Mutations in the skeletal muscle alpha-actin gene in patients with actin myopathy and nemaline myopathy.

Muscle contraction results from the force generated between the thin filament protein actin and the thick filament protein myosin, which causes the thick and thin muscle filaments to slide past each other. There are skeletal muscle, cardiac muscle, smooth muscle and non-muscle isoforms of both actin and myosin. Inherited diseases in humans have been associated with defects in cardiac actin (dilated cardiomyopathy and hypertrophic cardiomyopathy), cardiac myosin (hypertrophic cardiomyopathy) and non-muscle myosin (deafness). Here we report that mutations in the human skeletal muscle alpha-actin gene (ACTA1) are associated with two different muscle diseases, 'congenital myopathy with excess of thin myofilaments' (actin myopathy) and nemaline myopathy. Both diseases are characterized by structural abnormalities of the muscle fibres and variable degrees of muscle weakness. We have identified 15 different missense mutations resulting in 14 different amino acid changes. The missense mutations in ACTA1 are distributed throughout all six coding exons, and some involve known functional domains of actin. Approximately half of the patients died within their first year, but two female patients have survived into their thirties and have children. We identified dominant mutations in all but 1 of 14 families, with the missense mutations being single and heterozygous. The only family showing dominant inheritance comprised a 33-year-old affected mother and her two affected and two unaffected children. In another family, the clinically unaffected father is a somatic mosaic for the mutation seen in both of his affected children. We identified recessive mutations in one family in which the two affected siblings had heterozygous mutations in two different exons, one paternally and the other maternally inherited. We also identified de novo mutations in seven sporadic probands for which it was possible to analyse parental DNA.

The prevention of neurogenetic disease.

A significant number of major neurogenetic diseases have been defined at the molecular level in recent years, making it possible to determine precisely the genotype for familial Alzheimer's disease, Huntington's disease, Machado-Joseph disease, dominantly inherited ataxia, Charcot-Marie-Tooth disease, myotonic muscular dystrophy, Duchenne-Becker muscular dystrophy, familial amyotrophic lateral sclerosis, and neurofibromatosis. This information has made it possible to identify the abnormal genotype of at-risk persons for these diseases and for at-risk pregnancies for several of them. Precise molecular diagnoses are thus possible using applied molecular markers. Prevention of disease can be achieved using these molecular markers with genetic counseling and appropriate family planning. Significant progress is being made in this regard with Tay-Sachs disease, Huntington's disease, the dominantly inherited ataxias, and the muscular dystrophies. Further, this molecular genotyping will be of indispensible value to families with these diseases when somatic cell gene therapy becomes available. The field of molecular neurogenetics is moving forward rapidly, and advances in gene identification for these diseases will lead in the near future to the means to prevent many of them.

In vitro study of cytotoxic factors against endothelium in childhood dermatomyositis.

In a previous report we showed that leukocytes from a group of patients with childhood dermatomyositis (CDM) were not cytotoxic toward cultured normal human skeletal muscle cells. Blood products from 11 patients with CDM and 12 age- and sex-matched controls were tested for cytotoxicity toward human endothelium using a chromium 51 assay. Mixed lymphocytes, monocytes, and serum alone or in combination did not produce endothelial cell death. The combination of serum and leukocytes, however, did produce some cytotoxic effects in three of 11 patients with CDM. We conclude that those factors tested in vitro are not responsible for the endothelial cell death but together may produce cytotoxic changes in some patients.

Partial biochemical maturation of aneurally cultured human skeletal muscle.

We studied cultures of human skeletal muscle in vitro and established standards for biochemical markers of cellular differentiation. DNA synthesis ceased at the time of fusion, implying the absence of fibroblasts. Myosin heavy-chain synthesis, creatine and pyruvate kinase activities, and isoenzymes of creatine kinase were measured serially over 36 days. Filamin and fibronectin proteins were identified in these cultures. Compared to chick muscle in culture, human skeletal muscle cells remained relatively immature. These data provide a basis for the study of diseased human muscle cells in culture.

Function changes in spinal muscular atrophy II and III. The DCN/SMA Group.

