Soluble endoglin versus sFlt-1/PlGF ratio: detection of preeclampsia, HELLP syndrome, and FGR in a high-risk cohort.

The angiogenic factors sFlt-1 and PlGF play an established role in the detection of preeclampsia (PE). Recent data suggest that sEng might contribute to the pathogenesis of PE. However, only a few studies so far have addressed its role.This monocentric cross-sectional study of high-risk pregnancies aims to compare the levels of sFlt-1/PlGF ratio and sEng depending on different placental-related adverse pregnancy outcomes. The statistical analysis takes into account Pearson's correlation coefficient between angiogenic factors, the area under the curve estimates (AUCs) for detection, and adjusted odds ratios (aOR) with 95% confidence intervals (95%-CIs). The analysis included 206 patients: 60 controls, 90 PE (59 EOPE, 35 LOPE), 94 FGR, and 35 HELLP cases. Some outcomes overlapped because FGR commonly complicated PE and HELLP syndrome. Serum levels of sFlt-1/PlGF and sEng correlated with each other. Higher levels were observed in HELLP syndrome and EOPE cases. AUCs for sFlt-1/PlGF ratio and sEng were, respectively, 0.915 (95%-Cl 0.87-0.96) and 0.872 (95%-Cl 0.81-0.93) in PE, 0.895 (95%-Cl 0.83-0.96) and 0.878 (95%-Cl 0.81-0.95) in HELLP syndrome, 0.891 (95%-Cl 0.84-0.94), and 0.856 (95%-Cl 0.79-0.92) in FGR.aORsfor sFlt-1/PlGF ratio and sEng were, respectively: 2.69 (95%-Cl 1.86-3.9) and 2.33 (95%-Cl 1.59-3.48) in PE, 2.38 (95%-Cl 1.64-3.44) and 2.28 (95%-Cl 1.55-3.4) in FGR, and 2.10 (95%-Cl 1.45-3.05) and 1.88 (95%-Cl 1.31-2.69) in HELLP syndrome. In addition, the aORs between sFlt-1/PlGF and sEng were very similar but higher for PE and FGR than HELLP syndrome.In conclusion,sEng performed similarly to sFlt-1/PlGF to detect placental dysfunctions.

RAS testing of liquid biopsy correlates with the outcome of metastatic colorectal cancer patients treated with first-line FOLFIRI plus cetuximab in the CAPRI-GOIM trial.

Background: Liquid biopsy is an alternative to tissue for RAS testing in metastatic colorectal carcinoma (mCRC) patients. Little information is available on the predictive role of liquid biopsy RAS testing in patients treated with first-line anti-EGFR monoclonal antibody-based therapy. Patients and methods: In the CAPRI-GOIM trial, 340 KRAS exon-2 wild-type mCRC patients received first-line cetuximab plus FOLFIRI. Tumor samples were retrospectively assessed by next generation sequencing (NGS). Baseline plasma samples were analyzed for KRAS and NRAS mutations using beads, emulsion, amplification, and magnetics digital PCR (BEAMing). Discordant cases were solved by droplet digital PCR (ddPCR) or deep-sequencing. Results: A subgroup of 92 patients with available both NGS data on tumor samples and baseline plasma samples were included in this study. Both NGS analysis of tumor tissue and plasma testing with BEAMing identified RAS mutations in 33/92 patients (35.9%). However, 10 cases were RAS tissue mutant and plasma wild-type, and additional 10 cases were tissue wild-type and plasma mutant, resulting in a concordance rate of 78.3%. Analysis of plasma samples with ddPCR detected RAS mutations in 2/10 tissue mutant, plasma wild-type patients. In contrast, in all tissue wild-type and plasma mutant cases, ddPCR or deep-sequencing analysis of tumor tissue confirmed the presence of RAS mutations at allelic frequencies ranging between 0.15% and 1.15%. The median progression-free survival of RAS mutant and wild-type patients according to tissue (7.9 versus 12.6 months; P = 0.004) and liquid biopsy testing (7.8 versus 13.8 moths; P < 0.001) were comparable. Similar findings were observed for the median overall survival of RAS mutant and wild-type patients based on tissue (22.1 versus 35.8 months; P = 0.016) and plasma (19.9 versus 35.8 months; P = 0.013) analysis. Conclusion: This study indicates that RAS testing of liquid biopsy results in a similar outcome when compared with tissue testing in mCRC patients receiving first-line anti-EGFR monoclonal antibodies.

