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Antibody-mediated rejection (ABMR) is an important cause of both short- and long-term injury to renal allografts. Transplant glomerulopathy (TG) is strongly associated with ABMR and reduced graft survival. Ultrastructural changes in early-stage ABMR include TG as a duplication of the glomerular basement membrane (GBM), which can be observed only by transmission electron microscopy (TEM). Low-vacuum scanning electron microscopy (LVSEM) is a new technique that allows comparatively inexpensive, rapid, and convenient observations with high magnification. We analyzed human renal transplants using LVSEM and evaluated the ultrastructural changes representing TG in ABMR. GBM duplication was more clearly visible in the LVSEM images than in the light microscopy (LM) images. In the ABMR group, the cg score of the Banff classification was higher in 54% (7/13) of specimens for LVSEM images than for LM images. And 4 specimens exhibited duplication of the GBM analyzed by LVSEM, but not by LM. In addition, three-dimensional ultrastructural changes, such as coarse meshwork structures of GBM, were observed in ABMR specimens. The ABMR group also exhibited ultrastructural changes in the peritubular capillary basement membranes. In conclusion, analyses of renal transplant tissues using LVSEM allows the identification of GBM duplication and ultrastructural changes of basement membranes at the electron microscopic level, and is useful for early-stage diagnosis of ABMR.

Introduction Transplantation of IC-2-engineered bone marrow-derived mesenchymal stem cell (BM-MSC) sheets (IC-2 sheets) was previously reported to potentially reduce liver fibrosis. Methods This study prepared IC-2-engineered cell sheets from multiple lots of BM-MSCs and examined the therapeutic effects of these cell sheets on liver fibrosis induced by carbon tetrachloride in mice. The predictive factors for antifibrotic effect on liver fibrosis were tried to identify in advance. Results Secreted matrix metalloproteinase (MMP)-14 was found to be a useful predictive factor to reduce liver fibrosis. Moreover, the cutoff index of MMP-14 for 30% reduction of liver fibrosis was 0.918 fg/cell, judging from univariate analysis and receiver operating curve analysis. In addition, MMP-13 activity and thioredoxin contents in IC-2 sheets were also inversely correlated with hepatic hydroxyproline contents. Finally, IC-2 was also found to promote MMP-14 secretion from BM-MSCs of elderly patients. Surprisingly, the values of secreted MMP-14 from BM-MSCs of elderly patients were much higher than those of young persons. Conclusion The results of this study suggest that the IC-2 sheets would be applicable to clinical use in autologous transplantation for patients with cirrhosis regardless of the patient's age., Highlights • IC-2- sheets from multiple lots of BM-MSCs ameliorate liver fibrosis in mice. • Secreted MMP-14 is a useful predictive marker to reduce liver fibrosis. • MMP-13 and thioredoxin in IC-2 sheets were also associated with liver fibrosis. • IC-2 also promotes MMP-14 secretion from BM-MSCs of elderly patients.

We previously reported that genotype-phenotype correlations in 12 missense variants causing severe insulin resistance, located in the second and third fibronectin type III (FnIII) domains of the insulin receptor (INSR), containing the α-β cleavage and part of insulin-binding sites. This study aimed to identify genotype-phenotype correlations in FnIII domain variants of IGF1R, a structurally related homolog of INSR, which may be associated with growth retardation, using the recently reported crystal structures of IGF1R. A structural bioinformatics analysis of five previously reported disease-associated heterozygous missense variants and a likely benign variant in the FnIII domains of IGF1R predicted that the disease-associated variants would severely impair the hydrophobic core formation and stability of the FnIII domains or affect the α-β cleavage site, while the likely benign variant would not affect the folding of the domains. A functional analysis of these variants in CHO cells showed impaired receptor processing and autophosphorylation in cells expressing the disease-associated variants, but not in those expressing the wild-type form or the likely benign variant. These results demonstrated genotype-phenotype correlations in the FnIII domain variants of IGF1R, which are presumably consistent with those of INSR and would help in the early diagnosis of patients with disease-associated IGF1R variants.

Juvenile granulosa cell tumor (JGCT), classified as a sex cord-stromal tumor, is a rare neoplasm. This is an instructive case report of JGCT accompanied by augmented interleukin (IL)-6 secretion. A 13-year-old girl with prolonged fever and delayed puberty was diagnosed with JGCT of the left ovary based on an imaging study and pathological investigation. Although it was not clear whether IL-6 was secreted from the tumor cells, her serum level of IL-6 was very high. After tumorectomy, the patient's symptoms immediately disappeared, her IL-6 level decreased, and she entered puberty. Therefore, augmented IL-6 secretion production induced by tumors should be considered a potential cause of prolonged fever and/or delayed puberty.

Transplantation of IC-2-engineered bone marrow-derived mesenchymal stem cell (BM-MSC) sheets (IC-2 sheets) was previously reported to potentially reduce liver fibrosis. This study prepared IC-2-engineered cell sheets from multiple lots of BM-MSCs and examined the therapeutic effects of these cell sheets on liver fibrosis induced by carbon tetrachloride in mice. The predictive factors for antifibrotic effect on liver fibrosis were tried to identify in advance. Consequently, secreted matrix metalloproteinase (MMP)-14 was found to be a useful predictive factor to reduce liver fibrosis. Moreover, the cutoff index of MMP-14 for 30% reduction of liver fibrosis was 0.918 fg/cell, judging from univariate analysis and receiver operating curve analysis. In addition, MMP-13 activity and thioredoxin contents in IC-2 sheets were also inversely correlated with hepatic hydroxyproline contents. Finally, IC-2 was also found to promote MMP-14 secretion from BM-MSCs of elderly patients. Surprisingly, the values of secreted MMP-14 from BM-MSCs of elderly patients were much higher than those of young persons. The results of this study suggest that the IC-2 sheets would be applicable to clinical use in autologous transplantation for patients with cirrhosis regardless of the patient’s age.

