村松 一洋

Joubert syndrome (JS) is rare recessive disorders characterized by the combination of hypoplasia/aplasia of the cerebellar vermis, thickened and elongated superior cerebellar peduncles, and a deep interpeduncular fossa which is defined by neuroimaging and is termed the molar tooth sign'. JS is genetically highly heterogeneous, with at least 29 disease genes being involved. To further understand the genetic causes of JS, we performed whole-exome sequencing in 24 newly recruited JS families. Together with six previously reported families, we identified causative mutations in 25 out of 30 (24+6) families (83.3%). We identified eight mutated genes in 27 (21+6) Japanese families, TMEM67 (7/27, 25.9%) and CEP290 (6/27, 22.2%) were the most commonly mutated. Interestingly, 9 of 12 CEP290 disease alleles were c.6012-12T>A (75.0%), an allele that has not been reported in non-Japanese populations. Therefore c.6012-12T>A is a common allele in the Japanese population. Importantly, one Japanese and one Omani families carried compound biallelic mutations in two distinct genes (TMEM67/RPGRIP1L and TMEM138/BBS1, respectively). BBS1 is the causative gene in Bardet-Biedl syndrome. These concomitant mutations led to severe and/or complex clinical features in the patients, suggesting combined effects of different mutant genes.

【目的】先天性中枢性肺胞性低換気症候群(congenital central hypoventilation syndrome;CCHS)の精神発達予後を明らかにする。【方法】学齢以上のCCHS17例について、臨床経過、呼吸管理、精神発達などについて後方視的に検討した。【結果】15例が出生直後に発症し、2例が遅発性だった。出生時の低酸素は8例に認め、これを含む12例が挿管管理をうけた。全例が生後1ヵ月〜1歳(中央値5.5ヵ月)時に気管切開術を受けた。挿管例はHirschsprung病などを合併する頻度も高くCCHSとして重症であった。マスク換気5例は発症時の全身状態が比較的良好で合併症も少なかった。気管切開例のうち4例がマスク換気に、1例がマスク換気と横隔膜ペーシングの併用に移行した。気管切開例の精神発達は正常から重度遅滞まで認め、気管切開の時期が遅いほど遅滞が重度になる傾向にあった。マスク換気例では全例で境界域〜中等度までの遅滞を認めた。またマスク換気例ではマスク装着のコンプライアンス等に問題があった。【結論】CCHSでは低酸素が精神発達に影響している可能性があり、神経保護の見地から気管切開による確実な呼吸管理を出生後早期から行う必要がある。(著者抄録)

Objective: To evaluate the psychological development of patients with congenital central hypoventilation syndrome (CCHS). Methods: We performed a questionnaire-based survey of 17 patients with CCHS aged over 7 years and assessed their clinical course, respiratory management, and psychological development. Results: CCHS was present at birth in 15 patients, of which eight presented with respiratory failure with a low Apgar score. Twelve patients required mechanical ventilation with intubation, and five received mask ventilation. All patients with intubation underwent tracheostomy between 1 and 12 months of age (median 5.5 months), and most of them had associated conditions such as Hirschsprung disease. Four of 12 patients with intubation were eventually switched to mask ventilation and one to diaphragm pacing and mask ventilation. The patients undergoing mask ventilation had relatively milder disease severity and had fewer complications than did the patients with intubation. The psychological development of patients who received tracheostomy ranged from normal to severe retardation. Retardation was more likely to be severe in patients who received tracheostomy in late infancy. All patients who received mask ventilation experienced borderline to moderate psychological retardation. This effect could be attributed to poor compliance with mask fitting. Conclusion: Out findings suggest that the psychological development of CCHS patients was influenced by hypoxia tracheostomy and strict respiratory management since the neonatal period were needed for neurological protection.

先天性中枢性低換気症候群(CCHS)は先天的な自律神経系の発生学的異常に基づく呼吸機能不全を呈する疾患である。多くは生後間もなく、睡眠中の低換気あるいは無呼吸で発症し人工呼吸管理を必要とする。Hirschsprung病、不整脈や神経堤腫瘍といった全身の合併症も生じる。診断には睡眠中のSpO2モニタリング、ポリソムノグラフィや二酸化炭素応答試験による臨床的検査を実施する。PHOX2Bを原因遺伝子とし、最終的には遺伝子診断で確定する。CCHSは在宅での適切な呼吸管理が生涯にわたり必要となる。欧米では乳幼児期は気管切開による人工呼吸管理が一般的で、これにより長期的な神経学的予後が改善されてきた。(著者抄録)

Static encephalopathy of childhood with neurodegeneration in adulthood (SENDA) is a recently established subtype of neurodegeneration with brain iron accumulation (NBIA)(1-3). By exome sequencing, we found de novo heterozygous mutations in WDR45 at Xp11.23 in two individuals with SENDA, and three additional WDR45 mutations were identified in three other subjects by Sanger sequencing. Using lymphoblastoid cell lines (LCLs) derived from the subjects, aberrant splicing was confirmed in two, and protein expression was observed to be severely impaired in all five. WDR45 encodes WD-repeat domain 45 (WDR45). WDR45 (also known as WIP14) is one of the four mammalian homologs of yeast Atgl 8, which has an important role in autophagy(4,5). Lower autophagic activity and accumulation of aberrant early autophagic structures were demonstrated in the LCLs of the affected subjects. These findings provide direct evidence that an autophagy defect is indeed associated with a neurodegenerative disorder in humans.

