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Aim: To discover derivatives of the anthelmintic drug levamisole, which has been reported to possess immune-modulatory properties, as treatments for amyotrophic lateral sclerosis (ALS), which has been suggested to be in part an autoimmune disease. Results: We have synthesized ten analogs of the racemic version of levamisole, tetramisole, as well as eleven analogs on a related system. All of the analogs have been tested for their ability to affect the response of five ALS-relevant cytokines. Conclusion: We have discovered a number of interesting derivatives that have encouraging cytokine response data and good metabolic stability, with the potential to have a positive impact on ALS either as single agents, or in combination.

Cell replacement therapy is emerging as an important approach in novel treatments for neurodegenerative diseases. Many problems remain, in particular improvements are needed in the survival of transplanted cells and increasing functional integration into host tissue. These problems arise because of immune rejection, suboptimal precursor cell type, trauma during cell transplantation, and toxic compounds released by dying tissues and nutritional deficiencies. We recently developed an ex vivo system to facilitate identification of factors contributing to the death of transplanted neuronal (photoreceptor) and showed 2.8-fold improvement in transplant cell survival after pretreatment with a novel glycopeptide (PKX-001). In this study, we extended these studies to look at cell survival, maturation, and functional integration in an in vivo rat model of rhodopsin-mutant retinitis pigmentosa causing blindness. We found that only when human photoreceptor precursor cells were preincubated with PKX-001 prior to transplantation, did the cells integrate and mature into cone photoreceptors expressing S-opsin or L/M opsin. In addition, ribbon synapses were observed in the transplanted cells suggesting they were making synaptic connections with the host tissue. Furthermore, optokinetic tracking and electroretinography responses in vivo were significantly improved compared to cell transplants without PKX-001 pre-treatment. These data demonstrate that PKX-001 promotes significant long-term stem cell survival in vivo, providing a platform for further investigation towards the clinical application to repair damaged or diseased retina.

Strabismus is a common condition, affecting 1-4% of individuals. Isolated strabismus has been studied in families with Mendelian inheritance patterns. Despite the identification of multiple loci via linkage analyses, no specific genes have been identified from these studies. The current study is based on a seven-generation family with isolated strabismus inherited in an autosomal dominant manner. A total of 13 individuals from a common ancestor have been included for linkage analysis, and a single linkage signal has been identified at chromosome 14q12 with a multipoint LOD score of 4.69. Disruption of this locus is known to cause FOXG1 syndrome (or congenital Rett syndrome; OMIM #613454 and *164874), in which 84% of affected individuals present with strabismus. With the incorporation of next generation sequencing and in-depth bioinformatic analyses, a 4bp non-coding deletion was prioritized as the top candidate for the observed strabismus phenotype. The deletion is predicted to disrupt regulation of FOXG1, which encodes a transcription factor of the Forkhead family. Suggestive of an auto-regulation effect, the disrupted sequence matches the consensus FOXG1 and Forkhead family transcription factor binding site and has been observed in previous ChIP-seq studies to be bound by Foxg1 in early mouse brain development. The findings of this study indicate that the strabismus phenotype commonly observed within FOXG1 syndrome is separable from the more severe syndromic characteristics. Future study of this specific deletion may shed light on the regulation of FOXG1 expression and may enhance our understanding of the mechanisms contributing to strabismus and FOXG1 syndrome.Author summaryEye misalignment, or strabismus, can affect up to 4% of individuals. When strabismus is detected early, intervention in young children based on eye patching and/or corrective lenses can be beneficial. In some cases, corrective surgeries are used to align the eyes, with many individuals requiring multiple surgeries over a lifetime. A better understanding of the causes of strabismus may lead to earlier detection as well as improved treatment options. Hippocrates observed that strabismus runs in families over 2,400 years ago, an early recognition of what we now recognize as a portion of cases arising from genetic causes. We describe a large family affected by strabismus and identify a single region on chromosome 14 that may be responsible. The region contains FOXG1, in which mutations are known to cause a severe syndrome, with 84% of affected individuals also having strabismus. We identify a 4bp deletion in the region that appears to auto-regulate when FOXG1 is active. Future study of this genetic alteration may enhance our understanding of the mechanisms of strabismus.

Leber congenital amaurosis (LCA) is a severe, genetically heterogeneous recessive eye disease in which ~ 35% of gene mutations are in-frame nonsense mutations coding for loss-of-function premature termination codons (PTCs) in mRNA. Nonsense suppression therapy allows read-through of PTCs leading to production of full-length protein. A limitation of nonsense suppression is that nonsense-mediated decay (NMD) degrades PTC-containing RNA transcripts. The purpose of this study was to determine whether inhibition of NMD could improve nonsense suppression efficacy in vivo. Using a high-throughput approach in the recessive cep290 zebrafish model of LCA (cep290;Q1223X), we first tested the NMD inhibitor Amlexanox in combination with the nonsense suppression drug Ataluren. We observed reduced retinal cell death and improved visual function. With these positive data, we next investigated whether this strategy was also applicable across species in two mammalian models: Rd12 (rpe65;R44X) and Rd3 (rd3;R107X) mouse models of LCA. In the Rd12 model, cell death was reduced, RPE65 protein was produced, and in vivo visual function testing was improved. We establish for the first time that the mechanism of action of Amlexanox in Rd12 retina was through reduced UPF1 phosphorylation. In the Rd3 model, however, no beneficial effect was observed with Ataluren alone or in combination with Amlexanox. This variation in response establishes that some forms of nonsense mutation LCA can be targeted by RNA therapies, but that this needs to be verified for each genotype. The implementation of precision medicine by identifying better responders to specific drugs is essential for development of validated retinal therapies.

Ocular coloboma is an uncommon, but often severe, sight-threatening condition that can be identified from birth. This congenital anomaly is thought to be caused by maldevelopment of optic fissure closure during early eye morphogenesis. It has been causally linked to both inherited (genetic) and environmental influences. In particular, as a consequence of work to identify genetic causes of coloboma, new molecular pathways that control optic fissure closure have now been identified. Many more regulatory mechanisms still await better understanding to inform on the development of potential therapies for patients with this malformation. This review provides an update of known coloboma genes, the pathways they influence and how best to manage the condition. In the age of precision medicine, determining the underlying genetic cause in any given patient is of high importance.

