Conselho Nacional de Desenvolvimento Científico e Tecnológico / Marcadores genéticos presentes no cromossomo Y, como os microssatélites (Y-STRs) e polimorfismos de único nucleotídeo (Y-SNPs) são utilizados na caracterização de linhagens masculinas, visto que são transmitidos às gerações seguintes sem alterações, a menos que ocorram mutações (Singh et al., 2011; Mitchell & Hammer, 1996; Butler, 2009). Por isso, esses marcadores são amplamente empregados em diversas situações, destacando-se o uso constante dos Y-STRs na genética forense por apresentarem alta capacidade de discriminar linhagens. Recentemente, foram descritos 13 marcadores com taxas de mutação substancialmente superiores àquelas verificadas para loci STR do cromossomo Y, denominados Rapidly Mutating (RM) Y-STRs (Ballantyne et al., 2010; Kayser et al., 2012). Devido às taxas de mutação elevadas, os RM-YSTRs apresentam maior eficiência na discriminação entre indivíduos proximamente relacionados, pertencentes à mesma linhagem patrilínea. O presente trabalho buscou aprofundar o conhecimento acerca das características populacionais e mutacionais dos loci RM-YSTRs em amostra do Rio de Janeiro, contribuindo com estudos desta natureza na população brasileira. Realizou-se a análise de 13 loci do cromossomo Y em 258 indivíduos do sexo masculino, compondo 129 pares de pais e filhos, nascidos no estado do Rio de Janeiro. O DNA das amostras foi extraído, conforme os protocolos vigentes na rotina do LDD-UERJ. As sequências genéticas de interesse foram amplificadas pela técnica de reação em cadeira da polimerase (PCR) através da realização de três PCR multiplex, cujos produtos de amplificação foram separados por eletroforese em sequenciador automático ABI-3500 (Applied Biosystems). Para os pares pai/filho que apresentaram haplótipos mutados, empregou-se a técnica de sequenciamento para confirmação das mutações. Os loci RM-YSTR geraram um poder de discriminação de 1,0 na amostra analisada, o que significa que todos os 129 indivíduos da amostra populacional apresentaram haplótipos diferentes para tais marcadores, com frequências de 0,0077 e diversidade haplotípica igual a 1. Além disso, foram obtidos valores elevados de diversidade gênica para os 13 marcadores. A análise de distância genética e os resultados de AMOVA baseados nos valores de Fst demonstraram que os RM-YSTR não indicam subdivisão populacional e traços ancestrais comuns. Tais valores estão associados às elevadas taxas de mutação encontradas, cuja média foi de 2,11 x 10-2. Foi possível observar que os loci RM-YSTR são muito discriminativos na amostra miscigenada analisada, além de terem maior capacidade de diferenciar indivíduos do que outros conjuntos de marcadores normalmente usados em estudos populacionais e análises forenses. Sendo assim, é possível concluir que os marcadores RM-YSTR são promissores para discriminar indivíduos da mesma linhagem patrilínea, visto que devido às suas elevadas taxas mutacionais e poder de discriminação, são capazes de diferenciar indivíduos de maneira mais eficiente do que os outros conjuntos de STR. Porém, é necessário maior número de estudos para melhor caracterização destes loci em diferentes populações. / Genetic markers on Y chromosome, as microsatellites (Y-STRs) and single nucleotide polymorphisms (Y-SNPs) are used for the characterization of male lineages, since they are fully transmitted to next generations unless mutations occurs (Singh et al., 2011; Mitchell & Hammer, 1996; Butler, 2009). Therefore, these markers are widely applied in several situations, highlighting the constant use of Y-STRs in the field of forensic genetics because of their high capacity of discriminate lineages. Recently, 13 rapidly mutating markers were described due to their highly mutation rates in comparison to other common Y-chromosome STRs, being called as Rapidly Mutating Y-STR (RM-YSTR) (Ballantyne et al., 2010; Kayser et al., 2012). As a result of their high mutation rates, RM-YSTRs display high efficiency in discriminating paternally related males. The present work aimed to deepen the knowledge about population and mutational RM-YSTR loci characteristics in Rio de Janeiro sample, and then, contribute to other studies with this purpose in Brazilian population. Y chromosome 13 STRs analysis was realized in 258 males born in Rio de Janeiro state, grouped in 129 fathers/sons pairs. The extraction of DNA from biological samples was performed according to routine protocols from LDD-UERJ. Target sequences were amplified by three polimerase chain reactions (PCR) and the amplicons were separated through electrophoresis on automated sequencer ABI-3500 (Applied Biosystems). When mutations were detected, they were confirmed by sequencing. Among the investigated sample, RM-YSTR loci showed a discrimination capacity of 1,0 which means that all 129 analyzed individuals have different haplotypes for these markers, displaying frequencies of 0,0077 and haplotype diversity of 1,0. Moreover, high values of genetic diversities were obtained for the 13 markers. Distance genetic analysis and AMOVA values based on Fst results did not show population substructure and common ancestral traits. These results are associated with high mutation rates found, with an average rate about 2,11 x 10-2. RM-YSTR showed to be very discriminative at this mixed sample, besides proving to be more discriminative than other markers commonly used in population studies and forensic analysis. Thus, it is possible to conclude that RM-YSTR markers are promising to discriminate individuals of the same male strain and due to their high mutation rates and discrimination capacity, they are able to differentiate individuals better than other common markers. Nevertheless, for a better characterization of these loci in different populations more studies are needed.

