Kreation und Einrichtung der transgenic Maus modelliert für Mecp2, verursacht Rett Syndrom

Das MeCP2 Protein ist ein transkriptonaler Repressor, der an methylierte Cytosine bindet und die Chromatinstruktur modifiziert. Mecp2 ist für 95% aller Rett-Syndrom-Fälle verantwortlich. Trotz der ubiquitären Expression von MeCP2 wird angenommen, dass es einzigartige Funktionen in Neuronen hat. Aber bis heute ist wenig bekannt über die genaue Rolle von MeCP2 in neuronalen Zellen und über den Mechanismus, durch den die Krankheit ausgelöst wird. Das Ziel dieser Arbeit war es, zwei transgene Mausmodelle zu generieren, die Mecp2 gekoppelt an EGFP als Reporter exprimieren. Dadurch erhofften wir uns eine genauere Charakterisierung von Mecp2 und eine angemessene funktionelle Analyse durchführen zu können. Das transgene Konstrukt wurde durch GET Rekombination eines BAC-Klones (B22804) hergestellt, der das gesamte Mecp2 -Gen der Maus mit flankierenden Sequenzen enthält. Das erste Mausmodell überexprimiert das Wildtyp Mecp2, welches mit EGFP am C-Terminus gekoppelt ist. Das zweite transgene Mausmodell überexprimiert eine verkürzte Form (eine bekannte humane Mutation) des Mecp2 Proteins, welchem das Kernlokalisierungssignal fehlt, und ebenfalls am C-Terminus mit EGFP gekoppelt ist. Die Untersuchung der transgenen Linien erlaubt eine zeitlich-räumliche Analyse des MeCP2-Proteins über das EGFP-Signal. Ausserdem sollte das transgene Mausmodell, welches die verkürzte Form des Proteins exprimiert, helfen, die Rolle des mutierten MeCP2 in der Pathogenese des Rett-Syndroms zu verstehen. Obwohl MeCP2 vorwiegend im Gehirn exprimiert wird, scheint es nicht in allen Zellen, inclusive der Gliazellen, exprimiert zu werden. Also wurden die Mecp2+/EGFP+ exprimierenden Gehirnzellen durch FACS (Fluorescence Activated Cell Sorting) isoliert und weiter über Immunfärbungen konnte mit verschiedenen Markern charakterisiert. Ein Ansatz über Transcriptomics mit Mecp2+/EGFP+ Zellpopulationen beider transgener Linien könnte die Regulation von bekannten Zielgenen bestätigen oder neue Zielgene identifizieren. Weitere Analysen die gemacht werden sollen, sind unter anderem Live cell imaging von Mecp2 Synapsen, die Analyse von Mecp2+/EGFP+ Zellen im respiratorischen Netzwerk während der Mausentwicklung und die Bestimmung der Größe der Neuronen, die das transgene Protein exprimieren

PIWI-like protein, HIWI2 is aberrantly expressed in retinoblastoma cells and affects cell-cycle potentially through OTX2

Abstract Retinoblastoma (RB), a childhood cancer, is caused by biallelic mutation of the RB1 gene, but its development is not clearly understood. Furthermore, the presence of a cancer stem cell subpopulation in RB might impact its treatment. PIWI protein, known for its role in stem cell self-renewal, is aberrantly expressed in cancers. We examined the role of the PIWI-like protein HIWI2 in RB and its effect on the stem cell markers in cells of the RB line, Y79. The expression of HIWI2 is significantly increased in Y79 compared with its level in HeLa and ARPE19 cells. The stem cell markers Oct-3/4, Nanog and Sox-2 were not altered upon HIWI2 knockdown in Y79 cells. Interestingly, OTX2 was significantly downregulated in the absence of HIWI2. Otx2 transcripts also decreased in HIWI2-silenced Y79 and ARPE19 cells. Moreover, silencing HIWI2 in Y79 accumulated the cells at G2–M phase and reduced the levels of proliferating cell nuclear antigen (PCNA) and the tumor suppressor, p16. Our results demonstrate that HIWI2 is aberrantly expressed in Y79 cells and silencing of HIWI2 downregulates OTX2, suggesting that HIWI2 might play a role in the progression of RB

Next-generation sequencing-based method shows increased mutation detection sensitivity in an Indian retinoblastoma cohort

