Functional analysis of selected ABCA4 and CNGB3 variants identified in retinal degeneration patients

This study conducted in vitro analyses to investigate the splicing effects of twenty ABCA4 variants and one CNGB3, which had been predicted in silico to cause aberrant mRNA splicing. To circumvent the problem of tissue availability, the method of in vitro exon trapping was employed, based on the work of Cepko et al., 55 which capitalizes on the highly conserved sequences flanking splice sites, and implements transmissible expression vectors to generate and recover genomic inserts.56, 57 Following replication and selection in E.coli, the pSPL3b vector containing the construct was transfected in transient cells, the RNA isolated, reverse transcribed, and RT-PCR amplified. Visualizing the products via gel electrophoresis allowed for an initial comparison of splicing activity between the normal allele and mutant variants. Of the twenty-one variant analyses, nine were completed within the allotted timeframe for this medical dissertation. Seven variants (three canonical, two intronic, and two exonic) demonstrated disrupted splicing behaviour in vitro in accordance with in silico predictions

Towards identifying the ADRP gene in a large South African family with retinitis pigmentosa

Bibliography: leaves 162-190.The present study was initiated with the aim of elucidating the molecular genetic basis of the RP phenotype segregating in a large SA family of British origin. The family is one of the largest pedigrees from which DNA is archived in the Department, and the pedigree structure and ADRP phenotype will be discussed in detail in chapter two

Bioinformatic and molecular approaches for the analysis of the retinal pigment epithelium (RPE) transcriptome

