MAP7D2 is a brain expressing X-linked maternal imprinted gene in humans

Increasing evidence suggests imprinted genes influence mouse and human behaviors and cognitive functions. Unlike autosomal imprinted genes, X-linked imprinted genes are expressed in a sex-dependent manner because of male hemizygosity. Therefore, these genes could directly affect sex-specific brain functions and sex-biased vulnerability to psychiatric disorders such as autism1. Comparing lymphoblastoid cell lines (LCL) and peripheral blood mononuclear cells (PBMC) from healthy adult male and females, we identified MAP7 domain containing 2 (MAP7D2) as the first human X-linked imprinted gene. Both in LCL and PBMC, MAP7D2 expression was significantly suppressed in males by maternal imprinting. In each female LCL clone, MAP7D2 was expressed higher in paternally derived allele and was affected by X-chromosome inactivation. In female PBMC, however, reactivation of maternal MAP7D2 alleles was observed. MAP7D2 was expressed specifically in the brain among human tissues with unique isoforms. These results predict a crucial role of MAP7D2 for human sex-dependent neurobiological traits

Development of a simple and highly sensitive mutation screening system by enzyme mismatch cleavage with optimized conditions for standard laboratories

金沢大学子どものこころの発達研究センターEfficient screening of unknown DNA variations is one of the substantive matters of molecular biology even today. Historically, SSCP and heteroduplex analysis (HA) are the most commonly used methods for detecting DNA variations everywhere in the world because of their simplicity. However, the sensitivity of these methods is not satisfactory for screening purpose. Recently, several new PCR-based mutation screening methods have been developed, but most of them require special instruments and adjustment of conditions for each DNA sequence to attain the maximum sensitivity; eventually becoming as inconvenient as old methods. Enzyme mismatch cleavage (EMC) is potentially an ideal screening method. With high-performance nucleases and once experimental conditions are optimized, it requires only conventional staff and conditions remain the same for each PCR product. In this study we tested four commercially available endonucleases for EMC and optimized the electrophoresis and developing conditions. We prepared 25 known DNA variations consisting of 18 single base substitutions (8 transitions and 10 transversions, including all possible sets of mismatches) and 7 small deletions or insertions. The combination of CEL nuclease, 12% PAGE and rapid silver staining can detect all types of mutations and achieved 100% sensitivity. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Development of a practical NF1 genetic testing method through the pilot analysis of five Japanese families with neurofibromatosis type 1

Objective: Mutation analysis of NF1, the responsible gene for neurofibromatosis type 1 (NF1), is still difficult due to its large size, lack of mutational hotspots, the presence of many pseudogenes, and its wide spectrum of mutations. To develop a simple and inexpensive NF1 genetic testing for clinical use, we analyzed five Japanese families with NF1 as a pilot study. Methods: Our original method, CEL endonuclease mediated heteroduplex incision with polyacrylamide gel electrophoresis and silver staining (CHIPS) was optimized for NF1 mutation screening, and reverse transcription polymerase chain reaction (RT-PCR) was performed to determine the effect of transcription. Also, we employed DNA microarray analysis to evaluate the break points of the large deletion. Results: A new nonsense mutation, p.Gln209*, was detected in family 1 and the splicing donor site mutation, c.2850+1G>T, was detected in family 2. In family 3, c.4402A>G was detected in exon 34 and the p.Ser1468Gly missense mutation was predicted. However mRNA analysis revealed that this substitution created an aberrant splicing acceptor site, thereby causing the p.Phe1457* nonsense mutation. In the other two families, type-1 and unique NF1 microdeletions were detected by DNA microarray analysis. Conclusions: Our results show that the combination of CHIPS and RT-PCR effectively screen and characterize NF1 point mutations, and both DNA and RNA level analysis are required to understand the nature of the NF1 mutation. Our results also suggest the possibility of a higher incidence and unique profile of NF1 large deletions in the Japanese population as compared to previous studies performed in Europe

