Current Eye Research, Early Online, 1–7, 2015 ! Informa Healthcare USA, Inc. ISSN: 0271-3683 print / 1460-2202 online DOI: 10.3109/02713683.2014.997884
ORIGINAL ARTICLE
Evaluation of Genetic Polymorphisms in Clusterin and Tumor Necrosis Factor-Alpha Genes in South Indian Individuals with Pseudoexfoliation Syndrome
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Sushil K. Dubey1, James F. Hejtmancik2, Subbaiah R. Krishnadas3, Rajendrababu Sharmila3, Aravind Haripriya4 and Periasamy Sundaresan1 1
Department of Genetics, Dr. G. Venkataswamy Eye Research Institute, Aravind Medical Research Foundation, Madurai, Tamil Nadu, India, 2Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health, Rockville, MD, USA, 3Glaucoma Clinic, Aravind Eye Hospital, Madurai, Tamil Nadu, India, and 4Intraocular Lens and Cataract Clinic, Aravind Eye Hospital, Madurai, Tamil Nadu, India
ABSTRACT Purpose: The aim of this study was to explore the potential association of genetic variants across clusterin (CLU) and tumor necrosis factor-alpha (TNF- ) genes in South Indian individuals with pseudoexfoliation syndrome (PEXS) and pseudoexfoliation glaucoma (PEXG). Materials and Methods: A total of 523 individuals including 299 unrelated cases (150 PEXS and 149 PEXG) and 224 age- and ethnically-matched healthy controls were recruited for genetic analysis. Six single-nucleotide polymorphisms (SNPs) including, five CLU SNPs (rs11136000, rs2279590, rs9331888, rs9331931, rs3087554) and one promoter SNP (rs1800629) of TNF- were genotyped in all study subjects. Genotyping of CLU SNPs were performed using the TaqMan allelic discrimination assay while TNF- SNP was genotyped using polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis. Association analysis was performed by determining the distributions of genotype and allele frequencies, Hardy–Weinberg equilibrium, and chi-square p values and odds ratios as implemented in the Golden Helix SNP & Variation Suite (SVS). Results: Five CLU SNPs did not show any significant differences in allele frequencies between patients and control subjects (rs3087554, p = 0.919, OR = 1.01, 95% CI: 0.77–1.33; rs2279590, p = 0.432, OR = 1.12, 95% CI: 0.84–1.51; rs9331931, p = 0.310, OR = 1.24, 95% CI: 0.81–1.89; rs11136000, p = 0.072, OR = 1.31, 95% CI: 0.97–1.76; rs9331888, p = 0.911, OR = 1.01, 95% CI: 0.78–1.31). The investigation of TNF- SNP established a significant association with PEXS and PEXG (p = 0.042, OR = 0.61, 95% CI: 0.38–0.99). However, this association did not remain significant after Bonferroni correction. Conclusions: Our data suggest that genetic variants in CLU and TNF-a genes do not play a major role in the development of PEXS and PEXG in the South Indian population. Keywords: CLU, pseudoexfoliation glaucoma, pseudoexfoliation syndrome, SNP, TNF-
INTRODUCTION
pathologic deposition of abnormal microfibrillar aggregates on both intra- and extra-ocular tissues. In the eye, these materials are primarily seen on tissues lining the aqueous-bathed surfaces of anterior segment.1 The origin of these abnormal microfibrils is unclear but they are believed to be multifocally
Pseudoexfoliation syndrome (PEXS) is an age-related systemic disorder of the extracellular matrix and the most common identifiable cause of secondary openangle glaucoma.1,2 PEXS is characterized by the
Received 7 July 2014; revised 4 December 2014; accepted 8 December 2014; published online 2 April 2015 Correspondence: Periasamy Sundaresan, Department of Genetics, Dr. G. Venkataswamy Eye Research Institute, Aravind Medical Research Foundation, No 1, Anna Nagar, Madurai, Tamil Nadu, India. E-mail: [email protected]
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produced by various ocular tissues including the corneal endothelium, iris, non-pigmented ciliary epithelium, lens epithelium and trabecular meshwork.3 PEXS-associated glaucoma (pseudoexfoliation glaucoma, PEXG) is characterized by rapid progression, high resistance to medical therapy and poorer prognosis than the more common primary open-angle glaucoma.4 Progressive accumulation of pseudoexfoliation (PEX) material on the trabecular meshwork presumably causes obstruction of aqueous out flow and correlates with elevated intraocular pressure (IOP) and severity of optic nerve damage.5,6 Hence, PEXG represents PEXS accompanied by outflow obstruction causing