Design and Progress of Child Health Assessments at Community Support Centers in the Birth and Three-Generation Cohort Study of the Tohoku Medical Megabank Project.

The Tohoku Medical Megabank Project (TMM) has been conducting a birth and three-generation cohort study (the BirThree Cohort Study). We recruited 73,529 pregnant women and their family members for this cohort study, which included 23,143 newborns and 9,459 of their siblings. We designed and are in the process of conducting three-step health assessments for each newborn at approximately ages of 5, 10 and 16. These health assessments are administered at seven community support centers. Trained genome medical research coordinators conduct physical examinations of and collect biological specimens from each participant. The Sendai Children's Health Square has been established as the headquarters for these child health assessments and is utilized to accumulate knowledge that can facilitate the proper practice of child health assessments. We designed all the relevant health assessments facilities to allow parents and their children to participate in the health assessments concomitantly. Our centers serve as places where child participants and their parents can feel at ease as a result of the implementation of safety measures and child hospitality measures. The TMM BirThree Cohort Study is in the process of conducting strategically detailed health assessments and genome analysis, which can facilitate studies concerning the gene-environment interactions relevant to noncommunicable diseases. Through these operations, our study allows for a significant depth of data to be collected in terms of the number of biospecimens under study and the comprehensiveness of both basic and clinical data alongside relevant family information.

Trans-homophilic interaction of CADM1 promotes organ infiltration of T-cell lymphoma by adhesion to vascular endothelium.

The initial step of organ infiltration of malignant cells is the interaction with host vascular endothelial cells, which is often mediated by specific combinations of cell adhesion molecules. Cell adhesion molecule 1 (CADM1) is overexpressed in adult T-cell leukemia/lymphoma (ATL) and provides a cell-surface diagnostic marker. CADM1 promotes the adhesion of ATL cells to vascular endothelial cells and multiple organ infiltration in mice. However, its binding partner on host cells has not yet been identified. In this study, we show that CADM1 promotes transendothelial migration of ATL cells in addition to the adhesion to vascular endothelial cells. Moreover, CADM1 enhances liver infiltration of mouse T-cell lymphoma cells, EL4, after tail vein injection, whereas a CADM1 mutant lacking adhesive activity did not. Among the known CADM1-binding proteins expressed in primary endothelial cells, only CADM1 and CADM4 could induce morphological extension of ATL cells when plated onto glass coated with these proteins. Furthermore, CADM1-mediated liver infiltration of EL4 cells was canceled in conventional and vascular endothelium-specific Cadm1 knockout mice, whereas it was not canceled in Cadm4 knockout mice. These results suggest that CADM1 on host vascular endothelial cells is required for organ infiltration of ATL and other T-cell lymphomas expressing CADM1.

The return of individual genomic results to research participants: design and pilot study of Tohoku Medical Megabank Project.

Certain large genome cohort studies attempt to return the individual genomic results to the participants; however, the implementation process and psychosocial impacts remain largely unknown. The Tohoku Medical Megabank Project has conducted large genome cohort studies of general residents. To implement the disclosure of individual genomic results, we extracted the potential challenges and obstacles. Major challenges include the determination of genes/disorders based on the current medical system in Japan, the storage of results, prevention of misunderstanding, and collaboration of medical professionals. To overcome these challenges, we plan to conduct multilayer pilot studies, which deal with different disorders/genes. We finally chose familial hypercholesterolemia (FH) as a target disease for the first pilot study. Of the 665 eligible candidates, 33.5% were interested in the pilot study and provided consent after an educational "genetics workshop" on the basic genetics and medical facts of FH. The genetics professionals disclosed the results to the participants. All positive participants were referred to medical care, and a serial questionnaire revealed no significant psychosocial distress after the disclosure. Return of genomic results to research participants was implemented using a well-prepared protocol. To further elucidate the impact of different disorders, we will perform multilayer pilot studies with different disorders, including actionable pharmacogenomics and hereditary tumor syndromes.

Design and Progress of Oral Health Examinations in the Tohoku Medical Megabank Project.

