Nesta tese são apresentados um novo modelo estatístico para cálculo de probabilidade de paternidade e sua implementação em software. O modelo proposto utiliza o genótipo como informação básica, em contraste com outros modelos que usam alelos. Por esta diferença, o modelo proposto resulta mais abrangente, mas que, sob certas restrições, reproduz os resultados dos modelos que usam alelos. Este modelo foi implementado em um software que recebe descrições da genealogia e dos marcadores em uma linguagem dedicada a isso e constrói uma rede bayesiana para cada marcador. O usuário pode definir livremente a genealogia e os marcadores. O cálculo da probabilidade de paternidade é feito, sobre as redes construídas, por um software para inferência em redes bayesianas e a probabilidade de paternidade combinada considerando todos os marcadores é calculada, resultando em um \"índice de paternidade. / This thesis presents a novel statistical model for calculation of the probability of paternity and its implementation as a software. The proposed model uses genotype as basic information. Other models use alleles as basic information. As a result the proposed model is broader, in the sense that, under certain constraints the results from the other models are reproduced. The software implementation receives pedigree and markers data, in a specifically designed language, as input and builds one bayesian network for each marker. The user can freely define any pedigree and any marker. Paternity probabilities for each locus are calculated, from the built networks, by a software for inference on Bayesian Networks and these probabilities are combined into a single \"paternity index\".

Nesta tese são apresentados um novo modelo estatístico para cálculo de probabilidade de paternidade e sua implementação em software. O modelo proposto utiliza o genótipo como informação básica, em contraste com outros modelos que usam alelos. Por esta diferença, o modelo proposto resulta mais abrangente, mas que, sob certas restrições, reproduz os resultados dos modelos que usam alelos. Este modelo foi implementado em um software que recebe descrições da genealogia e dos marcadores em uma linguagem dedicada a isso e constrói uma rede bayesiana para cada marcador. O usuário pode definir livremente a genealogia e os marcadores. O cálculo da probabilidade de paternidade é feito, sobre as redes construídas, por um software para inferência em redes bayesianas e a probabilidade de paternidade combinada considerando todos os marcadores é calculada, resultando em um \"índice de paternidade. / This thesis presents a novel statistical model for calculation of the probability of paternity and its implementation as a software. The proposed model uses genotype as basic information. Other models use alleles as basic information. As a result the proposed model is broader, in the sense that, under certain constraints the results from the other models are reproduced. The software implementation receives pedigree and markers data, in a specifically designed language, as input and builds one bayesian network for each marker. The user can freely define any pedigree and any marker. Paternity probabilities for each locus are calculated, from the built networks, by a software for inference on Bayesian Networks and these probabilities are combined into a single \"paternity index\".

<p>The objective of the study was to conduct an internal validation on miniSTR systems and apply it to cases received from the South African Missing Persons Task Team (SAMPTT). This was prompted by the fact that miniSTR systems have been shown to out perform some of the commercial kits available in the time of the study and provide an alternative to mtDNA when analysing degraded DNA from skeletal remains and that the DNA extracted from skeletal remains received from the SAMPTT would be degraded due to the remains generally being fragmented or charred and buried for many years. The miniSTR loci chosen for validation comprised the Combined DNA Index System (CODIS) thirteen core loci and were arranged into four triplexes and one uniplex.</p>

<p>The objective of the study was to conduct an internal validation on miniSTR systems and apply it to cases received from the South African Missing Persons Task Team (SAMPTT). This was prompted by the fact that miniSTR systems have been shown to out perform some of the commercial kits available in the time of the study and provide an alternative to mtDNA when analysing degraded DNA from skeletal remains and that the DNA extracted from skeletal remains received from the SAMPTT would be degraded due to the remains generally being fragmented or charred and buried for many years. The miniSTR loci chosen for validation comprised the Combined DNA Index System (CODIS) thirteen core loci and were arranged into four triplexes and one uniplex.</p>

