Primary aldosteronism (PA) accounts for 5-10% of all hypertension. One of the major causes of PA is sporadic formation of aldosterone-producing adenomas (APAs). These benign tumours develop in the cortical region of adrenal glands and autonomously secrete excessive amounts of aldosterone. This hormone increases sodium retention and water reabsorption by the kidneys, leading to high blood pressure. Landmark discoveries of somatic mutations in APAs led to better understanding of molecular mechanisms causing autonomous aldosterone secretion. The first mutations were found in KCNJ5, followed by ATP1A1, ATP2B3 and CACNA1D, all encoding cation-channels or transporters. Several in vitro studies showed disruption of cellular ion-balance leading to the phenotype of hyper-aldosterone secretion from APAs. Following our lab's discovery of initial four somatic mutations by whole exome sequencing, over 30 single-base change mutations have been reported in the CACNA1D gene, which encodes the a1 subunit of an L-type Ca2+ channel (LTCC), CaV1.3. Initial and several subsequent mutations cause electrophysiological gain-of-function with increased activation and/or slowed inactivation of CaV1.3. Prior to the discovery of these mutations, L-type Ca2+ channels were not considered important in regulation of aldosterone production. In the first part of my thesis, I investigated two of the mutations and showed that the gain-of-function results in increased aldosterone secretion from an adrenocortical carcinoma cell line, H295R, when transiently transfected with the mutants. I also showed that CaV1.3 can play a role in physiological aldosterone secretion, finding that CYP11B2 expression is reduced by 50% in the adrenals of CaV1.3 knockout mice. The discovery of mutations in CACNA1D led to a drug discovery challenge award from a pharmaceutical company in which high-throughput screening of CaV1.3-expressing cells was undertaken against the company's 1.8M compound library. I identified the adrenal isoforms of the channel's alpha and beta subunits (CACNA1D and CACNB2), and helped development of the stable HEK293 cell line used for screening. This led to 3 tool compounds (A, B & C) that were selective antagonists for CaV1.3 over another family member of the ion channels in high-throughput electrophysiological experiments using IonWorks Barracuda and QPatch platforms. I showed compound B to effectively inhibit aldosterone secretion in both H295R and primary adrenal cells isolated from a normal adrenal. This finding is a significant step in developing compound B further into a CaV1.3-selective drug for treating PA patients without cardiovascular side effects as in the case of existing dihydropyridine class of Ca2+ channel blockers. The second part of my thesis focused on genotyping and whole exome sequencing of 59 APAs from 52 patients, in order to identify further genes underlying primary aldosteronism. Mutations in previously reported genes were identified in 34 of the APAs (57.6%). CACNA1D was the most commonly mutated gene (20.3%) in this cohort, but not KCNJ5 (16.9%) as previously reported. This variation in the frequencies observed is perhaps due to the different methods used for screening PA. For example, many of our patients were detected by renin measurement in resistant hypertension, and their APA identified by a unique PET-CT (using C11 metomidate), in place of adrenal vein sampling. In addition to this, novel somatic mutation was found in a gene not encoding an ion channel, however, this protein was previously linked to cell-cell adhesion and tumour suppression. The gene identified is CADM1, a cell adhesion molecule 1, and the mutation found leads to substitution of uncharged by negatively charged amino acid in the single transmembrane domain of this cell surface protein. The likely significance of this discovery was greatly enhanced when we ascertained that one of the 'private' somatic mutations found on whole exome sequencing of APAs in Munich was in fact a similar substitution in the adjacent amino acid of the membrane-spanning domain. High expression of CADM1 in zona glomerulosa (ZG) was found, the site of aldosterone synthesis in the adrenal cortex and in the APAs, as well as the aldosterone producing cell clusters (APCCs) within the ZG. In vitro experiments using H295R cells showed both mutations in CADM1 lead to 10-20 fold upregulation of CYP11B2 transcription, on qPCR, resulting in 2-4-fold increase of aldosterone secretion, compared to the wild-type CADM1. Despite the introduction of a negative charge into the transmembrane domain, both mutants could translocate to the cell surface. The evidence to date, points to the loss of cell-cell adhesion in the presence of mutant CADM1 as the cause of uncontrolled aldosterone synthesis. Silencing of CADM1 in H295R cells revealed downregulation of aldosterone synthesis and secretion. Transcriptome analysis by RNAseq, of H295R cells expressing wild-type or mutant CADM1 or silenced CADM1 showed a large number of differentially expressed genes. Mutant CADM1 upregulated genes involved in steroidogenesis and ACTH response pathways. A possible role of CADM1 was found to be in the regulation of inter-cell communication via gap junction protein, connexin-43 (Cx43). This was upregulated with higher expression on plasma membrane in the CADM1 silenced cells. TSG101, a protein involved in lysosomal degradation of Cx43 was downregulated in the absence of CADM1 and possibly the mechanism for increased Cx43 expression. Also, immunostaining of adrenal sections showed internalised para-nuclear staining localisation of Cx43 in the ZG, APAs and APCCs, regions with high CADM1 expression compared to membranous localisation of Cx43 in ZF. In contrast to the common and numerous mutations in CACNA1D, mutations in CADM1 are rare. Nonetheless, they may enhance our understanding of the functional significance of glomerular structure of the outer zone of adrenal cortex, where cell-cell adhesion and intercellular communication appear critical for the regulation of aldosterone secretion.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19567号 / 医博第4074号 / 新制||医||1013(附属図書館) / 32603 / 京都大学大学院医学研究科医学専攻 / (主査)教授 武田 俊一, 教授 小川 誠司, 教授 野田 亮 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Yes / Tenri Foundation

