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.

Haploid vererbte Polymorphismen des Y-Chromosoms sind wichtige diagnostische Werkzeuge der forensischen Genetik und verwandter Disziplinen, insbesondere der Anthropologie. Geschlechtsspezifität und uniparentaler Erbgang der Merkmale ermöglichen eine Reihe von Untersuchungen, die mit autosomalen Markern erfolglos bleiben müssen. Kurze tandem-repetitive STR-Sequenzen, die polymorphen Marker der Wahl im forensischen Labor, sind auch auf dem Y-Chromosom nachzuweisen. Aufgrund der rekombinationsfreien, paternalen Vererbung des größten Teils des Y-Chromosoms werden locus-spezifische Allele hier en bloc, als hochinformativer Haplotyp, vererbt. Die forensische Untersuchung profitiert insbesondere auf dem Gebiet der Untersuchung biologischer Spuren von der Y-chromosomalen Diagnostik: vor allem in Vergewaltigungsfällen kann die männliche DNA-Fraktion der Abstrichpräparate unabhängig von der weiblichen des Opfers untersucht und individualisiert werden. Bei der Abstammungsuntersuchung wird in solchen Fällen die Y-chromosomale Analyse empfohlen, in denen der Vater (eines männlichen Kindes) nicht zur Verfügung steht und paternale Verwandte an seiner statt untersucht werden müssen. Von den 14 evaluierten Y-chromosomalen STR-Systemen sind 9 für die forensische Praxis ausgewählt und empfohlen worden. Sie bilden den sogenannten "minimal haplotype", der heute international für die o. g. Analysen verwendet und von der zuständigen Fachgesellschaft (International Society of Forensic Genetics) in ihren Richtlinien empfohlen wird. Wegen der immensen Haplotyp-Variabilität und des uniparentalen Erbgangs ist die Frequenzbestimmung, und damit die Entscheidungsfindung für rechtsmedizinische Gutachter, nur über den Zugang zu großen Datenbanken möglich. Zu diesem Zweck wurde an der Charité die "European Y-STR Haplotype Reference Database" eingerichtet, die Frequenzabfragen auf Grundlage des aktuellen Datenmaterials online ermöglicht (http://ystr.charite.de). Aufgrund des uniparentalen Erbmodus und der im Vergleich zu Autosomen verringerten effektiven Zahl von Y-Chromosomen in der Population muß mit einem meßbaren Einfluß genetischer Drift auf die Y-STR-Haplotyp-Verteilung in der Population gerechnet werden. Mit Hilfe der AMOVA (Analysis of Molecular Variance) - Methode konnten genetische Distanzen für eine repräsentative Auswahl von über 50 weltweit verteilter Populationen berechnet werden. Der AMOVA-Test ist unentbehrlich für die Überprüfung der Eignung von Referenzdatenbanken als Grundlage der Frequenzbestimmung von Y-STR-Haplotypen. / There are a number of merits that qualify the Y chromosome as a special forensic genetic tool: the male specificity for most of its length, the absence of recombination which provides unambiguous male lineage's and the small effective population size that tends to create population specific allele distributions of the Y chromosomes. Particularly in cases of rape and other sexual assault as well as in kinship testing, Y-STR haplotyping can help to close informativity gaps. Since the main goal of forensic genetics is individualization of persons or lineages of descent an analytical strategy for the male chromosome must enable the expert to differentiate between the majority of unrelated haplotypes. For this to achieve the choice of the sequence type and its variability (i.e. its mutation rate) as well as the number of individual sequences to be used for profiling is crucial. We have introduced a STR profile for the Y-chromosome consisting of 11 microsatellite sequences which is both informative for individualization purposes as well as for a genetic distance analysis of populations. The technical simplicity of the approach led to a rapid introduction of the technique in many of the forensic labs world-wide. Intense international collaboration facilitates the generation of large haplotype reference databases, most of them are online available and searchable (Europe: http://ystr.charite.de and USA: http://www.ystr.org/usa/). By use of haplotype specific parameters such as the molecular distance (which equals the minimum number of mutational steps separating two haplotypes) and the largest available haplotype databases a Bayesian approach to evaluate Y-STR haplotype matches has been proposed. Directly inspired by our work are the recommendations of the International Society of Forensic Genetics (ISFG). These guidelines state some basic principles on forensic analysis using Y-STR polymorphisms: the use of sequenced allelic ladders, the application of a repeat-based nomenclature and the use of suitable haplotype reference databases for statistical evaluation of matches. Still a matter of research , but of the utmost interest is the potential of the Y-chromosome analysis to unravel the ethnological background of a given male profile. A dual approach - that using Y-STRs as well as Y-SNPs - probably renders the maximum amount of information about the descent of a male lineage typed in a forensic specimen.