Marina Grandis

Predictors of response to rituximab in patients with neuropathy and anti–myelin associated glycoprotein immunoglobulin M

Abstract  We evaluated the efficacy and safety of rituximab in an open‐label, uncontrolled study of 13 patients with polyneuropathy associated with antibodies to myelin‐associated glycoprotein (MAG) and correlated the response to therapy with clinical and laboratory features. One year after rituximab therapy, anti‐MAG immunoglobulin M (IgM) titers were significantly reduced. At that time, eight patients (62%) had improved in both the inflammatory neuropathy cause and treatment (INCAT) sensory sumscore and the Medical Research Council sumscore for muscle strength and seven of them also in the INCAT disability score. The improvement in the mean INCAT sensory sumscore was significant at 12 months and correlated with lower anti‐MAG antibody at entry and at follow‐up. This study suggests that rituximab may be efficacious in patients with anti–MAG associated neuropathy and particularly on sensory impairment and in those with moderately elevated antibody titers. These findings suggest that antibody reduction below a critical level may be necessary to achieve clinical improvement.

Different cellular and molecular mechanisms for early and late-onset myelin protein zero mutations

Mutations in the gene MPZ, encoding myelin protein zero (MPZ), cause inherited neuropathies collectively called Charcot-Marie-Tooth type 1B (CMT1B). Based on the age of onset, clinical and pathological features, most MPZ mutations are separable into two groups: one causing a severe, early-onset, demyelinating neuropathy and a second, causing a late-onset neuropathy with prominent axonal loss. To investigate potential pathomechanisms underlying the two phenotypes, we transiently transfected HeLa cells with two late-onset (T95M, H10P) and two early-onset (H52R, S22_W28 deletion) mutations and analyzed their effects on intracellular protein trafficking, glycosylation, cell viability and intercellular adhesion. We found that the two late-onset mutations were both transported to the cell membrane and moderately reduced MPZ-mediated intercellular adhesion. The two early-onset mutations caused two distinct abnormalities. H52R was correctly glycosylated and trafficked to the plasma membrane, but strongly affected intercellular adhesion. When co-expressed with wild-type MPZ (wtMPZ), a functional dominant negative effect was observed. Alternatively, S22_W28 deletion was retained within the cytoplasm and reduced both adhesion caused by wtMPZ and cellular viability. Since the same trafficking patterns were observed in transfected murine Schwann cells, they are not an artifact of heterologous cell expression. Our results suggest that at least some late-onset mutations cause a partial loss of function in the transfected cells, whereas multiple abnormal gain of function pathways can result in early-onset neuropathy. Further characterization of these pathways will lead to a better understanding of the pathogenesis of CMT1B and a rational basis for treating these debilitating inherited neuropathies.

Mycophenolate mofetil in dysimmune neuropathies: A preliminary study

Mycophenolate mofetil (MM) is an immunosuppressant that has been used successfully for preventing the rejection of renal, heart, or liver transplants and for the therapy of immune-mediated diseases.4 It shows modest side effects and has a lower risk for late malignancies compared with other immunosuppressive drugs.1 Recently, MM has been used also for the treatment of myasthenia gravis, polymyositis, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), and multifocal motor neuropathy (MMN).2,7,10 We treated four patients with possible, probable, or definite MMN11 and two with CIDP8 (Table 1) who were on large doses of intravenous immunoglobulins (IVIg), with the hope of reducing or withdrawing IVIg while maintaining a satisfactory and stable clinical state. Four patients were also receiving other immunomodulating agents. Patients received oral MM in a dosage of 1 g twice daily, for an average of 9 months (range, 6–12 months). Prior to MM treatment, all patients underwent several attempts to reduce IVIg dosage, resulting in clinical worsening, even in subjects receiving other immunomodulating agents. Patients were evaluated at baseline and each month thereafter, using the Medical Research Council (MRC) sumscore6 and the Immune Neuropathy Course and Treatment (INCAT) arm and leg disability scores.5 The values of the INCAT scale at baseline ranged from 0 to 3 (mean, 1.5). Full blood count, renal and liver function, and serum amylase levels were monitored every 2 months. Side effects were recorded and graded according to the Common Terminology Criteria for Adverse Events.9 All the patients provided informed consent to participate in the study. Table 1 summarizes the details of each patient. In five patients we did not observe any worsening in MRC sumscore or INCAT disability scale despite a significant reduction in IVIg dosage. On average, before starting MM treatment patients received 2g/kg every 4 weeks. After 6 months the mean dosage of IVIg was 0.75 g/kg every 4 weeks. In patients 1 and 2, IVIg infusion was reduced by 50% after 2 months and discontinued after 4 months. After 1 year of therapy with MM, IVIg treatment remains discontinued. In patient 4, the addition of MM allowed a reduction in IVIg dose to 50% after 4 months. In patient 3, IVIg dose was reduced by 25% but only for 4 months; symptoms relapse then required a return to the previous IVIg schedule. In patients 5 and 6, who had CIDP, IVIg dosage was reduced by 50%. After follow-up ranging from 6 to 12 months there was no deterioration in the MRC sumscore or in the INCAT disability scale in patients 1, 2, 4, 5, and 6; indeed, in patients 1 and 2 we observed a minimal improvement of overall disability within 2 months of starting MM. Finally, azathioprine (patients 3, 5, and 6) and cyclophosphamide (patient 2) were discontinued after 3 months of combined IVIg and MM treatment. Patients 1 and 4 discontinued MM because of loss of appetite, weight loss, abdominal pain, and increase of amylase levels (grade 1 in both cases). Patient 5 developed mild leukopenia (grade 1). Recently, MM has been introduced for the treatment of immune-mediated neuromuscular disorders,2,3 but the findings in only one patient with MMN and nine patients with CIDP have been published, to our knowledge, with conflicting results.7,10 We found that MM, in patients with MMN and CIDP, is effective in reducing IVIg requirement and in replacing other, more toxic, drugs. Only one patient with MMN relapsed, after 4 months, perhaps because of longer duration of disease, as previously observed in another series of patients.10 In fact, axonal impairment, more than immune-mediated de-

