Photoreceptor degeneration: genetic and mechanistic dissection of a complex trait.

The retina provides exquisitely sensitive vision that relies on the integrity of a uniquely vulnerable cell, the photoreceptor (PR). The genetic and mechanistic causes of retinal degeneration due to PR cell death--which occurs in conditions such as retinitis pigmentosa and age-related macular degeneration--are being successfully dissected. Over one hundred loci, some containing common variants but most containing rare variants, are implicated in the genetic architecture of this complex trait. This genetic heterogeneity results in equally diverse disease mechanisms that affect almost every aspect of PR function but converge on a common cell death pathway. Although genetic and mechanistic diversity creates challenges for therapy, some approaches--particularly gene-replacement therapy--are showing considerable promise.

Selective Determination of Entecavir in the Presence of its Oxidative Degradate by Spectrophotometric and Chromatographic Methods.

BACKGROUND: Entecavir (ENT) is an antiretroviral agent prescribed for the treatment of the hepatitis B virus(HBV) and human immunodeficiency virus(HIV). OBJECTIVE: Development and validation of three simple, sensitive, selective, and precise methods for determination of ENT in the presence of its oxidative degradation product (ENT deg.). METHOD: The first method was based on second derivative (D2) spectrophotometry through measuring the peak amplitude of D2 spectra at 293.6 nm. The second one is mean centering of the ratio spectra (MCR), which enabled measurement of the peak amplitude at 280.0 nm. The third method was HPLC, where ENT was separated from ENT deg. using a Zobrax C18 column and methanol:water (30:70, v/v) with pH 3 as a mobile phase. The three developed methods were validated according to the International Conference on Harmonization guidelines. RESULTS: Linearity range of ENT was 5.00-50.00 µg/mL for both D2 and MCR. However, higher sensitivity was achieved using HPLC (1.00-50.00 µg/mL). Accuracy of ENT were 100.60 ± 0.547%, 101.55 ± 1.2071%, and 100.61 ± 1.207% for D2, MCR, and HPLC methods, respectively, and precision was within 1.280. CONCLUSIONS: The developed methods were successfully applied for the determination of ENT in Tecavir® tablets without interference from ENT deg. They showed no significant difference in comparison with the official method and they can be applied in the quality analysis of ENT with high selectivity, accuracy, and precision. HIGHLIGHTS: ENT was quantified using two spectrophotometric (D2 and MCR) methods and an HPLC method in presence of ENT deg. The proposed methods were applied to analysis of ENT tablets with high selectivity, sensitivity, and accuracy.

Mutation spectrum of EYS in Spanish patients with autosomal recessive retinitis pigmentosa.

Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal dystrophies characterised ultimately by the loss of photoreceptor cells. We have recently identified a new gene(EYS) encoding an ortholog of Drosophila space maker (spam) as a commonly mutated gene in autosomal recessive RP. In the present study, we report the identification of 73 sequence variations in EYS, of which 28 are novel. Of these, 42.9% (12/28) are very likely pathogenic, 17.9% (5/28)are possibly pathogenic, whereas 39.3% (11/28) are SNPs. In addition, we have detected 3 pathogenic changes previously reported in other populations. We are also presenting the characterisation of EYS homologues in different species, and a detailed analysis of the EYS domains, with the identification of an interesting novel feature: a putative coiled-coil domain.Majority of the mutations in the arRP patients have been found within the domain structures of EYS. The minimum observed prevalence of distinct EYS mutations in our group of patients is of 15.9% (15/94), confirming a major involvement of EYS in the pathogenesis of arRP in the Spanish population. Along with the detection of three recurrent mutations in Caucasian population, our hypothesis of EYS being the first prevalent gene in arRP has been reinforced in the present study.

Biallelic mutation of protocadherin-21 (PCDH21) causes retinal degeneration in humans.

PURPOSE: To describe the clinical findings and mutations in affected members of two families with an autosomal recessive retinal dystrophy associated with mutations in the protocadherin-21 (PCDH21) gene. METHODS: A full genome scan of members of two consanguineous families segregating an autosomal recessive retinal dystrophy was performed and regions identical by descent identified. Positional candidate genes were identified and sequenced. All patients had a detailed ophthalmic examination, including electroretinography and retinal imaging. RESULTS: Affected members of both families showed identical homozygosity for an overlapping region of chromosome 10q. Sequencing of a candidate gene, PCDH21, showed two separate homozygous single-base deletions, c.337delG (p.G113AfsX1) and c.1459delG (p.G487GfsX20), which were not detected in 282 control chromosomes. Affected members of the two families first reported nyctalopia in late teenage years and retained good central vision until their late 30s. No color vision was detected in any proband. The fundus appearance included the later development of characteristic circular patches of pigment epithelial atrophy at the macula and in the peripheral retina. CONCLUSIONS: Biallelic mutations in the photoreceptor-specific gene PCDH21 cause recessive retinal degeneration in humans.

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

PURPOSE: To 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 METHODS: A 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. RESULTS: Three 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. CONCLUSIONS: This 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.

Novel CHST6 nonsense and missense mutations responsible for macular corneal dystrophy.

