Howard Feit

Autosomal-dominant distal myopathy associated with a recurrent missense mutation in the gene encoding the nuclear matrix protein, matrin 3.

Distal myopathies represent a heterogeneous group of inherited skeletal muscle disorders. One type of adult-onset, progressive autosomal-dominant distal myopathy, frequently associated with dysphagia and dysphonia (vocal cord and pharyngeal weakness with distal myopathy [VCPDM]), has been mapped to chromosome 5q31 in a North American pedigree. Here, we report the identification of a second large VCPDM family of Bulgarian descent and fine mapping of the critical interval. Sequencing of positional candidate genes revealed precisely the same nonconservative S85C missense mutation affecting an interspecies conserved residue in the MATR3 gene in both families. MATR3 is expressed in skeletal muscle and encodes matrin 3, a component of the nuclear matrix, which is a proteinaceous network that extends throughout the nucleus. Different disease related haplotype signatures in the two families provided evidence that two independent mutational events at the same position in MATR3 cause VCPDM. Our data establish proof of principle that the nuclear matrix is crucial for normal skeletal muscle structure and function and put VCPDM on the growing list of monogenic disorders associated with the nuclear proteome.

Vocal cord and pharyngeal weakness with autosomal dominant distal myopathy: clinical description and gene localization to 5q31.

Distal myopathy refers to a heterogeneous group of disorders in which the initial manifestations are weakness and atrophy of the hands and feet. We report a family segregating an autosomal dominant distal myopathy, with multiple affected individuals in whom vocal cord and pharyngeal weakness may accompany the distal myopathy, without involvement of the ocular muscles. To our knowledge, this pedigree displays a distinct distal myopathy with the added features of pharyngeal and vocal cord dysfunction (VCPDM) that has not been previously reported. We mapped the MPD2 gene for VCPDM to chromosome 5q within a 12-cM linkage interval between markers D5S458 and D5S1972 in a large pedigree (a maximum LOD score of 12.94 at a recombination fraction of 0 for D5S393) and combined genome screening and DNA pooling successfully adapted to fluorescent markers. This technique provides for the possibility of fully automated genome scans.

COMPARISON OF THE ISOELECTRIC AND MOLECULAR WEIGHT PROPERTIES OF TUBULIN SUBUNITS

Abstract— Brain tubulin subunits were separated by a combination of isoelectric focusing and electrophoresis in the presence of sodium dodecyl sulfate (SDS) using a two‐dimensional polyacrylamide slab gel technique. Isoelectric focusing separated tubulin subunits into two major groups of bands, such that the more acidic group corresponded to the α subunit and the less acidic group corresponded to the β subunit. In addition, isoelectric focusing resolved the β subunit into two subspecies which differed slightly in isoelectric properties but were the same apparent molecular weight. The a subunit was resolved into many subspecies that appear to differ from each other by both apparent molecular weight and isoelectric properties.

Polyradiculopathy in sarcoidosis.

We present three new and 14 retrospective cases of polyradiculopathy in sarcoidosis. Of these, 71% had weakness and 59% areflexia of the lower extremities, and 35% had sphincter dysfunction. Cases often were associated with central nervous system sarcoidosis. All cases involved thoracolumbar or lumbosacral roots, except a single case of cervical polyradiculopathy. Of 14 treated patients, nine improved with corticosteroids, laminectomy, or both. Polyradiculopathy complicating sarcoidosis: (1) is uncommon; (2) primarily involves thoracic and lumbar roots; (3) may arise from contiguous, hematogenous, or gravitational nerve root sleeve seeding; (4) may be asymptomatic; and (5) may improve with corticosteroids. Differential diagnosis of weakness in patients with sarcoidosis should include nerve root involvement from the primary process by direct sarcoid involvement.

Anomalous electrophoretic properties of brain filament protein subunits.

Introduction Filaments 10 nm in diameter are prominent constituents of the cytoskeleton of neuronal and glial cells. In nerve cells, the 10 nm filaments are called neurofilaments*, although similar filaments are found in a variety of other cell types and are generally referred to as tonofilaments or intermediate sized filaments so as to distinguish them from the filamentous forms of actin and myosin. Recently, 10 nm filaments have been isolated from myelinated bovine axonsZ, 21 and chicken smooth muscle 1,1z and from both of these sources, a polypeptide with a mol. wt. of 50,000 has been identified as the major constituent. An alternate approach to the identification of the protein subunits of the neurofilament was recently reported by Hoffman and Lasek 9. These workers took advantage of the electron microscope autoradiographic evidence that the slow component of axoplasmic flow contains mainly neurofilaments and neurotubules 3,4. Following injection of a radioactive amino acid precursor into the ventral horn of the spinal cord, a peak of radioactive protein corresponding to the slow component of axoplasmic flow was isolated from the ventral roots and analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The radioactivity was associated with tubulin and three other polypeptides: a major component at 68,000 and two minor components at 160,000 and 212,000. It was shown that these three polypeptides migrated down the axon at a velocity slightly slower than tubulin and were tentatively identified as components of a single structure, the 10 nm neurofilament. Thus, different methodologies have identified different polypeptides as constituents of the neurofilament. Direct isolation of brain filaments resulted in a single 50,000 mol. wt. subunit2,21; in contrast, analysis of axoplasmic flow identified three components with tool. wt. of 68,000, 160,000 and 212,0009. In this report, we show that the major component of a fraction enriched in brain filaments has an apparent tool.

