A genome-wide association study reveals a quantitative trait locus for calf mortality on chromosome 9 in Japanese Black cattle.

Calf mortality is a major problem affecting cattle production. To identify genetic variants associated with calf mortality in Japanese Black cattle, we evaluated calf mortality as a categorical trait using a threshold model and conducted a GWAS. We identified two SNPs between 32 549 297 and 32 606 924 bp on bovine chromosome 9 that were significantly associated with calf mortality from 61 to 180 days after birth. The SNP showing the highest association was localized at a region 624 bp downstream of exon 4 of the anti-silencing function 1A histone chaperone gene (ASF1A) that promotes DNA damage repair, and the null mice, which exhibit pre- and postnatal lethality. This association was also detected using the breeding value of 334 sires. The frequency of the risk allele in Japanese Black cattle from locations across Japan was 0.013; although the frequency of ASF1A risk allele was low, it is widespread in the Japanese Black cattle population. Thus, it may be necessary to routinely monitor the cattle population for the presence of this allele.

Direct DNA sequencing from colony: analysis of multiple deletions of mitochondrial genome.

We have developed a rapid and efficient nucleotide sequencing technique, named the colony direct sequencing method, which combines both the conventional cloning method for picking up a single gene and the polymerase chain reaction (PCR) method for amplifying the gene directly from a colony. In the present study, the colony direct PCR product was used both for identification of the DNA insert and for nucleotide sequencing by an automated DNA analysis system. A nucleotide sequence of 300 to 400 bp could be determined within 13 h after picking the bacterial colonies on LB medium plates. We applied this method to sequencing of junctional regions of multiple deleted mtDNAs in two siblings with inherited recurrent myoglobinuria. Mitochondrial DNA fragments with deletions were amplified by PCR and then cloned into plasmids. Among 48 white colonies propagated on LB medium plates, nine different clones were identified by PCR directly from colonies. Determination of six different junctional sequences disclosed involvement of directly repeated sequences of 2 to 12 bp in length on each side of the deletions. We believe that the colony direct sequencing method will be a powerful tool in molecular genetics for identification of a single gene among polymorphic DNAs.

Autosomal dominant hyaline body myopathy presenting as scapuloperoneal syndrome: clinical features and muscle pathology.

Hyaline bodies are rare subsarcolemmal aggregates in type 1 fibers of the skeletal muscle, stain pale pink with hematoxylin-eosin and pale green with the modified Gomori trichrome, and lack reactivity for glycogen and oxidative enzymes. We report clinical findings of autosomal-dominant hyaline body myopathy in seven members in four generations and muscle biopsy findings in two of them. Slowly progressive muscle weakness and atrophy developed with scapuloperoneal distribution; age at onset was from the first to the fifth decade. Muscle biopsy showed subsarcolemmal hyaline bodies in approximately 20% of type 1 fibers. Hyaline bodies showed myofibrillar ATPase activity after acid pre-incubation. Immunohistochemically, they stained intensely with myosin heavy chain (slow), but not with myosin heavy chain (fast). Ultrastructurally, they consisted of granules sometimes in linear array, filaments, and amorphous materials. These findings suggest that hyaline bodies may be products of degeneration of myosin heavy chain (slow).

Lymphatic transport of cholesterol in normocholesterolemic rats treated with pravastatin, an inhibitor of HMG-CoA reductase.

Lymphatic absorption and transport of cholesterol and triacylglycerols were examined in rats treated with pravastatin, an inhibitor of 3-hydroxy-3-methyglutaryl-CoA (HMG-CoA) reductase. Pravastatin-treatment for 1, 7 and 28 days did not affect the recovery of cholesterol and triacylglycerols during 24 h after the lipid administration: the recovery was 52-59% and 82-93% for cholesterol and triacylglycerols, respectively. Rats treated with pravastatin for 28 days had a higher lymphatic recovery of the lipids during 3-6 h after the lipid administration than did control rats. Pravastatin treatment did not affect the ratio of phospholipid to cholesterol in the gut mucosa, the fatty acid composition of the lymph and mucosal lipids. We concluded that an inhibitor of HMG-CoA reductase would exert no adverse effect on absorption of fat-soluble nutrients by gut.

Multiplicity of abnormal dystrophin in Becker muscular dystrophy. A Becker muscular dystrophy gene frequently produced two smaller sizes of dystrophin.

