Human Genome Center Laboratory of Genome Database Laboratory of Sequence Analysis ゲノムデータベース分野シークエンスデータ情報処理分野

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Professor Minoru Kanehisa, Ph.D. 教授（委嘱）理学博士金久實 Assistant Professor Toshiaki Katayama, M.Sc. 助教理学修士片山俊明 Assistant Professor Shuichi Kawashima, M.Sc. 助教理学修士川島秀一 Lecturer Tetsuo Shibuya, Ph.D. 講師理学博士渋谷哲朗 Assistant Professor Michihiro Araki, Ph.D. 助教薬学博士荒木通啓

Since the completion of the Human Genome Project, high-throughput experimental projects have been initiated for uncovering genomic information in an extended sense, including transcriptomics, proteomics metabolomics, glycomics, and chemical genomics. We are developing a new generation of databases and computational technologies, beyond the traditional genome databases and sequence analysis tools, for making full use of these divergent and ever-increasing amounts of data, especially for medical and pharmaceutical applications.

1. KEGG DRUG and KEGG DISEASE natural product information in the context of plant and other genomes. We also develop Minoru Kanehisa KEGG DISEASE (http://www.genome.jp/kegg/ disease/), a new resource for understanding KEGG is a database of biological systems that molecular mechanisms of human diseases, integrates genomic, chemical, and systemic func- where molecular networks involving disease tional information. It is widely used as a refer- genes are represented as KEGG pathway maps ence knowledge base for understanding higher- or, when such details are not known, simply by order functions and utilities of the cell or the or- a list of diseases genes and other lists of mole- ganism from genomic information. Although the cules including environmental factors. basic components of the KEGG resource are developed in Kyoto University, this Laboratory in 2. KEGG OC: Automatic assignments of the Human Genome Center is responsible for orthologs and paralogs in complete the applied areas of KEGG, especially in medi- genomes cal and pharmaceutical sciences. We develop KEGG DRUG (http://www.genome.jp/kegg/ Toshiaki Katayama, Shuichi Kawashima, drug/), a chemical structure based information Akihiro Nakaya and Minoru Kanehisa resource for all approved drugs in the world, integrating target information in the context of The increase in the number of complete KEGG pathways, efficacy information in the genomeshasprovidedcluestogainusefulin- context of hierarchical drug classifications, and sights to understand the evolution of the gene 117 universe. Among the KEGG suites of databases, Currently, It contains 2,452,094 sequence cata- the GENES database contains more than 4.2 mil- logues (779,490 contigs and 1,672,604 singletons) lion genes from over 1,000 organisms as of Feb- in 62 plants. 25% of the sequences are assigned ruary 2009. Sequence similarities among these K numbers. EGENES is available at http:// genes are calculated by all-against-all SSEARCH www.genome.jp/kegg/plant/pln_list.html. comparison and stored them in the SSDB database. Based on those databases, the ORTHOL- 4. KEGG API: SOAP/WSDL interface for the OGY database has been manually constructed to KEGG system store the relationships among the genes sharing the same biological function. However, in this Shuichi Kawashima, Toshiaki Katayama and strategy, only the well known functions can be Minoru Kanehisa used for annotation of newly added genes, thus the number of annotated genes is limited. To KEGG is a suite of databases and associated overcome this situation, we developed a fully software, integrating our current knowledge of automated procedure to find candidate ortholo- molecular interaction/reaction pathways and gous clusters including whose current functional other systemic functions (PATHWAY and BRITE annotation is anonymous. The method is based databases), the information about the genomic on a graph analysis of the SSDB database, treat- space (GENES database), and information about ing genes as nodes and Smith-Waterman se- the chemical space (LIGAND and DRUG data- quence similarity scores as weight of edges. The bases). To facilitate large-scale applications of cluster is found by our heuristic method for the KEGG system programmatically, we have finding quasi-cliques, but the SSDB graph is too been developing and maintaining the KEGG large to perform quasi-clique finding at a time. API as a stable SOAP/WSDL based web service. Therefore, we introduce a hierarchy (evolution- The KEGG API is available at http://www. ary relationship) of organisms and treat the genome.jp/kegg/soap/. SSDB graph as a nested graph. The automatic decomposition of the SSDB graph into a set of 5. KEGG DAS: Comprehensive repository for quasi-cliques results in the KEGG OC (Ortholog community genome annotation Cluster) database. We built a system that performs automatic update of the ortholog cluster, Toshiaki Katayama, Mari Watanabe and which can be run weekly basis. As a result, we Minoru Kanehisa obtained 669,043 clusters including 403,752 sin- gleton clusters from 3,721,464 protein coding KEGG DAS is an advanced genome database genes. Among them, only 2,309 clusters were system providing DAS (Distributed Annotation shared across kingdoms and other clusters were System) service for all bacterial organisms in the kingdom specific. The automatic classification of GENOME database in KEGG. Currently, KEGG our ortholog clusters largely consistent with the DAS contains over 8 million annotations as- manually curated ORTHOLOGY database. A signed to the genome sequences of 817 organ- web interface to search and browse genes in isms (increased from 615 organisms in last year). clusters is made available at http://oc.kegg.jp/. The KEGG DAS server provides gene annotations linked to the KEGG PATHWAY and 3. EGENES: A database for expressed se- LIGAND databases. In addition to the coding quence tag indices of plant species genes, information of non-coding RNAs predicted using Rfam database is also provided to Shuichi Kawashima, Yuki Moriya, Toshiaki fill the annotation of the intergenic regions of Tokimatsu , Susumu Goto and Minoru the genomes. The KEGG DAS service is avail- Kanehisa able at http://das.hgc.jp/.

EGENES is a knowledge-based database for 6. Full-Arthropods: Constructing full length efficient analysis of plant expressed sequence cDNA of pathogenetic arthropods tags (ESTs), which was recently added to the KEGG PLANT. It links plant genomic informa- Toshiaki Katayama, Shuichi Kawashima, Miho tion with higher order functional information in Usui, Hiroyuki Wakaguri, Eri Kibukawa, KEGG. The genomic information in EGENES is Masahide Sasaki , Kazuhisa Hiranuka , a collection of EST contigs constructed from as- Ryuichiro Maeda, Yutaka Suzuki, Sumio sembled plant ESTs by using EGassembler. The Sugano and Junichi Watanabe EST indices are automatically annotated with the KEGG Orthology identifiers (K numbers) by Anopheles mosquito, tsetse fly, tsutsugamushi KEGG Automatics Annotation Server (KAAS). -mite, dust mite are arthropods which are 118 known as medically important because these and adults using the vector trapper method and either transmit various infectious disease includ- sequenced the both ends of 11,520 cDNA clones. ing malaria, Japanese river fever, or cause al- Cleaning, clustering and assembling of the row lergy such as asthma and dermatitis. Because of sequences produced 3,031 contigs and 4,281 sin- serious medical problems they cause, their gletons. 1,797 of the total unique 7,312 se- genomes are being extensively analyzed re- quences were assigned KEGG Orthology by cently. We have produced libraries of the four KAAS system. More than 30% of the sequences organisms and are constructing their databases showed significant matches to KEGG GENES for the functional genome analysis. Full- database, which includes well characterized Der Arthropods is available on the site http://ful- f group 1 allergens. We predicted 1,109 peptides larth.hgc.jp/ longer than 20 amino acids from the 3,031 contigs. Some of the peptides are predicted to con- 7. Full-Entamoeba: a database for the full tain the 9-mer peptides with strong affinities to length cDNA library of Entamoeba MHC class II by NetMHC 3.0. We expect that these in silico analyses will pave the way toward Toshiaki Katayama , Kazushi Hiranuka , prediction of allergens from D. farinae. Masahiro Kumagai, Yutaka Suzuki, Sumio Sugano, Atsushi Toyoda, Asao Makioka, 9. HiGet and SSS: Search engines for the Junichi Watanabe large-scale biological databases

Entamoeba histolytica is a protozoan parasite Toshiaki Katayama, Shuichi Kawashima, which predominantly infects humans and other Kazuhiro Ohi, Kenta Nakai and Minoru primates and causes amebiasis. E. histolytica is Kanehisa estimated to infect about 50 million people worldwide and amebiasis is estimated to cause Recently, the number of entries in biological 70,000 deaths per year. Full-Entamoeba, a data- databases is exponentially increasing year by base for full-length cDNAs from a human para- year. For example, there were 10,106,023 entries site, E. histolytica, and a reptilian parasite, E. in- in the GenBank database in the year 2000, which vadens, has been produced. The full-length has now grown to 98,868,465 (Release 169＋ cDNA libraries were produced using the oligo- daily updates). In order for such a vast amount capping method from trophozoites of each spe- of data to be searched at a high speed, we have cies cultivated axenically. A total of 5,000 5’end- developed a high performance database entry one-pass sequences of cDNAs from the two spe- retrieval system, named HiGet. For this purpose, cies were compared with the non-redundant da- the system is constructed on the HiRDB, a com- tabase of DDBL/GenBank/EMBL using BLAST mercial ORDBMS (Object-oriented Relational and TBLASTX programs. These clones are avail- Database Management System) developed by able for further analysis and experiments. Full- Hitachi, Ltd. HiGet can perform full text search Entamoeba database is available at http://ful- on various biological databases including Gen- lent.hgc.jp/ Bank, RefSeq, UniProt, Prosite, OMIM and PDB. Additional advantage of the HiGet system is the 8. Analysis of sequence catalogs of the capability of a field specific search, which en- house dust mite Dermatophagoides fari- ables users to narrow down the number of re- nae sults, especially useful for collecting sequences of their specific needs. We have also developed Shuichi Kawashima, Atsushi Toyoda, Junichi a sequence similarity search (SSS) service to find Watanabe, Sadao Nogami and Minoru homologous sequences with various algorithms Kanehisa including BLAST, FASTA, SSEARCH, TRANS, and EXONERATE. This variety of options is The house dust mite is a cosmopolitan guest unique among the public services and users can in human habitation and the multicellular or- select an appropriate method to search similar ganism that is one of the most closely associated sequences according to their query. Because al- with our life. It is now well established that the gorithms such as TRANS and EXONERATE are dust mites are major allergens causing bronchial highly time consuming, the SSS service is back- asthma, allergic rhinitis and atopic dermatitis. ended by the distributed computing environ- Dermatophagoides farinae (American house dust ment with the Sun Grid Engine in our super mite) and D. pteronissinus (European house dust computer system. HiGet and SSS services are mite) are two most common species in the tem- available at http://higet.hgc.jp/ and http://sss. perate zone. We produced the cDNA libraries of hgc.jp/ respectively. our D. farinae sample containing young nymphs 119

10. Linear-time protein 3-D structure search- results. In other words, the actual computation ing algorithm time of our linear-expected-time algorithm is not influenced by the difference of query lengths, in Tetsuo Shibuya contrast to previous algorithms.

Finding similar structures from 3-D structure 11. Fast hinge detection algorithm in protein databases of proteins (or other molecules) is be- structures coming one of the most important issues in the post-genomic molecular biology. To compare 3- Tetsuo Shibuya D structures of two molecules, biologists mostly use the RMSD (root mean square deviation) as Analysis of conformational changes is one of the similarity measure. The RMSD is one of the the keys to the understanding of protein func- most fundamental similarity measures used in tions and interactions. For the analysis, we often various fields, such as computer vision and ro- compare two protein structures, taking flexible botics, for comparing two sets of coordinates. In regions like hinge domains into consideration. this research, we propose new theoretically and The RMSD (Root Mean Square Deviation) is the practically fast algorithms for the basic problem most popular measure for comparing two pro- of finding all the substructures of structures in a tein structures, but it is only for rigid structures structure database of chain molecules (such as without hinge domains. In this research, we pro- proteins), whose RMSDs to the query are within pose a new measure called RMSDh (Root Mean a given constant threshold. The best-known Square Deviation considering hinges) and its worst-case time complexity for the problem is O variant RMSDh(k) for comparing two flexible (N log m), where N is the database size and m proteins with hinge domains. We also propose is the query size. The previous best-known ex- novel efficient algorithms for computing them, pected time complexity for the problem is also which can detect the hinge positions at the same O (N log m). In this research, we propose a new time. The RMSDh is suitable for cases where breakthrough linear-expected-time algorithm. It there is one small hinge domain in each of the is not only a theoretically significant improve- two target structures. The new algorithm for ment over previous algorithms, but also a prac- computing the RMSDh runs in linear time, tically faster algorithm, according to computa- whichissameasthetimecomplexityforcom- tional experiments. We also propose a series of puting the RMSD and is faster than any of pre- preprocessing algorithms that enable faster que- vious algorithms for hinge detection. The ries, though there have been no known indexing RMSDh(k) is designed for comparing structures algorithm whose query time complexity is better with more than one hinge domain. The RMSDh than the above O (N log m) bound. One is an O (k) measure considers at most k small hinge do- (N log2 N )-time and O (N log N )-space pre- mains, i.e., the RMSDh(k) value should be small processing algorithm with expected query time if the two structures are similar except for at complexity of O (m＋N /m 0.5). Another is an O (N most k hinge domains. To compute the value, log N )-time and O (N )-space preprocessing algo- we propose an O (kn 2)-time and O (n)-space al- rithm with expected query time complexity of O gorithm based on a new dynamic programming (N /m 0.5＋m log (N /m)). technique. We also test our measures against We also extend the above linear-time algo- both flexible protein structures and non-flexible rithm into an algorithm with expected query protein structures, and show that the hinge po- time complexity of O (m＋N /m 1－ε), where ε is sitions can be correctly detected by our algo- an arbitrary small constant such that 0＜ε＜1. rithms. We furthermore extend the above linear-time algorithm so that it can deal with insertions and 12. Fast flexible protein structure alignment deletions. We checked the performance of our linear- Kohichi Suematsu and Tetsuo Shibuya expected-time algorithm through computational experiments over the whole PDB database. The The Hinge Detection Algorithm described in experiments show that our algorithm is much section 11 only considered rigid hinge points, faster than the previous algorithms. For exam- but the hinges are sometimes bends a little by it- ple, our algorithm is 3.6 to 28 times faster than self, which sometimes leads to inaccurate pre- previously known algorithms, to search for simi- diction of hinge positions. Thus we incorporated lar substructures whose RMSDs are within 1Å the notion ‘bending hinge’ to detect such hinge to queries of ordinary lengths. The experiments positions. We developed a very efficient heuris- also show that there is consistency between the tic algorithm for finding such bending hinges, as above theoretical results and the experimental the exact algorithm for this problem requires ex- 120 ponential time. For the algorithm, we developed a detailed score matrix for comparing local 15. Color space-DNA sequence mapping structures based on the naïve Bayse learning. alignment algorithm

13. Protein function prediction based on 3-D Ben Hachimori and Tetsuo Shibuya structure motifs Applied Biosystems’s SOLiD system encode Chia-Han Chu, Hiroki Sakai, and Tetsuo the DNA sequence into a sequence of data type Shibuya called the color space, where one of 4 fluores- cent colors is assigned to each two adjacent Protein functions are said to be determined by base’s 16 pattern orderings. However, there its 3-D structures, but not all functions have have been known no algorithm that aligns/ been known to be related to some 3-D structure maps the color-space sequence to the DNA se- motifs. The geometric suffix tree, a data struc- quence with consideration of the difference be- ture for indexing 3-D protein structures, which tween the experimental error and the actual mu- is also developed by us, enables comprehen- tation. We developed an alignment algorithm sively enumeration of all the possible structural that distinguishes the experimental error and ac- motifs among given set of proteins. We are de- tual DNA mutation to align the color-space data veloping a new algorithm based on the support against ordinary DNA sequences. Moreover, we vector machine that decides protein’s function computed the optimal score table for the align- from the 3-D structure of a protein. This algo- ment based on the actual E. coli data. rithm utilizes all the possible 3-D motifs found by using the geometric suffix tree. 16. Genotype clustering based on hidden Markov models 14. Suffix array construction with a lazy scheme Ritsuko Onuki, Tetsuo Shibuya, and Minoru Kanehisa Ben Hachimori and Tetsuo Shibuya Haplotype clustering is important for gene The suffix array is one of the most important mapping of human disease. Although its impor- indexing data structures for alphabet strings, in- tance for the analysis, it is difficult to obtain cluding DNA sequences, RNA sequences, pro- haplotype data from present experiment for its tein sequences, web pages, Medline database, cost and error rate. Instead of haplotypes, geno- and so on. But even the most sophisticated algo- types are much easier to obtain. In this work, rithm for constructing the suffix array requires a we propose a new method for clustering geno- lot of time. We developed a new efficient lazy types. In the algorithm, we first infer the multi- algorithm that computes the suffix array only ple haplotype candidates from the genotype, afterwegetthequery.Bydoingso,wehaveto and next we calculate the distance between the compute only the necessary part of the suffix ar- genotypes based on the results of the haplotype ray. We developed a lazy algorithm based on inference. Then we perform genotype clustering the Schurmann-Stoye algorithm, which is more based on the distances. We evaluated our algo- efficient than both Boyer-Moore algorithm and rithm by applying our algorithm against several other suffix tree-based algorithms in case the actual genotype data. number of queries is limited.

