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Human macular degeneration is generally characterized by a gradual deterioration in central vision accompanied by structural pathologic conditions such as atrophy of the retinal pigment epithelium (RPE), accumulation of lipofuscin in the RPE, and regional loss of photoreceptors.1,2 Stargardt disease 3 (STGD3) is an early-onset autosomal dominant form of atrophic macular degeneration that results from a 5-bp deletion mutation, and possibly other mutations, in the ELOVL4 gene (elongation of very-long-chain fatty acids [LCFAs]), which maps to 6q14.3–6 The deletion causes a frameshift and loss of a 51-amino-acid fragment at the C terminus including a dilysine (KXKXX) targeting signal, creating a premature stop codon and leading to synthesis of an aberrant ELOVL4 peptide.5 The wild-type ELOVL4 protein is found in the endoplasmic reticulum (ER), which is the site of very-LCFA biosynthesis.7 However, the intracellular trafficking of the truncated form is defective and appears to be sequestered in aggresomes in a complex that also includes mislocalized wild-type ELOVL4.5–14 The importance of ELOVL4 protein is implied by its evolutionary conservation. Human ELOVL4 encodes a protein of 314 amino acids with approximately 35% amino acid identity to members of the ELO protein family in yeast,5 and orthologues have been identified in other species, including Caenorhabditis elegans, zebrafish, and chicken.15–18 The cellular function of ELOVL4 is unknown, but homology with yeast proteins suggests that it is an ER-bound transmembrane protein associated with LCFA synthesis.5 LC polyunsaturated fatty acids (PUFAs) from the n-6 and n-3 classes are essential for brain and retinal development and for visual function.19 An important role for docosahexaenoic acid (DHA) within the retina is suggested by its high levels and active conservation in this tissue.5,20,21 ELOVL4 is expressed in rod and cone photoreceptor cells and is probably involved in one of the elongation steps necessary for fatty acid biosynthesis.5,20–23 The essential role of ELOVL4 in polyunsaturated fatty acids (PUFA) synthesis is also suggested by preliminary evidence of improved visual function in human STGD3 disease with supplementation of DHA.24 Another recent study found that phenotypic severity in a family with STGD3 disease correlates with dietary fatty acid intake, and that a high dietary intake of DHA may partially ameliorate the maculopathy.25 We characterized heterozygous animals in a mouse model with a targeted deletion of Elovl4, to further understand the role of this gene in macular degeneration.

A 52-year-old subject harboring an RS1 gene W112C mutation presented with a prominent and asymmetric tapetal-like retinal sheen. Transient ERG responses were smaller and slower in the eye with the more extensive sheen, an association that, to our knowledge, had not been previously reported. An ON-pathway dysfunction explained the abnormalities of the transient but not those of the flicker ERGs. Although in vitro studies showed that the W112C mutant retinoschisin is present only in the cellular fraction and is not secreted, disease expression was remarkably mild, consistent with the notion of the existence of genetic and/or epigenetic disease modifiers.

To identify the gene responsible for a complex ocular phenotype of late-onset macular degeneration, long anterior zonules (LAZ), and elevated intraocular pressure (IOP) and to study its expression.Ocular examination, visual field, fluorescein angiography, and electrophysiology testing were performed. One affected individual was treated with vitamin A. DNA from 55 family members (UM:H389) was used for linkage, mapping, and mutation analysis. Linkage analysis of macular degeneration and LAZ phenotypes was performed independently. Mutations in candidate genes were screened by sequencing. mRNA expression of CTRP5 and MFRP, which are bicistronic genes, was studied by semiquantitative RT-PCR (qRT-PCR) in various human tissues. CTRP5 expression was also evaluated by in situ hybridization.Affected members had LAZ detectable by the third decade and/or macular degeneration by the fourth to fifth decade. A six-month treatment with vitamin A shortened dark adaptation considerably in one affected member. Both conditions mapped independently with zero recombination to 11q23, with maximum lod scores of 3.31 for macular degeneration and 5.41 for LAZ. The same CTRP5 missense mutation was identified in all affected individuals. Retinal pigment epithelium (RPE) and ciliary epithelium (CE) showed highest CTRP5 transcript expression, which was also true for MFRP. CTRP5 tissue expression was confirmed by in situ hybridization.A single locus at 11q23 is implicated in a complex ocular phenotype involving RPE and CE, tissues of neuroectodermal origin. All individuals with either LAZ and/or macular degeneration carry the same CTRP5 S163R mutation, which is transmitted in autosomal dominant manner.