Discomfort and adaptation in psoriatic patients: an inchoate supportive care trial.

INTRODUCTION: Psoriasis produces functional and aesthetic damages which cause psychosocial discomfort and require new adjustment factors. AIMS: We describe the correlation among discomfort and personality factors as traits, coping, defense mechanism (DM) and resilience in Ps (skin psoriasis) and PsA (psoriatic arthritis) patients. MATERIALS AND METHODS: A random sample, age between 45-60, with 1:1 female/male, includes 3 groups each of 90 subjects: - single psoriasis (Ps, average 50.11, SD 4.9928) - Ps and psoriatic arthritis (PsA, average 50.61, SD 4.8765) - controls (C, average 50.0 and SD 4.8019). We evaluated traits by the 16PF-5, coping by Cope Scale, defence mechanism (DM) by DMRS, dysmorphophobia by BDDE and resilience by CR-RISC scales. Statistical analysis was performed by SPSS 14 version, p < 0.05 and t, F, W and P tests and clinical analysis. RESULTS: We recorded a reduction in traits and coping and an increase in DM, dysmorphophobia and resilience. PsA patients showed a greater distress and adjustment factors than Ps alone. Tests showed a valid significance (p < 0.00001), an effect size from 0.30 to > 0.50 in comparison patients-C and η2 = 1.4 in comparison Ps-PsA. In all patients- controls OR, PAR and NNT registered very high values. DISCUSSION: Reduction in autonomy and imperfections cause an internal damage. The adaptation process searches coping factors (to integrate traits) when lesions appear. It selects DM when stressful dynamics require a further adaptation. Finally it results in adequate resilience when damages and dysmorphophobia are offset by traits, coping and DM. CONCLUSIONS: Clinical signs increase more then traits in severity, coping decrease in resources, resilience and DM grow in use and dismorphophobia just increases in intensity.

Female lichen sclerosus genitalis: discomfort and adaptation.

INTRODUCTION: Female genitalis lichen sclerosus (FGLS) occurs on skin and mucous membranes and shows inflammatory lesions, chronic atrophic, itching and pain. These physical damages produce a decline in sex and a resulting relational couple discomfort. AIM: We describe the discomfort and the relationship between physical and psychological damage. MATERIALS AND METHODS: A random sample, between 35-55 years (average 44.55, SD 6.00) includes 2 groups of 55 subjects: - the first with LSAG diagnosis since 24 months - the latter with controls. We evaluated personality by the MMPI-2, discomfort by short SF-36, couple relationship by ISS, coping by Cope and resilience by CR-RISC scales. Statistical analysis was performed by SPSS 14 version. P-values <0.05, 95% confidence limits, t, F, W and P tests and two by two table, compare two rates and Cohort/RTC were employed. RESULTS: Discomfort makes same personality factors changed. 50 FGLS recorded higher scores than 50 controls in MMIP-2, ISS and CD-RISC scales, lower in ISF-36 and COPE scale. Statistical tests show a valid significance (p<0.00001), a 94.3% of exposed with outcome, an etiological fraction in exposed of 96,15% and a 26 risk / prevalence ratio. DISCUSSION: The psychological distress arises as a result of somatic damage and reduces expression and thought of self. Lesion and pain produce depression, anxiety, negative emotionality and decline of sex. High values of t, F and W tests indicate a good validity of results. CONCLUSIONS: The psychological distress grows from somatic damages and, in progressive way, it reduce expressions and thoughts of ego in increasingly tight limits: through successive stages, it involves social relations, couple relationships and individual identity.

High-resolution microarray analysis unravels complex Xq28 aberrations in patients and carriers affected by X-linked blue cone monochromacy.

