Mutações inativadoras em homozigose no gene do transdutor de sinal e ativador de transcrição 5B (STAT5B) causam insensibilidade ao hormônio de crescimento associada a disfunção imunológica grave que se manifesta na forma de infecções exacerbadas e de repetição, pneumonia intersticial linfocítica e outros eventos autoimunes. A caracterização do fenótipo destas mutações em heterozigose não foi realizada previamente. Dois pacientes descritos com mutação em homozigose na STAT5B (c.424_427del / p.L142RfsX19) são irmãos brasileiros naturais de Criciúma - Santa Catarina, sem consanguinidade conhecida na família. Houve também o relato de dois outros casos semelhantes na cidade, já falecidos, sugerindo que mutações na STAT5B pudessem ser relativamente frequentes nesta região. Os objetivos deste estudo foram investigar a frequência da mutação c.424_427del da STAT5B na população de Criciúma, avaliar a existência de efeito fundador e caracterizar o efeito da mutação c.424_427del da STAT5B em heterozigose sobre o fenótipo antropométrico e hormonal. Para investigar a frequência desta mutação em Criciúma, 1192 indivíduos da população foram genotipados. Foram identificados sete indivíduos heterozigotos, caracterizando uma frequência alélica mínima de 0,29% (intervalo de confiança 95%: 0,08 a 0,5%), significativamente mais alta que a frequência de outras variantes patogênicas da STAT5B descritas em bases de dados públicas. Utilizando-se o equilíbrio de Hardy-Weinberg, foi possível estimar a incidência de casos de homozigotos para o alelo mutado em um a cada 40 anos. No entanto, utilizando-se a maior frequência possível de acordo com o intervalo de confiança, esta incidência poderia atingir um a cada 13 anos. Além disso, foram estudados os pais dos dois casos relatados como semelhantes aos pacientes homozigotos para mutações na STAT5B e estes pais eram portadores da mutação c.424_427del da STAT5B em heterozigose. Para avaliar o efeito fundador, foram analisados dois marcadores próximos à mutação c.424_427del da STAT5B nos pacientes homozigotos para a mesma, em 33 indivíduos heterozigotos de sete famílias independentes e em 53 indivíduos controles. O mesmo haplótipo estava presente nos pacientes homozigotos para a mutação e em todos os heterozigotos, enquanto em apenas 9,4% dos controles (p < 0,001), apontando a probabilidade de que a mutação c.424_427del nas diferentes famílias tenha sido herdada de um antepassado em comum. Para avaliar o efeito da mutação c.424_427del da STAT5B em heterozigose, foram comparados em conjunto os 33 indivíduos heterozigotos e os 38 familiares não portadores em relação à altura e a alguns exames laboratoriais (gerais e hormonais). Os indivíduos heterozigotos foram significativamente mais baixos, com uma redução na altura de 0,6 desvios-padrão (p= 0,006). Também apresentaram redução significativa dos desvios-padrão de fator de crescimento insulina-símile 1 (IGF-1) e da proteína 3 de ligação a fator de crescimento insulina-símile (IGFBP-3), sem alteração em outros exames. Esses achados mostram que as mutações na STAT5B em heterozigose causam um impacto negativo significativo na altura, mais leve que o visto em pacientes com mutações em homozigose, com altura dentro da variação normal. Esse resultado favorece a hipótese de que variantes patogênicas raras em heterozigose contribuem para a variabilidade da altura normal / Homozygous inactivating mutations in signal transducer and activator of transcription 5B gene (STAT5B) cause growth hormone insensitivity associated with signs of severe immune dysfunction, such as recurrent infections, lymphoid interstitial pneumonia and other autoimmune events. The phenotypic characterization of these mutations in heterozygous state has not been accomplished previously. Two patients with a homozygous STAT5B mutation (c.424_427del / p.L142RfsX19) are Brazilian brothers born in the city of Criciúma, Santa Catarina, and there is not known consanguinity in their family. Moreover, there was a report about two similar cases in this city, already deceased, suggesting that STAT5B mutations could be relatively frequent in this region. The objectives of this study were to evaluate the frequency of STAT5B c.424_427del mutation in Criciúma, to assess the existence of the founder effect and to characterize the effect of heterozygous STAT5B c.424_427del mutation on anthropometric and hormonal phenotypes. To evaluate the frequency of this mutation in Criciúma, 1192 individuals from the population were genotyped. Seven heterozygous individuals were identified, which characterized a minimum allele frequency of 0.29% (95% confidence interval: 0.08 to 0.5%), significantly higher than the frequency of other pathogenic variants described in public databases. By using the Hardy-Weinberg law, it was possible to estimate the incidence of cases of individuals homozygous for this mutation at one every 40 years. However, by using the highest possible frequency according to the confidence