Chronic recurrent multifocal osteomyelitis (CRMO) is a rare, pediatric, autoinflammatory disease characterized by bone pain due to sterile osteomyelitis, and is often accompanied by psoriasis or inflammatory bowel disease. There are two syndromic forms of CRMO, Majeed syndrome and DIRA, for which the genetic cause is known. However, for the majority of cases, the genetic basis is unknown. Via whole-exome sequencing and linkage analysis, we determined the most likely causative mutations in four families. While the mutations are in three different genes – FBLIM1, PLCG2 and PIP; all three genes are involved in Fcγ signaling and osteoclast activation. In a large cohort of 61 individuals with CRMO, we performed gene and pathway based association analysis using the 1000 genomes participants of European ancestry as controls. One gene from the family-based analyses, ANO6, was significantly enriched for rare variants in our cohort of cases. ANO6 is involved in P2RX7- mediated inflammasome activation and in the regulation of bone mineralization. While no pathways were enriched for rare variants in the CRMO cohort after genome-wide correction, four pathways were significantly enriched for rare variants in the control samples, indicating a protective effect of the variants. The second most significant pathway, activation of chaperone genes by XBP1s, is relevant to CRMO pathogenesis as XBP1s is a transcription factor that attenuates ER stress, and regulates the expression of genes involved in RANKL signaling and bone remodeling. An association analysis using a larger set of cases followed by functional validation of candidate genes is necessary to confidently declare the mutations isolated in the work presented here to be pathogenic. Our preliminary findings suggest that mutations in genes involved in both the inflammatory response and bone remodeling underlie the pathogenesis of CRMO.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21254号 / 医博第4372号 / 新制||医||1029(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山田 亮, 教授 Shohab YOUSSEFIAN, 教授 小杉 眞司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Craniosynostosis is a disorder characterized by the premature fusing of cranial sutures in an infant. Premature closure of these sutures can lead to detrimental consequences on the development of a child. The two broad categories of craniosynostosis are classified as syndromic and nonsyndromic. Nonsyndromic craniosynostosis involves only the fusion of one or more sutures, whereas syndromic craniosynostosis involves other abnormalities throughout the body of the affected individual. Two of the families analyzed in this study were of the syndromic nature, and known FGFR mutations were discovered. However, phenotypical features documented in association with these mutations differed from our individuals. Two families affected with nonsyndromic sagittal synostosis were also analyzed. Within one of these families, three candidate mutations were identified as possible disease causing mutations. These mutations were found in the genes ITGAV, SLC30A9, and BAMBI. Here we analyze the function of these proteins and determine the significance of the role they may play in nonsyndromic craniosynostosis.

Novel technologies for genome analysis have provided almost unlimited opportunities to uncover structural gene variants behind human disorders. Whole exome sequencing (WES) is especially useful for understanding rare Mendelian conditions, because it reduces the requirements for a priori clinical data, and can be applied on a small number of patients. However, supporting functional data on the effect of specific gene variants are often required to power these findings. A variety of methods and biological model systems exists for this purpose. Among those, induced pluripotent stem cells (iPSCs), which are capable of self-renewal and differentiation, stand out as an alternative to animal models. In papers I and II we took advantage of WES to identify gene variants underlying autosomal recessive pure hair and nail ectodermal dysplasia (AR PHNED) as well as autosomal dominant familial visceral myopathy (FVM). We identified a homozygous variant c.821T>C (p.Phe274Ser) in the KRT74 gene as the causative mutation in AR PHNED, supported by the fact that Keratin-74 was undetectable in hair follicles of an affected family member. In a family segregating FVM we found a heterozygous tandem base substitution c.806_807delinsAA (p.(Gly269Glu)) in the ACTG2 gene in the affected members. This novel variant is associated with a broad range of visceral symptoms and a variable age of onset. In Paper III we explored the similarity between clonally derived iPSC lines originating from a single parental fibroblast line and we highlighted the necessity to use lines originating from various donors in disease modeling because of biological variation. Paper IV focused on how the genomic integrity of iPSCs is affected by the choice of reprogramming methods. We described several novel cytogenetic rearrangements in iPSCs and we identified a chromosome 5q duplication as a candidate aberration for growth advantage. In summary, this doctoral thesis brings novel findings on unreported disease-causing variants, as supported by extensive genetic analysis and functional data. A novel molecular mechanism behind AR PHNED is presented and the phenotypic spectrum associated with FVM is expanded. In addition, the thesis brings novel understanding of benefits and limitations of the iPSC technology to be considered for disease modeling.