HRG4 (UNC119) mutation found in cone-rod dystrophy causes retinal degeneration in a transgenic model.

PURPOSE: To investigate the function and pathogenicity of HRG4, a photoreceptor synaptic protein homologous to the Caenorhabditis elegans neuroprotein UNC119. METHODS: HRG4 was screened for mutations in patients with various retinopathies, and a transgenic mouse model was constructed and analyzed based on a mutation found. RESULTS: A heterozygous premature termination codon mutation was found in a 57-year-old woman with late-onset cone-rod dystrophy. In some transgenic mice carrying the identical mutation, age-dependent fundus lesions developed accompanied by electroretinographic changes consistent with defects in photoreceptor synaptic transmission (depressed b-wave, normal c-wave), and retinal degeneration occurred with marked synaptic and possible transsynaptic degeneration. CONCLUSIONS: HRG4, the only synaptic protein known to be highly enriched in photoreceptor ribbon synapses, is now shown to be pathogenic when mutated.

Localization of HRG4, a photoreceptor protein homologous to Unc-119, in ribbon synapse.

PURPOSE: To characterize further HRG4, a novel photoreceptor protein recently identified by subtractive cDNA cloning, by sequence analysis and immunolocalization. METHODS: The rat homolog of HRG4, RRG4 was expressed and used to prepare an antibody. The antibody was used in Western blot analysis, and immunofluorescent localization at the light and electron microscopic levels of HRG4-RRG4 protein. The HRG4-RRG4 sequence was also analyzed for homologies. RESULTS: HRG4-RRG4 showed 57% homology with unc-119, a Caenorhabditis elegans neuroprotein causing defects in locomotion, feeding, and chemosensation when mutated. By Western blot analysis, the HRG4-RRG4 protein was demonstrable only in retina and was soluble in nature. Immunofluorescence microscopic study of human and rat retinas, using the HRG4-RRG4 antibody, and other rod and cone photoreceptor-specific antibodies showed that the HRG4-RRG4 protein is localized in the outer plexiform layer of the retina in the synaptic termini of rod and cone photoreceptors. Electron microscopic immunolocalization showed the protein in the cytoplasm and on the presynaptic membranes of the photoreceptor synapses. CONCLUSIONS: The homology to unc-119 and localization to the photoreceptor synapse are suggestive of a function for HRG4-RRG4 in photoreceptor neurotransmission. HRG4 is the first photoreceptor-enriched synaptic protein to be reported, suggesting that its function may be unique to the specialized ribbon synapses formed between photoreceptors and the horizontal and bipolar cells of the retina.

Inherited retinal degeneration: basic FGF induces phagocytic competence in cultured RPE cells from RCS rats.

In RCS rats, the retinal pigment epithelium (RPE) is defective in phagocytosis of photoreceptor membranes. We have previously shown reduced expression of basic fibroblast growth factor (bFGF) in the RPE of 7-10-day-old RCS rats. This study using primary RPE cultures from rats of this age demonstrates that the phagocytic defect in the mutant RPE can be overcome by treatment with bFGF, by a mechanism involving gene transcription and that normal RPE phagocytosis, also requiring transcription, is blocked by a bFGF neutralizing antibody. The combined data point to a role for bFGF in the normal mechanism of RPE phagocytosis and the RCS defect.

Mammalian retinal pigment epithelial cells in vitro respond to the neurokines ciliary neurotrophic factor and leukemia inhibitory factor.

We examined whether primary cultures of rat retinal pigment epithelial (RPE) cells and RPE cells of an immortalized rat cell line, BPEI-1, would be responsive to the neurokines ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF), which are known to be potent trophic factors for neuronal cells. Primary RPE cell cultures were characterized by indirect immunofluorescence and exhibited positive immunoreactivity for RET-PE2, a monoclonal antibody that recognizes RPE cells, and for the intermediate filaments cytokeratin and vimentin. The survival of cultured RPE cells in serum-free defined medium in the presence of CNTF or LIF was investigated during a 0- to 5-day period. Both CNTF and LIF, at concentrations of 1-50 ng/mL (4-200 pM), markedly enhanced RPE cell survival. Bromodeoxyuridine labelling of RPE cells revealed an increased mitotic activity in cell cultures treated with either CNTF or LIF in comparison to untreated serum-free cultures. Increases in cell survival and proliferation after neurokine treatment were also observed with the BPEI-1 cell line. However, in comparison to the primary RPE cultures, LIF was more effective than CNTF in promoting survival of the cell line over a 5-day treatment period. These studies demonstrate that the neurokines CNTF and LIF are potent trophic factors for mammalian RPE cells in vitro and may serve as candidate therapeutic agents in degenerative conditions that affect the retina and RPE.

