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This study was undertaken to investigate the prevalence of sequence variants in LCA5 in patients with Leber congenital amaurosis (LCA), early-onset retinal dystrophy (EORD), and autosomal recessive retinitis pigmentosa (arRP); to delineate the ocular phenotypes; and to provide an overview of all published LCA5 variants in an online database. Patients underwent standard ophthalmic evaluations after providing informed consent. In selected patients, optical coherence tomography (OCT) and fundus autofluorescence imaging were possible. DNA samples from 797 unrelated patients with LCA and 211 with the various types of retinitis pigmentosa (RP) were screened by Sanger sequence analysis of all LCA5 exons and intron/exon junctions. Some LCA patients were prescreened by APEX technology or selected based on homozygosity mapping. In silico analyses were performed to assess the pathogenicity of the variants. Segregation analysis was performed where possible. Published and novel LCA5 variants were collected, amended for their correct nomenclature, and listed in a Leiden Open Variation Database (LOVD). Sequence analysis identified 18 new probands with 19 different LCA5 variants. Seventeen of the 19 LCA5 variants were novel. Except for two missense variants and one splice site variant, all variants were protein-truncating mutations. Most patients expressed a severe phenotype, typical of LCA. However, some LCA subjects had better vision and intact inner segment/outer segment (IS/OS) junctions on OCT imaging. In two families with LCA5 variants, the phenotype was more compatible with EORD with affected individuals displaying preserved islands of retinal pigment epithelium. One of the families with a milder phenotype harbored a homozygous splice site mutation; a second family was found to have a combination of a stop mutation and a missense mutation. This is the largest LCA5 study to date. We sequenced 1,008 patients (797 with LCA, 211 with arRP) and identified 18 probands with LCA5 mutations. Mutations in LCA5 are a rare cause of childhood retinal dystrophy accounting for ∼2% of disease in this cohort, and the majority of LCA5 mutations are likely null. The LCA5 protein truncating mutations are predominantly associated with LCA. However, in two families with the milder EORD, the LCA5 gene analysis revealed a homozygous splice site mutation in one and a stop mutation in combination with a missense mutation in a second family, suggesting that this milder phenotype is due to residual function of lebercilin and expanding the currently known phenotypic spectrum to include the milder early onset RP. Some patients have remaining foveal cone structures (intact IS/OS junctions on OCT imaging) and remaining visual acuities, which may bode well for upcoming treatment trials.

PurposeTo identify patients with autosomal recessive retinal dystrophy caused by mutations in the gene, retinal dehydrogenase 12 (RDH12), and to report the associated phenotype.MethodsAfter giving informed consent, all patients underwent full clinical evaluation. Patients were selected for mutation analysis based upon positive results from the Asper Ophthalmics Leber congenital amaurosis arrayed primer extansion (APEX) microarray screening, linkage analysis, or their clinical phenotype. All coding exons of RDH12 were screened by direct Sanger sequencing. Potential variants were checked for segregation in the respective families and screened in controls, and their pathogenicity analyzed using in silico prediction programs.ResultsScreening of 389 probands by the APEX microarray and/or direct sequencing identified bi-allelic mutations in 29 families. Seventeen novel mutations were identified. The phenotype in these patients presented with a severe early-onset rod-cone dystrophy. Funduscopy showed severe generalized retinal pigment epithelial and retinal atrophy, which progressed to dense, widespread intraretinal pigment migration by adulthood. The macula showed severe atrophy, with pigmentation and yellowing, and corresponding loss of fundus autofluorescence. Optical coherence tomography revealed marked retinal thinning and excavation at the macula.ConclusionsRDH12 mutations account for approximately 7% of disease in our cohort of patients diagnosed with Leber congenital amaurosis and early-onset retinal dystrophy. The clinical features of this disorder are highly characteristic and facilitate candidate gene screening. The term RDH12 retinopathy is proposed as a more accurate description.

