No description available.

No description available.

No description available.

The relatively low cost of Next-Generation Sequencing (NGS) has enabled researchers to generate large amounts of sequencing data in order to identify disease-causing mutations and to assemble simple genomes. However, NGS has inherent limitations due to the short DNA read lengths and high error rate associated with the technique. The short read lengths of NGS prevent the assembly of genomes with long stretches of repetitive DNA, and the high error rate prevents the accurate detection of rare mutations in heterogeneous populations such as tumors and microbiomes. I have co-developed new NGS methods to overcome these challenges. In order to increase the effective read length of NGS reads, local de novo assembly of short reads into long contigs can be achieved through the use of Paired-End Restriction-site Associated DNA Sequencing (RAD-PE-Seq). With the RAD-PE method, I sequenced a stickleback fosmid and generated contigs with an N50 length of 480 nucleotides. In order to eliminate false-positive mutations caused by the high error rate of NGS, the Paired-End Low Error Sequencing (PELE-Seq) method was developed, which uses numerous quality control measures during the sequencing library preparation and data analysis steps in order to effectively eliminate sequencing errors. Control testing of the PELE-Seq demonstrates that the method completely eliminates false-positive mutations at sequencing read depths below 20,000X coverage, compared to a ~20% false-positive rate obtained with previous methods. The high accuracy of the PELE-Seq method allows for the detection of ultra-rare mutations in a genome, which was previously impossible with NGS. This dissertation includes previously published and unpublished co-authored material.

Orientador: Hercílio Martelli Júnior / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-18T03:14:48Z (GMT). No. of bitstreams: 1 Aquino_SibeleNascimentode_M.pdf: 1254396 bytes, checksum: 23d834d1395b6d0cfe104c6231785589 (MD5) Previous issue date: 2011 / Resumo: Fissuras do lábio e/ou palato (FL/P) representam uma das anomalias congênitas mais comuns em humanos. A etiologia das FL/PNS é complexa e envolve a participação de inúmeros genes e fatores ambientais. Diversos estudos têm investigado genes relacionados a síndromes, que apresentam FL/P em seu espectro clínico, e/ou que são expressos durante o desenvolvimento do lábio e/ou palato. O objetivo deste estudo foi verificar se novos polimorfismos contidos nos genes relacionados ao desenvolvimento do lábio e palato, incluindo TGF?3, MSX1, MYH9 e JAG2, podem contribuir para a etiologia das FL/PNS. Seis regiões polimórficas foram genotipadas por PCR-RFLP (reação em cadeia da polimerase associada à análise de polimorfismo de fragmentos de restrição enzimática) em amostras de DNA proveniente de 367 pacientes com FL/PNS (grupo caso) e de 413 indivíduos não afetados (grupo controle). No grupo caso, 54% foram do gênero masculino e 46% do feminino, com idade média de 19,1 ± 14,9 anos e prevalência de indivíduos feodermas (42,5%) e leucodermas (42%). As fissuras lábio-palatinas (FLP) foram predominantes (54%), seguidas pela fissura labial (FL) (24%) e fissura palatina (FP) (22%). Do total de seis polimorfismos analisados neste estudo, apenas um foi confirmado nessa população: rs1057744 do gene JAG2. Para este locus polimórfico, o alelo A e o genótipo GA foi mais comum, no grupo controle e caso, não sendo encontrada diferença estatística significante. Para esse polimorfismo, a análise em um modo dominante ou recessivo também não mostrou diferenças estatísticas significantes. Assim, demonstrou-se que os polimorfismos rs34019007 e rs4252315, do gene TGF?3, rs62636562, do gene MSX1, rs11549910 e rs11549909, do gene MYH9 não foram confirmados. O polimorfismo rs1057744 do gene JAG2, embora confirmado, não apresentou associação significante com FL/PNS na população avaliada / Abstract: Cleft lip and/or cleft palate (CL/P) is one of the most common congenital anomaly in humans. NSCL/P etiology is complex and involves the participation of numerous genes and environmental factors. Several studies have investigated genes related to syndromes that have CL/P in their clinical and/or which are expressed during the development of lip and palate. The aim of this study was to determine whether polymorphisms contained genes related to the development of lip and/or palate, including TGF?3, MSX1, MYH9 and JAG2 can contribute to the etiology of NSCL/P. Six polymorphic regions were genotyped by PCR-RFLP (restriction fragment length polymorphism-polymerase chain reaction) in DNA samples from 367 patients affected by NSCL/P (experimental group) and 413 clinically normal subjects (control group). In the affected group, 54% were male and 46% female, mean age 19.1 ± 14.9 years and the prevalence of mixed black individuals (42.5%) and Caucasian (42%). The clefts of the lip with or without cleft palate (CLP) were predominant (54%), followed by cleft lip (FL) (24%) and cleft palate FP (22%). Out of 6 probable polymorphisms, only one was confirmed in this population: rs1057744 gene JAG2. For this polymorphic locus, the A allele and the genotype was slightly more common in the contr ol and experimental, without statistically signific ant differences. For this polymorphism , the analysis in a dominant or recessive mode also showed no statistically significant difference s between the group. This study demonstrated that the polymorphisms rs34019007 and rs4252315, present in the gene TGF?3, rs62636562, in the MSX1 gene, rs11549910 and rs11549909, in the MYH9 gene we re not confirmed. The rs1057744 polymorphism in JAG2 gene was confirmed in this study but not significantly associated with NSCL/P in the Brazilian population / Mestrado / Patologia / Mestre em Estomatopatologia

