The role of TXNIP in cancer: a fine balance between redox, metabolic, and immunological tumor control.

Thioredoxin-interacting protein (TXNIP) is commonly considered a master regulator of cellular oxidation, regulating the expression and function of Thioredoxin (Trx). Recent work has identified that TXNIP has a far wider range of additional roles: from regulating glucose and lipid metabolism, to cell cycle arrest and inflammation. Its expression is increased by stressors commonly found in neoplastic cells and the wider tumor microenvironment (TME), and, as such, TXNIP has been extensively studied in cancers. In this review, we evaluate the current literature regarding the regulation and the function of TXNIP, highlighting its emerging role in modulating signaling between different cell types within the TME. We then assess current and future translational opportunities and the associated challenges in this area. An improved understanding of the functions and mechanisms of TXNIP in cancers may enhance its suitability as a therapeutic target.

Predicting progression-free survival after systemic therapy in advanced head and neck cancer: Bayesian regression and model development.

Background: Advanced head and neck squamous cell carcinoma (HNSCC) is associated with a poor prognosis, and biomarkers that predict response to treatment are highly desirable. The primary aim was to predict progression-free survival (PFS) with a multivariate risk prediction model. Methods: Experimental covariates were derived from blood samples of 56 HNSCC patients which were prospectively obtained within a Phase 2 clinical trial (NCT02633800) at baseline and after the first treatment cycle of combined platinum-based chemotherapy with cetuximab treatment. Clinical and experimental covariates were selected by Bayesian multivariate regression to form risk scores to predict PFS. Results: A 'baseline' and a 'combined' risk prediction model were generated, each of which featuring clinical and experimental covariates. The baseline risk signature has three covariates and was strongly driven by baseline percentage of CD33+CD14+HLADRhigh monocytes. The combined signature has six covariates, also featuring baseline CD33+CD14+HLADRhigh monocytes but is strongly driven by on-treatment relative change of CD8+ central memory T cells percentages. The combined model has a higher predictive power than the baseline model and was successfully validated to predict therapeutic response in an independent cohort of nine patients from an additional Phase 2 trial (NCT03494322) assessing the addition of avelumab to cetuximab treatment in HNSCC. We identified tissue counterparts for the immune cells driving the models, using imaging mass cytometry, that specifically colocalized at the tissue level and correlated with outcome. Conclusions: This immune-based combined multimodality signature, obtained through longitudinal peripheral blood monitoring and validated in an independent cohort, presents a novel means of predicting response early on during the treatment course. Funding: Daiichi Sankyo Inc, Cancer Research UK, EU IMI2 IMMUCAN, UK Medical Research Council, European Research Council (335326), Merck Serono. Cancer Research Institute, National Institute for Health Research, Guy's and St Thomas' NHS Foundation Trust and The Institute of Cancer Research. Clinical trial number: NCT02633800.

Mutations in SPATA13/ASEF2 cause primary angle closure glaucoma.

Current estimates suggest 50% of glaucoma blindness worldwide is caused by primary angle-closure glaucoma (PACG) but the causative gene is not known. We used genetic linkage and whole genome sequencing to identify Spermatogenesis Associated Protein 13, SPATA13 (NM_001166271; NP_001159743, SPATA13 isoform I), also known as ASEF2 (Adenomatous polyposis coli-stimulated guanine nucleotide exchange factor 2), as the causal gene for PACG in a large seven-generation white British family showing variable expression and incomplete penetrance. The 9 bp deletion, c.1432_1440del; p.478_480del was present in all affected individuals with angle-closure disease. We show ubiquitous expression of this transcript in cell lines derived from human tissues and in iris, retina, retinal pigment and ciliary epithelia, cornea and lens. We also identified eight additional mutations in SPATA13 in a cohort of 189 unrelated PACS/PAC/PACG samples. This gene encodes a 1277 residue protein which localises to the nucleus with partial co-localisation with nuclear speckles. In cells undergoing mitosis SPATA13 isoform I becomes part of the kinetochore complex co-localising with two kinetochore markers, polo like kinase 1 (PLK-1) and centrosome-associated protein E (CENP-E). The 9 bp deletion reported in this study increases the RAC1-dependent guanine nucleotide exchange factors (GEF) activity. The increase in GEF activity was also observed in three other variants identified in this study. Taken together, our data suggest that SPATA13 is involved in the regulation of mitosis and the mutations dysregulate GEF activity affecting homeostasis in tissues where it is highly expressed, influencing PACG pathogenesis.

