Natural and artificial selection cause changes in certain regions of the genome resulting in selection signatures. Thus, is expected to identify genes associated with the traits under selection in such regions. Selection signatures may be identified using different methodologies, and some are based on detecting of contiguous sequences of homozygous identical-by-descent haplotypes, called runs of homozygosity (ROH), or estimating fixation index (FST) of genomic windows that indicates genetic differentiation. In our study, we aimed to identify selection signatures in a paternal broiler line and to investigate the genes annotated in these regions as well as the biological phenomena involved. For such purpose, ROH and FST-based analysis were performed using whole genome sequence of twenty eight chickens from two different generations. ROH analysis identified homozygous regions of short and moderate length. Analyzing ROH patterns it was observed regions commonly shared among some animals and changes in ROH abundance and length between the two generations. The results also suggests that WGS outperforms SNPchip data, however the number of individuals analyzed must be properly chosen. FST-based analysis revealed genetic differentiation in some genomic windows. Annotation of the consensus regions of ROH and FST windows counted for many genes of which some were previously associated with traits of economic interest, such as APOB, IGF1, IGFBP2, POMC, PPARG, and ZNF423. Overrepresentation analysis of the genes resulted in biological terms of skeletal muscle, matrilin proteins, adipose tissue, hyperglycemia and diabetes, Salmonella infections and tyrosine. Therefore, suggested that ancient and recent selection in TT line acted over regions affecting performance traits. / Identificação de assinaturas de seleção envolvidas com características de desempenho em uma linhagem paterna de frangos de corte Seleção natural e artificial causam mudanças em determinadas regiões do genoma, resultando em assinaturas de seleção. Assim, espera-se identificar genes associados às características sob seleção nessas regiões. Assinaturas de seleção podem ser identificadas usando diferentes metodologias, e algumas delas são baseadas na detecção de sequências contíguas de haplótipos homozigotos idênticos por descendência, chamados segmentos de homozigose (ROH), ou na estimativa do índice de fixação (FST) de janelas genômicas que indicam diferenciação genética. Em nosso estudo, objetivamos identificar assinaturas de seleção em uma linha paterna de frangos de corte e investigar os genes anotados nessas regiões, bem como os fenômenos biológicos envolvidos. Para tal propósito, as análises de ROH e FST foram realizadas usando dados de sequenciamento completo do genoma (WGS) de vinte e oito aves de duas gerações diferentes. A análise de ROH identificou regiões em homozigose de comprimentos curto e moderado. Analisando os padrões de ROH, foram observadas regiões comumente compartilhadas entre alguns animais e mudanças na abundância e no comprimento da ROH entre as duas gerações. Os resultados também sugerem que dados de WGS apresentam vantagens sobre os dados de SNPchip, porém o número de indivíduos analisados deve ser levado em consideração. A análise baseada em FST revelou diferenciação genética em algumas janelas genômicas. Anotação das regiões consenso de ROH e das janelas FST identificaram diversos genes, alguns dos quais já foram associados a características de interesse econômico, como APOB, IGF1, IGFBP2, POMC, PPARG e ZNF423. Análises de enriquecimento dos genes resultaram em termos biológicos de músculo esquelético, proteínas matrilinas, tecido adiposo, hiperglicemia e diabetes, infecções por Salmonella e tirosina. Portanto, sugerimos que a seleção ocorrida durante várias gerações na linha TT atuou sobre as regiões que afetam as características de desempenho destes animais.

Submitted by Priscila Arrigucci Bernardes (p.arrigucci@yahoo.com.br) on 2018-07-25T18:53:46Z No. of bitstreams: 1 Tese_Priscila_Arrigucci_Bernardes.pdf: 3025957 bytes, checksum: 866a988a470ab68070a7e7a922fc2993 (MD5) / Approved for entry into archive by Karina Gimenes Fernandes null (karinagi@fcav.unesp.br) on 2018-07-26T10:33:53Z (GMT) No. of bitstreams: 1 bernardes_pa_dr_jabo.pdf: 3025957 bytes, checksum: 866a988a470ab68070a7e7a922fc2993 (MD5) / Made available in DSpace on 2018-07-26T10:33:53Z (GMT). No. of bitstreams: 1 bernardes_pa_dr_jabo.pdf: 3025957 bytes, checksum: 866a988a470ab68070a7e7a922fc2993 (MD5) Previous issue date: 2018-06-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Dentre as informações fornecidas pelas metodologias que utilizam marcadores do tipo polimorfismo de nucleotídeo único (SNPs), as de segmentos de homozigose (ROH) e de desequilíbrio de ligação têm colaborado para estudos de aplicação direta da informação genômica em populações de bovinos de corte, como em estudos de associação com cobertura ampla do genoma, de seleção genômica e de estrutura da população, dentre outros. Atualmente a imputação vem sendo utilizada principalmente para reduzir custos com a genotipagem dos animais e pode ser utilizada combinando informações genômicas de diferentes