The course of spinal muscular atrophy (SMA) is not well established except for those patients whose age of onset is before 6 months and who achieve only "sit with support" as their maximum function (Werdnig-Hoffmann disease or SMA I). This study shows that there is another group of SMA patients whose age of onset and maximum function achieved can be used as prognostic guides. Fifty percent of SMA patients who could walk without assistance and whose onset was prior to age 2 years lost the ability to walk independently by age 12. Fifty percent of SMA patients who walked and whose onset was between 2 and 6 years of age lost walking ability by age 44 years. Fifty percent of SMA patients who could walk with assistance as their best function ever achieved lost this ability by age 7 years, unrelated to age of onset; none could walk with assistance after age 14 years. Seventy-five percent of SMA patients who developed the ability to sit independently as their best function were still sitting after age 7 years independent of age of onset; 50% of this group could sit independently after age 14 years. Eighty-five percent of SMA patients who could walk could not negotiate stairs without holding onto a rail. They could raise their hands above the head; however, as they lost walking ability, they lost this function as well. Only one SMA patient whose maximum function was sitting independently could get to the sitting position on his own. Only two of these patients could hold their hands above their heads. All patients with SMA lose function over time. This function loss occurs slowly and is related primarily to maximum function achieved; knowledge of age of onset provides helpful information, especially for predicting the loss of independent walking.

Cognitive dysfunction as the major presenting feature of Becker's muscular dystrophy.

We report four patients, currently aged 15, 17, 19, and 42 years, with X-linked dystrophinopathy who presented with mental retardation (IQ range, 60-68) and psychiatric disturbance in the absence of muscle weakness. All patients had elevated serum creatine kinase and dystrophic changes on muscle biopsy. There were alterations in the size and abundance of dystrophin on immunohistochemistry and immunoblotting in all cases, consistent with a molecular diagnosis of Becker's muscular dystrophy. Two patients had deletions of the dystrophin gene on DNA analysis. These findings suggest that Becker's muscular dystrophy may be associated with a predominantly neuropsychiatric presentation and that dystrophinopathy should be considered in the differential diagnosis of unexplained cognitive or psychiatric disturbance in males. Serum creatine kinase may provide an adequate screening test in this clinical situation.

Mutations of the slow muscle alpha-tropomyosin gene, TPM3, are a rare cause of nemaline myopathy.

The alpha-tropomyosin-3 (TPM3) gene was screened in 40 unrelated patients with nemaline myopathy (NM). A single compound heterozygous patient was identified carrying one mutation that converts the stop codon to a serine and a second splicing mutation that is predicted to prevent inclusion of skeletal muscle exon IX. TPM3 mutations are a rare cause of NM, probably accounting for less than 3% of cases. The severity of cases with TPM3 mutations may vary from severe infantile to late childhood onset, slowly progressive forms.

Myogenes and myotubes.

Investigation of the congenital myopathies has been limited by a lack of knowledge regarding basic mechanisms involved in normal myogenesis of human muscle and the relative rarity of these diseases. A newly recognized family of regulatory genes has been shown to be necessary for myogenesis to proceed to formation of normal mature muscle. It is likely that investigation of patients with one or more types of structural myopathy may show that abnormalities of the regulatory basic helix-loop-helix (bHLH) genes may be responsible for disease.

Dystrophin and DNA diagnosis in a large pediatric muscle clinic.

Eighty-seven unrelated patients from a large muscle clinic setting were analyzed by DNA for deletions in the dystrophin gene for diagnosis of Duchenne/Becker muscular dystrophy. The clinical phenotype of the patient population included 72% Duchenne, 13% Becker, and 15% outlier patients. Dystrophin gene deletions were detected in 61% of these patients, and disease phenotype was predicted by DNA with an accuracy of 95%. While DNA did not confirm diagnosis in all patients, dystrophin analysis of muscle biopsies, when available, predicted a disease phenotype. In the 66 patients in which muscle biopsies were available for analysis, the results of the dystrophin analysis agreed with actual clinical phenotype with 86% accuracy. Less agreement between dystrophin and clinical phenotype predictions were found in the Becker patient population. We suggest that, in at least 61% of Duchenne/Becker patients, DNA analysis provides a rapid and accurate diagnosis. DNA is less invasive and less expensive than biopsy and may allow family risk assessment. Therefore, DNA analysis may become the first recommended laboratory procedure for Duchenne/Becker diagnosis, and muscle biopsy with dystrophin analysis may become necessary only for those patients with undetectable gene mutations.

Long-term mechanical ventilation in infants with neuromuscular disease.

Seven children with neuromuscular disease were placed on mechanical ventilation before the age of 2 years. The outcome for these patients was variable and did not correlate with primary diagnosis. There appeared to be a high correlation between the incidence of electrocardiogram changes and death. Our experience demonstrates that there was a high mortality rate (57%) in neuromuscular patients supported by mechanical ventilation before the age of 2 years.

Decreased creatine kinase activity in cultured Duchenne dystrophic muscle cells.

