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BackgroundRett syndrome (RTT) is a characteristic neurological disease presenting with regressive loss of neurodevelopmental milestones. Typical RTT is generally caused by abnormality of methyl-CpG binding protein 2 (MECP2). Our objective to investigate the genetic landscape of MECP2-negative typical/atypical RTT and RTT-like phenotypes using whole exome sequencing (WES).MethodsWe performed WES on 77 MECP2-negative patients either with typical RTT (n=11), atypical RTT (n=22) or RTT-like phenotypes (n=44) incompatible with the RTT criteria.ResultsPathogenic or likely pathogenic single-nucleotide variants in 28 known genes were found in 39 of 77 (50.6%) patients. WES-based CNV analysis revealed pathogenic deletions involving six known genes (including MECP2) in 8 of 77 (10.4%) patients. Overall, diagnostic yield was 47 of 77 (61.0 %). Furthermore, strong candidate variants were found in four novel genes: a de novo variant in each of ATPase H+transporting V0 subunit A1 (ATP6V0A1), ubiquitin-specific peptidase 8 (USP8) and microtubule-associated serine/threonine kinase 3 (MAST3), as well as biallelic variants in nuclear receptor corepressor 2 (NCOR2).ConclusionsOur study provides a new landscape including additional genetic variants contributing to RTT-like phenotypes, highlighting the importance of comprehensive genetic analysis.

Hutchinson–Gilford progeria syndrome (HGPS) is an extremely rare genetic disorder that shows a characteristic progeria phenotype. We conducted a questionnaire survey of 1173 tertiary hospitals in Japan and reviewed the academic reports, to identify the characteristics of Asian patients with classical HGPS. As a result, four Japanese patients were identified; this was estimated to account for approximately two-third of the prevalence in Japan. Three Asian patients who had definitively been diagnosed with classical HGPS were identified in the literature; in total, the clinical characteristics of seven patients were evaluated. Most of the clinical phenotypes of Asian patients were essentially similar to those of patients of other ethnicities, such as sclerodermatous skin, growth failure, loss of scalp hair or severe complications of cardiovascular and cerebral ischemic disease. In conclusion, to circumvent or minimalize severe vascular complication, an early diagnosis, careful observation and, promisingly, new intervention with farnesylation inhibitors may improve the prognosis of classical HGPS patients.

abstractRotavirus gastroenteritis is a highly contagious, acute viral disease that imposes a significant health burden worldwide. In Japan, rotavirus vaccines have been commercially available since 2011 for voluntary vaccination, but vaccine coverage and effectiveness have not been evaluated. In the absence of a vaccination registry in Japan, vaccination coverage in the general population was estimated according to the number of vaccines supplied by the manufacturer, the number of children who received financial support for vaccination, and the size of the target population. Patients with rotavirus gastroenteritis were identified by reviewing the medical records of all children who consulted 6 major hospitals in Saga Prefecture with gastroenteritis symptoms. Vaccination status among these patients was investigated by reviewing their medical records or interviewing their guardians by telephone. Vaccine effectiveness was determined using a screening method. Vaccination coverage increased with time, and it was 2-times higher in municipalities where the vaccination fee was supported. In the 2012/13 season, vaccination coverage in Saga Prefecture was 14.9% whereas the proportion of patients vaccinated was 5.1% among those with clinically diagnosed rotavirus gastroenteritis and 1.9% among those hospitalized for rotavirus gastroenteritis. Thus, vaccine effectiveness was estimated as 69.5% and 88.8%, respectively. This is the first study to evaluate rotavirus vaccination coverage and effectiveness in Japan since vaccination began.

