Emerin deficiency at the nuclear membrane in patients with Emery-Dreifuss muscular dystrophy.

Mutations in the STA gene at the Xq28 locus have been found in patients with X-linked Emery-Dreifuss muscular dystrophy (EDMD). This gene encodes a hitherto unknown protein named 'emerin'. To elucidate the subcellular localization of emerin, we raised two antisera against synthetic peptide fragments predicted from emerin cDNA. Using both antisera, we found positive nuclear membrane staining in skeletal, cardiac and smooth muscles in the normal controls and in patients with neuromuscular diseases other than EDMD. In contrast, a deficiency in immunofluorescent staining of skeletal and cardiac muscle from EDMD patients was observed. A 34 kD protein is immunoreactive with the antisera--the protein is equivalent to that predicted for emerin. Together, our findings suggest the specific deficiency of emerin in the nuclear membrane of muscle cells in patients with EDMD.

Mutations in the integrin alpha7 gene cause congenital myopathy.

The basal lamina of muscle fibers plays a crucial role in the development and function of skeletal muscle. An important laminin receptor in muscle is integrin alpha7beta1D. Integrin beta1 is expressed throughout the body, while integrin alpha7 is more muscle-specific. To address the role of integrin alpha7 in human muscle disease, we determined alpha7 protein expression in muscle biopsies from 117 patients with unclassified congenital myopathy and congenital muscular dystrophy by immunocytochemistry. We found three unrelated patients with integrin alpha7 deficiency and normal laminin alpha2 chain expression. To determine if any of these three patients had mutations of the integrin alpha7 gene, ITGA7, we cloned and sequenced the full-length human ITGA7 cDNA, and screened the patients for mutations. One patient had splice mutations on both alleles; one causing a 21-bp insertion in the conserved cysteine-rich region, and the other causing a 98-bp deletion. A second patient was a compound heterozygote for the same 98-bp deletion, and had a 1-bp frame-shift deletion on the other allele. A third showed marked deficiency of ITGA7 mRNA. Clinically, these patients showed congenital myopathy with delayed motor milestones. Our results demonstrate that mutations in ITGA7 are involved in a form of congenital myopathy.

Dysferlin, a novel skeletal muscle gene, is mutated in Miyoshi myopathy and limb girdle muscular dystrophy.

Miyoshi myopathy (MM) is an adult onset, recessive inherited distal muscular dystrophy that we have mapped to human chromosome 2p13. We recently constructed a 3-Mb P1-derived artificial chromosome (PAC) contig spanning the MM candidate region. This clarified the order of genetic markers across the MM locus, provided five new polymorphic markers within it and narrowed the locus to approximately 2 Mb. Five skeletal muscle expressed sequence tags (ESTs) map in this region. We report that one of these is located in a novel, full-length 6.9-kb muscle cDNA, and we designate the corresponding protein 'dysferlin'. We describe nine mutations in the dysferlin gene in nine families; five are predicted to prevent dysferlin expression. Identical mutations in the dysferlin gene can produce more than one myopathy phenotype (MM, limb girdle dystrophy, distal myopathy with anterior tibial onset).

Absence of extraocular muscle pathology in Duchenne's muscular dystrophy: role for calcium homeostasis in extraocular muscle sparing.

Duchenne muscular dystrophy (DMD) is characterized by clinical weakness and progressive necrosis of striated muscle as a consequence of dystrophin deficiency. While all skeletal muscle groups are thought to be affected, enigmatically, the extraocular muscles (EOM) appear clinically unaffected. Here we show that dystrophin deficiency does not result in myonecrosis or pathologically elevated levels of intracellular calcium ([Ca2+]i) in EOM. At variance with a previous report, we find no evidence for dystrophin-related protein/utrophin up-regulation in EOM. In vitro experiments demonstrate that extraocular muscles are inherently more resistant to necrosis caused by pharmacologically elevated [Ca2+]i levels when compared with pectoral musculature. We believe that EOM are spared in DMD because of their intrinsic ability to maintain calcium homeostasis better than other striated muscle groups. Our results indicate that modulating levels of [Ca2+]i in muscle may be of potential therapeutic use in DMD.

MKBP, a novel member of the small heat shock protein family, binds and activates the myotonic dystrophy protein kinase.