painéis. Para que dados imputados sejam utilizados de forma eficiente, é necessário que a imputação tenha sido implementada de forma que todos os animais tenham seus genótipos inferidos com elevada acurácia. No entanto, esta é verificada apenas se houver o genótipo real para avaliar a confiabilidade do genótipo imputado. Dessa maneira, os objetivos deste trabalho foram: (1) estudar a imputação de painéis comercial e customizados de baixa densidade para painéis de alta densidade (Illumina e Affymetrix), assim como para um painel combinado (Illumina + Affymetrix) para bovinos da raça Nelore, e estudar o desequilíbrio de ligação e conformação de blocos de haplótipos antes e após a imputação; (2) estudar estratégias para predição da acurácia de imputação, utilizando redes neurais artificiais e regressão linear múltipla; (3) estudar os segmentos de homozigose e, com isso, a endogamia presente em uma população de bovinos da raça Nelore, assim como identificar os genes presentes nos segmentos de homozigose mais frequentes na população. Os estudos de ROH foram realizados com utilização de informações de 34 touros de diferentes linhagens e suas progênies, totalizando 809 animais genotipados da raça Nelore com informação de 509.107 SNPs (Illumina). Para as análises de imputação e de predição da acurácia de imputação foram utilizados os mesmos animais, sendo que 93 destes também foram genotipados com o painel “Axion Genome-Wide BOS 1 Array Plate”. A partir dos resultados das análises de imputação demonstrou-se que o uso combinado de painéis pode ser uma alternativa para aumentar a densidade e o número de bloco de haplótipos, aumentando a probabilidade de obter um marcador próximo a um QTL de interesse. Além disso, essa estratégia indica que a escolha de SNPs em comum entre os painéis de alta densidade (Ilumina e Affymetrix) pode ser utilizada para customizar um painel de menor densidade, permitindo elevar a acurácia de imputação do painel da Illumina e Affymetrix. Na análise de predição da acurácia de imputação, a utilização de redes neurais artificiais foi mais eficiente comparada ao modelo de regressão linear múltipla, podendo ser utilizada com esta finalidade. A partir dos resultados das análises de ROH observou-se que a população encontra-se com baixo nível de endogamia, no entanto os reprodutores apresentaram maior valor de endogamia comparado a progênie, o qual somado a presença de segmentos de homozigose mais longos nestes animais podem indicar que tenha ocorrido intensa utilização de poucos reprodutores nas gerações mais recentes em algumas famílias. / Among all the information provided by methodologies that use single nucleotide polymorphism (SNPs), the runs of homozygosity (ROH) and linkage disequilibrium have been used for studies that explore genomic information in beef cattle population, as the genome-wide association, genomic selection, the structure of population and others. Nowadays, the imputation is used in these studies to reduce genomic costs and this also can be used combining genomic information from different panels. The animals used to be imputed should present genotypes inferred with high accuracy to allow the use imputed genotypes in other studies. However, the accuracy is verified only if there is a real genotype to evaluate the imputed genotype. Therefore, this study aimed: (1) Evaluate imputation of commercial and customized low density panels to high density panels (Illumina and Affymetrix), as well as to a combined panel (Illumina + Affymetrix) in Nelore beef cattle, and estimating linkage disequilibrium and haplotype blocks conformation to high density panels individually and after imputation; (2) Study a strategy to predict imputation accuracy using artificial neural network and linear regression; (3) Study runs of homozygosity and inbreeding in a populations from Nelore beef cattle, as well as identify genes present in ROH with high frequency in population. For ROH studies were used 34 bulls from different lines and the progeny, totalizing 809 Nelore animals genotyped with information of 509.107 SNPs (Illumina). The imputation analysis and imputation accuracy prediction used the same animals, wherein 93 were also genotyped with Axion Genome-Wide BOS 1 Array Plate. The imputation analysis demonstrates that the combined panels used from different panels can be considered due to increasing the density and number of haplotype blocks, increasing the probability to find a marker close to an important QTL. Furthermore, this strategy indicates that the choice for common SNPs between high-density panels Illumina and Affymetrix to customize a lower density panel can increase the imputation accuracy to Illumina and Affymetrix. The prediction of imputation accuracy analysis showed that the neural network is more efficient compared to linear regression, and could be used for this purpose. The results from ROH analysis showed low population inbreeding, however the sires presented higher inbreeding compared to progenies and longer runs of homozigosity, which suggest that has occurred intense use of few sires in recent generations in some families. / FAPESP: 2015/25096-6 e 2016/22940-3.

No description available.

