Visualization of the spatial positioning of the SNRPN, UBE3A, and GABRB3 genes in the normal human nucleus by three-color 3D fluorescence in situ hybridization

The three-dimensional (3D) structure of the genome is organized non-randomly and plays a role in genomic function via epigenetic mechanisms in the eukaryotic nucleus. Here, we analyzed the spatial positioning of three target regions; the SNRPN, UBE3A, and GABRB3 genes on human chromosome 15q11.2–q12, a representative cluster of imprinted regions, in the interphase nuclei of B lymphoblastoid cell lines, peripheral blood cells, and skin fibroblasts derived from normal individuals to look for evidence of genomic organization and function. The positions of these genes were simultaneously visualized, and all inter-gene distances were calculated for each homologous chromosome in each nucleus after three-color 3D fluorescence in situ hybridization. None of the target genes were arranged linearly in most cells analyzed, and GABRB3 was positioned closer to SNRPN than UBE3A in a high proportion of cells in all cell types. This was in contrast to the genomic map in which GABRB3 was positioned closer to UBE3A than SNRPN. We compared the distances from SNRPN to UBE3A (SU) and from UBE3A to GABRB3 (UG) between alleles in each nucleus, 50 cells per subject. The results revealed that the gene-to-gene distance of one allele was longer than that of the other and that the SU ratio (longer/shorter SU distance between alleles) was larger than the UG ratio (longer/shorter UG distance between alleles). The UG distance was relatively stable between alleles; in contrast, the SU distance of one allele was obviously longer than the distance indicated by the genome size. The results therefore indicate that SNRPN, UBE3A, and GABRB3 have non-linear and non-random curved spatial positioning in the normal nucleus, with differences in the SU distance between alleles possibly representing epigenetic evidence of nuclear organization and gene expression

Follow-up nationwide survey on predictive genetic testing for late-onset hereditary neurological diseases in Japan

A follow-up nationwide survey on predictive genetic testing for late-onset neurological diseases in Japan was conducted. A questionnaire was sent to 89 institutional members of the Japan's National Liaison Council for Clinical Sections of Medical Genetics, and was returned by 60 (67.4%). A total of 301 clients with an interest in predictive testing were accumulated from April 2006 to March 2011. The greatest interest was shown for spinocerebellar degeneration (SCD, n = 110), followed by myotonic dystrophy type 1 (DM1, n = 69), Huntington's disease (HD, n = 52) and familial amyloid polyneuropathy (FAP, n = 35). The ratios of clients who actually underwent predictive testing were: SCD, 21.8%; DM1, 39.1%; HD, 26.9%; and FAP, 74.3%, indicating that predictive testing was conducted very cautiously for untreatable neurological diseases in Japan. Clinical geneticists were predominantly involved in genetic counseling, whereas the participation of non-medical doctor (non-MD) staff, including nurses, clinical psychologists and genetic counselors, was not common. Lack of non-MD counseling staff was one of the most serious issues in conducting predictive testing, which has not been improved since the previous survey performed in 2006. Institutional arrangements, such as revision of medical insurance system regarding genetic testing and counseling, might be necessary to resolve this issue.ArticleJOURNAL OF HUMAN GENETICS. 58(8):560-563 (2013)journal articl

Follow-up nationwide survey on predictive genetic testing for late-onset hereditary neurological diseases in Japan

A follow-up nationwide survey on predictive genetic testing for late-onset neurological diseases in Japan was conducted. A questionnaire was sent to 89 institutional members of the Japan's National Liaison Council for Clinical Sections of Medical Genetics, and was returned by 60 (67.4%). A total of 301 clients with an interest in predictive testing were accumulated from April 2006 to March 2011. The greatest interest was shown for spinocerebellar degeneration (SCD, n = 110), followed by myotonic dystrophy type 1 (DM1, n = 69), Huntington's disease (HD, n = 52) and familial amyloid polyneuropathy (FAP, n = 35). The ratios of clients who actually underwent predictive testing were: SCD, 21.8%; DM1, 39.1%; HD, 26.9%; and FAP, 74.3%, indicating that predictive testing was conducted very cautiously for untreatable neurological diseases in Japan. Clinical geneticists were predominantly involved in genetic counseling, whereas the participation of non-medical doctor (non-MD) staff, including nurses, clinical psychologists and genetic counselors, was not common. Lack of non-MD counseling staff was one of the most serious issues in conducting predictive testing, which has not been improved since the previous survey performed in 2006. Institutional arrangements, such as revision of medical insurance system regarding genetic testing and counseling, might be necessary to resolve this issue.ArticleJOURNAL OF HUMAN GENETICS. 58(8):560-563 (2013)journal articl

