Allelic Expression Imbalance in the Human Retinal Transcriptome and Potential Impact on Inherited Retinal Diseases

Inherited retinal diseases (IRDs) are often associated with variable clinical expressivity (VE) and incomplete penetrance (IP). Underlying mechanisms may include environmental, epigenetic, and genetic factors. Cis-acting expression quantitative trait loci (cis-eQTLs) can be implicated in the regulation of genes by favoring or hampering the expression of one allele over the other. Thus, the presence of such loci elicits allelic expression imbalance (AEI) that can be traced by massive parallel sequencing techniques. In this study, we performed an AEI analysis on RNA-sequencing (RNA-seq) data, from 52 healthy retina donors, that identified 194 imbalanced single nucleotide polymorphisms(SNPs) in 67 IRD genes. Focusing on SNPs displaying AEI at a frequency higher than 10%, we found evidence of AEI in several IRD genes regularly associated with IP and VE (BEST1, RP1, PROM1, and PRPH2). Based on these SNPs commonly undergoing AEI, we performed pyrosequencing in an independent sample set of 17 healthy retina donors in order to confirm our findings. Indeed, we were able to validate CDHR1, BEST1, and PROM1 to be subjected to cis-acting regulation. With this work, we aim to shed light on differentially expressed alleles in the human retina transcriptome that, in the context of autosomal dominant IRD cases, could help to explain IP or VE.Peer reviewe

CDHR1 mutations in retinal dystrophies

We report ophthalmic and genetic findings in patients with autosomal recessive retinitis pigmentosa (RP), cone-rod dystrophy (CRD) or cone dystrophy (CD) harboring potential pathogenic variants in the CDHR1 gene. Detailed ophthalmic examination was performed in seven sporadic and six familial subjects. Mutation screening was done using a customized next generation sequencing panel targeting 105 genes implicated in inherited retinal disorders. In one family, homozygosity mapping with subsequent candidate gene analysis was performed. Stringent filtering for rare and potentially disease causing variants following a model of autosomal recessive inheritance led to the identification of eleven different CDHR1 variants in nine index cases. All variants were novel at the time of their identification. In silico analyses confirmed their pathogenic potential. Minigene assays were performed for two non-canonical splice site variants and revealed missplicing for the mutant alleles. Mutations in CDHR1 are a rare cause of retinal dystrophy. Our study further expands the mutational spectrum of this gene and the associated clinical presentation

Characterization of Retinal Structure in ATF6-Associated Achromatopsia.

PurposeMutations in six genes have been associated with achromatopsia (ACHM): CNGA3, CNGB3, PDE6H, PDE6C, GNAT2, and ATF6. ATF6 is the most recent gene to be identified, though thorough phenotyping of this genetic subtype is lacking. Here, we sought to test the hypothesis that ATF6-associated ACHM is a structurally distinct form of congenital ACHM.MethodsSeven genetically confirmed subjects from five nonconsanguineous families were recruited. Foveal hypoplasia and the integrity of the ellipsoid zone (EZ) band (a.k.a., IS/OS) were graded from optical coherence tomography (OCT) images. Images of the photoreceptor mosaic were acquired using confocal and nonconfocal split-detection adaptive optics scanning light ophthalmoscopy (AOSLO). Parafoveal cone and rod density values were calculated and compared to published normative data as well as data from two subjects harboring CNGA3 or CNGB3 mutations who were recruited for comparative purposes. Additionally, nonconfocal dark-field AOSLO images of the retinal pigment epithelium were obtained, with quantitative analysis performed in one subject with ATF6-ACHM.ResultsFoveal hypoplasia was observed in all subjects with ATF6 mutations. Absence of the EZ band within the foveal region (grade 3) or appearance of a hyporeflective zone (grade 4) was seen in all subjects with ATF6 using OCT. There was no evidence of remnant foveal cone structure using confocal AOSLO, although sporadic cone-like structures were seen in nonconfocal split-detection AOSLO. There was a lack of cone structure in the parafovea, in direct contrast to previous reports.ConclusionsOur data demonstrate a near absence of cone structure in subjects harboring ATF6 mutations. This implicates ATF6 as having a major role in cone development and suggests that at least a subset of subjects with ATF6-ACHM have markedly fewer cellular targets for cone-directed gene therapies than do subjects with CNGA3- or CNGB3-ACHM

