Chloride ion transport and overexpression of TMEM16A in a guinea-pig asthma model.

BACKGROUND: TMEM16A, a Ca-activated Cl channel, regulates various physiological functions such as mucin secretion. However, the role of TMEM16A in hyper-secretion in asthma is not fully understood. OBJECTIVE: The aim of this study is to evaluate Cl ion transport via TMEM16A and determine the localization of TMEM16A in a guinea-pig asthma model. METHODS: Guinea-pigs were sensitized with ovalbumin (OVA) i.p. on Days 1 and 8. On Day 22, we assessed OVA challenge-induced Cl ion transport in the sensitized tracheas ex vivo in an Ussing chamber, compared with the non-sensitized tracheas. We then examined the effect of T16Ainh-A01, a TMEM16A inhibitor, on the increase in Cl ion transport. The tracheal epithelium was immunostained with an anti-TMEM16A antibody. Epithelial cells from guinea-pig tracheas were cultured at the air-liquid interface in the presence of IL-13 for in vitro study. We studied the effect of TMEM16A inhibitors on Ca-dependent agonist, uridine triphosphate (UTP)-induced increases in Cl ion transport in the cultured cells. The cells were immunostained with an anti-TMEM16A antibody, an anti-MUC5AC antibody and an anti-α-tubulin antibody. RESULTS: OVA challenge induced an increase in short circuit current within 1 min in the OVA-sensitized tracheas but not in the non-sensitized tracheas, which was inhibited by pretreatment of T16Ainh-A01. Sensitized tracheas showed goblet cell metaplasia with more positive TMEM16A immunostaining, particularly in the apical portion compared with the non-sensitized tracheas. The in vitro UTP-induced increase in Cl ion transport was strongly inhibited by pretreatment with T16Ainh-A01, benzbromarone, and niflumic acid. TMEM16A was positively immunostained at the apical portion and in the MUC5AC-positive area in IL-13-induced goblet cell metaplasia. CONCLUSIONS: Antigen challenge and Ca-dependent agonist treatment increased Cl ion transport via the overexpression of TMEM16A in goblet cell metaplasia in a guinea-pig asthma model. TMEM16A inhibitors may be useful for the treatment of hyper-secretion in asthma.

Neural damage associated with atopic diathesis: a nationwide survey in Japan.

BACKGROUND: We previously reported the occurrence of myelitis in patients with atopic disorders (atopic myelitis [AM]). To uncover the spectrum of neural damage associated with atopy, we conducted a cross-sectional nationwide survey of AM and atopy-related peripheral neuritis (APN), including Churg-Strauss syndrome (CSS), in individuals with atopic diathesis. METHOD: Cases with AM diagnosed between 1996 and 2006 and cases with APN between 2000 and 2006 were collected from all over Japan. Detailed data on 109 patients with AM and 133 patients with APN were collated. RESULTS: Patients with APN showed a preponderance of women, higher age at onset, and greater eosinophil counts than patients with AM. Patients with AM most commonly showed cervical cord involvement, whereas patients with APN preferentially exhibited mononeuritis multiplex predominantly affecting the lower limbs. Among patients with AM, motor weakness and muscle atrophy were significantly more frequent in those with bronchial asthma than in those with other atopic disorders. Patients with APN who met the criteria for CSS showed a higher age at onset, higher frequencies of systemic organ involvement, and greater disability than those who did not. Abnormalities suggesting peripheral nervous system involvement were seen in 25.7% of patients with AM, whereas 18.8% of patients with APN had abnormalities indicating CNS involvement. Multiple logistic regression analyses revealed that atopic dermatitis increased the risk of myelitis, whereas high age at onset and bronchial asthma decreased that risk. CONCLUSIONS: Atopy-related neural inflammation multifocally affects CNS and peripheral nervous system tissues. Both preceding atopic disorders and age seem to influence the distribution of neural damage.

Serum VEGF increases in diabetic polyneuropathy, particularly in the neurologically active symptomatic stage.