Publications

Kanehisa, M., Araki, M., Goto, S., Hattori, M., Okuda, S., Yamada, T., Hamajima, M., Itoh, M., Hirakawa, M., Itoh, M., Katayama, T., Katayama, T., Bork, P., Goto, S. and Kanehisa, Kawashima,S.,Okuda,S.,Tokimatsu,T.,and M. KEGG Atlas mapping for global analysis Yamanishi, Y. KEGG for linking genomes to of metabolic pathways. Nucleic Acids Research life and the environment. Nucleic Acids Re- 36: W423-426, 2008. search 36: D480-D484, 2008. Wakaguri, H., Suzuki, Y., Katayama, T., Kawashima, S., Pokarowski, P., Pokarowska, M., Kawashima, S., Kibukawa, E., Hiranuka, K., Kolinski, A., Katayama, T., Kanehisa, M. Sasaki,M.,Sugano,S.andWatanabe,J.Full- AAindex: amino acid index database, progress Malaria/Parasites and Full-Arthropods: data- report 2008. Nucleic Acids Research 36: D202-D base of full-length cDNAs of parasites and ar- 205, 2008. thropods, update 2009. Nucleic Acids Research 121

37: D520-D525, 2008. Transactions on Computational Biology and Bioin- Yamanishi, Y., Araki, M., Gutteridge, A., Honda, formatics, to appear. W. and Kanehisa, M. Prediction of drug-target Shibuya, T., Searching Protein 3-D Structures in interaction networks from the integration of Linear Time, Proc. 13th Annual International chemical and genomic spaces. Bioinformatics Conference on Research in Computational Molecu- 24: i232-i240, 2008. lar Biology (RECOMB 2009), 2009, to appear. Takarabe,M.,Okuda,S.,Itoh,M.,Tokimatsu,T., Shibuya, T., Linear-Time Algorithm for Search- Goto, S. and Kanehisa, M. Network analysis ing Protein 3-D Structures, IPSJ SIG Notes SI- of adverse drug interactions. Genome Informat- GAL 123-4, 2009, to appear. ics 20: 252-259, 2008. Suematsu, K., Shibuya, T., Flexible Protein Hashimoto, K., Yoshizawa, A.C., Okuda, S., Alignment of 3D-Structures Allowing Dy- Kuma,K.,Goto,S.andKanehisa,M.Therep- namic Transformation, ISPSJ SIG Notes SIG- ertoire of desaturases and elongases reveals BIO 12-12, 2008, pp. 87-94. fatty acid variations in 56 eukaryotic genomes. 本多渉，田辺麻央，矢野亜津子，金久實．バイオ J. Lipid Res. 49: 183-191 (2008). インフォマティクス・システムバイオロジーと Shibuya, T., Fast Hinge Detection Algorithms KEGG，生化学，８０Ã：１０９４―１１１１，２００８ for Flexible Protein Structures, IEEE/ACM 122

Human Genome Center Laboratory of DNA Information Analysis DNA情報解析分野

Professor Satoru Miyano, Ph.D. 教授理学博士宮野悟 Associate Professor Seiya Imoto, Ph.D. 准教授博士（数理学）井元清哉 Assistant Professor Masao Nagasaki, Ph.D. 助教博士（理学）長正朗 Project Lecturer Rui Yamaguchi, Ph.D. 特任講師博士（理学）山口類 Project AssistantProfessor Yoshinori Tamada, Ph.D. 特任助教博士（情報学）玉田嘉紀

The recent advances in biomedical research have been producing large-scale, ultra-high dimensional, ultra-heterogeneous data. Due to these post-genomic research progresses, our current mission is to create computational strategy for systems biology and medicine towards translational bioinformatics. With this mission, we have been developing computational methods for understanding life as system and applying them to practical issues in medicine and biology.

1. Computational Systems Biology rules based on hybrid functional Petri net with extension (HFPNe), which is suitable for graphi- a. Systematic reconstruction of TRANSPATH cal representation and simulation of biological data into Cell System Markup Language processes. In these modeling rules, each Petri net element is incorporated with Cell System Masao Nagasaki, Ayumu Saito, Chen Li, Euna Ontology (CSO) to enable semantic interoper- Jeong, Satoru Miyano ability of models. As a formal ontology for biological pathway modeling with dynamics, CSO Many biological repositories store information also defines biological terminology and corre- based on experimental study of the biological sponding icons. By combining HFPNe with the processes within a cell, such as protein-protein CSO features, we made a method for transform interactions, metabolic pathways, signal trans- ing TRANSPATH data to simulation-based se- duction pathways, or regulations of transcrip- mantically valid models. The results are en- tion factors and miRNA. Unfortunately, it is dif- coded into a biological pathway format, Cell ficult to directly use such information when System Markup Language (CSML), which eases generating simulation-based models. Thus, mod- the exchange and integration of biological data eling rules for encoding biological knowledge and models. By using the 16 modeling rules, into system-dynamics-oriented standardized for- 97％ of the reactions in TRANSPATH are con- mats would be very useful for full understand- verted into simulation-based models represented ing of cellular dynamics at the system level. We in CSML. This reconstruction demonstrated that selected the TRANSPATH database, a manually it is possible to use our rules to generate quanti- curated high-quality pathway database, which tative models from static pathway descriptions. provides a rich source of cellular events in humans, mice, and rats, curated from over 31,500 b. Finding optimal Bayesian network given a papers. In this work, we defined 16 modeling super-structure 123

Eric Perrier, Seiya Imoto, Satoru Miyano spacemodel,weprovidedanapproachtouse the technical replicates of gene expression pro- Conventional approaches for learning Baye- files, which are often measured in duplicate or sian network structure from data have disad- triplicate. The use of technical replicates is im- vantages in terms of complexity and lower accu- portant for achieving highly-efficient inference racy of their results. However, a recent empiri- of gene networks with short time-course data. cal study has shown that a hybrid algorithm im- The potential of the proposed method were proves sensitively accuracy and speed: it learns demonstrated through the time-course analysis askeletonwithanindependencytest(IT)ap- of the gene expression profiles of human umbili- proach and constrains on the directed acyclic cal vein endothelial cells undergoing growth graphs considered during the search-and-score factor deprivation-induced apoptosis. phase. Subsequently, we defined the structural constraint by introducing the concept of super- d. Predicting differences in gene regulatory structure S , which is an undirected graph that systems by state space models restricts the search to networks whose skeleton is a subgraph of S . We developed a super- Rui Yamaguchi, Seiya Imoto, Mai Yamauchi, structure constrained optimal search (COS): its Masao Nagasaki, Ryo Yoshida1, Teppei Shima- n time complexity is upper bounded by O（γm), mura, Yosuke Hatanaka, Kazuko Ueno, To- 1 where γm＜2 depends on the maximal degree m moyuki Higuchi , Noriko Gotoh, Satoru Miy- of S . Empirically, complexity depends on the ano average degree m’ and sparse structures allow larger graphs to be calculated. Our algorithm is We developed a statistical method to predict faster than an optimal search by several orders differentially regulated genes of case and control and even finds more accurate results when samples from time-course gene expression data given a sound super-structure. Practically, S can by leveraging unpredictability of the expression be approximated by IT approaches; significance patterns from the underlying regulatory system level of the tests controls its sparseness, enabling inferred by a state space model. The proposed to control the trade-off between speed and accu- method can screen out genes that show different racy. For incomplete super-structures, a greedily patterns but generated by the same regulations post-processed version (COS＋) still enables to in both samples, since these patterns can be pre- significantly outperform other heuristic searches. dicted by the same model. Our strategy consists of three steps. Firstly, a gene regulatory system c. Statistical inference of transcriptional is inferred from the control data by a state space module-based gene networks from time model. Then the obtained model for the under- course gene expression profiles by using lying regulatory system of the control sample is state space models used to predict the case data. Finally, by assess- ing the significance of the difference between Osamu Hirose, Ryo Yoshida1, Seiya Imoto, Rui case and predicted-case time-course data of each Yamaguchi, Tomoyuki Higuchi1,D.Stephen gene, we are able to detect the unpredictable Charnock-Jones2, Cristin Print3, Satoru Miy- genes that are the candidate as the key differ- ano; 1Institute of Statistical Mathematics, ences between the regulatory systems of case 2Cambridge University, 3University of Auck- and control cells. We illustrate the whole proc- land ess of the strategy by an actual example, where human small airway epithelial cell gene regula- We developed a novel method based on the tory systems were generated from novel time state space model to identify the transcriptional courses of gene expressions following treatment modules and module-based gene networks si- with(case)/without(control) the drug gefitinib, multaneously. The state space model has the po- an inhibitor for the epidermal growth factor re- tential to infer large-scale gene networks, e.g. of ceptor tyrosine kinase. Finally, in gefitinib re- order 103, from time-course gene expression pro- sponse data we succeeded in finding unpredict- files. Particularly, we succeeded in identification able genes that are candidates of the specific tar- of a cell cycle system by using the gene expres- gets of gefitinib. We also discussed differences sion profiles of Saccharomyces cerevisiae in which in regulatory systems for the unpredictable the length of the time-course and number of genes. The proposed method would be a prom- genes were 24 and 4382, respectively. However, ising tool for identifying biomarkers and drug when analyzing shorter time-course data, e.g. of target genes. length 10 or less, the parameter estimations of the state space model often fail due to overfit- e. Bayesian learning of biological pathways ting. To extend the applicability of the state on genomic data assimilation 124

