CRB1 Research Articles

Threatened fishes of the world: Pseudophoxinus egridiri (Karaman, 1972) (Cyprinidae)

Common name: Egirdir akca cicek baligi (Tr), Orient minnow (En), Egridir pfrille (Al), Veron d’oriantal (Fr), Amarante di oriente (It), Daska Turko (Is) (Artuz, 2005). Conservation status: Critically Endangered CR B1 ab (i,ii,iv,v) + 2 ab (i,ii,iv,v) (Crivelli and Erk’akan 2006). Identification: D III 8, A III 7, Lateral line scales 43–54, pored lateral line scales 2, pharyngeal teeth 5-4, gill-rakers 7–9 (10), total number of vertebra 35–36 (rarely 34 or 37) (Bogutskaya 1992). Distribution: P. egridiri is restricted to the tributaries of Lake Egirdir in Central Anatolia, Turkey. Abundance: Rare populations in Lake Egirdir (Kuru et al. 2001). Habitat and ecology: It occurs in temperate lentic biotopes and streams with plentiful vegetation. Feeds especially on insect larvae (Geldiay and Balik 2007). Reproduction: There is no information on the reproduction of this species. Threats: Introduction of some exotic fishes [in 1955 Sander lucioperca, in 1970 Gambusia affinis, in 1994 Carassius auratus, in 1996 Tinca tinca, in 1999 Knipowitschia caucasica, in 2002 Atherina boyeri (Kesici et al. 2006)] to the lake in addition to habitat loss, eutrophication and pollution, (Fricke et al. 2007). Conservations action: No conservation actions have been implemented. Conservation recommendations: Detailed information on biology and ecology of this species is required. After introduction of Sander lucioperca, P. egridiri disappeared from the area. However the species has been recorded from some of the streams connected to the lake. Predation by S. lucioperca must be controlled and limited the permit survival of the species. Remarks: This species is endemic with a restricted distribution in Turkey.

CRB1Gene Mutations Are Associated with Keratoconus in Patients with Leber Congenital Amaurosis

To present an association of mutations in the CRB1 gene with keratoconus in patients with Leber congenital amaurosis (LCA). Sixteen patients with genotyped LCA (having the CRB1, CRX, RetGC, RPE65, and AIPL1 mutations) were recruited from one ophthalmology practice and examined for the presence of keratoconus. Corneal topography, visual acuity, and slit lamp biomicroscopic examination were performed in all cases. The mean age of the patients was 34.5 years (range, 13-74). Visual acuities ranged from 20/40 to light perception. Corneal topography was successfully collected in 15 of the cases. Five of the 16 cases had slit lamp and/or topographic features consistent with keratoconus. One patient had a clinical picture that was keratoglobus-like. Of these six cases, four had a CRB1 mutation and two had a CRX mutation. Of the three subjects with the CRX mutation, one had keratoconus, one had the keratoglobus-like presentation, and one was normal. Our cohort represents 14 separate, unrelated families. Only one family comprised multiple members with LCA. These were three affected brothers, one with keratoconus, all with CRB1 mutations. Although the results cannot exclude other gene mutations, they suggest that LCA patients with a CRB1 mutation may have a particular susceptibility to keratoconus.

Genetic heterogeneity in two consanguineous families segregating early onset retinal degeneration: The pitfalls of homozygosity mapping

Retinitis pigmentosa is the most common form of hereditary retinal degeneration, with a worldwide prevalence of 1 in 4,000. At least 28 genes and loci have been implicated in nonsyndromic autosomal recessive retinitis pigmentosa. Here we report two extended and highly consanguineous families segregating early onset retinitis pigmentosa. Despite the consanguinity in both families, we found allelic heterogeneity in one of them, in which affected individuals were compound heterozygotes for two different mutations of the CRB1 gene. In the second family we found evidence for locus heterogeneity. A novel homozygous mutation of RDH12 was found in only 14 of 17 affected individuals in this family. Our data indicate that in the other affected individuals the disease is caused by a different gene/s. These findings demonstrate that while homozygosity mapping is an efficient tool for identification of the underlying mutated genes in inbred families, both locus and allelic heterogeneity may occur even within the same consanguineous family. These observations should be taken into account, especially when studying common and heterogeneous recessive genetic conditions.

