Multitissular involvement in a family with LMNA and EMD mutations: Role of digenic mechanism?

BACKGROUND: Mutations in the EMD and LMNA genes, encoding emerin and lamins A and C, are responsible for the X-linked and autosomal dominant and recessive forms of Emery-Dreifuss muscular dystrophy (EDMD). LMNA mutations can also lead to several other disorders, collectively termed laminopathies, involving heart, fat, nerve, bone, and skin tissues, and some premature ageing syndromes. METHODS: Fourteen members of a single family underwent neurologic, electromyographic, and cardiologic assessment. Gene mutation and protein expression analyses were performed for lamins A/C and emerin. RESULTS: Clinical investigations showed various phenotypes, including isolated cardiac disease (seven patients), axonal neuropathy (one patient), and a combination of EDMD with axonal neuropathy (two patients), whereas five subjects remained asymptomatic. Genetic analyses identified the coincidence of a previously described homozygous LMNA mutation (c.892C-->T, p. R298C) and a new in-frame EMD deletion (c.110-112delAGA, p. delK37), which segregate independently. Analyses of the contribution of these mutations showed 1) the EMD codon deletion acts in X-linked dominant fashion and was sufficient to induce the cardiac disease, 2) the combination of both the hemizygous EMD and the homozygous LMNA mutations was necessary to induce the EDMD phenotype, 3) emerin was present in reduced amount in EMD-mutated cells, and 4) lamin A/C and emerin expression was most dramatically affected in the doubly mutated fibroblasts. CONCLUSIONS: This highlights the crucial role of lamin A/C-emerin interactions, with evidence for synergistic effects of these mutations that lead to Emery-Dreifuss muscular dystrophy as the worsened result of digenic mechanism in this family.

Molecular etiology of idiopathic cardiomyopathy.

Idiopathic cardiomyopathy (ICM) is a primary cardiac disorder associated with abnormalities of ventricular wall thickness, size of ventricular cavity, contraction, relaxation, conduction and rhythm. Over the past two decades, molecular genetic analyses have revealed that mutations in the various genes cause ICM and such information concerning the genetic basis of ICM enables us to speculate the pathogenesis of this heterogeous cardiac disease. This review focuses on the molecular pathogenesis, i.e., genetic abnormalities and functional alterations due to the mutations especially in sarcomere/cytoskeletal components, in three characteristic features of ICM, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM) and restrictive cardiomyopathy (RCM). Understanding the functional abnormalities of the sarcomere/cytoskeletal components, in ICM, has unraveled the function of these components not only as a contractile unit but also as a pivot for transduction of biochemical signals.

Involvement of polyamines in evening primrose extract-induced apoptosis in Ehrlich ascites tumor cells.

We previously demonstrated that evening primrose extract (EPE) induced apoptosis and inhibited the DNA synthesis in Ehrlich ascites tumor cells (EATC) and suggested that EPE-induced inhibition of the growth of EATC are via at least two pathway differentially modulated by reactive oxygen species, notably intracellular peroxides. These are (a) the EPE-induced apoptosis pathway which is dependent on increases in hydrogen peroxide and (b) the EPE-induced inhibition of cell proliferation which is hydrogen peroxide independent. In this study, EPE brought about a significant decrease in intracellular polyamine levels. Furthermore, the addition of polyamines reversed the EPE-induced decrease in cell viability and suppressed the EPE-induced increase in intracellular hydrogen peroxides. However, the addition of polyamines did not reverse EPE-induced decrease in DNA synthesis and phosphorylation of Rb protein, and EPE-induced translocation of AIF. These results suggest the involvement of polyamines in the EPE-induced apoptosis pathway which is dependent on increase in hydrogen peroxide.

Nuclear envelope alterations in fibroblasts from patients with muscular dystrophy, cardiomyopathy, and partial lipodystrophy carrying lamin A/C gene mutations.

Mutations in LMNA, the gene that encodes nuclear lamins A and C, cause up to eight different diseases collectively referred to as "laminopathies." These diseases affect striated muscle, adipose tissue, peripheral nerve, and bone, or cause features of premature aging. We investigated the consequences of LMNA mutations on nuclear architecture in skin fibroblasts from 13 patients with different laminopathies. Western-blotting showed that none of the mutations examined led to a decrease in cellular levels of lamin A or C. Regardless of the disease, we observed honeycomb nuclear structures and nuclear envelope blebs in cells examined by immunofluorescence microscopy. Concentrated foci of lamin A/C in the nucleoplasm were also observed. Only mutations in the head and tail domains of lamins A and C significantly altered the nuclear architecture of patient fibroblasts. These results confirm that mutations in lamins A and C may lead to a weakening of a structural support network in the nuclear envelope in fibroblasts and that nuclear architecture changes depend upon the location of the mutation in different domains of lamin A/C.

