The genetics of primary congenital glaucoma

One of the objectives of the molecular biological study of glaucoma is to establish how the disease develops as a result of the production of aberrant gene products. Many of the genes associated with glaucoma code for proteins which are likely to be directly or indirectly involved in the development and/or function of cells within the trabecular meshwork. The identification of specific defects in these genes is likely to lead to a better understanding of the mechanisms involved in PCG and glaucoma in general and to the development of alternative therapies to surgery. The CYP1B1 gene in particular, which is a linked to congenital glaucoma, and is expressed in the trabecular meshwork, codes for a member of the cytochrome P450 group of proteins. These iron binding proteins constitute a family of enzymes involved in the processes of xenobiotic metabolism, growth, and development. The discovery of the CYP1B1 gene in PCG emphases the importance of abnormalities in the molecular structure of proteins expressed in cells of the trabecular network as a cause of PCG. The identification of specific genetic defects leads to the possibility of more widespread screening for PCG especially in affected families and hence, the possibility of the identification of asymptomatic carriers of the disease. Early identification of 'at risk' parents may then enable earlier detection of PCG and intervention in the infant.

The isolation, characterization, and application of WRKY genes as useful molecular markers in tropical trees

The improvement of tropical tree crops using conventional breeding methods faces challenges due to the length of time involved. Thus, like most crops, there is an effort to utilize molecular genetic markers in breeding programs to select for desirable agronomic traits. Known as marker assisted breeding or marker assisted selection, genetic markers associated with a phenotype of interest are used to screen and select material reducing the time necessary to evaluate candidates. As the focus of this research was improving disease resistance in tropical trees, the usefulness of the WRKY gene superfamily was investigated as candidates for generating useful molecular genetic markers. WRKY genes encode plant-specific transcriptional factors associated with regulating plants' responses to both biotic and abiotic stress. ^ One pair of degenerate primers amplified 48 WRKY gene fragments from three taxonomically distinct, economically important, tropical tree crop species: 18 from Theobroma cacao L., 21 from Cocos nucifera L. and 9 from Persea americana Mill. Several loci from each species were polymorphic because of single nucleotide substitutions present within a putative non-coding region of the loci. Capillary array electrophoresis-single strand conformational polymorphism (CAE-SSCP) mapped four WRKY loci onto a genetic linkage map of a T. cacao F2 population segregating for resistance to witches' broom disease. Additionally, PCR primers specific for four T. cacao loci successfully amplified WRKY loci from 15 members of the Byttneriae tribe. A method was devised to allow the reliable discrimination of alleles by CAE-SSCP using only the mobility assigned to the sample peaks. Once this method was validated, the diversity of three WRKY loci was evaluated in a germplasm collection of T. cacao . One locus displayed high diversity in the collection, with at least 18 alleles detected from mobility differences of the product peaks. The number of WRKY loci available within the genome, ease of isolation by degenerate PCR, codominant segregation demonstrated in the F2 population, and usefulness for screening germplasm collections and closely related wild species demonstrates that the WRKY superfamily of genes are excellent candidates for developing a number of genetic molecular markers for breeding purposes in tropical trees. ^

Molecular markers and human history: A tale of two haplogroup systems

To chronicle demographic movement across African Asian corridors, a variety of molecular (sequence analysis, restriction mapping and denaturing high performance liquid chromatography etc.) and statistical (correspondence analysis, AMOVA, calculation of diversity indices and phylogenetic inference, etc.) techniques were employed to assess the phylogeographic patterns of mtDNA control region and Y chromosomal variation among 14 sub-Saharan, North African and Middle Eastern populations. The patterns of genetic diversity revealed evidence of multiple migrations across several African Asian passageways as well within the African continent itself. The two-part analysis uncovered several interesting results which include the following: (1) a north (Egypt and Middle East Asia) to south (sub-Saharan Africa) partitioning of both mtDNA and Y chromosomal haplogroup diversity, (2) a genetic diversity gradient in sub-Saharan Africa from east to west, (3) evidence in favor of the Levantine Corridor over the Horn of Africa as the major genetic conduit since the Last Glacial Maximum, (4) a substantially higher mtDNA versus Y chromosomal sub-Saharan component in the Middle East collections, (5) a higher representation of East versus West African mtDNA haplotypes in the Arabian Peninsula populations versus no such bias in the Levant groups and lastly, (6) genetic remnants of the Bantu demographic expansion in sub-Saharan Africa. ^