Muscle cells are frequently subjected to severe conditions caused by heat, oxidative, and mechanical stresses. The small heat shock proteins (sHSPs) such as alphaB-crystallin and HSP27, which are highly expressed in muscle cells, have been suggested to play roles in maintaining myofibrillar integrity against such stresses. Here, we identified a novel member of the sHSP family that associates specifically with myotonic dystrophy protein kinase (DMPK). This DMPK-binding protein, MKBP, shows a unique nature compared with other known sHSPs: (a) In muscle cytosol, MKBP exists as an oligomeric complex separate from the complex formed by alphaB-crystallin and HSP27. (b) The expression of MKBP is not induced by heat shock, although it shows the characteristic early response of redistribution to the insoluble fraction like other sHSPs. Immunohistochemical analysis of skeletal muscle cells shows that MKBP localizes to the cross sections of individual myofibrils at the Z-membrane as well as the neuromuscular junction, where DMPK has been suggested to be concentrated. In vitro, MKBP enhances the kinase activity of DMPK and protects it from heat-induced inactivation. These results suggest that MKBP constitutes a novel stress-responsive system independent of other known sHSPs in muscle cells and that DMPK may be involved in this system by being activated by MKBP. Importantly, since the amount of MKBP protein, but not that of other sHSP family member proteins, is selectively upregulated in skeletal muscle from DM patients, an interaction between DMPK and MKBP may be involved in the pathogenesis of DM.

Integrins (alpha7beta1) in muscle function and survival. Disrupted expression in merosin-deficient congenital muscular dystrophy.

Mutations in genes coding for dystrophin, for alpha, beta, gamma, and delta-sarcoglycans, or for the alpha2 chain of the basement membrane component merosin (laminin-2/4) cause various forms of muscular dystrophy. Analyses of integrins showed an abnormal expression and localization of alpha7beta1 isoforms in myofibers of merosin-deficient human patients and mice, but not in dystrophin-deficient or sarcoglycan-deficient humans and animals. It was shown previously that skeletal muscle fibers require merosin for survival and function (Vachon, P.H., F. Loechel, H. Xu, U.M. Wewer, and E. Engvall. 1996. J. Cell Biol. 134:1483-1497). Correction of merosin deficiency in vitro through cell transfection with the merosin alpha2 chain restored the normal localization of alpha7beta1D integrins as well as myotube survival. Overexpression of the apoptosis-suppressing molecule Bcl-2 also promoted the survival of merosin-deficient myotubes, but did not restore a normal expression of alpha7beta1D integrins. Blocking of beta1 integrins in normal myotubes induced apoptosis and severely reduced their survival. These findings (a) identify alpha7beta1D integrins as the de facto receptors for merosin in skeletal muscle; (b) indicate a merosin dependence for the accurate expression and membrane localization of alpha7beta1D integrins in myofibers; (c) provide a molecular basis for the critical role of merosin in myofiber survival; and (d) add new insights to the pathogenesis of neuromuscular disorders.

Effect of artificial (CTG) repeat expansion on the expression of myotonin protein kinase (MtPK) in COS-1 cells.

A major challenge in the study of a new genetic entity called triplet-repeat disease is to identify the role of triplet repeats in the pathogenesis of the disease. We have developed a strategy to demonstrate the effect in the 3'-untranslated end of the (CTG) repeats in myotonic dystrophy gene (MtPK) and found that repeat expansion (CTG46) causes a slight decrease in the translation rate of MtPK cDNA which correlates with the finding in patients with myotonic dystrophy of a low amount of MtPK protein in muscle. These results provide an important clue for characterizing the genetic abnormality in other triplet-repeat diseases.

Immunoblot analysis of dystrophin-related protein (DRP).

Polyclonal antibodies against the carboxy-terminal portion of dystrophin-related protein (DRP), the putative autosomal gene product which shares sequence homology with dystrophin, show the clear expression of DRP in mouse fetal muscle and in cultured human muscle cells, but not in mature mouse or human muscle. DRP has the same molecular mass as X-linked dystrophin and is recovered from the membrane fraction, but is associated with membranes more loosely than dystrophin.

Calcium is essential for both the membrane binding and lytic activity of pore-forming protein (perforin) from cytotoxic T-lymphocyte.

The influence of Ca on the membrane binding and lytic activity of lymphocyte pore-forming protein (perforin) was studied. In the absence of Ca, perforin did not bind to the target membranes and did not support lysis of the target cells. In contrast, in the presence of Ca perforin was able to bind to the cell membrane (Km greater than 0.2 mM). Almost all the perforin molecules bind to the membrane within 1 min at 0 degrees C. The addition of EDTA abolished the binding, indicating that the effects of Ca on the membrane binding are reversible. On the other hand, the perforin-mediated lysis of target cells was temp-dependent and also required the presence of Ca in the reaction mixture (Km = 0.05 mM). The difference between the Km values for the membrane binding and lytic activity suggests the presence of two distinct Ca-requiring steps in perforin-mediated target cell lysis.

Apolipoprotein B is a major perforin inhibitor protein in human serum.

