the patients affected by MFN2 mutations have diverse inheritance patterns including (1) dominant heterozygotes (most common), (2) recessive homozygotes, and (3) compound dominant heterozygotes. Previous reports13 suggested that the patients with compound heterozygosity had more severe neuropathy, and their phenotypes are consistent with additive dominant negative effect of MFN2 mutation. Axonal motor sensory neuropathy with more severe lower-extremity than upper-extremity involvement provides the predominant clinical phenotype of CMT2A2. However, observations of patients with MFN2 mutations with peripheral neuropathy and myelopathy (hereditary motor sensory neuropathy type V),14 or peripheral neuropathy and optic neuropathy (hereditary motor sensory neuropathy type VI),15 expand the clinical presentations. In addition, clinical involvement of the central nervous system (CNS) is supported by magnetic resonance imaging (MRI) of brain abnormalities in some affected persons.3,16
Two relatively distinct, temporal onset severity groups appear to distinguish different MFN2 mutations: (1) adult or adolescent onset, typically mild and insidious and (2) early-severe onset, often before age 5 years, with rapid progression.3 These 2 distinct phenotypes have tended to run in specific families, with optic atrophy occurring in more severely affected persons. However, 1 detailed kindred evaluation17 suggested that, within individual families, clinically affected status may be very mild and may require detailed neurologic and electrophysiologic examination to identify affected status. Nonetheless, most reports have highlighted the variability in expression between and not within CMT2A2 families.
With institutional research board approval and informed patient consent, we undertook kindred evaluation and genetic analysis in a large American kindred of Northern European and Cherokee American Indian descent. Clinical examinations, clinical history evaluation, and MFN2 exon sequencing were performed within this large family. We identified an MFN2 DNA base alteration 436 C→T, changing amino acid sequence of leucine to phenylalanine at codon position 146 CTC→TTC (Figure 1). This base alteration had not been previously reported, to our knowledge, and 800 controls were sequenced to exclude the possibility of rare polymorphism. To examine whether this compound heterozygous status may cause the observed clinical variable expression, the entire MFN2 open reading frame was sequenced. The kindred and clinical features were extracted and medical records reviewed; the earliest known affected person (designated as I-1) had a Cherokee American Indian mother and father coming from England in the 1600s, but their affected status is unknown.
A whole sural nerve biopsy of the proband (VI-9) was examined. Teased nerve fibers and paraffin- and epoxy-embedded semithin sections were studied by techniques previously described,18 including nerve morphometric and electron microscopy. Qualitative mitochondrial structural features were reviewed in the patient and in comparison with 21 healthy examined persons and 100 consecutively obtained diseased sural nerve biopsy specimens in longitudinal and transverse section at varied magnification under electron microscopy. The diseased and healthy control electron micrographs were collected as part of an earlier study.
All examined patients clinically confirmed with neuropathy (n = 10) were shown to have the Leu146Phe mutation (Figure 1). None of the clinically unaffected persons (n = 5) had the mutation, nor did the healthy screened controls. Compound heterozygote status was not found. The clinical symptoms, including ages of clinical onset, involvement of the CNS, and optic atrophy, are summarized in the Table. The clinical features among affected persons were demonstrated with illustrative cases highlighting largely 3 categories of clinical presentations: (1) optic atrophy and CNS disease with mild neuropathy; (2) early-onset, rapid-progression severe neuropathy; and (3) late-onset, slow-progression mild neuropathy. Hearing loss, scoliosis, tremor, and parkinsonism did not occur.
We describe a new MFN2 amino acid alteration Leu146Phe within the GTPase domain of the MFN2 protein. This mutation appears pathogenic for a large CMT2A2 American kindred with markedly varied clinical phenotypes. To our knowledge, to date, this mutation has not been described among the many identified European-origin CMT2 families, raising the question of a unique American mutation arising from this kindred with Cherokee American Indian ancestry. The broad phenotypic spectrum seen included (1) optic atrophy and CNS involvement with mild neuropathy; (2) early-onset, rapidly progressive severe neuropathy; and (3) late-onset, slowly progressive mild sensory neuropathy. Compound heterozygous mutations were not found and therefore did not account for the most severely affected persons with toddler onset, as has been described previously.13 The study supports the belief that MFN2 mutations do not necessarily associate with a particular discrete phenotype, as previously observed.3 Small kindred size from earlier studies did not emphasize that there could be a significant clinical variability within 1 family with all affected carrying the same mutation. In counseling families with MFN2 gene mutations (OMIM *608507), it is important to provide understanding that a relatively mildly affected parent may give rise to a severely affected child; conversely, a severely affected parent with motor neuropathy may give rise to a child with mild neuropathy. Both situations were observed here; mildly affected parent IV-8 had severely affected child V-7. Parent V-18 had severe neuropathy, whereas her children, VI-9 and VI-10, had mild neuropathy but with blindness from optic atrophy.