Landscape genetics of Phaedranassa herb. (Amaryllidaceae) in Ecuador

Speciation can be understood as a continuum occurring at different levels, from population to species. The recent molecular revolution in population genetics has opened a pathway towards understanding species evolution. At the same time, speciation patterns can be better explained by incorporating a geographic context, through the use of geographic information systems (GIS). Phaedranassa (Amaryllidaceae) is a genus restricted to one of the world’s most biodiverse hotspots, the Northern Andes. I studied seven Phaedranassa species from Ecuador. Six of these species are endemic to the country. The topographic complexity of the Andes, which creates local microhabitats ranging from moist slopes to dry valleys, might explain the patterns of Phaedranassa species differentiation. With a Bayesian individual assignment approach, I assessed the genetic structure of the genus throughout Ecuador using twelve microsatellite loci. I also used bioclimatic variables and species geographic coordinates under a Maximum Entropy algorithm to generate distribution models of the species. My results show that Phaedranassa species are genetically well-differentiated. Furthermore, with the exception of two species, all Phaedranassa showed non-overlapping distributions. Phaedranassa viridiflora and P. glauciflora were the only species in which the model predicted a broad species distribution, but genetic evidence indicates that these findings are likely an artifact of species delimitation issues. Both genetic differentiation and nonoverlapping geographic distribution suggest that allopatric divergence could be the general model of genetic differentiation. Evidence of sympatric speciation was found in two geographically and genetically distinct groups of P. viridiflora. Additionally, I report the first register of natural hybridization for the genus. The findings of this research show that the genetic differentiation of species in an intricate landscape as the Andes does not necessarily show a unique trend. Although allopatric speciation is the most common form of speciation, I found evidence of sympatric speciation and hybridization. These results show that the processes of speciation in the Andes have followed several pathways. The mixture of these processes contributes to the high biodiversity of the region.

Reverse Engineering of Modified Genes by Bayesian Network Analysis Defines Molecular Determinants Critical to the Development of Glioblastoma

In this study we have identified key genes that are critical in development of astrocytic tumors. Meta-analysis of microarray studies which compared normal tissue to astrocytoma revealed a set of 646 differentially expressed genes in the majority of astrocytoma. Reverse engineering of these 646 genes using Bayesian network analysis produced a gene network for each grade of astrocytoma (Grade I–IV), and ‘key genes’ within each grade were identified. Genes found to be most influential to development of the highest grade of astrocytoma, Glioblastoma multiforme were: COL4A1, EGFR, BTF3, MPP2, RAB31, CDK4, CD99, ANXA2, TOP2A, and SERBP1. All of these genes were up-regulated, except MPP2 (down regulated). These 10 genes were able to predict tumor status with 96–100% confidence when using logistic regression, cross validation, and the support vector machine analysis. Markov genes interact with NFkβ, ERK, MAPK, VEGF, growth hormone and collagen to produce a network whose top biological functions are cancer, neurological disease, and cellular movement. Three of the 10 genes - EGFR, COL4A1, and CDK4, in particular, seemed to be potential ‘hubs of activity’. Modified expression of these 10 Markov Blanket genes increases lifetime risk of developing glioblastoma compared to the normal population. The glioblastoma risk estimates were dramatically increased with joint effects of 4 or more than 4 Markov Blanket genes. Joint interaction effects of 4, 5, 6, 7, 8, 9 or 10 Markov Blanket genes produced 9, 13, 20.9, 26.7, 52.8, 53.2, 78.1 or 85.9%, respectively, increase in lifetime risk of developing glioblastoma compared to normal population. In summary, it appears that modified expression of several ‘key genes’ may be required for the development of glioblastoma. Further studies are needed to validate these ‘key genes’ as useful tools for early detection and novel therapeutic options for these tumors.