Dystrophin is a muscle cytoskeletal protein with a molecular mass (MM) of approximately 420 kDa and an isoelectric point (pI) of approximately 5.5, which is abnormal in size and/or abundance in Becker muscular dystrophy (BMD). We investigated the abnormality of dystrophin molecule in muscles biopsied from 23 BMD patients using the two-dimensional gel electrophoresis (TDGE). We found 7 protein spots which reacted specifically with the monoclonal anti-dystrophin antibody (mAb) A1C raised against N-terminal domain of the normal dystrophin. These spots were focused on the two-dimensional gel at the same position as the normal dystrophin (#1), at the position with MM approximately 480 kDa/pI approximately 5.35 (#2), the position with MM approximately 400-330 kDa/pI approximately 5.51-5.47 (#3), the position with MM approximately 300 kDa/pI approximately 5.4 (#4), the position with MM approximately 235-250 kDa/pI approximately 5.53-5.5 (#5), the position with MM approximately 165 kDa/pI approximately 6.0 (#6), and the position with MM approximately 160 kDa/pI approximately 5.75 (#7). These spots were classified into five patterns in individuals, that is, #1 alone in 3 patients, #3 alone in 1, the combination of #3 and 5 in 17, the combination of #1, 3 and 5 in 1 and the combination of #1, 2, 4, 6 and 7 in 1. The combination of #3 and 5 was observed in 17 of 23 patients (75%).(ABSTRACT TRUNCATED AT 250 WORDS)

Cytoplasmic body and mitochondrial DNA deletion.

A patient with chronic progressive external ophthalmoplegia (CPEO) who had abundant cytoplasmic bodies in muscle fibers and a deletion of mitochondrial DNA is reported. The patient was a 26-year-old male suffering from ophthalmoplegia from age 21. He had a marfanoid skeletal abnormality and perceptive hearing loss, but had neither retinopathy, ataxia, nor dementia. In the mitochondria isolated from the biopsied skeletal muscle, NADH-ubiquinone oxidoreductase activity was slightly decreased, succinate-cytochrome c reductase activity was slightly increased, and cytochrome c oxidase activity remained normal. Southern blot analysis of the muscle DNA identified heteroplasmy composed of a normal-sized mitochondrial DNA and a mutant mitochondrial DNA with a 4.2-kilobase deletion. The PCR plus S1 analysis showed that the deletion extended from nucleotide position 7860 +/- 60 to 12,090 +/- 70. The histological studies of the biopsied muscle revealed ragged-red fibers and cytochrome c oxidase-negative fibers in 15.7% and 18.6% of the muscle fibers, respectively. Other conspicuous histological change was abundant cytoplasmic bodies surrounded by clusters of abnormal mitochondria. The cytoplasmic bodies were found preferentially in type 1 fibers, and exclusively in cytochrome c oxidase-negative fibers and in ragged-red fibers. Focal existence of cytoplasmic bodies in muscle fibers with abnormal mitochondria suggests that segregated distribution of the abnormal mitochondria with deleted mitochondrial DNA is involved in the pathogenesis of cytoplasmic bodies.

Immunostaining of dystrophin and utrophin in skeletal muscle of dystrophinopathies.

Immunostaining of biopsied skeletal muscle of 4 Duchenne (DMD), 12 Becker muscular dystrophy (BMD) and 3 DMD carriers' was performed using monoclonal antibodies against dystrophin and utrophin. In DMD, dystrophin-negative staining was observed except for revertant fibers which showed different stain patterns for each antibody. In 7 BMDs, there was faint/patchy stain in cases of deletion between exons 45-52, while in one case there was deletion between exons 12-17 and no stain was noted relevant to the deletion site. Moreover, in 2 cases of undetectable deletion, antibodies which recognize a terminal portion of the C-terminal domain revealed the absent stain. In DMD, the utrophin-positive fibers corresponded to dystrophin-negative fibers. In BMD, this relationship did not necessarily occur in each fiber. In DMD carriers, a cluster of dystrophin-negative fibers which was positive for utrophin were prominent. In dystrophinopathy, the immunostaining of dystrophin and utrophin is useful, in combination with dystrophin gene analysis to make a definite diagnosis.

Progressive external ophthalmoplegia and myositis.