SUMMARYCell replacement therapy is emerging as an important approach in novel treatments for neurodegenerative diseases. Many problems remain, in particular improvements are needed in the survival of transplanted cells and increasing functional integration into host tissue. These problems arise because of immune rejection, suboptimal precursor cell type, trauma during cell transplantation, toxic compounds released by dying tissues and nutritional deficiencies. We recently developed an ex vivo system to facilitate identification of factors contributing to the death of transplanted neuronal (photoreceptor) and showed 2.8-fold improvement in transplant cell survival after pre-treatment with a novel glycopeptide (PKC-100). In this study we extended these studies to look at cell survival, maturation and functional integration in an in vivo rat model of rhodopsin-mutant retinitis pigmentosa causing blindness. We found that only when human photoreceptor precursor cells (PPCs) were pre-incubated with PKX-100 prior to transplantation, did the cells integrate and mature into cone photoreceptors expressing S-opsin or L/M opsin. In addition, ribbon synapses were observed in the transplanted cells suggesting they were making synaptic connections with the host tissue. Furthermore, optokinetic tracking and electroretinography responses in vivo were significantly improved compared to cell transplants without PKX-100 pre-treatment. These data demonstrate that PKX-100 promotes significant long-term stem cell survival in vivo, providing a platform for further investigation towards the clinical application to repair damaged or diseased retina.

// Miriam S. Butler 1, *, # , Mani Roshan-Moniri 1, *, # , Michael Hsing 1, *, # , Desmond Lau 2, *, # , Ari Kim 1 , Paul Yen 1 , Marta Mroczek 1 , Mannan Nouri 1 , Scott Lien 1 , Peter Axerio-Cilies 1 , Kush Dalal 1 , Clement Yau 1 , Fariba Ghaidi 1 , Yubin Guo 1 , Takeshi Yamazaki 1 , Sam Lawn 1 , Martin E. Gleave 1 , Cheryl Y. Gregory-Evans 3 , Lawrence P. McIntosh 2, * , Michael E. Cox 1, * , Paul S. Rennie 1, * and Artem Cherkasov 1, * 1 Vancouver Prostate Centre and the Department of Urologic Sciences, University of British Columbia, Vancouver, BC V6H 3Z6, Canada 2 Department of Biochemistry and Molecular Biology, Department of Chemistry, Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z3, Canada 3 Department of Ophthalmology and Visual Sciences, Eye Care Centre, University of British Columbia, Vancouver, BC V5Z 3N9, Canada * These authors contributed equally to this work # Co-first authors Correspondence to: Michael E. Cox, email: mcox@prostatecentre.com Artem Cherkasov, email: acherkasov@prostatecentre.com Keywords: prostate cancer, ERG, rational drug design, small molecule inhibitor, TMPRSS2-ERG Received: July 29, 2016 Accepted: April 04, 2017 Published: April 15, 2017 ABSTRACT Genomic alterations involving translocations of the ETS-related gene ERG occur in approximately half of prostate cancer cases. These alterations result in aberrant, androgen-regulated production of ERG protein variants that directly contribute to disease development and progression. This study describes the discovery and characterization of a new class of small molecule ERG antagonists identified through rational in silico methods. These antagonists are designed to sterically block DNA binding by the ETS domain of ERG and thereby disrupt transcriptional activity. We confirmed the direct binding of a lead compound, VPC-18005, with the ERG-ETS domain using biophysical approaches. We then demonstrated VPC-18005 reduced migration and invasion rates of ERG expressing prostate cancer cells, and reduced metastasis in a zebrafish xenograft model. These results demonstrate proof-of-principal that small molecule targeting of the ERG-ETS domain can suppress transcriptional activity and reverse transformed characteristics of prostate cancers aberrantly expressing ERG. Clinical advancement of the developed small molecule inhibitors may provide new therapeutic agents for use as alternatives to, or in combination with, current therapies for men with ERG-expressing metastatic castration-resistant prostate cancer.

The fovea is an anatomical specialization of the central retina containing closely packed cone-photoreceptors providing an area of high acuity vision in humans and primates. Despite its key role in the clarity of vision, little is known about the molecular and cellular basis of foveal development, due to the absence of a foveal structure in commonly used laboratory animal models. Of the amniotes the retina in birds of prey and some reptiles do exhibit a typical foveal structure, but they have not been studied in the context of foveal development due to lack of availability of embryonic tissue, lack of captive breeding programs, and limited genomic information. However, the genome for the diurnal bifoveate reptile species Anolis carolinensis (green anole) was recently published and it is possible to collect embryos from this species in captivity. Here, we tested the feasibility of using the anole as a model to study foveal development. Eyes were collected at various stages of development for histological analysis, immunofluorescence, and apoptosis. We show that at embryonic stage (ES) 10 there is peak ganglion cell density at the incipient central foveal region and a single row of cone photoreceptor nuclei. At ES17 the foveal pit begins to form and at this stage there are 3-4 rows of cone nuclei. Post-hatching a further increase in cone density and lengthening of inner and outer segments is observed. A yellowish pigment was seen in the adult central foveal region, but not in the temporal fovea. At ES14 Pax6 was localized across the entire retina, but was more prominent in the ganglion cell layer (GCL) and the part of the inner nuclear layer (INL) containing amacrine cell bodies. However, at ES17 Pax6 expression in the ganglion cells of the central retina was markedly reduced. Bioinformatic analysis revealed that 86% of human candidate foveal hypoplasia genes had an orthologous gene or DNA sequence in the green anole. These findings provide the first insight into foveal morphogenesis in the green anole and suggest that it could be a very useful model for investigating the molecular signals driving foveal development, and thus inform on human foveal development and disease.

Retinitis pigmentosa (RP) is a group of inherited neurological disorders characterized by rod photoreceptor cell death, followed by secondary cone cell death leading to progressive blindness. Currently, there are no viable treatment options for RP. Due to incomplete knowledge of the molecular signaling pathways associated with RP pathogenesis, designing therapeutic strategies remains a challenge. In particular, preventing secondary cone photoreceptor cell loss is a key goal in designing potential therapies. In this study, we identified the main drivers of rod cell death and secondary cone loss in the transgenic S334ter rhodopsin rat model, tested the efficacy of specific cell death inhibitors on retinal function, and compared the effect of combining drugs to target multiple pathways in the S334ter and P23H rhodopsin rat models. The primary driver of early rod cell death in the S334ter model was a caspase-dependent process, whereas cone cell death occurred though RIP3-dependent necroptosis. In comparison, rod cell death in the P23H model was via necroptotic signaling, whereas cone cell loss occurred through inflammasome activation. Combination therapy of four drugs worked better than the individual drugs in the P23H model but not in the S334ter model. These differences imply that treatment modalities need to be tailored for each genotype. Taken together, our data demonstrate that rationally designed genotype-specific drug combinations will be an important requisite to effectively target primary rod cell loss and more importantly secondary cone survival.