The Genetics of Success: How Single-Nucleotide Polymorphisms Associated With Educational Attainment Relate to Life-Course Development.

A previous genome-wide association study (GWAS) of more than 100,000 individuals identified molecular-genetic predictors of educational attainment. We undertook in-depth life-course investigation of the polygenic score derived from this GWAS using the four-decade Dunedin Study (N = 918). There were five main findings. First, polygenic scores predicted adult economic outcomes even after accounting for educational attainments. Second, genes and environments were correlated: Children with higher polygenic scores were born into better-off homes. Third, children's polygenic scores predicted their adult outcomes even when analyses accounted for their social-class origins; social-mobility analysis showed that children with higher polygenic scores were more upwardly mobile than children with lower scores. Fourth, polygenic scores predicted behavior across the life course, from early acquisition of speech and reading skills through geographic mobility and mate choice and on to financial planning for retirement. Fifth, polygenic-score associations were mediated by psychological characteristics, including intelligence, self-control, and interpersonal skill. Effect sizes were small. Factors connecting DNA sequence with life outcomes may provide targets for interventions to promote population-wide positive development.

Molecular analysis of evolutionary relationships of Phaseolus dumosus with the gene pool of common bean

Valuable genetic variation for bean breeding programs is held within the common bean secondary gene pool which consists of Phaseolus albescens, P. coccineus, P. costaricensis, and P. dumosus. However, the use of close relatives for bean improvement is limited due to the lack of knowledge about genetic variation and genetic plasticity of many of these species. Characterisation and analysis of the genetic diversity is necessary among beans' wild relatives; in addition, conflicting phylogenies and relationships need to be understood and a hypothesis of a hybrid origin of P. dumosus needs to be tested. This thesis research was orientated to generate information about the patterns of relationships among the common bean secondary gene pool, with particular focus on the species Phaseolus dumosus. This species displays a set of characteristics of agronomic interest, not only for the direct improvement of common bean but also as a source of valuable genes for adaptation to climate change. Here I undertake the first comprehensive study of the genetic diversity of P. dumosus as ascertained from both nuclear and chloroplast genome markers. A germplasm collection of the ancestral forms of P. dumosus together with wild, landrace and cultivar representatives of all other species of the common bean secondary gene pool, were used to analyse genetic diversity, phylogenetic relationships and structure of P. dumosus. Data on molecular variation was generated from sequences of cpDNA loci accD-psaI spacer, trnT-trnL spacer, trnL intron and rps14-psaB spacer and from the nrDNA the ITS region. A whole genome DArT array was developed and used for the genotyping of P. dumosus and its closes relatives. 4208 polymorphic markers were generated in the DArT array and from those, 742 markers presented a call rate >95% and zero discordance. DArT markers revealed a moderate genetic polymorphism among P. dumosus samples (13% of polymorphic loci), while P. coccineus presented the highest level of polymorphism (88% of polymorphic loci). At the cpDNA one ancestral haplotype was detected among all samples of all species in the secondary genepool. The ITS region of P. dumosus revealed high homogeneity and polymorphism bias to P. coccineus genome. Phylogenetic reconstructions made with Maximum likelihood and Bayesian methods confirmed previously reported discrepancies among the nuclear and chloroplast genomes of P. dumosus. The outline of relationships by hybridization networks displayed a considerable number of interactions within and between species. This research provides compelling evidence that P. dumosus arose from hybridisation between P. vulgaris and P. coccineus and confirms that P. costaricensis has likely been involved in the genesis or backcrossing events (or both) in the history of P. dumosus. The classification of the specie P. persistentus was analysed based on cpDNA and ITS sequences, the results found this species to be highly related to P. vulgaris but not too similar to P. leptostachyus as previously proposed. This research demonstrates that wild types of the secondary genepool carry a significant genetic variation which makes this a valuable genetic resource for common bean improvement. The DArT array generated in this research is a valuable resource for breeding programs since it has the potential to be used in several approaches including genotyping, discovery of novel traits, mapping and marker-trait associations. Efforts should be made to search for potential populations of P. persistentus and to increase the collection of new populations of P. dumosus, P. albescens and P. costaricensis that may provide valuable traits for introgression into common bean and other Phaseolus crops.

