Altered expression of β-galactosidase-1-like protein 3 (Glb1l3) in the retinal pigment epithelium (RPE)-specific 65-kDa protein knock-out mouse model of Leber’s congenital amaurosis

Purpose: In this study, we investigated the expression of the gene encoding beta-galactosidase (Glb)-1-like protein 3 (Glb1l3), a member of the glycosyl hydrolase 35 family, during retinal degeneration in the retinal pigment epithelium (RPE)-specific 65-kDa protein knockout (Rpe65(-/-)) mouse model of Leber congenital amaurosis (LCA). Additionally, we assessed the expression of the other members of this protein family, including beta-galactosidase-1 (Glb1), beta-galactosidase-1-like (Glb1l), and beta-galactosidase-1-like protein 2 (Glb1l2).Methods: The structural features of Glb1l3 were assessed using bioinformatic tools. mRNA expression of Glb-related genes was investigated by oligonucleotide microarray, real-time PCR, and reverse transcription (RT) -PCR. The localized expression of Glb1l3 was assessed by combined in situ hybridization and immunohistochemistry.Results: Glb1l3 was the only Glb-related member strongly downregulated in Rpe65(-/-) retinas before the onset and during progression of the disease. Glb1l3 mRNA was only expressed in the retinal layers and the RPE/choroid. The other Glb-related genes were ubiquitously expressed in different ocular tissues, including the cornea and lens. In the healthy retina, expression of Glb1l3 was strongly induced during postnatal retinal development; age-related increased expression persisted during adulthood and aging.Conclusions: These data highlight early-onset downregulation of Glb1l3 in Rpe65-related disease. They further indicate that impaired expression of Glb1l3 is mostly due to the absence of the chromophore 11-cis retinal, suggesting that Rpe65 deficiency may have many metabolic consequences in the underlying neuroretina

Germline mutations in retinoma patients: Relevance to low-penetrance and low-expressivity molecular basis

PURPOSE: To study phenotype-genotype correlation in patients who have retinoma, which is a benign tumor resembling the post irradiation regression pattern of retinoblastoma (RB). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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

Differential neuroglycan C expression during retinal degeneration in Rpe65−/− mice

PURPOSE: An increased mRNA expression of the genes coding for the extracellular matrix proteins neuroglycan C (NGC), interphotoreceptor matrix proteoglycan 2 (IMPG2), and CD44 antigen (CD44) has been observed during retinal degeneration in mice with a targeted disruption of the Rpe65 gene (Rpe65-/- mouse). To validate these data, we analyzed this differential expression in more detail by characterizing retinal NGC mRNA isoform and protein expression during disease progression. METHODS: Retinas from C57/Bl6 wild-type and Rpe65-/- mice, ranging 2 to 18 months of age, were used. NGC, IMPG2, and CD44 mRNA expression was assessed by oligonucleotide microarray, quantitative PCR, and in situ hybridization. Retinal NGC protein expression was analyzed by western blot and immunohistochemistry. RESULTS: As measured by quantitative PCR, mRNA expression of NGC and CD44 was induced by about 2 fold to 3 fold at all time points in Rpe65-/- retinas, whereas initially 4 fold elevated IMPG2 mRNA levels progressively declined. NGC and IMPG2 mRNAs were expressed in the ganglion cell layer, the inner nuclear layer, and at the outer limiting membrane. NGC mRNA was also detected in retinal pigment epithelium cells (RPE), where its mRNA expression was not induced during retinal degeneration. NGC-I was the major isoform detected in the retina and the RPE, whereas NGC-III was barely detected and NGC-II could not be assessed. NGC protein expression was at its highest levels on the apical membrane of the RPE. NGC protein levels were induced in retinas from 2- and 4-month-old Rpe65-/- mice, and an increased amount of the activity-cleaved NGC ectodomain containing an epidermal growth factor (EGF)-like domain was detected. CONCLUSIONS: During retinal degeneration in Rpe65-/- mice, NGC expression is induced in the neural retina, but not in the RPE, where NGC is expressed at highest levels

