Cytogenetics is a study on the genetic considerations associated with structural and functional aspects of the cells with reference to chromosomal inclusions. Chromosomes are structures within the cells containing body's information in the form of strings of DNA. When atypical version or structural abnormality in one or more chromosomes prevails, it is defined as chromosomal aberrations (CA) depicting certain genetic pathogeny (known as genetic disorders). The present study assumes the presence of normal and abnormal chromosomal sets in varying proportions in the cytogenetic complex ; and, stochastical mixture theory is invoked to ascertain the information redundancy as a function of fractional abnormal chromosome population. This bioinformatic measure of redundancy is indicated as a track-parameter towards the progression of genetic disorder, for example, the growth of cancer. Lastly, using the results obtained, conclusions are enumerated, inferences are outlined and directions for future studies are considered. / by Jagadeshwari Karri. / Thesis (M.S.C.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Sjèogren's syndrome (S) is a chronic autoimmune disease characterized by ocular and oral dryness and primarily affects post menopausal women. In the present study we investigated the time course of lymphocytic infiltration, apoptosis, caspase-3 activity and different cytokines levels in the lacrimal glands of both genetically predisposed and control mice to elucidate immunopathological mechanism leading to dry eye. The results of our experiments showed that ovariectomy accelerated pathological findings of SS by increasing lympocytic infiltration, cytokine production, lacrimal gland cell death and cleaved caspase-3 activity, and these effects were more pronounced and persistent in the genetically predisposed mouse model of SS. In addition, we observed that lymphocytic infiltration occurred earlier compared to apoptosis which may perpetuate immune mediated destruction of lacrimal epithelial cells. Furthermore, treatment with physioloigical doses of 17-B Estradiol (E2) or DIhydrotestosterone (DHT) prevented all these pathological events observed after ovariectomy. / by Safinaz Mostafa. / Thesis (M.S.)--Florida Atlantic University, 2011. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2011. Mode of access: World Wide Web.

Inherited retinal dystrophies encompass a broad group of genetic disorders affecting visual functions in as high as 1 in 3,000 individuals around the world. Common symptoms include loss of central, periphery, or night visions, and in severe cases progression to complete blindness. Syndromic forms also exist involving abnormalities in other parts of the body. Currently, more than 250 genes representing a wide variety of functional roles have been shown to be responsible for the disease phenotypes. Moreover, mutations in the same gene sometimes cause different phenotypes while mutations in multiple genes can give rise to the same clinical subtype, further demonstrating the level of complexity in these disorders. Such genetic heterogeneity has substantially complicated the process of pinpointing precise genetic causes underlying these conditions. The goal of my thesis research is to clarify the genetic causes underlying retinal dystrophies, with a primary focus on phenotypes resembling ABCA4-associated diseases and retinitis pigmentosa in both syndromic and non-syndromic forms. Recent advances in the next-generation sequencing (NGS), the high-throughput, ‘deep’ sequencing technology, have enabled several novel genes to be identified, or found new mutations in known genes. Nevertheless, a substantial fraction of unsolved cases still remain. The primary work in this thesis involves utilizing NGS, particularly whole-exome sequencing, to identify disease-causal mutations in families where at least one parent and affected or unaffected siblings are available. Determining all genetic variation underlying retinal diseases is necessary for precise molecular genetic diagnosis and improved prognosis of these conditions. The first part of my thesis highlights the complexity in genetic inheritance of diseases caused by mutations in the ABCA4 gene. In a substantial fraction of Stargardt Disease cases with only one mutation in the ABCA4 coding region, deep sequencing of the entire locus identified the second mutation in the intronic region of the gene in 10% of cases. The genetic heterogeneity of ABCA4 was further demonstrated by the identification of 4 different pathogenic ABCA4 mutations and 4 phenotypes in a single family. These findings epitomized