interval, this incidence could reach one every 13 years. Additionally, the parents of the two reported cases who were similar to patients with homozygous STAT5B mutations were genotyped and these parents were heterozygous for STAT5B c.424_427del mutation. To assess the founder effect, two markers near the mutation were analyzed in the two boys homozygous for STAT5B c.424_427del mutation, in 33 heterozygous individuals from seven unrelated families and in 53 control individuals. The same haplotype was present in the homozygous boys and in all heterozygous individuals, while in only 9,4% control individuals (p < 0,001), pointing to the probability that STAT5B c.424_427del mutation in different families has been inherited from a common ancestor. To study the effects of heterozygous STAT5B c.424_427del mutation, 33 heterozygous individuals were compared to 38 non-carrier relatives on height and some laboratorial tests. Heterozygous individuals were significantly shorter than their noncarrier relatives, with a height reduction of 0.6 standard deviation scores (p= 0,006). Furthermore, they had a significant reduction in insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) standard deviation scores, without differences in the other exams. These findings show that heterozygous STAT5B mutations cause a significant negative impact on height, milder than the effect seen in patients with homozygous mutations, with height within the normal range. This result favors the hypothesis that rare pathogenic variants in heterozygous state contribute to normal height variability

Mutações inativadoras em homozigose no gene do transdutor de sinal e ativador de transcrição 5B (STAT5B) causam insensibilidade ao hormônio de crescimento associada a disfunção imunológica grave que se manifesta na forma de infecções exacerbadas e de repetição, pneumonia intersticial linfocítica e outros eventos autoimunes. A caracterização do fenótipo destas mutações em heterozigose não foi realizada previamente. Dois pacientes descritos com mutação em homozigose na STAT5B (c.424_427del / p.L142RfsX19) são irmãos brasileiros naturais de Criciúma - Santa Catarina, sem consanguinidade conhecida na família. Houve também o relato de dois outros casos semelhantes na cidade, já falecidos, sugerindo que mutações na STAT5B pudessem ser relativamente frequentes nesta região. Os objetivos deste estudo foram investigar a frequência da mutação c.424_427del da STAT5B na população de Criciúma, avaliar a existência de efeito fundador e caracterizar o efeito da mutação c.424_427del da STAT5B em heterozigose sobre o fenótipo antropométrico e hormonal. Para investigar a frequência desta mutação em Criciúma, 1192 indivíduos da população foram genotipados. Foram identificados sete indivíduos heterozigotos, caracterizando uma frequência alélica mínima de 0,29% (intervalo de confiança 95%: 0,08 a 0,5%), significativamente mais alta que a frequência de outras variantes patogênicas da STAT5B descritas em bases de dados públicas. Utilizando-se o equilíbrio de Hardy-Weinberg, foi possível estimar a incidência de casos de homozigotos para o alelo mutado em um a cada 40 anos. No entanto, utilizando-se a maior frequência possível de acordo com o intervalo de confiança, esta incidência poderia atingir um a cada 13 anos. Além disso, foram estudados os pais dos dois casos relatados como semelhantes aos pacientes homozigotos para mutações na STAT5B e estes pais eram portadores da mutação c.424_427del da STAT5B em heterozigose. Para avaliar o efeito fundador, foram analisados dois marcadores próximos à mutação c.424_427del da STAT5B nos pacientes homozigotos para a mesma, em 33 indivíduos heterozigotos de sete famílias independentes e em 53 indivíduos controles. O mesmo haplótipo estava presente nos pacientes homozigotos para a mutação e em todos os heterozigotos, enquanto em apenas 9,4% dos controles (p < 0,001), apontando a probabilidade de que a mutação c.424_427del nas diferentes famílias tenha sido herdada de um antepassado em comum. Para avaliar o efeito da mutação c.424_427del da STAT5B em heterozigose, foram comparados em conjunto os 33 indivíduos heterozigotos e os 38 familiares não portadores em relação à altura e a alguns exames laboratoriais (gerais e hormonais). Os indivíduos heterozigotos foram significativamente mais baixos, com uma redução na altura de 0,6 desvios-padrão (p= 0,006). Também apresentaram redução significativa dos desvios-padrão de fator de crescimento insulina-símile 1 (IGF-1) e da proteína 3 de ligação a fator de crescimento insulina-símile (IGFBP-3), sem alteração em outros exames. Esses achados mostram que as mutações na STAT5B em heterozigose causam um impacto negativo significativo na altura, mais leve que o visto em pacientes com mutações em homozigose, com altura dentro da variação normal. Esse resultado favorece a hipótese de que variantes patogênicas raras em heterozigose contribuem para a variabilidade da altura normal / Homozygous inactivating