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Biomedical subjects

Andrew G Engel

Publications and source records attributed to Andrew G Engel.

At least 37 records · Page 2Linked to original sources
PubMed2004

Are MuSK antibodies the primary cause of myasthenic symptoms?

OBJECTIVE: To investigate the morphologic, electrophysiologic, and molecular correlates of muscle-specific tyrosine kinase-seropositive [MuSK(+)] myasthenia gravis (MG). BACKGROUND: Anti-MuSK antibodies are detected in some of acetylcholine receptor-seronegative [AChR(-)] patients with MG with prominent facial, bulbar, and respiratory muscle involvement. The morphologic and electrophysiologic correlates of MuSK(+) MG have not been investigated to date. METHODS: Immunohistochemistry, electron microscopy, and in vitro electrophysiology studies were performed on an intercostal muscle specimen of a patient with MuSK(+) MG and in control subjects. MUSK was directly sequenced, and the nucleotide changes were traced with allele-specific PCR in control subjects. RESULTS: A man aged 34 years has had facial weakness since childhood and progressive bulbar and respiratory muscle weakness and intermittent diplopia since age 21 years. He has thin temporalis and masseter muscles, a high-arched palate, and an atrophic tongue. EMG shows a 36% decrement in facial muscles. His mother has similar facial features. His endplates (EPs) show no AChR or MuSK deficiency, but the amplitudes of the miniature EP potentials and currents are reduced to 35% and 55% of normal, respectively. EP ultrastructure is well preserved, but some junctional folds immunostain faintly for immunoglobulin G. Mutation analysis of MUSK reveals one rare and two common DNA polymorphisms. CONCLUSIONS: 1) The circulating anti-muscle-specific tyrosine kinase antibodies caused neither muscle-specific tyrosine kinase nor acetylcholine receptor deficiency at the endplates; 2) the reduced intercostal miniature endplate potential and current amplitudes were not accounted for by acetylcholine receptor deficiency; 3) the faint immunoglobulin G deposits at the endplates may or may not represent anti-muscle-specific tyrosine kinase antibodies; and 4) the anti-muscle-specific tyrosine kinase antibodies may not be the primary cause of myasthenic symptoms in this patient.

Adult↗
PubMed2004

Choline acetyltransferase structure reveals distribution of mutations that cause motor disorders.

Choline acetyltransferase (ChAT) synthesizes acetylcholine in neurons and other cell types. Decreases in ChAT activity are associated with a number of disease states, and mutations in ChAT cause congenital neuromuscular disorders. The crystal structure of ChAT reported here shows the enzyme divided into two domains with the active site in a solvent accessible tunnel at the domain interface. A low-resolution view of the complex with one substrate, coenzyme A, defines its binding site and suggests an additional interaction not found in the related carnitine acetyltransferase. Also, the preference for choline over carnitine as an acetyl acceptor is seen to result from both electrostatic and steric blocks to carnitine binding at the active site. While half of the mutations that cause motor disorders are positioned to affect enzyme activity directly, the remaining changes are surprisingly distant from the active site and must exert indirect effects. The structure indicates how ChAT is regulated by phosphorylation and reveals an unusual pattern of basic surface patches that may mediate membrane association or macromolecular interactions.

Amino Acid Sequence↗
PubMed2004

Mutations in myotilin cause myofibrillar myopathy.