the extremely complex mutational spectrum underlying the ABCA4-associated diseases and suggested thorough sequencing of variations in the entire genomic locus, including copy number variant analysis. In the second part of my thesis, exome-sequencing has led to findings of phenotypic expansions in known disease gene, and in one case the precise molecular diagnosis resulted in an immediate treatment. A family with 2 affected siblings presented novel phenotype of a macular dystrophy caused by mutations in CRB1. In another family where 9 members were affected with late-onset BEM, a mutation was found in CRX given incomplete penetrance. In one family with an affected adult, two well-documented mutations in MMACHC - a gene causal for a potentially debilitating disorder of cobalamin deficiency, were found to segregate with bull’s eye maculopathy (BEM) and minimal systemic features in the proband. Early diagnosis in this patient resulted in hydroxycobalamin treatment for her condition, and possibly an improvement of her systemic prognosis. Together, these findings revealed that clinical phenotype can be very divergent from those described, and only genetic testing can unequivocally determine the cause of a disease. The third part of my thesis work highlights first-time discovery, and co-discovery of new genes associated with retinal diseases. A new form of syndromic RP was investigated in a family presenting a previously undescribed constellation of phenotypic features. Exome sequencing analysis of 3 affected siblings and their unaffected parents revealed deleterious mutations in the RDH11 gene. In another family where 2 affected siblings presented with a remarkably similar phenotype, no mutations in RDH11 were detected. However, analysis of absence of heterozygosity revealed causal mutations in the CWC27 gene. In the search for novel genes in cone-rod dystrophy cases negative of ABCA4 mutations, WES identified new rare, deleterious mutations in RAB28 in two families of Spanish descent. These findings revealed novel genetic causes underlying hereditary retinal diseases, and demonstrated the effectiveness of WES analysis in rare disease gene discovery. In summary, this work represents a comprehensive mutational analysis of inherited retinal dystrophies with complex genotype and phenotype correlations, utilizing next-generation DNA sequencing in large study cohorts. The power of whole-exome sequencing for gene discovery was well demonstrated by unequivocally solving close to 50% of all patients examined in this study. Establishing precise correlations between genotype and clinical phenotype is important for facilitating patient care, counseling, and therapeutic intervention for inherited diseases.

Submitted by Suzana Dias (suzana.dias@famerp.br) on 2018-10-22T17:00:05Z No. of bitstreams: 1 CristianiCortez_tese.pdf: 982360 bytes, checksum: e268e8163d7c58538dd0c1134e9b0121 (MD5) / Made available in DSpace on 2018-10-22T17:00:05Z (GMT). No. of bitstreams: 1 CristianiCortez_tese.pdf: 982360 bytes, checksum: e268e8163d7c58538dd0c1134e9b0121 (MD5) Previous issue date: 2017-02-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP / Down syndrome (DS) results from failure in chromosomal segregation during maternal meiosis in about 90-95 % of the cases. The advanced maternal age at conception is considered the major risk factor for DS, however, many mothers of DS individuals are young, suggesting the existence of other etiological factors. Studies suggest that abnormal folate metabolism may cause hypomethylation of DNA pericentromeric, resulting in chromosomal nondisjunction. Moreover, genetic polymorphisms involved in folate pathway have been appointed as maternal risk factors for DS. Objectives: We compared the global DNA methylation between mothers of individuals with DS and mothers of individuals without the syndrome. We also investigated the impact of 18 polymorphisms involved in folate metabolism, and folate, homocysteine (Hcy) and methylmalonic acid (MMA) concentrations on the global DNA methylation. Finally, we evaluated the influence of thymidylate synthase (TYMS) 28-base pair (bp) repeats, TYMS 1494del6, DNA methyltransferase 3B (DNMT3B) -579G>T, DNMT3B -149C>T and DNMT3B -283T>C polymorphisms as maternal risk factors for DS and the association between these polymorphisms and the concentrations of folate, Hcy and MMA. Methods: One hundred and five mothers of individuals with free trisomy 21 and 185 mothers of individuals without the syndrome were included in this study. LINE-1 and Alu methylation were quantified by pyrosequencing. The TYMS 28-bp repeats