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Human Mendelian disease in Saudi Arabia is both significant and challenging. Next-generation sequencing (NGS) has resulted in important discoveries of the genetic variants responsible for inherited disease. However, the success of clinical genomics using NGS requires accurate and consistent identification of rare genome variants. Rarity is one very important criterion for pathogenicity. Here we describe a model to detect variants by analyzing allele frequencies of a Saudi population. This work will enhance the opportunity to improve variant calling workflow to gain robust frequency estimates in order to better detect rare and unusual variants which are frequently associated with inherited disease.

Entre as muitas aplicações das tecnologias de identificação biológica humana, estão as finalidades forenses. O objetivo desta pesquisa foi verificar frequências alélicas de Short Tandem Repeat (STR) e os parâmetros estatísticos de interesse em genética de populações e forense para desenvolver o primeiro banco de dados populacional de DNA na Faculdade de Odontologia de Bauru, Universidade de São Paulo, (FOB/USP) para futuros usos forenses. Frequências alélicas de 15 locos autossômicos e do marcador de gênero amelogenina foram determinadas utilizando amostras de 200 μL de saliva doados por 296 alunos de graduação da FOB/USP, com idade ≥ 18 anos, após aprovação ética. Os testes laboratoriais foram feitos com kits comerciais. Resultados e parâmetros estatísticos foram obtidos por meio de programas clássicos: GeneMapper-ID-X, MS Excel 2002 versão 10.6871.6870, GenAlEx 6.5 e Arlequin 3.5, comparando quatro populações (brasileira, portuguesa, norte-americana e a população deste estudo). Os locos mais polimórficos foram D18S51 (17 alelos) e FGA (15 alelos), seguidos pelo D21S11 (13 alelos) e os menos polimórficos foram D16S539 e TH01 (7 alelos cada). A análise comparativa com amostra da população brasileira proveniente de estudos anteriores (n > 100.000) pelo teste goodness of fit X2 index não mostrou diferenças significativas entre estes grupos (p = 0,9999). Outros parâmetros estatísticos foram calculados comparando as populações: local (deste estudo), portuguesa e norte-americana. A análise de variância molecular (AMOVA) entre as três populações, entre as pessoas da mesma população e para cada pessoa de cada população mostrou que existe uma elevada variância individual (99%), que esta variância é mantida uniformemente entre as pessoas da mesma amostra/região (1%) e entre as três populações estudadas (0%). O estudo confirmou o elevado grau de polimorfismo e a alta heterozigosidade (96,5%) da população. Houve diferença significativa quanto ao gênero (79,7% mulheres) quando comparado à população brasileira em geral (50,4%), explicada pelas características do corpo discente da FOB/USP composto por 80,6% de pessoas do gênero feminino. Interessante foi a observação de uma microvariante alélica no loco D18S51, fora da escada padrão e da escala de abrangência do kit, correspondente ao alelo 29, ainda não definida na base de dados internacional (STRBase, atualizada em 07/08/2015). Esta microvariante deverá ser confirmada por testes familiares e sequenciamento de DNA para verificar a possibilidade de outra ocorrência familiar ou duplicação de nucleotídeos. No futuro, os dados obtidos neste estudo devem ser incorporados ao banco de dados da população brasileira e podem ser considerados como referência genética da população regional, ajudando a elucidar casos forenses. Após a confirmação, a potencial nova microvariante alélica contribuirá para a base de dados internacional STRBase. / There are many ways of applying biological human identification technologies, among these are forensic applications. The objective of this study was to verify allele frequencies for 15 autosomal short tandem repeat (STR) loci and develop the first human DNA population database at the Bauru School of Dentistry, University of São Paulo (FOB/USP), for future forensic uses. Allele frequencies for these STR loci and an amelogenin gender marker were determined using 200 μL samples of saliva donated by 296 undergraduates from FOB/USP who were ≥ 18 years old at the time of the sample collect after signing a consent form with ethical approval. For laboratory tests, commercial kits were used. Results and statistical parameters were obtained using the following software: GeneMapper IDX (version 1.5), MS Excel 2002 (version 10.6871.6870), GenAlEx (version 6.5) and Arlequin (version 3.5) to compare four populations (Brazil, Portugal, U.S. and this study population). Results indicated that the most polymorphic loci were D18S51 (17 alleles) and FGA (15 alleles), followed by D21S11 (13 alleles); the least polymorphic loci were D16S539 and TH01 (7 alleles each). Various Brazilian populations (n > 100,000) from other studies were compared with this studys Brazilian population using a goodness-of-fit chi-squared test, and no significant differences in these frequencies were observed between these two population groups (p = 0.9999). Other forensic and population genetic statistical parameters were calculated comparing this studys population with Portugal and U.S. populations. For example, an analysis of molecular variance (AMOVA) among all populations, among people of the same population and for each person for each population, showed that people have high individual variance (99%) and that this variance is maintained evenly between people of the same sample/region (1%) and among the three populations studied (0%). This study reinforced the conclusion of other allele frequency population studies for the 15 autosomal STR loci tested, confirming high polymorphic grade and high heterozygosity (96.5%). There were significantly more women in the study (79.7%) when compared to the general Brazilian population (50.4%) since the student body of FOB/USP is 80.6% female. Interestingly, an off-ladder D18S51 allele micro-variance corresponding to the allele 29, not yet identified, was found which should be confirmed using paternity and sequencing tests to verify the possibility of either familial occurrence or nucleotide duplication. In the future, due to the small differences found, the parameters obtained in this study should be incorporated into the Brazilian population database and be considered for a regional population genetic prototype database potentially aiding forensic cases with comparisons and calculations. After confirmation, the potentially new micro variant allele found could be included in the international STRBase.

