Rds Gene Research Articles

Genotype Pattern Mining for Pairs of Interacting Variants Underlying Digenic Traits.

Some genetic diseases (“digenic traits”) are due to the interaction between two DNA variants, which presumably reflects biochemical interactions. For example, certain forms of Retinitis Pigmentosa, a type of blindness, occur in the presence of two mutant variants, one each in the ROM1 and RDS genes, while the occurrence of only one such variant results in a normal phenotype. Detecting variant pairs underlying digenic traits by standard genetic methods is difficult and is downright impossible when individual variants alone have minimal effects. Frequent pattern mining (FPM) methods are known to detect patterns of items. We make use of FPM approaches to find pairs of genotypes (from different variants) that can discriminate between cases and controls. Our method is based on genotype patterns of length two, and permutation testing allows assigning p-values to genotype patterns, where the null hypothesis refers to equal pattern frequencies in cases and controls. We compare different interaction search approaches and their properties on the basis of published datasets. Our implementation of FPM to case-control studies is freely available.

A natural short pathway synthesizes roquefortine C but not meleagrin in three different Penicillium roqueforti strains

The production of mycotoxins and other secondary metabolites in Penicillium roqueforti is of great interest because of its long history of use in blue-veined cheese manufacture. In this article, we report the cloning and characterization of the roquefortine gene cluster in three different P. roqueforti strains isolated from blue cheese in the USA (the type strain), France, and the UK (Cheshire cheese). All three strains showed an identical roquefortine gene cluster organization and almost identical (98-99%) gene nucleotide sequences in the entire 16.6-kb cluster region. When compared with the Penicillium chrysogenum roquefortine/meleagrin seven-gene cluster, the P. roqueforti roquefortine cluster contains only four genes (rds, rdh, rpt, and gmt) encoding the roquefortine dipeptide synthetase, roquefortine D dehydrogenase, roquefortine prenyltransferase, and a methyltransferase, respectively. Silencing of the rds or rpt genes by the RNAi strategy reduced roquefortine C production by 50% confirming the involvement of these two key genes in roquefortine biosynthesis. An additional putative gene, orthologous of the MFS transporter roqT, is rearranged in all three strains as a pseudogene. The same four genes and a complete (not rearranged) roqT, encoding a MFS transporter containing 12 TMS domains, occur in the seven-gene cluster in P. chrysogenum although organized differently. Interestingly, the two "late" genes of the P. chrysogenum roquefortine/meleagrin gene cluster that convert roquefortine C to glandicoline B and meleagrin are absent in the P. roqueforti four-gene cluster. No meleagrin production was detected in P. roqueforti cultures grown in YES medium, while P. chrysogenum produces meleagrin in these conditions. No orthologous genes of the two missing meleagrin synthesizing genes were found elsewhere in the recently released P. roqueforti genome. Our data suggest that during evolution, the seven-gene cluster present in P. chrysogenum, and probably also in other glandicoline/meleagrin producing fungi, has been trimmed down to a short cluster in P. roqueforti leading to the synthesis of roquefortine C rather than meleagrin as a final product.

Structural characterization of the second intra-discal loop of the photoreceptor tetraspanin RDS

Vertebrate photoreceptors contain a unique tetraspanin protein known as 'retinal degeneration slow' (RDS). Mutations in the RDS gene have been identified in a variety of human retinal degenerative diseases, and more than 70% of these mutations are located in the second intra-discal (D2) loop, highlighting the importance of this region. Here we examined the conformational and thermal stability properties of the D2 loop of RDS, as well as interactions with ROM-1, a non-glycosylated homolog of RDS. The RDS D2 loop was expressed in Escherichiacoli as a fusion protein with maltose binding protein (MBP). The fusion protein, referred to as MBP-D2, was purified to homogeneity. Circular dichroism spectroscopy showed that the wild-type (WT) D2 loop consists of approximately 21% α-helix, approximately 20% β-sheet and approximately 59% random coil. D2 loop fusion proteins carrying disease-causing mutations in RDS (e.g. R172W, C214S, N244H/K) were also examined, and conformational changes were observed (compared to wild-type D2). In particular, the C150S, C214S and N244H proteins showed significant reductions in α-helicity. However, the thermal stability of the mutants was unchanged compared to wild-type, and all the mutants were capable of interacting with ROM-1, indicating that this functional aspect of the isolated D2 loop remained intact in the mutants despite the observed conformational changes. An I-TASSER model of the RDS D2 loop predicted a structure consistent with the circular dichroism experiments and the structure of the conserved region of the D2 loop of other tetraspanin family members. These results provide significant insight into the mechanism of RDS complex formation and the disease process underlying RDS-associated retinal degeneration.