We previously reported genotype-phenotype correlations in 12 missense variants causing severe insulin resistance, located in the second and third fibronectin type III (FnIII) domains of the insulin receptor (INSR), containing the α-β cleavage and part of insulin-binding sites. This study aimed to identify genotype-phenotype correlations in FnIII domain variants of IGF1R, a structurally related homolog of INSR, which may be associated with growth retardation, using the recently reported crystal structures of IGF1R. A structural bioinformatics analysis of five previously reported disease-associated heterozygous missense variants and a likely benign variant in the FnIII domains of IGF1R predicted that the disease-associated variants would severely impair the hydrophobic core formation and stability of the FnIII domains or affect the α-β cleavage site, while the likely benign variant would not affect the folding of the domains. A functional analysis of these variants in CHO cells showed impaired receptor processing and autophosphorylation in cells expressing the disease-associated variants but not in those expressing the wild-type form or the likely benign variant. These results demonstrated genotype-phenotype correlations in the FnIII domain variants of IGF1R, which are presumably consistent with those of INSR and would help in the early diagnosis of patients with disease-associated IGF1R variants.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disease causing renal cysts. Reports on kidney cyst infection in children are rare despite cyst infections being important complications of ADPKD. Here, we report a case of a child without any medical history who had a urinary tract infection with sepsis at 7 months. Leukocyturia persisted despite antibiotic therapy because the infection was treatment-resistant. Initial ultrasound and contrast computed tomography were inconclusive because cysts could not be detected clearly, and a family history of renal cysts was not determined. Subsequently, history of paternal renal cysts, thick walls in infectious cystic lesions on diffusion-weighted magnetic resonance imaging (MRI), and multiple small lesions with high signals on T2-weighted imaging in both kidneys became apparent. Upon diagnosis of ADPKD with cyst infection, antibiotic therapy was switched from cefotaxime to trimethoprim/sulfamethoxazole to achieve better cyst penetration, which successfully resolved the infection. In this patient, MRI was effective for clear visualization and diagnosis of infectious lesions and small cysts in undiagnosed ADPKD with cyst infection. Administering antibiotics with better cyst penetration is important. Trimethoprim/sulfamethoxazole is an option for use in children. This is the first case report that describes ADPKD with cyst infection in an infant in detail.

Background Treatment costs for children with growth hormone (GH) deficiency are subsidized by the government in Japan if the children meet clinical criteria, including height limits (boys: 156.4 cm; girls: 145.4 cm). However, several funding programs, such as a subsidy provided by local governments, can be used by those who exceed the height limits. In this study, we explored the impacts of financial support on GH treatment using this natural allocation. Methods A retrospective analysis of 696 adolescent patients (451 boys and 245 girls) who reached the height limits was conducted. Associations between financial support and continuing treatment were assessed using multiple logistic regression analyses adjusting for age, sex, height, growth velocity, bone age, and adverse effects. Results Of the 696 children in the analysis, 108 (15.5 %) were still eligible for financial support. The proportion of children who continued GH treatment was higher among those who were eligible for support than among those who were not (75.9 % vs. 52.0 %, P

Aims The aim of this study was to clarify the significance of previously reported susceptibility variants in the development of autoimmune Type 1 diabetes in non-white children. Tested variants included rs2290400, which has been linked to Type 1 diabetes only in one study on white people. Haplotypes at 17q12-q21 encompassing rs2290400 are known to determine the susceptibility of early-onset asthma by affecting the expression of flanking genes. Methods We genotyped 63 variants in 428 Japanese people with childhood-onset autoimmune Type 1 diabetes and 457 individuals without diabetes. Possible association between variants and age at diabetes onset was examined using age-specific quantitative trait locus analysis and ordered-subset regression analysis. Results Ten variants, including rs2290400 in GSDMB, were more frequent among the people with Type 1 diabetes than those without diabetes. Of these, rs689 in INS and rs231775 in CTLA4 yielded particularly high odds ratios of 5.58 (corrected P value 0.001; 95% CI 2.15–14.47) and 1.64 (corrected P value 5.3 × 10-5; 95% CI 1.34–2.01), respectively. Age-specific effects on diabetes susceptibility were suggested for rs2290400; heterozygosity of the risk alleles was associated with relatively early onset of diabetes, and the allele was linked to the phenotype exclusively in the subgroup of age at onset ≤ 5.0 years. Conclusions The results indicate that rs2290400 in GSDMB and polymorphisms in INS and CTLA4 are associated with the risk of Type 1 diabetes in Japanese children. Importantly, cis-regulatory haplotypes at 17q12-q21 encompassing rs2290400 probably determine the risk of autoimmune Type 1 diabetes predominantly in early childhood.

The etiology of idiopathic short stature (ISS) and Leri-Weill dyschondrosteosis (LWD) in European patients is known to include SHOX mutations and copy-number variations (CNVs) involving SHOX and/or the highly evolutionarily conserved non-coding DNA elements (CNEs) flanking the gene. However, the frequency and types of SHOX abnormalities in non-European patients and the clinical importance of mutations in the CNEs remains to be clarified. Here, we performed systematic molecular analyses of SHOX for 328 Japanese patients with ISS or LWD. SHOX abnormalities accounted for 3.8% of ISS and 50% of LWD cases. CNVs around SHOX were identified in 16 cases, although the ~47 kb deletion frequently reported in European patients was absent in our cases. Probably damaging mutations and benign/silent substitutions were detected in four cases, respectively. Although CNE-linked substitutions were detected in 15 cases, most of them affected poorly conserved nucleotides and were shared by unaffected individuals. These results suggest that the frequency and mutation spectrum of SHOX abnormalities are comparable between Asian and European patients, with the exception of a European-specific downstream deletion. Furthermore, this study highlights the clinical importance and genetic heterogeneity of the SHOX-flanking CNVs, and indicates a limited clinical significance of point mutations in the CNEs.