A infertilidade é um problema de saúde pública com um significativo impacto social, econômico e psicológico. Em todo o mundo, a incidência da infertilidade entre a população geral é estimada em 10-15%. Cerca de 50% da infertilidade dos casais são de origem masculina. Em mais da metade dos homens inférteis, a causa da infertilidade é desconhecida (idiopática). Etiologicamente, a infertilidade masculina apresenta causas genéticas e não genéticas. Dentre as causas genéticas mais conhecidas temos mutação do receptor de andrógenos, mutação do gene regulador da condutibilidade transmembrana da fibrose cística (CFTR), anomalias cromossômicas clássicas, anomalias meióticas, microdeleções do cromossomo Y, etc. As anomalias cromossômicas são encontradas com muito mais frequência em homens inférteis, com uma incidência de 4-16% em relação à incidência de 0,4% na população fértil. Estudos mostram que as CNVs também podem estar relacionadas com a infertilidade masculina, especificamente com a falha na espermatogênese. CNVs encontradas tanto no cromossomo Y quanto nos cromossomos autossômicos também foram associadas a possíveis falhas na espermatogênese. Um outro fator que também pode estar envolvido com a infertilidade masculina é a expressão desregulada dos miRNAs. O presente trabalho teve como objetivo promover a análise em larga escala da distribuição de CNVs e do perfil transcricional dos miRNAs em amostras de biopsias testiculares de paciente com azoospermia. Para o estudo das CNVs nós utilizamos a metodologia do CytoScan HDTM da Affymetrix. O perfil transcricional de miRNAs nos indivíduos estudados foi avaliado por meio da tecnologia de microarranjos também da plataforma Affymetrix. Para estas analises montamos dois grupos de estudo (Parada de Maturação (MA) de Células Germinativas e Síndrome de Células Sertoli Only (SCOS)) e um grupo controle (azoospermia obstrutiva e espermatogênese normal). Através das análises das CNVs nós encontramos 94 CNVs nos cromossomos autossômicos e sexuais, 35 (37%) CNVs foram classificadas como benignas, 24 (23%) como potencialmente benignas, sete CNVs (7,4%) como patogênicas e sete foram classificadas como potencialmente patogênica. Todas as CNVs classificadas como patogênica estão presentes no cromossomo Y, cinco CNVs são do tipo duplicação e duas do tipo deleção. A CNV do tipo duplicação foi encontrada no paciente MA e a CNV do tipo deleção foi encontrada no paciente SCOS. As CNVs se sobrepõem e quando analisadas em conjunto (formando uma única CNV de cada condição) elas apresentam um tamanho parecido. Estas CNVs apresentam genes envolvidos na espermatogênese. As CNVs classificadas como potencialmente patogênicas estavam presentes nos cromossomos autossômicos e cromossomo X. Nestas CNVs estavam presentes genes que foram associados com a falha na espermatogênese. A análise da expressão dos miRNAs revelou um perfil transicional muito mais alterado nos pacientes com SCOS. As duas condições apresentaram miRNAs exclusivos, mas também compartilharam: 30 miRNAs. Nós identificamos duas famílias de miRNAs (miR449 e miR34) diferencialmente expressos nas duas condições e que apresentam expressão preferencial no testículo. Nossos resultados mostram que alterações no número de copias (CNVs) no cromossomo Y levam a infertilidade masculina e CNVs nos cromossomos autossômicos e X podem levar a infertilidade masculina. As alterações do tipo deleção podem levar a uma falha na espermatogênese maior que as alterações do tipo duplicação. A expressão diferencial dos miRNAs em tecido testicular de pacientes com diferenças histopatológicas (SCOS e MA) apresentam um padrão de expressão de miRNAs diferentes devido ao tipo de células germinativas que eles apresentam no tecido epitelial do testículo. / Infertility is a public health problem with significant social, economic and psychological impact. Worldwide, the incidence of infertility in the general population is estimated at 10- 15%. Approximately 50% of infertility of couples is of male origin. In more than half of infertile men, the cause of infertility is unknown (idiopathic). Etiologically, male infertility has genetic and non-genetic causes. Among the best known genetic causes we found the mutation of the androgen receptor, the cystic fibrosis transmembrane conductance regulator (CFTR), classic chromosomal abnormalities, meiotic abnormalities and microdeletions of the Y chromosome. Chromosomal abnormalities are found much more frequently in infertile men, with an incidence of 4-16% in the incidence of 0.4% in the fertile population. Studies show that CNVs can also be related to male infertility, specifically in the failure of spermatogenesis. CNVs found in both the Y and autosomes chromosomes were also associated with possible failures in spermatogenesis. Another factor that may also be involved in male infertility is the deregulated expression of miRNAs. This work aimed to promote the analysis of large-scale distribution of CNVs and the transcriptional profile of miRNAs in testicular biopsy samples from patients with azoospermia. For the study of CNV we used the CytoScan HDTM Affymetrix methodology and the transcriptional profile of miRNAs in the samples was assessed by means of microarray technology from Affymetrix platform. For these analyzes we set up two study groups (Stop Maturation (MA) of Germ Cells and Sertoli Cell