Developmental defects and neuromuscular alterations due to mitofusin 2 gene (MFN2) silencing in zebrafish: a new model for Charcot-Marie-Tooth type 2A neuropathy

The development of new animal models is a crucial step in determining the pathological mechanism underlying neurodegenerative diseases and is essential for the development of effective therapies. We have investigated the zebrafish (Danio rerio) as a new model to study CMT2A, a peripheral neuropathy characterized by the selective loss of motor neurons, caused by mutations of mitofusin 2 gene. Using a knock-down approach, we provide evidence that during embryonic development, mitofusin 2 loss of function is responsible of several morphological defects and motility impairment. Immunohistochemical investigations, revealing the presence of severe alterations in both motor neurons and muscles fibres, indicated the central role played by MFN2 in axonal and neuromuscular development. Finally, we demonstrated the ability of human MFN2 to balance the downregulation of endogenous mfn2 in zebrafish, further supporting the conserved function of the MFN2 gene. These results highlight the essential role of mitofusin 2 in the motor axon development and demonstrate the potential of zebrafish as a suitable and complementary platform for dissecting pathogenetic mechanisms of MFN2 mutations in vivo.     

Sensory manifestations in Charcot-Marie-Tooth disease

Involvement of sensory nerves in Charcot-Marie-Tooth (CMT) disease is well known, however, sensory symptoms are usually overlooked. To assess the frequency and features of sensory symptoms in a cohort of patients with CMT, we investigated in a prospective study 52 consecutive CMT patients, diagnosed on the basis of clinical, neurophysiological, and genetic features and classified in CMT type 1 (CMT1) (20 patients, including 14 with CMT1A) and CMT type 2 (CMT2) (32 patients). Positive sensory symptoms were reported by 28 patients (54%), including neuropathic pain in 6 patients. Pain, either neuropathic or nociceptive, was present in 29 patients (56%) and in 15 patients as a main symptom. Positive sensory symptoms were present in 24 of 32 CMT2 patients (75%) and in 4 of 20 CMT1 patients (20%) (p < 0.001); there was a presenting manifestation in 11/32 CMT2 patients vs. 1/20 in CMT1 patients (p = 0.018), and one of the main features in 6/32 CMT2 patients vs. 1/20 CMT1 patients. Frequency of positive sensory symptoms in CMT1A patients was similar to that of the entire CMT1 group. Within the CMT2 group, patients with positive sensory symptoms as a main or onset feature (11 patients) had significantly later onset (median 57 vs. 25 years; p = 0.042) and less severely impaired motor action potentials than CMT2 patients without positive sensory symptoms (8 patients). Nociceptive pain was especially frequent in CMT1A patients (10/14, 71%). Sensory manifestations in CMT seem more frequent than previously thought, especially in CMT2; however, their frequency may be different in the genetic subtypes of the disease and/or an expression of phenotypic variability. Sensory symptoms, and in particular pain, may represent an important issue in the management of CMT patients, especially in a physical medicine approach.     

Co-occurrence of amyotrophic lateral sclerosis and Charcot-Marie-Tooth disease type 2A in a patient with a novel mutation in the mitofusin-2 gene

Mitofusin-2 gene (MFN2) mutations cause Charcot-Marie-Tooth type 2A (CMT2A), sometimes complicated by additional features such as optic atrophy, hearing loss, upper motor neuron signs and cerebral white-matter abnormalities. Here we report, for the first time, the occurrence of motor neuron disease, consistent with amyotrophic lateral sclerosis (ALS), in a 62-year-old woman affected by early-onset slowly progressive CMT2A, due to a novel MFN2 mutation. After age 60, rate of disease progression changed and she rapidly developed generalised muscle wasting, weakness, and fasciculations, together with dysarthria and dysphagia. Clinical features, EMG findings, and fast progression were consistent with ALS superimposed on CMT.     