PurposeTo report the clinical phenotype in patients with a retinal dystrophy associated with novel mutations in the MER tyrosine kinase (MERTK) gene.MethodsA consanguineous family of Middle Eastern origin was identified, and affected members underwent a full clinical evaluation. Linkage analysis was performed using the Affymetrix 50K chip. Regions of homozygosity were identified. The positional candidate genes protocadherin 21 (PCDH21), retinal G protein-coupled receptor (RGR), and MERTK were polymerase chain reaction (PCR) amplified and sequenced. Long-range PCR was performed to characterize the deletion. Two hundred and ninety-two probands with autosomal recessive, childhood onset, retinal dystrophies were analyzed using the Asper Ophthalmics Leber congenital amaurosis chip to screen for known MERTK mutations.ResultsAnalysis of a 50K-Affymetrix whole genome scan identified three regions of homozygosity on chromosomes 2 and 10. Screening of the candidate gene MERTK showed a possible deletion of exon 8. Long-range PCR identified a ~9 kb deletion within MERTK that removes exon 8. Screening of DNA from a panel of Saudi Arabian patients with autosomal recessive retinitis pigmentosa identified a second consanguineous family with the same mutation. One patient with a known MERTK mutation (p.R651X) was identified using the Asper Ophthalmics Leber congenital amaurosis chip. Further screening of the gene identified a second novel splice site mutation in intron 1. The phenotype associated with these identified MERTK mutations is of a childhood onset rod-cone dystrophy with early macular atrophy. The optical coherence tomography (OCT) appearance is distinctive with evidence of debris beneath the sensory retina.ConclusionsMutations in MERTK are a rare cause of retinal dystrophy. Non homologous recombination between Alu Y repeats near or within disease genes may be an important cause of retinal dystrophies.

PurposeTo describe the clinical findings and mutations in affected members of two families with an autosomal recessive retinal dystrophy associated with mutations in the protocadherin-21 (PCDH21) gene.MethodsA full genome scan of members of two consanguineous families segregating an autosomal recessive retinal dystrophy was performed and regions identical by descent identified. Positional candidate genes were identified and sequenced. All patients had a detailed ophthalmic examination, including electroretinography and retinal imaging.ResultsAffected members of both families showed identical homozygosity for an overlapping region of chromosome 10q. Sequencing of a candidate gene, PCDH21, showed two separate homozygous single-base deletions, c.337delG (p.G113AfsX1) and c.1459delG (p.G487GfsX20), which were not detected in 282 control chromosomes. Affected members of the two families first reported nyctalopia in late teenage years and retained good central vision until their late 30s. No color vision was detected in any proband. The fundus appearance included the later development of characteristic circular patches of pigment epithelial atrophy at the macula and in the peripheral retina.ConclusionsBiallelic mutations in the photoreceptor-specific gene PCDH21 cause recessive retinal degeneration in humans.

Several analyses and case studies are conducted to explain the different mechanisms that lead to complete congenital stationary night blindness in humans. The results reveal that the various recessive mutations taking place in the gene TRPM1 and the functions of the ON bipolar cells act as the major factors for the onset of the disorder.

Background: Inherited cataract is a clinically important and genetically heterogeneous cause of visual impairment. Typically, it presents at an early age with or without other ocular/systemic signs and lacks clear phenotype-genotype correlation rendering both clinical classification and molecular diagnosis challenging. Here we have utilized trio-based whole exome sequencing to discover mutations in candidate genes underlying autosomal dominant cataract segregating in three nuclear families. Results: In family A, we identified a recurrent heterozygous mutation in exon-2 of the gene encoding γD-crystallin (CRYGD; c.70C > A, p.Pro24Thr) that co-segregated with ‘coralliform’ lens opacities. Families B and C were found to harbor different novel variants in exon-2 of the gene coding for gap-junction protein α8 (GJA8; c.20T > C, p.Leu7Pro and c.293A > C, p.His98Pro). Each novel variant co-segregated with disease and was predicted in silico to have damaging effects on protein function. Conclusions: Exome sequencing facilitates concurrent mutation-profiling of the burgeoning list of candidate genes for inherited cataract, and the results can provide enhanced clinical diagnosis and genetic counseling for affected families. [ABSTRACT FROM AUTHOR]