Heat shock-regulated expression of calreticulin in retinal pigment epithelium.

Calreticulin is a major Ca2+ binding protein in the endoplasmic reticulum of non-muscle cells. In this report we show that calreticulin protein is strongly induced by heat shock. Activation and attenuation of the heat shock transcriptional response is caused by heat shock factor that binds to 5'-flanking sequences of heat shock responsive genes, the heat shock element. The smallest stretch of DNA that shows detectable binding of heat shock factor in vitro contains a two-sequence unit nGAAnnTTCn which exists in the 5'-flanking region of calreticulin DNA (5'-gGAAccCAGcgTTC-3'). The present data provide direct evidence that calreticulin expression can be modulated by heat shock. Thus, our results strengthen the hypothesis that calreticulin, in addition to its function as a cellular Ca2+ store, is a multifunctional protein which performs at least some of its functions from the lumen of the ER.

Kinetics of rod outer segment phagocytosis by cultured retinal pigment epithelial cells. Relationship to cell morphology.

PURPOSE: To study phenotypic variation in primary cultures of rat retinal pigment epithelium (RPE) and to correlate cell morphology with rates of binding and ingestion of rod outer segments (ROS). METHOD: Replicate cultures were prepared using RPE cell sheets isolated with Dispase from Royal College of Surgeons normal (RCS rdy+ p+) and dystrophic (RCS p+) rats. Retinal pigment epithelial morphology was analyzed, and phagocytosis was assessed by fluorescence microscopy in cultures fixed at 2-hour intervals from 3 to 19 hours after continuous incubations with fluorescein isothiocyanate (FITC)-stained ROS. RESULTS: A wide range of RPE cell size, shape, and pigmentation was present at confluence; however, distinct morphologic subtypes were recognized, defined as types 1 to 3, and studied separately. In both normal and dystrophic cultures, the extent and rate of ROS binding varied with RPE phenotype. In normal cultures, highly spread pigmented binucleate cells (type 3) bound and rapidly ingested multiple ROS per cell starting at 3 hours and reached a peak at 9 hours. Lightly pigmented daughter cells (type 2) bound and ingested far fewer ROS per cell than did type 3 RPE, which had not divided. Patches of hexagonally packed cells with in vivo morphology (type 1) bound large numbers of ROS per cell only after prolonged (9- to 11-hour) incubations and ingested them synchronously. Comparison of normal versus dystrophic RPE subtypes 1 to 3 revealed the known ingestion defect in all three mutant phenotypes but indicated delayed ROS binding in type 2 and type 3 cells as well. CONCLUSIONS: Kinetics of ROS binding and ingestion differ markedly among phenotypic variants of RPE cells typically found in primary cultures at confluence. Thus, accurate quantitation requires comparison of equivalent microscopic fields or like RPE subtypes, and the heterogeneous responses of various RPE subtypes should be considered when interpreting phagocytic data obtained from entire cultures at a particular time.

Analysis of basic fibroblast growth factor in rats with inherited retinal degeneration.

In RCS rats, photoreceptors degenerate between postnatal days 20 and 60, secondary to a genetic defect expressed in the neonatal retinal pigmented epithelium (RPE). Previous work has shown delay of the photoreceptor degeneration in this model by intraocular injection of basic fibroblast growth factor (bFGF). Evidence is presented here, from bFGF immunostaining and Northern analysis of bFGF mRNA, for reduced bFGF expression in uncultured RPE of dystrophic RCS pups. It is also shown that in the mutant eyes angiogenesis in the underlying choroid, which normally occurs between postnatal days 7 and 10, is markedly delayed, with irregular distribution of vessels, consistent with a reduction in this known angiogenesis factor. Mutational analysis of the bFGF transcript and gene by denaturing gradient gel electrophoresis and Southern analysis did not, however, reveal abnormalities in the coding sequence of this gene in RCS rats.

Immunolocalization of X-arrestin in human cone photoreceptors.

