Sylvia W. Y. Chiang

HTRA1 variants in exudative age-related macular degeneration and interactions with smoking and CFH.

PURPOSE Mapping the genes for age-related macular degeneration (AMD) had not been successful until recent genome-wide association studies revealed Tyr402His in CFH and rs11200638 in HTRA1 as AMD-related genetic variants. This study was conducted to identify other critical factors in HTRA1 that are associated with exudative AMD. METHODS The promoter, splice regions, and coding exons of HTRA1 were sequenced in 163 patients with exudative AMD and 183 sex- and age-matched control subjects. Also documented were the CFH genotype and smoking status. RESULTS Four significant SNPs were found in the promoter and the first exon of HTRA1: rs11200638 (-625G>A), rs2672598 (-487T>C), rs1049331 (102C>T, Ala34Ala), and rs2293870 (108G>T, Gly36Gly) with respective P = 1.7 x 10(-14), 3.0 x 10(-10), 3.7 x 10(-12), and 3.7 x 10(-12). Among them, rs11200638 is the most significant associated SNP with a high odds ratio (OR) of 7.6 (95% CI: 3.94-14.51). One risk haplotype block across the promoter and exon 1, ACCTT, significantly predisposes to AMD (P = 6.68 x 10(-14)). In both models, significant independent additive effects were identified with smoking and rs800292 (184G>A, Val62Ile) of CFH. Smoking and rs11200638 (HTRA1) combined caused a 15.7-fold increased risk, whereas combined rs800292 and rs11200638 caused a 23.3-fold increased risk. An extremely high population attributable risk (PAR) of 78% was also found. CONCLUSIONS A high impact of the additive effect of CFH and HTRA1 in the development of exudative AMD was shown. The HTRA1-smoking additive effect found in this study further suggests the importance of this environmental risk factor in AMD.

Longitudinal Profile of Retinal Ganglion Cell Damage after Optic Nerve Crush with Blue-Light Confocal Scanning Laser Ophthalmoscopy

PURPOSE To investigate the long-term longitudinal profile of retinal ganglion cell (RGC) damage after optic nerve crush with a new technique for in vivo imaging of RGCs. METHODS A blue-light confocal scanning laser ophthalmoscope (bCSLO; 460 nm excitation, 490 nm detection) was used to image Thy-1 CFP mice aged 6 to 9 months (n = 5) before optic nerve crush, weekly after crush for 3 weeks, and at weeks 10 and 50 after optic nerve crush. A sham procedure was performed in the contralateral eye, and it was imaged as a control. Corresponding retinal areas before and after optic nerve crush were compared, and the fluorescent spots were counted manually. The longitudinal profile of RGC degeneration was modeled and compared with one-phase and two-phase exponential decay equations. RESULTS A significant and progressive loss of fluorescent spots was found after optic nerve crush with 18.6% +/- 2.3%, 11.3% +/- 3.4%, 8.8% +/- 5.3%, 4.2% +/- 3.1%, and 3.3% +/- 2.1% of Thy-1-expressing RGCs remaining at weeks 1, 2, 3, 10, and 50, respectively, after optic nerve crush (P < 0.001; n = 5). There was no change in the fluorescence density in the contralateral control (P = 0.893). Two-phase exponential decay (y = 0.03 + 0.83e(-)(2.78t) + 0.14e(-)(0.30t)) was a better fit than one-phase exponential decay (y = 0.94e(-)(1.93t) + 0.06; P = 0.003) equations, with half-lives of fast phase and slow phase of 1.7 days and 16.3 days, respectively. CONCLUSIONS The longitudinal profile of RGC degeneration after optic nerve crush is characterized by a two-phase exponential decay model. bCSLO imaging provides an efficient and noninvasive approach to the longitudinal study of progressive RGC damage.