We reported a senile male patient with progressive external ophthalmoplegia (PEO) and myositis. The ophthalmoplegia was severe, but other neuromuscular features were nearly normal. Muscle enzymes in serum were moderately elevated. Autoimmune, endocrinological or malignant diseases were not observed during the previous 4 years. Pathology of non-weak limb muscles biopsied twice was consistent with active inflammatory myopathy. The ragged-red or cytochrome c oxidase-negative fibers, which are a hallmark of mitochondrial myopathy with PEO, were not increased in comparison with age-matched control muscles. Analysis of mitochondrial DNA in muscle by the Southern blot method did not reveal any deletions. It was concluded that the inflammatory myopathy, myositis clinically localized at the ocular muscles, is an important and distinct disorder in PEO.

[Immunohistochemical localization of chymase; a mast cell marker and clinical significance in diseased human skeletal muscle].

UNLABELLED: In the advanced stage of dystrophinopathy, cardiac dysfunction is a serious complication for prognosis. Recently, an angiotensin converting enzyme (ACE), which converts angiotensin (A) 1 to A 2, has been reported to be effective for cardiac insufficiency. The A 2 is produced more dominantly in the path via the production of a neutral serine protease, chymase (MW 25,000), secreted from the mast cell. We have observed localization of chymase in diseased human skeletal muscle tissues, and evaluated its clinical significance. The frozen muscle biopsied specimens from 91 neuromuscular disorders (muscular dystrophies, inflammatory myopathies and neurogenic muscular disorders) were stained by using monoclonal antibody against the chymase, and the positive cells in a whole sectional field were counted. In the serial sections, we also performed routine histochemistry and immunostainings of immunological markers (CD4, CD8 and others) as well as the apoptotic proteins for comparison. RESULTS: The chymase-positive mast cells were scattered mainly in the endomysium, partly in the perimysium and around small vessels. Although the positivity was not disease specific, more numerous strongly positive cells were observed in dystrophinopathy and inflammatory myopathies, but less in myotonic dystrophy and neurogenic muscle disorders. In the normal control muscle, however, strongly positive cells appeared less frequently than in the above mentioned diseased muscles. The chymase-positive cells partly corresponded to the ubiquitin-positive ones, but perforin, granzyme A, Fas and Bcl-2 did not. In conclusion, the chymase-positive mast cell may play a primary or secondary role in the diseased muscle, and their more abundant appearance in dystrophinopathy and some other myopathies suggest the effectiveness of an ACE blocker, an anti-chymase drug.

[Immunostaining of anti-Fas IgG1 antibody in diseased human muscle].

Immunostaining of the Fas antigen using the anti-Fas IgG1 antibody was performed on biopsied human diseased muscles. The immunostaining showed negative results in the control muscles. In dystrophinopathy [DMD and BMD], positivity was seen mainly in type 2 fibers with no correlation to the opaque fibers and histochemical Ca2+ loading fibers in DMD. In DMD carriers, a relative correlation was seen between dystrophin-negative and Fas-positive fibers. In distal myopathy with rimmed vacuoles, fibers with positive staining in the vacuoles but negative in their membranes were seen at high frequency. In FSH, a very low frequency of positivity was seen. And in myotonic dystrophy, positivity was seen in the type 2 fibers containing the internal nuclei. In inflammatory myopathies, strong positivity was seen in the medium size fibers, and mild to moderate positivity in the fibers facing the perimysium. In neurogenic muscular disorders, fibers with concave borders or highly atrophic fibers showed Fas-positivity. In conclusion, there was no disease-specific Fas reaction in the human pathologic muscle samples, but the high positivity was apparent in some myopathies. This fact of Fas antigen would reflect a pathologic state in the skeletal muscle.

[Expression of MHC and cell adhesion molecules in muscular sarcoidosis].

Biopsied skeletal muscles from 5 patients with muscular sarcoidosis (nodular type; 1, and myopathic type; 4) were immunocytochemically examined. All biopsies presented granulomatous changes. Atrophic or regenerating muscle fibers adjacent to granuloma demonstrated compression or ischemic changes. In the center of the granuloma, CD68+ epitheloid cells and giant cells, and CD4+ T cells were localized. At the periphery of the granuloma, CD4+ T cells, CD8+ T cells, CD20+ B cells, and CD68+ macrophages were found. Expression of HLA-A,B,C was diffuse in the muscle fibers. Expression of HLA-DR and ICAM-1 was more prominent near the granuloma or perifascicular fibers, and that of LFA-3 was moderate in those lesions. VCAM-1 was expressed in endothelial cells and macrophages near the granuloma. Those findings indicate that interferon-gamma or TNF-alpha produced by infiltrating inflammatory cells may induce expression of these immunologic markers or adhesion molecules. Immunocytochemical differences between the nodular and myopathic forms of sarcoidosis are not evident, but either localization or abundance of granuloma in muscle bulks is relevant to weakness or atrophy of clinically affected muscle.

