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author:("selden, Duygu")
1.  DPAGT1 myasthenia and myopathy 
Neurology  2014;82(20):1822-1830.
To investigate patients with DPAGT1 (UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase 1)-associated myasthenic syndrome.
We performed exome and Sanger sequencing, determined glycoprotein expression in patient muscles, assessed pathogenicity of the mutant proteins by examining their expression and enzymatic activity in transfected cells, evaluated structural changes in muscle and the neuromuscular junction, and examined electrophysiologic aspects of neuromuscular transmission in vitro.
Patients 1 and 2, 16 and 14 years of age, had progressive fatigable weakness since infancy and are intellectually disabled. Patient 3, a less severely affected brother of patient 1, also has autistic features. Each patient harbors 2 novel heteroallelic mutations in DPAGT1, an enzyme subserving protein N-glycosylation. Patients 1 and 3 harbor Met1Leu, which reduces protein expression, and His375Tyr, which decreases enzyme activity. Patient 2 carries Val264Met, which abolishes enzyme activity, and a synonymous Leu120Leu mutation that markedly augments exon skipping, resulting in some skipped and infrequent nonskipped alleles. Therefore, the nonskipped allele rescues the phenotype. Intracellular microelectrode studies indicate combined pre- and postsynaptic defects of neuromuscular transmission with evidence for somatic mosaicism in patient 2. Structural studies reveal hypoplastic endplates, fiber-type disproportion, tubular aggregates, and degeneration of muscle fiber organelles resulting in autophagocytosis.
DPAGT1 myasthenia affects multiple parameters of neuromuscular transmission, causes fiber-type disproportion and an autophagic myopathy, and can be associated with intellectual disability. We speculate that hypoglycosylation of synapse-specific proteins causes defects in central as well as motor synapses.
PMCID: PMC4035711  PMID: 24759841
2.  PREPL deficiency with or without cystinuria causes a novel myasthenic syndrome 
Neurology  2014;82(14):1254-1260.
To investigate the genetic and physiologic basis of the neuromuscular symptoms of hypotonia-cystinuria syndrome (HCS) and isolated PREPL deficiency, and their response to therapy.
We performed molecular genetic, histochemical, immunoblot, and ultrastructural studies, investigated neuromuscular transmission in vitro in a patient with isolated PREPL deficiency, and evaluated the effect of pyridostigmine in this patient and in 3 patients with the HCS.
HCS is caused by recessive deletions involving the SLC3A1 and PREPL genes. The major clinical features of HCS are type A cystinuria, growth hormone deficiency, muscle weakness, ptosis, and feeding problems. The proband with isolated PREPL deficiency had myasthenic symptoms since birth and a positive edrophonium test but no cystinuria. She and 1 of 3 patients with HCS responded transiently to pyridostigmine during infancy. The proband harbors a paternally inherited nonsense mutation in PREPL and a maternally inherited deletion involving both PREPL and SLC3A1; therefore, the PREPL deficiency determines the phenotype. We detected no PREPL expression in the patient's muscle and endplates. Electrophysiology studies revealed decreased quantal content of the endplate potential and reduced amplitude of the miniature endplate potential without endplate acetylcholine receptor deficiency or altered endplate geometry.
Isolated PREPL deficiency is a novel monogenic disorder that causes a congenital myasthenic syndrome with pre- and postsynaptic features and growth hormone deficiency. The myasthenic symptoms in PREPL deficiency with or without cystinuria may respond to pyridostigmine in early life. We attribute the myasthenia to abrogated interaction of PREPL with adaptor protein 1.
PMCID: PMC4001208  PMID: 24610330
Current opinion in neurology  2008;21(5):585-589.
Purpose of review
The aim of this communication is to provide an up-to-date overview of myofibrillar myopathies (MFMs).
Recent findings
The most important recent advance in the MFMs has been the identification of mutation Bag3 (Bcl-2-associated athanogene-3) as a new cause of MFM. Although, the typical clinical manifestations of MFMs are slowly progressive weakness, the patients with Bag3opathy may have had a rapidly progressive and more severe phenotype.
Several MFM disease genes have recently been recognized. The identified disease proteins (desmin, αB-crystallin, myotilin, Zasp, filamin C, and Bag3) interact with components or with chaperones of the Z-disk. In each case the molecular defect leads to a largely stereotyped cascade of structural perturbation of the muscle fiber architecture.
PMCID: PMC4151125  PMID: 18769253
Myofibrillar myopathy; desmin; αB-crystallin; myotilin; Zasp; filamin C; Bag3; FHL1
4.  GFPT1-myasthenia 
Neurology  2013;81(4):370-378.
To identify patients with GFPT1-related limb-girdle myasthenia and analyze phenotypic consequences of the mutations.