AIM: To identify the relationship between vascular endothelial growth factor (VEGF) and diabetic polyneuropathy (DPN). METHODS: Two hundred and twenty diabetic patients participated, 113 with DPN and 107 without DPN. All patients were also classified according to the four stages of DPN (no neuropathy: stage 0; asymptomatic neuropathy: stage 1; symptomatic neuropathy: stage 2; disabling neuropathy: stage 3). Serum VEGF concentration was measured using an enzyme-linked immunosorbent assay (ELISA) and levels between the patients with and without DPN and also between the different stages of DPN, were compared. RESULTS: The mean serum VEGF level in all patients was 264.6 +/- 218.8 pg/ml. The mean serum VEGF level was higher in patients with DPN (310.1 +/- 224.3 pg/ml) than in the patients without DPN (216.5 +/- 204.0 pg/ml, P = 0.0014). Serum VEGF was higher in the 'symptomatic' stage (stage 2, 364.8 +/- 225.9 pg/ml) in comparison with the 'asymptomatic' (stage 1, 256.7 +/- 224.4 pg/ml, P = 0.015) and 'disabling' (stage 3, 180.3 +/- 109.4 pg/ml, P = 0.042) stages. The mean serum VEGF level in patients with diabetic retinopathy (261.1 +/- 210.6 pg/ml) and in patients with diabetic nephropathy (241.5 +/- 185.7 pg/ml) was not increased. CONCLUSIONS: The serum VEGF level is increased in patients with DPN, particularly in patients in the neurologically active 'symptomatic' stage.

Genetically confirmed patients with merosin-deficient congenital muscular dystrophy in China.

We report a family and a single patient in China involved with merosin-deficient congenital muscular dystrophy (MDC1A) with typical clinical symptoms. Pathological analysis of biopsied skeletal muscle showed dystrophic changes with proliferated fibrotic tissue elements as the predominant finding. Immunohistochemical analysis demonstrated the complete absence of the laminin alpha2 chain (merosin) around muscle fibers. In patient 1, a double mutation, c.[9101_9104dupAACA:3412G>A] p.[H3035QfsX4:V1138M] was detected, whereas her parents and another sibling were heterozygous carriers. Patient 2 had a novel homozygous nonsense mutation, c.2907C>A (p.Cys969X), in exon 21. The genotype-phenotype correlation of Chinese children with novel merosin-deficient congenital muscular dystrophy is reported.

Autoantibodies against glutamate receptor epsilon2-subunit detected in a subgroup of patients with reversible autoimmune limbic encephalitis.

We investigated the presence of autoantibodies against glutamate receptor (GluR) epsilon2 in serum and cerebrospinal fluid (CSF) samples from 12 consecutive patients with acute encephalitis/encephalopathy by immunoblotting using recombinant GluR epsilon2 as antigen. In 4 patients, IgM autoantibodies against GluR epsilon2 were detected in CSF in the early phase of the disease but were not detectable after several months. Seizures and psychiatric symptoms were noted during the acute phase of the disease in these 4 patients, who showed various degrees of residual amnesia. Immunotherapy was performed on 3 patients (patients 1, 3 and 4), and they showed marked improvements. Immunohistochemistry using these patients' sera showed that immunoreactivity is specifically detected in the cytoplasm of rat hippocampal and cortical neurons. The clinical features and neuroimaging findings of patients with IgM autoantibodies against GluR epsilon2 in CSF resemble those of patients with reversible autoimmune limbic encephalitis.

Increased serum high mobility group box-1 level in Churg-Strauss syndrome.

Churg-Strauss syndrome (CSS) is a rare form of systemic vasculitis occurring in patients with asthma and hypereosinophilia; however, its mechanisms involved in the severe tissue inflammation with vasculitis are poorly understood. High mobility group box 1 (HMGB1) protein, originally identified as a DNA binding protein, also has potent pro-inflammatory and proangiogenic properties. In this study, we hypothesized that HMGB1 might be associated with CSS, and examined serum HMGB1 levels and compared those of asthma patients and healthy volunteers. We also investigated HMGB1 expression in the lesion, and eosinophil HMGB1 amount in CSS patients. We found that the serum HMGB1 levels in CSS patients were significantly higher than those of asthma patients and healthy volunteers. Eosinophils in the CSS lesion expressed HMGB1 and HMGB1 level in eosinophils from CSS patients was significantly higher than that of asthma patients, while there was no significant difference in HMGB1 levels in peripheral mononuclear cells. The serum HMGB1 level in CSS patients decreased after the steroid therapy, and showed significant positive correlations with several molecules, including soluble interleukin-2 receptor, soluble thrombomodulin, and eosinophil cationic protein in sera. We propose that HMGB1 might contribute to the pathogenesis of CSS.

Mechanomyographic determination of post-activation potentiation in myopathies.