Identification of MICA alleles with a long Leu-repeat in the transmembrane region and no cytoplasmic tail due to a frameshift-deletion in exon 4.

MHC class I chain-related gene A (MICA) is located close to HLA-B gene and expressed in epithelial cells. The MICA gene is reported to be highly polymorphic as are the classical class I genes. To further assess the polymorphism in the MICA gene, we analyzed a total of 60 HLA-homozygous cells for the sequences spanning exons 2-6. In the analysis, four new MICA alleles were identified and six variations were recognized in exon 6. MICA*017, which was identified in three HLA-B57 homozygous cells (DBB, DEM and WIN), differed from MICA*002 in exon 3 and had a guanine deletion at the 3' end of exon 4. MICA*015 identified in an HLA-B45 homozygous cell (OMW) also had the same deletion that causes a frameshift mutation resulting in complete change of the transmembrane region and premature termination in the cytoplasmic tail; these alleles have a long hydrophobic leucine-rich region instead of the alanine repeat in the transmembrane region and terminate at the second position in the cytoplasmic domain. The frameshift deletion was found only in HLA-B45- or -B57-positive panels tested, suggesting a strong linkage disequilibrium between the deletion and B45 or B57. MICA*048, which was different in exon 5 from MICA*008, was identified in an HLA-B61 homozygous cell (TA21), while MICA*00901 identified in HLA-B51 homozygous cells (LUY and KT2) was distinguished from MICA*009 by exon 6.

Molecular etiology of idiopathic cardiomyopathy in Asian populations.

BACKGROUND: Idiopathic cardiomyopathy was by definition a disease of unknown etiology and there are two major clinical forms, hypertrophic cardiomyopathy and dilated cardiomyopathy. Recent molecular genetic analyses have now revealed that mutations in genes for sarcomere cause hypertrophic cardiomyopathy leading to a hypothesis of hypertrophic cardiomyopathy as sarcomeropathy. On the other hand, mutations in genes for Z-disc component cause dilated cardiomyopathy speculating that dilated cardiomyopathy is cytoskeletopathy at least in part. METHODS: A large panel of Asian patients and families with hypertrophic cardiomyopathy or dilated cardiomyopathy was analyzed for gene abnormalities in all exons and adjacent introns of the known disease-related genes and in a part of several candidates of novel disease-related genes. RESULTS: Mutations in the genes for sarcomere were found in 47% of familial cases and 14% of sporadic cases of hypertrophic cardiomyopathy and there were locus and allelic differences in clinical phenotypes of hypertrophic cardiomyopathy patients. In contrast, only a few patients with dilated cardiomyopathy were identified for mutations in the known disease-causing genes. Mutations in the gene for titin, a giant molecule linking Z-disc with sarcomere components, were found in hypertrophic cardiomyopathy patients. CONCLUSIONS: The molecular etiologies of cardiomyopathy can be identified in about half of hypertrophic cardiomyopathy and a small part of dilated cardiomyopathy, suggesting that there are several novel disease-causing genes. Identification of titin mutation in hypertrophic cardiomyopathy indicate that hypertrophic cardiomyopathy is in part considered as the cytoskeletopathy.

Identification, characterization, and functional analysis of heart-specific myosin light chain phosphatase small subunit.

Myosin light chain phosphatase consists of three subunits, a 38-kDa catalytic subunit, a large 110-130-kDa myosin binding subunit, and a small subunit of 20-21 kDa. The catalytic subunit and the large subunit have been well characterized. The small subunit has been cloned and studied from smooth muscle, but little is known about its function and specificity in the other muscles such as cardiac muscle. In this study, cDNAs for heart-specific small subunit isoforms, hHS-M(21), were isolated and characterized. Evidence was obtained from an analysis of genome to suggest that the small subunit was the product of the same gene as the large subunit. Using permeabilized renal artery preparation and permeabilized cardiac myocytes, it was shown that the small subunit increased sensitivity to Ca(2+) in muscle contraction. It was also shown using an overlay assay that hHS-M(21) bound the large subunit. Mapping experiments demonstrated that the binding domain and the domain involved in the increasing Ca(2+) sensitivity mapped to the same N-terminal region of hHS-M(21). These observations suggest that the heart-specific small subunit hHS-M(21) plays a regulatory role in cardiac muscle contraction by its binding to the large subunit.

