Autosomal recessive ocular albinism associated with a functionally significant tyrosinase gene polymorphism.

Autosomal recessive ocular albinism (AROA) is a disorder characterized by reduced pigmentation of the retina and iris, hypoplastic fovea, variably reduced visual acuity and nystagmus. Pigmentation of the skin and hair is normal, but is usually slightly lighter than in unaffected sibs. We analysed 12 unrelated patients with AROA, and found that two had abnormalities of the tyrosinase (TYR) gene. These two patients were each a compound heterozygote for a different pathologic mutant allele and an allele containing a 'normal' polymorphism, Arg402Gln, which results in a tyrosinase polypeptide with reduced thermal stability. In these patients, AROA thus appears to represent a clinically mild form of OCA1, with a fixed visual deficit resulting from low tyrosinase activity during fetal development but with normal pigmentation of the skin and hair postnatally.

Dystrophin expression in the human retina is required for normal function as defined by electroretinography.

We have studied retinal function by electroretinography in five Becker and six Duchenne muscular dystrophy patients. All had abnormal electroretinograms with a markedly reduced amplitude for the b-wave in the dark-adapted state. Using three antisera raised to different domains of dystrophin, we identified dystrophin in the outer plexiform layer of human retina. The retinal dystrophin is present in multiple isoforms as the result of alternative splicing. The localization of dystrophin to the outer plexiform layer coincident with the abnormal b-wave suggests that dystrophin is required for normal retinal electrophysiology.

Mutations in NYX, encoding the leucine-rich proteoglycan nyctalopin, cause X-linked complete congenital stationary night blindness.

During development, visual photoreceptors, bipolar cells and other neurons establish connections within the retina enabling the eye to process visual images over approximately 7 log units of illumination. Within the retina, cells that respond to light increment and light decrement are separated into ON- and OFF-pathways. Hereditary diseases are known to disturb these retinal pathways, causing either progressive degeneration or stationary deficits. Congenital stationary night blindness (CSNB) is a group of stable retinal disorders that are characterized by abnormal night vision. Genetic subtypes of CSNB have been defined and different disease actions have been postulated. The molecular bases have been elucidated in several subtypes, providing a better understanding of the disease mechanisms and developmental retinal neurobiology. Here we have studied 22 families with 'complete' X-linked CSNB (CSNB1; MIM 310500; ref. 4) in which affected males have night blindness, some photopic vision loss and a defect of the ON-pathway. We have found 14 different mutations, including 1 founder mutation in 7 families from the United States, in a novel candidate gene, NYX. NYX, which encodes a glycosylphosphatidyl (GPI)-anchored protein called nyctalopin, is a new and unique member of the small leucine-rich proteoglycan (SLRP) family. The role of other SLRP proteins suggests that mutant nyctalopin disrupts developing retinal interconnections involving the ON-bipolar cells, leading to the visual losses seen in patients with complete CSNB.

Mutation of a nuclear receptor gene, NR2E3, causes enhanced S cone syndrome, a disorder of retinal cell fate.

Hereditary human retinal degenerative diseases usually affect the mature photoreceptor topography by reducing the number of cells through apoptosis, resulting in loss of visual function. Only one inherited retinal disease, the enhanced S-cone syndrome (ESCS), manifests a gain in function of photoreceptors. ESCS is an autosomal recessive retinopathy in which patients have an increased sensitivity to blue light; perception of blue light is mediated by what is normally the least populous cone photoreceptor subtype, the S (short wavelength, blue) cones. People with ESCS also suffer visual loss, with night blindness occurring from early in life, varying degrees of L (long, red)- and M (middle, green)-cone vision, and retinal degeneration. The altered ratio of S- to L/M-cone photoreceptor sensitivity in ESCS may be due to abnormal cone cell fate determination during retinal development. In 94% of a cohort of ESCS probands we found mutations in NR2E3 (also known as PNR), which encodes a retinal nuclear receptor recently discovered to be a ligand-dependent transcription factor. Expression of NR2E3 was limited to the outer nuclear layer of the human retina. Our results suggest that NR2E3 has a role in determining photoreceptor phenotype during human retinogenesis.

Polymorphisms in C2, CFB and C3 are associated with progression to advanced age related macular degeneration associated with visual loss.