X-arrestin is a recently identified retina-specific gene of unknown function. Affinity-purified anti-peptide antibody to human X-arrestin was prepared, and used in Western blot analysis of human retinal proteins and for immunohistochemistry on human retinal sections. By Western blot analysis, the antibody specifically bound to an approximately 47 kDa protein, and by indirect immunofluorescence specifically labeled cone photoreceptors with greatest intensity in their outer segments. In single and double label experiments, the localization of X-arrestin immunoreactivity was compared with immunolabeling patterns obtained with antibodies to red/green cone opsin, rhodopsin, and S-antigen. The results showed that X-arrestin is expressed in red-, green- and blue-sensitive cones in the human retina.

Cloning of the cDNA for a novel photoreceptor protein.

A subtractive cDNA cloning strategy was used to isolate a 1381-base pair human retina-specific cDNA, human retinal gene 4 (HRG4), which hybridized to a 1.4-kilobase message in the retina and encoded a 240-amino acid acidic protein with a calculated molecular mass of 26,964 Da. The proximal 1/4 of the conceptual protein sequence was rich in glycine (18%) and proline (20%), had a predicted secondary structure of turns, and showed a loose similarity (19-24%) to various alpha-collagen sequences, while the distal 2/4 consisted of a mixture of alpha-helices, beta-sheets, and turns. Genomic Southern analysis with HRG4 showed cross-hybridizing sequences in six different species, and HRG4 was 92% homologous with a 1264-base pair rat cDNA (rat retinal gene 4; RRG4) at the protein level. The region of 100% identity between the two sequences corresponded to the distal 3/4 of the protein sequence consisting of mixed secondary structures, suggesting a functionally important domain. In vitro transcription and translation corroborated the open reading frames corresponding to HRG4 and RRG4 in the cDNAs. Expression of HRG4 in the retina was localized to the photoreceptors by in situ hybridization. Developmentally, RRG4 began to be highly expressed around postnatal day 5 in the rat outer retina when the photoreceptors begin to differentiate and rapidly increased in expression to reach the mature adult level by postnatal day 23. No diurnal fluctuation in expression of RRG4 was seen.

X-arrestin: a new retinal arrestin mapping to the X chromosome.

We have been using a differential cDNA cloning approach to isolate human retina-specific and retina-enriched genes [1]. A 1,314 bp cDNA was isolated by this approach, representing a highly retina-specific message encoding a 388 amino acid protein showing 58%, 50%, and 49% homology to bovine beta-arrestin, and bovine and human retinal arrestin (S-antigen), respectively. Chromosomal mapping localized this new arrestin gene to the proximal long arm of the X chromosome, hence it was named X-arrestin. In situ hybridization demonstrated its expression in the inner and outer segments and the inner plexiform regions of the retina.

Spontaneously arising immortal cell line of rat retinal pigmented epithelial cells.

A continuous cell line of rat retinal pigment epithelium (RPE), named BPEI-1, has been established and characterized. Sheets of pure RPE cells, uncontaminated by choroidal or neural retinal cell types, were isolated from eyes of 7-day-old Long Evans rats and established in primary culture. The primary RPE cells became extensively spread and grew slowly for approximately 1 month, at which time a colony of small rapidly dividing cells spontaneously appeared. Following trypsinization, most of the typical primary RPE cells did not survive and were quickly outnumbered by the smaller cells, which gave rise to a cell line that was grown continuously for several hundred generations. When growing at the maximal rate in media containing 20% FBS (doubling time 18 h), the cells were fibroblastic and nearly devoid of pigment, but were capable of morphologic transition back to a pigmented, epithelioid form when cultured under low serum conditions. Evidence that these cells originated from RPE included specific immunolabeling with antibodies to cellular retinaldehyde binding protein and cytokeratin, negative GFAP immunoreactivity, and demonstration of avid phagocytosis of isolated rod outer segments by these cells. Partial characterization of choroidal cells eliminated the latter cells as possible contaminants which could have given rise to the cell line. The BPEI-1 cell line, and other rat RPE cell lines currently being developed from pigmented normal (LE, RCS rdy+p+) and retinal dystrophic (RCS p+) rats should facilitate biochemical and molecular biological approaches to study of RPE cell function in health and disease.

Double fluorescent vital assay of phagocytosis by cultured retinal pigment epithelial cells.