Identification of a high incidence region for retroviral vector integration near exon 1 of the LMO2 locus

Therapeutic retroviral vector integration near the oncogene LMO2 is thought to be a cause of leukemia in X-SCID gene therapy trials. However, no published studies have evaluated the frequency of vector integrations near exon 1 of the LMO2 locus. We identified a high incidence region (HIR) of vector integration using PCR techniques in the upstream region close to the LMO2 transcription start site in the TPA-Mat T cell line. The integration frequency of the HIR was one per 4.46 × 10(4 )cells. This HIR was also found in Jurkat T cells but was absent from HeLa cells. Furthermore, using human cord blood-derived CD34(+ )cells we identified a HIR in a similar region as the TPA-Mat T cell line. One of the X-linked severe combined immunodeficiency (X-SCID) patients that developed leukemia after gene therapy had a vector integration site in this HIR. Therefore, the descriptions of the location and the integration frequency of the HIR presented here may help us to better understand vector-induced leukemogenesis

BRASSICA オヨビ RAPHANUS ゾク ノ ニホンサン ザイライ ヒンシュ ノ アブラナカ コクハンビョウキン ALTERNARIA BRASSICAE ニ タイスル テイコウセイ ヒョウカ

BrassicaおよびRaphanus属5種138品種を用い,アブラナ科黒斑病菌A. brassicaeによる接種試験を行った。実生葉へA. brassicaeを接種し,4日後の接種部位あるいは接種葉の状態から,発病度を5段階(0-4)で評価した。その結果,無病徴(発病度0)から,極めて軽微な病徴(発病度1),接種部位における病斑(発病度2),接種部位を越えた病斑(発病度3),接種葉が枯れる(発病度4)までの幅広い病徴発現の差が認められた。発病度0はB. rapa Pekinensis Groupより2品種,B. rapa Rapifera GroupおよびR. sativusよりそれぞれ1品種ずつの計4品種で見出された。A total of 138 cultivar seedlings of Brassica spp. and Raphanus sativus were inoculated with the fungal pathogen Alternaria brassicae. Four days after inoculation, disease severity on the seedlings was judged with five criteria (0-4) according to the incidence of lesions. Lesions of various levels, from undetectable (disease severity 0) until withering (disease severity 4), were shown on the seedling depending on the cultivars. As disease severity 0, two cultivars from B. rapa Pekinensis Group, and one cultivar each from B. rapa Rapifera Group and R. sativus were found

Two random amplified polymorphic DNA (RAPD) markers linked to CMV-B2 resistance gene (Creb-2) in melon cultivar Yamatouri were cloned and sequenced to design sequence characterized amplified region (SCAR) markers for detection of CMV-B2 resistance gene (Creb-2) in melon. SCOPE14 derived from OPE-14 yielded a single DNA band at 541 bp, while SCAPB05 derived from APB-05, yielded a single DNA band at 1,046 bp, respectively. Segregation of SCOPE14 and SCAPB05 markers in bulk of F2 plants demonstrated that they were co-segregated with RAPD markers from which the SCAR markers were originated. Furthermore, results of SCAR analysis in diverse melons showed SCAPB05 primers obtained a single 1,046 bp linked to Creb-2 in resistant cultivars Sanuki-shirouri and Kohimeuri. However, SCOPE14 failed to detect Creb-2 in diverse melons. Results of this study revealed that SCAR analysis not only confirmed melons that had been clearly scored for resistance to CMV-B2 by RAPD markers, but also clarified the ambiguous resistance results obtained by the RAPD markers. Key words: Cucumis melo L., Creb-2, RAPD, SCAR