In order to assess the long-term impact of the Great East Japan Earthquake on the oral health of disaster victims and to evaluate gene-environmental interactions in the development of major oral diseases and oral-systemic associations, the oral part of two large-scale genome cohort studies by the Tohoku Medical Megabank Organization (ToMMo), including the Community-based cohort (CommCohort) study and the Birth and Three-Generation cohort (BirThree) study, have been conducted. The study population comprised 32,185 subjects, including 16,886 participants in the CommCohort study and 15,299 participants in the BirThree cohort study, recruited from 2013 to 2017. The oral studies consist of a questionnaire regarding oral hygiene behavior, clinical examinations by dentists, and oral plaque and saliva sampling for microbiome analyses, which were carried out at seven community support centers in Miyagi prefecture. The median age of all participants was 55.0 years, and 66.1% of participants were women. Almost all participants reported that they brushed their teeth more than once a day. The median number of present teeth was 27.0, and the decayed, missing and filled tooth number was 16.0, with a significant difference according to age and sex. The median periodontal pocket and clinical attachment level was 2.48 mm and 4.00 mm, respectively. Periodontal parameters increased significantly according to age, except for the accumulation of dental calculus. The oral part of these extensive cross-sectional studies provides a unique and important platform for future studies on oral health and diseases that elicit through interactions with systemic diseases, lifestyles, life events and genetic backgrounds, and contributes to researches clarifying the long-term effects of disasters on oral health.

A training and education program for genome medical research coordinators in the genome cohort study of the Tohoku Medical Megabank Organization.

BACKGROUND: Genome cohort studies are used to analyze interactions between genetic and environmental factors, providing valuable information for personalized healthcare. Large-scale and long-term cohort studies require a number of specially trained personnel, of whom those involved in obtaining informed consent play a vital role, especially during the initial phase of such studies. The Japanese Society of Human Genetics (JSHG) previously established a certification system for genome medical research coordinators (GMRCs) responsible for obtaining written consent via face-to-face explanation. Meanwhile, in the Tohoku Medical Megabank Organization (ToMMo), GMRCs are expected to play important roles not only in obtaining informed consent and conducting various assessments, but also in communicating with participants throughout the long-term follow-up. Based on the JSHG program, we therefore developed a specific education and training program for ToMMo GMRCs consisting of 17 lectures, one practical training session on the informed consent procedure, and written and interview examinations. Re-education workshops aimed at self-improvement are also carried out following certification. In this study, we evaluated the education and training program in terms of overall understanding, usefulness, and satisfaction using an anonymous questionnaire. METHODS: An anonymous questionnaire addressing each aspect of the education and training program (understanding, usefulness, and satisfaction) was distributed among 152 qualified ToMMo GMRCs. Responses were received from 94 participants (61.8%). RESULTS: There was a significant association between the level of overall understanding of lectures and medical qualification (nurse or clinical laboratory technologist), but not with age or educational background. The level of understanding and overall usefulness were lower in sessions related to genetics and epidemiology than those dealing with ToMMo practices. In the re-education workshops, GMRCs showed a preference for and hoped to learn more about both background knowledge and research progress in the ToMMo. CONCLUSIONS: The results of our questionnaire suggest that not all ToMMo GMRCs are able to understand everything during the initial education and training program, especially in terms of genomic medicine. Continuous re-education is therefore vital in improving knowledge, skills and motivation, and preparing GMRCs for a specialist role in community-based personalized healthcare.

Regional genetic differences among Japanese populations and performance of genotype imputation using whole-genome reference panel of the Tohoku Medical Megabank Project.

