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1.  Differential dimerization of variants linked to enhanced S-Cone Sensitivity Syndrome (ESCS) located in the NR2E3 ligand-binding domain 
Human mutation  2015;36(6):599-610.
NR2E3 encodes the photoreceptor-specific nuclear hormone receptor that acts as a repressor of cone-specific gene expression in rod photoreceptors, and as an activator of several rod-specific genes. Recessive variants located in the ligand-binding domain (LBD) of NR2E3 cause enhanced short wavelength sensitive- (S-) cone syndrome (ESCS), a retinal degeneration characterized by an excess of Scones and non-functional rods. We analyzed the dimerization properties of NR2E3 and the effect of disease-causing LBD missense variants by bioluminescence resonance energy transfer (BRET2) protein interaction assays. Homodimerization was not affected in presence of p.A256V, p.R039G, p.R311Q and p.R334G variants, but abolished in presence of p.L263P, p.L336P, p.L353V, p.R385P and p.M407K variants. Homology modeling predicted structural changes induced by NR2E3 LBD variants. NR2E3 LBD variants did not affect interaction with CRX, but with NRL and rev-erbα/NR1D1. CRX and NRL heterodimerized more efficiently together, than did either with NR2E3. NR2E3 did not heterodimerize with TLX/NR2E1 and RXRα/NR2C1. The identification of a new compound heterozygous patient with detectable rod function, who expressed solely the p.A256V variant protein, suggests a correlation between LBD variants able to form functional NR2E3 dimers and atypical mild forms of ESCS with residual rod function.
PMCID: PMC5014385  PMID: 25703721
NR2E3; retinal degeneration; genotype-phenotype correlation; photoreceptor development
2.  Congenital Nystagmus Gene FRMD7 Is Necessary for Establishing a Neuronal Circuit Asymmetry for Direction Selectivity 
Neuron  2016;89(1):177-193.
Neuronal circuit asymmetries are important components of brain circuits, but the molecular pathways leading to their establishment remain unknown. Here we found that the mutation of FRMD7, a gene that is defective in human congenital nystagmus, leads to the selective loss of the horizontal optokinetic reflex in mice, as it does in humans. This is accompanied by the selective loss of horizontal direction selectivity in retinal ganglion cells and the transition from asymmetric to symmetric inhibitory input to horizontal direction-selective ganglion cells. In wild-type retinas, we found FRMD7 specifically expressed in starburst amacrine cells, the interneuron type that provides asymmetric inhibition to direction-selective retinal ganglion cells. This work identifies FRMD7 as a key regulator in establishing a neuronal circuit asymmetry, and it suggests the involvement of a specific inhibitory neuron type in the pathophysiology of a neurological disease.
Video Abstract
•FRMD7 is required for the horizontal optokinetic reflex in mice as in humans•Horizontal direction selectivity is lost in the retina of FRMD7 mutant mice•Asymmetry of inhibitory inputs to horizontal DS cells is lost in FRMD7 mutant mice•FRMD7 is expressed in ChAT-expressing cells in the retina of mice and primates
Yonehara et al. show that FRMD7, a gene that is defective in human congenital nystagmus, is required in the mouse retina to establish spatially asymmetric inhibitory inputs from starburst cells to horizontal direction-selective ganglion cells.
PMCID: PMC4712192  PMID: 26711119
3.  Enrichment of pathogenic alleles in the brittle cornea gene, ZNF469, in keratoconus 
Human Molecular Genetics  2014;23(20):5527-5535.
Keratoconus, a common inherited ocular disorder resulting in progressive corneal thinning, is the leading indication for corneal transplantation in the developed world. Genome-wide association studies have identified common SNPs 100 kb upstream of ZNF469 strongly associated with corneal thickness. Homozygous mutations in ZNF469 and PR domain-containing protein 5 (PRDM5) genes result in brittle cornea syndrome (BCS) Types 1 and 2, respectively. BCS is an autosomal recessive generalized connective tissue disorder associated with extreme corneal thinning and a high risk of corneal rupture. Some individuals with heterozygous PRDM5 mutations demonstrate a carrier ocular phenotype, which includes a mildly reduced corneal thickness, keratoconus and blue sclera. We hypothesized that heterozygous variants in PRDM5 and ZNF469 predispose to the development of isolated keratoconus. We found a significant enrichment of potentially pathologic heterozygous alleles in ZNF469 associated with the development of keratoconus (P = 0.00102) resulting in a relative risk of 12.0. This enrichment of rare potentially pathogenic alleles in ZNF469 in 12.5% of keratoconus patients represents a significant mutational load and highlights ZNF469 as the most significant genetic factor responsible for keratoconus identified to date.
PMCID: PMC4168824  PMID: 24895405
4.  The cataract and glucosuria associated monocarboxylate transporter MCT12 is a new creatine transporter 
Human Molecular Genetics  2013;22(16):3218-3226.