No description available.

A comprehensive molecular and phenotypic analysis of congenital and childhood cataractRachel L. Gillespie; The University of Manchester, Doctor of Philosophy, 2015Congenital and childhood cataract (CCC) is estimated to affect 3.5-6 per 10,000 children under 16 years in developed countries - a major cause of lifelong visual impairment. It is estimated that 25-50% of CCC cases are caused by genetic mutations. CCC demonstrates extreme heterogeneity with more than 100 associated genes, and may occur as an isolated anomaly of the eye (non-syndromic) or as a manifestation of a multisystem condition (syndromic). Limitations of conventional sequencing technologies have precluded precise genetic diagnosis and limited understanding of the epidemiological basis of the condition. Next generation sequencing (NGS) technologies have revolutionised the approach to the study of human disease. The aim of this research was to conduct a comprehensive molecular and phenotypic analysis of CCC using NGS.A disease-targeted NGS assay was designed to screen, in parallel, 115 genes associated with all forms of CCC. DNA from 36 patients, randomly selected from the study cohort, underwent cataract-targeted NGS. Putative cataract-causing variants were identified in 75% of individuals; 85% of non-syndromic patients and 63% of syndromic CCC patients. Cataract-targeted NGS was able to efficiently delineate disease sub-type and in some cases identified rare syndromic forms of the condition. These findings were envisaged to alter care and management of CCC patients demonstrating the potential clinical utility of the test. In a subset of cases, NGS identified CCC was a manifestation of an inborn error of metabolism. A number of these conditions were eligible for preventative treatment emphasizing the importance of early diagnosis. A strategic approach to the identification of novel recessive causes of CCC was also undertaken. Affected children from seven consanguineous families underwent pre-screening by cataract-targeted NGS to delineate those with mutations in known genes. Mutation negative patients underwent autozygosity-guided whole exome sequencing (WES) analysis. This strategic approach to disease gene discovery led to the identification three novel cataract-causing candidate genes, TRIM8, CCDC13 and GRWD1. It also led to the association of EIF2B2, known to cause adult-onset leukoencephalopathy with vanishing white matter (VWM) disease featuring pre-senile onset cataract, with childhood-onset cataract. This work demonstrated that cataract-targeted NGS offers an efficient and unbiased means of pre-screening, however, causation is difficult to assign to novel disease genes in the absence of experimental evidence. Correspondingly, in vitro analysis of a missense variant in HMX1 demonstrated the deleterious effect of the mutation on protein function. This work confirmed HMX1 as the cause of a rare oculoauricular phenotype and expanded the class of disease-causing mutations in this gene. In conclusion, this study has demonstrated that NGS is effective in the study of CCC and has provided a platform for future studies in to the genetic aetiology of the condition, as well as the molecular mechanisms underlying lens transparency and human development. The work adds to the increasing body of evidence that augurs an era of personalised genomic medicine in ophthalmology that will foresee improved patient outcomes attributable to the implementation of a stratified approach to medicine.