Variable phenotypic expressivity in a Swiss family with autosomal dominant retinitis pigmentosa due to a T494M mutation in the PRPF3 gene

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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

PAX6 aniridia and interhemispheric brain anomalies

Purpose: To report the clinical and genetic study of patients with autosomal dominant aniridia

RAX and anophthalmia in humans: Evidence of brain anomalies

PURPOSE: To report the clinical and genetic study of two families of Egyptian origin with clinical anophthalmia. To further determine the role of the retina and anterior neural fold homeobox gene (RAX) in anophthalmia and associated cerebral malformations. METHODS: Three patients with clinical anophthalmia and first-degree relatives from two consanguineous families of Egyptian origin underwent full ophthalmologic, general and neurologic examination, and blood tests. Cerebral magnetic resonance imaging (MRI) was performed in the index cases of both families. Genomic DNA was prepared from venous leukocytes, and direct sequencing of all the exons and intron-exon junctions of RAX was performed after PCR amplification. RESULTS: Clinical bilateral anophthalmia was observed in all three patients. General and neurologic examinations were normal; obesity and delay in psychomotor development were observed in the isolated case. Orbital MRI showed a hypoplastic orbit with present but rudimentary extraocular muscles and normal lacrimal glands. Cerebral MRI showed agenesis of the optic nerves, optic tracts, and optic chiasma. In the index case of family A, the absence of the frontal and sphenoidal sinuses was also noted. In the index case of family B, only the sphenoidal sinus was absent, and there was significant cortical atrophy. The three patients carried a novel homozygous c.543+3A>G mutation (IVS2+3A>G) in RAX. Parents were healthy heterozygous carriers. No mutations were detected in orthodenticle homeobox 2 (OTX2), ventral anterior homeobox 1 (VAX1), or sex determining region Y-box 2 (SOX2). CONCLUSIONS: This is the first report of a homozygous splicing RAX mutation associated with autosomal recessive bilateral anophthalmia. To our knowledge, only two isolated cases of anophthalmia, three null and one missense case affecting nuclear localization or the DNA-binding homeodomain, have been found to be caused by compound heterozygote RAX mutations. A novel missense RAX mutation was identified in three patients with bilateral anophthalmia and a distinct systemic and neurologic phenotype. The mutation potentially affects splicing of the last exon and is thought to result in a protein that has an aberrant homeodomain and no paired-tail domain. Functional consequences of this change still need to be characterized

Compound Heterozygous VSX2 Mutation Causing Bilateral Anophthalmia in a Consanguineous Egyptian Family

Purpose: To report the clinical and genetic study of a child with bilateral anophthalmia. Methods: A 14-year-old Egyptian boy, born from consanguineous parents, underwent a general and a full ophthalmological examination. Mutation screen of the A/M genes with recessive inheritance was done stepwise and DNA was analyzed by Sanger sequencing. Results: Bilateral anophthalmia, arachnodactyly of the feet and high arched palate were observed on general examination. The parents were first cousins and healthy. Sequencing analysis revealed a novel compound heterozygous mutation in one of the copy of exon 2 of VSX2 and a possible deletion of at least exon 2 on the other allele. Conclusions: A compound heterozygous VSX2 mutation associated with anophthalmia was identified in a patient from an Egyptian consanguineous family. This report brings the number of VSX2 mutation in anophthalmia/microphthalmia (A/M) to 13. Functional consequences of the reported changes still need to be characterized, as well as the percentage of A/M caused by mutations in the VSX2 gene. This family also shows that despite consanguinity, heterozygous mutations can also happen and one should not restrict the molecular analysis to homozygous mutations

Overexpression of a mutant form of TGFBI/BIGH3 induces retinal degeneration in transgenic mice

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Cellular degeneration in the retina of transgenic animals suggest that perturbation of the transforming growth factor beta (TGFbeta) 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

Enrichment of pathogenic alleles in the brittle cornea gene, ZNF469, in keratoconus

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

Enrichment of pathogenic alleles in the brittle cornea gene, ZNF469, in keratoconus

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 dat