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Purpose: Retinitis pigmentosa (RP; MIM 268000) is a hereditary disease characterized by poor night vision and progressive loss of photoreceptors, eventually leading to blindness. This degenerative process primarily affects peripheral vision due to the loss of rods. Autosomal recessive RP (arRP) is clinically and genetically heterogeneous. It has been associated with mutations in different genes, including CRB1 (crumbs homolog 1). The aim of this study was to determine the causative gene in a Tunisian patient with arRP born to non-consanguineous parents., Methods: Four accessible family members were included. They underwent full ophthalmic examination with best-corrected Snellen visual acuity, fundus photography and fluorescein angiography. Haplotype analysis was used to evaluate homozygosity in the family to 20 arRP loci. All exons and intron-exon junctions of candidate genes not excluded by haplotype analysis were PCR amplified and directly sequenced., Results: The proband was a 43-year-old female patient. Best-corrected visual acuity was 20/63 (right eye) and 20/80 (left eye). Visual loss began during the third decade. Funduscopic examination and fluorescein angiography revealed typical advanced RP changes with bone spicule-like pigment deposits in the posterior pole and the midperiphery along with retinal atrophy, narrowing of the vessels, and waxy optic discs. Haplotype analysis revealed homozygosity with microsatellite markers D1S412 and D1S413 on chromosome 1q31.3. These markers flanked CRB1. Our results excluded linkage of all the other arRP loci/genes tested. Sequencing of the 12 coding exons and splice sites of CRB1 disclosed a homozygous missense mutation in exon 7 at nucleotide c. 2291G>A, resulting in an arginine to histidine substitution (p.R764H)., Conclusions: R764H is a novel mutation associated with CRB1-related arRP. Previously, an R764C mutation was reported. Extending the mutation spectrum of CRB1 with additional families is important for genotype-phenotype correlations and characterization of the scope of mutation.

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.

Purpose: To report the clinical and genetic study of patients with autosomal dominant aniridia., Methods: 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., Results: 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., Conclusions: 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.

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.

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.

Purpose: To study phenotype-genotype correlations in 65 retinoblastoma patients, who were seen between March 2004 and January 2006 and to report undescribed retinoblastoma 1 (RB1) mutations identified in ten additional patients in whom mutations were detected before 2004., Methods: Complete ophthalmic examinations were performed in all patients and their parents. DNA was extracted from peripheral blood leukocytes, and the RB1 gene was screened by denaturing high-performance liquid chromatography and direct sequencing of the promoter and all the exons., Results: Seven cases were familial, 30 were sporadic bilateral, and 28 were sporadic unilateral. Screening of the RB1 gene resulted in the identification of four mutations in the familial cases (57%), 22 in the sporadic bilateral cases (73%), and three in the sporadic unilateral cases (10.7%). Twenty-two mutations, were single-base substitutions (76%). Of these mutations, 68% were of the nonsense type (15 cases). Ten patients with bilateral retinoblastoma in whom ten mutations were detected in a non-systematic approach between 1995 and 1998 were added to our recent series. In total, ten novel mutations were identified, including four single base substitutions, four small deletions and two small duplications. These are g.39445G>A, g.41924A>G, g.56851A>G, g.156795T>G, g.41983delT, g.44699_44706delAGCAGTTC, g.73788_73789delAA, g.78253delA, g.2157dupC, and g.2179_2183dupGGACC. Two patients had dysmorphic features associated with 13q14 large deletions., Conclusions: The detection rates of 73% in the sporadic bilateral cases and of 10.7% in the sporadic unilateral cases in our series are in accordance with recently published literature. Our pattern of mutations confirms the predominantly gene-inactivating mutations, i.e. single-base non-sense mutations and splice site mutations.

Purpose: To investigate the location and tissue-specificity of the pathologic keratoepithelin (KE) deposition in a patient with a keratoepithelinopathy (KEP), TGFBI/BIGH3-related corneal dystrophy., Methods: An autopsy was performed in a patient with lattice type I corneal dystrophy (LCDI) after authorization was obtained from the family. Mutation screening in TGFBI/BIGH3 was done on the patient several years ago. Eighteen different tissues or organs, including brain, heart, lung, kidney, liver, lymph nodes, spleen, aorta, esophagus, bone marrow, urinary bladder (including a papillary urothelial carcinoma), samples of a metastatic squamous cell carcinoma, adrenal gland, parathyroid gland, muscle, prostate, and cornea were investigated, and sections from the tissues were labeled with KE2 rabbit TGFBI/BIGH3 antiserum., Results: The patient, diagnosed with LCDI and Alzheimer's disease, died at 79 years of age from a complicated chronic obstructive lung disease. Mutation analysis showed the classical Arg124Cys mutation in exon 4 of TGFBI/BIGH3, associated with LCDI. Except for the cornea, immunostaining with KE2 antisera did not reveal any deposits in any of the 17 other organs analyzed., Conclusions: Pathologic deposits caused by KE accumulation were only observed in the cornea and in no other tissue or organ in this patient. These results suggest a cornea-specific mechanism in the aggregation of KE. Further studies need to be done to investigate whether the degradation of mutated KE generates cornea-specific fragments that aggregate or whether the clearing of normal fragments is different in affected corneas, which then leads to aggregation.