OBJECTIVE: There is a need to provide an index of muscle contractility in evaluating myopathies especially in the clinical setting. This study was conducted to investigate if the mechanomyogram post-activation potentiation (MMG-PAP) can be used as an index of muscle contractile force potentiation (force-PAP), if it differs between normal and myopathic muscles, and if it can reflect abnormalities in muscle fiber anatomy. METHODS: The correlation between MMG-PAP and force-PAP was evaluated in 12 normal subjects after maximum voluntary contraction (MVC) of the biceps brachii muscle. The same method was then applied to study MMG-PAP in 16 patients with myopathies, 16 disease and 25 normal controls. Mean fiber diameters and the proportions of type 1 and 2 fibers in biopsied biceps brachii muscle were determined and compared with MMG-PAP values. RESULTS: There was a significant positive correlation between force-PAP (197 +/- 148%) and MMG-PAP (135 +/- 68%) immediately after MVC (P < 0.05). The mean MMG-PAP in myopathies (66 +/- 53%) was significantly lower than those of the disease (128 +/- 34%; P < 0.005) and normal controls (120 +/- 56%; P < 0.005). Patients with non-dystrophic myopathies, including those with myositis, had significantly lower MMG-PAP values (38 +/- 20%; P < 0.005) than those with muscular dystrophy (148 +/- 23%). MMG-PAP did not clearly correlate with either type 2 fiber atrophy or type 2 fiber disproportion based on muscle biopsy analysis of myopathic patients. CONCLUSIONS: This study shows that MMG-PAP can be used as an index of muscle contractility and that it is significantly lower in non-dystrophic myopathies compared to normal subjects. MMG-PAP does not seem to reflect abnormal muscle fiber anatomy. SIGNIFICANCE: MMG-PAP may become a valuable non-invasive tool in augmenting routine clinical electrophysiologic studies especially in evaluating muscle contractility in myopathies.

Clinical investigation: increased serum stromal derived factor 1 alpha levels in pulmonary tuberculosis.

Pulmonary tuberculosis, a granulomatous disease, has few serological markers for its activity. Recently, an increased plasma level of stromal derived factor 1 alpha (SDF-1alpha), which can induce strong chemotaxis of cells through its receptor CXCR4, was detected in patients with tuberculosis. In this study we investigated serum SDF-1alpha levels and CXCR4 expression on peripheral blood mononuclear cells (PBMCs). Fifty-five active tuberculosis patients, 30 resolved tuberculosis patients, 27 acute bronchitis patients and 8 healthy volunteers were examined. Histological expression of SDF-1alpha in the tuberculosis lesion and CXCR4 expression of PBMCs were also analysed. Serum SDF-1alpha levels in active tuberculosis patients were significantly higher than other groups. The sensitivity and specificity for the diagnosis of active tuberculosis was 88.5% and 85.3% (cutoff value = 650 pg/ml), respectively. CXCR4 expression levels on PBMCs showed a significant negative correlation with serum SDF-1alpha levels. Inflammatory cells including multinuclear giant cells in the lesion expressed SDF-1alpha. Measurement of serum SDF-1alpha could be a useful screening marker for the identification of active pulmonary tuberuculosis. We propose that interaction of SDF-1alpha and CXCR4 might be involved in the pathogenesis of pulmonary tuberculosis.

Neurologic manifestations of Kanzaki disease.

We describe the neurologic findings in a patient with alpha-N-acetylgalactosaminidase deficiency (Kanzaki disease). Clinical and electrophysiologic studies revealed sensory-motor polyneuropathy, and sural nerve pathology showed decreased density of myelinated fibers with axonal degeneration. The patient had mildly impaired intellectual function with abnormal brain MRI and sensory-neuronal hearing impairment with repeated episodes of vertigo attacks. These findings suggest that Kanzaki disease may develop neurologic complications in the CNS and peripheral nervous system.

Influence of human T lymphotrophic virus type I on diffuse pan-bronchiolitis.