Background: Recent studies suggest potential roles of immune response in the pathophysiology of epilepsy. Anti-seizure medications (ASMs) are known to have side effects of drug eruption caused by immune responses. A few reports in adults have demonstrated disappearance of seizures after an ASM drug eruption episode. In this paper, we described 2 cases of childhood absence epilepsy (CAE) who showed seizure disappearance after ethosuximide (ESM) drug eruption, suggesting the possibility that the epilepsy disappears due to immune responses to ASM. Case presentation: Case 1 was an 8-year-old girl diagnosed with CAE. She was treated with valproate acid (VPA) initially, and then ESM was administered as an additional treatment. Her epileptic seizure disappeared 4 days after initiation of ESM. However, drug eruption appeared 1 week after the administration of ESM. Even after discontinuation of ESM administration, she maintains no seizure after the drug eruption. Case 2 was a 5-year-old boy diagnosed as CAE. He was treated with VPA initially, and ESM was administered additionally. Drug eruption appeared 1 month after the administration of ESM. Even after ESM was terminated, he maintained seizure freedom after the appearance of eruption. Conclusions: Epileptic seizures may have been suppressed due to the immune responses caused by ASM eruption. Further studies are needed to elucidate the pathophysiologic effects of drug eruption on epilepsy through immune responses.

Niemann‐Pick disease type C (NPC) is a lysosomal storage disease characterized by abnormal accumulation of free cholesterol and glycolipids. Here, we established induced pluripotent stem cell (iPSC) lines from NPC patients. Hepatocyte‐like cells (HLCs) and neural progenitors derived from the iPSC lines accumulated cholesterol and displayed impaired autophagy and ATP production. A molecular signature related to lipid metabolism was also impaired in the NPC‐iPSC‐derived HLCs. These findings indicate that iPSC‐derived cells can phenocopy human NPC. We also newly found that 2‐hydroxypropyl‐γ‐cyclodextrin (HPGCD) could reduce the cholesterol accumulation and restore the functional and molecular abnormalities in the NPC patient‐derived cells, and do so more effectively than 2‐hydroxypropyl‐β‐cyclodextrin treatment. In addition, NPC model mice showed an improved liver status and prolonged survival with HPGCDs. Thus, iPSC lines derived from patient cells are powerful tools to study cellular models of NPC, and HPGCD is a potential new drug candidate for future treatment of this disease. StemCells2015;33:1075–1088

Purpose:Expression of imprinted genes is regulated by DNA methylation of differentially methylated regions (DMRs). Beckwith–Wiedemann syndrome is an imprinting disorder caused by epimutations of DMRs at 11p15.5. To date, multiple methylation defects have been reported in Beckwith–Wiedemann syndrome patients with epimutations; however, limited numbers of DMRs have been analyzed. The susceptibility of DMRs to aberrant methylation, alteration of gene expression due to aberrant methylation, and causative factors for multiple methylation defects remain undetermined.Methods:Comprehensive methylation analysis with two quantitative methods, matrix-assisted laser desorption/ionization mass spectrometry and bisulfite pyrosequencing, was conducted across 29 DMRs in 54 Beckwith–Wiedemann syndrome patients with epimutations. Allelic expressions of three genes with aberrant methylation were analyzed. All DMRs with aberrant methylation were sequenced.Results:Thirty-four percent of KvDMR1–loss of methylation patients and 30% of H19DMR–gain of methylation patients showed multiple methylation defects. Maternally methylated DMRs were susceptible to aberrant hypomethylation in KvDMR1–loss of methylation patients. Biallelic expression of the genes was associated with aberrant methylation. Cis-acting pathological variations were not found in any aberrantly methylated DMR.Conclusion:Maternally methylated DMRs may be vulnerable to DNA demethylation during the preimplantation stage, when hypomethylation of KvDMR1 occurs, and aberrant methylation of DMRs affects imprinted gene expression. Cis-acting variations of the DMRs are not involved in the multiple methylation defects.Genet Med 16 12, 903–912.

Prostaglandin H synthase (PHS) is a key enzyme in the synthesis of prostaglandins (PGs). Recently, enhanced expression of PHS-2 in brain tumors and the correlation between the PHS-2 level and the histopathological grade of glioma has been reported. Furthermore, in vitroinhibition of glioma cell growth by a specific PHS-2 inhibitor, NS398, has been demonstrated. It has also been shown that prostaglandin E2(PGE2) contributes to colon carcinogenesis by binding to the prostaglandin E receptor subtype EP1. We therefore evaluated the effects of NS398 and two EP1 antagonists, SC51089 and AH6809, on glioma cell lines. To evaluate mechanisms of NS398's action, two glioma cell lines, a PHS-2–positive cell line (KMG4) and a PHS-2-deficient cell line (A172), were used. NS398 inhibited both the anchorage-dependent and -independent growth of glioma cell lines regardless of PHS-2 expression, suggesting that some PHS-2-independent mechanisms underlie the antineoplastic effect of NS398. However, the antineoplastic effect was attenuated by the addition of PGE2, which is one of the main products of PHS, suggesting the predominant mechanism is PHS-dependent. The EP1 antagonists, SC51089 and AH6809, inhibited the growth of glioma cell lines in vitro. Furthermore, NS398 or SC51089 slowed tumor growth in vivo, which was assessed using KMG4 tumor xenografts on SCID mice. PHS-2 inhibitors and EP1 antagonists might be useful in the prevention and/or treatment of glioma.

