Maurice K. H. Yap

PAX6 Haplotypes Are Associated with High Myopia in Han Chinese

Background The paired box 6 (PAX6) gene is considered as a master gene for eye development. Linkage of myopia to the PAX6 region on chromosome 11p13 was shown in several studies, but the results for association between myopia and PAX6 were inconsistent so far. Methodology/Principal Findings We genotyped 16 single nucleotide polymorphisms (SNPs) in the PAX6 gene and its regulatory regions in an initial study for 300 high myopia cases and 300 controls (Group 1), and successfully replicated the positive results with another independent group of 299 high myopia cases and 299 controls (Group 2). Five SNPs were genotyped in the replication study. The spherical equivalent of subjects with high myopia was ≤−8.0 dioptres. The PLINK package was used for genetic data analysis. No association was found between each of the SNPs and high myopia. However, exhaustive sliding-window haplotype analysis highlighted an important role for rs12421026 because haplotypes containing this SNP were found to be associated with high myopia. The most significant results were given by the 4-SNP haplotype window consisting of rs2071754, rs3026393, rs1506 and rs12421026 (P = 3.54×10−10, 4.06×10−11 and 1.56×10−18 for Group 1, Group 2 and Combined Group, respectively) and the 3-SNP haplotype window composed of rs3026393, rs1506 and rs12421026 (P = 5.48×10−10, 7.93×10−12 and 6.28×10−23 for the three respective groups). The results remained significant after correction for multiple comparisons by permutations. The associated haplotyes found in a previous study were also successfully replicated in this study. Conclusions/Significance PAX6 haplotypes are associated with susceptibility to the development of high myopia in Chinese. The PAX6 locus plays a role in high myopia.

Association of PAX6 Polymorphisms with High Myopia in Han Chinese Nuclear Families

PURPOSE The paired box 6 (PAX6) gene is critical to eye development. Based on prior linkage evidence, this study was conducted to investigate the association of PAX6 polymorphisms with high myopia in a Han Chinese population. METHODS Tag single nucleotide polymorphisms (tSNPs) in the PAX6 locus were selected based on HapMap data, and other polymorphisms in its functional regions were also identified. Both tSNPs and identified variants were genotyped in 164 nuclear families with 170 highly myopic (spherical equivalent < -6.0 D in both eyes) offspring. The linkage disequilibrium pattern of SNPs was established in the parental group (n = 328). Family-based association tests were performed using family-based association testing (FBAT) and genetic association computer analyses. RESULTS Single marker analysis of SNPs rs3026390 and rs3026393 showed significant association with high myopia as a qualitative trait in dominant and recessive models (P = 0.0014 and P = 0.0011, respectively). For rs3026393, the genotype relative risk was 2.57 for G/T and 2.22 for T/T with reference to G/G. Significantly increased transmission was demonstrated for the haplotypes carrying allele T of rs3026393 in the additive and dominant models (P < 0.0070), whereas significantly decreased transmission was found for haplotypes carrying allele G of rs3026393 in the recessive model (P = 0.0173). Preferential transmission of single alleles and haplotypes remained significant after correction for multiple comparisons. CONCLUSIONS This study demonstrates the association of PAX6 variants with susceptibility to high myopia. The PAX6 locus may contain polymorphisms playing a role in high myopia in southern Han Chinese.