Characterization of the human nebulette gene: a polymorphism in an actin-binding motif is associated with nonfamilial idiopathic dilated cardiomyopathy.

Idiopathic dilated cardiomyopathy (IDC) is characterized by a thin-walled heart with systolic dysfunction of unknown etiology. Because abnormalities in genes for cytoskeletal proteins related to Z-disc function have recently been reported to cause IDC, genomic organization of the gene for nebulette, a novel actin-binding Z-disc protein, was determined and its sequence variations were searched for in Japanese patients with IDC and healthy controls. The nebulette gene consists of 28 exons, and four sequence variations leading to amino acid replacement (Gln187His, Met351Val, Asn654Lys, and Thr728Ala) were identified in the patients. These variations were also found in the healthy controls and hence they were polymorphisms and not disease-specific mutations. Frequencies of Gln187His, Met351Val, and Thr728Ala variants were similar in the patients and controls. However, the frequency of homozygotes for Lys at codon 654, a variant at a relatively conserved residue in an actinbinding motif, was significantly increased in nonfamilial IDC patients (n=106) as compared with healthy control subjects (n=331) (7.54% vs 1.21%, OR=6.25, P=0.002, 95% CI=1.92-20.29), while this association was not found in familial IDC patients (n=24). These observations suggest that the nebulette polymorphism in the actin-binding motif was a novel genetic marker of susceptibility to nonfamilial IDC.

Epithelioid haemangioendothelioma of the lung in a dog.

Epithelioid haemangioendothelioma of the lung was found in a 9-year-old female dog. The tumour occurred bilaterally in the form of multiple, discrete, small nodular lesions with a similar histological appearance. The lesions were characterized by a hypocellular sclerotic core surrounded by a more cellular peripheral zone, from which the tumour tissue extended into the adjacent alveolar spaces and bronchioles in a micropolypoid manner, filling their lumina. In addition, invasion of the pulmonary vessels was frequently observed within, around, and at a distance from the nodular neoplastic lesions. Most of the tumour cells had abundant eosinophilic cytoplasm, large, round or oval nuclei, and occasional intracytoplasmic vacuoles containing red blood cells. By means of immunolabelling, factor VIII-related antigen, a marker for endothelial cells, was detected within the cytoplasm of a small proportion (< 5%) of the tumour cells. This appears to be the first report of such a tumour in an animal other than man.

Transmural myocardial infarction caused by thromboembolism associated with mitral insufficiency in a dog.

A 10-year-old dog, which had been treated for mitral insufficiency, died suddenly. Transmural myocardial infarction secondary to thromboembolic occlusion of the subsinuosal interventricular branch of the left circumflex artery was noted in the posterior lower half of the left ventricular wall, involving the interventricular septum and a part of right ventricular wall. The mitral valve leaflets were markedly thickened (valvular endocardiosis). These were a patchy area of jet lesion and several mural thrombi on the left-atrial endocardium. The embolus in the subsinuosal interventricular branch was composed of mostly platelets and fibrin, showing the same histologic features as those of the mural thrombi on the left-atrial endocardium. From these findings, it was concluded that dislodgement of part of the mural thrombi on the left-atrial endocardium caused thromboembolism of the subsinuosal interventricular branch.

Fatal rupture of the brachiocephalic artery in a dog.

A case of arterial rupture that caused sudden death in a 3-year-old dog is presented. Rupture of the brachiocephalic artery was located just distal to the origin of the left common carotid artery. Histological examination of the vessel wall revealed necrosis of the media.

MICA gene polymorphism in Takayasu's arteritis and Buerger's disease.