Case Report: Autofluorescence Imaging and Phenotypic Variance in a Sibling Pair with Early-Onset Retinal Dystrophy Due to Defective CRB1 Function

Purpose: To phenotype two siblings with autosomal recessive early-onset retinal dystrophy due to CRB1 mutations. Methods: Autofluorescence (AF) imaging, high resolution optical coherence tomography (OCT), and full-field electroretinography (ERG) were performed. The results of DNA sequencing from polymerase chain reaction (PCR) products of the CRB1 gene were obtained from hospital records. Results: Two siblings, 14 years old and 17 years old, were compound heterozygotes for 749 del Ser and C948Y mutations in the gene encoding CRB1. AF imaging documented the preservation of retinal pigment epithelium (RPE) along the arterioles. High-resolution OCT showed abnormally thick retinae with increased lamination. Conclusion: Leber congenital amaurosis caused by CRB1 is a unique form of early-onset retinal dystrophy because it spares the para-arteriolar RPE and causes abnormal retinal lamination with thickening. These findings, detectable with AF imaging and high-resolution OCT, can be combined with electrophysiology and genetic testing to molecularly classify retinal degenerations efficiently.

Identification of Novel Mutations in Patients with Leber Congenital Amaurosis and Juvenile RP by Genome-wide Homozygosity Mapping with SNP Microarrays

Leber congenital amaurosis (LCA) and juvenile retinitis pigmentosa (RP) cause severe visual impairment early in life. Thus far, mutations in 13 genes have been associated with autosomal recessive LCA and juvenile RP. The purpose of this study was to use homozygosity mapping to identify mutations in known LCA and juvenile RP genes. The genomes of 93 consanguineous and nonconsanguineous patients with LCA and juvenile RP were analyzed for homozygous chromosomal regions by using SNP microarrays. This patient cohort was highly selected, as mutations in the known genes had been excluded with the LCA mutation chip, or a significant number of LCA genes had been excluded by comprehensive mutation analysis. Known LCA and juvenile RP genes residing in the identified homozygous regions were analyzed by sequencing. Detailed ophthalmic examinations were performed on the genotyped patients. Ten homozygous mutations, including seven novel mutations, were identified in the CRB1, LRAT, RPE65, and TULP1 genes in 12 patients. Ten patients were from consanguineous marriages, but in two patients no consanguinity was reported. In 10 of the 12 patients, the causative mutation was present in the largest or second largest homozygous segment of the patient's genome. Homozygosity mapping using SNP microarrays identified mutations in a significant proportion (30%) of consanguineous patients with LCA and juvenile RP and in a small number (3%) of nonconsanguineous patients. Significant homozygous regions which did not map to known LCA or juvenile RP genes and may be instrumental in identifying novel disease genes were detected in 33 patients.

Clinical and Molecular Genetics of Leber's Congenital Amaurosis: A Multicenter Study of Italian Patients

