Rebecca Croxen

Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria

The gene products involved in mammalian mitochondrial DNA (mtDNA) maintenance and organization remain largely unknown. We report here a novel mitochondrial protein, Twinkle, with structural similarity to phage T7 gene 4 primase/helicase and other hexameric ring helicases. Twinkle colocalizes with mtDNA in mitochondrial nucleoids. Screening of the gene encoding Twinkle in individuals with autosomal dominant progressive external ophthalmoplegia (adPEO), associated with multiple mtDNA deletions, identified 11 different coding-region mutations co-segregating with the disorder in 12 adPEO pedigrees of various ethnic origins. The mutations cluster in a region of the protein proposed to be involved in subunit interactions. The function of Twinkle is inferred to be critical for lifetime maintenance of human mtDNA integrity.

Mutation of the acetylcholine receptor ε-subunit promoter in congenital myasthenic syndrome

Congenital myasthenic syndrome comprises a heterogeneous group of inherited disorders of neuromuscular transmission. Acetylcholine receptor (AChR) deficiency is the most common form of congenital myasthenic syndrome and in most cases results from mutations within the coding region of the AChR ε subunit. However, studies in mice have established that synapse‐specific expression of AChR is dependent on a sequence contained within the AChR‐subunit promoter regions, termed an N‐box. We describe a consanguineous family in which 2 of 7 siblings had clinical and electromyographic features consistent with AChR deficiency. Muscle biopsy demonstrated low AChR numbers, establishing the disorder as postsynaptic. Single‐strand conformational polymorphism analysis identified an abnormal conformer in the AChR ε‐subunit gene promoter of the patients. DNA sequence and restriction endonuclease analysis shows that the disorder cosegregates with recessive inheritance of a single point mutation, a transition (C→T) in the N‐box of the ε‐subunit promoter. Analysis of an intercostal biopsy from 1 of the patients showed a dramatic reduction in ε‐subunit mRNA levels compared with disease and normal controls. This is the first evidence in humans that an N‐box mutation can lead to disruption of ε‐subunit transcription, resulting in the loss of adult AChR synthesis and the clinical phenotype of AChR‐deficiency congenital myasthenic syndrome. Ann Neurol 1999;45:439–443

Acetylcholine receptor delta subunit mutations underlie a fast-channel myasthenic syndrome and arthrogryposis multiplex congenita.

Limitation of movement during fetal development may lead to multiple joint contractures in the neonate, termed arthrogryposis multiplex congenita. Neuromuscular disorders are among the many different causes of reduced fetal movement. Many congenital myasthenic syndromes (CMSs) are due to mutations of the adult-specific epsilon subunit of the acetylcholine receptor (AChR), and, thus, functional deficits do not arise until late in gestation. However, an earlier effect on the fetus might be predicted with some defects of other AChR subunits. We studied a child who presented at birth with joint contractures and was subsequently found to have a CMS. Mutational screening revealed heteroallelic mutation within the AChR delta subunit gene, delta 756ins2 and delta E59K. Expression studies demonstrate that delta 756ins2 is a null mutation. By contrast, both fetal and adult AChR containing delta E59K have shorter than normal channel activations that predict fast decay of endplate currents. Thus, delta E59K causes dysfunction of fetal as well as the adult AChR and would explain the presence of joint contractures on the basis of reduced fetal movement. This is the first report of the association of AChR gene mutations with arthrogryposis multiplex congenita. It is probable that mutations that severely disrupt function of fetal AChR will underlie additional cases.

Recessive inheritance and variable penetrance of slow-channel congenital myasthenic syndromes

Background Slow-channel congenital myasthenic syndromes (SCCMS) typically show dominant inheritance. They are caused by missense mutations within the subunits of muscle nicotinic acetylcholine receptors (AChR) that result in prolonged ion channel activations. SCCMS mutations within the AChR &agr; subunit are located in various functional domains, whereas fully described mutations in AChR non-&agr; subunits have, thus far, been located only in the M2 channel-lining domain. The authors identified and characterized two &egr;-subunit mutations, located outside M2, that underlie SCCMS in three kinships. In two of the three kinships, the syndrome showed an atypical inheritance pattern. Methods These methods included clinical diagnosis, mutation detection, haplotype analysis, and functional expression studies using single-channel recordings of mutant AChR transiently transfected into HEK293 cells. Results The authors identified two SCCMS mutations in the AChR &egr; subunit, &egr;L78P and &egr;L221F. Both mutations prolonged ACh-induced ion channel activations. &egr;L78P is present in a consanguineous family and appears to be pathogenic only when present on both alleles, and &egr;L221F shows variable penetrance in one of the two families that were identified harboring this mutation. Conclusion SCCMS mutations may show a recessive inheritance pattern and variable penetrance. A diagnosis of SCCMS should not be ruled out in cases of CMS with an apparent recessive inheritance pattern.

Genes at the junction – candidates for congenital myasthenic syndromes

The neuromuscular junction is the site of several myasthenic (mys, muscle; aesthenia, weakness) disorders of autoimmune and genetic origin. The acquired autoimmune conditions are mainly adult-onset and caused by antibodies to specific neuronal and muscle ion channels, but can occur neonatally due to placental transfer of maternal antibodies. This review focuses on the rarer genetic conditions, called congenital myasthenic syndromes (CMS), that often present at birth. Mutations have yet to be characterized for familial infantile myasthenia, acetylcholinesterase deficiency and ACh-receptor deficiency; but genes encoding both structural and functional NMJ protiens should be considered. Other syndromes have recently been shown to involve defects in the functioning of the ACh receptor itself. In particular, eight different mutations have been reported in cases of the slow channel syndrome, a dominant condition associated with point mutations that generate single amino acid changes within the ACh receptor and result in prolonged channel activations. These investigations are providing new insights into the structure and function of the ACh receptor. Further studies of CMS should pave the way for analysis and treatment of disorders involving other synapses in the peripheral and central nervous system.