Creatine transport has been assigned to creatine transporter 1 (CRT1), encoded by mental retardation associated SLC6A8. Here, we identified a second creatine transporter (CRT2) known as monocarboxylate transporter 12 (MCT12), encoded by the cataract and glucosuria associated gene SLC16A12. A non-synonymous alteration in MCT12 (p.G407S) found in a patient with age-related cataract (ARC) leads to a significant reduction of creatine transport. Furthermore, Slc16a12 knockout (KO) rats have elevated creatine levels in urine. Transport activity and expression characteristics of the two creatine transporters are distinct. CRT2 (MCT12)-mediated uptake of creatine was not sensitive to sodium and chloride ions or creatine biosynthesis precursors, breakdown product creatinine or creatine phosphate. Increasing pH correlated with increased creatine uptake. Michaelis–Menten kinetics yielded a Vmax of 838.8 pmol/h/oocyte and a Km of 567.4 µm. Relative expression in various human tissues supports the distinct mutation-associated phenotypes of the two transporters. SLC6A8 was predominantly found in brain, heart and muscle, while SLC16A12 was more abundant in kidney and retina. In the lens, the two transcripts were found at comparable levels. We discuss the distinct, but possibly synergistic functions of the two creatine transporters. Our findings infer potential preventive power of creatine supplementation against the most prominent age-related vision impaired condition.
PMCID: PMC3723308  PMID: 23578822
5.  Intravitreal chemotherapy for vitreous seeding in retinoblastoma: Recent advances and perspectives 
Saudi Journal of Ophthalmology  2013;27(3):147-150.
For decades intravitreal chemotherapy (IViC) remained virtually banished from the therapeutic armamentarium against retinoblastoma, except as a heroic attempt of salvage before enucleation in only eyes with refractory vitreous seeding. Very recently, we have initiated a reappraisal of this route of administration by (1) profiling eligibility criteria, (2) describing a safety-enhanced injection procedure, (3) adjusting the tumoricidal dose of melphalan, and (4) reporting an unprecedented efficacy in terms of tumor control of vitreous seeding. Since then, intravitreal chemotherapy is being progressively implemented worldwide with great success, but still awaits formal validation by the ongoing prospective phase II clinical trial. As far as preliminary results are concerned, IViC appears to achieve complete vitreous response in 100% of the 35 newly recruited patients irrespective of the previous treatment regimen, including external beam radiotherapy and/or intra-arterial melphalan. In other words, vitreous seeding, still considered as the major cause of primary and secondary enucleation, can now be controlled by IViC. However, sterilization of vitreous seeding does not necessarily translate into eye survival, unless the retinal source of the seeds receives concomitant therapy. In conclusion, IViC, an unsophisticated and cost-effective treatment, is about to revolutionize the eye survival prognosis of vitreous disease in advanced retinoblastoma.
PMCID: PMC3770220  PMID: 24227979
Intravitreal chemotherapy; Melphalan; Retinoblastoma; Vitreous seeding
6.  Mutations in NR2E3 can cause dominant or recessive retinal degenerations in a same family 
Human mutation  2009;30(3):342-351.
NR2E3 (PNR), a nuclear receptor specifically expressed in photoreceptors, represses cone-specific genes and activates several rod-specific genes. In humans, mutations in NR2E3 have been associated with the recessively inherited enhanced short wavelength sensitive (S-) cone syndrome (ESCS) and, recently, with autosomal dominant retinitis pigmentosa (adRP). In the present work, we describe two additional families affected by adRP that carry a heterozygous c.166G>A (p.G56R) mutation in the NR2E3 gene. Functional analysis determined dominant negative activity of the p.G56R mutant protein as the molecular mechanism of adRP. Interestingly, in one pedigree, the most common causal variant for ESCS (p.R311Q) co-segregated with the adRP-linked p.G56R mutation, and, the compound heterozygotes exhibited an ESCS-like phenotype, which in one of the 2 cases was strikingly “milder” than the patients carrying the p.G56R mutation alone. Impaired repression of cone-specific genes by the corepressors atrophin-1 (dentatorubral-pallidoluysian atrophy DRPLA gene product) and atrophin-2 (RERE repeat protein) appeared to be a molecular mechanism mediating the beneficial effect of the p.R311Q mutation. Finally, the functional dominance of the p.R311Q to the p.G56R mutation is discussed.
PMCID: PMC3658139  PMID: 19006237
retinal degeneration; transcriptional regulation; cofactor assembly; corepressor binding; NR2E3; photoreceptor-specific nuclear receptor; PNR
7.  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.
PMCID: PMC3659006  PMID: 23642083
Brittle cornea syndrome; Corneal rupture; Blue sclera; ZNF469; PRDM5
8.  Trilateral retinoblastoma: neuroimaging characteristics and value of routine brain screening on admission 
Journal of Neuro-Oncology  2012;109(3):535-544.