mutations in signal transducer and activator of transcription 5B gene (STAT5B) cause growth hormone insensitivity associated with signs of severe immune dysfunction, such as recurrent infections, lymphoid interstitial pneumonia and other autoimmune events. The phenotypic characterization of these mutations in heterozygous state has not been accomplished previously. Two patients with a homozygous STAT5B mutation (c.424_427del / p.L142RfsX19) are Brazilian brothers born in the city of Criciúma, Santa Catarina, and there is not known consanguinity in their family. Moreover, there was a report about two similar cases in this city, already deceased, suggesting that STAT5B mutations could be relatively frequent in this region. The objectives of this study were to evaluate the frequency of STAT5B c.424_427del mutation in Criciúma, to assess the existence of the founder effect and to characterize the effect of heterozygous STAT5B c.424_427del mutation on anthropometric and hormonal phenotypes. To evaluate the frequency of this mutation in Criciúma, 1192 individuals from the population were genotyped. Seven heterozygous individuals were identified, which characterized a minimum allele frequency of 0.29% (95% confidence interval: 0.08 to 0.5%), significantly higher than the frequency of other pathogenic variants described in public databases. By using the Hardy-Weinberg law, it was possible to estimate the incidence of cases of individuals homozygous for this mutation at one every 40 years. However, by using the highest possible frequency according to the confidence interval, this incidence could reach one every 13 years. Additionally, the parents of the two reported cases who were similar to patients with homozygous STAT5B mutations were genotyped and these parents were heterozygous for STAT5B c.424_427del mutation. To assess the founder effect, two markers near the mutation were analyzed in the two boys homozygous for STAT5B c.424_427del mutation, in 33 heterozygous individuals from seven unrelated families and in 53 control individuals. The same haplotype was present in the homozygous boys and in all heterozygous individuals, while in only 9,4% control individuals (p < 0,001), pointing to the probability that STAT5B c.424_427del mutation in different families has been inherited from a common ancestor. To study the effects of heterozygous STAT5B c.424_427del mutation, 33 heterozygous individuals were compared to 38 non-carrier relatives on height and some laboratorial tests. Heterozygous individuals were significantly shorter than their noncarrier relatives, with a height reduction of 0.6 standard deviation scores (p= 0,006). Furthermore, they had a significant reduction in insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) standard deviation scores, without differences in the other exams. These findings show that heterozygous STAT5B mutations cause a significant negative impact on height, milder than the effect seen in patients with homozygous mutations, with height within the normal range. This result favors the hypothesis that rare pathogenic variants in heterozygous state contribute to normal height variability

Short noncoding RNAs play roles in regulating nearly every biological process, in nearly every organism, yet the exact function and importance of these molecules remains a subject of some debate. In order to gain a better understanding of the contexts in which these regulators have evolved, I have undertaken a variety of approaches to study the evolutionary history of the components that make up these pathways, in the form of two main research efforts. In the first chapter, I have used a combination of population genetics and molecular evolution techniques to show that proteins involved in the piRNA pathway are rapidly evolving, and that different components of the pathway seem to be evolving rapidly on different timescales. These rapidly evolving piRNA pathway proteins can be loosely separated into two groups. The first group appears to evolve quickly at the species level, perhaps in response to transposons that invade across species lines, while the second group appears to evolve quickly at the level of individual populations, perhaps in response to transposons that are paternally present yet novel to the maternal genome. In the second chapter of my research, I have used molecular evolution techniques and carefully devised controls to show that the binding sites of well-conserved miRNAs are among the most slowly changing short motifs in the genome, consistent with a conserved function for these short RNAs in regulatory pathways that are ancient and extremely slow to change. I have additionally discovered a major flaw in an existing approach to motif turnover calculations, which may lead to systematic biases in the published literature toward the false inference of increased regulatory complexity over time. I have implemented a revised approach to motif turnover that addresses this flaw.

