Characterization of the Asian myopathy patients with VCP mutations.

BACKGROUND AND PURPOSE: Mutations in the valosin-containing protein (VCP) gene are known to cause inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD) and familial amyotrophic lateral sclerosis (ALS). Despite an increasing number of clinical reports, only one Asian family with IBMPFD has been described. METHODS: To characterize patients with VCP mutations, we screened a total of 152 unrelated Asian families who were suspected to have rimmed vacuolar myopathy. RESULTS: We identified VCP mutations in seven patients from six unrelated Asian families. Five different missense mutations were found, including a novel p.Ala439Pro substitution. All patients had adult-onset progressive muscle wasting with variable involvement of axial, proximal, and distal muscles. Two of seven patients were suggested to have mild brain involvement including cerebellar ataxia, and only one showed radiological findings indicating a change in bone. Findings from skeletal muscle indicated mixed neurogenic and myogenic changes, fibers with rimmed vacuoles, and the presence of cytoplasmic and nuclear inclusions. These inclusions were immunopositive for VCP, ubiquitin, transactivation response DNA-binding protein 43, and also histone deacetylase 6 (HDAC6), of which function is regulated by VCP. Evidence of early nuclear and mitochondrial damage was also characteristic. CONCLUSIONS: Valosin-containing protein mutations are not rare in Asian patients, and gene analysis should be considered for patients with adult-onset rimmed vacuolar myopathy with neurogenic changes. A wide variety of central and peripheral nervous system symptoms coupled with rare bone abnormalities may complicate diagnosis.

Primary collagen VI deficiency is the second most common congenital muscular dystrophy in Japan.

OBJECTIVES: To determine the frequency of primary collagen VI deficiency in congenital muscular dystrophy (CMD) in Japan and to establish the genotype-phenotype correlation. METHODS: We performed immunohistochemistry for collagen VI in muscles from 362 Japanese patients with CMD, and directly sequenced the three collagen VI genes, COL6A1, COL6A2, and COL6A3, in patients found to have collagen VI deficiency. RESULTS: In Japan, primary collagen VI deficiency accounts for 7.2% of congenital muscular deficiency. Among these patients, five had complete deficiency (CD) and 29 had sarcolemma-specific collagen VI deficiency (SSCD). We found two homozygous and three compound heterozygous mutations in COL6A2 and COL6A3 in all five patients with CD, and identified heterozygous missense mutations or in-frame small deletions in 21 patients with SSCD in the triple helical domain (THD) of COL6A1, COL6A2, and COL6A3. All mutations in SSCD were sporadic dominant. No genotype-phenotype correlation was seen. CONCLUSION: Primary collagen VI deficiency is the second most common CMD after Fukuyama type CMD in Japan. Dominant mutations located in the N-terminal side from the cysteine residue in the THD of COL6A1, COL6A2, and COL6A3 are closely associated with SSCD.

Patterns of dystrophin gene deletion in Egyptian Duchenne/Becker muscular dystrophy patients.

Large variations in the proportion of intragenic deletion in the dystrophin gene have been observed in different populations. Although dystrophin gene deletion was extensively studied all over the world, only few studies were done on Egyptian population and there was no account on the dystrophin gene duplication. In this study, we present our results on the pattern of deletion of the dystrophin gene together with the usage of quantitative polymerase chain reaction (PCR) as a method for duplication analysis within the dystrophin gene in Egyptian patients. Forty one Duchene/Becker muscular dystrophy patients were included in this study. The diagnosis was based on detailed clinical assessment, serum creatine kinase (CK) level, neurophysiologic study and muscle biopsy for histopathological analysis. DNA was extracted from ten milliliter peripheral blood according to basic protocol, and multiplex polymerase chain reaction for dystrophin gene using both Chamberlin and Beggs sets of primers amplifying eighteen exons covering the two main dystrophin gene hot spots. In addition primers from Abbs set were used when it was necessary to check the exon borders. DNA from cases with no detectable deletion was analyzed for dystrophin gene duplication using quantitative PCR technique. We had a percentage of 61.1% deletion which is higher than data from previous Egyptian studies and most of the deletion was localized in the major hotspot region between exons 44 and 52 and we had 5% of the cases with duplication. Our results were compared with previous studies from Egypt and with studies from different populations especially with data recorded in the Middle East and North Africa.

A new diagnostic test for VLCAD deficiency using immunohistochemistry.