We report the case of a 13-month-old girl who suffered frequent vomiting, intractable diarrhea, hyperlactatemia, and liver dysfunction. Although the symptoms were treatment-resistant, administration of an enteral nutrition formula containing medium-chain triglycerides reduced her weight loss, vomiting, and diarrhea. Immunostaining of mitochondrial respiratory chain (MRC) complexes of her colonic mucosa confirmed the diagnosis of MRC complex I deficiency. This case shows that this disease should be included in the differential diagnosis in patients with hyperlactatemia and intractable, cryptogenic gastrointestinal symptoms. In addition, the mucosa of the affected gastrointestinal organ should be analyzed with immunostaining or electron microscopy for MRC complexes.

Cholesterol ester storage disease (CESD) is an autosomal recessive disorder caused by deficient lysosomal acid lipase (LAL) activity, resulting in cholesteryl ester (CE) accumulation. CESD patients have liver disease associated with mixed dyslipidemia leading to liver failure. We here report the case of an 11-year-old male CESD patient with a novel mutation who had the chief complaint of massive hepatomegaly. The patient's liver reached to his pelvis, and his spleen was 2 cm below the costal margin. The patient had elevated serum liver enzymes and mixed dyslipidemia. The liver biopsy tissue showed characteristic CESD pathology, which included microvesicular steatosis, mild fibrosis and foamy macrophages. Electron microscopy showed a remnant cleft of CE crystals, and dried blood spot testing showed reduced LAL activity. We identified compound heterozygous mutations in the LIPA gene in this patient, namely, c.607G>C and c.791T>C. The former mutation was previously reported only in a Japanese patient, whereas the latter mutation is novel. The findings of this study suggest that LIPA gene mutations in Japanese CESD patients are different from those in Western patients. Although CESD is rare, it is likely that many patients are unrecognized or misdiagnosed, and thus the possibility of CESD should be considered in patients with hepatosplenomegaly and dyslipidemia.

Pseudoautosomal region 1 (PAR1) contains SHOX, in addition to seven highly conserved non-coding DNA elements (CNEs) with cis-regulatory activity. Microdeletions involving SHOX exons 1-6a and/or the CNEs result in idiopathic short stature (ISS) and Leri-Weill dyschondrosteosis (LWD). Here, we report six rare copy-number variations (CNVs) in PAR1 identified through copy-number analyzes of 245 ISS/LWD patients and 15 unaffected individuals. The six CNVs consisted of three microduplications encompassing SHOX and some of the CNEs, two microduplications in the SHOX 3'-region affecting one or four of the downstream CNEs, and a microdeletion involving SHOX exon 6b and its neighboring CNE. The amplified DNA fragments of two SHOX-containing duplications were detected at chromosomal regions adjacent to the original positions. The breakpoints of a SHOX-containing duplication resided within Alu repeats. A microduplication encompassing four downstream CNEs was identified in an unaffected father-daughter pair, whereas the other five CNVs were detected in ISS patients. These results suggest that microduplications involving SHOX cause ISS by disrupting the cis-regulatory machinery of this gene and that at least some of microduplications in PAR1 arise from Alu-mediated non-allelic homologous recombination. The pathogenicity of other rare PAR1-linked CNVs, such as CNE-containing microduplications and exon 6b-flanking microdeletions, merits further investigation.

SummaryObjective The type I insulin-like growth factor I receptor (IGF1R) plays an important role in growth. We aimed to evaluate the detailed mechanism underlying the effect of IGF1R on human growth. Patients and Methods We have performed sequence analysis of IGF1R in 55 patients with SGA short stature in Japan, since 2004, and identified novel heterozygous nonsense mutations in 2 patients: an 8-year-old Japanese boy (case 1), with a birthweight of 2228 g (−3·3 SDS) and height of 46 cm (−2·1 SDS), and a 3-year-old Japanese girl (case 2), with a birthweight of 2110 g (−3·0 SDS) and height of 44·3 cm (−2·8 SDS). Both patients had a short stature (−3·2 SDS, −3·1 SDS). We determined the protein expression of mutated IGF1R, assessed the effect of the endoplasmic reticulum-associated degradation (ERAD) pathway on mutated IGF1R, assessed the dominant-negative effect of IGF1R and performed quantitative RT-PCR analysis of IGF1R mRNA expression in whole blood cells. Results Two novel heterozygous nonsense mutations (case 1: p.Q1250X and case 2: p.W1249X) were identified. Although these mutations did not affect blood IGF1R mRNA levels, they significantly decreased the expression of IGF1R protein in transiently transfected cells. Treatment with the proteasome inhibitor MG132 showed significantly increased IGF1R protein. Conclusions Heterozygous nonsense mutations affecting the C-terminal region (p.Q1250X, p.W1249X) of IGF1R decreased the expression of IGF1R through the ERAD pathway. Our study revealed the importance of the C-terminal region and the dosage of this receptor for growth.

Background: Early predictors of status epilepticus (SE)-associated mortality and morbidity have not been systematically studied in children, considerably impeding the identification of patients at risk. Objectives: To determine reliable early predictors of SE-associated mortality and morbidity and identify the etiology of SE-associated sequelae in Japanese children. Methods: We conducted a prospective multicenter study of clinical findings and initial laboratory data acquired at SE onset, and assessed outcomes at the last follow-up examination. In-hospital death during the acute period and neurological sequelae were classified as poor outcomes. Results: Of the 201 children who experienced their first SE episode, 16 exhibited poor outcome that was most commonly associated with acute encephalopathy. Univariate analysis revealed that the following were associated with poor outcomes: young age (⩽24months); seizure duration >90min; seizure intractability (failure of the second anticonvulsive drug); biphasic seizures; abnormal blood glucose levels ( 250mg/dL); serum aspartate aminotransferase (AST) ⩾56U/L; and C-reactive protein (CRP) levels >2.00mg/dL. Multivariate analysis revealed that young age, seizure intractability, abnormal blood glucose levels, and elevated AST and CRP levels were statistically significant. Conclusions: Young age and seizure intractability were highly predictive of poor outcomes in pediatric SE. Moreover, abnormal blood glucose levels and elevated AST and CRP levels were predictors that might be closely associated with the etiology, especially acute encephalopathy and severe bacterial infection (sepsis and meningitis) in Japanese children.