Identification and characterization of the VAX2 p.Leu139Arg variant: possible involvement of VAX2 in cone dystrophy.

OBJECTIVE: This study was undertaken with the objective to investigate the potential involvement of VAX2 in retinal degeneration. METHODS: A cohort of macular and cone dystrophy patients (n = 70) was screened for variant identification. Polymerase chain reaction (PCR) products were purified using ExoSAP-IT. Direct sequencing of PCR products was performed using BigDye 3.1 on the ABI 3730 DNA Analyzer and analyzed using DNASTAR software tool. Search for known variant was performed using the following platforms: 1000 Genomes Project, Ensembl, UCSC, ExAc, and dbSNP. The VAX2 mutants were generated using the GeneArt® Site-Directed Mutagenesis kit. In vitro analysis was performed in hTERTRPE-1 (RPE-1) cell line. Cells were photographed using a Zeiss AXIOVERT S100 microscope. Images were analyzed using Photoshop CS4 software. RESULTS: Here, we report the identification of a heterozygous non-synonymous variant (c.416T>G; p.Leu139Arg) in one cone dystrophy proband. Functional characterization of this variant in vitro revealed an aberrant phenotype seen as protein mislocalization to cytoplasm/nucleus and aggregates undergoing degradation or forming aggresomes. The cellular phenotype suggests protein loss-of-function. Analysis of the VAX2 p.Leu139Met, a variant present in the normal population, showed a phenotype similar to the wild-type, further supporting the hypothesis for the Leucine 139 to Arginine change to be damaging. CONCLUSIONS: This study raises the interesting possibility for evaluating VAX2 as a candidate gene for cone dystrophy.

EYS Is a Protein Associated with the Ciliary Axoneme in Rods and Cones.

PURPOSE: Mutations in the EYS gene are a common cause of autosomal recessive retinitis pigmentosa (arRP), yet the role of the EYS protein in humans is presently unclear. The aim of this study was to investigate the isoform structure, expression and potential function of EYS in the mammalian retina in order to better understand its involvement in the pathogenesis of arRP. METHODS: To achieve the objective, we examined the expression of mRNA transcripts of EYS isoforms in human tissues and cell lines by RT-PCR. We also investigated the localisation of EYS in cultured cells and retinal cryo-sections by confocal fluorescence microscopy and Western blot analysis. RESULTS: RT-PCR analysis confirmed that EYS has at least four isoforms. In addition to the previously reported EYS isoforms 1 and 4, we present the experimental validation of two smaller variants referred to as EYS isoforms 2 and 3. All four isoforms are expressed in the human retina and Y79 cells and the short variants were additionally detected in the testis. Immunofluorescent confocal microscopy and Western blot analysis revealed that all EYS isoforms preferentially localise to the cytoplasm of Y79 and HeLa cells. Moreover, an enrichment of the endogenous protein was observed near the centrosomes in Y79 cells. Interestingly, EYS was observed at the ciliary axoneme in Y79 ciliated cells. In macaque retinal cryosections, EYS was found to localise in the region of the photoreceptor ciliary axoneme in both rods and cones as well as in the cytoplasm of the ganglion cells. CONCLUSION: The results obtained in this study lead us to speculate that, in photoreceptor cells, EYS could be a protein involved in maintaining the stability of the ciliary axoneme in both rods and cones. The variability of its isoform structure suggests that other roles are also possible and yet to be established.

