Mohamed F. El-Ashry

EYS, encoding an ortholog of Drosophila spacemaker, is mutated in autosomal recessive retinitis pigmentosa.

Using a positional cloning approach supported by comparative genomics, we have identified a previously unreported gene, EYS, at the RP25 locus on chromosome 6q12 commonly mutated in autosomal recessive retinitis pigmentosa. Spanning over 2 Mb, this is the largest eye-specific gene identified so far. EYS is independently disrupted in four other mammalian lineages, including that of rodents, but is well conserved from Drosophila to man and is likely to have a role in the modeling of retinal architecture.

The effect of phacoemulsification cataract surgery on the measurement of retinal nerve fiber layer thickness using optical coherence tomography.

Purpose: To evaluate whether the measurement of retinal nerve fiber layer (RNFL) thickness using optical coherence tomography (OCT) changes after cataract surgery. Methods: This prospective, randomized, observational, cross-sectional clinical study included 24 eyes of 24 patients who underwent phacoemusification with implant. All patients had no preexisting retinal or optic nerve pathology or other media opacities that might influence the RNFL thickness. The classification and the grading of cataract were based on the Lens Opacities Classification System III (LOCS III). All eyes were scanned immediately before cataract surgery with OCT using Fast RNFL program. All eyes had circular scans around the optic disk with a diameter of 3.4 mm. The OCT was repeated 4 weeks after surgery when the patients returned for postoperative check using the same programme. Results: The mean preoperative RNFL thickness was 84.9 ± 16.5. The postoperative mean RNFL thickness was 93.0 ± 17.6. The pre-and postoperative values of the mean RNFL and signal/noise ratio (SNR) are statistically significant (P < 0.05). Conclusions: Lens opacities may affect the image quality of OCT scans used to measure RNFL thickness as indicated by preoperative low SNR. Cataract extraction results in an apparent increase of the RNFL thickness.

Identification of novel mutations in the ortholog of Drosophila eyes shut gene (EYS) causing autosomal recessive retinitis pigmentosa.

PURPOSE Recently, a novel gene was cloned for autosomal recessive retinitis pigmentosa (arRP), EYS, on 6q12. This study was conducted to determine the spectrum and frequency of EYS mutations in 195 unrelated patients with autosomal recessive and autosomal dominant RP (adRP). METHODS All cases had a complete ophthalmic examination, and the clinical diagnosis of RP was based on visual acuity, fundus photography, and electroretinography findings. The DNA extracted from all participants was subjected to molecular genetic analysis entailing amplification of the coding regions and exon-intron boundaries of EYS by polymerase chain reaction, followed by direct sequencing. Bioinformatics analysis was undertaken to study the effect of the identified mutations on protein structure and function. RESULTS Eleven novel missense, nonsense, and splice site mutations were identified within EYS in 10 unrelated arRP patients, with probable allele frequency of 11%. However, no mutations were observed in the adRP panel. In addition, 53 single-nucleotide polymorphisms (SNPs) were found, of which 12 were previously unreported. Bioinformatics analyses revealed that all mutations were highly conserved across other species and/or involved important domains on protein structure. Intrafamilial phenotypic variability was also observed in a family with double heterozygous mutations. CONCLUSIONS This is the first report of molecular genetic analysis of EYS in a cohort of unrelated British and Chinese patients with RP. The results further the initial hypothesis that EYS is a major causative gene for recessive RP and emphasize the role of different types of mutations in disrupting the function of EYS.

A clinical and molecular genetic study of Egyptian and Saudi Arabian patients with primary congenital glaucoma (PCG).