Purpose: Retinoblastoma (Rb) is the most common primary intraocular cancer of childhood and one of the major causes of blindness in children. India has the highest number of patients with Rb in the world. Mutations in the RB1 gene are the primary cause of Rb, and heterogeneous mutations are distributed throughout the entire length of the gene. Therefore, genetic testing requires screening of the entire gene, which by conventional sequencing is time consuming and expensive. Methods: In this study, we screened the RB1 gene in the DNA isolated from blood or saliva samples of 50 unrelated patients with Rb using the TruSight Cancer panel. Next-generation sequencing (NGS) was done on the Illumina MiSeq platform. Genetic variations were identified using the Strand NGS software and interpreted using the StrandOmics platform. Results: We were able to detect germline pathogenic mutations in 66% (33/50) of the cases, 12 of which were novel. We were able to detect all types of mutations, including missense, nonsense, splice site, indel, and structural variants. When we considered bilateral Rb cases only, the mutation detection rate increased to 100% (22/22). In unilateral Rb cases, the mutation detection rate was 30% (6/20). Conclusions: Our study suggests that NGS-based approaches increase the sensitivity of mutation detection in the RB1 gene, making it fast and cost-effective compared to the conventional tests performed in a reflex-testing mode

Genetic studies in a patient with X-linked retinoschisis coexisting with developmental delay and sensorineural hearing loss

<p><i>Background</i>: In this study, we present a juvenile retinoschisis patient with developmental delay, sensorineural hearing loss, and reduced axial tone. X-linked juvenile retinoschisis (XLRS) is a retinal dystrophy, most often not associated with systemic anomalies and also not showing any locus heterogeneity. Therefore it was of interest to understand the genetic basis of the condition in this patient.</p> <p><i>Materials and methods: RS1</i> gene screening for XLRS was performed by Sanger sequencing. Whole genome SNP 6.0 array analysis was carried out to investigate gross chromosomal aberrations that could result in systemic phenotype. In addition, targeted next generation sequencing (NGS) was employed to determine any possible involvement of X-linked syndromic and non-syndromic mental retardation genes. This NGS panel consisted of 550 genes implicated in several other rare inherited diseases.</p> <p><i>Results: RS1</i> gene screening revealed a pathogenic hemizygous splice site mutation (c.78+1G>T), inherited from the mother. SNP 6.0 array analysis did not indicate any significant chromosomal aberrations that could be disease-associated. Targeted resequencing did not identify any mutations in the X-linked mental retardation genes. However, variations in three other genes (<i>NSD1, LARGE</i>, and <i>POLG</i>) were detected, which were all inherited from the patient’s unaffected father.</p> <p><i>Conclusions</i>: Taken together, <i>RS1</i> mutation was found to segregate with retinoschisis phenotype while none of the other identified variations were co-segregating with the systemic defects. Hereby, we infer that the multisystemic defects harbored by the patient are a rare coexistence of XLRS, developmental delay, sensorineural hearing loss, and reduced axial tone reported for the first time in the literature.</p

Understanding variable disease severity in X-linked retinoschisis: Does RS1 secretory mechanism determine disease severity?

<div><p>X-linked retinoschisis (XLRS) is a retinal degenerative disorder caused by mutations in <i>RS1</i> gene leading to splitting of retinal layers (schisis) which impairs visual signal processing. Retinoschisin (RS1) is an adhesive protein which is secreted predominantly by the photoreceptors and bipolar cells as a double-octameric complex. In general, XLRS patients show wide clinical heterogeneity, presenting practical challenges in disease management. Though researchers have attempted various approaches to offer an explanation for clinical heterogeneity, the molecular basis has not been understood yet. Therefore, this study aims at establishing a link between the phenotype and genotype based on the molecular mechanism exerted by the mutations. Twenty seven XLRS patients were enrolled, of which seven harboured novel mutations. The mutant constructs were genetically engineered and their secretion profiles were studied by <i>in vitro</i> cell culture experiments. Based on the secretory profile, the patients were categorized as either secreted or non-secreted group. Various clinical parameters such as visual acuity, location of schisis, foveal thickness and ERG parameters were compared between the two groups and control. Although the two groups showed severe disease phenotype in comparison with control, there was no significant difference between the two XLRS groups. However, the secreted group exhibited relatively severe disease indications. On the other hand molecular analysis suggests that most of the <i>RS1</i> mutations result in intracellular retention of retinoschisin. Hence, clinical parameters of patients with non-secreted profile were analyzed which in turn revealed wide variability even within the group. Altogether, our results indicate that disease severity is not merely dependent on secretory profile of the mutations. Thus, we hypothesize that intricate molecular detail such as the precise localization of mutant protein in the cell as well as its ability to assemble into a functionally active oligomer might largely influence disease severity among XLRS patients.</p></div

List of XLRS patients and their clinical characteristics.

<p>List of XLRS patients and their clinical characteristics.</p

Representative clinical images and data of XLRS patients.

<p>(A) Fundus exhibiting spoke wheel pattern like schisis at the macula (indicated by arrow). (B) Optical coherence tomography showing splitting of the inner retinal layers. (C) Electroretinogram showing reduced waveforms of rod and cone responses, a negative b-wave pattern noted on standard combined response (circled).</p

<i>RS1</i> mutations identified in the study and their secretion profiles.

<p><i>RS1</i> mutations identified in the study and their secretion profiles.</p