Yuriko Katsushima

Three Families with Autosomal Dominant Nephrogenic Diabetes Insipidus Caused by Aquaporin-2 Mutations in the C-Terminus

The vasopressin-regulated water channel aquaporin-2 (AQP2) is known to tetramerize in the apical membrane of the renal tubular cells and contributes to urine concentration. We identified three novel mutations, each in a single allele of exon 4 of the AQP2 gene, in three families showing autosomal dominant nephrogenic diabetes insipidus (NDI). These mutations were found in the C-terminus of AQP2: a deletion of G at nucleotide 721 (721 delG), a deletion of 10 nucleotides starting at nucleotide 763 (763-772del), and a deletion of 7 nucleotides starting at nucleotide 812 (812-818del). The wild-type AQP2 is predicted to be a 271-amino acid protein, whereas these mutant genes are predicted to encode proteins that are 330-333 amino acids in length, because of the frameshift mutations. Interestingly, these three mutant AQP2s shared the same C-terminal tail of 61 amino acids. In Xenopus oocytes injected with mutant AQP2 cRNAs, the osmotic water permeability (Pf) was much smaller than that of oocytes with the AQP2 wild-type (14%-17%). Immunoblot analysis of the lysates of the oocytes expressing the mutant AQP2s detected a band at 34 kD, whereas the immunoblot of the plasma-membrane fractions of the oocytes and immunocytochemistry failed to show a significant surface expression, suggesting a defect in trafficking of these mutant proteins. Furthermore, coinjection of wild-type cRNAs with mutant cRNAs markedly decreased the oocyte Pf in parallel with the surface expression of the wild-type AQP2. Immunoprecipitation with antibodies against wild-type and mutant AQP2 indicated the formation of mixed oligomers composed of wild-type and mutant AQP2 monomers. Our results suggest that the trafficking of mutant AQP2 is impaired because of elongation of the C-terminal tail, and the dominant-negative effect is attributed to oligomerization of the wild-type and mutant AQP2s. Segregation of the mutations in the C-terminus of AQP2 with dominant-type NDI underlies the importance of this domain in the intracellular trafficking of AQP2.

Evaluation of a New Rapid Antigen Test Using Immunochromatography for Detection of Human Metapneumovirus in Comparison with Real-Time PCR Assay

ABSTRACT A new rapid human metapneumovirus (hMPV) detection kit using immunochromatography (SAS hMPV test) was compared to real-time PCR for 224 nasal swab specimens, 96.4% of which were obtained from children of <15 years of age. The overall sensitivity and specificity were 82.3% and 93.8%, respectively, suggesting that this test is useful for pediatricians to diagnose hMPV infection in a clinical setting.

Increase in cell-surface localization of parathyroid hormone receptor by cytoskeletal protein 4.1G.

The cell-surface localization of GPCRs (G-protein-coupled receptors) has emerged as one of critical factors of the GPCR-mediated signal transduction. It has been reported that the C-termini of GPCRs contain the sequences for sorting the receptors to cell surface. In the present study, we have searched for proteins that interact with the C-terminus of PTH (parathyroid hormone)/PTH-related protein receptor (PTHR) by using the yeast two-hybrid system, and identified a cytoskeletal protein 4.1G (generaltype 4.1 protein) as an interactant with the C-terminus. Immunohistochemical study revealed that both PTHR and 4.1G were co-localized on plasma membranes, when they were transiently expressed in COS-7 cells. When 4.1G or the C-terminal domain of 4.1G (4.1G-CTD), a dominant-negative form of 4.1G, was co-expressed with PTHR in COS-7 cells, 4.1G, but not 4.1G-CTD, facilitated the cell-surface localization of PTHR, determined by cell-surface biotinylation assay. PTH-(1-34) caused phosphorylation of ERK (extracellular-signal-regulated kinase) 1/2 in PTHR-expressed cells mainly mediated through EGF (epidermal growth factor) receptor. The phosphorylation was enhanced by the expression of 4.1G, but not 4.1G-CTD. PTH-(1-34) elevated [Ca2+]i (intracellular Ca2+ concentration) independent of EGF receptor activation, and the elevation was enhanced by the expression of 4.1G, but not 4.1G-CTD. These data indicate that 4.1G facilitates the cell-surface localization of PTHR through its interaction with the C-terminus of the receptor, resulting in the potentiation of PTHR-mediated signal transduction.