BACKGROUND: Genotype imputation from single-nucleotide polymorphism (SNP) genotype data using a haplotype reference panel consisting of thousands of unrelated individuals from populations of interest can help to identify strongly associated variants in genome-wide association studies. The Tohoku Medical Megabank (TMM) project was established to support the development of precision medicine, together with the whole-genome sequencing of 1070 human genomes from individuals in the Miyagi region (Northeast Japan) and the construction of the 1070 Japanese genome reference panel (1KJPN). Here, we investigated the performance of 1KJPN for genotype imputation of Japanese samples not included in the TMM project and compared it with other population reference panels. RESULTS: We found that the 1KJPN population was more similar to other Japanese populations, Nagahama (south-central Japan) and Aki (Shikoku Island), than to East Asian populations in the 1000 Genomes Project other than JPT, suggesting that the large-scale collection (more than 1000) of Japanese genomes from the Miyagi region covered many of the genetic variations of Japanese in mainland Japan. Moreover, 1KJPN outperformed the phase 3 reference panel of the 1000 Genomes Project (1KGPp3) for Japanese samples, and IKJPN showed similar imputation rates for the TMM and other Japanese samples for SNPs with minor allele frequencies (MAFs) higher than 1%. CONCLUSIONS: 1KJPN covered most of the variants found in the samples from areas of the Japanese mainland outside the Miyagi region, implying 1KJPN is representative of the Japanese population's genomes. 1KJPN and successive reference panels are useful genome reference panels for the mainland Japanese population. Importantly, the addition of whole genome sequences not included in the 1KJPN panel improved imputation efficiencies for SNPs with MAFs under 1% for samples from most regions of the Japanese archipelago.

The Health History of First-Degree Relatives' Dyslipidemia Can Affect Preferences and Intentions following the Return of Genomic Results for Monogenic Familial Hypercholesterolemia.

Genetic testing is key in modern healthcare, particularly for monogenic disorders such as familial hypercholesterolemia. This Tohoku Medical Megabank Project study explored the impact of first-degree relatives' dyslipidemia history on individual responses to familial hypercholesterolemia genomic results. Involving 214 participants and using Japan's 3.5KJPN genome reference panel, the study assessed preferences and intentions regarding familial hypercholesterolemia genetic testing results. The data revealed a significant inclination among participants with a family history of dyslipidemia to share their genetic test results, with more than 80% of participants intending to share positive results with their partners and children and 98.1% acknowledging the usefulness of positive results for personal health management. The study underscores the importance of family health history in genetic-testing perceptions, highlighting the need for family-centered approaches in genetic counseling and healthcare. Notable study limitations include the regional scope and reliance on questionnaire data. The study results emphasize the association between family health history and genetic-testing attitudes and decisions.

Aberrations of a cell adhesion molecule CADM4 in renal clear cell carcinoma.

Renal clear cell carcinoma (RCCC) is the most frequent subpopulation of renal cell carcinoma and is derived from the proximal uriniferous tubules. We have previously reported that an actin-binding protein, 4.1B/DAL-1, is expressed in renal proximal tubules, whereas it is inactivated in 45% of RCCC by promoter methylation. In the lung and several epithelial tissues, 4.1B is shown to associate with a tumor suppressor protein, CADM1, belonging to the immunoglobulin-superfamily cell adhesion molecules. Here, we demonstrate by immunohistochemistry that another member of the CADM-family protein, CADM4, as well as 4.1B is expressed specifically in human proximal tubules, while CADM1 and 4.1N, another member of the 4.1 proteins, are expressed in the distal tubules. Immunoprecipitation analysis coupled with Western blotting revealed that CADM4 associated with 4.1B, while CADM1 associated with 4.1N in the lysate from normal human kidney, implicating that a cascade of CADM4 and 4.1B plays an important role in normal cell adhesion of the proximal tubules. On the other hand, CADM4 expression was lost or markedly reduced in 7 of 10 (70%) RCC cell lines and 28 of 40 (70%) surgically resected RCCC, including 10 of 16 (63%) tumors with T1a. CADM4 expression was more preferentially lost in RCCC with vascular infiltration (p = 0.04), suggesting that loss of CADM4 is involved in tumor invasion. Finally, introduction of CADM4 into an RCC cell line, 786-O, dramatically suppressed tumor formation in nude mice. These findings suggest that CADM4 is a novel tumor suppressor candidate in RCCC acting with its binding partner 4.1B.

Expression of a splicing variant of the CADM1 specific to small cell lung cancer.