Objective To describe the clinical presentation and genotype of subjects with aniridia with a particular focus on foveal hypoplasia. Design Prospective cohort study. Participants Thirty-three Canadian participants with aniridia and of various ethnic backgrounds residing in British Columbia. Methods Full ophthalmic examinations and posterior segment spectral domain-optical coherence tomography (SD-OCT) imaging were performed. Foveal hypoplasia was graded independently by 2 staff ophthalmologists. PAX6 sequencing was performed and chromosomal 11p anomalies investigated. Candidate gene and single-nucleotide polymorphism sequencing in genes functionally related to PAX6 were also studied. Results Best corrected visual acuities in the cohort ranged from 0.0 logMAR to no light perception. Total absence of iris tissue was seen in the majority (42 of 66 eyes). In those in whom SD-OCT was possible, foveal hypoplasia was seen in the majority (45 of 56 eyes, 80%). Molecular genetic defects involving PAX6 were identified in 30 participants (91%), including 4 novel PAX6 mutations (Gly18Val; Ser65ProfsX14; Met337ArgfsX18; Ser321CysfsX34) and 4 novel chromosome 11p deletions inclusive of PAX6 or a known PAX6 regulatory region. Conclusions The number of PAX6 mutations associated with aniridia continues to increase. Variable foveal architecture despite nearly identical anterior segment disease in 4 participants with an Ex9 ELP4-Ex4 DCDC1 deletion suggested that molecular cues causing variation in disease in the posterior segment differ from those at play in the anterior segment. Results in 3 patients without identifiable PAX6 mutations and a review of the literature suggest that such cases be described as phenocopies rather than actual cases of the syndrome of aniridia.

The acquisition of an invasive phenotype by epithelial cells occurs through a loss of cellular adhesion and polarity, heralding a multistep process that leads to metastatic dissemination. Since its characterization in 1995, epithelial-mesenchymal transition (EMT) has been closely linked to the metastatic process. As a defining aspect of EMT, loss of cell adhesion through downregulation of E-cadherin is carried out by several transcriptional repressors; key among them the SNAI family of transcription factors. Here we identify for the first time that Lyn kinase functions as a key modulator of SNAI family protein localization and stability through control of the Vav-Rac1-PAK1 (Vav-Rac1-p21-activated kinase) pathway. Accordingly, targeting Lyn in vitro reduces EMT and in vivo reduces metastasis of primary tumors. We also demonstrate the clinical relevance of targeting Lyn as a key player controlling EMT; patient samples across many cancers revealed a strong negative correlation between Lyn and E-cadherin, and high Lyn expression in metastatic tumors as well as metastasis-prone primary tumors. This work reveals a novel pancancer mechanism of Lyn-dependent control of EMT and further underscores the role of this kinase in tumor progression.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the progressive degeneration of upper and lower motor neurons. Cell death in ALS and in general was previously believed to exist as a dichotomy between apoptosis and necrosis. Most research investigating cell death mechanisms in ALS was conducted before the discovery of programmed necrosis thus did not use selective cell death pathway-specific markers. Recently, a new form of programmed cell death, termed "necroptosis", has been characterized and has been recently implicated in ALS as a primary mechanism driving motor neuron cell death in different forms of ALS. The present review is aimed at summarizing cell death pathways that are currently implicated in ALS and highlighting the emerging evidence on necroptosis as a major driver of motor neuron cell death.

Recent evidence has implicated the transmembrane co-receptor neuropilin-1 (NRP1) in cancer progression. Primarily known as a regulator of neuronal guidance and angiogenesis, NRP1 is also expressed in multiple human malignancies, where it promotes tumor angiogenesis. However, non-angiogenic roles of NRP1 in tumor progression remain poorly characterized. In this study, we define NRP1 as an androgen-repressed gene whose expression is elevated during the adaptation of prostate tumors to androgen-targeted therapies (ATTs), and subsequent progression to metastatic castration-resistant prostate cancer (mCRPC). Using short hairpin RNA (shRNA)-mediated suppression of NRP1, we demonstrate that NRP1 regulates the mesenchymal phenotype of mCRPC cell models and the invasive and metastatic dissemination of tumor cells in vivo. In patients, immunohistochemical staining of tissue microarrays and mRNA expression analyses revealed a positive association between NRP1 expression and increasing Gleason grade, pathological T score, positive lymph node status and primary therapy failure. Furthermore, multivariate analysis of several large clinical prostate cancer (PCa) cohorts identified NRP1 expression at radical prostatectomy as an independent prognostic biomarker of biochemical recurrence after radiation therapy, metastasis and cancer-specific mortality. This study identifies NRP1 for the first time as a novel androgen-suppressed gene upregulated during the adaptive response of prostate tumors to ATTs and a prognostic biomarker of clinical metastasis and lethal PCa. Refereed/Peer-reviewed

The Pax6 transcription factor is essential for development of the brain, eye, olfactory and endocrine systems. Haploinsufficiency of PAX6 in humans and mice causes the congenital condition aniridia, with defects in each of these organs and systems. Identification of the PAX6 transcription networks driving normal development is therefore critical in understanding the pathophysiology observed with loss-of-function defects. Here we have focused on identification of the downstream targets for Pax6 in the developing iris and ciliary body, where we used laser capture microdissection in mouse eyes from E12.5-E16.5, followed by chromatin immunoprecipitation, promoter-reporter assays and immunohistochemistry. We identified 6 differentially expressed genes between wildtype and Pax6 heterozygous mouse tissues and demonstrated that Bmp4, Tgfβ2, and Foxc1 were direct downstream targets of Pax6 in developing iris/ciliary body. These results improve our understanding of how mutations in Bmp4, Tgfβ2, and Foxc1 result in phenocopies of the aniridic eye disease and provide possible targets for therapeutic intervention.

Many different types of pathologies can arise in the central nervous system (CNS), such as neurodegeneration. The incidence of neurodegenerative diseases continues to increase, yet the pathogenesis underlying most neurodegenerative diseases, notably in amyotrophic lateral sclerosis (ALS), remains elusive. Neuronal support cells, or glia, are known to play a crucial role in ALS. Microglia are the resident immune cells of the CNS and also have neurotrophic support functions. These cells have a disease-modifying function in ALS, yet this role is not well understood. A likely reason for this is that the intact CNS is particularly challenging to access for investigation in patients and in most animal models, which has impeded research in this field. The zebrafish is emerging as a robust model system to investigate cells in vivo, and offer distinct advantages over other vertebrate models for investigating neurodegenerative diseases. Live imaging in vivo is a powerful technique to characterize the role of dynamic cells such as microglia during neurodegeneration, and zebrafish provide a convenient means for live imaging. Here, we discuss the zebrafish as a model for live imaging, provide a brief overview of available high resolution imaging platforms that accommodate zebrafish, and describe our own in vivo studies on the role of microglia during motor neuron degeneration. Live in vivo imaging is anticipated to provide invaluable advancements to defining the pathogenesis underlying neurodegenerative diseases, which may in turn allow for more specifically targeted therapeutics.