No description available.

No description available.

Protein product tumor suppressor PALB2 gene plays a major role in pathway of DNA repair of double-strand breaks throught the homologous recombination mechanism. Significance of its pathogenic variants in hereditary forms of breast cancer in BRCA1/2- negative patients in families with multiple breast cancers may be in the Czech Republic comparable with the BRCA2 gene. A role of the PALB2 gene in sporadic breast cancer occurence, which represent 90 - 95 % of all cancers, is still unknown. This thesis focuses on inactivation pathway of tumor suppressor PALB2 in the sporadic breast cancer by a mechanism of allelic loss detecting by loss of heterozygosity (LOH) of corresponding microsatellite markers and hypermethylation of promoter region as the most common mechanisms of inactivation tumor suppressors in early tumorigenesis. In a group of 51 nonselected patients with sporadic breast cancer we found four samples with PALB2 locus allelic loss. These samples were analyzed for somatic mutations. No mutation was found. There is no evidence of promotor hypermethylation in any of the samples. Our data suggest a role of the PALB2 gene inactivation in a minority group of sporadic breast cancers.

Endemic Burkitt lymphoma (eBL) is the most common pediatric cancer in malaria-endemic equatorial Africa and nearly always contains Epstein-Barr virus (EBV), unlike sporadic Burkitt Lymphoma (sBL) that occurs with a lower incidence in developed countries. Despite this increased burden the study of eBL has lagged. Additionally, while EBV was isolated from an African Burkitt lymphoma tumor 50 years ago, however, the impact of viral variation in oncogenesis is just beginning to be fully explored. In my thesis research, I focused on investigating molecular genetics of the endemic form of this lymphoma with a particular emphasis on the role of the virus and its variation in pathogenesis using novel sequencing and bioinformatic strategies. First, we sought to understand pathogenesis by investigating transcriptomes using RNA sequencing (RNAseq) from 30 primary eBL tumors and compared to sBL tumors. BL tumor samples were prospectively obtained from 2009 until 2012 in Kenya. Within eBL tumors, minimal expression differences were found based on anatomical presentation site, in-hospital survival rates, and EBV genome type; suggesting that eBL tumors are homogeneous without marked subtypes. The outstanding difference detected using surrogate variable analysis was the significantly decreased expression of key genes in the immunoproteasome complex in eBL tumors carrying type 2 EBV compared to type 1 EBV. Secondly, in comparison to previously published pediatric sBL specimens, the majority of the expression and pathway differences were related to the PTEN/PI3K/mTOR signaling pathway and was correlated most strongly with EBV status rather than the geographic designation. Moreover, the common mutations were observed significantly less frequently in eBL tumors harboring EBV type 1, with mutation frequencies similar between tumors with EBV type 2 and without EBV. In addition to the previously reported genes, we identified a set of new genes mutated in BL. Overall, these suggested that EBV, particularly EBV type 1, supports BL oncogenesis alleviating the need for particular driver mutations in the human genome. Second, we sought to comprehensively define sequence variations of EBV across the viral genome in eBL tumor cells and normal infections, and correlate variations with clinical phenotypes and disease risk. We investigated the whole genome sequence of EBV from primary tumors (N=41) and plasma from eBL patients (N=21) as well as EBV in the blood of healthy children (N=29) within the same malaria endemic region. We conducted a genome wide association analysis study with viral genomes of healthy kids and BL kids. Furthermore, we found that the frequencies of EBV types among healthy kids were at equal levels while they were skewed in favor of type 1 (70%) among eBL kids. To pinpoint the fundamental divergence between viral genome subtypes, type 1 and type 2, we constructed phylogenetic trees comparing to all public EBV genomes. The pattern of variation defined the substructures correlated with the subtypes. This investigation not only deciphers the puzzling pathogenic differences between subtypes but also helps to understand how these two EBV types persist in the population at the same time. Overall, this research provides insight into the molecular underpinning of eBL and the role of EBV. It further provides the groundwork and means to unravel the complexity of EBV population structure and provide insight into the viral variation that may influence oncogenesis and outcomes in eBL and other EBV-associated diseases. In addition, genomic and mutational analyses of Burkitt lymphoma tumors identify key differences based on viral content and clinical outcomes suggesting new avenues for the development of prognostic molecular biomarkers and therapeutic interventions.