Association of IGF1 Gene Haplotypes With High Myopia in Chinese Adults

OBJECTIVE To investigate the association of high myopia with common single-nucleotide polymorphisms (SNPs) in the IGF1, IGFBP3, and IGFBP4 genes in a Chinese population. METHODS For our case-control study, we recruited 600 unrelated participants: 300 case participants with high myopia (-8.00 diopters or less) and 300 emmetropic controls (within ±1.00 diopter). Twenty-one tag SNPs were selected from these candidate genes and were genotyped. Genotype data were analyzed by logistic regression. Multiple comparisons were corrected by running 15 000 permutations of case-control status. RESULTS Although we did not find any significant association for IGF1 SNPs using single-marker analysis, we identified many windows with haplotype frequencies significantly different between case participants and control participants using a variable-sized sliding-window strategy. In particular, the most significant association was shown by a 3-SNP window consisting of rs12423791, rs7956547, and rs5742632 (omnibus test: asymptotic P(asym) = 3.70 × 10(-9) and empirical P(emp) = 6.67 × 10(-5)). There were 3 high-risk haplotypes: CAC (P(asym) = 2.35 × 10(-6); odds ratio [OR], 5.06), GAT (P(asym) = 3.56 × 10(-4); OR, 3.18), and GGC (P(asym) = 2.25 × 10(-3); OR, 25.10). There was 1 protective haplotype: GAC (P(asym) = 8.43 × 10(-4); OR, 0.63). On the other hand, our single-marker and haplotype analyses did not show any significant association for IGFBP3 and IGFBP4. CONCLUSIONS IGF1 haplotypes are associated with genetic susceptibility to high myopia in Chinese adults, whereas IGFBP3 and IGFBP4 are unlikely to be important in the genetic predisposition to high myopia. CLINICAL RELEVANCE IGF1 is associated with high myopia, and identifying the causal variants is important for understanding the underlying mechanisms.

Identification of myopia-associated WNT7B polymorphisms provides insights into the mechanism underlying the development of myopia

Myopia can cause severe visual impairment. Here, we report a two-stage genome-wide association study for three myopia-related traits in 9,804 Japanese individuals, which was extended with trans-ethnic replication in 2,674 Chinese and 2,690 Caucasian individuals. We identify WNT7B as a novel susceptibility gene for axial length (rs10453441, Pmeta=3.9 × 10(-13)) and corneal curvature (Pmeta=2.9 × 10(-40)) and confirm the previously reported association between GJD2 and myopia. WNT7B significantly associates with extreme myopia in a case-control study with 1,478 Asian patients and 4,689 controls (odds ratio (OR)meta=1.13, Pmeta=0.011). We also find in a mouse model of myopia downregulation of WNT7B expression in the cornea and upregulation in the retina, suggesting its possible role in the development of myopia.

Association of High Myopia with Crystallin Beta A4 (CRYBA4) Gene Polymorphisms in the Linkage-Identified MYP6 Locus

Background Myopia is the most common ocular disorder worldwide and imposes tremendous burden on the society. It is a complex disease. The MYP6 locus at 22 q12 is of particular interest because many studies have detected linkage signals at this interval. The MYP6 locus is likely to contain susceptibility gene(s) for myopia, but none has yet been identified. Methodology/Principal Findings Two independent subject groups of southern Chinese in Hong Kong participated in the study an initial study using a discovery sample set of 342 cases and 342 controls, and a follow-up study using a replication sample set of 316 cases and 313 controls. Cases with high myopia were defined by spherical equivalent ≤ -8 dioptres and emmetropic controls by spherical equivalent within ±1.00 dioptre for both eyes. Manual candidate gene selection from the MYP6 locus was supported by objective in silico prioritization. DNA samples of discovery sample set were genotyped for 178 tagging single nucleotide polymorphisms (SNPs) from 26 genes. For replication, 25 SNPs (tagging or located at predicted transcription factor or microRNA binding sites) from 4 genes were subsequently examined using the replication sample set. Fisher P value was calculated for all SNPs and overall association results were summarized by meta-analysis. Based on initial and replication studies, rs2009066 located in the crystallin beta A4 (CRYBA4) gene was identified to be the most significantly associated with high myopia (initial study: P = 0.02; replication study: P = 1.88e-4; meta-analysis: P = 1.54e-5) among all the SNPs tested. The association result survived correction for multiple comparisons. Under the allelic genetic model for the combined sample set, the odds ratio of the minor allele G was 1.41 (95% confidence intervals, 1.21-1.64). Conclusions/Significance A novel susceptibility gene (CRYBA4) was discovered for high myopia. Our study also signified the potential importance of appropriate gene prioritization in candidate selection.