We purified the high-molecular-weight perforin inhibitor protein from normal human serum using DEAE-cellulose, HPLC-gel filtration and hydroxylapatite chromatography. This protein was shown to be identical to the serum apolipoprotein B-100 in terms of amino acid composition and the sequence of the digested peptides. This inhibitor protein not only inhibits the membrane binding activity of perforin but also the pore insertion activity of membrane-bound perforin.

Changes in pre-mRNA splicing factors during neural differentiation in P19 embryonal carcinoma cells.

Alternative RNA splicing can be regulated in a highly cell- and tissue-specific or developmentally specific manner. In neurons, the functions of many gene products, such as those of trk genes are regulated by alternative splicing. In this paper the mechanism of neural-specific RNA splicing is investigated using trk genes as models. First, we confirm the splicing patterns of trk transcripts during neural differentiation of P19 embryonal carcinoma (EC) cells. The full-length form of trk B was expressed in the neuronal state. In contrast, both the full-length and truncated forms of trk C were expressed constitutively in all differentiation states. However, two alternatively spliced forms with either 42- or 117-nucleotide insertions in the tyrosine kinase domain were detected only in the neuronal state. Thus, the expression of functional trk B and C was found to be regulated by alternative splicing during neural differentiation. To examine the molecular basis of neural-specific splicing, and how splicing regulation is modulated in different neurons. The expression of a number of general splicing factors was studied. The mRNA levels of the splicing factors ASF/SF2, U2AF SF3a, p54nrb and PTB was found to decrease rapidly during differentiation. In contrast, Nova, an RNA-binding protein was expressed in the neuronal state. We also found that the levels of two SR proteins, members of a family of splicing factors, increased in the neuronal state. These results suggest that the stoichiometric balance among some splicing factors, including SR proteins, may be associated with the alternative splicing of trk transcripts during differentiation.

Expression of a novel human myotonin protein kinase (MtPK) cDNA clone which encodes a protein with a thymopoietin-like domain in COS cells.

A full-length cDNA of human myotonin protein kinase (MtPK) was cloned and expressed in COS-1 cells. MtPK is recovered from the cytosolic fraction of the COS extract as a 70 kDa protein, which coincides with the size deduced from the predicted amino acid sequence. The sequence has a significant homology to thymopoietin, a peptide hormone of the thymus. Biochemical characteristics of MtPK expressed in COS-1 cells and its expression in rat tissues are investigated.

Laminin alpha2 chain-null mutant mice by targeted disruption of the Lama2 gene: a new model of merosin (laminin 2)-deficient congenital muscular dystrophy.

Using the gene targeting technique, we have generated a new mouse model of congenital muscular dystrophy (CMD), a null mutant for the laminin alpha2 chain. These homozygous mice, designated dy3Kldy3K, are characterized by growth retardation and severe muscular dystrophic symptoms and die by 5 weeks of age. Light microscopy revealed that muscle fiber degeneration in these mice begins no later than postnatal day 9. In degenerating muscles, considerable amounts of TUNEL positive nuclei were detected as well as DNA laddering, suggesting increased apoptotic cell death was involved in the process of muscle fiber degeneration.

Prominent expression of glial cell line-derived neurotrophic factor in human skeletal muscle.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to exert neurotrophic effects on motor neurons as well as mesencephalic dopaminergic neurons. Because GDNF promotes survival of motor neurons in vivo and in vitro and rescues motor neurons from naturally occurring cell death, the potential use of GDNF for treatment of motor neuron diseases has been a major focus of recent research. The expression of GDNF in humans, however, has not been fully examined. In the present study, we examined the expression of GDNF in adult human muscle by Northern blot, reverse transcriptase polymerase chain reaction (RT-PCR), and immunohistochemical analyses to address physiological roles of GDNF in humans. Northern blot analysis demonstrated high expression of GDNF mRNA in human skeletal muscle when compared to that of mouse. Intense GDNF immunoreactivity was observed in the vicinity of plasma membranes of skeletal muscle, particularly at neuromuscular junctions. GDNF immunoreactivity was also observed within the axons and surrounding Schwann cells of peripheral nerves. However, RT-PCR detected expression of GDNF mRNA only in skeletal muscle, and not within the anterior horn cells of human spinal cord. These results suggest that GDNF is produced by skeletal muscle and taken up at the nerve terminals for retrograde transport by axons. Thus, GDNF in human skeletal muscle may be involved in promoting motor neuron survival as a target-derived neurotrophic factor.

Epilepsy and mental retardation in a subset of early onset 4q35-facioscapulohumeral muscular dystrophy.