PURPOSE. To describe hereditary macular degeneration observed in the cynomolgus monkey (Macaca fascicularis), which shares phenotypic features with age-related macular degeneration in humans, and to test the involvement of candidate gene loci by mutation screening and linkage analysis. METHODS. Ophthalmic examinations with fundus photography, fluorescein angiography (FA), indocyanine green angiography (IA), electroretinography (ERG), and histologic studies were performed on both affected and unaffected monkeys in the pedigree. The monkey orthologues of the human ABCA4, VMD2, EFEMP1, TIMP3, and ELOVL4 genes were cloned and screened for mutations by single-strand conformation polymorphism (SSCP) analysis or denaturing high-performance liquid chromatography (DHPLC) and direct sequencing in six affected and five unaffected monkeys from the pedigree and in six unrelated, unaffected monkeys. Subsequently, 13 human macular degeneration loci including these five genes were analyzed to test for linkage with the disease. Nineteen affected and seven unaffected monkeys in the pedigree were analyzed by using human microsatellite markers linked to the 13 loci. RESULTS. Yellowish white spots were observed in the macula and fovea centralis, and in some cases the spots scattered to the peripheral retina along the blood vessels. FA showed hyperfluorescence corresponding to the dots except in the foveola. No anomalies were found by IA and ERG. Histologic studies demonstrated that the spots were drusen. Mutation analysis of the ABCA4, VMD2, EFEMP1, TIMP3, and ELOVL4 genes identified a few sequence variants, but none of them segregated with the disease. Linkage analysis with markers linked to these five genes and an additional eight human macular degeneration loci failed to establish linkage. Haplotype analysis excluded the involvement of the 13 candidate loci for harboring the gene associated with macular degeneration in the monkeys. CONCLUSIONS. Significant homology was identified between monkey and human orthologues of the five macular degeneration genes. Thirteen loci associated with macular degeneration in humans or harboring macular degeneration genes were excluded as causal of early-onset macular degeneration in the monkeys. It is likely that none of these loci, but rather a novel gene, is involved in causing the observed phenotype in this monkey pedigree. (Invest Ophthalmol Vis Sci. 2005;46: 683‐691) DOI:10.1167/iovs.04-1031