Using denaturing HPLC for SNP discovery and genotyping, and establishing the linkage disequilibrium pattern for the all- trans -retinol dehydrogenase ( RDH8 ) gene

AbstractAll-trans-retinol dehydrogenase (RDH8) is a visual cycle enzyme that reduces all-trans-retinal to all-trans-retinol. As part of an on-going effort to map genes involved in complex eye diseases, myopia in particular, using association studies, single nucleotide polymorphisms (SNPs) were identified and linkage disequilibrium (LD) pattern was established within and around the RDH8 gene. We used denaturing high-performance liquid chromatography (DHPLC) to screen SNPs in four DNA pools each consisting of DNA from five individuals and genotyped the identified SNPs in 150 Chinese subjects from Hong Kong. Fifteen SNPs were identified: seven were common with the minor allele frequency >0.05 and ten were novel. Common SNPs were included in LD and haplotype analysis using the ASSOCIATE and EH programmes. Four SNPs in the 3′ region exhibited significant LD and formed a haplotype block, while three common SNPs in the 5′ region did not exhibit useful LD. The LD pattern around the RDH8 gene suggested that one SNP from the 3′region and two to three SNPs from the 5′ region were needed in association studies involving RDH8. Our results demonstrated the efficiency of DHPLC in screening SNPs when coupled with DNA pooling strategy and in genotyping SNPs.

Genetic susceptibility to refractive error: association of vasoactive intestinal peptide receptor 2 (VIPR2) with high myopia in Chinese.

Myopia is the most common ocular disease worldwide. We investigated the association of high myopia with the common single nucleotide polymorphisms (SNPs) of five candidate genes – early growth response 1 (EGR1), v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS), jun oncogene (JUN), vasoactive intestinal peptide (VIP), and vasoactive intestinal peptide receptor 2 (VIPR2). We recruited 1200 unrelated Chinese subjects with 600 cases (spherical equivalent ≤−8.00 diopters) and 600 controls (spherical equivalent within ±1.00 diopter). A discovery sample set was formed from 300 cases and 300 controls, and a replication sample set from the remaining samples. Tag SNPs were genotyped for the discovery sample set, and the most significant haplotypes and their constituent SNPs were followed up with the replication sample set. The allele and haplotype frequencies in cases and controls were compared by logistic regression adjusted for sex and age to give P a values, and multiple comparisons were corrected by permutation test to give P aemp values. Odd ratios (OR) were calculated accordingly. In the discovery phase, EGR1, JUN and VIP did not show any significant association while FOS and VIPR2 demonstrated significant haplotype association with high myopia. In the replication phase, the haplotype association for VIPR2 was successfully replicated, but not FOS. In analysis combining both sample sets, the most significant association signals of VIPR2 were the single marker rs2071625 (P a = 0.0008, P aemp = 0.0046 and OR = 0.75) and the 4-SNP haplotype window rs2071623-rs2071625-rs2730220-rs885863 (omnibus test, P a = 9.10e-10 and P aemp = 0.0001) with one protective haplotype (GGGG: P aemp = 0.0002 and OR = 0.52) and one high-risk haplotype (GAGA: P aemp = 0.0027 and OR = 4.68). This 4-SNP haplotype window was the most significant in all sample sets examined. This is the first study to suggest a role of VIPR2 in the genetic susceptibility to high myopia. EGR1, JUN, FOS and VIP are unlikely to be important in predisposing humans to high myopia.

Evaluation of Proteoglycan Gene Polymorphisms as Risk Factors in the Genetic Susceptibility to High Myopia