The genus Populus is ideally suited to investigate questions related to the interplay between an individual’s environmental history and its capacity to respond to external stimuli. In order to dissect the influence of individual history on subsequent plant responses, transcriptome level changes due to water deficit were assessed in clonal populations of Populus hybrids. Results indicate variation in the drought transcriptomes of genetically identical clones originating from different locations can be shaped by the individual history of the clone. Additionally, yearly variations in drought transcriptome patterns showed specific trends associated with a clonal population that were not related to an unknown influence at a location, nor with the biological source of cuttings. Despite these sources of transcriptome variation, a common shared response was identified across all populations. The findings hint at the influence of the environment and epigenetic factors in the dynamic regulation of transcriptome level responses in clonal individuals.

The genus Populus is ideally suited to investigate questions related to the interplay between an individual’s environmental history and its capacity to respond to external stimuli. In order to dissect the influence of individual history on subsequent plant responses, transcriptome level changes due to water deficit were assessed in clonal populations of Populus hybrids. Results indicate variation in the drought transcriptomes of genetically identical clones originating from different locations can be shaped by the individual history of the clone. Additionally, yearly variations in drought transcriptome patterns showed specific trends associated with a clonal population that were not related to an unknown influence at a location, nor with the biological source of cuttings. Despite these sources of transcriptome variation, a common shared response was identified across all populations. The findings hint at the influence of the environment and epigenetic factors in the dynamic regulation of transcriptome level responses in clonal individuals.

Indiana University-Purdue University Indianapolis (IUPUI) / Reliable detection of low-frequency single nucleotide variants (SNVs) carries great significance in many applications. In cancer genetics, the frequencies of somatic variants from tumor biopsies tend to be low due to contamination with normal tissue and tumor heterogeneity. Circulating tumor DNA monitoring also faces the challenge of detecting low-frequency variants due to the small percentage of tumor DNA in blood. Moreover, in population genetics, although pooled sequencing is cost-effective compared with individual sequencing, pooling dilutes the signals of variants from any individual. Detection of low frequency variants is difficult and can be cofounded by multiple sources of errors, especially next-generation sequencing artifacts. Existing methods are limited in sensitivity and mainly focus on frequencies around 5%; most fail to consider differential, context-specific sequencing artifacts. To face this challenge, we developed a computational and experimental framework, RareVar, to reliably identify low-frequency SNVs from high-throughput sequencing data. For optimized performance, RareVar utilized a supervised learning framework to model artifacts originated from different components of a specific sequencing pipeline. This is enabled by a customized, comprehensive benchmark data enriched with known low-frequency SNVs from the sequencing pipeline of interest. Genomic-context-specific sequencing error model was trained on the benchmark data to characterize the systematic sequencing artifacts, to derive the position-specific detection limit for sensitive low-frequency SNV detection. Further, a machine-learning algorithm utilized sequencing quality features to refine SNV candidates for higher specificity. RareVar outperformed existing approaches, especially at 0.5% to 5% frequency. We further explored the influence of statistical modeling on position specific error modeling and showed zero-inflated negative binomial as the best-performed statistical distribution. When replicating analyses on an Illumina MiSeq benchmark dataset, our method seamlessly adapted to technologies with different biochemistries. RareVar enables sensitive detection of low-frequency SNVs across different sequencing platforms and will facilitate research and clinical applications such as pooled sequencing, cancer early detection, prognostic assessment, metastatic monitoring, and relapses or acquired resistance identification.