Chorioretinal atrophy and choroidal neovascularization are sight-threatening complications that frequently result in permanent loss of central vision in patients with many macular disorders including age-related macular degeneration (AMD). The molecular pathogenesis of these severely disabling and poorly treatable conditions is not well understood. Recent evidence suggests that chorioretinal atrophy in patients with various retinal dystrophies can be traced to a wide array of mutations in photoreceptor-specific genes.1-4 Evidence tying choroidal neovascular disease to these same or other mutations in photoreceptor-specific genes has been weaker, although rare patients with familial age-related macular degeneration manifest both nonexudative atrophic and neovascular exudative phenotypes in association with sequence variants in the photoreceptor-specific gene ABCA4.5 Most other macular degenerative disorders studied to date with prominent neovascular exudative complications, including Sorsby dystrophy, Doyne honeycomb dystrophy, and Best vitelliform dystrophy, are caused by genes that are predominantly expressed in the retinal pigment epithelium and not in photoreceptors.6-9 Among dystrophies with neovascular features, Sorsby macular dystrophy10,11 stands out as the only late-onset autosomal dominant dystrophy in which choroidal neovascularization and disciform maculopathy are key defining clinical features, although atrophic features are also encountered. Mutations in the gene for tissue inhibitor of metalloproteinase (TIMP)-3, which is expressed primarily in the retinal pigment epithelium, were reported to cause this exudative disease.6 Most families and patients described with late-onset exudative phenotype either have been linked to a TIMP3 locus or carry a mutation in the TIMP3 gene, except for a family that we described earlier.12-14 We recently identified another large family (SUNY901) whose members have an unusual late-onset maculopathy with exudative and atrophic features. After excluding most of the previously described macular disease loci including the loci for exudative fundus dystrophies and AMD,5,7,15-17 we mapped the disease locus to a 9-centimorgan (cM) interval on chromosome 6. Mutation analysis of the RDS/peripherin, GUCA1A, and GUCA1B genes contained within the interval revealed a missense mutation resulting in a Tyr141Cys substitution in the RDS/peripherin gene product. Comparison of haplotypes between SUNY901 and a separate family (BCM-AD033) with the identical Tyr141Cys mutation suggests a possible common ancestral origin.18 Identification of Tyr141Cys establishes an association between an unusual exudative and nonexudative autosomal dominant late-onset macular degeneration with mutation in the photoreceptor-specific RDS/peripherin gene. Mutations in this essential outer segment structural gene have been associated with a wide array of central and peripheral human retinal dystrophies, but not typically the exudative phenotypes.19,20

PURPOSE. The gene elongation of very long chain fatty acids-4 (ELOVL4) has been shown to underlie phenotypically heterogeneous forms of autosomal dominant macular degeneration. In this study, the extent of evolutionary conservation and the existence and localization of retinal expression of this gene was investigated across a wide variety of species. METHODS. Southern blot analysis of genomic DNA and bioinformatic analysis using the human ELOVL4 cDNA and protein sequences, respectively, were performed to identify species in which ELOVL4 orthologues and/or homologues are present. Retinal RNA and protein extracts derived from different species were assessed by Northern hybridization and immunoblot techniques to assess evolutionary conservation of gene expression. Immunohistochemical analysis of tissue sections prepared from various mammalian retinas was performed to determine the distribution of ELOVL4 and homologous proteins within specific retinal cell layers. RESULTS. The existence of ELOVL4 sequence orthologues and homologues was confirmed by both Southern blot analysis and in silico searches of protein sequence databases. Phylogenetic analysis places ELOVL4 among a large family of known and putative fatty acid elongase proteins. Northern blot analysis revealed the presence of multiple transcripts corresponding to ELOVL4 homologues expressed in the retina of several different mammalian species. Conserved proteins were also detected among retinal extracts of different mammals and were found to localize predominantly to the photoreceptor cell layer within retinal tissue preparations. CONCLUSIONS. The ELOVL4 gene is highly conserved throughout evolution and is expressed in the photoreceptor cells of the retina in a variety of different species, which suggests that it plays a critical role in retinal cell biology.

ELOVL4, elongation factor of very long chain fatty acids-4, is known to be responsible for autosomal dominant macular degeneration and Stargardt-like macular degeneration. In this study, we cloned the monkey homologue of ELOVL4 and determined the cellular and tissue distribution of the gene product. Sequence analysis of the monkey ELOVL4 gene revealed a high degree of homology between human and monkey. The cloned full-length cDNA of monkey ELOVL4 encoded 314 amino acids, the same length as human and two amino acids longer than mouse. The monkey ELOVL4 conserved the characteristics typical of the super family of ELO enzymes involved in the metabolism of membrane-bound fatty acid elongation. Real-time quantitative PCR demonstrated that the monkey ELOVL4 gene was highly expressed in restricted tissue-specific fashion, not only in the retina but also in the skin (90% of retina) and thymus (111% of retina). Immunohistochemical analysis detected signals predominantly in the photoreceptor layer of the monkey retina.