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18156号 / 医博第3876号 / 新制||医||1003(附属図書館) / 31014 / 京都大学大学院医学研究科医学専攻 / (主査)教授 野田 亮, 教授 武藤 学, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Yes / Currently, out of the 82 US FDA-approved targeted therapies for adult cancer treatments, only three are approved for use in children irrespective of their genomic status. Apart from leukemia, only a handful of genomic-based trials involving children with solid tumors are ongoing. Emerging genomic data for pediatric solid tumors may facilitate the development of precision medicine in pediatric patients. Here, we provide an up-to-date review of all reported genomic aberrations in the eight most common pediatric solid tumors with whole-exome sequencing or whole-genome sequencing data (from cBioPortal database, Pediatric Cancer Genome Project, Therapeutically Applicable Research to Generate Effective Treatments) and additional non-whole-exome sequencing studies. Potential druggable events are highlighted and discussed so as to facilitate preclinical and clinical research in this area. / Seed Grant of Strategic Research Theme for Cancer, The University of Hong Kong of AKSC. VWY Lui is funded by the Research Grant Council, Hong Kong (#17114814, #17121616, General Research Fund; T12–401/13-R, Theme-based Research Scheme), and the Start-up Fund, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong. W Piao is funded by the Faculty Postdoctoral Fellowship Scheme, Faculty of Medicine, the Chinese University of Hong Kong.

Next-Generation Sequencing has opened the doors to nearly limitless amounts of genomic data, but the clinical utility of this data is not yet clear. From examining at sequencing data of known familial cancer genes in hereditary cancer patients, the NCGENES study found a clear molecular diagnosis in about 5% of patients and an uncertain molecular result in about 15% of patients. The remaining 80% of hereditary cancer patients received a negative result for the screening of known cancer genes. These latter patients were followed up by whole exome sequencing analysis, and the data was used to perform a research sweep to potentially identify mutation(s) in gene(s) that have yet to be clearly associated with their phenotype. Hereditary breast cancer has a relatively well-established set of susceptibility genes, yet a large percentage of the molecular etiology is still unknown. There are many genes that are good candidates for breast cancer genes based on their protein's function, but they may not actually contribute to breast cancer susceptibility. The ClinGen consortium is aiming to establish the clinical validity of gene-disease associations so that clinicians and patients can better interpret and utilize sequencing results. Six breast cancer susceptibility genes were evaluated using the ClinGen clinical validity framework with the goal of both evaluating the genes already on hereditary breast cancer panels and evaluating genes not yet widely tested to determine if there is enough evidence to support their role in disease to warrant widespread testing. These genes have varying levels of evidence supporting their role in breast cancer susceptibility. The variants in each of the six genes were compared between a cancer patient cohort and a non-cancer patient cohort enrolled in the NCGENES whole exome sequencing study. One likely pathogenic variant and several variants of unknown significance were identified in various genes, and the burden of variants in cancer cases versus controls was evaluated, although the controls were not matched to the cancer cohort in any way. Research sweeps were performed for patients with VUSs to ensure that there were no other mutations in genes that would better fit the phenotype. This thesis presents a method for evaluating gene-disease associations and for utilizing whole exome sequencing data to pinpoint a molecular diagnosis in hereditary breast cancer patients. Overall, it was found that the ClinGen method of evaluating clinical validity of gene-disease associations could be helpful when determining if variants are pathogenic or benign. A new gene, RINT1, was found to have enough evidence to be moderately associated with hereditary breast cancer and it was subsequently added to the diagnostic list so that all cancer patients will now be screened for RINT1 variants. In addition, it was found that two of the genes currently on the diagnostic list, RAD51C and RAD51D, have "disputed" evidence with respect to breast cancer susceptibility. Interestingly, they have much more evidence for an association with ovarian cancer, so if variants are found in these genes, the patient's phenotype should be considered when evaluating them. It was also shown that PALB2, an established breast cancer susceptibility gene, indeed is definitively associated with breast cancer, and the NCGENES cancer patients have more truncating variants than the controls, further validating the clinical validity assertion. Finally, an ovarian cancer patient with two interesting variants, one in SLX4 and one in GEN1, were evaluated. Studies showed that knocking out both of these genes' pathways was highly destructive to the cell. A VUS was found in each of these genes, and it was hypothesized that perhaps these two variants together may be sufficient to contribute to this patient's cancer susceptibility.