PurposeTo undertake mutation screening of cytochrome P4501B1 (CYP1B1, OMIM 601771) and myocilin (MYOC, OMIM 601652) genes in Egyptian and Saudi Arabian patients with primary congenital glaucoma (PCG). Patients and MethodsA clinical and molecular genetic study was performed on 11 Egyptian and Saudi Arabian patients with PCG. Clinical diagnosis was confirmed by slit lamp biomicroscopy, gonioscopy, measurement of intraocular pressure, and corneal diameter. The coding regions of CYP1B1 and MYOC genes were amplified by polymerase chain reaction for all affected subjects. Direct sequence analysis was performed to search for sequence alterations. Haplotype analysis and genotype/phenotype correlation were carried out. ResultsThree CYP1B1 mutations were identified in 5 PCG patients (45.4%) of which 2 were novel (homozygous E173K and heterozygous N498D) and the third (G61E) had previously been reported. In addition 10 single nucleotide polymorphisms were identified in CYP1B1 and MYOC genes of which 2 were novel. However, no pathologic changes in either of the genes were detected in the remaining 6 patients. ConclusionsThis is the first report of molecular genetic analysis of PCG in the Egyptian population in which 2 novel mutations have been identified. It is possible that these mutations are specific to this population and may lead to alterations in the protein structure encoded by the gene. Patients with no mutations in the screened genes may have mutations in genes yet to be identified.

Genetic Analysis of FAM46A in Spanish Families with Autosomal Recessive Retinitis Pigmentosa: Characterisation of Novel VNTRs

Retinitis pigmentosa (RP) is a group of retinal dystrophies characterised primarily by rod photoreceptor cell degeneration. Exhibiting great clinical and genetic heterogeneity, RP be inherited as an autosomal dominant (ad) and recessive (ar), X‐linked (xl) and digenic disorder. RP25, a locus for arRP, was mapped to chromosome 6p12.1‐q14.1 where several retinal dystrophy loci are located. A gene expressed in the retina, FAM46A, mapped within the RP25 locus, and computational data revealed its involvement in retinal signalling pathways. Therefore, we chose to perform molecular evaluation of this gene as a good candidate in arRP families linked to the RP25 interval. A comprehensive bioinformatic and retinal tissue expression characterisation of FAM46A was performed, together with mutation screening of seven RP25 families.

A Novel Genetic Study of Chinese Families with Autosomal Recessive Retinitis Pigmentosa

Autosomal recessive retinitis pigmentosa (arRP) is the commonest form of RP worldwide. To date 22 loci have been implicated in the pathogenesis of this disease; however none of these loci independently account for a significant proportion of recessive RP. Linkage studies of arRP in consanguineous families have been mainly based on homozygosity mapping, but this strategy cannot be applied in the case of non‐consanguineous families. Therefore, we implemented a systematic approach for identifying the disease locus in three non‐consanguineous Chinese families with arRP. Initially, linkage analysis using SNPs/microsatellite markers or mutation screening of known arRP genes excluded all loci/genes except RP25 on chromosome 6. Subsequently a whole genome scan for the three families using the 10K GeneChip Mapping Array was performed, in order to identify the possible disease locus. To the best of our knowledge this is the first report on the utilisation of the 10K GeneChip to study linkage in non‐consanguineous Chinese arRP. This analysis indicates that the studied families are probably linked to the RP25 locus, a well defined arRP locus in other populations. The identification of another ethnic group linked to RP25 is highly suggestive that this represents a major locus for arRP.

Linkage Validation of RP25 Using the 10K GeneChip Array and Further Refinement of the Locus by New Linked Families

Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of retinal dystrophies, characterised by rod photoreceptor cell degeneration with autosomal recessive RP (arRP) as the commonest form worldwide. To date, a total of 26 loci have been reported for arRP, each having a prevalence of 1–5%, except for the RP25 locus which was identified as the genetic cause of 14% of arRP cases in Spain. In order to validate the original linkage of RP25, we undertook a total genome scan using the 10K GeneChip mapping array on three of the previously linked families. The data obtained supported the initial findings of linkage. Additionally, linkage analysis in 18 newly ascertained arRP families was performed using microsatellite markers spanning the chromosome 6p12.1‐q15 interval. Five out of the 18 families showed suggestive evidence of linkage to RP25, hence supporting the high prevalence of this locus in the Spanish population. Furthermore, the finding of a crossover in one of these families is likely to have refined the disease interval from the original 16 cM to only a 2.67 cM region between D6S257 and D6S1557.