Autosomal Recessive Retinitis Pigmentosa Due ToABCA4Mutations: Clinical, Pathologic, and Molecular Characterization

Autosomal recessive retinitis pigmentosa (ARRP) is a genetically heterogeneous condition characterized by progressive loss of retinal photoreceptor cells. In order to gain new insights into the pathogenesis of ARRP, we evaluated the morphological, biochemical, and gene expression changes in eyes from a human donor with ARRP due to mutations in the ABCA4 gene. Eyes were obtained postmortem from a donor with end-stage retinitis pigmentosa. The coding sequences of the RDS, RHO, and ABCA4 genes were screened for disease-causing mutations. Morphological changes in different regions of the retina were examined histologically, and levels of lipofuscin-associated bisretinoids were measured. Gene expression was examined in retinal/choroidal tissue using microarray analysis, and all parameters were compared to those in unaffected control donors. Genetic analysis of the donor's DNA identified two mutations in the ABCA4 gene, IVS14+1G > C and Phe1440del1 cT, each on a separate allele. Morphological evaluation revealed complete loss of the outer nuclear layer, remodeling of the inner retina, loss of retinal vasculature, and regional neovascularization. The retinal pigment epithelium and choriocapillaris exhibited regional preservation. Microarray analysis revealed loss of photoreceptor cell-associated transcripts, with preservation of multiple genes expressed specifically in inner retinal neurons. The persistence of transcripts expressed by inner retinal neurons suggests that despite significant plasticity that occurs during retinal degeneration, bipolar cells and ganglion cells remain at least partially differentiated. Findings from this study suggest that some forms of therapy currently under investigation may have benefit even in advanced retinal degeneration.

Gene mapping and mutation screeneng in candidate genes in a Chinese family of autosomal dominant retinitis pigmentosa

We wanted to find the gene defect in a Chinese pedigree with autosomal dominant form of retinitis pigmentosa (ADRP). A small Chinese family with retinitis pigmentosa was collected. The genetic analysis of the family suggested an autosomal dominant pattern. Microsatellite (STR) markers tightly linked to candidate genes for ADRP were selected for linkage analysis. We got a maximum LOD score of 0.87 between markers D19S210 and D19S418. Precursor mRNA-processing factor (PRPF) 31, 3, 8, rhodopsin (RHO), peripherin 2 (PRPH2 or RDS), rod outer segment protein 1 (ROM1), neural retina leucine zipper (NRL), cone-rod homeobox-containing (CRX), inosine-5-prime-monophosphate dehydrogenase, type I (IMPDH1) and retinitis pigmentosa 1 (RPI) were amplified by polymerase chain reaction (PCR) and screened by direct sequencing. One new sequence variation was found. It was the missence mutation c.148G > C (D50H) occurred in exon 1 of RDS gene which existed in all the effected individuals and one unaffected family member. The DNA sequence variation didn't cosegregate with the RP disease. We considered this transition was one new polymorphism which we speculate involved in the pathogenesis of ADRP and increased the risk of ADRP. Further study should be conducted to confirm the causative gene of this family.

