MiR-204 is responsible for inherited retinal dystrophy associated with ocular coloboma.

Ocular developmental disorders, including the group classified as microphthalmia, anophthalmia, and coloboma (MAC) and inherited retinal dystrophies, collectively represent leading causes of hereditary blindness. Characterized by extreme genetic and clinical heterogeneity, the separate groups share many common genetic causes, in particular relating to pathways controlling retinal and retinal pigment epithelial maintenance. To understand these shared pathways and delineate the overlap between these groups, we investigated the genetic cause of an autosomal dominantly inherited condition of retinal dystrophy and bilateral coloboma, present in varying degrees in a large, five-generation family. By linkage analysis and exome sequencing, we identified a previously undescribed heterozygous mutation, n.37 C > T, in the seed region of microRNA-204 (miR-204), which segregates with the disease in all affected individuals. We demonstrated that this mutation determines significant alterations of miR-204 targeting capabilities via in vitro assays, including transcriptome analysis. In vivo injection, in medaka fish (Oryzias latipes), of the mutated miR-204 caused a phenotype consistent with that observed in the family, including photoreceptor alterations with reduced numbers of both cones and rods as a result of increased apoptosis, thereby confirming the pathogenic effect of the n.37 C > T mutation. Finally, knockdown assays in medaka fish demonstrated that miR-204 is necessary for normal photoreceptor function. Overall, these data highlight the importance of miR-204 in the regulation of ocular development and maintenance and provide the first evidence, to our knowledge, of its contribution to eye disease, likely through a gain-of-function mechanism.

Mutations in PRDM5 in brittle cornea syndrome identify a pathway regulating extracellular matrix development and maintenance.

Extreme corneal fragility and thinning, which have a high risk of catastrophic spontaneous rupture, are the cardinal features of brittle cornea syndrome (BCS), an autosomal-recessive generalized connective tissue disorder. Enucleation is frequently the only management option for this condition, resulting in blindness and psychosocial distress. Even when the cornea remains grossly intact, visual function could also be impaired by a high degree of myopia and keratoconus. Deafness is another common feature and results in combined sensory deprivation. Using autozygosity mapping, we identified mutations in PRDM5 in families with BCS. We demonstrate that regulation of expression of extracellular matrix components, particularly fibrillar collagens, by PRDM5 is a key molecular mechanism that underlies corneal fragility in BCS and controls normal corneal development and maintenance. ZNF469, encoding a zinc finger protein of hitherto undefined function, has been identified as a quantitative trait locus for central corneal thickness, and mutations in this gene have been demonstrated in Tunisian Jewish and Palestinian kindreds with BCS. We show that ZNF469 and PRDM5, two genes that when mutated cause BCS, participate in the same regulatory pathway.

Discovery and functional analysis of a retinitis pigmentosa gene, C2ORF71.

Retinitis pigmentosa is a genetically heterogeneous group of inherited ocular disorders characterized by progressive photoreceptor cell loss, night blindness, constriction of the visual field, and progressive visual disability. Homozygosity mapping and gene expression studies identified a 2 exon gene, C2ORF71. The encoded protein has no homologs and is highly expressed in the eye, where it is specifically expressed in photoreceptor cells. Two mutations were found in C2ORF71 in human RP patients: A nonsense mutation (p.W253X) in the first exon is likely to be a null allele; the second, a missense mutation (p.I201F) within a highly conserved region of the protein, leads to proteosomal degradation. Bioinformatic and functional studies identified and validated sites of lipid modification within the first three amino acids of the C2ORF71 protein. Using morpholino oligonucleotides to knockdown c2orf71 expression in zebrafish results in visual defects, confirming that C2ORF71 plays an important role in the development of normal vision. Finally, localization of C2ORF71 to primary cilia in cultured cells suggests that the protein is likely to localize to the connecting cilium or outer segment of photoreceptor cells.

ZNF469 frequently mutated in the brittle cornea syndrome (BCS) is a single exon gene possibly regulating the expression of several extracellular matrix components.