Les dystrophies musculaires des ceintures (ou limb-girdle muscular dystrophy, LGMD) sont un groupe hétérogène de dystrophies musculaires chez l’adulte et sont définies par une atrophie et une faiblesse progressive qui surviennent dans les muscles proximaux. Chez une cohorte canadienne-française, nous avons précédemment décrit une nouvelle forme récessive, désignée LGMD2L et marquée par une atrophie asymétrique du quadriceps, que nous avions cartographiée au chromosome 11p12-p13 grâce à des analyses de liaison. L’objectif de ce projet de thèse était de raffiner l’intervalle candidat, puis d’identifier et de caractériser le gène muté responsable de la LGMD2L. Grâce à une cartographie par homozygotie de polymorphismes de nucléotide simple (SNPs) réalisée sur une grande famille consanguine, nous avons redéfini l’intervalle candidat à une région du chromosome 11p14.3-p15.1. Par séquençage de l’ADN génomique et complémentaire au gène Anoctamine 5 (ANO5) inclus dans cet intervalle, nous avons identifié trois mutations, chez autant de familles: une substitution créant un site d’épissage aberrant, une insertion d’un nucléotide et une mutation faux-sens. Les deux premières mutations étaient associées à une hausse de la dégradation de l’ARN messager médiée par une troncation prématurée. Nous avons également identifié des mutations ANO5 chez une seconde dystrophie musculaire de type distal cartographiant au même locus que la LGMD2L, nommée MMD3, et dont la manifestation initiale était une faiblesse des mollets, mais qui pouvait progresser vers une atrophie des quadriceps. Une réparation membranaire défective avait été observée chez les fibroblastes de deux patients MMD3, suggérant un rôle pour ANO5 dans ce mécanisme. La localisation et la fonction d’ANO5 dans le muscle sont inconnues, mais cette protéine fait partie d’une famille conservée de protéines à huit domaines transmembranaires, les Anoctamines, dont certains membres sont des transporteurs chloriques activés par le calcium. Les résultats de nos études d’immunofluorescence suggèrent qu’ANO5 se localise peu au sarcolemme, mais plutôt à une structure intracellulaire qui suit la ligne Z des myofibrilles. De façon étonnante, cette localisation était préservée chez un patient LGMD2L porteur homozygote de la mutation d’épissage, en dépit du fait que cette dernière était considérée comme une mutation nulle. Néanmoins, nous avons identifié un épissage alternatif de l’exon 15 qui se produisait sur une proportion des transcrits porteurs de la mutation d’épissage, ce qui rétablirait le cadre de lecture, soulignant la complexité de la régulation de l’épissage d’ANO5 et laissant croire que la LGMD2L pourrait être causée par une perte de fonction partielle, et non complète, d’ANO5. Des études subséquentes par des groupes européens ont montré que les anoctaminopathies 5 sont une cause fréquente de dystrophies musculaires des ceintures chez l’adulte. Notre découverte de mutations au gène Anoctamine 5 a mis en évidence une nouvelle classe de protéines importantes pour la biologie du muscle et a ouvert la voie à de nouvelles pistes pour étudier les mécanismes par lesquels un défaut de réparation membranaire progresse en une dystrophie musculaire. / Limb-girdle muscular dystrophies (LGMD) encompass a broad spectrum of muscular dystrophies in which the initial weakness arises in proximal muscles. We previously described in French-Canadian (FC) families a new form of LGMD characterized by asymmetrical quadriceps femoris atrophy, named LGMD2L, which we mapped to chromosome 11p12-p13 using linkage analyses. The objectives of this thesis project were to refine the candidate interval, identify and characterize the LGMD2L gene. Using single nucleotide polymorphisms (SNPs) homozygosity mapping in a large consanguineous family, we narrowed down the LGMD2L candidate interval to a region on chromosome 11p14.3-p15.1, and identified three mutations in the Anoctamin 5 (ANO5) gene located in the interval. These mutations consisted of a missense, a one-bp duplication and a splice site mutation. We demonstrated that the latter two triggered the nonsense-mediated RNA decay pathway. In addition, we identified ANO5 mutations in cases affected by a non-dysferlin Miyoshi muscular dystrophy mapped also to chromosome 11, termed MMD3. In two MMD3 families of European descent, patients presented with calf weakness as the initial symptoms, sometimes evolving to quadriceps atrophy. Fibroblasts from one MMD3 family were shown to be defective for membrane repair. ANO5 localization and function in muscle are unknown, but it is a member of the conserved Anoctamin family of proteins with eight transmembrane domains, of which some function as calcium-activated chloride channel. Our immunofluorescence studies on longitudinal muscle sections suggest that ANO5 is not importantly localized to the sarcolemma, but rather to a structure following the Z-line. To our surprise, this localization was preserved for a LGMD2L patient homozygous for the splice site mutation, previously considered as a null mutation. By studying the splicing isoforms in this patient, we observed that skipping of exon 15 occurs on a proportion of transcripts, in addition to the aberrant splicing caused by the mutation. This alternative splicing event would recover the reading frame, thus underlining the complexity of ANO5 splicing and suggesting that LGMD2L could be the consequence of a partial, rather than complete, loss-of-function. Subsequent studies by other groups have shown that anoctaminopathies 5 are a common cause of adult-onset LGMD. Our discovery of ANO5 mutations has shed light on a new class of proteins important for the muscle biology and opened new research avenues to study how defective membrane repair progresses into muscular dystrophies.

