ß-Sarcoglycan gene transfer decreases fibrosis and restores force in LGMD2E mice.

Limb-girdle muscular dystrophy type 2E (LGMD2E) results from mutations in the ß-sarcoglycan (SGCB) gene causing loss of functional protein and concomitant loss of dystrophin-associated proteins. The disease phenotype is characterized by muscle weakness and wasting, and dystrophic features including muscle fiber necrosis, inflammation and fibrosis. The Sgcb-null mouse recapitulates the clinical phenotype with significant endomysial fibrosis providing a relevant model to test whether gene replacement will be efficacious. We directly addressed this question using a codon optimized human ß-sarcoglycan gene (hSGCB) driven by a muscle-specific tMCK promoter (scAAVrh74.tMCK.hSGCB). Following isolated limb delivery (5 × 10(11) vector genome (vg)), 91.2% of muscle fibers in the lower limb expressed ß-sarcoglycan, restoring assembly of the sarcoglycan complex and protecting the membrane from Evans blue dye leakage. Histological outcomes were significantly improved including decreased central nucleation, normalization of muscle fiber size, decreased macrophages and inflammatory mononuclear cells, and an average of a 43% reduction in collagen deposition in treated muscle compared with untreated muscle at end point. These measures correlated with improvement of tetanic force and resistance to eccentric contraction. In 6-month-old mice, as indicated by collagen staining, scAAVrh74.tMCK.hSGCB treatment reduced fibrosis by 42%. This study demonstrates the potential for gene replacement to reverse debilitating fibrosis, typical of muscular dystrophy, thereby providing compelling evidence for movement to clinical gene replacement for LGMD2E.

AAV1.NT-3 gene therapy attenuates spontaneous autoimmune peripheral polyneuropathy.

The spontaneous autoimmune peripheral polyneuropathy (SAPP) model in B7-2 knockout non-obese diabetic mice shares clinical and histological features with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). Secondary axonal loss is prominent in the progressive phase of this neuropathy. Neurotrophin 3 (NT-3) is an important autocrine factor supporting Schwann cell survival and differentiation and stimulates neurite outgrowth and myelination. The anti-inflammatory and immunomodulatory effects of NT-3 raised considerations of potential efficacy in the SAPP model that could be applicable to CIDP. For this study, scAAV1.tMCK.NT-3 was delivered to the gastrocnemius muscle of 25-week-old SAPP mice. Measurable NT-3 levels were found in the serum at 7-week postgene delivery. The outcome measures included functional, electrophysiological and histological assessments. At week 32, NT-3-treated mice showed increased hind limb grip strength that correlated with improved compound muscle action potential amplitude. Myelinated fiber density was 1.9 times higher in the NT-3-treated group compared with controls and the number of demyelinated axons was significantly lower. The remyelinated nerve fiber population was significantly increased. These improved histopathological parameters from scAAV1.tMCK.NT-3 treatment occurred in the setting of reduced sciatic nerve inflammation. Collectively, these findings suggest a translational application to CIDP.

Plasmapheresis eliminates the negative impact of AAV antibodies on microdystrophin gene expression following vascular delivery.

Duchenne muscular dystrophy is a monogenic disease potentially treatable by gene replacement. Use of recombinant adeno-associated virus (AAV) will ultimately require a vascular approach to broadly transduce muscle cells. We tested the impact of preexisting AAV antibodies on microdystrophin expression following vascular delivery to nonhuman primates. Rhesus macaques were treated by isolated limb perfusion using a fluoroscopically guided catheter. In addition to serostatus stratification, the animals were placed into one of the three immune suppression groups: no immune suppression, prednisone, and triple immune suppression (prednisone, tacrolimus, and mycophenolate mofetil). The animals were analyzed for transgene expression at 3 or 6 months. Microdystrophin expression was visualized in AAV, rhesus serotype 74 sero-negative animals (mean: 48.0 ± 20.8%) that was attenuated in sero-positive animals (19.6 ± 18.7%). Immunosuppression did not affect transgene expression. Importantly, removal of AAV binding antibodies by plasmapheresis in AAV sero-positive animals resulted in high-level transduction (60.8 ± 18.0%), which is comparable with that of AAV sero-negative animals (53.7 ± 7.6%), whereas non-pheresed sero-positive animals demonstrated significantly lower transduction levels (10.1 ± 6.0%). These data support the hypothesis that removal of AAV binding antibodies by plasmapheresis permits successful and sustained gene transfer in the presence of preexisting immunity (natural infection) to AAV.