To further clarify the HLA-linked genes susceptible to arterio-vasculitis of unknown etiology, Takayasu's arteritis and Buerger's disease, polymorphism in the MICA gene, a newly identified gene near the HLA-B gene and expressed in epithelial cell lineage, was investigated. Polymerase chain reaction (PCR)-DNA conformation polymorphism (DCP) analysis and subsequent sequencing of the MICA gene have revealed that there are 5 MICA alleles which are different in the number of a GCT repeat in exon 5: MICA alleles MICA-1.1, -1.2, -1.3 and -1.4 have 9, 6, 5 and 4 GCT repeats, respectively, and MICA-1.5 has 5 GCT repeats with a 1 bp frameshift insertion in the repeat. MICA genotyping data in 81 Japanese patients with Takayasu's arteritis, 38 Japanese patients with Buerger's disease, and 160 healthy Japanese controls showed that MICA-1.2 and -1.4 were significantly associated with Takayasu's arteritis and Buerger's disease, respectively. Because MICA-1.2 and -1.4 were in strong linkage disequilibria with HLA-B52 and -B54 in the Japanese populations, respectively, we have compared the odds ratio (OR) of the risk to the diseases for individuals having both or each of the disease-associated MICA and HLA-B alleles. It was found that MICA-1.2 gave a significantly high OR of risk to Takayasu's arteritis in the absence of HLA-B52, suggesting that the HLA-linked gene susceptible to Takayasu's arteritis is mapped near the MICA gene. In contrast, MICA-1.4 gave a significantly high OR of risk to Buerger's disease only in the presence of HLA-B54, suggesting that the HLA-linked gene susceptible to Buerger's disease is linked to the HLA-B54-MICA-1.4 haplotype, and may be differently mapped from that to Takayasu's arteritis.

Analysis of Cdc2 and Cyclin D1 Expression in Breast Cancer by Immunoblotting.

Cyclins and cyclin-dependent kinases may reflect the status of cell proliferation in cancer tissues. The authors sought to determine whether cdc2 and cyclin D1 are expressed in breast cancer and are useful as prognostic factors. Accumulation of cdc2 and cyclin D1 proteins was examined in 88 cases of breast cancer using immunoblotting techniques and correlations with clinicopathological factors and prognoses were investigated. Cdc2 and cyclin D1 proteins were observed in 27.3 % and 75.0 % of breast cancers studied, respectively. The incidence of lymph node metastasis was significantly high in cdc2/cyclin D1-double positive group and low in double negative group. On the other hand, the incidence of estrogen receptor (ER) negative cases was significantly higher in the cdc2-positive/cyclin D1-negative group. Relapse-free survival times of cdc2-positive cases were significantly shorter than those of Cdc2-negative cases. The relapse-free survival times of cyclin D1-positive cases also tended to be poorer than those of cyclin D1-negative cases. Multivariate analyses revealed cdc2 as the second most significant of the prognostic variables, following lymph node status. The three-year relapse-free survival rate of cdc2/cyclin D1-double positive cases was 58.9%, whereas that of cdc2/cyclin D1-doublue negative cases was 100%. Cdc2 and cyclin D1 represent the status of cell proliferation in breast cancer, and may be useful in breast cancer assessment.

Prospective evaluation of skin surface electropotentials in Japanese patients with suspicious breast lesions.

The biofield breast examination (BBE) is a new, noninvasive and cost-effective method for diagnosing breast lesions currently undergoing multicenter evaluation in the USA and Europe. The test analyzes subtle differences in electrical potential caused by dysregulated epithelial proliferation. This report summarizes a prospective evaluation of BBE in a population of 101 patients with suspicious breast lesions scheduled either for open surgical biopsy or fine needle aspiration biopsy. Of the 101 patients included in the study, 49 were found to have a breast malignancy and 52 were found to have a benign breast lesion. BBE correctly identified 44 of 49 biopsy-proven cancers (sensitivity=90%) and correctly indicated no cancer in 31 of 52 biopsy-proven benign cases (specificity=60%). Sensitivity increased to 95% for cancers less than 2.5 cm in size. These results indicate that BBE may be an effective adjunctive test to help to resolve abnormalities discovered by physical examination or other screening methods.

[Non-invasive monitoring of cerebral oxygenation by NIR spectrophotometry (clinical trial)].

A Shimazu OM-100A near infrared spectrophotometer was used to monitor the cerebral oxygenated hemoglobin (Oxy-Hb), deoxygenated hemoglobin (Deoxy-Hb) and total hemoglobin (Total-Hb) in 3 patients with a) massive hemorrhage, b) ruptured aneurysm in the abdominal aorta, or c) hypercapnia. The hematocrit value of the patient with massive hemorrhage decreased rapidly to 18 %; the Total-Hb and Oxy-Hb also decreased significantly. Red blood cell transfusion raised the Oxy-Hb level, indicating an improvement in cerebral oxygenation. In the patient with a ruptured aneurysm, changes in blood flow brought about by clamping or declamping of the abdominal aorta were immediately reflected by corresponding changes in Total-Hb and Oxy-Hb. Oxy-Hb increased significantly and correlated well with the PETCO2 value in the patient with hypercapnia. Thus, NIR spectrophotometry is a useful non-invasive tool which can monitor metabolic and hemodynamic changes in the brain in various pathological conditions.