In recent years the field of retinal gene discovery has experienced an explosion. An estimated 10,000-30,000 genes are thought to be expressed in the human retina (Shimizu-Matsumoto et al., 1997), with nearly 5,000 retinal transcripts catalogued from expressed sequence tag (EST) datasets (Bortoluzzi et al., 2000) and over 26,000 different transcripts represented in retina serial analysis of gene expression (SAGE) libraries (Sharon et al, 2002). A wide variety of methods have been developed and utilized to define novel retinal genes, particularly in an effort to identify genes targeted in human retinopathies. To date there are 144 diseases of the retina described that have been mapped to human chromosomal locations, and an additional 41 human disorders involving some form of retinal dystrophy for which the genetic map locations have yet to be determined (RetNet Retinal Information Networkhttp://www.sph.uth.tmc.edu/retnet/;Online Mendelian Inheritance in Man (OMIM)http://www.ncbi.nlm.nih.gov/omim/). Despite recent efforts to discover novel genes by analyzing increasingly greater volumes of information arising from human genomic sequence data organization and expression studies, the disease genes for less than one-half of these conditions have at present been cloned. While some of these disorders may be attributed to allelic heterogeneity, a significant number of genes that account for retinal degenerative phenotypes remain to be defined.

We have analyzed 145 RS families for mutations in the XLRS1 gene and have sought clinical correlation of phenotype severity with the type and location of mutation in a subset of these families. Some of the RS families exhibited consistency of either severe or mild clinical phenotypes in multiple affected male members. Intrafamilial variability of clinical phenotypes was encountered in affected males of several other RS families.

PURPOSE To present a case of retinal pattern dystrophy and the genetic analysis identifying the molecular basis of the disease. METHODS A 46-year-old man with a 6-year history of vision loss of the right eye. A clinical exam revealed pattern dystrophy of the retina bilaterally, with more involvement of the right eye. RESULTS Molecular diagnostic analysis of the retinal degeneration slow (RDS)/peripherin gene showed a novel change at nucleotide position 665 (G665C) that alters the amino acid at position 222 from cysteine to serine (C222S). CONCLUSION This study describes an RDS/peripherin mutation in a case of retinal pattern dystrophy, which is the first identification of this mutation to our knowledge.

Best vitelliform macular dystrophy (VMD2) is an autosomal dominant dystrophy with a juvenile age of onset. Mutations in the Bestrophin gene were shown in patients affected with VMD2. In a mutation study, we made three new and interesting observations. First, we identified possible mutation hotspots within the gene, suggesting that particular regions of the protein have greater functional significance than others. Second, we described a 2-bp deletion in a part of the gene where mutations have not previously been reported; this mutation causes a frameshift and subsequent premature termination of the protein. Finally, we have evidence that some mutations are associated with variable expression of the disease, suggesting the involvement of other factors or genes in the disease phenotype.

The Usher Syndromes are a group of autosomal recessive disorders characterized by congenital sensorineural deafness and progressive pigementory retinopathy. Several types of Usher syndromes (Ush) have been distinguished by age of onset, rate of progression and severity of clinical symptoms.

Purpose We identified and examined four generations of a family with coexisting autosomal dominant zonular cataracts and sutural opacities and sought to determine their genetic basis. Methods Twenty-four of the 48 members in the family were examined. Systemic and ocular histories were obtained, and a detailed ophthalmic examination was performed. From each individual, 20 ml of blood was drawn for linkage studies with microsatellite markers in regions to which zonular cataracts had previously been localized (chromosomes 1, 2, and 16). Results Individuals of the first generation were reportedly asymptomatic. Several members of the second generation had morphologically identical zonular cataracts. Affected members of the third generation showed morphologic heterogeneity, with the zonular opacity varying from a uniform lamella to a segregation of dots. A high degree of consanguinity in the second generation suggested recessive inheritance with a pseudodominant inheritance pattern. However, examination of one member of the asymptomatic first generation disclosed senile cataractous changes superimposed on a faint zonular cataract enclosing sutural opacities and a pulverulent fetal nucleus. The latter findings were reconfirmed to be present in affected members of all generations, suggesting an autosomal dominant mode of inheritance. Initial efforts at linkage analysis excluded the gene locus causing this cataract from the Duffy, haptoglobin, and gamma-crystallin regions. Conclusions The cataract in this family is both phenotypically and genetically distinct from previously described and mapped cataracts.

