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Null Retinoschisin-Protein Expression from an RS1 C354del1-Ins18 Mutation Causing Progressive and Severe XLRS in a Cross-Sectional Family Study

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Null Retinoschisin-Protein Expression from an RS1 C354del1-Ins18 Mutation Causing Progressive and Severe XLRS in a Cross-Sectional Family Study Null Retinoschisin-Protein Expression from an RS1 c354del1-ins18 Mutation Causing Progressive and Severe XLRS in a Cross-Sectional Family Study Camasamudram Vijayasarathy,1 Lucia Ziccardi,1 Yong Zeng,1 Nizar Smaoui,2 Rafael C. Caruso,2 and Paul A. Sieving1,3 PURPOSE. To explore the retinoschisin (RS1) protein biochem- -linked juvenile retinoschisis (XLRS) is a highly penetrant ical phenotype from an RS1 exon-5 deletion/insertion frame- Xrecessive retinal dystrophy characterized by early-onset shift mutation in a family with X-linked retinoschisis (XLRS) central visual loss from bilateral foveo-macular cystic cavities and describe the clinical and electrophysiological features. involving the inner retina1 and additional retinal layers.2,3 Ap- proximately half the affected males also exhibit peripheral METHODS. Six XLRS males underwent ophthalmic examination retinoschisis.4 XLRS is one of the more common causes of RS1 and electroretinogram (ERG) recording. The gene was juvenile macular degeneration in males, with estimated preva- sequenced. Mutant RS1-RNA and protein expression were as- lence at 1:5,000 to 1:25,000.5–7 sessed by transfecting COS-7 cells with minigene constructs. The RS1 gene contains six exons and encodes a 24-kDa RESULTS. All six males carried the RS1 c354del1-ins18 mutation secreted retinoschisin (RS1) protein, that includes a conserved in which an 18-bp insertion replaced nucleotide 354, duplicat- discoidin domain8 homologous to proteins implicated in cell ing the adjacent upstream intron 4-to-exon 5 junction and adhesion and cell–cell interactions.9 RS1 is highly expressed in creating a premature termination codon downstream. Analysis photoreceptor cells and also in neurons of the inner reti- 10–12 indicated normal pre-mRNA splicing producing mRNA tran- na. More than 150 mutations have been described across 13 scripts. Truncated RS1 protein was expressed transiently but the small RS1 gene. The majority are missense mutations in exons 4 to 6; deletions, insertions, and nonsense and splice site was degraded rapidly by a proteasomal pathway rather than by 6–8,14–16 nonsense-mediated mRNA decay. Two boys, 1.5 and 5 years of mutations have also been reported. age, had foveal cysts and minimal peripheral schisis, and re- The XLRS phenotype is quite variable. The most common clinical finding is bilateral foveomacular schisis.17 Retinal fun- tained near-normal scotopic b-wave amplitude and normal ERG dus examination and optical coherence tomography (OCT) waveforms. The 5-year-old’s ERG was diminished when re- scans of older affected males often demonstrate flattening and peated 3 years later. Four older XLRS relatives 32 to 45 years coalescence of the foveal schisis cavities.18 The retinal pigment old had substantial b-wave loss and strongly electronegative epithelium (RPE) may show granularity and atrophy in later ERGs; three had overt macular atrophy. Cross-sectional family ages.18,19 The full-field electroretinogram (ERG) in XLRS gen- analysis showed the b-/a-wave amplitude ratio as inversely erally shows a greater loss of the positive-going b-wave than of related to age in the six males. the negative-going a-wave,20–23 although relative b-wave pres- 6,24 CONCLUSIONS. The c354del1-ins18 mutation caused an RS1-null ervation has also been described. As the b-wave from bipo- biochemical phenotype and a progressive clinical phenotype lar cells lies postsynaptic to the photoreceptor a-wave, the ERG in a 5-year-old boy, whereas the older XLRS relatives had provides evidence of inner retinal dysfunction possibly involv- macular atrophy and marked ERG changes. The phenotypic ing the photoreceptor synapse. heterogeneity with age by cross-sectional study of this family Genotype–phenotype correlations can help to elucidate mutation argues that XLRS disease is not stationary and raises molecular genetic mechanisms underlying macular degenera- questions regarding factors involved in progression. (Invest tion induced by RS1 mutations. In this cross-sectional study, we describe the clinical features of an XLRS family with an RS1 Ophthalmol Vis Sci. 2009;50:5375–5383) DOI:10.1167/iovs.09- exon 5 deletion/insertion mutation (c354del1-ins18). The ERG 3839 changes observed across three years in a 5-year-old affected boy and the ERG differences between the younger versus middle-age affected males raise questions about the nature of From the 1Section for Translational Research in Retinal and Mac- disease progression in XLRS. ular Degeneration, National Institute on Deafness and Other Commu- nication Disorders, the 2Ophthalmic Genetics and Visual Function Branch, National Eye Institute, and the 3Office of the Director, National METHODS Eye Institute, National Institutes of Health, Bethesda, Maryland. Supported by the Intramural Program of the National Institutes of Subjects Health and National Institute of Diseases of Childhood Diseases (NIH/ We studied six affected males