Major myelin protein gene (P0) mutation causes a novel form of axonal degeneration.

Mutations in the major peripheral nervous system (PNS) myelin protein, myelin protein zero (MPZ), cause Charcot‐Marie‐Tooth Disease type 1B (CMT1B), typically thought of as a demyelinating peripheral neuropathy. Certain MPZ mutations, however, cause adult onset neuropathy with minimal demyelination but pronounced axonal degeneration. Mechanism(s) for this phenotype are unknown. We performed an autopsy of a 73‐year‐old woman with a late‐onset neuropathy caused by an H10P MPZ mutation whose nerve conduction studies suggested severe axonal loss but no demyelination. The autopsy demonstrated axonal loss and reorganization of the molecular architecture of the axolemma. Segmental demyelination was negligible. In addition, we identified focal nerve enlargements containing MPZ and ubiquitin either in the inner myelin intralaminar and/or periaxonal space that separates axons from myelinating Schwann cells. Taken together, these data confirmed that a mutation in MPZ can cause axonal neuropathy, in the absence of segmental demyelination, thus uncoupling the two pathological processes. More important, it also provided potential molecular mechanisms as to how the axonal degeneration occurred: either by disruption of glial–axon interaction by protein aggregates or by alterations in the molecular architecture of internodes and paranodes. This report represents the first study in which the molecular basis of axonal degeneration in the late‐onset CMT1B has been explored in human tissue. J. Comp. Neurol. 498:252–265, 2006.

HSPB1 and HSPB8 in inherited neuropathies: study of an Italian cohort of dHMN and CMT2 patients.

Mutations in the small heat‐shock protein 27 kDa protein 1 (HSPB1) and 22 kDa protein 8 (HSPB8) genes were associated with distal hereditary motor neuropathy (dHMN) and with the axonal form of Charcot‐Marie‐Tooth disease type 2 (CMT2). Here we report the clinical and molecular evaluation of an Italian dHMN and CMT2 cohort to establish HSPB1 and HSPB8 mutation occurrence and associated clinical features. One hundred and sixty‐seven patients with dHMN or CMT2 were studied. HSPB1 and HSPB8 exons 1 and 3 molecular analysis was carried out through DHPLC and direct sequencing of each variant chromatogram. HSPB8 exon 2 was analyzed by direct sequencing. Four mutations in five unrelated dHMN patients and four mutations in four unrelated CMT2 cases were found in HSPB1. The p.Arg136Leu mutation was found in two patients with different phenotypes. Electroneurographical follow‐up study in a dHMN patient revealed that sensory impairment occurred with disease progression. The HSPB1 mutation frequency was 8% in dHMN and 4% in CMT2 patients. The significant HSPB1 mutation frequency in both phenotypes indicates its relevance in the pathogenesis of these neuropathies. Recent literature data suggest a continuum between dHMN and CMT2. We confirm this finding in our cohort, proposing a definite relationship between these disorders.

Impairment of PMP22 transgenic Schwann cells differentiation in culture: implications for Charcot-Marie-Tooth type 1A disease

Charcot-Marie-Tooth type 1A (CMT1A) is a hereditary demyelinating neuropathy due to an increased genetic dosage of the peripheral myelin protein 22 (PMP22). The mechanisms leading from PMP22 overexpression to impairment of myelination are still unclear. We evaluated expression and processing of PMP22, viability, proliferation, migration, motility and shaping properties, and ability of forming myelin of PMP22 transgenic (PMP22(tg)) Schwann cells in culture. In basal conditions, PMP22(tg) Schwann cells, although expressing higher PMP22 levels than control ones, show normal motility, migration and shaping properties. Addition of forskolin to the media induces an additional stimulation of PMP22 expression and results in an impairment of cells migration and motility, and a reduction of cell area and perimeter. Similarly, co-culturing transgenic Schwann cells with neurons causes an altered cells differentiation and an impairment of myelin formation. In conclusion, exposure of PMP22(tg) Schwann to the axon or to axonal-mimicking stimuli significantly affects the transition of transgenic Schwann cells to the myelinating phenotype.

