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Biomedical subjects

A G Engel

Publications and source records attributed to A G Engel.

At least 37 records · Page 2Linked to original sources
PubMed1999

Congenital myasthenic syndrome caused by a mutation in the Ets-binding site of the promoter region of the acetylcholine receptor epsilon subunit gene.

Forty-two missense, truncation, or splice-site mutations of the acetylcholine receptor (AChR) subunit genes have been reported to date in patients with congenital myasthenic syndromes. Here we report a homozygous mutation, epsilon-155G --> A, in the promoter region of the AChR epsilon subunit gene that converts the Ets-binding site of the promoter region from CGGAA to CAGAA. The asymptomatic parents and brother are heterozygous and an affected sister is homozygous for epislon-155G --> A. The Ets-binding site mediates synapse specific expression of the AChR epsilon subunit gene. An identical G-to-A mutation in the mouse Ets-binding site was previously shown to decrease the binding affinity of the Ets-binding site for the GA binding protein, a transactivating factor for the Ets-binding site, and to reduce the synapse specific expression of the epsilon subunit. The decreased synaptic expression of the epsilon subunit readily accounts for the congenital myasthenic phenotype.

Adolescent↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1999

Congenital end-plate acetylcholinesterase deficiency caused by a nonsense mutation and an A-->G splice-donor-site mutation at position +3 of the collagenlike-tail-subunit gene (COLQ): how does G at position +3 result in aberrant splicing?

Congenital end-plate acetylcholinesterase (AChE) deficiency (CEAD), the cause of a disabling myasthenic syndrome, arises from defects in the COLQ gene, which encodes the AChE triple-helical collagenlike-tail subunit that anchors catalytic subunits of AChE to the synaptic basal lamina. Here we describe a patient with CEAD with a nonsense mutation (R315X) and a splice-donor-site mutation at position +3 of intron 16 (IVS16+3A-->G) of COLQ. Because both A and G are consensus nucleotides at the +3 position of splice-donor sites, we constructed a minigene that spans exons 15-17 and harbors IVS16+3A-->G for expression in COS cells. We found that the mutation causes skipping of exon 16. The mutant splice-donor site of intron 16 harbors five discordant nucleotides (at -3, -2, +3, +4, and +6) that do not base-pair with U1 small-nuclear RNA (snRNA), the molecule responsible for splice-donor-site recognition. Versions of the minigene harboring, at either +4 or +6, nucleotides complementary to U1 snRNA restore normal splicing. Analysis of 1,801 native splice-donor sites reveals that presence of a G nucleotide at +3 is associated with preferential usage, at positions +4 to +6, of nucleotides concordant to U1 snRNA. Analysis of 11 disease-associated IVS+3A-->G mutations indicates that, on average, two of three nucleotides at positions +4 to +6 fail to base-pair, and that the nucleotide at +4 never base-pairs, with U1 snRNA. We conclude that, with G at +3, normal splicing generally depends on the concordance that residues at +4 to +6 have with U1 snRNA, but other cis-acting elements may also be important in assuring the fidelity of splicing.

Acetylcholinesterase↗
PubMed1999

Myopathy, myasthenic syndrome, and epidermolysis bullosa simplex due to plectin deficiency.

Plectin, an intermediate filament linking protein, is normally associated with the sarcolemma, nuclear membrane, and intermyofibrillar network in muscle, and with hemisdesmosomes in skin. A 20-year-old female with epidermolysis bullosa simplex since birth had progressive ocular, facial, limb, and trunkal weakness and fatigability since age 9, fivefold CK elevation, a 25% decrement with myopathic motor unit potentials and increased electrical irritability on electromyography, and no anti-acetylcholine receptor (AChR) antibodies. Plectin expression was absent in muscle and severe plectin deficiency was noted in skin. Morphologic studies revealed necrotic and regenerating fibers and a wide spectrum of ultrastructural abnormalities: large accumulations of heterochromatic and lobulated nuclei, rare apoptotic nuclei, numerous cytoplasmic and few intranuclear nemaline rods, disarrayed myofibrils, thick-filament loss, vacuolar change, and pathologic alterations in membranous organelles. Many endplates (EPs) had an abnormal configuration with chains of small regions over the fiber surface and a few displayed focal degeneration of the junctional folds. The EP AChR content was normal. In vitro electrophysiologic studies showed normal quantal release by nerve impulse, small miniature EP potentials, and fetal as well as adult AChR channels at the EP. Our findings support the notion that plectin is essential for the structural integrity of muscle and skin, and for normal neuromuscular transmission.