Animal traits differ not only in mean, but also in variation around the mean. For instance, one sire’s daughter group may be very homogeneous, while another sire’s daughters are much more heterogeneous in performance. The difference in residual variance can partially be explained by genetic differences. Models for such genetic heterogeneity of environmental variance include genetic effects for the mean and residual variance, and a correlation between the genetic effects for the mean and residual variance to measure how the residual variance might vary with the mean. The aim of this thesis was to develop a method based on double hierarchical generalized linear models for estimating genetic heteroscedasticity, and to apply it on four traits in two domestic animal species; teat count and litter size in pigs, and milk production and somatic cell count in dairy cows. The method developed is fast and has been implemented in software that is widely used in animal breeding, which makes it convenient to use. It is based on an approximation of double hierarchical generalized linear models by normal distributions. When having repeated observations on individuals or genetic groups, the estimates were found to be unbiased. For the traits studied, the estimated heritability values for the mean and the residual variance, and the genetic coefficients of variation, were found in the usual ranges reported. The genetic correlation between mean and residual variance was estimated for the pig traits only, and was found to be favorable for litter size, but unfavorable for teat count.

Beef cattle are continuously selected for different traits and the success in improving these traits has been remarkable. However, for certain traits, it is essential not only to improve the average performance, but also to control the variation around the mean. There is evidence that residual variance may be under genetic control, which opens the possibility of selecting for uniformity. In this sense, the objectives of the present dissertation were: 1) to investigate the extent of genetic heterogeneity of residual variance at the pedigree level in birth weight (BW), weaning weight (WW), yearling weight (YW), foot angle (FA), and claw set (CS) in American Angus cattle; 2) to compare the results of different genetic heterogeneity models; 3) to evaluate the effectiveness of Box-Cox transformation in continuous traits; and 4) to address limitations and explore alternative solutions for implementing genetic parameters for residual variance in genetic evaluations. The first study investigated the genetic heterogeneity of residual variances for BW, WW, and YW. Three models were compared: a homoscedastic residual variance model (M1), a double hierarchical generalized linear model (DHGLM, M2), and a genetically structured environmental variance model (MCMC, M3). The results showed significant genetic heterogeneity of residual variances in growth traits, suggesting the possibility of selection for uniformity. The genetic coefficient of variation for residual variance ranged from 0.90 to 0.92 in M2 and 0.31 to 0.38 in M3 for BW, 0.64 in M2 and 0.01 to 0.29 in M3 for WW, and 0.67 to 0.63 in M2 and 0.25 to 0.31 in M3 for YW. Low heritability estimates for residual variance were found, particularly in M2 (0.08 for BW, 0.06 for WW, and 0.09 for YW). The study identified both negative and positive genetic correlations between mean and residual variance, depending on the trait and data transformation. Negative correlations suggest the potential to increase trait means while decreasing residual variance. However, positive correlations indicate that the genetic response to selection for uniformity may be limited unless a selection index is used. Data transformation reduced skewness but did not eliminate genetic heterogeneity of residual variances. The Bayesian approach provided higher estimates of additive genetic variance for residual variance compared to DHGLM. Overall, the findings indicate the potential to reduce variability through selection and lay the groundwork for incorporating uniformity of growth traits into breeding goals. The second study focused on the genetic heterogeneity of residual variance for two foot conformation traits, FA and CS. Using 45,667 phenotypic records collected between 2009 and 2021, three models were compared: a traditional homoscedastic residual variance model (M1), a DHGLM (M2), and a genetically structured environmental variance model (M3). Results showed that heritability estimates for FA and CS means were within expected ranges, although lower