The 2-min walk test is sufficient for evaluating walking abilities in sporadic inclusion body myositis.

Sporadic inclusion body myositis causes progressive functional loss due to declining muscle strength. Although the underlying cause is unknown, clinical trials are underway to improve strength and function. Selection of appropriate outcome measures is critical for the success of these trials. The 6-min walk test has been the de facto standard for assessing function in neuromuscular disease; however, the optimal walking test has not been determined in this disease. In this study, 67 individuals with sporadic inclusion body myositis completed a battery of quantitative strength and functional tests including timed walking tests, patient-reported outcomes, and other tasks. The 2-min and 6-min walk tests are highly correlated to each other (r=0.97, p<0.001) and to all lower extremity strength, patient-reported, and functional measures in this population. All subjects completed the 2-min walk test, but 7% of subjects were unable to walk the full 6-min of the 6-min walk test due to fatigue. The 2-min walk test demonstrates similar correlation to all outcomes compared to the 6-min walk test, is less fatiguing and better tolerated. Results suggest that the 2-min walk test is a better alternative to tests of longer duration. Further research is needed to determine longitudinal changes on this outcome.

Lack of toxicity of alpha-sarcoglycan overexpression supports clinical gene transfer trial in LGMD2D.

BACKGROUND: Alpha-sarcoglycan (alpha-SG) deficiency (limb-girdle muscular dystrophy [LGMD] type 2D) is the most common form of sarcoglycan-LGMD. No treatment is currently available. Prior studies suggest that overexpression of alpha-SG via adeno-associated virus (AAV)-mediated gene transfer results in poorly sustained gene expression related to transgene toxicity. These findings potentially preclude gene therapy as a treatment approach for LGMD2D. METHODS: The human alpha-SG gene (halpha-SG) was directly transferred to the tibialis anterior muscle of 4- to 5-week-old alpha-SG KO mice using AAV, type 1. The gene was placed under control of either the ubiquitously expressed cytomegalovirus (CMV) promoter or muscle specific promoters that included desmin, muscle creatine kinase (MCK), and its further modification, truncated MCK (tMCK). Low (3 x 10(9) vg) and high (3 x 10(10) vg) doses of AAV1.halpha-SG were administered. RESULTS: Sustained gene expression was observed irrespective of promoters at 6 and 12 weeks post gene transfer. Quantitation of alpha-SG gene expression by fiber counts yielded similar levels of myofiber transduction for both MCK promoters (60 to 70%), while 34% of fibers were transduced with the DES promoter. There was a trend toward lower expression at the 12-week time point with the CMV promoter. Western blot analysis revealed alpha-SG overexpression using CMV and both the MCK promoters. CONCLUSION: Our data demonstrate robust and sustained adeno-associated virus type 1 alpha-sarcoglycan gene expression under control of muscle creatine kinase promoters, without evidence of cytotoxicity. These findings support the use of gene therapy as a potential treatment approach for limb-girdle muscular dystrophy type 2D.

Orthopedic outcomes of long-term daily corticosteroid treatment in Duchenne muscular dystrophy.