PURPOSE: The goal of this study was to develop the first vital assay system for in vitro analysis of phagocytosis of rod outer segments (ROS) by normal retinal pigment epithelial (RPE) cells and for study of the phagocytic defect in RPE of the Royal College of Surgeons (RCS) rat with inherited retinal degeneration. Required features included ability to directly visualize and quantitate the phagocytic process in living RPE cultures, and capability for subsequent quantitative analysis after fixation of the cells at any chosen time after incubation with ROS. METHODS: A double fluorescent method was designed, based on the process of phagosome-lysosome fusion. For vital staining of lysosomes, confluent cultures of rat RPE cells were incubated with sulforhodamine (SR), a red fluorescent lysosomotropic dye. SR-stained cultures were challenged with isolated rat ROS tagged with fluorescein isothiocyanate (FITC), a green fluorescent probe. RESULTS: This method was used to observe all phases of the phagocytic process in the living cells and the kinetics of ROS binding, ingestion, and phagosome-lysosome fusion were determined. Control studies showed no differences in binding, ingestion, or digestion of unstained versus FITC-stained ROS. Additionally, the phagocytic defect in dystrophic RCS rat RPE cells was confirmed using this technique. CONCLUSIONS: This relatively simple new method is useful in that it uses inexpensive, readily available reagents, it enables real-time analysis of phagocytosis experiments, and it does not require termination of the cultures for analysis of phagocytic ability.

Mitral valve prolapse and body habitus in children.

Mitral valve prolapse has generally been associated in adults with a thin body habitus. However, prior studies used biased samples or limited anthropometric measures. In addition, no information has been available on the subjective assessment of body habitus and diagnosis of mitral valve prolapse, especially in children. We conducted a cross-sectional study on 813 children with uniform assessment of anthropometric measures and mitral valve prolapse. Consistent with research conducted on adults, those subjects with mitral valve prolapse were lighter, thinner, and had, on average, lower values for several, quantifiable anthropometric parameters with the exception of height. However, the subjective assessment showed that while the assessment did not differ by diagnosis, those subjects with mitral valve prolapse were never described as fat. These data support an association between mitral valve prolapse and slender body habitus and extends it to children, thus underscoring the clinical importance that a thin body habitus may be a marker for mitral valve prolapse throughout the age span. This association may partly explain the observed genetic distribution of mitral valve prolapse.

Mitral valve prolapse: associations with symptoms and anxiety.

Mitral valve prolapse has been studied extensively in the adult population, but less is known about it in children. Therefore, 813 children between 9 and 14 years of age were examined by a team of cardiologists and technicians. The children also responded to a questionnaire concerning the presence of symptoms and the What I Think and Feel anxiety instrument. The prevalence of mitral valve prolapse using auscultatory criteria was 4.2% (6.2% for girls, 2.3% for boys). Of those with mitral valve prolapse, 85% had a solitary click, 9% had a click and systolic murmur, and 6% had multiple clicks. Children with auscultatory mitral valve prolapse were less likely to have symptoms than those free of cardiac abnormalities. No difference in average anxiety scores was detected between the two groups. It is concluded that auscultatory mitral valve prolapse is common in children and not accompanied by an increased likelihood of symptoms or anxiety.

Innocent murmurs and third heart sounds in Black schoolchildren.

Normal auscultatory findings were studied during a heart survey in which 12 050 Black schoolchildren, aged 2 to 18 years, were examined by cardiologists. Physiological third heart sounds were detected in 96 per cent of children, innocent systolic murmurs in 72 per cent, and innocent mid-diastolic murmurs in 0.27 per cent. The term 'innocent systolic murmur" was used for vibratory systolic murmurs (70%) and pulmonary ejection systolic murmurs (4.2%) but distinct separation of these two murmurs was often difficult. Vibratory systolic murmurs were present throughout the age range. Important features in differentiating innocent systolic murmurs from those caused by mild organic heart disease included the intonation, site of maximal intensity, timing in systole, and behaviour with postural change. Innocent mid-diastolic murmurs are short murmurs occurring immediately after the third heart sound in children, with no supportive evidence of organic heart disease.

Prevalence of congenital heart disease in black schoolchildren of Soweto, Johannesburg.