PURPOSE. To investigate the relationship between high myopia and single nucleotide polymorphisms (SNPs) in six proteoglycan genes: aggrecan (ACAN), fibromodulin (FMOD), decorin (DCN), lumican (LUM), keratocan (KERA), and epiphycan (EPYC). These genes were selected for study because they are involved in induced myopia in animals and/or are within the human MYP3 locus identified by linkage analysis of families with high myopia. METHODS. Two groups of Chinese subjects were studied: group 1 (300 cases and 300 controls) and group 2 (356 cases and 354 controls). Cases were high myopes with spherical equivalent (SE) ≤ -8.00 D, and controls had SE between +1.0 and -1.0 D. From these candidate genes, 60 tagging SNPs were selected. First, 12 DNA pools were each constructed from 50 samples of the same phenotype from group 1 subjects and were tested for association with the SNPs. Second, putatively positive SNPs were confirmed by individual genotyping of group 1 subjects. Finally, positive results were replicated in group 2 subjects. RESULTS. Of the 58 SNPs successfully screened by DNA pooling, 8 ACAN SNPs passed the threshold of P ≤ 0.10 (nested ANOVA) and were then genotyped in the individual samples. Haplotypes rs3784757 and rs1516794 showed significant association with high myopia. However, the positive result could not be replicated in the second subject group. CONCLUSIONS. These six proteoglycan genes were not associated with high myopia in these Chinese subjects and hence are unlikely to be important in the genetic predisposition to high myopia.

Systematic Investigation of the Relationship between High Myopia and Polymorphisms of the MMP2, TIMP2, and TIMP3 Genes by a DNA Pooling Approach

PURPOSE This study examined the relationship between high myopia and three myopia candidate genes--matrix metalloproteinase 2 (MMP2) and tissue inhibitor of metalloproteinase-2 and -3 (TIMP2 and TIMP3)--involved in scleral remodeling. METHODS Recruited for the study were unrelated adult Han Chinese who were high myopes (spherical equivalent, ≤ -6.0 D in both eyes; cases) and emmetropes (within ±1.0 D in both eyes; controls). Sample set 1 had 300 cases and 300 controls, and sample set 2 had 356 cases and 354 controls. Forty-nine tag single-nucleotide polymorphisms (SNPs) were selected from these candidate genes. The first stage was an initial screen of six case pools and six control pools constructed from sample set 1, each pool consisting of 50 distinct subjects of the same affection status. In the second stage, positive SNPs from the first stage were confirmed by genotyping individual samples forming the DNA pools. In the third stage, positive SNPs from stage 2 were replicated, with sample set 2 genotyped individually. RESULTS Of the 49 SNPs screened by DNA pooling, three passed the lenient threshold of P < 0.10 (nested ANOVA) and were followed up by individual genotyping. Of the three SNPs genotyped, two TIMP3 SNPs were found to be significantly associated with high myopia by single-marker or haplotype analysis. However, the initial positive results could not be replicated by sample set 2. CONCLUSIONS MMP2, TIPM2, and TIMP3 genes were not associated with high myopia in this Chinese sample and hence are unlikely to play a major role in the genetic susceptibility to high myopia.

Genotyping Performance Assessment of Whole Genome Amplified DNA with Respect to Multiplexing Level of Assay and Its Period of Storage

Whole genome amplification can faithfully amplify genomic DNA (gDNA) with minimal bias and substantial genome coverage. Whole genome amplified DNA (wgaDNA) has been tested to be workable for high-throughput genotyping arrays. However, issues about whether wgaDNA would decrease genotyping performance at increasing multiplexing levels and whether the storage period of wgaDNA would reduce genotyping performance have not been examined. Using the Sequenom MassARRAY iPLEX Gold assays, we investigated 174 single nucleotide polymorphisms for 3 groups of matched samples: group 1 of 20 gDNA samples, group 2 of 20 freshly prepared wgaDNA samples, and group 3 of 20 stored wgaDNA samples that had been kept frozen at −70°C for 18 months. MassARRAY is a medium-throughput genotyping platform with reaction chemistry different from those of high-throughput genotyping arrays. The results showed that genotyping performance (efficiency and accuracy) of freshly prepared wgaDNA was similar to that of gDNA at various multiplexing levels (17-plex, 21-plex, 28-plex and 36-plex) of the MassARRAY assays. However, compared with gDNA or freshly prepared wgaDNA, stored wgaDNA was found to give diminished genotyping performance (efficiency and accuracy) due to potentially inferior quality. Consequently, no matter whether gDNA or wgaDNA was used, better genotyping efficiency would tend to have better genotyping accuracy.