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Purpose Missense mutations in the splicing factor gene PRPF31 cause a dominant form of retinitis pigmentosa (RP11) with reduced penetrance. Missense mutations in PRPF31 have previously been shown to cause reduced protein solubility, suggesting insufficiency of functional protein as the disease mechanism. Here we examine in further detail the effect of the A216P mutation on splicing function. Methods Splicing activity was assayed using an in vivo assay in transfected mammalian cells with rhodopsin (RHO) and transducin (GNAT1) splicing templates. Pull-down assays were used to study the interaction between PRPF31 and one of its cognate partners in the spliceosome, PRPF6. Results Splicing of RHO intron 3 and GNAT1 introns 3–5 mini-gene templates was inefficient with both spliced and unspliced products clearly detected. Assays using the RHO minigene template revealed a direct negative effect on splicing efficiency of the mutant. However, no effect of the mutation on splicing efficiency could be detected using the longer GNAT1 minigene template or using a full-length RHO transcript, splicing of which had an efficiency of 100%. No unspliced RHO transcripts could be detected in RNA from human retina. Pull-down assays between PRPF31 and PRPF6 proteins showed a stronger interaction for the mutant than wild type, suggesting a mechanism for the negative effect. Conclusions Splicing of full-length RHO is more efficient than splicing of the minigene, and assays using a full-length template more accurately mimic splicing in photoreceptors. The RP11 missense mutations exert their pathology mainly via a mechanism based on protein insufficiency due to protein insolubility, but there is also a minor direct negative effect on function.

PURPOSE. Myopia is a common complex trait that affects up to 60% of some populations. Its development is influenced by multiple genes and environmental factors. PAX6 and SOX2 are genes with fundamental roles in ocular growth and development, and they have been linked with myopia in a recent linkage study. The authors investigated the roles of PAX6 and SOX2 in common myopia as part of a broader association study of refractive error. METHODS. Five hundred ninety-six persons from the 1958 British Birth Cohort, a nationally representative population, were randomly selected from the outer tertiles of the refractive error (RE) distribution and were genotyped using 25 tagSNPs across PAX6 and 3 tagSNPs across SOX2 and their putative control regions. This experiment had 80% power to exclude either gene contributing more than 10% of the variance of refractive error. RESULTS. All SNPs were in Hardy-Weinberg equilibrium, and the genotyping failure rate was less than 5%. Accounting for multiple testing, no significant association (P < 0.05) was found between any of the SNPs or haplotypes and refractive error. CONCLUSIONS. PAX6 and SOX2 are obvious candidates in RE genetic studies because of their biological roles and prior linkage studies. The present findings strongly suggest refractive error is not directly affected in this population by variants in either gene or by their known promoters/enhancers. The authors suggest that neither PAX6 nor SOX2 should be prioritized in the international search for genetic modifiers of refractive error. Their findings contribute to broader understanding of the pathophysiology of refractive error and highlight the critical role of replication in genetic research on complex disorders.

Retinitis pigmentosa (RP) is a group of retinal dystrophies characterised primarily by rod photoreceptor cell degeneration. Exhibiting great clinical and genetic heterogeneity, RP be inherited as an autosomal dominant (ad) and recessive (ar), X-linked (xl) and digenic disorder. RP25, a locus for arRP, was mapped to chromosome 6p12.1-q14.1 where several retinal dystrophy loci are located. A gene expressed in the retina, FAM46A, mapped within the RP25 locus, and computational data revealed its involvement in retinal signalling pathways. Therefore, we chose to perform molecular evaluation of this gene as a good candidate in arRP families linked to the RP25 interval. A comprehensive bioinformatic and retinal tissue expression characterisation of FAM46A was performed, together with mutation screening of seven RP25 families. Herein we present 4 novel sequence variants, of which one is a novel deletion within a low complexity region close to the initiation codon of FAM46A. Furthermore, we have characterised for the first time a coding tandem variation in the Caucasian population. This study reports on bioinformatic and moleculardata for the FAM46A gene that may give a wider insight into the putative function of this gene and its pathologic relevance to RP25 and other retinal diseases mapping within the 6q chromosomal interval.

To report clinical and genetic findings in a white British family with autosomal recessive cornea plana (CNA2) with a negative history for consanguinity. To look for evidence of a common ancestry with previously reported Finnish CNA2 patients by studying haplotypes.Clinical examination and direct sequencing of the keratocan (KERA) gene was performed in two siblings affected with CNA2 and one unaffected parent. We also studied 22 single nucleotide polymorphisms distributed in the KERA genomic region by direct sequencing in this family as well as in one additional Finnish patient with CNA2 and 24 white British control subjects.Both siblings had the homozygous c.740AG mutation leading to a p.N247S amino acid change originally reported as the founder mutation in 35 Finnish families. Genetic characterization of genomic regions surrounding the gene revealed large linkage disequilibrium, but the presence of shared extended haplotypes between affected individuals from Finland and the United Kingdom is consistent with a recent common ancestor.This is the first description of recessive cornea plana in a white British family and it is the second report on the p.N247S change in the KERA gene. Extended haplotype analysis suggests that the two geographically remote occurrences of the c.740AG mutation may have a common origin.

Leber congenital amaurosis (LCA) is the most early and severe form of all inherited retinal dystrophies, responsible for congenital blindness. The genetic heterogeneity of LCA has been accepted for a long time but it turned out to be largely higher than all odds. So far, 11genes have been mapped on human chromosomes and eight identified. i) the retinal specific guanylate cyclase gene (GUCY2D, retGC1; 17p13.1; LCA1; MIM 600179), ii) the gene encoding the 65-kD protein specific to the retinal pigment epithelium (RPE65; 1p31; LCA2; MIM180069), iii) the cone-rod homeobox-containing gene (CRX; 19q13.3; LCA7; MIM 60225), iv) the gene encoding the arylhydrocarbon receptor interacting protein-like 1 (AIPL1; 17p13.1; LCA; MIM 604392), v) the gene encoding the retinitis pigmentosa GTPase regulator-interacting protein 1 (RPGRIP1; 14q11; LCA6; MIM 605446), vi) the human homologue of the drosophila melanogater crumbs gene (CRB1; 1q31; LCA8; MIM 604210), vii) the gene encoding the tubby-like protein 1 (TULP1; 6q21.3; LCA10; MIM 602280), viii) the retinol dehydrogenase 12 (RDH12; 14q24; LCA11; MIM 608830), ix) LCA3 (14q24; MIM 604232), x) LCA5 (6q11-16; MIM 604537) and xi) LCA9 (1p36; MIM608553).

To evaluate mutations in the transforming-growth-factor-beta-induced (TGFBI) gene in patients of Czech origin with autosomal dominant corneal dystrophies.The coding sequence of the TGFBI gene was analysed in 22 affected Czech individuals from 7 apparently unrelated families. Comparison of phenotype to genotype was performed.A H626P mutation, previously only described in a family with a variant of lattice corneal dystrophy (LCD), was detected in one family with superficial geographic corneal opacities. Light microscopy of 2 samples obtained following either a prior superficial keratectomy or keratoplasty showed amyloid but no fuchsinophilic deposits. In a family with LCD type I, an R124C mutation was identified. The R124L mutation was shown to be causative of Reis-Bucklers corneal dystrophy in 2 families. A family with Thiel-Behnke corneal dystrophy exhibited an R555Q mutation. In 2 families with granular corneal dystrophy type I, the typical R555W change was identified.The phenotype of the family with the H626P mutation differed from the phenotype previously reported for this change.

Autosomal recessive congenital hereditary endothelial dystrophy (CHED2) is a severe and rare corneal disorder that presents at birth or shortly thereafter, characterized by corneal opacification and nystagmus. Recently the gene for CHED2 was identified and seven different mutations in the SLC4A11 gene were reported. Here, we report seven novel mutations and two previously identified mutations in families from India and the United Kingdom with recessive CHED. The novel changes include two nonsense (p.Trp240X; p.Gln800X) three missense (p.Glu143Lys; p.Cys386Arg; p.Arg755Trp) and two splice site mutations (c.2240+1GA; c.2437-1GA). Interestingly, the c.2398CT (p.Gln800X) and c.2437-1GA identified in two affected siblings represent the first compound heterozygous mutations in the SLC4A11 gene.

3 páginas, 2 figuras, 1 tabla.-- et al., Retinal photoreceptor dystrophies (RD) are a highly heterogeneous group of genetic disorders of the retina, representing the most frequently inherited form of visual handicap, affecting approximately 1.5 million people world wide. To date, more than 40 genetic loci have been implicated in RD. One of them, the CORD2 locus, for an autosomal dominant form of cone-rod dystrophy (CRD), maps to chromosome 19q and has previously been reported in a single large family of British origin. We now report a new family with severe early onset CRD, phenotypically very similar to the British family, which also maps to 19q, but is of Greek origin. Haplotype data of the Greek family showed no recombination between and including markers D19S219 and D19S246 and linkage analysis gave a lod score of 2.7 (at theta=0) with marker D19S412, confirming the data obtained in the British family., M Papaioannou is a fellow of the National Greek Scholarship Foundation. Drs D Bessant and A Payne are funded by the Medical Research Council of the UK (grant No G9301094).

