The Molecular Genetics Research Of Two Retinitis Pigmentosa Pedigrees

Retinitis pigmentosa is a set of hereditary retinal diseases affecting about 1 in 3500people worldwide,and considered to be the main cause of adult blindness.Patients withretinitis pigmentosa usually show the early onset of night blindness followed by aprogressive loss of the visual field.As a result,the grossly constricted central visioncauses irreversible blindness,often after midlife.In addition,perivascular pigmentation,attenuation of the retinal arterioles,and pale appearance of the optic disk are commonfindings during a fundus examination.To date,there is still no efficient prevention andtherapy for retinitis pigmentosa.Retinitis pigmentosa exhibits extreme heterogeneity both clinically and genetically.Itcan be inherited most frequently (50-60%) as an autosomal recessive trait,followed by anautosomal dominant pattern (30-40%) and an X-linked (5-15%) pattern.A small proportionof retinitis pigmentosa cases present with non-Mendelian inheritance patterns,such asdigenic or mitochondrial inheritance patterns.In some cases,different mutations in the samegene may involve in different inheritance traits.To date,a number of loci or genesresponsible for retinitis pigmentosa have been reported,but for about 60% of RP cases,thegenetic basis has not been found.Localization and identification of more pathogenic genesfor retinitis pigmentosa is crucial for genetic diagnosis,understanding the molecularmechanism by which the disease develops,and for finding efficient therapeutic methods.In this thesis project,we studied two Chinese retinitis pigmentosa families,including oneautosomal dominant family and one autosomal recessive family.By linkage and halpotypeanalyses,we localized the pathogenic genes onto specific chromosomal locations,and thenused direct DNA sequence analysis to find the disease-causing mutations and to demonstrate ifthe mutations co-segregate with the disease in the family.The High Resolution Melt (HRM)analysis was used to verify if these alterations exist in normal controls.In the first section of the thesis,we started our studies from a Chinese family withautosomal recessive RP from Shandong Province.Linkage analysis linked the diseasecausing gene of the arRP family to the RP26 locus,and DNA sequence analysis revealedtwo compound heterozygous mutations in the newly identified RP26 disease-causing gene, CERKL.One mutation is c.156₁57insT in exon 1,and the other is c.758delT in exon 5.The affected male individuals in the family inherited both mutations,whereas twounaffected sisters inherited normal alleles from their parents.The two mutations werenot detected in more than 100 unrelated normal controls by HRM.Both mutations are predicted to cause frameshift of CERKL protein.Thec.156₁57insT mutation introduces a stop codon at position 53 of CERKL,causing aprematurely truncated CERKL protein with only 52 amino acids.Mutation c.758delTcauses frameshift and substitutes 6 amino acids 253-METDRI-258 by abnormal RKQTES,and produces a truncated CERKL protein with only 257 amino acid residues.To date,there are only three RP causing CERKL mutations that have been reported,and all of them were identified in consanguineous families.The two novel CERKLmutations identified in this project are from a non-consanguineous Chinese family,andrepresent the first report that compound heterozygous mutations of CERKL cause retinitispigmentosa.These studies expand the spectrum of CERKL mutations causing autosomalrecessive retinitis pigmentosa.In order to usderstand the mechanism by which mutations c.156 157insT andc.758delT in CERKL causing retinitis pigmentosa,we generate the compound mutationsby the method of mutagenesis,fusing the wildtype and two mutant CERKLs withpEGFP-C1 vector,and transfected them to HeLa cell line.We investigated if the twomutations could alterate the normal subcellular localization of CERKL protein.Asignificant difference was identified between the two mutant CERKL proteins and thenormal wild type protein.Altered subcellular localization may be a mechanism the twomutations causing retinitis pigmentosa.We also investigated a Chinese family with autosomal dominant retinitis pigmentosa,inthe second section of the thesis.The patients of this family display some unique character ofsymptom of RP,such as early onset,and combining with cataract.Linkage analysis excludedall RP loci except for RP4,sequencing of the whole coding area of RHO revealed a Pro347Leumutation in exon 5 of this gene.We further performed gene diagnosis study for twoasymptomatic young members in the RP family.Our studies expand the clinical spectrum ofp.P347L mutation in RHO,and may help to understand the correlationship between thegenotypes of RHO mutations and phenotypes of retinitis pigmentosa.