BACKGROUND: Muscle pathology is often unhelpful in elucidating the specific underlying abnormality in patients with metabolic myopathy with rhabdomyolysis, including very-long chain acyl-CoA dehydrogenase (VLCAD) deficiency. Biochemical analyses require large amounts of biopsy samples for each enzyme assay. OBJECTIVE: To develop a more efficient diagnostic method for VLCAD deficiency. METHODS: The authors performed immunohistochemical analysis using an antibody to VLCAD on muscles from 344 patients (226 men and 118 women) without a specific diagnosis who had at least one of the following symptoms: myoglobinuria, high CK level, muscle pain, muscle stiffness, sudden infant death syndrome, and Reye-like syndrome. RESULTS: Immunoreactivity to VLCAD was absent or markedly reduced in 13 patients. Biochemical analyses confirmed that all these patients had low enzymatic activity and reduced amount of protein. They all had the myopathic phenotype. The authors identified homozygous or compound heterozygous mutations in all of them. All recombinant proteins had reduced enzymatic activity except for 128G>A (G43D) and 796C>G (P266A) mutants, indicating that they are neutral polymorphisms. CONCLUSIONS: The new screening method for the detection of VLCAD deficiency using an immunohistochemical technique identified 13 new Japanese patients with VLCAD deficiency.

A neonatal form of glycogen storage disease type IV.

We report of an infant with neonatal glycogen storage disease type IV (GSD IV) who was examined for severe hypotonia and cardiomyopathy. On the muscle biopsy there were many fibers with diastase-resistant polyglucosan bodies. Glycogen branching enzyme (GBE1) activity in the muscle was markedly reduced. The infant had a homozygous single nucleotide deletion in the open reading frame of GBE1 gene.

A novel form of autophagic vacuolar myopathy with late-onset and multiorgan involvement.

The authors report a 41-year-old man with a novel form of adult-onset autophagic vacuolar myopathy (AVM) with multiple organ involvement including eyes, heart, liver, lung, kidney, and skeletal muscle. The vacuolar membranes had sarcolemmal features similar to vacuoles in Danon disease, X-linked myopathy with excessive autophagy, and infantile AVM. Lysosome associated membrane protein-2, absent in Danon disease, was present. Defined by distinct clinical features, this disease constitutes the fourth entity in the group of autophagic vacuolar myopathy in which the vacuolar membranes have features of sarcolemma.

Mutation in the caveolin-3 gene causes a peculiar form of distal myopathy.

The authors describe a patient with sporadic distal myopathy associated with reduced caveolin-3 in muscle fibers in which the muscle atrophy was restricted to the small muscles of the hands and feet. Gene analysis disclosed a heterozygous 80 G-->A substitution in the caveolin-3 gene that was identical to that of reported cases of elevated serum creatine kinase. This patient further demonstrated possible clinical heterogeneity of myopathies with mutations in the caveolin-3 gene.

The significance of type 1 fiber atrophy (hypotrophy) in childhood neuromuscular disorders.

To determine the incidence of selective type 1 fiber atrophy (hypotrophy) and its possible significance in various muscle diseases of childhood, we reviewed 2212 muscle biopsies from children which we had examined in the past 20 years histochemically with ATPase staining. Type 1 fiber atrophy was seen in a variety of neuromuscular disorders, but predominantly in congenital myopathies, including all patients with congenital fiber type disproportion myopathy (20 patients), central core disease (12 patients) and multicore disease (four patients). Although type 1 fiber atrophy was not a constant feature in nemaline myopathy and myotubular myopathy, all patients with these diseases had abnormal fiber type distribution which included type 1 fiber predominance both with and without type 2B fiber deficiency. Together with abnormal fiber type distribution, type 1 fiber atrophy was a common finding in childhood neuromuscular disorders, especially congenital myopathies.

A mitochondrial encephalo-myo-neuropathy with a nucleotide position 3271 (T-C) point mutation in the mitochondrial DNA.