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is caused by mutations in the CYP21A2 gene. The residual enzyme activity is strongly associated with the phenotype. We describe a rare case of CAH with a rare CYP21A2 mutation. The patient was a one-year-old Japanese boy. At 16 days old, he was referred to our hospital because of elevated serum 17-OH-progesterone (17-OHP) levels in neonatal screening. The compound heterozygous mutations (IVS2-13 A/C>G, and p.E431K) in CYP21A2 were identified at 2 months old, and we diagnosed non-classical CAH, since he did not have significant physical signs (pigmentation and salt-wasting). However, his body weight decreased, and his serum 17-OHP level (99.5 ng/mL) was elevated at 3 months old. Steroid replacement therapy was started at 3 months old. Our patient's clinical course resembled simple virilizing (SV) CAH, but classification was difficult because the patient showed increased renin activity indicating an aldosterone deficiency, and late onset of symptoms. While the IVS 2-13 A/C>G mutation is common in the classical form of CAH, p.E431K is a rare point mutation. Functional analysis revealed that the residual enzyme activity of p.E431L was 5.08±2.55% for 17-OHP and 4.12±2.37% for progesterone, which is consistent with SV CAH. p.E431 is localized in the L-helix near the heme-binding site. The mutation might interfere with heme binding, leading to deactivation of CYP21A2. This report showed that CYP21A2 p.E431 has an important effect on enzyme activity.

The primary goal of the Genetics and Neuroendocrinology of Short Stature International Study (GeNeSIS) was to assess the safety and effectiveness of Humatrope®, a GH preparation, in the treatment of pediatric patients with short stature. We report our findings in the GH-treated Japanese pediatric population focusing on the incidence of type 2 diabetes (T2D) and occurrence of neoplasms. A total of 2,345 Japanese patients were assessed for safety. During a mean observation period of 3.2 yr, T2D occurred in 3 patients (0.13%) and slowly progressive insulin-dependent diabetes mellitus (SPIDDM) related to underlying mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) in 1 patient (0.04%). Neoplasms were reported in 13 patients (0.56%), including 1 patient with brain tumor (germinoma) and 5 with craniopharyngiomas (4 recurrences); the remainder were benign, typically dermatological, neoplasms. The incidence of diabetes mellitus determined in the study did not differ from previous reports in GH-treated pediatric patients, and there was no apparent increase in the risk of new neoplastic lesions or malignant tumors.

Background A preference for calorie-dense food in men seems to be closely linked with a considerably higher incidence of obesity in adulthood for men than women, but it is not clear in which life stage the gender differences in food preference begin to appear. In order to clarify this, a picture choice method has been developed that is designed to evaluate food preferences or interests in children based on their subjective choices. Methods In total, 486 children aged 6–12 years were enrolled. To evaluate food interest, children were instructed to choose any 10 from 36 pictures in the panel showing 10 different foods and 26 other things. The number of foods chosen was set as the food interest score. For food preference, they were also instructed to choose any 10 from 36 pictures in the other panel depicting 36 different foods. For the 10 foods chosen, Japanese food score, energy density, fat energy content, and saturated fatty acid score were calculated. These indices were compared for sex, age group and body mass index. Results Indices reflecting food interest or fat preference were significantly higher in boys than girls both in the 7–9- and 10–12-year-old age groups. Positive correlations were found between food interest score and energy density, fat energy content, and saturated fatty acid score. Conclusions Using the picture choice method, definite gender differences in food preference were identified in early elementary school children. This information could be useful for dietary therapy in childhood obesity.

Background A KCNE1 polymorphism, D85N, causes long QT syndrome (LQTS) with a decrease in the slowly activating delayed-rectifier K+ channel current (IKs). We examined impacts of D85N polymorphism on KCNE1 protein stability and functions, and tested the ability of various drugs to modify them. Methods KCNE1-D85N or the wild-type protein was coexpressed in COS7 cells with KCNQ1 to form K+ channels. Expression, degradation, and intracellular localization of KCNE1 proteins, as well as the currents conferred by KCNQ1/KCNE1 complexes, were determined using immunoblots, immunofluorescence, and patch-clamp techniques. Results The protein level of KCNE1-D85N was lower than that of the wild-type, in spite of the comparable levels of their mRNA. KCNE1-D85N was highly ubiquitinated and rapidly degraded as compared to the wild-type; a proteasome inhibitor, MG132, inhibited its degradation and increased its steady-state level. Both KCNE1-D85N and the wild-type proteins were co-immunoprecipitated with KCNQ1. Immunofluorescent signals of KCNE1-D85N accumulated in the endoplasmic reticulum and Golgi apparatus, with reduced levels on the cell membrane. Patch-clamp experiments demonstrated that the membrane current corresponding to IKs was much smaller in cells expressing KCNE1-D85N than in those expressing the wild-type. Verapamil (0.5–10 μM) increased the protein level of KCNE1-D85N, decreased its ubiquitination, slowed its degradation, and enhanced KCNQ1/KCNE1-D85N channel currents. Pretreatment with amiodarone abolished these effects of verapamil. Conclusion KCNE1-D85N is less stable than the wild-type protein, and is rapidly degraded through the ubiquitin-proteasome system. Verapamil may be of a therapeutic value in LQTS patients via preventing degradation of KCNE1-D85N.

The aim of this study was to assess changes in quality of life (QoL) in Japanese children with GH deficiency (GHD) after 12 mo of GH treatment or with idiopathic short stature (ISS) after 12 mo without treatment. Children with GHD were treated with GH after enrollment. Outcome measures included the parent-rated Child Behavior Checklist (CBCL), the Youth Self-Report Form (YSR), and height standard deviation scores (SDS). Total CBCL scores significantly decreased in children with GHD (n = 152, mean change (standard deviation [SD]) = –3.42 [11.21]) and ISS (n = 129, mean change = –4.82 [10.09]) after 12 mo (p < 0.001). Total YSR scores (mean change = –9.21 [14.07]) and height SDS (mean change = 0.35 [0.38]) significantly decreased in children with GHD (p < 0.001), but were unchanged in children with ISS. The change in total YSR score was significantly correlated with the change in height SDS in children with GHD (r = –0.516, p = 0.003). Our findings demonstrate that GH treatment can improve QoL in Japanese children with GHD. The correlation between the changes in total YSR score and height SDS demonstrated that increased height resulted in improved QoL.