OBJECTIVE: To document the effects of long-term daily corticosteroid treatment on a variety of orthopedic outcomes in boys with Duchenne muscular dystrophy. METHODS: We reviewed the charts of 159 boys with genetically confirmed dystrophinopathies followed at the Ohio State University Muscular Dystrophy Clinic between 2000 and 2003. Charts were reviewed for ambulation status, type and duration of steroid treatment (if any), and orthopedic complications including presence and location of long bone fractures, vertebral compression fractures, and the presence and degree of scoliosis. RESULTS: The cohort consisted of 143 boys (16 boys with Becker dystrophy were excluded); 75 had been treated with steroids for at least 1 year, whereas 68 boys had never been treated or had received only a brief submaximal dose. The mean duration of daily steroid treatment was 8.04 years. Treated boys ambulated independently 3.3 years longer than the untreated group (p < 0.0001) and had a lower prevalence of scoliosis than the untreated group (31 vs 91%; p < 0.0001). The average scoliotic curve was also milder in the treated group (11.6 degrees) compared with the untreated group (33.2 degrees; p < 0.0001). Vertebral compression fractures occurred in 32% of the treated group, whereas no vertebral fractures were discovered in the steroid naive group (p = 0.0012). Long bone fractures were 2.6 times greater in steroid-treated patients. CONCLUSIONS: Although boys with Duchenne muscular dystrophy on long-term corticosteroid treatment have a significantly decreased risk of scoliosis and an extension of more than 3 years' independent ambulation, they are at increased risk of vertebral and lower limb fractures compared with untreated boys.

Coexisting muscular dystrophies and epilepsy in children.

Muscular dystrophies are composed of a variety of genetic muscle disorders linked to different chromosomes and loci and associated with different gene mutations that lead to progressive muscle atrophy and weakness. Fukuyama congenital muscular dystrophy is frequently associated with partial and generalized epilepsy and congenital brain anomalies, including cobblestone complex and other neuronal migration defects. We report generalized convulsive epilepsy in a boy with normal brain magnetic resonance imaging and Duchenne muscular dystrophy with deletion of dystrophin gene, and we report absence epilepsy with normal brain magnetic resonance imaging in another boy with limb girdle muscular dystrophy with partial calpain deficiency. We, therefore, review coexisting muscular dystrophies and epilepsy in children. In addition to Fukuyama congenital muscular dystrophy, partial or generalized epilepsy has also been reported in the following types of muscular dystrophies, including Duchenne/Becker dystrophy, facioscapulohumeral dystrophy, congenital muscular dystrophy with partial and complete deficiency of laminin alpha2 (merosin) chain, and limb girdle muscular dystrophy with partial calpain deficiency.

Pilot trial of etanercept in the treatment of inclusion-body myositis.

Inclusion-body myositis (IBM) is an inflammatory muscle disease that has proven resistant to treatment. Tumor necrosis factor molecules have been detected in muscle biopsies from patients with IBM. Etanercept is a TNFalpha receptor fusion protein that binds and inactivates tumor necrosis factor. Nine patients were treated with etanercept at a dose of 25 mg, two times a week for an average of 17 +/- 6.1 months. Each patient was evaluated using quantitative strength testing. Their data were compared to two different control groups. The first control group consisted of patients who participated in trials of beta-interferon-1A and had received placebo. There was no significant difference. The second control group was a natural history cohort of IBM patients. There was no statistically significant difference between the treated group and the natural history group at 6 and 12 months when looking at elbow flexors, or 6 months when looking at hand grip. In the treated patients there was a small but significant improvement (p = 0.002) in handgrip at 12 months.

NT-3 promotes nerve regeneration and sensory improvement in CMT1A mouse models and in patients.