The human X chromosome contains ∼ 1600 genes, about 15% of which have been associated with a specific genetic condition, mainly affecting males. Blue cone monochromacy (BCM) is an X-linked condition caused by a loss-of-function of both the OPN1LW and OPN1MW opsin genes. The cone opsin gene cluster is composed of 2-9 paralogs with 99.8% sequence homology and is susceptible to deletions, duplications, and mutations. Current diagnostic tests employ polymerase chain reaction (PCR)-based technologies; however, alterations remain undetermined in 10% of patients. Furthermore, carrier testing in females is limited or unavailable. High-resolution X chromosome-targeted CGH microarray was applied to test for rearrangements in males with BCM and female carriers from three unrelated families. Pathogenic alterations were revealed in all probands, characterized by sequencing of the breakpoint junctions and quantitative real-time PCR. In two families, we identified a novel founder mutation that consisted of a complex 3-kb deletion that embraced the cis-regulatory locus control region and insertion of an additional aberrant OPN1MW gene. The application of high-resolution X-chromosome microarray in clinical diagnosis brings significant advantages in detection of small aberrations that are beyond the resolution of clinically available aCGH analysis and which can improve molecular diagnosis of the known conditions and unravel previously unrecognized X-linked diseases.

Melainomania: a study on 100 subjects.

AIM: The melainomania is a psychological and skin syndrome and it expresses the obsessive need of tanned skin in a complete and constant way. We searched for the psycho-patogenetic factors of the syndrome and the psychological and skin ends point useful to evaluate the psychic and skin damages. METHODS: Seventy-two women and 28 men, aged 38-56 years, who were used to a constant photo exposure have been examined. We have considered the psychosocial damages by Rorschach e Machover tests and by psychologist-subject interviews, skin damages by Glogau signs and by echography. Furthermore, the subjects have been submitted to a psychotherapeutic treatment. RESULTS: The tests and the interviews have underlined obsessive lines, dependence, bodily misperception, low index of reality. The Spearman correlations among clinical signs and echographic findings shows high significance. The psychotherapy has reduced the uneasiness in 66 women and 24 men. Each event that produces uneasiness requires a rationalization process that individualizes in successive phases logical justifications for the lived uneasiness and for the perceived consequences. The rational psycho mechanics is a new method that has identified the phase of the thought-deviation and it has planned the corrective therapy. CONCLUSION: The psychological tests, the interviews, the clinical signs and the ultrasound have allowed us to appraise the psychic and skin damages, while the method of the rational psycho mechanics seems to have brought a satisfactory therapeutic result.

Confirmatory tests in the diagnosis of primary aldosteronism.

Primary aldosteronism is the most common form of secondary hypertension and patients with hyperaldosteronism are more prone to premature cardiovascular complications compared to essential hypertensives. The diagnostic flow-chart for the diagnosis of PA is performed in three steps: a) screening; b) confirmation; and c) subtype differentiation. Instead of proceeding directly to subtype classification, the recently published Endocrine Society Guidelines recommend that patients with a positive ARR should undergo a confirmatory test, in order to definitively confirm or exclude the diagnosis of PA. The Guidelines recognize four testing procedures: oral sodium loading, saline infusion, fludrocortisone suppression, and captopril challenge. Herein we discuss the diagnostic protocols for these confirmatory tests and highlight both the advantages and contraindications and we discuss studies in which these confirmatory tests have been compared.

Predicting the pathogenicity of RPE65 mutations.

To assist in distinguishing disease-causing mutations from nonpathogenic polymorphisms, we developed an objective algorithm to calculate an "estimate of pathogenic probability" (EPP) based on the prevalence of a specific variation, its segregation within families, and its predicted effects on protein structure. Eleven missense variations in the RPE65 gene were evaluated in patients with Leber congenital amaurosis (LCA) using the EPP algorithm. The accuracy of the EPP algorithm was evaluated using a cell-culture assay of RPE65-isomerase activity The variations were engineered into plasmids containing a human RPE65 cDNA and the retinoid isomerase activity of each variant was determined in cultured cells. The EPP algorithm predicted eight substitution mutations to be disease-causing variants. The isomerase catalytic activities of these RPE65 variants were all less than 6% of wild-type. In contrast, the EPP algorithm predicted the other three substitutions to be non-disease-causing, with isomerase activities of 68%, 127%, and 110% of wild-type, respectively. We observed complete concordance between the predicted pathogenicities of missense variations in the RPE65 gene and retinoid isomerase activities measured in a functional assay. These results suggest that the EPP algorithm may be useful to evaluate the pathogenicity of missense variations in other disease genes where functional assays are not available.

