Genetic and physical mapping of the RP17 locus on 17q identified a 3.6-megabase candidate region that includes the gene encoding carbonic anhydrase IV (CA4), a glycosylphosphatidylinositol-anchored protein that is highly expressed in the choriocapillaris of the human eye. By sequencing candidate genes in this region, we identified a mutation that causes replacement of an arginine with a tryptophan (R14W) in the signal sequence of the CA4 gene at position ؊5 relative to the signal sequence cleavage site. This mutation was found to cosegregate with the disease phenotype in two large families and was not found in 36 unaffected family members or 100 controls. Expression of the mutant cDNA in COS-7 cells produced several findings, suggesting a mechanism by which the mutation can explain the autosomal dominant disease. In transfected COS-7 cells, the R14W mutation (i) reduced the steadystate level of carbonic anhydrase IV activity expressed by 28% due to a combination of decreased synthesis and accelerated turnover; (ii) led to up-regulation of immunoglobulin-binding protein, double-stranded RNA-regulated protein kinase-like ER kinase, and CCAAT͞enhancer-binding protein homologous protein, markers of the unfolded protein response and endoplasmic reticulum stress; and (iii) induced apoptosis, as evidenced by annexin V binding and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining, in most cells expressing the mutant, but not the WT, protein. We suggest that a high level of expression of the mutant allele in the endothelial cells of the choriocapillaris leads to apoptosis, leading in turn to ischemia in the overlying retina and producing autosomal dominant retinitis pigmentosa.unfolded protein response ͉ choriocapillaris ͉ annexin V ͉ terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining ͉ endoplasmic reticulum stress

Summary The cause of autosomal-dominant retinitis pigmentosa (adRP), which leads to loss of vision and blindness, was investigated in families lacking a molecular diagnosis. A refined locus for adRP on Chr17q22 (RP17) was delineated through genotyping and genome sequencing, leading to the identification of structural variants (SVs) that segregate with disease. Eight different complex SVs were characterized in 22 adRP-affected families with >300 affected individuals. All RP17 SVs had breakpoints within a genomic region spanning YPEL2 to LINC01476. To investigate the mechanism of disease, we reprogrammed fibroblasts from affected individuals and controls into induced pluripotent stem cells (iPSCs) and differentiated them into photoreceptor precursor cells (PPCs) or retinal organoids (ROs). Hi-C was performed on ROs, and differential expression of regional genes and a retinal enhancer RNA at this locus was assessed by qPCR. The epigenetic landscape of the region, and Hi-C RO data, showed that YPEL2 sits within its own topologically associating domain (TAD), rich in enhancers with binding sites for retinal transcription factors. The Hi-C map of RP17 ROs revealed creation of a neo-TAD with ectopic contacts between GDPD1 and retinal enhancers, and modeling of all RP17 SVs was consistent with neo-TADs leading to ectopic retinal-specific enhancer- GDPD1 accessibility. qPCR confirmed increased expression of GDPD1 and increased expression of the retinal enhancer that enters the neo-TAD. Altered TAD structure resulting in increased retinal expression of GDPD1 is the likely convergent mechanism of disease, consistent with a dominant gain of function. Our study highlights the importance of SVs as a genomic mechanism in unsolved Mendelian diseases.

RPGR exon ORF15 variants are one of the most frequent causes for inherited retinal disorders (IRDs), in particular retinitis pigmentosa. The low sequence complexity of this mutation hotspot makes it prone to indels and challenging for sequence data analysis. Whole-exome sequencing generally fails to provide adequate coverage in this region. Therefore, complementary methods are needed to avoid false positives as well as negative results. In this study, next-generation sequencing (NGS) was used to sequence long-range PCR amplicons for an IRD cohort of African ancestry. By developing a novel secondary analysis pipeline based on de novo assembly, we were able to avoid the miscalling of variants generated by standard NGS analysis tools. We identified pathogenic variants in 11 patients (13% of the cohort), two of which have not been reported previously. We provide a novel and alternative end-to-end secondary analysis pipeline for targeted NGS of ORF15 that is less prone to false positive and negative variant calls.

We investigated the use of amniocentesis performed at eight to 14 weeks' gestation as a possible alternative to chorionic villus sampling. Patients, methods, and results Samples of amniotic fluid were taken from 40 gestation, and the mean time to the cells being harvested was 12 6 days. In contrast only 17 (68%) of the 25 samples taken at eight to 11 weeks yielded a result. One sample taken at 13 weeks' gestation yielded a female karyotype, whereas the fetal parts revealed a male karyotype; the sample was subsequently identified as maternal urine. The mean volume of amniotic fluid obtained was 13 9 ml (range 1-40 ml). CommentAll 15 samples taken at 12-14 weeks' gestation yielded a result. The mean time to cells being harvested in this group (12-6 days) compared favourably with the current mean of 11 days for the samples obtained routinely at [16][17][18][19] weeks that are processed by our laboratory. Culture of all the 5 ml aliquots obtained at 12-14 weeks was successful. Thus a 10 ml sample would provide two cultures, which are necessary for the interpretation of equivocal results and in case of microbial infection.In one case, a urine sample was obtained at 13 weeks' gestation from an obese patient in whom imaging was poor. In a clinical environment sampling would not have been attempted, and this patient would have been recalled later.Our results show that amniocentesis from as early as 12 weeks' gestation can provide sufficient material for cytogenetic diagnosis and could be offered as an alternative to current methods of prenatal diagnosis. Furthermore, the procedure could be carried out by doctors already familiar with the technique, using existing resources. Patients must, however, be advised that the risks of this procedure are unknown. Preliminary reports from the United States suggest that early amniocentesis is safer than chorionic villus sampling.24 Further evaluation, preferably by means of a randomised trial, is urgently needed. We are continuing our investigation of amniocentesis before 12 weeks with the aim of bringing the procedure forward into the first trimester of pregnancy.We acknowledge contributions to the study from Mr N Fisk, Mr P Reginald, Mr M Michel, and Mrs R Rebello.