CADM1, a member of the immunoglobulin superfamily cell adhesion molecule, acts as a tumor suppressor in a variety of human cancers. CADM1 is also ectopically expressed in adult T-cell leukemia (ATL), conferring an invasive phenotype characteristic to ATL. Therefore, CADM1 plays dual roles in human oncogenesis. Here, we investigate the roles of CADM1 in small cell lung cancer (SCLC). Immunohistochemistry demonstrates that 10 of 35 (29%) primary SCLC tumors express CADM1 protein. Western blotting and RT-PCR analyses reveal that CADM1 is significantly expressed in 11 of 14 SCLC cells growing in suspension cultures but in neither of 2 SCLC cells showing attached growth to plastic dishes, suggesting that CADM1 is involved in anchorage-independent growth in SCLC. In the present study, we demonstrate that SCLC expresses a unique splicing variant of CADM1 (variant 8/9) containing additional extracellular fragments corresponding to exon 9 in addition to variant 8, a common isoform in epithelia. Variant 8/9 of CADM1 is almost exclusively observed in SCLC and testis, although this variant protein localizes along the membrane and shows similar cell aggregation activity to variant 8. Interestingly, both variant 8/9 and variant 8 of CADM1 show enhanced tumorigenicity in nude mice when transfected into SBC5, a SCLC cell lacking CADM1. Inversely, suppression of CADM1 expression by shRNA reduced spheroid-like cell aggregation of NCI-H69, an SCLC cell expressing a high amount of CADM1. These findings suggest that CADM1 enhances the malignant features of SCLC, as is observed in ATL, and could provide a molecular marker specific to SCLC.

Transcriptional regulation of the CADM1 gene by retinoic acid during the neural differentiation of murine embryonal carcinoma P19 cells.

CADM1 is a multifunctional cell adhesion molecule expressed predominantly in the nerve system, testis and lung. The expression of the Cadm1 gene is induced during the neural differentiation of murine embryonal carcinoma P19 cells by treatment with retinoic acid (RA). Here, we show that the suppression of CADM1 expression using RNAi interfered with P19 cell aggregation and reduced cell populations expressing MAP2 after RA treatment. Nonaggregated P19 cells were not differentiated into neurons, suggesting that CADM1 participates in the aggregate formation and neuronal differentiation of P19 in vitro. A luciferase assay of a series of deletion mutants of the CADM1 promoter localized an RA-responsive cis-acting element to an approximately 90-bp fragment upstream of the translational start site. This element contains a putative binding site for transcription factor Sp1, named Sp1-binding site-1 (Sp1BS-1). Sp1BS-1 and adjacent Sp1-binding sites (Sp1BS-2 and Sp1BS-3) showed enhanced transcriptional activity by RA. Moreover, a chromatin immunoprecipitation showed that RA receptor (RAR)α was associated with a DNA fragment containing Sp1BS-1, whereas suppression of RARα expression using siRNA reduced the responsiveness of the CADM1 promoter to RA. These results suggest that Sp1 plays a critical role in RA-induced CADM1 expression through possible interaction with RARα in the neural differentiation of P19.

A Pilot Study for Return of Individual Pharmacogenomic Results to Population-Based Cohort Study Participants.

Introduction: Pharmacogenomic (PGx) testing results provide valuable information on drug selection and appropriate dosing, maximization of efficacy, and minimization of adverse effects. Although the number of large-scale, next-generation-sequencing-based PGx studies has recently increased, little is known about the risks and benefits of returning PGx results to ostensibly healthy individuals in research settings. Methods: Single-nucleotide variants of three actionable PGx genes, namely, MT-RNR1, CYP2C19, and NUDT15, were returned to 161 participants in a population-based Tohoku Medical Megabank project. Informed consent was obtained from the participants after a seminar on the outline of this study. The results were sent by mail alongside sealed information letter intended for clinicians. As an exception, genetic counseling was performed for the MT-RNR1 m.1555A > G variant carriers by a medical geneticist, and consultation with an otolaryngologist was encouraged. Questionnaire surveys (QSs) were conducted five times to evaluate the participants' understanding of the topic, psychological impact, and attitude toward the study. Results: Whereas the majority of participants were unfamiliar with the term PGx, and none had undergone PGx testing before the study, more than 80% of the participants felt that they could acquire basic PGx knowledge sufficient to understand their genomic results and were satisfied with their potential benefit and use in future prescriptions. On the other hand, some felt that the PGx concepts or terminology was difficult to fully understand and suggested that in-person return of the results was desirable. Conclusions: These results collectively suggest possible benefits of returning preemptive PGx information to ostensibly healthy cohort participants in a research setting.