elevated intra-ocular pressure. Approximately 60% of the eyes with PEXS develop PEXG within 15 years of diagnosis.2 Thus, while there might be some additional components (genetic or environmental) to PEXG, certainly every PEXG patient can be counted as having PEXS. Genetic studies also supported that both PEXS and PEXG have overlapping, if not necessarily identical, genetic risk factors.7,8 The average worldwide prevalence rate of PEXS is 10–20% of the general population over 60 years of age.9 Population-based studies in South India have reported the overall prevalence of 3–6% among subjects older than 40 years.10–12 Thorleifsson et al.7 performed a genome-wide association study in Icelandic and Swedish populations and identified a strong association between PEXS and three common single-nucleotide polymorphisms (SNPs) in lysyl oxidase-like 1 (LOXL1; OMIM: 153456) gene. Subsequently, these findings were replicated in multiple populations globally, which established LOXL1 as a major genetic risk factor for PEXS and PEXG.13–27 However, the risk associated alleles of these LOXL1 SNPs are common and relatively more prevalent – up to 88% – among the general populations. In some populations, while the frequency of risk allele is high, the disease penetrance is low.7,16,18 Moreover, recent studies demonstrated the reversal of LOXL1 risk allele in some populations21–27 as well as lack of impact of two non-synonymous variants (R141L and G153D) on amine oxidase activity of LOXL1 protein.28 Taken together, these data suggest that common SNPs in LOXL1 are not the causative variants and additional as yet unrevealed genetic and environmental factors might be involved in the development of PEXS and PEXG. Previous studies in some populations have reported association of SNP allele in the clusterin (CLU) and tumor necrosis factor-alpha (TNF- ) genes with PEXS and/or PEXG.29–32 CLU is an extracellular multifunctional protein that primarily acts as a molecular chaperone, preventing the precipitation and aggregation of misfolded extracellular proteins.33 Lower levels of CLU protein in aqueous humor as well as down regulation of CLU mRNA in many
anterior segment tissues has been detected in eyes with the PEX syndrome.34 CLU is also a major component of PEX material.35 Burdon et al.29 reported a nominal association between rs3087554 of CLU and PEXS in Australian Blue Mountains Eye Study cohort. Subsequently, Krumbiegel et al.30 genotyped five SNPs of CLU in PEXS, PEXG and controls from Europeans and reported that allelic variant of rs2279590 is a risk factor for the development of PEXS in German patients but not in Italian patients. SNP rs3087554 alleles, which showed association with PEXS in Australians, were not associated with PEXS in either German or Italian populations. Interestingly, rs2279590 has shown association with Alzheimer disease in Caucasian and Asian populations.36 Therefore, it would be of interest to know whether CLU SNPs associated with Alzheimer’s are also associated with PEX. Studies associating CLU gene with PEXS and PEXG have not been conducted previously in Asian populations. A functional SNP (rs1800629) at position –308 in the promoter region of TNF- gene, characterized by G to A substitution, is associated with higher TNF-a production.37 TNF-a, a pleiotropic proinflammatory cytokine, regulates the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs).38 Both MMPs and TIMPs are required for proper turnover of extracellular matrix components.38 Deposition of abnormal extracellular matrix components in PEXS may be due to reduced turnover of matrix components. Interestingly, decreased level of MMP-2 and increased level of both TIMP-1 and TIMP-2 have been detected in the aqueous humor of the PEX patients, suggesting the involvement of TNF-a in PEXS and PEXG.39,40 Association studies of rs1800629 with PEXG have been done in few populations which showed contradictory results.31,32,41,42 Therefore, further replication studies in different populations are essential for a comprehensive understanding of the role of rs1800629 in PEX pathogenesis. Both CLU and TNF- genes have not been characterized in PEX patients of South Indian ethnic background. The purpose of this study was to evaluate the possible association of genetic variants in CLU and TNF- genes with PEXS and PEXG in a South Indian population.