Charcot-Marie-Tooth disease type 1A: morphological phenotype of the 17p duplication versus PMP22 point mutations

Charcot-Marie-Tooth disease type 1A (CMT1A) or hereditary motor and sensory neuropathy type Ia (HMSN type Ia) is an autosomal dominant demyelinating polyneuropathy, which may result from duplications as large as 1.5 Mb on chromosome 17p11.2–p12 encompassing the gene for the peripheral myelin protein PMP22, or from point mutations in this gene. In general, it is not possible to distinguish, by clinical and neurophysiological criteria, the cases associated with the duplication mutation from those associated with point mutations of the PMP22 gene, although the latter tend to be more severe. In this study we demonstrated that the two genotypes exhibit different morphological characteristics. In the PMP22 duplicated cases the mean g-ratio (axon diameter versus fibre diameter) is significantly lower than normal, while in cases of PMP22 point mutations nearly all myelinated fibres have an extremely high g-ratio. In cases with point mutations, onion bulbs are abundantly present from an early age, whereas onion bulbs in the duplicated cases develop gradually in the first years of life. Increase in total transverse fascicular area is most pronounced in the point mutation cases. The differences in pathology between these two very different types of mutations involving the same gene likely reflect differences in pathogenesis and may offer clues in understanding the function of PMP22.     

Molecular genetics of autosomal-dominant axonal Charcot-Marie-Tooth disease

The autosomal-dominant axonal peripheral neuropathies comprise a genetically heterogeneous group of disorders that are clinically subsumed under Charcot-Marie-Tooth disease type 2 (CMT2). A significant increase in the number of genes underlying major forms of CMT2 has improved the classification of specific CMT phenotypes. The molecular dissection of cellular functions of the related gene products has only begun and detailed pathophysiological models are still missing, but already the biological scope of genes linked to CMT2 is more diversified than CMT1. The known CMT2 genes present key players in these pathways and will likely prove as powerful tools in identifying eventual future targets for therapeutic intervention.     

A novel mutation in the dynamin 2 gene in a Charcot-Marie-Tooth type 2 patient: clinical and pathological findings

Mutations in dynamin 2 (DNM2) have been associated with autosomal dominant centronuclear myopathy, dominant intermediate Charcot-Marie-Tooth (CMT) type B and CMT2. Here, we report a novel DNM2 mutation in the Pleckstrin homology domain of DNM2 (p.K559del) in a patient with an axonal length-dependent sensorimotor polyneuropathy predominantly affecting the lower limbs. Neuropathy is associated with congenital cataracts, ophthalmoparesis, ptosis and neutropenia. There was no evidence of a skeletal myopathy on EMG or muscle biopsy. We suggest that this constellation of clinical features can help the diagnosis and selection of patients for direct DNM2 genetic analysis.     

Pattern shift visual evoked potentials in Charcot-Narie-Tooth disease,HMSN type I

Summary Pattern shift visual evoked potentials were investigated in 9 subjects with Charcot-Marie-Tooth disease, who were classified as HMSN I on clinical, genetic and electromyographic criteria. The latency of the second positive peak was increased in 7 eyes of 5 subjects. These results suggest that patients with HMSN I the optic nerves may be affected more frequently than suspected on clinical examinations.

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Zusammenfassung Visuelle, durch Schachbrettmusterumkehr evozierte Potentiale wurden bei 9 Patienten mit einer neuralen Muskelatrophie untersucht. Die Patienten wurden entsprechend genetischer, klinischer und elektromyographischer Befunde als Typ HMSN I klassifiziert. Die Latenzen des zweiten positiven Gipfels der visuell evozierten Potentiale waren in 7 Augen von 5 Patienten eindeutig pathologisch verlängert. Aufgrund dieser Ergebnisse ist es wahrscheinlich, daß der N. opticus in einem höheren Prozentsatz bei Patienten mit einer neuralen Muskelatrophie, Typ HMSN I, betroffen ist als klinisch vermutet werden kann.

     

X-linked dominant Charcot-Marie-Tooth disease: nerve biopsies allow morphological evaluation and detection of connexin32 mutations (Arg15Trp, Arg22Gln)

X-linked Charcot-Marie-Tooth neuropathy (CMTX) is caused by mutations in the connexin32 gene on Xq13. Because of overlapping morphological and clinical data, CMTX patients often meet the criteria of autosomal-dominant CMT2, the neuronal type of CMT. Hence, it might be useful to analyse the connexin32 gene in suspected CMT2 patients when there is no male-to-male transmission. We selected a cohort of 30 patients who were considered having CMT2 on the basis of previous clinical and histopathological evaluation. DNA was extracted from paraffin-embedded sural nerve biopsy samples and screened for connexin32 mutations to verify the possible diagnosis of CMTX. In 2 patients mutations were found corresponding to amino acid substitutions of arginine for tryptophan in codon 15 and arginine for glutamine in codon 22 of connexin32. This study illustrates that archival material allows genetic classification of suspected CMT cases. Furthermore, there is additional proof that connexin32 mutations represent the underlying genetic defect in some cases of predominantly neuronal CMT. Received: 30 December 1997 / Revised, accepted: 16 February 1998     