The molecular mechanisms of neuronal morphology and synaptic vesicle transport have been largely elusive, and only a few of the molecules involved in these processes have been identified. Here, we developed a novel morphology-based gene trap method, which is theoretically applicable to all cell lines, to easily and rapidly identify the responsible genes. Using this method, we selected several genetrapped clones of rat pheochromocytoma PC12 cells, which displayed abnormal morphology and distribution of synaptic vesicle-like microvesicles (SLMVs). We identified several genes responsible for the phenotypes and analyzed three genes in more detail. The first gene was BTB/POZ domain-containing protein 9 (Btbd9), which is associated with restless legs syndrome. The second gene was cytokine receptor-like factor 3 (Crlf3), whose involvement in the nervous system remains unknown. The third gene was single-stranded DNA-binding protein 3 (Ssbp3), a gene known to regulate head morphogenesis. These results suggest that Btbd9, Crlf3, and Ssbp3 regulate neuronal morphology and the biogenesis/transport of synaptic vesicles. Because our novel morphology-based gene trap method is generally applicable, this method is promising for uncovering novel genes involved in the function of interest in any cell lines. © The Author(s).

先天性中枢性肺胞低換気症候群(以下CCHS)は睡眠時に低換気を呈する稀な疾患である。2005年に小児慢性特定疾患治療研究事業において新たにCCHSが対象となり、疫学的動態の把握が可能となった。日本での疾患状況の正確な把握を目的に小慢事業登録データの検討を行った。2005〜2009年の5年間で登録総数562例中新規登録は170名、人工呼吸管理施行例73%、気管切開施行例51%。発症時期の大半は生後早期であるが、診断までには時間を要する例が多かった。治癒例はなく多くは経過不変であった。日本でのCCHSの統計的な調査は、2006年に長谷川らが行った全国アンケート調査が唯一である。今回は小慢事業登録データを活用した疫学調査であり、日本の疾患状況の把握には有用であった。今後、CCHSに関しての詳細な調査を改めて行い、適切な診断及び管理方法の検討と、長期予後、合併症や併存障害を評価していくことが極めて重要であると考えられる。(著者抄録)

The molecular mechanisms of neuronal morphology and synaptic vesicle transport have been largely elusive, and only a few of the molecules involved in these processes have been identified. Here, we developed a novel morphology-based gene trap method, which is theoretically applicable to all cell lines, to easily and rapidly identify the responsible genes. Using this method, we selected several gene-trapped clones of rat pheochromocytoma PC12 cells, which displayed abnormal morphology and distribution of synaptic vesicle-like microvesicles (SLMVs). We identified several genes responsible for the phenotypes and analyzed three genes in more detail. The first gene was BTB/POZ domain-containing protein 9 (Btbd9), which is associated with restless legs syndrome. The second gene was cytokine receptor-like factor 3 (Crlf3), whose involvement in the nervous system remains unknown. The third gene was single-stranded DNA-binding protein 3 (Ssbp3), a gene known to regulate head morphogenesis. These results suggest that Btbd9, Crlf3, and Ssbp3 regulate neuronal morphology and the biogenesis/transport of synaptic vesicles. Because our novel morphology-based gene trap method is generally applicable, this method is promising for uncovering novel genes involved in the function of interest in any cell lines.-Hashimoto, Y., Muramatsu, K., Kunii, M., Yoshimura, S., Yamada, M., Sato, T., Ishida, Y., Harada, R., Harada, A. Uncovering genes required for neuronal morphology by morphology-based gene trap screening with a revertible retrovirus vector. FASEB J. 26, 4662-4674 (2012). www.fasebj.org

Delirious behavior associated with influenza usually has an onset within a few days after fever and lasts <24 hours. As we encountered several patients with 2009 H1N1 influenza who presented with late-onset and long-standing delirious behavior, we retrospectively evaluated the clinical, radiologic, and laboratory features to elucidate the possible pathophysiology. This information was collected on 5 previously healthy patients (2 boys and 3 girls, aged 10-15 years) with 2009 H1N1 influenza who presented with late onset (>3 days after fever) and long-standing (>48 hours) delirious behavior. Each exhibited mild to moderate drowsiness between the episodes of delirious behavior. Electroencephalography was normal except for 1 patient with high voltage and slow activity bilaterally in the occipital regions. Brain MRI was normal. The outcome was excellent with no neurologic sequel in 4 of the 5 patients. In all 5 patients, autoantibodies against N-methyl-D-aspartate type glutamate receptor were elevated or positive in cerebrospinal fluid or serum; the autoantibody levels normalized in the 3 patients who had follow-up studies. This study indicates that 2009 H1N1 influenza has a tendency to cause late-onset and long-standing delirious behavior, at least in Japanese children. Mild autoimmune-mediated encephalitis should be considered as an underlying cause. Pediatrics 2012;129:e1068-e1071