自閉症に関連するX染色体上の刷り込み遺伝子の同定

金沢大学附属病院X染色体上の刷り込み遺伝子は自閉症関連遺伝子として注目されている。平成17年度はDNAメチル化を指標としたスクリーニングを行い29個の候補遺伝子が挙げられた。平成18年度は発現解析アレイを用いて更なる候補遺伝子を検討した。正常女性由来のEBウイルス株化リンパ球を作成し、株化の初期段階で限界希釈・クローニングすることにより、父親由来X染色体が活性化しているクローンと母親由来X染色体が活性化しているクローンとに分離した。各々複数個のクローンを得、これらを混合したRNAをマイクロアレイ解析を用いて全ゲノムに関して発現比較した。この結果、X染色体上に位置する遺伝子で、父親由来のX染色体が活性化しているクローンで発現量が5倍以上高いものが25個、母親由来のX染色体が活性化しているクローンで発現量が5倍以上高いものが7個得られた。17年度の研究と合わせて計61個の候補遺伝子が得られた事となる。これらの候補遺伝子に関して各々特異的なPCRプライマーを設計し、リアルタイムPCRにて発現量の差を定量化した。この結果、最終的に3つのX染色体上の遺伝子、CDR1,RPS6KA6,NAP1L2が母親由来X染色体での発現量よりも父親由来X染色体での発現量が有意に高いことが証明された。すなわちこれらの遺伝子は母性刷り込みをもつX染色体上の遺伝子である。CDR1は小脳に特異的に発現する遺伝子であり、自閉症者が小脳に形態学的異常を持つことからも関連が注目される。またRPS6KA6はmTORパスウエイに関連するプロテインキナーゼであるが、mTORパスウエイは自閉症の合併率が高い結節性硬化症における中心的な細胞内情報伝達経路であり、この遺伝子の自閉症との関連は興味深い。NAP1L2は神経管の発生に重要な遺伝子である。遺伝子の5‘末端側にCpGアイランドがありDNAメチル化を受けるが、刷り込み現象との関連が注目される。以上、今回の萌芽研究において自閉症関連遺伝子として極めて興味深い3つの遺伝子が新規に同定された。これらの解析結果は現在医学雑誌に投稿準備中である。研究課題/領域番号:17659310, 研究期間(年度):2005 – 2006出典：「自閉症に関連するX染色体上の刷り込み遺伝子の同定」研究成果報告書　課題番号17659310（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-17659310/)を加工して作

Intermittent X-linked thrombocytopenia with a novel WAS gene mutation

X-linked thrombocytopenia (XLT) is caused by mutations in the WAS gene and characterized by thrombocytopenia with minimal or no immunodeficiency. Patients with XLT usually exhibit persistent thrombocytopenia, and intermittent thrombocytopenia has been described only in two families. Here, we report a patient with intermittent XLT carrying a novel missense mutation (Ala56Thr). He showed residual expression of Wiskott-Aldrich syndrome protein in the lymphocytes and platelets. There appeared to be an association between normal platelet numbers and a post infectious state. Our findings further support the importance of analysis of Wiskott-Aldrich syndrome protein in male patients who exhibit fluctuating courses of thrombocytopenia. Pediatr Blood Cancer 2014;61:746-748. © 2013 Wiley Periodicals, Inc

A case of acute encephalopathy with hemophagocytic lymphohistiocytosis and clonal T-cell expansion

We report on a 9-year-old boy who presented with acute encephalopathy and hemophagocytic lymphohistiocytosis (HLH). The patient was referred to our hospital because of fever, seizures, and decreased consciousness. He showed moderately elevated levels of proinflammatory cytokines in the cerebrospinal fluid and plasma, and clonal expansion of highly activated CD8 + T cells in the peripheral blood. These CD8 + T cells were found to be larger cells that stained positive for T-cell receptor Vβ13.6, and decreased shortly after steroid therapy. Our findings suggest that his acute encephalopathy was likely a clinical manifestation of HLH, and that immunophenotypic analysis may be helpful for early recognition of HLH in such rare encephalopathy. © 2011 The Japanese Society of Child Neurology

Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 deficiency

金沢大学医薬保健研究域医学系The first known human case of heme oxygenase-1 (HO-1) deficiency is presented in this report. The patient is a six-year-old boy with severe growth retardation. He has been suffering from persistent hemolytic anemia characterized by marked erythrocyte fragmentation and intravascular hemolysis, with paradoxical increase of serum haptoglobin and low bilirubin. An abnormal coagulation/fibrinolysis system, associated with elevated thrombomodulin and von Willebrand factor, indicated the presence of severe, persistent endothelial damage. Electron microscopy of renal glomeruli revealed detachment of endothelium, with subendothelial deposition of an unidentified material. Iron deposition was noted in renal and hepatic tissue. Immunohistochemistry of hepatic tissue and immunoblotting of a cadmium- stimulated Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) revealed complete absence of HO-1 production. An LCL derived from the patient was extremely sensitive to hemin-induced cell injury. Sequence analysis of the patient\u27s HO-1 gene revealed complete loss of exon-2 of the maternal allele and a two-nucleotide deletion within exon3 of the paternal allele. Growth retardation, anemia, iron deposition, and vulnerability to stressful injury are all characteristics observed in recently described HO-1 targeted mice. This study presents not only the first human case of HO-1 deficiency but may also provide clues to the key roles played by this important enzyme in vivo

A custom magnetoencephalography device reveals brain connectivity and high reading/decoding ability in children with autism

A subset of individuals with autism spectrum disorder (ASD) performs more proficiently on certain visual tasks than may be predicted by their general cognitive performances. However, in younger children with ASD (aged 5 to 7), preserved ability in these tasks and the neurophysiological correlates of their ability are not well documented. In the present study, we used a custom child-sized magnetoencephalography system and demonstrated that preserved ability in the visual reasoning task was associated with rightward lateralisation of the neurophysiological connectivity between the parietal and temporal regions in children with ASD. In addition, we demonstrated that higher reading/decoding ability was also associated with the same lateralisation in children with ASD. These neurophysiological correlates of visual tasks are considerably different from those that are observed in typically developing children. These findings indicate that children with ASD have inherently different neural pathways that contribute to their relatively preserved ability in visual tasks