Post-zygotic genetic variation has previously been shown in healthy individuals and linked to various disorders. The definition of post-zygotic or somatic variation is the existence of genetically distinct populations of cells in a subject derived from a single zygote. Structural changes in the human genome are a major type of inter-individual genetic variation and copy number variation (CNV), involving changes in the copy number of genes, are one of the best studied category of structural genetic changes. In paper I we reported a pair of healthy female monozygotic (MZ) twins discordant for aneuploidy of chromosomes X and Y, contributing to the delineation of the frequency of somatic variation in MZ twins. It also illustrates the plasticity of the genome for tolerating large aberrations in healthy subjects. In paper II we showed age-related accumulation of copy number variation in the nuclear genomes in vivo for both megabase- and kilobase-range variants. Using age-stratified MZ twins and single-born subjects, we detected megabase-range aberrations in 3.4% of people ≥60 years old but not in individuals younger than 55 years. Moreover, the longitudinal analysis of subjects with aberrations suggests that the aberrant cell clones are not immortalized and disappear from circulation. We also showed that sorted blood cells display different genomic profiles.  The detected recurrent rearrangements are candidates for common age-related defects in blood cells. This work might help to describe the cause of an age-related decline in the number of cell clones in the blood, which is one of the hallmarks of immunosenescence. In paper III we described a variable number tandem repeat (VNTR) ~4 kb upstream of the IFNAR1 gene, which was somatically variable.  We detected 14 alleles displaying inter- and intra-individual variation. Further analyses indicated strong clustering of transcription factor binding sites within this region, suggesting an enhancer. This putative VNTR-based enhancer might influence the transcriptional regulation of neighboring cytokine receptor genes and the pathways they are involved in. These three studies stress the importance of research on post-zygotic variation in genetics. Furthermore, they emphasize that biobanks should consider sampling of multiple tissues to better address this issue in the genetic studies.

Magister Scientiae - MSc / The objective of the study was to conduct an internal validation on miniSTR systems and apply it to cases received from the South African Missing Persons Task Team (SAMPTT). This was prompted by the fact that miniSTR systems have been shown to out perform some of the commercial kits available in the time of the study and provide an alternative to mtDNA when analysing degraded DNA from skeletal remains and that the DNA extracted from skeletal remains received from the SAMPTT would be degraded due to the remains generally being fragmented or charred and buried for many years. The miniSTR loci chosen for validation comprised the Combined DNA Index System (CODIS) thirteen core loci and were arranged into four triplexes and one uniplex. / South Africa

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18161号 / 医博第3881号 / 新制||医||1003(附属図書館) / 31019 / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊達 洋至, 教授 杉田 昌彦, 教授 中山 健夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The determination and application of Analytical Threshold (AT) is a vital part of the forensic Deoxyribonucleic Acid (DNA) internal validation process. AT is the relative fluorescence unit (RFU) signal at which allelic peaks can be confidently distinguished from baseline noise. Several methods of calculating AT are currently being implemented within the forensic DNA community. These methods may utilize DNA negative sample data, DNA positive sample data, or both in their calculations. In this study, two of the DNA positive-based AT calculation techniques were chosen for assessment and comparison. The simplified upper limit approach (ULA) and the cost-based approach. ATs were calculated for each dye channel using a dilution series of 3 single source DNA samples ranging from 0.05-0.8ng. The ATs calculated via the cost-based approach consistently exhibited lower values than those determined via the ULA. As a result, the incidence of allelic drop-out exhibited by these AT values was also consistently lower, with an equivalent or only marginally increased incidence of baseline noise drop-in. These results indicated that the cost-based approach may be a more effective and practical method of calculating AT than the ULA, particularly in the analysis of low DNA template samples.