Trilateral retinoblastoma (TRb) is a rare disease associating intraocular retinoblastoma with intracranial primitive neuroectodermal tumor. Treatment is difficult and prognosis is poor. This multicenter study evaluates clinical findings and MR imaging characteristics of associated intracranial tumors in Rb patients. Clinical data of 17 patients (16 TRb and 1 quadrilateral Rb patients) included time intervals between Rb and TRb diagnosis and presence of baseline brain-imaging (BBI). Two reviewers reviewed all images individually and one reviewer per center evaluated their images. Consensus was reached during a joint scoring session. Studies were reviewed for tumor location, size and imaging characteristics (signal intensity (SI) on T1- and T2-weighted images, enhancement pattern and cystic appearance). Of 18 intracranial tumors, 78 % were located in the pineal gland and 22 % suprasellar. All tumors showed well-defined borders with mostly heterogenous enhancement (72 %) and isointense SI on T1- (78 %) and T2-weighted images (72 %) compared to gray matter. The majority of pineal TRbs showed a cystic component (57 %). TRb detected synchronously with the intraocular tumors on BBI (n = 7) were significantly smaller (P = 0.02), and mainly asymptomatic than TRb detected later on (n = 10). Overall, 5-year-survival of TRb patients detected on BBI was 67 % (95 % CI 29–100 %) compared to 11 % (95 % CI 0–32 %) for the group with delayed diagnosis. TRb mainly develops in the pineal gland and frequently presents with a cystic appearance that could be misinterpreted as benign pineal cysts. Routine BBI in all newly diagnosed Rb patients can detect TRb at a subclinical stage.
PMCID: PMC3434888  PMID: 22802019
Trilateral retinoblastoma; Pineoblastoma; MR imaging; Pediatric oncology; Head and neck
9.  The PROM1 Mutation p.R373C Causes an Autosomal Dominant Bull's Eye Maculopathy Associated with Rod, Rod–Cone, and Macular Dystrophy 
Sequence variation in PROM1 should be considered in patients presenting with bull's eye maculopathy.
To characterize in detail the phenotype of five unrelated families with autosomal dominant bull's eye maculopathy (BEM) due to the R373C mutation in the PROM1 gene.
Forty-one individuals of five families of Caribbean (family A), British (families B, D, E), and Italian (family C) origin, segregating the R373C mutation in PROM1, were ascertained. Electrophysiological assessment, fundus autofluorescence (FAF) imaging, fundus fluorescein angiography (FFA), and optical coherence tomography (OCT) were performed in available subjects. Mutation screening of PROM1 was performed.
The R373C mutant was present heterozygously in all affected patients. The age at onset was variable and ranged between 9 and 58 years, with most of the individuals presenting with reading difficulties. Subjects commonly had a mild to moderate reduction in visual acuity except for members of family C who experienced markedly reduced central vision. The retinal phenotype was characterized by macular dystrophy, with retinal pigment epithelial mottling in younger subjects, progressing to typical BEM over time, with the development of macular atrophy in older patients. In addition, all members of family C had typical features of RP. The electrophysiological findings were variable both within and between families.
Mutations in PROM1 have been described to cause a severe form of autosomal recessive RP in two families of Indian and Pakistani descent. The results of this study have demonstrated that a distinct redundant PROM1 mutation (R373C) can also produce an autosomal dominant, fully penetrant retinopathy, characterized by BEM with little inter- and intrafamilial variability, and retinal dystrophy with variable rod or rod–cone dysfunction and marked intra- and interfamilial variability, ranging from isolated maculopathy without generalized photoreceptor dysfunction to maculopathy associated with very severe rod–cone dysfunction.
PMCID: PMC2941169  PMID: 20393116
10.  Alterations of the 5′Untranslated Region of SLC16A12 Lead to Age-Related Cataract 
The authors identified a new genetic factor, SLC16A12, which encodes a monocarboxylate transporter, as involved in age-related cataract. Sequence alterations in its 5′untranslated region affect translational efficiency, a potential mechanism to challenge homeostasis within the lens.
Knowledge of genetic factors predisposing to age-related cataract is very limited. The aim of this study was to identify DNA sequences that either lead to or predispose for this disease.
The candidate gene SLC16A12, which encodes a solute carrier of the monocarboxylate transporter family, was sequenced in 484 patients with cataract (134 with juvenile cataract, 350 with age-related cataract) and 190 control subjects. Expression studies included luciferase reporter assay and RT-PCR experiments.
One patient with age-related cataract showed a novel heterozygous mutation (c.-17A>G) in the 5′untranslated region (5′UTR). This mutation is in cis with the minor G-allele of the single nucleotide polymorphism (SNP) rs3740030 (c.-42T/G), also within the 5′UTR. Using a luciferase reporter assay system, a construct with the patient's haplotype caused a significant upregulation of luciferase activity. In comparison, the SNP G-allele alone promoted less activity, but that amount was still significantly higher than the amount of the common T-allele. Analysis of SLC16A12 transcripts in surrogate tissue demonstrated striking allele-specific differences causing 5′UTR heterogeneity with respect to sequence and quantity. These differences in gene expression were mirrored in an allele-specific predisposition to age-related cataract, as determined in a Swiss population (odds ratio approximately 2.2; confidence intervals, 1.23–4.3).
The monocarboxylate transporter SLC16A12 may contribute to age-related cataract. Sequences within the 5′UTR modulate translational efficiency with pathogenic consequences.
PMCID: PMC2904002  PMID: 20181839
11.  The IC3D Classification of the Corneal Dystrophies 
Cornea  2008;27(Suppl 2):S1-83.
The recent availability of genetic analyses has demonstrated the shortcomings of the current phenotypic method of corneal dystrophy classification. Abnormalities in different genes can cause a single phenotype, whereas different defects in a single gene can cause different phenotypes. Some disorders termed corneal dystrophies do not appear to have a genetic basis.