BACKGROUND: Age related macular degeneration (AMD) is a leading cause of blindness. AMD is a complex disorder caused by genetic and environmental factors in which single nucleotide polymorphisms (SNPs) in the genes CFH and LOC387715/HTRA1/ARMS2 have prognostic importance for progression to advanced AMD (with visual loss). CFH may also have a pharmacogenetic role by affecting treatment response to widely used nutritional supplements. This paper examines other AMD susceptibility genes to determine if these genotypes influenced disease progression and treatment response. METHODS: Three cohorts, totalling 3137 individuals, were genotyped for SNPs in 13 genes previously published to be associated with advanced AMD (other than CFH and LOC387715/ARMS2/HTRA1). Those genes found associated were then evaluated for their involvement in disease progression. Interactions between the genes and with AREDS (Age-Related Eye Disease Study) nutritional supplements were investigated. RESULTS: Positive independent associations were noted in SNPs in the genes C2 (p = 0.0001, odds ratio (OR) 0.35, 95% confidence interval (CI) 0.2 to 0.6), CFB (p = 0.0001, OR 0.35, 95% CI 0.2 to 0.6), C3 (p = 0.0001, OR 3.91, 95% CI 1.94 to 7.88), APOE (epsilon4, p = 0.01, OR 0.50, 95% CI 0.29 to 0.86) and VEGFA (p = 0.01, OR 2.23, 95% CI 1.06 to 4.68). C2/CFB and C3 were independently related to progression from early/intermediate to advanced AMD with OR 0.32 (95% CI 0.14 to 0.73) and 3.32 (95% CI 1.46 to 7.59), respectively. Gene-gene and pharmacogenetic interactions were not observed. No preferential associations were observed with geographic atrophy or choroidal neovascularisation. CONCLUSION: This study provides insights into the genetic pathogenesis of AMD. Five genes have now been shown to be independently involved in progression from intermediate disease (before vision loss has occurred) to advanced disease in which blindness is frequent.

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.

Genetic and phenotypic heterogeneity in pattern dystrophy.

BACKGROUND: The pattern dystrophies (PD) represent a clinically heterogeneous family of inherited macular diseases frequently caused by mutations in the peripherin/RDS gene. Most previous studies have detailed the clinical findings in single families, making it difficult to derive data from which progression and visual outcome can be generalised. METHODS: Families were ascertained and clinically evaluated including angiography and electrophysiology where appropriate. RESULTS: In each of the six families with autosomal dominant PD, a mutation in the peripherin/RDS gene was identified, including a novel Cys250Phe variant. These data suggest that the condition is characterised by the accumulation of yellow to grey subretinal flecks, followed by pigmentary change accompanied by patches of chorioretinal atrophy. Subsequently, 50% (16/32) of individuals with PD developed poor central vision because of chorioretinal geographic atrophy or subretinal neovascularisation. The risk of these complications appears to increase with age. CONCLUSION: PD should not necessarily be considered a benign condition. Instead, patients should be counselled that there is a significant chance of losing central vision in their later years. Some elderly patients with probands showing PD may be misdiagnosed with age related macular degeneration owing to the phenotypic similarities between these conditions in the advanced state.

Molecular genetic heterogeneity in autosomal dominant drusen.

OBJECTIVE: Autosomal dominant drusen is of particular interest because of its phenotypic similarity to age related macular degeneration. Currently, mutation R345W of EFEMP1 and, in a single pedigree, linkage to chromosome 6q14 have been causally related to the disease. We proposed to investigate and quantify the roles of EFEMP1 and the 6q14 locus in dominant drusen patients from the UK and USA. DESIGN: Molecular genetic analysis. PARTICIPANTS: Ten unrelated families and 17 young drusen patients. MAIN OUTCOME MEASURES: Exons 1 and 2 of EFEMP1 were characterised by 5' rapid amplification of cDNA ends and direct sequencing. Exons 1-12 of EFEMP1 were then investigated for mutation by direct sequencing. A HpaII restriction digest test was constructed to detect the EFEMP1 R345W mutation. Marker loci spanning the two dominant drusen linked loci were used to generate haplotype data. RESULTS: Only seven of the 10 families (70%) and one of the 17 sporadic patients (6%) had the R345W mutation. The HpaII restriction digest test was found to be a reliable and quick method for detecting this. No other exonic or splice site mutation was identified. Of the three families without EFEMP1 mutation, two were linked to the 2p16 region. CONCLUSIONS: EFEMP1 R345W accounts for only a proportion of the dominant drusen phenotype. Importantly, other families linked to chromosome 2p16 raise the possibility of EFEMP1 promoter sequence mutation or a second dominant drusen gene at this locus. Preliminary haplotype data suggest that the disease gene at the 6q14 locus is responsible for only a minority of dominant drusen cases.