This study reexamines the nuclear microsatellite analysis by Rooney et al. (1999a) of Bering-Chukchi-Beaufort Seas bowhead whales (Balaena mysticetus) to determine if this population underwent a genetic bottleneck as a result of 19th and early 20th Century commercial whaling. This investigation used more accurate laboratory techniques to score alleles, had a larger sample size that was divided into two groups (mainland Alaska and St. Lawrence Island (SLI)), and used a moderately different set of microsatellite loci which are more variable and thus, more informative. The results corroborate the findings of Rooney et al. (1999a) for mainland Alaska showing no evidence of a genetic bottleneck. However, the SLI data analyses provide conflicting conclusions. The Wilcoxon test is significant for a heterozygote excess (p = 0.042) suggesting that a genetic bottleneck has occurred. This is not substantiated by the exact tests of each locus or the table-wide sign test. There is a possibility that a bottleneck has occurred, but due to the small sample size this is not a definitive conclusion and warrants reanalysis with a larger sample size.

Background: The limited within-breed genetic heterogeneity and an enrichment of disease-predisposing alleles have made the dog a very suitable model for the identification of genes associated with risk for specific diseases. Canine mammary cancer is an example of such a disease. However, the underlying inherited risk factors for canine mammary tumours (CMTs) are still largely unknown. In this study, 52 single nucleotide polymorphisms (SNPs) in ten human cancer-associated genes were genotyped in two different datasets in order to identify genes/alleles associated with the development of CMTs. The first dataset consisted of English Springer Spaniel (ESS) CMT cases and controls. ESS is a dog breed known to be at increased risk of developing CMTs. In the second dataset, dogs from breeds known to have a high frequency of CMTs were compared to dogs from breeds with a lower occurrence of these tumours. Results: We found significant associations to CMT for SNPs and haplotypes in the estrogen receptor 1 (ESR1) gene in the ESS material (best P-Bonf = 0.021). A large number of SNPs, among them several SNPs in ESR1, showed significantly different allele frequencies between the high and low risk breed groups (best P-Bonf = 8.8E-32, best P-BPerm = 0.076). Conclusions: The identification of CMT-associated SNPs in ESR1 in two independent datasets suggests that this gene might be involved in CMT development. These findings also support that CMT may serve as a good model for human breast cancer research.

This study reexamines the nuclear microsatellite analysis by Rooney et al. (1999a) of Bering-Chukchi-Beaufort Seas bowhead whales (Balaena mysticetus) to determine if this population underwent a genetic bottleneck as a result of 19th and early 20th Century commercial whaling. This investigation used more accurate laboratory techniques to score alleles, had a larger sample size that was divided into two groups (mainland Alaska and St. Lawrence Island (SLI)), and used a moderately different set of microsatellite loci which are more variable and thus, more informative. The results corroborate the findings of Rooney et al. (1999a) for mainland Alaska showing no evidence of a genetic bottleneck. However, the SLI data analyses provide conflicting conclusions. The Wilcoxon test is significant for a heterozygote excess (p = 0.042) suggesting that a genetic bottleneck has occurred. This is not substantiated by the exact tests of each locus or the table-wide sign test. There is a possibility that a bottleneck has occurred, but due to the small sample size this is not a definitive conclusion and warrants reanalysis with a larger sample size.