To identify the molecular basis of Leber's congenital amaurosis (LCA) in a cohort of Italian patients and to perform genotype-phenotype analysis. DNA samples from 95 patients with LCA were analyzed by using a microarray chip containing disease-associated sequence variants in eight LCA genes. In addition, all patients in whom no mutations were identified by microarray were subjected to sequence analysis of the CEP290 gene. Patients with mutations identified underwent a detailed ophthalmic evaluation. Disease-causing mutations were identified in 28% of patients, and twelve novel variants were identified. Mutations occurred more frequently in the RPE65 (8.4%), CRB1 (7.4%), and GUCY2D (5.2%) genes. Mutations in CEP290 were found in only 4.2% of the patients analyzed. Clinical assessment of patients carrying RPE65 or CRB1 mutations revealed the presence of retained visual capabilities in the first decade of life. RPE65 mutations were almost always associated with normal macular thickness, as assessed by optical coherence tomography (OCT), whereas CRB1 mutations were associated with reduced retinal thickness and a coarsely laminated retina. Fundus autofluorescence was mostly observed in patients with RPE65 and GUCY2D mutations and was not elicitable in patients carrying CRB1. RPE65 gene mutations represented a significant cause of LCA in the Italian population, whereas GUCY2D and CEP290 mutations had a lower frequency than that found in other reports. This finding suggests that the genetic epidemiology of LCA in Italy is different from that reported in the United States and in northern European countries. Autofluorescence in patients with RPE65 mutations was more frequently associated with preserved retinal thickness, which suggests that these mutations are not associated with progression of retinal degeneration. Therefore, normal retinal thickness (identified with OCT) and fundus autofluorescence may be the means with which to identify patients with LCA who carry RPE65 mutations, which are expected to be a potential gene therapy target in the near future.

Genetic testing for retinal dystrophies and dysfunctions: benefits, dilemmas and solutions

Human retinal dystrophies have unparalleled genetic and clinical diversity and are currently linked to more than 185 genetic loci. Genotyping is a crucial exercise, as human gene-specific clinical trials to study photoreceptor rescue are on their way. Testing confirms the diagnosis at the molecular level and allows for a more precise prognosis of the possible future clinical evolution. As treatments are gene-specific and the 'window of opportunity' is time-sensitive; accurate, rapid and cost-effective genetic testing will play an ever-increasing crucial role. The gold standard is sequencing but is fraught with excessive costs, time, manpower issues and finding non-pathogenic variants. Therefore, no centre offers testing of all currently 132 known genes. Several new micro-array technologies have emerged recently, that offer rapid, cost-effective and accurate genotyping. The new disease chips from Asper Ophthalmics (for Stargardt dystrophy, Leber congenital amaurosis [LCA], Usher syndromes and retinitis pigmentosa) offer an excellent first pass opportunity. All known mutations are placed on the chip and in 4 h a patient's DNA is screened. Identification rates (identifying at least one disease-associated mutation) are currently approximately 70% (Stargardt), approximately 60-70% (LCA) and approximately 45% (Usher syndrome subtype 1). This may be combined with genotype-phenotype correlations that suggest the causal gene from the clinical appearance (e.g. preserved para-arteriolar retinal pigment epithelium suggests the involvement of the CRB1 gene in LCA). As approximately 50% of the retinal dystrophy genes still await discovery, these technologies will improve dramatically as additional novel mutations are added. Genetic testing will then become standard practice to complement the ophthalmic evaluation.

A G1103R Mutation in CRB1 is Co-Inherited with High Hyperopia and Leber Congenital Amaurosis

Purpose: To identify the genetic basis of recessive inheritance of high hyperopia and Leber congenital amaurosis (LCA) in a family of Middle Eastern origin. Materials and methods: The patients were examined using standard ophthalmic techniques. DNA samples were obtained and genetic linkage was carried out using polymorphic markers flanking the known genes and loci for LCA. Exons were amplified and sequenced. Results: All four members of this family affected by LCA showed high to extreme hyperopia, with average spherical refractive errors ranging from +5.00 to +10.00. Linkage was obtained to 1q31.3 with a maximal LOD score of 5.20 and a mutation found in exon 9 of the CRB1 gene, causing a G1103R substitution at a highly conserved site in the protein. CRB1 is a vertebrate homolog of the Drosophila crumbs gene, which is required for photoreceptor morphogenesis, and has been associated with either retinitis pigmentosa (RP) or LCA. This sequence variant has previously been reported as a compound heterozygote in one sporadic LCA patient. Conclusion: Although hyperopia has been associated with LCA, it is typically moderate and variable between patients with the same mutation. In addition, some CRB1 mutations can be associated with either RP or LCA. We have shown that hyperopia and LCA are linked to the mutant CRB1 gene itself and are not dependent on unlinked modifiers.