The effect of age on human cone and rod ganzfeld electroretinograms.

The ganzfeld-evoked electroretinogram (ERG) is the most effective measure of the mass electrical response of cones and rods; however, all variables affecting the various components of the ERG have not been identified. The scotopic b-wave amplitude has been appreciated to be age-dependent, but little or no information exists in the literature regarding age dependency for other components. Linear regression analysis against age and multiple regression analysis against age and log intensity of the stimulus were performed on ERG responses from 24 prospectively normal subjects. Significant age dependency was found for scotopic rod-mediated b-wave amplitude, scotopic mixed rod-and cone-mediated bx-wave amplitude, and for photopic cone-mediated and b-wave amplitude. No significant age correlation was found for dark-adapted cone a-wave amplitude, scotopic cone-mediated x-wave amplitude, scotopic a-wave amplitude from mixed rod and cone responses to bright stimuli, or for implicit times for any scotopic or photopic responses. These findings indicate that for clinical patient evaluation, age-corrected normal ranges derived from linear or multiple regression coefficients should be used for rod- and cone-mediated b-wave response, whereas normal ranges derived from mean and standard deviation are more appropriate for scotopic a-wave amplitude, scotopic cone-mediated x-wave amplitude, and for all implicit times.

A histopathologic study of a choroideremia carrier.

We have examined eyes from a heterozygote (carrier) of choroideremia, an X-linked disease. Gross examination revealed irregular pigmentation at the level of the retinal pigment epithelium (RPE) except at the posterior pole, and islands of well defined depigmentation of 1-4 mm in diameter in the midperiphery. The optic nerve and retinal blood vessels appeared normal, and there was minimal pigment migration into the retina. Histopathologic examination showed normal photoreceptors in the posterior and anterior fundus, but the outer segments were short or absent in much of the equatorial region. Little gliosis was noted in areas of retinal atrophy. The RPE was abnormal, with irregular thickness and pigmentation associated with variable lipofuscin content from one RPE cell to another, as shown by fluorescence microscopy. There were areas of profound atrophy in the equatorial region, with abrupt transitions between relatively normal RPE and photoreceptors, and retina devoid of RPE and photoreceptors. Bruch's membrane was thickened to a greater extent than is common in age-related change. The choriocapillaris was normal in areas with normal photoreceptors, except for widening of the intercapillary pillars. In those regions with abnormal photoreceptors, choroidal capillaries were fewer in number, had reduced luminal diameter, and fenestrae were sparse. In some areas of intense atrophy, there were no choroidal capillaries. The findings are compatible with the primary defect residing in the RPE. The Lyon hypothesis of X-chromosome inactivation and mosaicism could explain the irregularity of change and areas of intense atrophy, but abrupt demarcation between grossly abnormal, and relatively well preserved retina also occurs in hemizygotes (affected males).

Sperm abnormalities in retinitis pigmentosa.

PURPOSE: To determine the fatty acid composition of erythrocytes and sperm, along with the functional characteristics of sperm, in patients with retinitis pigmentosa. Sperm and retinal cells share important homologies. Both are rich in the highly polyunsaturated fatty acid, docosahexaenoic acid (DHA, 22:6[n-3]), and both contain a structural component called the axoneme. Low concentrations of DHA in the retina of monkeys are known to cause visual impairment. Because blood levels of DHA in retinitis pigmentosa patients are less than normal, reduced DHA in the retina might contribute to the visual impairment characteristic of this disease. This study was conducted on the hypothesis that the sperm of retinitis pigmentosa patients might be abnormal and that these abnormalities might infer similar lipid and structural abnormalities of the retina. METHODS: The lipid composition of erythrocytes and sperm (fatty acids and sterols) and sperm function were analyzed in 26 patients with retinitis pigmentosa and in 8 healthy men. RESULTS: The sperm of patients with retinitis pigmentosa had a much lower DHA concentration, a lower desmosterol-to-cholesterol ratio, reduced motility, abnormal structure, and lower sperm counts compared with that in normal subjects. Usher's II patients exhibited the most pronounced reductions of DHA in sperm. Sperm DHA concentration was positively correlated to sperm motility, to sperm count, and to the desmosterol-to-cholesterol ratio. Lower erythrocyte DHA was also observed in retinitis pigmentosa patients. CONCLUSIONS: These results indicate that the sperm of patients with retinitis pigmentosa, particularly those with Usher's II, have an abnormal lipid composition that is associated with reduced motility. The possibility exists that these patients might have similar abnormalities in the DHA biochemistry of the retina. Sperm biochemistry and function may be a marker for this disease. A clinical trial of DHA in retinitis pigmentosa is suggested for future study.