The purpose of this study was to develop a new classification system for corneal dystrophies, integrating up-to-date information on phenotypic description, pathologic examination, and genetic analysis.
The International Committee for Classification of Corneal Dystrophies (IC3D) was created to devise a current and accurate nomenclature.
This anatomic classification continues to organize dystrophies according to the level chiefly affected. Each dystrophy has a template summarizing genetic, clinical, and pathologic information. A category number from 1 through 4 is assigned, reflecting the level of evidence supporting the existence of a given dystrophy. The most defined dystrophies belong to category 1 (a well-defined corneal dystrophy in which a gene has been mapped and identified and specific mutations are known) and the least defined belong to category 4 (a suspected dystrophy where the clinical and genetic evidence is not yet convincing). The nomenclature may be updated over time as new information regarding the dystrophies becomes available.
The IC3D Classification of Corneal Dystrophies is a new classification system that incorporates many aspects of the traditional definitions of corneal dystrophies with new genetic, clinical, and pathologic information. Standardized templates provide key information that includes a level of evidence for there being a corneal dystrophy. The system is user-friendly and upgradeable and can be retrieved on the website
PMCID: PMC2866169  PMID: 19337156
corneal dystrophy; inherited corneal disease; genetic corneal disease; corneal histopathology; gene; mutation; key reference; eponym; epithelial basement membrane dystrophy; epithelial recurrent erosion dystrophy; subepithelial mucinous corneal dystrophy; Meesmann corneal dystrophy; Lisch epithelial corneal dystrophy; gelatinous drop-like corneal dystrophy; Grayson-Wilbrandt corneal dystrophy; lattice corneal dystrophy; lattice gelsolin type dystrophy; granular corneal dystrophy 1; granular corneal dystrophy 2; Avellino corneal dystrophy; Reis-Bücklers corneal dystrophy; Thiel-Behnke corneal dystrophy; macular corneal dystrophy; Schnyder corneal dystrophy; Schnyder crystalline corneal dystrophy; congenital stromal corneal dystrophy; fleck corneal dystrophy; posterior amorphous corneal dystrophy; central cloudy dystrophy of François; pre-Descemet corneal dystrophy; Fuchs endothelial corneal dystrophy; posterior polymorphous corneal dystrophy; congenital hereditary endothelial dystrophy 1; congenital hereditary endothelial dystrophy 2; X-linked endothelial corneal dystrophy
12.  Retinal Stem Cells Transplanted into Models of Late Stages of Retinitis Pigmentosa Preferentially Adopt a Glial or a Retinal Ganglion Cell Fate 
To characterize the potential of newborn retinal stem cells (RSCs) isolated from the radial glia population to integrate the retina, this study was conducted to investigate the fate of in vitro expanded RSCs transplanted into retinas devoid of photoreceptors (adult rd1 and old VPP mice and rhodopsin-mutated transgenic mice) or partially degenerated retina (adult VPP mice) retinas.
Populations of RSCs and progenitor cells were isolated either from DBA2J newborn mice and labeled with the red lipophilic fluorescent dye (PKH26) or from GFP (green fluorescent protein) transgenic mice. After expansion in EGF+FGF2 (epidermal growth factor+fibroblast growth factor), cells were transplanted intravitreally or subretinally into the eyes of adult wild-type, transgenic mice undergoing slow (VPP strain) or rapid (rd1 strain) retinal degeneration.
Only limited migration and differentiation of the cells were observed in normal mice injected subretinally or in VPP and rd1 mice injected intravitreally. After subretinal injection in old VPP mice, transplanted cells massively migrated into the ganglion cell layer and, at 1 and 4 weeks after injection, harbored neuronal and glial markers expressed locally, such as β-tubulin-III, NeuN, Brn3b, or glial fibrillary acidic protein (GFAP), with a marked preference for the glial phenotype. In adult VPP retinas, the grafted cells behaved similarly. Few grafted cells stayed in the degenerating outer nuclear layer (ONL). These cells were, in rare cases, positive for rhodopsin or recoverin, markers specific for photoreceptors and some bipolar cells.
These results show that the grafted cells preferentially integrate into the GCL and IPL and express ganglion cell or glial markers, thus exhibiting migratory and differentiation preferences when injected subretinally. It also appears that the retina, whether partially degenerated or already degenerated, does not provide signals to induce massive differentiation of RSCs into photoreceptors. This observation suggests that a predifferentiation of RSCs into photoreceptors before transplantation may be necessary to obtain graft integration in the ONL.
PMCID: PMC2823590  PMID: 17197566
13.  Variable phenotypic expressivity in a Swiss family with autosomal dominant retinitis pigmentosa due to a T494M mutation in the PRPF3 gene 
Molecular Vision  2010;16:467-475.
To characterize the clinical, psychophysical, and electrophysiological phenotypes in a five-generation Swiss family with dominantly inherited retinitis pigmentosa caused by a T494M mutation in the Precursor mRNA-Processing factor 3 (PRPF3) gene, and to relate the phenotype to the underlying genetic mutation.