First insights into the expression of VAX2 in humans and its localization in the adult primate retina.

VAX2 is a transcription factor specifically expressed in the ventral region of the prospective neural retina in vertebrates and is required for ventral eye specification. Despite its extensive analysis in vertebrates, the biological role of VAX2 in the human is presently unclear. This study was undertaken to investigate VAX2 in humans aiming to gain new knowledge into its involvement in retinal function. Here, we report VAX2 gene expression and protein localization in cultured cells and adult retina. RT-PCR experiments indicated that VAX2 is enriched in neuronal tissues. Moreover, we identified a novel isoform most abundantly expressed in the retina. We termed the known transcript (NM_012476) isoform-1, and the newly identified transcript as isoform-2. Analysis of protein localization in cultured cells revealed that isoform-1 localizes to the nucleus and isoform-2 is widely expressed within the cell; partial co-localization of isoform-2 and actin filaments was also observed. In nonhuman primate retina VAX2 was seen either in the nuclear or in the cytoplasmic compartment depending on the retinal cell type. In addition, a noteworthy enrichment of the signal was observed in the outer segment of cone photoreceptors. Overall, this study provides the first insights into the expression of VAX2 in humans and its localization in the adult primate retina. Moreover, preliminary characterization of alternative variants suggests an involvement of VAX2 in multiple cellular pathways. Our findings raise the interesting possibility for further investigation of VAX2 in the retina in health and disease.

Genetic association and stress mediated down-regulation in trabecular meshwork implicates MPP7 as a novel candidate gene in primary open angle glaucoma.

BACKGROUND: Glaucoma is the largest cause of irreversible blindness affecting more than 60 million people globally. The disease is defined as a gradual loss of peripheral vision due to death of Retinal Ganglion Cells (RGC). The RGC death is largely influenced by the rate of aqueous humor production by ciliary processes and its passage through the trabecular meshwork (TM) in the anterior part of the eye. Primary open angle glaucoma (POAG), the most common subtype, is a genetically complex disease. Multiple genes and many loci have been reported to be involved in POAG but taken together they explain less than 10 % of the patients from a genetic perspective warranting more studies in different world populations. The purpose of this study was to perform genome-wide search for common variants associated with POAG in an east-Indian population. METHODS: The study recruited 746 POAG cases and 697 controls distributed into discovery and validation cohorts. In the discovery phase, genome-wide genotype data was generated on Illumina Infinium 660 W-Quad platform and the significant SNPs were genotyped using Illumina GGGT assay in the second phase. Logistic regression was used to test association in the discovery phase to adjust for population sub-structure and chi-square test was used for association analysis in validation phase. Publicly available expression dataset for trabecular meshwork was used to check for expression of the candidate gene under cyclic mechanical stress. Western blot and immunofluorescence experiments were performed in human TM cells and murine eye, respectively to check for expression of the candidate gene. RESULTS: Meta-analysis of discovery and validation phase data revealed the association of rs7916852 in MPP7 gene (p = 5.7x10(-7)) with POAG. We have shown abundant expression of MPP7 in the HTM cells. Expression analysis shows that upon cyclic mechanical stress MPP7 was significantly down-regulated in HTM (Fold change: 2.6; p = 0.018). MPP7 protein expression was also found to be enriched in the ciliary processes of the murine eye. CONCLUSION: Using a genome-wide approach we have identified MPP7 as a novel candidate gene for POAG with evidence of its expression in relevant ocular tissues and dysregulation under mechanical stress possibly mimicking the disease scenario.

TOPORS, a Dual E3 Ubiquitin and Sumo1 Ligase, Interacts with 26 S Protease Regulatory Subunit 4, Encoded by the PSMC1 Gene.