Association between Serum Biotin Levels and Cedar Pollinosis in Japanese Schoolchildren.

Biotin is a water-soluble B complex vitamin and coenzyme of five types of carboxylase and plays crucial roles in fatty acid, glucose, and amino acid metabolism. Nutritional biotin deficiency and defective enzymes essential for biotin metabolism cause inflammatory diseases such as eczema-like dermatitis and Crohn's disease; however, little is known about the pathophysiological roles of biotin. This study investigated the relationship between biotin metabolism and human allergic sensitization and diseases by measuring serum levels of biotin, total immunoglobulin E (IgE) and allergen-specific IgEs in more than 400 Japanese schoolchildren aged 6 to 12. The prevalence of allergic diseases, and environmental and life-style factors were also examined by a questionnaire. Like total IgE, serum biotin levels of children showed a log-normal distribution. Meanwhile, Spearman's rank correlation analysis showed weak but significant positive associations between serum biotin levels and total IgE (rho=0.147, p=0.0029) as well as allergen-specific IgEs against egg whites (rho=0.215, p=0.00013), cedar pollen (rho=0.176, p=0.00036), and cat dander (rho=0.130, p=0.0085). Furthermore, mean serum biotin levels in children with cedar pollinosis, but not with other allergic diseases such as asthma and allergic rhinitis, were significantly higher than in those without (p=0.0015). These results suggest a correlation between serum biotin levels and the development of cedar pollinosis. Further prospective studies are needed to evaluate the causal relationship between biotin metabolism and cedar pollen sensitization and pollinosis development.

Construction and integration of three de novo Japanese human genome assemblies toward a population-specific reference.

The complete human genome sequence is used as a reference for next-generation sequencing analyses. However, some ethnic ancestries are under-represented in the reference genome (e.g., GRCh37) due to its bias toward European and African ancestries. Here, we perform de novo assembly of three Japanese male genomes using > 100× Pacific Biosciences long reads and Bionano Genomics optical maps per sample. We integrate the genomes using the major allele for consensus and anchor the scaffolds using genetic and radiation hybrid maps to reconstruct each chromosome. The resulting genome sequence, JG1, is contiguous, accurate, and carries the Japanese major allele at most loci. We adopt JG1 as the reference for confirmatory exome re-analyses of seven rare-disease Japanese families and find that re-analysis using JG1 reduces total candidate variant calls versus GRCh37 while retaining disease-causing variants. These results suggest that integrating multiple genomes from a single population can aid genome analyses of that population.

Japonica Array NEO with increased genome-wide coverage and abundant disease risk SNPs.

Ethnic-specific SNP arrays are becoming more important to increase the power of genome-wide association studies in diverse population. In the Tohoku Medical Megabank Project, we have been developing a series of Japonica Arrays (JPA) for genotyping participants based on reference panels constructed from whole-genome sequence data of the Japanese population. Here, we designed a novel version of the SNP array for the Japanese population, called Japonica Array NEO (JPA NEO), comprising a total of 666,883 markers. Among them, 654,246 tag SNPs of autosomes and X chromosome were selected from an expanded reference panel of 3,552 Japanese, 3.5KJPNv2, using pairwise r2 of linkage disequilibrium measures. Additionally, 28,298 markers were included for the evaluation of previously identified disease risk markers from the literature and databases, and those present in the Japanese population were extracted using the reference panel. Through genotyping 286 Japanese samples, we found that the imputation quality r2 and INFO score in the minor allele frequency bin >2.5-5% were >0.9 and >0.8, respectively, and >12 million markers were imputed with an INFO score >0.8. From these results, JPA NEO is a promising tool for genotyping the Japanese population with genome-wide coverage, contributing to the development of genetic risk scores.

