京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19545号 / 理博第4205号 / 新制||理||1603(附属図書館) / 32581 / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 村山 美穂, 教授 幸島 司郎, 教授 伊谷 原一 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20789号 / 医博第4289号 / 新制||医||1025(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 大森 孝一, 教授 高田 穣 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Rare diseases represent a heterogeneous group of more than ~7000 different diseases, affecting 3,5-5,9% of the global population. Most rare diseases are genetic, but causal genes are known only in some of them. Many patients with rare diseases remain without a diagnosis, which is crucial for genetic counseling, prevention, and treatment. With the development of new methods of genome analysis, decreasing cost of sequencing, and increasing knowledge of the human genome, a new concept for identifying disease-causing genes was established. It is based on comparing the patient's genetic variability with the genetic variability of the general population. This dissertation describes next-generation sequencing technologies (NGS), bioinformatic analysis of acquired data and their applications in the elucidation of molecular underpinnings of rare genetic diseases. These procedures have led to the identification and characterization of causal genes and gene mutations in autosomal dominant tubulointerstitial kidney disease (SEC61A1, MUC1), autosomal dominant neuronal ceroid lipofuscinosis (CLN6, DNAJC5), neurodegenerative disease of unknown etiology (VPS15), Acadian variant of Fanconi syndrome (NDUFAF6) and spinal muscular atrophy (SMN1). The application of novel genome analysis techniques increased the...

Polycystic kidney disease is a severe genetic disease occurring in both adult and pediatric patients. The basic characteristic of this disease is the development and progressive enlargement of renal cysts gradually replacing functional kidney tissue. This leads to renal failure in many patients. However, renal cysts may also occur in a number of other diseases, including multisystem syndromes. This complicates differential diagnosis in some patients. In our study, we first focused on the diagnosis and characterization of genotypic-phenotypic relationships in patients with polycystic disease arising in childhood, later we extended our study to adult patients and patients with unclear clinical diagnosis. At the same time, we expanded the portfolio of analyzed disorders to a number of diseases in which the phenotype of polycystic kidneys may occur, and noncystic diseases as well. During our project, massive parallel sequencing was used to analyze 149 patients - 128 with cystic and 21 with noncystic clinically diagnosed nephropathies. At the same time, the findings were verified by Sanger sequencing in 176 relatives of our probands. Mutation detection reached 59% in cystic patients, and 43% in non-cystic patients, respectively. In many patients, molecular genetic analysis revealed a different etiology...

BK polyomavirus (BKV) is associated with urinary apparatus pathogenesis in kidney transplant recipient. Immune suppression triggers BKV reactivation that potentially causes polyomavirus associated nephropathy (PVAN), a major post-transplant problem causes graft rejection. Antiviral immunity plays the key role in limiting the viral replication but selection by the immune system or antivirals may cause the evolvement of escape variants with higher fitness. Mutation in VP1, the major capsid protein can allow BKV to escape neutralizing antibodies. In an attempt to detect co-infection and minor variants, BKV VP1 genomic region was amplified by PCR and analysed by deep sequencing from plasma samples of four kidney transplant recipients. BKV genotype I and IV was identified in patients and each patient was detected with one BKV genotype. Multiple point mutations and subsequent changes in amino acid were detected in majority, three out of four, of the patients.

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Chapter 1. The family Pteronarcyidae (Plecoptera) is a highly studied group of stoneflies and very important to a wide variety of aquatic studies. Several phylogenies have been proposed for this group recent decades, however there is little congruence between the various topologies. The present study revises the phylogeny of the group by combining molecular data from mitochondrial cytochrome oxidase subunit II, ribosomal subunit 12S, ribosomal subunit 16S, and nuclear loci ribosomal subinit 18S and Histone H3, with published morphological data in a parsimony-based total evidence analysis. The analysis produced a well-supported phylogeny with novel relationships within the genus Pteronarcys. Maximum Likelihood and Bayesian analyses produced topologies congruent with parsimony analysis. Character mapping revealed several homoplasious morphological characters that were previously thought to be homologous. Chapter 2. Phylogeographic studies in aquatic insects provide valuable insights into mechanisms that shape the genetic structure of aquatic communities. Yet studies that include broad geographic areas are uncommon for this group. We conducted a broad scale phylogeographic analysis of P. badia across western North America. In order to allow us to generate a larger mitochondrial data set, we used 454 seqeuncing to reconstruct the complete mitochondrial genome in the early stages of the project. Our analysis reveals what appears to be a complex history of isolation and multiple invasions among some lineages. The study provides evidence of multiple glacial refugia and suggests that historical climactic isolations have been important mechanisms in determining genetic structure of insects in western North America. Our ability to generate a large mitochondrial data set through mitochondrial genome reconstruction greatly improved nodal support of our mitochondrial gene tree, and allowed us to make stronger inference of relationships between lineages and timing of divergence events.