Made available in DSpace on 2015-04-01T14:16:04Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1809210 bytes, checksum: e118d10e8ea156df56a7871608a59d7b (MD5) Previous issue date: 2014-02-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Human identification is based on analyzing DNA through present throughout the genome molecular markers. These markers are transmitted from parents to offspring by heredity. STR markers are currently the most commonly used genetic markers in Forensic Genetics due to their high polymorphism, high reproducibility, possibility of being amplified by PCR in multiple copies in a single reaction, and minute quantities of DNA (1ng). The DNA test that allows individualization of the people is essential tool to the solution of forensic human identification cases, sex crimes, crime scenes (including or excluding suspects), mass disasters, and its result is presented in statistical calculations that consider allele frequency of markers used. So it is important to know the allele frequencies presented in the regional population so that the results are the most reliable possible. In this study , 15 autossomal markers (loci) STR or microsatellite (CSF1PO, D13S317, D16S539, D18S51, D19S433, D21S11, D2S1338, D3S1358, D5S818, D7S820, D8S1179, FGA, TH01, and VWA TPOX) were studied in 766 unrelated individuals paraibanos, demonstrating a tri population - hybrid formed Africans (25.86 %), Amerindian (6.81 %) and Europeans (67.33 %). The most informative were D21S11 and FGA, and were less informative TPOX, D7S820 and D13S317. The results are important for a database with allele frequencies found in Paraiba population can serve as a useful basis for calculating forensic practice in the State of Paraíba. / A identificação humana está baseada na análise do DNA através de marcadores moleculares presente em todo o genoma. Estes marcadores são transmitidos de pais para filhos por hereditariedade. Atualmente os marcadores STR são os marcadores genéticos mais utilizados em Genética Forense devido ao seu elevado polimorfismo, alta reprodutibilidade, possibilidade de serem amplificados por PCR em inúmeras cópias numa só reação e em mínimas quantidades de DNA (1ng). O exame de DNA que permite a individualização das pessoas é ferramenta indispensável à solução de casos forenses de identificação humana, crimes sexuais, locais de crime (incluindo ou excluindo suspeitos), desastres em massa, e tem seu resultado apresentado em cálculos estatísticos que consideram a frequência alélica dos marcadores usados. Por isso é importante o conhecimento das frequências alélicas apresentadas na população regional de forma que os resultados sejam os mais fidedignos possíveis. Neste trabalho, 15 marcadores autossômicos (loci) STR ou microssatélites (CSF1PO, D13S317, D16S539, D18S51, D19S433, D21S11, D2S1338, D3S1358, D5S818, D7S820, D8S1179, FGA, TH01, TPOX e vWA) foram estudados em 766 indivíduos paraibanos não aparentados, demonstrando uma população tri - hibrida, formada de africanos (25,86%), ameríndios (6,81%) e europeus (67,33%). Os mais informativos foram D21S11 e FGA, e os menos informativos foram TPOX, D7S820 e D13S317. Os resultados são importantes para que um banco de dados com as frequências alélicas encontradas na população paraibana possa servir de base de cálculo útil para prática forense no Estado da Paraíba.

