Objective:

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.

Methods:

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.

Results:

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.

Conclusions:

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.

Background

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.

Methods

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.

Results

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.

Discussion

Our observations should spur controlled prospective clinical trials of albuterol in these as well as other CMS.

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.

Objective

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.

Methods

We performed histochemical, immunocytochemical, electron microscopy and molecular genetic studies in a cohort of 12 patients affected by this disease.

Results

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.

Interpretation

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.

SUMMARY

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.

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.

Objective

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.

Methods

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.

Results

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.

Interpretation

We conclude mutation in Bag3 defines a novel severe autosomal dominant childhood muscular dystrophy.

Objective

Detailed analysis of phenotypic and molecular genetic aspects of Dok-7 myasthenia in 16 patients.

Methods

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.

Results

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.

Interpretation

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.