Eine Exkursion der gemeinsamen Baukommission von VDB und dbv nach Rotterdam und Umgebung

Wenn in den vergangenen Jahren die Anforderungen an neue Bibliotheksbauten diskutiert wurden, dann fielen zumeist Begriffe wie „Lernort“ oder „Dritter Ort“. Für die bauliche Entwicklung von Bibliotheken sind dies ohne Frage wichtige Themen. Die Arbeit der gemeinsamen Baukommission von dbv und VDB verdeutlichte jedoch den Bedarf, künftig auch weitere Aspekte näher zu betrachten. Initiativ dazu reiste die gemeinsame Baukommission im Oktober 2019 mit Kolleg*innen fast aller Bundesländer in die Niederlande, um dort unterschiedliche Bibliotheksneu- und -umbauten der letzten Jahre unter jeweils verschiedenen Fragestellungen zu untersuchen

The need for widely available genomic testing in rare eye diseases: an ERN-EYE position statement.

BACKGROUND: Rare Eye Diseases (RED) are the leading cause of visual impairment and blindness for children and young adults in Europe. This heterogeneous group of conditions includes over 900 disorders ranging from relatively prevalent disorders such as retinitis pigmentosa to very rare entities such as developmental eye anomalies. A significant number of patients with RED have an underlying genetic etiology. One of the aims of the European Reference Network for Rare Eye Diseases (ERN-EYE) is to facilitate improvement in diagnosis of RED in European member states. MAIN BODY: Technological advances have allowed genetic and genomic testing for RED. The outcome of genetic testing allows better understanding of the condition and allows reproductive and therapeutic options. The increase of the number of clinical trials for RED has provided urgency for genetic testing in RED. A survey of countries participating in ERN-EYE demonstrated that the majority are able to access some forms of genomic testing. However, there is significant variability, particularly regarding testing as part of clinical service. Some countries have a well-delineated rare disease pathway and have a national plan for rare diseases combined or not with a national plan for genomics in medicine. In other countries, there is a well-established organization of genetic centres that offer reimbursed genomic testing of RED and other rare diseases. Clinicians often rely upon research-funded laboratories or private companies. Notably, some member states rely on cross-border testing by way of an academic research project. Consequently, many clinicians are either unable to access testing or are confronted with long turnaround times. Overall, while the cost of sequencing has dropped, the cumulative cost of a genomic testing service for populations remains considerable. Importantly, the majority of countries reported healthcare budgets that limit testing. SHORT CONCLUSION: Despite technological advances, critical gaps in genomic testing remain in Europe, especially in smaller countries where no formal genomic testing pathways exist. Even within larger countries, the existing arrangements are insufficient to meet the demand and to ensure access. ERN-EYE promotes access to genetic testing in RED and emphasizes the clinical need and relevance of genetic testing in RED

Rhodopsin F45L Allele Does Not Cause Autosomal Dominant Retinitis Pigmentosa in a Large Caucasian Family

Purpose: To ascertain the potential pathogenicity of a retinitis pigmentosa (RP)-causing RHO F45L allele in a family affected by congenital achromatopsia (ACHM). Methods: Case series/observational study that included two patients with ACHM and 24 extended family members. Molecular genetic analysis was performed to identify RHO F45L carrier status in the family and a control population. An adaptive optics scanning light ophthalmoscope (AOSLO) was used to image the photoreceptor mosaic and assess rod and cone structure. Spectral domain optical coherence tomography (SD-OCT) was used to examine retinal lamination. Comprehensive clinical testing included acuity, color vision, and dilated fundus examination. Electroretinography was used to assess rod and cone function. Results: Five carriers of the RHO F45L allele alone (24-80 years) and three carriers in combination with a heterozygous CNGA3 mutant allele (10-64 years) were all free of the classic symptoms and signs of RP. In heterozygous carriers of both mutations, SD-OCT showed normal retinal thickness and intact outer retinal layers; rod and cone densities were within normal limits on AOSLO. The phenotype in two individuals affected with ACHM and harboring the RHO F45L allele was indistinguishable from that previously reported for ACHM. Conclusions: The RHO F45L allele is not pathogenic in this large family; hence, the two ACHM patients would unlikely develop RP in the future. Translational Relevance: The combined approach of comprehensive molecular analysis of individual genomes and noninvasive cellular resolution retinal imaging enhances the current repertoire of clinical diagnostic tools, giving a substantial impetus to personalized medicine