PI3K/akt, JAK/STAT and MEK/ERK pathway inhibition protects retinal ganglion cells via different mechanisms after optic nerve injury

Recently we unexpectedly found that PI3K/akt, JAK/STAT and MEK/ERK pathway inhibitors enhanced retinal ganglion cell (RGC) survival after optic nerve (ON) axotomy in adult rat, a phenomenon contradictory to conventional belief that these pathways are pro‐survival. In this study we showed that: (i) the RGC protection was pathway inhibition‐dependent; (ii) inhibition of PI3K/akt and JAK/STAT, but not MEK/ERK, activated macrophages in the eye, (iii) macrophage removal from the eye using clodronate liposomes significantly impeded PI3K/akt and JAK/STAT inhibition‐induced RGC survival and axon regeneration whereas it only slightly affected MEK/ERK inhibition‐dependent protection; (iv) in the absence of recruited macrophages in the eye, inhibition of PI3K/akt or JAK/STAT did not influence RGC survival; and (v) strong PI3K/akt, JAK/STAT and MEK/ERK pathway activities were located in RGCs but not macrophages after ON injury. In retinal explants, in which supply of blood‐derived macrophages is absent, MEK/ERK inhibition promoted RGC survival whereas PI3K/akt or JAK/STAT inhibition had no effect on RGC viability. However, MEK/ERK inhibition exerted opposite effects on the viability of purified adult RGCs at different concentrations in vitro, suggesting that this pathway may be bifunctional depending on the level of pathway activity. Our data thus demonstrate that inhibition of the PI3K/akt or JAK/STAT pathway activated macrophages to facilitate RGC protection after ON injury whereas the two pathways per se did not modulate RGC viability under the injury conditions (in the absence of the pathway activators). In contrast, the MEK/ERK pathway inhibition protected RGCs via macrophage‐independent mechanism(s).

Associations of the C2-CFB-RDBP-SKIV2L locus with age-related macular degeneration and polypoidal choroidal vasculopathy.

PURPOSE To investigate the associations of the C2-CFB-RDBP-SKIV2L region with neovascular age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV). DESIGN Cross-sectional, case-control association study. PARTICIPANTS A Chinese case-control group of 200 neovascular AMD patients, 233 PCV patients, and 275 control subjects. METHODS An association analysis was performed of the C2-CFB-RDBP-SKIV2L locus with both neovascular AMD and PCV in a Chinese population using 19 haplotype-tagging single nucleotide polymorphisms (SNPs) and 6 previously reported SNPs across the C2-CFB-RDBP-SKIV2L region. All SNPs were genotyped using the TaqMan genotyping technology (TaqMan; Applied Biosystems [ABI], Foster City, CA). MAIN OUTCOME MEASURES Allele and haplotype frequencies of the SNPs in the C2-CFB-RDBP-SKIV2L region. RESULTS The SKIV2L SNPs rs429608 and rs453821 were significantly associated with neovascular AMD (P = 7.39 × 10(-5); odds ratio [OR], 0.22; 95% confidence interval [CI], 0.10-0.50; and P = 0.001; OR, 0.38; 95% CI, 0.21-0.70, respectively), whereas borderline associations were detected for C2 rs547154 (P = 0.002) and RDBP rs760070 (P = 0.003). Conditional haplotype analysis revealed that SKIV2L rs429608 could account fully for the global haplotype association identified in this region. The association of SKIV2L rs429608 with neovascular AMD remained significant after adjusting for CFH rs800292 and HTRA1 rs11200638. No individual SNP or haplotype was associated significantly with PCV. CONCLUSIONS In this concurrent investigation of the associations of the entire C2-CFB-RDBP-SKIV2L region with neovascular AMD and PCV, the results suggested that SKIV2L is a likely causal gene for neovascular AMD, conferring a significant protective effect independent of CFH and HTRA1. These data do not support a significant role of this region in PCV, suggesting different molecular mechanisms between neovascular AMD and PCV.

Differentiation of exudative age-related macular degeneration and polypoidal choroidal vasculopathy in the ARMS2/HTRA1 locus.