Les dystrophies musculaires des ceintures (ou limb-girdle muscular dystrophy, LGMD) sont un groupe hétérogène de dystrophies musculaires chez l’adulte et sont définies par une atrophie et une faiblesse progressive qui surviennent dans les muscles proximaux. Chez une cohorte canadienne-française, nous avons précédemment décrit une nouvelle forme récessive, désignée LGMD2L et marquée par une atrophie asymétrique du quadriceps, que nous avions cartographiée au chromosome 11p12-p13 grâce à des analyses de liaison. L’objectif de ce projet de thèse était de raffiner l’intervalle candidat, puis d’identifier et de caractériser le gène muté responsable de la LGMD2L. Grâce à une cartographie par homozygotie de polymorphismes de nucléotide simple (SNPs) réalisée sur une grande famille consanguine, nous avons redéfini l’intervalle candidat à une région du chromosome 11p14.3-p15.1. Par séquençage de l’ADN génomique et complémentaire au gène Anoctamine 5 (ANO5) inclus dans cet intervalle, nous avons identifié trois mutations, chez autant de familles: une substitution créant un site d’épissage aberrant, une insertion d’un nucléotide et une mutation faux-sens. Les deux premières mutations étaient associées à une hausse de la dégradation de l’ARN messager médiée par une troncation prématurée. Nous avons également identifié des mutations ANO5 chez une seconde dystrophie musculaire de type distal cartographiant au même locus que la LGMD2L, nommée MMD3, et dont la manifestation initiale était une faiblesse des mollets, mais qui pouvait progresser vers une atrophie des quadriceps. Une réparation membranaire défective avait été observée chez les fibroblastes de deux patients MMD3, suggérant un rôle pour ANO5 dans ce mécanisme. La localisation et la fonction d’ANO5 dans le muscle sont inconnues, mais cette protéine fait partie d’une famille conservée de protéines à huit domaines transmembranaires, les Anoctamines, dont certains membres sont des transporteurs chloriques activés par le calcium. Les résultats de nos études d’immunofluorescence suggèrent qu’ANO5 se localise peu au sarcolemme, mais plutôt à une structure intracellulaire qui suit la ligne Z des myofibrilles. De façon étonnante, cette localisation était préservée chez un patient LGMD2L porteur homozygote de la mutation d’épissage, en dépit du fait que cette dernière était considérée comme une mutation nulle. Néanmoins, nous avons identifié un épissage alternatif de l’exon 15 qui se produisait sur une proportion des transcrits porteurs de la mutation d’épissage, ce qui rétablirait le cadre de lecture, soulignant la complexité de la régulation de l’épissage d’ANO5 et laissant croire que la LGMD2L pourrait être causée par une perte de fonction partielle, et non complète, d’ANO5. Des études subséquentes par des groupes européens ont montré que les anoctaminopathies 5 sont une cause fréquente de dystrophies musculaires des ceintures chez l’adulte. Notre découverte de mutations au gène Anoctamine 5 a mis en évidence une nouvelle classe de protéines importantes pour la biologie du muscle et a ouvert la voie à de nouvelles pistes pour étudier les mécanismes par lesquels un défaut de réparation membranaire progresse en une dystrophie musculaire. / Limb-girdle muscular dystrophies (LGMD) encompass a broad spectrum of muscular dystrophies in which the initial weakness arises in proximal muscles. We previously described in French-Canadian (FC) families a new form of LGMD characterized by asymmetrical quadriceps femoris atrophy, named LGMD2L, which we mapped to chromosome 11p12-p13 using linkage analyses. The objectives of this thesis project were to refine the candidate interval, identify and characterize the LGMD2L gene. Using single nucleotide polymorphisms (SNPs) homozygosity mapping in a large consanguineous family, we narrowed down the LGMD2L candidate interval to a region on chromosome 11p14.3-p15.1, and identified three mutations in the Anoctamin 5 (ANO5) gene located in the interval. These mutations consisted of a missense, a one-bp duplication and a splice site mutation. We demonstrated that the latter two triggered the nonsense-mediated RNA decay pathway. In addition, we identified ANO5 mutations in cases affected by a non-dysferlin Miyoshi muscular dystrophy mapped also to chromosome 11, termed MMD3. In two MMD3 families of European descent, patients presented with calf weakness as the initial symptoms, sometimes evolving to quadriceps atrophy. Fibroblasts from one MMD3 family were shown to be defective for membrane repair. ANO5 localization and function in muscle are unknown, but it is a member of the conserved Anoctamin family of proteins with eight transmembrane domains, of which some function as calcium-activated chloride channel. Our immunofluorescence studies on longitudinal muscle sections suggest that ANO5 is not importantly localized to the sarcolemma, but rather to a structure following the Z-line. To our surprise, this localization was preserved for a LGMD2L patient homozygous for the splice site mutation, previously considered as a null mutation. By studying the splicing isoforms in this patient, we observed that skipping of exon 15 occurs on a proportion of transcripts, in addition to the aberrant splicing caused by the mutation. This alternative splicing event would recover the reading frame, thus underlining the complexity of ANO5 splicing and suggesting that LGMD2L could be the consequence of a partial, rather than complete, loss-of-function. Subsequent studies by other groups have shown that anoctaminopathies 5 are a common cause of adult-onset LGMD. Our discovery of ANO5 mutations has shed light on a new class of proteins important for the muscle biology and opened new research avenues to study how defective membrane repair progresses into muscular dystrophies.