[Expression of the heat shock protein 70 in inflammatory myopathies].

Heat shock protein 70 (HSP 70) expression was immunohistochemically observed in diseased muscle fibers of 35 patients with dermatomyositis (DM) and 7 with polymyositis (PM). In DM, HSP 70 was localized in the sarcoplasm of type 1 fibers adjacent to the small vessels showing deposits of complement components in 13 patients and in the atrophic fibers at perifascicular regions in 7. HSP 70 was also expressed more preferentially in the small vessels rather than in the sarcoplasm in 13 DM patients. In PM, the expression of HSP 70 was blurred in all fibers including non-necrotic fibers invaded by T cells. In conclusion, HSP 70 is likely more frequently to be expressed in the sarcoplasm of DM than PM due to probable ischemic insults.

[Acquired unilateral blepharoptosis by wearing contact-lenses in young adult women].

Four young unmarried women developed unilateral non-fluctuating blepharoptosis by wearing contact-lenses. Past and family histories were unremarkable. Blepharoptosis insidiously occurred within a few years after wearing lenses. Contralateral lid was quite normal. No abnormalities was observed in pupils, extraocular muscles, ocular positioning and other systems. So far recognized causes of blepharoptosis were ruled out through extensive clinical and laboratory observations. Improvement was insufficient even after wearing lenses off. Pathogenesis is probably due to repeated minor trauma to the levator palpebral muscle and its tendon secondary to frequent wearing on/off contact-lenses.

[Corticosteroid responsive inflammatory myopathy with autoantibody against Golgi apparatus].

A 51-year-old woman of progressive myopathy predominantly involving proximal groups of limb-muscle was reported. Serum enzymes originating from the skeletal muscle always remained within normal range, and a positive autoantibody against Golgi apparatus and the SS-A (Ro) antibody in serum were noted. In muscle biopsy performed twice, extensive degenerating/regenerating fibers and sparce inflammatory cells between connective tissue elements in light microscopy, and disrupted lamellar structure of Golgi apparatus in electron microscopy were observed. Corticosteroid therapy was markedly effective. In the present case of a "so-called" limb-girdle syndrome, humoral autoantibodies, especially against Golgi apparatus, could induce a metabolic disturbance in the muscle fiber.

[Clinico-pathological analysis of vesiculotubular myopathy of adult onset].

The patient was a 50-year-old house wife. There were complicated consanguineous marriages in the family tree. Since 30 years of age, she had suffered from progressive limb muscle weakness, but without myalgia and myasthenia. At present, she was wheelchair-bound. Physical examinations showed obesity, congenital livedo racemosa, epicanthus palpebrae and left renal defect. Neurologically, facial, anterior cervical, and iliopsoas muscles were well preserved, but others were severely involved. Laboratory examinations revealed mildly elevated myogenic serum enzymes, and myogenic changes on needle EMG. In her muscle biopsy from the left rectus femoris muscle, there were no inflammatory changes, but marked variations of the fiber size as well as adipose tissue replacement were recognized. Strickingly, basophilic masses located in the center of the sarcoplasm were present in about 10% of the fibers. Histochemically, the masses were present in both type 1 and 2 fibers, and exhibited almost similar stained patterns to the tubular aggregates, but were dystrophin-, GRP78- and clathrin-positive. Under electron microscopy, the masses consisted of aggregates of the vesiculotubular structure, measuring approximately from 60 nm to more than 6 microns in diameter, which were continuous with T system/sarcoplasmic reticulum and were clearly segregated from myofilaments. This is a chronic progressive muscular disorder of adult onset with the peculiar pathological finding of vesiculotubular structure.

[Immunostaining of anti-Bcl-2 antibody in diseased human muscles].