International audience; Mutations in KIF14 have previously been associated with either severe, isolated or syndromic microcephaly with renal hypodysplasia (RHD). Syndromic microcephaly-RHD was strongly reminiscent of clinical ciliopathies, relating to defects of the primary cilium, a signalling organelle present on the surface of many quiescent cells. KIF14 encodes a mitotic kinesin, which plays a key role at the midbody during cytokinesis and has not previously been shown to be involved in cilia-related functions. Here, we analysed four families with fetuses presenting with the syndromic form and harbouring biallelic variants in KIF14. Our functional analyses showed that the identified variants severely impact the activity of KIF14 and likely correspond to loss-of-function mutations. Analysis in human fetal tissues further revealed the accumulation of KIF14-positive midbody remnants in the lumen of ureteric bud tips indicating a shared function of KIF14 during brain and kidney development. Subsequently, analysis of a kif14 mutant zebrafish line showed a conserved role for this mitotic kinesin. Interestingly, ciliopathy-associated phenotypes were also present in mutant embryos, supporting a potential direct or indirect role for KIF14 at cilia. However, our in vitro and in vivo analyses did not provide evidence of a direct role for KIF14 in ciliogenesis and suggested that loss of kif14 causes ciliopathy-like phenotypes through an accumulation of mitotic cells in ciliated tissues. Altogether, our results demonstrate that KIF14 mutations result in a severe syndrome associating microcephaly and RHD through its conserved function in cytokinesis during kidney and brain development.

Zebrafish have been used to investigate motor neuron degeneration, including as a model system to examine the pathogenesis of amyotrophic lateral sclerosis (ALS). The use of zebrafish for this purpose has some advantages over other in vivo model systems. In the current paper, we show that bisphenol A (BPA) exposure in zebrafish embryos results in motor neuron degeneration with affected motor function, reduced motor axon length and branching, reduced neuromuscular junction integrity, motor neuron cell death and the presence of activated microglia. In zebrafish, motor axon length is the conventional method for estimating motor neuron degeneration, yet this measurement has not been confirmed as a valid surrogate marker. We also show that reduced motor axon length as measured from the sagittal plane is correlated with increased motor neuron cell death. Our preliminary timeline studies suggest that axonopathy precedes motor cell death. This outcome may have implications for early phase treatments of motor neuron degeneration.

Retinal disease is the major cause of irreversible blindness in developed countries. Transplantation of photoreceptor precursor cells (PPCs) derived from human embryonic stem cells (hESCs) is a promising and widely applicable approach for the treatment of these blinding conditions. Previously, it has been shown that after transplantation into the degenerating retina, the percentage of PPCs that undergo functional integration is low. The factors that inhibit PPC engraftment remain largely unknown, in part, because so many adverse factors could be at play during in vivo experiments. To advance our knowledge in overcoming potential adverse effects and optimize PPC transplantation, we have developed a novel ex vivo system. Harvested neural retina was placed directly on top of cultured retinal pigment epithelial (RPE) cells from a number of different sources. To mimic PPC transplantation into the subretinal space, hESC-derived PPCs were inserted between the retinal explant and underlying RPE. Explants cocultured with hESC-derived RPE maintained normal gross morphology and viability for up to 2 weeks, whereas the explants cultured on ARPE19 and RPE-J failed by 7 days. Furthermore, the proportion of PPCs expressing ribbon synapse-specific proteins BASSOON and RIBEYE was significantly higher when cocultured with hESC-derived RPE (20% and 10%, respectively), than when cocultured with ARPE19 (only 6% and 2%, respectively). In the presence of the synaptogenic factor thrombospondin-1 (TSP-1), the proportion of BASSOON-positive and RIBEYE-positive PPCs cocultured with hESC-derived RPE increased to ∼30% and 15%, respectively. These data demonstrate the utility of an ex vivo model system to define factors, such as TSP-1, which could influence integration efficiency in future in vivo experiments in models of retinal degeneration.

Achromatopsia is a progressive autosomal recessive retinal disease characterized by early loss of cone photoreceptors and later rod photoreceptor loss. In most cases, mutations have been identified in CNGA3, CNGB3, GNAT2, PDE6C or PDE6H genes. Owing to this genetic heterogeneity, mutation-independent therapeutic schemes aimed at preventing cone cell death are very attractive treatment strategies. In pde6c(w59) mutant zebrafish, cone photoreceptors expressed high levels of receptor-interacting protein kinase 1 (RIP1) and receptor-interacting protein kinase 3 (RIP3) kinases, key regulators of necroptotic cell death. In contrast, rod photoreceptor cells were alternatively immunopositive for caspase-3 indicating activation of caspase-dependent apoptosis in these cells. Morpholino gene knockdown of rip3 in pde6c(w59) embryos rescued the dying cone photoreceptors by inhibiting the formation of reactive oxygen species and by inhibiting second-order neuron remodelling in the inner retina. In rip3 morphant larvae, visual function was restored in the cones by upregulation of the rod phosphodiesterase genes (pde6a and pde6b), compensating for the lack of cone pde6c suggesting that cones are able to adapt to their local environment. Furthermore, we demonstrated through pharmacological inhibition of RIP1 and RIP3 activity that cone cell death was also delayed. Collectively, these results demonstrate that the underlying mechanism of cone cell death in the pde6c(w59) mutant retina is through necroptosis, whereas rod photoreceptor bystander death occurs through a caspase-dependent mechanism. This suggests that targeting the RIP kinase signalling pathway could be an effective therapeutic intervention in retinal degeneration patients. As bystander cell death is an important feature of many retinal diseases, combinatorial approaches targeting different cell death pathways may evolve as an important general principle in treatment.

Nonsense mutations leading to premature stop codons are common occurring in approximately 12% of all human genetic diseases. Thus, pharmacological nonsense mutation suppression strategies would be beneficial to a large number of patients if the drugs could be targeted to the affected tissues at the appropriate time. Here, we used nonsense suppression to manipulate Pax6 dosage at different developmental times in the eye of the small eye (Pax6Sey/+; G194X) mouse model of aniridia. Efficacy was assessed by functional assays for visual capacity, including electroretinography and optokinetic tracking (OKT), in addition to histological and biochemical studies. Malformation defects in the Pax6Sey/+ postnatal eye responded to topically delivered nonsense suppression in a dose- and time-dependent manner. Elevated levels of Mmp9, a direct downstream target of Pax6 in the cornea, were observed with the different treatment regimens. The lens capsule was particularly sensitive to Pax6 dosage, revealing a potential new role for Pax6 in lens capsule maintenance and development. The remarkable capacity of malformed ocular tissue to respond postnatally to Pax6 dosage in vivo demonstrates that the use of nonsense suppression could be a valuable therapeutic approach for blinding diseases caused by nonsense mutations.