Retinitis pigmentosa-related gene researches in China

Objective To review retinitis pigmentosa (RP)-related gene researches in China. Methods RP genes and RP gene therapy as key words were searched from those online databases including China Knowledge Resource Integrated Database of China National Knowledge Infrastructure (CNKI), National Center for Biotechnology Information (NCBI), Human Gene Mutation Database (HGMD), and Human Genome Variation Society (HGVS). Related papers written by authors from China or other countries,published from 1991 to 2011 were collected for analysis. Results Overall 60 RP-related genes have been identified now. Chinese researchers from 29 hospitals and research institutes confirmed 17 RP-related gene changes (239 mutations, 131 of them are pathogenic) in 300 RP families and 1572 sporadic RP patients from 1991 to 2011, as reported in 66 papers. Mutations in rhodopsin (RHO) gene, NR2E3 gene and RP1 gene were found in 2.0％, 2.9％ and 1.0％ of Chinese RP patients respectively. The relationship between genotypes and clinical phenotypes were investigated in 15 papers, including RHO gene, RDS gene, RP1 and RPGR or RP3 gene in ADRP families. Gene therapy for RP has been investigated in 4 papers. Seven hundred and eighteen papers about RP gene-mutation screening have been published from other countries,and more than 2000 variants were identified which including 352 disease-causing mutations. Mutations of RHO gene, RDS and RP1 were found in 25％-50％, 8％ and 5％-10％ of ADRP patients in Caucasian populations. RPGR mutations were found in 70％-80％ of XLRP patients in Caucasian populations, and 50％-60％ of mutations are identified in open reading frame (ORF) 15. Totally 391 papers about gene therapy for RP have been published. Conclusions Seventeen RP-related genes have been studied and 131pathogenic mutations were found in China. Considering Chinese population, our RP research is still behind other countries. Key words: Retinitis pigmentosa; Gene; Mutation; Therapy

Peripherin/rds co-distributes with putative binding partners in basal rod outer segment disks

The retinal degeneration slow protein peripherin/rds, (a.k.a. RDS), encoded by the rds gene, is essential for the formation, organization and maintenance of outer segments (Arikawa et al., 1992; Connell et al., 1991; Connell and Molday, 1990; Kedzierski et al., 2001; Molday, 1998). In humans, any one of over 160 mutations within this gene results in a broad variety of late onset progressive retinal dystrophies characterized by abnormal photoreceptor structure (Dryja et al., 1997; Keen and Inglehearn, 1996; Kohl et al., 1998). The pathogenic mechanism(s) underlying peripherin/rds mediated retinal dystrophies are unknown, however new insight has been gained in our understanding of peripherin/rds function based upon recent structural studies identifying the C-terminal domain of this protein as homologous to structures called intrinsically disordered domains (IDD) (Dyson, 2005; Edrington, 2007b; Ritter, 2005). IDDs are unstructured domains that become structurally stabilized by interacting with one or with several binding partners (Uversky et al., 2008). Structural flexibility imparts multi-functionality to the protein and binding partners act as regulators of its function (Lakoucheva et al., 2002; Vucetic et al., 2007). In humans, numerous mutations in the rds C-terminus result in rod/cone or cone/rod dystrophies. In a murine model of retinitis pigmentosa, deletion of codon 307 in the rds gene results in severely dysmorphic outer segments (OSs) in mice (McNally et al., 2002). The rds 307 del mouse provides the first in vivo evidence for a correlation between intrinsic disorder and retinopathy. In these mice disruption of the peripherin/rds C-terminus results in a gene product with a more intrinsically ordered C-terminal domain characterized by an increase in α-helical content.

A 350 bp region of the proximal promoter of Rds drives cell-type specific gene expression

RDS (retinal degeneration slow) is a photoreceptor-specific tetraspanin protein required for the biogenesis and maintenance of rod and cone outer segments. Mutations in the Rds gene are associated with multiple forms of rod- and cone-dominant retinal degeneration. To gain more insight into the mechanisms underlying the regulation of this gene, the identification of regulatory sequences within the promoter of Rds was undertaken. A 3.5 kb fragment of the 5′ flanking region of the mouse Rds gene was isolated and binding sites for Crx, Otx2, Nr2e3, RXR family members, Mef2C, Esrrb, NF1, AP1, and SP1 in addition to several E-boxes, GC-boxes and GAGA-boxes were identified. Crx binding sequences were conserved in all mammalian species examined. Truncation expression analysis of the Rds promoter region in Y-79 retinoblastoma cells showed maximal activity in the 350 bp proximal promoter region. We also show that inclusion of more distal fragments reduced promoter activity to the basal level, and that the promoter activities are cell-type and direction specific. Co-transfection with Nrl increased promoter activity, suggesting that this gene positively regulates Rds expression. Based on these findings, a relatively small fragment of the Rds promoter may be useful in future gene transfer studies to drive gene expression in photoreceptors.