Only Syndrome (SCOS)) and compared them to a control group (obstructive azoospermia, normal spermatogenesis). Through analysis of CNVs, we found 94 CNVs in sexual and autosomes chromosomes, 35 (37%) were classified as benign CNVs, 24 (23%) as a potentially benign seven CNVs (7.4%) as pathogenic and 7 were classified as potentially pathogenic. All CNVs classified as pathogenic are present on the Y chromosome, five CNVs are of duplication type and two are deletion type. The duplication type CNV was found in MA patients and deletion type CNV was found in SCOS patient. We identified that CNVs overlap and when analyzed jointed - as a single CNV of each condition - they have a similar size. These CNVs have genes involved in spermatogenesis. CNVs classified as potentially pathogenic were present in autosomes and in the X chromosome. In these CNVs were present genes that were associated with failure in spermatogenesis. The analysis of the expression of miRNAs revealed a transitional profile much more altered in patients with SCOS. The two conditions presented exclusive miRNAs, but shared 30 miRNAs differentially expressed when compared to the control group. We identify two families of miRNAs (miR449 and miR34) which exhibit preferential expression in testis as differentially expressed in both conditions. Our results show that changes in the number of copies (CNVs) on the Y chromosome lead to male infertility and CNVs in autosomes and X chromosomes may lead to male infertility. The deletion type changes can lead to a failure of spermatogenesis greater than the duplication type changes. The differential expression of miRNAs in patients with testicular tissue histopathologic differences (SCOS and MA) has a different pattern of miRNA expression due to the type of germ cells they present in epithelial tissue of the testis.

<p>Many of our closest relatives, the primates, are endangered and could be extinct in a near future. To increase the knowledge of non-human primate genomes, and at the same time acquire information on our own genomic evolution, studies using high-throughput technologies are applied, which raises the demand for large amounts of high quality DNA.</p><p>In study I and II, we evaluated the multiple displacement amplification (MDA) technique, a whole genome amplification method, on a wide range of DNA sources, such as blood, hair and semen, by comparing MDA products to genomic DNA as templates for several commonly used genotyping methods. In general, the genotyping success rate from the MDA products was in concordance with the genomic DNA. The quality of sequences of the mitochondrial control region obtained from MDA products from blood and non-invasively collected semen samples was maintained. However, the readable sequence length was shorter for MDA products.</p><p>Few studies have focused on the genetic variation in the nuclear genes of non-human primates. In study III, we discovered 23 new single nucleotide polymorphisms (SNPs) in the Y-chromosome of the chimpanzee. We designed a tag-microarray minisequencing assay for genotyping the SNPs together with 19 SNPs from the literature and 45 SNPs in the mitochondrial DNA. Using the microarray, we were able to analyze the population structure of wild-living chimpanzees.</p><p>In study IV, we established 111 diagnostic nucleotide positions for primate genera determination. We used sequence alignments of the nuclear epsilon globin gene and apolipoprotein B gene to identify positions for determination on the infraorder and Catarrhini subfamily level, respectively, and sequence alignments of the mitochondrial 12S rRNA (MT-RNR1) to identify positions to distinguish between genera. We designed a microarray assay for immobilized minisequencing primers for genotyping these positions to aid in the forensic determination of an unknown sample.</p>

Our ability to map and sequence whole genomes is one of the most important developments in biological science. It will provide us with an unprecedented insight into the genetic background of phenotypic traits, such as disease resistance, reproduction and growth and also makes it feasible to study the processes of genome evolution. The main focus of this thesis has been to develop a linkage map of the horse (Equus caballus) genome. A secondary aim was to expand the number of physically mapped genes in the horse, allowing comparative analyses with data from the human genome map. Finally, attempts were made to identify single nucleotide polymorphisms (SNPs) on the horse Y chromosome. The development of a genome map relies on the information generated by both linkage and cytogenetical studies. To integrate genetical and physical assignments in the very early phase of equine genome map construction, 19 polymorphic microsatellite markers were isolated from lambda phage clones which, in parallel, were physically assigned to chromosomes by fluorescent in situ hybridization (FISH). The microsatellites were simultaneously mapped by linkage analysis in a Swedish reference pedigree. A first primary male autosomal linkage map of the domestic horse was constructed by segregation analysis of 140 genetic markers within eight half-sib families with, in total, 263 offspring. One hundred markers were arranged into 25 linkage groups, 22 of which could be assigned physically to 18 different chromosomes. The total map