To identify the disease-causing mutations in two large Bulgarian Romani (Gypsy) pedigrees: one with autosomal dominant retinitis pigmentosa (adRP) with partial penetrance and the other with severe X-linked RP (xlRP).Detailed clinical investigations were undertaken and genomic DNA was extracted from blood samples. DNA was analyzed by PCR amplification with gene-specific primers and direct genomic sequencing.Analysis of the complete coding sequence of PRPF31 in the adRP family led to the identification of a new heterozygous splice site mutation IVS6+1GT. RPGR mutation screening in affected male individuals in the X-linked RP family identified a hemizygous c.ORF15+652_653delAG mutation. Interestingly this mutation was found in a homozygous state in one severely affected female from the family.In this first report of molecular genetic analysis of retinitis pigmentosa in Romani families, we describe a novel PRPF31 mutation and present the first case of a homozygous mutation in the RPGR gene in a female individual.

PURPOSE. To determine ( 1) clinical features that distinguish maculopathy due to the R345W substitution in fibulin- 3 from other forms of inherited or early- onset drusen, ( 2) the phenotypic variability, and ( 3) the extent of retinal disease in those with a positive molecular diagnosis.METHODS. Affected individuals underwent ophthalmic examination, digital color fundus photography, fundus autofluorescence ( AF) imaging, and psychophysical testing with automated photopic and dark- adapted perimetry and fine matrix mapping. Blood samples were taken for DNA extraction and screening for the R345W mutation in fibulin- 3. Patients were subsequently divided into mutation- positive and - negative groups, to compare the identified phenotypic findings in these two sets of subjects.RESULTS. Twenty- nine subjects from 19 families were ascertained with inherited or early- onset drusen. Twenty- four ( 83%) subjects from 15 families were found to harbor the R345W fibulin- 3 mutation. Peripapillary deposition and a radial distribution of macular drusen were consistent, distinguishing signs in the mutation- positive group. Subretinal neovascular membrane ( SRNVM) was a rare occurrence, affecting only 1 of 48 eyes, whereas hyperpigmentation and atrophy of the retinal pigment epithelium ( RPE) were common in older mutationpositive patients. Increased AF corresponding to the drusen was detected in both the mutation- positive and - negative groups. The phenotype in the group of patients positive for the R345W mutation was extremely variable, with evidence of interocular, intrafamilial, and interfamilial variability in visual loss, natural history, ophthalmoscopic findings, autofluorescence imaging, and psychophysical data. The novel finding of nonpenetrance was observed in a 62- year- old asymptomatic, mutation- positive man. The findings from detailed perimetry performed on a subset of subjects were consistent with the presence of widespread retinal dysfunction not isolated to the macula.CONCLUSIONS. Marked inter- and intrafamilial variation associated with the fibulin- 3 R345W mutation in terms of retinal appearance, severity, progression, and nonpenetrance were identified. It was noted that SRNVM is a rare occurrence in R345W fibulin- 3 maculopathy. These findings are helpful for advice regarding prognosis and for genetic counseling. The findings established that the presence of peripapillary deposit is highly likely to indicate that a patient carries the R345W mutation.

Similar retinitis pigmentosa (RP) phenotypes can result from mutations affecting different rhodopsin regions, and distinct amino acid substitutions can cause different RP severity and progression rates. Specifically, both the R135L and R135W mutations (cytoplasmic end of H3) result in diffuse, severe disease (class A), but R135W causes more severe and more rapidly progressive RP than R135L. The P180A and G188R mutations (second intradiscal loop) exhibit a mild phenotype with regional variability (class B1) and diffuse disease of moderate severity (class B2), respectively. Computational and in vitro studies of these mutants provide molecular insights into this phenotypic variability.

To report a large deletion that encompasses more than 90% of PRPF31 gene and two other neighboring genes in their entirety in an adRP pedigree that appears to show only the typical clinical features of retinitis pigmentosa.To identify PRPF31 mutation in a dominant RP family (ADRP2) previously linked to the RP11 locus, the 14 exons of PRPF31 were screened for mutations by direct sequencing. To investigate the possibility of a large deletion, microsatellite markers near PRPF31 gene were analyzed by non-denaturing PAGE.Initial screening of PRPF31 gene in the ADRP2 family did not reveal an obvious mutation. A large deletion was however suspected due to lack of heterozygosity for nearly all PRPF31 intragenic single nucleotide polymorphysm (SNPs). In order to estimate the size of the deletion, SNPs and microsatellite markers spanning and flanking PRPF31 were analyzed in the entire ADRP2 family. Haplotype analysis with the above markers suggested a deletion of approximately 30 kb that included the putative promoter region of a novel gene OSCAR, the entire genomic content of genes NDUFA3, TFPT and more than 90% of PRPF31 gene. Sequence analysis of the region flanking the potential deletion showed a high presence of Alu elements implicating Alu mediated recombination as the mechanism responsible for this event.This mutation provides evidence that haploinsufficiency rather than aberrant function of mutated proteins is the cause of disease in these adRP patients with mutations in PRPF31 gene.

To identify the disease gene in five Spanish families with autosomal recessive retinitis pigmentosa (arRP) linked to the RP25 locus. Two candidate genes, EEF1A1 and IMPG1, were selected from the region between D6S280 and D6S1644 markers where the families are linked. The genes were selected as good candidates on the basis of their function, tissue expression pattern, and/or genetic data.A molecular genetic study was performed on DNA extracted from one parent and one affected member of each studied family. The coding exons, splice sites, and the 5' UTR of the genes were amplified by polymerase chain reaction (PCR). For mutation detection, direct sequence analysis was performed using the ABI 3100 automated sequencer. Segregation of an IMPG1 single nucleotide polymorphism (SNP) in all the families studied was analyzed by restriction enzyme digest of the amplified gene fragments.In total, 15 SNPs were identified of which 7 were novel. Of the identified SNPs, one was insertion, two were deletions, five were intronic, six were missense, and one was located in the 5' UTR. These changes, however, were also identified in unaffected members of the families and/or 50 control Caucasians. The examined known IMPG1 SNP was not segregating with the disease phenotype but was correlating with the genetic data in all families studied.Our results indicate that neither EEF1A1 nor IMPG1 could be responsible for RP25 in the studied families due to absence of any pathogenic variants. However, it is important to notice that the methodology used in this study cannot detect larger deletions that lie outside the screened regions or primer site mutations that exist in the heterozygous state. A role of both genes in other inherited forms of RP and/or retinal degenerations needs to be elucidated.

PURPOSE. Posterior polymorphous corneal dystrophy (PPCD) is an autosomal dominant disorder, affecting both the corneal endothelium and Descemet's membrane. In the Czech Republic, PPCD is one of the most prevalent corneal dystrophies. The purpose of this study was to determine the chromosomal locus of PPCD in two large Czech families, by using linkage analysis. METHODS. Linkage analysis was performed on 52 members of two Czech families with PPCD and polymorphic microsatellite markers and lod scores were calculated. The candidate gene VSX1 was also screened for mutations. RESULTS. Significant lod scores were obtained with microsatellite markers on chromosome 20. Linkage analysis delineated the Czech PPCD locus to a 2.7-cM locus on chromosome 20, region p11.2, between flanking markers D20S48 and D20S139, which excluded VSX1 as the disease-causing gene in both families. In addition, the exclusion of VSX1 was confirmed by sequence analysis. CONCLUSIONS. This study reports the localization of PPCD in patients of Czech origin to chromosome 20 at p11.2. Linkage data and sequence analysis exclude VSX1 as causative of PPCD in two Czech families. This refined locus for PPCD overlaps the congenital hereditary endothelial dystrophy (CHED1) disease interval, and it is possible that these corneal dystrophies are allelic.

