Search PubMed⌕ Search

Biomedical subjects

Andrew G Engel

Publications and source records attributed to Andrew G Engel.

At least 19 recordsLinked to original sources
PubMed2006

Reconstitution of paired T cell receptor alpha- and beta-chains from microdissected single cells of human inflammatory tissues.

We describe a strategy to "revive" putatively pathogenic T cells from frozen specimens of human inflammatory target organs. To distinguish pathogenic from irrelevant bystander T cells, we focused on cells that were (i) clonally expanded and (ii) in direct morphological contact with a target cell. Using CDR3 spectratyping, we identified clonally expanded T cell receptor (TCR) beta-chains in muscle sections of patients with inflammatory muscle diseases. By immunohistochemistry, we identified those Vbeta-positive T cells that fulfilled the morphological criteria of myocytotoxicity and isolated them by laser microdissection. Next, we identified coexpressed pairs of TCR alpha- and beta-chains by a multiplex PCR protocol, which allows the concomitant amplification of both chains from single cells. This concomitant amplification had not been achieved previously in histological sections, mainly because of the paucity of available anti-alpha-chain antibodies and the great heterogeneity of the alpha-chain genes. From muscle tissue of a patient with polymyositis, we isolated 64 T cells that expressed an expanded Vbeta1 chain. In 23 of these cells, we identified the corresponding alpha-chain. Twenty of these 23 alpha-chains were identical, suggesting antigen-driven selection. After functional reconstitution of the alphabeta-pairs, their antigen-recognition properties could be studied. Our results open avenues for combined analysis of the full TCR alpha- and beta-chain repertoire in human inflammatory tissues.

Autoimmunity↗
PubMed2006

Recent advances in Cys-loop receptor structure and function.

Throughout the nervous system, moment-to-moment communication relies on postsynaptic receptors to detect neurotransmitters and change the membrane potential. For the Cys-loop superfamily of receptors, recent structural data have catalysed a leap in our understanding of the three steps of chemical-to-electrical transduction: neurotransmitter binding, communication between the binding site and the barrier to ions, and opening and closing of the barrier. The emerging insights might be expected to explain how mutations of receptors cause neurological disease, but the opposite is generally true. Namely, analyses of disease-causing mutations have clarified receptor structure-function relationships as well as mechanisms governing the postsynaptic response.

Acetylcholine↗
PubMed2006

The human adult subtype ACh receptor channel has high Ca2+ permeability and predisposes to endplate Ca2+ overloading.

Slow-channel congenital myasthenic syndrome, caused by mutations in subunits of the endplate ACh receptor (AChR), results in prolonged synaptic currents and excitotoxic injury of the postsynaptic region by Ca2+ overloading. The Ca2+ overloading could be due entirely to the prolonged openings of the AChR channel or could be abetted by enhanced Ca2+ permeability of the mutant channels. We therefore measured the fractional Ca2+ current, defined as the percentage of the total ACh-evoked current carried by Ca2+ ions (Pf), for AChRs harbouring the alphaG153S or the alphaV249F slow-channel mutation, and for wild-type human AChRs in which Pf has not yet been determined. Experiments were performed in transiently transfected GH4C1 cells and human myotubes with simultaneous recording of ACh-evoked whole-cell currents and fura-2 fluorescence signals. We found that the Pf of the wild-type human endplate AChR was unexpectedly high (Pf approximately 7%), but neither the alphaV249F nor the alphaG153S mutation altered Pf. Fetal human AChRs containing either the wild-type or the mutated alpha subunit had a much lower Pf (2-3%). We conclude that the Ca2+ permeability of human endplate AChRs is higher than that reported for any other human nicotinic AChR, with the exception of alpha7-containing AChRs (Pf > 10%); and that neither the alphaG153S nor the alphaV249F mutations affect the Pf of fetal or adult endplate AChRs. However, the intrinsically high Ca2+ permeability of human AChRs probably predisposes to development of the endplate myopathy when opening events of the AChR channel are prolonged by altered AChR-channel kinetics.