MATERIALS AND METHODS Study Population The study protocol was approved by the Institutional Review Board of Aravind Eye Hospital and was carried out in accordance with the tenets of the Declaration of Helsinki. Informed consent was obtained from all study participants after explaining the nature of the study. 299 unrelated cases (PEXS, Current Eye Research
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CLU and TNF-a Gene Variants in Pseudoexfoliation 150; PEXG, 149) and 224 age- and ethnicity-matched controls were enrolled for this study. All participants underwent a complete ophthalmic examination including visual acuity, slit lamp biomicroscopy, tonometric assessment of IOP, gonioscopy and retinal examination. PEXS cases were diagnosed as those with clinical evidence of PEX material on pupillary margin and/or anterior lens capsule, IOP 519 mm Hg and no glaucomatous optic atrophy and visual field defects. PEXG were defined as the presence of PEX material, open iridocorneal angles, IOP 22 mm Hg in either eye, typical glaucomatous optic disc changes (defined as a thinning or notching of neuroretinal rim, vertical cup-to-disc ratio 40.7 or cup-to-disc ratio asymmetry 0.3 between both eyes) and typical visual field defects in computed perimetry. Control subjects had no evidence of PEX material, IOP 519 mm Hg, no glaucomatous changes in optic disc (no thinning or notching of neuroretinal rim, cup-to-disc ratio range was 0.3–0.5 or cup-to-disc ratio asymmetry 50.2 between both eyes), normal visual fields and had no medical or family history of glaucoma.
Genotyping Venous blood samples (5 ml) were collected from all participants and total genomic DNA was isolated by salt precipitation method.43 Five CLU SNPs (rs11136000, rs2279590, rs9331888, rs9331931, rs3087554) were selected for this study as these SNPs are in a large loose LD block that covers most of CLU gene. The same SNPs were accessed for association with PEXS and PEXG in a previous study.30 Promoter SNP of TNF- , rs1800629, was selected based on its association with PEXG in some populations.31,32 CLU SNPs were genotyped using pre-developed TaqMan SNP genotyping assays (Applied Biosystems, Foster City, CA). Assays were performed according to manufacturer’s instructions and each 5 ml reaction mixture was prepared in a 384well plate, containing 2.5 ml TaqMan universal polymerase chain reaction (PCR) master mix (2), 0.125 ml of SNP genotyping assay (40), 2 ml of DNA (20 ng/ ml) and 0.375 ml MilliQ water. Reactions were amplified using standard thermal cycling conditions and analyzed on an ABI 7900 HT Fast real-time PCR system (Applied Biosystems, Foster City, CA). SNP rs1800629 of TNF- were genotyped by PCR-based restriction fragment length polymorphism (RFLP) approach as described earlier42 with certain modifications in PCR parameter and RFLP. PCR, using gradient thermo cycler (ASTEC, Fukuoka, Japan), was performed in total volume of 20 ml, containing 1 PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, and 0.001% gelatin), 100 ng of genomic DNA, 0.5 pmol of each primer, 200 mM of dNTPs (Medox !
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Biotech India Pvt. Ltd, Chennai, India) and 1 U of Taq DNA polymerase (Sigma, Saint Louis, MO). Thermal cycling conditions were 5 min at 95 C, followed by 36 cycles (30 s at 95 C, 30 s for primer annealing at 62 C and 45 s at 72 C) and a final extension for 7 min at 72 C. PCR amplicon (13 ml) was restriction digested at 37 C overnight with 10 U of Bsp19I restriction enzyme (Fermentas, India) and electrophoresed in a 3% (wt/ vol) agarose gels. Amplicons carrying the A allele remained uncut (212 bp), while amplicons carrying the G allele were digested into two fragments of 192 bp and 20 bp.
Statistical Analysis The observed genotype frequencies for each SNP were tested for Hardy–Weinberg equilibrium (HWE) in both cases and controls and the difference between the observed and expected frequencies was tested for significance using Fisher exact test. 2 and Fisher exact tests were used to examine the genotypic and allelic associations of all the SNPs, as implemented in the Golden Helix SVS software suite 7 (Golden Helix, Bozeman, MT). Call rates, odds ratios (ORs), and confidence intervals (CIs) were also calculated using the same program. A p50.05 was considered statistically significant in hypothesis testing and 95% CIs were used to describe the estimation of unknown parameters. p Values were corrected for multiple testing using a Bonferroni correction. Power was estimated as described by Purcell et al.44 assuming a high-risk allele frequency and prevalence of 0.15, genotype relative risks for both heterozygotes and homozygous risk alleles of 2, a significant p at 0.05, and D’ = 1. Logistic regression analysis was done after adjusting for age and sex to assess the association of CLU and TNF- SNPs with PEXS and PEXG.