PURPOSE. To study the relationship between macular cone structure, fundus autofluorescence (AF), and visual function in patients with Stargardt disease (STGD). METHODS. High-resolution images of the macula were obtained with adaptive optics scanning laser ophthalmoscopy (AOSLO) and spectral domain optical coherence tomography in 12 patients with STGD and 27 age-matched healthy subjects. Measures of retinal structure and AF were correlated with visual function, including best-corrected visual acuity, color vision, kinetic and static perimetry, fundus-guided microperimetry, and full-field electroretinography. Mutation analysis of the ABCA4 gene was completed in all patients. RESULTS. Patients were 15 to 55 years old, and visual acuity ranged from 20/25-20/320. Central scotomas were present in all patients, although the fovea was spared in three patients. The earliest cone spacing abnormalities were observed in regions of homogeneous AF, normal visual function, and normal outer retinal structure. Outer retinal structure and AF were most normal near the optic disc. Longitudinal studies showed progressive increases in AF followed by reduced AF associated with losses of visual sensitivity, outer retinal layers, and cones. At least one disease-causing mutation in the ABCA4 gene was identified in 11 of 12 patients studied; 1 of 12 patients showed no disease-causing ABCA4 mutations. CONCLUSIONS. AOSLO imaging demonstrated abnormal cone spacing in regions of abnormal fundus AF and reduced visual function. These findings provide support for a model of disease progression in which lipofuscin accumulation results in homogeneously increased AF with cone spacing abnormalities, followed by heterogeneously increased AF with cone loss, then reduced AF with cone and RPE cell death.

Usher syndrome consists of a group of autosomal recessive diseases characterized by congenital sensorineural hearing loss and the progressively blinding disorder, retinitis pigmentosa. In 1914 Usher was the first to describe the hereditary nature of this syndrome (1). Usher syndrome is the most common cause of deaf-blindness, causing at least 50% of all reported cases of combined deafness and blindness in developed countries. Its prevalence in the general population ranges from 3 to 5 per 100,000 and estimates of gene frequency variy from 1/70 to 1/100 (2). The Diagnosis of Usher syndrome is generally made following ophthalmoscopic examination of congenitally deaf patients. In Usher syndrome patients patients retinitis pigmentosa is the most important ocular finding while sensorineural hearing loss is the most frequent non-ocular abnormality.

Objective To describe clinical molecular testing for hereditary retinal degenerations,highlighting results, interpretation, and patient education. Methods Mutation analysis of 8 retinal genes was performed by dideoxy sequencing.Pretest and posttest genetic counseling was offered to patients. The laboratory report listed results and provided individualized interpretation. Results A total of 350 tests were performed. The molecular basis of disease was determined in 133 of 266 diagnostic tests; the disease-causing mutations were not identified in the remaining 133 diagnostic tests. Predictive and carrier tests were requested for 9 and 75 nonsymptomatic patients with known familial mutations, respectively. Conclusions Molecular testing can confirm a clinical diagnosis, identify carrier status, and confirm or rule out the presence of a familial mutation in nonsymptomatic at-risk relatives. Because causative mutations cannot be identified in all patients with retinal diseases, it is essential that patients are counseled before testing regarding the benefits and limitations of this emerging diagnostic tool. Clinical Relevance The molecular definition of the genetic basis of disease provides a unique adjunct to the clinical care of patients with hereditary retinal degenerations.