A survey conducted by cardiologists in Soweto, Johannesburg, provided an opportunity of assessing the frequency of congenital heart disease in black schoolchildren. Among 12,050 schoolchildren aged 2 to 18 years, 48 had a congenital heart defect, yielding a prevalence of 3.9 per 1000. Only in 2- to 6-year-old children did the prevalence exceed that of rheumatic heart disease. The distribution of the types of defects was largely similar to that reported in other surveys with a predominance (52%) of ventricular septal defects. Two unusual findings were the unexplained absence of persistent ductus arteriosus in these children, and the detection of 5 children with situs inversus (1 in 2410). In all but one child, the congenital heart defect was first discovered during the survey. Despite the limitations of a prevalence study, it can be concluded that congenital heart disease is at least as common in this South African black community as in Caucasians.

Four year follow-up of black schoolchildren with non-ejection systolic clicks and mitral systolic murmurs.

In 1972 we conducted a survey of 12,050 urban Black schoolchildren and detected 168 (prevalence rate of 14 per 1,000) with a non-ejection systolic click (NESC), a late systolic murmur, or both. The etiology of the mitral valve abnormality was unknown but we considered that a significant proportion might have early rheumatic heart disease. The auscultatory features four years later of 139 of the original 168 subjects as well as those of 139 age- and sex-matched controls are presented in this study. No cardiac abnormality was detected in as many as 55 of the subjects. Five children now had pansystolic murmurs but the mitral regurgitation was assessed as mild in four. Twenty-five (17.9 per cent) of the controls, 23 of whom had NESCs, had auscultatory features compatible with mitral valve prolapse. These findings do not support our earlier suggestion that a large number of the 1972 subjects have mild rheumatic heart disease. The results are in accord with other studies which have indicated that auscultatory features compatible with mitral valve prolapse are common in "normals" and also that the prognosis of the specific "billowing mitral leaflet syndrome" is generally benign.

Non-ejection systolic clicks and mitral systolic murmurs in black schoolchildren of Soweto, Johannesburg.

A survey was conducted on 12 050 Black schoolchildren, aged 2 to 18 years, in the South Western Townships of Johannesburg (Soweto), and the prevalence of non-ejection systolic clicks and late systolic murmurs was determined. One or both of these auscultatory findings were detected in 168 children, yielding a prevalence rate of 13-99 per 1000 in the school population. A female preponderance of 1-9:1 was present and there was a strong linear increase in prevalence with age, with a peak rate of 29-41 per 1000 in 17-year-old children. A non-ejection click was the only abnormal auscultatory finding in 123 children (73%) and a mitral systolic murmur in 8 (5%), whereas in 37 (22%) both these findings were present. Of the latter 37 children, the murmur was late systolic in 32; in 5 it was early systolic. Auscultation in different postures was important in the detection of both non-ejection clicks and mitral systolic murmurs. Experience in the detection of these auscultatory findings influenced the frequency with which they were heard. Electrocardiographic abnormalities compatible with those previously described in the billowing mitral leaflet syndrome were present in 11 of 158 children. The aetiology of these auscultatory findings in this community remains unknown. In the same survey, a high prevalence rate of rheumatic heart disease was recorded and the epidemiology of the non-ejection clicks and these mitral systolic murmurs showed similarties to that of rheumatic heart disease. Though the specific billowing mitral leaflet syndrome almost certainly accounts for some of these auscultatory findings, a significant proportion may have early rheumatic heart disease. Further elucidation of this problem is necessary.

Epidemiology of rheumatic heart disease in black shcoolchildren of Soweto, Johannesburg.

A survey to determine the prevalence of rheumatic heart disease (R.H.D.) in Black children was conducted in the creeches and primary schools of the South Western Townships of Johannesburg (Soweto). A total of 12 050 Black children were examined by 10 cardiologists in May to October 1972. The overal prevalence rate of R.H.D. was 6.9 per 1000, with a peak rate of 19.2 per 1000 in children of the seventh school grade. The maximal age incidence was 15-18 years and there was a female preponderance of 1 6:1. A rise in prevalence occurred with increasing family size. Most children (92%) were asymptomatic, and in 82.5% R.H.D. was diagnosed for the first time during the school survey. The commonest valve lesion was mitral regurgitation, which was present in 93% and occurred as an isolated lesion in 47.5%. Lancefield's group A beta-haemolytic streptococcus was isolated from the throats of 52 per 1000 Soweto children. The auscultatory features of a non-ejection systolic click and late systolic murmur were prevalent (13.9 per 1000) and had several epidemiological factors in common with R.H.D. A comprehensive preventative campaign is urgently needed in South Africa, directed at both primary and secondary prophylaxis of R.H.D. The socioeconomic status of the community must be improved if optimal prevention is to be achieved.