The Screening Of The Gene Mutation Of The X-linked Juvenile Retinoschisis

Juvenile retinoschisis is a rare X-linked inherited disorder, mainly affects bilateral retina, and leads to the splitting of the retina between the nerve fiber and ganglion cell layers. The patients have typically a spoke-like schisis in the macular fovea, and in 50% of cases bilateral inferotemporal retinoschisis. The electroretinogram is beneficial in the diagnosis of juvenile retinoschisis. The a-wave can be of normal or nearly normal amplitude in this disorder, whereas the amplitude of the b-wave is appreciably reduced, giving a decrease in the proportion of b/a. The disease is transmitted exclusively in males, females are only carriers. The cloned retinoschisis gene (RS1) maps to the distal short arm of the X chromosome (Xp22.2). In 1997, the gene responsible for X-linked juvenile retinoschisis was identified by positional cloning , it consists of six exons and encodes a 224-amino-acid protein containing a hydrophobic leader sequence with a consensus signal peptidase cleavage site which mediates the transportation of the protein. RS1 is expressed on the surface of rod and cone photoreceptor cells and also on bipolar cells, but not on Müller cells, ganglion cells or inner limiting membrane. This protein is thought to play an important role in cell adhesion, cell-cell interaction or phospholipid binding on membrane surfaces that maintains the integrity of the retina. It has been verified that XLRS is caused by mutation of RS1. The Retinoschisis Consortium has screened the gene for mutations in 234 familial and sporadic retinoschisis cases and identified 82 different mutations in 214. Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) combined with silver-staining is undertaken to screen the six exons of RS1 in two retinoschisis pedigrees.Method Genomic DNA from RS pedigrees was isolated from peripheral EDTA-blood by standard phenol-chloroform method, and the purity of the DNA was determined. The coding sequences of the RS1 gene were PCR amplified using the primers designed in the introns. PCR products were detected on the 1.5% agarose gel , then were ran on polyacrylamide gel by SSCP method at low current for about 3 hours ,and selected the normal ssDNA and dsDNA as control spontaneously . The DNA bands were demonstrated by silver-staining .Result The six exons of RS1 were amplified by PCR. The products were 195bp ,178bp ,178bp ,218bp, 313bp and 414bp respectively. The size of PCR products on the agarose gel matched the expected size. The annealing temperature of each PCR was designed at 56℃, 54℃, 60℃, 60℃, 65 ℃ and 54℃ . No abnormal shifted SSCP bands were observed in exon 1,2,3,4,and an abnormal band in exon 5 and 6 respectively in two RS patients of the first pedigree. 2 abnormal bands were also observed in exon 5 in the RS patient and his sister of the second pedigree. These show that there are mutations in these exons which are the same type, and the sister is a carrier.Conclusion RS1 is the cause of the RS in these 2 pedigrees. The finding is of help with hereditary consultation. SSCP can be used to screen the RS1.