Gene delivery to mitotic and postmitotic photoreceptors Via compacted DNA nanoparticles results in improved phenotype in a mouse model of retinitis pigmentosa

The purpose of the present study was to test the therapeutic efficiency and safety of compacted-DNA nanoparticle-mediated gene delivery into the subretinal space of a juvenile mouse model of retinitis pigmentosa. Nanoparticles containing the mouse opsin promoter and wild-type mouse Rds gene were injected subretinally into mice carrying a haploinsufficiency mutation in the retinal degeneration slow (rds(+ or -)) gene at postnatal day (P)5 and 22. Control mice were either injected with saline, injected with uncompacted naked plasmid DNA carrying the Rds gene, or remained untreated. Rds mRNA levels peaked at postinjection day 2 to 7 (PI-2 to PI-7) for P5 injections, stabilized at levels 2-fold higher than in uninjected controls for both P5 and P22 injections, and remained elevated at the latest time point examined (PI-120). Rod function (measured by electroretinography) showed modest but statistically significant improvement compared with controls after both P5 and P22 injections. Cone function in nanoparticle-injected eyes reached wild-type levels for both ages of injections, indicating full prevention of cone degeneration. Ultrastructural examination at PI-120 revealed significant improvement in outer segment structures in P5 nanoparticle-injected eyes, while P22 injection had a modest structural improvement. There was no evidence of macrophage activation or induction of IL-6 or TNF-alpha mRNA in P5 or P22 nanoparticle-dosed eyes at either PI-2 or PI-30. Thus, compacted-DNA nanoparticles can efficiently and safely drive gene expression in both mitotic and postmitotic photoreceptors and retard degeneration in this model. These findings, using a clinically relevant treatment paradigm, illustrate the potential for application of nanoparticle-based gene replacement therapy for treatment of human retinal degenerations.-Cai, X., Conley, S. M., Nash, Z., Fliesler, S. J., Cooper, M. J., Naash, M. I. Gene delivery to mitotic and postmitotic photoreceptors via compacted DNA nanoparticles results in improved phenotype in a mouse model of retinitis pigmentosa.

A Partial Structural and Functional Rescue of a Retinitis Pigmentosa Model with Compacted DNA Nanoparticles

Previously we have shown that compacted DNA nanoparticles can drive high levels of transgene expression after subretinal injection in the mouse eye. Here we delivered compacted DNA nanoparticles containing a therapeutic gene to the retinas of a mouse model of retinitis pigmentosa. Nanoparticles containing the wild-type retinal degeneration slow (Rds) gene were injected into the subretinal space of rds+/− mice on postnatal day 5. Gene expression was sustained for up to four months at levels up to four times higher than in controls injected with saline or naked DNA. The nanoparticles were taken up into virtually all photoreceptors and mediated significant structural and biochemical rescue of the disease without histological or functional evidence of toxicity. Electroretinogram recordings showed that nanoparticle-mediated gene transfer restored cone function to a near-normal level in contrast to transfer of naked plasmid DNA. Rod function was also improved. These findings demonstrate that compacted DNA nanoparticles represent a viable option for development of gene-based interventions for ocular diseases and obviate major barriers commonly encountered with non-viral based therapies.