Eleven affected patients were ascertained for phenotypic and genotypic characterization. Ophthalmologic evaluations included color vision testing, Goldmann perimetry, and digital fundus photography. Some patients had autofluorescence imaging, Optical Coherence Tomography, and ISCEV-standard full-field electroretinography. All affected patients had genetic testing.
The age of onset of night blindness and the severity of the progression of the disease varied between members of the family. Some patients reported early onset of night blindness at age three, with subsequent severe deterioration of visual acuity, which was 0.4 in the best eye after their fifties. The second group of patients had a later onset of night blindness, in the mid-twenties, with a milder disease progression and a visual acuity of 0.8 at age 70. Fundus autofluorescence imaging and electrophysiological and visual field abnormalities also showed some degree of varying phenotypes. The autofluorescence imaging showed a large high-density ring bilaterally. Myopia (range: −0.75 to −8) was found in 10/11 affected subjects. Fundus findings showed areas of atrophy along the arcades. A T494M change was found in exon 11 of the PRPF3 gene. The change segregates with the disease in the family.
A mutation in the PRPF3 gene is rare compared to other genes causing autosomal dominant retinitis pigmentosa (ADRP). Although a T494M change has been reported, the family in our study is the first with variable expressivity. Mutations in the PRPF3 gene can cause a variable ADRP phenotype, unlike in the previously described Danish, English, and Japanese families. Our report, based on one of the largest affected pedigree, provides a better understanding as to the phenotype/genotype description of ADRP caused by a PRPF3 mutation.
PMCID: PMC2842095  PMID: 20309403
14.  Germline mutations in retinoma patients: Relevance to low-penetrance and low-expressivity molecular basis 
Molecular Vision  2009;15:771-777.
To study phenotype-genotype correlation in patients who have retinoma, which is a benign tumor resembling the post irradiation regression pattern of retinoblastoma (RB).
We selected patients who had retinoma and positive family history for RB and patients who had retinoma in one eye and either retinoma or RB in the other eye. The study included 22 patients with available DNA: 18 from 11 families and four sporadic cases. DNA was extracted from peripheral blood leukocytes. The RB1 gene was screened by DHPLC and direct sequencing of the promoter and all the exons.
We identified 17 occurrences of 11 distinct germline mutations in two sporadic and in 15 familial cases (nine families). The 11 identified mutations were located in exons 1, 10,11,13,14, and 19 to 23. Four of the identified mutations were not previously reported, including g.64407delT, g.153236A>T, g.156743delTCTG, and g.162078delA. Eight out the 11 mutations were truncating and three were nontruncating (missense). There was no correlation between the type of mutation and the number of tumor foci per eye (RB or retinomas). Highly heterogeneous intrafamilial expressivity was observed.
To our knowledge, this study is the largest series of mutations of consecutive retinoma patients. The present data suggest that the type of inherited mutations underlying retinoma is undistinguishable from RB related ones, i.e., largely dominated by truncating mutants. This finding is in contrast with the RB1 genotypic spectrum of mutations associated with low-penetrance RB, i.e., nontruncating mutants. The molecular mechanism underlying low-penetrance and attenuated expressivity (retinomas) appeared to be distinct.
PMCID: PMC2671583  PMID: 19390654
15.  PAX6 aniridia and interhemispheric brain anomalies 
Molecular Vision  2009;15:2074-2083.
To report the clinical and genetic study of patients with autosomal dominant aniridia.
We studied ten patients with aniridia from three families of Egyptian origin. All patients underwent full ophthalmologic, general and neurological examination, and blood drawing. Cerebral magnetic resonance imaging was performed in the index case of each family. Genomic DNA was prepared from venous leukocytes, and direct sequencing of all the exons and intron–exon junctions of the Paired Box gene 6 (PAX6) was performed after PCR amplification. Phenotype description, including ophthalmic and cerebral anomalies, mutation detection in PAX6 and phenotype-genotype correlation was acquired.
Common features observed in the three families included absence of iris tissue, corneal pannus with different degrees of severity, and foveal hypoplasia with severely reduced visual acuity. In Families 2 and 3, additional findings, such as lens dislocation, lens opacities or polar cataract, and glaucoma, were observed. We identified two novel (c.170-174delTGGGC [p.L57fs17] and c.475delC [p.R159fs47]) and one known (c.718C>T [p.R240X]) PAX6 mutations in the affected members of the three families. Systemic and neurological examination was normal in all ten affected patients. Cerebral magnetic resonance imaging showed absence of the pineal gland in all three index patients. Severe hypoplasia of the brain anterior commissure was associated with the p.L57fs17 mutation, absence of the posterior commissure with p.R159fs47, and optic chiasma atrophy and almost complete agenesis of the corpus callosum with p.R240X.
We identified two novel PAX6 mutations in families with severe aniridia. In addition to common phenotype of aniridia and despite normal neurological examination, absence of the pineal gland and interhemispheric brain anomalies were observed in all three index patients. The heterogeneity of PAX6 mutations and brain anomalies are highlighted. This report emphasizes the association between aniridia and brain anomalies with or without functional impact, such as neurodevelopment delay or auditory dysfunction.