Fetal borderline cerebral ventriculomegaly: clinical significance and management.

AIM: The aim of this study was to evaluate the clinical significance and the management of fetal borderline lateral cerebral ventriculomegaly. METHODS: Fetuses with a sonographic diagnosis of lateral cerebral ventriculomegaly isolated or associated to other fetal malformations were followed monthly and a review of the English-language literature was made. RESULTS: Of 86 fetuses analyzed, 30 suffered from hydrocephaly (monolateral, bilateral), 56 showed also several other malformations . Chromosomal aberrations is possible also in case of isolated hydrocephaly. Neurological sequelae, mostly a mild to moderate delay in cognitive and/or motor development, is present in 10% of the cases. CONCLUSIONS: In most cases, isolated borderline lateral cerebral ventriculomegaly has no consequence. However, the risk of cerebral maldevelopment, delayed neurological development and, possibly, chromosomal aberrations is increased. The optimum management of these cases remains uncertain.

Functionally rodless mice: transgenic models for the investigation of cone function in retinal disease and therapy.

Two genetically engineered strains of mice were used to characterize murine cone function electroretinographically, without interference of rod-driven responses: (1) mice with a deletion of the gene for the rod transducin alpha-subunit (transducin alpha-/-), and (2) mice with rod arrestin deleted (arrestin -/-). In the first three months of age, both strains have a normal complement of rods and normal rod structure, but transducin alpha-/- mice have no rod-driven responses to light, while rod-driven activity of arrestin -/- mice can be suppressed by a single intense flash for hours. In response to intense flashes the electroretinograms of these strains of mice showed a readily identifiable, pure-cone a-wave of approximately 10 microV saturating amplitude. A 530 nm background that saturates rod responses of wild type mice was found to desensitize the b-wave responses of mice of both transgenic lines, whether the b-waves were driven by photons captured by M- or UV-cone pigments. The desensitizing effect of the 530 nm background on UV-pigment driven responses provides new evidence in support of the hypothesis of functional co-expression of the M-pigment in cones expressing primarily the UV-pigment.

Genotype-phenotype correlations and differential diagnosis in autosomal dominant macular disease.

In the past few years, great progress has been made in the understanding of macular diseases. A number of disease-causing genes have been cloned, and numerous loci for other conditions have been mapped. The purpose of this article is to provide an overview of the current understanding of the genotype-phenotype correlations in autosomal dominant macular diseases with an emphasis on differential diagnostic issues. Whenever possible, the molecular correlates have been reviewed and the implications for age-related macular degeneration have been discussed. The many similarities of these diseases to age-related macular degeneration of the atrophic or exudative type, which can be misleading in elderly subjects, have also been addressed. While some conditions yield disease truly confined to the macula, others show widespread retinal involvement on functional testing. Clear-cut genotype-phenotype correlations are possible only for some forms of macular diseases. To further complicate the diagnostic process, there is a considerable degree of clinical overlap between many of them, making the differential diagnostic process potentially challenging. Functional testing, careful assessment of family history and extensive family work-up are essential in differentiating at the clinical level most, but not all, of these disease entities. Awareness of all of these conditions is required to direct correctly diagnostic investigations, to formulate an accurate prognosis, and for proper genetic counseling.

Pigment epithelium-derived factor supports normal Müller cell development and glutamine synthetase expression after removal of the retinal pigment epithelium.