Gene discovery using massively parallel sequencing has focused on phenotypes diagnosed postnatally such as well-characterized syndromes or intellectual disability, but is rarely reported for fetal disorders. We used family-based whole-exome sequencing in order to identify causal variants for a recurrent pattern of an undescribed lethal fetal congenital anomaly syndrome. The clinical signs included intrauterine growth restriction (IUGR), severe microcephaly, renal cystic dysplasia/agenesis and complex brain and genitourinary malformations. The phenotype was compatible with a ciliopathy, but not diagnostic of any known condition. We hypothesized biallelic disruption of a gene leading to a defect related to the primary cilium. We identified novel autosomal recessive truncating mutations in KIF14 that segregated with the phenotype. Mice with autosomal recessive mutations in the same gene have recently been shown to have a strikingly similar phenotype. Genotype-phenotype correlations indicate that the function of KIF14 in cell division and cytokinesis can be linked to a role in primary cilia, supported by previous cellular and model organism studies of proteins that interact with KIF14. We describe the first human phenotype, a novel lethal ciliary disorder, associated with biallelic inactivating mutations in KIF14. KIF14 may also be considered a candidate gene for allelic viable ciliary and/or microcephaly phenotypes.

We proposed to optimize the retinal differentiation protocols for human embryonic stem cells (hESCs) by improving cell handling. To improve efficiency, we first focused on the production of just one retinal precursor cell type (photoreceptor precursor cells [PPCs]) rather than the production of a range of retinal cells. Combining information from a number of previous studies, in particular the use of a feeder-free culture medium and taurine plus triiodothyronine supplements, we then assessed the values of using size-controlled embryoid bodies (EBs) and negative cell selection (to remove residual embryonic antigen-4-positive hESCs). Using size-controlled 1000 cell EBs, significant improvements were made, in that 78% CRX+ve PPCs could be produced in just 17 days. This could be increased to 93% PPCs through the added step of negative cell selection. Improved efficiency of PPC production will help in efforts to undertake shorter and larger preclinical studies as a prelude to future clinical trials.

Mesenchymal stem cells (MSCs) have well-established paracrine effects that are proving to be therapeutically useful. This potential is based on the ability of MSCs to secrete a range of neuroprotective and anti-inflammatory molecules. Previous work in our laboratory has demonstrated that intravenous injection of MSCs, treated with superparamagnetic iron oxide nanoparticle fluidMAG-D resulted in enhanced levels of glial-derived neurotrophic factor, ciliary neurotrophic factor, hepatocyte growth factor and interleukin-10 in the dystrophic rat retina. In this present study we investigated whether the concentration of fluidMAG-D in cell culture media affects the secretion of these four molecules in vitro. In addition, we assessed the effect of fluidMAG-D concentration on retinoschisin secretion from genetically modified MSCs. ELISA-assayed secretion of these molecules was measured using escalating concentrations of fluidMAG-D which resulted in MSC iron loads of 0, 7, 120, or 274 pg iron oxide per cell respectively. Our results demonstrated glial-derived neurotrophic factor and hepatocyte growth factor secretion was significantly decreased but only at the 96 hour’s time-point whereas no statistically significant effect was seen with ciliary neurotrophic factor secretion. Whereas no effect was observed on culture media concentrations of retinoschisin with increasing iron oxide load, a statistically significant increase in cell lysate retinoschisin concentration (p = 0.01) was observed suggesting that increasing fluidMAG-D concentration did increase retinoschisin production but this did not lead to greater secretion. We hypothesize that higher concentrations of iron-oxide nanoparticle fluidMAG-D have an effect on the innate ability of MSCs to secrete therapeutically useful molecules and also on secretion from genetically modified cells. Further work is required to verify these in vitro finding using in vivo model systems.

Genomic alterations involving translocations of the ETS-related gene ERG occur in approximately half of prostate cancer cases. These alterations result in aberrant, androgen-regulated production of ERG protein variants that directly contribute to disease development and progression. This study describes the discovery and characterization of a new class of small molecule ERG antagonists identified through rational in silico methods. These antagonists are designed to sterically block DNA binding by the ETS domain of ERG and thereby disrupt transcriptional activity. We confirmed the direct binding of a lead compound, VPC-18005, with the ERG-ETS domain using biophysical approaches. We then demonstrated VPC-18005 reduced migration and invasion rates of ERG expressing prostate cancer cells, and reduced metastasis in a zebrafish xenograft model. These results demonstrate proof-of-principal that small molecule targeting of the ERG ETS domain can suppress transcriptional activity and reverse transformed characteristics of prostate cancers aberrantly expressing ERG. Clinical advancement of the developed small molecule inhibitors may provide new therapeutic agents for use as alternatives to, or in combination with, current therapies for men with ERG-expressing metastatic castration-resistant prostate cancer. Citation Format: Miriam S. Butler, Mani Roshan-Moniri, Michael Hsing, Desmond Lau, Ari Kim, Paul Yen, Marta Mroczek, Mannan Nouri, Scott Lien, Peter Axerio-Cilies, Kush Dalal, Clement Yau, Fariba Ghaidi, Yubin Guo, Takeshi Yamazaki, Sam Lawn, Martin Gleave, Cheryl Y. Gregory-Evans, Lawrence P. McIntosh, Paul S. Rennie, Artem Cherkasov, Michael E. Cox. Discovery and characterization of small molecules targeting the DNA-binding ETS domain of ERG in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 130. doi:10.1158/1538-7445.AM2017-130