Novel functional ε‐subunit polypeptide generated by a single nucleotide deletion in acetylcholine receptor deficiency congenital myasthenic syndrome

Acetylcholine receptor (AChR) deficiency is a recessively inherited congenital myasthenic syndrome in which fatigable muscle weakness results from impaired neuromuscular transmission caused by reduced AChR numbers. In mature muscle, AChRs consist of α2βδ together with the adult‐specific ε subunit. We have identified a deletion of the first nucleotide in exon 12 of the AChR ε‐subunit gene (ε1267delG) and demonstrate its recessive inheritance segregates with disease in 6 unrelated cases of AChR deficiency. In addition, we found that both healthy and AChR‐deficient muscle contain a population of AChR ε‐subunit mRNA transcripts that retain intron 11. We investigated the possible consequences of combining this mutation with the alternative mRNA species through AChR expression studies in human embryonic kidney cells and Xenopus oocytes. ε1267delG generates a polypeptide that lacks M4 and is not detected in surface AChR, whereas retention of intron 11 in the RNA transcript restores the reading frame, conserves M4, and generates a polypeptide that is incorporated into functional surface AChR, although at a reduced level, consistent with the disease phenotype. Our results indicate that for some AChR deficiency mutations located between M3 and M4, the retention of intron 11 in the ε‐subunit mRNA transcripts may rescue adult AChR function.

Mapping of autosomal dominant progressive external ophthalmoplegia to a 7-cM critical region on 10q24.

Objective: To map the gene responsible for autosomal dominant progressive external opthalmoplegia. Background: The pathogenesis of progressive external ophthalmoplegia (PEO) can be associated with multiple deletions of mitochondrial DNA (mtDNA). PEO may show autosomal dominant (adPEO) or autosomal recessive (arPEO) patterns of inheritance, indicating that the genetic defect has a Mendelian basis and most likely involves a nuclear gene encoding a protein that interacts with the mitochondrial genome. adPEO is heterogeneous genetically, and thus far disease loci have been identified on chromosomes 3 and 10. The locus on chromosome 10q23-q25 was assigned by linkage analysis in a single Finnish family. Methods: Samples from a large Pakistani family with adPEO, in which clinical symptoms are bilateral ptosis, limitations of eye movements, and varying degrees of proximal muscle weakness, were collected. Muscle biopsy and mtDNA rearrangement analysis was used to confirm the diagnosis. Genomewide linkage analysis was set up using a set of 391 microsatellite markers. Results: The muscle biopsy from an affected member showed ragged red fibers, increased succinic dehydrogenase staining, lack of cytochrome oxidase activity, and multiple deletions of mtDNA. The disease locus was mapped to 10q23.31-q25.1 by linkage analysis, and a maximum lod score of 5.72 was obtained with D10S1267. Conclusion: By analysis of meiotic recombinations in affected individuals, the critical region was restricted to the 7-cM interval between D10S198 and D10S1795.

Myasthenia gravis in a woman with congenital AChR deficiency due to ε-subunit mutations

A reduction in the number of acetylcholine receptors (AChR) on the postsynaptic membrane is characteristic of MG. This may be inherited (AChR deficiency syndrome) or acquired (MG). The authors report two sisters with AChR deficiency caused by heteroallelic mutations in the AChR epsilon-subunit gene. The younger sister developed MG at 34 years. This unusual case raises the possibility that genetic defects of the AChR might be a factor in the etiology of autoimmune MG.

Mutation in the AChR ion channel gate underlies a fast channel congenital myasthenic syndrome

Background: Most congenital myasthenic syndromes (CMS) have postsynaptic defects from mutations within the muscle acetylcholine receptor (AChR). Mutations underlying the slow channel syndrome cause a “gain of function” and usually show dominant inheritance, whereas mutations underlying AChR deficiency or the fast channel syndrome cause a “loss of function” and show recessive inheritance. Objective: To characterize the disease mechanism underlying an apparently dominantly inherited CMS that responds to IV edrophonium. Methods: DNA from CMS patients was analyzed for mutations by single-strand conformation polymorphism analysis, DNA sequence analysis, and restriction endonuclease digestion. Functional analysis of mutations was by α-bungarotoxin binding studies and by patch clamp analysis of mutant AChR expressed in human embryonic kidney cells. Results: Analysis of muscle biopsies from father and son in an affected kinship showed normal endplate morphology and AChR number but severely reduced miniature endplate potentials. DNA analysis revealed that each harbors a single missense mutation in the AChR α-subunit gene, αF256L. Expression studies demonstrate this mutation underlies a fast channel phenotype with fewer and shorter ion channel activations. The major effect of αF256L, located within the M2 transmembrane domain, is on channel gating, both reducing the opening and increasing the closure rate. Conclusions: Mutation αF256L results in fast channel kinetics. Expression studies suggest a dominant-negative effect within the AChR pentamer, severely compromising receptor function.

Structural Abnormalities of the AChR Caused by Mutations Underlying Congenital Myasthenic Syndromes

Abstract: The objective was to define the molecular mechanisms underlying congenital myasthenic syndromes (CMS) by studying mutations within genes encoding the acetylcholine receptor (AChR) and related proteins at the neuromuscular junction. It was found that mutations within muscle AChRs are the most common cause of CMS. The majority are located within the ε‐subunit gene and result in AChR deficiency.