In conditions in which the retinal pigment epithelium (RPE) is dystrophic, carries a genetic mutation, or is removed physically, Müller cells undergo degenerative changes that contribute to the retinal pathology. We previously demonstrated that pigment epithelium-derived factor (PEDF), a glycoprotein secreted by the RPE cells with neuroprotective and differentiation properties, protects against photoreceptor degeneration induced by RPE removal. The purpose of the present study was to analyze the putative gliosupportive activity of PEDF on Müller cells of RPE-deprived retinas and assess whether protection of Müller cells was correlated with improved photoreceptor outer segment assembly. Eyes were dissected from Xenopus laevis tadpoles, and the RPE was removed before culturing in medium containing purified PEDF, PEDF plus anti-PEDF, or medium alone. Control eyes matured with an adherent RPE or in medium containing PEDF plus nonimmune serum. Müller cell ultrastructure was examined. Glial fibrillary acidic protein (GFAP) and glutamine synthetase were localized immunocytochemically, and the corresponding protein levels were quantified. In control retinas, Müller cells were structurally intact and formed adherens junctions with neighboring photoreceptors. In addition, they did not express GFAP, whereas glutamine synthetase expression was high. RPE removal dramatically altered the ultrastructure and biosynthetic activity of Müller cells; Müller cells failed to form adherens junctions with photoreceptors and glutamine synthetase expression was suppressed. PEDF prevented the degenerative glial response; Müller cells were ultrastructurally normal and formed junctional complexes with photoreceptors. PEDF also preserved the expression of glutamine synthetase at near-normal levels. The morphogenetic effects of PEDF were blocked by the anti-PEDF antibody. Our study documents the glioprotective effects of PEDF and suggests that maintenance of the proper Müller cell ultrastructure and expression of glutamine synthetase may be necessary to support the proper assembly of photoreceptor outer segments.

Identification of the gene that, when mutated, causes the human obesity syndrome BBS4.

Bardet-Biedl syndrome (BBS, MIM 209900) is a heterogeneous autosomal recessive disorder characterized by obesity, pigmentary retinopathy, polydactyly, renal malformations, mental retardation, and hypogenitalism. The disorder is also associated with diabetes mellitus, hypertension, and congenital heart disease. Six distinct BBS loci map to 11q13 (BBS1), 16q21 (BBS2), 3p13-p12 (BBS3), 15q22.3-q23 (BBS4), 2q31 (BBS5), and 20p12 (BBS6). Although BBS is rare in the general population (<1/100,000), there is considerable interest in identifying the genes causing BBS because components of the phenotype, such as obesity and diabetes, are common. We and others have demonstrated that BBS6 is caused by mutations in the gene MKKS (refs. 12,13), mutation of which also causes McKusick-Kaufman syndrome (hydrometrocolpos, post-axial polydactyly, and congenital heart defects). MKKS has sequence homology to the alpha subunit of a prokaryotic chaperonin in the thermosome Thermoplasma acidophilum. We recently identified a novel gene that causes BBS2. The BBS2 protein has no significant similarity to other chaperonins or known proteins. Here we report the positional cloning and identification of mutations in BBS patients in a novel gene designated BBS4.

Reliability assessment of a rod photoreceptor outer segment grading system.

The purpose of this study was to assess the reliability of a rod photoreceptor outer segment (PR-OS) grading system based on the analysis of 1 microm thick retinal sections obtained from Xenopus laevis whole-eye organ cultures. Digitally captured images, representative of the entire spectrum of rod PR-OS organization levels, were selected and coded numerically. A total of 102 individual rod PR-OS profiles were graded according to a six-step classification scheme based on the percentage of rod PR-OS membrane organization. Unweighted (exact agreement) and weighted kappa (kappa) coefficients (for use with ordered categorical rating scales) were calculated. Differences between kappa coefficients were tested for by chi-square analysis. To investigate the intra- and inter-rater variability and the possible presence of an interaction of the measurements with time, a repeated-measures analysis of variance was performed. The overall unweighted and weighted intra-rater kappa coefficients were 0.78 and 0.92, respectively. The overall unweighted and weighted inter-rater kappa coefficients were 0.73 and 0.90, respectively. There was no significant difference between raters or between first and second reading, nor was interaction between raters and time of rating documented. Individual kappa coefficients were equivalent both between raters and between sessions. Intra- and inter-rater agreement was within one step in 100% of cases. The estimated values of the kappa coefficients are consistent with a good to excellent degree of reliability and reproducibility of this rod PR-OS grading system. This system will be useful in the assessment of rod PR-OS morphology in studies of photoreceptor physiology and pathology.

Positional cloning of a novel gene on chromosome 16q causing Bardet-Biedl syndrome (BBS2).