Immunostaining of Bcl-2 protein which represses apoptosis was performed on 178 biopsied human pathologic muscles and 10 control muscles by the ABC method using two monoclonal anti-Bcl-2 antibodies. Bcl-2 in control muscles was positive mainly in nuclear membrane and cytoplasm in type 2 fibers (especially type 2B fibers), and negative in type 1 fibers. In myopathies, it was not expressed in type 2C (regenerating) fibers, and its expression in atrophic fibers such as forming pyknotic nuclear clumps was strong. In inflammatory myopathies, expression was observed in infiltrating lymphocytes, and especially in dermatomyositis in atrophic fibers facing perimysium. In mitochondrial myopathies, the positivity was observed only in type 2 ragged-red fibers. In muscles of neurogenic disorders, both small angulated fibers and atrophic grouped fibers were strongly positive. Western blot analysis using anti-Bcl-2 antibody showed a single band at 26 kDa in control and diseased skeletal muscles. Compared to immunostaining of Fas antigen in serial sections, both Bcl-2 and Fas were expressed in same atrophic fibers in distal myopathy with rimmed vacuoles. In myotonic dystrophy, they were often expressed in type 2 fibers containing internal nucleus. In carriers of Duchenne dystrophy, Fas-positive but Bcl-2 negative fibers were observed in same dystrophin-negative fibers. In conclusion, expression of Bcl-2 in skeletal muscles might suggest that Bcl-2 plays a role on surviving muscle fibers.

[HTLV-I associated myelopathy (HAM) and sarcoid myopathy].

A 65-year-old house-wife developed dirty erythematous rash on her face in April, 1989. Almost simultaneously, she complained of muscle soreness and weakness on both lower extremities. Pathological findings of the skin biopsy at that time was consistent with those of sarcoidosis with moderate inflammatory cell infiltration. In December, 1989, when she was admitted to our hospital, her lower extremities were paretic with marked spasticity, and mild bladder dysfunction was noted. HTLV-I antibody titers in serum and cerebrospinal fluid were significantly elevated. Biopsied limb skeletal muscle revealed the findings of the sarcoid myopathy with small inflammatory cell infiltration in endomysium. HLA haplotypes showed A24, B7, BW61, CW7, CW8, DR1 and DR4 which show relatively common types of those in HAM. Corticosteroid treatments including the methylprednisolone pulse therapy healed the skin lesion, but did not improve her neurological signs. Paraplegia and urinary disturbance were progressive. It is concluded that the inflammatory sarcoid myopathy with HAM in this patient may be caused by a common abnormal immunological background.

[A mother and her son with autosomal dominant bulbar spinal muscular atrophy].

We reported a 49-year-old mother and her 28-year-old son with autosomal dominantly inherited bulbar spinal muscular atrophy (AD-BSMA). They showed progressive bulbar paresis, muscle wasting and weakness dominant in the proximal groups of limb muscles, and finger tremor. Onset of illness was in adult life. In laboratory examinations, elevated creatine kinase in serum and neurogenic changes either in EMG or muscle biopsy were noted. The son had neither gynecomastia nor abnormal sexual hormone levels which were observed in the sex-linked recessive bulbar spinal muscular atrophy (SR-BSMA). Elongation due to the CAG repeats at the androgen receptor gene of the X chromosome in SR-BSMA was not detected. In conclusion, it is clear that AD-BSMA is different from SR-BSMA on the basis of clinical and genetical aspects.

[Quantitative skeletal muscle pathology of aging regarding ragged-red fibers and cytochrome c oxidase-negative fibers].

A statistical analysis of mitochondrial abnormality of aging in human skeletal muscle fibers was performed. Sixty one muscle samples were obtained from patients with acute medical illness autopsied strictly within 2 hours after death, or with orthopedic or surgical diseases biopsied with informed consents. The patients aged from 16 to 89, averaging 58 +/- 21 years in males and 21 to 92, averaging 55 +/- 20 years in females. Sections were stained by modified Gomori's trichrome, succinate dehydrogenase and cytochrome c oxidase-negative [CCO(-)] fibers approximately in 10,000 fibers in each muscle were evaluated. Both RRF and CCO (-) fibers were not observed below the fourth decade, but sequentially increased with age, especially after the seventh decade. The incidence of CCO (-) fibers was higher than that of RRF. RRF did not necessarily correspond to CCO (-) fibers. The present quantitative pathological result is a useful tool to evaluate the mitochondrial function in fresh human skeletal muscles by the age.