RESULTS The present study group comprised 299 PEX patients and 224 age- and ethnicity- matched healthy control subjects. The mean age was 65.0 ± 7.0 years (range, 45– 80) in PEXS cases, 67.0 ± 7.6 years (range, 46–95) in PEXG cases and 66.0 ± 6.2 years (range, 55–92) in controls. There were no significant differences found among the means of these three groups (p = 0.134, PEXS versus control; p = 0.188, PEXG versus control). The observed genotype frequencies of CLU and TNF- SNPs were in HWE in both cases and control groups. The distribution of genotype and allele frequencies of PEX cases and controls are shown in Table 1. Statistical analysis was performed to establish the association of these SNPs with PEXS and PEXG after adjusting for age and sex (Table 2). After controlling for the effects of age and sex, logistic
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TABLE 1 Distribution of genotype and allele frequencies of CLU and TNF- SNPs in PEXG, PEXS and controls in a South Indian cohort. SNP (gene) rs3087554 (CLU)
Genotype frequency (%) Study subjects (N) PEXG (149) PEXS (150) PEXG + PEXS (299) Control (222)
rs2279590 (CLU)
68 59 127 100
(45.6) (39.3) (42.5) (45.0)
TC 66 75 141 93
CC PEXG (147) PEXS (150) PEXG + PEXS (297) Control (224)
rs9331931 (CLU) Curr Eye Res Downloaded from informahealthcare.com by University of Victoria on 04/25/15 For personal use only.
TT
81 90 171 122
(55.1) (60.0) (57.6) (54.5)
rs11136000 (CLU)
119 111 230 183
(79.9) (74.0) (76.9) (82.1)
57 51 108 86
rs9331888 (CLU)
82 90 172 115
(55.0) (60.0) (57.5) (51.3)
29 38 67 36
rs1800629 (TNF-a)
49 42 91 64
(32.9) (28.0) (30.4) (28.6)
57 51 108 93
127 128 255 172
(87.0) (88.3) (87.6) (81.9)
9(6.1) 9 (6.0) 18(6.0) 16(7.1)
(19.4) (25.3) (22.4) (16.1)
(38.3) (34.0) (36.1) (41.5)
71 77 148 113
(47.6) (51.3) (49.5) (50.4)
GA 17 16 33 35
(11.6) (11.0) (11.4) (16.7)
202 193 395 293
1(0.7) 1 (0.7) 2 (0.7) 4 (1.8)
219 231 450 330
267 260 527 402
(19.5) (20.7) (20.1) (21.0)
221 231 452 323
169 161 330 241
(74.5) (77.0) (75.8) (73.7)
75 69 144 118
(25.5) (23.0) (24.2) (26.3) C
(89.6) (86.7) (88.1) (90.1)
31 40 71 44
(10.4) (13.3) (11.9) (9.9) T
(74.2) (77.0) (75.6) (72.1)
77 69 146 125
(25.8) (23.0) (24.4) (27.9) G
(56.7) (53.7) (55.2) (53.8)
129 139 268 207
G 271 272 543 379
(32.2) (35.7) (33.9) (34.0) T
C
AA 2 (1.4) 1 (0.7) 3(1.0) 3 (1.4)
96 107 203 151
C
GG 29 31 60 47
(67.8) (64.3) (66.1) (66.0)
G
TT 10(6.7) 9 (6.0) 19(6.4) 16(7.2)
C
C
CC
CG
GG PEXG (146) PEXS (145) PEXG + PEXS (291) Control (210)
(38.8) (34.0) (36.4) (38.4)
(10.1) (10.7) (10.4) (13.1)
T
TT
CT
CC PEXG (149) PEXS (150) PEXG + PEXS (299) Control (224)
15 16 31 29
GC
CC PEXG (149) PEXS (150) PEXG + PEXS (299) Control (224)
(44.3) (50.0) (47.1) (41.9)
CC
CT
GG PEXG (149) PEXS (150) PEXG + PEXS (299) Control (223)
Allele frequency (%)
(43.3) (46.3) (44.8) (46.2) A
(92.8) (93.8) (93.3) (90.2)
21 18 39 41
(7.2) (6.2) (6.7) (9.8)
PEXG, pseudoexfoliation glaucoma; PEXS, pseudoexfoliation syndrome.