The Atlantic cod (Gadus morhua L.) fishery is of great economic importance to the Faroese economy. There are two separately managed cod stocks around the Faroe Islands, the Faroe Plateau and the Faroe Bank cod. Both have experienced dramatic decreases in size and informed management decisions are vital for both stock viability and exploitation. The stocks are geographically isolated by an 800 m deep channel and water temperatures are on average 1 – 2 ºC higher on the Faroe Bank than on the Faroe Plateau. There are clear phenotypic differences between the stocks; in particular, the markedly higher growth rate for the Faroe Bank cod has caught public and scientific attention. There is continuing debate regarding the relative importance of genetics and environmental contributions to the contrasting phenotypes. Analyses of reproductive parameters (field data and experimental captive spawnings) as well as analyses of microsatellite and single nucleotide polymorphism (SNP) markers were undertaken to better resolve the issue. Field data as well as data from experimental captive spawnings provided evidence of reproductive differences between Faroe Plateau and Faroe Bank cod. Peak spawning occurred earlier on the Faroe Plateau than on the Faroe Bank and this difference in timing of spawning was maintained in captivity. In particular, differences in sizes of eggs (average diameters of 1.40 and 1.30 mm for Faroe Plateau and Faroe Bank cod eggs, respectively) and indirect evidence of greater volumes spawned by the Faroe Bank females suggested stock differences with respect to egg size – egg number trade-off. It was hypothesised that the strategy adopted by cod on the Faroe Bank, with a higher number of smaller eggs, evolved in response to a more hostile environment (bare seabed and higher exposure to predators) experienced by early life stages in this area. Experimental captive spawnings with Faroe Bank cod showed a large interfamily skew in survival rates of cod eggs and fry. Egg size was identified as a useful indicator of survival rates in the egg stage, but egg survival rates could not be used to predict viability in later developmental stages, thus highlighting the importance of employing some sort of genetic monitoring of cod fry to ensure sufficient family representation in the progeny. While no tank effect was evident concerning fry survival, a significant tank effect was identified concerning body sizes of fry. Microsatellite data were analysed using large sample sizes of Faroe Plateau and Faroe Bank cod with the Faroe Plateau divided into two locations, Faroe Plateau North-East and Faroe Plateau West (cod from each of the two were known to belong to separate spawning grounds). Two Norwegian coastal cod samples were included as outlier populations. While no genetic differentiation was detected between the two Faroe Plateau locations, these analyses revealed a detectable, albeit relatively modest, degree of genetic differentiation between cod from the Faroe Plateau and the Faroe Bank (FST = 0.0014 and 0.0018; DJost_EST = 0.0027 and 0.0048; P < 0.0001 and P < 0.001 for the Faroe Plateau North-East – Faroe Bank and the Faroe Plateau West – Faroe Bank comparisons). These values were several times smaller than those between Faroese and Norwegian coastal cod (pairwise FST and DJost_EST values in the range of 0.0061 – 0.0137 and 0.0158 – 0.0386, respectively). Despite recent reductions in census population sizes for Faroe Plateau and, particularly, Faroe Bank cod, genetic diversity estimates were comparable to the ones observed for Norwegian coastal cod and there was no evidence of significant genetic bottlenecks. Lastly, data for one of the markers (Gmo132) indicated genotype-dependent vertical distribution of cod (as investigated for Faroe Plateau North-East cod). Contrary to some previously published studies, analysis of SNPs of two candidate genes for adaptive divergence, the hemoglobin gene Hb-ß1 and the transferrin gene Tf1, failed to detect differentiation between samples of Faroe Plateau and