distance contained within linkage groups was 679 cM. The presented map provides an important framework for future genome mapping in the horse. Our contribution to the comparative horse genome map, was the presentation of map data for 12 novel genes using FISH and somatic cell hybrid mapping. AD chromosomal assignments except one were in agreement with human-horse Zoo-FISH data. The exception concerned the CLU gene which was mapped by synteny to ECA2 while human-horse Zoo-FISH data predicted that it would be located on ECA9. The level of SNPs on the horse Y chromosome was also investigated by DNA sequencing and denaturing high performance liquid chromatography (DHPLC) of Y chromosome-specific fragments derived mainly from BAC clone subcloning. The amount of genetic variability was found to be very low, consistent with low male effective population size.

Many of our closest relatives, the primates, are endangered and could be extinct in a near future. To increase the knowledge of non-human primate genomes, and at the same time acquire information on our own genomic evolution, studies using high-throughput technologies are applied, which raises the demand for large amounts of high quality DNA. In study I and II, we evaluated the multiple displacement amplification (MDA) technique, a whole genome amplification method, on a wide range of DNA sources, such as blood, hair and semen, by comparing MDA products to genomic DNA as templates for several commonly used genotyping methods. In general, the genotyping success rate from the MDA products was in concordance with the genomic DNA. The quality of sequences of the mitochondrial control region obtained from MDA products from blood and non-invasively collected semen samples was maintained. However, the readable sequence length was shorter for MDA products. Few studies have focused on the genetic variation in the nuclear genes of non-human primates. In study III, we discovered 23 new single nucleotide polymorphisms (SNPs) in the Y-chromosome of the chimpanzee. We designed a tag-microarray minisequencing assay for genotyping the SNPs together with 19 SNPs from the literature and 45 SNPs in the mitochondrial DNA. Using the microarray, we were able to analyze the population structure of wild-living chimpanzees. In study IV, we established 111 diagnostic nucleotide positions for primate genera determination. We used sequence alignments of the nuclear epsilon globin gene and apolipoprotein B gene to identify positions for determination on the infraorder and Catarrhini subfamily level, respectively, and sequence alignments of the mitochondrial 12S rRNA (MT-RNR1) to identify positions to distinguish between genera. We designed a microarray assay for immobilized minisequencing primers for genotyping these positions to aid in the forensic determination of an unknown sample.

The pantherine lineage of cats diverged from the remainder of modern Felidae less than 11 million years ago. This clade consists of the five big cats of the genus Panthera, the lion, tiger, jaguar, leopard, and snow leopard, as well as the closely related clouded leopard, which diverged from Panthera approximately 6 million years ago. A significant problem exists with respect to the precise phylogeny of these highly threatened great cats. Within the past four years, despite multiple publications on the subject, no two studies have reconstructed the phylogeny of Panthera with the same topology, showing particular discordance with respect to sister-taxa relationships to the lion and the position of the enigmatic snow leopard. The evolutionary relationship among these cats remains unresolved partially due to their recent and rapid radiation 3-5 million years ago, individual speciation events occurring within less than 1 million years, and probable introgression between lineages following their divergence. We assembled a 47.6 kb dataset using novel and published DNA sequence data from the autosomes, both sex chromosomes and the mitochondrial genome. This dataset was analyzed both as a supermatrix and with respect to individual partitions using maximum likelihood and Bayesian phylogeny inference. Since discord may exist among gene segments in a multilocus dataset due to their unique evolutionary histories, inference was also performed using Bayesian estimation of species trees (BEST) to form a robust consensus topology. Incongruent topologies for autosomal loci indicated phylogenetic signal conflict within the corresponding segments. We resequenced four mitochondrial and three nuclear gene segments used in recent attempts to reconstruct felid phylogeny. The newly generated data was combined with available GenBank sequence data from all published studies to highlight phylogenetic disparities stemming either from the amplification of a mitochondrial to nuclear translocation event, or errors in species identification. We provide an alternative, highly supported interpretation of the evolutionary history of the pantherine lineage using 39 single-copy regions of the felid Y chromosome and supportive phylogenetic evidence from a revised mitochondrial partition. These efforts result in a highly corroborated set of species relationships that open up new avenues for the study of speciation genomics and understanding the historical events surrounding the origin of the members of this lineage.

