Bilateral cerebellar cysts and cerebral white matter lesions with cortical dysgenesis: Expanding the phenotype of LAMB1 gene mutations.

LAMB1 gene analysis should be considered for intellectually disabled patients with cerebellar cysts, white matter signal change, and cortical malformation. Muscular involvement is absent, in contrast to the α-dystroglycanopathy types of congenital muscular dystrophies.

Tuning glycosidase inhibition through aglycone interactions: pharmacological chaperones for Fabry disease and GM1 gangliosidosis.

Competitive inhibitors of either α-galactosidase (α-Gal) or ß-galactosidase (ß-Gal) with high affinity and selectivity have been accessed by exploiting aglycone interactions with conformationally locked sp(2)-iminosugars. Selected compounds were profiled as potent pharmacological chaperones for mutant lysosomal α- and ß-Gal associated with Fabry disease and GM(1) gangliosidosis.

Attenuation of ganglioside GM1 accumulation in the brain of GM1 gangliosidosis mice by neonatal intravenous gene transfer.

A single intravenous injection with 4 x 10(7) PFU of recombinant adenovirus encoding mouse beta-galactosidase cDNA to newborn mice provided widespread increases of beta-galactosidase activity, and attenuated the development of the disease including the brain at least for 60 days. The beta-galactosidase activity showed 2-4 times as high a normal activity in the liver and lung, and 50 times in the heart. In the brain, while the activity was only 10-20% of normal, the efficacy of the treatment was distinct. At the 30th day after the injection, significant attenuation of ganglioside GM1 accumulation in the cerebrum was shown in three out of seven mice. At the 60th day after the injection, the amount of ganglioside GM1 was above the normal range in all treated mice, which was speculated to be the result of reaccumulation. However, the values were still definitely lower in most of the treated mice than those in untreated mice. In the histopathological study, X-gal-positive cells, which showed the expression of exogenous beta-galactosidase gene, were observed in the brain. It is noteworthy that neonatal administration via blood vessels provided access to the central nervous system because of the incompletely formed blood-brain barrier.

Absence of mutations in the beta-catenin and adenomatous polyposis coli genes in papillary and follicular thyroid carcinomas.

beta-Catenin has multiple functions both in intercellular adhesion and in signal transduction. As a signaling molecule, mutations in exon 3 of the beta-catenin gene stabilize this protein in the cytoplasm. Subsequently, accumulated beta-catenin protein translocates to nuclei with T-cell factor-4, and upregulates transcriptional activity of the target genes involved in carcinogenesis. Mutations in exon 3 of the beta-catenin gene have been detected in various carcinomas. We examined immunolocalization of beta-catenin protein and mutations in the beta-catenin and adenomatous polyposis coli (APC) genes in papillary carcinoma (25 cases), follicular carcinoma (two cases), and benign thyroid tumor (29 cases). We detected no mutation in exon 3 of the beta-catenin gene in both malignant and benign thyroid tumors by polymerase chain reaction (PCR) and direct sequencing. No mutations in the mutation cluster region of APC were found in any tumor samples analyzed. Immunohistochemically, beta-catenin showed membranous localization in most specimens. These results suggest that mutations of the beta-catenin and APC genes are rare and that activation of the Wnt signaling pathway may not contribute to pathogenesis in human papillary and follicular thyroid carcinomas.

Angiotensinogen gene variation and hypertension in a cohort study in Japanese.

Many recent case-control studies have suggested a significant relationship between M235T (the substitution of threonine for methionine at position 235 codon) polymorphism of the angiotensinogen (AGT) gene and hypertension. To investigate whether the M235T polymorphism of AGT gene affects the incidence of hypertension, a retrospective cohort study was performed among Japanese workers. The subjects were Japanese workers at an occupational site in Shimane Prefecture in Japan. The baseline data were set at the received regular health examination in 1992, and a retrospective cohort study was performed for analyzing the incidence of hypertension in 1998. The rates of M235M (MM), M235T (MT) and T235T (TT) genotypes were 4%, 32% and 64%, respectively. The relative risks of MT and TT against MM for the incidence of hypertension by single variance analysis were 1.47 [95% confidence interval (CI) 0.50 - 4.33] and 1.35 (95% CI 0.47 - 3.90), respectively. The relative risks of MT and TT against MM for the incidence of hypertension, adjusted for sex, age, body mass index, fasting glucose and cigarette smoking, drinking and exercise in 1992, were 1.49 (95% CI 0.49 - 4.53) and 1.25 (95% CI 0.42 3.74), respectively. The data from this study suggest that the M235T polymorphism of AGT gene has a weak role in the manifestation of hypertension. Further comprehensive studies are needed to resolve this issue.