Molecular genetic basis of tapetoretinal degeneration

The review on problem tapetoretinal degeneration (TD) which represents serious enough and incurable disease revealed with frequency 1 : 3500-5000 in general population is presented. The most often reason of occurrence TD are mutations in RHO, RDS and RPE65 genes. The precise interrelation of pigmentary degenerations of a retina and mutations in genes RHO, RDS and RPE65 will allow to develop approaches of DNA--diagnostics of hereditary dystrophies of a retina so frequently meeting in clinical practice of the ordinary ophthalmologist, and also to pass at medical genetics consultation from probability estimations of risk of disease to unequivocal. Also the molecular analysis of genes changes in the providing correct functioning of photoreceptors and pigmentary epithelium of a retina and determining pathological changes at TD, will allow to approach to understanding of the physiological and pathological processes proceeding in a retina and by that will serve becoming and development pathogenic to caused therapy TD closer.

Phenotypic Intrafamilial Variability Associated with S212G Mutation in the RDS/Peripherin Gene

To describe an Italian family in which two separate phenotypes (retinitis pigmentosa and adult onset vitelliform macular dystrophy) are associated with an identical mutation (S212G) in the peripherin/RDS gene. This mutation has already been reported in patients with retinitis pigmentosa, but it has never been previously detected in association with adult onset vitelliform macular dystrophy. A 38-year-old woman complained of bilateral mild metamorphopsias and on ophthalmologic examination she showed the clinical phenotype of adult onset vitelliform macular dystrophy. Her 62-year-old mother was clinically diagnosed with a retinitis pigmentosa, with a severe clinical course. In both patients, molecular genetic analysis revealed a 874A-->G transition in the exon 2 of the RDS gene leading to the amino acid change of S212G. Peripherin/RDS S212G mutation may have damaging effects on the formation and stability of the photoreceptors' disk structure and may be associated with different clinical phenotypes, even in the same family. Intrafamilial phenotypic variability has been reported for other RDS mutations; this supports the possible influence of modifier genes or environmental factors in the clinical expression of RDS gene variants. Moreover, it suggests that in patients with retinal degeneration and peripherin/RDS mutation, caution should be taken both in using molecular genetic results to predict the clinical course of the disease and in offering genetic counseling.

An intramembrane glutamic acid governs peripherin/rds function for photoreceptor disk morphogenesis.

Peripherin/rds (P/rds), the product of the retinal degeneration slow (rds) gene, is a tetraspanin protein that plays a pivotal role for photoreceptor outer segment (OS) structure and is involved in a broad spectrum of inherited retinal degenerations. P/rds interacts with the homologous protein rom-1, previously proposed to regulate P/rds function. The authors examined the significance of an intramembrane glutamic acid conserved in all P/rds proteins (and many other tetraspanins) but absent in all rom-1 orthologs. The authors performed isosteric glutamine substitution of the conserved glutamate at position 276, in the fourth transmembrane domain of bovine P/rds, and expressed E276Q P/rds in COS-1 cells and in transgenic mouse photoreceptors of rds +/+, -/+, and -/- backgrounds. Western blot, immunoprecipitation, and sedimentation analyses were used to assess protein structure and interactions. Microscopy and electroretinography were used to characterize transgenic protein localization and retinal photoreceptor structure and function. E276Q P/rds was expressed, assembled, and properly localized in photoreceptor OSs of transgenic mice. In contrast to wild-type (WT) P/rds, however, this mutant did not rescue the OS structural defects observed in rds -/- and -/+ mice. Moreover, E276Q expression did not prevent the retinal degeneration that occurred as a consequence of OS disruption. E276 plays a critical role in P/rds support of photoreceptor OS structure. This finding provides a molecular rationale for asymmetry in P/rds and rom-1 function and for rom-1 regulation of P/rds activity. These findings also suggest that ionizable intramembrane residues may serve regulatory roles for tetraspanin proteins more generally.