7 páginas, 2 figuras, 5 tablas.-- et al., [Purpose]: To investigate the clinical features of subjects with glaucoma with the E50K mutation in the optineurin (OPTN) gene and to compare the onset, severity, and clinical course of these patients with a control group of subjects with glaucoma without this mutation. [Methods]: The phenotype of well-characterized subjects from Moorfields Eye Hospital, London, who had been identified as carrying the OPTN E50K mutation was examined. A wide range of structural, psychophysical, and demographic factors were then compared with those in a control group of subjects with glaucoma without this mutation. [Results]: Eleven subjects with glaucoma with the E50K mutation (nine in two families and two sporadic cases) were studied. All 11 subjects had normal tension glaucoma (NTG), with presenting and highest IOP of 15.3 ± 3.0 and 16.5 ± 2.5 mm Hg (±SD) on diurnal testing. Compared with 87 NTG control subjects who did not have this mutation, subjects with E50K presented at a younger age (40.8 ± 15 years, P = 0.0001) and had more advanced optic disc cupping (mean cup-disc ratio ± SD 0.86 ± 0.1, P = 0.001) and smaller neuroretinal rim area (±SD; 0.5 ± 0.28 mm2, P = 0.001) at diagnosis. The rate of filtration surgery performed for progressive visual field loss in those with and without the E50K mutation was 72.7% and 25.3%, respectively (P = 0.003), and all subjects with E50K were found to have progressing visual fields. In addition, seven E50K mutation-carrying individuals in two families (age range, 23–58 years) presented with normal optic discs and visual fields and, as yet, no signs of glaucoma. [Conclusions]: In this study, subjects with glaucoma who had the OPTN E50K mutation were found to have NTG that appeared to be more severe than that in a control group of subjects with NTG without this mutation. The findings emphasize the importance of early detection and treatment of glaucoma in such individuals, to minimize visual loss., Supported in part by the International Glaucoma Association, Moorfields Special Trustees, and the Glaucoma Research Foundation. MS and TR are supported by National Eye Institute Grants EY009947 and EY014959, GB by the RNIB and AHC by the IGA and the Bluff Field Charitable Trust.

8 páginas, 3 figuras, 1 tabla.-- et al., In this study the mechanism of nuclear importation of the splicing factor PRPF31 is examined and the impact of two disease-linked mutations, A194E and A216P, assessed. Using pull-down assays with GST-tagged importin proteins, we demonstrate that His-tagged PRPF31 interacts with importin β1 for translocation to the nucleus, with no requirement for importin α1. The A194E and A216P mutations have no affect on this interaction. Fluorescence recovery after photobleaching (FRAP) was used to estimate the rate of movement of EGFP-tagged PRPF31 into the nuclei of live cells. The kinetics indicated a two-component recovery process; a fast component with τ ∼ 6 s and a slow component with τ ∼ 80 s. The mutations affected neither component. We conclude that the two mutations have no negative effect on interaction with the nuclear importation machinery. Reduced mutant protein solubility resulting in an insufficiency of splicing activity in cells with a very high metabolic demand remains the most likely explanation for the disease pathology in ADRP patients., This work was supported by the British Retinitis Pigmentosa Society, the Wellcome Trust, Fight for Sight and by a Value in People award to SEW from University College London.

To identify the disease gene in 6 Spanish families with autosomal recessive retinitis pigmentosa linked to the RP25 locus, mutation screening of 4 candidate genes, KHDRBS2, PTP4A1, KIAA1411 and OGFRL1, was undertaken based on their expression or functional relevance to the retina. Twenty-six single nucleotide polymorphisms were identified, of which 14 were novel. Even though no pathological mutations were detected, these genes however remain as good candidates for other retinal degenerations mapping to the same chromosomal region.

Aims: To identify the underlying mutations in our British families and sporadic patients with different types of corneal dystrophies (CDs) and to establish a phenotype-genotype correlation. Methods: Twenty-nine patients, 9 sporadic and 20 patients from 7 families were subjected to both clinical and genetic examination. Slit lamp examination was performed for all patients who participated in the study to assess their corneal phenotype. Genomic DNA was extracted from 10 ml venous blood, and the BIGH3 gene was amplified exon by exon to perform heteroduplex analysis. Exons that displayed double bands were then analysed by direct bi-directional sequencing and restriction digest analyses. Results: Clinically our patients showed three distinct phenotypes of CD: 16 with Thiel-Behnke corneal dystrophy or corneal dystrophy of Bowman layer type 2 (CDB2), 8 with granular CD (GCD), and 5 with lattice CD type I (LCDI). Three different missense mutations have been detected in the coding region of BIGH3 gene, R555Q, in 16 CDB2 patients, R555W in 8 GCD patients, and R124C in 5 LCDI patients. These mutations were the same as to those previously reported in patients from other ethnic origins. Also,we identified seven nucleotide substitutions that did not change the amino acid sequence of the encoded protein of which four were novel. Conclusions: In our patients of British origin, each phenotype of CD has been linked to a particular point mutation of the BIGH3 gene. Our study also highlights the importance of codons 124 and 555 as mutation hot spots in the BIGH3 gene in the British population.

PURPOSE. To investigate the genetic basis of autosomal dominant vitreoretinochoroidopathy (ADVIRC), a rare, inherited retinal dystrophy that may be associated with defects of ocular development, including nanophthalmos. METHODS. A combination of linkage analysis and DNA sequencing in five families was used to identify disease-causing mutations in VMD2. The effect of these mutations on splicing was assessed using a minigene system. RESULTS. Three pathogenic sequence alterations in VMD2 were identified in five families with nanophthalmos associated with ADVIRC. All sequences showed simultaneous missense substitutions and exon skipping. CONCLUSIONS. VMD2 encodes bestrophin, a transmembrane protein located at the basolateral membrane of the RPE, that is also mutated in Best macular dystrophy. We support that each heterozygous affected individual produces three bestrophin isoforms consisting of the wild type and two abnormal forms: one containing a missense substitution and the other an inframe deletion. The data showed that VMD2 mutations caused defects of ocular patterning, supporting the hypothesized role for the RPE, and specifically VMD2, in the normal growth and development of the eye.

The human lens is a unique tissue continually evolving and ever increasing in size primarily formulated by placement of newly differentiated fibre cells in concentric lamellae. This is analogous to an onion skin arrangement. This differentiation triggers loss of both nucleus and other cell organelles. Furthermore, the main factors for maintaining lens clarity are the tight and ordered mutual binding of the fibre cells and the packing of their intracellular building blocks termed crystallins. It is known that lens transparency is a crucial trade-off between the concentration of these macromolecules and their hydration. Disruption of normal levels of hydration can lead to opacity. Cataract, opacification of the eye lens, is the commonest cause of blindness in the world. During infancy and early childhood it frequently results in visual impairment or blindness. In children, it can cause an irreversible amblyopia. Nearly half of all congenital cataracts are normally characterised as familial and they are perceived to be a key feature if not the predominant feature of about 200 genetic diseases.1–3 Recent research has paved the way for a better understanding of the underlying mechanisms of inherited forms of cataract. Inherited cataract is clinically heterogeneous, and, thus far, more than 14 distinct loci in humans have been identified, for 11 phenotypically distinct forms of autosomal dominant congenital cataracts (ADCC).4,5 Progress has been made in elucidating cataract causing mutations in human genes encoding the transparent intracellular lens proteins (crystallins), membrane gap junction proteins (connexins), water channel proteins (aquaporins), developmental transcription factors FOXE3 ,6 EYA1,7 PAX6 ,8 MAF ,9 and PITX3. 10 Based on this insight, it can be deduced that cataracts exhibit marked phenotypic and genotypic heterogeneity. Here we report a number of interesting findings about the PITX3 gene: (1) we have found mutations in …

Objectives: To describe the phenotype of Leber congenital amaurosis (LCA) in 26 probands with mutations in aryl hydrocarbon receptor interacting protein-like 1 protein (AIPL1) and compare it with phenotypes of other LCA-related genes. To describe the electroretinogram (ERG) in heterozygote carriers. Methods: Patients with AIPL1-related LCA were identified in a cohort of 303 patients with LCA by polymerase chain reaction single-strand confirmational polymorphism mutation screening and/or direct sequencing. Phenotypic characterization included clinical and ERG evaluation. Seven heterozygous carrier parents also underwent ERG testing. Results: Seventeen homozygotes and 9 compound heterozygotes were identified. The W278X mutation was most frequent (48% of alleles). Visual acuities ranged from light perception to 20/400. Variable retinal appearances, ranging from near normal to varying degrees of chorioretinal atrophy and intraretinal pigment migration, were noted. Atrophic and/or pigmentary macular changes were present in 16 (80%) of 20 probands. Keratoconus and cataracts were identified in 5 (26%) of 19 patients, all of whom were homozygotes. The ERG of a parent heterozygote carrier revealed significantly reduced rod function, while ERGs for 6 other carrier parents were normal. Conclusions: The phenotype of LCA in patients with AIPL1 mutations is relatively severe, with a maculopathy in most patients and keratoconus and cataract in a large subset. Rod ERG abnormalities may be present in heterozygous carriers of AIPL1 mutations. Clinical Relevance: Understanding and recognizing the phenotype of LCA may help in defining the course and severity of the disease. Identifying the gene defect is the first step in preparation for therapy since molecular diagnosis in LCA will mandate the choice of treatment.