The D355V Mutation Decreases EGR2 Binding to an Element within the Cx32 Promoter

Mutations in the early growth response 2 (EGR2) gene are associated with some forms of Charcot--Marie--Tooth disease (CMT) and other demyelinating neuropathies. These mutations modify the EGR2 binding to specific DNA sequences suggesting a role in the transcriptional control of myelination-specific genes. Here we show that the D355V mutation, associated with a CMT case combining axonal and demyelinating abnormalities, reduces three times the affinity of EGR2 to its consensus sequence and ten times its affinity to a sequence in the human Cx32 promoter. These findings could indicate that this EGR2 mutation leads to the development of CMT1 through the transcriptional deregulation of Cx32 gene.

Outcome measures and rehabilitation treatment in patients affected by Charcot-Marie-Tooth neuropathy: a pilot study.

Objective: We evaluated the sensitivity of various rehabilitation and lung function scales to detect differences between people with Charcot‐Marie‐Tooth (CMT) disease and healthy controls. We also studied whether these measurements are sensitive to disclose changes in patients with CMT disease after rehabilitative treatment. Design: Eight patients with different types of CMT participated in the study. Data were gathered at baseline; at the end of the treadmill training, stretching, respiratory, and proprioceptive exercise (TreSPE) treatment period; and after a washout period of 6 mos. The following instruments were used for data collection: Medical Research Council scale for lower limb strength; Tinetti Balance Scale; Physical Performance Battery; ankle angle, oxygen consumption, and lung function tests; peak treadmill velocity and slope; time to walk 6 m; and CMT Neuropathy Score. The participants underwent TreSPE treatment twice every week for 8 wks. Results: All rehabilitative measures were significantly worse in subjects with CMT disease than in healthy controls. Lung function was always normal except for the maximum expiratory pressure and maximum inspiratory pressure. No dropouts or worsening in any of the different outcome measures were observed after TreSPE. The ankle angle and the time to walk 6 m were the only measures that significantly improved after treatment. Conclusions: The rehabilitative outcome measures used in this protocol are useful in detecting clinical impairment in people with CMT disease. Lung function tests were confirmed to be minimally abnormal in patients with CMT disease. The TreSPE treatment, besides being safe and well tolerated, induced some training effects in CMT neuropathy, in the absence of lung function amelioration and effort tolerance. Follow‐up showed that CMT patients should be treated at least twice every year because a regression of all outcome measures to the baseline state was found after a 6‐mo washout period.

Severe Neuropathy After Diphtheria-Tetanus-Pertussis Vaccination in a Child Carrying a Novel Frame-Shift Mutation in the Small Heat-Shock Protein 27 Gene

Mutations in small heat-shock protein 27 and small heat-shock protein 22 genes were found in association with Charcot-Marie-Tooth disease type 2 and distal hereditary motor neuropathy. We searched for mutations in small heat-shock protein 27 gene in an Italian family with peripheral neuropathy and intrafamilial phenotypic variability. A novel heterozygous frame-shift mutation c.476_477delCT was found while point mutations in most genes associated with hereditary neuropathies were ruled out. In the proband, who showed a severe early onset peripheral neuropathy, an independent pathogenetic effect on the peripheral nervous system secondary to the tetanus toxoid injection may be supposed. This is the first truncating nonsense mutation in the small heat-shock protein 27 gene identified so far and the clinical, neurophysiologic, and neuropathological findings are discussed.

Clinical features and molecular modelling of novel MPZ mutations in demyelinating and axonal neuropathies

Mutations in the myelin protein zero (MPZ) gene have been associated with different Charcot–Marie–Tooth disease (CMT) phenotypes, including classical demyelinating CMT1B and the axonal form of the disease (CMT2). The MPZ role in the pathogenesis of both demyelinating and axonal inherited neuropathies was evaluated in the Italian population by screening a cohort of 214 patients with CMT1 or CMT2. A MPZ mutation frequency of 7.9% in demyelinating cases and of 4.8% in axonal cases was observed. In the total cohort (264 patients), including those with mutations in other genes, a mutation frequency of 5.8% (7/121) in demyelinating cases and 4.2% (6/143) in axonal cases was found. Three novel MPZ mutations, two missense (p.Ser111Cys, p.Thr124Ala) and one frameshift (p.Tyr145fs) were found, and a molecular modelling approach was used to test the effects of these mutations on the protein structure. Electrostatic distribution changes within the protein, caused by the amino acid substitution, fit in with phenotypes presented by patients herein described. Our findings suggest that the clinical features associated with MPZ mutations depend partly on the nature of amino acid change and that molecular modelling may provide useful support, based on effects on secondary and tertiary protein structure, to predict the phenotype associated with MPZ mutations.