The structural and functional organization of the adult mouse small intestinal epithelium lends itself to studying both the regulation and integration of cellular proliferation, differentiation, and death programs. The epithelium contains four principal cell types: absorptive enterocytes (comprising Ͼ80% of the total population), enteroendocrine cells, mucus-producing goblet cells, and Paneth cells. All four lineages are derived from a multipotent stem cell that is functionally anchored near the base of each of the small intestine's 1.1 million crypts of Lieberkü hn (1-4). Cell division is confined to these crypts (5). Enterocytes, enteroendocrine, and goblet cells migrate out of the crypt and up an adjacent villus. Migration is highly ordered and associated with terminal differentiation. Cell death occurs near the villus tip where cells are exfoliated into the lumen (6, 7). Proliferation, differentiation, and death take place in a spatially well-organized continuum that extends from the crypt to the apex of a villus. This sequence is completed rapidly (2-5 days in the case of enterocytes, enteroendocrine, and goblet cells; Refs. 1 and 8 -10) and is recapitulated throughout the lifespan of the mouse.The Paneth cell lineage differs from the others in a number of notable ways. It is the only lineage that executes its terminal differentiation program during a downward migration from the stem cell zone to the crypt base (11). It is the longest lived lineage, and the only one that exists entirely within the proliferative compartment. Each crypt contains 30 -50 mature Paneth cells that survive for 18 -23 days before degenerating and undergoing phagocytosis by their neighbors (11-13). Paneth cell age correlates with position in the crypt; the most mature cells are located at or near the crypt base (2). The size of the Paneth cell's apical secretory granules also correlates with age; larger granules are produced by older cells (2, 11).The function of the Paneth cell has not yet been clearly defined. Residency at the crypt base places this lineage in a position to release products from its apical granules that could affect establishment and/or maintenance of the stem cell's niche or influence the properties of the stem cell's descendants. A number of factors exported by Paneth cells could regulate epithelial proliferation and differentiation programs. They include tumor necrosis factor-␣ (14), guanylin (15), and epidermal growth factor (16). Two Paneth cell products have been implicated as modifiers of adenoma formation in mice heterozygous for a mutation in the adenomatous polyposis coli gene, Apc Min (17). Production of matrilysin, a matrix metalloprotein-

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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.

IMPORTANCEThe mechanisms behind the phenotypic variability and reduced penetrance in autosomal recessive Stargardt disease (STGD1), often a blinding disease, are poorly understood. Identification of the unknown disease modifiers can improve patient and family counseling and provide valuable information for disease management.OBJECTIVE To assess the association of incompletely penetrant ABCA4 alleles with sex in STGD1.DESIGN, SETTING, AND PARTICIPANTS Genetic data for this cross-sectional study were obtained from 2 multicenter genetic studies of 1162 patients with clinically suspected STGD1. Unrelated patients with genetically confirmed STGD1 were selected. The data were collected from June 2016 to June 2019, and post hoc analysis was performed between July 2019 and January 2020.MAIN OUTCOMES AND MEASURES Penetrance of reported mild ABCA4 variants was calculated by comparing the allele frequencies in the general population (obtained from the Genome Aggregation Database) with the genotyping data in the patient population (obtained from the ABCA4 Leiden Open Variation Database). The sex ratio among patients with and patients without an ABCA4 allele with incomplete penetrance was assessed.RESULTS A total of 550 patients were included in the study, among which the mean (SD) age was 45.7 (18.0) years and most patients were women (311 [57%]). Five of the 5 mild ABCA4 alleles, including c.5603A>T and c.5882G>A, were calculated to have incomplete penetrance. The women to men ratio in the subgroup carrying c.5603A>T was 1.7 to 1; the proportion of women in this group was higher compared with the subgroup not carrying a mild allele (difference, 13%; 95% CI, 3%-23%; P = .02). The women to men ratio in the c.5882G>A subgroup was 2.1 to 1, and the women were overrepresented compared with the group carrying no mild allele (difference, 18%; 95% CI, 6%-30%; P = .005).CONCLUSIONS AND RELEVANCE This study found an imbalance in observed sex ratio among patients harboring a mild ABCA4 allele, which concerns approximately 25% of all patients with STGD1, suggesting that STGD1 should be considered a polygenic or multifactorial disease rather than a disease caused by ABCA4 gene mutations alone. The findings suggest that sex should be considered as a potential disease-modifying variable in both basic research and clinical trials on STGD1.