polymorphism was performed by Polymerase Chain Reaction (PCR) using difference in the size of fragments; TYMS 1494del6 and DNMT3B -579G>T polymorphisms were analyzed by PCR followed by enzymatic digestion; and real-time polymerase chain reaction allelic discrimination was used for the genotyping of DNMT3B -149C>T and -283T>C polymorphisms. Data from the other polymorphisms were obtained from previously published articles by the research group. Plasma MMA and Hcy concentrations were determined by liquid chromatography-tandem mass spectrometry and serum folate by chemiluminescence. Results: LINE-1 methylation was lower in DS mothers than control mothers. Mothers with TCN2 776 CG and GG genotype present high Alu methylation and BHMT 742 GA and AA genotype were associated with low Alu methylation. Moreover, serum folate concentration was a predictor of LINE-1 methylation. Increased maternal risk for DS was associated with TYMS 3R/3R and DNMT3B -149TT/-283TC genotypes. In relation to metabolites, low Hcy concentration was observed in mothers with DNMT3B -149CT/-283CC genotypes when compared with the other combined genotypes. Conclusions: Reduced methylation of the LINE-1 sequence is a maternal risk factor for SD, as well as the TYMS 3R/3R genotype and the DNMT3B -149CT/-283CC combined genotypes. TCN2 776 CG and GG and BHMT 742 GA and AA genotypes modulate the Alu methylation. Serum folate is a predictor of the LINE-1 methylation and Hcy concentration is modulated by DNMT3B -149CT/-283CC combined genotypes in the studied population. / A síndrome de Down (SD) resulta de falhas na segregação cromossômica durante a meiose materna em cerca de 90-95 % dos casos. Embora a idade materna seja um fator de risco bem estabelecido, o nascimento de indivíduos com SD de mães jovens indica a existência de outros fatores etiológicos. Estudos sugerem que o metabolismo anormal do folato pode causar hipometilação do DNA pericentromérico, favorecendo a não disjunção cromossômica. Além disso, polimorfismos em genes envolvidos no metabolismo do folato tem sido apontados como fatores de risco materno para a SD. Objetivos: Comparar a metilação global do DNA entre mães de indivíduos com SD e mães controle; avaliar a influência de 18 polimorfismos em genes envolvidos no metabolismo do folato e das concentrações de folato, homocisteína (Hcy) e ácido metilmalônico (MMA) na metilação do DNA; investigar a contribuição dos polimorfismos timidilato sintase (TYMS) repetição 28 pb, TYMS 1494del6, DNA metiltransferase 3B (DNMT3B) -149C>T, DNMT3B -283T>C e DNMT3B -579G>T na modulação do risco materno para a SD e a associação entre esses polimorfismos e as concentrações de folato, Hcy e MMA. Casuística e Métodos: Foram incluídas 105 mães de indivíduos com trissomia livre do cromossomo 21 e 185 mães de indivíduos sem a síndrome. A metilação das sequências LINE-1 e Alu foram quantificadas por meio de pirosequenciamento. A análise do polimorfismo TYMS repetição 28 pb foi realizada por meio da reação em cadeia da polimerase (PCR) por diferença de tamanho de fragmentos; os polimorfismos TYMS 1494del6 e DNMT3B -579G>T foram analisados por PCR seguida de digestão enzimática; e a PCR em tempo real foi utilizada para a genotipagem dos polimorfismos DNMT3B -149C>T e DNMT3B -283T>C. Os dados dos demais polimorfismos foram obtidos de artigos publicados previamente pelo grupo de pesquisa. O folato sérico foi quantificado por quimioluminescência, e Hcy e MMA plasmáticos foram determinados por cromatografia líquida/espectrometria de massas sequencial. Resultados: A metilação da sequência LINE-1 foi menor em mães de indivíduos com SD quando comparadas com as mães controle. Os genótipos TCN2 776 CG e GG foram associados com elevada metilação da sequência Alu, enquanto baixa metilação dessa sequência foram observadas em mães com os genótipos BHMT 742 GA e AA. O folato foi um preditor da metilação da sequência LINE-1. Mães com os genótipos TYMS 3R/3R ou DNMT3B -149TT/-283TC apresentaram maior risco de ter um filho com SD. Em relação aos metabólitos, baixa concentração de Hcy foi observada em mães com os genótipos DNMT3B -149CT/-283CC quando comparados com os demais genótipos combinados. Conclusões: A metilação reduzida da sequência LINE-1 é um fator de risco materno para a SD, assim como o genótipo TYMS 3R/3R e os genótipos combinados DNMT3B -149TT/-283TC. Os genótipos TCN2 776 CG e GG e BHMT 742 GA e AA modulam a metilação da sequência Alu. O folato sérico é um preditor da metilação da sequência LINE-1 e a concentração de Hcy é modulada pelos genótipos combinados DNMT3B -149CT/-283CC na população estudada.