L'Indonésie a été l'objet de la dispersion Austronésienne qui a débuté il y a environ 5000 ans depuis Taiwan, se propager à travers les Philippines et l'Indonésie, puis toucher l'Océanie à l'est, et à Madagascar à l'ouest. Malgré de nombreuses recherches en génétique sur la dispersion Austronésienne vers l'est, il y a très peu de données sur la dispersion vers l'ouest, laissant sans réponse de nombreuses questions, liées notamment au peuplement de Madagascar. Reposant sur l'analyse des données culturelles et biologiques, les populations d'Indonésie semblent avoir joué un rôle majeur dans la colonisation de Madagascar, le premier millénaire de notre ère. Cependant, le peu de populations Indonésiennes étudiées à ce jour n'a pas permis jusqu'à présent d'identifier la population indonésienne source. Dans ce présent travail, j'ai réalisé des études en génétique des populations de 12 populations Indonésiennes, qui à priori devraient éclairer l'histoire des migrations austronésiennes dans l'Océan Indien. Parmi elles sont inclus le Ma'anyan du sud-est de Bornéo qui sont les plus proches linguistiquement des Malgaches. En utilisant différents marqueurs génétiques, ma recherche a amélioré nos connaissances de la diversité génétique Indonésienne, et du lien génétique entre l'Indonésie et Madagascar. Résultats L'analyse des marqueurs uniparentaux (chr-Y et ADNmt) suggère que les Malgaches proviennent de plusieurs régions d'Indonésie, avec un lien privilégié avec le sud-est de Bornéo, le sud de Sulawesi et les îles de la Sonde. Etonnamment, les Ma'anyan partagent un nombre limité de lignées paternelles et maternelles avec les Malgaches, malgré leur proximité linguistique. Par ailleurs, en combinant l'analyse de fréquences des SNPs et l'analyse haplotypique à partir des données autosomales, il a été confirmé que la diversité génétique des Ma'anyan ne correspond pas à l'ancestralité asiatique des Malgaches. Cependant, en centrant l'analyse sur les populations du sud-est de Bornéo, l'origine de l'ancestralité asiatique des Malgaches est ancrée dans la population Banjar, un mélange de population Ma'anyan et Malaise, résultat des activités commerciales de l'empire Malais dans le sud-est de Bornéo, qui se sont poursuivies à travers l'océan Indien. Par ailleurs nos résultats ont aussi permis d'accroitre notre compréhension de la diversité génétique de l'Indonésie en identifiant (1) une nouvelle composante génétique austronésienne présente chez les Ma'anyan, et retrouvée à faible fréquence à travers l'Asie du Sud-Est, suggérant une plus grande complexité du modèle d'expansion austronésien dans la région et (2) le rôle joué par les nomades de la mer dans la structuration de la diversité génétique et les échanges entre populations dans l'Indonésie, soulignant l'histoire génétique complexe de populations suivant un mode de vie nomade. / Indonesia hosts a wide range of linguistic, ethnic and genetic diversity, comprising ~600 ethnic groups and 700 living languages. Indonesia has facilitated the last substantial wave of human migration was the Austronesian dispersal ~5,000 years ago, which is thought to have originated in Taiwan. Its influence spread through Philippines and Indonesia, ultimately impacting a wide geographical area, from Remote Oceania in the east and to Madagascar in the west. Despite considerable genetic research on the eastward Austronesian expansion, there is little equivalent research on the western edge, leaving major issues unresolved regarding the settlement of Madagascar. Based on cultural and biological studies, it has been suggested that Indonesian peoples played a major role in the colonization of Madagascar from around the mid-first millennium CE (Current Era). However, poor geographical coverage of Indonesian populations has prevented the Indonesian source populations from being identified. Here, I performed human population genetic studies on 12 new Indonesian populations, which were a priori expected to shed light on the westward migration of Austronesians across the Indian Ocean. This includes the Ma'anyan ethnic group from Southeast Borneo, who are the closest linguistic siblings to modern Malagasy. Using different genetic markers (Y-chromosome SNPs, mitochondrial DNA and genome-wide SNPs), my research has improved the description of Indonesian genetic diversity, and investigated the genetic links between Indonesia and Madagascar. Results Uniparental markers (Y-chromosome and mtDNA) analyses suggest that Malagasy derive from multiple regional sources in Indonesia, with a focus on southeastern Borneo, southern Sulawesi and the Lesser Sunda islands. Interestingly, the Ma'anyan share limited paternal and maternal lineages with the Malagasy, despite