Adult↗
PubMed2006

Slow-channel mutation in acetylcholine receptor alphaM4 domain and its efficient knockdown.

OBJECTIVE: To identify the genetic basis of a slow-channel myasthenic syndrome, characterize functional properties of the mutant receptor, and selectively silence the mutant allele. METHODS: We performed nutation analysis, cloning, and patch-clamp analysis of the functional properties of the mutant receptor; screening for a small interfering RNA with check plasmid; and assessed of the efficacy of small interfering RNA at the messenger RNA, protein, and functional levels. RESULTS: We traced the cause of a slow-channel myasthenic syndrome to a C418W mutation in the M4 domain of the acetylcholine receptor alpha subunit. The mutation is the first one to occur spontaneously in an M4 domain of the receptor, and it is positioned within a stripe of hydrophobic residues facing the lipid bilayer. Kinetic analysis shows that alphaC418W enhances the channel opening equilibrium constant 26-fold without altering agonist affinity. Using a check plasmid as a screening tool, we identified a small interfering RNA that markedly suppresses the mutant but not the wild-type allele at the messenger RNA, protein, and functional levels. INTERPRETATION: alphaC418W occurring in humans causes a slow-channel syndrome by enhancing the relative stability of the channel open state. Efficient and selective knockdown of the mutant allele holds promise of therapeutic gene silencing.

Adult↗
PubMed2005

IBM-type inclusions in a patient with slow-channel syndrome caused by a mutation in the AChR epsilon subunit.

We report a patient with a slow-channel congenital myasthenic syndrome who carries a novel slow-channel mutation in the epsilon subunit of the acetylcholine receptor and has tubulofilamentous inclusion bodies, in skeletal muscle of the type observed in hereditary and sporadic inclusion body myositis. Ultrastructural analysis of a muscle specimen obtained at the age of 9 years showed an endplate myopathy typical of the slow-channel syndrome. Twenty years later, a second muscle specimen again showed the endplate myopathy as well numerous nuclear and cytoplasmic tubulofilamentous inclusion bodies. Molecular genetic studies revealed a novel valine to phenylalanine mutation (epsilonV259F) in the M2 domain of the acetylcholine receptor. Coexistence of the slow-channel syndrome with a feature of IBM has not been observed before.

Acetylcholine↗
PubMed2005

Sporadic late onset nemaline myopathy.

OBJECTIVE: To review the clinicopathologic features and outcome of sporadic late onset nemaline myopathy (SLONM). BACKGROUND: Non-HIV-related SLONM is an uncommon disease of undefined etiology. METHODS: This study is based on clinical, EMG, histochemical, immunocytochemical, and electron microscopy evaluation, and long-term follow-up of 14 patients observed at the Mayo Clinic between 1975 and 2003. RESULTS: The disease presented between 43 and 81 years and evolved subacutely. The weakness was predominantly proximal in 11, equal proximally and distally in 3, and asymmetric in 4; dysphagia was a symptom in 6. The EMG showed myopathic features with fibrillations but the serum CK level at the time of initial examination or reevaluation was normal or below the Mayo Clinic's range of normal values for sex and age at the time of the assay. Seven patients had an associated monoclonal gammopathy. On light microscopy, the nemaline structures were best identified in 3-mum-thick frozen sections stained trichromatically or immunostained for alpha-actinin or myotilin. Electron microscopy done in 12 cases identified the rods in all and revealed additional structural abnormalities. Seven patients with monoclonal gammopathy were followed for 1 to 5 years; five died of respiratory failure. Five patients without monoclonal gammopathy were followed for 4 to 23 years and none died of the disease. Immunotherapy in eight patients was of uncertain benefit. CONCLUSIONS: 1) Subacutely evolving weakness after age 40, normal to low CK level, myopathic EMG with fibrillations, and often a monoclonal gammopathy are clues for the diagnosis of sporadic late onset nemaline myopathy. 2) The diagnosis is confirmed by visualizing the rods in trichrome or immunostained cryosections. 3) An associated monoclonal gammopathy heralds an unfavorable prognosis.