In a prior study, a S163R mutation in the complement-1q tumor necrosis factor-related protein 5 (CTRP5/ C1QTNF5) was reported to be associated with early-onset long anterior zonules (LAZ) and late-onset retinal degeneration (L-ORD). The ocular tissues involved in the phenotype are the retinal pigment epithelium (RPE) in the posterior segment and ciliary epithelium (CE) and lens in the anterior segment. The purpose of this study was to characterize the spatial and temporal expression of the mouse Ctrp5 gene, determine tissue and subcellular localization, and study the effect of the S163R mutation.The expression of the Ctrp5 gene in the mouse was studied by quantitative (q)RT-PCR and in situ hybridization. CTRP5 protein expression and distribution were studied by Western blot analysis, immunohistochemistry, and immunoelectron microscopy. Cellular location of wild-type and mutant CTRP5 in MDCK and COS-7 cells was determined by immunofluorescence and immunoblot analysis.A significant level of Ctrp5 expression was detected in the adult mouse in the ciliary body (CB) and RPE, and expression started at a very early stage of embryogenesis. Immunohistochemical analysis showed CTRP5 protein in the apical processes of the RPE and forming a hexagonal lattice associated with the RPE lateral membranes. In the ciliary body, CTRP5 was localized to the apical aspects of the CE, the region between the bilayered ciliary epithelial cells. The membrane association of CTRP5 in the RPE and CE was further confirmed by immunoelectron microscopy. Furthermore, cultured cells were used to show that the CTRP5 is a secretory protein and that its secretion is impaired by the S163R mutation.CTRP5, a secretory and membrane-associated protein, is localized to the lateral and apical membranes of the RPE and CB. Impaired secretion of the mutant protein may underlie the pathophysiology of L-ORD and LAZ.

PURPOSE. Complement factor H (CFH) is a component of the mammalian complement system, which regulates the alternative pathway of complement activation and protects the host cell from inappropriate complement activation. CFH is a key regulator of innate immunity, and CFH deficiency leads to membranoproliferative glomerulonephritis type II. A variation in human CFH, Y402H, has been shown to be associated with an increased risk for age-related macular degeneration. The authors describe studies on the spatial and temporal expression of the CFH gene and localization of this protein in ocular tissues to gain insight into its role in the eye. METHODS. CFH expression in human and mouse tissues was studied by quantitative RT-PCR and Western blot analysis, and localization of CFH was studied by immunohistochemical analysis followed by fluorescence microscopy. RESULTS. In human and mouse, CFH expression was found to be similar to the highest level of expression in the liver. In ocular tissue, CFH was detected in the distalmost optic nerve (3 mm) cut from the scleral surface of the eyeball, sclera, RPE-choroid, retina, lens, and ciliary body. In mouse, Cfh expression was observed from early embryonic stages, and in the eye its expression increased with age. CONCLUSIONS. A significant level of CFH expression is maintained in different ocular tissues during development and aging. Sustained high levels of CFH expression in eye tissues suggest that this protein may play a role in protecting these tissues from indiscriminate complement activation and inflammatory insult.

PURPOSE. To develop and apply microarray-based resequencing technology to detect sequence alterations in multiple autosomal recessive retinal disease genes on a single high-throughput platform. METHODS. Oligonucleotides corresponding to both strands of the target exons and the flanking intron sequences of 29,214 bp from 11 genes associated with autosomal recessive retinitis pigmentosa (arRP) were tiled on 20 25-m microarrays (arRP-I arrays). A total of 155 exons were amplified from 35 arRP patient DNA samples, with each sample being sequenced on an arRP-I chip by hybridization. RESULTS. With the arRP-I arrays, 97.6% of the tiled sequence were determined with more than 99% accuracy and reproducibility. Of the 2.4% unread sequence, 89.5% involved stretches of G or C. In analyzing the 903,140-bp sequence from the 35 patient samples, 506 sequence changes have been detected in which 386 are previously reported alterations, and 120 are novel. In addition to four known causative mutations, six novel sequence changes that are potentially pathogenic were observed. Additional analysis is needed to determine whether these changes are responsible for arRP in these patients. CONCLUSIONS. The use of microarray for sequencing is a novel approach, and the arRP-I chip is the first successful application of this technology for determining sequence alteration in multiple disease-related genes. These arrays can be used for highthroughput genotyping of patients with relevant retinal conditions. In addition, these arrays offer a unique opportunity to interrogate complex patterns of inheritance due to the involvement of more than one gene by screening multiple genes on a single platform. (Invest Ophthalmol Vis Sci. 2005;46