BACKGROUND: Xenografts from patients with Charcot-Marie-Tooth type 1A (CMT1A) have shown delayed myelination and impaired regeneration of nude mice axons passing through the grafted segments. Neurotrophin-3 (NT-3), an important component of the Schwann cell (SC) autocrine survival loop, could correct these deficiencies. OBJECTIVE: To assess the efficacy of NT-3 treatment in preclinical studies using animal models of CMT1A and to conduct a double-blind, placebo-controlled, randomized, pilot clinical study to assess the efficacy of subcutaneously administered NT-3 in patients with CMT1A. METHODS: Nude mice harboring CMT1A xenografts and Trembler(J) mice with a peripheral myelin protein 22-point mutation were treated with NT-3, and the myelinated fiber (MF) and SC numbers were quantitated. Eight patients received either placebo (n = 4) or 150 microg/kg NT-3 (n = 4) three times a week for 6 months. MF regeneration in sural nerve biopsies before and after treatment served as the primary outcome measure. Additional endpoint measures included the Mayo Clinic Neuropathy Impairment Score (NIS), electrophysiologic measurements, quantitative muscle testing, and pegboard performance. RESULTS: The NT-3 treatment augmented axonal regeneration in both animal models. For CMT1A patients, changes in the NT-3 group were different from those observed in the placebo group for the mean number of small MFs within regeneration units (p = 0.0001), solitary MFs, (p = 0.0002), and NIS (p = 0.0041). Significant improvements in these variables were detected in the NT-3 group but not in the placebo group. Pegboard performance was significantly worsened in the placebo group. NT-3 was well tolerated. CONCLUSION: Neurotrophin-3 augments nerve regeneration in animal models for CMT1A and may benefit patients clinically, but these results need further confirmation.

Correlating phenotype and genotype in the periodic paralyses.

BACKGROUND: Periodic paralyses and paramyotonia congenita are rare disorders causing disabling weakness and myotonia. Mutations in sodium, calcium, and potassium channels have been recognized as causing disease. OBJECTIVE: To analyze the clinical phenotype of patients with and without discernible genotype and to identify other mutations in ion channel genes associated with disease. METHODS: The authors have reviewed clinical data in patients with a diagnosis of hypokalemic periodic paralysis (56 kindreds, 71 patients), hyperkalemic periodic paralysis (47 kindreds, 99 patients), and paramyotonia congenita (24 kindreds, 56 patients). For those patients without one of the classically known mutations, the authors analyzed the entire coding region of the SCN4A, KCNE3, and KCNJ2 genes and portions of the coding region of the CACNA1S gene in order to identify new mutations. RESULTS: Mutations were identified in approximately two thirds of kindreds with periodic paralysis or paramyotonia congenita. The authors found differences between the disorders and between those with and without identified mutations in terms of age at onset, frequency of attacks, duration of attacks, fixed proximal weakness, precipitants of attacks, myotonia, electrophysiologic studies, serum potassium levels, muscle biopsy, response to potassium administration, and response to treatment with acetazolamide. CONCLUSIONS: Hypokalemic periodic paralysis, hyperkalemic periodic paralysis, and paramyotonia congenita may be distinguished based on clinical data. This series of 226 patients (127 kindreds) confirms some clinical features of this disorder with notable exceptions: In this series, patients without mutations had a less typical clinical presentation including an older age at onset, no changes in diet as a precipitant, and absence of vacuolar myopathy on muscle biopsy.

Nonsystemic vasculitic neuropathy: insights from a clinical cohort.