In many inherited diseases, the same phenotype can be produced both by single-base changes and by large deletions, or in some cases by duplications. Routine high-throughput sequencing can now detect small mutations relatively easily in a diagnostic setting, but deletions and duplications in the 50-500-kb region remain a more difficult problem. We have explored the application of array-CGH to the detection of such changes on a set of 20 samples consisting of patients with eye diseases associated with changes on chromosome 6p25 together with unaffected individuals, as well as two samples from tuberous sclerosis 2 (TSC2)-affected patients. We developed a microarray consisting of degenerate oligonucleotide primer (DOP)-PCR products from 260 human genomic clones, including BACs, PACs, and cosmids. In a masked study, chromosome changes in patients with iris hypoplasia (duplication) and Axenfeld-Rieger syndrome (deletion) were unequivocally distinguished from controls. Of the 20 6p25 samples analyzed, 19 were analyzed correctly (10 duplication cases, two deletions, and seven normals), while one individual failed to give a result because of poor hybridization. The extent of the duplication or deletion estimated was similar to that obtained by independent and much more time-consuming FISH experiments. On the other hand, deletions in the two TSC2-affected samples, previously mapped by DNA molecular combing, were not detected on the array, possibly due to the repeat content of that region. Excluding the 16p13 cosmids, consistent results were obtained from all other cosmid clones; the potential for producing affordable disease-specific diagnostic microarray as an adjunct to diagnosis is discussed.

PURPOSE: Autosomal dominant optic atrophy (ADOA) is characterized by primary degeneration of retinal ganglion cells and atrophy of the optic nerve. The OPA1 gene encodes a 960-amino-acid protein. In the current study the temporal and spatial localization of OPA1 were examined in developing and adult murine ocular tissues and the adult human eye. Because the Bst/+ mouse has been postulated as a model of ADOA, the mOPA1 expression in the Bst/+ retina was also examined. METHODS: A polyclonal antibody generated against a C-terminal peptide of OPA1 was used to assess by immunohistochemistry the expression of mOPA1 in the wild-type embryonic and postnatal mouse ocular tissues and the Bst/+ retina. Western blot analyses of total proteins from a panel of adult human tissues were used to examine the expression of human OPA1, and spatial localization was assessed by immunohistochemistry. RESULTS: The ocular expression of mOPA1 begins at E15 in the inner retina in a location corresponding to that of the subsequently developing ganglion cell layer (GCL) and peaks between postnatal day (P)0 and P1 in the retina and the optic nerve. There is a sharp decline in mOPA1 expression after P2, but it is expressed at a basal level until at least P12 in the GCL, inner plexiform layer (IPL), and inner nuclear layer (INL) of the retina as well as in the optic nerve. In the adult Bst/+ retina, mOPA1 is strongly expressed in the GCL and IPL and weakly in the INL. In the adult human eye, OPA1 is expressed in the GCL, IPL, INL, and outer plexiform layer (OPL) of the retina and in the optic nerve, where it is observed only in the myelinated region. CONCLUSIONS: OPA1 is not restricted to the GCL of the mammalian retina, and its expression extends into the IPL, INL, and OPL. OPA1 is distinctly expressed in the myelinated region beyond the lamina cribrosa in the human optic nerve, whereas its expression is weaker in the mouse optic nerve. In the Bst/+ mouse retina, despite the structural defects, mOPA1 expression is comparable to that observed in the wild-type adult mouse retina. These observations suggest a wider role for OPA1 than previously anticipated., Supported by the Wellcome Trust and the Birth Defects Foundation, United Kingdom.

9 páginas, 6 figuras, 2 tablas.-- et al., Congenital cataract is a leading cause of visual disability in children. Inherited isolated (non-syndromic) cataract represents a significant proportion of cases and the identification of genes responsible for inherited cataract will lead to a better understanding of the mechanism of cataract formation at the molecular level both in congenital and age-related cataract. Crystallins are abundantly expressed in the developing human lens and represent excellent candidate genes for inherited cataract. A genome-wide search of a five-generation family with autosomal dominant lamellar cataract demonstrated linkage to the 17p12–q11 region. Screening of the CRYBA1/3 gene showed a 3 bp deletion, which resulted in a G91del mutation within the tyrosine corner, that co-segregated with disease and was not found in 96 normal controls. In order to understand the molecular basis of cataract formation, the mutant protein was expressed in vitro and its unfolding and refolding characteristics assessed using far-UV circular dichroism spectroscopy. Defective folding and a reduction in solubility were found. As the wild-type protein did not refold into the native conformation following unfolding, a corresponding CRYBB2 mutant was genetically engineered and its refolding characteristics analysed and compared with wild-type CRYBB2. Its biophysical properties support the hypothesis that removal of the glycine residue from the tyrosine corner impairs the folding and solubility of β-crystallin proteins. This study represents the first comprehensive description of the biophysical consequences of a mutant β-crystallin protein that is associated with human inherited cataract., The work at the Department of Molecular Genetics, Institute of Ophthalmology was supported by a Wellcome Trust grant 063969/Z/01.

6 páginas, 3 figuras, 2 tablas.-- et al., [Aims]: To report a Bangladeshi family displaying intrafamilial phenotypic heterogeneity of lattice corneal dystrophy type I (LCDI) and to identify the causative mutation. [Methods]: Molecular genetic analysis was performed on DNA extracted from all members of the family. Exons of BIGH3gene were amplified by polymerase chain reaction. Gene mutation and polymorphisms were identified by heteroduplex and sequence analyses. Segregation of the mutation in the family was confirmed by restriction digestion of amplified gene fragments. [Results]: A heterozygous C T transition at the first nucleotide position of codon 124 of the BIGH3gene was detected in the three affected members and not in the unaffected members of the family. [Conclusions]: This is the first report of BIGH3gene mutation in a Bangladeshi family with phenotypic heterogeneity. This study confirms that BIGH3gene screening should be undertaken for proper classification of corneal dystrophy, especially in the absence of histopathological examination.

Lattice corneal dystrophy type I (LCDI) is a relatively common corneal dystrophy which can cause severe visual impairment. Recent studies have suggested a genetic localisation for the disease to chromosome 5q. Independent genetic linkage analysis in a six generation LCDI pedigree confirmed linkage to the 5q region bounded by marker loci IL9 and D5S436 suggesting genetic homogeneity. A maximum two point lod score of 7.51 (theta = 0.03) was obtained with marker D5S393. Multipoint and haplotype data positioned the disease between loci D5S393 and D5S396 corresponding to a genetic distance of 2cM, thus refining linkage sufficiently to allow for physical mapping of this disorder.

PURPOSE. To investigate whether the incomplete penetrance phenotype characteristic of adRP families linked to chromosome 19q13.4 (RP11) with mutations in the PRPF31 gene is due to differentially expressed wild-type alleles in symptomatic and asymptomatic individuals. METHODS. Real-time quantitative RT-PCR was performed on RNA from lymphoblastoid cell lines derived from a large adRP family (RP856/AD5) that segregates an 11bp deletion in exon 11 of PRPF31. The mRNA levels from only the wild-type allele of PRPF31 were assayed using a probe designed across the deletion. The Mann–Whitney U test was used to compare the median mRNA copy numbers of the symptomatic with the asymptomatic carriers of the mutant PRPF31 allele. The PRPF31 protein levels from symptomatic and asymptomatic individuals were also assayed by Western blot analysis using an antibody specific to the wild-type PRPF31 protein. RESULTS. The use of cell lines was validated by the observation that cell transformation did not alter PRPF31 expression in the cell lines compared with nucleated blood cells and donor retinas. A significant difference in wild-type PRPF31 mRNA levels was observed between symptomatic and asymptomatic individuals (P 0.001) and was supported by Western blot analysis of the PRPF31 protein. CONCLUSIONS. Partial penetrance in RP11 could be due to the coinheritance of a PRPF31 gene defect and a low-expressed wild-type allele. This study revealed a potential avenue for future therapy in that it appears the moderate overexpression of wild-type PRPF31 may prevent clinical manifestation of the disease. (Invest Ophthalmol Vis Sci. 2003;44:4204 – 4209) DOI:10.1167/iovs.03-0253 R etinitis pigmentosa is a clinically and genetically heterogeneous disorder characterized by progressive degeneration of the peripheral retina leading to night blindness and visual field loss. Clinical manifestations include pigment deposition in the retina and attenuation of retinal blood vessels, with later depigmentation or atrophy of the RPE. ERG abnormalities are recordable in the early stages with attenuation of rod and cone responses. In advanced RP both rod and cone ERG responses are extinguished. 1 With an incidence of approximately 1 in 3500, RP can be inherited as an X-linked, autosomal dominant or autosomal recessive condition. 1