The Tetraspanin Protein Peripherin-2 Forms a Complex with Melanoregulin, a Putative Membrane Fusion Regulator

Peripherin-2, the product of the rds gene, is a tetraspanin protein. In this study, we show that peripherin-2 forms a complex with melanoregulin (MREG), the product of the Mreg locus. Genetic studies suggest that MREG is involved in organelle biogenesis. In this study, we explore the role of this protein in processes associated with the formation of disk membranes, specialized organelles of photoreceptor rod cells. MREG antibodies were generated and found to be immunoreactive with a 28 kDa protein in retinal extracts, bovine OS, ARPE-19 cells, and rat RPE. MREG colocalized with peripherin-2 in WT (CB6F1/J) and in rds+/- retinas. Western blots of serial tangential sections confirmed the close association of these two proteins within the IS and basal outer segment of rods. Immunoprecipitation (IP) of OS extracts showed formation of a complex between MREG and peripherin-2-ROM-1 hetero-oligomers. This interaction was confirmed with pulldown analyses in which the GST-PerCter protein selectively pulled down His-MREG and His-MREG selectively pulled down PerCter. Biacore analysis using peptide inhibitors and per-2 truncation mutant studies allowed us to map the MREG binding site on per-2 to the last five residues of the C-terminus (Gln341-Gly346), and kinetic data predicted a KD of 80 nM for PerCter-MREG binding. Finally, the effect of MREG on photoreceptor specific membrane fusion was assayed using a disk-plasma membrane cell free assay. Preincubation of target membranes with MREG resulted in a dose-dependent inhibition of fusion with an IC50 in the submicromolar range. Collectively, these results suggest that this newly identified protein regulates peripherin-2 function.

Clinical Findings in a Multigeneration Family With Autosomal Dominant Central Areolar Choroidal Dystrophy Associated With an Arg195Leu Mutation in the Peripherin/RDS Gene

To characterize clinical findings associated with a mutation in codon 195 (Arg195Leu) of the peripherin/RDS gene in a large multigeneration family of European decent. Sixteen members from 2 generations underwent ophthalmologic examination, including best-corrected visual acuity, examination of the anterior segments, and inspection of the ocular fundus after pharmacologic mydriasis. All affected family members underwent Farnsworth Panel-D15 color testing. Five selected family members with early stages of the disease underwent multifocal electroretinography. Full-field electroretinography was performed in 2 family members with more advanced fundus changes. Finally, former patients' records and fundus images were analyzed to determine the course of the disease in affected individuals. Nine family members in 2 generations were diagnosed as having autosomal dominant central areolar choroidal dystrophy. The family demonstrated an age-dependent increase of central granular fundus abnormalities with progressive development of geographic atrophy. Interindividual phenotypic variability was apparent and ranged from predominantly drusenlike depositions to single perifoveal pigment clumps. Age of onset of visual disturbances varied between 27 and 48 years. All individuals who manifested signs of disease were found to carry an Arg195Leu mutation in the peripherin/RDS gene. Age of onset, progression of the disease, and characteristic fundus abnormalities share similarities to previous reports on families with central areolar choroidal dystrophy associated with peripherin/RDS gene mutations in codons 172, 142, and 195, respectively. However, striking variability in individual phenotypic findings and age of onset in our family suggests that additional factors that modify the defined peripherin/RDS gene mutation Arg195Leu likely influence the severity of the disease. Caution should be advised in predicting the clinical course and severity of the disease based solely on a specific mutation in the peripherin/RDS gene.

An eye on cone outer segments

![Graphic][1] Cone lamellae fail to form when Rds is absent (right).The retinal degeneration slow ( Rds ) gene is required in rod photoreceptor cells for morphogenesis of outer segments (OSs) and cell survival. On page [59][2], Farjo et al. report that cone photoreceptor cells also

RNAi-based suppression and replacement ofrds-peripherin in retinal organotypic culture

