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.

Characterization of SPATA5-related encephalopathy in early childhood.

Mutations in SPATA5 have recently been shown to result in a phenotype of microcephaly, intellectual disability, seizures, and hearing loss in childhood. Our aim in this report is to delineate the SPATA5 syndrome as a clinical entity, including the facial appearance, neurophysiological, and neuroimaging findings. Using whole-exome sequencing and Sanger sequencing, we identified three children with SPATA5 mutations from two families. Two siblings carried compound heterozygous mutations, c.989_991del (p.Thr330del) and c.2130_2133del (p.Glu711Profs*21), and the third child had c.967T>A (p.Phe323Ile) and c.2146G>C (p.Ala716Pro) mutations. The three patients manifested microcephaly, psychomotor retardation, hypotonus or hypertonus, and bilateral hearing loss from early infancy. Common facies were a depressed nasal bridge/ridge, broad eyebrows, and retrognathia. Epileptic spasms or tonic seizures emerged at 6-12 months of age. Interictal electroencephalography showed multifocal spikes and bursts of asynchronous diffuse spike-wave complexes. Augmented amplitudes of visually evoked potentials were detected in two patients. Magnetic resonance imaging revealed hypomyelination, thin corpus callosum, and progressive cerebral atrophy. Blood copper levels were also elevated or close to the upper normal levels in these children. Clinical delineation of the SPATA5-related encephalopathy should improve diagnosis, facilitating further clinical and molecular investigation.

Conformationally-locked C-glycosides: tuning aglycone interactions for optimal chaperone behaviour in Gaucher fibroblasts.

A series of conformationally locked C-glycosides based on the 3-aminopyrano[3,2-b]pyrrol-2(1H)-one (APP) scaffold has been synthesized. The key step involved a totally stereocontrolled C-Michael addition of a serine-equivalent C-nucleophile to tri-O-benzyl-2-nitro-D-galactal, previously published by the authors. Stereoselective transformations of the Michael adduct allowed us the synthesis of compounds with mono- or diantennated aglycone moieties and different topologies. In vitro screening showed highly selective inhibition of bovine liver ß-glucosidase/ß-galactosidase and specific inhibition of human ß-glucocerebrosidase among lysosomal glycosidases for compounds bearing palmitoyl chains in the aglycone, with a marked dependence of the inhibition potency upon their number and location. Molecular dynamics simulations highlighted the paramount importance of an optimal orientation of the hydrophobic substituent to warrant efficient non-glycone interactions, which are critical for the binding affinity. The results provide a rationale for the strong decrease of the inhibition potency of APP compounds on going from neutral to acidic pH. The best candidate was found to behave as pharmacological chaperone in Gaucher fibroblasts with homozygous N370S and F213I mutations, with enzyme activity enhancements similar to those encountered for the reference compound Ambroxol.

A new type of familial central diabetes insipidus caused by a single base substitution in the neurophysin II coding region of the vasopressin gene.

We studied the genetic basis of familial neurohypophyseal diabetes insipidus in a Japanese family. The members had polyuria and a deficiency of plasma vasopressin (AVP). Polymerase chain reaction (PCR) amplified exons of the AVP-neurophysin-II gene were subcloned and sequenced. Exons 1 and 3 were normal, but nucleotide 1884 Guanine (G) in exon 2 was substituted with Thymine (T), which induced a substitution of glycine (Gly) for valine (Val). To examine the presence of this mutation in the affected subjects, we designed two mutated primers. One of them induced a new endonuclease restriction site in the PCR fragments from normal, and the other induced a new endonuclease restriction site from patients with the mutation. DNA fragments from two affected members of this family were amplified with this primer, and the PCR products were digested by endonuclease and resolved by electrophoresis. The results indicated that these subjects had both normal and mutant alleles, indicating that the mutation was heterozygous. We concluded that this mutation caused neurohypophyseal diabetes insipidus in this family.

Instability of expressed Cu/Zn superoxide dismutase with 2 bp deletion found in familial amyotrophic lateral sclerosis.

The mutant Cu/Zn superoxide dismutase (SOD1) associated with familial amyotrophic lateral sclerosis (FALS) with a 2 bp deletion was produced in two protein expression systems. The mutant SOD1, expressed as a fusion protein in E. coli, had immunoreactivity to an anti-human SOD1 antibody but no SOD activity. It was more susceptible to proteolysis and its immunoreactivity decreased more rapidly than the wild type. The mutant SOD1, expressed in Cos1 cells, was not detected by either SOD activity staining or Western blot analysis, although expression of its mRNA was confirmed. These results suggest that the mutant SOD1 is seriously unstable in mammalian cells.

