Purchase this article with an account.
Leera J. D'Souza, Candice Craig, Hong He, Ji-Yun Lee, Christian Antolik, Shibo Li, Xinjing Wang; X-Linked Juvenile Retinoschisis with Novel Exonic Deletions in the RS1 Gene. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4532.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
X-linked juvenile retinoschisis (XLRS) is the most common inherited retinal dystrophy occurring in males. XLRS causes early-onset macular degeneration due to the formation of foveo-macular cavities in the inner retina. It is a highly penetrant disorder and is caused by mutations in the RS1 gene, which encodes the secreted protein retinoschisin. This protein is assembled and secreted from photoreceptors and bipolar cells as a homo-oligomeric protein complex. Over 170 distinct mutations comprising of missense, nonsense, insertions/deletions and splice site mutations have been described of which approximately 10% are single exon deletions. In the present study, we report the identification of two novel exonic deletions in the RS1 gene of patients suffering from XLRS.
Patients were diagnosed with XLRS at the National Eye Institute clinic. Peripheral blood specimens were obtained and the DNA was extracted using the Gentra Puregene kit supplied by Qiagen. Coding sequences and ~25bp of flanking intronic sequences were amplified by PCR using primers located in the intronic region. The PCR products were then directly sequenced using the Sanger sequencing methodology. Array CGH was performed for the patients whose PCR failed to amplify the target exon despite repeated attempts, suspecting an exonic deletion. The Human CGH 3x720K Whole-Genome Tiling array was used and the data analysis was carried out using the NimbleGen systems software package from Roche. To determine the exact location of the breakpoint causing deletions, primer walking was carried out by the long range PCR methodology and the junction fragments were then directly sequenced using the Sanger sequencing methodology.
Among the two patients, patient #1 had exon 1 PCR failure and patient #2 had exon 4 and exon 5 PCR failures in the RS1 gene. We then successfully mapped the candidate regions within RS1 gene locus by array CGH. The junction fragments of the exonic deletions were then identified by primer walking using long range PCRs. The location of breakpoints was confirmed by Sanger sequencing of the junction fragments. Furthermore the inheritance of the mutant allele was confirmed by long range PCR on maternal DNA.
In the present study, we have sequenced the junction fragments in patients with exonic deletions and have identified the breakpoints at the nucleotide level. This indicates that both of the deletion mutations result in a non-functioning allele. Therefore, this study provides molecular confirmation for the clinical diagnosis of XLRS in these patients. It also reveals a new mechanism that generated disease causing mutations in the RS1 gene locus.
This PDF is available to Subscribers Only