Establishment Of Haplotype Pattern In The RDH8 And HGF Genes, And The Family-based Association Study With Highmyopia

Myopia is a very common ocular refractive condition in which the parallel rays is focused in front of retina with accommodation at zero level. Myopia has a high prevalence all over the world particularly in Asian populations and has become a public health problem in modern society. Not like low to moderate myopia with refractive error lower than -6 Diopters (D) which is normally an inconvenience due to spectacle wear without causing serious ocular complications, high myopia, typically classed as in excess of - 6 D, could result in severe ocular morbidity, visual impairment and even blindness.Myopia is a complex trait for which multiple genes, environment and interaction of genes and environment have been all implicated. Evidences were strong that high myopia had an obvious genetic background. Several studies have reported genetic linkage between high myopia and some chromosome regions (7q36, 12q21-23, 18p11). However, common high myopia (not syndromic one) is thought to be multifactorial or polygeneic and the power of conventional pedigree-based linkage analysis is limited whereas genetic association study is currently thought to be the most powerful approach to map the susceptibility loci. Transmission disequilibrium test (TDT) developed by Spielman et al. and its extension methods are the effective approach to detect the association of traits and disease susceptibility genes with modest impact on disease since it can eliminate the spurious linkage due to the population admixture, heterogeneity, or stratification in conventional case-control association study.RDH8, one of the important enzymes in visual cycle, is a potential candidate for myopia susceptibility as previous studies suggested the retinoic acid might be a mediator between visual signal and compensatory eye growth. And HGF, a multifunctional cytokine, is also found to be a potential candidate for myopia, as it is closely related to the functions of MMPs and TIMPs, and more importantly, animal genetic experiments indicate the HGF is a potential gene responsible for eye size growth. A critical part of the association study is the collection and selection of markers on the basis of characterizing the LD patterns incandidate genes beforehand. In this study, we used denaturing high performance liquid chromatography (DHPLC) coupled with DNA pooling strategy to identify and genotype the SNPs to establish the LD/haplotype pattern in the candidate genes of RDH8 and HGF. Based on the LD pattern established, we selected the SNP markers for association study in Chinese population. The family-based association study (FBAT) was carried out in a group of high myopia nuclear families to test the association of the RDH8 and HGF genes with high myopia.1. SNP discovery and genotyping, and establishing the linkage disequilibrium pattern for the RDH8 and HGF genesBlood samples were obtained with written consent from 20 university students and used in the screening stage of the study. Touch-down PCR was used to amplify the genes' exons and their immediate flanking regions, and non-coding sequences about 2.5-5 kb upstream of the start codon and downstream of the stop codon. In the screening stage, 4 DNA pools were constructed with each consisting of equal amounts from 5 individuals for DHPLC analysis. The samples showing potential SNP signals by DHPLC were tested and confirmed with direct sequencing.Of the 15 SNPs identified in the RDH8, 12 were transitions (7 A/G and 5 C/T transitions), 2 transversions, and 1 was a two-base insertion/deletion. Only 3 SNPs were located in coding regions while the rest were in non-coding regions with 9 found upstream of the start codon and 3 downstream of the stop codon. One coding SNP was a synonymous transition (8117C> T or RDH8E61) in exon 6. The other two coding SNPs were adjacent to each other in the same codon (202) located in exon 5 (7826T>C and 7827G>A, or RDH8E5a and RDH8E5b, respectively).For the HGF, altogether 18 SNPs were identified in 11 fragments. Among these 18 SNPs, 2 were in the 5' flanking region, 9 in intron regions flanking e...