The significance of the ubiquitin-proteasome system (UPS) for protein degradation has been highlighted in the context of neurodegenerative diseases, including retinal dystrophies. TOPORS, a dual E3 ubiquitin and SUMO1 ligase, forms a component of the UPS and selected substrates for its enzymatic activities, such as DJ-1/PARK7 and APOBEC2, are important for neuronal as well as retinal homeostasis, respectively. TOPORS is ubiquitously expressed, yet its mutations are only known to result in autosomal dominant retinitis pigmentosa. We performed a yeast two-hybrid (Y2H) screen of a human retinal cDNA library in order to identify interacting protein partners of TOPORS from the retina, and thus begin delineating the putative disease mechanism(s) associated with the retina-specific phenotype resulting from mutations in TOPORS. The screen led to isolation of the 26 S protease regulatory subunit 4 (P26s4/ PSMC1), an ATPase indispensable for correct functioning of UPS-mediated proteostasis. The interaction between endogenous TOPORS and P26s4 proteins was validated by co-immuno-precipitation from mammalian cell extracts and further characterised by immunofluorescent co-localisation studies in cell lines and retinal sections. Findings from hTERT-RPE1 and 661W cells demonstrated that TOPORS and P26s4 co-localise at the centrosome in cultured cells. Immunofluorescent staining of mouse retinae revealed a strong P26s4 reactivity at the interface between retinal pigmented epithelium (RPE) layer and the photoreceptors outer segments (OS). This finding leads us to speculate that P26s4, along with TOPORS, may have a role(s) in RPE phagocytosis, in addition to contributing to the overall photoreceptor and retinal homeostasis via the UPS.

Disease-causing mutations in BEST1 gene are associated with altered sorting of bestrophin-1 protein.

Mutations in BEST1 gene, encoding the bestrophin-1 (Best1) protein are associated with macular dystrophies. Best1 is predominantly expressed in the retinal pigment epithelium (RPE), and is inserted in its basolateral membrane. We investigated the cellular localization in polarized MDCKII cells of disease-associated Best1 mutant proteins to study specific sorting motifs of Best1. Real-time PCR and western blots for endogenous expression of BEST1 in MDCK cells were performed. Best1 mutant constructs were generated using site-directed mutagenesis and transfected in MDCK cells. For protein sorting, confocal microscopy studies, biotinylation assays and statistical methods for quantification of mislocalization were used. Analysis of endogenous expression of BEST1 in MDCK cells revealed the presence of BEST1 transcript but no protein. Confocal microscopy and quantitative analyses indicate that transfected normal human Best1 displays a basolateral localization in MDCK cells, while cell sorting of several Best1 mutants (Y85H, Q96R, L100R, Y227N, Y227E) was altered. In contrast to constitutively active Y227E, constitutively inactive Y227F Best1 mutant localized basolaterally similar to the normal Best1 protein. Our data suggest that at least three basolateral sorting motifs might be implicated in proper Best1 basolateral localization. In addition, non-phosphorylated tyrosine 227 could play a role for basolateral delivery.

A Study into the Evolutionary Divergence of the Core Promoter Elements of PRPF31 and TFPT.

Mutations in PRPF31 have been implicated in retinitis pigmentosa, a blinding disease caused by degeneration of rod photoreceptors. The disease mechanism in the majority of cases is haploinsufficiency. Crucially, attempts at generation of animal models of disease have proved unsuccessful, yielding animals with a visual phenotype that does not mirror human disease. This suggests that, in these animals, the transcriptional regulation of PRPF31 is different to humans and compared to other species. Study of the evolution of the PRPF31 core promoter has important implications for our understanding of human disease, as disease phenotype is modified by differentially expressed alleles in the population. PRPF31 lies in a head-to-head arrangement with TFPT, a gene involved in cellular apoptosis. The two genes were shown to share common regulatory elements in the human genome. In this study, the core promoters of PRPF31 and TFPT were characterised by dual-luciferase reporter assay using genomic DNA from the green monkey, domestic dog and house mouse. It was found that the core promoters were conserved between human and monkey. In dog, the TFPT core promoter was conserved, but different PRPF31 gene architecture meant the gene was controlled by a long-range promoter lying some 2000bp from the transcription start site. There was very low level of conservation (<20%) of the PRPF31 5' region between mouse and human. It was shown that mouse populations did not show variable Prpf31 expression levels, revealing a potential explanation for the lack of phenotype observed in the Prpf31 knock-out mouse model.