Human T lymphotrophic virus type-I (HTLV-I), a human retrovirus, infects CD4(+) lymphocytes and is thought to modify their function and a possible association with pulmonary diseases has also been suggested. However, little is known about the influence of HTLV-I on diffuse pan-bronchiolitis (DPB), a chronic inflammatory lung disease with infiltration of lymphocytes and hyperplasia of the bronchus-associated lymphoid tissue. In this study, 35 DPB patients with and without HTLV-I infection were examined. HTLV-I positive DPB patients were likely to have a larger affected area with lower FEV(1). The CD3(+)/CD25(+) lymphocyte percentage was significantly higher in the BALF of HTLV-I positive patients than in negative patients. MIP-1 alpha, IP-10 and levels in BALF were also significantly higher in HTLV-I positive patients than in negative patients. The levels of MCP-1 and IL-8 were not significantly different. In HTLV-I positive patients, the MIP-1 alpha and IP-10 levels showed a significant positive correlation with the percentage of CD3(+)/CD25 lymphocytes. BALF cells of all HTLV-I positive DPB patients showed expression of p40(tax) mRNA. We suggest that HTLV-I infection may modify DPB pathogenesis via activation of T cells. We also found that the frequency of ATL development in HTLV-I positive DPB patients was significantly higher than in all HTLV-I positive patients (OR = 8.22, 95% CI = 2.61-25.9, P < 0.01). The levels of TGF-beta in patients who developed ATL were significantly lower than in patients who did not develop ATL. Sensitivity and specificity were 80% and 85.7%, respectively (cut-off = 20 pg/ml). We also propose that these features should be taken into consideration in the treatment of DPB in HTLV-I infected individuals.

Highly accumulated platelet vascular endothelial growth factor in coagulant thrombotic region.

BACKGROUND: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific potent mitogen that induces angiogenesis and microvascular hyperpermeability. Recently, it has been reported that megakaryocytes and platelets contain VEGF in their cytoplasm. OBJECTIVES: To elucidate and confirm the bioactivity and role of VEGF in platelets (platelet VEGF), which may be closely related to vascular thrombosis and atherosclerosis. METHODS: The VEGF localization in megakaryocytes on bone marrow smears was analyzed by immunofluorescence and confocal laser scanning microscopic analysis. The intracellular VEGF expressed in platelets was determined by flow cytometric analysis. Platelet-rich plasma and washed platelets were used to analyze the secretion of VEGF during platelet aggregation by thrombin or gelatinase A (matrix metalloproteinase-2) stimulation. Immunohistochemical studies for VEGF in the thrombotic region were performed. RESULTS AND CONCLUSIONS: Megakaryocytes and platelets are a very rich source of circulating VEGF. Gelatinase A, which is closely associated with vascular remodeling, enhances the VEGF levels released from platelets. VEGF was clearly detected in the fibrin nets of a thrombus. Taken together, platelet VEGF is bioactive as a direct angiogenic growth factor, and may play a very important role in wound healing and atherosclerosis in conjunction with other platelet cytokines such as platelet-derived growth factor, platelet-derived endothelial cell growth factor, transforming growth factor (TGF)-alpha, and TGF-beta.

Gap junction protein beta 1 (GJB1) mutations and central nervous system symptoms in X-linked Charcot-Marie-Tooth disease.

OBJECTIVES: To clarify the clinical variability, including central nervous system (CNS) involvement, in X-linked Charcot-Marie-Tooth disease (CMTX) patients. MATERIAL AND METHODS: We clinically, pathologically and genetically studied six CMTX patients with distinct symptoms and four different GJB1 mutations. RESULTS: One patient with Val63Ile had deafness, low intelligence, saccadic eye movement, upper extremity distal dominant muscle weakness and normal sensation. Another patient with Glu186Lys had severe sensorineural deafness at the age of 6 years, but did not develop muscle weakness until the age of 20 years. Two patients with Arg22Gln had typical CMT1A-like clinical features, no CNS symptoms and obvious onion bulb formations. Two siblings with deletion of the entire GJB1 gene had mild to moderate lower extremity muscle weakness and sensory disturbance without CNS involvement. CONCLUSION: These findings suggest that some gain of function mutations of GJB1 may be related to CNS symptoms because the patients with GJB1 deletion only had peripheral neuropathy, although other unknown associated factors may contribute to their clinical phenotypes.

The clinical and pathological features of peripheral neuropathy accompanied with HTLV-I associated myelopathy.

We describe the clinical and pathological studies in HTLV-I associated myelopathy (HAM)/tropical spastic paraparesis (TSP) patients with peripheral neuropathy as proven by sural nerve biopsy. Sural nerve pathology in HAM/TSP patients revealed that the most common type of pathologic change is a combination of both demyelination and remyelination and axonal degeneration and regeneration, and this change is modified by the complications. The pathologic changes were correlated with neither the duration of disease nor human T lymphotropic virus type I (HTLV-I) proviral load. This study suggests that peripheral nerves could be involved in HAM/TSP.