PURPOSE differentiate the associations of exudative age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV) with the ARMS2/HTRA1 locus. METHODS The entire ARMS2 sequence was sequenced and HTRA1 rs11200638 genotyped in 568 unrelated Chinese individuals: 156 exudative AMD patients, 164 PCV patients, and 248 controls. A meta-analysis was performed to examine the effects of rs10490924 and rs11200638 at the ARMS2/HTRA1 locus in PCV. RESULTS In total, 31 polymorphisms in ARMS2 were identified. Significant associations with both exudative AMD and PCV were observed in 11 of them and HTRA1 rs11200638, with different genotypic distributions between exudative AMD and PCV (P < 0.001). After adjusting for rs11200638, ARMS2 rs10490924 remained significantly associated with exudative AMD (P = 0.011), but not with PCV (P = 0.077). Meta-analysis showed consistent allelic associations of rs10490924 and rs11200638 with PCV in different study populations. CONCLUSIONS There is a strong and consistent association of the ARMS2/HTRA1 locus with both exudative AMD and PCV, suggesting the two disorders share, at least partially, similar molecular mechanisms. Different effect sizes indicate the existence of additional genetic and environmental factors affecting them to different extents.

Compound Heterozygosity of Two Novel Truncation Mutations in RP1 Causing Autosomal Recessive Retinitis Pigmentosa

Purpose. To evaluate the phenotypic effects of two novel frameshift mutations in the RP1 gene in a Chinese pedigree of autosomal recessive retinitis pigmentosa (ARRP). Methods. Family members of a proband with ARRP were screened for RP1, RHO, NR2E3, and NRL mutations by direct sequencing. Detected RP1 mutations were genotyped in 225 control subjects. Since one family member with the RP1 deletion mutation in exon 2 was found to have age-related macular degeneration (AMD) but not RP, exons 2 and 3 of RP1 were screened in 120 patients with exudative AMD. Major AMD-associated SNPs in the HTRA1 and CFH genes were also investigated. Results. Two novel frameshift mutations in RP1, c.5_6delGT and c.4941_4942insT, were identified in the pedigree. They were absent in 225 control subjects. Family members who were compound heterozygous for the nonsense mutations had early-onset and severe RP, whereas those with only one mutation did not have RP. No mutations in RHO, NR2E3, and NRL were identified in the pedigree. Subject I:2 with AMD carried both at-risk genotypes at HTRA1 rs11200638 and CFH rs800292. No mutation in RP1 exons 2 and 3 was identified in 120 AMD patients. Conclusions. This report is the first to associate ARRP with compound heterozygous nonsense mutations in RP1. Identification of the nonsense-mediated mRNA decay (NMD)-sensitive mutation c.5_6delGT provided further genetic evidence that haploinsufficiency of RP1 is not responsible for RP. The authors propose four classes of truncation mutations in the RP1 gene with different effects on the etiology of RP.

Novel and homozygous BEST1 mutations in Chinese patients with Best vitelliform macular dystrophy.

Purpose: The purpose of this study was to investigate the BEST1 gene mutations in Chinese patients with Best vitelliform macular dystrophy (BVMD). Methods: Twenty-six subjects from 7 Chinese families with BVMD and 100 unrelated healthy Chinese subjects without a family history of BVMD were screened for mutations in the BEST1 gene by direct sequencing. The subjects underwent complete ophthalmologic examination and BEST1 gene screening. Results: Six novel missense mutations (Thr2Asn, Leu75Phe, Ser144Asn, Arg255Trp, Pro297Thr, and Asp301Gly) and 1 previously reported mutation (Arg218Cys) were identified. Each family was found to have a unique BEST1 mutation that segregated with the disease. Two of the six novel mutations are located within the four previously reported common mutation clusters within the BEST1 gene. One family with patients having homozygous Leu75Phe mutations did not have the more severe BVMD phenotype. None of the patients with mutations was identified among the 100 healthy control subjects. Conclusion: A large number of unique novel missense mutations was found in Chinese patients with BVMD, suggesting considerable interethnic differences between the mutation sites in the BEST1 gene in different populations. The few truncating BEST1 mutations and the lack of a more severe phenotype in homozygous patients suggest that the missense BEST1 mutation may produce a dominant negative effect on wild-type BEST1 gene.