Submitted by LETÍCIA BORGES JOAQUIM null (leticiajoaquim@gmail.com) on 2016-03-18T19:56:09Z No. of bitstreams: 1 DissertaçãoLeticia.pdf: 1246720 bytes, checksum: 72c61040420200f71667f56d8d424fec (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-03-22T12:39:49Z (GMT) No. of bitstreams: 1 joaquim_lb_me_jabo.pdf: 1246720 bytes, checksum: 72c61040420200f71667f56d8d424fec (MD5) / Made available in DSpace on 2016-03-22T12:39:49Z (GMT). No. of bitstreams: 1 joaquim_lb_me_jabo.pdf: 1246720 bytes, checksum: 72c61040420200f71667f56d8d424fec (MD5) Previous issue date: 2016-02-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os painéis de marcadores de alta densidade têm demonstrado a sua funcionalidade nos estudos de estrutura da população e conservação genética. Esses painéis permitem avaliar similaridades no padrão do desequilíbrio de ligação em toda população, assim como informações sobre parentesco da população. Segmentos de homozigose são utilizados como indicativo da estrutura da população e fornecem informações sobre o histórico demográfico e eventos de endogamia da mesma. O objetivo deste trabalho foi estudar a estrutura genômica de uma linhagem sintética base Landrace por meio de (i) análises de desequilíbrio de ligação; (ii) estimação do coeficiente de endogamia utilizando dados genômicos e de pedigree; (iii) análise do número e tamanho de segmentos de homozigose e (iv) determinação da estratificação da população. Foram utilizados registros de 300 fêmeas e 25 machos de uma linhagem fêmea sintética base Landrace genotipados com o painel Illumina PorcineSNP60 v2 BeadChip. A edição dos dados foi realizada no programa PLINK v.1.9 para remoção de marcadores SNPs (“Single Nucleotide Polymorphism”) que falharam em mais de 10% das amostras (“call rate”) e amostras que falharam em mais de 10% dos marcadores. A extensão do desequilíbrio de ligação foi avaliada entre todos os pares SNPs adjacentes presentes nos cromossomos autossômicos por meio da medida r2. O coeficiente de endogamia foi calculado usando registros de pedigree e de dados genômicos. Os segmentos de homozigose foram detectados para os cromossomos autossômicos com o programa computacional PLINK v.1.9, considerando pelo menos 50 SNPs homozigotos dentro de tamanho mínimo de 1.000 Kb por animal, permitindo um SNP heterozigoto e um SNP faltante/perdido dentro de uma janela de 50 SNPs. Os segmentos de homozigose detectados foram utilizados para cálculo do coeficiente de endogamia genômica e como indicativo do histórico da população. A estratificação da população foi avaliada utilizando análises de componentes principais e pelo modelo de ancestralidade por metodologia bayesiana aplicado no programa STRUCTURE. Na análise de ancestralidade foram testados valores de K (número de “clusters”) variando de um a oito usando período de burn-in de 1.000 iterações e Cadeia de Markov e Monte Carlo de 10.000 iterações. As análises foram repetidas dez vezes para cada K e a determinação do melhor número para K foi estimada utilizando a estatística Delta K. O valor médio de r² encontrado para todos os SNPs adjacentes que estão a uma distância menor que 100 Kb foi de 0,291 ± 0,312. Os coeficientes de endogamia médios obtidos a partir dos segmentos de homozigose e de registros de pedigree foram de 0,119 e 0,00011, respectivamente. A baixa correlação (r<0,04) encontrada entre os coeficientes de endogamia pode ser explicada pelo efeito de recombinação gênica sobre aqueles estimados a partir dos segmentos de homozigose que não é considerada nas estimativas obtidas à partir de registros de pedigree e devido aos erros de identificação dos animais no pedigree. A identificação de um grande número de longos segmentos de homozigose pode ser indicativa de endogamia recente na população estudada. O estudo da estratificação da amostra indicou que a mesma estaria dividida em duas populações (k=2), sendo que a separação pode ser explicada pelo cruzamento entre as raças ocidentais e orientais utilizadas na formação da linhagem. Os dados de genotipagem permitiram concluir que a população estudada possui endogamia recente, sugerindo-se que seja priorizado acasalamento entre indivíduos menos aparentados para assegurar a manutenção da diversidade genética. / High-density single nucleotide polymorphism (SNP) panels have been used in genomic studies, such as population structure and conservation genetic studies. These panels allow to assess similarities in the patterns of linkage disequilibrium across populations and to estimate relatedness between populations. Runs of homozygosity are used as indicative of population structure and it provides information about demographic history and recent inbreeding. The aim of this study was to describe the genomic structure of a synthetic Landrace line by (i) linkage disequilibrium (LD) analyses; (ii) inbreeding estimates through pedigree and genomic data; (iii) analysing the number and length of runs of homozygosity (ROH); and (iv) determination of population structure. A total of 300 females and 25 males from synthetic Landrace line were genotyped using Illumina PorcineSNP60 v2 BeadChip. Data editing was performed using PLINK v.1.9. The SNPs and samples with a call rate lower than 0.90 were excluded from the data set. Only the autosomal chromosomes were considered for LD an ROH analyses. The LD between all pairs of SNPs were measured by the means of the genotype correlation coefficient (r²) and it determined the decay of LD with physical distance. The coefficient of inbreeding was calculated using genomic and pedigree data. ROH were detected using PLINK v.1.9 considering the follow parameters: a minimum ROH of 50 SNPs with a minimum length of 1000 (Kb), one heterozygous SNP and one missing SNP were allowed within the sliding window of 50 SNPs. The individual genomic inbreeding and population history were identified from estimated ROH. The population structure was evaluated using principal component analysis and Bayesian admixture model. The number of clusters (K) was tested from two to eight considering a burning period of 1,000 followed by 10,000 Markov chain Monte Carlo repetitions and replicated ten times for each K. The best K was estimated using the Delta K statistic. For all SNPs adjacent less than 100 kilobase (kb) apart, the average r2 was 0.291 ± 0.312. The average inbreeding coefficient, calculated by ROH and pedigree analyses, were 0.119 and 0.00011, respectively. The low correlation between the inbreeding coefficients can be justified by the genetic recombination effect over those estimated from runs of homozygosity that were no considered on the estimates from the traditional pedigree. The high number of long ROH is an evidence of recent inbreeding in this population. The population structure analysis revealed K=2 was the best number of clusters and separation. This result can be explained by the crossbreeding between the Eastern and Western breeds used in the formation of the line. The genotyping data helped confirm that the inbreeding in the studied population is recent, which suggests that mating between individuals less related occurred to ensure the maintenance of genetic diversity.