BACKGROUND: Nonsystemic vasculitic neuropathy (NSVN) is an uncommon disorder. Few series with small numbers of patients have been reported. The prognosis and treatment of patients presenting with NSVN remain uninvestigated. The authors sought to address these issues by assembling a large retrospective cohort with extended follow-up. METHODS: All nerve biopsies performed over 20 years were reviewed; cases with definite, probable, or possible vasculitis were segregated for clinical correlation. Patients satisfying clinical criteria for NSVN at presentation were selected. Clinicopathologic, treatment, and outcome measures were analyzed in patients followed for > or = 6 months. RESULTS: A total of 48 patients (30 women, 18 men) with a median of 63 months of follow-up were identified. Most patients (85%) had extensive, overlapping involvement of multiple nerves. Only one had a symmetric polyneuropathy. Most neuropathies (96%) were painful. In 96%, nerve damage was distally accentuated, but most had concurrent proximal weakness. Diagnostic sensitivity was 58% for superficial peroneal nerve/peroneus brevis muscle biopsy and 47% for sural nerve biopsy. Combination corticosteroid/cytotoxic therapy was more effective than corticosteroid monotherapy in inducing remission and improving disability, with trends toward reduced relapses and chronic pain. Treatment with cyclophosphamide for >6 months decreased the relapse rate, which was 46% for all patients. Disease/treatment-related mortality was 10%. Six percent developed cutaneous involvement. Although chronic pain persisted in 60% of survivors, 80% had good outcomes. CONCLUSIONS: NSVN nearly always presents as an asymmetric, distally accentuated, painful, sensorimotor polyneuropathy. Risks for systemic spread and death are small, and, aside from pain, neurologic prognosis is unexpectedly good. Although this was not a randomized controlled trial, combination therapy produced the best outcome in this cohort.

Randomized, double-blind, placebo-controlled trial of albuterol in facioscapulohumeral dystrophy.

BACKGROUND/OBJECTIVES: Animal and human studies suggest that beta(2)-adrenergic agonists exert anabolic effects on muscles, inducing and preventing atrophy after a variety of insults. Based on data from an open-label trial of albuterol in 15 patients with facioscapulohumeral dystrophy (FSHD), the authors conducted a randomized, double-blind, placebo-controlled trial of sustained-release albuterol in this disease. METHODS: Ninety patients were randomized to three groups: placebo; 8.0 mg albuterol twice daily; or 16.0 mg albuterol twice daily. Patients were treated for 1 year with assessments at baseline and weeks 13, 26, and 52. The primary outcome was the 52-week change in global strength by maximum voluntary isometric contraction testing (MVICT). Secondary outcomes included changes at 52 weeks in strength by manual muscle testing (MMT), grip strength, functional testing, and muscle mass assessed by dual energy x-ray absorptiometry (DEXA). RESULTS: Eighty-four patients completed the study. The mean changes in composite MVICT scores were not significantly different between the groups (mean +/- SD: placebo 0.20 +/- 0.91; low dose -0.04 +/- 0.84; high dose 0.08 +/- 0.98). Similarly, there were no differences in the mean MMT change (placebo 0.04 +/- 0.16; low dose -0.03 +/- 0.13; high dose 0.00 +/- 0.15). Grip improved in both treatment groups compared to placebo (placebo -0.53 +/- 4.13, low dose +1.90 +/- 3.34 [p = 0.02], high dose +1.70 +/- 4.13 [p = 0.03]). The high-dose group had a significant increase in lean mass by DEXA (+1.57 +/- 1.71 kg) compared to placebo (0.25 +/- 2.24; p = 0.007). Albuterol was well tolerated; side effects included cramps, tremors, insomnia, and nervousness. CONCLUSIONS: Although albuterol did not improve global strength or function in patients with FSHD, it did increase muscle mass and improve some measures of strength.

Novel and recurrent mutations in lamin A/C in patients with Emery-Dreifuss muscular dystrophy.

Emery-Dreifuss muscular dystrophy (EDMD) is characterized by slowly progressive muscle wasting and weakness; early contractures of the elbows, Achilles tendons, and spine; and cardiomyopathy associated with cardiac conduction defects. Clinically indistinguishable X-linked and autosomal forms of EDMD have been described. Mutations in the STA gene, encoding the nuclear envelope protein emerin, are responsible for X-linked EDMD, while mutations in the LMNA gene encoding lamins A and C by alternative splicing have been found in patients with autosomal dominant, autosomal recessive, and sporadic forms of EDMD. We report mutations in LMNA found in four familial and seven sporadic cases of EDMD, including seven novel mutations. Nine missense mutations and two small in-frame deletions were detected distributed throughout the gene. Most mutations (7/11) were detected within the LMNA exons encoding the central rod domain common to both lamins A/C. All of these missense mutations alter residues in the lamin A/C proteins conserved throughout evolution, implying an essential structural and/or functional role of these residues. One severely affected patient possesed two mutations, one specific to lamin A that may modify the phenotype of this patient. Mutations in LMNA were frequently identified among patients with sporadic and familial forms of EDMD. Further studies are needed to identify the factors modifying disease phenotype among patients harboring mutations within lamin A/C and to determine the effect of various mutations on lamin A/C structure and function.