EVALUATION OF GENOTYPE–PHENOTYPE ASSOCIATIONS IN LEBER CONGENITAL AMAUROSIS

To describe the clinical phenotypes associated with various genotypes known to cause Leber congenital amaurosis (LCA). One hundred ten LCA patients were screened for various probable disease-causing gene sequence variations. Those patients with a probable disease-causing sequence variation in one of six genotypes were recalled for a follow-up examination. Evaluations included assessment of visual acuity, slit-lamp biomicroscopy, and dilated fundus examination. When possible, Goldmann perimetry was also performed. Of the 37 LCA patients with suspected disease-causing sequence variations, 7 had an AIPL1 variation, 8, a CRB1 variation, 2, a CRX variation, 4, a GUCY2D variation, 11, an RPE65 variation, and 5, an RPGRIP1 variation. Across the 6 genotypes, we observed a wide range of visual acuities from 20/40 to no light perception. The widest range of vision was noted for patients with a CRB1 or RPE65 variation. Younger patients with an AIPL1 or RPGRIP1 variation were found to have severely reduced vision. Drusenlike deposits were more selectively observed in patients with mutations in the AIPL1, CRB1, RPE65, and RPGRIP1 genes, whereas focal regions of peripheral chorioretinal atrophy were observed only in patients with AIPL1 or RPE65 variations. Neurologic, intellectual, or psychomotor developmental delay was noted in 8.1% of our cohort. There was considerable overlap of phenotypic expression in six genetic subtypes in our LCA cohort. However, phenotypic trends were noted in our patients' visual acuities and posterior segment findings within genotypes. These findings have practical value for genetic screening strategies for LCA patients based upon phenotype as well as for counseling patients on their visual prognosis.

Clinical phenotypes in carriers of Leber congenital amaurosis mutations

To determine the clinical phenotypes in carriers with probable disease-causing sequence variations in 1 of 6 genes established to cause Leber congenital amaurosis (LCA). Observational prospective comparative study. Thirty carriers with various probable disease-causing sequence variations in 1 of 6 genes known to cause LCA. After the establishment of various disease-causing sequence variations in 37 (33.6%) of 110 patients with LCA, we examined a number of carriers who were either parents or offspring and who were willing to participate in our study. Evaluations included assessment of visual acuity, slit-lamp biomicroscopy, dilated fundus examination, and full-field electroretinogram (ERG) measurements. Dilated fundus examination and full-field ERGs. Of the 30 carriers with probable disease-causing sequence variations for LCA, 5 (16.7%) carriers had an AIPL1 variation, 4 (13.3%) CRB1, 0 (0%) CRX, 5 (16.7%) GUCY2D, 9 (30%) RPE65, and 7 (23.3%) carriers had a RPGRIP1 variation. Twenty-nine (96.7%) carriers had 20/20 or better visual acuity in their better seeing eye with correction. Drusenlike deposits were more selectively observed in carriers with mutations in the AIPL1, CRB1, RPE65, and RPGRIP1 genes, whereas mild peripheral chorioretinal atrophy was only observed in AIPL1 and RPE65 carriers. A reduced dark-adapted isolated rod ERG response and/or maximal combined cone and rod response was recorded in carriers with mutations in the AIPL1, GUCY2D, and RPGRIP1 genes. A reduced light-adapted ERG response to a single-flash and/or 32-Hz flicker was recorded in carriers with mutations in the AIPL1, CRB1, GUCY2D, and RPGRIP1 genes. Overall, our cohort of LCA carriers did not describe significant subjective visual difficulties, including nyctalopia and/or photosensitivity. The variation of phenotypic expression in carriers among 5 LCA genotypes indicates that there is considerable phenotypic overlap. However, phenotypic trends were noted in carriers' fundus findings and ERG responses for each genetic subtype. Observations of phenotypic associations with specific disease-causing sequence variations in carriers have potential practical value for molecular screening strategies of patients with LCA.