Leber Congenital Amaurosis (LCA) and Early Childhood Onset Severe Retinal Dystrophy are clinically and genetically heterogeneous retinal disorders characterised by visual impairment and nystagmus from birth or early infancy. We investigated the prevalence of sequence variants in AIPL1 in a large cohort of such patients (n = 392) and probed the likelihood of disease-causation of the identified variants, subsequently undertaking a detailed assessment of the phenotype of patients with disease-causing mutations. Genomic DNA samples were screened for known variants in the AIPL1 gene using a microarray LCA chip, with 153 of these cases then being directly sequenced. The assessment of disease-causation of identified AIPL1 variants included segregation testing, assessing evolutionary conservation and in silico predictions of pathogenicity. The chip identified AIPL1 variants in 12 patients. Sequencing of AIPL1 in 153 patients and 96 controls found a total of 46 variants, with 29 being novel. In silico analysis suggested that only 6 of these variants are likely to be disease-causing, indicating a previously unrecognized high degree of polymorphism. Seven patients were identified with biallelic changes in AIPL1 likely to be disease-causing. In the youngest subject, electroretinography revealed reduced cone photoreceptor function, but rod responses were within normal limits, with no measurable ERG in other patients. An increasing degree and extent of peripheral retinal pigmentation and degree of maculopathy was noted with increasing age in our series. AIPL1 is significantly polymorphic in both controls and patients, thereby complicating the establishment of disease-causation of identified variants. Despite the associated phenotype being characterised by early-onset severe visual loss in our patient series, there was some evidence of a degree of retinal structural and functional preservation, which was most marked in the youngest patient in our cohort. This data suggests that there are patients who have a reasonable window of opportunity for gene therapy in childhood. [ABSTRACT FROM AUTHOR]

Purpose: To report novel variants and characterize the phenotype associated with the autosomal recessive retinal dystrophy caused by mutations in the lecithin retinol acyltransferase (LRAT) gene., Methods: A total of 149 patients with Leber's congenital amaurosis (LCA) or early onset retinal dystrophy were screened for mutations in LCA-associated genes using an arrayed-primer extension (APEX) genotyping microarray (Asper Ophthalmics). LRAT sequencing was subsequently performed in this 148-patient panel. Patients identified with mutations underwent further detailed phenotyping., Results: APEX analysis identified one patient with a previously reported homozygous LRAT mutation. Sequencing of the panel identified three additional patients with novel homozygous LRAT mutations in exon 2. All four patients had severe progressive nyctalopia, visual field constriction, and photophilia in childhood. Visual acuity ranged from 0.22 logMAR to hand motion. Funduscopy revealed severe retinal pigment epithelial atrophy and minimal retinal pigmentation. Asteroid hyalosis and macular epiretinal fibrosis were frequent. All demonstrated reduced fundus autofluorescence. Optical coherence tomography identified disrupted retinal lamination, outer-retinal debris, and an unidentifiable photoreceptor layer in two cases. Full-field electroretinograms were undetectable or showed severe rod-cone dysfunction. Photopic perimetry revealed severe visual field constriction. Dark-adapted perimetry demonstrated markedly reduced photoreceptor sensitivity. Dark-adapted spectral sensitivity measurements identified functioning rods in two of three patients. All three had severely reduced L- and M-cone sensitivity and poor color discrimination., Conclusions: LRAT mutations cause a severe, early childhood onset, progressive retinal dystrophy. Phenotypic similarities to the retinal dysfunction associated with RPE-specific protein 65 kDa mutations, another visual cycle gene, suggest that LRAT deficiency may show a good response to novel therapies.