Brittle cornea syndrome (BCS; MIM 229200) is an autosomal recessive generalized connective tissue disorder caused by mutations in ZNF469 and PRDM5. It is characterized by extreme thinning and fragility of the cornea that may rupture in the absence of significant trauma leading to blindness. Keratoconus or keratoglobus, high myopia, blue sclerae, hyperelasticity of the skin without excessive fragility, and hypermobility of the small joints are additional features of BCS. Transcriptional regulation of extracellular matrix components, particularly of fibrillar collagens, by PRDM5 and ZNF469 suggests that they might be part of the same pathway, the disruption of which is likely to cause the features of BCS. In the present study, we have performed molecular analysis of a cohort of 23 BCS affected patients on both ZNF469 and PRDM5, including those who were clinically reported previously [1]; the clinical description of three additional patients is reported in detail. We identified either homozygous or compound heterozygous mutations in ZNF469 in 18 patients while, 4 were found to be homozygous for PRDM5 mutations. In one single patient a mutation in neither ZNF469 nor PRDM5 was identified. Furthermore, we report the 12 novel ZNF469 variants identified in our patient cohort, and show evidence that ZNF469 is a single exon rather than a two exon gene.

The primordial growth disorder 3-M syndrome connects ubiquitination to the cytoskeletal adaptor OBSL1.

3-M syndrome is an autosomal-recessive primordial growth disorder characterized by significant intrauterine and postnatal growth restriction. Mutations in the CUL7 gene are known to cause 3-M syndrome. In 3-M syndrome patients that do not carry CUL7 mutations, we performed high-density genome-wide SNP mapping to identify a second locus at 2q35-q36.1. Further haplotype analysis revealed a 1.29 Mb interval in which the underlying gene is located and we subsequently discovered seven distinct null mutations from 10 families within the gene OBSL1. OBSL1 is a putative cytoskeletal adaptor protein that localizes to the nuclear envelope. We were also able to demonstrate that loss of OBSL1 leads to downregulation of CUL7, implying a role for OBSL1 in the maintenance of CUL7 protein levels and suggesting that both proteins are involved within the same molecular pathway.

Missense mutations in a retinal pigment epithelium protein, bestrophin-1, cause retinitis pigmentosa.

Bestrophin-1 is preferentially expressed at the basolateral membrane of the retinal pigmented epithelium (RPE) of the retina. Mutations in the BEST1 gene cause the retinal dystrophies vitelliform macular dystrophy, autosomal-dominant vitreochoroidopathy, and autosomal-recessive bestrophinopathy. Here, we describe four missense mutations in bestrophin-1, three that we believe are previously unreported, in patients diagnosed with autosomal-dominant and -recessive forms of retinitis pigmentosa (RP). The physiological function of bestrophin-1 remains poorly understood although its heterologous expression induces a Cl--specific current. We tested the effect of RP-causing variants on Cl- channel activity and cellular localization of bestrophin-1. Two (p.L140V and p.I205T) produced significantly decreased chloride-selective whole-cell currents in comparison to those of wild-type protein. In a model system of a polarized epithelium, two of three mutations (p.L140V and p.D228N) caused mislocalization of bestrophin-1 from the basolateral membrane to the cytoplasm. Mutations in bestrophin-1 are increasingly recognized as an important cause of inherited retinal dystrophy.

Biallelic mutation of BEST1 causes a distinct retinopathy in humans.

We describe a distinct retinal disorder, autosomal-recessive bestrophinopathy (ARB), that is consequent upon biallelic mutation in BEST1 and is associated with central visual loss, a characteristic retinopathy, an absent electro-oculogram light rise, and a reduced electroretinogram. Heterozygous mutations in BEST1 have previously been found to cause the two dominantly inherited disorders, Best macular dystrophy and autosomal-dominant vitreoretinochoroidopathy. The transmembrane protein bestrophin-1, encoded by BEST1, is located at the basolateral membrane of the retinal pigment epithelium in which it probably functions as a Cl(-) channel. We sequenced BEST1 in five families, identifying DNA variants in each of ten alleles. These encoded six different missense variants and one nonsense variant. The alleles segregated appropriately for a recessive disorder in each family. No clinical or electrophysiological abnormalities were identified in any heterozygotes. We conducted whole-cell patch-clamping of HEK293 cells transfected with bestrophin-1 to measure the Cl(-) current. Two ARB missense isoforms severely reduced channel activity. However, unlike two other alleles previously associated with Best disease, cotransfection with wild-type bestrophin-1 did not impair the formation of active wild-type bestrophin-1 channels, consistent with the recessive nature of the condition. We propose that ARB is the null phenotype of bestrophin-1 in humans.

A synonymous codon variant in two patients with autosomal recessive bestrophinopathy alters in vitro splicing of BEST1.