Expression of PRPF31 and TFPT: regulation in health and retinal disease.

PRPF31, a gene located at chromosome 19q13.4, encodes the ubiquitous splicing factor PRPF31. The gene lies in a head-to-head arrangement with TFPT, a poorly characterized gene with a role in cellular apoptosis. Mutations in PRPF31 have been implicated in autosomal dominant retinitis pigmentosa (adRP), a frequent and important cause of blindness worldwide. Disease associated with PRPF31 mutations is unusual, in that there is often non-penetrance of the disease phenotype in affected families, caused by differential expression of PRPF31. This study aimed to characterize the basic promoter elements of PRPF31 and TFPT. Luciferase reporter constructs were made, using genomic DNA from an asymptomatic individual with a heterozygous deletion of the entire putative promoter region. Fragments were tested by the dual-luciferase reporter assay in HeLa and RPE-1 cell lines. A comparison was made between the promoter regions of symptomatic and asymptomatic mutation-carrying individuals. A patient (CAN493) with adRP was identified, harbouring a regulatory region mutation; both alleles were assayed by the dual-luciferase reporter assay. Luciferase assays led to the identification of core promoters for both PRPF31 and TFPT; despite their shared gene architecture, the two genes appear to be controlled by slightly different regulatory regions. One functional polymorphism was identified in the PRPF31 promoter that increased transcriptional activation. The change was not, however, consistent with the observed symptomatic-asymptomatic phenotypes in a family affected by PRPF31-adRP. Analysis of the mutant promoter fragment from CAN493 showed a >50% reduction in promoter activity, suggesting a disease mechanism of functional haploinsufficiency-the first report of this disease mechanism in adRP.

The absence of dystrophin brain isoform expression in healthy human heart ventricles explains the pathogenesis of 5' X-linked dilated cardiomyopathy.

BACKGROUND: In X-linked dilated cardiomyopathy due to dystrophin mutations which abolish the expression of the M isoform (5'-XLDC), the skeletal muscle is spared through the up-regulation of the Brain (B) isoform, a compensatory mechanism that does not appear to occur in the heart of affected individuals. METHODS: We quantitatively studied the expression topography of both B and M isoforms in various human heart regions through in-situ RNA hybridization, Reverse-Transcriptase and Real-Time PCR experiments. We also investigated the methylation profile of the B promoter region in the heart and quantified the B isoform up regulation in the skeletal muscle of two 5'-XLDC patients. RESULTS: Unlike the M isoform, consistently detectable in all the heart regions, the B isoform was selectively expressed in atrial cardiomyocytes, but absent in ventricles and in conduction system structures. Although the level of B isoform messenger in the skeletal muscle of 5'-XLDC patients was lower that of the M messenger present in control muscle, it seems sufficient to avoid an overt muscle pathology. This result is consistent with the protein level in XLDC patients muscles we previously quantified. Methylation studies revealed that the B promoter shows an overall low level of methylation at the CG dinucleotides in both atria and ventricles, suggesting a methylation-independent regulation of the B promoter activity. CONCLUSIONS: The ventricular dilatation seen in 5'-XLDC patients appears to be functionally related to loss of the M isoform, the only isoform transcribed in human ventricles; in contrast, the B isoform is well expressed in heart but confined to the atria. Since the B isoform can functionally replace the M isoform in the skeletal muscle, its expression in the heart could potentially exert the same rescue function. Methylation status does not seem to play a role in the differential B promoter activity in atria and ventricles, which may be governed by other regulatory mechanisms. If these mechanisms could be deduced, de-silencing of the B isoform may represent a therapeutic strategy in 5'-XLDC patients.