The ultrasonic coagulating and cutting system injures nerve function.

BACKGROUND: Although, many surgeons have used ultrasonic coagulating and cutting systems and shears (LCS) when carrying out endoscopic thyroidectomies and parathyroidectomies, in our experience some patients had temporary paralysis of the recurrent laryngeal nerve (RLN) after these operations. We had sometimes noticed that the heat of the blade of the LCS was increased just after use; therefore, we designed an RLN model, and investigated the damage to the RLN which could be attributed to the heat of the LCS. METHODS: We investigated the effects of the heat of the LCS blade on rat femoral and sciatic nerves, using temperature measurement, histological examination and evoked electromyography. RESULTS: The temperature of the LCS blade exceeded 150 degrees C after 30 seconds when it was not used for cutting. When we used the LCS to cut rat muscle or fat tissue, the temperature of the blade exceeded 100 degrees C after 20 seconds. There was no damage to the nerve histologically when the LCS was used for less than 20 seconds at a distance of 3 mm. Electrophysiological study showed that touching the nerve with the LCS blade after only 5 seconds of use resulted in damage to the nerve. CONCLUSIONS: Our results suggest that the RLN should not be touched directly with the blade just after it has been used, and that it is possible to use the LCS at a distance of 3 mm from the RLN for less than 20 seconds at level 3. In order to maintain these distances, the RLN must be endoscopically visualized during surgery of the neck.

Increased inactivation of nitric oxide is involved in impaired coronary flow reserve in heart failure.

Recent evidence suggests that increased inactivation of endothelium-derived nitric oxide (NO) by oxygen free radical (OFR) formation is involved in the pathogenesis of endothelial dysfunction in heart failure (HF). However, it is unclear whether increased OFR limits coronary flow reserve in HF. To test this hypothesis, we examined the effects of antioxidant therapy on coronary flow reserve in a canine model of tachycardia-induced HF. The flow reserve (percent increase in coronary blood flow) to adenosine or to 20-s ischemia was less and OFR formation (electron-spin resonance spectroscopy) in myocardial tissues was greater in HF dogs than in controls. Immunohistochemical staining of 4-hydroxy-2-nonenal, an OFR-induced lipid peroxide, was detected in coronary microvessels of HF dogs. Intracoronary infusion of a cell-permeable OFR scavenger, tiron, suppressed OFR formation and improved the vasodilating capacity to adenosine or brief ischemia in HF dogs but not in controls. A NO synthesis inhibitor, N(G)-monomethyl-L-arginine (L-NMMA), diminished the beneficial effects of tiron in HF dogs. Vasodilation to sodium nitroprusside was similar between control and HF dogs, and no change in its response was noted with tiron or tiron + L-NMMA in either group. In summary, antioxidant treatment with tiron improved coronary flow reserve by increasing NO bioactivity in HF dogs. Thus increased OFR formation may impair coronary flow reserve in HF by reducing NO bioactivity.

HTLV-I proviral load correlates with progression of motor disability in HAM/TSP: analysis of 239 HAM/TSP patients including 64 patients followed up for 10 years.

To clarify clinical and laboratory findings that may be related to the pathomechanism of HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), we analyzed these findings in 239 patients with HAM/TSP, including 64 patients followed up for 10 years after their first examinations, with special interest in the HTLV-I proviral load in peripheral blood mononuclear cells (PBMCs). The proviral load in PBMCs did not differ in terms of modes of HTLV-I transmission. However, the proviral load in patients with age of disease onset greater than 65 years tended to be higher than those with a younger age of onset. In the 64 patients followed up for 10 years, the clinical symptoms deteriorated in 36 patients (56%), unchanged in 26 patients (41%), and improved in 2 patients (3%). HTLV-I proviral load also appeared to be related to the deterioration of motor disability in these patients. To our knowledge, the present study is the first longitudinal study concerning the relationship between the clinical course of HAM/TSP and HTLV-I proviral load. It is suggested that HTLV-I proviral load is related to the progression of motor disability and is an important factor to predict prognosis of patients with HAM/TSP.

Expression of vascular endothelial growth factor in tuberculous meningitis.