regression analysis revealed that none of the CLU SNPs were associated with PEXS and PEXG (rs3087554, p = 0.919, OR = 1.01; rs2279590, p = 0.432, OR = 1.12; rs9331931, p = 0.310, OR = 1.24; rs11136000, p = 0.072, OR = 1.31; rs9331888, p = 0.911, OR = 1.01). However, rs1800629 of TNF- showed a marginal association with PEXS and PEXG after controlling for age and sex (p = 0.042, OR = 0.61, 95% CI: 0.38–0.99; Table 2), which did not remain statistically significant after Bonferroni correction (data not shown). Association tests of specific inheritance models also gave similar results as no significant association was seen under a dominant or recessive model (Table 2). While analysis of the above SNPs did not provide any support for association of CLU and TNF- with either PEXS or PEXG, the study did not have sufficient power to exclude lower levels of association. Assuming an OR = 2 with an allelic association model, the sample set used for this study was predicted to
have about 80% power to detect association with rs1800629, 73% power with rs3087554, 87% with rs2279590 and rs11136000, 92% power with rs11136000, and 54% power with rs9331888. Similarly, the 95% CIs for the ORs range from 0.38 to 0.99 for rs1800629 to 0.81–1.89 for rs9331931 (Table 2), suggesting that any association that might exist would lie within these bounds.
DISCUSSION Here we have examined possible association between SNPs in the CLU and TNF- genes with PEXS and PEXG in a South Indian population. In contrast to previous reports28–31 in other populations, the present study demonstrates that CLU and TNF- genes are not the major risk factors for either PEXS or PEXG in the South Indian population. While the relatively Current Eye Research
CLU and TNF-a Gene Variants in Pseudoexfoliation
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TABLE 2 Association of CLU and TNF- SNPs with PEXG + PEXS after controlling for the effects of age and sex and analysis under specific models. SNP rs3087554
rs2279590
rs9331931
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rs11136000
rs9331888
rs1800629
Minor allele
Cases (PEXG + PEXS)
Controls
P_Age & sex*
OR (95% CI)*
Model
p Valuey
OR (95% CI)
C
33.9%
34.0%
0.919
1.01 (0.77–1.33)
Genotypic Dominant Recessive
0.410 0.558 0.340
0.90 (0.63–1.28) 0.77 (0.45–1.32)
Genotypic Dominant Recessive
0.744 0.478 0.620
1.13 (0.80–1.61) 0.84 (0.42–1.68)
Genotypic Dominant Recessive
0.112 0.152 0.233
0.73 (0.47–1.12) 0.37 (0.07–2.03)
Genotypic Dominant Recessive
0.371 0.159 0.721
1.28 (0.91–1.82) 0.88 (0.44–1.76)
Genotypic Dominant Recessive
0.893 0.644 0.797
1.09 (0.75–1.60) 0.94 (0.61–1.45)
Genotypic Dominant Recessive
0.203 0.074 0.686
1.56 (0.95–2.57) 0.72 (0.14–3.60)
T
C
T
G
A
24.2%
11.9%
24.4%
44.8%
6.7%
26.3%
9.9%
27.9%
46.2%
9.8%
0.432
0.310
0.072
0.911
0.042
1.12 (0.84–1.51)
1.24 (0.81–1.89)
1.31 (0.97–1.76)
1.01 (0.78–1.31)
0.61 (0.38–0.99)
OR, odds ratio; PEXG, pseudoexfoliation glaucoma; PEXS, pseudoexfoliation syndrome; SNP, single-nucleotide polymorphism. The asterisk that p values and odds ratios (ORs) with 95% confidence intervals (CI) were obtained from logistic regression analysis of SNPs with PEXG + PEXS after controlling for the effects of age and sex. yChi-square test was used to calculate genotypic p value, dominant and recessive model p values.