BACKGROUND AND OBJECTIVE: The term myofibrillar myopathy (MFM) is a noncommittal term for a pathologic pattern of myofibrillar dissolution associated with accumulation of myofibrillar degradation products and ectopic expression of multiple proteins. Ultrastructural studies implicate the Z-disk as the site of the initial pathologic change, and mutations in two Z-disk-related proteins, desmin and alphaB-crystallin, have been identified in a minority of patients with MFM. The authors' objective was to determine whether mutations in myotilin, a key Z-disk component and the disease protein in limb-girdle muscular dystrophy (LGMD) 1A, are another cause of MFM. METHODS: The authors used histochemical, immunocytochemical, ultrastructural, and mutation analysis. RESULTS: The authors detected four missense mutations in 6 of 57 patients with MFM in the serine-rich exon 2 of MYOT, where the two previously identified LGMD1A mutations are located. Three mutations were novel, and one had been previously identified in LGMD1A. Each patient had evidence for neuropathy, and at least three kinships had associated cardiomyopathy. Distal weakness greater than proximal weakness was present in three patients. Except for minor differences, the morphologic features were similar to those in other patients with MFM. CONCLUSIONS: 1) Mutations in myotilin cause MFM; 2) exon 2 of MYOT is a hotspot for mutations; 3) peripheral neuropathy, cardiomyopathy, and distal weakness greater than proximal weakness are part of the spectrum of myotilinopathy; 4) not all cases of myotilinopathy have a limb-girdle phenotype; and 5) the molecular basis of the majority of MFM cases remains to be discovered.

Age of Onset↗
PubMed2004

Myofibrillar myopathy: clinical, morphological and genetic studies in 63 patients.

The term myofibrillar myopathy (MFM) was proposed in 1996 as a non-committal term for a pathological pattern of myofibrillar dissolution associated with accumulation of myofibrillar degradation products and ectopic expression of multiple proteins that include desmin, alphaB-crystallin (alphaBC), dystrophin and congophilic amyloid material. Subsequent studies revealed dominant mutations in desmin and alphaBC in some MFM patients, and clinical differences between kinships. We here review the clinical, structural and genetic features of 63 unrelated patients diagnosed as having MFM at the Mayo Clinic between 1977 and 2003. The age of onset was 54 +/- 16 years (mean +/- SD). Weakness was both proximal and distal in 77% and proximal only in 13%. Cardiomyopathy was diagnosed in 16%. Electro myography revealed a myopathic pattern associated with abnormal electrical irritability; 13 patients had abnormal nerve conduction studies but four of these had long-standing diabetes. The abnormal muscle fibres are best identified in trichrome-stained sections as harbouring amorphous, granular or pleomorphic hyaline structures, and vacuoles containing membranous material. The hyaline structures are strongly congophilic. Semiquantitative analysis in each case indicates that among the abnormal fibres, an average of 90, 75, 75, 70 and 70% abnormally express myotilin, desmin, alphaBC, dystrophin and beta-amyloid precursor protein, respectively. Therefore, immunostains for these proteins, and especially for myotilin, are useful adjuncts in the diagnosis of MFM. Electron microscopy shows progressive myofibrillar degeneration commencing at the Z-disk, accumulation of degraded filamentous material and entrapment of dislocated membranous organelles in autophagic vacuoles. In all patients, we searched for mutations in desmin and alphaBC, as well as in telethonin, a Z-disk-associated protein, or in syncoilin, which together with plectin links desmin to the Z-disk. Two of the 63 patients carry truncation mutations in the C-terminal domain of alphaBC, four carry missense mutations in the head or tail region of desmin, and none carries a mutation in syncoilin or telethonin. Thus, MFM is morphologically distinct but genetically heterogeneous. Further advances in defining the molecular causes of MFM will probably come from linkage studies of informative kinships or from systematic search for mutations in proteins participating in the intricate network supporting the Z-disk.

Adolescent↗
PubMed2004

Novel truncating RAPSN mutations causing congenital myasthenic syndrome responsive to 3,4-diaminopyridine.

Rapsyn is essential for clustering the acetylcholine receptor at the postsynaptic membrane of the neuromuscular junction. Direct sequencing of RAPSN in two children with congenital myasthenic syndromes with no mutation in any of the AChR subunits identified two heterozygous recessive mutations in each: a previously characterized N88K mutation in both, and a second frameshifting mutation in Patient (Pt) 1 and a nonsense mutation in Pt 2. An intercostal muscle biopsy in Pt 1 revealed decreased AChRs per endplate and decreased amplitude of the miniature endplate potential, predicted consequences of rapsyn deficiency. Clinically, both children manifested with hypomotility in utero, fatigable ocular and limb weakness since birth, decreased strength during viral illness, decremental response on electromyography, and absence of AChR antibodies. Pt 1, however, had a more severe clinical course with recurrent episodes of respiratory failure, contractures, and craniofacial malformations. In both patients, treatment with pyridostigmine was of some benefit, but the addition of 3,4-diaminopyridine led to significant clinical improvement. Thus, rapsyn deficiency predicting similar consequences at the cellular level can result in phenotypes with marked differences in severity of symptoms, risk of respiratory failure, and presence of contractures and craniofacial malformations.