Les ataxies forment un groupe de maladies neurodégénératives qui sont caractérisées par un manque de coordination des mouvements volontaires. Mes travaux ont porté sur une forme d'ataxie à début tardif (LOCA), après l’âge de 50 ans. Les principales caractéristiques cliniques sont: atrophie cérébelleuse à l’IRM (88%), dysarthrie (81%), atrophie du lobe frontal (50%) et nystagmus (52%). La ségrégation dans les familles de cette ataxie est en faveur d’une transmission récessive. Afin d'identifier le gène responsable de LOCA, nous avons recruté 38 patients affectés d'une forme tardive d'ataxie, issus du SLSJ, des Cantons de l’Est ou d’autres régions du Québec. Un premier criblage du génome a été effectué avec des marqueurs microsatellites sur une famille clé. Une analyse de liaison paramétrique nous a suggéré une liaison au chromosome 13 (4.4Mb). Une recherche d’un haplotype partagé entre 17 familles LOCA a diminué la taille de l'intervalle candidat à 1.6Mb, mais l’haplotype s’est avéré fréquent dans la population canadienne-française. Un second criblage du génome avec des marqueurs SNP nous a permis d’évaluer par cartographie d’homozygotie la possibilité qu’une mutation fondatrice partagée dans des sous-groupes de malades. Plusieurs stratégies d'analyse ont été effectuées, entre autre par regroupement régional. Aucun loci candidats ne fut identifié avec confiance. Nous avons donc combiné les données de génotypage avec le séquençage exomique afin d'identifier le gène responsable. L'analyse de six individus atteints nous a permis d'obtenir une liste de variants rare contenant quatre gènes potentiels. Cette analyse doit se poursuivre pour identifier le gène responsable de LOCA. / Ataxias are a heterogeneous group of neurodegenerative diseases and are characterized by a lack of voluntary movements. My Master's project was on a late-onset ataxia (LOCA), after 50 years of age. The main clinical features are: cerebellar atrophy on MRI (88%), dysarthria (81%), frontal lobe atrophy (50%) and nystagmus (52%). Disease segregation in the family is suggestive of a recessive transmission. In order to identify the causal gene of LOCA, we have recruited 38 patients affected by a late-onset ataxia, originated from SLSJ, Eastern townships or other region in Quebec. A first genome scan was done with microsatellite markers on an informative family. Parametric linkage analysis suggested linkage on chromosome 13 (4.4Mb). Haplotype sharing analysis on 17 families reduced the candidate interval to 1.6Mb, but this haplotype was found to be frequent in the French-Canadian population. A second genome scan with SNPs markers allowed us to performed homozygozity mapping and look for founder mutations in subgroup of patients. Many strategies were performed, including regional clustering. No candidate loci were identified with confidence. We decided to combine the genotyping analysis results with exome sequencing to uncover the causative gene. The analysis on six affected individuals allowed us to obtain a rare variants list with four putative genes. More analysis is needed to identify the gene responsible for LOCA.

No description available.

Craniofacial development is a tightly regulated event that requires expression of many genes at a precise space-temporal specificity. Interference in the regulation of such genes and their pathways is known to lead to abnormal phenotypes affecting the face and cranium. In this manner, regulation of these pathways is further complicated by interaction between genetic and environmental factors such that disturbance to either may result in craniofacial malformation, as orofacial clefts. Despite several at-risk loci have been identified, they do not completely explain the high heritability observed for the orofacial clefts and many questions remain open. For example, concerning the orofacial clefts transcriptome, the gene pathways which may be dysregulated and the affected cellular processes are still poorly understood. Further, if there is gene expression dysregulation in orofacial clefts, the causes leading to that need to be elucidated, such as the investigation of epigenetic factors. Also, since the multifactorial contribution makes environment relevant to this malformation, epigenetic and epigenomic differences in orofacial clefts should clarified. At last, rare syndromic forms of orofacial clefts with still unknown molecular cause and mechanisms should be elucidated in order to better understand craniofacial development and their impact in non-syndromic forms. Therefore, the main objective of this study was to investigate the molecular mechanisms involved in the aetiology of orofacial clefts, which was focused in gene expression and epigenetic analysis in non-syndromic cleft lip and/or palate (NSCL/P) as well as genetic, gene expression, animal modelling and epigenetics in Richieri-Costa-Pereira Syndrome (RCPS), a rare autosomal recessive syndromic form of orofacial cleft. We found significant transcriptome differences in NSCL/P in comparison to controls, revealing the BRCA1-dependent DNA damage repair pathway as compromised in NSCL/P cells leading to DNA damage accumulation. Next, we studied the potential of DNA methylation in those cells and found a slight but significant increase of BRCA1 promoter DNA methylation in NSCL/P cells and a distinct DNA methylation distribution, point to a possible epigenetic contribution in this phenomenon. We also evaluated the contribution of DNA methylation in 8q24.21 region, one of the most replicated regions in NSCL/P Genome-wide association studies and found no significant differences in our sample. Attempting to investigate DNA methylation in NSCL/P in an epigenomic level, we analysed methylomes and found 578 methylation variable positions in NSCL/P, highly enriched in regulatory regions and in relevant gene pathways for craniofacial development as Epithelial-Mesenchymal Transition pathway. We also studied effect of DNA methylation in familial NSCL/P displaying incomplete penentrance and found a significant increase of CDH1 promoter hypermethylation