Ryo Yoshida1, Masao Nagasaki, Rui Yama- models do not infer directionality in these con- guchi, Seiya Imoto, Satoru Miyano, Tomoyuki nections. Thus, a priori biological knowledge is Higuchi1 required. However, in pathological cases, no a priori biological information is available. To Mathematical modeling and simulation, based overcome these problems, we present the non- on biochemical rate equations, provide us a rig- linear vector autoregressive (NVAR) model. We orous tool for unraveling complex mechanisms have applied the NVAR model to estimate non- of biological pathways. To proceed to simulation linear gene regulatory networks based entirely experiments, it is an essential first step to find on gene expression profiles obtained from DNA effective values of model parameters, which are microarray experiments. We showed the results difficult to measure from in vivo and in vitro ex- obtained by NVAR through several simulations periments. Furthermore, once a set of hypotheti- and by the construction of three actual gene cal models has been created, any statistical crite- regulatory networks (p53, NF-κB, and c-Myc) rion is needed to test the ability of the con- for HeLa cells. structed models and to proceed to model revi- sion. We developed a new statistical technology g. Fast grid layout algorithm for biological towards data-driven construction of in silico bio- networks with sweep calculation logical pathways. The method starts with a knowledge-based modeling with hybrid func- Kaname Kojima, Masao Nagasaki, Satoru Miy- tional Petri net. It then proceeds to the Bayesian ano learning of model parameters for which experimental data are available. This process exploits Properly drawn biological networks are of quantitative measurements of evolving bio- great help in the comprehension of their charac- chemical reactions, e.g. gene expression data. teristics. The quality of the layouts for retrieved Another important issue that we consider is sta- biological networks is critical for pathway data- tistical evaluation and comparison of the con- bases. However, since it is unrealistic to manu- structed hypothetical pathways. For this pur- ally draw biological networks for every re- pose, we have developed a new Bayesian trieval, automatic drawing algorithms are essen- information-theoretic measure that assesses the tial. Grid layout algorithms handle various bio- predictability and the biological robustness of in logical properties such as aligning vertices hav- silico pathways. ing the same attributes and complicated positional constraints according to their subcellular f. Modeling nonlinear gene regulatory net- localizations; thus, they succeed in providing works from time series gene expression biologically comprehensible layouts. However, data existing grid layout algorithms are not suitable for real-time drawing, which is one of requisites André Fujita, João Ricardo Sato5, Humberto for applications to pathway databases, due to Miguel Garay-Malpartida5 , Mari Cleide their high-computational cost. In addition, they Sogayar5, Carlow Eduardo Ferreira5, Satoru do not consider edge directions and their result- Miyano; 5University of São Paulo ing layouts lack traceability for biochemical reactions and gene regulations, which are the In cells, molecular networks such as gene most important features in biological networks. regulatory networks are the basis of biological We devised a new calculation method termed complexity. Therefore, gene regulatory networks sweep calculation and reduced the time com- have become the core of research in systems bi- plexity of the current grid layout algorithms ology. Understanding the processes underlying through its encoding and decoding processes. the several extracellular regulators, signal trans- We conduct ed practical experiments by using duction, protein-protein interactions, and differ- 95 pathway models of various sizes from ential gene expression processes requires de- TRANSPATH and showed that our new grid tailed molecular description of the protein and layout algorithm is much faster than existing gene networks involved. To understand better grid layout algorithms. For the cost function, we these complex molecular networks and to infer introduced a new component that penalizes un- new regulatory associations, we developed a desirable edge directions to avoid the lack of statistical method based on vector autoregres- traceability in pathways due to the differences sive models and Granger causality to estimate in direction between in-edges and out-edges of nonlinear gene regulatory networks from time each vertex. series microarray data. Most of the models available in the literature assume linearity in the inference of gene connections; moreover, these 125 h. Estimation of nonlinear gene regulatory also classified as the top seven most informative networks via L1 regularized NVAR from genes for the prostate cancer genesis process by time series gene expression data our discriminant analysis. Moreover, among the identified genes we found classically known Kaname Kojima, André Fujita, Teppei Shima- biomarkers and genes which are closely related mura, Seiya Imoto, Satoru Miyano to tumoral prostate, such as KLK3 and KLK2 and several other potential ones. We have dem- Recently, nonlinear vector autoregressive onstrated that changes in functional connectivity (NVAR) model based on Granger causality was may be implicit in the biological process which proposed to infer nonlinear gene regulatory net- renders some genes more informative to dis- works from time series gene expression data. criminate between normal and tumoral condi- Since NVAR requires a large number of parame- tions. Using the proposed method, namely, ters due to the basis expansion, the length of MLDA, in order to analyze the multivariate time series microarray data is insufficient for ac- characteristic of genes, it was possible to capture curate parameter estimation and we need to the changes in dependence networks which are limit the size of the gene set strongly. To ad- related to cell transformation. dress this limitation, we employed L1 regularization technique to estimate NVAR. Under L1 j. Rule-based reasoning for system dynam- regularization, direct parents of each gene can ics in cell systems be selected efficiently even when the number of parameters exceeds the number of data samples. Euna Jeong, Masao Nagasaki, Satoru Miyano We can thus estimate larger gene regulatory networks more accurately than those from existing A system-dynamics-centered ontology, called methods. Through the simulation study, we the Cell System Ontology (CSO), has been de- verified the effectiveness of the proposed veloped for representation of diverse biological method by comparing its limitation in the num- pathways. Many of the pathway data based on ber of genes to that of the existing NVAR. The the ontology have been created from databases proposed method was also applied to time se- via data conversion or curated by expert biolo- ries microarray data of Human hela cell cycle. gists. It is essential to validate the pathway data which may cause unexpected issues such as se- i. Multivariate gene expression analysis re- mantic inconsistency and incompleteness. This veals functional connectivity changes be- paper discusses three criteria for validating the tween normal/tumoral prostates pathway data based on CSO as follows: (1) structurally correct models in terms of Petri André Fujita, Luciana Rodrigues Gomes5, João nets, (2) biologically correct models to capture Ricardo Sato6, Rui Yamaguchi, Carlos Edu- biological meaning, and (3) systematically cor- ardo Thomaz7, Mari Cleide Sogayar5, Satoru rect models to reflect biological behaviors. Si- Miyano; 6Universidade Federal do ABC, 7Cen- multaneously, we have investigated how logic- tro Universitário da FEI based rules can be used for the ontology to extend its expressiveness and to complement the Principal Component Analysis (PCA) com- ontology by reasoning, which aims at qualifying bined with the Maximum-entropy Linear Dis- pathway knowledge. Finally, we show how the criminant Analysis (MLDA) was applied in or- proposed approach helps exploring dynamic der to identify genes with the most discrimina- modeling and simulation tasks without prior tive information between normal and tumoral knowledge. prostatic tissues. Data analysis was carried out using three different approaches, namely: (i) dif- k. A novel strategy to search conserved tran- ferences in gene expression levels between nor- scription factor binding sites among coex- mal and tumoral conditions from a univariate pressing genes in human point of view; (ii) in a multivariate fashion using MLDA; and (iii) with a dependence network ap- Yosuke Hatanaka, Masao Nagasaki, Rui Yam- proach. Our results show that malignant trans- aguchi, Takeshi Obayashi, Kazuyuki Numata, formationintheprostatictissueismorerelated André Fujita, Teppei Shimamura, Yoshinori to functional connectivity changes in their de- Tamada, Seiya Imoto, Kengo Kinoshita, Kenta pendence networks than to differential gene ex- Nakai, Satoru Miyano pression. The MYLK, KLK2, KLK3, HAN11, LTF, CSRP1 and TGM4 genes presented signifi- We reported various transcription factor bind- cant changes in their functional connectivity being sites (TFBSs) conserved among co-expressed tween normal and tumoral conditions and were genes in human promoter region using expres- 126 sion and genomic data. Assuming similar pro- sion models is investigated to analyze data with moter structure induces similar transcriptional complex structure. We introduced radial basis regulation, hence induces similar expression functions with hyperparameter that adjusts the profile, we compared the promoter structure amount of overlapping basis functions and similarities between co-expressed genes. Com- adopts the information of the input and re- prehensive TF binding site predictions for all sponse variables. By using the radial basis func- human genes were conducted for 19,777 pro- tions, we constructed nonlinear regression mod- moter regions around the transcription start site els with help of the technique of regularization. (TSS) given from DBTSS and promoter similar- Crucial issues in the model building process are ity search were conducted among coexpressing the choices of a hyperparameter, the number of genes data provided from newly developed basis functions and a smoothing parameter. We COXPRESdb. Combination of Position Weight present information-theoretic criteria for evaluat- Matrix (PWM) motif prediction and bootstrap ing statistical models under model misspecifica- method, 7,313 genes have at least one statisti- tion both for distributional and structural as- cally significant conserved TFBS. We also ap- sumptions. We used real data examples and plied basket method analysis for seeking combi- Monte Carlo simulations to investigate the prop- natorial activities of those conserved TFBSs. erties of the proposed nonlinear regression modeling techniques. The simulation results showed l. Simulation analysis for the effect of light- that our nonlinear modeling performs well in dark cycle on the entrainment in circadian various situations, and clear improvements were rhythm obtained for the use of the hyperparameter in the basis functions. Natumi Mitou8, Yuto Ikegami8,HiroshiMat- suno8, Satoru Miyano, Shin-ichi T. Inouye8; b. The GC and window-averaged DNA curva- 8Yamaguchi University ture profile of secondary metabolite gene cluster in Aspergillus fumigatus genome Circadian rhythms of the living organisms are 24hr oscillations found in behavior, biochemistry Jin Hwan Do, Satoru Miyano and physiology. Under constant conditions, the rhythms continue with their intrinsic period An immense variety of complex secondary length, which are rarely exact 24hr. In this pa- metabolites is produced by filamentous fungi in- per, we examine the effects of light on the phase cluding Aspergillus fumigatus, a main inducer of of the gene expression rhythms derived from invasive aspergillosis. The identification of fun- the interacting feedback network of a few clock gal secondary metabolite gene cluster is essen- genes, taking advantage of a computer simula- tial for the characterization of fungal secondary tion with Cell Illustrator. The simulation results metabolism in terms of genetics and biochemis- suggested that the interacting circadian feedback try through recombinant technologies such as network at the molecular level is essential for gene disruption and cloning. Most of the predic- phase dependence of the light effects, observed tion methods for secondary metabolite gene in mammalian behavior. Furthermore, the simu- cluster severely depend on homology searches. lation reproduced the biological observations However, homology-based approach has intrin- that the range of entrainment to shorter or sic limitation to unknown or novel gene cluster. longer than 24hr light-dark cycles is limited, We analyzed the GC and window-averaged centering around 24hr. Application of our model DNA curvature profile of 26 secondary metabo- to inter-time zone flight successfully demon- lite gene clusters in the A. fumigatus genome to strated that 6 to 7 days are required to recover find out potential conserved features of secon- from jet lag when traveling from Tokyo to New dary metabolite gene cluster. Fifteen secondary York. metabolite gene clusters showed a conserved pattern in window-averaged DNA curvature 2. Statistical and Computational Knowledge profile, that is, the DNA regions including sec- Discovery ondary metabolic signature genes such as polyketide synthase, nonribosomal peptide syn- a. Nonlinear regression modeling via regular- thase, and/or dimethylallyl tryptophan synthase ized radial basis function networks consisted of window-averaged DNA curvature values lower than 0.18 and these DNA regions Tomohiro Ando9, Sadanori Konishi10,Seiya were at least 20 kb. Forty percent of secondary Imoto; 9Keio University, 10Kyushu University metabolite gene clusters with this conserved pattern were related to severe regulation by a tran- The problem of constructing nonlinear regres- scription factor, LaeA. Our result could be used 127 for identification of other fungal secondary me- that is the first all-in-one web service for analy- tabolite gene clusters, especially for secondary sis of exon array data to detect transcripts that metabolite gene cluster that is severely regulated have significantly different splicing patterns in by LaeA or other proteins with similar function two cells, e.g. normal and cancer cells. Exon- to LaeA. Miner can perform the following analyses: (1) data normalization, (2) statistical analysis based c. ExonMiner: Web service for analysis of on two-way ANOVA, (3) finding transcripts GeneChip exon array data with significantly different splice patterns, (4) efficient visualization based on heatmaps and bar- Kazuyuki Numata, Ryo Yoshida1, Masao Na- plots, and (5) meta-analysis to detect exon level gasaki, Ayumu Saito, Seiya Imoto, Satoru Miy- biomarkers. We implemented ExonMiner on the ano supercomputer system of Human Genome Cen- ter in order to perform genome-wide analysis Some splicing isoform-specific transcriptional for more than 300,000 transcripts in exon array regulations are related to disease. Therefore, de- data, which has the potential to reveal the aber- tection of disease specific splice variations is the rant splice variations in cancer cells as exon first step for finding disease specific transcrip- level biomarkers. ExonMiner is well suited for tional regulations. Affymetrix Human Exon 1.0 analysis of exon array data and does not require ST Array can measure exon-level expression any installation of software except for internet profiles that are suitable to find differentially ex- browsers. The URL of ExonMiner is http://ae. pressed exons in genome-wide scale. However, hgc.jp/exonminer. Users can analyze full dataset exon array produces massive datasets that are of exon array data within hours by high-level more than we can handle and analyze on per- statistical analysis with sound theoretical basis sonal computer. We have developed ExonMiner that finds aberrant splice variants as biomarkers.

Publications

1. Ando,T.,Konishi,S.,Imoto,S.Nonlinearre- ing sites among coexpressing genes in hu- gression modeling via regularized radial ba- man. Genome Informatics. 20: 212-221, 2008. sis function networks. Journal of Statistical 7. Hirose, O., Yoshida, R., Imoto, S., Yama- Planning and Inference. 138 (11): 3616-3633, guchi, R., Higuchi, T., Charnock-Jones, D.S., 2008. Print, C., Miyano, S. Statistical inference of 2. Brazma, A., Miyano, S., Akutsu, T. Proceed- transcriptional module-based gene networks ings of the 6th Asia-Pacific Bioinformatics from time course gene expression profiles by Conference (APBC 2008). Imperial College using state space models. Bioinformatics. 24 Press, 2008. (7): 932-942, 2008. 3. Do, J.H., Miyano, S. The GC and window- 8. Hirose, O., Yoshida, R., Yamaguchi, R., averaged DNA curvature profile of secon- Imoto, S., Higuchi, T., Miyano, S. Analyzing dary metabolite gene cluster in Aspergillus time course gene expression data with bio- fumigatus genome. Applied Microbiology logical and technical replicates to estimate and Biotechnology. 80 (5): 841-847, 2008. gene networks by state space models. Proc. 4. Fujita, A., Gomes, L.R., Sato, J.R., Yama- 2nd Asia International Conference on Mod- guchi, R., Thomaz, C.E., Sogayar, M.C., Miy- elling & Simulation, 940-946, 2008. (AMS ano, S. Multivariate gene expression analysis 2008: Refereed conference) reveals functional connectivity changes be- 9. Jeong, E., Nagasaki, M., Miyano, S. Rule- tween normal/tumoral prostates. BMC Sys- based reasoning for system dynamics in cell tems Biology. 2: 106, 2008. systems. Genome Informatics. 20: 25-36, 5. Fujita, A., Sato, J.R., Garay-Malpartida, H. 2008. M., Sogayar, M.C., Ferreira, C.E., Miyano, S. 10. Kitakaze, H., Kanda, M., Nakatsuka, H., Modeling nonlinear gene regulatory net- Ikeda, N., Matsuno, H., Miyano, S. Predic- works from time series gene expression tion of fragile points for robustness checking data. J. Bioinformatics and Computational of cell systems. IEICE TRANSACTIONS on Biology. 6 (5): 961-979, 2008. Information and Systems D. J91-D (9): 2404- 6. Hatanaka, Y., Nagasaki, M., Yamaguchi, R., 2417, 2008. Obayashi, T., Numata, K., Fujita, A., Shima- 11. Knapp, E.-W., Benson, G., Holzhutter, H.-G., mura, T., Tamada, Y., Imoto, S., Kinoshita, Kanehisa, M., Miyano, S. (Eds.) Genome In- K.,Nakai,K.,Miyano,S.Anovelstrategyto formatics. 20, 2008. search conserved transcription factor bind- 12. Kojima, K., Fujita, A., Shimamura, T., Imoto, 128

S., Miyano, S. Estimation of nonlinear gene Saito, S., Imoto, S., Miyano, S. ExonMiner: regulatory networks via L1 regularized Web service for analysis of GeneChip exon NVAR from time series gene expression array data. BMC Bioinformatics. 9: 494, 2008. data. Genome Informatics. 20: 37-51, 2008. 18. Numata, K., Imoto, S., Miyano, S. Partial 13.Kojima,K.,Nagasaki,M.,Miyano,S.Fast order-based Bayesian network learning algo- grid layout algorithm for biological net- rithm for estimating gene networks. Proc. works with sweep calculation. Bioinformat- IEEE 8th International Symposium on Bioin- ics. 24 (12): 1426-1432, 2008. formatics & Bioengineering, IEEE Computer 14. Mito, N., Ikegami, Y., Matsuno, H., Miyano, Society, 357-360, 2008. (BIBM 2008: Refereed S., Inouye, S. Simulation analysis for the ef- conference) fect of light-dark cycle on the entrainment in 19. Perrier, E., Imoto, S., Miyano, S. Finding op- circadian rhythm. Genome Informatics. 21: timal Bayesian network given a super- 212-223, 2008. structure. J. Machine Learning Research. 9: 15. Nagasaki, M., Saito, A., Chen, L., Jeong, E., 2251-2286, 2008. Miyano,S.Systematicreconstructionof 20. Yamaguchi, R., Imoto, S., Yamauchi, M., Na- TRANSPATH data into Cell System Markup gasaki, M., Yoshida, R., Shimamura, T., Language. BMC Systems Biology. 2: 53, Hatanaka, Y., Ueno, K., Higuchi, T., Gotoh, 2008. N., Miyano, S. Predicting differences in gene 16. Niida, A., Smith, A.D., Imoto, S., Tsutsumi, regulatory systems by state space models. S., Aburatani, H., Zhang, M.Q., Akiyama, T. Genome Informatics. 21: 101-113, 2008. Integrative bioinformatics analysis of tran- 21. Yoshida, R., Nagasaki, M., Yamaguchi, R., scriptional regulatory programs in breast Imoto, S., Miyano, S., Higuchi, T. Bayesian cancer cells. BMC Bioinformatics. 9: 404, learning of biological pathways on genomic 2008. data assimilation. Bioinformatics. 24(22): 17. Numata, K., Yoshida, R., Nagasaki, M., 2592-2601, 2008. 129

Human Genome Center Laboratory of Molecular Medicine Laboratory of Genome Technology ゲノムシークエンス解析分野シークエンス技術開発分野

Professor Yusuke Nakamura, M.D., Ph.D. 教授医学博士中村祐輔 Associate Professor Toyomasa Katagiri, Ph.D. 准教授医学博士片桐豊雅 Associate Professor Yataro Daigo, M.D., Ph.D. 准教授医学博士醍醐弥太郎 Assistant Professor Ryuji Hamamoto, Ph.D. 助教理学博士浜本隆二 Assistant Professor Koichi Matsuda, M.D., Ph.D. 助教医学博士松田浩一 Assistant Professor Hitoshi Zembutsu, M.D., Ph.D. 助教医学博士前佛均

The major goal of our group is to identify genes of medical importance, and to develop new diagnostic and therapeutic tools. We have been attempting to isolate genes involving in carcinogenesis and also those causing or predisposing to various diseases as well as those related to drug efficacies and adverse reactions. By means of technologies developed through the genome project including a high- resolution SNP map, a large-scale DNA sequencing, and the cDNA microarray method, we have isolated a number of biologically and/or medically important genes, and are developing novel diagnostic and therapeutic tools.

