Sarah Finlayson

Mutations in DPAGT1 cause a limb-girdle congenital myasthenic syndrome with tubular aggregates.

Congenital myasthenic syndromes are a heterogeneous group of inherited disorders that arise from impaired signal transmission at the neuromuscular synapse. They are characterized by fatigable muscle weakness. We performed whole-exome sequencing to determine the underlying defect in a group of individuals with an inherited limb-girdle pattern of myasthenic weakness. We identify DPAGT1 as a gene in which mutations cause a congenital myasthenic syndrome. We describe seven different mutations found in five individuals with DPAGT1 mutations. The affected individuals share a number of common clinical features, including involvement of proximal limb muscles, response to treatment with cholinesterase inhibitors and 3,4-diaminopyridine, and the presence of tubular aggregates in muscle biopsies. Analyses of motor endplates from two of the individuals demonstrate a severe reduction of endplate acetylcholine receptors. DPAGT1 is an essential enzyme catalyzing the first committed step of N-linked protein glycosylation. Our findings underscore the importance of N-linked protein glycosylation for proper functioning of the neuromuscular junction. Using the DPAGT1-specific inhibitor tunicamycin, we show that DPAGT1 is required for efficient glycosylation of acetylcholine-receptor subunits and for efficient export of acetylcholine receptors to the cell surface. We suggest that the primary pathogenic mechanism of DPAGT1 mutations is reduced levels of acetylcholine receptors at the endplate region. These individuals share clinical features similar to those of congenital myasthenic syndrome due to GFPT1 mutations, and their disorder might be part of a larger subgroup comprising the congenital myasthenic syndromes that result from defects in the N-linked glycosylation pathway and that manifest through impaired neuromuscular transmission.

Congenital myasthenic syndromes due to mutations in ALG2 and ALG14

Congenital myasthenic syndromes are a heterogeneous group of inherited disorders that arise from impaired signal transmission at the neuromuscular synapse. They are characterized by fatigable muscle weakness. We performed linkage analysis, whole-exome and whole-genome sequencing to determine the underlying defect in patients with an inherited limb-girdle pattern of myasthenic weakness. We identify ALG14 and ALG2 as novel genes in which mutations cause a congenital myasthenic syndrome. Through analogy with yeast, ALG14 is thought to form a multiglycosyltransferase complex with ALG13 and DPAGT1 that catalyses the first two committed steps of asparagine-linked protein glycosylation. We show that ALG14 is concentrated at the muscle motor endplates and small interfering RNA silencing of ALG14 results in reduced cell-surface expression of muscle acetylcholine receptor expressed in human embryonic kidney 293 cells. ALG2 is an alpha-1,3-mannosyltransferase that also catalyses early steps in the asparagine-linked glycosylation pathway. Mutations were identified in two kinships, with mutation ALG2p.Val68Gly found to severely reduce ALG2 expression both in patient muscle, and in cell cultures. Identification of DPAGT1, ALG14 and ALG2 mutations as a cause of congenital myasthenic syndrome underscores the importance of asparagine-linked protein glycosylation for proper functioning of the neuromuscular junction. These syndromes form part of the wider spectrum of congenital disorders of glycosylation caused by impaired asparagine-linked glycosylation. It is likely that further genes encoding components of this pathway will be associated with congenital myasthenic syndromes or impaired neuromuscular transmission as part of a more severe multisystem disorder. Our findings suggest that treatment with cholinesterase inhibitors may improve muscle function in many of the congenital disorders of glycosylation.