Muscle cells were cultured from six patients with Duchenne muscular dystrophy and nine normal subjects. Protein and myosin content and pyruvate kinase (PK) activity were similar in normal and Duchenne muscular dystrophy cultures. Creatine kinase (CK) activity was lower in Duchenne muscular dystrophy cultures and the isoenzyme distribution indicated MB-CK was significantly lower, while BB-CK was significantly higher in later Duchenne muscular dystrophy cultures. This abnormal isoenzyme pattern suggested aberrant or impaired maturation of Duchenne muscular dystrophy myotubes in vitro.

Muscle fatigue in spinal muscular atrophy.

We previously reported that patients with spinal muscular atrophy do not lose muscle strength over time as measured quantitatively. However, we noted that many patients with spinal muscular atrophy suffer from what they call fatigue. We wondered if we could measure fatigue during a single maximal voluntary contraction, whether fatigue might increase with time, independent of muscle strength, and whether increasing fatigue might correlate with loss of function in some patients. We measured fatigue during a single maximal voluntary contraction in a cohort of patients having spinal muscular atrophy using quantitative strength testing. We included only patients with spinal muscular atrophy aged 5 years or older, so they could follow instructions regarding muscle contraction, and who were followed for at least 2 years. Seventy-six children with spinal muscular atrophy and 24 untrained individuals, aged 5 to 57 years (mean, 16.8 years), were studied. There was no discernible abnormal fatigue in patients with spinal muscular atrophy compared to untrained controls using our methodology. Thus, spinal muscular atrophy may not be associated with fatiguability. Moreover, spinal muscular atrophy does not appear to cause progressive muscle fatigue with age or loss of function. It is possible that fatigue was undetectable by our methods. An alternative explanation is that what patients describe as fatigue may be caused by factors outside the neuromuscular system. Such factors may include chronic respiratory insufficiency with hypoventilation and carbon dioxide retention as well as chronic malnutrition and negative nitrogen balance.

Carbamazepine trial for Lesch-Nyhan self-mutilation.

Anticonvulsants may reduce the self-mutilation of acquired sensory neuropathy, and one report described sensory neuropathy in an older patient with Lesch-Nyhan syndrome. We performed nerve and muscle biopsies on four patients with Lesch-Nyhan syndrome and initiated an uncontrolled pilot trial to see if carbamazepine would reduce the self-mutilation in these patients. All of the boys had clinical features typical of Lesch-Nyhan syndrome, and the diagnosis was confirmed in each by enzyme analysis. No specific abnormalities were identified in either nerve or muscle. Nevertheless, self-mutilation and the need for constant restraint diminished in all four patients, though in one the effect was only transient. Two patients had increased self-mutilation when carbamazepine was stopped, then improved a second time when treatment was restarted. Sensory neuropathy was not confirmed, so any effect of carbamazepine is likely to be on the central nervous system.

Prospective analysis of strength in spinal muscular atrophy. DCN/Spinal Muscular Atrophy Group.

Spinal muscular atrophy is a genetic disorder of the motor neurons that causes profound hypotonia, severe weakness, and often fatal restrictive lung disease. Patients with spinal muscular atrophy present a spectrum of disease from the most severe infantile-onset type, called Werdnig-Hoffmann disease (type 1), associated with a mortality rate of up to 90%, to a late-onset mild form (type 3), wherein patients remain independently ambulatory throughout adult life. Although many clinicians agree that patients with spinal muscular atrophy lose motor abilities with age, it is unknown whether progressive weakness occurs in all patients with spinal muscular atrophy. We present here results of the first prospective study of muscle strength in patients with spinal muscular atrophy. There was no loss in muscle strength as determined by a quantitative muscle test during the observation period. However, motor function diminished dramatically in some patients with spinal muscular atrophy. Explanations for this loss of function could not be determined from our data. Decrease in motor function could be caused by factors other than loss of strength. Therefore, it is not clear from our results whether spinal muscular atrophy is a neurodegenerative disease. We conclude that treatment trials in spinal muscular atrophy should be designed with consideration of the natural history of strength and motor function in this disorder.

Pulmonary function in spinal muscular atrophy.

We present the first prospective study on pulmonary function in spinal muscular atrophy patients. Seventy-seven spinal muscular atrophy patients, ages 5 to 18 years, from three centers, were studied with regard to forced vital capacity, using height as a predictor. Patients were categorized into four motor function categories. The highest-functioning group had normal or near-normal values, and those who sat with support had the lowest values. Those with intermediate function had intermediate values. Forced vital capacity was studied longitudinally in 40 spinal muscular atrophy patients for 1.1 to 4.4 years. Eighty-eight percent of patients grew in height, but only 35% showed an increase in height-adjusted forced vital capacity percent. In those patients with the least function, 100% lost height-adjusted forced vital capacity over time. In those patients with the highest function, 57% lost height-adjusted forced vital capacity. In addition, the basic forced vital capacity, not correlated to height, decreased in 43% of cases. These pulmonary function alterations appear to be important determinants for function and survival in spinal muscular atrophy patients.