1. Genes playing significant roles in human Piao, Chizu Tanikawa, Motoko Unoki, Masa- cancer nori Yoshimatsu, Shinya Hayami, and Yusuke Nakamura Toyomasa Katagiri, Yataro Daigo, Hidewaki Nakagawa, Hitoshi Zembutsu, Koichi Matsuda, (1) Lung cancer Ryuji Hamamoto, Sachiko Dobashi, Tomomi Ueki, Chikako Fukukawa, Eiji Hirota, Meng- DLX5 (distal-less homeobox 5) Lay Lin, Jae-Hyun Park, Yosuke Harada, Sa- toshi Nagayama, Toshihiko Nishidate, Arata We found that distal-less homeobox 5 (DLX5) Shimo, Masahiko Ajiro, Jung-Won Kim, Tat- gene, a member of the human distal-less ho- suya Kato, Daizaburo Hirata, Koji Ueda, At- meobox transcriptional factor family was over- sushi Takano, Nobuhisa Ishikawa, Koji Taka- expressed in the great majority of lung cancers. hashi, Takumi Yamabuki, Nagato Sato, Northern blot and immunohistochemical analy- Nguyen Minh-Hue, Ryohei Nishino, Junkichi ses detected expression of DLX5 only in pla- Koinuma, Daiki Miki, Ken Masuda, Masato centa among 23 normal tissues examined. Im- Aragaki, Dragomira Nikolaeva Nikolova, Sa- munohistochemical analysis showed that posi- toko Uno, Yoichiro Kato, Kenji Tamura, Kotoe tive immunostaining of DLX5 was correlated Kashiwaya, Masayo Hosokawa, Shingo Ashida, with tumor size (pT classification; P ＝0.0053) Su-Youn Chung, Motohide Uemura, Lianhua and poorer prognosis of non-small cell lung can- 130 cer patients (P ＝0.0045). It was also shown to be of endogenous DTL/RAMP protein in breast an independent prognostic factor (P ＝0.0415). cancer cells; nuclear localization was observed in Treatment of lung cancer cells with small inter- cells at interphase and the protein was concen- fering RNAs for DLX5 effectively knocked down trated at the contractile ring in cytokinesis proc- its expression and suppressed cell growth. These ess. The expression level of DTL/RAMP protein data implied that DLX5 is useful as a target for became highest at G(1)/S phases, whereas its the development of anticancer drugs and cancer phosphorylation level was enhanced during mi- vaccinesaswellasforaprognosticbiomarkerin totic phase. Treatment of breast cancer cells, T47 clinic. D and HBC4, with small-interfering RNAs against DTL/RAMP effectively suppressed its ECT2 (epithelial cell transforming sequence expression and caused accumulation of G(2)/M 2) cells, resulting in growth inhibition of cancer cells. We further demonstrate the in vitro phos- We screened for genes that were frequently phorylation of DTL/RAMP through an interac- overexpressed in the tumors through gene ex- tion with the mitotic kinase, Aurora kinase-B pression profile analyses of 101 lung cancers (AURKB). Interestingly, depletion of AURKB ex- and 19 esophageal squamous cell carcinomas pression with siRNA in breast cancer cells re- (ESCC) by cDNA microarray consisting of duced the phosphorylation of DTL/RAMP and 27,648 genes or expressed sequence tags. In this decreased the stability of DTL/RAMP protein. process, we identified epithelial cell transform- These findings imply important roles of DTL/ ing sequence 2 (ECT2) as a candidate. Northern RAMP in growth of breast cancer cells and sug- blot and immunohistochemical analyses de- gest that DTL/RAMP might be a promising mo- tected expression of ECT2 only in testis among leculartargetfortreatmentofbreastcancer. 23 normal tissues. Immunohistochemical stain- ing showed that a high level of ECT2 expression (3) Renal cancer was associated with poor prognosis for patients with NSCLC (P ＝0.0004) as well as ESCC (P ＝ TMEM22 (transmembrane protein 22) 0.0088). Multivariate analysis indicated it to be an independent prognostic factor for NSCLC (P In order to clarify the molecular mechanism ＝0.0005). Knockdown of ECT2 expression by involved in renal carcinogenesis, and to identify small interfering RNAs effectively suppressed molecular targets for development of novel lung and esophageal cancer cell growth. In ad- treatments of renal cell carcinoma (RCC), we dition, induction of exogenous expression of previously analyzed genome-wide gene expres- ECT2 in mammalian cells promoted cellular in- sion profiles of clear-cell types of RCC by cDNA vasive activity. ECT2 cancer-testis antigen is microarray. Among the transcativated genes, we likely to be a prognostic biomarker in clinic and herein focused on functional significance of a potential therapeutic target for the develop- TMEM22 (transmembrane protein 22), a trans- ment of anticancer drugs and cancer vaccines membrane protein, in cell growth of RCC. for lung and esophageal cancers. Northern blot and semi-quantitative RT-PCR analyses confirmed up-regulation of TMEM22 in (2) Breast Cancer a great majority of RCC clinical samples and cell lines examined. Immunocytochemical analysis DTL/RAMP (denticleless/RA-regulated nuclear validated its localization at the plasma mem- matrix associated protein) brane. We found an interaction between TMEM 22 and RAB37 (Ras-related protein Rab-37), To investigate the detailed molecular mecha- which was also up-regulated in RCC cells. Inter- nism of mammary carcinogenesis and discover estingly, knockdown of either of TMEM22 or novel therapeutic targets, we previously ana- RAB37 expression by specific siRNA caused sig- lysed gene expression profiles of breast cancers. nificant reduction of cancer cell growth. Our re- We here report characterization of a significant sults imply that the TMEM22/RAB37 complex is role of DTL/RAMP (denticleless/RA-regulated likely to play a crucial role in growth of RCC nuclear matrix associated protein) in mammary and that inhibition of the TMEM22/RAB37 ex- carcinogenesis. Semiquantitative RT-PCR and pression or their interaction should be novel northern blot analyses confirmed upregulation therapeutic targets for RCC. of DTL/RAMP in the majority of breast cancer cases and all of breast cancer cell lines exam- (4) Synovial sarcoma ined. Immunocytochemical and western blot analyses using anti-DTL/RAMP polyclonal anti- FZD10 (Frizzled homologue 10) body revealed cell-cycle-dependent localization 131

We previously reported that Frizzled homo- pancreatic cancer probably through its protein- logue 10 (FZD10), a member of the Wnt signal ase inhibitory activity, and it is a promising mo- receptor family, was highly and specifically lecular target for development of new therapeu- upregulated in synovial sarcoma and played tic strategies for PDAC. critical roles in its cell survival and growth. We investigated a possible molecular mechanism of C2orf18 (ANTBP) the FZD10 signaling in synovial sarcoma cells. We found a significant enhancement of phos- Through our genome-wide gene expression phorylation of the Dishevelled (Dvl)2/Dvl3 profiles of microdissected PDAC cells, we here complex as well as activation of the Rac1-JNK identified a novel gene C2orf18 as a molecular cascade in synovial sarcoma cells in which FZD target for PDAC treatment. Transcriptional and 10 was overexpressed. Activation of the FZD10- immunohistochemical analysis validated its Dvls-Rac1 pathway induced lamellipodia forma- overexpression in PDAC cells and limited ex- tion and enhanced anchorage-independent cell pression in normal adult organs. Knockdown of growth. FZD10 overexpression also caused the C2orf18 by small-interfering RNA in PDAC cell destruction of the actin cytoskeleton structure, lines resulted in induction of apoptosis and sup- probably through the downregulation of the pression of cancer cell growth, suggesting its es- RhoA activity. Our results have strongly im- sential role in maintaining viability of PDAC plied that FZD10 transactivation causes the acti- cells. We showed that C2orf18 was localized in vation of the non-canonical Dvl-Rac1-JNK path- the mitochondria and it could interact with ade- way and plays critical roles in the develop- nine nucleotide translocase 2 (ANT2), which is ment/progression of synovial sarcomas. involved in maintenance of the mitochondrial membrane potential and energy homeostasis, (5) Pancreatic cancer and was indicated some roles in apoptosis. These findings implicated that C2orf18, termed CST6 (Cystatin 6) ANT2-binding protein (ANT2BP), might serve as a candidate molecular target for pancreatic Pancreatic ductal adenocarcinoma (PDAC) cancer therapy. shows the worst mortality among the common malignancies and development of novel thera- (6) Prostate cancer pies for PDAC through identification of good molecular targets is an urgent issue. Among STC2 (stanniocalcin 2) dozens of over-expressing genes identified through our gene-expression profile analysis of Prostate cancer is usually androgen-dependent PDAC cells, we here report CST6 (Cystatin 6 or and responds well to androgen ablation therapy E/M) as a candidate of molecular targets for based on castration. However, at a certain stage PDAC treatment . Reverse transcriptase- some prostate cancers eventually acquire a polymerase chain reaction (RT-PCR) and immu- castration-resistant phenotype where they pro- nohistochemical analysis confirmed over- gress aggressively and show very poor response expression of CST6 in PDAC cells, but no or to any anticancer therapies. To characterize the limited expression of CST6 was observed in nor- molecular features of these clinical castration- mal pancreas and other vital organs. Knock- resistant prostate cancers, we previously ana- down of endogenous CST6 expression by small lyzed gene expression profiles by genome-wide interfering RNA attenuated PDAC cell growth, cDNA microarrays combined with microdissec- suggesting its essential role in maintaining vi- tion and found dozens of trans-activated genes ability of PDAC cells. Concordantly, constitutive in clinical castration-resistant prostate cancers. expression of CST6 in CST6-null cells promoted Among them, we report the identification of a their growth in vitro and in vivo.Furthermore, new biomarker, stanniocalcin 2 (STC2), as an the addition of mature recombinant CST6 in cul- overexpressed gene in castration-resistant pros- ture medium also promoted cell proliferation in tate cancer cells. Real-time polymerase chain re- a dose-dependent manner, whereas recombinant action and immunohistochemical analysis con- CST6 lacking its proteinase-inhibitor domain firmed overexpression of STC2, a 302-amino- and its non-glycosylated form did not. Over- acid glycoprotein hormone, specifically in cas- expression of CST6 inhibited the intracellular ac- trationresistant prostate cancer cells and aggres- tivity of cathepsin B, which is one of the puta- sive castration-naïve prostate cancers with high tive substrates of CST6 proteinase inhibitor and Gleason scores (8-10). The gene was not ex- can intracellularly function as a pro-apoptotic pressed in normal prostate, nor in most indolent factor. These findings imply that CST6 is likely castration-naïve prostate cancers. Knockdown of to involve in the proliferation and survival of STC2 expression by short interfering RNA in a 132 prostate cancer cell line resulted in drastic at- transcripts, only 87 (31.9％) were previously re- tenuation of prostate cancer cell growth. Concor- ported as upregulated in microarray studies us- dantly, STC2 overexpression in a prostate cancer ing bulk cancer tissues and normal ovarian tis- cell line promoted prostate cancer cell growth, sues for analysis. CHMP4C (chromatinmodify- indicating its oncogenic property. These findings ing protein 4C) was frequently overexpressed in suggest that STC2 could be involved in aggres- ovarian carcinoma tissue, but not expressed in sive phenotyping of prostate cancers, including the normal human tissues used as a control. Our castration-resistant prostate cancers, and that it data should contribute to an improved under- should be a potential molecular target for devel- standing of tumorigenesis in ovarian cancer, and opment of new therapeutics and a diagnostic aid in the development of diagnostic tumor biomarker for aggressive prostate cancers. markers and molecular-targeting therapy for patients with the disease. (7) Thyroid cancer (9) Proteomics In order to clarify the molecular mechanism involved in thyroid carcinogenesis and to iden- To screen for glycoproteins showing aberrant tify candidate molecular targets for diagnosis sialylation patterns in sera of cancer patients and treatment, we analyzed genome-wide gene and apply such information for biomarker iden- expression profiles of 18 papillary thyroid carci- tification, we performed SELDI-TOF MS analysis nomas with a microarray representing 38,500 coupled with lectin-coupled ProteinChip arrays genes in combination with laser microbeam mi- (Jacalin or SNA) using sera obtained from lung crodissection. We identified 243 transcripts that cancer patients and control individuals. Our ap- were commonly up-regulated and 138 tran- proach consisted of three processes, (1) removal scripts that were down-regulated in thyroid car- of 14 abundant proteins in serum, (2) enrich- cinoma. Among these 243 transcripts identified, ment of glycoproteins with lectin-coupled Prote- only 71 transcripts were reported as up- inChip arrays, and (3) SELDI-TOF MS analysis regulated genes in previous microarray studies, with acidic glycoprotein-compatible matrix. We in which bulk cancer tissues and normal thyroid identified 41 protein peaks showing significant tissues were used for the analysis. We further differences (P ＜0.05) in the peak levels between selected genes that were overexpressed very the cancer and control groups using the Jacalin- commonly in thyroid carcinoma, though were and SNA- ProteinChips. Among them, we iden- not expressed in the normal human tissues ex- tified loss of Neu5Ac （α2, 6) Gal/GalNAc amined. Among them, we focused on the regu- structure in apolipoprotein C-III (apoC-III) in lator of G-protein signaling 4 (RGS4)and cancer patients through subsequent MALDI- knocked-down its expression in thyroid cancer QIT-TOF MS/MS. Furthermore, subsequent vali- cells by small-interfering RNA. The effective dation experiments using an additional set of 60 down-regulation of its expression levels in thy- lung adenocarcinoma patients and 30 normal roid cancer cells significantly attenuated viabil- controls demonstrated that there is a higher fre- ity of thyroid cancer cells, indicating the signifi- quency of serum apoC-III with loss of α2, 6- cant role of RGS4 in thyroid carcinogenesis. Our linkage Neu5Ac residues in lung cancer patients data should be helpful for a better understand- compared to controls. Our results have demon- ing of the tumorigenesis of thyroid cancer and strated that lectin-coupled ProteinChip technol- could contribute to the development of diagnos- ogy allows the high-throughput and specific rec- tic tumor markers and molecular-targeting ther- ognition of cancer-associated aberrant glycosyla- apy for patients with thyroid cancer. tions, and implied a possibility of its applicability to studies on other diseases. (8) Ovarian cancer (10) Chemosensitivity We aimed to clarify the molecular mechanisms involved in ovarian carcinogenesis, and to Breast Cancer identify candidate molecular targets for its diagnosis and treatment. The genome-wide gene ex- Neoadjuvant chemotherapy with docetaxel for pression profiles of 22 epithelial ovarian carcino- advanced breast cancer can improve the radical- mas were analyzed with a microarray represent- ity for a subset of patients, but some patients ing 38,500 genes, in combination with laser mi- suffer from severe adverse drug reactions with- crobeam microdissection. A total of 273 com- out any benefit. To establish a method for pre- monly up-regulated transcripts and 387 down- dicting responses to docetaxel, we analyzed regulated transcripts were identified in the ovar- gene expression profiles of biopsy materials ian carcinoma samples. Of the 273 up-regulated from 29 advanced breast cancers using a cDNA 133 microarray consisting of 36,864 genes or ESTs, 7.2％, P ＜0.001, among those requiring＞or＝49 after enrichment of cancer cell population by la- mg per week). The use of a pharmacogenetic al- ser microbeam microdissection. Analyzing eight gorithm for estimating the appropriate initial PR (partial response) patients and twelve pa- dose of warfarin produces recommendations tients with SD (stable disease) or PD (progres- that are significantly closer to the required sta- sive disease) response, we identified dozens of ble therapeutic dose than those derived from a genes that were expressed differently between clinical algorithm or a fixed-dose approach. The the ‘responder (PR)’ and ‘non-responder (SD or greatest benefits were observed in the 46.2％ of PD)’ groups. We further selected the nine ‘pre- the population that required 21 mg or less of dictive’ genes showing the most significant dif- warfarin per week or 49 mg or more per week ferences and established a numerical prediction for therapeutic anticoagulation. scoring system that clearly separated the responder group from the non-responder group. (2) Genotype of CYP2D6 and selection of ad- This system accurately predicted the drug re- juvant hormonal therapy with tamoxifen sponses of all of nine additional test cases that for breast cancer patients were reserved from the original 29 cases. More- over, we developed a quantitative PCR-based Authors: Kazuma Kiyotani1, Taisei Mushi- prediction system that could be feasible for rou- roda1, Mitsunori Sasa2, Yoshimi Bando3, Ikuko tine clinical use. Our results suggest that the Sumitomo2, Naoya Hosono4, Michiaki Kubo4, sensitivity of an advanced breast cancer to the Yusuke Nakamura1,5, and Hitoshi Zembutsu5: neoadjuvant chemotherapy with docetaxel could 1Laboratory for Pharmacogenetics, SNP Re- be predicted by expression patterns in this set of search Center, The Institute of Physical and genes. Chemical Research (RIKEN), 2Department of Surgery, Tokushima Breast Care Clinic, 3De- 2. Pharmacogenomics partment of Molecular and Environmental Pa- thology, Institute of Health Biosciences, The (1) Warfarin maintenance-dose requirements University of Tokushima Graduate School, 4Laboratory for genotyping, SNP Research The International Warfarin Pharmacogenetics Center, The Institute of Physical and Chemical Consortium Research (RIKEN), 5Laboratory of Molecular Medicine, Human Genome Center, Institute of Genetic variability among patients plays an Medical Science, The University of Tokyo important role in determining the dose of warfarin that should be used when oral anticoagula- The clinical outcomes of breast cancer patients tion is initiated, but practical methods of using treated with tamoxifen may be influenced by genetic information have not been evaluated in the activity of cytochrome P450 2D6 (CYP2D6) a diverse and large population. We developed enzyme because tamixifen is metabolized by and used an algorithm for estimating the appro- CYP2D6 to its active forms of antiestrogenic me- priate warfarin dose that is based on both clini- tabolite, 4-hydroxytamoxifen and endoxifen. We cal and genetic data from a broad population investigated the predictive value of the base. Clinical and genetic data from 4043 pa- CYP2D6 ＊10 allele, which decreased CYP2D6 ac- tients were used to create a dose algorithm that tivity, for clinical outcomes of patients that re- was based on clinical variables only and an al- ceived adjuvant tamoxifen monotherapy after gorithm in which genetic information was surgical operation on breast cancer. Among 67 added to the clinical variables. In a validation patients examined, those homozygous for the cohort of 1009 subjects, we evaluated the poten- CYP2D6 ＊10 alleles revealed a significantly tial clinical value of each algorithm by calculat- higher incidence of recurrence within 10 years ing the percentage of patients whose predicted after the operation (P ＝0.0057; odds ratio, 16.63; dose of warfarin was within 20％ of the actual 95％ confidence interval, 1.75-158.12), compared stable therapeutic dose; we also evaluated other with those homozygous for the wild-type clinically relevant indicators. In the validation CYP2D6 ＊1 alleles. The elevated risk of recur- cohort, the pharmacogenetic algorithm accu- rence seemed to be dependent on the number of rately identified larger proportions of patients CYP2D6 ＊10 alleles (P ＝0.0031 for trend). Cox who required 21 mg of warfarin or less per proportional hazard analysis demonstrated that week and of those who required 49 mg or more the CYP2D6 genotype and tumor size were in- per week to achieve the target international nor- dependent factors affecting recurrence-free sur- malized ratio than did the clinical algorithm vival. Patients with the CYP2D6 ＊10/＊10 geno- (49.4％ vs. 33.3％, P ＜0.001, among patients re- type showed a significantly shorter recurrence- quiring＜or＝21 mg per week; and 24.8％ vs. free survival period (P ＝0.036; adjusted hazard 134 ratio, 10.04; 95％ confidence interval, 1.17-86.27) compared to those with a score of 0 (P ＝ compared to patients with CYP2D6 ＊1/＊1after 0.0000057; odds ratio [OR], 7.00; 95％ CI [confi- adjustment of other prognosis factors. The pre- dence interval], 2.95-16.59). This prediction sys- sent study suggests that the CYP2D6 genotype tem correctly classified 69.2％ of severe leuko- should be considered when selecting adjuvant penia / neutropenia and 75.7％ of non- hormonal therapy for breast cancer patients. leukopenia/neutropenia into the respective cate- gories, indicating that SNPs in ABCC2 and (3) Genotype of drug metabolism/transporter SLCO1B3 may predict the risk of leukopenia/ genes and Docetaxel-induced leukopenia/ neutropenia induced by docetaxel chemother- neutropenia apy.