Purpose X-linked retinoschisis (XLRS) is juvenile-onset macular degeneration caused by haploinsufficiency of the extracellular cell adhesion protein retinoschisin (RS1). RS1 mutations can lead to either a non-functional protein or the absence of protein secretion, and it has been established that extracellular deficiency of RS1 is the underlying cause of the phenotype. Therefore, we hypothesized that an ex vivo gene therapy strategy could be used to deliver sufficient extracellular RS1 to reverse the phenotype seen in XLRS. Here, we used adipose-derived, syngeneic mesenchymal stem cells (MSCs) that were genetically modified to secrete human RS1 and then delivered these cells by intravitreal injection to the retina of the Rs1h knockout mouse model of XLRS. Methods MSCs were electroporated with two transgene expression systems (cytomegalovirus (CMV)-controlled constitutive and doxycycline-induced Tet-On controlled inducible), both driving expression of human RS1 cDNA. The stably transfected cells, using either constitutive mesenchymal stem cell (MSC) or inducible MSC cassettes, were assayed for their RS1 secretion profile. For single injection studies, 100,000 genetically modified MSCs were injected into the vitreous cavity of the Rs1h knockout mouse eye at P21, and data were recorded at 2, 4, and 8 weeks post-injection. The control groups received either unmodified MSCs or vehicle injection. For the multiple injection studies, the mice received intravitreal MSC injections at P21, P60, and P90 with data collection at P120. For the single- and multiple-injection studies, the outcomes were measured with electroretinography, optokinetic tracking responses (OKT), histology, and immunohistochemistry. Results Two lines of genetically modified MSCs were established and found to secrete RS1 at a rate of 8 ng/million cells/day. Following intravitreal injection, RS1-expressing MSCs were found mainly in the inner retinal layers. Two weeks after a single injection of MSCs, the area of the schisis cavities was reduced by 65% with constitutive MSCs and by 83% with inducible MSCs, demonstrating improved inner nuclear layer architecture. This benefit was maintained up to 8 weeks post-injection and corresponded to a significant improvement in the electroretinogram (ERG) b-/a-wave ratio at 8 weeks (2.6 inducible MSCs; 1.4 untreated eyes, p

The 2p15p16.1 microdeletion syndrome has a core phenotype consisting of intellectual disability, microcephaly, hypotonia, delayed growth, common craniofacial features, and digital anomalies. So far, more than 20 cases of 2p15p16.1 microdeletion syndrome have been reported in the literature; however, the size of the deletions and their breakpoints vary, making it difficult to identify the candidate genes. Recent reports pointed to 4 genes (XPO1, USP34, BCL11A, and REL) that were included, alone or in combination, in the smallest deletions causing the syndrome. Here, we describe 8 new patients with the 2p15p16.1 deletion and review all published cases to date. We demonstrate functional deficits for the above 4 candidate genes using patients’ lymphoblast cell lines (LCLs) and knockdown of their orthologs in zebrafish. All genes were dosage sensitive on the basis of reduced protein expression in LCLs. In addition, deletion of XPO1, a nuclear exporter, cosegregated with nuclear accumulation of one of its cargo molecules (rpS5) in patients’ LCLs. Other pathways associated with these genes (e.g., NF-κB and Wnt signaling as well as the DNA damage response) were not impaired in patients’ LCLs. Knockdown of xpo1a, rel, bcl11aa, and bcl11ab resulted in abnormal zebrafish embryonic development including microcephaly, dysmorphic body, hindered growth, and small fins as well as structural brain abnormalities. Our multifaceted analysis strongly implicates XPO1, REL, and BCL11A as candidate genes for 2p15p16.1 microdeletion syndrome.

Tissue fusion is an essential morphogenetic mechanism in development, playing a fundamental role in developing neural tube, palate and the optic fissure. Disruption of genes associated with the tissue fusion can lead to congenital malformations, such as spina bifida, cleft lip/palate and ocular coloboma. For instance, the Pax2 transcription factor is required for optic fissure closure, although the mechanism of Pax2 action leading to tissue fusion remains elusive. This lack of information defining how transcription factors drive tissue morphogenesis at the cellular level is hampering new treatments options. Through loss- and gain-of-function analysis, we now establish that pax2 in combination with vax2 directly regulate the fas-associated death domain (fadd) gene. In the presence of fadd, cell proliferation is restricted in the developing eye through a caspase-dependent pathway. However, the loss of fadd results in a proliferation defect and concomitant activation of the necroptosis pathway through RIP1/RIP3 activity, leading to an abnormal open fissure. Inhibition of RIP1 with the small molecule drug necrostatin-1 rescues the pax2 eye fusion defect, thereby overcoming the underlying genetic defect. Thus, fadd has an essential physiological function in protecting the developing optic fissure neuroepithelium from RIP3-dependent necroptosis. This study demonstrates the molecular hierarchies that regulate a cellular switch between proliferation and the apoptotic and necroptotic cell death pathways, which in combination drive tissue morphogenesis. Furthermore, our data suggest that future therapeutic strategies may be based on small molecule drugs that can bypass the gene defects causing common congenital tissue fusion defects.

The molecular signaling leading to cell death in hereditary neurological diseases such as retinal degeneration is incompletely understood. Previous neuroprotective studies have focused on apoptotic pathways; however, incomplete suppression of cell death with apoptosis inhibitors suggests that other mechanisms are at play. Here, we report that different signaling pathways are activated in rod and cone photoreceptors in the P23H rhodopsin mutant rat, a model representing one of the commonest forms of retinal degeneration. Up-regulation of the RIP1/RIP3/DRP1 axis and markedly improved survival with necrostatin-1 treatment highlighted necroptosis as a major cell-death pathway in degenerating rod photoreceptors. Conversely, up-regulation of NLRP3 and caspase-1, expression of mature IL-1β and IL-18 and improved cell survival with N-acetylcysteine treatment suggested that inflammasome activation and pyroptosis was the major cause of cone cell death. This was confirmed by generation of the P23H mutation on an Nlrp3-deficient background, which preserved cone viability. Furthermore, Brilliant Blue G treatment inhibited inflammasome activation, indicating that the 'bystander cell death' phenomenon was mediated through the P2RX7 cell-surface receptor. Here, we identify a new pathway in cones for bystander cell death, a phenomenon important in development and disease in many biological systems. In other retinal degeneration models different cell-death pathways are activated, which suggests that the particular pathways that are triggered are to some extent genotype-specific. This also implies that neuroprotective strategies to limit retinal degeneration need to be customized; thus, different combinations of inhibitors will be needed to target the specific pathways in any given disease.

The fovea is an anatomical specialization of the vertebrate retina critical for high spatial visual acuity and refined color vision. In mammals, the fovea is present only in humans and primates. It is characterized as a cone photoreceptor-rich region, with a central rod-free avascular zone containing elongated cone photoreceptors and Muller cell processes. A clearer understanding of the molecular mechanisms implicated in foveal development and the consequences of abnormalities of this process is emerging. This will help to explain why phenotypic variability occurs within specific developmental diseases. In addition, although human foveal development begins mid gestation, it continues after birth, thus providing a window of opportunity to remodel the abnormal foveal architecture through pharmacological approaches in young patients otherwise destined for poor vision.