in a multigeneration Caucasian Hispanic NIDCD). XLRS family (Fig. 1) including two young affected boys (VI.5, proband Submitted for publication April 9, 2009; accepted July 30, 2009. and brother VI.6), 5 and 1.5 years old, and four older maternal male Disclosure: C. Vijayasarathy, None; L. Ziccardi, None; Y. Zeng, None; N. Smaoui, None; R.C. Caruso, None; P.A. Sieving, None relatives, 32 to 45 years old. The study protocols were approved by the The publication costs of this article were defrayed in part by page National Institutes of Health Institutional Review Board, consonant charge payment. This article must therefore be marked “advertise- with the tenets of the Declaration of Helsinki, and the subjects or their ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. guardians gave informed consent. Corresponding author: Paul A. Sieving, Section for Translational Research in Retinal and Macular Degeneration, National Institute on Clinical Examination Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD 20892; [email protected]. The subjects were examined by fundus biomicroscopy and indirect ophthalmoscopy. Best-corrected Snellen visual acuity, Goldman kinetic Investigative Ophthalmology & Visual Science, November 2009, Vol. 50, No. 11 Copyright © Association for Research in Vision and Ophthalmology 5375 5376 Vijayasarathy et al. IOVS, November 2009, Vol. 50, No. 11 FIGURE 1. Pedigree of the XLRS family. Arrow: the proband. perimetry (Haag-Streit, Bern, Switzerland) and optical coherence to- Site-Directed Mutagenesis mography (Stratus OCT 3; Carl Zeiss Meditec, Dublin, CA) were per- formed. The central visual absolute luminance threshold was measured For downstream cloning, the EcoRI site was introduced by four silent after 30 minutes of dark adaptation with a Goldmann-Weekers adap- point mutations at nucleotides (NT)-309–314 in exon 4 of RS1 cDNA. tometer (Haag-Streit). Sense and antisense oligomers that differed from the wild-type se- quence at four sites (C309G, C312T, A313T, and G314C) were chem- ically synthesized. These synthesized fragments were introduced into a ERG Recording Tag4A-RS1 vector (Quick Change Site-Directed Mutagenesis kit; Strat- agene). The clones were verified by DNA sequencing. The creation of ERG responses were elicited by full-field flash stimuli (UTAS 2000; LKC the EcoRI site altered neither the RS1-protein sequence nor the expres- Technologies, Gaithersburg, MD, and Espion 1, Diagnosys Inc., Lowell, sion and localization. MA) after pupil dilation (phenylephrine hydrochloride 2.5% and tropi- camide 1%) and 30 minutes of dark adaptation. Burian-Allen bipolar RS1 Minigene Expression Vectors corneal ERG electrodes (Hansen Ophthalmic Instruments, Iowa City, IA) were placed after topical corneal anesthesia (proparacaine hydro- The RS1 minigene included RS1 genomic introns 4 and 5 in frame with chloride 0.5%). Dark-adapted, rod-mediated, and combined rod-plus- exons 1–6 of RS1 cDNA in the pCMV Tag 4A expression vector. cone ERG responses were recorded. Cone-mediated responses were Genomic DNA from normal control males and XLRS-affected males of then elicited after a 10-minute light adaption. ERG responses were this family was PCR-amplified to generate fragments encoding intron-4 amplified at 0.3 to 300 Hz, digitized, and stored. ERG a-wave and and -5 sequences, and the corresponding flanking exon sequences. b-wave amplitudes (␮V) and implicit times (milliseconds) were com- Primers sequences were: intron 4 with flanking sequences: forward: pared with those in 120 unaffected subjects. 5Ј-ctgaattctcaaggctttgggtaagcagg-3Ј and reverse: 5Ј-gggtgcgagctgaagt- tgg-3Ј; and intron 5 with flanking sequences forward: 5Ј-gagagccagcac- ctgcgg-3Ј and reverse: 5Ј-gctcgagtcaggcacacttgctgacgcac-3Ј. PCR Amplification and Mutation Detection A central segment corresponding to intron 4 with the flanking Genomic DNA was isolated from peripheral blood leukocytes. Exons 1 exon-4 and -5 sequences was introduced between the EcoRI and to 6 and the intron–exon boundaries of RS1 were amplified by PCR BamHI sites. Likewise, intron 5 and its flanking exon-5 and -6 se- with a combination of primer sets, as previously described,8 and were quences were cloned between the BamHI and XhoI sites. The mutant sequenced (Prism 310 Genetic Analyzer; Applied Biosystems, Inc. minigene contained the exon-5 deletion/insertion mutation at NT-354. [ABI], Foster City, CA). DNA sequence of all constructs was verified at each step. Cell Culture and Transfection Expression Vectors Functionality of the wild-type (WT) and mutant (MUT) [c354del1- RS1 cDNA (NM 000330) was amplified from human retinal total RNA ins18] RS1 minigenes was studied. COS-7 cells were grown in DMEM using RT-PCR and cloned into the pCR II-TOPO vector (Invitrogen, with 10% FBS. A day before transfection, the cells were plated at Carlsbad, CA).25 The entire RS1 cDNA coding sequence was further 400,000 cells per 10-cm culture dish. These were transiently trans- PCR-amplified using the NotI primed sense primer (5Ј-gcggccgcgccac- fected the next day using 18 ␮L of transfection reagent (FuGene 6; catgtcacgcaagatagaaggctt-3Ј) and the XhoI primed antisense primer Roche Applied Science, Indianapolis, IN) and 6 ␮g of pCMV-Tag 4A (5Ј-ctcgagtcaggcacacttgctgacgcac-3Ј).
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