The long noncoding RNA Vax2os1 controls the cell cycle progression of photoreceptor progenitors in the mouse retina.

Long noncoding RNAs (lncRNAs) are emerging as regulators of many basic cellular pathways. Several lncRNAs are selectively expressed in the developing retina, although little is known about their functional role in this tissue. Vax2os1 is a retina-specific lncRNA whose expression is restricted to the mouse ventral retina. Here we demonstrate that spatiotemporal misexpression of Vax2os1 determines cell cycle alterations in photoreceptor progenitor cells. In particular, the overexpression of Vax2os1 in the developing early postnatal mouse retina causes an impaired cell cycle progression of photoreceptor progenitors toward their final committed fate and a consequent delay of their differentiation processes. At later developmental stages, this perturbation is accompanied by an increase of apoptotic events in the photoreceptor cell layer, in comparison with control retinas, without affecting the proper cell layering in the adult retina. Similar results are observed in mouse photoreceptor-derived 661W cells in which Vax2os1 overexpression results in an impairment of the cell cycle progression rate and cell differentiation. Based on these results, we conclude that Vax2os1 is involved in the control of cell cycle progression of photoreceptor progenitor cells in the ventral retina. Therefore, we propose Vax2os1 as the first example of lncRNA that acts as a cell cycle regulator in the mammalian retina during development.

Vax2 regulates retinoic acid distribution and cone opsin expression in the vertebrate eye.

Vax2 is an eye-specific homeobox gene, the inactivation of which in mouse leads to alterations in the establishment of a proper dorsoventral eye axis during embryonic development. To dissect the molecular pathways in which Vax2 is involved, we performed a transcriptome analysis of Vax2(-/-) mice throughout the main stages of eye development. We found that some of the enzymes involved in retinoic acid (RA) metabolism in the eye show significant variations of their expression levels in mutant mice. In particular, we detected an expansion of the expression domains of the RA-catabolizing enzymes Cyp26a1 and Cyp26c1, and a downregulation of the RA-synthesizing enzyme Raldh3. These changes determine a significant expansion of the RA-free zone towards the ventral part of the eye. At postnatal stages of eye development, Vax2 inactivation led to alterations of the regional expression of the cone photoreceptor genes Opn1sw (S-Opsin) and Opn1mw (M-Opsin), which were significantly rescued after RA administration. We confirmed the above described alterations of gene expression in the Oryzias latipes (medaka fish) model system using both Vax2 gain- and loss-of-function assays. Finally, a detailed morphological and functional analysis of the adult retina in mutant mice revealed that Vax2 is necessary for intraretinal pathfinding of retinal ganglion cells in mammals. These data demonstrate for the first time that Vax2 is both necessary and sufficient for the control of intraretinal RA metabolism, which in turn contributes to the appropriate expression of cone opsins in the vertebrate eye.

Identification and expression analysis of novel Jakmip1 transcripts.

Janus kinase and microtubule interacting protein 1, (Jakmip1) conserved in vertebrates and predominantly expressed in neural tissues, was identified for its ability to bind Tyk2, a member of the Janus kinase (Jak) family of non-receptor tyrosine kinases. Recently Jakmip1 was also identified as an interacting partner of GABA(B)R1 and as a regulatory protein of GABA(B)R2 mRNA. We have confirmed that this gene is highly expressed in brain and retina tissues and it is also present at lower levels in other tissues. We have identified four new transcripts of 2975 bp, 1743 bp, 2189 bp and 2420 bp respectively, named Jakmip1B, Jakmip1C, Jakmip1D and Jakmip1E. The involvement of the Janus kinase pathway in the development of mouse retina and in the control of survival and proliferation of human retinal ganglion cells, together with the restricted Jakmip1 gene expression pattern, may suggest this gene is a putative candidate for neuro-degenerative and retinal diseases. For this reason, a mutation analysis of the Jakmip1 gene in a panel of 50 unrelated patients with retinitis pigmentosa has been performed, revealing no pathogenic mutations.