Glaucoma, the leading cause of irreversible blindness world wide, affecting about 70 million people,1,2 is characterised by progressive loss of optic nerve axons and visual field damage. As the condition is insidious, the diagnosis is often missed and the disease detected only later when patients have severe and irreversible visual impairment. Adult primary open angle glaucoma (POAG) is a major form of glaucoma world wide. Most POAG in white and Afro-Caribbean populations is of the high tension glaucoma (HTG) type, with raised intraocular pressure (IOP) being a major contributory factor for visual loss.3–6 Normal tension glaucoma (NTG) is another important subtype of POAG in which typical glaucomatous cupping of the optic nerve head and visual field loss are present, but IOPs are consistently within the statistically normal population range. This accounts for about a third of all patients with POAG.4–7 Although the proportion of cases of glaucoma with a genetic basis has not been precisely defined, an increasing body of evidence derived from a range of populations indicates that glaucoma has a substantial heritable basis. It has been estimated that 20%–60% of patients with the disease have a family history, and under-reporting of a family history has been well documented in glaucoma.8–11 In 1997, myocilin ( MYOC , MIM 601652), located on chromosome 1q25,12 was the first POAG gene to be characterised and found to be mutated in patients with juvenile and adult onset POAG.13 Subsequent studies found that MYOC mutations account for fewer than 5% of cases of adult POAG,13–17 with lower frequencies of MYOC mutations in Chinese and Japanese populations compared to white populations.16,18 Rezaie et al 19 recently identified a second POAG gene, optineurin ( OPTN , MIM 602432) in the GLC1E interval on chromosome …

Since the first forkhead (Fox) gene was identified, the importance of this family of transcription factors has increased steadily with the discoveries of the diverse range of developmental processes that they regulate in eukaryotes. Among other processes, the Fox factors are important in the establishment of the body axis and the development of tissues from all three germ layers. In this article, we present some of the recent data on this gene family with reference to selected phenotypes observed in patients and model organisms, and the sensitivity of developmental processes to alterations in forkhead gene dosage.

6 páginas, 4 figuras, 3 tablas.-- Licence Creative Commons, attribution, Non-commercial licence.-- et al., [AIM]: To phenotype and genetically map the disease locus in a family presenting with autosomal dominant microcornea, rod-cone dystrophy, cataract, and posterior staphyloma. [METHODS]: Six affected and three unaffected members of the pedigree were examined. All individuals provided a history and underwent a full clinical examination with A-scan and B-scan ultrasonography and electrophysiological testing where appropriate. PCR based microsatellite marker genotyping using a positional candidate gene approach was then performed on DNA samples extracted from venous blood provided by each subject. [RESULTS]: The disorder is inherited as an autosomal dominant trait with variable expressivity and has a complex phenotype. Affected individuals had bilateral microcornea, pulverulent-like lens opacities, a rod-cone dystrophy and posterior staphyloma (MRCS). Using a positional candidate gene approach, the authors have evidence suggestive of linkage of this disorder to a region on 11q13 within the nanophthalmos 1 (NNO1) genetic interval. The small family size militates against achieving a LOD score of 3, but the haplotype data and the position of the putative MRCS locus within a known nanophthalmos locus are suggestive of linkage. A candidate gene within this region (ROM1) was screened and no mutations were found in affected members of the family. [CONCLUSION]: This rare developmental disorder has some phenotypic similarities to nanophthalmos and possibly maps to a locus within the genetic interval encompassing the NNO1 locus. Screening of candidate genes within this region continues.

This study investigates the functional consequences of two mutations, A194E and A216P, in the splicing factor gene PRPF31 linked to autosomal dominant retinitis pigmentosa (RP11). Using a yeast complementation assay, we demonstrate that introduction of the human A216P mutation into the yeast orthologue PRP31p results in only partial rescue of growth at the restrictive temperature, indicating that splicing function is not fully restored. An in vivo assay of splicing function in human cells using a bovine rod opsin splicing template did not detect any defect in splicing efficiency or accuracy attributable to either mutation, suggesting that neither has a dominant negative effect on splicing. However, western analysis and immunofluorescence microscopy of mammalian cells transfected with PRPF31 revealed that both mutations substantially hinder translocation of the protein into the nucleus. The overall effect may thus be an insufficiency in splicing function, which is revealed only under conditions of elevated splicing demand. With the need to replenish disc proteins on a daily basis, such conditions will exist in rod photoreceptors and this may underlie the disease pathology, This work was supported by a programme grant from the Wellcome Trust and by project grants from the UK Medical Research Council and the Foundation Fighting Blindness.

8 páginas, 3 figuras, 1 tabla.-- et al., As part of our project to generate a catalogue of genes with potential relevance to human retinal disease, we have cloned a transcript abundantly expressed in the human retina and testis. Analysis of the deduced 1322 amino acid protein sequence demonstrates that it encodes a novel WD repeat protein, termed WDR17. The N-terminal moiety of the WDR17 protein is predicted to consist of at least 12 conserved WD repeats that likely adopts a β-propeller-like structure. Homology searches with the C-terminal region revealed no similarity to known or hypothetical proteins. However, putative orthologous ESTs with 82–91% identity to the human cDNA were found in several mammalian species including rodents, pig and cattle, suggesting that WDR17 represents an evolutionarily novel subtype of WD repeat proteins with unique function(s) in higher eukaryotes. Temporal expression analysis in the murine eye showed that transcription of WDR17 begins prenatally, suggesting a functional role of the protein in the early stages of retinal development. Human WDR17 maps to the same chromosomal interval as the locus for autosomal recessive retinitis pigmentosa (RP29) on 4q34, making it a candidate for this disease gene. Sequencing of the entire coding region of WDR17 in an affected patient of the original RP29 pedigree has not revealed any disease-causing sequence variations likely excluding WDR17 as the gene underlying RP29., This work was supported by a grant from the Bundesministerium für Bildung und Forschung (BMBF) under 01KW9921/0.