Prospective study of spinal muscular atrophy before age 6 years. DCN/SMA Group.

Spinal muscular atrophy (SMA) is a common neuromuscular disorder of childhood, associated with a high mortality rate during the first 2 years of life. Most practitioners expect patients with SMA to follow a progressive course with loss of muscle strength and function over 2-10 years. Counselling sessions with parents frequently emphasize the high mortality rate and risk for respiratory failure. The progressive nature of SMA has been attributed to the loss of motor neurons. Fifty-eight children, ages 6 years and younger, were examined between January, 1987, and April, 1992, as part of a large, multicenter collaborative study of SMA. Muscle function was evaluated at regular intervals using a standardized protocol that was demonstrated to be reliable. We determined a prevalence of 56% for tongue fasciculations, a prevalence of 22% for facial weakness, and persistent deep tendon reflexes in one patient. Improved motor function and acquired milestones during the study were documented. This work should contribute toward a better understanding of the natural history of SMA.

Prolonged myasthenic syndrome after one week of muscle relaxants.

A child developed severe, generalized muscle weakness which persisted for 6 weeks, after receiving muscle relaxants for 1 week while requiring ventilator support. Electrodiagnostic studies indicated a presynaptic disorder of the neuromuscular junction which improved with high-frequency stimulation, similar to findings in Lambert-Eaton syndrome. Muscle specimens exhibited neurogenic targetoid fiber atrophy. Ultrastructure of the neuromuscular junction indicated terminal axon degeneration and atrophy with depletion of the secretory vesicles. Most reported patients with post-ventilator paresis have received steroids and muscle relaxants; muscle weakness commonly has been brief and attributed to steroids. We believe that this reversible myasthenic syndrome probably represents neurotoxicity due to high doses of steroidal nondepolarizing blocking agents; however, available data are insufficient to resolve this controversy.

Current status of Duchenne muscular dystrophy.

Duchenne muscular dystrophy is the second most common lethal genetic disorder in humans. With the advent of molecular genetic technology, the definition of this disease has been modified to include an abnormality of dystrophin in muscle--a dystrophinopathy. Accurate genetic counseling is possible using methods of deletion detection and linkage analysis. Treatment of this type of muscular dystrophy may soon mean the routine use of steroids and later include direct injection of an artificial gene for dystrophin.

Intramuscular hematopoiesis in hypotonic infants with type 1 muscle fiber dysmaturation.

Three term infants had severe hypotonia, a maturation defect of type 1 muscle fibers, and extramedullary hematopoiesis (EMH) in quadriceps muscle specimens obtained within one month of birth. Although predominantly myelopoietic, signs of inflammatory myopathy were absent. One patient had congenital myopathy with maturation arrest of type 1 fibers, another had transient maturation delay of type 1 fibers, and the third patient was subsequently classified as having spinomuscular atrophy. Extramedullary hematopoiesis was demonstrated in normal muscle obtained from young fetuses, but not in muscle obtained at autopsy from infants representing the third trimester and first postnatal month, or in muscle biopsy specimens from 15 other hypotonic infants with type 1 fiber size disproportion. We conclude that EMH in muscle of hypotonic infants is an abnormal persistence of a fetal state that is associated with delayed muscle maturation with diverse origins. Extramedullary hematopoiesis in muscle may indicate hypoxia, but signs of perinatal asphyxia in these babies were inconclusive. Low blood flow due to inactivity or an unidentified product of immature muscle may promote intramuscular EMH, but there is no evidence to suggest that myelopoiesis is injurious to muscle fibers. Intramuscular EMH should be distinguished from inflammation.

Muscle maturation delay in infantile myotonic dystrophy.

Specimens of the quadriceps femoris muscle from four infants with neonatal myotonic dystrophy had features of muscle fiber immaturity. Delayed establishment of major fiber subtypes and persistent myotubes, seen in the youngest infant, resolved in a repeated specimen obtained at the age of 4 months. Prominence of satellite cells, central nuclei, acid phosphatase activity sites, and Golgi zones diminished with age at biopsy. All four infants had type 1 fiber-size disproportion. These results substantiate the concept of delayed muscle fiber maturation in myotonic dystrophy.