This study assessed the effectiveness and safety of growth hormone (GH; Humatrope(®)) therapy in Japanese children with GH deficiency (GHD) or Turner syndrome (TS) enrolled in the Genetics and Neuroendocrinology of Short Stature International Study (GeNeSIS). GeNeSIS is an open-label, multinational, multicenter, observational study conducted in 30 countries. In this interim report, there were 1129 GH treatment-naive children with GHD, with a mean chronological age (± standard deviation) of 8.75 (3.32) years, and 90 girls with TS, with a mean chronological age of 8.93 (3.67) years. The mean height standard deviation score (SDS) increased from -2.73 (0.63) SD and -2.71 (0.63) SD at study entry to -2.22 (0.68) SD and -2.20 (0.60) SD after 1 year of treatment in the GHD and TS groups, respectively. In both groups, mean height SDS increased further with each year of treatment to 4 years; however, the magnitude of change in height SDS declined with time. The mean insulin-like growth factor-I SDS increased from below the mean of the reference population at study entry to a level similar to (GHD group) or higher than (TS group) the mean of the reference population during the 4-year treatment period. The incidence of serious adverse events (AEs), treatment-related AEs, and AEs related to glucose intolerance was low in both groups (0.1% to 3.0%). In conclusion, GH treatment in Japanese children with GHD or TS resulted in increased growth over a 4-year treatment period with a favorable safety profile; however, the improvements in growth declined with time.

Pseudohypoaldosteronism type 1 (PHA1) is a rare genetic disease characterized by resistance to aldosterone, and the renal form of PHA1 is associated with heterozygous inactivating mutations in NR3C2, which encodes mineralocorticoid receptor (MR). Here we report a case of renal PHA1 due to a novel frameshift mutation in NR3C2. A 10-day-old Japanese male infant, born at 39 weeks gestation (birth weight, 2,946 g), was admitted to our hospital because of lethargy and vomiting, with a 6.7% weight loss since birth. Laboratory test results were: Na+, 132 mEq/L; K+, 6.6 mEq/L; Cl+, 93 mEq/L. Both plasma aldosterone level and plasma renin activity were markedly elevated at diagnosis, 2,940 ng/dL (normal range: 26.9-75.8 ng/dL) and 560 ng/mL/h (normal range 3.66-12.05 ng/mL/h), respectively. Direct sequence analysis of NR3C2 revealed a novel heterozygous mutation (c.3252delC) in the patient and his father. The mutation causes a frameshift starting at amino acid I 963 within the C terminal ligand-binding domain of MR and results in a putative abnormal stop codon at amino acid 994, with an extension of 10 amino acids compared to normal MR. We performed cell culture experiments to determine the levels of mutant NR3C2 mRNA and MR, and evaluate the effects of the mutation on MR response to aldosterone. The mutation decreased the expression of MR, but not NR3C2 mRNA, and led to decreased MR function, with no dominant negative effect. These results provide important information about MR function and NR3C2 mutation in PHA1.

Aim: Mother-to-child transmission (MTCT) is the major transmission pathway of hepatitis C virus (HCV) in children. However, its risk factors remain unsettled for introduction of putative intervention. Methods: Pregnant women screened for HCV and MTCT in children born to antibody-positive mothers were prospectively studied in Tottori, Japan. Results: Among 41 856 screened women, 188 (0.45%) were HCV antibody-positive, of whom 61% had detectable HCV RNA. While 10 of the 34 children (29%) born to high viral load (HVL: ≥6.0 × 105 IU/mL) mothers were infected, none born to RNA-detectable but non-HVL mothers were infected (P

Type I insulin-like growth factor receptor (IGF-IR) is widely expressed across many cell types in fetal and postnatal tissues. The activation of this receptor after the binding of secreted IGF-I and IGF-II promotes cell differentiation and proliferation. IGF-IR has an important role in normal fetal and postnatal growth and development. IGF-IR gene anomalies presenting with intrauterine and postnatal growth retardation have recently been reported in some families. Familial short stature with IGF-IR gene anomaly is considered rare, and the clinical condition and features remain unknown. IGF-IR gene anomaly such as heterozygous IGF-IR mutation or haploinsufficiency of the IGF-IR gene should be investigated in those patients presenting with 1) low birth weight and birth height (IGF-IR gene anomaly.

Background: Patients with Turner syndrome (TS) are prone to having metabolic abnormalities, such as obesity, dyslipidemia, hypertension, hyperinsulinemia and type 2 diabetes mellitus, resulting in increased risks of developing atherosclerotic diseases. Objective: To determine the effect of growth hormone (GH) therapy on serum cholesterol levels in prepubertal girls with TS enrolled in the Turner syndrome Research Collaboration (TRC) in Japan. Patients and methods: Eighty-one girls with TS were enrolled in the TRC, and their total cholesterol (TC) levels before GH therapy were compared with reported levels of healthy school-aged Japanese girls. TC levels after 1, 2 and 3 yr of GH treatment were available for 28 of the 81 patients with TS. GH was administered by daily subcutaneous injections, 6 or 7 times/wk, with a weekly dose of 0.35 mg/kg body weight. Results: Baseline TC levels revealed an age-related increase in TS that was in contrast to healthy girls showing unchanged levels. During GH therapy, TC decreased significantly after 1 yr of GH treatment and remained low thereafter. Conclusions: Girls with untreated TS showed an age-related increase in TC that was a striking contrast to healthy girls, who showed unchanged levels. GH therapy in girls with TS brought about a favorable change in TC that indicates the beneficial impact of GH on atherogenic risk.