To the Editor:We thank Dr. Siiri Veromann (Veromann 2002xTheoretical considerations regarding the study “Alpha-B crystallin gene (CRYAB) mutation causes dominant congenital posterior polar cataract in humans.”. Veromann, S. Am J Hum Genet. 2002; 71: 684–685Abstract | Full Text | Full Text PDF | PubMed | Scopus (3)See all References2002 [in this issue]) for the interest shown in our article (Berry et al. 2001xAlpha-B crystallin gene (CRYAB) mutation causes dominant congenital posterior polar cataract in humans. Berry, V, Francis, P, Reddy, MA, Collyer, D, Vithana, E, MacKay, I, Dawson, G, Carey, AH, Moore, A, Bhattacharya, SS, and Quinlan, RA. Am J Hum Genet. 2001; 69: 1141–1145Abstract | Full Text | Full Text PDF | PubMed | Scopus (170)See all References2001). Dr. Veromann has highlighted the fact that it is difficult to explain why a mutation in CRYAB should give rise to a discrete opacity at the posterior pole of the lens, since α-crystallin continues to be synthesized throughout life. Dr. Veromann suggests that the posterior polar phenotype may occur secondary to an effect on the regression of the primary vitreous.The mechanisms by which precise genetic mutations give rise to a specific lens phenotype are ill understood. We know that mutations in different genes may give rise to an identical lens phenotype, and we also know that different mutations within the same gene may give rise to very different patterns of lens opacification. Posterior polar cataract is itself genetically heterogeneous (Richards et al. 1984xCongenital cataract possibly linked to haptoglobin. Richards, J, Maumanee, IH, Rowe, S, and Lovrien, EW. Cytogenet Cell Genet. 1984; 37: 570See all References1984; Ionides et al. 1997xA locus for autosomal dominant posterior polar cataract on chromosome 1p. Ionides, AC, Berry, V, Mackay, DS, Moore, AT, Bhattacharya, SS, and Shiels, A. Hum Mol Genet. 1997; 6: 47–51Crossref | PubMed | Scopus (68)See all References1997; Yamada et al. 2000xAn autosomal dominant posterior polar cataract locus maps to human chromosome 20p12-q12. Yamada, K, Tomita, H, Yoshiura, K, Kondo, S, Wakui, K, Fukushima, Y, Ikegawa, S, Nakamura, Y, Amemiya, T, and Niikawa, N. Eur J Hum Genet. 2000; 8: 535–539Crossref | PubMed | Scopus (19)See all References2000). Such genetic and allelic heterogeneity is common in other inherited eye disorders, particularly in retinal dystrophies (Briggs et al. 2001xMutations in ABCR (ABCA4) in patients with Stargardt macular degeneration or cone-rod degeneration. Briggs, CE, Rucinski, D, Rosenfeld, PJ, Hirose, T, Berson, EL, and Dryja, TP. Invest Ophthalmol Vis Sci. 2001; 42: 2229–2236PubMedSee all References2001; Sohocki et al. 2001xPrevalence of mutations causing retinitis pigmentosa and other inherited retinopathies. Sohocki, MM, Daiger, SP, Bowne, SJ, Rodriquez, JA, Northrup, H, Heckenlively, JR, Birch, DG, Mintz-Hittner, H, Ruiz, RS, Lewis, RA, Saperstein, DA, and Sullivan, LS. Hum Mutat. 2001; 17: 42–51Crossref | PubMed | Scopus (144)See all References2001). It remains a challenge to identify the mechanism by which specific mutations give rise to regional opacification within the lens.In our article, we suggested that the CRYAB mutation could give rise to posterior polar cataract by two possible mechanisms: either by impaired chaperone-like function or by increased tendency of the mutant polypeptides to aggregate as a direct effect of abnormal α-crystallin structure. Studies of the temporal and spatial distribution of the αA- and αB-crystallin in the human lens suggest that they are expressed ubiquitously, but their expression varies at different time points during human lens development. For instance, αB-crystallin is expressed in the lens placode at Carnegie stage 13, but by stage 15 the lens vesicle is intensely positive for both αA- and αB-crystallin (Oguni et al. 1994xOntogeny of alpha-crystallin subunits in the lens of human and rat embryos. Oguni, M.T., Setogawa, T, Hashimoto, R, Tanaka, O, Shinohara, H, and Kato, K. Cell Tissue Res. 1994; 276: 151–154Crossref | PubMedSee all References1994). The effects of the mutant protein could be manifested early in human lens development, but, of course, αB-crystallin is also widely expressed in many other cell types, including muscle, epithelial, and endothelial cells, although the lens is the tissue that expresses the highest physiological concentrations of these proteins. It is conceivable, therefore, that the reported mutation in CRYAB could have produced other clinical symptoms, and we were aware of this possibility. Indeed, cardiac muscle expresses high levels of αB-crystallin (Kato et al. 1991xTissue distribution and developmental profiles of immunoreactive αB crystallin in the rat non-lenticular tissues determined with a sensitive immunoassay system. Kato, K, Shinohara, H, Kurobe, N, Inaguma, Y, Shimizu, K, and Ohshima, K. Biochim Biophys Acta. 1991; 1074: 201–208Crossref | PubMed | Scopus (146)See all References1991), but the family history and clinical analysis failed to identify any cardiac problems. We suggest, therefore, that the effects of the reported mutation are most likely restricted to the lens.Impaired chaperone function and/or the increased tendency of the mutant αB-crystallin polypeptide to aggregate may affect the internal structure of lens fibers so that opacification occurs. The subcapsular posterior region of the lens is the one where α-crystallin polypeptides are most highly phosphorylated, at least in the bovine lens (Chiesa et al. 1989xDifferential synthesis and phosphorylation of the alpha-crystallin A and B chains during bovine lens fiber cell differentiation. Chiesa, R, McDermott, MJ, and Spector, A. Curr Eye Res. 1989; 8: 151–158Crossref | PubMed | Scopus (24)See all References1989). It is also a lens region where there are functionally important cytoskeletal elements that utilize αB-crystallin to function properly (Quinlan and Prescott, in pressxSee all Referencesin press). Phosphorylation modulates the oligomerization status and function of small heat-shock proteins and their interaction with the cytoskeleton, and so it is possible to rationalize a localized cataract at the lens posterior as the characteristic phenotypic manifestation of this CRYAB mutation.Dr. Veromann suggests another mechanism to account for the position of the lens opacity. The hypothesis states that the mutation impairs regression of the primary vitreous and that this may result in posterior polar cataract. Lack of regression of the primary vitreous (or persistent hyperplastic primary vitreous [PHPV]) is associated with posterior polar cataract in humans, but the disorder is usually unilateral and associated with other developmental abnormalities of the eye. A family with autosomal recessive inherited nonsyndromic bilateral PHPV has shown linkage to a 13-cM region on chromosome 10 (Khaliq et al. 2001xLocus for autosomal recessive nonsyndromic persistent hyperplastic primary vitreous. Khaliq, S, Hameed, A, Ismail, M, Anwar, K, Leroy, B, Payne, AM, Bhattacharya, SS, and Mehdi, SQ. Invest Ophthalmol Vis Sci. 2001; 42: 2225–2228PubMedSee all References2001). However, in this family, posterior polar cataract was not a consistent feature. The association between PHPV and posterior polar cataract has been reported in mice (Colitz et al. 2000xPersistent hyperplastic tunica vasculosa lentis and persistent hyperplastic primary vitreous in transgenic line TgN3261Rpw. Colitz, CM, Malarkey, DE, Woychik, RP, and Wilkinson, JE. Vet Pathol. 2000; 37: 422–427Crossref | PubMedSee all References2000), but, again, there are other developmental abnormalities, including persistent tunica vasculosa lentis, detached retina, and anterior segment abnormalities. We have been unable to find any literature relating to a role for αB-crystallin in the regression of the primary vitreous, but, given the widespread expression αB-crystallin transcripts in the developing eye, it remains a possibility that it may influence regression of the primary vitreous. However, given the current state of knowledge of its function, it is difficult to identify a plausible biological mechanism by which a mutation in αB-crystallin would result in failure of regression of the hyaloid system. Furthermore, in the family in our study, all affected members were examined carefully after pupil dilatation, and none showed any evidence of persistence of the hyaloid system.Our knowledge of the relationship between genotype and phenotype in different forms of inherited cataract is at an early stage. In the family in our study, we can only speculate how mutations in αB-crystallin give such a localized opacity. Although Dr. Veromann has suggested another possible mechanism, the evidence for this is weak at present.

8 páginas, 5 figuras, 1 tabla.-- et al., cDNA and genomic clones encoding guanylate cyclase activating proteins (GCAP1 and GCAP2) in the Japanese puffer fish (Fugu rubripes) were identified by probing, respectively, a retinal cDNA library and a whole genomic cosmid library with human GCAP1 and GCAP2 cDNA probes. Clones were identified as GCAP1 and GCAP2 on the basis of amino acid identity with the equivalent frog sequences and their placement into GCAP1 and GCAP2 clades within a GCAP phylogenetic tree. The Fugu genes have an identical four exon/three intron structure to GCAP1 and GCAP2 genes from other vertebrates but the introns are smaller, with the result that the four exons spread over approximately 1 kb of DNA in each case. The two genes are separated on to separate cosmids. However, the results of Southern analysis of the cosmids and of genomic DNA are consistent with a tail-to-tail gene arrangement, as in other species, but with a surprisingly large intergenic separation of around 18.7 kb. Recombinant Fugu GCAP1 failed to activate human retinal guanylate cyclase (retGC) in vitro although CD spectroscopy shows that the protein is folded with a similar secondary structure to that of human GCAP1. The failure to activate may be due therefore to a lack of molecular compatibility in this heterologous assay system., This work was supported by grant number 055252 from the Wellcome Trust.

Mutations in the forkhead transcription factor gene FOXC1 on 6p25 cause a range of ocular developmental abnormalities, with associated glaucoma. However, FOXC1 mutations have not been found in all similarly affected pedigrees mapping to this interval. This study was undertaken to investigate the potential role of 6p25 rearrangements in causing such phenotypes.Two large families with autosomal dominant iris hypoplasia and early-onset glaucoma, 21 probands with Axenfeld-Rieger phenotypes not attributable to PITX2 mutations, and 7 individuals with documented 6p25 cytogenetic rearrangements, were investigated by genotyping and fluorescence in situ hybridization, with markers and probes from the 6p25 region.Interstitial 6p25 duplications were present in the unrelated families with iris hypoplasia, whereas an interstitial 6p25 deletion was identified in one Axenfeld-Rieger pedigree. Larger cytogenetic rearrangements, leading to trisomy or monosomy of the 6p25 region, resulted in microcornea and Rieger syndrome phenotypes, respectively. All the rearrangements encompassed FOXC1, increasing or decreasing the number of FOXC1 copies present, and appeared to correlate with the phenotypes observed.These findings represent the first example of both interstitial duplications and deletions cosegregating with a human developmental disorder that is attributable to altered dose of transcription factor. The data presented provide additional evidence for the pathogenicity of altered gene dosage of FOXC1 and suggest that a common mechanism is responsible for rearrangements of 6p25.