De novo intrachromosomal gene conversion from OPN1MW to OPN1LW in the male germline results in Blue Cone Monochromacy

X-linked cone dysfunction disorders such as Blue Cone Monochromacy and X-linked Cone Dystrophy are characterized by complete loss (of) or reduced L- and M- cone function due to defects in the OPN1LW/OPN1MW gene cluster. Here we investigated 24 affected males from 16 families with either a structurally intact gene cluster or at least one intact single (hybrid) gene but harbouring rare combinations of common SNPs in exon 3 in single or multiple OPN1LW and OPN1MW gene copies. We assessed twelve different OPN1LW/MW exon 3 haplotypes by semi-quantitative minigene splicing assay. Nine haplotypes resulted in aberrant splicing of ≥20% of transcripts including the known pathogenic haplotypes (i.e. ‘LIAVA’, ‘LVAVA’) with absent or minute amounts of correctly spliced transcripts, respectively. De novo formation of the ‘LIAVA’ haplotype derived from an ancestral less deleterious ‘LIAVS’ haplotype was observed in one family with strikingly different phenotypes among affected family members. We could establish intrachromosomal gene conversion in the male germline as underlying mechanism. Gene conversion in the OPN1LW/OPN1MW genes has been postulated, however, we are first to demonstrate a de novo gene conversion within the lineage of a pedigree

In Vivo Analysis of Disease-Associated Point Mutations Unveils Profound Differences in mRNA Splicing of Peripherin-2 in Rod and Cone Photoreceptors

Point mutations in peripherin-2 (PRPH2) are associated with severe retinal degenerative disorders affecting rod and/or cone photoreceptors. Various disease-causing mutations have been identified, but the exact contribution of a given mutation to the clinical phenotype remains unclear. Exonic point mutations are usually assumed to alter single amino acids, thereby influencing specific protein characteristics;however, they can also affect mRNA splicing. To examine the effects of distinct PRPH2 point mutations on mRNA splicing and protein expression in vivo, we designed PRPH2 minigenes containing the three coding exons and relevant intronic regions of human PRPH2. Minigenes carrying wild type PRPH2 or PRPH2 exon 2 mutations associated with rod or cone disorders were expressed in murine photoreceptors using recombinant adeno-associated virus (rAAV) vectors. We detect three PRPH2 splice isoforms in rods and cones: correctly spliced, intron 1 retention, and unspliced. In addition, we show that only the correctly spliced isoform results in detectable protein expression. Surprisingly, compared to rods, differential splicing leads to lower expression of correctly spliced and higher expression of unspliced PRPH2 in cones. These results were confirmed in qRT-PCR experiments from FAC-sorted murine rods and cones. Strikingly, three out of five cone disease-causing PRPH2 mutations profoundly enhanced correct splicing of PRPH2, which correlated with strong upregulation of mutant PRPH2 protein expression in cones. By contrast, four out of six PRPH2 mutants associated with rod disorders gave rise to a reduced PRPH2 protein expression via different mechanisms. These mechanisms include aberrant mRNA splicing, protein mislocalization, and protein degradation. Our data suggest that upregulation of PRPH2 levels in combination with defects in the PRPH2 function caused by the mutation might be an important mechanism leading to cone degeneration. By contrast, the pathology of rod-specific PRPH2 mutations is rather characterized by PRPH2 downregulation and impaired protein localization

Informal Caregiving in Amyotrophic Lateral Sclerosis (ALS): A High Caregiver Burden and Drastic Consequences on Caregivers’ Lives

