Image_1_Detection of DNA Double Strand Breaks by γH2AX Does Not Result in 53bp1 Recruitment in Mouse Retinal Tissues.pdf

<p>Gene editing is an attractive potential treatment of inherited retinopathies. However, it often relies on endogenous DNA repair. Retinal DNA repair is incompletely characterized in humans and animal models. We investigated recruitment of the double stranded break (DSB) repair complex of γH2AX and 53bp1 in both developing and mature mouse neuroretinas. We evaluated the immunofluorescent retinal expression of these proteins during development (P07-P30) in normal and retinal degeneration models, as well as in potassium bromate induced DSB repair in normal adult (3 months) retinal explants. The two murine retinopathy models used had different mutations in Pde6b: the severe rd1 and the milder rd10 models. Compared to normal adult retina, we found increased numbers of γH2AX positive foci in all retinal neurons of the developing retina in both model and control retinas, as well as in wild type untreated retinal explant cultures. In contrast, the 53bp1 staining of the retina differed both in amount and character between cell types at all ages and in all model systems. There was strong pan nuclear staining in ganglion, amacrine, and horizontal cells, and cone photoreceptors, which was attenuated. Rod photoreceptors did not stain unequivocally. In all samples, 53bp1 stained foci only rarely occurred. Co-localization of 53bp1 and γH2AX staining was a very rare event (< 1% of γH2AX foci in the ONL and < 3% in the INL), suggesting the potential for alternate DSB sensing and repair proteins in the murine retina. At a minimum, murine retinal DSB repair does not appear to follow canonical pathways, and our findings suggests further investigation is warranted.</p

Bestrophin 1 – Phenotypes and Functional Aspects in Bestrophinopathies

<div><p></p><p>This is to review the current state of knowledge on the functional and clinical aspects of bestrophin 1, a prominent member of a family of proteins involved in the control and properties of the light peak of the EOG. Initially human bestrophin 1 gene (<i>BEST1</i>) mutations were identified to underlie Best vitelliform macular dystrophy (VMD), a dominantly inherited, juvenile-onset form of macular degeneration. In the recent past the phenotypical spectrum of retinal disorders associated with <i>BEST1</i> mutations has been extended and the term bestrophinopathies was coined. The physiological role of bestrophin 1 is still not completely understood but has been linked to the generation of a transepithelial chloride current by controlling voltage-dependent calcium channels (VDCC). Dysfunction of bestrophin 1 may result in abnormal ion and fluid transport by the retinal pigment epithelium (RPE) disturbing and even disrupting direct interactions between the RPE and the photoreceptors.</p></div

Quantification of cone distribution.

<p>(<b>A–D</b>) S-cone (green) and L/M-cone (red) distribution and quantification in wild-type and mutated canine <i>RPE65^−/−</i> retinae. Box blots display the respective distributions of S-cones and L/M-cones in the 4 regions defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086304#pone-0086304-g001" target="_blank">Figure 1</a>. Statistical analysis was done by a Mann Whitney rank sum test, significant values are marked with asterisks * p<0.001. Bar diagrams show the percentage loss of both cone types compared healthy and affected animal medians in the analyzed regions. Both types of cones are significantly reduced in all analyzed regions. L/M-cones in <i>RPE65^−/−</i> retinae are not only reduced but look different in shape, thus appearing worm-like. Wild type (WT), scale 25 µm.</p

Orientation in the canine eye.

<p>(<b>A</b>) Funduscopy of a <i>RPE65^−/−</i> dog. (<b>B</b>) Extracted eye after euthanasia. The superior part of the eye determines the tapetum lucidum (t). The red marked area corresponds to the retinal flatmount used in the cone opsin expression analysis. The grey marked areas are used for cyrosections used in the bipolar cell sprouting analysis and the numbers correspond to regions shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086304#pone-0086304-g003" target="_blank">Figure 3</a> und 4. (<b>C</b>) Retinal flatmount for cone analysis. Every single quadrat stands for one microscope image (see material and methods). The retina was divided into 4 different regions. The numbers correspond to the regions shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086304#pone-0086304-g002" target="_blank">Figure 2</a>. p = papilla, t = tapetum lucidum.</p

Analysis of sprouting events of rod bipolar cells.

<p>(<b>A</b>) Bar diagrams of the 4 different analyzed regions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086304#pone-0086304-g001" target="_blank">Figure 1B</a>) compared wild type (n = 12 images) and RPE65^−/− (n = 24 images). In regions 1–3 there are no essential differences between wild type and mutated dogs. Region 4 displays a statistical significant discrepancy between wild type and <i>RPE65^−/−</i> dogs (* p<0,001, student t-test). (<b>B</b>) High resolution confocal images for the analysis. The images are projections of the 4 considered images of a z-stack. The ribbon synapses are marked with CtBP2 (red) and the rod bipolar cells are marked with PKC α (green). The counted sprouting events are marked with white circles. Wild type (WT), outer nuclear layer (ONL), outer plexiform layer (OPL), scale 20 µm.</p

List of antibodies used in this study.