PURPOSE: Autosomal recessive bestrophinopathy (ARB) is a newly defined retinal dystrophy caused by biallelic mutations in bestrophin-1 (BEST1) and is hypothesized to represent the null bestrophin-1 phenotype in humans. The aim was to determine whether a synonymous BEST1 variant, c.102C>T, identified in two unrelated ARB patients, alters pre-mRNA splicing of the gene. Additionally a detailed phenotypic characterization of this distinctive condition is presented for both patients. METHODS: BEST1 was analyzed by direct sequencing. Patients underwent standard ophthalmic assessment. In silico and in vitro analysis using a minigene system was performed to assess whether a synonymous variant identified, c.102C>T p.Gly34Gly, alters pre-mRNA splicing of BEST1. RESULTS: Both ARB patients harbored either proven (patient 1; c.102C>T p.Gly34Gly and c.572T>C p.Leu191Pro) or presumed (patient 2; c.102C>T p.Gly34Gly and c.1470_1471delCA, p.His490GlnfsX24) biallelic mutations in BEST1 and were found to have phenotypes consistent with ARB. In vitro analysis of the synonymous variant, c.102C>T p.Gly34Gly, demonstrated it to introduce a cryptic splice donor site 52 nucleotides upstream of the actual splice donor site. CONCLUSIONS: The novel BEST1 variant identified, c.102C>T p.Gly34Gly, alters pre-mRNA splicing in vitro and is potentially pathogenic. In vivo this splicing variant is predicted to lead to the production of an mRNA transcript with a premature termination codon (p.Glu35TrpfsX11) that is predicted to be degraded by NMD.

Mutations in GDF6 are associated with vertebral segmentation defects in Klippel-Feil syndrome.

Klippel-Feil syndrome (KFS) is a congenital disorder of spinal segmentation distinguished by the bony fusion of anterior/cervical vertebrae. Scoliosis, mirror movements, otolaryngological, kidney, ocular, cranial, limb, and/or digit anomalies are often associated. Here we report mutations at the GDF6 gene locus in familial and sporadic cases of KFS including the recurrent missense mutation of an extremely conserved residue c.866T>C (p.Leu289Pro) in association with mirror movements and an inversion breakpoint downstream of the gene in association with carpal, tarsal, and vertebral fusions. GDF6 is expressed at the boundaries of the developing carpals, tarsals, and vertebrae and within the adult vertebral disc. GDF6 knockout mice are best distinguished by fusion of carpals and tarsals and GDF6 knockdown in Xenopus results in a high incidence of anterior axial defects consistent with a role for GDF6 in the etiology, diversity, and variability of KFS.

Developmental and tissue expression of Xenopus laevis RPGR.

PURPOSE: The present study examined the developmental and tissue expression of the retinitis pigmentosa GTPase regulator (RPGR) gene in Xenopus laevis. METHODS: The cDNA for X. laevis RPGR (XRPGR) was isolated from adult eye mRNA by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. The deduced peptide sequence was aligned with RPGR orthologues. Gene expression was examined by whole-mount in situ hybridization and RT-PCR. The localization of XRPGR in X. laevis photoreceptor cells and XTC-2 cells was determined by immunostaining. RESULTS: The XRPGR(ex1-19) isoform encodes a protein of 727 amino acids containing an RCC1 domain and a C-terminal isoprenylation anchorage motif. It shares 33% to 41% amino acid identity with human, mouse, and dog RPGR. The C-terminal exon of the alternatively spliced RPGR(ORF15) isoform is also conserved across species. XRPGR is expressed at the earliest stages of X. laevis development and persists into adulthood, where expression is highest in the eye. XRPGR is expressed in presumptive eye fields (stages 18 to 22), becoming largely restricted to the central retina (stages 28 to 40). XRPGR protein colocalizes with beta-tubulin at the X. laevis ciliary axoneme and with gamma-tubulin at centrosomes in XTC-2 cells. CONCLUSIONS: XRPGR is widely expressed throughout development but shows highest expression after the appearance of the eye primordium and persists in the eye into adulthood. The data are consistent with XRPGR expression in a single microtubular organelle-the centriole or basal body and associated ciliary transitional zone found in modified sensory cilia of photoreceptors and motile cilia.

Inherited eye disease: cause and late effect.

Molecular genetics has provided relatively few insights into late-onset eye disorders, but epidemiological data indicate that genetic factors are important in some late-onset eye disorders that cause major health burdens. Much clinical genetic research is based on the belief that developmental and late-onset disorders are not necessarily the result of defects in different genes, but are often caused by different mutations in the same collection of genes. Thus, mutations that either abolish or radically change gene function might cause early-onset disorders, whereas more-subtle changes in gene expression might underlie late-onset diseases. We present arguments and examples that indicate that this principle might be a fruitful guide to investigating the causes of late-onset eye disorders.

