Novel congenital myasthenic syndromes associated with defects in quantal release.

BACKGROUND: Most congenital myasthenic syndromes are caused by defects in postsynaptic or synaptic basal lamina-associated proteins; congenital myasthenic syndromes (CMSs) associated with presynaptic defects are uncommon. Here, the authors describe clinical, electrophysiologic, and morphologic features of two novel and highly disabling CMSs, one determined by presynaptic and the other determined by combined presynaptic and postsynaptic defects. METHODS: Microelectrode, single channel patch clamp, immunocytochemical, [(125)I]alpha-bungarotoxin binding, and quantitative electron microscopy studies of endplates were performed. Candidate genes were directly sequenced. RESULTS: Patient 1, a 7-year-old boy, had severe myasthenic symptoms since infancy. Patient 2, a 48-year-old man, had delayed motor milestones and became progressively weaker after age 2 years. Both used wheelchairs and had a 30-50% EMG decrement on 2-Hz stimulation. Evoked quantal release was reduced to approximately 25% of normal in both. In Patient 2, the synaptic response to acetylcholine was further compromised by degeneration of the junctional folds with concomitant loss of the acetylcholine receptor (AChR). A search for mutations in components of the synaptic vesicle release complex and in other candidate proteins failed to identify the molecular basis of the two syndromes. CONCLUSIONS: Combined clinical, morphologic, and in vitro electrophysiologic findings define two novel congenital myasthenic syndromes. The molecular basis of these syndromes awaits discovery.

Spectrum of splicing errors caused by CHRNE mutations affecting introns and intron/exon boundaries.

BACKGROUND: Mutations in CHRNE, the gene encoding the muscle nicotinic acetylcholine receptor epsilon subunit, cause congenital myasthenic syndromes. Only three of the eight intronic splice site mutations of CHRNE reported to date have had their splicing consequences characterised. METHODS: We analysed four previously reported and five novel splicing mutations in CHRNE by introducing the entire normal and mutant genomic CHRNEs into COS cells. RESULTS AND CONCLUSIONS: We found that short introns (82-109 nucleotides) favour intron retention, whereas medium to long introns (306-1210 nucleotides) flanking either or both sides of an exon favour exon skipping. Two mutations are of particular interest. Firstly, a G-->T substitution at the 3' end of exon 8 predicts an R286M missense mutation, but instead results in skipping of exon 8. In human genes, a mismatch of the last exonic nucleotide to U1 snRNP is frequently compensated by a matching nucleotide at intron position +6. CHRNE intron 8 has a mismatch at position +6, and accordingly fails to compensate for the exonic mutation at position -1. Secondly, a 16 bp duplication, giving rise to two 3' splice sites (g.IVS10-9_c.1167dup16), results in silencing of the downstream 3' splice site. This conforms to the scanning model of recognition of the 3' splice site, which predicts that the first "ag" occurring after the branch point is selected for splicing.

Congenital myasthenic syndrome caused by low-expressor fast-channel AChR delta subunit mutation.

OBJECTIVE: To determine the molecular basis of a disabling congenital myasthenic syndrome (CMS) observed in two related and one unrelated Arab kinship. BACKGROUND: CMS can arise from defects in presynaptic, synaptic basal lamina-associated, or postsynaptic proteins. Most CMS are postsynaptic, and most reside in the AChR epsilon subunit; only two mutations have been reported in the AChR delta subunit to date. METHODS: Cytochemistry, electron microscopy, alpha-bungarotoxin binding studies, microelectrode and patch-clamp recordings, mutation analysis, mutagenesis, and expression studies in human embryonic kidney cells were employed. RESULTS: Endplate studies showed AChR deficiency, fast decaying, low-amplitude endplate currents, and abnormally brief channel opening events. Mutation analysis revealed a novel homozygous missense mutation (deltaP250Q) of the penultimate proline in the first transmembrane domain (TMD1) of the AChR delta subunit. Expression studies indicate that deltaP250Q (1) hinders delta/alpha subunit association during early AChR assembly; (2) hinders opening of the doubly occupied closed receptor (A(2)R); and (3) speeds the dissociation of acetylcholine from A(2)R. Mutagenesis studies indicate that deltaP250L also has fast-channel effects, whereas epsilon P245L and epsilon P245Q, identical mutations of the corresponding proline in the epsilon subunit, have mild slow-channel effects. CONCLUSIONS: deltaP250Q represents the third mutation observed in the AChR delta subunit. The severe phenotype caused by deltaP250Q is attributed to endplate AChR deficiency, fast decay of the synaptic response, and lack of compensatory factors. That the penultimate prolines in TMD1 of the delta and epsilon subunits exert a reciprocal regulatory effect on the length of the channel opening bursts reveals an unexpected functional asymmetry between the two subunits.