Ocular coloboma is a developmental anomaly of the eye that results from incomplete closure of the optic fissure, occurring 5–7 weeks post conception. Congenital colobomata are important causes of childhood visual impairment and blindness. This defect typically affects the iris, cornea, ciliary body, zonules, retina, choroid and optic nerve. Colobomata can be seen in isolation and in combination with an impressive number of multisystem disorders. As yet, no effective treatment is available for this condition and management is only supportive. Based upon animal studies of coloboma, mendelian genetic disorders and chromosomal abnormalities, the molecular and genetic mechanisms that regulate optic fissure morphogenesis are now being discovered. Defining the genes and environmental factors involved in coloboma formation will aid in the development of potential therapies for patients with this malformation. This review explores the progress in understanding the clinical and molecular aspects of ocular coloboma.

Background and purpose: RANTES is an inflammatory chemokine with a critical role in T-lymphocyte activation and proliferation. Its effects are mediated through G protein-coupled heptahelical receptors (GPCRs). We show for the first time that RANTES activates the orphan G protein-coupled receptor 75 (GPR75). Experimental approach: To identify a ligand for GPR75 we have used three different and independent methods, namely luciferase assay, bioluminescence assay and IP3 accumulation assay. Key results: Treatment of cells expressing GPR75 with subnanomolar concentrations of RANTES led to stimulation of the luciferase activity in a reporter-gene assay, an increase in inositol trisphosphate, and intracellular Ca2+. The latter effect was blocked by the phospholipase-C inhibitor (PLC) U73122 indicating that Gq proteins mediate GPR75 signaling. RANTES enhanced the phosphorylation of AKT and mitogen-activated protein kinase (MAPK) in GPR75-transfected cells and this effect was blocked by the PLC inhibitor U73122 and the phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin. The hippocampal cell line HT22, which expresses GPR75 endogenously, but not the other known RANTES receptors, was used to study the effects of RANTES and GPR75 on neuronal survival. Treatment of HT22 cells with RANTES significantly reduced the neurotoxicity of amyloid-β peptides, by activating PLC and PI3K. Conclusions and implications: This demonstrate clearly and undoubtedly the ability of RANTES to act on GPR75. Defects in the RANTES/GPR75-signaling pathway may contribute to neuroinflammatory and neurodegenerative processes as observed in Alzheimer's disease. British Journal of Pharmacology (2006) 149, 490–497. doi:10.1038/sj.bjp.0706909

Syndromic Hirschsprung disease has been associated with mutations in ZFHX1B, a Smad-interacting transcriptional repressor protein. Tissue in situ hybridization has demonstrated strong expression of ZFHX1B in the developing eye, suggesting that some mutations in this gene may cause visual loss. However, none of the reported mutations have been associated with an ocular phenotype. We describe a patient with Down syndrome and Hirschsprung disease with high myopia and ocular coloboma affecting the iris and retina. In addition to trisomy 21, a novel, de novo heterozygous A to G transition in exon 8 of the ZFHX1B gene was identified, which results in a R953G amino acid substitution. This abnormality was not seen in a screen of 200 chromosomes from ethnically matched, normal controls. The arginine residue at position 953 is an extremely conserved amino acid throughout evolution. This is the first report associating Hirschsprung disease and severe eye defects with a specific genetic mutation and is the first report of a mutation in ZFHX1B causing a developmental ocular anomaly.

Familial exudative vitreoretinopathy (FEVR) is an inherited blinding disorder of the retinal vascular system. Autosomal dominant FEVR is genetically heterogeneous, but its principal locus, EVR1, is on chromosome 11q13-q23. The gene encoding the Wnt receptor frizzled-4 (FZD4) was recently reported to be the EVR1 gene, but our mutation screen revealed fewer patients harboring mutations than expected. Here, we describe mutations in a second gene at the EVR1 locus, low-density-lipoprotein receptor–related protein 5 (LRP5), a Wnt coreceptor. This finding further underlines the significance of Wnt signaling in the vascularization of the eye and highlights the potential dangers of using multiple families to refine genetic intervals in gene-identification studies.

We have determined that the human SEPW1 (selenoprotein W) gene maps to chromosome 19q13.3, spans approximately 6.3 kb and comprises six exons, in contrast to the previously published five exons. The gene lacks canonical TATA and CAAT boxes, but has numerous Sp1 consensus binding sites upstream of multiple transcription start sites. SEPW1 is expressed in all of the 22 tissues assayed, and shows highest expression in skeletal muscle and heart. Additionally, we have also identified a retroprocessed SEPW1 pseudogene, SEPW1P, which maps to chromosome 1p34-35.

Premature termination codons (PTCs) are caused by nonsense mutations and this leads to either degradation of the mutant mRNA template by nonsense-mediated decay (NMD) or the production of a non-functional, truncated polypeptide. PTCs contribute significantly to inherited human diseases including ocular disorders. Nonsense suppression therapy allows readthrough of PTCs, thereby rescuing the production of a full-length functional protein. In this review, we highlight the mechanisms that are involved in discriminating normal translation termination from premature termination codons; the current understanding of nonsense-mediated mRNA decay models (NMD); the association and crosstalk between PTC and the underlying dynamic NMD process; and the suppression therapies that have been employed in nonsense-medicated ocular disease models. Defining the mechanistic complexity of PTC and NMD will be important to improve treatments of the numerous genetic disorders caused by PTC mutations.

Cone--rod homeobox (CRX), a paired-like homeobox transcription factor, plays a major role in photoreceptor development and maintenance of the retina. Fifteen different mutations in the CRX gene have been identified as a cause of blinding retinal dystrophy. As a step towards characterizing the underlying pathophysiology of disease, temporal and spatial gene expression patterns during human and mouse eye development were investigated for CRX and for downstream retinally expressed genes, postulated to be transactivated by CRX. We found that human CRX was expressed at 10.5 weeks post-conception (p.c.). This was significantly later than observed in mouse development. Immunocytochemistry in human retina showed that CRX protein was not detected until >4 weeks later at 15 weeks p.c., implying that it would be unable to transactivate PDEB, IRBP and arrestin, which were all expressed before 15 weeks. These data therefore eliminate CRX as the major transcriptional activator of these three genes from a wide group of retinal genes that can be transactivated by CRX in vitro. Additionally, PDEB was expressed 2 weeks before CRX whereas murine Pdeb was expressed after Crx, highlighting a potential difference for the role of PDEB in human eye development. Previous data had shown CRX expression in the adult human retina to be photoreceptor-specific; however, we demonstrate that this gene is also expressed in the inner nuclear layer (INL) of the human and mouse retina by in situ hybridization and immunocytochemistry. INL localization of murine Crx was confirmed in rd/rd,cl mice, as in this mouse model the photoreceptors are absent. We have found important differences in the temporal expression of this gene in human and mouse retina, although spatial expression of the CRX gene appears to be conserved. In addition, downstream targets of CRX in vitro might not represent in vivo function during development. These data support concerns about the extent to which we can extrapolate from rodent models regarding embryonic development and disease pathophysiology.