Cysteinyl leukotrienes (cysLTs; LTC4, LTD4, and LTE4) are important mediators in asthma. LTC4is metabolized to less active LTE4via LTD4by glutamyl transpeptidases and dipeptidases. The enzymatic conversion of LTC4to LTE4may be a regulatory mechanism in asthma. We examined the conversion rate of LTC4to LTE4in children it to asthma, and compared it to that in controls. After serum specimens from children with asthma were incubated with exogenous LTC4for 30 minutes at 37°C, the levels of LTC4and LTE4were measured by high-performance liquid chromatography. Conversion rate of LTC4to LTE4was significantly lower in children with asthma (67.6 ± 4.8 LTE4ng/mg of protein per minute) than controls (86.8 ± 4.4 LTE4ng/mg of protein per minute). The result indicates that the conversion rate of LTC4to LTE4may contribute to the pathogenesis of childhood asthma.

Interleukin (IL)-4, IL-10, and IL-13, Th2 cell–derived cytokines, play major roles in the pathophysiology of allergic diseases. These cytokines up-regulate or down-regulate the production of arachidonic acid metabolites. In this study, we have investigated the effect of IL-4, IL-10, IL-13, and other cytokines on A23187-stimulated synthesis of leukotriene (LT) B4 in human polymorphonuclear leukocytes (PMNs). Production of LTB4 was measured by specific radioimmunoassay and high performance liquid chromatography. Messenger RNA (mRNA) expression of cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase (5-LO), and LTA4 hydrolase, which were involved in the synthesis of LTB4, was determined by reverse transcription–polymerase chain reaction and Northern blot analysis. Protein synthesis of their enzymes was determined by Western blot analysis. IL-4 and IL-13 enhanced A23187-stimulated LTB4 synthesis and increased mRNA expression and protein synthesis of LTA4hydrolase, but not those of cPLA2 or 5-LO. These results indicate that IL-4 and IL-13 transcriptionally or post-transcriptionally up-regulate the synthesis of LTB4, a potent chemotactic factor to PMNs, at the enzyme level of LTA4 hydrolase, and this up-regulation mechanism may participate in the development of allergic inflammation.

Background. BLEIA ™ “EIKEN” Helicobacter pylori antigen (B[EIA]) is based on the bioluminescent enzyme immunoassay (BLEIA) method that was newly developed with high sensitivity in detecting Helicobacter pylori (H. pylori) antigen in feces. Methods. In the project for H. pylori screening and treatment in Saga Prefecture in 2019, 141 students received the stool H. pylori antigen test as a secondary test. For 141 students, a comparative test was conducted between B (EIA) and extracorporeal diagnostic agents that were marketed in Japan as of 2019. The detection performance of H. pylori ATCC43504 standard strain and H. pylori antigen in commercial human fecal specimens were conducted. Results. The comparison of B (EIA) with Quick Chaser TMH. pylori (Q [IC]) revealed positive and negative concordance ratios of B (EIA) to Q (IC) of 100.0% (110/110) and 71.0% (22/31), respectively. A comparative test was conducted between B (EIA) and extracorporeal diagnostic agents that were marketed in Japan as of 2019, and B (EIA) was most sensitive on “detecting H. pylori antigen of ATCC43504 standard strain” and “detecting H. pylori antigen in commercial human fecal specimens,” compared with other kits. Nine dissociated specimens that were negative for Q (IC) and positive for B (EIA) were confirmed. The measured value of B (EIA) in the dissociation samples were 1.3–87.4 cutoff index in the range that can be evaluated as negative by other fecal H. pylori antigen test kits, all the dissociation samples were H. pylori antigen-positive cases, and finally the cause of result divergence was presumed as false negative due to insufficient sensitivity of Q (IC). Conclusion. B (EIA) that is based on the BLEIA method, which applies firefly luciferase luminescence, is more sensitive than stool antigen test kits that are currently marketed in Japan and is very useful in diagnosing H. pylori infection, especially in situations where noninvasive tests are preferred, such as in children.