Mitochondrial DNA deletion associated with the reduction of adenine nucleotides in human atrium and atrial fibrillation.

BACKGROUND: Structural changes in the number, size, and shape of mitochondria (mt) have been observed in the atrial muscles of patients with atrial fibrillation (AF) and of animals with rapid atrial pacing, however, it is not known whether the mitochondrial function is impaired in human atrium with AF. MATERIALS AND METHODS: We determined adenine nucleotides concentrations and mtDNA deletions in 26 human right atria obtained at the time of cardiac surgery, using HPLC and PCR amplification, and studied the relationship between mtDNA deletions and clinical manifestations, the haemodynamic parameters of the patients and adenine nucleotide concentrations in their atrium. RESULTS: The age and the prevalence of AF were significantly higher in the patients with a mtDNA deletion of 7.4 kb than in those without a deletion; there were no significant differences regarding haemodynamic parameters between the two groups. The concentrations of ATP, ADP, AMP and total adenine nucleotides in the right atrium were significantly lower in the patients with mtDNA deletions than the patients without a deletion. In a gender- and diseased-matched population, the mtDNA deletion was still significantly associated with age and a decreased concentration of adenine nucleotides in the atrium. Using quantitative PCR analysis, the proportion of mtDNA deletion to normal mtDNA of the atrium, was estimated to be 0.3-2% in four cases. CONCLUSION: These results suggest that the deletion of mtDNA associated with ageing or AF can lead to a bioenergetic deficiency due to an impaired ATP synthesis in the human atrium; however, no conclusion can be made whether mtDNA deletion were the result or the cause of an impaired ATP synthesis, ageing, hemodynamic deterioration, or AF.

Epigenetic silencing of PEG3 gene expression in human glioma cell lines.

Genomic imprinting, the phenomenon in which alleles of genes are expressed differentially depending on their parental origins, has important consequences for mammalian development, and disturbance of normal imprinting leads to abnormal embryogenesis and some inherited diseases and is also associated with various cancers. In the context of screening for novel imprinted genes on human chromosome 19q13.4 with mouse A9 hybrids, we identified a maternal allele-specific methylated CpG island in exon 1 of paternally expressed imprinted gene 3 (PEG3), a gene that exhibits paternal allele-specific expression. Because PEG3 expression is downregulated in some gliomas and glioma cell lines, despite high-level expression in normal brain tissues, we investigated whether the loss of PEG3 expression is related to epigenetic modifications involving DNA methylation. We found monoallelic expression of PEG3 in all normal brain tissues examined and five of nine glioma cell lines that had both unmethylated and methylated alleles; the remaining four glioma cell lines exhibited gain of imprinting with hypermethylated alleles. In addition, treatment of glioma cell lines with the DNA demethylating agent 5-aza-2'-deoxycytidine reversed the silencing of PEG3 biallelically. In this article, we report that the epigenetic silencing of PEG3 expression in glioma cell lines depends on aberrant DNA methylation of an exonic CpG island, suggesting that PEG3 contributes to glioma carcinogenesis in certain cases.

Regulation of cigarette smoke-induced cytochrome P4501A1 gene expression in osteogenic disorder Shionogi rat liver and in lung by large ascorbic acid dose.