Degeneration of anterior horn cell in neuronal type of Charcot-Marie-Tooth disease (hereditary motor and sensory neuropathy type II): A Golgi study

Summary A morphological study using the Golgi impregnation method was carried out on the anterior horn cells at cervical (C), thoracic (Th), and lumbar (L) levels of the spinal cord in a patient with neuronal type of Charcot-Maire-Tooth disease (hereditary motor and sensory neuropathy type II) and an age-matched control. The present study demonstrated an uneven cell body surface, loss of cells (particularly large cells), loss of dendrites, reduced dendritic extent and an irregular surface and shape of dendrites at the C and L levels. In contrast, hematoxylin and eosin and Klüver-Barrera staining showed only simple atrophy or no change. The Th level of the patient showed none of these changes. Our results suggest that the degeneration or loss of dendrites of anterior horn cells by the Golgi staining method, which is most severe at the L level, is closely related to clinical findings such as muscle atrophy and weakness in neuronal type of Charcot-Marie-Tooth disease.     

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.     

Phenotypic variation of a new P0 mutation in genetically identical twins

We have identified a new point mutation in the myelin protein zero (P0) gene in two genetically identical twins with a demyelinating neuropathy. The G to A transition at nucleotide position 382 caused an aspartic acid to asparagine substitution in exon 3. Moreover, we found clear clinical differences which were most evident at an early age. These observations suggest that the expression of this P0 mutation may be susceptible to external, non-genetic influences that may act early in the course of the disease to alter the phenotype. Received: 4 August 1998 Received in revised form: 29 October 1998 Accepted: 19 January 1999     

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.     

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.     

Autonomic function in hereditary motor and sensory neuropathy (Charcot-Marie-Tooth disease)

Autonomic tests of heart rate and blood pressure control using both invasive and noninvasive techniques were performed on 11 patients with hereditary motor and sensory neuropathy (HMSN) types 1, and 4 patients with HMSN type 2. The results were compared with those of 76 control subjects. No significant difference was found between the patient and control groups. Impairment of sweating on the extremities was found in patients with HMSN types 1 and 2, consistent with distal degeneration of sympathetic fibers in peripheral nerves, but there were no abnormalities of cardiovascular reflex control mechanisms.     

线粒体融合蛋白2基因新突变导致的遗传性运动感觉性神经病6型一家系

目的 报道1个遗传性运动感觉性神经病6型家系的临床表现、病理改变以及基因突变特点。方法 先证者男性,15岁。患者5岁出现双下肢无力,症状进行性加重,伴随出现双足跟腱挛缩;11岁开始出现慢性进行性视力下降;12岁出现双手肌肉萎缩,无肢体麻木。周围神经传导速度检查显示诱发电位未能引出或波幅显著下降,感觉神经较运动神经改变更明显。视诱发电位提示双眼P100潜伏期均延长,波幅正常。眼底照相提示视神经萎缩,视网膜电图正常。患者母亲7岁时开始出现走路费力,10岁出现视力下降。对先证者进行腓肠神经活体组织检查。对先证者及其母亲进行线粒体融合蛋白2( MFN2)基因测序,100名健康人作为正常对照。结果 腓肠神经病理改变主要为有髓神经纤维显著减少,电镜检查发现个别有髓神经纤维出现洋葱球样结构和再生簇结构,个别神经纤维的轴索内可见线粒体聚集和空泡化。先证者和母亲的MFN2基因第19号外显子存在c.2218T＞C杂合突变,导致MFN2第740位的色氨酸由精氨酸替代(W740R)。100名健康对照没有发现该突变。结论 MFN2基因c.2218T＞C突变导致了遗传性运动感觉性神经病6型,其视力下降多出现在脊神经损害之后,周围神经可以存在髓鞘损害。     