in M2. Despite low heritability estimates for residual variance (0.07 for FA and 0.05 for CS in M2), significant genetic coefficients of variation were found, suggesting that selection on trait mean would also influence residual variance. Positive genetic correlations between mean and residual variance in M2 and M3 indicate that selection for uniformity is feasible, but may require additional strategies such as selection indices. The study highlights the potential of FA and CS as indicators for breeding programs aimed at improving production uniformity in beef cattle. Our findings suggest that selection for uniformity in growth and foot score traits in beef cattle may be limited by low heritability of residual variance and moderate to high positive genetic correlations between mean and residual variance. This was observed for most of the traits studied. To overcome these challenges, further research is needed, particularly to explore genomic information to improve the prediction accuracy of estimated breeding values (EBV) for residual variance. Although studies of uniformity using genomic data are limited, they have shown improved EBV accuracy for residual variance. Additionally, alternative methods for measuring uniformity, such as different uniformity or resilience indicators, should be considered, especially with advances in digital phenotyping. Precision livestock farming technologies that allow for extensive data collection on various production traits should be integrated into the development of new uniformity indicators. This dissertation provides valuable insights into the genetic heterogeneity of residual variance in American Angus cattle and highlights the complexity of selecting for uniformity while improving mean traits. Continued research with larger data sets, genomic information, and further methodological refinement will be critical to advance these findings to improve uniformity and productivity in beef cattle breeding. / Doctor of Philosophy / Uniformity in livestock breeding refers to the goal of reducing variability in certain traits within a livestock population to achieve more consistent and predictable outcomes. This is particularly important for traits that affect productivity, economic efficiency, animal welfare, and product quality. By achieving greater uniformity, producers can optimize management practices, improve marketability, and enhance the overall efficiency of animal production systems. Residual variance refers to the variation in traits that is not explained by known genetic or environmental factors. Recent research suggests that residual variance may be under genetic control, meaning that it is possible to select animals that not only have desirable traits, but also have less variability in those traits. Therefore, this dissertation investigates the genetic control of residual variance that may allow selection for uniformity in traits. The research focused on American Angus cattle and aimed to 1) investigate genetic heterogeneity of residual variance in traits, such as birth weight, weaning weight, yearling weight, foot angle, and claw set; 2) compare different genetic models; 3) evaluate the effectiveness of data transformations; and 4) address limitations in genetic evaluations. The first study examined genetic heterogeneity in growth traits using three models. It revealed significant genetic variability, suggesting the potential for selection for uniformity. The study found both positive and negative genetic correlations between trait means and residual variance, indicating varying potential for reducing variance while improving trait means. Data transformations reduced skewness but did not eliminate genetic heterogeneity. A Bayesian approach provided higher estimates of genetic variance than other methods. The second study focused on foot conformation traits with over 45,000 records. The study showed that despite low heritability for residual variance, there was significant genetic variation, indicating the possibility of altering residual variance through selection. Positive genetic correlations suggested that additional strategies, such as selection indices, may be needed to achieve uniformity in practice. Overall, the findings highlight the complexity of selecting for uniformity while improving average traits and underscore the need for further research, particularly using genomic data, to improve prediction accuracy. Integrating precision livestock farming technologies could help develop new indicators of uniformity, improving productivity and uniformity in beef cattle breeding.