Neural crest is a vertebrate-specific population of embryonic precursor cells thought to have been essential in vertebrate evolution. During development, a group of naïve ectoderm cells are induced to become neural crest and then undergo series of developmental events to give rise to diverse derivatives. Failure of these events often leads to malfunction of neural crest derived tissues and organs. This thesis focuses on the genetic regulation of two events during neural crest development, induction and differentiation. Neural crest induction refers to the specification of ectoderm cells to the neural crest lineage. It is believed that combinatorial activity of transcription factors governs neural crest induction, but the function of specific transcription factors in this process are not yet clear. The AP-2 family of transcription factors is implicated in control of neural crest development, but whether there is a cell autonomous role of AP-2 transcription factors in neural crest induction has remained uncertain. Here I show that in zebrafish, two AP-2 family members, Tfap2a and Tfap2c, are required redundantly for neural crest induction, and that this requirement is cell autonomous. Failure of neural crest induction in the zebrafish embryos that are devoid of Tfap2a and Tfap2c is not caused by defects in cell survival or cell proliferation, but rather appears to result from a failure neural crest cell fate specification. Simultaneous knockdown of Tfap2a and Tfap2c is one of the only known genetic manipulations that result in failure of neural crest induction. Thus the Tfap2a/c double knockdown embryos will be useful for further studies on the emergence of neural crest during both development and evolution. The second section of my thesis concerns differentiation of neural crest derived zebrafish melanophores. This study reveals that Tfap2a and another AP-2 family member, Tfap2e, redundantly and autonomously regulate melanophore differentiation. This is the first report on the function of Tfap2e in any animal. Given that the expression of AP-2 transcription factors is tightly associated with the metastasis potential of human melanoma, my study reinforces the view that cancer cells co-opt regulatory pathways employed in embryonic development.

Pharmacogenetics is the study of genomic-guided individualized drug prescription that plays an important role in preventing the severe adverse effects of drugs, decreasing the time and cost of therapeutic choices, and directing healthcare professionals to choose medications that are effective and safe. It is noteworthy that this approach becomes highly beneficial in patients suffering from chronic diseases or disorders, since these conditions may require multiple and long term pharmacological therapies, as in children with autism spectrum disorder (ASD). However, public acceptance is a major challenge when implementation of pharmacogenetics merges into clinical practice. The purpose of this study is a) to investigate, among small cohort group of children with ASD, several genetic variants of enzymes that influence the metabolism of commonly prescribed drugs to treat ASD and b) to inspect the knowledge of, attitude towards and future expectations with regards to pharmacogenetics among parents of children with ASD. A group of 15 school-aged participants with ASD were recruited for the study. Approximately 5 ml of venous blood was drawn for each participant to analyze the genotype of enzymes implicated in drug metabolism via pharmacogenetics testing. Thereafter, the parents of these children attended a training session to help them gain a better understanding of the pharmacogenetics results depicted in the drug panel results. A pre-training and post-training survey was conducted to assess the knowledge of, attitude towards and future expectations of pharmacogenetics among the children’s parents.