Authors: Kazuma Kiyotani1, Taisei Mushi- (4) HLA genotype and Nevirapine (NVP)- roda1, Michiaki Kubo2, Hitoshi Zembutsu3, induced skin rash Yuichi Sugiyama4, and Yusuke Nakamura1,3: 1Laboratory for Pharmacogenetics, SNP Re- Authors: Soranun Chantarangsu1,2 ,Taisei search Center, The Institute of Physical and Mushiroda1, Surakameth Mahasirimongkol5, Chemical Research (RIKEN), 2Laboratory for Sasisopin Kiertiburanakul3, Somnuek Sungkan- genotyping, SNP Research Center, The Insti- uparph3, Weerawat Manosuthi6, Woraphot tute of Physical and Chemical Research Tantisiriwat7, Angkana Charoenyingwattana4, (RIKEN), 3Laboratory of Molecular Medicine, Thanyachai Sura3, Wasun Chantratita2,and Human Genome Center, Institute of Medical Yusuke Nakamura1 : 1Research Group for Science, The University of Tokyo, 4Department Pharmacogenomics , RIKEN , Center for of Molecular Pharmacokinetics, Graduate Genomic Medicine Departments of 2Pathology, School of Pharmaceutical Sciences, The Uni- 3Medicine, Faculty of Medicine, 4Department of versity of Tokyo Pharmacy, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand, 5Center for In- Despite long-term clinical experience with do- ternational Cooperation, Department of Medi- cetaxel, unpredictable severe adverse reactions cal Sciences, 6Bamrasnaradura Infectious Dis- remain an important determinant for limiting eases Institute, Ministry of Public Health, 7De- the use of the drug. To identify a genetic factor partment of Preventive Medicine, Faculty of (s) determining the risk of docetaxel-induced Medicine, Srinakharinwirot University, Nak- leukopenia/neutropenia, we selected subjects ornnayok, Thailand who received docetaxel chemotherapy from samples recruited at BioBank Japan, and con- We investigated a possible involvement of dif- ducted a case-control association study. We ferences in human leukocyte antigens (HLA) in genotyped 84 patients, 28 patients with grade 3 the risk of nevirapine (NVP)-induced skin rash or 4 leukopenia/neutropenia, and 56 with no among HIV-infected patients by a step-wise toxicity (patients with grade 1 or 2 were ex- case-control association study. We first geno- cluded), for a total of 79 single nucleotide poly- typed by a sequence-based HLA typing method morphisms (SNPs) in seven genes possibly infor the HLA-A, HLA-B, HLA-C, HLA-DRB1, volved in the metabolism or transport of this HLA-DQB1,andHLA-DPB1 in the first set of drug: CYP3A4, CYP3A5, ABCB1, ABCC2, SLCO1 samples consisted of 80 samples from patients B3, NR1I2,andNR1I3. Since one SNP in ABCB with NVP-induced skin rash and 80 samples 1,fourSNPsinABCC2,fourSNPsinSLCO1B3, from NVP-tolerant patients. Subsequently, we and one SNP in NR1I2 showed a possible asso- verified HLA alleles that showed a possible as- ciation with the grade 3 leukopenia/neutropenia sociation in the first screening using an addi- (P -value of＜0.05), we further examined these tional set of samples consisting of 67 cases with 10 SNPs using 29 additionally obtained patients, NVP-induced skin rash and 105 controls. An 11 patients with grade 3/4 leukopenia/neutro- HLA-B ＊3505 allele revealed a significant associa- penia, and 18 with no toxicity. The combined tion with NVP-induced skin rash in the first and analysis indicated a significant association of rs second screenings. In the combined data set, the 12762549 in ABCC2 (P ＝0.00022) and rs11045585 HLA-B ＊3505 allele was observed in 17.5％ of the in SLCO1B3 (P ＝0.00017) with docetaxel- patients with NVP-induced skin rash compared induced leukopenia/neutropenia. When patients with only 1.1％ observed in NVP-tolerant pa- were classified into three groups by the scoring tients [odds ratio (OR)＝18.96; 95％ confidence system based on the genotypes of these two interval (CI)＝4.87-73.44, Pc＝4.6×10] and 0.7％ SNPs, patients with a score of 1 or 2 were in general Thai population (OR＝29.87; 95％ CI shown to have a significantly higher risk of ＝5.04-175.86, Pc＝2.6×10). The logistic regres- docetaxel-induced leukopenia/neutropenia as sion analysis also indicated HLA-B ＊3505 to be 135 significantly associated with skin rash with OR 0501 and HLA-DPA1 ＊0202-DPB1 ＊0301, OR＝ of 49.15 (95％ CI＝6.45-374.41, P ＝0.00017). We 1.45 and 2.31, respectively) and protective suggest that strong association between the haplotypes (HLA-DPA1 ＊0103-DPB1 ＊0402 and HLA-B ＊3505 and NVP-induced skin rash pro- HLA-DPA1 ＊0103-DPB1 ＊0401, OR＝0.52 and vides a novel insight into the pathogenesis of 0.57, respectively). Our findings demonstrated drug-induced rash in the HIV-infected popula- that genetic variations in the HLA-DP locus are tion. On account of its high specificity (98.9％) strongly associated with the risk of persistent in- in identifying NVP-induced rash, it is possible fection of hepatitis B virus. to utilize the HLA-B ＊3505 as a marker to avoid a subset of NVP-induced rash, at least in Thai (2) Idiopathic pulmonary fibrosis (IPF) population. Authors: Taisei Mushiroda1, Sukanya Wattana- 3. Common diseases pokayakit2, Atsushi Takahashi3 , Toshihiro Nukiwa4, Shoji Kudoh5, Takashi Ogura6,Hi- (1) Chronic hepatitis B royuki Taniguchi7, Michiaki Kubo8, Naoyuki Kamatani3, Yusuke Nakamura1,9,andthePir- Authors: Yoichiro Kamatani1,2, Sukanya Wat- fenidone Clinical Study Group4: 1Laboratory tanapokayakit3, Hidenori Ochi4,5, Takahisa for Pharmacogenetics, Institute of Physical and Kawaguchi4 , Atsushi Takahashi4 , Naoya Chemical Research (RIKEN), 2Laboratory for Hosono4, Michiaki Kubo4, Tatsuhiko Tsunoda4, Cardiovascular Diseases, Institute of Physical Naoyuki Kamatani4 , Hiromitsu Kumada6 , and Chemical Research (RIKEN), 3Laboratory Aekkachai Puseenam7 , Thanyachai Sura7 , of Statistical Analysis, Institute of Physical and Yataro Daigo2, Kazuaki Chayama4,5,Wasun Chemical Research (RIKEN), 4Department of Chantratita8, Yusuke Nakamura1,4, and Koichi Respiratory Oncology and Molecular Medicine, Matsuda1: 1Laboratory of Molecular Medicine, Institute of Development, Aging, and Cancer, Human Genome Center, Institute of Medical Tohoku University, 5Fourth Department of In- Science, The University of Tokyo, 2Department ternal Medicine, Nippon Medical School, 6De- of Medical Genome Sciences, Graduate School partment of Respiratory Medicine, Kanagawa of Frontier Sciences, The Universtiy of Tokyo, Cardiovascular and Respiratory Center, 7De- 3Center for International Cooperation, Depart- partment of Respiratory Medicine and Allergy, ment of Medical Sciences, Ministry of Public Tosei General Hospital, Aichi, 8Laboratory for Health, Thailand, 4Center for Genomic Medi- genotyping, Institute of Physical and Chemical cine, RIKEN, 5Department of Medicine and Research (RIKEN), 9Laboratory of Molecular Molecular Science, Division of Frontier Medi- Medicine, Institute of Medical Science, Univer- cal Science, Programs for Biomedical Research, sity of Tokyo Graduate School of Biomedical Sciences, Hiro- shima University, 6Department of Hepatology, In order to identify a gene (s) susceptible to Toranomon Hospital, 7Department of Medicine, idiopathic pulmonary fibrosis (IPF), we con- Faculty of Medicine and 8Virology and Molecu- ducted a genome-wide association (GWA) study lar Microbiology Unit, Department of Pathol- by genotyping 159 patients with IPF and 934 ogy, Faculty of Medicine, Ramathidi Hospital, controls for 214,508 tag single-nucleotide poly- Mahidol University, Thailand morphisms (SNPs). We further evaluated selected SNPs in a replication sample set (83 cases Chronic hepatitis B is a serious infectious liver and 535 controls) and found a significant asso- disease that often progresses to liver cirrhosis ciation of an SNP in intron 2 of the TERT gene and hepatocellular carcinoma; however, clinical (rs2736100), which encodes a reverse transcrip- outcomes after viral exposure enormously vary tase that is a component of a telomerase, with among individuals . Through a two-step IPF; a combination of two data sets revealed a p genome-wide association study using 786 Japa- value of 2.9×10 (-8) (GWA, 2.8×10 (-6); replica- nese chronic hepatitis B patients and 2,201 con- tion, 3.6×10 (-3)). Considering previous reports trols, here we identified a significant association indicating that rare mutations of TERT are of chronic hepatitis B with 11 SNPs in a region found in patients with familial IPF, we suggest including HLA-DPA1 and HLA-DPB1 genes. that the common genetic variation within TERT These associations were validated in two Japa- may contribute to the risk of sporadic IFP in the nese and one Thai cohorts consisting of 1,300 Japanese population. cases and 2,100 controls (combined P ＝6.34× 10－39 and 2.31×10－38,OR＝0.57 and 0.56, respec- (3) Schizophrenia tively). Subsequent analyses revealed disease susceptible haplotypes (HLA-DPA1 ＊0202-DPB1 ＊ Authors: Elitza T Betcheva1, Taisei Mushi- 136

roda2, Atsushi Takahashi3, Michiaki Kubo4, Medical Science, The University of Tokyo Sena K Karachanak5, Irina T Zaharieva6,Ra- doslava V Vazharova5, Ivanka I Dimova5,Vi- The development of molecular psychiatry in hra K Milanova6, Todor Tolev7, George Kirov8, the last few decades identified a number of can- Michael J Owen8, Michael C O’Donovan8, didate genes that could be associated with Naoyuki Kamatani3, Yusuke Nakamura9,and schizophrenia. A great number of studies often Draga I Toncheva5: 1Laboratory for Cardiovas- result with controversial and non-conclusive cular Diseases, SNP Research Center, The In- outputs. However, it was determined that each stitute of Physical and Chemical Research of the implicated candidates would independ- (RIKEN), 2Laboratory for Pharmacogenetics, ently have a minor effect on the susceptibility to SNP Research Center, The Institute of Physical that disease. Herein we report results from our and Chemical Research (RIKEN), 3Laboratory replication study for association using 255 Bul- of Statistical Analysis, SNP Research Center, garian patients with schizophrenia and schizoaf- The Institute of Physical and Chemical Re- fective disorder and 556 Bulgarian healthy con- search (RIKEN), 4Laboratory for Genotyping, trols. We have selected from the literatures 202 SNP Research Center, The Institute of Physical single nucleotide polymorphisms (SNPs) in 59 and Chemical Research (RIKEN), 5Department candidate genes, which previously were impli- of Medical Genetics, Medical Faculty, Medical cated in disease susceptibility, and we have University, Sofia, Bulgaria, 6Department of genotyped them. Of the 183 SNPs successfully Psychiatry, Aleksandrovska Hospital, Medical genotyped, only 1 SNP, rs6277 (C957T) in the University, Sofia, Bulgaria, 7Department of DRD2 gene (P ＝0.0010, odds ratio＝1.76), was Psychiatry, Dr Georgi Kisiov Hospital, Rad- considered to be significantly associated with nevo, Bulgaria, 8Department of Psychological schizophrenia after the replication study using Medicine, Cardiff University, School of Medi- independent sample sets. Our findings support cine, Henry Wellcome Building, Heath Park, one of the most widely considered hypotheses Cardiff, UK, 9Laboratory of Molecular Medi- for schizophrenia etiology, the dopaminergic hy- cine, Human Genome Center, Institute of pothesis.

Publications

1. Hosono, N., Kubo, M., Tsuchiya, Y., Sato, Y., Nakamura, Y. and Furukawa, Y. En- H., Kitamoto, T., Saito, S., Ohnishi, Y. and hanced expression of RAD51AP1 is involved Nakamura, Y. Multiplex PCR-based real- in the growth of intrahepatic cholangiocarci- time Invader assay (mPCR-RETINA): a noma cells. Clin Cancer Res. 14: 1333-1339, novel SNP-based method for detecting alle- 2008 lic asymmetries within copy number vari- 5. M. Kato, F. Miya, Y. Kanemura, T. Tanaka, ation regions. Hum. Mutation 29: 182-189, Y. Nakamura, and T. Tsunoda: Recombina- 2008. tion rates of genes expressed in human tis- 2. Onouchi,Y.,Gunji,T.,Burns,J.C.,Shimizu, sues. Hum. Mol. Genet. 17: 577-586, 2008. C., Newburger, J.W., Yashiro, M., Naka- 6. Leung, A.A.C., Wong, V.C.L., Yang, L.C., mura, Yo., Yanagawa, H., Wakui, K., Chan, P.L., Daigo, Y., Nakamura, Y., Qi, R. Fukushima, Y., Kishi, F., Hamamoto, K., Z., Miller, L., Liu, E. T.-K., Wang, L.D. J.-L., Terai,M.,Sato,Y.,Ouchi,K.,Saji,T.,Nariai, S.Law,Tsao,W.andLung,M.L.Frequent A., Kaburagi, Y., Yoshikawa, T., Suzuki, K., decreased expression of candidate tumor Tanaka, T., Nagai, T., Cho, H., Fujino, A., suppressor gene, DEC1, and its anchorage- Sekine, A., Nakamichi, R., Tsunoda, T., independent growth properties and impact Kawasaki, T., Nakamura, Yu. and Hata, A. on global gene expression in esophageal car- A functional polymorphism in ITPKC is as- cinoma. Int. J. Cancer. 122: 587-594, 2008. sociated with Kawasaki disease susceptibil- 7. Shimo, A., Tanikawa, C., Nishidate, T., Mat- ity and formation of coronary artery aneu- suda, K., Lin, M.-L., Park, J.-H., Ohta, T., rysms. Nat. Genet. 40: 35-42, 2008. Hirata, K., Fukuda, M., Nakamura, Y. and 3. Silva, F.P., Hamamoto, R., Kunizaki, M., Katagiri,T.InvolvementofKIF2C/MCAK Tsuge, M., Nakamura, Y. and Furukawa, Y. overexpression in mammary carcinogenesis. Enhanced methyltransferase activity of Cancer Sci. 99: 62-70, 2008. SMYD3 by the cleavage of its N-terminal re- 8. Uemura, M., Tamura, K., Chung, S., Honma, gion in human cancer cells. Oncogene. 27: S., Okuyama, A., Nakamura, Y. and Naka- 2686-2692, 2008. gawa, H.A novel 5-steroid reductase (SRD5 4. Obama, K., Satoh, S., Hamamoto, R., Sakai, A3, type-3) is overexpressed in hormone- 137