PMCID: PMC2765237  PMID: 19862335
16.  Overexpression of a mutant form of TGFBI/BIGH3 induces retinal degeneration in transgenic mice 
Molecular Vision  2008;14:1129-1137.
Despite ubiquitous expression of the keratoepithelin (KE) protein encoded by the transforming growth factor beta induced/beta induced gene human clone 3 (TGFBI/BIGH3) gene, corneal dystrophies are restricted to the cornea, and no other tissues are affected. We investigated the role of TGFBI/BIGH3 in Groenouw corneal dystrophies by generating transgenic mice overexpressing TGFBI/BIGH3 containing the R555W mutation.
Transgenic animals expressing the Groenouw mutation of human TGFBI/BIGH3 were generated using lentiviral vectors. The line expressed TGFBI/BIGH3 containing the R555W mutation under the control of the phosphoglycerate kinase (PGK) promoter. Expression of the transgene was monitored by Southern and western blotting and by RT–PCR. Electroretinogram analysis was performed and four mice were subjected to complete necroscopy.
Transgene expression was observed in different organs although without specific expression in the cornea. The overall morphology of the transgenic animals was not severely affected by KE overexpression. However, we observed an age-dependent retinal degeneration both functionally and histologically. Female-specific follicular hyperplasia in the spleen and increased levels of lipofuscin in the adrenal gland were also seen in transgenic animals.
Cellular degeneration in the retina of transgenic animals suggest that perturbation of the transforming growth factor beta (TGFβ) family regulation may affect photoreceptor survival and may induce possible accelerated aging in several tissues. No corneal phenotype could be observed, probably due to the lack of transgene expression in this tissue.
PMCID: PMC2429980  PMID: 18568131
17.  Three new PAX6 mutations including one causing an unusual ophthalmic phenotype associated with neurodevelopmental abnormalities 
Molecular Vision  2007;13:511-523.
The PAX6 gene was first described as a candidate for human aniridia. However, PAX6 expression is not restricted to the eye and it appears to be crucial for brain development. We studied PAX6 mutations in a large spectrum of patients who presented with aniridia phenotypes, Peters' anomaly, and anterior segment malformations associated or not with neurological anomalies.
Patients and related families were ophthalmologically phenotyped, and in some cases neurologically and endocrinologically examined. We screened the PAX6 gene by direct sequencing in three groups of patients: those affected by aniridia; those with diverse ocular manifestations; and those with Peters' anomaly. Two mutations were investigated by generating crystallographic representations of the amino acid changes.
Three novel heterozygous mutations affecting three unrelated families were identified: the g.572T>C nucleotide change, located in exon 5, and corresponding to the Leucine 46 Proline amino-acid mutation (L46P); the g.655A>G nucleotide change, located in exon 6, and corresponding to the Serine 74 Glycine amino-acid mutation (S74G); and the nucleotide deletion 579delG del, located in exon 6, which induces a frameshift mutation leading to a stop codon (V48fsX53). The L46P mutation was identified in affected patients presenting bilateral microphthalmia, cataracts, and nystagmus. The S74G mutation was found in a large family that had congenital ocular abnormalities, diverse neurological manifestations, and variable cognitive impairments. The 579delG deletion (V48fsX53) caused in the affected members of the same family bilateral aniridia associated with congenital cataract, foveal hypolasia, and nystagmus. We also detected a novel intronic nucleotide change, IVS2+9G>A (very likely a mutation) in an apparently isolated patient affected by a complex ocular phenotype, characterized primarily by a bilateral microphthalmia. Whether this nucleotide change is indeed pathogenic remains to be demonstrated. Two previously known heterozygous mutations of the PAX6 gene sequence were also detected in patients affected by aniridia: a de novo previously known nucleotide change, g.972C>T (Q179X), in exon 8, leading to a stop codon and a heterozygous g.555C>A (C40X) recurrent nonsense mutation in exon 5. No mutations were found in patients with Peters' anomaly.
We identified three mutations associated with aniridia phenotypes (Q179X, C40X, and V48fsX53). The three other mutations reported here cause non-aniridia ocular phenotypes associated in some cases with neurological anomalies. The IVS2+9G>A nucleotide change was detected in a patient with a microphthalmia phenotype. The L46P mutation was detected in a family with microphthalmia, cataract, and nystagmus. This mutation is located in the DNA-binding paired-domain and the crystallographic representations of this mutation show that this mutation may affect the helix-turn-helix motif, and as a consequence the DNA-binding properties of the resulting mutated protein. Ser74 is located in the PAX6 PD linker region, essential for DNA recognition and DNA binding, and the side chain of the Ser74 contributes to DNA recognition by the linker domain through direct contacts. Crystallographic representations show that the S74G mutation results in no side chain and therefore perturbs the DNA-binding properties of PAX6. This study highlights the severity and diversity of the consequences of PAX6 mutations that appeared to result from the complexity of the PAX6 gene structure, and the numerous possibilities for DNA binding. This study emphasizes the fact that neurodevelopmental abnormalities may be caused by PAX6 mutations. The neuro-developmental abnormalities caused by PAX6 mutations are probably still overlooked in the current clinical examinations performed throughout the world in patients affected by PAX6 mutations.