Evidence of genetic heterogeneity in MRCS (microcornea, rod-cone dystrophy, cataract, and posterior staphyloma) syndrome.

PURPOSE: To present the detailed phenotype of a subject with MRCS (microcornea, retinal dystrophy, cataract, and posterior staphyloma) syndrome and to investigate the underlying molecular genetic basis. DESIGN: Interventional case report. METHODS: Clinical examination, electrophysiologic assessment, B-scan ultrasonography, and mutation screening of the gene VMD2. The protocol of the study was approved by the local ethics committee and informed consent was obtained. RESULTS: A 12-year-old boy was identified with bilateral microcornea, rod-cone dystrophy, congenital cataracts, and posterior staphylomata associated with high myopia (MRCS). Mutation screening failed to identify disease-causing sequence variants in VMD2, the gene associated with MRCS syndrome. All previous subjects have had pathogenic VMD2 sequence alterations. CONCLUSIONS: We present a further report of the MRCS syndrome and provide evidence in support of genetic heterogeneity in this phenotype.

Mutations of VMD2 splicing regulators cause nanophthalmos and autosomal dominant vitreoretinochoroidopathy (ADVIRC).

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 in-frame 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.

Brittle cornea syndrome: recognition, molecular diagnosis and management.

Brittle cornea syndrome (BCS) is an autosomal recessive disorder characterised by extreme corneal thinning and fragility. Corneal rupture can therefore occur either spontaneously or following minimal trauma in affected patients. Two genes, ZNF469 and PRDM5, have now been identified, in which causative pathogenic mutations collectively account for the condition in nearly all patients with BCS ascertained to date. Therefore, effective molecular diagnosis is now available for affected patients, and those at risk of being heterozygous carriers for BCS. We have previously identified mutations in ZNF469 in 14 families (in addition to 6 reported by others in the literature), and in PRDM5 in 8 families (with 1 further family now published by others). Clinical features include extreme corneal thinning with rupture, high myopia, blue sclerae, deafness of mixed aetiology with hypercompliant tympanic membranes, and variable skeletal manifestations. Corneal rupture may be the presenting feature of BCS, and it is possible that this may be incorrectly attributed to non-accidental injury. Mainstays of management include the prevention of ocular rupture by provision of protective polycarbonate spectacles, careful monitoring of visual and auditory function, and assessment for skeletal complications such as developmental dysplasia of the hip. Effective management depends upon appropriate identification of affected individuals, which may be challenging given the phenotypic overlap of BCS with other connective tissue disorders.

Functional characterization of bestrophin-1 missense mutations associated with autosomal recessive bestrophinopathy.

PURPOSE: Autosomal recessive bestrophinopathy (ARB) is a retinal dystrophy affecting macular and retinal pigmented epithelium function resulting from homozygous or compound heterozygous mutations in BEST1. In this study we characterize the functional implications of missense bestrophin-1 mutations that cause ARB by investigating their effect on bestrophin-1's chloride conductance, cellular localization, and stability. METHODS: The chloride conductance of wild-type bestropin-1 and a series of ARB mutants were determined by whole-cell patch-clamping of transiently transfected HEK cells. The effect of ARB mutations on the cellular localization of bestrophin-1 was determined by confocal immunofluorescence on transiently transfected MDCK II cells that had been polarized on Transwell filters. Protein stability of wild-type and ARB mutant forms of bestrophin-l was determined by the addition of proteasomal or lysosomal inhibitors to transiently transfected MDCK II cells. Lysates were then analyzed by Western blot analysis. RESULTS: All ARB mutants investigated produced significantly smaller chloride currents compared to wild-type bestrophin-1. Additionally, co-transfection of compound heterozygous mutants abolished chloride conductance in contrast to co-transfections of a single mutant with wild-type bestrophin-l, reflecting the recessive nature of the condition. In control experiments, expression of two dominant vitelliform macular dystrophy mutants was shown to inhibit wild-type currents. Cellular localization of ARB mutants demonstrated that the majority did not traffic correctly to the plasma membrane and that five of these seven mutants were rapidly degraded by the proteasome. Two ARB-associated mutants (p.D312N and p.V317M) that were not trafficked correctly nor targeted to the proteasome had a distinctive appearance, possibly indicative of aggresome or aggresome-like inclusion bodies. CONCLUSIONS: Differences in cellular processing mechanisms for different ARB associated mutants lead to the same disease phenotype. The existence of distinct pathogenic disease mechanisms has important ramifications for potential gene replacement therapies since we show that missense mutations associated with an autosomal recessive disease have a pathogenic influence beyond simple loss of function.