Early-onset, rapidly progressive familial tauopathy with R406W mutation.

An early-onset and rapidly progressive familial tauopathy with R406W mutation is described. The patient was a 47-year-old man who first presented with psychiatric symptoms followed by overt dementia at age 52 and died 1 year later. Postmortem study revealed tangle-associated neuronal degeneration, accentuated in the medial temporal lobe. R406W mutation was determined by sequence analysis and immunocytochemically with anti-mutant tau antibody.

Dog liver glutathione S-transferase and its strong immunoreactivity with rat transferase-P(7-7).

Dog liver cytosolic glutathione S-transferases (GSTs) were investigated to characterize their properties in comparison with rat liver transferases. Dog liver GSTs after the glutathione affinity column chromatography showed three subunit bands on SDS-polyacrylamide gel electrophoresis. These three subunits, designated as Yd1 (mol.wt 26,000), Yd2 (mol.wt 27,000) and Yd3 (mol.wt 28,000), were distinctly different from rat liver GST subunits, i.e. Ya(1) (mol.wt 26,500), Yb1(3)/Yb2(4) (mol.wt 27,500) and Yc(2) (mol.wt 28,500). Western blot analysis revealed that Yd1, Yd2 and Yd3 were immunoreacted with anti-rat GST 7-7, 1-1 and 3-3 antibodies, respectively. Four transferase activity fractions, I (pH greater than 7.63), II (pH 6.92), III (pH 5.80) and IV (pH 5.65), were obtained from affinity purified GSTs by chromatofocusing. Each fraction exhibited a characteristic substrate specificity. GST-II, III and IV were all strongly immunoreacted with anti-rat GST 7-7 antibody by immunoblotting, thus suggesting the occurrence of the heterogeneity of transferases immunologically related to rat GST subunit 7 in dog liver. Immunohistochemical examination showed that transferases immunoreacted with anti-GST 7-7 antibody have diffusely distributed throughout the lobule, while enzymes related to subunit 3 have been localized in a narrow range of cells around the central vein. These data suggest that GSTs immunologically associated with rat transferase subunit 7 may be major forms in dog liver.

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.

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.

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.

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.

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.

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.

Hypoxia-induced ABR change and heat shock protein expression in the pontine auditory pathway of young rabbits.

The auditory brainstem response (ABR) was compared with the immunohistochemical expression of heat shock protein (HSP-72) and microtubule-associated protein 2 (MAP-2) of the brainstem auditory pathway in young rabbits subjected to hypoxic stress. Severe hypoxia for 2 h produced significant prolongation and decreased amplitude of the later component of ABR. HSP-72 expression was distinctly increased in the cochlear nucleus, but there was less induction in the inferior colliculus under severe hypoxia. MAP-2 immunostaining of neuropiles in the inferior collicular nucleus was decreased slightly after severe-long hypoxia, but cytoplasmic staining did not change. The present ABR change, which was produced by brainstem hypoxia-ischemia and acidosis, may be due to the neural cytoarchitectural derangement and less induction of stress proteins in the upper brainstem.

A splicing mutation in the hydroxymethylbilane synthase gene in a Japanese family with acute intermittent porphyria.

OBJECTIVES: Acute intermittent porphyria (AIP) is an autosomal dominant inherited disease caused by a decreased activity of hydroxymethylbilane synthase (HMBS). As far as the gene abnormalities of the HMBS, many different mutations have been reported. In this work, we investigated the presence of mutations in a Japanese family with AIP. DESIGN AND METHODS: A 44-year-old Japanese male and nine members of his family were investigated. All of them were screened by traditional biochemical markers. Mutational analysis was performed using polymerase chain reaction-single strand conformation polymorphism method followed by DNA sequencing. A reliable restriction enzyme cleavage assay was established for the pedigree analysis. RESULTS: The mutation was a splicing mutation, a C to G transversion at position -3 of the acceptor site of intron 11 of the HMBS gene, resulting in the exon 12 skipping. The patient is heterozygous for the mutation, and his father appeared to be the source of the mutant allele. This mutation created a new cleavage site of the Nla III restriction enzyme and could be screened by a amplified fragment from genomic DNA with digestion. Using this cleavage assay, an asymptomatic carrier in the family was definitively identified. CONCLUSIONS: This mutation was first found among Japanese AIP patients, but happened to be the same as reported previously from Europe. A similarity of gene abnormality may suggest that those European and Japanese AIP families have a common ancestor. Molecular investigations on the family members should be applied not only for more accurate diagnosis, but also for understanding the molecular genetic heterogeneity underlying this dominantly inherited enzymopathy.

