Remodeling of the human retina in choroideremia: rab escort protein 1 (REP-1) mutations

PURPOSE: To characterize in detail the disease expression in choroideremia (CHM), a blinding X-linked disease of the retina caused by loss-of-function mutations in Rab Escort Protein 1 (REP-1). CHM is readily diagnosed in the clinic and by molecular testing but has lacked an animal model to test hypotheses and therapeutics. The recent report of a mouse model for CHM prompts the need for reassessment of the human disease in anticipation of treatment initiatives. METHODS: CHM hemizygotes with REP-1 mutations, spanning an age range of 7 decades, were studied with in vivo microscopy by optical coherence tomography. RESULTS: The disease expression was complex. Earliest stages involved a thickening of the retina that was otherwise normally laminated. Loss of photoreceptors, either independent or associated with retinal pigment epithelium (RPE) depigmentation, was followed by disorganization and further thickening of the retina with interlaminar bridges. The dysmorphic retina then slowly thinned over decades. Laminopathy occurred first in more peripheral rod-rich regions and later in the cone-rich fovea. CONCLUSIONS: The CHM disease sequence involves detectable retinal thickening, which may be due to Müller cell activation and hypertrophy from photoreceptor stress. Photoreceptor degeneration, RPE depigmentation, and retinal remodeling follow. The results represent in vivo evidence in humans for retinal remodeling and provide a marker for the earliest stage of this response to genetic retinal disease. For CHM and other candidate human retinopathies considered for therapy, there is now a framework for making informed decisions about timing, retinal location, and potential value of treatment.     

Cytochrome P450 1B1 gene mutations in Japanese patients with primary congenital glaucoma(1)

PURPOSE: To report a novel missense mutation and DNA polymorphism of the CYP1B1gene in Japanese patients with primary congenital glaucoma. METHODS: A series of 11 unrelated patients with primary congenital glaucoma was examined. Patients were followed in the Kagoshima University Hospital between 1979 and 1998. DNA was extracted from leukocytes of the patients, their families, and unrelated healthy individuals. Amplicons spanning the coding regions of the CYP1B1 gene were examined by direct sequencing and enzyme-restriction detection. RESULTS: In the 11 unrelated patients, besides the previously reported insertional mutation (1620 ins G), a novel missense mutation was identified at codons 444 to replace arginine with glutamine (R444Q) in one patient. The novel missense mutation cosegregated in the relevant family as an autosomal recessive pattern and was not found in other patients or control individuals. In addition, five polymorphic sites were found at codons 48, 119, 330, 432, and 449. These polymorphic alleles did not cosegregate with the disease, and they were found in healthy individuals as well. CONCLUSIONS: Approximately 20% of Japanese patients with primary congenital glaucoma may be affected by mutations in the CYP1B1 gene. Further studies are justified to explore whether a relationship exists between the phenotypic expressivity of the disease and the type of mutation.     

Novel mutations in the GRK1 gene in Japanese patients With Oguchi disease

PURPOSE: To report novel mutations in the GRK1 gene in Japanese patients with Oguchi disease. DESIGN: Observational case report. METHODS: Two unrelated Japanese patients with Oguchi disease were examined. After informed consent was obtained, the coding regions of SAG and GRK1 were analyzed by direct sequencing. RESULTS: Although no mutation was found in SAG, two novel homozygous mutations in GRK1, c.1079 del T and c.1408-1412 CCCCC to CCC, were identified. Both mutations are expected to generate null alleles of GRK1. CONCLUSIONS: The authors found two different novel mutations in Japanese patients. The results indicate that a considerable number of GRK1 mutations exist in the Japanese population.     

Screening for mutations in the IMPDH1 gene in Japanese patients with autosomal dominant retinitis pigmentosa

PURPOSE: To determine the presence and frequency of mutations in the IMPDH1 gene in Japanese patients with autosomal dominant retinitis pigmentosa (ADRP), and to characterize the clinical characteristics of patients with the Lys238Arg mutation in the IMPDH1 gene. DESIGN: Case reports and results of DNA analysis. METHODS: All 14 coding exons of the IMPDH1 gene were directly sequenced in 96 unrelated patients with ADRP. The clinical features were determined by visual acuity, slit-lamp biomicroscopy, and kinetic visual field tests. RESULTS: Two novel mutations, a Leu227Pro and Lys238Arg, in the IMPDH1 gene were identified in two unrelated families with ADRP. The clinical features associated with the Lys238Arg mutation were an early-onset and severe retinal degeneration. CONCLUSIONS: The most commonly reported Asp226Asn mutation was not found in the Japanese population, instead two novel mutations were found. These findings suggest that mutations of the IMPDH1 gene cause ADRP in the Japanese population.     