13 páginas, 2 figuras, 3 tablas.-- et al., Human chromosome Xp11.3–Xp11.23 encompasses the map location for a growing number of diseases with a genetic basis or genetic component. These include several eye disorders, syndromic and nonsyndromic forms of X-linked mental retardation (XLMR), X-linked neuromuscular diseases and susceptibility loci for schizophrenia, type 1 diabetes, and Graves' disease. We have constructed an ∼2.7-Mb high-resolution physical map extending from DXS8026 to ELK1, corresponding to a genetic distance of ∼5.5 cM. A combination of chromosome walking and sequence-tagged site (STS)-content mapping resulted in an integrated framework and transcript map, precisely positioning 10 polymorphic microsatellites (one of which is novel), 16 ESTs, and 12 known genes (RP2, PCTK1, UHX1, UBE1, RBM10, ZNF157, SYN1, ARAF1, TIMP1, PFC, ELK1, UXT). The composite map is currently anchored with 89 STSs to give an average resolution of ∼1 STS every 30 kb. By a combination of EST database searches and in silico detection of UniGene clusters within genomic sequence generated from this template map, we have mapped several novel genes within this interval: a Na+/H+ exchanger (SLC9A7), at least two zincfinger transcription factors (KIAA0215 and Hs.68318), carbohydrate sulfotransferase-7 (CHST7), regucalcin (RGN), inactivation-escape-1 (INE1), the human ortholog of mouse neuronal protein 15.6, and four putative novel genes. Further genomic analysis enabled annotation of the sequence interval with 20 predicted pseudogenes and 21 UniGene clusters of unknown function. The combined PAC/BAC transcript map and YAC scaffold presented here clarifies previously conflicting data for markers and genes within the Xp11.3–Xp11.23 interval and provides a powerful integrated resource for functional characterization of this clonally unstable, yet gene-rich and clinically significant region of proximal Xp., We thank the Guide Dogs for the Blind Association (D. L. T.) and The Wellcome Trust (A. J. H.) for funding this research. A. M. was supported by a grant from the German Ministry for Education and Research.

5 páginas, 3 figuras, 2 tablas.-- et al., [Purpose]: To genetically map the gene causing isolated X linked cataract in a large European pedigree. [Methods]: Using the patient registers at Birmingham Women’s Hospital, UK, we identified and examined 23 members of a four generation family with nuclear cataract. Four of six affected males also had complex congenital heart disease. Pedigree data were collated and leucocyte DNA extracted from venous blood. Linkage analysis by PCR based microsatellite marker genotyping was used to identify the disease locus and mutations within candidate genes screened by direct sequencing. [Results]: The disease locus was genetically refined to chromosome Xp22, within a 3 cM linkage interval flanked by markers DXS9902 and DXS999 (Zmax=3.64 at q=0 for marker DXS8036). [Conclusions]: This is the first report of a locus for isolated inherited cataract on the X chromosome. The disease interval lies within the Nance-Horan locus suggesting allelic heterogeneity. The apparent association with congenital cardiac anomalies suggests a possible new oculocardiac syndrome., This work is supported by a grant from the Wellcome trust (053416/Z/98).

Macular corneal dystrophy (MCD) is a rare corneal dystrophy that is characterized by abnormal deposits in the corneal stroma, keratocytes, Descemet's membrane, and endothelium, accompanied by progressive clouding. It has been classified into three immunophenotypes--MCD types I, IA, and II--according to the serum level of sulfated keratan sulfate (KS) and immunoreactivity of the corneal tissue. Recently, mutations in a new carbohydrate sulfotransferase gene (CHST6) encoding corneal glucosamine N-acetyl-6-sulfotransferase (C-GlcNac-6-ST) have been identified as the cause of MCD. Mutation screening of the CHST6 gene has been undertaken to identify the underlying mutations in five unrelated British families with MCD.DNA was extracted from venous blood obtained from all participants, and the coding region of CHST6 was amplified by polymerase chain reaction (PCR). The PCR products were analyzed by direct sequencing and restriction enzyme digestion. Enzyme-linked immunosorbent assay (ELISA) was performed to assess the presence of KS in serum from the probands of MCD-affected families participating in the study.Six novel missense mutations--four homozygous and two compound heterozygous--were identified in the CHST6 gene. The ELISA showed that the disease in all patients participating in the study was of MCD type I, including the subtype IA.These novel mutations are thought to result in loss of corneal sulfotransferase function, which would account for the MCD phenotype.

5 páginas, 2 figuras, 2 tablas.-- et al., Normal tension glaucoma (NTG) is a major form of glaucoma, associated with intraocular pressures that are within the statistically normal range of the population. OPA1, the gene responsible for autosomal dominant optic atrophy represents an excellent candidate gene for NTG, as the clinical phenotypes are similar and OPA1 is expressed in the retina and optic nerve. Eighty-three well-characterized NTG patients were screened for mutations in OPA1 by heteroduplex analysis and bi-directional sequencing. Sequences found to be altered in NTG subjects were examined for variations in 100 population controls. A second cohort of 80 NTG patients and 86 population controls was subsequently screened to determine whether the initial findings could be replicated. A single nucleotide polymorphism (SNP) on intervening sequence (IVS) 8 (IVS8 + 4 C/T) was found to be strongly associated with the occurrence of NTG in both cohorts (L2=7.97, P=0.005 in the first cohort, L2=9.93, P=0.002 in the second cohort; odds ratio 3.1 (95% CI: 1.8-5.6). A second SNP (IVS8 + 32 T/C) appeared to be associated with disease in the first cohort (L2=4.71, P=0.030), but this finding could not be replicated in the second cohort. In the combined cohort, the compound at-risk genotype IVS8 + 4 C/T, + 32 T/C was strongly associated with the occurrence of NTG (L2=22.04, P=0.00001 after correcting for testing four genotypes). These results indicate that polymorphisms in the OPA1 gene are associated with NTG and may be a marker for the disease., The work was supported in part by the International Glaucoma Association and Moorfields Special Trustees, and an equipment grant from the Glaucoma Research Foundation. Dr Aung is supported by the National Medical Research Council of Singapore and the Singapore National Eye Centre, Mr Brice by the Royal National Institute for the Blind and Dr Child by the Bluff-Field Charitable Trust and the IGA.

Item does not contain fulltext Inherited retinal dystrophies represent the most important cause of vision impairment in adolescence, affecting approximately 1 out of 3000 individuals. Mutations of the photoreceptor-specific gene ABCA4 (ABCR) are a common cause of retinal dystrophy. A number of mutations have been repeatedly reported for this gene, notably the 2588G>C mutation which is frequent in both patients and controls. Here we ascertained the frequency of the 2588G>C mutation in a total of 2343 unrelated random control individuals from 11 European countries and 241 control individuals from the US, as well as in 614 patients with STGD both from Europe and the US. We found an overall carrier frequency of 1 out of 54 in Europe, compared with 1 out of 121 in the US, confirming that the 2588G>C ABCA4 mutation is one of the most frequent autosomal recessive mutations in the European population. Carrier frequencies show an increasing gradient in Europe from South-West to North-East. The lowest carrier frequency, 0 out of 199 (0%), was found in Portugal; the highest, 11 out of 197 (5.5%), was found in Sweden. Haplotype analysis in 16 families segregating the 2588G>C mutation showed four intragenic polymorphisms invariably present in all 16 disease chromosomes and sharing of the same allele for several markers flanking the ABCA4 locus in most of the disease chromosomes. These results indicate a single origin of the 2588G>C mutation which, to our best estimate, occurred between 2400 and 3000 years ago.

The RP9 form of autosomal dominant retinitis pigmentosa (adRP) maps to a locus on human chromosome 7p14. We now report two different disease associated mutations in a previously unidentified human gene, the mouse orthologue of which has been characterised by its interaction with the Pim-1 oncogene. In the original linked family we identified the missense mutation H137L. A second missense mutation, D170G, was found in a single RP patient. The putative RP9 gene appears to be expressed in a wide range of tissues, but its function is unknown and a pathogenic mechanism remains to be determined.

Mutations in the gene for guanylate cyclase–activating protein-1 (GCAP1) (GUCA1A) have been associated with autosomal dominant cone dystrophy (COD3). In the present study, a severe disease phenotype in a large white family was initially shown to map to chromosome 6p21.1, the location of GUCA1A. Subsequent single-stranded conformation polymorphism analysis and direct sequencing revealed an A464G transition, causing an E155G substitution within the EF4 domain of GCAP1. Modeling of the protein structure shows that the mutation eliminates a bidentate amino acid side chain essential for Ca2+ binding. This represents the first disease-associated mutation in GCAP1, or any neuron-specific calcium-binding protein within an EF-hand domain, that directly coordinates Ca2+. The functional consequences of this substitution were investigated in an in vitro assay of retinal guanylate cyclase activation. The mutant protein activates the cyclase at low Ca2+ concentrations but fails to inactivate at high Ca2+ concentrations. The overall effect of this would be the constitutive activation of guanylate cyclase in photoreceptors, even at the high Ca2+ concentrations of the dark-adapted state, which may explain the dominant disease phenotype.