It is important to understand the entire spectrum of somatic variants to gain more insight into mutations that occur in different cancers for development of better diagnostic, prognostic and therapeutic tools. This thesis outlines our work in understanding somatic variant calling, improving the identification of somatic variants from whole genome and whole exome platforms and identification of biomarkers for lung cancer. Integrating somatic variants from whole genome and whole exome platforms poses a challenge as variants identified in the exonic regions of the whole genome platform may not be identified on the whole exome platform and vice-versa. Taking a simple union or intersection of the somatic variants from both platforms would lead to inclusion of many false positives (through union) and exclusion of many true variants (through intersection). We develop the first framework to improve the identification of somatic variants on whole genome and exome platforms using a machine learning approach by combining the results from two popular somatic variant callers. Testing on simulated and real data sets shows that our framework identifies variants more accurately than using only one somatic variant caller or using variants from only one platform. Short tandem repeats (STRs) are repetitive units of 2-6 nucleotides. STRs make up approximately 1% of the human genome and have been traditionally used as genetic markers in population studies. We conduct a series of in silico analyses using the exome data of 32 individuals with lung cancer to identify 103 STRs that could potentially serve as cancer diagnostic markers and 624 STRs that could potentially serve as cancer predisposition markers. Overall these studies improve the accuracy in identification of somatic variants and highlight the association of STRs to lung cancer. / Ph. D.

La première partie de nos travaux a consisté au séquençage de génomes complets de trois souches pathogènes et de trois souches de portage de Staphylococcus lugdunensis pour les comparer aux 15 génomes complets disponibles sur NCBI. Aucun déterminant génétique associé au contexte de virulence ou de portage de S. lugdunensis n’a été identifié. Cependant, nous avons mis en évidence la présence d’éléments génétiques mobiles et des variations dépendantes des complexes clonaux,définis par MultiLocus Sequence Typing, au sein de loci potentiellement associés à la virulence. Des variations ont été observées dans un locus homologue à celui de Staphylococcus aureus codant le système de sécrétion Ess/type VII (SST7). Nous avons mis en évidence huit organisations génétiques chez cette espèce présentant pourtant une structure de population clonale. La seconde partie de nos travaux a consisté à la caractérisation phénotypique et moléculaire du SST7 chez S. lugdunensis par la formation d’un mutant de délétion du gène essC codant une protéine essentielle à la sécrétion. Nos résultats suggèrent que le SST7 serait impliqué dans la translocation de protéines prédites in silico comme impliquées dans la virulence. Néanmoins, dans des modèles de cytotoxicité cellulaire et d’infection du nématode Caenorhabditis elegans, aucune atténuation de la virulence n’a été observée chez la souche mutante malgré une perte de sa capacité à lyser les erythrocytes, comparativement à la souche sauvage. Nos travaux ont également permis de développer et d’évaluer le pouvoir discriminant de trois nouvelles méthodes de typage constituant des outils très prometteurs pour l’épidémiologie moléculaire des infections à S. lugdunensis. / The first part of this study consisted in whole genome sequencing of three pathogenic and three carriage strains of S. lugdunensis and comparison with the 15 genomes available in the NCBI. No genetic determinant was associated to the pathogenic or carriage context. However, we have highlighted the presence of mobile genetic elements and MultiLocus Sequence Typing clonal complex dependent variations within loci potentially associated with virulence. Variations wereobserved in the ess locus homologous to that of Staphylococcus aureus encoding the type VII secretion system (T7SS). We showed eight genetic organizations in this species with a clonal population structure. The second part of our work consisted in a phenotypic and molecular characterization of T7SS in S. lugdunensis by construction of a deletion essC gene mutant. This gene encodes a protein requiredfor protein secretion. Our results suggest that T7SS could be involved in translocation of proteins predicted as implicated in virulence in silico. Nevertheless, no virulence attenuation was observed in cells cytotoxicity assay and Caenorhabditis elegans virulence assays between wild-type and mutant strains which yet has lost the ability to lyse erythrocytes. We also developed and evaluated discriminating power of three new typing methods, which are very promising tools for the molecular epidemiology of S. lugdunensis infections.