Growth charts are essential and universally used for evaluating growth and development of children in both clinical settings and in public health examinations (1, 2). We previously reported the growth standards for Japanese children with percentile values based on the year 2000 national survey data (3), which were established by the lambda-mu-sigma (LMS) method (4). These standards have been widely used mainly in public health examinations. In clinical practices, Japanese physicians preferably assess growth with standard deviation (SD) scores, because many physicians feel that percentiles are not suitable for monitoring children with extreme growth retardation. Considering this, we created practical growth charts with mean and SD values, based on the criteria of the national medical aid program for specific pediatric chronic diseases by using the eye-fitting method (5). Although these charts have been widely used in clinical settings, they do not reflect the correct distributions of height and weight for Japanese children, especially the weight chart. Weight is not usually distributed normatively, but the practical weight chart was made with the assumption of a normal distribution. To this end, we saw the need for growth standards that can be used appropriately both for clinical and public health purposes. Therefore, we reanalyzed the previously reported growth standard charts with percentile values (3) and constructed the growth standards with mean and SD values for Japanese children, which would be applicable not only for clinical practices but also for public health examinations.

Sugihara S, Ogata T, Kawamura T, Urakami T, Takemoto K, Kikuchi N, Takubo N, Tsubouchi K, Horikawa R, Kobayashi K, Kasahara Y, Kikuchi T, Koike A, Mochizuki T, Minamitani K, Takaya R, Mochizuki H, Nishii A, Yokota I, Kizaki Z, Mori T, Shimura N, Mukai T, Matsuura N, Fujisawa T, Ihara K, Kosaka K, Kizu R, Takahashi T, Matsuo S, Hanaki K, Igarashi Y, Sasaki G, Soneda S, Teno S, Kanzaki S, Saji H, Tokunaga K, Amemiya S, and The Japanese Study Group of Insulin Therapy for Childhood and Adolescent Diabetes (JSGIT). HLA-class II and class I genotypes among Japanese children with Type 1A diabetes and their families. Objective: To determine the HLA-DRB1, DQB1, DPB1, A, C, and B genotypes among Japanese children with autoimmune type 1 diabetes. Methods: Four hundred and thirty patients who were GADAb and/or IA-2Ab-positive (Type 1A) were recruited from 37 medical centers as part of a nationwide multicenter collaborative study. DNA samples from 83 siblings of the children with Type 1A diabetes and 149 parent–child trios were also analyzed. A case-control study and a transmission disequilibrium test (TDT) were then performed. Results: The susceptible and protective DRB1 and DQB1 alleles and haplotypes were confirmed. DPB1 alleles unique to the Japanese population and those common to multiple ethnic groups were also present. A linkage disequilibrium (LD) analysis showed both susceptible and protective haplotypes. The TDT did not reveal any alleles that were transmitted preferentially from the mother or father to children with Type 1A. Homozygosity for DRB1∗09:01-DQB1∗03:03 and heterozygosity for DRB1∗04:05-DQB1∗04:01 and DRB1∗08:02-DQB1∗03:02 were associated with an extremely high risk of Type 1A. A comparison of children with Type 1A and their parents and siblings suggested a dose effect of susceptible DRB1-DQB1 haplotypes and an effect of protective alleles on immunological pathogenesis. DRB1∗09:01 appeared to be strongly associated with an early onset in preschool children with Type 1A diabetes. Conclusions: This study demonstrated the characteristic association of HLA-class II and class I genes with Type 1A diabetes among Japanese children. A TDT did not reveal the genomic imprinting of HLA-class II and class I genes in Type 1A diabetes.

Aim: The lack of a nationwide survey on hepatitis B virus (HBV) and hepatitis C virus (HCV) infections in Japan led us to investigate the epidemiological profiles of these infections among Japanese children. Methods: We conducted a questionnaire survey of children (

We attempted to clarify the effects of cyclohexenonic long-chain fatty alcohol (CHLFA) on the alterations of type 2 diabetes-induced nephropathy. Forty-week-old male Goto-Kakizaki (GK) and Wistar rats were divided into four groups of 6 to 8 animals. Group A consisted of eight Wistar rats and served as an age-matched control group. Group B (7 GK rats) received no treatment and served as a diabetic group. Group C (6 GK rats) was treated daily with low-dose CHLFA (2 mg/ kg/body weight, subcutaneously) for 30 weeks, and Group D (6 GK rats) with high-dose CHLFA (8 mg/kg/body weight) for 30 weeks. At the end of the treatment period, urinary protein excretion, blood chemistry, renal histological, and immunohistological analyses were conducted. Although CHLFA administration did not influence serum glucose or insulin levels, it reversed diabetes-induced increases in urinary protein excretion and serum creatinine. Light microscopically, CHLFA treatment ameliorated the otherwise elevated glomerular sclerotic scores in the diabetic group.Immunohistochemically, increased expression of desmin and decreased expression of rat endothelial cell antigen-1 in the group with untreated diabetes both showed a reversal to control levels in the high-dose CHLFA treatment group. In conclusion, CHLFA may ameliorate type 2 diabetes-induced nephropathy.

Specific immunoassay systems for intact human osteocalcin (I-OC) and its 26-residue propeptide have been newly developed to assess their usefulness as biochemical markers of bone metabolism. Using human cultured osteoblastic periosteal cells, we monitored 24 h secretion of these molecules from the osteoblastic cells and also examined the deposition of Ca, P, and I-OC on the extracellular matrix. At day 5, both I-OC and its propeptide were secreted by osteoblastic cells in a concentration-dependent manner by treatment with 1,25-(OH)2D3. This propeptide was not detected in the serum of adult subjects but was detected in the serum of normal children, which confirmed this in vitro result of propeptide secretion. The secretion of I-OC into medium transiently decreased at day 11, when the rapid accumulation of I-OC, Ca, and P, namely mineralization, was observed on the extracellular matrix of osteoblastic cells, although secretion of the propeptide constantly increased throughout the culture period. Therefore, the ratio of the amount of propeptide to I-OC in the supernatant markedly increased when mineralization started. These data demonstrate the superior specificity of propeptide as a marker of osteoblastic function in vitro compared with I-OC and that monitoring the changes in propeptide to I-OC ratios in the culture supernatant may be useful for predicting the timing of mineralization on the extracellular matrix of osteoblastic cells.