[A case of Corynebacterium jeikeium septicemia].

We report a case of Corynebacterium jeikeium septicemia associated with malignant lymphoma. The patient is a 58-year-old male who was diagnosed as malignant lymphoma on August 1992. May 15, 1993, he was admitted to our hospital because of oliguria, abdominal flatulence and vomiting which developed a few days before admission. Anticancer regimen were started. In the middle of July, white blood cell (WBC) count dropped to 100/mm3 and body temperature rose to 39 degrees C. He was been treated with Ceftazidime and Piperacillin. C. jeikeium was recovered from blood culture. Antibiotics were switched to minocycline and vancomycin. He died of septic shock and pneumonia. Autopsy revealed the presence of the colonies of Rods. Which were morphologically compatible with C. jeikeium were observed in lung tissue and in the small pulmonary vessels.

Current status of type 2 (non-insulin-dependent) diabetic subjects on dialysis therapy in Japan.

According to a national survey of dialysis patients in Japan conducted by the Japanese Society for Dialysis Therapy, there were 1,033 patients on dialysis in the Shiga area which has a population of about 1.2 million. Of these 1,033 dialysis patients 140 were the result of diabetic nephropathy. From four hospitals affiliated to Shiga University of Medical Science the medical records of 90 diabetic subjects on dialysis therapy were reviewed and various clinical parameters were analysed and compared with those of patients with chronic glomerulonephritis. Since only one patient had Type 1 (insulin-dependent) diabetes, the remaining 89 with Type 2 (non-insulin-dependent) diabetes were used for this study. The significantly different variables between patients with Type 2 diabetes and chronic glomerulonephritis were age (60.4 vs 54.6 years, p < 0.05), BMI (22.4 vs 20.6 kg/m2, p < 0.001), cardiothoracic ratio (56.4 vs 53.3%, p < 0.001), mean blood pressure (110 vs 117 mmHg, p < 0.05), serum creatinine (9.0 vs 11.5 mg/dl, p < 0.001), serum urea-N (98.2 vs 115.5 mg/dl, p < 0.001), serum total protein (6.0 vs 6.5 g/dl, p < 0.001) and serum albumin (3.5 vs. 3.9 g/dl, p < 0.001). Serum levels of cholesterol and triglyceride were not significantly different between two groups, though the prevalence of electrocardiogram abnormalities, oedema, neuropathy, myocardial infarction and cerebrovascular diseases was significantly higher in the Type 2 diabetic group. These results suggested that Type 2 diabetic patients with end-stage renal disease were older, more malnourished, fluid overloaded and multi-morbid as a result of vasculopathy and neuropathy.(ABSTRACT TRUNCATED AT 250 WORDS)

Dihydropyridine-sensitive calcium current mediates neurotransmitter release from bipolar cells of the goldfish retina.

The release of neurotransmitter is evoked by activation of the Ca current (ICa) at presynaptic terminals. Though multiple types of ICa have been reported in various cells, little is known about the properties of presynaptic ICa in the vertebrate CNS. The aim of this article is to identify the type of ICa involved in the release of neurotransmitter from retinal bipolar cells. Bipolar cells with a large axon terminal were isolated enzymatically from the goldfish retina, and studied by the following techniques: (1) recordings of ICa in the whole-cell recording configuration, (2) visualization of intracellular free Ca2+ concentration ([Ca2+]i) with the Fura-2 imaging system, and (3) real-time electrophysiological bioassay of released excitatory amino acid transmitter by a voltage-clamped horizontal cell isolated from the catfish retina. The only ICa found in bipolar cells was the high-voltage-activated, dihydropyridine-sensitive type. This result supports the recent study by Heidelberger and Matthews (1992). When ICa was activated by a short depolarizing pulse, a rapid increase of [Ca2+]i was restricted to the axon terminal. A much slower and smaller increase of [Ca2+]i was sometimes observed at the cell body, probably due to the diffusion of intracellular free Ca2+ from the axon terminal. The increase of [Ca2+]i was completely suppressed by nicardipine, suggesting that Ca2+ entered through dihydropyridine-sensitive Ca channels located mainly at the axon terminal. Activating ICa of the bipolar cell evoked a transmitter-induced current in the excitatory amino acid probe (i.e., the catfish horizontal cell). Both currents were suppressed concomitantly by nifedipine but not by omega-conotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)