A new locus for autosomal dominant congenital cataracts maps to chromosome 3.

PURPOSE: To map a gene for cataracts in a family with congenital nuclear and sutural cataracts and to examine candidate genes in the linked region. METHODS: A large family with autosomal dominant congenital nuclear and sutural cataracts was identified and characterized. A genome-wide screen was conducted with a set of markers spaced at 10- to 15-cM intervals, and linkage was assessed using standard LOD score analysis. RESULT: Fifteen (15) affected individuals were identified. This form of congenital cataracts maps to a 12-cM region on chromosome 3q21.2-q22.3 between markers D3S3674 and D3S3612, with a maximum multipoint LOD score of 6.94 at D3S1273. The crystallin gene, CRYGS, was excluded as a candidate gene for this locus. CONCLUSIONS: There are now more than 12 different genetic loci that cause congenital cataracts. The most recent locus to be identified is on chromosome 3q21.2-q22.3, in a family with congenital nuclear and sutural cataracts.

Expression defect of ornithine aminotransferase gene in gyrate atrophy.

A generalized deficiency in the mitochondrial enzyme, ornithine aminotransferase (OAT: EC 2.6.1.13), is the hallmark of gyrate atrophy (GA), a hereditary degenerative disease of the choroid and retina of the eye that leads to blindness. A human OAT cDNA, previously constructed and characterized in our laboratory, and anti-human OAT antibody were used as probes to examine the OAT gene, mRNA and protein of GA patients. A blot analysis of the genomic DNAs, RNAs and proteins of 14 GA patients identified a case with a partial heterozygous deletion of the functional OAT gene located on chromosome 10, no detectable OAT mRNA, and a barely detectable level of OAT antibody-reactive protein. The rest of the cases showed grossly normal OAT gene, mRNA, and variably reduced levels of OAT protein. A restriction fragment length polymorphism (RFLP) was identified in the functional OAT gene sequence with EcoRI which may be useful for prenatal diagnosis of GA. RFLPs were also identified in the OAT-related gene sequences located on the X chromosome with Hind III and Pst I which may potentially show linkage to X-linked retinitis pigmentosa locus. The finding of an OAT gene, mRNA, and protein defect in a GA case constitutes the first real demonstration of the molecular genetic defect of OAT in GA.

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.

mdxCv3 mouse is a model for electroretinography of Duchenne/Becker muscular dystrophy.

PURPOSE: To identify an animal model for the abnormal scotopic electroretinogram found in a majority of Duchenne and Becker muscular dystrophy patients. METHODS: Ganzfeld electroretinograms were recorded in dark-adapted normal C57BL/6 mice, and two strains of mice with different X-linked muscular dystrophy mutations (mdx and mdxCv3). Responses for the right eye were averaged and the amplitudes and implicit times of the a-wave and b-wave were measured. The electroretinogram was digitally filtered to extract the oscillatory potentials. Statistical analyses included one-way analysis of variance and the Scheffé S test. RESULTS: While the electroretinogram in mdx was normal, in mdxCv3 the scotopic b-wave was markedly reduced and the oscillatory potentials were delayed, similar to changes observed in Duchenne and Becker muscular dystrophy patients. Some of the mdxCv3 animals demonstrated negative configuration electroretinograms, with the b-wave amplitude reduced compared to that of the a-wave. CONCLUSIONS: Abnormalities found in the electroretinograms of Duchenne and Becker muscular dystrophy patients led to the identification of dystrophin in human retina and the discovery that dystrophin is required for normal retinal electrophysiology. These results indicate that mdxCv3 is a model for elucidating the role of dystrophin in retina and suggest that dystrophin isoforms, consisting of only the C-terminal domains of the full-length protein, may be important to the development of normal retinal electrical potentials.