in penetrant cases in comparison to non-penetrants. Finally, by the use of different sequencing strategies and identity-by-descent analysis we mapped the mutation region of RCPS to EIF4A3 5\'UTR/promoter and found a complex structure of expanded repeats in RCPS patients leading to EIF4A3 downregulation. We were also able to validate the phenotypes using an animal modelling strategy in zebrafish. Because those repeats are CG rich, we investigated whether they were submitted to DNA hypermethylation in RCPS patients as a cause for EIF4A3 hypomorphism, however we found no evidence of methylation increase in RCPS. In conclusion, we were able to associate dysregulated pathways to NSCL/P susceptibility and DNA methylation differences to both non-familial and familial NSCLP. Besides, we were able to identify the genetic cause of RCPS, which now can be molecularly diagnosed. Altogether, our results add to the understanding of craniofacial development and the aetiology of orofacial clefts / O desenvolvimento craniofacial é um evento finamente regulado que requer a expressão de muitos genes em uma precisão espaço-temporal específica. A interferência na regulação de tais genes e suas respectivas vias é sabidamente causadora de fenótipos que afetam a face e o crânio. Neste sentido, a regulação destas vias é decorrente da interação entre fatores genéticos e ambientais, de tal forma que a perturbação de quaisquer destes fatores pode resultar em malformações craniofaciais, como as fissuras orofaciais. Apesar dos muitos loci de risco já identificados, estes não explicam completamente a alta herdabilidade observadas nas fissuras orofaciais e muitas questões permanecem em aberto. Por exemplo, em relação ao transcriptoma em fissuras orofaciais, as vias genéticas que podem estar desreguladas, assim como processos celulares afetados em decorrência, são ainda pouco compreendidos. Além disso, se há desregulação na expressão de genes em fissuras orofaciais, as causas que levam a essas diferenças necessitam ser elucidadas, como, por exemplo, por meio da investigação de fatores epigenéticos. Também, uma vez que o componente multifatorial torna a influência do ambiente relevante para esta malformação, diferenças epigenéticas e epigenômicas nas fissuras orofaciais devem ser melhor compreendidas. Por fim, formas raras e sindrômicas de fissuras orofaciais sem elucidação de causa moleculares devem ser estudadas para que melhor se compreenda o desenvolvimento craniofacial e o impacto destes mecanismos moleculares em formas não-sindrômicas. Portanto, nosso objetivo principal neste estudo foi investigar os mecanismos moleculares envolvidos na etiologia das fissuras orofaciais, com o foco na análise de expressão gênica e epigenètica em fissuras de lábio-palatinas não-sindrômicas (FL/P NS) e também o estudo genético, de expressão gênica, modelagem animal e epigenética na Síndrome de Richieri-Costa-Pereira (RCPS), uma forma sindrômica e autossômica recessiva de fissura orofacial. Nós encontramos diferenças significantes no transcriptoma de FL/P NS em comparação com controles, que revelaram o comprometimento da via do BRCA1 no reparo ao dano de DNA e o acúmulo de dano de DNA em células FL/P NS. Em seguida, nós estudamos o potencial da metilação de DNA nestas células e encontramos um pequeno, porém significante, aumento de metilação de DNA no promotor do BRCA1 e uma distribuição diferente de metilação, apontando para uma possível contribuição epigenética na desregulação do gene. Nós também avaliamos a contribuição da metilação de DNA na região 8q24.21, uma das mais associadas às FL/P NS por meio de Genome-wide association studies, porém não encontramos diferenças significantes na nossa amostra. Com o intuito de investigar a metilação de DNA em FL/P NS em uma escala epigenômica, nós analisamos o perfil de metilomas e encontramos 578 sítios diferencialmente metilados nas FL/P NS, altamente enriquecidos em regiões regulatórias e em vias relevantes para o desenvolvimento craniofacial como a via de Transição Epitélio-Mesenquimal. Nós também estudamos o efeito da metilação de DNA em casos famílias de FL/P NS com penetrância incompleta e encontramos um aumento significativo de metilação do promotor do CDH1 nos casos penetrantes em comparação aos não-penetrantes. Por último, por meio de diferentes estratégias de sequenciamento e análise de segregação de haplótipos nós mapeamos a mutação de RCPS na região 5\'UTR/promotor do EIF4A3 e encontramos uma estrutura complexa de expansão de repetições nos pacientes RCPS, ocasionando a diminuição da expressão do EIF4A3. Nós também reproduzimos fenótipos comparáveis aos da RCPS por meio de modelo animal em zebrafish. Uma vez que tais repetições são ricas em CG, nós investigamos se estas poderiam ser submetidas à metilação de DNA em pacientes RCPS como uma causa para a redução dos transcritos do EIF4A3, porém não encontramos evidências de aumento de metilação em RCPS. Em conclusão, nós conseguimos associar vias gênicas desreguladas à susceptibilidade para as FL/P NS e diferenças de metilação de DNA tanto em casos familiais como não-familiais de FL/P NS. Além disso, identificamos a causa genética de RCPS, sendo que a síndrome pode ser agora diagnosticada molecularmente. Em conjunto, nossos resultados adicionam ao conhecimento do desenvolvimento craniofacial e na etiologia das fissuras orofaciais