Congenital myasthenic syndrome associated with episodic apnea and sudden infant death.

The sudden infant death syndrome has multiple etiologies. Some congenital myasthenic syndromes can cause sudden infant death syndrome by apnea, but the frequency of this etiology is unknown. We report here a young patient with sudden respiratory crises culminating in apnea followed by recovery, against a background of no or variable myasthenic symptoms without dyspnea. One sib without myasthenic symptoms and one sib who only had mild ptosis died previously during febrile episodes. Studies reported by us elsewhere traced the proband's illness to mutations in choline acetyltransferase. Here, we describe in detail the morphologic investigations and electrophysiologic findings, which point to a presynaptic defect in acetylcholine resynthesis or vesicular filling, in the proband. Analysis of DNA from a sib who previously died of sudden infant death syndrome revealed the same choline acetyltransferase mutation. Thus, mutations in choline acetyltransferase may be a cause of sudden infant death syndrome as, theoretically, could other presynaptic myasthenic disorders.

Three novel COLQ mutations and variation of phenotypic expressivity due to G240X.

OBJECTIVE: To determine the molecular basis and consequences of endplate (EP) acetylcholinesterase (AChE) deficiency. BACKGROUND: The EP species AChE is an asymmetric enzyme consisting of a tail subunit composed of three collagenic strands (ColQ), each attached to a tetramer of catalytic subunits. The tail subunit is essential for insertion of AChE into the synaptic basal lamina. Human EP AChE deficiency is caused by mutations in COLQ. The authors report three novel COLQ mutations in eight kinships. METHODS: Immunocytochemistry, electron microscopy, microelectrode recordings, mutation analysis, and expression studies in COS cells were employed. RESULTS: Two mutations (275insC and Q211X) were heterozygous in one patient. EP studies in this patient revealed no EP AChE, small nerve terminals, reduced presynaptic membrane length, as well as abnormally low-evoked quantal release. The third mutation (G240X) was homozygous in six Palestinian Arab families of the same tribe and in an Iraqi Jewish patient. Expression studies of the three mutations in COS cells indicate that each abrogates formation of insertion competent asymmetric AChE. Although the three mutations have identical predicted consequences at the EP, their phenotypic expressivity varies as regards age at onset, rate of progression, and severity of symptoms. CONCLUSIONS: 1) After mutations in the AChR epsilon subunit, mutations in COLQ are emerging as second most common cause of congenital myasthenic syndromes. 2) A founder effect is likely for G240X in the Palestinian Arab families. 3) That mutations predicting total absence of AChE from the EP have variable phenotypic expressivity suggests that modifying genes or environmental factors can partially compensate for EP AChE deficiency.

Immunohistochemical demonstration of nerve fibers in the synovial fold of the human cervical facet joint.

The role of the intra-articular synovial fold as a source of facet joint pain is unclear, because the nature of nociceptive innervation in lumbar synovial folds is controversial, and there have been no such studies in cervical synovial folds. The present study aimed to demonstrate the presence of nerve fibers including nociceptive fibers in synovial folds of human cervical facet joints using immunohistochemistry. Synovial folds of cervical facet joints removed from patients undergoing cervical spine laminoplasty were analyzed immunohistochemically using antibodies to protein gene product 9.5, beta III-tubulin, substance P and calcitonin gene-related peptide. Many nerve fibers immunoreactive for protein gene product 9.5 and beta III-tubulin were demonstrated both around blood vessels and as free fibers in the stroma of the synovial fold. Also. immunostaining showed the presence of free nerve fibers immunoreactive for substance P and calcitonin gene-related peptide in the stroma. The presence of putative nociceptive fibers in cervical synovial folds supports a possible role for these structures as a source of cervical facet joint pain.

Choline acetyltransferase mutations cause myasthenic syndrome associated with episodic apnea in humans.