4-Aminopyridine↗
PubMed2003

C-terminal and heparin-binding domains of collagenic tail subunit are both essential for anchoring acetylcholinesterase at the synapse.

The collagen-tailed form of acetylcholinesterase (A(12)-AChE) appears to be localized at the neuromuscular junction in association with the transmembrane dystroglycan complex through binding of its collagenic tail (ColQ) to the proteoglycan perlecan. The heparan sulfate binding domains (HSBD) of ColQ are thought to be involved in anchoring ColQ to the synaptic basal lamina. The C-terminal domain (CTD) of ColQ is also likely involved, but there has been no direct evidence. Mutations in COLQ cause endplate AChE deficiency in humans. Nine previously reported and three novel mutations are in CTD of ColQ, and most CTD mutations do not abrogate formation of A(12)-AChE in transfected COS cells. Patient endplates, however, are devoid of AChE, suggesting that CTD mutations affect anchoring of ColQ to the synaptic basal lamina. Based on our observations that purified AChE can be transplanted to the heterologous frog neuromuscular junction, we tested insertion competence of nine naturally occurring CTD mutants and two artificial HSBD mutants. Wild-type human A(12)-AChE inserted into the frog neuromuscular junction, whereas six CTD mutants and two HSBD mutants did not. Our studies establish that the CTD mutations indeed compromise anchoring of ColQ and that both HSBD and CTD are essential for anchoring ColQ to the synaptic basal lamina.

Acetylcholinesterase↗
PubMed2003

A frameshifting mutation in CHRNE unmasks skipping of the preceding exon.

A frameshifting 7 bp deletion (epsilon553del7) in exon 7 of CHRNE encoding the acetylcholine receptor epsilon subunit, observed in seven congenital myasthenic syndrome patients, enhances expression of an aberrantly spliced transcript that skips the preceding 101 bp exon 6. To recapitulate the aberrant splicing, we cloned the entire CHRNE spanning 12 exons and 11 introns and expressed it in COS cells. Scanning mutagenesis revealed that epsilon553del7 does not disrupt an exonic splicing enhancer. Inhibition of protein synthesis and of nonsense-mediated mRNA decay (NMD) by anisomycin shows that even wild-type CHRNE produces an exon 6-skipped transcript, and that even epsilon553del7-CHRNE yields a normally spliced transcript. Both transcripts, however, are degraded by NMD due to a premature stop codon. In contrast, the normally spliced transcript from wild-type CHRNE and the exon 6-skipped transcript from epsilon553del7-CHRNE carry no premature stop codon and hence are immune to NMD. Optimization of splicing signals for exon 6 prevents it being skipped even in the presence of anisomycin and/or epsilon553del7, indicating that inherently weak splicing signals for exon 6 account for its skipping. We suggest that a similar mechanism probably operates in other genes in skipping of remote exons. The presence of weak splicing signals for exon 6 also prompted us to search for mutations in exon 6 that disrupt an exonic splicing enhancer. Indeed, we found that epsilonEF157V and epsilonE154X in exon 6, observed in two other patients, caused aberrant splicing of exon 6.

Adolescent↗
PubMed2003

Treatment of slow-channel congenital myasthenic syndrome with fluoxetine.

The authors found that fluoxetine significantly shortens at 5 microM/L and nearly normalizes at 10 microM/L the prolonged opening bursts of slow-channel congenital myasthenic syndrome (SCCMS) acetylcholine receptors (AChR) expressed in fibroblasts. Prompted by this observation, they treated two SCCMS patients allergic to quinidine with up to 80 to 120 mg of fluoxetine per day over 3 years (serum fluoxetine + norfluoxetine levels 8 to 11 microM/L). Both patients showed marked subjective and objective improvement by quantitative muscle strength testing and electromyography.