La pratique du mariage entre apparentés au sein de la population libanaise, favorisée par des raisons sociales, religieuses, géographiques et aussi politiques, a vu apparaître des sous-groupes de populations de taille plus ou moins réduite, parfois à la limite d’isolats génétiques. Ceci a engendré une augmentation de la prévalence des maladies autosomiques récessives fréquentes mais aussi et surtout rares. Parmi ces dernières, les chondrodysplasies ont retenu notre attention. Elles sont caractérisées par un retard statural dû à un défaut du processus d’ossification endochondale, qui est responsable de la croissance des os longs. Au cours de ces dernières décennies, plus de 230 gènes responsables d’environ 400 maladies osseuses constitutionnelles ont été identifiés. Cependant, les bases moléculaires d'une centaine de dysplasies osseuses restent, à ce jour, inconnues. L’identification de gènes codant pour des protéines de nature extrêmement variée a contribué à la compréhension du mécanisme complexe d’ossification endochondrale. Mon travail de thèse, réalisé en cotutelle entre l’équipe de recherche « Bases moléculaires et physiopathologiques des chondrodysplasies » de l’hôpital Necker enfants-malades, à Paris en France et l’Unité de Génétique Médicale (UGM) de l’Université Saint-Joseph au Liban, a consisté à identifier des gènes impliqués dans des dysplasies osseuses autosomiques récessives dans quatre familles libanaises consanguines. Dans ce cadre, différentes stratégies ont été adoptées. La première a été une stratégie d’intersection des variations détectées par le séquençage de l’exome de deux patients, atteints d’une forme sévère de dysplasie spondylodysplastique létale et issus de deux familles libanaises consanguines et non apparentées (Familles A et B). Nous avons identifié une mutation homozygote du gène MAGMAS (NM_016069, p.Asn76Asp) (Mitochondria-associated granulocyte macrophage CSF-signaling molecule) à l’origine de la maladie dans les deux familles A et B. MAGMAS est une protéine associée à la mitochondrie et impliquée dans la régulation de l’import actif des protéines vers la matrice mitochondriale. Par immunohistochimie, nous avons montré que MAGMAS est spécifiquement exprimée au niveau de l’os et de la zone hypertrophique du cartilage. MAGMAS, ayant une fonction cruciale pour la survie, est très conservé entre les espèces. Après avoir généré des souches de levures exprimant une copie normale ou mutée du gène humain MAGMAS, nous avons validé l’effet délétère de la mutation p.Asn76Asp, i) sur la croissance des levures, en montrant que les souches portant le gène humain muté présentent un caractère thermosensible, ii) sur la fonction d’import des protéines vers la matrice mitochondriale, qui est altérée dans les souches mutées et iii) sur la stabilité de la protéine. Nous avons également observé un effet de la mutation sur la morphologie des mitochondries et des peroxysomes des cellules de levures, suggérant une induction de l’autophagie dans les souches de levures portant la mutation p.Asn76Asp. L’identification de mutations de MAGMAS dans une dysplasie osseuse sévère, permet d’attribuer à cette protéine un rôle spécifique dans le processus complexe d’ossification endochondrale. La deuxième stratégie a été une combinaison, au sein d’une même famille, d’une stratégie de cartographie par homozygotie et du séquençage de l’exome d’un seul patient. Cette approche a été utilisée dans une famille consanguine avec 3 enfants atteints porteurs d’une dysplasie rhizomélique (Famille C). Nous avons identifié une mutation homozygote du gène NWD1 (NACHT and WD repeat domain containing 1) (NM_001007525, p.Cys1376Tyr) responsable de la maladie dans cette famille C. Ce gène code pour une protéine ayant des domaines WD répétés qui lui confèrent un rôle dans divers mécanismes comme la transduction de signal, la régulation de la transcription, le transport vésiculaire et le contrôle du cycle cellulaire. (...) / Social, religious, geographic and political reasons have favored the consanguineous marriage in the Lebanese population. This led to an increase in the prevalence of autosomal recessive disorders, especially the rare entities including chondrodysplasias. This group of diseases is due to an impairment of the endochondral ossification process. Causative mutations have now been identified in over 230 different genes in more than 400 unique skeletal phenotypes. However, the genetic basis of over 100 different entities remains to be determined. My PhD research project, held between the research group « Bases moléculaires et physiopathologiques des chondrodysplasies » of Necker enfants-malades hospital (INSERM U781, PARIS, France) and the Medical Genetics Unit of Saint-Joseph University (Lebanon), aims to identify genes involved in autosomal recessive skeletal dysplasias in four consanguineous Lebanese families. Different strategies were carried out: the first consists in overlapping data from whole exome sequencing of two patients affected by a new lethal type of spondylodysplastic dysplasia and issued from two consanguineous unrelated Lebanese families (Families A and B). Here, we report a homozygous missense mutation in the Mitochondria-associated granulocyte macrophage CSF-signaling gene (MAGMAS: NM_016069, p.Asn76Asp) in this severe skeletal dysplasia. MAGMAS, also referred to as PAM16, is a mitochondria-associated protein, involved in pre-proteins import into mitochondria and essential for cell growth and development. We demonstrate that MAGMAS is expressed in trabecular bone and cartilage at early developmental stages underlining its specific role in skeletogenesis. We also give strong evidence of the deleterious effect of the identified mutation on the stability of the protein, its in-vivo activity and the viability of yeast strains. We also show that the mutation is able to induce autophagy in yeast cells. Reporting deleterious MAGMAS mutation in a skeletal dysplasia supports a key and specific role for this mitochondrial protein in ossification. Additional studies would be of interest to further understand the specific role of magmas in ossification. The second strategy was to combine, in a consanguineous family, homozygosity mapping with whole exome sequencing of one of the patients. This strategy was undertaken in family C with 3 patients affected by a rhizomelic dysplasia. It allowed us to identify a homozygous missense mutation in the NWD1 gene (NACHT and WD repeat domain containing 1: NM_001007525, p.Cys1376Tyr) as responsible for the skeletal dysplasia in this family. NWD1 belongs to a large group of WD-repeat domain-containing proteins that are involved in different physiological mechanisms such as signal transduction, transcription regulation, vesicular transport and cell cycle control. (...)