Choline acetyltransferase (ChAT; EC ) catalyzes the reversible synthesis of acetylcholine (ACh) from acetyl CoA and choline at cholinergic synapses. Mutations in genes encoding ChAT affecting motility exist in Caenorhabditis elegans and Drosophila, but no CHAT mutations have been observed in humans to date. Here we report that mutations in CHAT cause a congenital myasthenic syndrome associated with frequently fatal episodes of apnea (CMS-EA). Studies of the neuromuscular junction in this disease show a stimulation-dependent decrease of the amplitude of the miniature endplate potential and no deficiency of the ACh receptor. These findings point to a defect in ACh resynthesis or vesicular filling and to CHAT as one of the candidate genes. Direct sequencing of CHAT reveals 10 recessive mutations in five patients with CMS-EA. One mutation (523insCC) is a frameshifting null mutation. Three mutations (I305T, R420C, and E441K) markedly reduce ChAT expression in COS cells. Kinetic studies of nine bacterially expressed ChAT mutants demonstrate that one mutant (E441K) lacks catalytic activity, and eight mutants (L210P, P211A, I305T, R420C, R482G, S498L, V506L, and R560H) have significantly impaired catalytic efficiencies.

Ulnar groove plasty for friction neuropathy at the elbow.

In this paper, we report on a new operation for ulnar neuropathy caused by friction at the elbow. The operation consists of ulnar groove plasty proximal to the cubital tunnel. The ulnar nerve is replaced into this reconstructed groove. Patients are relieved of discomfort, and motor and sensory functions are recovered. The nerve is kept stable throughout the full range of elbow motion and showed neither irritation nor adhesion. Friction ulnar neuropathy is traditionally treated by anterior transpositon or medial epicondylectomy. The ulnar nerve may become entrapped in scar tissue after these operations. We believe that this anatomical position is optimum for the nerve and that this procedure is essential for treatment of friction neuropathy.

Electrodiagnosis in entrapment neuropathy by the arcade of Struthers.

Two cases of high ulnar nerve neuropathy are reported. Lesions were localized at the midarm level by electrophysiologic studies. In the first case, the lesion was found mainly to be a prolonged neurapraxia, and neurolysis was effective. The ulnar nerve was swollen 1 cm in length under the arcade of Struthers. After neurolysis, the palsy recovered rapidly. In the other case, the lesion seemed to be a mild injury to the myelin sheath. Delayed segmental conduction velocity and partial conduction block were found at the midarm level. The paresis improved slightly during the 11-month followup without any treatment, but the electrophysiologic studies were unchanged. In both cases, physical examination did not distinguish the lesions from cubital tunnel syndrome. Electrophysiologic examination proved to be effective as a diagnostic procedure. In the presence of ulnar neuropathy, the upper arm segment should be included in a routine nerve conduction study to screen for the rare but important entrapment neuropathy caused by the arcade of Struthers.

Acetylcholine receptor M3 domain: stereochemical and volume contributions to channel gating.

By defining the functional defect in a congenital myasthenic syndrome (CMS), we show that the third transmembrane domain (M3) of the muscle acetylcholine receptor governs the speed and efficiency of gating of its channel. The clinical phenotype of this CMS results from the mutation V285I in M3 of the alpha subunit, which attenuates endplate currents, accelerates their decay and causes abnormally brief acetylcholine-induced single-channel currents. Kinetic analysis of engineered alpha V285I receptors demonstrated a predominant effect on channel gating, with abnormally slow opening and rapid closing rates. Analysis of site-directed mutations revealed stereochemical and volume-dependent contributions of alpha V285 to channel gating. Thus, we demonstrate a functional role for the M3 domain as a key component of the nicotinic acetylcholine receptor channel-gating mechanism.

Human endplate acetylcholinesterase deficiency caused by mutations in the collagen-like tail subunit (ColQ) of the asymmetric enzyme.

In skeletal muscle, acetylcholinesterase (AChE) exists in homomeric globular forms of type T catalytic subunits (ACHET) and heteromeric asymmetric forms composed of 1, 2, or 3 tetrameric ACHET attached to a collagenic tail (ColQ). Asymmetric AChE is concentrated at the endplate (EP), where its collagenic tail anchors it into the basal lamina. The ACHET gene has been cloned in humans; COLQ cDNA has been cloned in Torpedo and rodents but not in humans. In a disabling congenital myasthenic syndrome, EP AChE deficiency (EAD), the normal asymmetric species of AChE are absent from muscle. EAD could stem from a defect that prevents binding of ColQ to ACHET or the insertion of ColQ into the basal lamina. In six EAD patients, we found no mutations in ACHET. We therefore cloned human COLQ cDNA, determined the genomic structure and chromosomal localization of COLQ, and then searched for mutations in this gene. We identified six recessive truncation mutations of COLQ in six patients. Coexpression of each COLQ mutant with wild-type ACHET in SV40-transformed monkey kidney fibroblast (COS) cells reveals that a mutation proximal to the ColQ attachment domain for ACHET prevents association of ColQ with ACHET; mutations distal to the attachment domain generate a mutant approximately 10.5S species of AChE composed of one ACHET tetramer and a truncated ColQ strand. The approximately 10.5S species lack part of the collagen domain and the entire C-terminal domain of ColQ, or they lack only the C-terminal domain, which is required for formation of the triple collagen helix, and this likely prevents their insertion into the basal lamina.