Purpose: To investigate the prevalence of sequence variants in the gene SPATA7 in patients with Leber congenital amaurosis (LCA) and autosomal recessive, severe, early-onset retinal dystrophy (EORD) and to delineate the ocular phenotype associated with SPATA7 mutations., Methods: Patients underwent standard ophthalmic evaluation after providing informed consent. One hundred forty-one DNA samples from patients with LCA and EORD had been analyzed for mutations by using a microarray, with negative results. One additional patient underwent SPATA7 screening due to a region of autozygosity surrounding this gene. A further patient was screened who had a compatible ocular phenotype. The entire SPATA7 coding sequence was assayed, including the intron-exon junctions, by using a combination of direct DNA sequencing and high-resolution melting screening., Results: Screening of SPATA7 identified several known and novel single-nucleotide polymorphisms (SNPs). Affected individuals from five unrelated families were identified to have coding changes. Clinical features demonstrated a severe infantile onset retinal dystrophy, similar to Leber congenital amaurosis. The retina had widespread retinal pigment epithelial atrophy, with minimal pigment migration into the neurosensory retina. Fundus autofluorescence imaging showed a parafoveal annulus of increased autofluorescence. High-definition optical coherence tomography showed preservation of the inner segment/outer segment junction at the fovea., Conclusions: Mutations in SPATA7 are a rare cause of childhood retinal dystrophy accounting for 1.7% of disease in this cohort. Affected patients present in infancy with severe visual loss, but may have some preservation of the photoreceptor structure in the central retina.

Purpose: Mutations of C2ORF71 have recently been reported to be associated with autosomal recessive (AR) retinitis pigmentosa (RP) in humans and with visual defects in zebrafish. C2ORF71 is located on 2p23.2 and encodes a 1288-amino-acid protein of unknown function, predominately expressed in the photoreceptors. The study was conducted to determine the prevalence of mutations in C2ORF71 in a cohort of probands with AR retinal degeneration and to detect coding sequence variation in controls., Methods: A combination of high-resolution DNA melting (HRM) analysis and automated DNA sequencing was used to screen for C2ORF71 in 286 affected unrelated individuals. Among them, 95 subjects had Leber congenital amaurosis, and 191 had AR RP. In a similar fashion, 151 European and 40 South Asian control DNAs were screened., Results: Overall, 40 DNA sequence variants were detected, with 17 novel polymorphisms found in the control subjects (8 missense, 7 synonymous, and 2 other). Importantly, 11 novel sequence variants (6 missense and 5 synonymous) in 20 alleles were detected in the cohort of patients but not in the controls. Only one proband was a compound heterozygote but segregation analysis revealed her unaffected father to be homozygous for one of the putative mutations., Conclusions: C2ORF71 is a highly polymorphic gene (average heterozygosity of coding region in controls: 2.118 × 10(-3)) with many rare variants that confound mutation detection. Further analysis will determine the spectrum of retinal disease caused by mutations in C2ORF71 and distinguish true pathogenic alleles from the high background of polymorphism elucidating the role of this rare cause of RP in the visual process.

Purpose: To identify patients with autosomal recessive retinal dystrophy caused by mutations in the gene, retinal dehydrogenase 12 (RDH12), and to report the associated phenotype., Methods: After giving informed consent, all patients underwent full clinical evaluation. Patients were selected for mutation analysis based upon positive results from the Asper Ophthalmics Leber congenital amaurosis arrayed primer extansion (APEX) microarray screening, linkage analysis, or their clinical phenotype. All coding exons of RDH12 were screened by direct Sanger sequencing. Potential variants were checked for segregation in the respective families and screened in controls, and their pathogenicity analyzed using in silico prediction programs., Results: Screening of 389 probands by the APEX microarray and/or direct sequencing identified bi-allelic mutations in 29 families. Seventeen novel mutations were identified. The phenotype in these patients presented with a severe early-onset rod-cone dystrophy. Funduscopy showed severe generalized retinal pigment epithelial and retinal atrophy, which progressed to dense, widespread intraretinal pigment migration by adulthood. The macula showed severe atrophy, with pigmentation and yellowing, and corresponding loss of fundus autofluorescence. Optical coherence tomography revealed marked retinal thinning and excavation at the macula., Conclusions: RDH12 mutations account for approximately 7% of disease in our cohort of patients diagnosed with Leber congenital amaurosis and early-onset retinal dystrophy. The clinical features of this disorder are highly characteristic and facilitate candidate gene screening. The term RDH12 retinopathy is proposed as a more accurate description.