Adult↗
PubMed1999

Mutation causing congenital myasthenia reveals acetylcholine receptor beta/delta subunit interaction essential for assembly.

We describe a severe postsynaptic congenital myasthenic syndrome with marked endplate acetylcholine receptor (AChR) deficiency caused by 2 heteroallelic mutations in the beta subunit gene. One mutation causes skipping of exon 8, truncating the beta subunit before its M1 transmembrane domain, and abolishing surface expression of pentameric AChR. The other mutation, a 3-codon deletion (beta426delEQE) in the long cytoplasmic loop between the M3 and M4 domains, curtails but does not abolish expression. By coexpressing beta426delEQE with combinations of wild-type subunits in 293 HEK cells, we demonstrate that beta426delEQE impairs AChR assembly by disrupting a specific interaction between beta and delta subunits. Studies with related deletion and missense mutants indicate that secondary structure in this region of the beta subunit is crucial for interaction with the delta subunit. The findings imply that the mutated residues are positioned at the interface between beta and delta subunits and demonstrate contribution of this local region of the long cytoplasmic loop to AChR assembly.

Acetylcholinesterase↗
PubMed1998

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.

Acetylcholinesterase↗
PubMed1998

Quinidine normalizes the open duration of slow-channel mutants of the acetylcholine receptor.

Quinidine is a long-lived open-channel blocker of the wild-type endplate acetylcholine receptor (AChR). To test the hypothesis that quinidine can normalize the prolonged channel opening events of slow-channel mutants of human AChR, we expressed wild-type AChR and five well characterized slow-channel mutants of AChR in HEK 293 cells and monitored the effects of quinidine on acetylcholine-induced channel currents. Quinidine shortens the longest component of channel opening burst (tau3b) of both wild-type and mutant AChRs in a concentration-dependent manner, and 5 microM quinidine reduces tau3b of the mutant AChRs to that of wild-type AChRs in the absence of quinidine. Because this concentration of quinidine is attainable in clinical practice, the findings predict a therapeutic effect for quinidine in the slow-channel congenital myasthenic syndrome.

Cholinergic Antagonists↗
PubMed1998

T-cell heterogeneity in muscle lesions of inclusion body myositis.

In the muscle lesions of inclusion body myositis (IBM), two populations of T cells can morphologically be distinguished, T cells that invade muscle fibers and T cells that remain in interstitial areas. Using combined immunohistochemistry and RT-PCR, we analysed the TCR expressed by these distinct T cell populations. In three of eight IBM muscle specimens, the autoinvasive T cells were stained with one of the available mAbs: anti-Vbeta5.3 in patient 1, anti-Vbeta5.2 in patient 2, and anti-Vbeta3 in patient 3. The corresponding TCR Vbeta mRNAs were amplified by RT-PCR and the PCR products were cloned and sequenced. In all three specimens, the TCRs expressed by the autoinvasive T cells were clonally restricted. Furthermore, in patient 1 two different mAbs labelled two distinct populations of T cells: an autoaggressive population of CD8 + Vbeta5.3 + cells that invaded muscle fibers and a noninvasive interstitial population of CD8 - Vbeta5.1 + cells. TCR sequence analysis revealed that the noninvasive Vbeta5.1 + T cells were clonally diverse, whereas the autoinvasive Vbeta5.3 + T cells were clonally restricted.