Les syndromes myélodysplasiques (SMD) forment un groupe de pathologies clonales de la cellule souche hématopoïétique (CSH) caractérisées par une hématopoïèse inefficace. La présence d’au moins une anomalie génétique (anomalie cytogénétique ou mutation somatique) est observée dans plus de 90% des cas. Ainsi, plusieurs clones moléculaires pouvaient coexister au moment du diagnostic de la maladie. Dans les SMD avec délétion du chromosome 5 (del(5q)), il a récemment été montré que les anomalies étaient présentes dès le stade de la CSH. Dans les SMD, la pénétrance des anomalies génétiques décrites est incomplète. De plus, peu de choses sont actuellement connues sur l’ordre d’apparition des mutations et leur impact fonctionnel sur les différents clones moléculaires dans le cas des SMD non-del(5q). Grâce au séquençage d’exome entier (WES) de patients ne présentant aucune mutation dans les gènes décrits dans les SMD, nous avons décrit l’existence de mutations dans les gènes BCOR et BCORL1, chez respectivement 4,2% et 0,8% des patients. Les mutations du gène BCOR arrivent tardivement au cours de l’évolution de la maladie et affectent le pronostic des patients. Des approches à l’échelle unicellulaire nous ont également permis d’observer que la majeure partie des mutations identifiées chez les patients sont retrouvées dès le stade CD34+CD38-. Chez les patients, plusieurs clones moléculaires coexistent à ce stade. De plus, les mutations des gènes de l’épissage et de la régulation épigénétique sont fréquemment acquises en premier dans les cellules hématopoïétiques les plus immatures des patients porteurs de SMD. Nous avons observé que certaines mutations, acquises secondairement, sont réparties inégalement dans les différents compartiments hématopoïétiques et peuvent avoir un impact sur la différenciation hématopoïétique. Enfin, nous montrons que la répartition des clones moléculaires évolue au cours du temps. En réponse au traitement par Lenalidomide, on observe également une évolution rapide de l’architecture clonale qui peut être liée au statut de réponse des patients. Ces résultats tendent à confirmer l’hétérogénéité génétique mais aussi fonctionnelle des SMD. Nous avons pu identifier de nouvelles mutations impliquées secondairement dans la physiopathologie des SMD. Il existe une dominance clonale précoce dans les SMD du fait de l’acquisition de toutes les mutations dans les cellules hématopoïétiques immatures. Cependant, les différentes populations hématopoïétiques peuvent présenter des génotypes différents. Enfin cette architecture est variable au cours de l’évolution de la maladie. / Myelodysplastic syndromes (MDS) are a group of clonal disorders of the hematopoietic stem cell (HSC) characterized by ineffective hematopoiesis. At least one genetic abnormality (cytogenetic abnormality or somatic mutation) is observed in more than 90% of cases. Thus, it has been observed several molecular clones which could coexist at diagnosis of the disease. In MDS with deletion of chromosome 5 (del (5q)), it has recently been shown that defects were present in the HSC. In MDS, the penetrance of genetic abnormalities described is incomplete. In addition, little is currently known about the order of appearance of mutations and their functional impact on different molecular clones in the case of non-del (5q) MDS. Through the whole exome sequencing (WES) of patients without mutation in the genes described in MDS, we described the existence of mutations in genes BCOR and BCORL1, in respectively 4.2% and 0.8% of patients. Mutations in the gene BCOR were acquired lately during the course of the disease and affect the prognosis of patients. Approaches at the single cell level have also allowed us to observe that most of the mutations identified in patients are found at the immature differentiation stage CD34+CD38-. In patients, several molecular clones could coexist at this stage. In addition, mutations in gene splicing and epigenetic regulation are frequently first acquired in the most immature hematopoietic cells of MDS patients. We found that certain mutations, acquired in a second time, are distributed unevenly in different hematopoietic compartment and may have an impact on hematopoietic differentiation. Finally, we showed that the distribution of molecular clones evolves over time. In response to treatment with Lenalidomide, it has also been observed a rapid evolution of clonal architecture that can be linked to patient response status. These results tend to confirm the genetic but also functional heterogeneity in MDS. We have identified new mutations involved in the pathogenesis of MDS. We observed an early clonal dominance in MDS because of the acquisition of all mutations in immature hematopoietic cells. However, different hematopoietic populations can have different genotype. Finally, the architecture of mutations could be modifying during the course of the disease.