We performed genetic analysis, histochemical, immunoblot, and ultrastructural studies and in vitro electrophysiologic analysis of neuromuscular transmission.
We identified 16 recessive mutations in GFPT1 in 11 patients, of which 12 are novel. Ten patients had slowly progressive limb-girdle weakness responsive to cholinergic agonists with onset between infancy and age 19 years. One patient (no. 6) harbored a nonsense mutation and a second mutation that disrupts the muscle-specific GFPT1 exon. This patient never moved in utero, was apneic and arthrogrypotic at birth, and was bedfast, tube-fed, and barely responded to therapy at age 6 years. Histochemical studies in 9 of 11 patients showed tubular aggregates in 6 and rimmed vacuoles in 3. Microelectrode studies of intercostal muscle endplates in 5 patients indicated reduced synaptic response to acetylcholine in 3 and severely reduced quantal release in patient 6. Endplate acetylcholine receptor content was moderately reduced in only one patient. The synaptic contacts were small and single or grape-like, and quantitative electron microscopy revealed hypoplastic endplate regions. Numerous muscle fibers of patient 6 contained myriad dilated and degenerate vesicular profiles, autophagic vacuoles, and bizarre apoptotic nuclei. Glycoprotein expression in muscle was absent in patient 6 and reduced in 5 others.
GFPT1-myasthenia is more heterogeneous than previously reported. Different parameters of neuromuscular transmission are variably affected. When disruption of muscle-specific isoform determines the phenotype, this has devastating clinical, pathologic, and biochemical consequences.
PMCID: PMC3772836  PMID: 23794683
5.  New horizons for congenital myasthenic syndromes 
During the past 5 years an increasing number of patients were diagnosed with congenital myasthenic syndromes (CMS) and a number of novel syndromes were recognized and investigated. This presentation focuses on the CMS caused by defects in choline acetyltransferase, novel fast-channel syndromes that hinder isomerization of the acetylcholine receptor from the closed to the open state, the consequences of deleterious mutations in the intermediate filament linker plectin, altered neuromuscular transmission in a centronuclear myopathy, and two recently identified CMS caused by congenital defects in glycosylation.
PMCID: PMC3546605  PMID: 23278578
congenital myasthenic syndromes; acetylcholine receptor; fast-channel syndromes; choline acetyltransferase; plectin; centronuclear myopathy; GFPT1; DPAGT1
6.  Reducing bodies and myofibrillar myopathy features in FHL1 muscular dystrophy 
Neurology  2011;77(22):1951-1959.
Some pathologic features of the FHL1 myopathies and the myofibrillar myopathies (MFMs) overlap; we therefore searched for mutations in FHL1 in our cohort of 50 patients with genetically undiagnosed MFM.
Mutations in FHL1 were identified by direct sequencing. Polymorphisms were excluded by using allele-specific PCR in 200 control subjects. Structural changes in muscle were analyzed by histochemistry, immunocytochemistry, and electron microscopy.
We detected 2 novel and 1 previously identified missense mutation in 5 patients. Patients 1–4 presented before age 30, display menadione–nitro blue tetrazolium–positive reducing bodies, and harbor mutations in the FHL1 LIM2 domain. Patient 5 presented at age 75 and has no reducing bodies, and his mutation is not in a LIM domain. The clinical features include progressive muscle weakness, hypertrophied muscles, rigid spine, and joint contractures, and 1 patient also has peripheral neuropathy. High-resolution electron microscopy reveals the reducing bodies composed of 13-nm tubulofilaments initially emanating from Z-disks. At a more advanced stage, abundant reducing bodies appear in the cytoplasm and nuclei with concomitant myofibrillar disintegration, accumulation of cytoplasmic degradation products, and aggregation of endoplasmic reticulum and sarcotubular profiles.
FHL1 dystrophies can be associated with MFM pathology. Mutations in the LIM2 domain are associated with reducing bodies composed of distinct tubulofilaments. A mutation extraneous to LIM domains resulted in a mild late-onset phenotype with MFM pathology but no reducing bodies.
PMCID: PMC3235356  PMID: 22094483
Muscle & nerve  2011;44(5):789-794.
Congenital myasthenic syndromes (CMS) are disabling but treatable disorders. Anticholinesterase therapy is effective in most, but is contraindicated in endplate (EP) acetylcholinesterase (AChE) deficiency, the slow-channel syndrome, Dok-7 myasthenia, β2-laminin deficiency, and is not useful in CMS due to defects in MuSK, agrin, and plectin. EP AChE, Dok-7 and β2-laminin deficiencies respond favorably to ephedrine but ephedrine can no longer be prescribed in the US.