Diagnosis of Duchenne dystrophy by enhanced detection of small mutations.

OBJECTIVE: To determine whether detection of small mutations of the dystrophin gene can be increased using an enhanced method of single-strand conformation polymorphism analysis. BACKGROUND: Usual methods of DNA analysis for Duchenne dystrophy cannot identify mutations in one-third of cases. Muscle biopsy, with its inherent risks and added liability for patients with Duchenne dystrophy, becomes the sole method of diagnosis. Even with a tissue diagnosis of dystrophin deficiency, many families are excluded from carrier detection and prenatal diagnosis. METHODS: Genomic DNA from a cohort of 93 patients with Duchenne dystrophy without identifiable gene mutations was screened for mutations. In each case, 22 kilobases of genomic DNA were scanned, including all 79 exons of the dystrophin gene, adjacent intronic regions, and six alternative exons 1. RESULTS: Sixty-eight (73%) had small mutations, including 34 nonsense mutations, 27 microdeletions and insertions, and 7 splice site mutations. No missense mutations were found. One nonsense mutation in exon 59 was detected in four patients. Most mutations were new; 54 of 62 different small mutations have not been reported. Mutations were found throughout the gene: 24% in the first quartile, 31% in the second, 16% in the third, and 29% in the fourth. CONCLUSIONS: A highly sensitive single-strand conformation polymorphism method substantially increased detection of small dystrophin gene mutations and made it possible to diagnose approximately 90% of patients with Duchenne dystrophy by DNA analysis. These findings, combined with cost savings and safety issues, provide compelling reasons to consider DNA analysis as the initial diagnostic test for the suspected dystrophin-deficient patient.

Treatment approaches for Guillain-Barré syndrome and chronic inflammatory demyelinating polyradiculoneuropathy.

GBS and CIDP are important treatable forms of acquired peripheral neuropathies. GBS is a heterogeneous disorder representing at least five different entities. Three are predominantly motor: AIDP, AMSAN, and AMAN. Fisher syndrome and acute panautonomic neuropathy are other variants. Treatment for all of these conditions is the same and includes either plasma exchange or intravenous immunoglobulin. There is no indication that Guillain-Barré patients respond to corticosteroids. At the present time, it is uncertain if CIDP represents one or more disorders. Evidence favors a syndrome composed of more than one entity accounting for (1) clinical variations from subject-to-subject, ranging from symmetrical to focal neurologic deficits; (2) course variations from slowly progressive to step-wise, to relapsing; and, (3) laboratory variations in nerve conduction studies, spinal fluid protein, and nerve biopsy findings. CIDP patients respond to corticosteroids in contrast to those with GBS. CIDP improves with intravenous immunoglobulin and plasma exchange, paralleling the findings in GBS. Specific regimens of treatment for both GBS and CIDP are presented in this article and considerations that might influence one treatment regimen over another are discussed.

High mitochondrial DNA T8993G mutation (<90%) without typical features of Leigh's and NARP syndromes.