Four Japanese male patients with juvenile retinoschisis: only three have mutations in the RS1 gene

PURPOSE: To describe the clinical phenotypes of four unrelated Japanese male patients with juvenile retinoschisis and to investigate occurrences of mutations in the RS1 gene. DESIGN: Observational case series and experimental study. METHODS: Fundus examinations, fluorescein angiography, and single-flash electroretinography (ERG) were carried out. In one patient, optical coherence tomography (OCT) was performed. The coding regions of the RS1 gene that encodes retinoschisin were amplified by polymerase chain reaction (PCR). The PCR products were purified and directly sequenced. RESULTS: The four affected patients showed cystoid- or wheel-like foveal changes with a little or no fluorescein leakage and negative b-wave patterns in both eyes. The OCT images of foveal retinoschisis disclosed that splitting occurs in the putative fibers of Henle. In three patients, we identified three different missense mutations (p.S73P, p.Y89C, p.R209C) in the functionally important discoidin domain of the RS1 gene. The p.S73P mutation has not been previously reported. In contrast, no nucleotide substitutions were detected in the fourth patient whose parents were unrelated and asymptomatic. No other member of this family for three generations has had juvenile retinoschisis. CONCLUSION: Because serine 73 is conserved in the mouse ortholog and other discoidin proteins, the proline 73 allele is therefore very likely to encode a defective retinoschisin. Although the inheritance pattern is uncertain in the patient without the RS1 mutation, the clinical and ERG findings were indistinguishable from those of patients with RS1 mutations. This finding points to the genetic heterogeneity of juvenile retinoschisis.     

ABCA4 gene mutations in Japanese patients with Stargardt disease and retinitis pigmentosa

PURPOSE: To evaluate photoreceptor cell-specific adenosine triphosphate (ATP)-binding cassette transporter (ABCA4) gene mutations in Japanese patients with Stargardt disease (STGD) and the correlation of these mutations to clinical phenotypes. METHODS: Serum was obtained from 10 unrelated Japanese patients with STGD and 96 unrelated Japanese patients with autosomal recessive retinitis pigmentosa (arRP). All 50 ABCA4 gene exons of the patients with STGD were screened for mutations by a combination of single-strand conformation polymorphism analysis and polymerase chain reaction (PCR) direct-sequencing techniques. By restriction enzyme digestion, primer extension analysis, and PCR direct sequencing techniques, the patients with arRP were screened for three segregated, presumably null ABCA4 gene mutations observed in Japanese patients with STGD. RESULTS: Three novel, presumably null mutations of the ABCA4 gene, IVS7-45_952delinsTCTGACC, IVS12+2T-->G, and 1894delA, were identified. The Arg2149stop mutation that had been found in a white patient with STGD in a prior study was also found in a Japanese patient. Two arRP-affected siblings and two unrelated patients with STGD were found to be homozygous for the same IVS12+2T-->G mutation, and three other arRP-affected siblings were carriers of the IVS12+2T-->G mutation and/or the IVS7-45_952delinsTCTGACC mutation. These three siblings with arRP showed only atrophic degeneration in the macula early after the onset of the disease, and STGD had been diagnosed. CONCLUSIONS: Three novel ABCA4 gene mutations were identified in Japanese patients with STGD and arRP. Mutations in the ABCA4 gene can cause panretinal degeneration that changes its clinical appearance from STGD to arRP over time.     