Retinitis pigmentosa (RP) is a genetically heterogeneous disorder characterized by progressive degeneration of the peripheral retina leading to night blindness and loss of visual fields. With an incidence of approximately 1 in 4000, RP can be inherited in X-linked, autosomal dominant or autosomal recessive modes. The RP13 locus for autosomal dominant RP (adRP) was placed on chromosome 17p13.3 by linkage mapping in a large South African adRP family. Using a positional cloning and candidate gene strategy, we have identified seven different missense mutations in the splicing factor gene PRPC8 in adRP families. Three of the mutations cosegregate within three RP13 linked families including the original large South African pedigree, and four additional mutations have been identified in other unrelated adRP families. The seven mutations are clustered within a 14 codon stretch within the last exon of this large 7 kb transcript. The altered amino acid residues at the C-terminus exhibit a high degree of conservation across species as diverse as humans, Arabidopsis and trypanosome, suggesting that some functional significance is associated with this part of the protein. These mutations in this ubiquitous and highly conserved splicing factor offer compelling evidence for a novel pathway to retinal degeneration.

Usher syndrome (USH) is a combination of a progressive pigmentary retinopathy, indistinguishable from retinitis pigmentosa, and some degree of sensorineural hearing loss. USH can be subdivided in Usher type I (USHI), type II (USHII) and type III (USHIII), all of which are inherited as autosomal recessive traits. The three subtypes are genetically heterogeneous, with six loci so far identified for USHI, three for USHII and only one for USHIII. Mutations in a novel gene, USH2A, encoding the protein usherin, have recently been shown to be associated with USHII. The gene encodes a protein with partial sequence homology to both laminin epidermal growth factor and fibronectin motifs. We analysed 35 British and one Pakistani Usher type II families with at least one affected member, for sequence changes in the 20 translated exons of the USH2A gene, using heteroduplex analysis and sequencing. Probable disease-causing mutations in USH2A were identified in 15 of 36 (41.7%) Usher II families. The most frequently encountered mutation (11/15 families or 11/18 mutated alleles) was del2299G in exon 13, resulting in a frameshift and premature stop codon. Other mutations include insertions and point mutations, of which two are previously unreported. Five different polymorphisms were also detected. Our results indicate that mutations in this gene are responsible for disease in a large proportion of British Usher type II patients. Moreover, if screening for mutations in USH2A is considered, it is sensible to screen for the del2299G mutation first.

Nearly 100 mutations in the rhodopsin gene have been described in association with autosomal dominant retinitis pigmentosa (ADRP), autosomal recessive retinitis pigmentosa (ARRP) and congenital stationary night blindness (CSNB), a nonprogressive disease. Additionally, mutations in the rhodopsin gene have been observed in patients with sectorial retinitis pigmentosa (SRP). We set out to determine the prevalence of rhodopsin mutations in sectorial RP, and the nature of the ocular phenotype associated with each mutation. Separately, we have also studied the biochemical phenotype of some of these mutant opsin by expression studies in COS cells.

5 páginas, 2 figuras, 2 tablas.-- et al., Dominant optic atrophy (DOA) is a hereditary optic neuropathy characterised by decreased visual acuity, colour vision deficits, centro-coecal scotoma and optic nerve pallor. The gene OPA1, encoding a dynamin-related GTPase, has recently been identified within the genetic linkage interval for the major locus for DOA on chromosome 3q28 and shown to harbour genetic aberrations segregating with disease in DOA families. The prevalence of the disorder in Denmark is reported to be the highest of any geographical location, suggestive of a founder effect. In order to establish the genetic basis of disease in a sample of 33 apparently unrelated Danish families, we screened DNA from affected members for OPA1 gene mutations by heteroduplex analysis and direct sequencing. A novel identical mutation in exon 28 (2826delT) was associated with DOA in 14 pedigrees and led to a frameshift and abnormal OPA1 protein -COOH terminus. Haplotype analysis of a region of ~1 Mb flanking the OPA1 gene using eight polymorphic markers revealed a common haplotype shared by all 14 patients; this haplotype was markedly over-represented compared with ethnically matched controls. Statistical analysis confirmed significant linkage disequilibrium with DOA over ~600 kb encompassing the disease mutation. We have therefore demonstrated that the relatively high frequency of DOA in Denmark is attributable to a founder mutation responsible for ~42% of the examined families and suggest that presymptomatic screening for the (2826delT) mutation may facilitate diagnosis and genetic counselling in a significant proportion of DOA patients of Danish ancestry., This work was funded by the Wellcome Trust UK project grant 056047 (D.L.T., S.S.B., M.V.). C.A. is a fellow of the Deutsche Forschungsgemeinschaft.

Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal diseases which affects approximately 1 in 3500 people worldwide. RP is initially characterized by night blindness followed by progressive degeneration of the retina, often culminating in legal or complete blindness in the later decades of life (Heckenlively et al., 1997). To date 11 autosomal dominant (adRP), 13 autosomal recessive (arRP), 5 X-linked (xlRP), and one digenic form of retinitis pigmentosa have been identified (RetNet). While several disease-associated genes have been identified, the majority of genes remain unknown. Recently we identified the gene and mutations responsible for the RP1 form of autosomal dominant retinitis pigmentosa located on chromosome 8q (Sullivan et al., 1999; Pierce et al., 1999, Guillonneau et al., 1999).

The aim of this work was to identify NRL mutations in a panel of 200 autosomal dominant retinitis pigmentosa (adRP) families. All samples were subjected to heteroduplex analysis of the three exons of the NRL gene, and HphI restriction digest analysis of exon 2 (to identify the S50T mutation). Families found to have the S50T mutation, and six additional larger pedigrees (which had previously been excluded from the other nine adRP loci) underwent linkage analysis using polymorphic markers located in the region of 14q11. HphI restriction analysis followed by direct sequencing of the amplified NRL exon 2 product demonstrated the presence of the NRL S50T sequence change in three adRP families. Comparison of marker haplotypes in affected individuals from these families with those of affected members of the original 14q11 linked family revealed a common disease haplotype for markers within the adRP locus. Recombination events observed in these families define an adRP critical interval of 14.9 cM between D13S72 and D14S1041. Linkage analysis enabled all six of the larger adRP pedigrees to be excluded from the 14q11 locus. The NRL S50T mutation represents another example of a ‘founder effect’ in a dominantly inherited retinal dystrophy. Identification of such ‘founder effects’ may greatly simplify diagnostic genetic screening and lead to better prognostic counselling. The exclusion of several adRP families from all ten adRP loci indicates that at least one further adRP locus remains to be found.

Autosomal dominant optic atrophy (ADOA) is the most prevalent hereditary optic neuropathy resulting in progressive loss of visual acuity, centrocoecal scotoma and bilateral temporal atrophy of the optic nerve with an onset within the first two decades of life. The predominant locus for this disorder (OPA1; MIM 165500) has been mapped to a 1.4-cM interval on chromosome 3q28-q29 flanked by markers D3S3669 and D3S3562 (ref. 3). We established a PAC contig covering the entire OPA1 candidate region of approximately 1 Mb and a sequence skimming approach allowed us to identify a gene encoding a polypeptide of 960 amino acids with homology to dynamin-related GTPases. The gene comprises 28 coding exons and spans more than 40 kb of genomic sequence. Upon sequence analysis, we identified mutations in seven independent families with ADOA. The mutations include missense and nonsense alterations, deletions and insertions, which all segregate with the disease in these families. Because most mutations probably represent null alleles, dominant inheritance of the disease may result from haploinsufficiency of OPA1. OPA1 is widely expressed and is most abundant in the retina. The presence of consensus signal peptide sequences suggests that the product of the gene OPA1 is targeted to mitochondria and may exert its function in mitochondrial biogenesis and stabilization of mitochondrial membrane integrity.

Leber congenital amaurosis (LCA) is the most severe form of inherited retinal dystrophy and the most frequent cause of inherited blindness in children. LCA is usually inherited in an autosomal recessive fashion, although rare dominant cases have been reported. One form of LCA, LCA4, maps to chromosome 17p13 and is genetically distinct from other forms of LCA, We recently identified the gene associated with LCA4, AIPL1 (aryl-hydrocarbon interacting protein-like 1) and identified three mutations that were the cause of blindness in five families with LCA, In this study, AIPL1 was screened for mutations in 512 unrelated probands with a range of retinal degenerative diseases to determine if AIPL1 mutations cause other forms of inherited retinal degeneration and to determine the relative contribution of AIPL1 mutations to inherited retinal disorders in populations worldwide. We identified 11 LCA families whose retinal disorder is caused by homozygous or compound heterozygous AIPL1 mutations. We also identified affected individuals in two apparently dominant families, diagnosed with juvenile retinitis pigmentosa or dominant cone-rod dystrophy, respectively, who are heterozygous for a 12-bp AIPL1 deletion. Our results suggest that AIPL1 mutations cause approximately 7% of LCA worldwide and may cause dominant retinopathy, (C) 2000 Academic Press.