Identification of a novel locus for autosomal dominant primary open angle glaucoma on 4q35.1-q35.2.

PURPOSE: Primary open angle glaucoma is the most prevalent type of glaucoma and the leading cause of irreversible blindness worldwide. The genetic basis is poorly understood. Of 14 loci associated with this disease, only two genes have been identified, accounting for approximately 4% of cases. The authors investigated the genetic cause of primary open angle glaucoma in a large four-generation family with an apparent autosomal dominant mode of inheritance. METHODS: Twenty-three family members underwent comprehensive phenotyping by a single ophthalmologist, and the MYOC gene was sequenced in all affected family members for whom DNA was available. Parametric genomewide linkage analysis was performed on 10 affected family members and one unaffected family member. Within the critical region, mutation analysis of candidate genes LRP2BP, CYP4V2, and UFSP2 was carried out by direct sequencing. RESULTS: No mutations were identified in MYOC. Genomewide linkage analysis generated one significant LOD score of 3.1 (maximum affected-only LOD score of 2.8) centered on chromosome 4 at 4q35.1-q35.2, a critical region that does not contain any of the previously reported primary open angle glaucoma loci. A 1.866-Mb (7.2 cM) region was identified containing 17 known or hypothetical genes. No mutations were identified in the candidate genes LRPB2BP, CYP4V2, and UFSP2. CONCLUSIONS: This study identifies a new primary open angle glaucoma locus, GLC1Q, in a region on chromosome 4 not previously associated with glaucoma.

Left-sided embryonic expression of the BCL-6 corepressor, BCOR, is required for vertebrate laterality determination.

Oculofaciocardiodental (OFCD) syndrome is an X-linked male lethal condition encompassing cardiac septal defects, as well as ocular and dental anomalies. The gene mutated in OFCD syndrome, the BCL-6 corepressor (BCOR), is part of a transcriptional repression complex whose transcriptional targets remain largely unknown. We reviewed cases of OFCD syndrome and identified patients exhibiting defective lateralization including dextrocardia, asplenia and intestinal malrotation, suggesting that BCOR is required in normal laterality determination. To study the function of BCOR, we used morpholino oligonucleotides (MOs) to knockdown expression of xtBcor in Xenopus tropicalis, thus creating an animal model for OFCD syndrome. The resulting tadpoles had cardiac and ocular features characteristic of OFCD syndrome. Reversed cardiac orientation and disorganized gut patterning were seen when MOs were injected into the left side of embryos, demonstrating a left-sided requirement for xtBcor in lateral determination in Xenopus. Ocular defects displayed no left-right bias and included anterior and posterior segment disorders such as microphthalmia and coloboma. Expression of xtPitx2c was shown to be downregulated when xtBcor was depleted. This identifies a pathway in which xtBcor is required for lateral specification, a process intrinsically linked to correct cardiac septal development.

Mapping of deletion and translocation breakpoints in 1q44 implicates the serine/threonine kinase AKT3 in postnatal microcephaly and agenesis of the corpus callosum.

Deletions of chromosome 1q42-q44 have been reported in a variety of developmental abnormalities of the brain, including microcephaly (MIC) and agenesis of the corpus callosum (ACC). Here, we describe detailed mapping studies of patients with unbalanced structural rearrangements of distal 1q4. These define a 3.5-Mb critical region extending from RP11-80B9 to RP11-241M7 that we hypothesize contains one or more genes that lead to MIC and ACC when present in only one functional copy. Next, mapping of a balanced reciprocal t(1;13)(q44;q32) translocation in a patient with postnatal MIC and ACC demonstrated a breakpoint within this region that is situated 20 kb upstream of AKT3, a serine-threonine kinase. The murine orthologue Akt3 is required for the developmental regulation of normal brain size and callosal development. Whereas sequencing of AKT3 in a panel of 45 patients with ACC did not demonstrate any pathogenic variations, whole-mount in situ hybridization confirmed expression of Akt3 in the developing central nervous system during mouse embryogenesis. AKT3 represents an excellent candidate for developmental human MIC and ACC, and we suggest that haploinsufficiency causes both postnatal MIC and ACC.