New type of mutations in three spanish families with choroideremia

PURPOSE: Choroideremia (CHM) is an X-linked ophthalmic disease. The gene associated with CHM (REP-1) encodes a ubiquitously expressed protein that is indispensable for the posttranslational activation of retina-specific Rab protein. Different mutations, including large genomic rearrangements involving the REP-1 gene, are responsible for CHM, but they all cause the protein to be truncated or absent. The authors screened 20 Spanish families with clinical diagnoses of CHM to determine the molecular cause of the disease. METHODS: First, the authors performed haplotype analyses to determine whether the disease is linked to the REP-1 gene. In families in whom the disease segregated with the CHM locus (n = 14), mutational screening of the REP-1 gene was performed. RESULTS: In 13 of the 14 families in which the phenotype segregated with the CHM locus, the authors identified the mutation associated with the disease. Eight different molecular defects that led to truncation and one that led to complete absence of the REP-1 protein were found in nine families and one family, respectively. Furthermore, the authors identified a novel type of mutation in the REP-1 gene in three families. This novel type of mutation did not result in a truncated or absent protein. Rather, these patients lost different parts of the REP-1 mRNA in-frame that in all the cases encode a conserved protein domain implicated in the interaction with Rab proteins. CONCLUSIONS: Based on the different mutations found, the authors propose a four-step protocol for the molecular diagnosis of CHM.     

Novel cytochrome P4501B1 (CYP1B1) gene mutations in Japanese patients with primary congenital glaucoma

PURPOSE: To investigate CYP1B1 gene mutations in Japanese patients with primary congenital glaucoma (PCG). METHODS: Sixty-five unrelated Japanese patients with PCG were screened by PCR-single-strand conformational polymorphism (SSCP) analysis followed by direct sequencing. No patients were offspring of consanguineous marriages, a common occurrence among patients in previous reports. PCG haplotypes were constructed with intragenic polymorphisms in affected individuals. Three-dimensional atomic structures of human CYP1B1 and four mutant CYP1B1 sequences representing missense mutations were assembled using homology modeling and were regularized by an energy-minimization procedure. RESULTS: Eleven novel mutations, including seven definite and four probable mutations, were detected in 13 (20%) of the 65 unrelated patients. Of the seven definite mutations, three were predicted to truncate the CYP1B1 open reading frame. The other four were missense mutations (Asp192Val, Ala330Phe, Val364Met, and Arg444Gln), all located in conserved core structures determining proper folding and heme-binding ability of cytochrome P450 molecules. Molecular modeling demonstrated that two of four mutations in positions 330 and 364 were structurally neutral, but Arg444Gln caused significant structural change. Of the four probable mutations, three were missense (Val198Ile, Val320Leu, and Glu499Gly); the other was a base substitution in the noncoding region of exon 1. CONCLUSIONS: The 11 varied CYP1B1 mutations found in 13 unrelated Japanese patients with sporadic occurrence of PCG represent an allelic heterogeneity and may be unique to a specific population.     

OPA1 gene mutations in Japanese patients with bilateral optic atrophy unassociated with mitochondrial DNA mutations at nt 11778, 3460, and 14484

PURPOSE: To report mutations in the OPA1 gene in Japanese patients with bilateral optic atrophy unassociated with mitochondrial DNA mutations at nt 11778, 3460, and 14484. METHODS: Twelve unrelated patients with bilateral optic atrophy and 100 healthy controls were examined. Each exon of the OPA1 gene was amplified by polymerase chain reaction (PCR). All PCR products were sequenced. RESULTS: Of the 12 patients, 2 had nonsense mutations of the OPA1 gene (nt 1039G --> T and nt 1096C --> T, leading to Glu347Stop and Arg366Stop, respectively). These nonsense mutations were not found in the 100 healthy controls. Two of the patients had silent mutations of OPA1 gene (nt 1177T --> G and nt 1923G --> A causing no amino acid change). CONCLUSIONS: The mutations (Glu347Stop and Arg366Stop) of the OPA1 gene are involved in the pathogenesis of bilateral optic atrophy in Japanese patients.     