To determine the neuronal function of genes in vivo, the neuron-specific deletion of a target gene in animals is required. Tau, a microtubule-associated protein, is expressed abundantly in neurons but scarcely in glias and other tissues. Therefore, to generate mice that express Cre recombinase in neurons, we inserted Cre recombinase into the tau locus. By crossing these tau-Cre mice with ROSA26 lacZ reporter mice, we observed Cre recombinase activity in the neurons from most of the central nervous system, but not in glias nor in non-neuronal tissues. This neuronal-specific activity appeared during embryogenesis. We further crossed tau-Cre mice with rab8 'floxed' mice, and showed that the recombination was nearly complete in the brain, but incomplete or non-detectable in other tissues. Thus, tau-Cre knockin mouse is a useful tool for studying the neuronal function of a gene in vivo. (c) 2008 Elsevier Inc. All rights reserved.

Lung fibroblasts are a major source of several cytokines including CC chemokine eotaxin. We aimed to study the regulation of eotaxin-1/ CCL11 production by dexamethasone and analyze its molecular mechanisms in human lung fibroblasts. Normal human lung fibroblast cells were exposed to IL-4 (40 ng/ml) and/or dexamethasone (10(-6)-10(-9) M), and eotaxin in RNA expression and production was evaluated. Mechanisms of transcriptional regulation were assessed by Western blotting and dual luciferase assay for eotaxin promoter. The effects of dexamethasone on suppressor of cytokine signaling (SOCS)-1 and eotaxin mRNA expression in the cells transfected with expression vector (pAcGFP1 -C1) or short interfering RNA (siRNA) for SOCS-1 were also investigated. Within 24 hours, dexamethasone inhibited IL-4-induced eotaxin mRNA expression and protein production, while eotaxin production was markedly increased at 48 and 72 hours after coincubation with IL-4 and dexamethasone. IL-4-induced eotaxin promoter activity was inhibited by dexamethasone at 8 hours, but enhanced at 48 hours after coincubation. Dexamethasone suppressed SOCS-1 mRNA expression but enhanced IL-4-induced STAT6 phosphorylation at 36 to 48 hours after coincubation. Enhanced expression of eotaxin mRNA by dexamethasone 48 hours after coincubation was completely diminished in the cells transfected with either expression vector or siRNA for SOCS-1. These results indicated that dexamethasone, depending on the exposure duration, can either inhibit or enhance IL-4-induced expression and production of eotaxin in the lung fibroblasts. The mechanisms of later enhanced production may depend on the prolonged transcriptional activity of the eotaxin gene, in part due to inhibition of SOCS-1 expression.

To investigate the neuronal function of genes in vivo, a neuron-specific and inducible gene targeting system is desirable. In this study, we generated a knockin mouse line that expresses a fusion protein consisting of the Cre recombinase and the progesterone receptor (CrePR) in neurons. The neuron-specific expression of CrePR was attained by inserting CrePR gene into the tau locus, because T is expressed strongly in neurons but scarcely in glias and other tissues. By crossing this knockin mouse line (tau(CrePR)) with ROSA26 lacZ reporter mouse line (R26R), we observed that the anti progesterone RU486 could induce recombinase activity of the CrePR specifically in neurons. Thus, tau(CrePR) knockin line is a useful tool for studying neuronal gene functions.

BACKGROUND. Familial hemophagocytic lymphohistiocytosis HLH (FHL) is fatal, unless patients are rescued with hematopoietic stem cell transplantation (SCT). Although the molecular identification of FHL now is possible at least in part from perforin gene study, many cases escape detection or never are tested due to the lack of specific hallmarks, making diagnosis difficult. To the authors' knowledge, it remains to be determined whether persistently low natural killer cell (NK) activity and a high incidence of central nervous system (CNS) disease increase the probability of FHL. METHODS. The authors analyzed 42 HLH patients age < 2 years, 13 of whom developed overt CNS disease and 5 of whom demonstrated persistently deficient NK activity (Group 1). The remaining 24 patients had no CNS disease and had NK activity of moderate decrease to within the normal range (Group 2). RESULTS, In Group 1, CNS symptoms were detected in 6 cases within 1 month and between 4.5-9 months in 6 other patients. In these cases, spotty lesions demonstrating a high T2 signal in the white matter were noted on brain magnetic resonance imaging. The survival was significantly poor for patients in Group I unless they were rescued with SCT, which was performed in 5 of the 13 patients with CNS disease and in all 5 patients with persistent NK activity deficiency. SCT was successful in 9 patients, with no CNS sequelae reported after the transplantation. Conversely, the prognosis of the 24 patients in Group 2 was better and only 1 patient required SCT. CONCLUSIONS. Very young HLH patients (age < 2 years) who are at high risk of fatal FHL with persistently deficient NK activity and/or overt CNS disease require appropriate SCT to reverse CNS disease and achieve a complete cure. Cancer2002; 94.3023-31. (C) 2002 American Cancer Society.