Extensive mutational heterogeneity presents a significant barrier to the development of therapeutics for RDS-peripherin-linked autosomal-dominant retinitis pigmentosa (RP), for which more than 50 disease-related mutations have been identified to date. Mutation-independent suppression, using RNA interference (RNAi), together with simultaneous expression of a replacement rds gene (r-rds, which has been altered to escape suppression but nevertheless encodes wild-type protein) has been explored in COS-7 cells and mouse retinal explants. The efficacy of small interfering and short hairpin RNAs (si/shRNAs) silencing mouse rds, and the function of r-rds (containing degenerate substitutions in the RNAi target sequence) were analyzed at transcript (RT-PCR) and protein (ELISA) levels in COS-7 cells. "Dual-" and "triple-expression" constructs carrying the shRNA suppressor and the marker EGFP with or without the r-rds cassette were electroporated in vitro into retinal explants from 1-day-old pups. The retinae were dissociated at day 14, and transduced cells were FACS-sorted using the coexpressed EGFP marker and analyzed by RT-PCR. si/shRNAs decreased rds mRNA and protein expression by up to 82%, while r-rds was protected from suppression in COS-7 cells. Similarly, efficient RNAi-mediated suppression of endogenous rds was detected in retinal explants, while concomitant rescue of r-rds was also achieved. These data validate the concept of RNAi-based suppression coupled with replacement technology for the development of therapies targeting RDS-linked autosomal-dominant RP, and suggest that such approaches could potentially be used for other autosomal-dominant diseases with similarly extensive intragenic heterogeneity.

AUTOSOMAL DOMINANT PATTERN DYSTROPHY

To describe the clinical features of and identify the mutation responsible for an autosomal dominant pattern dystrophy occurring in a three-generation family. Five affected family members underwent clinical examination and additional testing including intravenous fluorescein angiography where indicated. Mutation screening of the peripherin/RDS gene was performed. Visual acuity ranged from 20/20 to counting fingers. All patients who reported vision loss noted the onset after the age of 40 years. Predominantly perifoveal, discrete, retinal pigment epithelial changes were present in all patients. Two patients had extensive peripheral yellowish flecks, and one had an atrophic macular scar. Mutation screening of the complete peripherin/RDS coding sequence and exon/intron boundaries revealed a novel splice site mutation. A three-generation family with an autosomal dominant pattern dystrophy arising from a previously unreported splice site mutation in the RDS gene is described.

Cellular Mechanisms of Retinal Degenerations: RPE65, ABCA4, RDS, and Bicarbonate Transporter Genes as Examples

Cellular Mechanisms of Retinal Degenerations: RPE65, ABCA4, RDS, and Bicarbonate Transporter Genes as Examples

Cone–Rod Dystrophy, Intrafamilial Variability, and Incomplete Penetrance Associated with the R172W Mutation in the Peripherin/ RDS Gene

To determine the underlying molecular genetic basis of a retinal dystrophy identified in a 5-generation family, and to examine the phenotype and degree of intrafamilial variability. Family genetic study. Nine affected individuals from a nonconsanguineous British family. Ophthalmologic examination, color vision testing, fundus photography, autofluorescence imaging, and electrophysiological assessment were performed. The clinical notes of 2 additional deceased affected family members were also reviewed. Blood samples were taken for DNA extraction, with linkage analysis being performed, and subsequent mutation screening of the peripherin/RDS gene. Linkage analysis established a disease interval on chromosome 6p, which harbored the retinal candidate gene, peripherin/RDS. The 3 coding exons of the peripherin/RDS gene were subsequently screened for mutations in affected and unaffected family members. A nonconservative missense substitution, Arg172Trp (R172W), segregated uniquely in all affected subjects. The majority of subjects carrying the R172W peripherin/RDS mutation complained of reduced central vision starting in the second or third decade, with subsequent gradual deterioration of visual acuity and color vision. Three affected individuals complained of nyctalopia. A range of macular appearances were seen, varying from a typical granular appearance to extensive macular atrophy. Autofluorescence imaging in the majority of individuals identified a highly characteristic speckled macular appearance. All affected subjects had abnormal pattern electroretinograms (ERGs) consistent with macular dysfunction and 4 subjects showed additional full-field ERG abnormalities, providing evidence of generalized retinal dysfunction. There was marked variation in the clinical phenotype in those individuals who carried the R172W peripherin/RDS mutation, ranging from severe cone-rod dystrophy to asymptomatic individuals with normal retinal function. The Arg172Trp (R172W) peripherin/RDS mutation has been previously reported to cause a fully penetrant progressive macular dystrophy with high intrafamilial and interfamilial consistency of phenotype. This is the first report describing marked intrafamilial variation associated with this mutation, including nonpenetrance. These findings are clinically important in relation to advice on prognosis and accurate genetic counseling.