The effects of a large ascorbic acid dose on cytochrome P4501A1 gene expression induced by cigarette smoke exposure was studied in Osteogenic Disorder Shionogi rats, which lack ascorbic acid biosynthesis. The rats were divided into four groups and were administered either a minimal amount (4 mg/day, 4S and 4C) or a large amount (40 mg/day, 40S and 40C) of ascorbic acid. The 4S group and 40S group were daily exposed to cigarette smoke for 2 hours, while the 4C group and 40C group were not. At the end of the 25-day experiment, the rats were killed. The cytochrome P4501A1 mRNA level both in the liver and lung was measured by a competitive reverse transcription-polymerase chain reaction method. When a minimal amount of ascorbic acid was administered, the cytochrome P4501A1 mRNA increased in the liver of the cigarette smoke-exposed group (4S) compared with the control group (4C). On the other hand, when a large amount of ascorbic acid was administered, this increase was not observed in the cigarette smoke-induced group (40S) in liver. On the other hand, in lung, an increased mRNA level in 4S group was not decreased by large ascorbic acid administration (40S). This is the first direct mRNA-level evidence of the effects of a large ascorbic acid dose on the gene expression stimulated by cigarette smoke.

Serum-soluble c-kit levels during mobilization of peripheral blood stem cells correlate with stem cell yield.

c-Kit is expressed in hematopoietic stem cells and plays an important role in hematopoiesis. In 16 patients with malignancies, serum-soluble c-Kit levels and the expressions of c-KIT messenger RNA (mRNA) and protein in peripheral blood mononuclear cells were analyzed serially during 26 courses of peripheral blood stem cell (PBSC) mobilization after granulocyte colony-stimulating factor administration following chemotherapy for PBSC harvest. Serum-soluble c-Kit levels were significantly lower in patients than in controls (179.7+/-17.7 arbitrary units [AU]/mL versus 274.5+/-18.9 AU/mL; P < .001), decreasing after chemotherapy (167.7+/-18.2 AU/mL), increasing from day 14, and peaking at day 19 (193.3+/-16.4 AU/mL). The numbers of both c-Kit+ cells and CD34+ cells and granulocyte-macrophage colony-forming units in peripheral blood peaked at day 17, following the peak of the expression of c-KIT mRNA. Serum-soluble c-Kit levels showed a significant positive correlation with the numbers of CD34+ cells in both peripheral blood and leukapheresis products (r = 0.553, P < .01, and r = 0.640, P < .001, respectively) and changed at higher levels in patients with large numbers of PBSCs versus patients with small numbers of PBSCs (P < .05). Serum-soluble c-Kit may reflect the capacity for hematopoiesis after chemotherapy and may be useful in predicting the number of PBSCs that can be mobilized and harvested after mobilization, as well as for monitoring the timing for PBSC harvest.

A novel interstitial deletion of KAL1 in a Japanese family with Kallmann syndrome.

We identified a novel interstitial deletion that spanned from exons 5 to 10 of KAL1 in two Japanese brothers with X-linked Kallmann syndrome (KS; MIM no. 308700). Both brothers had hypogonadism, unilateral renal agenesis, and disturbance of the sense of smell, but they had no other neurological manifestations, including mental disturbance. Their mother was confirmed to be an asymptomatic carrier, by use of a comparative multiplex polymerase chain reaction (PCR) analysis. The present patients are further examples of patients with KS without mental disturbance caused by a mutation confined to KAL1.

Allele frequency of human endothelial nitric oxide synthase gene polymorphism in abdominal aortic aneurysm.

OBJECTIVE: This study was performed to examine the role of the endothelial constitutive NO synthase (ecNOS) gene in patients with abdominal aortic aneurysm (AAA). METHODS: We determined the distributions of polymorphism in intron 4 of the ecNOS (ecNOS4) gene, amplified by polymerase chain reaction, and compared the allele frequencies between subjects with abdominal aortic aneurysms (AAAs) and healthy individuals. PATIENTS: Fifty-eight patients with AAAs and 410 race-matched healthy controls were studied. RESULTS: Two alleles of the ecNOS4 gene, containing 4 (a-allele) and 5 (b-allele) repeats, were identified. We found that the a-allele frequency of this gene was significantly higher in the surgical than in the non-surgical group. CONCLUSION: The results of this study suggest that the a-allele of the ecNOS4 gene is indicative of the need for surgery for AAA. Analysis of the alleles of the ecNOS4 gene polymorphism could provide useful information concerning the clinical course of AAA progression.