BIGH3 mutation in a Bangladeshi family with a variable phenotype of LCDI

AbstractAims To report a Bangladeshi family displaying intrafamilial phenotypic heterogeneity of lattice corneal dystrophy type I (LCDI) and to identify the causative mutation.Methods Molecular genetic analysis was performed on DNA extracted from all members of the family. Exons of BIGH3gene were amplified by polymerase chain reaction. Gene mutation and polymorphisms were identified by heteroduplex and sequence analyses. Segregation of the mutation in the family was confirmed by restriction digestion of amplified gene fragments.Results A heterozygous C → T transition at the first nucleotide position of codon 124 of the BIGH3gene was detected in the three affected members and not in the unaffected members of the family.Conclusions This is the first report of BIGH3gene mutation in a Bangladeshi family with phenotypic heterogeneity. This study confirms that BIGH3gene screening should be undertaken for proper classification of corneal dystrophy, especially in the absence of histopathological examination.

A clinical and molecular genetic study of autosomal-dominant stromal corneal dystrophy in British population.

Aims: To identify the underlying mutations in our British families and sporadic patients with different types of corneal dystrophies (CDs) and to establish a phenotype-genotype correlation. Methods: Twenty-nine patients, 9 sporadic and 20 patients from 7 families were subjected to both clinical and genetic examination. Slit lamp examination was performed for all patients who participated in the study to assess their corneal phenotype. Genomic DNA was extracted from 10 ml venous blood, and the BIGH3 gene was amplified exon by exon to perform heteroduplex analysis. Exons that displayed double bands were then analysed by direct bi-directional sequencing and restriction digest analyses. Results: Clinically our patients showed three distinct phenotypes of CD: 16 with Thiel-Behnke corneal dystrophy or corneal dystrophy of Bowman layer type 2 (CDB2), 8 with granular CD (GCD), and 5 with lattice CD type I (LCDI). Three different missense mutations have been detected in the coding region of BIGH3 gene, R555Q, in 16 CDB2 patients, R555W in 8 GCD patients, and R124C in 5 LCDI patients. These mutations were the same as to those previously reported in patients from other ethnic origins. Also,we identified seven nucleotide substitutions that did not change the amino acid sequence of the encoded protein of which four were novel. Conclusions: In our patients of British origin, each phenotype of CD has been linked to a particular point mutation of the BIGH3 gene. Our study also highlights the importance of codons 124 and 555 as mutation hot spots in the BIGH3 gene in the British population.

Molecular genetic analysis of two functional candidate genes in the autosomal recessive retinitis pigmentosa, RP25, locus

Purpose: To identify the disease gene in five Spanish families with autosomal recessive retinitis pigmentosa (arRP) linked to the RP25 locus. Two candidate genes, EEF1A1 and IMPG1, were selected from the region between D6S280 and D6S1644 markers where the families are linked. The genes were selected as good candidates on the basis of their function, tissue expression pattern, and/or genetic data. Methods: A molecular genetic study was performed on DNA extracted from one parent and one affected member of each studied family. The coding exons, splice sites, and the 5′ UTR of the genes were amplified by polymerase chain reaction (PCR). For mutation detection, direct sequence analysis was performed using the ABI 3100 automated sequencer. Segregation of an IMPG1 single nucleotide polymorphism (SNP) in all the families studied was analyzed by restriction enzyme digest of the amplified gene fragments. Results: In total, 15 SNPs were identified of which 7 were novel. Of the identified SNPs, one was insertion, two were deletions, five were intronic, six were missense, and one was located in the 5′ UTR. These changes, however, were also identified in unaffected members of the families and/or 50 control Caucasians. The examined known IMPG1 SNP was not segregating with the disease phenotype but was correlating with the genetic data in all families studied. Conclusions: Our results indicate that neither EEF1A1 nor IMPG1 could be responsible for RP25 in the studied families due to absence of any pathogenic variants. However, it is important to notice that the methodology used in this study cannot detect larger deletions that lie outside the screened regions or primer site mutations that exist in the heterozygous state. A role of both genes in other inherited forms of RP and/or retinal degenerations needs to be elucidated.