PMCID: PMC2649307  PMID: 17417613
18.  Lentiviral Gene Transfer of Rpe65 Rescues Survival and Function of Cones in a Mouse Model of Leber Congenital Amaurosis 
PLoS Medicine  2006;3(10):e347.
RPE65 is specifically expressed in the retinal pigment epithelium and is essential for the recycling of 11-cis-retinal, the chromophore of rod and cone opsins. In humans, mutations in RPE65 lead to Leber congenital amaurosis or early-onset retinal dystrophy, a severe form of retinitis pigmentosa. The proof of feasibility of gene therapy for RPE65 deficiency has already been established in a dog model of Leber congenital amaurosis, but rescue of the cone function, although crucial for human high-acuity vision, has never been strictly proven. In Rpe65 knockout mice, photoreceptors show a drastically reduced light sensitivity and are subject to degeneration, the cone photoreceptors being lost at early stages of the disease. In the present study, we address the question of whether application of a lentiviral vector expressing the Rpe65 mouse cDNA prevents cone degeneration and restores cone function in Rpe65 knockout mice.
Methods and Findings
Subretinal injection of the vector in Rpe65-deficient mice led to sustained expression of Rpe65 in the retinal pigment epithelium. Electroretinogram recordings showed that Rpe65 gene transfer restored retinal function to a near-normal pattern. We performed histological analyses using cone-specific markers and demonstrated that Rpe65 gene transfer completely prevented cone degeneration until at least four months, an age at which almost all cones have degenerated in the untreated Rpe65-deficient mouse. We established an algorithm that allows prediction of the cone-rescue area as a function of transgene expression, which should be a useful tool for future clinical trials. Finally, in mice deficient for both RPE65 and rod transducin, Rpe65 gene transfer restored cone function when applied at an early stage of the disease.
By demonstrating that lentivirus-mediated Rpe65 gene transfer protects and restores the function of cones in the Rpe65−/− mouse, this study reinforces the therapeutic value of gene therapy for RPE65 deficiencies, suggests a cone-preserving treatment for the retina, and evaluates a potentially effective viral vector for this purpose.
In theRpe65-/- mouse model of Leber congenital amaurosis, injection of a lentiviral vector expressing the Rpe65 mouse cDNA was able to prevent cone degeneration and restore cone function.
Editors' Summary
Leber congenital amaurosis (LCA) is the name of a group of hereditary diseases that cause blindness in infants and children. Changes in any one of a number of different genes can cause the blindness, which affects vision starting at birth or soon after. The condition was first described by a German doctor, Theodore Leber, in the 19th century, hence the first part of the name; “amaurosis” is another word for blindness. Mutations in one gene called retinal pigment epithelium-specific protein, 65 kDa (RPE65)—so called because it is expressed in the pigment epithelium, a cell layer adjacent to the light-sensitive cells, and is 65 kilodaltons in size—cause about 10% of cases of LCA. The product of this gene is essential for the recycling of a substance called 11-cis-retinal, which is necessary for the light-sensitive rods and cones of the retina to capture light. If the gene is abnormal, the sensitivity of the retina to light is drastically reduced, but it also leads to damage to the light-sensitive cells themselves.
Why Was This Study Done?
Potentially, eyes diseases such as this one could be treated by gene therapy, which works by replacing a defective gene with a normal functional one, usually by putting a copy of the normal gene into a harmless virus and injecting it into the affected tissue—in this case, the eye. The researchers here wanted to see whether expressing wild-type RPE65 using a particular type of gene vector that can carry large pieces of DNA transcript—a lentiviral vector—could prevent degeneration of cone cells and restore cone function in a mouse model of this type of LCA—mice who had had this Rpe65 gene genetically removed.
What Did the Researchers Do and Find?
Injection of the normal gene into the retina of Rpe65-deficient mice led to sustained expression of the protein RPE65 in the retinal pigment epithelium. Electrical recordings of the activity of the eyes in these mice showed that Rpe65 gene transfer restored retinal function to a near-normal level. In addition, Rpe65 gene transfer completely prevented cone degeneration until at least four months, an age at which almost all cones have degenerated in the untreated Rpe65-deficient mice.
What Do These Findings Mean?
These findings suggest that it is theoretically possible to treat this type of blindness by gene therapy. However, because this study was done in mice, many other steps need to be taken before it will be clear whether the treatment could work in humans. These steps include a demonstration that the virus is safe in humans, and experiments to determine what dose of virus would be needed and how long the effects of the treatment would last. Another question is whether it would be necessary (or even possible) to treat affected children during early childhood or when children start losing vision.
Additional Information.
Please access these Web sites via the online version of this summary at
The Foundation for Retinal Research has detailed information on Leber's congenital amaurosis
Contact a Family is a UK organization that aims to put families of children with illnesses in touch with each other
The Foundation for Fighting Blindness funds research into, and provides information about many types of blindness, including Leber's congenital amaurosis
This Web site provides information on gene therapy clinical trials, including those dedicated to cure eye diseases
This foundation provides information on diseases leading to blindness, including Leber's congenital amaurosis
PMCID: PMC1592340  PMID: 17032058
19.  Eight previously unidentified mutations found in the OA1 ocular albinism gene 
BMC Medical Genetics  2006;7:41.