Adult↗
PubMed2005

Mutations in ZASP define a novel form of muscular dystrophy in humans.

Myofibrillar myopathy (MFM) is a morphologically distinct disorder in which disintegration of the Z-disk and then of the myofibrils is followed by abnormal accumulation of multiple proteins. Mutations in desmin, alphaB-crystallin, and myotilin, all Z-disk-related proteins, cause MFM in the minority of cases. ZASP (a Z-band alternatively spliced PDZ motif-containing protein) is another Z-disk-associated protein, and targeted deletion of ZASP in mouse causes skeletal and cardiac myopathy. We therefore searched for mutations in ZASP in 54 MFM patients and detected 3 heterozygous missense mutations in 11. Their age at onset was 44 to 73 years. Dominant inheritance was apparent in seven patients, cardiac involvement in three, and signs of peripheral neuropathy in five. Most patients had proximal and distal weakness, but in six, the weakness was greater distally than proximally. Ten carried either of two mutations in exon 6 (A147T and A165V) at or within a motif important in linking ZASP to the Z-disk; one carried a missense mutation in exon 9 (R268C). We conclude that (1) mutations in ZASP cause stereotyped MFM pathology; (2) cardiomyopathy, distal more than proximal weakness, and neuropathy are in the spectrum of zaspopathy; and (3) mutations in ZASP define a novel form of autosomal dominant muscular dystrophy in humans.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Commonality of TRIM32 mutation in causing sarcotubular myopathy and LGMD2H.

Sarcotubular myopathy (OMIM 268950) is a rare autosomal recessive myopathy first described in two Hutterite brothers from South Dakota and in two non-Hutterite brothers from Germany. We report that sarcotubular myopathy (STM) is caused by mutation in TRIM32, the gene encoding the tripartite motif-containing protein 32. TRIM32 was found to be the gene mutated in limb girdle muscular dystrophy type 2H (LGMD2H [OMIM 254110]), a disorder that has been confined to the Hutterite population. The TRIM32 mutation found in the STM patients is identical to the causative mutation for LGMD2H (D487N), Haplotype analysis shows that the disease chromosomes share common ancestry.

Adult↗
PubMed2005

Current understanding of congenital myasthenic syndromes.

Investigation of congenital myasthenic syndromes (CMSs) disclosed a diverse array of molecular targets at the motor endplate. Clinical, electrophysiologic and morphologic studies paved the way for detecting CMS-related mutations in proteins such as the acetylcholine receptor, acetylcholinesterase, choline acetyltransferase, rapsyn, MuSK and Na(v)1.4. Analysis of electrophysiologic and biochemical properties of mutant proteins expressed in heterologous systems contributed crucially to defining the molecular consequences of the observed mutations and resulted in improved therapy of different CMSs. Recent crystallography studies of choline acetyltransferase and homology structural models of the acetylcholine receptor are providing further clues to how point mutations alter protein function.

Acetylcholinesterase↗
PubMed2005

Splicing abnormalities in congenital myasthenic syndromes.

A total of 173 mutations has been reported to date in eight genes in congenital myasthenic syndromes. Sixteen intronic and five exonic mutations in three genes affect pre-mRNA splicing. Eight of these are of particular interest, and are reviewed in this article. An A-to-G mutation at intron position +3 results in exon skipping only when there are mismatched nucleotides to U1 snRNA at positions +4 to +6. Similarly, a mutation at the last nucleotide of an exon causes exon skipping when a nucleotide at position +6 is not complementary to U1 snRNA. We observe the similar compensation mechanisms for mismatches to U1 snRNA at 179,917 native human splice donor sites. A 7-bp deletion in CHRNE exon 7 causes skipping of the preceding 101-bp exon 6. We found in general that the nonsense-mediated altered splicing of a remote exon (NASRE) is mediated by inherent weak splicing signals flanking the skipped exon and degradation of a normally spliced transcript by the nonsense-mediated mRNA decay (NMD). A 16-bp duplication spanning the CHRNE intron 10/exon 11 boundary generates two copies of 3' splice sites, and the downstream copy is exclusively silenced. Analysis of a series of artificial mutants conforms to the scanning model of recognition of the 3' splice site that predicts that the first 'ag' more than 13 nucleotides downstream of the branch point is selected for splicing. Splicing mutations may be more frequent than suspected, and one must always be aware of possible splicing abnormalities when analyzing human mutations.