Adult↗
PubMed2003

Myasthenic syndrome caused by mutation of the SCN4A sodium channel.

In a myasthenic syndrome associated with fatigable generalized weakness and recurrent attacks of respiratory and bulbar paralysis since birth, nerve stimulation at physiologic rates rapidly decremented the compound muscle action potential. Intercostal muscle studies revealed no abnormality of the resting membrane potential, evoked quantal release, synaptic potentials, acetylcholine receptor channel kinetics, or endplate ultrastructure, but endplate potentials depolarizing the resting potential to -40 mV failed to excite action potentials. Pursuing this clue, we sequenced SCN4A encoding the skeletal muscle sodium channel (Nav1.4) and detected two heteroallelic mutations involving conserved residues not present in 400 normal alleles: S246L in the S4/S5 cytoplasmic linker in domain I, and V1442E in the S3/S4 extracellular linker in domain IV. The genetically engineered V1442E-Na channel expressed in HEK cells shows marked enhancement of fast inactivation close to the resting potential, and enhanced use-dependent inactivation on high-frequency stimulation; S246L is likely a benign polymorphism. The V1442E mutation in SCN4A defines a novel disease mechanism and a novel phenotype with myasthenic features.

Adult↗
PubMed2003

E-box mutations in the RAPSN promoter region in eight cases with congenital myasthenic syndrome.

Myogenic determination factors are basic helix-loop-helix proteins that govern specification and differentiation of muscle cells, and bind to the E-box consensus sequence CANNTG in promoter regions of muscle-specific genes. No E-box mutation has been reported to date. RAPSN encodes rapsyn, a 43 kDa postsynaptic peripheral membrane protein that clusters the nicotinic acetylcholine receptor at the motor endplate. Transcriptional regulation mechanisms of RAPSN have not been studied. We here report two novel E-box mutations in the RAPSN promoter region in eight congenital myasthenic syndrome patients. Patient 1 carries -27C-->G that changes an E-box at -27 to -22 from CAGCTG to GAGCTG. An allele harboring -27C-->G is not transcribed in patient's muscle. Patients 2-8 are of Oriental Jewish stock of Iraqi or Iranian origin with facial malformations, and harbor -38A-->G that changes another E-box at -40 to -35 from CAACTG to CAGCTG, which does not affect the consensus CANNTG sequence. Haplotype analysis shows that -38A-->G arises from a common founder. For each mutation, position +1 represents the major transcriptional start site that we determine to be 172 nucleotides upstream of the translational start site. Electrophoretic mobility shift assays reveal that -38A-->G gains, and -27C-->G looses, binding affinity for different components of nuclear extracts of C2C12 myotubes. Luciferase reporter assays show that both -38A-->G and -27C-->G attenuate reporter gene expression in C2C12 myotubes, and that -27C-->G additionally attenuates reporter gene expression in MyoD- or myogenin-transfected HEK cells. The -27C-->G mutation also markedly attenuates the enhancer activity of an E-box on an SV40 promoter. Impaired transcriptional activities of the RAPSN promoter region predict reduced rapsyn expression and endplate acetylcholine receptor deficiency.

Adolescent↗
PubMed2003

Myofibrillar myopathy caused by novel dominant negative alpha B-crystallin mutations.

We here report the second and third mutations in alphaB-crystallin causing myofibrillar myopathy. Two patients had adult-onset muscle weakness. Patient 1 had cervical, limb girdle, and respiratory muscle weakness and died of respiratory failure. Patient 2 had proximal and distal leg muscle weakness. Both had myopathic electromyogram with abnormal electrical irritability and muscle biopsy findings of myofibrillar myopathy and mild denervation. Myofibrillar disintegration begins at the Z-disk and results in abnormal local expression of desmin, alphaB-crystallin, dystrophin, neural cell adhesion molecule (NCAM), and CDC2 kinase. Seven to 8% of nuclei display early apoptotic changes. Both patients carry a truncating mutation in the C-terminal region of alphaB-crystallin (464delCT in Patient 1 and Q151X in Patient 2) which is crucial for the solubilization and chaperone functions of the molecule. cDNA analysis shows the same mutations and no alternatively spliced transcripts. Immunoblots of muscle demonstrate increased expression of wild-type and reduced expression of the mutant protein. Immunoblots under nondenaturing conditions show that the mutant protein forms lower than normal molecular weight multimeric complexes with wild type. We conclude that (1) despite its reduced expression, the mutant protein exerts a dominant negative effect; (2) mutations in alphaB-crystallin are an infrequent cause of myofibrillar myopathy; (3) alphaB-crystallin-related myopathies display phenotypic heterogeneity.