Antibodies, Monoclonal↗
PubMed1998

Quinidine sulfate therapy for the slow-channel congenital myasthenic syndrome.

The slow-channel congenital myasthenic syndrome (SCCMS) is caused by gain of function mutations in subunits of the end-plate acetylcholine receptor (AChR). The mutations prolong the opening episodes of the AChR channel, leading to a depolarization block and an end-plate myopathy. Because levels of quinidine sulfate attainable in clinical practice shorten the opening episodes of genetically engineered mutant SCCMS receptors in vitro, we tested the notion that the drug can be of benefit in SCCMS. We treated 6 SCCMS patients with quinidine sulfate in an open-label trial, using objective clinical measures of muscle strength and repetitive stimulation studies as end points. One patient became allergic to quinidine after 7 days. The remaining patients tolerated the drug well and after 30 days of continuous therapy showed statistically significant improvement in muscle strength and in decrement of the compound muscle action potential elicited by rapid rates of stimulation.

Action Potentials↗
PubMed1998

Amyloid myopathy: an underdiagnosed entity.

Amyloidosis can involve multiple organs, including kidney, heart, peripheral nerve, skin, joints, and skeletal muscle, but rarely presents as a myopathy. We studied 13 adults with muscle weakness for between 3 months and 4 years in whom the diagnosis of systemic amyloidosis was unsuspected before or until just before the time of the muscle biopsy. All muscle specimens demonstrated congophilic deposits around blood vessels and muscle fibers, some necrotic and regenerating fibers, and signs of mild denervation. Immunostains in 10 patients revealed immunoglobulin amyloidosis in 7 and gelsolin amyloidosis in 1. Apolipoprotein E co-localized with the congophilic deposits in all 10, and a C-terminal epitope of the beta-amyloid precursor protein was detected in 6. The frequency of the diagnosis of amyloid myopathy increased 10-fold when we adopted the fluorescent Congo red stain as a routine procedure in assessing muscle biopsy specimens.

Adult↗
PubMed1998

Myasthenic syndromes in Turkish kinships due to mutations in the acetylcholine receptor.

We report and functionally characterize five new mutations of the acetylcholine receptor (AChR) in 11 Turkish patients with recessive congenital myasthenic syndromes (CMS) belonging to six families. All mutations are in the epsilon-subunit gene. Parental consanguinity is present in three families. The disease cosegregates with homozygous mutations in five families and with two different heteroallelic mutations in one family. Four mutations are frameshifting, predicting truncation of the epsilon subunit, and one occurs at a splice donor site. Expression of each frameshifting mutation and the likely transcripts of the splice-site mutation in human embryonic kidney 293 cells shows that each mutation is a null mutation. The findings support the notion that loss-of-function mutations of the acetylcholine receptor causing CMS are concentrated in the epsilon subunit, and that such mutations are a frequent cause of CMS.

Base Sequence↗
PubMed1998

Mode switching kinetics produced by a naturally occurring mutation in the cytoplasmic loop of the human acetylcholine receptor epsilon subunit.

We describe the genetic and kinetic defects in a congenital myasthenic syndrome caused by heteroallelic mutations of the acetylcholine receptor (AChR) epsilon subunit gene. The mutations are an in-frame duplication of six residues in the long cytoplasmic loop (epsilon1254ins18) and a cysteine-loop null mutation (epsilonC128S). The epsilon1254 ins18 mutation causes mode switching in the kinetics of receptor activation in which three modes activate slowly and inactivate rapidly. The epsilon1245ins18-AChR at the endplate shows abnormally brief activation episodes during steady state agonist application and appears electrically silent during the synaptic response to acetylcholine. The phenotypic consequences are endplate AChR deficiency, simplification of the postsynaptic region, and compensatory expression of fetal AChR that restores electrical activity at the endplate and rescues the phenotype.

Acetylcholine↗