[Genetic analysis of rhodopsin and peripherin genes in patients with autosomal dominant retinitis pigmentosa (adRP) in Polish families].
OBJECTIVE
The aim of that study was to identify the mutations in rhodopsin and peripherin genes in Polish families with autosomal dominant form of retinitis pigmentosa and determine the population polymorphism in both genes in adRP families.


METHODS
We performed ERG, visual acuity, Goldman visual fields, intraocular pressure measurements and fundoscopy in all the patients included in the study. On the basis of disease history, the families pedigree was made and the mode of inheritance was analyzed. The molecular analysis of DNA for each family with adRP was conducted. Genomic DNA was obtained from leucocytes by phenol-chloroform procedure according to Maniatis protocol. DNA was amplified by the PCR reaction in a volume of 50 microl containing 100 ng/microl of genomic DNA, water, Cetus buffer pH 8.4 (1 n Tris, 1 n MgCl, 1 n KCl, 2% gelatin), 0.25 microM of each primer, 200 microM of each of dATP, dTTP, dCTP, and dGTP and 2.5 U Taq polymerase (Promega). For amplification of rhodopsin gene 30 cycles of PCR were carried out. Each cycle consisting of denaturation at 95 degrees C for 5 min, annealing: at 58 degrees C (exon 1), 63 degrees C (exon 2 and 3), 68 degrees C (exon 4) and 2 min extension at 72 degrees C min. For amplification of peripherin gene 30 cycles of PCR were carried out with annealing at 60 degrees C. The entire PCR product was in electrophoresis on 8% PAA. The PCR-RFLP PCR-HD PCR-SSCP and analysis of polymorphism (CA)n dinucleotide repetition was performed.


RESULTS
Molecular study demonstrated, that mutations in rhodopsin gene were cause of retinitis pigmentosa in case of two families. In any study families mutations in peripherin gene were not identified. Two kinds of bases polymorphism were identified: restriction fragments length polymorphism (RFLP) in rhodopsin gene in exon 1 and 3 and single strand conformation polymorphism (SSCP) in exon 1 and 3 in rhodopsin gene and in exon 3 in peripherin gene. The confirmed mutations in rhodopsin gene, cosegregation with adRP, whereas two kinds of population polymorphism did not correlate with clinical symptoms. Natural polymorphism appeared to be a frequent feature in rhodopsin gene while a less frequent feature in peripherin gene.


CONCLUSIONS
Genetic investigations in patients with adRP allow to confirm the diagnosis and evaluate the prognosis. The mutation in rhodopsin gene should be confirmed in directly sequencing reaction in next study.
Brzeziańska
Ewa
E
Zakładu Tyreologii Instytutu Endokrynologii Uniwersytetu Medycznego w Lodzi.
Zdzieszyńska
Małgorzata
M
Goś
Roman
R
Lewiński
Andrzej
A
D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000235:genetics; D005091:Exons / Q000235:genetics; D005136:Eye Proteins / Q000235:genetics*; D006239:Haplotypes / Q000235:genetics; D006801:Humans; D007381:Intermediate Filament Proteins / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000235:genetics*; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins / Q000235:genetics*; D010375:Pedigree; D064531:Peripherins; D017354:Point Mutation / Q000235:genetics; D011044:Poland; D011110:Polymorphism, Genetic / Q000235:genetics; D012174:Retinitis Pigmentosa / Q000208:ethnology* / Q000235:genetics*; D012243:Rhodopsin / Q000235:genetics*
D017931:DNA Primers; D005136:Eye Proteins; D007381:Intermediate Filament Proteins; D008562:Membrane Glycoproteins; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; C578994:PRPH protein, human; D064531:Peripherins; C120042:RP1 protein, human; D012243:Rhodopsin