their linguistic connection. Furthermore, combining SNP frequency and haplotype-based analyses from autosomal genome-wide data, it was confirmed that the genetic diversity of the Ma'anyan does not match the Asian ancestry of the Malagasy. However, by focusing on Southeast Borneo populations, strong support was found for an origin of the Asian ancestry of Malagasy among the people of Banjar, an admixed population of Ma'anyan and Malay, likely resulting from trading activities by the Malay Empire in Southeast Borneo, and later continuing across the Indian Ocean arena. These results increase our understanding of genetic diversity across Indonesia by 1) identifying the unique and undiscovered Austronesian genetic component carried by the Ma'anyan, which occurs at low levels across Island Southeast Asia and suggests a more complex model for the Austronesian expansion in this region, and 2) describing the role played by sea-nomads in structuring genetic diversity and exchanges in central Indonesia, thus revealing the complex genetic history of populations living this rare nomadic lifestyle.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Marcadores genéticos presentes no cromossomo Y, como os microssatélites (Y-STRs) e polimorfismos de único nucleotídeo (Y-SNPs) são utilizados na caracterização de linhagens masculinas, visto que são transmitidos às gerações seguintes sem alterações, a menos que ocorram mutações (Singh et al., 2011; Mitchell & Hammer, 1996; Butler, 2009). Por isso, esses marcadores são amplamente empregados em diversas situações, destacando-se o uso constante dos Y-STRs na genética forense por apresentarem alta capacidade de discriminar linhagens. Recentemente, foram descritos 13 marcadores com taxas de mutação substancialmente superiores àquelas verificadas para loci STR do cromossomo Y, denominados Rapidly Mutating (RM) Y-STRs (Ballantyne et al., 2010; Kayser et al., 2012). Devido às taxas de mutação elevadas, os RM-YSTRs apresentam maior eficiência na discriminação entre indivíduos proximamente relacionados, pertencentes à mesma linhagem patrilínea. O presente trabalho buscou aprofundar o conhecimento acerca das características populacionais e mutacionais dos loci RM-YSTRs em amostra do Rio de Janeiro, contribuindo com estudos desta natureza na população brasileira. Realizou-se a análise de 13 loci do cromossomo Y em 258 indivíduos do sexo masculino, compondo 129 pares de pais e filhos, nascidos no estado do Rio de Janeiro. O DNA das amostras foi extraído, conforme os protocolos vigentes na rotina do LDD-UERJ. As sequências genéticas de interesse foram amplificadas pela técnica de reação em cadeira da polimerase (PCR) através da realização de três PCR multiplex, cujos produtos de amplificação foram separados por eletroforese em sequenciador automático ABI-3500 (Applied Biosystems). Para os pares pai/filho que apresentaram haplótipos mutados, empregou-se a técnica de sequenciamento para confirmação das mutações. Os loci RM-YSTR geraram um poder de discriminação de 1,0 na amostra analisada, o que significa que todos os 129 indivíduos da amostra populacional apresentaram haplótipos diferentes para tais marcadores, com frequências de 0,0077 e diversidade haplotípica igual a 1. Além disso, foram obtidos valores elevados de diversidade gênica para os 13 marcadores. A análise de distância genética e os resultados de AMOVA baseados nos valores de Fst demonstraram que os RM-YSTR não indicam subdivisão populacional e traços ancestrais comuns. Tais valores estão associados às elevadas taxas de mutação encontradas, cuja média foi de 2,11 x 10-2. Foi possível observar que os loci RM-YSTR são muito discriminativos na amostra miscigenada analisada, além de terem maior capacidade de diferenciar indivíduos do que outros conjuntos de marcadores normalmente usados em estudos populacionais e análises forenses. Sendo assim, é possível concluir que os marcadores RM-YSTR são promissores para discriminar indivíduos da mesma linhagem patrilínea, visto que devido às suas elevadas taxas mutacionais e poder de discriminação, são capazes de diferenciar indivíduos de maneira mais eficiente do que os outros conjuntos de STR. Porém, é necessário maior número de estudos para melhor caracterização destes loci em diferentes populações. / Genetic markers on Y chromosome, as microsatellites (Y-STRs) and single nucleotide polymorphisms (Y-SNPs) are used for the characterization of male lineages, since they are fully transmitted to next generations unless mutations occurs (Singh et al., 2011; Mitchell & Hammer, 1996; Butler, 2009). Therefore, these markers are widely applied in several situations, highlighting the constant use of Y-STRs in the field of forensic genetics