Ocular albinism type 1 (OA1) is an X-linked ocular disorder characterized by a severe reduction in visual acuity, nystagmus, hypopigmentation of the retinal pigmented epithelium, foveal hypoplasia, macromelanosomes in pigmented skin and eye cells, and misrouting of the optical tracts. This disease is primarily caused by mutations in the OA1 gene.
The ophthalmologic phenotype of the patients and their family members was characterized. We screened for mutations in the OA1 gene by direct sequencing of the nine PCR-amplified exons, and for genomic deletions by PCR-amplification of large DNA fragments.
We sequenced the nine exons of the OA1 gene in 72 individuals and found ten different mutations in seven unrelated families and three sporadic cases. The ten mutations include an amino acid substitution and a premature stop codon previously reported by our team, and eight previously unidentified mutations: three amino acid substitutions, a duplication, a deletion, an insertion and two splice-site mutations. The use of a novel Taq polymerase enabled us to amplify large genomic fragments covering the OA1 gene. and to detect very likely six distinct large deletions. Furthermore, we were able to confirm that there was no deletion in twenty one patients where no mutation had been found.
The identified mutations affect highly conserved amino acids, cause frameshifts or alternative splicing, thus affecting folding of the OA1 G protein coupled receptor, interactions of OA1 with its G protein and/or binding with its ligand.
PMCID: PMC1468396  PMID: 16646960
20.  Phenotypic and molecular assessment of seven patients with 6p25 deletion syndrome: Relevance to ocular dysgenesis and hearing impairment 
BMC Medical Genetics  2004;5:17.
Thirty-nine patients have been described with deletions involving chromosome 6p25. However, relatively few of these deletions have had molecular characterization. Common phenotypes of 6p25 deletion syndrome patients include hydrocephalus, hearing loss, and ocular, craniofacial, skeletal, cardiac, and renal malformations. Molecular characterization of deletions can identify genes that are responsible for these phenotypes.
We report the clinical phenotype of seven patients with terminal deletions of chromosome 6p25 and compare them to previously reported patients. Molecular characterization of the deletions was performed using polymorphic marker analysis to determine the extents of the deletions in these seven 6p25 deletion syndrome patients.
Our results, and previous data, show that ocular dysgenesis and hearing impairment are the two most highly penetrant phenotypes of the 6p25 deletion syndrome. While deletion of the forkhead box C1 gene (FOXC1) probably underlies the ocular dysgenesis, no gene in this region is known to be involved in hearing impairment.
Ocular dysgenesis and hearing impairment are the two most common phenotypes of 6p25 deletion syndrome. We conclude that a locus for dominant hearing loss is present at 6p25 and that this locus is restricted to a region distal to D6S1617. Molecular characterization of more 6p25 deletion patients will aid in refinement of this locus and the identification of a gene involved in dominant hearing loss.
PMCID: PMC455682  PMID: 15219231
22.  Classification and Management of Seeds in RetinoblastomaEllsworth Lecture Ghent August 24th 2013 
Ophthalmic Genetics  2014;35(4):193-207.
Retinoblastoma has the unique capacity to accelerate its own intra-ocular propagation by adopting semi-solid or even liquid growth properties through seeding. Until recently, the presence of any degree of seeding was mostly incompatible with successful conservative management, due to the multiresistant nature of the seeds. Surprisingly, this well-recognized retinoblastoma behavior has not undergone any detailed description of seeding patterns and anatomic sites. In this paper, we describe the phenotypic variability of seeds across the four possible intraocular seeding compartments and classify them into three fundamental types: namely dust, spheres, and clouds. We also provide an overview of the different therapeutic strategies developed for seeding, with special attention to intravitreal chemotherapy as the treatment of choice for vitreous and retro-hyaloid seeding. Finally, we propose criteria to enable assessment of the response to treatment by reporting seed regression patterns, as well as a clinical grading system for the retinal toxicity observed following intravitreal melphalan.
PMCID: PMC4245997  PMID: 25321846
Classification; intravitreal chemotherapy; retinoblastoma; seeding
23.  Differential diagnosis of leukocoria and strabismus, first presenting signs of retinoblastoma 
Leukocoria in infants is always a danger signal as retinoblastoma, a malignant retinal tumor, is responsible for half of the cases in this age group. More common signs should also be considered suspicious until proved otherwise, such as strabismus, the second most frequent sign of retinoblastoma. Less frequent manifestations are inflammatory conditions resistant to treatment, hypopyon, orbital cellulitis, hyphema or heterochromia. Other causal pathologies, including persistent hyperplastic primary vitreous (PHPV), Coats’ disease, ocular toxocariasis or retinopathy of prematurity, may also manifest the same warning signs and require specialized differential diagnosis. Members of the immediate family circle are most likely to notice the first signs, the general practitioner, pediatrician or general ophthalmologist the first to be consulted. On their attitude will depend the final outcome of this vision and life-threatening disease. Early diagnosis is vital.
PMCID: PMC2704541  PMID: 19668520
leukocoria; strabismus; retinoblastoma; Coats’ disease; persistent hyperplastic primary vitreous (PHPV)

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