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that causes progressive autonomy loss and need for care. This does not only affect patients themselves, but also the patients’ informal caregivers (CGs) in their health, personal and professional lives. The big efforts of this multi-center study were not only to evaluate the caregivers’ burden and to identify its predictors, but it also should provide a specific understanding of the needs of ALS patients’ CGs and fill the gap of knowledge on their personal and work lives. Using standardized questionnaires, primary data from patients and their main informal CGs (n = 249) were collected. Patients’ functional status and disease severity were evaluated using the Barthel Index, the revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) and the King’s Stages for ALS. The caregivers’ burden was recorded by the Zarit Burden Interview (ZBI). Comorbid anxiety and depression of caregivers were assessed by the Hospital Anxiety and Depression Scale. Additionally, the EuroQol Five Dimension Five Level Scale evaluated their health-related quality of life. The caregivers’ burden was high (mean ZBI = 26/88, 0 = no burden, ≥24 = highly burdened) and correlated with patients’ functional status (rp = −0.555, p < 0.001, n = 242). It was influenced by the CGs’ own mental health issues due to caregiving (+11.36, 95% CI [6.84; 15.87], p < 0.001), patients’ wheelchair dependency (+9.30, 95% CI [5.94; 12.66], p < 0.001) and was interrelated with the CGs’ depression (rp = 0.627, p < 0.001, n = 234), anxiety (rp = 0.550, p < 0.001, n = 234), and poorer physical condition (rp = −0.362, p < 0.001, n = 237). Moreover, female CGs showed symptoms of anxiety more often, which also correlated with the patients’ impairment in daily routine (rs = −0.280, p < 0.001, n = 169). As increasing disease severity, along with decreasing autonomy, was the main predictor of caregiver burden and showed to create relevant (negative) implications on CGs’ lives, patient care and supportive therapies should address this issue. Moreover, in order to preserve the mental and physical health of the CGs, new concepts of care have to focus on both, on not only patients but also their CGs and gender-associated specific issues. As caregiving in ALS also significantly influences the socioeconomic status by restrictions in CGs’ work lives and income, and the main reported needs being lack of psychological support and a high bureaucracy, the situation of CGs needs more attention. Apart from their own multi-disciplinary medical and psychological care, more support in care and patient management issues is required

Ten years of external quality assessment (EQA) of Neisseria gonorrhoeae antimicrobial susceptibility testing in Europe elucidate high reliability of data

BACKGROUND: Confidence in any diagnostic and antimicrobial susceptibility testing data is provided by appropriate and regular quality assurance (QA) procedures. In Europe, the European Gonococcal Antimicrobial Susceptibility Programme (Euro-GASP) has been monitoring the antimicrobial susceptibility in Neisseria gonorrhoeae since 2004. Euro-GASP includes an external quality assessment (EQA) scheme as an essential component for a quality-assured laboratory-based surveillance programme. Participation in the EQA scheme enables any problems with the performed antimicrobial susceptibility testing to be identified and addressed, feeds into the curricula of laboratory training organised by the Euro-GASP network, and assesses the capacity of individual laboratories to detect emerging new, rare and increasing antimicrobial resistance phenotypes. Participant performance in the Euro-GASP EQA scheme over a 10 year period (2007 to 2016, no EQA in 2013) was evaluated. METHODS: Antimicrobial susceptibility category and MIC results from the first 5 years (2007-2011) of the Euro-GASP EQA were compared with the latter 5 years (2012-2016). These time periods were selected to assess the impact of the 2012 European Union case definitions for the reporting of antimicrobial susceptibility. RESULTS: Antimicrobial susceptibility category agreement in each year was ≥91%. Discrepancies in susceptibility categories were generally because the MICs for EQA panel isolates were on or very close to the susceptibility or resistance breakpoints. A high proportion of isolates tested over the 10 years were within one (≥90%) or two (≥97%) MIC log2 dilutions of the modal MIC, respectively. The most common method used was Etest on GC agar base. There was a shift to using breakpoints published by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) in the latter 5 years, however overall impact on the validity of results was limited, as the percentage categorical agreement and MIC concordance changed very little between the two five-year periods. CONCLUSIONS: The high level of comparability of results in this EQA scheme indicates that high quality data are produced by the Euro-GASP participants and gives confidence in susceptibility and resistance data generated by laboratories performing decentralised testing.The study was funded by the European Centre for Disease Prevention and Control (Framework Contract No. ECDC/2013/015). The funding body contributed to the design of the study, the interpretation of the data and to the writing of the manuscript.S