Mutations in GFPT1 that underlie limb-girdle congenital myasthenic syndrome result in reduced cell-surface expression of muscle AChR

Mutations in GFPT1 underlie a congenital myasthenic syndrome (CMS) characterized by a limb-girdle pattern of muscle weakness. Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is a key rate-limiting enzyme in the hexosamine biosynthetic pathway providing building blocks for the glycosylation of proteins and lipids. It is expressed ubiquitously and it is not readily apparent why mutations in this gene should cause a syndrome with symptoms restricted to muscle and, in particular, to the neuromuscular junction. Data from a muscle biopsy obtained from a patient with GFPT1 mutations indicated that there were reduced endplate acetylcholine receptors. We, therefore, further investigated the relationship between identified mutations in GFPT1 and expression of the muscle acetylcholine receptor. Cultured myotubes derived from two patients with GFPT1 mutations showed a significant reduction in cell-surface AChR expression (Pt1 P < 0.0001; Pt2 P = 0.0097). Inhibition of GFPT1 enzymatic activity or siRNA silencing of GFPT1 expression both resulted in reduced AChR cell-surface expression. Western blot and gene-silencing experiments indicate this is due to reduced steady-state levels of AChR α, δ, ε, but not β subunits rather than altered transcription of AChR-subunit RNA. Uridine diphospho-N-acetylglucosamine, a product of the hexosamine synthetic pathway, acts as a substrate at an early stage in the N-linked glycosylation pathway. Similarity between CMS due to GFPT1 mutations and CMS due to DPAGT1 mutations would suggest that reduced endplate AChR due to defective N-linked glycosylation is a primary disease mechanism in this disorder.

Clinical features of congenital myasthenic syndrome due to mutations in DPAGT1

Background A newly defined congenital myasthenic syndrome (CMS) caused by DPAGT1 mutations has recently been reported. While many other CMS-associated proteins have discrete roles localised to the neuromuscular junction, DPAGT1 is ubiquitously expressed, modifying many proteins, and as such is an unexpected cause of isolated neuromuscular involvement. Methods We present detailed clinical characteristics of five patients with CMS caused by DPAGT1 mutations. Results Patients have prominent limb girdle weakness and minimal craniobulbar symptoms. Tubular aggregates on muscle biopsy are characteristic but may not be apparent on early biopsies. Typical myasthenic features such as pyridostigmine and 3, 4- diaminopyridine responsiveness, and decrement on repetitive nerve stimulation are present. Conclusions These patients mimic myopathic disorders and are likely to be under-diagnosed. The descriptions here should facilitate recognition of this disorder. In particular minimal craniobulbar involvement and tubular aggregates on muscle biopsy help to distinguish DPAGT1 CMS from the majority of other forms of CMS. Patients with DPAGT1 CMS share similar clinical features with patients who have CMS caused by mutations in GFPT1, another recently identified CMS subtype.

DOK7 congenital myasthenic syndrome in childhood: Early diagnostic clues in 23 children

Mutations in DOK7 are a common cause of congenital myasthenia. Treatment with ephedrine or salbutamol is effective, but diagnosis is often delayed. The aim of our study was to find early clues to the diagnosis of DOK7 congenital myasthenic syndrome. We included 23 children of 20 families. Onset of symptoms ranged from birth to age 3 years. 13 presented at birth with feeding difficulties, 11 with stridor (documented vocal cord palsy in 7), 3/11 with hypotonia/poor head control. Weakness was more pronounced proximally in all, axial in early presenting infants. Muscle biopsy showed non-specific features in 15/16, type 1 fibre predominance in 14/16, areas devoid of oxidative enzyme activity in 7/16. Muscle imaging was normal in 8/10, 2/10 showed mild non-specific changes. A diagnostic clue suggesting CMS rather than myopathy was the discrepancy between muscle imaging or histology findings compared with the degree of weakness. Repetitive nerve stimulation and stimulation single fibre electromyography were pathological in 9/17 and 13/14, respectively. In conclusion, stridor and feeding difficulties at birth or progressive weakness despite normal milestones in infancy point to the diagnosis and should lead to neurophysiological and genetic investigation. Fatigability can be absent or easily missed in the first years of life.

Identification of DPAGT1 as a new gene in which mutations cause a congenital myasthenic syndrome.