because of their high capacity of discriminate lineages. Recently, 13 rapidly mutating markers were described due to their highly mutation rates in comparison to other common Y-chromosome STRs, being called as Rapidly Mutating Y-STR (RM-YSTR) (Ballantyne et al., 2010; Kayser et al., 2012). As a result of their high mutation rates, RM-YSTRs display high efficiency in discriminating paternally related males. The present work aimed to deepen the knowledge about population and mutational RM-YSTR loci characteristics in Rio de Janeiro sample, and then, contribute to other studies with this purpose in Brazilian population. Y chromosome 13 STRs analysis was realized in 258 males born in Rio de Janeiro state, grouped in 129 fathers/sons pairs. The extraction of DNA from biological samples was performed according to routine protocols from LDD-UERJ. Target sequences were amplified by three polimerase chain reactions (PCR) and the amplicons were separated through electrophoresis on automated sequencer ABI-3500 (Applied Biosystems). When mutations were detected, they were confirmed by sequencing. Among the investigated sample, RM-YSTR loci showed a discrimination capacity of 1,0 which means that all 129 analyzed individuals have different haplotypes for these markers, displaying frequencies of 0,0077 and haplotype diversity of 1,0. Moreover, high values of genetic diversities were obtained for the 13 markers. Distance genetic analysis and AMOVA values based on Fst results did not show population substructure and common ancestral traits. These results are associated with high mutation rates found, with an average rate about 2,11 x 10-2. RM-YSTR showed to be very discriminative at this mixed sample, besides proving to be more discriminative than other markers commonly used in population studies and forensic analysis. Thus, it is possible to conclude that RM-YSTR markers are promising to discriminate individuals of the same male strain and due to their high mutation rates and discrimination capacity, they are able to differentiate individuals better than other common markers. Nevertheless, for a better characterization of these loci in different populations more studies are needed.

A infertilidade é um problema de saúde pública com um significativo impacto social, econômico e psicológico. Em todo o mundo, a incidência da infertilidade entre a população geral é estimada em 10-15%. Cerca de 50% da infertilidade dos casais são de origem masculina. Em mais da metade dos homens inférteis, a causa da infertilidade é desconhecida (idiopática). Etiologicamente, a infertilidade masculina apresenta causas genéticas e não genéticas. Dentre as causas genéticas mais conhecidas temos mutação do receptor de andrógenos, mutação do gene regulador da condutibilidade transmembrana da fibrose cística (CFTR), anomalias cromossômicas clássicas, anomalias meióticas, microdeleções do cromossomo Y, etc. As anomalias cromossômicas são encontradas com muito mais frequência em homens inférteis, com uma incidência de 4-16% em relação à incidência de 0,4% na população fértil. Estudos mostram que as CNVs também podem estar relacionadas com a infertilidade masculina, especificamente com a falha na espermatogênese. CNVs encontradas tanto no cromossomo Y quanto nos cromossomos autossômicos também foram associadas a possíveis falhas na espermatogênese. Um outro fator que também pode estar envolvido com a infertilidade masculina é a expressão desregulada dos miRNAs. O presente trabalho teve como objetivo promover a análise em larga escala da distribuição de CNVs e do perfil transcricional dos miRNAs em amostras de biopsias testiculares de paciente com azoospermia. Para o estudo das CNVs nós utilizamos a metodologia do CytoScan HDTM da Affymetrix. O perfil transcricional de miRNAs nos indivíduos estudados foi avaliado por meio da tecnologia de microarranjos também da plataforma Affymetrix. Para estas analises montamos dois grupos de estudo (Parada de Maturação (MA) de Células Germinativas e Síndrome de Células Sertoli Only (SCOS)) e um grupo controle (azoospermia obstrutiva e espermatogênese normal). Através das análises das CNVs nós encontramos 94 CNVs nos cromossomos autossômicos e sexuais, 35 (37%) CNVs foram classificadas como benignas, 24 (23%) como potencialmente benignas, sete CNVs (7,4%) como patogênicas e sete foram classificadas como potencialmente patogênica. Todas as CNVs classificadas como patogênica estão presentes no cromossomo Y, cinco CNVs são do tipo duplicação e duas do tipo deleção. A CNV do tipo duplicação foi encontrada no paciente MA e a CNV do tipo deleção foi encontrada no paciente SCOS. As CNVs se sobrepõem e quando analisadas em conjunto (formando uma única CNV de cada condição) elas