refractory prostate cancer. Cancer Sci. 99: 81- Nakamura,Y.andZembutsu,H.Impactof 86, 2008. CYP2D6 ＊10 on recurrence-free survival in 9. Kamatani, Y., Matsuda, K., Ohishi, T., Oht- breast cancer patients receiving adjuvant ta- subo, S., Yamazaki, K., Iida, A., Hosono, N., moxifen therapy. Cancer Sci. 99: 995-999, Kubo, M., Yumura, W., Nitta, K., Katagiri, 2008. T., Kawaguchi, Y., Kamatani, N. and Naka- 17. Kato, T., Sato, N., Takano, A., Miyamoto, mura, Y. Identification of a significant asso- M.,Nishimura,H.,Tsuchiya,E.,Kondo,S., ciationofanSNPinTNXBwithSLEin Nakamura, Y. and Daigo, Y. Activation of Japanese population. J. Hum. Genet. 53: 64- Placenta-Specific Transcription Factor Distal- 73, 2008. less Homeobox 5 Predicts Clinical Outcome 10. Fukukawa, C., Hanaoka, H., Nagayama, S., in Primary Lung Cancer Patients. Clin. Can- Tsunoda, T., Toguchida, J., Endo, K., Naka- cer Res. 14: 2363-2370, 2008. mura,Y.andKatagiri,T.Radioimmunother- 18. Tenesa, A., Farrington, S.M., Prendergast, J. apy of human synovial sarcoma using a G.,Porteous,M.E.,Walker,M.,Haq,N.,Bar- monoclonal antibody against FZD10. Cancer netson, R.A., Theodoratou, E., Cetnarskyj, Sci. 99: 432-440, 2008. R., Cartwright, N., Semple, C., Clark, A.J., 11. Brunet, J., Pfaff, A.W., Abidi, A., Unoki, M., Reid, F.J., Smith, L.A., Kavoussanakis, K., Nakamura, Y., Guinard, M., Klein, J.-P., Koessler, T., Pharoah, P.D., Buch, S., Schaf- Candolfi, E. and Mousli, M. Toxoplasma mayer, C., Tepel, J., Schreiber, S., Völzke, H., gondii exploits UHRF1 and induces host cell Schmidt, C.O., Hampe, J., Chang-Claude, J., cycle arrest at G2 to enable its proliferation. Hoffmeister, M., Brenner, H., Wilkening, S., Cell Microbiol. 10: 908-920, 2008. Canzian, F., Capella, G., Moreno, V., Deary, 12. Kato, N., Miyata, T., Tabara, Y., Katsuya, T., I.J., Starr, J.M., Tomlinson, I.P., Kemp, Z., Yanai, K., Hanada, H., Kamide, K., Nakura, Howarth, K., Carvajal-Carmona, L., Webb, J., Kohara, K., Takeuchi, F., Mano, H., Yasu- E., Broderick, P., Vijayakrishnan, J., Houl- nami, M., Kimura, A., Kita, Y., Ueshima, H., ston, R.S., Rennert, G., Ballinger, D., Rozek, Nakayama, T., Soma, M., Hata, A., Fujioka, L., Gruber, S.B., Matsuda, K., Kidokoro, T., A., Kawano, Y., Nakao, K., Sekine, A., Nakamura, Y., Zanke, B.W., Greenwood, C. Yoshida, T., Nakamura, Y., Saruta, T., Ogi- M., Rangrej, J., Kustra, R., Montpetit, A., hara, T., Sugano, S., Miki, T. and Tomoike, Hudson, T.J., Gallinger, S., Campbell, H. and H. High-Density Association Study and Dunlop, M.G. Genome-wide association scan Nomination of Susceptibility Genes for Hy- identifies a colorectal cancer susceptibility pertension in the Japanese National Project. locus on 11q23 and replicates risk loci at 8q Hum. Mol. Genet. 17: 617-627, 2008. 24 and 18q21. Nat. Genet. 40: 631-637, 2008. 13. Oishi, T., Iida, A., Otsubo, S., Kamatani, Y., 19. Mototani H., Iida, A., Nakajima, M., Fu- Usami, M., Takei, T., Uchida, K., Tsuchiya, ruichi, T., Miyamoto, Y., Tsunoda, T., Sudo, K., Saito, S., Ohnishi, Y., Tokunaga, K., A., Kotani, A., Uchida, K., Ozaki, K., Nitta, K., Kawaguchi, Y., Kamatani, N., Ko- Tanaka, Y., Nakamura, Y., Tanaka, T., No- chi, Y., Shimane, K., Yamamoto, K., Naka- toya, K. and Ikegawa, S.A functional SNP in mura,Y.,Yumura,W.andMatsuda,K.A EDG2 increases susceptibility to knee os- functional SNP in the NKX2.5-binding site teoarthritis in Japanese. Hum. Mol. Genet. of ITPR3 promoter is associated with sus- 17: 1790-1797, 2008. ceptibility to Systemic Lupus Erythematosus 20. Mizukami, Y., Kono, K., Daigo, Y., Takano, in Japanese population. J. Hum. Genet. 53: A., Tsunoda, T., Kawaguchi, Y., Nakamura, 151-162, 2008. Y. and Fujii, H. Detection of novel Cancer- 14. Daigo, Y. and Nakamura Y. From cancer Testis antigen-specific T-cell responses in genomics to thoracic oncology: discovery of TIL, regional lymph nodes and PBL in pa- new biomarkers and therapeutic targets for tients with esophageal squamous cell carci- lung and esophageal carcinoma. (Review noma. Cancer Sci. 99: 1448-1454, 2008. Article) General Thoracic and Cardiovascu- 21. Mushiroda, T., Wattanapokayakit, S., Taka- lar Surgery 56: 43-53, 2008. hashi, A., Nukiwa, T., Kudoh, S., Ogura, T., 15. Kiyotani, K., Mushiroda, T., Kubo, M., Zem- Taniguchi, H., Pirfenidone Clinical Study butsu, H., Sugiyama, Y. and Nakamura, Y. Group., Kubo, M., Kamatani, N. and Naka- Association of genetic polymorphisms in mura Y.A genome-wide association study SLCO1B3 and ABCC2 with docetaxel- identifies an association of a common vari- induced leukopenia. Cancer Sci. 99: 967-972, ant in TERT with susceptibility to idiopathic 2008. pulmonary fibrosis. J. Med. Genet. 45: 654- 16. Kiyotani, K., Mushiroda, T., Sasa, M., Bando, 656, 2008. Y., Sumitomo, I., Hosono, N., Kubo, M., 22. Hosokawa, M., Kashiwaya, K., Furihara, M., 138

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terferon therapy for chronic hepatitis C. Gas- Pettinotti, G. HJURP, a key CENP-A-partner troenterology., in press, 2009. for maintenance and deposition of CENP-A 66. Dunleavy, E.M., Roche, D., Tagami, H., La- at centromeres at late telophase/G1. Cell., in coste, N., Ray-Gallet, D., Nakamura, Y., press, 2009. Daigo, Y., Nakatani, Y. and Almouzni- 142

Human Genome Center Laboratory of Functional Genomics ゲノム機能解析分野

Visiting Professor Gregory Mark Lathrop, Ph.D. 客員教授理学博士グレゴリー・マーク・ラスロップ Associate Professor Ryo Yamada, M.D., Ph.D. 准教授医学博士山田亮

Genetic heterogeneity of human beings is one of the most important targets of post-genomic research. Genome-wide association studies are being actively carried out using the genetic polymorphism markers to identify disease-related loci. We focus on the development of new methods to interpret the heterogeneity and to map the disease-associated loci and collaborate with research groups for data- mining of their genetic epidemiology studies.

1. The development of new methods to map the beginning and also attempt to improve the disease-associated loci with genetic poly- interpretation of data of GWA studies as a morphisms. whole.

Ryo Yamada a. Test statistics correction for data of structured population Genome-wide association (GWA) studies are resulting in many useful findings. The scale of Because the genetic epidemiology studies on such studies is increasing along with rapid pro- complex genetic traits target relatively weak fac- gress in genotyping technology. This increase in tors, which means sample size of them should scale necessarily increases the degree of depend- be more than thousands and subsequently ence among individual tests in GWA studies. makes idealistic random sampling from homo- The inter-test dependence is problematic be- geneous population impossible. The test statis- cause almost all the conventional statistical tics of the studies in the heterogeneous popula- methods assume independence among multiple tion, in other words, structured population, tests. Besides the multiple sources of inter-test tends to give false positive results. One of the dependency, the variable inflation of test statis- methodstocorrecttheincreaseinthefalseposi- tics due to biased sampling from structured tives is genomic control method for chi-square population is one of the unavoidable conse- distribution. We modify the genomic control quences of enlarged sample size. These prob- method so that it could correct the Fisher’s exact lems that complicate the interpretation of data test statistics. of GWA studies are mutually related and there is no straight-forward solution of them all to- b. Characterization of exact 2×3 test for SNP gether. We decompose the difficulty into parts, case-control association test data i.e., the problem of linkage disequilibrium (LD), population structure, multiple genetic models, The 2×3 contingency table test of SNP data is study design and characterize their problem and the basic unit of genome-wide association stud- propose solution of the individual problems at ies. We investigate the factors to affect the dis- 143 crepancy between the asymptotic test and the to quantify the heterogeneity and complexity of exact test for 2×3 contingency tables. population of DNA sequence with SNPs so that various researches based on genetic heterogene- c. Geometric evaluation of SNP contingency ity. table tests. b. Geometric expression of haplotype popu- The 2×3 SNP contingency table tests are de- lations scribed in the context of geometry and characterize various tests for 2×3 tables and define Haplotypes are consisted of alleles of multiple tests fit for biological models by interpreting ta- markers. We attempt to deal the haplotype data bles in the context of geometry. from combination theory standpoint and investigated the utility of polyhedral handling of the 2. The development of new methods to inter- combinatorial aspects of haplotypes. pret the genetic heterogeneity. 3. Collaboration with genetic epidemiology Ryo Yamada research groups.

As a compound in nature, the DNA sequence Gregory Mark Lathrop and Ryo Yamada is under pressure to maximize the heterogeneity of the sequence. Under the most random condi- Besides the development of new methods to tion, all bases of the sequence would be poly- analyze genetic polymorphism data in the con- morphic, and all bases and all sets of bases are text of population genetics and genetic statistics, mutually independent. At the other extreme, un- we collaborate with multiple research groups in der the least random condition, all DNA mole- and out of the IMS-UT, including Kyoto Univer- cules would be clones. In living organisms, the sity, Kyoto, The University of Tokyo Hospital, number of polymorphic sites in the DNA se- Tokyo, Laboratory for Autoimmune Diseases, quence is limited due to the requirements for re- CGM, RIKEN, Yokohama, National Hospital Or- production and as a result of selection and ge- ganization Sagamihara National Hospital, Sa- netic drift, against which opposite forces act to gamihara, and The Centre National de Géno- increase heterogeneity (e.g., mutation and re- typage, Evry, France, for the interpretation of combination). A major research target following genetic epidemiology data with the conventional the completion of the genome sequence is the statistical methods. investigation of intra-species variations, among which diallelic single nucleotide polymorphisms 4. Public distribution of population genetics are the most common. and genetic association study tools. a. Quantitation of linkage disequilibrium of Ryo Yamada multiple markers Because the designs of genetic epidemiology Genetic variations within a population give studies have been changing, the analysis tools rise to LD, and the use of the genetic history of have to be updated all the time. The number of the population and LD mapping is a very prom- genetic epidemiology study groups is much ising method for identifying genetic back- more than the groups on genetic statistics in the grounds of various phenotypes. LD is a measure world and also in Japan. We opened the web of inter-marker dependence. Although the inter- site that distributes basic tool of linkage dise- marker dependence exist among any set of quilibrium mapping for public use. This distri- markers, only the pair-wise inter-marker de- bution is supported by the grant from Japan So- pendence is utilized for quantitation of the ge- ciety for the Promotion of Science on the permu- netic heterogeneity and for genetic epidemiol- tation test. ogy studies usually. We develop a new method Web-site URL: http://func-gen.hgc.jp/

Publications

Gotoh N, Yamada R, Matsuda F, Yoshimura N, 146, 2008 and Iida T. Manganese Superoxide Dismutase Nakayama-Hamada M, Suzuki A, Furukawa H, Gene (SOD2) Polymorphism and Exudative Yamada R, and Yamamoto K. Citrullinated fi- Age-related Macular Degeneration in the brinogen inhibits thrombin-catalyzed fibrin Japanese Population. Am J Ophthalmol 146: polymerization. J Biochem 144: 393-8, 2008 144

Okada Y, Mori M, Yamada R, Suzuki A, Kobay- Okada Y, Matsuda K, Kamatani Y, Mori M, ashi K, Kubo M, Nakamura Y, and Yamamoto Shimane K, Hirabayashi Y Takahashi A, K. SLC22A4 Polymorphism and Rheumatoid Tsunoda T, Miyatake A, Kubo M, Kamatani Arthritis Susceptibility: A Replication Study in N, Nakamura Y, and Yamamoto K. Functional a Japanese Population and a Metaanalysis. J SNPs in CD244 increase the risk of rheuma- Rheumatol 35: 1273-8, 2008 toid arthritis in a Japanese population. Nat Shimane K, Kochi Y, Yamada R, Okada Y, Genet 40: 1224-9, 2008 Suzuki A, Miyatake A, Kubo M, Nakamura Y, Yamada R. Primer: SNP-associated studies and and Yamamoto K. A single nucleotide poly- what they can teach us. Nat Clin Pract Rheu- morphism in the IRF5 promoter region is as- matol 4: 210-7, 2008 sociated with susceptibility to rheumatoid ar- Yamada R, and Okada Y. An optimal dose-effect thritis in the Japanese patients. Ann Rheum mode trend test for SNP genotype tables. Dis. (in press) Genet Epidemiol 33: 114-27, 2009 Suzuki A, Yamada R, Kochi Y, Sawada T, 145

Human Genome Center Laboratory of Functional Analysis In Silico 機能解析イン・シリコ分野

Professor Kenta Nakai, Ph.D. 教授理学博士中井謙太 Associate Professor Kengo Kinoshita, Ph.D. 准教授理学博士木下賢吾

The mission of our laboratory is to conduct computational ( “in silico”) studies on the functional aspects of genome information. Roughly speaking, genome information represents what kind of proteins/RNAs are synthesized on what conditions. Thus, our study includes the structural analysis of molecular function of each gene product as well as the analysis of its regulatory information, which will lead us to the understanding of its cellular role represented by the networks of inter-gene interaction.