This study demonstrates that persons with MFN2 mutations having optic atrophy and paraventricular white matter T2 signal changes may be considered to have multiple sclerosis when the neuropathy symptom is mild or unrecognized. Our proband's sister, VI-10, had bilateral optic atrophy and paraventricular MRI abnormalities that predated identification of her neuropathy. This led to initiation of interferon β-1a for presumed multiple sclerosis. In her case, the absence of CSF oligoclonal bands, no gadolinium enhancement on MRI brain lesion, insidious progression of optic atrophy without MRI brain progression, and absence of upper motor neuron signs would all suggest against the diagnosis of multiple sclerosis. The identification of her MFN2 mutation and electrophysiologic confirmation of her neuropathy led to the diagnosis of her CMT2A2. Of additional note is that she developed complex partial seizures years into her disease. This may be incidental because seizures have not been reported in other MFN2 mutation cases; however, alteration of consciousness with fatal encephalopathy is described.19 Evaluation of her brother (VI-9), who also has optic neuropathy and peripheral neuropathy, assisted in identification of the pathogenic cause in her case. The brother (VI-9) had no clinical stigmata of CNS disease with normal brain and spinal cord imaging, but his evoked potentials were consistent with spinal cord involvement. Because MFN2 is ubiquitously expressed, including in the mitochondria of brain and spinal cord, the pathologic involvements of these tissues is predicted and has been previously reported.3,4,20 In the evaluation of a patient with optic atrophy, a family history of axonal length-dependent neuropathy should not be considered an incidental occurrence, and MFN2 mutations need to be considered.
A limited number of electron microscopy reports4,12 have suggested abnormal shape, size, aggregation, and cristae of the mitochondria in patients with MFN2 mutation. However, we did not observe abnormal shape, size, aggregation, and cristae of the mitochondria in our studied case, despite the loss of large myelinated fibers. The mitochondria appeared evenly distributed in the axons in our case, and the cristae and other mitochondrial features in Schwann cell and axonal cytoplasms were indistinguishable from equivalently prepared healthy controls and patients with diseased sural nerve biopsy specimens4 (Figures 4 and 5).
This study expands the genetic causes of CMT2A2. The optic neuropathy and brain imaging findings may lead to major consideration of multiple sclerosis when neuropathy is overlooked or mild. The fact that there is a range of severity and presentation in a single large family is important. Specifically, in genetic counseling, a parent with mild disease may give rise to a child with severe early-onset of disease or a person with blindness. Last, mutations of MFN2 may exert their molecular pathogenic effect without clear structural mitochondrial abnormalities compared with normal and diseased biopsy specimens.
Correspondence: Christopher J. Klein, MD, Mayo Clinic, 200 First St SW, Mayo Clinic, Rochester, MN 55905 (firstname.lastname@example.org).
Accepted for Publication: February 17, 2011.
Author Contributions:Study concept and design: Klein and Dyck. Acquisition of data: Klein, Engelstad, and Cunningham. Analysis and interpretation of data: Klein, Kimmel, Pittock, and Wu. Drafting of the manuscript: Klein and Kimmel. Critical revision of the manuscript for important intellectual content: Klein, Pittock, Engelstad, Cunningham, Wu, and Dyck. Statistical analysis: Klein. Obtained funding: Klein. Administrative, technical, and material support: Klein and Kimmel. Study supervision: Klein and Dyck.
Financial Disclosure: Dr Pittock and Mayo Clinic have a financial interest in the technology titled “Aquaporin-4 Autoantibody as a Cancer Marker,” which has been licensed to a commercial entity, but no royalties have been received. In addition, he is an inventor of technology titled “Aquaporin-4 Binding Autoantibodies in Patients With Neuromyelitis Optic Impair Glutamate Transport by Down-Regulating EAAT2.” Mayo Clinic has filed a nonprovisional patent application for this technology. Also, he received a research grant from Alexion Pharmaceuticals for his investigator-initiated study titled “An Open Label Study of Eculizumab in NMO.” Dr Pittock is a consultant in the Department of Laboratory Medicine and Pathology. In his role as codirector of the Neuroimmunology Laboratory, he has no additional intellectual property related to any tests performed on a service basis in the laboratory. He receives no royalties from the sale of these tests when used for patients. Mayo Collaborative Services, Inc, does receive revenue for conducting these tests.
Funding/Support: This work was funded by grant K08 NS065007 from the National Institutes of Health and a Mayo Foundation Neuroscience Development award (Dr Klein).
Additional Contributions: We thank the family for their participation and Mr Larry Witt for coordinating the DNA library used in this work.