<p>List of antibodies used in this study.</p

Scatter plot of correlation between sprouting events and ONL thickness.

<p>In <i>RPE65^−/−</i> dogs, the highest number of sprouting events as well as the thinnest ONL are present in region 4. There is a strong correlation between sprouting events and ONL thickness (correlation coefficient R = 0.8333 for wild type and R = 0.8374 for <i>RPE65^−/−</i> dogs).</p

Cone distribution in healthy and affected animals.

<p>(<b>A</b>) Absolute mean S-cone distribution [10³ cones per mm²] in wild-type retina (n = 2). (<b>B</b>) Absolute mean S-cone distribution [10³ cones per mm2] in <i>RPE65^−/−</i> dogs (n = 4). (<b>C</b>) Relative mean S-cone loss [%] in the affected animals compared to healthy tissue. (<b>D</b>) Absolute mean L/M-cone distribution [10⁴ cones per mm²] in wild-type retina (n = 2). (<b>E</b>) Absolute mean L/M-cone distribution [10⁴ cones per mm²] in <i>RPE65^−/−</i> dogs (n = 4). (<b>F</b>) Relative mean LM-cone [%] loss in the affected animals compared to health tissue. p =  papilla.</p

Characterization of a family with X-linked retinitis pigmentosa and variable expressivity in mutation carriers

Pedigree with three generations. Circles represent females and squares represent males. Slashed symbols indicate deceased family members. Filled black symbols denote family members with retinitis pigmentosa (RP), and circles with a dot indicate female mutation carriers who had no history of visual complaints. Horizontal bars designate family members whose genotype was determined by molecular genetic testing. Arrow marks the index patient 25085. The mutation c.2405_2406delAG in exon ORF15 of segregates with the disease in males, and shows variable heterozygote manifestation in females. Fundus pictures of three affected family members show typical pigmentations found in the peripheral retina of patients with RP. Patient age and gender are provided below each fundus photograph. Pattern of X-chromosome inactivation of selected female family members. None showed a unilateral X-inactivation at the AR-locus. The following abbreviations and symbols are used: control nonrandom X-inactivation (C-nr), control random X-inactivation (C-r), HpaII digestion (+), and no HpaII digestion (-).<p><b>Copyright information:</b></p><p>Taken from "Identification of novel mutations in X-linked retinitis pigmentosa families and implications for diagnostic testing"</p><p></p><p>Molecular Vision 2008;14():1081-1093.</p><p>Published online 06 Jun 2008</p><p>PMCID:PMC2426717.</p><p></p

Increasing the Yield in Targeted Next-Generation Sequencing by Implicating CNV Analysis, Non-Coding Exons and the Overall Variant Load: The Example of Retinal Dystrophies

<div><p>Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are major causes of blindness. They result from mutations in many genes which has long hampered comprehensive genetic analysis. Recently, targeted next-generation sequencing (NGS) has proven useful to overcome this limitation. To uncover “hidden mutations” such as copy number variations (CNVs) and mutations in non-coding regions, we extended the use of NGS data by quantitative readout for the exons of 55 RP and LCA genes in 126 patients, and by including non-coding 5′ exons. We detected several causative CNVs which were key to the diagnosis in hitherto unsolved constellations, e.g. hemizygous point mutations in consanguineous families, and CNVs complemented apparently monoallelic recessive alleles. Mutations of non-coding exon 1 of <i>EYS</i> revealed its contribution to disease. In view of the high carrier frequency for retinal disease gene mutations in the general population, we considered the overall variant load in each patient to assess if a mutation was causative or reflected accidental carriership in patients with mutations in several genes or with single recessive alleles. For example, truncating mutations in <i>RP1</i>, a gene implicated in both recessive and dominant RP, were causative in biallelic constellations, unrelated to disease when heterozygous on a biallelic mutation background of another gene, or even non-pathogenic if close to the C-terminus. Patients with mutations in several loci were common, but without evidence for di- or oligogenic inheritance. Although the number of targeted genes was low compared to previous studies, the mutation detection rate was highest (70%) which likely results from completeness and depth of coverage, and quantitative data analysis. CNV analysis should routinely be applied in targeted NGS, and mutations in non-coding exons give reason to systematically include 5′-UTRs in disease gene or exome panels. Consideration of all variants is indispensable because even truncating mutations may be misleading.</p></div