Tumor suppressor CADM1 is involved in epithelial cell structure.

The tumor suppressor, CADM1, is involved in cell adhesion and preferentially inactivated in invasive cancer. We have previously reported that CADM1 associates with an actin-binding protein, 4.1B/DAL-1, and a scaffold protein, membrane protein palmitoylated 3 (MPP3)/DLG3. However, underlying mechanism of tumor suppression by CADM1 is not clarified yet. Here, we demonstrate that MPP1/p55 and MPP2/DLG2, as well as MPP3, interact with both CADM1 and 4.1B, forming a tripartite complex. We then examined cell biological roles of CADM1 and its complex in epithelia using HEK293 cells. Among MPP1-3, MPP2 is recruited to the CADM1-4.1B complex in the early process of adhesion in HEK293 cells. By suppression of CADM1 expression using siRNA, HEK293 lose epithelia-like structure and show flat morphology with immature cell adhesion. 4.1B and MPP2, as well as E-cadherin and ZO-1, are mislocalized from the membrane by depletion of CADM1 in HEK293. Mislocalization of MPP2 is also observed in several cancer cells lacking CADM1 expression with the transformed morphology. These findings suggest that CADM1 is involved in the formation of epithelia-like cell structure with 4.1B and MPP2, while loss of its function could cause morphological transformation of cancer cells.

Establishment of Integrated Biobank for Precision Medicine and Personalized Healthcare: The Tohoku Medical Megabank Project.

The Tohoku Medical Megabank (TMM) project was established to provide creative reconstruction of the Tohoku area that suffered from a huge earthquake and ensuing tsunami (the Great East Japan Earthquake, GEJE). TMM aims to establish two large-scale genome cohorts and an integrated biobank managing biospecimen and related information. It supports community medicine by establishing next-generation medical systems through a combination of the prospective genome cohort studies with a total of 150,000 participants and genomic medicine. The strategies for genome analyses in TMM are to develop an elaborate genome reference panel by means of high-fidelity Japanese whole-genome sequence, to design custom single nucleotide polymorphism (SNP) arrays based on the reference panel, and to obtain genotype data for all the TMM cohort participants subsequently. Disease-associated genomic information and omics data, including metabolomics and microbiome analysis, provide an essential platform for precision medicine and personalized healthcare (PHC). Ethical, legal, and social issues (ELSI) and education are important for implementing genomic medicine. The major considerations of ELSI regarding each participant of the cohort studies are the respect for the autonomy and the protection of privacies. Moreover, developing and provide human resources not only for the TMM project but also for the social implementation of precision medicine and PHC is required. We started a pilot study of the return of genomic results for familial hypercholesterolemia (FH) as a target disease. TMM aims to establish solid platforms that support precision medicine and PHC based on the genomic and omics information and environmental and lifestyle factors of the individuals, which is one of the most advanced medical care beyond the evidenced-based medicine in the near future.

3.5KJPNv2: an allele frequency panel of 3552 Japanese individuals including the X chromosome.

The first step towards realizing personalized healthcare is to catalog the genetic variations in a population. Since the dissemination of individual-level genomic information is strictly controlled, it will be useful to construct population-level allele frequency panels with easy-to-use interfaces. In the Tohoku Medical Megabank Project, we sequenced nearly 4000 individuals from a Japanese population and constructed an allele frequency panel of 3552 individuals after removing related samples. The panel is called the 3.5KJPNv2. It was constructed by using a standard pipeline including the 1KGP and gnomAD algorithms to reduce technical biases and to allow comparisons to other populations. Our database is the first large-scale panel providing the frequencies of variants present on the X chromosome and on the mitochondria in the Japanese population. All the data are available on our original database at https://jmorp.megabank.tohoku.ac.jp.

Identification of two novel breast cancer loci through large-scale genome-wide association study in the Japanese population.