Neuropathy, ataxia, and retinitis pigmentosa (NARP) syndrome and maternally inherited Leigh's syndrome have been associated with T8993G point mutations in the mitochondrial adenosine triphosphatase 6 gene. Typically, NARP syndrome is characterized by developmental delay, seizures, dementia, retinitis pigmentosa, ataxia, sensory neuropathy, and proximal weakness. Usually, there is a correlation between the percentage of mutated mitochondrial DNA and clinical severity, and when mutated mitochondrial DNA is > 90%, it is often seen with Leigh's syndrome. We now report a family with mitochondrial DNA T8993G mutation in eight living members, five with mutant mitochondrial DNA >90% and one with 20% mutant mitochondrial DNA. However, their clinical features include variable combinations of seizures, behavior problems, learning disability, mental retardation, sensorineural deafness, cerebellar ataxia, and proximal muscle weakness. No retinitis pigmentosa was found in all eight living members, including a 56-year-old grandmother. Only one dead female relative was diagnosed with Leigh's syndrome on the neuropathologic examination at age 22 years, when she died of an accident. High mitochondrial DNA T8993G mutation is not always associated with typical features of Leigh's and NARP syndromes.

Familial cerebellar ataxia with muscle coenzyme Q10 deficiency.

OBJECTIVE: To describe a clinical syndrome of cerebellar ataxia associated with muscle coenzyme Q10 (CoQ10) deficiency. BACKGROUND: Muscle CoQ10 deficiency has been reported only in a few patients with a mitochondrial encephalomyopathy characterized by 1) recurrent myoglobinuria; 2) brain involvement (seizures, ataxia, mental retardation), and 3) ragged-red fibers and lipid storage in the muscle biopsy. METHODS: Having found decreased CoQ10 levels in muscle from a patient with unclassified familial cerebellar ataxia, the authors measured CoQ10 in muscle biopsies from other patients in whom cerebellar ataxia could not be attributed to known genetic causes. RESULTS: The authors found muscle CoQ10 deficiency (26 to 35% of normal) in six patients with cerebellar ataxia, pyramidal signs, and seizures. All six patients responded to CoQ10 supplementation; strength increased, ataxia improved, and seizures became less frequent. CONCLUSIONS: Primary CoQ10 deficiency is a potentially important cause of familial ataxia and should be considered in the differential diagnosis of this condition because CoQ10 administration seems to improve the clinical picture.

Autonomic impairment in painful neuropathy.

OBJECTIVES: 1) To determine the degree and distribution and quantitate the severity of autonomic impairment in painful neuropathy (PN). 2) To assess the role of autonomic testing in evaluating PN. METHODS: The authors studied 92 patients with PN (60 women and 32 men, age 56.9 +/- 12.4 years) using: 1) autonomic reflex testing (ART), Quantitative Sudomotor Axon Reflex Test (QSART), cardiac-vagal, head-up tilt, and surface skin temperature; 2) autonomic symptoms questionnaire; 3) nerve conduction (NCS) and laboratory studies; 4) quantitative sensory testing; 5) skin biopsy; and 6) Composite Autonomic Symptoms Score (CASS) scale to grade ART results from 0 (normal) to 10 (autonomic failure). RESULTS: Autonomic involvement in PN had characteristic features. Main symptoms were pain, secretory and skin vasomotor signs, hypertension, and impotence. ART results were abnormal in 86 (93.5%) (CASS < 4), QSART in 67 (72.8%), cardiac-vagal index in 58 (63%), skin temperature in 51 (55.4%), orthostatic hypertension in 39 (42.3%), and family history of PN in 26 (21%) of patients. Group 1 (abnormal NCS) (n = 45) had more severe ART and sensory abnormalities than the Group 2 (normal NCS) (n = 47): 1) CASS 2.0 +/- 0.96 vs 1.55 +/- 0.88 (p < 0.01), cardiac-vagal index (p < 0.02), skin temperature (p < 0.02), hypertension (p < 0.03), cooling (p < 0.002), and vibration (p < 0.0005) thresholds. CONCLUSIONS: Autonomic symptoms in painful neuropathy are predominantly cholinergic and form a unique constellation of features that are distinct from other autonomic neuropathies.