Epidemic myalgia associated with human parechovirus type 3 infection among adults occurs during an outbreak among children: Findings from Yamagata, Japan, in 2011

BACKGROUND Based on our findings in Yamagata, Japan, in 2008, we reported that human parechovirus type 3 (HPeV3) could be associated with epidemic myalgia among adults, although HPeV3 is generally associated with infectious diseases in children. OBJECTIVES To clarify the relationship between community outbreaks among children and myalgia through the continued surveillance of HPeV3 infections. STUDY DESIGN In the summer season (June-August) of 2011, we collected 586 specimens from children with infectious diseases, and throat swabs, and stool and serum specimens from 5 patients with myalgia. We detected HPeV3 using virus isolation and reverse-transcription PCR, and carried out phylogenetic analysis. We also performed screening for HPeV3 using 309 stocked frozen specimens collected in 2008 for a comparison between 2008 and 2011 strains. RESULTS We detected HPeV3 in 59 children and isolated HPeV3 from all myalgia patients. Phylogenetic analysis indicated that the HPeV3 strains circulating in 2008 and 2011 could be clearly distinguished, apart from two strains. Further, we detected HPeV3 strains with identical nucleotide sequences from children and adults in 2008 and 2011, respectively. Two children belonging to one myalgia patient had upper respiratory infections prior to the onset of their fathers illness, and the HPeV3 isolates from these three patients had identical nucleotide sequences. CONCLUSIONS These findings suggest that HPeV3, circulating among children in the community, infects their household, including parents, a portion of whom may subsequently show symptoms of myalgia. Our observations in 2008 and 2011 strongly suggest that clinical consideration should be given to HPeV3 in children as well as in adults during summer seasons in which an HPeV3 outbreak occurs among the children in the community.

Epidemiology of parainfluenza virus types 1, 2 and 3 infections based on virus isolation between 2002 and 2011 in Yamagata, Japan.

To clarify the epidemiology of viral acute respiratory infections (ARIs), 305 human parainfluenza virus types 1 (HPIV1), 154 HPIV2 and 574 HPIV3 strains were isolated from 16,962 nasopharyngeal swabs obtained between 2002 and 2011 at pediatric clinics in Yamagata, Japan. The total isolation frequency for HPIV1–3 was 6.1%. Unlike HPIV1 infections, HPIV3 showed clear seasonality with yearly outbreaks in the spring–summer season. HPIV2 tended to appear biannually in autumn–winter. Although no reliable techniques for the laboratory diagnosis of these infections have been established, the present results suggest that HPIV1–3 are an important causative agent of ARIs in children.

Subacute thyroiditis in children: Patient report and review of the literature

Subacute thyroiditis is extremely rare during childhood. We treated a 10 year-old prepubertal girl who presented with typical clinical features of subacute thyroiditis. Considering this case and previous reports in children, we characterized the clinical features of this disorder as it occurs in childhood. In contrast to the predominance of subacute thyroiditis in adults, the incidence of subacute thyroiditis is lower than that of acute suppurative thyroiditis in children. This may cause difficulty with differential diagnosis, particularly when leukocytosis is present or the lesion is localized to the left lobe. Ultrasonography can be helpful in differentiation.