We report three members of a family, who exhibited a phenotype similar to 'myoclonus epilepsy with ragged-red fibers' but had a genotype usually associated with 'mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes'. The patients, a 48-year-old female, and her two sons, aged 21 and 19 respectively, presented with photo-reactive syncopal episodes, disturbances of gait and writing, dysarthria and finger tremor since the 3rd and 2nd decade of life, respectively, that were accompanied also by numbness and weakness of the extremities. Subsequently, cerebellar ataxia and myoclonus were also noted. Electromyography revealed both myogenic and neurogenic muscular changes, and nerve conduction studies demonstrated a sensory-motor neuropathy. Biopsy showed ragged-red fibers with strongly stained SDH-positive vessels in skeletal muscles, and a marked loss of myelinated fibers of the sural nerves. Mitochondrial (mt) DNA analyses of peripheral blood, muscles and nerves revealed that all members had a heteroplasmic np3271 (T-C) point mutation in the mitochondrial tRNA-Leu gene (UUR). This family is unique, in that all patients presented with a myoclonus epilepsy with ragged-red fibers-like phenotype and had a distinctive peripheral neuropathy, while the detected mtDNA 327l (T-C) mutation has been reported to date only in rare cases of mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes

Massive muscle cell degeneration in the early stage of merosin-deficient congenital muscular dystrophy.

Primary merosin-deficient congenital muscular dystrophy (CMD) is a severe form of congenital muscular disorder which is caused by mutations in the laminin alpha2 chain gene (LAMA2). The disease is characterized by marked dystrophic changes in skeletal muscles during early infancy, while little is known about the pathological process of the muscle fiber degeneration. Here, we report the immunohistochemical analysis of skeletal muscle in ten patients with primary merosin-deficient CMD using a panel of molecular markers for skeletal muscle proteins, cellular necrosis, and apoptosis. In the youngest patient (a 52 day old baby), prominent massive muscle cell degeneration occurred in association with the deposition of the C5-9 complement membrane attack complex (MAC). Most of the MAC-positive muscle fibers showed a severely deranged immunoreaction to dystrophin, dystroglycans, and other sarcolemmal proteins. In addition, we found scattered positive signals for apoptosis. Similar but milder changes were also observed in six other patients younger than 1 year. In the patients older than 3 years, muscle fibers positive for MAC and apoptotic signals were barely detectable. These findings imply that massive muscle fiber degeneration occurs in the very early stage of merosin-deficient CMD and may contribute to the severe dystrophic changes in muscle from early infancy.

A case of MERRF associated with chronic pancreatitis.

We report the first case to our knowledge of chronic pancreatitis associated with mitochondrial encephalopathy with the A8344G mitochondrial DNA (mtDNA) mutation. This 10-year-old-girl had suffered from recurrent abdominal pain with elevated serum amylase and lipase since the age of 6, and easy fatigability, tremor and astatic seizures since the age of 8. A biopsy of quadriceps muscle revealed ragged-red-fibers and cytochrome c oxidase deficiency. Analysis of mtDNA in peripheral blood identified an A8344G mutation in the mitochondrial tRNA(Lys) gene. Taken together with physical signs of myoclonic seizures and cerebellar dysfunction, we diagnosed her as myoclonic epilepsy with ragged-red fibers associated with chronic pancreatitis. Although no association between mitochondrial disease and pancreatitis has yet been established, this case suggests it is necessary to consider the participation of mitochondrial abnormality in the pathogenesis of recurrent pancreatitis.

Apoptotic muscle fiber degeneration in distal myopathy with rimmed vacuoles.

Rimmed vacuole formation, tubulofilamentous nuclear inclusions and muscle fiber atrophy are the characteristic pathological findings in distal myopathy with rimmed vacuoles (DMRV). Necrotic muscle fibers were few in number and did not appear to account for the muscle weakness, but the nuclear changes with myofibrillar degeneration followed by rimmed vacuole formation appeared to be the major reason for the muscle fiber atrophy in DMRV. To determine whether the nuclear change in DMRV was related to apoptosis, we examined 15 muscle biopsy specimens immunohistochemically, and 7 of them ultrastructurally. The characteristic tubulofilamentous nuclear inclusions were found in 4 and the typical fragmented apoptotic nuclei in 3 of the 7 muscle biopsy samples examined by electron microscopy. TUNEL-positive nuclei reflecting apoptotic DNA fragmentation were found in 13 of 15 biopsies ranging from a few to approximately 1.5% of myonuclei. Apoptosis-specific protein was expressed in the sarcoplasm of atrophic fibers in 13 biopsies both with or without rimmed vacuoles. These findings suggest that the apoptotic process plays a crucial role in myofibrillar degeneration followed by autophagocytosis, i.e., rimmed vacuole formation, in DMRV.

[Adult form of acid maltase deficiency presenting with pattern of muscle weakness resembling facioscapulohumeral dystrophy].