Base Sequence↗
PubMed2003

Congenital myasthenic syndromes: progress over the past decade.

Congenital myasthenic syndromes (CMS) stem from defects in presynaptic, synaptic basal lamina, and postsynaptic proteins. The presynaptic CMS are associated with defects that curtail the evoked release of acetylcholine (ACh) quanta or ACh resynthesis. Defects in ACh resynthesis have now been traced to mutations in choline acetyltransferase. A basal lamina CMS is caused by mutations in the collagenic tail subunit (ColQ) of the endplate species of acetylcholinesterase that prevent the tail subunit from associating with catalytic subunits or from becoming inserted into the synaptic basal lamina. Most postsynaptic CMS are caused by mutations in subunits of the acetylcholine receptor (AChR) that alter the kinetic properties or decrease the expression of AChR. The kinetic mutations increase or decrease the synaptic response to ACh and result in slow- and fast-channel syndromes, respectively. Most low-expressor mutations reside in the AChR epsilon subunit and are partially compensated by residual expression of the fetal type gamma subunit. In a subset of CMS patients, endplate AChR deficiency is caused by mutations in rapsyn, a molecule that plays a critical role in concentrating AChR in the postsynaptic membrane.

Humans↗
PubMed2003

Mutation causing severe myasthenia reveals functional asymmetry of AChR signature cystine loops in agonist binding and gating.

We describe a highly disabling congenital myasthenic syndrome (CMS) associated with rapidly decaying, low-amplitude synaptic currents, and trace its cause to a valine to leucine mutation in the signature cystine loop (cys-loop) of the AChR alpha subunit. The recently solved crystal structure of an ACh-binding protein places the cys-loop at the junction between the extracellular ligand-binding and transmembrane domains where it may couple agonist binding to channel gating. We therefore analyzed the kinetics of ACh-induced single-channel currents to identify elementary steps in the receptor activation mechanism altered by the alphaV132L mutation. The analysis reveals that alphaV132L markedly impairs ACh binding to receptors in the resting closed state, decreasing binding affinity for the second binding step 30-fold, but attenuates gating efficiency only about twofold. By contrast, mutation of the equivalent valine residue in the delta subunit impairs channel gating approximately fourfold with little effect on ACh binding, while corresponding mutations in the beta and epsilon subunits are without effect. The unique functional contribution of the alpha subunit cys-loop likely owes to its direct connection via a beta strand to alphaW149 at the center of the ligand-binding domain. The overall findings reveal functional asymmetry between cys-loops of the different AChR subunits in contributing to ACh binding and channel gating.

Acetylcholine↗
PubMed2003

Mechanistic diversity underlying fast channel congenital myasthenic syndromes.

A host of missense mutations in muscle nicotinic receptor subunits have been identified as the cause of congenital myasthenic syndromes (CMS). Two classes of CMS phenotypes have been identified: slow channel myasthenic syndromes (SCCMSs) and fast channel myasthenic syndromes (FCCMSs). Although both have similar phenotypic consequences, they are physiologic opposites. Expression of the FCCMS phenotype requires the missense mutation to be accompanied by a second mutation, either a null or a missense mutation, in the second allele encoding the same receptor subunit. This seemingly rare scenario has arisen with surprisingly high incidence over the past few years, and analyses of the syndromes have revealed a diverse array of mechanisms underlying the pathology. This review focuses on new mechanisms underlying the FCCMS.

Animals↗