Mutational analysis of TSC1 and TSC2 genes in Japanese patients with tuberous sclerosis complex.

We have surveyed the mutations of TSC1 and TSC2 from 38 (25 sporadic, 11 familial, and 2 unknown) Japanese patients with tuberous sclerosis complex. In 23 of 38 subjects, we detected 18 new mutations in addition to 4 mutations that had been previously reported. We also found 3 new polymorphisms. The mutations were not clustered on a particular exon in either of the genes. Seven TSC1 mutations found in 3 familial and 4 sporadic cases were on the exons (3 missense, 2 nonsense point mutations, a 1-base insertion, and a 2-bp deletion). Fifteen TSC2 mutations were found in 5 familial cases, 10 sporadic cases, and 1 unknown case. The 12 mutations were on the exons (8 missense, 1 nonsense point mutations, a 1-bp insertion, a 5-bp deletion, and a 4-bp replacement) and 3 point mutations were on the exon-intron junctions. Although the patients with TSC2 mutations tend to exhibit relatively severe mental retardation in comparison to those with TSC1 mutations, a genotype-phenotype correlation could not yet be established. The widespread distribution of TSC1/TSC2 mutations hinders the development of a simple diagnostic test, and the identification of individual mutations does not provide the prediction of prognosis.

Wild-type p53 gene transfer resulted in cell cycle arrest, but not apoptosis of newly established human malignant fibrous histiocytoma cell line.

We established a human malignant fibrous histiocytoma (MFH) cell line, MFH-ToE, from a tumor originally developed in the right thigh of a 78-year-old woman. The original tumor histologically consisted of histiocytic, fibroblastic and giant cells. The tumor cells showed immunoreactivity for vimentin and alpha-1-antichymotrypsin, and were positive for acid phosphatase and non-specific esterase, being compatible with MFH. Although the histology of the heterotransplanted tumor into nude mice was similar to that of the primary MFH, the population of giant cells gradually decreased along with the culture passages. Cytogenetic analysis revealed a highly aneuploid nature with varying numbers of chromosomes from 71 to 140. Chromosome 17 showed monosomy and exon 6 to 8 of p53 gene was not amplified by PCR, implying absence of p53 function. Adenovirus vector-mediated wild-type p53 gene was successfully transfected into the MFH-ToE, which showed up-regulation of P53 and P21, as well as gradual up-regulation of Bcl-2 protein. The transfection resulted in cell cycle arrest, but not apoptosis of the MFH-ToE cells. These results revealed unique properties of the MFH-ToE, which might be useful in further studies analyzing pathological and biological characteristics of MFH.

Novel TSC2 mutation in a patient with pulmonary tuberous sclerosis: lack of loss of heterozygosity in a lung cyst.

A Japanese patient with tuberous sclerosis (TSC), who manifested with multiple lung cysts and pneumothorax, is described. All exons of two TSC genes, TSC1 and TSC2, in peripheral blood leukocytes from the patient were analyzed by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). A novel T-to-G transition was found in exon 19 of TSC2 at nucleotide position 2168. This mutation caused an amino acid change, L717R. There was no such mutation in any other family members or in 100 normal Japanese. An automated sequencer-assisted quantitative analysis of normal and mutated SSCP-bands revealed no loss of heterozygosity (LOH) in the lung cyst tissue of the patient.

Serum soluble c-kit receptor and expression of c-kit protein and mRNA in acute myeloid leukemia.