We report a 61-year-old female patient with adult form of acid maltase deficiency showing many clinical similarities to facioscapulohumeral muscular dystrophy (FSHD). She developed difficulty in raising her right arm in her thirties followed by leg weakness. She had the typical features of FSHD, including bilateral scapular winging sparing the levator scapulae and deltoid muscles, and Beevor's sign. Muscle involvement was asymmetrical. Facial muscles were not affected, while the neck flexor was weak. No muscle shortening or joint contracture was observed. On muscle CT, the lumbar paravertebral, gluteal and thigh muscles were replaced by adipose tissue, while the rectus femoris, gracilis, and sartorius muscles were spared. Serum creatine kinase level was not elevated. Muscle biopsy showed some vacuoles and many granular inclusions with high acid phosphatase activity. Acid maltase activity was very low in both muscle and cultured skin fibroblasts. Absence of shortening of affected muscles appears to be the characteristic finding suggesting metabolic myopathies with minimal fibrosis, rather than FSHD.

[A patient with dermatomyositis and systemic sclerosis with preferential facioscapulohumeral muscle involvement and fatal cardiomyopathy].

We report a 23-year-old man suffering from an overlap syndrome of systemic scleroderma and dermatomyositis who died from severe dilated cardiomyopathy. Because his weakness involved predominantly muscles in the facio-scapulo-humeral regions, he was initially thought to have facioscapulohumeral muscular dystrophy (FSHD) at other hospitals. However, he had also Raynaud phenomenon and low voltages on electrocardiogram. His apparent facial weakness was mainly due to atrophic skin changes. Unlike FSHD, the deltoid and levator scapulae muscles were also atrophic. Deltoid muscle biopsy performed one year earlier at another hospital showed mild myopathic changes without inflammation, but there were scattered thick-walled endomysial capillaries, suggesting inflammatory myopathy. Biceps brachii muscle biopsy in our hospital showed marked inflammation with perifascicular atrophy. In this patient, the cardiac muscle involvement progressed together with the skeletal muscle inflammation before scleroderma became apparent.

Siblings of Schwartz-Jampel syndrome with abnormal muscle computed tomographic findings.

Schwartz-Jampel syndrome (SJS) is a disorder characterized by myotonia, joint contractures, skeletal abnormalities, facial dysmorphism and growth retardation. We present two boys of ages 4 and 8 years with SJS. Their clinical, electromyographic and histopathological findings were similar to those described, except for computed tomography (CT) images that revealed diffuse high attenuation in sternocleidomastoid muscles and low attenuation in the paraspinal, quadriceps, sartorius, soleus and gastrocnemius muscles. This is the first report describing abnormal muscle CT findings associated with SJS. Additional studies of muscle CT might help to improve understanding of the pathogenesis of SJS.

[Congenital nemaline myopathy with mitochondrial abnormalities. An adult case report].

We report a 42-year-old male suffering from congenital nemaline myopathy accompanied with mitochondrial abnormalities in his muscle biopsy. He had a dysmorphic face with a high-arched and narrow palate and slowly progressive generalized muscle weakness. He was still able to walk with a cane. CT showed symmetrical muscle atrophy and low densities in the thigh muscles, especially in the posterior compartment, and in the soleus muscles. Preferential posterior thigh involvement was unusual in congenital nemaline myopathy. The lumbar quadrate and paravertebral muscles were relatively well preserved; these muscles were reported to be severely involved in adult-onset nemaline myopathy patients. Muscle biopsy findings were consistent with nemaline myopathy; nemaline rods in approximately 10% of fibers, type 1 fiber atrophy, and type 2B fiber deficiency. In addition, ragged-red fibers were scattered and focal cytochrome c oxidase (CCO) deficiency was present. Formazan granules were large on succinate dehydrogenase stain. Many fibers with nemaline rods showed focal CCO deficiency. On electron microscopy, large (megaconial) mitochondria were lined regularly between Z lines. PCR and Southern blot analysis of muscle mitochondrial DNA revealed multiple deletions. It remains to be clarified whether mitochondrial abnormalities are primarily related to nemaline myopathy or secondarily induced phenomenon after a long-standing disease process.

Walker-Warburg syndrome is genetically distinct from Fukuyama type congenital muscular dystrophy.

A female patient who fulfilled the diagnostic criteria of Walker-Warburg syndrome had muscle biopsy finding of muscular dystrophy. There was normal expression of merosin (laminin alpha2 chain) and dystrophin and only slightly reduced dystrophin-associated glycoprotein expression. On genetic analysis, she had no specific haplotype, the common mutation of 3kb insertion, or point mutations in the Fukuyama-type congenital muscular dystrophy gene, suggesting that the two diseases are not genetically identical.