Association of C2 and CFB Polymorphisms with Anterior Uveitis

PURPOSE Association of rs800292 (I62V) in the complement factor H (CFH) gene with anterior uveitis (AU) was identified in our previous study. We proceeded to investigate whether polymorphisms of two tightly linked genes in the complement pathway, complement component 2 (C2) and complement factor B (CFB), are associated with AU. METHODS Five single-nucleotide polymorphisms (SNPs), rs1048709, rs537160, rs4151657, rs2072633 in CFB, and rs3020644 in C2, were examined using genotyping assays in 98 Chinese AU patients and 291 unrelated controls. Adjustments and stratifications were given for sex, clinical manifestations, and HLA-B27 status. RESULTS There were significant increases in the frequency of A allele and AA homozygosity for CFB-rs1048709 in AU patients compared with that of controls (P value after Bonferroni correction [P(corr)] = 2.67 × 10⁻⁴, P(corr) = 0.001, respectively). No association was found between AU and the other four SNPs after adjustment for multiple testing. Logistic regression analysis showed none of the 5 SNPs had significant interaction with sex. Stratified analyses showed that only rs1048709 was significantly associated with AU in HLA-B27-positive patients but not in HLA-B27-negative patients. No association was found in the 5 tested SNPs with clinical manifestations. A haplotype block across CFB (AATA) was significantly predisposed to AU with increased risk of 1.97 (P(corr) = 0.0005). Additive effect of CFB-rs1048709 and CFH-rs800292 was identified with an odds ratio of 7.48. CONCLUSIONS Our results revealed an association between AU and CFB-rs1048709. The influence on AU might differ depending on HLA-B27 status. The joint effect in CFB and CFH strengthens the concept that the complement system plays an important role in the pathogenesis of AU.

A novel mutation in CRYBB2 responsible for inherited coronary cataract.

PurposeTo study the molecular pathogenesis of a Chinese family with coronary form of cataract.MethodsOne Chinese three-generation family with inherited coronary cataract phenotype was recruited. Five affected and seven unaffected family members attended our study. Genome-wide linkage analysis was applied to map the disease loci, and two candidate genes from a locus on chromosome 1 and a locus on chromosome 22 were sequenced for mutation identification. Software at the Expasy proteomics server was utilized to predict the mutation effect on proteins.ResultsWhole genome linkage analysis indicated some regions on chromosome 1, 10, and 22, with LOD score values greater than 1. Within these loci, the GJA8 and CRYBB2 genes, located in the two loci with the highest LOD score of 1.51 on chromosomes 1 and 22, respectively, were sequenced. A novel mutation c.92C>G in exon 2 of CRYBB2 causing S31W was identified in all five patients. It was not found in 95 unrelated controls. This missense sequence alteration likely enhanced the local solubility. Around the mutation site, a lipocalin signature motif was predicted by ScanProsite.ConclusionsA novel disease-causing mutation S31W in CRYBB2 was identified in a Chinese cataract family. It is the first reported mutation for coronary cataract. Functional characterization should be carried out to evaluate the biological effects of this mutant.

Gender specific association of a complement component 3 polymorphism with polypoidal choroidal vasculopathy

Neovascular age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV) are leading causes of irreversible blindness in developed countries. In this study, we investigated the associations of haplotype-tagging single nucleotide polymorphisms (SNPs) in the complement component 3 (C3) gene with both neovascular AMD and PCV, and potential epistatic effects on C3. Eight tagging SNPs in C3 were genotyped in 708 unrelated study subjects: 200 neovascular AMD patients, 233 PCV patients and 275 controls. Among the eight C3 SNPs, rs17030 was associated with PCV after adjusted for gender and SNP-gender interaction (P = 0.008, OR = 2.94; 95% CI: 1.32-6.52). Moreover, an interaction between rs17030 and gender was identified in PCV (P = 0.02). After stratification by gender, the rs17030 G allele was found to confer an increased risk for PCV in male (P = 0.010, OR = 1.56) but not in female. The haplotype AG defined by the major alleles of rs17030 and rs344555 was also associated with PCV in male (P = 0.010, OR = 0.64). In contrast to PCV, none of the eight SNPs was significantly associated with neovascular AMD. This study shows an association of C3 rs17030 with PCV in male, indicating that C3 may have an epistatic effect with gender in the pathogenesis of PCV.