Faroe Bank cod analysed in this thesis. Of 3533 novel SNPs simultaneously discovered and genotyped by restriction-site associated DNA (RAD) sequencing, 58 showed evidence of genetic differentiation between Faroe Plateau North-East and Faroe Bank cod (P < 0.05). No single locus was fixed for different alleles between Faroe Plateau and Faroe Bank cod. A set of eight informative SNPs (FST values between Faroe Plateau and Faroe Bank samples > 0.25; P < 0.0005) were selected for validation in larger samples, that included cod from both Faroe Plateau areas and the Faroe Bank as well as Norwegian coastal and White Sea cod. Six out of the eight loci amplified successfully with a PCR-based method and there was 100 % concordance between genotypes of individuals screened by both techniques. Due to ascertainment bias, the SNPs should only be applied with caution in a broader geographical context. Nonetheless, these SNPs did confirm the genetic substructure suggested for Faroese cod by microsatellite analyses. While no genetic differentiation was evident between the two Faroe Plateau locations, significant genetic differentiation was evident between Faroe Plateau and Faroe Bank cod at five of the SNPs (FST values in the range of 0.0383 – 0.1914). This panel of five SNPs could confidently be used to trace groups of Faroe Plateau and Faroe Bank cod to their population of origin. In conclusion, multiple lines of evidence demonstrate that Faroe Plateau and Faroe Bank cod are truly two genetically distinct populations. While the findings contribute to a broader understanding of the biology and the genetics of Faroe Plateau and Faroe Bank cod, the novel SNPs developed may provide a valuable resource for potential future demands of i.e. genetic stock identification methods.

The complex demographic changes that underlie the expansion of anatomically modern humans out of Africa have important consequences on the dynamics of natural selection and our ability to detect it. In this thesis, I aimed to refine our knowledge on human population history using ancient genomes, and then used a climate-informed, spatially explicit framework to explore the interplay between complex demographies and selection. I first analysed a high-coverage genome from Upper Palaeolithic Romania from ~37.8 kya, and demonstrated an early diversification of multiple lineages shortly after the out-of-Africa expansion (Chapter 2). I then investigated Late Upper Palaeolithic (~13.3ky old) and Mesolithic (~9.7 ky old) samples from the Caucasus and a Late Upper Palaeolithic (~13.7ky old) sample from Western Europe, and found that these two groups belong to distinct lineages that also diverged shortly after the out of Africa, ~45-60 ky ago (Chapter 3). Finally, I used East Asian samples from ~7.7ky ago to show that there has been a greater degree of genetic continuity in this region compared to Europe (Chapter 4). In the second part of my thesis, I used a climate-informed, spatially explicit demographic model that captures the out-of-Africa expansion to explore natural selection. I first investigated whether the model can represent the confounding effect of demography on selection statistics, when applied to neutral part of the genome (Chapter 5). Whilst the overlap between different selection statistics was somewhat underestimated by the model, the relationship between signals from different populations is generally well-captured. I then modelled natural selection in the same framework and investigated the spatial distribution of two genetic variants associated with a protective effect against malaria, sickle-cell anaemia and β⁰ thalassemia (Chapter 6). I found that although this model can reproduce the disjoint ranges of different variants typical of the former, it is incompatible with overlapping distributions characteristic of the latter. Furthermore, our model is compatible with the inferred single origin of sickle-cell disease in most regions, but it can not reproduce the presence of this disorder in India without long-distance migrations.