We used albuterol, another sympathomimetic agent, to treat three patients with EP AChE deficiency and 15 with Dok-7 myasthenia. Response to therapy was evaluated by a 9-point questionnaire pertaining to activities of daily life.
Comparison of the pre- and post-treatment responses indicated a beneficial response to albuterol (p values <0.001) in both patient groups. The adverse effects of therapy were like those of ephedrine.
Our observations should spur controlled prospective clinical trials of albuterol in these as well as other CMS.
PMCID: PMC3196786  PMID: 21952943
Congenital myasthenic syndrome; Dok-7 myasthenia; Endplate AChE deficiency; Albuterol
8.  Further Observations in Congenital Myasthenic Syndromes 
During the past five years many patients suffering from congenital myasthenic syndromes (CMS) have been identified worldwide and novel causative genes and mutations have been discovered. The disease genes now include those encoding each subunit of the acetylcholine receptor (AChR), the ColQ part of acetylcholinesterase (AChE), choline acetyltransferase, Nav 1.4, MuSK, and Dok-7. Moreover, emerging genotype-phenotype correlations are providing clues for targeted mutation analysis. This review focuses on the recent observations in selected CMS.
PMCID: PMC3478107  PMID: 18567859
congenital myasthenic syndromes; acetylcholinesterase; choline acetyltransferase; acetylcholine receptor; Dok-7
9.  Infantile Muscular Dystrophy in Canadian Aboriginals is an αB-Crystallinopathy 
Annals of neurology  2011;69(5):866-871.
A recessively transmitted fatal hypertonic infantile muscular dystrophy has been described in Canadian Aboriginals. The affected infants present with progressive limb and axial muscle stiffness, develop severe respiratory insufficiency, and most die in the first year of life. We sought to determine the genetic basis of this disease.
We performed histochemical, immunocytochemical, electron microscopy and molecular genetic studies in a cohort of 12 patients affected by this disease.
Conventional histochemical and electron microscopy studies suggested myofibrillar myopathy (MFM). Therefore we searched for ectopic expression of multiple proteins typical of MFM. αB-crystallin (αBC) expression was absent from all fibers using a monoclonal antibody raised against the entire protein. However, a monoclonal antibody directed against the first 10 residues of αBC immunostained portions of abnormal fibers. Pursuing this clue, we searched for mutations in the gene for αBC (CRYAB) in available DNA samples of 8 patients. All harbor a homozygous deletion, c.60C, predicting a Ser to Ala change at codon 21 and a stop codon after 23 missense residues (p.Ser21AlafsX24). Clinically unaffected parents are heterozygous for this mutation.
The homozygous c.60delC in CRYAB pinpoints the genetic basis of the fatal infantile hypertonic muscular dystrophy of Canadian Aboriginals. MFMs are typically transmitted by dominant inheritance but in this disease the parental phenotype is rescued by limited expression of the highly truncated nonfunctional mutant gene product. The severe patient phenotype is due to homozygosity for the markedly hypomorphic allele.
PMCID: PMC3085857  PMID: 21337604
Neuromuscular disorders : NMD  2011;21(3):161-171.
Myofibrillar myopathies represent a group of muscular dystrophies with a similar morphologic phenotype. They are characterized by a distinct pathologic pattern of myofibrillar dissolution associated with disintegration of the Z-disk, accumulation of myofibrillar degradation products, and ectopic expression of multiple proteins and sometimes congophilic material. The clinical features of myofibrillar myopathies are more variable. These include progressive muscle weakness, that often involves or begins in distal muscles but limb-girdle or scapuloperoneal distributions can also occur. Cardiomyopathy and peripheral neuropathy are frequent associated features. EMG of the affected muscles reveals myopathic motor unit potentials and abnormal irritability often with myotonic discharges. Rarely, neurogenic motor unit potentials or slow nerve conductions are present. The generic diagnosis of myofibrillar myopathies is based on muscle biopsy findings in frozen sections. To date, all myofibrillar myopathy mutations have been traced to Z-disk associated proteins, namely, desmin, αB-crystallin, myotilin, ZASP, filamin C and Bag3. However, in the majority of the myofibrillar myopathy patients the disease gene awaits discovery.