DNA Mutational Analysis↗
PubMed2004

Subunit-specific contribution to agonist binding and channel gating revealed by inherited mutation in muscle acetylcholine receptor M3-M4 linker.

We trace the cause of congenital myasthenic syndromes in two patients to mutations in the epsilon subunit of the muscle acetylcholine receptor (AChR). Both patients harbour deletion of an asparagine residue in the epsilon subunit (epsilonN436del) at the C-terminus of the cytoplasmic loop linking the third (M3) and fourth (M4) transmembrane domains. The presence of a null mutation in the second allele of the epsilon subunit shows that epsilonN346del determines the phenotype. Endplate studies show markedly reduced expression of the epsilonN346del-AChR and compensatory accumulation of fetal gamma-AChR. Expression studies in HEK cells reveal decreased expression of epsilonN436del-AChR and abnormally brief channel openings. Thus, neuromuscular transmission is compromised by AChR deficiency, fast channel kinetics of the epsilonN346del-AChR and incomplete phenotypic rescue by gamma-AChR. Single-channel kinetic analysis shows that the epsilonN436del shortens channel openings by reducing stability of the diliganded receptor: rates of channel closing and of ACh dissociation are increased and the rate of channel opening is decreased. In addition to shortening the M3-M4 loop, epsilonN436del shifts a negatively charged aspartic acid residue adjacent to M4; the effects of epsilonN436del are shown to result from shortening of the M3-M4 loop and not from juxtaposition of a negative charge to M4. To determine whether the consequences of epsilonN346del are subunit-specific, we deleted residues that align with epsilonN436 in beta, delta and alpha subunits. Each deletion mutant reduces AChR expression, but whereas the beta and delta mutants curtail channel open duration, the alpha mutant strikingly prolongs open duration. Kinetic analysis reveals that the alpha mutant increases the stability of the diliganded receptor: rates of channel closing and of ACh dissociation are decreased and the rate of channel opening is increased. The overall studies reveal subunit asymmetry in the contributions of the M3-M4 loops in optimizing AChR activation through allosteric links to the channel and the agonist binding site.

Adult↗
PubMed2004

Structural abnormalities at neuromuscular synapses lacking multiple syntrophin isoforms.

The syntrophins are modular adapter proteins that function by recruiting signaling molecules to the cytoskeleton via their direct association with proteins of the dystrophin protein family. We investigated the physiological function of beta2-syntrophin by generating a line of mice lacking this syntrophin isoform. The beta2-syntrophin null mice show no overt phenotype, or muscular dystrophy, and form structurally normal neuromuscular junctions (NMJs). To determine whether physiological consequences caused by the lack of beta2-syntrophin were masked by compensation from the alpha-syntrophin isoform, we crossed these mice with our previously described alpha-syntrophin null mice to produce mice lacking both isoforms. The alpha/beta2-syntrophin null mice have NMJs that are structurally more aberrant than those lacking only alpha-syntrophin. The NMJs of the alpha/beta2-syntrophin null mice have fewer junctional folds than either parent strain, and the remaining folds are abnormally shaped with few openings to the synaptic space. The levels of acetylcholine receptors are reduced to 23% of wild type in mice lacking both syntrophin isoforms. Furthermore, the alpha/beta2-syntrophin null mice ran significantly shorter distances on voluntary exercise wheels despite having normal neuromuscular junction transmission as determined by micro-electrode recording of endplate potentials. We conclude that both alpha-syntrophin and beta2-syntrophin play distinct roles in forming and maintaining NMJ structure and that each syntrophin can partially compensate for the loss of the other.

Animals↗