Finger flexion sign for ulnar neuropathy.

We report a diagnostic sign of ulnar neuropathy. Function of the interossei is tested by asking the patient to hold a sheet of paper between the middle and ring fingers, by adducting the fingers while the examiner pulls it firmly away. The metacarpophalangeal joints will flex more on the affected side as the flexor tendons are recruited. This test can easily detect muscle weakness in the early stage of ulnar neuropathy, and is produced by a similar mechanism to that of Froment's sign.

Malignant peripheral nerve sheath tumors developing multifocally in the central nervous system in a patient with neurofibromatosis type 2.

We describe autopsy findings of multifocal malignant peripheral nerve sheath tumors (MPNSTs) appearing in the central nervous system in a 45-year-old Japanese female with neurofibromatosis type 2. Multiple MPNSTs were detected in both III and VIII, left IV, and V cranial nerves, and a number of nerve roots of the spinal cord. Neurofibromata were on the other hand evident on some nerve roots of the spinal cord and femoral and sciatic nerves. Our results suggest that a mutation of p53 gene may have played a role in the malignant transformation of nerve tumors in this patient since p53 protein was immunohistochemically detected in MPNST cells but not in tumor cells of the neurofibromata.

Elevated serum levels of soluble E- and L-selectin in patients with human T-cell lymphotropic virus type I-associated myelopathy.

We compared soluble E-selectin (sE-selectin) and L-selectin (sL- selectin) levels in sera and cerebrospinal fluid (CSF) of 30 patients with human T-lymphotropic virus type I (HTLV-I)-associated myelopathy (HAM), with those of 10 patients with the relapsing-remitting form of multiple sclerosis (MS), and 16 patients with other neurological diseases (OND). Serum levels of both sE-selectin and sL-selectin, as measured by enzyme-linked immunosorbent assay, were significantly elevated in patients with HAM, compared to patients with OND. In addition, serum levels of sL-selectin were significantly elevated in HAM patients compared to MS patients. No significant difference was found in CSF levels of sL-selectin between HAM patients and controls. However, HAM patients who had received blood transfusions had significantly higher CSF levels of sL-selectin than HAM patients without a past history of transfusions, suggesting that HAM patients with past history of transfusion have a more active immunological state in the central nervous system. sE-selectin was not detected in CSF of HAM patients and controls. This finding might be based on exaggerated inflammatory conditions following increased attachment of lymphocytes to activated endothelial cells in HAM patients.

Up-regulation of iNOS mRNA expression and increased production of NO in human monoblast cell line, U937 transfected by HTLV-I tax gene.

We investigated the mRNA expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO) in human T cell lymphotropic virus type I (HTLV-I) p40tax-transfected U937 cells, a human monoblast cell line. Transfection of HTLV-I p40tax U937 cells induced up-regulation of iNOS mRNA expression and subsequent NO production. Furthermore, interferon gamma (IFN-gamma) stimulation of HTLV-I p40tax-transfected U937 cells enhanced iNOS mRNA expression and NO production. The kinetics of iNOS mRNA expression and NO production indicated maximal effect at 24 and 48 hours, respectively, after culture with or without IFN-gamma. These findings suggest that HTLV-I p40tax can act as a transactivator of NO production in cells of Mo/M phi lineage. To what extent this mechanism may be involved in the pathogenesis of HTLV-I-associated diseases warrants further investigation.

Successful application of pentoxifylline in the treatment of HTLV-I associated myelopathy.

Fifteen patients with human T-cell lymphotropic virus type-I (HTLV-I)-associated myelopathy (HAM) were treated in an uncontrolled preliminary trial by oral administration of pentoxifylline (PTX). Motor function, neurological evaluation, immunological markers and parameters were evaluated after four weeks. In 13 of the 15 patients, motor disability, especially spasticity, improved substantially. PTX suppressed spontaneous proliferation of peripheral blood mononuclear cells in 14 of the 15 patients at four weeks. No adverse effect was observed. We concluded that PTX may be a safe and beneficial agent for the treatment of HAM.

[A case of malignant rheumatoid arthritis with transverse myelopathy and multiple lacunar infarction].