Objective To describe the clinical features of autosomal dominant cone–rod retinal dystrophy (CRD) in a British family mapping to chromosome 17p12-p13 (CORD6), with a heterozygous mutation (Glu837Asp/Arg838Ser) of GUCY2D . Design A prospective, clinical family survey. Patients Ten affected members of a family with autosomal dominant CRD. Methods Full clinical examinations were undertaken. Selected affected family members underwent electrophysiologic evaluation, scotopic static perimetry, dark adaptometry, and color vision assessment. Main outcome measures Clinical appearance and electroretinographic responses. Results Typical clinical and electroretinographic features of childhood-onset CRD were recorded. In addition, moderate myopia and pendular nystagmus were seen in affected individuals. Color vision assessment in the youngest affected individual showed no color discrimination on a tritan axis, but retention of significant red–green discrimination. Electronegative electroretinogram responses were seen on electrophysiology in the only young family member examined. Conclusions The phenotype associated with GUCY2D CRD is clinically distinct from that associated with other dominant CRD loci. Unusual electroretinographic responses may indicate that this mutation of GUCY2D is associated with early defects in photoreceptor synaptic transmission to second-order neurons.

Doyne honeycomb retinal dystrophy (DHRD) is a late-onset autosomal dominant disorder that causes degeneration of the retina and can lead to blindness. We have previously assigned DHRD to a 5-cM region of chromosome 2p16 between marker loci D2S2739 and D2S378. Using sequence-tagged sites (STSs), expressed sequence tags (ESTs) and polymorphic markers within the DHRD region, we have identified 18 yeast artificial chromosomes (YACs) encompassing the DHRD locus, spanning approximately 3 Mb. The YAC contig was constructed by STS content mapping of these YACs and incorporates 13 STSs, including four genes and six polymorphic marker loci. We also report the genetic mapping of two families with a dominant drusen phenotype to the DHRD locus, and genetic refinement of the disease locus to a critical interval flanked by microsatellite marker loci D2S2352 and D2S2251, a distance of approximately 700 kb. These studies exclude a number of candidate genes and provide a resource for construction of a transcriptional map of the region, as a prerequisite to identification of the DHRD disease-causing gene and genes for other diseases mapping in the region, such as Malattia leventinese and Carney complex.

Objective Little is known of the cytopathology of photoreceptors in human inherited retinal dystrophies that initially affect the central retina, including the macula. The current study sought to determine the cytologic features of dysfunctional cone and rod photoreceptors, as well as the pattern of degeneration of the cells in representative cases of central retinal dystrophy. Study design Comparative human tissue study. Materials Four human donor eyes with the following forms of central retinal dystrophy: cone-rod dystrophy (CRD), central areolar choroidal dystrophy, Bardet-Biedl syndrome, and cone dystrophy-cerebellar ataxia. The cytologic features of retinal photoreceptors in these eyes were compared with those in an eye with retinitis pigmentosa and six normal human eyes. Methods and outcome measures Immunocytochemistry and electron microscopy were used to evaluate the retinal histopathology in the donor eyes. Results Cone numbers were decreased in the case of CRD, particularly in the central and far peripheral retina, and both cone and rod outer segments were slightly shortened. Occasional degenerate cones had dense cytoplasm and pyknotic nuclei dislocated sclerad to the external-limiting membrane. The most prominent alteration in this retina was marked enlargement and distortion of the cone photoreceptor pedicles, which contained reduced numbers of synaptic vesicles. The retina with central areolar choroidal dystrophy contained a few cones with similarly abnormal synapses. However, comparable cone synapse abnormalities were not observed in the cases of Bardet-Biedl syndrome, cone dystrophy-cerebellar ataxia, retinitis pigmentosa, or in the normal retinas. Conclusions The functional consequences of the cone synapse abnormalities in CRD are not known but may correlate with the electroretinographic abnormalities documented in some cases of CRD. To our knowledge, comparable synapse changes have not been noted in either rods or cones in other forms of retinal dystrophy, including retinitis pigmentosa, suggesting that different cytopathologic mechanisms may be involved.

In this study, we report the identification and characterisation of a novel carbonic anhydrase related-protein. We have determined that the full length coding sequence of an anonymous expressed sequenced tag, D19S799E, encodes a novel carbonic anhydrase related-protein (CARP-2) that is 328 amino acids in length. This peptide exhibits between 23.1-28.8% amino acid identity with the seven active human carbonic anhydrase (CA) isozymes. Four substitutions of key amino acids in the catalytic domain of CAs (equivalent to His94Arg, His96Leu, His119Gln, and Thr199Ser) are likely to render CARP-2 inactive as a carbonic anhydrase. Northern blot analysis of 23 human tissues indicates that CARP2 is expressed abundantly in the brain with moderate expression also present in spinal cord and thyroid. D19S799E (and thus CARP2) has previously been localised close to the polymorphic marker D19S412 and the genes DBP and FUT1/FUT2 on 19q13. 3.

We thank the family members for their cooperation in this study. This work was supported by the Wellcome Trust (grant 041905), the Frost Charitable Trust, and the Foundation Fighting Blindness.

Congenital cataracts are an important cause of bilateral visual impairment in infants. Through genome-wide linkage analysis in a four-generation family of Irish descent, the disease-associated gene causing autosomal-dominant congenital nuclear cataract was mapped to chromosome 4p16.1. The maximum logarithm of odds (LOD) score was 2.62 at a recombination fraction θ=0, obtained for marker D4S432 physically close to the Wolfram gene (WFS1). By sequencing the coding regions and intron-exon boundaries of WFS1, we identified a DNA substitution (c.1385A-to-G) in exon 8, causing a missense mutation at codon 462 (E462G) of the Wolframin protein. This is the first report of a mutation in this gene causing an isolated nuclear congenital cataract. These findings suggest that the membrane trafficking protein Wolframin may be important for supporting the developing lens.

Genetic retinal diseases such as age-related macular degeneration and monogenic diseases such as retinitis pigmentosa account for some of the commonest causes of blindness in the developed world. Diverse genetic abnormalities and environmental causes have been implicated in triggering multiple pathological mechanisms such as oxidative stress, lipofuscin deposits, neovascularisation, and programmed cell death. In recent years, inflammation has also been highlighted although whether inflammatory mediators play a central role in pathogenesis or a more minor secondary role has yet to be established. Despite this, numerous interventional studies, particularly targeting the complement system, are underway with the promise of novel therapeutic strategies for these important blinding conditions.