apresentam um tamanho parecido. Estas CNVs apresentam genes envolvidos na espermatogênese. As CNVs classificadas como potencialmente patogênicas estavam presentes nos cromossomos autossômicos e cromossomo X. Nestas CNVs estavam presentes genes que foram associados com a falha na espermatogênese. A análise da expressão dos miRNAs revelou um perfil transicional muito mais alterado nos pacientes com SCOS. As duas condições apresentaram miRNAs exclusivos, mas também compartilharam: 30 miRNAs. Nós identificamos duas famílias de miRNAs (miR449 e miR34) diferencialmente expressos nas duas condições e que apresentam expressão preferencial no testículo. Nossos resultados mostram que alterações no número de copias (CNVs) no cromossomo Y levam a infertilidade masculina e CNVs nos cromossomos autossômicos e X podem levar a infertilidade masculina. As alterações do tipo deleção podem levar a uma falha na espermatogênese maior que as alterações do tipo duplicação. A expressão diferencial dos miRNAs em tecido testicular de pacientes com diferenças histopatológicas (SCOS e MA) apresentam um padrão de expressão de miRNAs diferentes devido ao tipo de células germinativas que eles apresentam no tecido epitelial do testículo. / Infertility is a public health problem with significant social, economic and psychological impact. Worldwide, the incidence of infertility in the general population is estimated at 10- 15%. Approximately 50% of infertility of couples is of male origin. In more than half of infertile men, the cause of infertility is unknown (idiopathic). Etiologically, male infertility has genetic and non-genetic causes. Among the best known genetic causes we found the mutation of the androgen receptor, the cystic fibrosis transmembrane conductance regulator (CFTR), classic chromosomal abnormalities, meiotic abnormalities and microdeletions of the Y chromosome. Chromosomal abnormalities are found much more frequently in infertile men, with an incidence of 4-16% in the incidence of 0.4% in the fertile population. Studies show that CNVs can also be related to male infertility, specifically in the failure of spermatogenesis. CNVs found in both the Y and autosomes chromosomes were also associated with possible failures in spermatogenesis. Another factor that may also be involved in male infertility is the deregulated expression of miRNAs. This work aimed to promote the analysis of large-scale distribution of CNVs and the transcriptional profile of miRNAs in testicular biopsy samples from patients with azoospermia. For the study of CNV we used the CytoScan HDTM Affymetrix methodology and the transcriptional profile of miRNAs in the samples was assessed by means of microarray technology from Affymetrix platform. For these analyzes we set up two study groups (Stop Maturation (MA) of Germ Cells and Sertoli Cell Only Syndrome (SCOS)) and compared them to a control group (obstructive azoospermia, normal spermatogenesis). Through analysis of CNVs, we found 94 CNVs in sexual and autosomes chromosomes, 35 (37%) were classified as benign CNVs, 24 (23%) as a potentially benign seven CNVs (7.4%) as pathogenic and 7 were classified as potentially pathogenic. All CNVs classified as pathogenic are present on the Y chromosome, five CNVs are of duplication type and two are deletion type. The duplication type CNV was found in MA patients and deletion type CNV was found in SCOS patient. We identified that CNVs overlap and when analyzed jointed - as a single CNV of each condition - they have a similar size. These CNVs have genes involved in spermatogenesis. CNVs classified as potentially pathogenic were present in autosomes and in the X chromosome. In these CNVs were present genes that were associated with failure in spermatogenesis. The analysis of the expression of miRNAs revealed a transitional profile much more altered in patients with SCOS. The two conditions presented exclusive miRNAs, but shared 30 miRNAs differentially expressed when compared to the control group. We identify two families of miRNAs (miR449 and miR34) which exhibit preferential expression in testis as differentially expressed in both conditions. Our results show that changes in the number of copies (CNVs) on the Y chromosome lead to male infertility and CNVs in autosomes and X chromosomes may lead to male infertility. The deletion type changes can lead to a failure of spermatogenesis greater than the duplication type changes. The differential expression of miRNAs in patients with testicular tissue histopathologic differences (SCOS and MA) has a different pattern of miRNA expression due to the type of germ cells they present in epithelial tissue of the testis.

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