腓骨肌萎缩症2A2A型家系的神经电生理及基因突变分析

目的探讨一个腓骨肌萎缩症(charcot-marie-tooth,CMT)家系的临床表现、神经电生理学和基因突变特点。方法收集家系先证者及其他成员的临床资料,对先证者进行神经电生理学检查和全外显子组基因测序,用Sanger测序技术对先证者及其家系进行突变位点验证。应用计算机软件预测突变位点氨基酸进化保守性和突变可能导致的蛋白质结构和功能变化,分析突变位点的性质。结果先证者儿童期发病,出现双下肢对称性肌肉无力伴跟腱反射消失及足部畸形,其母亲有类似症状。先证者神经电生理检查示运动和感觉神经纤维脱髓鞘及轴索性改变。基因检测发现先证者和母亲MFN2基因第11个外显子均检出c.1066A>G(p.T356A)杂合错义突变;先证者姐姐和父亲未检测到该突变。用PolyPhen-2和MutationTaster软件预测该突变为致病性,突变区域序列在不同物种间高度保守。结论儿童CMT2A2A患者的神经电生理、临床特点、发病机制及相关基因表型均有改变,此可为儿童CMT的临床诊断提供依据。     

Charcot-Marie-Tooth disease in northern Sweden: an epidemilogical and clinical study

One hundred four cases of Charcot-Marie-Tooth desease (CMT) in 52 families were identified within a defined area in northern Sweden corresponding to a prevalence rate of 20.1 cases per 100000. The distribution of cases was not uniform. The prevalence rate is compared with previous prevalences studies focusing on Charcot-Marie-Tooth disease or hereditary motor and sensory neurophaty (HMSN). Three patients were classified as the distal spinal muscular atrophy type of CMT and one patient was not possible to classfiy. For seventy-five patients, available to clinical examination within the study, data were collected as to age to onset, symnptoms, clinical findigs and degree of disability.     

Charcot-Marie-Tooth disease type 1B: marked phenotypic variation of the Ser78Leu mutation in five Italian families

Objectives – To describe clinical, electrophysiological and genetic data of five unrelated Sicilian pedigrees harbouring a heterozygous Ser78Leu mutation in the myelin protein zero (MPZ) extracellular domain. Materials and methods – Clinical, electrophysiological and genetic findings of 16 patients were reported. Polymorphic markers flanking the coding sequence of MPZ gene were also analysed. Results – A wide range of age at onset was observed in families 1 and 3, with a clinical heterogeneity, in terms of severity of the disease, within the same family (families 1 and 3), and among families. A markedly unsteady gait was a distinctive feature of many members of family 1. All patients in family 2 complained of severe cramps and painful paresthesia. Molecular genetic analysis showed that all affected subjects shared a common haplotype at three microsatellite loci D1S2858, D1S2624 and D1S484. Conclusions – Our study provides further evidence that phenotypic features of MPZ mutations can vary within and among different families. High frequency of Ser78Leu mutation in Sicily as well as the results of haplotype analyses suggest that the mutation may have been inherited from a common ancestor.     

Severe Charcot-Marie-Tooth neuropathy type 1A with 1-base pair deletion and frameshift mutation in the peripheral myelin protein 22 gene

A 27-year-old man with negative family history and both parents with normal neurological evaluation and motor nerve conduction velocities (MNCVs) showed onset of severe weakness of feet at 4 years of age. Subsequently he developed left equinovarus deformity, thoracic scoliosis, ulnar nerve enlargement, areflexia, distal hypesthesia and slowing of MNCVs for median and ulnar nerves (15–25 m/sec). Molecular genetic studies showed deletion of one nucleotide (G330) (codon 94) in exon 3 of the PMP22 gene associated with frameshift mutation. © 1997 John Wiley & Sons, Inc. Muscle Nerve 20: 1308–1310, 1997     

Behavioural profiling of a murine Charcot-Marie-Tooth disease type 1A model

Different features of motor behaviour were studied on a transgenic mouse model of Charcot-Marie-Tooth's disease (CMT). Mutants with 4 or 7 copies of the human PMP22 gene leading to a phenotype significantly close to CMT's disease type 1A were compared with control animals. The aim of the study was to validate this transgenic model and to characterise the impairments occurring in the various lines. Three main types of analysis were performed in 2-month-old mice without any peculiar visible deficit: (i) a study of standardised clinical tests (SHIRPA protocol) demonstrated that only a few motor deficits were expressed; (ii) a measurement of general spontaneous activity by means of a commercial video-tracking system was performed and revealed that the main spontaneous activities were identical in the three lines with, however, some slight localised modifications; and, (iii) by contrast, the three lines respond very differently to the footprints, grip strength, splay test and rotarod test. Even in lines with a significantly limited copy number of the transgene, we observed and quantified impairments. In conclusion, mutants of CMT1A seem to be a very pertinent model of this human pathology and will certainly be useful for therapeutic procedures and for theoretical studies on this disease.