Detection of the Human Coronavirus 229E, HKU1, NL63, and OC43 between 2010 and 2013 in Yamagata, Japan

The available literature on human coronaviruses (HCoVs) in Japan is limited to epidemiological studies conducted over a maximum of 1 year. We conducted a 4-year study of HCoVs by analyzing 4,342 respiratory specimens obtained in Yamagata, Japan, between January 2010 and December 2013. A pan-coronavirus reverse transcription-PCR screening assay was performed, and all HCoV-positive specimens were subsequently confirmed by sequencing of the PCR products. We detected in 332 (7.6%) HCoV strains during the study period, comprising 133 (3.1%) HCoV-NL63, 83 (1.9%) HCoV-HKU1, 78 (1.8%) HCoV-OC43, and 38 (0.9%) HCoV-229E strains. HCoV detection per year ranged from 3.5% to 9.7%. HCoVs were detected mainly in winter, with January (28.5%) and February (25.3%) 2011 and December 2012 (14.6%) being the only months in which HCoV-NL63 detection per month exceeded 10.0%. HCoV-HKU1 displayed clear biennial peaks in January (18.3%) and February (10.7%) 2010 and in February (18.8%) and March (14.7%) 2012. The peak detection of HCoV-OC43 was 13.6% in November 2010, while that of HCoV-229E was 10.8% in March 2013. Our results indicated that there may be annual variations in the circulation of individual HCoV strains. Further long-term surveillance is necessary to clarify HCoV prevalence and circulation patterns in Japan.

Molecular epidemiology of enterovirus 71 strains isolated from children in Yamagata, Japan, between 1990 and 2013

Enterovirus 71 infections have become a major public issue in the Asia-Pacific region due to the large number of fatal cases. To clarify the longitudinal molecular epidemiology of enterovirus 71 (EV71) in a community, we isolated 240 strains from children, mainly with hand-foot-and-mouth diseases, between 1990 and 2013 in Yamagata, Japan. We carried out a sequence analysis of the VP1 region (891 bp) using 223 isolates and identified six subgenogroups (B2, B4, B5, C1, C2 and C4) during the study period. Subgenogroups C1 and B2 were found only between 1990 and 1993 and have not reappeared since. In contrast, strains in subgenogroups C2, C4 and B5 appeared repeatedly with genomic variations. Recent reports from several local communities in Japan have suggested that identical predominant subgenogroup strains, which have also been found in the Asia-Pacific region, have been circulating in a wide area in Japan. However, it is likely that there is a discrepancy between the major subgenogroups circulating in the Asia-Pacific region and those in Europe. It is necessary to continue the analysis of the longitudinal epidemiology of EV71 in local communities, as well as on regional and global levels, to develop strategies against severe EV71 infections.

Characteristics of Mycoplasma pneumoniae infection identified on culture in a pediatric clinic

The appropriate choice of antibiotics against Mycoplasma pneumoniae infection has become difficult, as the prevalence of macrolide‐resistant M. pneumoniae has increased.

Chronological changes of mumps virus genotypes in Japan between 1999–2013

Abstract Background: The molecular epidemiology of mumps virus (MuV) has been carried out worldwide based on genotyping proposed by the World Health Organisation. However, longitudinal molecular epidemiological studies of MuV are still limited. Methods: This study carried out genotyping of MuVs isolated in Yamagata prefecture, which is located in northern Japan, between 1999–2013, using standard nomenclature based on the sequence analysis of the entire 316 nucleotides of the small hydrophobic (SH) gene. Results: During this 15-year period, 249 MuVs were isolated, with the majority of them belonging to genotype G. Phylogenetic analysis revealed that genotype G strains were divided into two distinct clusters 1 and 2, consisting of 178 and 47 strains, respectively. The cluster 1 strains were isolated every year since 2001, except for 2012. The cluster 2 strains first appeared in 2011 and were dominant in 2011 and 2012. The epidemic pattern of genotype G strains observed in Yamagata was similar to those in Kanagawa and Hyogo prefectures located in eastern and western Japan, respectively. Only one L, three H and one F genotype strains were isolated in 2001, 2004 and 2010, respectively. Almost every year several genotype B strains related to Japanese vaccine strains were isolated. Conclusions: These data demonstrated that the genotype G strains have been endemically perpetuating as the major type over a wide area of Japan since 2001, although the genotype G strains that emerged after 2011 differed from the earlier strains.