PURPOSE: To identify the underlying mutations in two unrelated British families with macular corneal dystrophy (MCD) by screening the carbohydrate sulfotransferase (CHST6) gene. DESIGN: Case reports and results of DNA analysis. METHODS: Two subjects from two British families with MCD were studied. The genetic status of CHST6 was determined for all members of these MCD families. In addition, sulfated keratan sulfate (KS) assay from the probands was also undertaken. CHST6 gene was amplified by polymerase chain reaction (PCR). The PCR products were analyzed by sequencing and restriction digestion. Enzyme-linked immunosorbent assay (ELISA) was performed to assess KS presence in serum. RESULTS: Four compound heterozygous mutations were identified, three of which are novel. The ELISA showed that the probands were of MCD type I. CONCLUSIONS: These novel mutations are expected to result in loss of CHST6 function, which would account for the MCD phenotype.

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.

Identification of novel mutations in the carbohydrate sulfotransferase gene (CHST6) causing macular corneal dystrophy.

PURPOSE: Macular corneal dystrophy (MCD) is a rare corneal dystrophy that is characterized by abnormal deposits in the corneal stroma, keratocytes, Descemet's membrane, and endothelium, accompanied by progressive clouding. It has been classified into three immunophenotypes--MCD types I, IA, and II--according to the serum level of sulfated keratan sulfate (KS) and immunoreactivity of the corneal tissue. Recently, mutations in a new carbohydrate sulfotransferase gene (CHST6) encoding corneal glucosamine N-acetyl-6-sulfotransferase (C-GlcNac-6-ST) have been identified as the cause of MCD. Mutation screening of the CHST6 gene has been undertaken to identify the underlying mutations in five unrelated British families with MCD. METHODS: DNA was extracted from venous blood obtained from all participants, and the coding region of CHST6 was amplified by polymerase chain reaction (PCR). The PCR products were analyzed by direct sequencing and restriction enzyme digestion. Enzyme-linked immunosorbent assay (ELISA) was performed to assess the presence of KS in serum from the probands of MCD-affected families participating in the study. RESULTS: Six novel missense mutations--four homozygous and two compound heterozygous--were identified in the CHST6 gene. The ELISA showed that the disease in all patients participating in the study was of MCD type I, including the subtype IA. CONCLUSIONS: These novel mutations are thought to result in loss of corneal sulfotransferase function, which would account for the MCD phenotype.

Molecular genetic study of Egyptian patients with macular corneal dystrophy.

AIM: To identify the underlying genetic defect in Egyptian patients with macular corneal dystrophy (MCD). METHODS: A clinical and molecular genetic study was performed on 11 patients from six families with MCD. Clinical diagnosis was confirmed by slit-lamp biomicroscopy and histopathological examination of corneal buttons following keratoplasty. The coding region of the carbohydrate sulfotransferase (CHST6) gene was amplified by polymerase chain reaction (PCR) in all affected subjects. This was followed by direct sequencing and restriction digest analyses. Enzyme-linked immunosorbent assay of antigenic keratan sulfate (KS) in patients' serum was also performed. RESULTS: Six homozygous mutations, of which three are novel, were identified within the coding region of CHST6 in six unrelated MCD families. The barely detectable level of antigenic KS in the serum of the affected individuals indicated that they all have MCD type I, including the subtype IA. CONCLUSIONS: This is the first report of a molecular genetic analysis of MCD in the Egyptian population. These data indicate the extensive allelic heterogeneity within CHST6 and further support its essential role in maintaining corneal transparency.

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.

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.

Mutations in TOPORS cause autosomal dominant retinitis pigmentosa with perivascular retinal pigment epithelium atrophy.

We report mutations in the gene for topoisomerase I-binding RS protein (TOPORS) in patients with autosomal dominant retinitis pigmentosa (adRP) linked to chromosome 9p21.1 (locus RP31). A positional-cloning approach, together with the use of bioinformatics, identified TOPORS (comprising three exons and encoding a protein of 1,045 aa) as the gene responsible for adRP. Mutations that include an insertion and a deletion have been identified in two adRP-affected families--one French Canadian and one German family, respectively. Interestingly, a distinct phenotype is noted at the earlier stages of the disease, with an unusual perivascular cuff of retinal pigment epithelium atrophy, which was found surrounding the superior and inferior arcades in the retina. TOPORS is a RING domain-containing E3 ubiquitin ligase and localizes in the nucleus in speckled loci that are associated with promyelocytic leukemia bodies. The ubiquitous nature of TOPORS expression and a lack of mutant protein in patients are highly suggestive of haploinsufficiency, rather than a dominant negative effect, as the molecular mechanism of the disease and make rescue of the clinical phenotype amenable to somatic gene therapy.

Exclusion of four candidate genes, KHDRBS2, PTP4A1, KIAA1411 and OGFRL1, as causative of autosomal recessive retinitis pigmentosa.

To identify the disease gene in 6 Spanish families with autosomal recessive retinitis pigmentosa linked to the RP25 locus, mutation screening of 4 candidate genes, KHDRBS2, PTP4A1, KIAA1411 and OGFRL1, was undertaken based on their expression or functional relevance to the retina. Twenty-six single nucleotide polymorphisms were identified, of which 14 were novel. Even though no pathological mutations were detected, these genes however remain as good candidates for other retinal degenerations mapping to the same chromosomal region.