Purpose: To report the clinical phenotype in patients with a retinal dystrophy associated with novel mutations in the MER tyrosine kinase (MERTK) gene., Methods: A consanguineous family of Middle Eastern origin was identified, and affected members underwent a full clinical evaluation. Linkage analysis was performed using the Affymetrix 50K chip. Regions of homozygosity were identified. The positional candidate genes protocadherin 21 (PCDH21), retinal G protein-coupled receptor (RGR), and MERTK were polymerase chain reaction (PCR) amplified and sequenced. Long-range PCR was performed to characterize the deletion. Two hundred and ninety-two probands with autosomal recessive, childhood onset, retinal dystrophies were analyzed using the Asper Ophthalmics Leber congenital amaurosis chip to screen for known MERTK mutations., Results: Analysis of a 50K-Affymetrix whole genome scan identified three regions of homozygosity on chromosomes 2 and 10. Screening of the candidate gene MERTK showed a possible deletion of exon 8. Long-range PCR identified a ~9 kb deletion within MERTK that removes exon 8. Screening of DNA from a panel of Saudi Arabian patients with autosomal recessive retinitis pigmentosa identified a second consanguineous family with the same mutation. One patient with a known MERTK mutation (p.R651X) was identified using the Asper Ophthalmics Leber congenital amaurosis chip. Further screening of the gene identified a second novel splice site mutation in intron 1. The phenotype associated with these identified MERTK mutations is of a childhood onset rod-cone dystrophy with early macular atrophy. The optical coherence tomography (OCT) appearance is distinctive with evidence of debris beneath the sensory retina., Conclusions: Mutations in MERTK are a rare cause of retinal dystrophy. Non homologous recombination between Alu Y repeats near or within disease genes may be an important cause of retinal dystrophies.

Purpose: To describe the clinical findings and mutations in affected members of two families with an autosomal recessive retinal dystrophy associated with mutations in the protocadherin-21 (PCDH21) gene., Methods: A full genome scan of members of two consanguineous families segregating an autosomal recessive retinal dystrophy was performed and regions identical by descent identified. Positional candidate genes were identified and sequenced. All patients had a detailed ophthalmic examination, including electroretinography and retinal imaging., Results: Affected members of both families showed identical homozygosity for an overlapping region of chromosome 10q. Sequencing of a candidate gene, PCDH21, showed two separate homozygous single-base deletions, c.337delG (p.G113AfsX1) and c.1459delG (p.G487GfsX20), which were not detected in 282 control chromosomes. Affected members of the two families first reported nyctalopia in late teenage years and retained good central vision until their late 30s. No color vision was detected in any proband. The fundus appearance included the later development of characteristic circular patches of pigment epithelial atrophy at the macula and in the peripheral retina., Conclusions: Biallelic mutations in the photoreceptor-specific gene PCDH21 cause recessive retinal degeneration in humans.

Purpose: Autosomal dominant cataracts are a clinically and genetically heterogeneous eye-lens disorder that usually present in childhood with symptoms of impaired vision. The purpose of this study was to map and identify the mutation underlying autosomal dominant nuclear punctate cataracts segregating in a six generation Caucasian pedigree., Methods: Genomic DNA was prepared from blood leucocytes, genotyping was performed using microsatellite markers, and LOD scores were calculated using the LINKAGE programs. Mutation detection was performed using direct sequencing and restriction fragment length analysis., Results: Significant evidence of linkage was obtained at marker D13S175 (LOD score [Z]=4.11, recombination fraction [theta]=0.0) and haplotyping indicated that the disease gene lay in the about 2 Mb physical interval between D13S1316 and D13S1236, which contained the gene for gap-junction protein a3 (GJA3) or connexin46. Sequencing of GJA3 detected a C->T transition in exon 2 that resulted in the gain of an Alu 1 restriction site and was predicted to cause a conservative substitution of proline to leucine at codon 59 (P59L). Restriction analysis confirmed that the novel Alu 1 site co-segregated with cataracts in the family but was not detected in a control panel of 170 normal unrelated individuals., Conclusions: The present study has identified a fifth mutation in GJA3, rendering this connexin gene one of the most common non-crystallin genes associated with autosomal dominant cataracts in humans.