The expanding roles of ABCA4 and CRB1 in inherited blindness.

Mutations in the ABCA4 gene cause Stargardt disease (STGD), most cases with autosomal recessive (ar) cone-rod dystrophy (CRD), and some cases with atypical ar retinitis pigmentosa (arRP). We found compound heterozygous ABCA4 mutations in two unrelated patients with STGD and homozygous splice site mutations in their 2nd and 4th degree cousins with RP. Some ABCA4 mutations display strong founder effects. In Dutch and German STGD patients, the 768G > T mutation is present in 8% and 0.6% of ABCA4 alleles respectively. Vice versa, the complex L541P;A1038V allele is found in 70% of ABCA4 alleles in German STGD patients but absent in Dutch patients. As approximately 70% of ABCA4 mutations are known, a microarray-based analysis of known ABCA4 gene variants allows routine DNA diagnostics in Caucasian patients. Mutations in the CRB1 gene underlie RP12, some cases with classic arRP, 55% of cases with RP and Coats-like exudative vasculopathy, and 13% of patients with Leber congenital amaurosis (LCA), rendering CRB1 a significant cause of autosomal recessive retinal dystrophy. Different combinations of mutations in ABCA4 or CRB1 can be correlated with disease severity, suggesting that small increments of protein activities in patients might have significant therapeutic effects. Mouse and Drosophila studies strongly suggest that both patient groups might benefit from reduced light exposure and therefore should be detected as early as possible using molecular techniques.

CRB1 is essential for external limiting membrane integrity and photoreceptor morphogenesis in the mammalian retina.

Mutations within the CRB1 gene have been shown to cause human retinal diseases including retinitis pigmentosa and Leber congenital amaurosis. We have recently identified a mouse model, retinal degeneration 8 (rd8) with a single base deletion in the Crb1 gene. This mutation is predicted to cause a frame shift and premature stop codon which truncates the transmembrane and cytoplasmic domain of CRB1. Like in Drosophila crumbs (crb) mutants, staining for adherens junction proteins known to localize to the external limiting membrane, the equivalent of the zonula adherens in the mammalian retina, is discontinuous and fragmented. Shortened photoreceptor inner and outer segments are observed as early as 2 weeks after birth, suggesting a developmental defect in these structures rather than a degenerative process. Photoreceptor degeneration is observed only within regions of retinal spotting, which is seen predominantly in the inferior nasal quadrant of the eye, and is caused by retinal folds and pseudorosettes. Photoreceptor dysplasia and degeneration in Crb1 mutants strongly vary with genetic background, suggesting that the variability in phenotypes of human patients that carry mutations in CRB1 may be due to interactions with background modifiers in addition to allelic variations. The Crb1rd8 mouse model will facilitate the analysis of Crb1 function in the neural retina and the identification of interacting factors as candidate retinal disease genes.

A new species of the genus <i>Chondrostoma</i> Agassiz, 1832 (Actinopterygii, Cyprinidae) from the Iberian Peninsula

A new species considered in the past to be included in Chodrostoma lemmingii is described on the basis of genetic and morphological characters. This new species inhabits tributaries of the Jandula River in the Guadalquivir basin, in southern Spain. It is distinguished from C. lemmingii by the following: 1) six to seven branched rays on the dorsal and anal fins, 2) a relatively small number of gill rakers ( x = 25.4), 3) a large eye placed very anterior on the head, 4) a high and narrow coronoid process on the dentary, 5) the palatine process of the maxilla posteriorly oriented and, 6) the presence of a diagnostic locus ( MDH-A ). The range of Chondrostoma oretanum sp. nov. is restricted to the Robledillo and Fresneda Rivers, two small tributaries of the Jandula River in the Guadalquivir basin on the Atlantic slope of the Iberian Peninsula. Following the criteria established by IUCN Red List Categories, we consider C. oretanum to be critically endangered (CR B1 + 2ce).

