Kaoru Nishiyama

Detection of mutations in the COL4A5 gene in over 90% of male patients with X-linked Alport's syndrome by RT-PCR and direct sequencing

X-linked Alports syndrome is caused by mutations in the COL4A5 gene encoding the type IV collagen alpha5 chain (alpha5[IV]). Polymerase chain reaction-single-str and conformation polymorphism (PCR-SSCP) on genomic DNA has previously been used to screen for mutations in the COL4A5 gene, but this method was relatively insensitive, with mutations detected in less than 50% of patients. Here, we report a systematic analysis of the entire coding region of the COL4A5 gene, using nested reverse-transcription-polymerase chain reaction (RT-PCR) and the direct sequence method using leukocytes. This study examines twenty-two unrelated Japanese patients with X-linked Alports syndrome showing abnormal expression of alpha5(IV) in the glomerular or epidermal basement membranes. Mutations that were predicted to be pathogenic were identified in 12 of the 13 male patients (92%) and five of the nine female patients (56%). Six patients had missense mutations, four had out-of-frame deletion mutations, three had nonsense mutations, and three had mutations causing exon loss of the transcript. The current study shows that nested RT-PCR and the direct sequence method using leukocytes are highly sensitive and offer a useful approach for systematic gene analysis in patients with X-linked Alports syndrome.

A Nerve Growth Factor‐Dependent Protein Kinase That Phosphorylates Microtubule‐Associated Proteins In Vitro: Possible Involvement of Its Activity in the Outgrowth of Neurites from PC12 Cells

We have established a subline of PC12 cells (PC12D) that extend neurites very quickly in response not only to nerve growth factor (NGF) but also to cyclic AMP (cAMP) in the same way as primed PC12 cells (NGF‐pretreated cells). When phosphorylation of brain microtubule proteins by extracts of these cells was monitored, two distinct kinase activities were found to be increased [from three‐ to eightfold in terms of phosphorylation of microtubule‐associated protein (MAP) 2] by a brief exposure of cells to NGF or to dibutyryl cAMP(dbcAMP). The effect of the combined stimulation with both NGF and dbcAMP was additive in terms of the phosphorylation of MAP2. The apparent molecular mass of the kinase activated by dbcAMP was 40 kDa, and this kinase appears to be cAMP‐dependent protein kinase. The molecular mass of the kinase activated by NGF was 50 kDa. The latter was activated to a measurable extent after 5 min of exposure of cells to NGF; it required Mg2+ for activity but not Mn2+ or Ca2+. This kinase appears to be distinct from previously reported kinases in PC12 cells, and it has been designated as NGF‐dependent MAP kinase, although its physiological substrates are not known at present. An inhibitor of protein kinases, K‐252a, selectively inhibited the outgrowth of neurites from PC12D cells in response to NGF but not to dbcAMP. When this inhibitor was added to the incubation medium of cells exposed simultaneously to NGF or dbcAMP, the increase in activity of the NGF‐dependent MAP kinase was selectively abolished. We isolated several mutant clones of PC12D cells that were deficient in the ability to induce neurites in response to either of the two stimulators. In these variant cells, the activity of the relevant protein kinase was decreased, in parallel with the deficiency in the neurite response to NGF or dbcAMP. These observations suggest that the NGF‐dependent MAP kinase may play an important role in the outgrowth of neurites from PC12 cells in response to NGF.

Characterization of two adenosine 3′,5′-monophosphate-dependent protein kinases from rat liver☆

Abstract Two adenosine 3′,5′-monophosphate (cyclic AMP)-dependent protein kinases are partially purified from rat liver soluble fraction by ammonium sulfate fractionation, followed by DEAE-Sephadex and hydroxylapatite column chromatography. Both kinases are dissociated by cyclic AMP into a common active protein kinase and regulatory proteins which differ from each other in their affinities for the cyclic nucleotide. Experiments with diisopropyl fluorophosphate as a protease inhibitor suggest that the two cyclic AMP-dependent protein kinases do not represent simply artifacts due to proteolysis during the isolation procedure.