The gene for facioscapulohumeral muscular dystrophy (FSHD) has been mapped to chromosome 4q35. In most patients with FSHD, a deletion of 3.3 kb tandemly repeated units within the EcoRI fragment that can be detected by probe p13E-11 is associated with the disease. To elucidate the relation between the phenotype and the genotype in FSHD, we examined 91 Japanese unrelated families with a clinical diagnosis of FSHD (140 patients, 205 healthy individuals). Of these, 78 families (86%) had 4q35-FSHD (127 patients), in which 20 patients (20/127, 16%) were classified as early onset FSHD. We found that all nine patients with the smallest EcoRI fragments (10 to 11 kb) were classified among the early onset group (9/20, 45%), and these patients showed a high frequency of both epilepsy (4/9, 44%) and mental retardation (8/9, 89%). In contrast, none of the remaining patients with 4q35-FSHD had epilepsy or mental retardation. We conclude that FSHD patients with a large gene deletion in the FSHD gene region tend to have a higher chance of showing severe clinical phenotypes with CNS abnormalities. This finding may have practical implications for genetic counseling, although the molecular genetic background remains unclear.

Congenital muscular dystrophy: Clinical and pathologic study of 50 patients with the classical (Occidental) merosin-positive form.

We studied 50 patients with the merosin-positive form of congenital muscular dystrophy (MP-CMD) clinically and pathologically. The frequency of MP-CMD in our laboratory was approximately one-half that of the Fukuyama type and one-sixth that of Duchenne muscular dystrophy. The early signs of MP-CMD included decreased fetal movement during pregnancy (14%) and poor suck (42%), floppiness (30%), and respiratory difficulty (16%) in early infancy. Eighty-six percent of the patients had delayed motor development. Ninety-two percent of the patients followed beyond age 4 years had learned to walk. The disease was relatively slowly progressive, except in six patients who rapidly lost ambulation. Almost all patients had normal IQ, except four who were mildly to moderately retarded. Of the patients examined by cranial CT/MRI, 24% showed cerebral atrophy and 11% had areas of white matter lucency. Muscle biopsy results in those younger than 5 years showed mild dystrophic changes consisting of variation in fiber size and scattered necrotic and regenerating fibers. In older children, there were additional chronic dystrophic changes, including fiber splitting (32%), moth-eaten appearance (32%), marked fatty replacement (46%), and abnormal fiber type distribution (59%). The manifestations of MP-CMD were generally milder and more slowly progressive than those of the Fukuyama type and merosin-negative form of congenital muscular dystrophy.

Dystrophinopathy in isolated cases of myopathy in females.

X-linked dystrophinopathy is the most common cause of isolated cases of myopathy in males. To investigate dystrophin abnormalities as a cause of myopathy in girls and women, we used dystrophin immunocytochemistry to study muscle biopsies from 505 girls and women with neuromuscular disease. Forty-six muscle biopsies showed a combination of fibers containing or lacking dystrophin; this mosaic immunostaining pattern denoted a carrier status. Twenty-one of 46 (45.6%) had a family history of Duchenne muscular dystrophy in males. Twenty-five of 46 (54.3%) were isolated cases, with no previous family history of neuromuscular disorder. The laboratory findings of the isolated cases were consistent with the familial cases; all showed myopathic histopathology and abnormal elevations of serum CK. The clinical presentations of the isolated cases varied but were consistent with the familial cases: 40% (10/25) of isolated cases showed proximal limb weakness before age 10, 24% (6/25) presented with myalgias or cramps, 24% (6/25) presented with incidental findings of grossly elevated CK levels, 8% (2/25) noted easy fatigue, and 4% (1/25) had slowly progressive proximal limb weakness beginning at age 45. From our data, the clinical criteria for consideration of an underlying dystrophinopathy in isolated female cases of myopathy are CK levels greater than 1,000 IU/l and myopathic histopathology. About 10% of the isolated cases of hyperCKemic myopathy (25/210) were proven by dystrophin analysis to have a dystrophinopathy as the cause of their disease (manifesting carriers of Duchenne dystrophy). However, we feel that this may be an underestimate. The correct diagnosis in these patients is imperative for appropriate genetic counseling to the patients and their families.

Tongue atrophy in facioscapulohumeral muscular dystrophy.

Involvement of the lingual muscle is considered one of the exclusion criteria of facioscapulohumeral muscular dystrophy (FSHD). In a series of 151 Japanese patients with 4q35-FSHD, seven patients (4.6%) had tongue atrophy with abnormal MRI findings and typical myogenic patterns of electromyography. All seven patients belong to a group of early-onset FSHD with large gene deletions on chromosome 4q35. Our result suggests that the patients with 4q35-FSHD could have myopathic tongue atrophy.