1. Tissue and developmental stage specific- mental stages. ity of trans-splicing in C. intestinalis 2. Improvement of the database of tunicate Nicolas Sierro, Shuang Li, Yutaka Suzuki1,Riu gene regulation Yamashita, and Kenta Nakai: 1Graduate School of Frontier Sciences, U. Tokyo Nicolas Sierro, Takehiro Kusakabe2,Yutaka Suzuki1, Riu Yamashita and Kenta Nakai: 2 Ciona intestinalis is a useful model organism to University of Hyogo analyze chordate development and genetics. However, unlike vertebrates, it shares a unique The database of tunicate gene regulation, mechanism called trans-splicing with lower eu- DBTGR, was first released in 2006 as a small da- karyotes. Our computational analysis of trans- tabase summarizing published information splicing in C. intestinalis showed that although about tunicate promoters and cis-regulatory re- the amount of non-trans-spliced and trans- gions. In 2008 it was extended to include gene spliced genes is usually equivalent, the expres- expression reporter constructs as well as a new sion ratio between the two groups varies signifi- genome browser providing all whole genome cantly with tissues and developmental stages. alignments between Ciona intestinalis and Ciona Among the seven tissues studied, the observed savignyi. The description of 81 gene expression ratios ranged from 2.53 in “gonad” to 19.53 in reporter vectors, as well as sample images of the “endostyle”, and during development they in- expression observed with them in Ciona is now creased from 1.68 at the “egg” stage to 7.55 at available, and the database provides users with the “juvenile” stage. We hypothesize that this contact information to the owners of these con- enrichment in trans-spliced mRNAs in early de- structs. With the new flexible genome browser velopmental stages might be related to the built in DBTGR, users have now access to two abundance of trans-spliced mRNAs in “gonad”. different genome alignments between C. intesti- To further investigate this phenomenon, we are nalis and C. savignyi, obtained with different al- currently analyzing a larger set of short 5’-EST gorithms. In addition, predicted binding sites for tags obtained from specific tissues and develop- the JASPAR core matrices, as well as regulatory 146 elements and binding sites reported in literature CpG-rich, but over a narrower range around the are also directly available. DBTGR is accessible TSSs. Furthermore, almost all promoters fall into at http://dbtgr.hgc.jp. the same category, whereas vertebrate promoters are divided into two classes in terms of 3. Promoter architecture analysis and predic- CpG. Comparison of the experimental result tion of expression with the genome of another ascidian species also supported our finding, leading to the first Alexis Vandenbon and Kenta Nakai definition of promoter-associated CpG islands in invertebrate organisms. Regulation of transcription is implemented through transcription factors (TFs) binding regu- 5. Computational verifications of gene regulatory regions in the neighborhood of genes. We latory networks in ascidian early develop- can make the assumption that genes showing ment similar expression profiles contain some shared structural patterns in their regulatory regions. Xuyang Yuan, Atsushi Kubo3, Yutaka Satou3, Until recently, these patterns were considered and Kenta Nakai: 3Kyoto University only on the level of presence or absence of specific transcription factor binding sites (TFBSs), The ascidian Ciona intestinalis has been useful but there is growing evidence that additional as a model system to explore chordate develop- structural patterns exist. Here we are focusing ment. Systematic gene knockdown experiments our attention not only on the presence of TFBSs, highly contributed to the depiction of the gene but also on their orientation and positioning regulatory network governing ascidian early de- with regard to the transcription start site and velopment. However, limitations of the experi- also between pairs of TFBSs. We developed an ment itself prevent the blueprint from giving approach for extracting such structural motifs further information regarding direct or indirect from promoter sequences and subsequently regulation. In this study, we are computation- combining them to make a promoter structure ally detecting direct target genes of each tran- model. We applied our model on a dataset of scription factor by scanning all promoter se- promoter sequences of muscle-specific genes of quences for its binding site. For representing the Caenorhabditis elegans and verified that our sequence specificity of transcription factors, we model is capable of distinguishing muscle- utilized positional weight matrices, of which expressed genes from genes not expressed in threshold values we need to set. We maximized muscle tissues based on the structure of their an over-representation index (ORI) value to find regulatory regions. We are further developing the optimum threshold. For trans-acting factors our model, and runs on Mus musculus datasets whose binding sites are unknown but have indicate that the approach is applicable in mam- orthologues with known binding sites, we are mals too. predicting them by the examination of orthologues. The regulation network of C. intestinalis 4. Characterization and definition of promo- transcription factor ZicL is consistent with the ter-associated CpG islands in ascidian data of a newly produced ChIP-chip experi- genomes ment. Using our method together with ChIP- chip data, we further expanded the original net- Kohji Okamura, Riu Yamashita, Koki Nishit- work to cover all 16,000 C. intestinalis genes. So suji2, Yutaka Suzuki1, Takehiro Kusakabe2,and that not only the kernel components of the regu- Kenta Nakai latory network making body plan, but also pe- ripheral components which actually make build- While CpG islands are often linked to a pro- ing block of the body are included. moter in mammals, their existence in invertebrates is unclear. Since there is a striking differ- 6. Pseudocounts for transcription factor bin- ence in DNA methylation pattern between ver- ding sites tebrates and invertebrates, which show global and fractional methylation, respectively, the Keishin Nishida, Martin Frith4, and Kenta function of methylation per se in the latter group Nakai: 4CBRC, AIST is also elusive. To address these questions, we performed determination of TSSs of ascidian To represent the sequence specificity of tran- genes by combination of the oligo-capping scription factors, the position weight matrix method and massive-scale cDNA sequencing. As (PWM) is widely used. In most cases, each ele- a result, we found characteristic features of as- ment is defined as a log likelihood ratio of a cidian promoters. They tend to be G＋C- and base appearing at a certain position, which is es- 147 timated from a finite number of known binding of 1st promoters. sites. To avoid bias due to this small sample size, a certain numeric value, called a pseudo- 8. Effects of Alu elements on global nucle- count, is usually allocated for each position, and osome positioning in the human genome its fraction according to the background base composition is added to each element. So far, Yoshiaki Tanaka, Riu Yamashita and Kenta there has been no consensus on the optimal Nakai pseudocount value. In this study, we simulated the sampling process by artificially generating Because chromatin can limit the accessibility binding sites based on observed nucleotide fre- of regulatory sites, understanding the genome quencies in a public PWM database, and then sequence-specific positioning of nucleosome is the generated matrix with an added pseudo- important for the analyses of transcription and count value was compared to the original fre- replication. It has been previously reported that quency matrix using various measures. Al- the 10-bp dinucleotide periodicities are strongly though the results were somewhat different be- associated with nucleosome positioning, but it is tween measures, in many cases, we could find unknown whether these features can affect in an optimal pseudocount value for each matrix. vivo nucleosome locations through the wholte These optimal values are independent of the genomes of all eukaryote. Fourier analysis to the sample size and are clearly anti-correlated with genome fragments indicates that these are not the information content of the original matrices, common in 16 eukaryotes, but the two primate- meaning that larger pseudocount vales are pref- specific periodicities (84-bp and 167-bp) are ob- erable for less conserved binding sites. As a sim- served. The 167 bp is similar with the sum of ple representative, we suggest the value of 0.8 the lengths of a nucleosome unit and its linker for practical uses. region. After masking Alu elements, these periodicities were greatly diminished. Therefore, we 7. Definition and analysis of alternative pro- next analyzed the distribution of nucleosomes in moters using a huge number of TSS infor- the vicinity of them. Using two independent mation large-scale sets of recently published nucleosome mapping data, we found that (1) there are Riu Yamashita, Yutaka Suzuki1, Hiroyuki one or two fixed slot(s) for nucleosome position- Wakaguri1, Sumio Sugano1,KentaNakai ing within the Alu element and (2) the positioning of neighboring nucleosomes seems to be in In order to support transcriptional studies, we phase, more or less, with the presence of Alu have constructed a database, DataBase of Tran- elements. Our study provides an important clue scriptional Start Sites (DBTSS: http://dbtss.hgc. to understanding the whole chromatin composi- jp), which includes a number of 5’-end se- tion of the primate genomes. quences produced by oligo-capping method. Re- cently, we have added 296.5 million tags from 9. Estimation and Comparison of minimal eight kinds of cells (15 kinds of experimental cellular function sets for bacteria and eu- conditions) using a SOLEXA sequencer. Here, karyotes we performed analysis of alternative promoters with these data. From these data, we obtained Yusuke Azuma and Kenta Nakai 75,918 promoters. These promoters could be classified into 36,251 gene regions and 39,667 in- A minimal cell, containing only necessary and tergenic regions. Former intragenic promoters sufficient components, has been estimated corresponded to 14,307 genes ,and 5,428 of them mostly by the reduction of the genome of a liv- have one promoter, and 8,879 genes have more ing cell. But the “minimal gene set” obtained by than one promoter. For each gene, we defined the former approach may be inaccurate due to the promoter with the largest number of tags as the effect of evolution. Thus, we tried to detect the ‘1st promoter’ and the 2nd highest promoter the minimal cellular function, instead. As cellu- as the ‘2nd promoter’. Between different cell lar functions, we used KEGG pathway maps. types, the average percentage of the discrepancy The minimal pathway maps were detected as a for 1st and 2nd promoters was 28.3％.Onthe combination of the conserved pathway maps other hand, we observed 9.6％ of difference for and the organism-specific pathway maps. The promoters expressed in the same cell types with conserved pathway maps are those containing different conditions. These results indicate that more orthologous genes in all pathway maps the expression ratio of promoters is conserved and are estimated by homology searches. They among cells. We also observed that 2nd promot- should be close to the minimal pathways but it ers preferentially occur in downstream regions is not sure whether they are organized to sus- 148 tain life from only external nutrients like living measureofgenecoexpressiontoretrievefunc- cells. Then, the organism-specific pathway maps tionally related genes more accurately, (ii) im- are detected as those that can synthesize com- plementing clickable maps for all gene networks pounds required for the conserved pathway for step-by-step navigation, (iii) applying Google maps from nutrients. The minimal pathway Maps API to create a single map for a large net- maps detected for bacteria agree well with the work, (iv) including information about protein- experimental essential genes. Most of the catabo- protein interactions, (v) identifying conserved lization pathways were selected as organism- patterns of coexpression and (vi) showing and specific pathways rather than conserved ones, connecting KEGG pathway information to iden- suggesting that they are adapted to each envi- tify functional modules. With these enhanced ronment. The minimal pathway maps of eukary- functions for gene network representation, otes contain more pathway maps for DNA re- ATTED-II can help researchers to clarify the pair than those of bacteria. In addition, there are functional and regulatory networks of genes in more links in the pathways of eukaryotes. Thus, Arabidopsis. it is likely that eukaryotes need to be more stable genetically. 12. PiSite: a database of protein interaction sites using multiple binding states in the 10. Development of new indices to evaluate PDB protein-protein interfaces: Assembling space volume, assembling space dis- M. Higurashi, T. Ishida, and K. Kinoshita tance, and global shape descriptor The vast accumulation of protein structural M. Maeda5 and K. Kinoshita: 5National Insti- data has now facilitated the observation of tute of Agrobiological Sciences many different complexes in the PDB for the same protein. Therefore, a single protein com- Protein-protein interaction is an initial step to plex is not sufficient to identify their interaction realize complex biological functions, therefore, sites, especially for proteins with multiple bind- understanding of the protein-protein interfaces ing states or different partners, such as hub pro- will give us a clue to predict the protein com- teins. Thus, we developed a database that pro- plex structures. For the purpose, efficient de- vides protein-protein interaction sites at the resi- scriptors of the interface and database analyses due level with consideration of multiple com- are important. In this study, we developed three plexes at the same time, by mapping the bind- new descriptors of protein-protein interfaces, ing sites of all complexes containing the same that is, assembling space volume, assembling protein in the PDB. We also implemented easy space distance, and global shape descriptor, by web-interfaces with an interactive viewer work- using Delaunay tessellation technique. The first ing with typical web-browsers, and the different two indexes enable us to evaluate how well the binding modes can be checked visually. protein interfaces are build up, and the third descriptor quantifies the complexity of the protein- 13. Discrimination between biological inter- protein interfaces. Systematic comparison with faces and crystal-packing contacts some existing descriptors, our indexes could elucidate the different aspects of the protein inter- Y. Tsuchiya, H. Nakamura7 and K. Kinoshita: faces. 7Osaka University

11. ATTED-II: a coexpression database for The quaternary structures of proteins are the Arabidopsis. bases of their physiological functions, and thus it is indispensable to know the biologically rele- T. Obayashi, S. Hayashi6,M.Saeki6,H.Ohta6, vant complexes of proteins to understand their K. Kinoshita: 6Tokyo Institute of Technology functions at the molecular level. The structures of proteins are usually determined by X-ray ATTED-II (http://atted.jp) is a database of crystallography, which could contain non- gene coexpression in Arabidopsis that can be biological interactions due to the nature of crys- used to design a wide variety of experiments, tals. Therefore, discrimination between biologi- including the prioritization of genes for func- cally relevant interfaces and artificial crystal- tional identification or for studies of regulatory packing contacts in crystal structures is re- relationships. Here, we report updates of quired. We developed a discrimination method ATTED-II that focus especially on functionalities between biological and non-biological interfaces, for constructing gene networks with regard to which evaluates protein-protein interfaces in the following points: (i) introducing a new terms of complementarities for hydrophobicity, 149 electrostatic potential and shape on the protein chines from the prediction results of the seven surfaces, and chooses the most probable biologi- independent predictors. As a result of our cal interfaces among all possible contacts in the evaluation, the meta approach achieved higher crystal. Our discrimination method achieved a prediction accuracy than previously developed good success rate, comparable to that of the con- methods. tact area-dependent discrimination. Subsequent detailed review of the discrimination results 16. A cavity with an appropriate size is the raised the success rate to 91.4％. basis of the PPIase activity

14. Effect of surface-to-volume ratio of pro- Teikichi Ikura8, Kengo Kinoshita, Nobutoshi teins on hydrophilic residues Ito8: 8Tokyo Medical and Dental University

M. Shirota, T. Ishida and K. Kinoshita Peptidyl-prolyl isomerases (PPIase) are important enzymes in biological systems, but the cata- The size of a protein has been shown to affect lytic mechanisms are not well understood. To both the amino acid composition and the resi- elucidate the essential amino acids for the enzy- due burial in the protein. To demonstrate that matic activities, we have carried out the similar- these effects are the results from the reduction ity search of atomic configurations of the active of surface regions relative to the volume in site of PPIase against the known protein struc- larger proteins, we examined the effect of tures, and found alpha amylase and prolyl en- surface-to-volume ratio (SVR), which is the ratio dopeptidase have the similar spatial arrange- between the accessible surface area and volume ment of atoms with PPIase active sites. Further- of a protein, to amino acid composition. The re- more, we proved experimentally that these production of several hydrophilic residues was teins actually have the PPIase activities, which more strongly correlated with SVR than with have not been considered at all. In addition, we protein size (i.e. the number of amino acids), created the similar hole in the barnase, which is which indicats that SVR directly affected the a enzyme to catalyze the ribonuclease activity aminoacidcomposition.Furthermore,thesehy- and does not have the PPIase activities, and drophilic residues also increased in buried frac- found that the mutated barnase exhibit the PPI- tionatthesametimeofthereduction.Thein- ase activity. These results indicate that the PPI- crease in burial was found to be accelerated ase activity can be realized by a hole with ap- compared with the decrease in occurrence as propriatesizeonthesurfaceofprotein. SVR decreased below SVR＝0.3Å－1 (approxi- mately protein size exceeded 132 residues) ex- 17. COXPRESdb: co-expressed gene data- cept for lysine, which was the most difficult for base for mouse and human. being buried. T. Obayashi, S. Hayashi6, M. Shibaoka6,M. 15. Prediction of disordered regions in pro- Saeki6,H.Ohta6,K.Kinoshita teins based on the meta approach A database of coexpressed gene sets can pro- Takashi Ishida and Kengo Kinoshita vide valuable information for a wide variety of experimental designs, such as targeting of genes Intrinsically disordered regions in proteins for functional identification, gene regulation have no unique stable structures without their and/or protein-protein interactions. Coexpre- partner molecules, thus these regions sometimes ssed gene databases derived from publicly avail- prevent high-quality structure determination. able GeneChip data are widely used in Arabi- Furthermore, proteins with disordered regions dopsis research, but platforms that examine co- are often involved in important biological proc- expression for higher mammals are rather lim- esses, and the disordered regions are considered ited. Therefore, we have constructed a new da- to play important roles in molecular interac- tabase, COXPRESdb (coexpressed gene data- tions. Therefore, identifying disordered regions base) (http://coxpresdb.hgc.jp), for coexpressed is important to obtain high-resolution structural gene lists and networks in human and mouse. information and to understand the functional Coexpression data could be calculated for 19 777 aspects of these proteins. Thus, we developed a and 21 036 genes in human and mouse, respec- new prediction method for disordered regions tively, by using the GeneChip data in NCBI in proteins based on the meta approach and im- GEO. COXPRESdb enables analysis of the four plemented a web-server for this prediction types of coexpression networks: (i) highly coex- method. The method predicts the disorder ten- pressed genes for every gene, (ii) genes with the dency of each residue using support vector ma- same GO annotation, (iii) genes expressed in the 150 same tissue and (iv) user-defined gene sets. able configurations of membrane molecules can When the networks became too big for the static generate heterogeneity, such as lipid rafts and pictureonthewebinGOnetworksorintissue transportsome regions, in the membrane. To re- networks, we used Google Maps API to visual- veal the bidirectional influences between pro- ize them interactively. COXPRESdb also proteins and surrounding lipids, we performed mo- vides a view to compare the human and mouse lecular dynamics simulations of biological mem- coexpression patterns to estimate the conserva- branes with and without proteins and choles- tion between the two species. terol, and compared those trajectories. As a result, alamethicin, a small transmembrane pep- 18. Influence of proteins and cholesterol on tide, was shown to reduce the whole membrane biological membranes analyzed by mo- undulation, in addition to decreasing local lecular dynamics membrane thickness according to the size of alamethicin’s hydrophobic region. On the con- Naoya Fujita, Takashi Ishida and Kengo Ki- trary, water accessibility of alamethicin and its noshita hydrogen bonds with lipids were different de- pending on the cholesterol availability. Further Protein-membrane interactions are fundamen- investigations with aquaporin are also being tal for both protein functions and membrane performed. properties. By means of these interactions, suit-