Role of platelet-activating factor acetylhydrolase gene mutation in Japanese childhood IgA nephropathy

Platelet-activating factor (PAF) is a potent mediator of inflammatory injury in renal diseases. PAF is degraded to inactive products by PAF acetylhydrolase. Recently, a point mutation (G to T transversion) of the PAF acetylhydrolase gene was observed at position 994, and this mutation was found to contribute to the variability in plasma PAF levels, with undetectable plasma PAF acetylhydrolase activity occurring in homozygous patients (TT genotype) and reduced levels of activity in heterozygous patients (GT genotype). Therefore, we investigated the effect of the PAF acetylhydrolase gene mutation on the pathogenesis and progression of immunoglobulin A (IgA) nephropathy. Genomic DNA was obtained from 89 children with IgA nephropathy and 100 controls. We identified the PAF acetylhydrolase gene mutation (G994T) by polymerase chain reaction. There was no significant difference in genotypic frequency between patients and controls. However, urinary protein excretion at the time of biopsy was significantly greater in patients with the GT/TT genotypes than in those with the GG genotype. The percentage of glomeruli with mesangial cell proliferation was significantly greater in patients with the GT/TT genotypes than in those with the GG genotype. These results indicate the PAF acetylhydrolase gene mutation may influence the degree of proteinuria and the extent of mesangial proliferation in the early stage of childhood IgA nephropathy.

C677T mutation in the MTHFR gene was not found in patients with frontoethmoidal encephalocele in East Java, Indonesia

Abstract Background : Frontoethmoidal encephalocele (FEE) is a neural tube defect (NTD) characterized by a congenital bone defect in the anterior cranium and herniation of the intracranial mass through the defect. The C677T mutation in the 5,10‐methylenetetrahydrofolate reductase gene (MTHFR) has been reported as a genetic risk factor for spina bifida. However, the role of the MTHFR in the pathogenesis of FEE remains to be clarified.

Screening for glucose-6-phosphate dehydrogenase deficiency using a modified formazan method: A pilot study on Filipino male newborns

Abstract Background : Glucose‐6‐phosphate dehydrogenase (G6PD) deficiency has increased prevalence rates in tropical Africa, tropical and subtropical Asia and some parts of the Mediterranean. Earlier studies on G6PD deficiency in the Philippines have shown prevalence rates of 4.5% to 25.7%.

Molecular basis of glucose-6-phosphate dehydrogenase deficiency among Filipinos

Abstract Background: Multiplex polymerase chain reaction (PCR) using multiple tandem forward primers and a common reverse primer (MPTP) was recently established as a comprehensive scanning system for mutations in X‐linked recessive diseases. In this report, MPTP was tested to scan for mutations of the glucose‐6‐phosphate dehydrogenase (G6PD) gene.

Glycogen phosphorylase kinase deficiency: A survey of enzymes in phosphorylase activating system

Abstract A four year-old Japanese boy with hepatomegaly and hypoglycemia has low activity of hepatic phosphorylase. A survey of enzymes involved in the phosphorylase activating system has revealed that liver phosphorylase kinase is deficient although adenosine 3′,5′-monophosphate (cyclic AMP)-dependent protein kinase and total phosphorylase measured in a mixture supplemented by rabbit muscle phosphorylase kinase show normal activities. The hormone receptor as well as adenyl cyclase system appears to be normal since cyclic AMP increases immediately after intravenous injection of glucagon. His muscle phosphorylase activating system is normal.

A comprehensive method to scan for point mutations of the glucose 6 phosphate dehydrogenase gene

SummaryWe have developed a fast and comprehensive method to scan for point mutations in a gene on X chromosome. A target region of the gene is first amplified. Then, using the amplified product as a template, PCR is carried out with multiple short-length forward primers arrayed in tandem in the scanned region, and a common reverse primer. The absence of amplified product defines the site of a mutation within a narrow region of the primer recognition site. To evaluate our method, point mutations in exon 12 of the human glucose-6-phosphate dehydrogenase (G6PD) gene were used as a model system. Out of 12 Singaporean G6PD-deficient patients, 6 cases were shown by the method to have a nucleotide change in this exon. Sequence analysis confirmed the presence of a nucleotide change in the region identified by our scanning. Thus, our method is accurate in localizing mutations within a narrow region, and allows large numbers of samples to be handled simultaneously.

High prevalence of Southeast Asian ovalocytosis in Malays with distal renal tubular acidosis

AbstractSoutheast Asian ovalocytosis (SAO) is a red blood cell abnormality common in malaria-endemic regions and caused by a 27 nt deletion of the band 3 protein gene. Since band 3 protein, also known as anion exchanger 1, is expressed in renal distal tubules, the incidence of SAO was examined in distal renal tubular acidosis (dRTA) in Malays in Kelantan, Malaysia. Twenty-two patients with dRTA and 50 healthy volunteers were examined for complication of SAO by both morphological and genetic analyses. SAO was identified in 18 of the 22 dRTA patients (81.8%), but only two of the 50 controls (4%). The incidence of SAO was significantly high in those with dRTA (p<0.001), indicating a dysfunctional role for band 3 protein/anion exchanger 1 in the development of dRTA.