The mouse NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) gene and its 5′-flanking region was cloned from a 129 mouse ES bacteriophage P1 genomic library. The gene contains 7 exons and 6 introns and is 11.3 kb in length. The transcription initiation site was mapped at 35 bases upstream from the ATG start codon. The nucleotide sequence of the 1.6 kb promoter region contains two TATA boxes and a number of potential regulatory elements including Sp1, CRE, GRE, AP1, AP2, NF-IL6 and estrogen receptor binding site. Studies of the promoter's activity showed that the first 400 nucleotides of 5′-flanking region efficiently drove the transcription of the luciferase reporter gene in U936 cells upon stimulation with a phorbol ester.

To determine anti-allergic effects of Koboku, a Chinese herbal medicine, we investigated its inhibitory action on the production of cysteinyl leukotrienes (LT) and LTB4, which are important chemical mediators in the pathogenesis of allergic diseases. A231 87-stimulated synthesis of cysteinyl LT and LTB4was measured by HPLC in the absence or presence of various concentrations of Koboku in rat basophilic leukemia-1 (RBL-1) cells. In a dose-dependent manner, Koboku inhibited synthesis of cysteinyl LT and LTB4by up to 92.3 and 100%, respectively. Immunoglobulin E-mediated release of cysteinyl LT and LTB4, which was measured by specific radioimmunoassay (RIA) was also inhibited by Koboku in RBL-2H3 cells. Sites of inhibition of Koboku in the metabolic pathway of LT synthesis were phospholipase A2(PLA2) and 5-lipoxygenase (5-LO), but not LTC4synthase or LTA4hydrolase. We conclude that the anti-allergic effect of Koboku may be attributable, at least in part, to the inhibition of LT synthesis.

There are substantial numbers of reports showing that leukotrienes (LTs) play important roles in adult asthma. No definite evidence has been demonstrated that LTs are involved in asthma attacks in children, although it is highly expected. In this report, we demonstrated that the levels of LTB4 and LTC4 but not thromboxane B2 (TXB2), a stable metabolite of TXA2, were significantly elevated in the bronchoalveolar lavage fluid, which was obtained from intubated and mechanically ventilated children with severe asthma attacks. This is direct evidence that LTB4 and LTC4 predominantly participate in asthma attacks in pediatric patients.

A 3-year-old boy developed idiopathic thrombocytopenic purpura (ITP) and Perthes' disease concurrently. Antiphospholipid antibodies were detected in the serum of the patient. Therefore, it Is possible that Perthes' disease in this patient might be one of the manifestations of ITP-associated antiphospholipid syndrome.

The cDNA for mouse NAD+ dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) was isolated from a lung cDNA library. The cDNA contains a 798 bp open reading frame that codes for a protein of 266 amino acids (Mr 28 775) which shares 87% identity with the human 15-PGDH protein. The regions that are believed to form the NAD+ binding site and the active site are conserved in the mouse and human enzymes. The authenticity of the mouse cDNA was confirmed by expression of an active 15-PGDH inEscherichia coli. Northern blot analysis demonstrated that 15-PGDH mRNA is expressed primarily in lung, intestine, stomach and liver.

The recent increase in macrolide-resistant Mycoplasma pneumoniaein Asia has become a continuing problem. A point-of-care testing method that can quickly detect M. pneumoniaeand macrolide-resistant mutations (MR mutations) is critical for proper antimicrobial use.

We report on a 13-year-old boy with temporal lobe epilepsy associated with left hippocampal sclerosis and a contralateral arachnoid cyst in the middle cranial fossa (ACMCF). Chronic intracranial recording from subdural grid electrodes showed the left medial temporal lobe to be the ictal onset zone. After left anterior temporal lobectomy with hippocampectomy, seizure control was improved. ACMCF was not considered the direct cause of epilepsy; instead the seizures were attributed to hippocampal sclerosis.