Two random amplified polymorphic DNA (RAPD) markers linked to CMV-B2 resistance gene (Creb-2) in melon cultivar Yamatouri were cloned and sequenced to design sequence characterized amplified region (SCAR) markers for detection of CMV-B2 resistance gene (Creb-2) in melon. SCOPE14 derived from OPE-14 yielded a single DNA band at 541 bp, while SCAPB05 derived from APB-05, yielded a single DNA band at 1,046 bp, respectively. Segregation of SCOPE14 and SCAPB05 markers in bulk of F2 plants demonstrated that they were co-segregated with RAPD markers from which the SCAR markers were originated. Furthermore, results of SCAR analysis in diverse melons showed SCAPB05 primers obtained a single 1,046 bp linked to Creb-2 in resistant cultivars Sanuki-shirouri and Kohimeuri. However, SCOPE14 failed to detect Creb-2 in diverse melons. Results of this study revealed that SCAR analysis not only confirmed melons that had been clearly scored for resistance to CMV-B2 by RAPD markers, but also clarified the ambiguous resistance results obtained by the RAPD markers.         Key words: Cucumis melo L., Creb-2, RAPD, SCA

The relationship between sleep-wake patterns and depression among institute-dwelling elderly with cognitive dysfunction

認知機能の低下と抑うつは,ともに睡眠・覚醒パターンの変調の原因となる.しかし,認知機能が低下した高齢者の場合,抑うつや睡眠障害を自覚症状から把握することは困難である.本研究では,抑うつ度の他覚的評価法として近年開発されたCornell Scale for Depression in Dementia(CSDD)を用い,療養型病院に入所していた高齢者39名(年齢84.1±8.7歳,改訂長谷川式簡易知能評価スケール8.6±6.2点,Barthel Index 43.2±30.3点)の59日間の睡眠指標との関連を検討した.その結果として,総睡眠時間とCSDD得点との間に負の相関が認められた(r=-0.423, p=0.007).以上の結果より,認知機能の低下した高齢者でも一般集団の場合と同様,抑うつ度と睡眠障害とが関連し,また,認知機能の低下した高齢者の睡眠援助において抑うつ度を考慮するには,CSDDが有効である可能性が示唆された.Cognitive dysfunction and depression are the most frequent psychiatric problems in the elderly population. However, it is difficult to assess the status of sleep disorder as well as depression among patients with cognitive dysfunction based on self-reported symptoms. The purpose of this study was to elucidate the relationship between sleep-wake patterns and depression among institute-dwelling elderly with cognitive dysfunction. The subjects were 39 elderly patients (age 84.1±8.7, HDS-R 8.6±6.2, Barthel Index 43.2±30.3) in a convalescent hospital. The Cornell Scale for Depression in Dementia (CSDD) was used to assess the status of depression of the elderly with cognitive dysfunction. Their 59-day sleep-wake patterns were monitored and recorded in the sleep log by nurses. The total sleep time (r=-0.423, p=0.007), the longest sleep episode at nighttime (r=-0.360, p=0.024), and intermittent awakening time at nighttime (r=0.329, p=0.041) were associated with depression. These results suggest a close relationship between depression and difficulty in sleep maintenance among the elderly with cognitive dysfunction as well as among the general population, and also that CSDD can be an efficient tool for the assessment of their depressive status. If this is true, it is important in making care plans for sleep disorders among elder patients with cognitive dysfunction to take into account the effects of depression on sleep-wake patterns

Identification of Four Novel Synonymous Substitutions in the X-Linked Genes Neuroligin 3 and Neuroligin 4X in Japanese Patients with Autistic Spectrum Disorder

Mutations in the X-linked genes neuroligin 3 (NLGN3) and neuroligin 4X (NLGN4X) were first implicated in the pathogenesis of X-linked autism in Swedish families. However, reports of mutations in these genes in autism spectrum disorder (ASD) patients from various ethnic backgrounds present conflicting results regarding the etiology of ASD, possibly because of genetic heterogeneity and/or differences in their ethnic background. Additional mutation screening study on another ethnic background could help to clarify the relevance of the genes to ASD. We scanned the entire coding regions of NLGN3 and NLGN4X in 62 Japanese patients with ASD by polymerase chain reaction-high-resolution melting curve and direct sequencing analyses. Four synonymous substitutions, one in NLGN3 and three in NLGN4X, were identified in four of the 62 patients. These substitutions were not present in 278 control X-chromosomes from unrelated Japanese individuals and were not registered in the database of Single Nucleotide Polymorphisms build 132 or in the Japanese Single Nucleotide Polymorphisms database, indicating that they were novel and specific to ASD. Though further analysis is necessary to determine the physiological and clinical importance of such substitutions, the possibility of the relevance of both synonymous and nonsynonymous substitutions with the etiology of ASD should be considered