There is substantial interest in the identification of genes underlying susceptibility to complex human diseases because of the potential utility of such genes in disease prediction and therapy. The complex age-related macular degeneration (AMD) is a prevalent cause of legal blindness in industrialized countries and predominantly affects the elderly population over 75 years of age. Although vision loss in AMD results from photoreceptor cell death in the central retina, the initial pathogenesis likely involves processes in the retinal pigment epithelium (RPE) (Liang and Godley, 2003). The goal of the current study was to identify and characterize genes specifically or abundantly expressed in the RPE in order to determine more comprehensively the transcriptome of the RPE. In addition, our aim was to assess the role of these genes in AMD pathogenesis. Towards this end, a bovine cDNA library enriched for RPE transcripts was constructed in-house using a PCR-based suppression subtractive hybridization (SSH) technique (Diatchenko et al., 1996, 1999), which normalizes for sequence abundance and achieves high enrichment for differentially expressed genes. CAP3 (Huang and Madan, 1999) was used to assemble the high quality sequences of all the 2379 ESTs into clusters or singletons. 1.2% of the 2379 RPE-ESTs contains vector sequences and was excluded from further analysis. 5% of the RPE-ESTs showed homology to multipe chromosomes and were not included in further assembly process. The rest of the ESTs (2245) were assembled into 175 contigs and 509 singletons, which revealed approximately 684 unique genes in the dataset. Out of the 684, 343 bovine RPE transcripts did not align to their human orthologues. A large fraction of clones were shown to include a considerable 3´untranslated regions of the gene that are not conserved between bovine and human. It is the coding regions that can be conserved between bovine and human and not the 3’ UTR (Sharma et al., 2002). Therefore, more sequencing from the cDNA library with reclustering of those 343 ESTs together with continuous blasting might reveal their human orthologoues. To handle the large volume of data that the RPE cDNA library project has generated a highly efficient and user-friendly RDBMS was designed. Using RDBMS data storage can be managed efficiently and flexibly. The RDBMS allows displaying the results in query-based form and report format with additional annotations, links and search functions. Out of the 341 known and predicted genes identified in this study, 2 were further analyzed. The RPE or/and retina specificity of these two clones were further confirmed by RT-PCR analysis in adult human tissues. Construction of a single nucleotide polymphism (SNP) map was initiated as a first step in future case/control association studies. SNP genotyping was carried out for one of these two clones (RPE01-D2, now known as RDH12). 12 SNPs were identified from direct sequencing of the 23.4-kb region, of which 5 are of high frequency. In a next step, comparison of allele frequencies between AMD patients and healthy controls is required. Completion of the expression analysis for other predicted genes identified during this study is in progress using real time RT-PCR and will provide additional candidate genes for further analyses. This study is expected to contribute to our understanding of the genetic basis of RPE function and to clarify the role of the RPE-expressed genes in the predisposition to AMD. It may also help reveal the mechanisms and pathways that are involved in the development of AMD or other retinal dystrophies.Es besteht ein grosses medizinisches Interesse an der Identifizierung von Genen, welche an der Entstehung komplexer, häufiger Krankheiten des Menschen beteiligt sind. Eine solche Krankheit ist die alters-korrelierte Makuladegeneration (AMD). Die AMD ist eine der häufigsten Ursachen für den Verlust der Sehfähigkeit im Alter von über 75 Jahren. Obwohl die Erblindung bei der AMD letztlich durch das Absterben von Photorezeptor-Zellen in der zentralen Retina bedingt wird, gibt es genügend Hinweise dafür, dass die Pathogenese der AMD ihren Ausgang vom retinalen Pigmentepithel (RPE) nimmt (Liang and Godley, 2003). Ziel dieser Arbeit war die Identifizierung und Charakterisierung von RPE-spezifischen Genen als Beitrag zur umfassenden Charakterisierung des RPE-Transkriptoms. Darüberhinaus war es Ziel der Arbeit, die mögliche Rolle der RPE-spezifischen Gene bei der Entstehung der AMD zu explorieren. Ausgangspunkt der Arbeit war eine RPE-spezifische, bovine cDNA Bibliothek, welche in der Arbeitsgruppe auf der Grundlage der SSH-Technik (Diatchenko et al, 1996, 1999) hergestellt worden war. Die SSH-Technik gestattet die Anreicherung von differentiell exprimierten Genen bei gleichzeitiger Normalisierung redundanter Sequenzen. Mit Hilfe des Software-Programms CAP3 (Huang and Madan, 1999) wurden insgesamt 2379 ESTs gruppiert und geordnet. 1,2% der 2379 RPE-ESTs enthielten Vektor Sequenzen und wurden daher von der weiteren Analyse ausgeschlossen. 5% der RPE-ESTs wiesen Homologien zu multiplen Chromosomen auf und wurden daher ebenfalls von der weiteren Analyse ausgeschlossen. Die übrigen 2245 ESTs wurden in 175 Contigs und 509 Singletons gruppiert, woraus sich Hinweise auf insgesamt 684 putative Einzelgene ergaben. 343 dieser 684 Klone zeigten jedoch keine Homologien zu humanen orthologen Sequenzen. Ursache für die fehlende Homologie muss in der grossen Zahl der Klone gesehen werden, bei welchen nur die 3´untranslatierten verglichen wurden. Im Gegensatz zu den kodierenden Sequenzabschnitten kommt es in den nicht-kodierenden Regionen in der Regel zu einer relativ raschen evolutionären Divergenz und damit zum Verlust der Homologie (Sharma et al, 2002). Durch zusätzliche Sequenzierung und Sequenzvergleiche der kodierenden Bereiche dieser 343 Klone lassen sich möglicherweise weitere RPE-spezifische Gene finden. Um die grosse Anzahl der im Rahmen des RPE-Projektes generierten Daten bearbeiten zu können wurde eine sehr effiziente und Benutzer-freundliche Datenbank auf Grundlage des RDBMS-Moduls etabliert. Dieses System gestattet die interaktive Bearbeitung der gespeicherten Daten im Query-Format. Darüberhinaus können die Daten in beliebiger Weise annotiert und verbunden werden. Nach Abzug der 343 nicht-homologen cDNA Klone von den 684 putativen Einzelsequenzen verblieben 341 Kandidaten-Sequenzen. 2 dieser Sequenzen wurden als putative neue RPE-spezifische Gene einer weiteren Analyse zugeführt. Dabei wurde zunächst die RPE- bzw. Retina-Spezifität dieser Kandidaten-Sequenzen mit Hilfe der RT-PCR Analyse bestätigt. Als Basis für zukünftige Fall-Kontroll- und Assoziationsstudien wurde eine SNP-Genotypisierung eines dieser zwei Klone (ursprüngliche Bezeichnung: RPE01-D2; derzeitige Bezeichnung: RDH12) durchgeführt. Die direkte Sequenzanalyse umfasste 23.4 kb und ergab insgesamt 12 SNPs, von denen sich 5 als hoch-informativ erwiesen. Auf dieser Grundlage können zukünftig Allel-Frequenzen zwischen Kontrollpersonen und AMD-Patienten ermittelt und verglichen werden. Zukünftig werden darüberhinaus real-time PCR Methoden zur Expressionsanalyse der verbliebenen Kandidaten-Klone eingesetzt. Zusammenfassend liefert die vorliegende Arbeit einen Beitrag zum Verständnis der genetischen Grundlagen der RPE-Funktionen und trägt zur Aufklärung der Rolle von RPE-spezifischen Genen bei der Disposition zur AMD bei. Zusätzlich ergaben sich Hinweise auf Kandidatengene, welche möglicherweise in der Pathogenese der AMD eine Rolle spielen