Phenotype of cytochrome P4501B1 gene (CYP1B1) mutations in Japanese patients with primary congenital glaucoma

AIM: To investigate the phenotypes associated with cytochrome P4501B1 gene (CYP1B1) mutations in Japanese patients with primary congenital glaucoma (PCG). METHODS: 66 Japanese patients with PCG were screened for sequence mutations in the CYP1B1 gene using single strand conformation polymorphism analysis followed by automated DNA sequencing. 11 cases had a CYP1B1 mutation in both alleles (the mutation group) and 21 cases did not have a CYP1B1 mutation (the "no mutation" group). The clinical features, such as age of onset, sex, intraocular pressure, and Descemet's membrane rupture, of the two groups were compared. RESULTS: The clinical symptoms and signs did not differ for the two groups. The mean age at onset was 1.7 months in the mutation group and 3.1 months in the no mutation group, and the male:female ratio was 6:5 in the mutation group and 19:2 in the no mutation group. Both of these differences were statistically significant. CONCLUSIONS: In clinically diagnosed cases of PCG, a subgroup shows a CYP1B1 gene mutation. Age at onset was earlier in PCG patients with CYP1B1 mutations than in patients without mutations. Women were more prevalent among patients with mutations than those without mutations.     

Novel mutations in Norrie disease gene in Japanese patients with Norrie disease and familial exudative vitreoretinopathy

PURPOSE: To search for mutations in the Norrie disease gene (NDP) in Japanese patients with familial exudative vitreoretinopathy (FEVR) and Norrie disease (ND) and to delineate the mutation-associated clinical features. METHODS: Direct sequencing after polymerase chain reaction of all exons of the NDP gene was performed on blood collected from 62 probands (31 familial and 31 simplex) with FEVR, from 3 probands with ND, and from some of their family members. The clinical symptoms and signs in the patients with mutations were assessed. X-inactivation in the female carriers was examined in three FEVR families by using leukocyte DNA. RESULTS: Four novel mutations-I18K, K54N, R115L, and IVS2-1G-->A-and one reported mutation, R97P, in the NDP gene were identified in six families. The severity of vitreoretinopathy varied among these patients. Three probands with either K54N or R115L had typical features of FEVR, whereas the proband with R97P had those of ND. Families with IVS2-1G-->A exhibited either ND or FEVR characteristics. A proband with I18K presented with significant phenotypic heterogeneity between the two eyes. In addition, affected female carriers in a family harboring the K54N mutation presented with different degrees of vascular abnormalities in the periphery of the retina. X-inactivation profiles indicated that the skewing was not significantly different between affected and unaffected women. CONCLUSIONS: These observations indicate that mutations of the NDP gene can cause ND and 6% of FEVR cases in the Japanese population. The X-inactivation assay with leukocytes may not be predictive of the presence of a mutation in affected female carriers.     

Novel mutations in the arrestin gene and associated clinical features in Japanese patients with Oguchi's disease

PURPOSE: Only 2 mutations in the arrestin gene have been previously reported to be associated with Oguchi's disease, a homozygous Asn309(1-bp del) mutation in Japanese families and a homozygous Arg193stop mutation in an Indian family. The aim of this article is to report 2 novel mutations in the arrestin gene in 2 Japanese patients with Oguchi's disease and to describe the clinical features with the mutations. DESIGN: Molecular genetic study and observational case report. PARTICIPANTS: Two unrelated Japanese patients with Oguchi's disease associated with novel arrestin mutations. METHODS: Genomic DNA was extracted from leukocytes of the peripheral blood, and exons 2 through 16 of the arrestin gene were amplified by polymerase chain reaction and directly sequenced. A complete ophthalmologic examination was performed, including best-corrected visual acuity, slit-lamp and fundus examinations, fundus photography, and electroretinography (ERG). MAIN OUTCOME MEASURES: Direct sequencing of the arrestin gene, evaluation of visual acuity, refraction, and ERG. RESULTS: Three arrestin gene mutations were identified in 2 patients. A compound heterozygous mutation, Arg175stop and Asn309(1-bp del), was identified in 1 patient with Oguchi's disease. The former mutation has not been reported, whereas the latter is known to be a frequent mutation in Oguchi's disease in Japanese families. In a second patient, another novel mutation was detected in the gene, a homozygous Arg292stop mutation. Both patients demonstrated characteristic features of Oguchi's disease, including night blindness, golden-yellow discoloration of the retina, absent rod ERG response, and "negative" type bright-flash ERG after 30 minutes of dark adaptation. CONCLUSIONS: The existence of 2 novel mutations of the arrestin gene in 2 unrelated Japanese patients strongly supports the previous data that arrestin gene mutations are associated with Oguchi's disease. All of the mutations in the arrestin gene that have been identified in Oguchi's disease are null mutations, indicating that only critical gene defects in the arrestin gene are associated with Oguchi's disease.