Morphological and molecular characterization of a new species of Atlantic stalked barnacle (Scalpelliformes: Pollicipedidae) from the Cape Verde Islands

[EN] The taxonomy of pedunculate cirripedes belonging to the genus Pollicipes has essentially remained unchanged since Charles Darwin described them in his exhaustive work on the Cirripedia. This genus includes three species of stalked barnacles: Pollicipes pollicipes in the north-eastern Atlantic, P. polymerus in the north-eastern Pacific and P. elegans in the central-eastern Pacific. However, a population genetics analysis of P. pollicipes suggested the presence of a putative cryptic species collected from the Cape Verde Islands in the central-eastern Atlantic. This study examines the morphology of these genetically divergent specimens and compares them with that of representative Atlantic samples of the biogeographically closely related P. pollicipes and with the poorly described P. elegans.

Molecular Mechanisms of Frontotemporal Lobar Degeneration

The aim of this thesis was to identify genetic factors involved in frontotemporal lobar degeneration (FTLD), a neurodegenerative disorder clinically characterised by a progressive change in personality, behaviour and language. FTLD is a genetically complex disorder and a positive family history is found in up to 40% of the cases. In 10-20% of the familial cases the disease can be explained by mutations in the gene encoding the microtubule associated protein tau (MAPT). In the first study we describe the clinical and neuropathological features of a Finnish family with FTLD caused by a mutation in MAPT. We also provide evidence that the pathogenic mechanism of this mutation is through altered splicing of MAPT transcripts. Recently, mutations in the gene encoding progranulin (PGRN) were identified as a major cause of FTLD. In the second study we describe a Swedish family with FTLD caused by a frameshift mutation in PGRN. We provide a clinical and neuropathological description of the family, as well as evidence that the pathogenicity of this mutation is through nonsense-mediated decay of the mutant mRNA transcripts and PGRN haploinsufficiency. In the third study we describe a novel PGRN splice site mutation and a previously described PGRN frameshift mutation, found in a mutation screen of 51 FTLD patients. We describe the clinical and neuropathological characteristics of the mutation carriers and demonstrate that haploinsufficiency is the pathogenic mechanism of the two mutations. In the fourth study we investigate the prevalence of PGRN and MAPT gene dosage alterations in 39 patients with FTLD. No gene dosage alterations were identified, indicating that variations in copy number of the PGRN and MAPT genes are not a common cause of disease, at least not in this FTLD patient collection.

Molecular Mechanism of the Response to Hydrogen Sulfide in C. elegans

Thesis (Ph.D.)--University of Washington, 2016-08

Molecular analysis of RNA1 and RNA2 sequences from a betanodavirus isolated from giant grouper (Epinephelus lanceolatus) in Australia

Betanodavirus infections have a significant impact through direct losses and trade restrictions for aquaculture sectors in Australia. The giant grouper, Epinephelus lanceolatus, is a high-value, fast-growing species with significant aquaculture potential. With subacute to chronic mortalities reported from a commercial aquaculture facility in northern Queensland, the viral nervous necrosis in the affected fish was confirmed using a RT-qPCR followed by virus isolation using the SSN-1 cell line. The RNA1 and RNA2 segments were sequenced and nucleotide sequences were compared with betanodavirus sequences from GenBank. Phylogenetic analysis revealed that both these sequences clustered with sequences representing red spotted grouper nervous necrosis virus genotype and showed high sequence identity to virus sequences affecting other grouper species. This is the first report confirming infection by betanodavirus in E. lanceolatus from Australia with successful isolation of the virus in a cell culture system, and analysis of nearly full length RNA1 and RNA2 sequences.