The pathogenesis of tuberculous meningitis is still unclear. Recently, vascular endothelial growth factor (VEGF) was found to be associated with inflammatory diseases and we found the increased serum level of VEGF in pulmonary tuberculosis. We hypothesized that VEGF might be associated with the pathogenesis of tuberculous meningitis and measured serum and cerebrospinal fluid (CSF) levels of VEGF in 28 patients with tuberculous meningitis and 31 non-tuberculous infectious meningitis patients (13 bacterial meningitis patients, eight fungal meningitis patients and 10 patients with viral meningitis) before therapy. We examined the CSF VEGF levels 3 months after in 12 tuberculous meningitis patients. The serum and CSF levels of VEGF were significantly higher in tuberculous meningitis than in other meningitis. The decrease in titer of CSF VEGF paralleled the clinical improvement of tuberculous meningitis. Immunohistochemical staining of autopsied brains demonstrated the presence of VEGF in the inflammatory mononuclear cells of the dense fibroconnective tissue both in the subarachnoid space and surrounding the vasculitis lesion. We found the expression of VEGF in tuberculous meningitis and think that VEGF reflects its activity.

Involvement of enhanced sensitivity of N-methyl-D-aspartate receptors in vulnerability of developing cortical neurons to methylmercury neurotoxicity.

The developing cortical neurons have been well documented to be extremely vulnerable to the toxic effect of methylmercury (MeHg). In the present study, a possible involvement of N-methyl-D-aspartate (NMDA) receptors in MeHg neurotoxicity was examined because the sensitivity of cortical neurons to NMDA neurotoxicity has a similar developmental profile. Rats on postnatal day 2 (P2), P16, and P60 were orally administered MeHg (10 mg/kg) for 7 consecutive days. The most severe neuronal damage was observed in the occipital cortex of P16 rats. When MK-801 (0.1 mg/kg), a non-competitive antagonist of NMDA, was administered intraperitoneally with MeHg, MeHg-induced neurodegeneration was markedly ameliorated. Furthermore, there was a marked accumulation of nitrotyrosine, a reaction product of peroxynitrite and L-tyrosine, after chronic treatment of MeHg in the occipital cortex of P16 rats. The accumulation of nitrotyrosine was also significantly suppressed by MK-801. In the present electrophysiological study, the amplitude of synaptic responses mediated by NMDA receptors recorded in cortical neurons of P16 rats was significantly larger than those from P2 and P60 rats. These observations strongly suggest that a generation of peroxynitrite through activation of NMDA receptors is a major causal factor for MeHg neurotoxicity in the developing cortical neurons. Furthermore, enhanced sensitivity of NMDA receptors may make the cortical neurons of P16 rats most susceptible to MeHg neurotoxicity.

Circulating levels of MMP-1, -2, -3, -9, and TIMP-1 are increased in POEMS syndrome.

The authors quantitatively measured levels of matrix metalloproteinases (MMP), tissue inhibitor of metalloproteinases (TIMP), and vascular endothelial growth factor (VEGF) in blood samples of POEMS syndrome. Circulating levels of MMP-1, -2, -3, -9, and TIMP-1 were more increased in patients with POEMS syndrome than in patients with other neurologic disorders or in healthy controls. Serum levels of VEGF and TIMP-1 were strongly correlated with each other. Increased circulating levels of MMP-1, -2, -3, -9, and TIMP-1 may lead to a better understanding the pathogenesis of POEMS syndrome.

Clinical phenotype in X-linked Charcot-Marie-Tooth disease with an entire deletion of the connexin 32 coding sequence.

To clarify the clinical phenotype and molecular mechanism in X-linked Charcot-Marie-Tooth disease (CMTX) patients with a deletion of the whole connexin 32 (Cx32) coding sequence, we studied a family with this deletion by electrophysiology, Southern blotting and quantitative PCR analyses. Two brothers with no copy of Cx32, 27 and 25 years old, showed steppage gait, moderate muscle atrophy and weakness, and mild sensory disturbance in the distal parts of the legs. The clinical phenotypes in these brothers were not different from those in patients with other types of severe Cx32 mutations. Their mother, with one copy of Cx32, showed very mild muscle weakness and sensory disturbance. An electrophysiological study showed a nonuniform demyelinating neuropathy with some aspects of an axonal-loss neuropathy. Sural nerve biopsy showed loss of myelinated fibers, many relatively thin myelin sheaths, clusters of small myelinated fibers, and some onion bulb formations. The present findings suggest that both a demyelinating process and an axonal involvement were present in the patients with total defect of Cx32 probably due to loss of the function mechanism of Cx32 as the underlying molecular mechanism, because a dominant negative effect theory is not applicable in these patients.