Purpose: Hereditary cataract is a clinically and genetically heterogeneous lens disorder that usually presents as a sight-threatening trait in childhood. The purpose of this study was to map and identify the mutation underlying an autosomal dominant form of coral-shaped cataract segregating in a three generation Caucasian pedigree., Methods: Genomic DNA was prepared from blood leucocytes, genotyping was performed using microsatellite markers, and LOD scores were calculated using the LINKAGE programs. Mutation detection was performed using direct sequencing and primer extension analysis. Following site-directed mutagenesis, mutant and wild type expression constructs were transfected into a human lens epithelial cell line (HLE B-3) and recombinant protein was detected by immunoblotting, imunofluorescence, and immunogold microscopy. Cell death was monitored by fluorescence activated cell sorting., Results: Significant evidence of linkage was detected at markers D2S371 (LOD score [Z]=3.81, recombination fraction [theta]=0) and D2S369 (Z=3.64, theta=0). Haplotyping indicated that the disease gene lay in the approximate 10 Mb physical interval between D2S1384 and D2S128, containing the gamma-crystallin gene (CRYGA-CRYGD) cluster on chromosome 2q33.3-q34. Sequencing of the CRYGA-CRYGD cluster identified a C->A transversion in exon 2 of CRYGD that was predicted to result in the non-conservative substitution of threonine for proline at amino-acid residue 23 (P23T) in the processed CRYGD protein. Transfection studies suggested that the P23T mutant was less soluble than its wild type counterpart when expressed in HLE B-3 cells., Conclusions: This study has identified an eighth type of cataract morphology associated with CRYGD and suggests that a CRYGD mutation may underlie the historically important "coralliform" cataract first reported in 1895.

Purpose: To investigate possible associations between sequence changes in the galactokinase gene (GALK1) and age-related cataract in a European population., Methods: Persons without lens opacities and persons with clinically significant age-related cataract were selected from those participating in the Collaborative Italian-American Clinical Trial of Nutritional Supplements and Age-Related Cataract or from those attending the Section of Ophthalmology of the University of Parma for cataract surgery. Type and severity of the opacities were assessed by slit-lamp and retro-illumination lens photographs. Mutations in GALK1 were identified by PCR amplification of individual exons and flanking sequences and sequencing using fluorescent terminator technology in an ABI 377 Prism or 3100 automated DNA sequencer., Results: DNA samples were obtained from 115 individuals with clear lenses and from 185 individuals with cataract (106 with any nuclear, 88 with any cortical, and 25 with any posterior sub capsular cataract). 157 of the 185 patients with cataract (85%) were age-matched with a control within an age range of plus or minus 1 year. SNPs causing amino acid changes in the galactokinase protein were identified in exon 4; I184M, 1/115 control versus 0/185 cataractous individuals, p=0.38, exon 6; G274D, 0/115 control versus 1/185 cataractous individuals, p>0.99, and exon 7; V338A, 0/115 control versus 1/185 cataractous individuals, p>0.99. Thus, there were no significant differences in the distribution of sequence alterations resulting in amino acid changes between control and cataractous individuals. Eighty samples showed a C to T transition 43 bases into intron 7 (46 cataracts and 34 controls). Testing the distribution of the intron 7 findings showed Hardy-Weinberg equilibrium for both cases (p=0.73) and controls (p=0.51). There was no difference in C/T distribution between cases and controls (p=0.27)., Conclusions: In this northern Italian population age-related cataract does not appear to be associated with GALK1 alleles. Since this is due to a lack of sequence changes in both affected and control individuals, this study cannot rule out the possibility of an association in other populations.