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Katsumata N.,National Health Research Institute | Shinagawa T.,National Health Research Institute | Horikawa R.,National Center for Child Health and Development | Fujikura K.,Sapporo City Institute of Public Health
Metabolism: Clinical and Experimental | Year: 2010

Congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency (21-OHD) is an autosomal recessive disorder caused by the defective CYP21A2 gene that leads to various degrees of impaired secretion of both cortisol and aldosterone. In the present study, we analyzed the CYP21A2 gene in a Japanese male patient with 21-OHD and functionally characterized the mutant CYP21A2 gene. The patient presented with hypoglycemia and a salt-losing crisis during the neonatal period, and was diagnosed as having the salt-wasting form of 21-OHD based on the clinical and laboratory findings. Analysis of the CYP21A2 gene revealed that the patient is homozygous for a novel C to A conversion at -9 position of intron 9 (IVS9-9C>A) and that his parents are heterozygous for the IVS9-9C>A mutation. Transient expression of the IVS9-9C>A mutant CYP21A2 gene in COS-1 cells demonstrated that the mutation creates an aberrant splice acceptor site at -7 position of intron 9 and totally inactivates the authentic splice acceptor site of intron 9, which results in complete deficiency of 21-hydroxylase activity and loss of immunoreactive 21-hydroxylase protein. Clinical presentations of the patient as the severe salt-wasting form of 21-OHD are in good agreement with these results of the expression study. In conclusion, the patient is a homozygote for the novel intronic IVS9-9C>A mutation, which affects messenger RNA splicing and totally inactivates 21-hydroxylase to give rise to clinically manifest classic salt-wasting 21-OHD. © 2010 Elsevier Inc. Source


Katsumata N.,National Health Research Institute | Ogawa E.,Tohoku University | Fujiwara I.,Tohoku University | Fujikura K.,Sapporo City Institute of Public Health
Metabolism: Clinical and Experimental | Year: 2010

Combined 17α-hydroxylase/17,20-lyase deficiency is caused by a defect of P450c17 that catalyzes both 17α-hydroxylase and 17,20-lyase reactions in adrenal glands and gonads. In the present study, we analyzed the CYP17A1 gene in a Japanese girl with 17α-hydroxylase/17,20-lyase deficiency. The patient was referred to us for clitoromegaly at the age of 3 years. The karyotype was 46,XY. The patient was diagnosed as having 17α-hydroxylase/17,20-lyase deficiency based on the clinical and laboratory findings. Analysis of the CYP17A1 gene revealed a compound heterozygous mutation. One mutation was a deletion of codon 53 or 54 encoding Phe (TTC) in exon 1 (ΔF54) on a maternal allele, which has been previously shown to partially abolish both 17α-hydroxylase and 17,20-lyase activities. The other was a novel missense mutation resulting in a substitution of Asn (AAC) for His (CAC) at codon 373 in exon 6 (H373N) on a paternal allele. Functional expression study demonstrated that the H373N mutation almost completely eliminates enzymatic activity. Previous studies have demonstrated that replacement of histidine by leucine at position 373 causes complete loss of both 17α-hydroxylase and 17,20-lyase activities with a defect in heme binding due to a global alteration of P450c17 structure, indicating the importance of H373 for P450c17 structure and function. Together, these results indicate that the patient is a compound heterozygote for the ΔF54 and H383N mutations and that these mutations inactivate both 17α-hydroxylase and 17,20-lyase activities and give rise to clinically manifest combined 17α-hydroxylase/17,20-lyase deficiency. © 2010 Elsevier Inc. All rights reserved. Source


Higashi T.,University of Shizuoka | Suzuki M.,University of Shizuoka | Hanai J.,Sapporo City Institute of Public Health | Inagaki S.,University of Shizuoka | And 3 more authors.
Journal of Separation Science | Year: 2011

Vitamin D deficiency in an infant is associated with a wide range of adverse health outcomes in later life. A method for the quantification of 25-hydroxyvitamin D3 [25(OH)D3, the best-established indicator of vitamin D status] in neonatal dried blood spots (DBSs) using LC/ESI-MS/MS has been developed and validated. The method employed two steps of derivatization, a Diels-Alder reaction with 4-phenyl-1,2,4-triazoline-3,5-dione followed by acetylation, to enhance the detectability of 25(OH)D3 in ESI-MS/MS and to separate 25(OH)D3 from 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3], a potent interfering metabolite. 25(OH)D3 was extracted from two DBS punches (3 mm in diameter, equivalent to 5.3μL of whole blood), purified using an Oasis HLB® cartridge, and subjected to derivatization prior to analysis with LC/ESI-MS/MS. 25-Hydroxyvitamin D4 was used as the internal standard. This method was reproducible (intra- and inter-assay RSDs, <6.9%) and accurate (analytical recovery, 95.2-102.7%), and the LOQ was 3.0 ng/mL. The developed method enabled specific quantification of 25(OH)D3 in neonatal DBSs and detection of vitamin D deficiency without interference from 3-epi-25(OH)D3. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source


Takashita E.,Japan National Institute of Infectious Diseases | Ejima M.,Japan National Institute of Infectious Diseases | Itoh R.,Japan National Institute of Infectious Diseases | Miura M.,Japan National Institute of Infectious Diseases | And 4 more authors.
Eurosurveillance | Year: 2014

Six influenza A(H1N1)pdm09 viruses were detected in Sapporo, Japan, between November and December 2013. All six viruses possessed an H275Y substitution in the neuraminidase protein, which confers cross-resistance to oseltamivir and peramivir. No epidemiological link among the six cases could be identified; none of them had received neuraminidase inhibitors before specimen collection. The haemagglutinin and neuraminidase genes of the six viruses were closely related to one another, suggesting clonal spread of a single resistant virus. Source


Kaneko H.,Fukushima Medical University | Aoki K.,Hokkaido University | Ohno S.,Hokkaido University | Ishiko H.,Mitsubishi Group | And 7 more authors.
Journal of Clinical Microbiology | Year: 2011

For 4 months from September 2008, 102 conjunctival swab specimens were collected for surveillance purposes from patients across Japan suspected of having epidemic keratoconjunctivitis (EKC). Human adenovirus (HAdV) DNA was detected in 61 samples by PCR, though the HAdV type for 6 of the PCR-positive samples could not be determined by phylogenetic analysis using a partial hexon gene sequence. Moreover, for 2 months from January 2009, HAdV strains with identical sequences were isolated from five conjunctival swab samples obtained from EKC patients in five different regions of Japan. For the analyses of the 11 samples mentioned above, we determined the nucleotide sequences of the entire penton base, hexon, and fiber genes and early 3 (E3) region, which are variable regions among HAdV types, and compared them to those of other HAdV species D strains. The nucleotide sequences of loops 1 and 2 in the hexons of all 11 samples showed high degrees of identity with those of the HAdV type 15 (HAdV-15) and HAdV-29 prototype strains. However, the fiber gene and E3 region sequences showed high degrees of identity with those of HAdV-9, and the penton base gene sequence showed a high degree of identity with the penton base gene sequences of HAdV-9 and -26. Moreover, the complete genome sequence of the 2307-S strain, which was isolated by viral culture from 1 of the 11 samples, was determined. The 2307-S strain was a recombinant HAdV between HAdV-9, -15, -26, -29, and/or another HAdV type; however, the recombination sites in the genome were not obvious. We propose that this virus is a novel intertypic recombinant, HAdV-15/29/H9, and may be an etiological agent of EKC. Copyright © 2011, American Society for Microbiology. All Rights Reserved. Source

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