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous autosomal recessive disorder with the primary clinical features of obesity, pigmented retinopathy, polydactyly, hypogenitalism, mental retardation and renal anomalies. Associated features of the disorder include diabetes mellitus, hypertension and congenital heart disease. There are six known BBS loci, mapping to chromosomes 2, 3, 11, 15, 16 and 20. The BBS2 locus was initially mapped to an 18 cM interval on chromosome 16q21 with a large inbred Bedouin kindred. Further analysis of the Bedouin population allowed for the fine mapping of this locus to a 2 cM region distal to marker D16S408. Physical mapping and sequence analysis of this region resulted in the identification of a number of known genes and expressed sequence tag clusters. Mutation screening of a novel gene (BBS2) with a wide pattern of tissue expression revealed homozygous mutations in two inbred pedigrees, including the large Bedouin kindred used to initially identify the BBS2 locus. In addition, mutations were found in three of 18 unrelated BBS probands from small nuclear families.

Lactose supports Muller cell protein expression patterns in the absence of the retinal pigment epithelium.

PURPOSE: We have previously shown that lactose, but not mannose, promotes the assembly of nascent photoreceptor outer segments in the absence of the retinal pigment epithelium (RPE). The purpose of the present study was to determine if, in addition to the improved outer segment assembly observed in the presence of lactose, biosynthetic changes in Muller cells could also be detected. METHODS: The RPE was removed from intact isolated Xenopus embryonic eyes that were allowed to complete differentiation in Niu-Twitty medium, Niu-Twitty with mannose, or Niu-Twitty with lactose. Control retinas matured in vitro with an adherent RPE. Retinal morphology was evaluated for organized folding of outer segment membranes and cell loss. In addition, the expression of three Muller cell proteins, glial fibrillary acidic protein (GFAP), cellular retinaldehyde-binding protein (CRALBP), and glutamine synthetase, was examined. RESULTS: In control retinas, GFAP is undetectable, CRALBP heavily immunolabels Muller cells, and radial patterns of glutamine synthetase immunoreactivity are present. In the absence of the RPE, Muller cells upregulate GFAP expression, CRALBP labeling is present at a slightly reduced level, and glutamine synthetase immunolabeling is negligible. Neither mannose nor lactose modify significantly the expression of CRALBP. Similarly, both compounds completely prevent the upregulation of GFAP. However, normal glutamine synthetase expression was observed only in the presence of lactose, but not in the presence of mannose. Statistical analyses of slot blot-based protein quantification confirmed our immunochemical results. CONCLUSIONS: In RPE-deprived retinas supplemented with lactose, Muller cells were morphologically normal. The proper photoreceptor outer segment morphogenesis observed under these conditions was uniquely associated with normal levels of glutamine synthetase expression. The exact significance of this finding with respect to photoreceptor outer segment morphogenesis is unknown. We suggest that glutamine synthetase may be a marker of Muller cell metabolic or structural integrity that may reflect the enhanced ability of these cells, in the presence of lactose, to support photoreceptor outer segment morphogenesis.

Pigment epithelium-derived factor supports normal development of photoreceptor neurons and opsin expression after retinal pigment epithelium removal.

Dysfunction of the retinal pigment epithelium (RPE), its loss, or separation from the underlying neural retina results in severe photoreceptor degeneration. Pigment epithelium-derived factor (PEDF) is a glycoprotein with reported neuroprotective and differentiation properties that is secreted in abundance by RPE cells. The "pooling" of PEDF within the interphotoreceptor matrix places this molecule in a prime physical location to affect the underlying neural retina. The purpose of this study was to analyze the morphogenetic activity of PEDF in a model of photoreceptor dysmorphogenesis induced by removal of the RPE. Eyes were dissected from embryonic Xenopus laevis, and the RPE was removed before culturing in medium containing PEDF, PEDF plus anti-PEDF antibodies, or medium alone. Control retinas were maintained with an adherent RPE. Light and electron microscopic analysis was used to examine retinal ultrastructure. Opsin was localized immunocytochemically and quantified as an index of outer segment membranous material and photoreceptor protein expression. Removal of the RPE resulted in an aberrant assembly of photoreceptor outer segments, loss of fine subcellular ultrastructure in photoreceptors, and a reduction in opsin protein levels when compared with control retinas. The addition of PEDF prevented the dysmorphic photoreceptor changes induced by RPE removal. In particular, photoreceptor ultrastructure, outer segment membrane assembly, and steady-state levels of opsin were equivalent to control conditions. Anti-PEDF antibodies completely blocked the morphogenetic activity of PEDF. These results indicate that PEDF is able to mimic the supportive role of the RPE on photoreceptors during the final stages of retinal morphogenesis.