Identification and functional validation of genomic boundaries in mammals

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 30-06-2014Eukaryotic genomes are divided into expression domains, which contain DNA coding sequences together with all the regulatory elements needed for their correct spatio-­‐ temporal expression pattern. Genomic boundaries, also known as insulators, flank these domains preventing undesirable crosstalk between the regulatory elements of neighboring domains. They employ various mechanisms and thus, are functionally rather than structurally defined. For this reason, in an attempt to find boundaries in a genome-­‐ wide unbiased fashion in mammals, we focused on identifying those loci where the presence of boundary function would be required to satisfy a biological need. For example, we hypothesized that adjacent genes with opposite expression patterns would need to be separated by boundaries to maintain the independency of their different expression domains. Also, boundaries could be found partitioning the chromatin into inactive heterochromatic and active euchromatic domains, impeding the deleterious effects the spread of the former would have on the latter. Finally, boundaries could also bracket clusters of co-­‐expressed genes to ensure their co-­‐regulation and co-­‐expression. Different algorithms, based on the analysis of gene expression data, were developed in order to explore these scenarios. The resulting evolutionarily conserved non-­‐coding putative insulator sequences were functionally validated using a number of assays. Their enhancer-­‐ blocking properties were evaluated in vitro in human cells in culture, and then in vivo by using transgenic zebrafish. Additionally, one of the most powerful elements was further tested for its ability to protect from chromosomal position effects in transgenic mice. The description and characterization of new genomic boundaries would shed some light into the way mammalian genomes are organized, as well as expand the repertoire of genetic tools that can be incorporated in heterologous constructs to improve the gene transfer technologies by preventing chromosomal position effects.Los genomas de eucariotas están divididos en dominios de expresión, que se definen como aquellas porciones del genoma que contienen uno o varios genes y todos los elementos reguladores necesarios para que que se expresen de acuerdo con un patrón espacio-­‐temporal concreto. Los aisladores genómicos, también llamados insulators, flanquean estos dominios y los protegen de la influencia no deseada de los elementos reguladores contenidos en los dominios vecinos. Existen diversos mechanismos de aislamiento, por lo que los insulators no se definen por una secuencia de ADN concreta, sino porque comparten una misma función. Así, para encontrar aisladores en el genoma de mamíferos de una forma no sesgada, nos propusimos identificar aquellas posiciones del genoma donde se requiere la presencia de función aisladora para satisfacer un problema biológico. Por ejemplo, genes adyacentes con perfiles de expresión completamente distintos deberían estar separados por aisladores que mantuviesen dominios de expresión independientes. Asimismo, cabe esperar la presencia de aisladores entre dominios silentes de heterocromatina y dominios activos de eucromatina. Aquí, impedirían los efectos perjudiciales que el avance de los primeros tendrían sobre los segundos. Finalmente, también podrían encontrarse aisladores flanqueando grupos de genes co-­‐expresados para asegurar su co-­‐regulación y, por tanto, co-­‐expresión. Basándonos en estos escenarios, se desarrollaron diversos algoritmos que usaban datos de expresión génica para predecir la presencia de aisladores. Como resultado de estos algoritmos, se obtuvo una serie de secuencias conservadas evolutivamente y no codificantes que se validaron funcionalmente empleando varios tests. La capacidad de bloqueo de enhancers se evaluó mediante ensayos in vitro en células humanas en cultivo primero, y luego in vivo mediante el uso de peces cebra transgénicos. Además, se analizó la capacidad de uno de los elementos más potentes para proteger de efectos de posición cromosomales en ratones transgénicos. La descripción y caracterización de nuevos aisladores genómicos no sólo sirve para entender mejor cómo se organizan los genomas de mamíferos. También es útil para ampliar el abanico de herramientas disponibles que se pueden usar en construcciones heterólogas para bloquear los efectos de posición cromosomales que se dan comúnmente en experimentos de transferencia genética

CRX controls retinal expression of the X-linked juvenile retinoschisis (RS1) gene

X-linked juvenile retinoschisis is a heritable condition of the retina in males caused by mutations in the RS1 gene. Still, the cellular function and retina-specific expression of RS1 are poorly understood. To address the latter issue, we characterized the minimal promoter driving expression of RS1 in the retina. Binding site prediction, site-directed mutagenesis, and reporter assays suggest an essential role of two nearby cone-rod homeobox (CRX)-responsive elements (CRE) in the proximal −177/+32 RS1 promoter. Chromatin immunoprecipitation associates the RS1 promoter in vivo with CRX, the coactivators CBP, P300, GCN5 and acetylated histone H3. Transgenic Xenopus laevis expressing a green fluorescent protein (GFP) reporter under the control of RS1 promoter sequences show that the −177/+32 fragment drives GFP expression in photoreceptors and bipolar cells. Mutating either of the two conserved CRX binding sites results in strongly decreased RS1 expression. Despite the presence of sequence motifs in the promoter, NRL and NR2E3 appear not to be essential for RS1 expression. Together, our in vitro and in vivo results indicate that two CRE sites in the minimal RS1 promoter region control retinal RS1 expression and establish CRX as a key factor driving this expression