Entre as muitas aplicações das tecnologias de identificação biológica humana, estão as finalidades forenses. O objetivo desta pesquisa foi verificar frequências alélicas de Short Tandem Repeat (STR) e os parâmetros estatísticos de interesse em genética de populações e forense para desenvolver o primeiro banco de dados populacional de DNA na Faculdade de Odontologia de Bauru, Universidade de São Paulo, (FOB/USP) para futuros usos forenses. Frequências alélicas de 15 locos autossômicos e do marcador de gênero amelogenina foram determinadas utilizando amostras de 200 μL de saliva doados por 296 alunos de graduação da FOB/USP, com idade ≥ 18 anos, após aprovação ética. Os testes laboratoriais foram feitos com kits comerciais. Resultados e parâmetros estatísticos foram obtidos por meio de programas clássicos: GeneMapper-ID-X, MS Excel 2002 versão 10.6871.6870, GenAlEx 6.5 e Arlequin 3.5, comparando quatro populações (brasileira, portuguesa, norte-americana e a população deste estudo). Os locos mais polimórficos foram D18S51 (17 alelos) e FGA (15 alelos), seguidos pelo D21S11 (13 alelos) e os menos polimórficos foram D16S539 e TH01 (7 alelos cada). A análise comparativa com amostra da população brasileira proveniente de estudos anteriores (n > 100.000) pelo teste goodness of fit X2 index não mostrou diferenças significativas entre estes grupos (p = 0,9999). Outros parâmetros estatísticos foram calculados comparando as populações: local (deste estudo), portuguesa e norte-americana. A análise de variância molecular (AMOVA) entre as três populações, entre as pessoas da mesma população e para cada pessoa de cada população mostrou que existe uma elevada variância individual (99%), que esta variância é mantida uniformemente entre as pessoas da mesma amostra/região (1%) e entre as três populações estudadas (0%). O estudo confirmou o elevado grau de polimorfismo e a alta heterozigosidade (96,5%) da população. Houve diferença significativa quanto ao gênero (79,7% mulheres) quando comparado à população brasileira em geral (50,4%), explicada pelas características do corpo discente da FOB/USP composto por 80,6% de pessoas do gênero feminino. Interessante foi a observação de uma microvariante alélica no loco D18S51, fora da escada padrão e da escala de abrangência do kit, correspondente ao alelo 29, ainda não definida na base de dados internacional (STRBase, atualizada em 07/08/2015). Esta microvariante deverá ser confirmada por testes familiares e sequenciamento de DNA para verificar a possibilidade de outra ocorrência familiar ou duplicação de nucleotídeos. No futuro, os dados obtidos neste estudo devem ser incorporados ao banco de dados da população brasileira e podem ser considerados como referência genética da população regional, ajudando a elucidar casos forenses. Após a confirmação, a potencial nova microvariante alélica contribuirá para a base de dados internacional STRBase. / There are many ways of applying biological human identification technologies, among these are forensic applications. The objective of this study was to verify allele frequencies for 15 autosomal short tandem repeat (STR) loci and develop the first human DNA population database at the Bauru School of Dentistry, University of São Paulo (FOB/USP), for future forensic uses. Allele frequencies for these STR loci and an amelogenin gender marker were determined using 200 μL samples of saliva donated by 296 undergraduates from FOB/USP who were ≥ 18 years old at the time of the sample collect after signing a consent form with ethical approval. For laboratory tests, commercial kits were used. Results and statistical parameters were obtained using the following software: GeneMapper IDX (version 1.5), MS Excel 2002 (version 10.6871.6870), GenAlEx (version 6.5) and Arlequin (version 3.5) to compare four populations (Brazil, Portugal, U.S. and this study population). Results indicated that the most polymorphic loci were D18S51 (17 alleles) and FGA (15 alleles), followed by D21S11 (13 alleles); the least polymorphic loci were D16S539 and TH01 (7 alleles each). Various Brazilian populations (n > 100,000) from other studies were compared with this studys Brazilian population using a goodness-of-fit chi-squared test, and no significant differences in these frequencies were observed between these two population groups (p = 0.9999). Other forensic and population genetic statistical parameters were calculated comparing this studys population with Portugal and U.S. populations. For example, an analysis of molecular variance (AMOVA) among all populations, among people of the same population and for each person for each population, showed that people have high individual variance (99%) and that this variance is maintained evenly between people of the same sample/region (1%) and among the three populations studied (0%). This study reinforced the conclusion of other allele frequency population studies for the 15 autosomal STR loci tested, confirming high polymorphic grade and high heterozygosity (96.5%). There were significantly more women in the study (79.7%) when compared to the general Brazilian population (50.4%) since the student body of FOB/USP is 80.6% female. Interestingly, an off-ladder D18S51 allele micro-variance corresponding to the allele 29, not yet identified, was found which should be confirmed using paternity and sequencing tests to verify the possibility of either familial occurrence or nucleotide duplication. In the future, due to the small differences found, the parameters obtained in this study should be incorporated into the Brazilian population database and be considered for a regional population genetic prototype database potentially aiding forensic cases with comparisons and calculations. After confirmation, the potentially new micro variant allele found could be included in the international STRBase.