Congenital myasthenic syndromes (CMS) are a group of inherited disorders that arise from impaired signal transmission at the neuromuscular synapse. They are characterized by fatigable muscle weakness. This is a heterogenous group of disorders with 15 different genes implicated in the development of the disease. Using whole‐exome sequencing we identified DPAGT1 as a new gene associated with CMS. DPAGT1 catalyses the first step of N‐linked protein glycosylation. DPAGT1 patients are characterized by weakness of limb muscles, response to treatment with cholinesterase inhibitors, and the presence of tubular aggregates on muscle biopsy. We showed that DPAGT1 is required for glycosylation of acetylcholine receptor (AChR) subunits and efficient export of AChR to the cell surface. We suggest that the primary pathogenic mechanism of DPAGT1‐associated CMS is reduced levels of AChRs at the endplate region. This finding demonstrates that impairment of the N‐linked glycosylation pathway can lead to the development of CMS.

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Muscle magnetic resonance imaging in congenital myasthenic syndromes

In this study we investigated muscle magnetic resonance imaging in congenital myasthenic syndromes (CMS).

Slow channel congenital myasthenic syndrome responsive to a combination of fluoxetine and salbutamol.

Introduction: Slow channel congenital myasthenic syndrome is a dominant disorder characterized by prolonged acetylcholine receptor ion‐channel activation. Methods: Molecular genetic techniques, electrophysiology, and binding studies in human embryonic kidney (HEK) 293 cells determined mutant function and expression levels. Patient response to treatment was measured by quantitative myasthenic gravis and Medical Research Council grade strength scores. Results: We report an unusual case due to heteroallelic mutations in CHRNE. The slow channel mutation, p.εS278del, is accompanied by a severe low‐expression mutation, p.εR217L, on the second allele. Expression studies and cosegregation of p.εS278del with the disorder in the patients offspring demonstrate robust expression of the p.εS278del mutation. The patient showed modest benefits from standard treatment with fluoxetine, but there was dramatic improvement when salbutamol was combined with fluoxetine. Conclusions: This case suggests that salbutamol, which is beneficial in some other congenital myasthenic syndromes, might also be considered in addition to fluoxetine in slow channel syndrome. Muscle Nerve, 2013

Characterisation of a novel cardiac phenotype in patients with GFPT1 or DPAGT1 mutations

Background Mutations in the GFPT1 and DPAGT1 genes, which encode enzymes associated with roles in protein N-linked glycosylation, have been recently identified in a rare subgroup of patients with congenital myasthenic syndromes (CMSs). These mutations are inherited in an autosomal recessive pattern, and the mechanism of impaired neuromuscular transmission may be acetylcholine receptor (AChR) deficiency due to impaired (AChR) subunit glycosylation. Aberrant protein glycosylation is also implicated in the development of severe cardiomyopathies in the congenital disorders of glycosylation, although the mechanisms responsible for cardiac involvement are unknown. We investigated whether patients with CMS and GFPT1 or DPAGT1 mutations also had evidence of a cardiac phenotype. Methods We performed comprehensive cardiovascular magnetic resonance (CMR) imaging at 1.5T (Avanto, Siemens), 31P spectroscopy at 3T (Tim Trio, Siemens) and echocardiography to evaluate cardiac structure and function in patients with GFPT1 (n = 2) and DPAGT1 (n = 2) mutations. The mean age of the participants was 45 (range 25-57) and two were male. Results Electrocardiography was abnormal in all, with abnormal repolarisation and deep S waves (n = 3) or marked left ventricular hypertrophy by voltage criteria (n = 1). Despite normal biventricular volumes and systolic function, GFPT1/DPAGT1 patients demonstrated late gadolinium enhancement suggestive of myocardial fibrosis (n = 4, mean proportion of enhanced myocardium > 5 SD above individual reference regions 3.2% +/-1.6, Figure 1), impaired energetics (n = 2) and diastolic dysfunction (n = 3). No patient had symptoms attributable to cardiovascular disease on structured interview. Conclusions Patients with GFPT1 or DPAGT1 mutations demonstrate a cardiac phenotype including abnormal electrocardiography, myocardial fibrosis, diastolic dysfunction and impaired energetics, despite normal systolic function. These findings may reflect incipient cardiomyopathy due to aberrant cardiac glycoprotein function. The reason for