Genome-wide association studies (GWAS) have successfully identified about 70 genomic loci associated with breast cancer. Owing to the complexity of linkage disequilibrium and environmental exposures in different populations, it is essential to perform regional GWAS for better risk prediction. This study aimed to investigate the genetic architecture and to assess common genetic risk model of breast cancer with 6,669 breast cancer patients and 21,930 female controls in the Japanese population. This GWAS identified 11 genomic loci that surpass genome-wide significance threshold of P < 5.0 × 10-8 with nine previously reported loci and two novel loci that include rs9862599 on 3q13.11 (ALCAM) and rs75286142 on 21q22.12 (CLIC6-RUNX1). Validation study was carried out with 981 breast cancer cases and 1,394 controls from the Aichi Cancer Center. Pathway analyses of GWAS signals identified association of dopamine receptor medicated signaling and protein amino acid deacetylation with breast cancer. Weighted genetic risk score showed that individuals who were categorized in the highest risk group are approximately 3.7 times more likely to develop breast cancer compared to individuals in the lowest risk group. This well-powered GWAS is a representative study to identify SNPs that are associated with breast cancer in the Japanese population.

Genome analyses for the Tohoku Medical Megabank Project towards establishment of personalized healthcare.

Personalized healthcare (PHC) based on an individual's genetic make-up is one of the most advanced, yet feasible, forms of medical care. The Tohoku Medical Megabank (TMM) Project aims to combine population genomics, medical genetics and prospective cohort studies to develop a critical infrastructure for the establishment of PHC. To date, a TMM CommCohort (adult general population) and a TMM BirThree Cohort (birth+three-generation families) have conducted recruitments and baseline surveys. Genome analyses as part of the TMM Project will aid in the development of a high-fidelity whole-genome Japanese reference panel, in designing custom single-nucleotide polymorphism (SNP) arrays specific to Japanese, and in estimation of the biological significance of genetic variations through linked investigations of the cohorts. Whole-genome sequencing from >3,500 unrelated Japanese and establishment of a Japanese reference genome sequence from long-read data have been done. We next aim to obtain genotype data for all TMM cohort participants (>150,000) using our custom SNP arrays. These data will help identify disease-associated genomic signatures in the Japanese population, while genomic data from TMM BirThree Cohort participants will be used to improve the reference genome panel. Follow-up of the cohort participants will allow us to test the genetic markers and, consequently, contribute to the realization of PHC.

Promoter methylation of DAL-1/4.1B predicts poor prognosis in non-small cell lung cancer.

PURPOSE: DAL-1/4.1B is an actin-binding protein originally identified as a molecule whose expression is down-regulated in lung adenocarcinoma. We have previously shown that a lung tumor suppressor, TSLC1, associates with DAL-1, suggesting that both proteins act in the same cascade. The purpose of this study is to understand the molecular mechanisms and clinical significance of DAL-1 inactivation in lung cancer. EXPERIMENTAL DESIGN: We studied aberration of the DAL-1 in 103 primary non-small cell lung cancers (NSCLC) and 18 lung cancer cells. Expression and allelic and methylation status of DAL-1 was examined by reverse transcription-PCR, microsatellite analysis, and bisulfite sequencing or bisulfite single-strand conformational polymorphism, respectively. RESULTS: Loss of DAL-1 expression was strongly correlated with promoter methylation in lung cancer cells, whereas DAL-1 expression was restored by a demethylating agent, 5-aza-2'-deoxycytidine. The DAL-1 promoter was methylated in 59 (57%) primary NSCLC tumors, 37% of which were associated with loss of heterozygosity around the DAL-1 on chromosomal region 18p11.3. In squamous cell carcinomas, DAL-1 methylation was observed in 9 of 10 tumors at stage I, whereas the incidence of methylation gradually increased in adenocarcinomas as they progressed [13 of 36 (36%), 4 of 12 (33%), 14 of 17 (82%), and 3 of 3 (100%) tumors at stages I, II, III, and IV, respectively; P = 0.0026]. Furthermore, in adenocarcinomas, disease-free survival and overall survival were significantly shorter in patients with tumors harboring the methylated DAL-1 (P = 0.0011 and P = 0.045, respectively). CONCLUSIONS: DAL-1 methylation is involved in the development and progression of NSCLC and provides an indicator for poor prognosis.