Mutation screening of Pakistani families with congenital eye disorders

To map the disease loci several Pakistani families suffering from autosomal recessive retinitis pigmentosa with preserved para-arteriolar retinal pigment epithelium and Leber congenital amaurosis (LCA) were analyzed. Analysis revealed close genetic linkage between the disease phenotype of some of the families (3330RP, 111RP and 010LCA) and the microsatellite markers on chromosome 1q31. Mutation screening of the candidate gene CRB1 revealed a G to A transversion in exon 7 in arRP family 330RP and a T to C substitution in another arRP family, 111RP. In exon 9 of the CRB1 gene a T to C transversion was found in the family suffering from LCA (010LCA). The LCA phenotype of another family (011LCA) in which the CRB1 locus was excluded, showed linkage with microsatellite markers D17S1294 and D17S796 on chromosome 17p13·1. The association of the candidate gene GUCY2D (17p13·1) with the disease phenotype was excluded as no disease-associated mutation was found in any of its exons. Mutation screening of another candidate gene, AIPL1 located in the same region, showed a novel homozygous C to A substitution in exon 2. These sequence changes are unique for the Pakistani families and some of these have not been reported previously.

Mutations in the CRB1 gene cause Leber congenital amaurosis.

To test the hypothesis that mutations in the CRB1 gene cause Leber congenital amaurosis (LCA) and, if so, to describe the ocular phenotype of patients with LCA who harbor CRB1 sequence variations. One hundred ninety probands with a clinical diagnosis of LCA were selected from a cohort of 233 probands ascertained in 5 different countries. The remaining 43 probands (18%) were excluded because they harbored sequence variations in previously identified LCA genes. One hundred ninety unrelated individuals with LCA were screened for coding sequence mutations in the CRB1 gene with single-strand conformation polymorphism analysis followed by automated DNA sequencing. Twenty-one of the 190 probands (9% of the total cohort of 233) and 2 (1.4%) of 140 controls harbored amino acid-altering sequence variations in the CRB1 gene (P =.003). In our cohort of patients with LCA, coding sequence variations were observed in the CRB1 gene more frequently than in any of the other 5 known LCA-associated genes. Likely disease-causing sequence variations have now been identified in 64 (28%) of 233 subjects in this cohort. Molecular diagnosis can confirm and clarify the diagnosis in an increasing fraction of patients with LCA. As genotype data accumulate, clinical phenotypes associated with specific mutations may be established. This will facilitate the counseling of patients regarding their visual prognosis and the likelihood of associated systemic anomalies.

CRB1 mutations may result in retinitis pigmentosa without para-arteriolar RPE preservation

Purpose: To report a new phenotype in retinitis pigmenotosa (RP) patients with CRB1 mutations at the RP12 locus. Patients: Thirty-seven patients from two Pakistani families with severe retinitis pigmentosa. Methods: Samples were screened with single-strand conformation polymorphism analysis followed by DNA sequencing of the coding sequence of the CRB1 gene. Results: Two novel CRB1 mutations were discovered. No patients had evidence of preservation of the para-arteriolar retinal pigment epithelium (PPRPE) that has been previously reported in all cases of RP associated with CRB1 mutations. Conclusions: Patients with severe autosomal recessive (or simplex) RP who lack the finding of PPRPE should not be excluded from molecular analysis of CRB1 purely because they lack the clinical feature of PPRPE. This report illustrates that RP at the RP12 locus is not clinically uniform. The absence of PPRPE cannot be used to exclude CRB1 as a potential molecular explanation for RP.

High temperature thermodynamic properties of some transition metal borides

The results of an experimental study of the thermodynamic properties of HfB 2, Nb 3B 2, NbB0.99, Nb 3B 4, NbB 1.96, TaB 1.97, CrB 0.99, Cr B1.99, Mo 2B, MoB 1.07, Mo 2B 5, W 2B, WB and W 2B 5 at high temperature are summarized. The temperature coefficients for the enthalpy and heat capacity are tabulated.