Publications

Chiba, H., Yamashita, R., Kinoshita, K. and space distance, and global shape descriptor. J. Nakai, K. Weak correlation between sequence Mol. Graph. Mod . 27: 706-711, 2009. conservation in promoter regions and in Miura,K.,Toh,H.,Hirakawa,H.,Sugii,M.,Mu- protein-coding regions of human-mouse rata, M., Nakai, K., Tashiro, K., Kuhara, S., orthologous gene pairs. BMC Genomics. 9: 152, Azuma, Y. and Shirai, M. Genome-wide 2008. analysis of Chlamydophila pneumoniae gene ex- Genome Information Integration Project and H- pression at the late stage of infection. DNA invitational 2 Consortium. The H-Invitational Res. 15 (2): 83-91, 2008. Database (H-InvDB), a comprehensive annota- Murakami,K.,Imanishi,T.,Gojobori,T.and tion resource for human genes and tran- Nakai, K. Two different classes of co- scripts. Nucl. Acids Res. 36: D793-D799, 2008. occurring motif pairs found by a novel visu- Hatada,I.,Morita,S.,Kimura,M.,Horii,T., alization method in human promoter regions. Yamashita, R. and Nakai, K. Genome-wide BMC Genomics. 9 (1): 112, 2008. demethylation during neural differentiation of Nishida,K.,Frith,M.andNakai,K.Pseudo- P19 embryonal carcinoma cells. J. Human counts for transcription factor binding sites. Genet. 53 (2): 185-191, 2008. Nucl. Acids Res. 37: 939-944, 2009; published Hatanaka, Y., Nagasaki, M., Yamaguchi, R., online on December 23, 2008. Obayashi, T., Numata, K., Imoto, S., Shima- Obayashi, T., Hayashi, S., Shibaoka, M., Saeki, mura, T., Kinoshita, K., Nakai, K. and Miy- M.,Ohta,H.andKinoshita,K.COXPRESdb:a ano, S. A novel strategy to search concerted database of coexpressed gene networks in transcription factor activities using gene ex- mammals. Nucleic Acids Res. 36: D77-82, 2008. pression profile and genomic data. Genome In- Obayashi, T., Hayashi, S., Saeki, M., Ohta, H., formatics. 20: 212-221, 2008. andKinoshita,K.ATTED-IIprovidescoex- Higurashi, M., Ishida, T. and Kinoshita, K. pressed gene networks for Arabidopsis. Nu- PiSite: a database of protein interaction sites cleic Acids Res. 37: D987-991, 2009. using multiple binding states in the PDB. Nu- Okamura, K. and Nakai, K. Retrotransposition cleic Acids Res. 37: D360-364, 2009. as a source of new promoters. Mol. Biol. Evol . Ikura, T., Kinoshita, K. and Ito, N. A cavity with 25 (6): 1231-1238, 2008. an appropriate size is the basis of the PPIase Sierro,N.,Makita,Y.,deHoon,M.andNakai, activity. Protein Eng Des Sel . 21: 83-89, 2008. K. DBTBS: a database of transcriptional regu- Ishida, T. and Kinoshita, K. Prediction of disor- lation in Bacillus subtilis containing upstream dered protein regions based on meta- intergenic conservation information. Nucl. Ac- approach. Bioinformatics. 24: 1344-1348, 2008. ids Res. 36: D93-D96, 2008. Maeda, M. and Kinoshita, K. Development of Sierro,N.,Li,S.,Suzuki,Y.,Yamashita,R.and new indices to evaluate protein-protein inter- Nakai, K. Spatial and temporal preferences for faces: Assembling space volume, assembling trans-splicing in Ciona intestinalis revealed by 151

EST-based gene expression analysis. Gene. perial College Press, London). vol. 21, pp. 188- 430: 44-49, 2009; available online on October 199, 2008. 21, 2008. Wakaguri, H., Yamashita, R., Suzuki, Y., Shirota, M., Ishida, T. and Kinoshita, K. Effects Sugano, S. and Nakai, K., DBTSS: DataBase of of surface-to-volume ratio of proteins on hy- Transcription Start Sites, progress report 2008. drophilic residues, decrease in occurrence and Nucl. Acids Res. 36: D97-D101, 2008. increase in buried fraction. Protein Sci. 17: Yamashita, R., Suzuki, Y., Takeuchi, N., Wak- 1596-1602, 2008. aguri, H., Ueda, T., Sugano, S. and Nakai, K. Tsuchihara, K., Suzuki, Y., Wakaguri, H., Irie, Comprehensive detection of human terminal T., Tanimoto, K., Hashimoto, S., Matsushima, oligo-pyrimidine (TOP) gene and analysis of K., Mizushima-Sugano, J., Yamashita, R., their characteristics. Nucl. Acids Res. 36 (11): Nakai, K., Bentley, D., Esumi, H. and Sugano, 3707-3715, 2008. S. Massive transcriptional start site analysis of Kinoshita,K.,Kono,H.,andYura,K.Prediction human genes in hypoxia cells. Nucl. Acids Res. of molecular interactions from 3D-structures: in press. from small ligands to large protein complexes. Tsuchiya, Y., Nakamura, H. and Kinoshita, K. Edited by Bujnicki, J. (Wiley and Sons, USA). Discrimination between biological interfaces in printing, 2009. and crystal-packing contacts. Compt Biol Chem. 伊倉貞吉，木下賢吾，伊藤暢聡．ペプチジルプロ 1: 99-113, 2008. リルイソメラーゼの構造・機能相関．蛋白質核 Vandenbon, A., Miyamoto, Y., Takimoto, N., 酸酵素．５４：１６７―１７２，２００９． Kusakabe, T. and Nakai, K. Markov chain- 木下賢吾．立体構造からのタンパク質機能予測： based promoter structure modeling for tissue- 現状と展望．遺伝子医学MOOK．１４号．in press specific expression pattern prediction. DNA 中井謙太，ポール・ホートン．第３章３．アミ Res. 15 (1): 3-11, 2008. ノ酸配列に基づくタンパク質の細胞内局在予 Vandenbon, A. and Nakai, K. Using simple 測．実験医学増刊 vol.２６¾：１１０６―１１１２，２００８． rules on presence and positioning of motifs 中井謙太．タンパク質のシステム生物学．猪飼・ for promoter structure modeling and tissue 伏見・卜部・上野川・中村・浜窪編．タンパク specific expression prediction. Genome Infor- 質の事典．朝倉書店．５７５―５７８，２００８． matics. Edited by Arthur, J. and Ng, S.-K. (Im- 152

Human Genome Center Department of Public Policy 公共政策研究分野

Associate ProfessorKaoriMuto,Ph.D.准教授保健学博士武藤香織 Project Assistant Professor Hyongoo Hong, Ph.D. 特任助教学術博士洪賢秀 Project Assistant Professor Ayako Kamisato 特任助教法学修士神里彩子

Department of Public Policy works for three major missions; public policy studies on translational research, its application to healthcare and its impact on social security; practical advices and survey for research projects to build public trust; and “minority-centered” scientific communication. We have conducted a comparative political study on stem cell research regarding homecare services for ALS in East Asia. We also supported for “BioBank Japan” project from ethical, legal and social standpoints and ended the first questionnaire survey. We held Sci/Art Café twice at the Medical Science Museum as one of the outreach activities.

1. A comparative political study on stem cell Under the Dean’s courageous decision, the research and genetic testing in East Asia IMSUT have established the Office of Research Ethics (ORE) for supporting research activities. Supported by Japan Bioindustry Association, Our department has main responsibility for we conducted a comparative study on research managing the ORE and our research ethics re- policy on stem cells to examine broader social view system, supported by Professor Hiroshi Ki- and cultural agendas on industrialization of yono of Division of Mucosal Immunology, Pro- stem cell research and genetic testing. We’ve in- fessor Kensuke Miyake of Division of Infectious terviewed main players in this area; the relevant Genetics, Professor Fumitaka Nagamura and Dr. authorities, bioindustry CEOs, physicians, aca- Makiko Tajima of Department of Clinical Trial demics and patients support groups. We also Safety Management, Professor Yasushi Kodama conducted literature reviews regarding regula- of Graduate School of Public Policy and Profes- tions. One of the key preliminary findings is the sor Akira Akabayashi of Graduate School of contrary regulative differences between South Medicine. After conducting our survey on past Korea and Japan. After the fabrication of Hwang ethical reviews and a comparative study on re- Woo-suk’s stem cell cloning and unethical hu- search ethics review system in the US, the UK man egg collection, bioethics law has been re- and South Korea, we checked our current prob- vised and the government seeks more strict lems which tend to stuck fluent research review regulation towards life science and healthcare. process so as to secure quality assurance of ethi- We’ve found some correlations in political op- cal discussions. Since February 3rd of 2009, Ay- tions on stem cell research and genetic testing in ako Kamisato has assumed main responsibility terms of regulations among in East Asia. on “bench consulting” regarding consent, research protocols and pre-review on research eth- 2. Establishment of Office of Research Ethics ics of all research involving human subjects. We (ORE) will start communication with other relevant di- visions on research ethics review founded by re- 153 search institutes and prepare for new study on and serums and the experience of watching the research ethics review and ethical governance DVD or the leaflet about the project overview. for future. We’ve found that participants who responded that they had forgotten the whole consent proc- 3. Ethical, legal and social support for “Bio- ess are not the elderly population. Furthermore, Bank Japan” project MCs explains that this project doesn’t have any plans to disclose personal genotyped data to For supporting “BioBank Japan” project, led each participant, but a certain amount of partici- by Professor Yusuke Nakamura of Laboratory of pants responded that they now want to see their Molecular Medicine of IMSUT, we’ve conducted own genotyped data or tentative research feed- three types of surveys and issued newsletters backs, while others are just satisfied with their for participants. By the end of 2007, the project contribution to genomic research without any has obtained 200,000 written consent forms by rewards. Even though participants should forget research coordinators called Medical Coordina- the fact that they gave consent for research, tors (MC). The project trained nurses or phar- MCs explain, encourage and appreciate partici- macists as MCs for obtaining free and fully in- pants at each time and participants recall their formed consent from participants. We con- will for contribution. ducted our questionnaire survey to participants To appreciate participants’ and MCs’ contri- of the BioBank Japan Project. Our data shows bution to the project, we had issued “BioBank that the younger participants thought that their newsletters” three times in 2007 for MCs and personal analyzed data should be disclosed. The participants. We will explore more methods and consent process had been well-worked out in opportunities to communicate with participants. advance and is fully complied with the govern- Because the current forms of BioBank newslet- ment ethical guidelines for genetic/genomic re- ters are available only for the sighted with good search. However, recent publications show that eyesight, we make efforts for personalized infor- the long and tedious consent process may not mation security to meet with disabilities of par- contribute to participants’ understanding the ticipants. overview of the research, may be unethical rather than ethical. If we long for “personalized 4. Sci/Art Café medicine”, we should think further about the construction of “personalized consent process” According to the 3rd Science and Technology and we have to change the relationship between Basic Plan (FY2006-FY2010), outreach activities participants and researchers, from one-time in- are promoted that aim for the sharing of public formed consent to long lasting public trust. needs through interactive communication be- Obtaining feedbacks from participants is also tween researchers and the public. As one of effective to keep incentives for participation and such outreach activities, we held our original prevent dropout of participants from research science café series called as “Sci/Art Café” twice process. We conducted three kinds of surveys to in 2008. Our original intent of “Sci/Art Café” is evaluate and improve the consent process and to promote communication between scientists explore what the project should do for public in- and those who don’t have regular communica- volvement; questionnaire surveys towards re- tion with science but love art. The 1st session search participants, a web-based questionnaire called “Rhythm generated by network” was survey towards all MCs and focus group inter- held in Shibuya during the 3rd World Rhythm views with chief MCs to triangulate the consent Summit, supported by Dr. Atsuko Takamatsu process. The preliminary results show that par- (Waseda Univ.), Dr. Shin-ichi Nakagawa ticipants are basically satisfied with the consent (RIKEN) and Dr. Hideaki Takeuchi (UT). The 2nd process and highly evaluate MCs’ attitudes to- session called “Doing science, doing art” was wards them. Most MCs also responded that held on October 8th at the Medical Science Mu- they have made their original efforts to make seumintheIMSUT,supportedbyDr.Hideo their explanation easier and understandable spe- Iwasaki (Waseda Univ.) and Dr. Yoichiro Mu- cifically towards the elderly. However, certain rakami (JST). We prepare for the 3rd session in amounts of participants have already forgotten next early summer 2009. about what for they have donated their DNA

Publications

1. Ishiyama I, Nagai A, Muto K, Tamakoshi A, gata Z. Relationship between Public Atti- Kokado M, Mimura K, Tanzawa T, Yama- tudes toward Genomic Studies Related to 154

Medicine and Their Level of Genomic Liter- 学的検査まで―．日米の医療―制度と倫理． acy in Japan. American Journal of Medical 杉田米行編．大阪大学出版会．２０３―２２４， Genetics. 146A (13). 696-706, 2008. ２００８． 2. 洪賢秀．韓国社会における子どもの「性保護」 10．武藤香織．DNA親子鑑定は「ふしだらな」女と性犯罪防止対策．比較法研究．７０号．性にとっての救済策か？．ジェンダー研究の２００９．印刷中．フロンティア第４巻テクノ／バイオ・ポリ 3. 神里彩子・成澤光編著．生殖補助医療生命ティクス―科学・医療・技術のいま．舘かお倫理と法―基本資料集３．信山社．２１―１２３，る編．作品社．２３８―２６４，２００８．２６２―３０８，２００８． 11．洪賢秀．研究用卵子提供の何が問題なのか― 4. 張瓊方．諸外国における生殖補助医療の規制韓国黄禹錫論文捏造事件を中心に―．ジェン状況と実施状況（台湾）．生殖補助医療生命ダー研究のフロンティア第４巻テクノ／バ倫理と法―基本資料集３．神里彩子・成澤光イオ・ポリティクス―科学・医療・技術のい編．信山社．３２３―３３４，２００８．ま．舘かおる編．作品社．１９６―２１４，２００８ 5. 大上泰弘・神里彩子・城山英明．イギリス及 12．張瓊方．生殖技術と台湾社会．ジェンダー研びアメリカにおける動物実験規制の比較分析究のフロンティア第４巻テクノ／バイオ・ ―日本の規制体制への示唆．社会技術研究論ポリティクス―科学・医療・技術のいま．舘文集５号．１３２―１４２，２００８．かおる編．作品社．２１５―２２２，２００８ 6. 大上泰弘・成廣孝・神里彩子・城山英明・打 13．三村恭子，小門穂，武藤香織，張瓊方，洪賢越綾子．日本における生命科学・技術者の動秀，柘植あづみ．女性にやさしい機械のつく物実験に関する意識―生命科学実験及び動物られ方―内診台を例にして．ジェンダー研究慰霊祭に関するアンケート調査の分析．ヒトのフロンティア第４巻テクノ／バイオ・ポと動物の関係学会誌２０号．６６―７３，２００８．リティクス―科学・医療・技術のいま．舘か 7. 大上泰弘・神里彩子・城山英明．イギリスにおる編．作品社．２２３―２４０，２００８おける動物の実験規制を支えている思考様 14．神里彩子．生殖補助医療をめぐる議論―その式．科学技術社会論研究５号．８４―９２，２００８．回顧と展望―．家永登編『生殖技術と家族 8．渡部麻衣子，上田昌文．人の必要を充足する À』．早稲田大学出版部．４２―７１，２００８．科学技術．福祉工学における開発現場の分 15．渡部麻衣子，上田昌文編訳．エンハンスメン析．科学技術社会研究．１３８―１５１，２００８．ト論争．身体・精神の増強と先端科学技術． 9．武藤香織．「脱医療化」する予測的な遺伝学社会評論社，２００８．的検査への日米の対応―遺伝病から栄養遺伝

Human Genome Center Laboratory of Genome Database Laboratory of Sequence Analysis ゲノムデータベース分野 シークエンスデータ情報処理分野

Human Genome Center Laboratory of Genome Database Laboratory of Sequence Analysis ゲノムデータベース分野シークエンスデータ情報処理分野