Mapping The Disease-causative Gene For Pedigree Li With Primary Open Angle Glaucoma In Zhongxian, Chongqing

Primary open angle glaucoma(POAG) is one of severe eye disease leading to blindness.Although its pathogenesis has not been understood completely,epidemiological survey found that genetic factors played an important role in POAG's development.A large pedigree affected by POAG in Zhongxian,Chongqing had been discovered, which had significantly Mendelian feature of autosomal dominant inheritance.This pedigree with clear structure,enough members and certain phenotype is an ideal subject to map the disease-causative gene(DCG) of POAG.Based on the above,we planned to regard Pedigree Li as the subject of the study and make comprehensive genetic investigation and strict phenotype-defining.And we intended to apply the strategy of "candidate loci cloning" and linkage analysis to screen the candidate genes and loci of POAG by means of short tandem repeat(STR),hoping to find the real DCG wrecking Pedigree Li and reinforce the causes spectrum of POAG and at the same time,to supply some useful information for the following functional study.The study was divided into the following five parts:1.Replenished the genetic investigation of Pedigree Li and confirmed its members' clinical information.2. Constructed and conserved the immortal cell strains of Pedigree Li by means of Epstein-Barr virus(EBV) transforming with the help of cyclosporin A(CsA),in order to ensure the sustainable application of the pedigree's genomic DNA(gDNA) in the future.3. Applied the strategy of "candidate loci cloning" and linkage analysis to screen the candidate genes and loci of POAG in OMIM in order to get significantly linked loci.4. Sequenced the coding region of CYP1B1 both in Pedigree Li and control group,in order to make clear whether the mutation of CYP1B1 co-segregate with the pedigree and is the real cause of the disease development.5.Increased the density of STR markers near D2S2259 and went on genotyping and linkage analysis,in order to narrow down the significantly linked region and verify whether CYP1B1 is the real DCG of Pedigree Li from the point of linkage mapping. The main results and conclusions are as follows:1.Pedigree Li is an ideal subject to map the disease-causative gene(DCG) of POAG with autosomal dominant inheritance.There were 34 members of 4 generations in actrual existence and 11 patients with POAG(8 males and 3 females).Members of the pedigree had high intraocular pressure(IOP)(greater than 21mmHg when the disease attacking), age-of-onset less than 40 years old(the male's average:26.5;the female's average:32.7), severly damaged optic nerve(vertical cup-disk ratio almost equal to 1.0) and serious loss of visual function(characteristic visual field loss of advanced stage).2.The successful rate of immortal cell strains' construction and reactivation for Pedigree Li was 91.2%(31 strains) and 87.1%(27 strains),respectively,by means of EBV transforming with the help of CsA.This method could conserve rare genetic materials with relatively simple and reliable results.3.The 34 gDNAs,corresponding to the 34 individuals of Pedigree Li,had good property to meet the standard of genotyping,which were extracted by salting-out method (11),Blood DNA Kit(20) and Oragene Saliva Purification Kit(3),respectively.Eight STR markers representing MYOC,OPTN,WDR36 and CYP1B1 were made to run the first screening experiment under the strategy of candidate loci cloning.The results of two-point linkage analysis showed that D2S367 and D2S2259 near CYP1B1 had significant LOD score(LODs),especially D2S2259,whose LODs(2.135) andθ(0.06) came up to the standard(LOD≥2.1,θ＜0.10) put forward by Lander and Kruglyak in 1995, suggesting that the DCG of Pedigree Li links to D2S2259 and it is likely to locate beside CYP1B1.The above results also exclude the possibility of linkage for MYOC(1q21-q31), OPTN(10p15-p14) and WDR36(5q22.1).4.Seven heterozygous mutations(342 G/C,441 G/A,494 G/C,1720 C/A,1933 G/C, 2087 G/A and 2198-2199 CT del) from 5 POAG patients and 1 normal relative were found by sequencing the coding region of CYP1B1 in 34 members of Pedigree Li and 30 normal controls.The mutation frequency was 17.65%of Pedigree Li,45.45%of the pedigree's patients and 4.35%of the pedigree's normal relatives.The above results suggest that the mutations of CYP1B1's coding region are not the real cause of Pedigree Li's pathology,but correlated with their phenotype.5.Verified 4 fine-mapping STR markers(D2S2369:LODs=4.584,θ=0.00; D2S2352:LODs=2.992,θ=0.00;D2S378:LODs=6.238,θ=0.00;D2S337:LODs=4.892,θ=0.00) to be the significantly linked with Pedigree Li and narrowed the linked region down to 2p15～2p16.3.This region was about 7cM or 7.5Mb and there were at least 12 genes expressing in the eye within it.The discovery is not only consistent with the study by Suriyapperuma SP,etc,in this year,but also smaller than theirs.