PMCID: PMC3052736  PMID: 21256014
Myofibrillar myopathies; Z-disk; desmin; αB-crystallin; myotilin; Zasp; Filamin C; Bag3
11.  What Have We Learned from the Congenital Myasthenic Syndromes 
The congenital myasthenic syndromes have now been traced to an array of molecular targets at the neuromuscular junction encoded by no fewer than 11 disease genes. The disease genes were identified by the candidate gene approach, using clues derived from clinical, electrophysiological, cytochemical, and ultrastructural features. For example, electrophysiologic studies in patients suffering from sudden episodes of apnea pointed to a defect in acetylcholine resynthesis and CHAT as the candidate gene (Ohno et al., Proc Natl Acad Sci USA 98:2017–2022–2001); refractoriness to anticholinesterase medications and partial or complete absence of acetylcholinesterase (AChE) from the endplates (EPs) has pointed to one of the two genes (COLQ and ACHET) encoding AChE, though mutations were observed only in COLQ. After a series of patients carrying mutations in a disease gene have been identified, the emerging genotype–phenotype correlations provided clues for targeted mutation analysis in other patients. Mutations in EP-specific proteins also prompted expression studies that proved pathogenicity, highlighted important functional domains of the abnormal proteins, and pointed to rational therapy.
PMCID: PMC3050586  PMID: 19688192
Congenital myasthenic syndromes; Acetylcholine esterase; Choline acetyltransferase; Acetylcholine receptor; Dok-7
12.  Mutation in BAG3 Causes Severe Dominant Childhood Muscular Dystrophy 
Annals of neurology  2009;65(1):83-89.
Myofibrillar myopathies (MFM) are morphologically distinct but genetically heterogeneous muscular dystrophies in which disintegration of Z disks and then of myofibrils is followed by ectopic accumulation of multiple proteins. Cardiomyopathy, neuropathy, and dominant inheritance are frequent associated features. Mutations in αB-crystallin, desmin, myotilin, Zasp, or filamin-C can cause MFM, and were detected in 32/85 patients of the Mayo MFM cohort. Bag3, another Z-disk associated protein, has antiapoptotic properties and its targeted deletion in mice causes fulminant myopathy with early lethality. We therefore searched for mutations in BAG3 in 53 unrelated MFM patients.
We searched for mutations in BAG3 by direct sequencing and excluded polymorphism using allele-specific PCR in relatives and 200 control subjects. We analyzed structural changes in muscle by histochemistry, immunocytochemistry and electron microscopy, examined mobility of the mutant Bag3 by nondenaturing electrophoresis, and searched for abnormal aggregation of the mutant protein in COS-7 cells.
We identified a heterozygous p.Pro209Leu mutation in three patients. All presented in childhood, had progressive limb and axial muscle weakness, and developed cardiomyopathy and severe respiratory insufficiency in their teens; two had rigid spines and one a peripheral neuropathy. Electron microscopy showed disintegration of Z disks, extensive accumulation of granular debris and larger inclusions, and apoptosis of 8% of the nuclei. On nondenaturing electrophoresis of muscle extracts, the Bag3 complex migrated faster in patient than control extracts, and expression of FLAG-labeled mutant and wild-type Bag3 in COS cells revealed abnormal aggregation of the mutant protein.
We conclude mutation in Bag3 defines a novel severe autosomal dominant childhood muscular dystrophy.
PMCID: PMC2639628  PMID: 19085932
13.  Dok-7 Myasthenia: Phenotypic and Molecular Genetic Studies in 16 Patients 
Annals of neurology  2008;64(1):71-87.
Detailed analysis of phenotypic and molecular genetic aspects of Dok-7 myasthenia in 16 patients.
We assessed our patients by clinical and electromyographic studies, by intercostal muscle biopsies for in vitro microelectrode analysis of neuromuscular transmission and quantitative electron microscopy EM of 409 end plates (EPs), and by mutation analysis, and expression studies of the mutants.
The clinical spectrum varied from mild static limb-girdle weakness to severe generalized progressive disease. The synaptic contacts were single or multiple, and some, but not all, were small. In vitro microelectrode studies indicated variable decreases of the number of released quanta and of the synaptic response to acetylcholine; acetylcholine receptor (AChR) channel kinetics were normal. EM analysis demonstrated widespread and previously unrecognized destruction and remodeling of the EPs. Each patient carries 2 or more heteroallelic mutations: 11 in genomic DNA, 7 of which are novel; and 6 identifiable only in complementary DNA or cloned complementary DNA, 3 of which are novel. The pathogenicity of the mutations was confirmed by expression studies. Although the functions of Dok-7 include AChR β-subunit phosphorylation and maintaining AChR site density, patient EPs showed normal AChR β-subunit phosphorylation, and the AChR density on the remaining junctional folds appeared normal.
First, the clinical features of Dok-7 myasthenia are highly variable. Second, some mutations are complex and identifiable only in cloned complementary DNA. Third, Dok-7 is essential for maintaining not only the size but also the structural integrity of the EP. Fourth, the profound structural alterations at the EPs likely contribute importantly to the reduced safety margin of neuromuscular transmission.
PMCID: PMC2570015  PMID: 18626973

Results 1-13 (13)