Cerebrovascular disease is a major cause of dementia in elderly individuals, especially Black/African Americans. Within my dissertation, we focused on two vascular morphologies that affect small vessels: brain arteriolosclerosis (B-ASC) and multi-vascular profiles (MVPs). B-ASC is characterized by degenerative thickening of the wall of brain arterioles. The risk factors, cognitive sequelae, and co-pathologies of B-ASC are not fully understood. To address this, we used multimodal data from the National Alzheimer’s Coordinating Center, Alzheimer’s Disease Neuroimaging Initiative, and brain-banked tissue samples from the University of Kentucky Alzheimer’s Disease Center (UK-ADC) brain repository. We analyzed two age at death groups separately: < 80 years and ≥ 80 years. Hypertension was a risk factor in the < 80 years at death group. In addition, an ABCC9 gene variant (rs704180), previously associated with aging-related hippocampal sclerosis, was associated with B-ASC in the ≥ 80 years at death group. With respect to cognition as determined by test scores, severe B-ASC was associated with worse global cognition in both age groups. With brain-banked tissue samples, we described B-ASC’s relationship to hippocampal sclerosis of aging (HS-Aging), a pathology characterized by neuronal cell loss in the hippocampal region not due to Alzheimer’s disease. We also studied MVPs, which are characterized by multiple small vessel lumens within a single vascular (Virchow-Robin) space. Little information exists on the frequency, risk factors, and co-pathologies of MVPs. Therefore, we used samples and data from the UK-ADC, University of Kentucky pathology department, and University of Pittsburgh pathology department to address this information. We only found MVPs to be correlated with age. Lastly, given the high prevalence of cerebrovascular disease and dementia in Black/African Americans, we discussed the challenges and considerations for studying Blacks/African Americans in these contexts.

The identification of disease genes (genes that when mutated cause human diseases) is an important and challenging problem. Proper diagnosis, prevention, as well as care for patients require an understanding of disease pathophysiology, which is best understood when the underlying causative gene(s) or genetic element(s) are identified. While the availability of the sequenced human genome helped to lead to the discovery of more than 1,900 disease genes, the rate of disease gene discovery is still occurring at a slow pace. The use of genetic linkage methods have successfully led to the identification of numerous disease genes. However, linkage studies are ultimately restricted by available meioses (clinical samples) which result in numerous candidate disease genes. This thesis addresses candidate gene prioritizations in disease gene discovery as applied toward a genetically heterogeneous disease known as Bardet-Biedl Syndrome (BBS). Specifically, the integration of various functional information and the development of a novel comparative genomic approach (Computational Orthologous Prioritization - COP) that led to the identification of BBS3 and BBS11. Functional data integration and application of the COP method may be helpful toward the identification of other disease genes.

Errata pasted onto back page. Bibliography: leaves 193-228.

Preimplantation genetic diagnosis is a technique used in the field of assisted reproduction. The technique is applied to embryos that have been created in vitro, in order to facilitate the selection of embryos according to particular genetic parameters. The use of preimplantation genetic diagnosis by prospective parents at high risk for having a child affected by a genetic disorder has facilitated the birth of unaffected children. Preimplantation genetic diagnosis has already been used for other purposes, such as screening for gender, and could in principle be used to screen for a wide range of genetic traits. The aim of this thesis is to provide good answers to the ethical questions provoked by the advent and continuing development of preimplantation genetic diagnosis. The thesis is divided into four parts. Part One provides a brief overview of the science of genetic selection. Part Two is centred on a discussion of two ethical principles. The principle of procreative liberty is based upon the idea that acts of interference in the reproductive lives of others should be avoided unless there is good justification for such acts. The principle of procreative beneficence is based upon the idea that prospective parents should select the child, of the possible children they could have, who is expected to have the best life. I will argue that the principle of procreative liberty should be applied to acts of interference in individuals� freedom to use preimplantation genetic diagnosis, while the principle of procreative beneficence should be applied to acts of selecting children. In Part Three, I will endorse a position that accords embryos a relatively low moral status, reject the arguments of the disability rights critique, argue that the eugenic aspects of preimplantation genetic diagnosis do not warrant much concern, and develop a framework for critically evaluating slippery slope arguments. Finally, in Part Four, specific applications of preimplantation genetic diagnosis will be examined in detail. Although each application raises unique ethical questions, this thesis aims to demonstrate that the consistent application of the principles and preliminary conclusions developed in Parts Two and Three provides the best means for determining how PGD should be used and which uses should be restricted.