Objective To describe the clinical phenotype of juvenile X-linked retinoschisis in patients with different mutations in the XLRS1 gene. Methods Thirty patients with 7 different XLRS1 mutations were examined. The genotype was determined by molecular genetics, which identified 6 known and 1 novel mutation (exon 5, 489 G→T). Ophthalmologic examination included full-field electroretinogram (ERG) recordings. Results The fundus appearance showed marked variations between, as well as within, families with different XLRS1 mutations. The ERG demonstrated typical reduction of B-wave amplitude, with relative A-wave preservation, causing a reduced B-A ratio in all affected males. The implicit time of the 30-Hz flicker ERG was prolonged in all patients examined. In a large family with a deletion of exon 1 and the promoter region, 12 affected males showed a phenotype ranging from moderate to severe vision impairment and a broad range of ERG abnormality, suggesting that additional factors may contribute to the disease severity. Conclusions Juvenile retinoschisis shows a wide variability in the phenotype between, as well as within, families with different genotypes. The ERG findings show reduced B-A ratios of dark-adapted recordings and prolonged implicit times of 30-Hz flicker response, which provide a useful clinical marker to confirm the clinical diagnosis. Clinical Relevance This study describes the wide variability in the phenotype in patients with juvenile retinoschisis and different mutations in the XLRS1 gene. The study emphasizes the importance of complementing the ophthalmologic examination with full-field ERG and molecular genetics in boys with visual failure of unknown etiology to determine the diagnosis early in the course of the disease.

Objective To describe the ophthalmic and genetic findings of a large kindred (UM:h389) with autosomal dominant hemorrhagic macular dystrophy. Methods The disease state of family members was documented by dilated fundus examination, electroretinography, color vision tests, fluorescein angiography, measurement of visual fields, biomicroscopy, gonioscopy, and intraocular pressure measurement. Linkage and haplotype analyses were carried out with markers flanking the Sorsby fundus dystrophy TIMP3 (tissue inhibitor of metalloproteinase 3) gene locus, and mutation analysis was carried out by screening exon 5 of the TIMP3 gene. Results This 4-generation pedigree with autosomal dominant hemorrhagic macular degeneration has visual symptoms beginning in the sixth decade of life. Several family members developed choroidal neovascular membrane formation in the macula of both eyes. The phenotype overlaps that of Sorsby fundus dystrophy. Some of the affected members have unusual zonularlike radial striations on the anterior lens capsule surface, and glaucoma or ocular hypertension has developed in 2 of them. Involvement of the TIMP3 gene was excluded by linkage, haplotype, and mutation analyses. Conclusions The phenotype of this family with autosomal dominant macular dystrophy overlaps that of Sorsby fundus dystrophy. Exclusion of the TIMP3 gene in this family indicates genetic heterogeneity for hemorrhagic macular dystrophy. Anterior segment anomalies may occur with this condition, but cosegregation has not yet been established. Clinical Relevance This study broadens the spectrum of hemorrhagic macular dystrophy by identifying a family in which the TIMP3 gene is not involved. Once the gene is cloned, we are eager to learn whether this gene may be involved in age-related macular degeneration.

Most of the legumes and cereals were found to contain lectins. Hemagglutinating activity differed when assayed at different temperatures (4, 18 and 37°C). Considerable variation was observed in hemagglutinating activity of different cultivars of a species. Lectin and β-oxalyl-amino-alanine (BOAA) content of different cultivars of Lathyrus sativus showed significant variation whereas tannin, trypsin inhibitor and total protein content did not vary much. Cooked foods were found to have lower levels of antinutrients as compared to raw foods. Dry heating did not inactivate the antinutrients. Soaking the seeds in water and processing effectively removed the antinutrients.