Widespread expression of alpha-synuclein and tau immunoreactivity in Hallervorden-Spatz syndrome with protracted clinical course.

Hallervorden-Spatz syndrome (HSS) is a rare autosomal recessive disorder clinically characterized by extrapyramidal signs and progressive dementia. In a typical case, the clinical symptoms become apparent during late childhood, and usually the course is protracted over a decade or more. We recently had an opportunity to study the brains of two cases of HSS with a clinical course of over 30 years. Case 1 was a 44-year-old female and case 2 was a 37-year-old male. Grossly, the brains showed severe fronto-temporal lobar atrophy with abundant spheroids and mild iron deposits in the globus pallidus, associated with features of motor neuron disease. In addition, there was diffuse sponginess in the atrophic cortex as well as widespread Alzheimer's neurofibrillary tangles (NFTs) and Lewy bodies (LBs) in the cortical and subcortical regions, including the spinal cord. Ultrastructurally, NFTs were composed of paired helical filaments, and LBs of central dense cores with radiating fibrils. Discrete immunostaining was demonstrated in NFTs and neuropil threads with various antibodies against phosphorylated tau, and in LBs with antibody against alpha-synuclein. In addition, diffuse, overlapping immunoreactivity of alpha-synuclein and phosphorylated tau was seen within the cytoplasm of many neurons. However, when LBs and NFTs coexisted within the same neurons, they were clearly segregated. The findings of our present cases as well as those reported in the literature may indicate that simultaneous and extensive occurrence of abnormal phosphorylation of tau and accumulation of alpha-synuclein may constitute cardinal pathological features of HSS with protracted clinical course.

Congenital myotonic dystrophy: report of paternal transmission.

A female neonate born to a healthy mother was hospitalized because of enlargement of the lateral ventricles, muscle weakness, irregular respiration, and poor sucking. Characteristic facial appearance such as high forehead and carp mouth were noted. The father had mild manifestations of adult type myotonic dystrophy, including muscle weakness of the extremities, percussion myotonia and atrophy of the facial muscles. PCR analysis and southern blot analysis revealed that CTG repeats in the myotonic dystrophy gene of the infant and the father were about 1000 and 400, respectively. This is a rare case showing paternally transmitted congenital myotonic dystrophy and seems to be the first report describing a neonate.

Intracranial hemorrhage in the full-term neonate and young infant: correlation of the location and outcome.

The cranial computed tomography (CT) and outcome for 13 full-term neonates and 12 young infants with intracranial hemorrhage (ICH) were studied. The full-term neonates had perinatal asphyxia or neurological signs such as seizures. All infants were breast-fed and showed bleeding diathesis. In the full-term neonates there was a high incidence of intraventricular hemorrhage (IVH) and hemorrhage around the falx. The location of the hemorrhage on CT and brain pathology suggested that the original site of IVH might be the choroid plexus vessels in the lateral ventricle or in the subependymal layer. On the other hand, the sites of ICH in infants were multifocal compared with those in full-term neonates. Subdural hemorrhage (SDH) was seen more frequently and IVH less frequently in infants than in full-term neonates. The cases with SDH frequently showed accompanying cerebral infarction followed by porencephaly. Thus, SDH with cerebral low density on CT may predict a poor prognosis.

Galactonojirimycin derivatives restore mutant human beta-galactosidase activities expressed in fibroblasts from enzyme-deficient knockout mouse.

Ten low molecular compounds analogous to galactose were screened for inhibition of human beta-galactosidase activity. Among them, 1-deoxy-galactonojirimycin and N-(n-butyl)-deoxy-galactonojirimycin showed an inhibitory effect at high concentrations. However, they restored mutant enzyme activities expressed in enzyme-deficient knockout mouse fibroblasts and human beta-galactosidosis fibroblasts at lower intracellular concentrations. This effect was more remarkable on G(M1)-gangliosidosis mutations (R201C, I51T, R201H, R457Q) than Morquio B disease mutations (W273L, Y83H). These low molecular compounds pass though the blood-brain barrier in mice. We hope that this new therapeutic approach will become clinically applicable in the near future.