Purpose To identify the genetic cause and describe the phenotype in four families with autosomal recessive retinitis pigmentosa (arRP) that can be associated with pseudocoloboma. Design Case series. Subjects Seven patients from four unrelated families with arRP of which three patients had bilateral early-onset macular pseudocoloboma. Methods We performed homozygosity mapping and whole-exome sequencing (WES) in five probands and two unaffected family members of four unrelated families. Subsequently, Sanger sequencing and segregation analysis were done in additional family members. We reviewed the medical history of individuals carrying IDH3A variants and performed additional ophthalmic examinations, including full-field electroretinography (ffERG), fundus photography, fundus autofluorescence imaging and optical coherence tomography. Main Outcome Measures IDH3A variants, age at diagnosis, visual acuity, fundus appearance, visual field, ffERG, fundus autofluorescence and OCT findings. Results We identified seven different variants in IDH3A in four unrelated families, i.e. five missense, one nonsense and one frameshift variant. All subjects developed symptoms early in life ranging from night blindness to decreased visual acuity and were diagnosed between the ages of one and 11 years. Four subjects with biallelic IDH3A variants displayed a typical arRP phenotype and three subjects were diagnosed with arRP and pseudocoloboma of the macula. Conclusions IDH3A variants were identified as a novel cause of typical arRP, in some individuals associated with macular pseudocoloboma. We observed both phenotypes in two siblings carrying the same compound heterozygous variants, which could be explained by variable disease expression and warrants caution when making assertions about genotype-phenotype correlations.

To investigate the prevalence of sequence variants in LCA5 in patients with Leber congenital amaurosis (LCA), early onset rod-cone dystrophy (EORD) and autosomal recessive retinitis pigmentosa (RP), to delineate the ocular phenotypes, and to provide an overview of all published LCA5 variants in an online database._Patients underwent standard ophthalmic evaluations after providing informed consent. In selected patients, optical coherence tomography (OCT) and fundus autofluorescence imaging was possible. DNA samples from 797 unrelated patients with LCA and 211 with the various types of RP were screened by Sanger sequence analysis of all LCA5 exons and intron/exon junctions. Some LCA patients were pre-screened by APEX technology or selected based on homozygosity mapping. In silico analyses were performed to assess the pathogenicity of the variants. Segregation analysis was performed where possible. Published and novel LCA5 variants were collected, amended for their correct nomenclature, and listed in a Leiden Open Variation Database (LOVD). Sequence analysis identified 18 new probands with 19 different LCA5 variants. Seventeen of the 19 LCA5 variants were novel. Except for two missense variants and one splice site variant, all variants were protein-truncating mutations. Most patients expressed a severe phenotype, typical of LCA. However, some LCA subjects had better vision and intact inner segment/outer segment (IS/OS) junctions on OCT imaging. In two families with LCA5 variants, the phenotype was more compatible with EORD with affected individuals displaying preserved islands of RPE. One of these milder families harbored a homozygous splice site mutation, a second family was found to have a combination of a stop mutation and a missense mutation. This is the largest LCA5 study to date. We sequenced 1008 patients (797 with LCA, 211 with arRP) and identified 18 probands with LCA5 mutations. Mutations in LCA5 are a rare cause of childhood retinal dystrophy accounting for ~2% of disease in this cohort and the majority of LCA5 mutations are likely null. The LCA5 protein truncating mutations are predominantly associated with LCA. However, in two families with the milder EORD, the LCA5 gene analysis revealed a homozygous splice site mutation in one and a stop mutation in combination with a missense mutation in a second family, suggesting that this milder phenotype is due to residual function of lebercilin and expanding the currently known phenotypic spectrum to include the milder early onset RP. Some patients have remaining foveal cone structures (intact IS/OS junctions on OCT imaging) and remaining visual acuities, which may bode well for upcoming treatment trials.

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.