Insulin-like growth factors (IGFs) are found in human circulation predominantly as part of a growth hormone (GH)-dependent complex of 125-150 kD, which is composed of three subunits: IGF-I or IGF-II, an acid stable IGF binding protein (IGFBP)-3, and an acid labile subunit (ALS). Although recent studies demonstrate that a number of cell types in culture secrete IGFs and IGFBP-3, very little is known with regard to the origin of circulating ALS. To test the hypothesis that human bone cells (HBCs), which produce abundant amounts of IGF-II and IGFBP-3, also produce ALS, we measured the IGF-I, IGF-II, IGFBP-3, and ALS levels using specific radioimmunoassays (RIAs) in the conditioned medium (CM) of untransformed normal HBCs and SaOS-2 osteosarcoma cells treated with various effectors (IGF-II, osteogenic protein-1 [OP-1, bone morphogenetic protein-7] and human GH) for 48 h. No detectable levels (< 3 ng/ml) of ALS were found in the CM of various HBC types under basal conditions. In contrast, CM collected from liver explants in culture contained significant amount of ALS prepared and assayed under identical conditions. The IGF-I level was also undetectable in the CM of various HBC types. In the IGF-II (3, 30 ng/ml)-treated HBC CM, the IGFBP-3 level was increased in a dose-dependent manner but neither IGF-I nor ALS could be detected. In the SaOS-2 cell culture, OP-1 (1, 100 ng/ml) increased both IGF-II and IGFBP-3 secretion but neither ALS nor IGF-I secretion. Treatment of HBCs with GH (1, 10, 100 ng/ml) had no significant effect on the secretion of either IGF-I, IGF-II, IGFBP-3, or ALS.(ABSTRACT TRUNCATED AT 250 WORDS)

Although associated factors are important for the occurrence of neural damage in neonatal hypoglycemia, they are not fully understood. Sixty patients with neonatal hypoglycemia were studied through a review of their medical records in Tottori University Hospital. The patients were classified into two main groups: Group I were patients who had mental retardation, developmental delay, cerebral palsy or epilepsy while Group II were those who were normal in their follow-up. Group I consisted of 12 patients while Group II consisted of 48 patients. The median gestational age was 38 weeks in Group I and 36.7 weeks in Group II. The frequencies of small for gestational age were similar in both groups. Blood glucose levels less than 15 mg/dl were more frequent in Group 1 (50.0%) than in Group 2 (14.6%) (P=0.015). Duration of hypoglycemia was longer in Group I (median, 14 h) than in Group II (median, 1.75 h) (p

Background/aims To describe the clinical and biological findings of two Japanese siblings with novel MPV17 gene mutations (c.451insC/c.509C > T) manifesting hepatic mitochondrial DNA depletion syndrome. Methods We observed these brothers and sought to determine the efficacy of treatment targeting respiratory chain complex II for the younger brother. Results A 3-month-old boy had presented with profound liver dysfunction, failure to thrive, and watery diarrhea. Although he was then placed on a carbohydrate-rich diet, his liver function thereafter fluctuated greatly in association with viral infections, and rapidly deteriorated to liver failure. He underwent liver transplantation at 17 months of age but died at 22 months of age. The younger brother, aged 47 months at the time of this writing, presented with liver dysfunction from 8 months of age. His transaminase levels also fluctuated considerably fluctuations in association with viral infections. At 31 months of age, treatment with succinate and ubiquinone was initiated together with a lipid-rich diet using ketone milk. Thereafter, his transaminase levels normalized and never fluctuated, and the liver histology improved. Conclusions These cases suggested that the clinical courses of patients with MPV17 mutations are greatly influenced by viral infections and that dietary and pharmaceutical treatments targeting the mitochondrial respiratory chain complex II may be beneficial in the clinical management of MPV17 mutant patients.

As gastrointestinal motility disorders are frequently reported in patients with diabetes, we attempted to clarify the effects of cyclohexenonic long-chain fatty alcohol in type 2 Goto-Kakizaki (GK) diabetic enteropathy. At 40 weeks of age male GK rats divided into three groups (treated with 0, 2 or 8 mg/kg of cyclohexenonic long-chain fatty alcohol; started at the age of 40 weeks). Age-matched male Wistar rats were used in this study. At 70 weeks of age the ileal functions were estimated by organ bath studies using 100 mM KCl and carbachol. The expression levels of muscarinic M(2) and M(3) receptor mRNAs in the ileum were investigated by real-time polymerase chain reaction (PCR). Treatment with cyclohexenonic long-chain fatty alcohol did not alter the diabetic status of the GK rats, i.e., body weight, serum glucose, and serum insulin levels, but significantly ameliorated diabetes-induced hypercontractility of the rat ileum caused by carbachol in a dose-dependent manner. Although there were no significant differences in the expression levels of muscarinic M(3) receptor mRNAs in any of the groups, cyclohexenonic long-chain fatty alcohol reversed the diabetes-induced up-regulation of intestinal muscarinic M(2) receptor mRNAs in treatment groups. These results indicate that cyclohexenonic long-chain fatty alcohol exerts its therapeutic effects on hypercontractility in the ileum of 70-week-old GK type 2 diabetic rats by ameliorating overexpression of muscarinic M(2) receptors.

We attempted to clarify the effects of cyclohexenonic long-chain fatty alcohol (N-hexacosanol) on nitric oxide synthase (NOS) in streptozotocin-induced diabetic nephropathy. After induction of experimental diabetes with streptozotocin, rats were maintained for 8 weeks with or without treatment by N-hexacosanol (8 mg/kg i.p. every day). Urinary albumin excretion, blood chemistry, immunoblot analysis, and real-time polymerase chain reactions (real-time PCR) of endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) were investigated. Although N-hexacosanol had no effects on serum glucose or insulin level, it normalized serum creatinine and urinary albumin excretion. N-hexacosanol was found to improve the diabetes-induced alterations in the eNOS, iNOS, and nNOS protein and their mRNA levels. Histologically, N-hexacosanol inhibited the progression to glomerular sclerosis. Our data suggest that N-hexacosanol improves diabetes-induced NOS alterations in the kidney, resulting in the amelioration of diabetic nephropathy.