Targeted disruption of Müller cell metabolism induces photoreceptor dysmorphogenesis.

Within the retina, the Müller cells and photoreceptors are in close physical proximity and are metabolically coupled. It is unknown, however, whether Müller cells affect photoreceptor differentiation and outer segment membrane assembly. The objective of this study was to determine whether targeted disruption of Müller cell metabolism would induce photoreceptor dysmorphogenesis. Intact isolated Xenopus laevis embryonic eyes were cultured in medium with or without Müller cell-specific inhibitors (i.e., alpha-aminoadipic acid and fluorocitrate). To assess Müller cell injury, the gross retinal morphology was examined along with immunocytochemical assessment of Müller cell-specific protein expression patterns. The steady-state levels of opsin were quantified to determine whether the Müller cell inhibitors negatively affected photoreceptor protein synthesis. Müller and photoreceptor cell ultrastructure was scrutinized and the organization of the outer segment membranes was graded. In control retinas, there was no swelling of Müller cell cytoplasm. Glial fibrillary acidic protein (GFAP) was undetectable, whereas glutamine synthetase was abundant. The steady-state level of opsin was high and photoreceptors elaborated properly folded outer segments. Exposure to both Müller cell-specific inhibitors induced swelling of Müller cell endfeet, cytoplasmic paling and alterations of Müller cell-specific protein expression patterns. The steady-state level of opsin in retinas exposed to alpha-aminoadipic acid was unchanged compared with control eyes, whereas, in eyes exposed to fluorocitrate, opsin levels were slightly reduced. The most significant finding was that targeted disruption of Müller cell metabolism adversely affected photoreceptor outer segment membrane assembly, causing dysmorphogenesis of nascent outer segments. These results suggest that the termination signal(s) necessary for proper outer segment folding were disrupted by targeted inhibition of Müller cells and support the hypothesis that Müller cells interact with photoreceptors through mechanisms that may regulate, at least in part, the assembly of photoreceptor outer segment membranes.

Disease expression of RP1 mutations causing autosomal dominant retinitis pigmentosa.

PURPOSE: To determine the disease expression in heterozygotes for mutations in the RP1 gene, a newly identified cause of autosomal dominant retinitis pigmentosa (adRP). METHODS: Screening strategies were used to detect disease-causing mutations in the RP1 gene, and detailed studies of phenotype were performed in a subset of the detected RP1 heterozygotes using electroretinography (ERG), psychophysics, and optical coherence tomography (OCT). RESULTS: Seventeen adRP families had heterozygous RP1 changes. Thirteen families had the Arg677ter mutation, whereas four others had one of the following: Pro658 (1-bp del), Ser747 (1-bp del), Leu762-763 (5-bp del), and Tyr1053 (1-bp del). In Arg677ter RP1 heterozygotes, there was regional retinal variation in disease, with the far peripheral inferonasal retina being most vulnerable; central and superior temporal retinal regions were better preserved. The earliest manifestation of disease was rod dysfunction, detectable as reduced rod ERG photoresponse maximum amplitude, even in heterozygotes with otherwise normal clinical, functional, and OCT cross-sectional retinal imaging results. At disease stages when cone abnormalities were present, there was greater rod than cone dysfunction. Patients with the RP1 frameshift mutations showed similarities in phenotype to those with the Arg677ter mutation. CONCLUSIONS: Earliest disease expression of RP1 gene mutations causing adRP involves primarily rod photoreceptors, and there is a gradient of vulnerability of retinopathy with more pronounced effects in the inferonasal peripheral retina. At other disease stages, cone function is also affected, and severe retina-wide degeneration can occur. The nonpenetrance or minimal disease expression in some Arg677ter mutation-positive heterozygotes suggests important roles for modifier genes or environmental factors in RP1-related disease.