We reported a 34-year-old woman with malignant rheumatoid arthritis (MRA) associated with transverse myelopathy and multiple lacunar infarction. She had suffered from MRA for 9 years, then developed sensory disturbance of left big toe and weakness of right lower limb. Neurological examination revealed the muscle weakness of right lower limb. Deep tendon reflexes were hyperactive in bilateral lower limbs. Babinski's sign was positive bilaterally. Superficial sensation was decreased below Th10 level on the left side. Urinary bladder and rectal disturbance were not present. Laboratory examination disclosed perinuclear antineutrophil cytoplasmic autoantibody (p-ANCA) and anti-nuclear antibody (ANA). Multiple lacunar infarction and syringomyelia were found by MRI studies. Histological examinations of skin ulcer biopsied at 17 years of age disclosed vasculitis. We speculated that vasculitis associated with MRA might cause the damage of central nervous system (CNS) in our case. The p-ANCA may accelerate the vasculitic changes in CNS.

Cubital tunnel reconstruction for ulnar neuropathy in osteoarthritic elbows.

We operated on 16 patients for ulnar neuropathy associated with osteoarthritis of the elbow. They were all male manual workers, with an average age of 51 years at the time of surgery. The severity of the symptoms was McGowan grade 1 in five patients, grade 2 in nine and grade 3 in two. The mean follow-up was 36 months. The operation consists of resecting the osteophytes around the postcondylar groove. The shallow and narrow cubital tunnel is made deep and wide and the ulnar nerve is replaced with its surrounding soft tissues in the enlarged groove. All patients were relieved of discomfort and all showed some improvement or full recovery of motor and sensory function. The ulnar nerve showed no evidence of irritation or adhesion. This procedure also allows early movement of the elbow after operation, because the subcutaneous tissues and muscles have not been detached.

Heightened transmigrating activity of CD4-positive T cells through reconstituted basement membrane in patients with human T-lymphotropic virus type I-associated myelopathy.

We investigated the transmigrating activity of peripheral blood T cells of patients with human T-lymphotropic virus type I-associated myelopathy (HAM) through reconstituted basement membrane. The transmigrating activity of CD4+ T cells of HAM patients was increased significantly in comparison to that of anti-HTLV-I-seropositive carriers and HTLV-I-seronegative controls. However, the migrating activity of CD8+ T cells was not significantly different in HAM patients and controls. The activity of aminopeptidase-N in peripheral blood T cells of HAM patients also was increased significantly, as compared to that of controls. In addition, HTLV-I proviral load in transmigrating CD4+ T cells of HAM patients was increased significantly (two- to eight-fold), compared to that in nontransmigrating CD4+ cells. By contrast, no significant difference in HTLV-I proviral load was found between transmigrating and nontransmigrating CD4+ cells of HTLV-I-seropositive carriers, although copy numbers of HTLV-I proviral load were very low in them. The heightened transmigrating activity of CD4+ cells from HAM patients through reconstituted basement membrane is based on the increased activity of aminopeptidase-N. Collectively, these findings suggest that HTLV-I-infected CD4+ T cells play an important role in the early stage of the pathogenesis of HAM.

Pentoxifylline down-regulates adhesion molecule expression and inflammatory cytokine production in cultured peripheral blood mononuclear cells from patients with HTLV-I-associated myelopathy.

To clarify if pentoxifylline (PTX) may have therapeutic potential for human T-cell lymphotropic virus type I (HTLV-I)-associated myelopathy (HAM), we investigated the in vitro effect of PTX on spontaneous proliferation of peripheral blood lymphocytes (SPP), as well as on the expression of adhesion molecules, such as lymphocyte function antigen-1 (LFA-1) and very late activation antigen-4 (VLA-4), and the production of inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and granulocyte-monocyte colony stimulating factor (GM-CSF), in cultured PBMC from 10 HAM patients, compared with control subjects. SPP in HAM patients was significantly suppressed in a dose-dependent manner with PTX. Using flow cytometry, PTX was found to down-regulate the expression of LFA-1 and VLA-4 on CD4+ and CD8+ T cells in HAM patients as well as control subjects. However, the fall in the expression of LFA-1 and VLA-4 on CD4+ T cell population in HAM patients was higher than that of control subjects. PTX caused a significant suppression of spontaneous production of TNF-alpha by cultured PBMC of HAM patients. It also caused a small but significant suppression GM-CSF and IFN-gamma production. Collectively, our results suggest that PTX might be therapeutically effective at critical points in the immunopathogenesis of HAM.