Philosophiae Doctor - PhD / Solute carrier transporters belonging to the major facilitator family of membrane transporter are increasingly being recognized as a possible mechanism to explain inter-individual variation in drug efficacy and response. Genetic factors are estimated to be responsible for approximately 15-30% of inter-individual variation in drug disposition and response. The aims of this study were to determine the minor allele frequencies of 78 previously identified single nucleotide polymorphisms in the pharmacogenomically relevant SLC22A1-3 and SLCO1B1 genes in the Admixed population of South Africa. Thereafter, to determine whether allele and genotype frequencies for these SNP were different from that reported for other African, Caucasian, and Asian populations. The inferred haplotypes from the genetic information possessed the potential to subsequently be used in future to design and interpret results of pharmacogenomic association studies involving these genes and their substrate drugs. Furthermore, to determine whether the Cape Admixed population harbour novel SNPs in the proximal promoter regions of SLC22A1- 3 and SLCO1B1-3 genes, that encodes hOCT1-3 and hOATP1 and hOATP3, respectively. SNaPshot™ multiplex single base mini-sequencing systems were developed and optimized for each of SLC22A1, SLC22A2, SLC22A3, and SLCO1B1 genes covering the previously identified 78 SNPs. These systems were then used to genotype the alleles of 130 healthy Cape Admixed subjects residing in Cape Town, South Africa. In addition, the proximal promoter regions of the SLC22A1-3 and SLCO1B1-3 genes of 96 of the participants were screened for novel SNPs by direct sequencing. The Cape Admixed subjects investigated displayed a lack of variation and were monomorphic for 78% of the SNPs screened. None of the SLC22A3 SNPs investigated was observed in this study. Sequencing of the proximal promoter regions of the SLC22 and SLCO genes did not reveal any novel SNPs in the 96 Cape Admixed subjects that were screened. This study highlights the fact that African populations do not have the same allele frequencies for SNPs in harmacogenomically relevant genes. Furthermore, the Cape Admixed and other African populations do not share all reduced-function variants of the SLC22A1-3 and SLCO1B1-3 genes with Caucasian and Asian populations. In addition, previously identified novel regulatory variants in SLC22A2 did not exhibit a significant effect on the ability of the promoter to drive transcription. However, it must be noted that these results were observed at 95% confidence interval, and that a 99% confidence interval the significance may increase theoretically. Additionally, it should be noted that more intensive studies are required to determine the potential effect these novel variants may well cause. This study lays the foundation for the design and interpretation of future pharmacogenomic association studies between the variant alleles of the SLC22A and SLCO genes in the Cape Admixed population, as well as optimizations for future expression, and more importantly, drug transport assays with respect to drug disposition and efficacy. / National Research Foundation (NRF) and the Medical Research Council (MRC)

Microarray technology is a promising approach for the simultaneous analysis of multiple single nucleotide polymorphisms (SNPs), which are the most abundant form of genetic variation. In this thesis enzyme-assisted microarray-based methods were developed to improve the accuracy and genotype discrimination power of the current methods for SNP genotyping. The improved technology was applied for analysing recessively inherited disease mutations, for Y-chromosomal SNPs in a population study, for an evolutionary analysis of SNPs in flycatchers and for multiplexed quantitative determination of SNP-allele frequencies in pooled DNA samples. A robust attachment chemistry for immobilising oligonucleotides on glass surface was established, based on an evaluation of eight covalent coupling methods. A four-colour fluorescence detection strategy, which enabled a multiplexed quantitative analysis for as little as 2% of a minority allele frequency in pooled samples was generated. Twenty-five Y-chromosomal SNPs were screened in a collection of 300 samples from five Finno-Ugric-speaking populations using minisequencing on microarrays. In these populations six distinct haplotypes were defined by the six SNPs that were polymorphic. Data from five microsatellite markers was combined with the SNP data, revealing shared Y-chromosomal haplotypes between the Finns and the Saami, indicating, in accordance with earlier data, at least two founding Y-chromosomal lineages in these populations. Database screening and subsequent validation of 125 potential SNPs in the highly repetitive type 1 interferon genes and genes coding for proteins in the interferon-related regulatory pathways revealed 25 informative SNPs in the Finnish and Swedish populations. These SNPs were included in a panel for microarray based genotyping that should find a variety of applications in genetic studies due to the important immunoregulatory functions of the IFN family. The significance of sex-chromosome evolution on speciation was investigated in two naturally hybridising flycatcher species (N=459) by analysing a panel of 20 SNPs using minisequencing on microarrays. A strong selection against gene flow across the species boundary of sex-linked genes was observed, as well as a sex-chromosomal influence on male plumage characteristics that have previously been shown to reinforce isolation in these birds. The results suggest a major role for sex-chromosome-mediated isolation of the two flycatcher species.