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Purpose: We examined the relationship between high myopia and common polymorphisms in four candidate genes: collagen, type XI, alpha 1 (COL11A1); collagen, type XVIII, alpha 1 (COL18A1); fibrillin 1 (FBN1); and procollagen-lysine 1,2-oxoglutarate 5-dioxygenase 1 (PLOD1). These genes were selected because rare pathogenic mutations in these genes cause disease syndromes that have myopia, usually high myopia, as one of the common presenting features., Methods: This study recruited 600 unrelated Han Chinese subjects including 300 cases with high myopia (spherical equivalent or SE≤-8.00 diopters) and 300 controls (SE within ±1.00 diopter). A total of 66 tag single nucleotide polymorphisms (SNPs) were selected for study from these four candidate genes. The study adopted a DNA pooling strategy with an initial screen of DNA pools to identify putatively positive SNPs and then confirmed the "positive" SNPs by genotyping individual samples forming the original DNA pools. DNA pools were each constructed by mixing equal amounts of DNA from 50 individuals with the same phenotype status. Six case pools were prepared from 300 cases and six control pools from 300 controls. Allele frequencies of DNA pools were estimated by analyzing the primer-extended products with denaturing high performance liquid chromatography and compared between case pools and control pools with nested ANOVA., Results: In the first stage, 60 SNPs from the 4 candidate genes were successfully screened using the DNA pooling approach. Of these, 6 SNPs showed a statistical significant difference in estimated allele frequencies between case pools and controls at p<0.10. In the second stage, these "positive" SNPs were followed up by individual genotyping, but failed to be confirmed via standard single-marker and haplotype analyses., Conclusions: Common polymorphisms in these four candidate genes (COL11A1, COL18A1, FBN1 and PLOD1) were unlikely to play important roles in the genetic susceptibility to high myopia.

Purpose: Choroideremia (CHM) is an X-linked retinal degenerative disorder caused by mutations in the CHM gene. The mutations result in malfunction of the Rab escort protein 1 (REP-1). In this study, mutational analysis of the CHM gene was performed on five Chinese families clinically diagnosed with CHM., Methods: Denaturing high performance liquid chromatography was used for mutation screening for all 15 exons and flanking intron regions of the CHM gene. Mutations were confirmed and characterized with DNA sequencing. Second samples were later collected for extraction of mRNA and proteins from leukocytes. A non-radioactive protein truncation test (PTT) was developed and used to characterize the truncating nature of the mutations. Immunoblot analysis of proteins extracted from leukocytes was also performed., Results: Five mutations were identified in these five families, each with one distinct mutation: three frameshift, one nonsense, and one splicing. Two of these were novel mutations: c.627dupA in exon 5 and c.703-1G>C in intron 5. The truncating nature of the mutations was experimentally proved by PTT for four families with second samples collected. In particular, c.703-1G>C spliced exon 5 directly to exon 7 and deleted the entire exon 6 from the transcript. Direct immunoblot analysis failed to detect REP-1 in males affected by CHM, but demonstrated its presence in female carriers and homozygous normal females., Conclusions: This is the first study reporting mutations in the CHM gene in Chinese families. Mutational analysis was performed at the DNA, mRNA and protein levels. Five truncating mutations were found, and two of these were novel.

Purpose: To test the association between myocilin gene (MYOC) polymorphisms and high myopia in Hong Kong Chinese by using family-based association study., Methods: A total of 162 Chinese nuclear families, consisting of 557 members, were recruited from an optometry clinic. Each family had two parents and at least one offspring with high myopia (defined as -6.00D or less for both eyes). All offspring were healthy with no clinical evidence of syndromic disease and other ocular abnormality. Genotyping was performed for two MYOC microsatellites (NGA17 and NGA19) and five tag single nucleotide polymorphisms (SNPs) spreading across the gene. The genotype data were analyzed with Family-Based Association Test (FBAT) software to check linkage and association between the genetic markers and myopia, and with GenAssoc to generate case and pseudocontrol subjects for investigating main effects of genetic markers and calculating the genotype relative risks (GRR)., Results: FBAT analysis showed linkage and association with high myopia for two microsatellites and two SNPs under one to three genetic models after correction for multiple comparisons by false discovery rate. NGA17 at the promoter was significant under an additive model (p=0.0084), while NGA19 at the 3' flanking region showed significant results under both additive (p=0.0172) and dominant (p=0.0053) models. SNP rs2421853 (C>T) exhibited both linkage and association under additive (p=0.0009) and dominant/recessive (p=0.0041) models. SNP rs235858 (T>C) was also significant under additive (p=4.0E-6) and dominant/recessive (p=2.5E-5) models. Both SNPs were downstream of NGA19 at the 3' flanking region. Positive results for these SNPs were novel findings. A stepwise conditional logistic regression analysis of the case-pseudocontrol dataset generated by GenAssoc from the families showed that both SNPs could separately account for the association of NGA17 or NGA19, and that both SNPs contributed separate main effects to high myopia. For rs2421853 and with C/C as the reference genotype, the GRR increased from 1.678 for G/A to 2.738 for A/A (p=9.0E-4, global Wald test). For rs235858 and with G/G as the reference, the GRR increased 2.083 for G/A to 3.931 for A/A (p=2.0E-2, global Wald test). GRR estimates thus suggested an additive model for both SNPs, which was consistent with the finding that, of the three models tested, the additive model gave the lowest p values in FBAT analysis., Conclusions: Linkage and association was shown between the MYOC polymorphisms and high myopia in our family-based association study. The SNP rs235858 at the 3' flanking region showed the highest degree of confidence for association.