Allelic variation in the VMD2 gene in best disease and age-related macular degeneration.

PURPOSE: To assess the allelic variation of the VMD2 gene in patients with Best disease and age-related macular degeneration (AMD). METHODS: Three hundred twenty-one AMD patients, 192 ethnically similar control subjects, 39 unrelated probands with familial Best disease, and 57 unrelated probands with the ophthalmoscopic findings of Best disease but no family history were screened for sequence variations in the VMD2 gene by single-strand conformation polymorphism (SSCP) analysis. Amplimers showing a bandshift were reamplified and sequenced bidirectionally. In addition, the coding regions of the VMD2 gene were completely sequenced in six probands with familial Best disease who showed no SSCP shift. RESULTS: Forty different probable or possible disease-causing mutations were found in one or more Best disease or AMD patients. Twenty-nine of these variations are novel. Of the 39 probands with familial Best disease, mutations were detected in all 39 (33 by SSCP and 6 by DNA sequencing). SSCP screening of the 57 probands with a clinical diagnosis of Best disease but no family history revealed 16 with mutations. Mutations were found in 5 of 321 AMD patients (1.5%), a fraction that was not significantly greater than in control individuals (0/192, 0%). CONCLUSIONS: Patients with the clinical diagnosis of Best disease are significantly more likely to have a mutation in the VMD2 gene if they also have a positive family history. These findings suggest that a small fraction of patients with the clinical diagnosis of AMD may actually have a late-onset variant of Best disease, whereas at the same time, a considerable fraction of isolated patients with the ophthalmoscopic features of Best disease are probably affected with some other macular disease.

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.

Screening of the gene encoding the alpha'-subunit of cone cGMP-PDE in patients with retinal degenerations.

PURPOSE: To screen the exons of the gene encoding the alpha'-subunit of cone cyclic guanosine monophosphate (cGMP>phosphodiesterase (PDE6C) for mutations in a group of 456 unrelated patients with various forms of inherited retinal disease, including cone dystrophy, cone-rod dystrophy, macular dystrophy, and simplex/multiplex and autosomal recessive retinitis pigmentosa. METHODS: The 22 exons of the PDE6C gene were screened for mutations either by denaturing gradient gel electrophoresis and single-strand conformation polymorphism electrophoresis (SSCP) or by SSCP alone; variants were sequenced directly. RESULTS: Although many sequence variants were found, none could be associated with disease. CONCLUSIONS: The results show that PDE6C was not the site of the amutations responsible for the types of inherited retinal degenerations analyzed in the large population of patients 'in the present study. The types of degeneration included those that predominantly affect cone-mediated function (cone and cone-rod dystrophies) or rod-mediated function (retinitis pigmentosa) or that have a predilection for disease in the macula (macular dystrophies).

An analysis of allelic variation in the ABCA4 gene.

PURPOSE: To assess the allelic variation of the ATP-binding transporter protein (ABCA4). METHODS: A combination of single-strand conformation polymorphism (SSCP) and automated DNA sequencing was used to systematically screen this gene for sequence variations in 374 unrelated probands with a clinical diagnosis of Stargardt disease, 182 patients with age-related macular degeneration (AMD), and 96 normal subjects. RESULTS: There was no significant difference in the proportion of any single variant or class of variant between the control and AMD groups. In contrast, truncating variants, amino acid substitutions, synonymous codon changes, and intronic variants were significantly enriched in patients with Stargardt disease when compared with their presence in subjects without Stargardt disease (Kruskal-Wallis P < 0.0001 for each variant group). Overall, there were 2480 instances of 213 different variants in the ABCA4 gene, including 589 instances of 97 amino acid substitutions, and 45 instances of 33 truncating variants. CONCLUSIONS: Of the 97 amino acid substitutions, 11 occurred at a frequency that made them unlikely to be high-penetrance recessive disease-causing variants (HPRDCV). After accounting for variants in cis, one or more changes that were compatible with HPRDCV were found on 35% of all Stargardt-associated alleles overall. The nucleotide diversity of the ABCA4 coding region, a collective measure of the number and prevalence of polymorphic sites in a region of DNA, was found to be 1.28, a value that is 9 to 400 times greater than that of two other macular disease genes that were examined in a similar fashion (VMD2 and EFEMP1).