To investigate the clinical role of the soluble form of c-kit receptor (s-kit) in patients with acute myeloid leukemia (AML), we determined the levels of serum s-kit and expression of c-kit antigens and mRNA in leukemic cells. The serum s-kit level was measured using ELISA assay in 30 AML patients and 20 normal controls. C-kit antigens of leukemic blasts were stained immunohistologically, and c-kit mRNA was detected by RT-PCR. The serum s-kit level in M1 and M2 were significantly increased (p<0.01) and that in M4 or M5 was significantly decreased (p<0.05) compared to that in the controls. In the comparisons among subtypes of FAB classification, M1 and M2 showed significantly higher levels than M4 or M5 (p<0.05 and p<0.01, respectively). Both expression of c-kit antigens and mRNA were observed in M0 (1/4), M1 (2/4) and M2 (6/8), but neither was observed in M4 or M5. The serum s-kit levels were correlated with the absolute number of AML blasts in peripheral blood (r=0.564, p<0.05). These results indicate that the serum s-kit level is related to the stage of differentiation of AML blasts in accordance with the expression of c-kit protein and mRNA in AML blasts, and is useful for assessment of leukemic cell burden.

Ataxia-telangiectasia without immunodeficiency: novel point mutations within and adjacent to the phosphatidylinositol 3-kinase-like domain.

Ataxia-telangiectasia (AT) is an autosomal recessive disorder characterized by progressive ataxia, telangiectasia, sinopulmonary infections, hypersensitivity to ionizing radiation, and combined immunodeficiency. Recently, the AT gene (ATM) was cloned and shown to be mutated in AT patients. In this report, mutation analysis of ATM was performed in a 24-year-old AT patient without immunodeficiency. ATM amplified with reverse transcriptase-polymerase chain reaction (RT-PCR) was screened with a ribonuclease (RNase) cleavage assay and auto-sequenced. This patient, a compound heterozygote, showed two mutations in ATM: one missense mutation leading to a Leu2656Pro substitution and the other to the truncation at codon 3047 (Arg-->ter). The latter mutation is within the phosphatidylinositol 3-kinase (PI 3-kinase)-like domain and the former is outside but close to the domain. The particular phenotype in our patient, no immunodeficiency, suggests incomplete functional loss of ATM protein. The clinical spectrum of AT caused by ATM mutations may be broader than previously thought. Further analysis of patients with similar phenotypes will make the relation between ATM genotype and phenotype clear.

Establishment and characteristics of a practical and useful astrocyte cell line transformed by a temperature-sensitive mutant of simian virus 40.

A practical mouse astrocyte cell line (A640-IG) was established by transformation with a temperature-sensitive mutant of simian virus 40 (SV40) and the relationship between the function of SV40 large T antigen and the growth and differentiation of A640-IG cells, which are most clearly dependent on temperature that ever established, was reported. A640-IG cells proliferated actively with expression of large T antigen when they were cultured at 33 degrees C. They had a fibroblast-like appearance, and displayed faint immunoreactivity with an antibody against glial fibrillary acidic protein (GFAP). However, when large T antigen expression ceased at 39 degrees C, the cells did not grow actively and differentiated into astrocytes as demonstrated by both their morphological and immunohistochemical characteristics. Differentiation into astrocytes was more obvious when the cells were plated on bacteriological dishes in high density. Western blotting confirmed immunohistochemical observations. A640-IG cells thus showed contrasting behaviour in terms of cell growth and differentiation depending on the temperature. This unique and practical astrocyte cell line is a useful model for investigating the mechanisms of astrocyte growth and differentiation.

Diagnosis of lymphoma in paraffin wax sections by nested PCR and immunohistochemistry.

AIMS: To investigate whether nested polymerase chain reaction (PCR) and immunohistochemistry can be used to diagnose malignant lymphoma. METHODS: Paraffin wax embedded tissue sections from 31 patients with malignant lymphoma were analysed by nested PCR and immunohistochemistry using standard protocols. RESULTS: Nested PCR amplification of 1 pg DNA confirmed monoclonality in B cell lymphoma; PCR amplification of 10 pg DNA confirmed monoclonality in T cell lymphoma. Twenty seven (87%) samples were diagnosed as malignant lymphoma by nested PCR, and 24 (77%) by immunohistochemistry. Seven samples were diagnosed as malignant lymphoma by nested PCR, but not by immunohistochemistry, whereas the use of both procedures gave a diagnosis of malignant lymphoma in all 31 samples. CONCLUSIONS: A combination of immunohistochemistry and nested PCR can be used to diagnose malignant lymphoma in routine paraffin wax embedded sections.