Natural antisense transcripts associated with genes involved in eye development.

Natural antisense transcripts (NATs) are a class of genes whose role in controlling gene expression is becoming more and more relevant. We describe the identification of eight novel mouse NATs associated with transcription factors (Pax6, Pax2, Six3, Six6, Otx2, Crx, Rax and Vax2) that play an important role in eye development and function. These newly identified NATs overlap with the mature processed mRNAs or with the primary unprocessed transcript of their corresponding sense genes, are predicted to represent either protein coding or non-coding RNAs and undergo extensive alternative splicing. Expression studies, by both RT-PCR and RNA in situ hybridization, demonstrate that most of these NATs, similarly to their sense counterparts, display a specific or predominant expression in the retina, particularly at postnatal stages. We found a significant reduction of the expression levels of one of these NATs, Vax2OS (Vax2 opposite strand) in a mouse mutant carrying the inactivation of Vax2, the corresponding sense gene. In addition, we overexpressed another NAT, CrxOS, in mouse adult retina using adeno-associated viral vectors and we observed a significant decrease in the expression levels of the corresponding sense gene, Crx. These results suggest that these transcripts are functionally related to their sense counterparts and may play an important role in regulating the molecular mechanisms that underlie eye development and function in both physiological and pathological conditions.

Identification and characterisation of the retinitis pigmentosa 1-like1 gene (RP1L1): a novel candidate for retinal degenerations.

Retinitis pigmentosa (RP) is the most common form of inherited retinopathy, with an approximate incidence of 1 in 3700 individuals worldwide. Mutations in the retinitis pigmentosa 1 (RP1) gene are responsible for about 5-10% cases of autosomal dominant RP. The RP1 gene is specifically expressed in the photoreceptor layers of the postnatal retina and encodes a predicted protein characterised by the presence of two doublecortin (DC) domains, known to be implicated in microtubule binding. We identified and characterised, both in human and in mouse, a novel mammalian gene, termed Retinitis Pigmentosa1-like1 (RP1L1), because of its significant sequence similarity to the RP1 gene product. The sequence homology between RP1 and RP1L1 was found to be mostly restricted to the DC domains and to the N-terminal region, including the first 350 amino acids. The RP1L1 gene was also found to be conserved in distant vertebrates, since we identified a homologue in Fugu rubripes (pufferfish). Similar to RP1, RP1L1 expression is restricted to the postnatal retina, as determined by semiquantitative reverse transcriptase-PCR and Northern analysis. The retina-specific expression and the sequence similarity to RP1 render RP1L1 a potential candidate for inherited retinal disorders.

Characterization of MPP4, a gene highly expressed in photoreceptor cells, and mutation analysis in retinitis pigmentosa.

Membrane-associated guanylate kinase (MAGUK) proteins are cell-cell contact organizing molecules that mediate targeting, clustering and anchoring of proteins at synapses and other cell junctions. MAGUK proteins may contain multiple protein-protein interaction motifs including PDZ, SH3 and guanylate kinase (GuK) domains. In this study, we performed a detailed analysis of the expression pattern of MPP4, a recently described member of the MAGUK protein family. We confirmed that this gene is highly expressed in retina, and demonstrate that it is also present, at lower levels, in brain. We identified a new retina specific isoform encoding a predicted protein lacking 71 amino acids. This protein region contains a newly identified L27 domain, another module playing a role in protein-protein interaction. By RNA in situ hybridization, Mpp4 expression was found to be localized to photoreceptor cells in postnatal retina. The MPP4 gene is localized to chromosome 2, in band 2q31-33, where a locus for autosomal recessive retinitis pigmentosa (RP26) has been mapped. Mutation analysis of the entire open reading frame of the MPP4 gene in a RP26 family revealed no pathologic mutations. In addition, we did not identify mutations in a panel of 300 unrelated patients with retinitis pigmentosa.