Heterogeneity of Tropomyosin and Actin in Normal and Diseased Muscle

Actin and tropomyosin in muscle samples from normal humans, from human fetuses between 12 and 17 gestational weeks, and from patients with a variety of neuromuscular disorders were studied with two-dimensional electrophoresis using isoelectric focusing with either a broad pH range (8.6-4.5) or a narrow pH range (5.9-3.8) for the first dimension and either SDS or SDS-urea for the second dimension. With the broad pH range, two brothers with Duchenne muscular dystrophy were noted to have a less acidic variant of alpha-tropomyosin in biceps muscle which was not found in biceps muscle from other patients or controls. Studies of 8 additional biopsy specimens from patients with Duchenne muscular dystrophy and comparison with both fetal and normal human muscle using the narrow pH range revealed multiple forms of actin and tropomyosin which varied from individual to individual. This heterogeneity appeared to be unrelated to the dystrophic state but also obscured the ability to detect a change in actin or tropomyosin which could be related to dystrophy.

The contractile proteins in muscular dystrophy: An analytical review and a new hypothesis

The reduced capacity for force generation that is characteristic of muscle fibers in Duchenne muscular dystrophy is a predictable consequence of the alteration of sarcomere length that has been reported in this form of muscle disease in the early stages. Evidence that the primary defect may be a disorder of the cross-bridge cycle itself is reviewed.

Physiological and Biochemical Characterization of Avian Dystrophic Muscle Reveals Alterations of Collagen

Avian muscular dystrophy’ was chosen for this investigation because the early, hence fundamental, changes related to the dystrophic process can be studied. This disorder is inherited as a semidominant trait which selectively involves fast-twitch fibers. In the lines used (413 homozygous dystrophic, 412 normal), the disorder is manifested clinically in the third week after hatching? Chemically skinned’ pectoral myofibers (MF) were obtained from normal and dystrophic chickens at seven weeks; the passive and active mechanical properties were determined using sinusoidal and step-length changes?’ At this stage of the disease, the Ca-activated isometric tension is unaltered (normal = 0.69 2 0.16 Mdyn/cmz, n = 9; dystrophic = 0.75 2 0.11 Mdyn/cm2, n = 15). Transient kinetic properties (apparent rate constants of cross-bridge cycles) are likewise unaltered, and the frequency analysis method shows a typical response of fast twitch fibers: and not that of slow twitch fibers. This method also reveals that the number of permanently attached (rigor-like) cross-bridges are not increased in avian dystrophy. Whereas cross-bridge properties seem to be unaltered, extracellular elements appear to be affected in dystrophy. The resting length-tension diagram is very steep, and dystrophic MF exhibit higher stiffness than normal (FIGURE 1). Much of this stiffness persists even after extraction with 0.6 M KI and/or 5 M guanidine-HC1 in 1% mercaptoethanol. We infer from this observation that the increased stiffness in dystrophic MF is not due to actin-myosin interactions or other cytoskeletal elements such as connectin (titin) or intermediate filaments. In order to determine the significance of collagen molecules on resting stiffness, normal and dystrophic MF were treated by protease-free collagenase (Clostridium histolyticium, Calbiochem-Behring), with the results shown in FIGURE 2. Although the digestion conditions are adequate to destroy the stiffness of tendon (also in FIGURE 2), this treatment has little effect on stiffness of dystrophic MF. A partial effect is observed on normal MF. We infer from these studies that dystrophic MF contain a form of collagen which is extensively cross-linked; hence less susceptible to the collagenase?8 Fujii and Murota’ reported that the number of NaBI-h-reducible cross-links of collagen is greater than normal in the pectoral muscle of dystrophic chicken.

Changes in Turbidity During Microtubule Assembly in Brain Extracts from Normal Chickens and Chickens with Muscular Dystrophy

When extracts of brain from normal and dystrophic chickens were incubated at 30 degrees C under conditions that favored microtubule assembly, the increase in turbidity of the extract from the dystrophic animal was approximately 50% less than the increase in the turbidity of the extract from the normal animal. In developmental studies using age-matched normal and dystrophic chickens this difference in turbidity was observed only with chickens 65 days ex-ovo or older. The extent of microtubule assembly, as determined by a sedimentation methodology, was a linear function of the total protein concentration in the extract and was equal in extracts from normal and dystrophic chickens. In contrast, the total increase in turbidity was much larger than could be accounted for by sedimenting microtubules and varied in a non-linear manner with the total protein concentration. There was no increase in turbidity when brain extracts that were prepared by centrifugation at 100,000 g X 40 min were incubated at 30 degrees C. The condensation reaction of the tubulin in these high speed extracts with microtubules was equal in the extracts from normal and dystrophic animals. These studies indicate that a developmentally regulated difference in brain extracts from normal and dystrophic chickens can be demonstrated by turbidimetric methods but this difference is unrelated to the formation of microtubules in these extracts. These studies support the concept that muscular dystrophy is also accompanied by a change in the central nervous system.