Background Decreased energy expenditure and increased food intake are principal causes for obesity. In the present study, genotypes of beta(3)-adrenergic receptor (beta(3)AR) and of melanocortin-4 receptor (MC4R), both of which are believed to have a close link to the cause of obesity, were analyzed and compared with phenotypes of childhood obesity. Methods Thirty-five obese children with moderate to severe obesity were enrolled. Direct sequencing of the MC4R coding region and pinpoint-polymerase chain reaction were used to detect genomic variation in the beta(3)AR gene using peripheral blood-derived DNA. Results Allele frequency of Trp64Arg variation in the beta(3)AR gene in the obese subjects was 0.16, which is comparable with that in the healthy general population in eastern Asia. Comparison of phenotypical characteristics did not show a significant difference between Trp/Trp and Trp/Arg subjects. It was notable that body height SD was significantly higher in the Trp/Trp than the Trp/Arg subjects (0.93 +/- 1.0 SD vs 0.07 +/- 1.3 SD, P= 0.03). Annual weight gains were far beyond a hypothetical fat gain in an Arg64 heterozygote with decreased energy consumption, suggesting increased food intake in childhood obesity. There was, however, no variation in the MC4R gene despite thorough sequencing of the entire coding region. Conclusions The Trp64Arg variation in the beta(3)AR gene has no relationship to the degree or the incidence of childhood obesity. The majority of childhood obesity can be characterized as tall stature, more rapid weight gain than that expected by decreased energy expenditure. Further investigation is necessary in regard to the increased food intake as a major cause of childhood obesity.

Previous studies have reported that the respiratory cycle of healthy newborns is more irregular during active sleep. This study aimed to apply non-linear analysis to examine the irregularity of respiratory movement in newborns at different sleep states. The respiratory movement signals from an abdominal band during quiet and active sleep were analyzed using approximate entropy (ApEn). The breathing interval of active sleep was significantly shorter than that of quiet sleep [1.30 (0.17) s vs. 1.58 (0.11) s; (P < 0.03)]. The ApEn of respiratory movements during active sleep were significantly larger than that during quiet sleep [0.785 (0.135) s vs. 0.678 (0.083) s; (P < 0.05)]. We found that the ApEn of respiratory movement in healthy newborns could detect irregularities in respiration during sleep.

Background Langerhans cell histiocytosis (LCH) is a proliferative disorder in which abnormal Langerhans cell (LC)-like cells (LCH cells) intermingle with inflammatory cells. Whether LCH is reactive or neoplastic remains a controversial matter. We recently described Merkel cell polyomavirus (MCPyV) as a possible causative agent of LCH and proposed interleukin-1 loop model: LCH is a reactive disorder with an underlying oncogenic potential and we now propose to test this theory by looking for acute markers of inflammation. We detected MCPyV-DNA in the peripheral blood cells of patients with high-risk organ-type (LCH-risk organ (RO) (+)) but not those with non–high-risk organ-type LCH (LCH-RO (−)); this difference was significant. LCH-RO (−) is further classified by its involvement of either a single organ system (SS-LCH) or multiple organ systems (MS-LCH). In patients with LCH-RO (−), MCPyV-DNA sequences were present in LCH tissues, and significant differences were observed between LCH tissues and control tissues associated with conditions such as dermatopathic lymphadenopathy and reactive lymphoid hyperplasia. Although MCPyV causes subclinical infection in nearly all people and 22 % of healthy adults will harbor MCPyV in their buffy coats, circulating monocytes could serve as MCPyV reservoirs and cause disseminated skin lesions. Methods Plasma sample from 12 patients with LCH-RO (−) (5 MS-LCH and 7 SS-LCH) and 5 non-LCH patients were analyzed by peptidomics. Mass spectrometry (MS) spectra were acquired and peptides exhibiting quantitative differences between MS-LCH and SS-LCH patients were targeted. Results One new candidate biomarker, m/z 3145 was selected and identified after obtaining a MS/MS fragmentation pattern using liquid chromatography-MS/MS. This peak was identified as a proteolytic fragment derived from inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4, [PDB: Q14624]). Conclusions Peptidomics of LCH have revealed that the level of acute-phase ITIH4 distinguishes MS-LCH-RO (−) from SS-LCH-RO (−). Acute-phase proteins serve non-specific, physiological immune functions within the innate immune system. LCH may be a reactive disorder with both underlying neoplastic potential of antigen presenting cells harboring BRAF mutations and hyper-immunity of other inflammatory cells against MCPyV infection. Among LCH-RO (−), MCPyV-DNA sequences were present in both MS-LCH tissues and SS-LCH tissues without significant differences. ITIH4 may show that LCH activity or LCH subtypes correlates with the systemic or localized reactions of MCPyV infection.

The Growject® database on human GH treatment in Turner syndrome was analyzed in the Turner Syndrome Research Collaboration, and the relationships of the frequencies of spontaneous breast development and spontaneous menarche with karyotype and GH treatment were investigated. One hundred and three cases started GH treatment with 0.5 IU/kg/ week (0.5 IU group), and their dose was increased to 0.35 mg/kg/wk midway through the treatment course. Another 109 cases started GH at a dose of 0.35 mg/kg/wk (0.35 mg group). Spontaneous breast development was observed in 77 (36.3%) of the 212 patients, and spontaneous menarche occurred in 31 patients (14.6%). The frequency of spontaneous breast development was significantly lower in patients with the 45,X karyotype and significantly higher in patients with a structural abnormality of the second X chromosome. The frequency of spontaneous menarche was significantly higher in patients with mosaicism characterized by X monosomy and a cellular line with no structural abnormality of the X chromosome. No significant differences in frequencies of spontaneous breast development and spontaneous menarche were observed between the two dose groups, indicating that GH treatment does not increase the frequency of spontaneous puberty.