small size of our study does not allow exclusion of low levels of association with these markers, these results strongly suggest that if such association exists, it is relatively weak or perhaps impacts only a small part of the population. This is in strong contrast to our previous results with LOXL1-related SNPs, which showed association of some SNPs with ORs as high as 15, and strong haplotypic association with an OR as low as 0.1.26 Indeed, a number of genetic association studies in multiple populations have convincingly shown that LOXL1-related polymorphisms are a major risk factor for the development of PEXS and PEXG.13–27 LOXL1 SNPs hold promise as potential markers for identification of individuals predisposed to this condition. However, several lines of evidence, including reversal of LOXL1 risk alleles in some populations,21–27 the high prevalence of LOXL1 risk alleles in general population, and reduced disease penetrance in some populations regardless of the high frequency of risk alleles,7,16,18 all suggest that LOXL1 variants themselves are not biologically causative. Other genetic and/or environmental factors are likely to contribute to the development of PEXS and PEXG. Genetic studies in some populations have reported association of SNPs tagging PEXS with CLU and TNF- . However, the strength of association of these markers was not as strong as that seen with the LOXL1 gene, suggesting that the CLU and TNF- gene variants are not major contributors to the development of PEXS. !
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Furthermore, some studies did not show association of these genes with PEXS. Therefore, independent replication studies are required to precisely evaluate the role of CLU and TNF- gene variants in PEX pathogenesis. CLU has shown the association with PEXS in Australian and German populations but not in an Italian cohort.29,30 A significant study by Ovodenko et al.35 identified CLU as a prominent component of PEX material. Zenkel et al.34 demonstrated a reduced expression of CLU in the anterior segment tissues and aqueous humor of PEX-affected eyes, which has been suggested to promote the aggregation and deposition of pseudoexfoliative material in the extracellular space. These findings strongly suggest a role for CLU in the development of PEXS. In this study, we evaluated the hypothesis that common genetic variants in CLU might be associated with the PEXS in a South Indian population. In the present study, genotypic and allelic analyses showed that common variants in CLU do not contribute significantly to the risk of PEXS. Additionally, logistic regression failed to identify significant association between CLU SNPs and PEXS and PEXG after controlling for the effects of age and/or sex as well as correcting for multiple testing (Table 2). Therefore, these data indicate that common variants in the CLU gene do not represent a strong genetic contributor in the development of PEXS and PEXG in a South Indian population.
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Previous studies36,45,46 have revealed association of rs11136000, rs2279590, and rs9331888 of CLU with Alzheimer disease. SNP rs2279590 has shown association with both PEX and Alzheimer disease. Aging is the principal risk factor for PEX, Alzheimer’s disease, age-related macular degeneration and age-related cataract. Alzheimer-like changes and SNPs associated with Alzheimer disease have been reported for other age-related eye disorders including age-related macular degeneration and age-related cataract.47,48 However, our study did not find association of any of these SNPs with PEX. An increased or decreased level of TNF-a has been shown to affect the activity and synthesis of MMPs and TIMPs, which play a key role in accumulation of abnormal extracellular PEX material.38,39 Genetic evaluation between PEX syndrome and rs1800629, a SNP associated with elevated TNF levels,37 showed conflicting outcomes in different populations. SNP rs1800629 has been found to be significantly associated with PEXG in Pakistani and Iranian individuals whereas in a European population of Caucasian descent this SNP did not show any association with PEXG.31,32,42 In another study conducted in Turkish population, this variant played a role as a possible protective factor against PEXG.41 In the present study, no significant associations were detected either in the genotype distribution or in allelic frequency of the TNF- –308 G4A polymorphism. Although, logistic regression analysis after controlling for age and sex identified a marginal association of rs1800629 with PEXS and PEXG (Table 2), this association disappeared after Bonferroni correction for multiple testing. Our results are in contradiction with two previous Asian studies, which have shown significant association of the SNP rs1800629 with PEXG.31,32 Possible explanations for these conflicting results may be due to different genetic makeup and varying genotype distributions among different ethnic groups. In conclusion, polymorphisms in CLU and TNF- genes are not major risk factors for the pathogenesis of PEXS and PEXG in the South Indian population. However, low levels of association of these SNPs with PEX cannot be ruled out, nor can the possibility that other, as yet unidentified functional variants in these genes are linked in the development of PEXS and PEXG. These factors make it challenging to draw a firm conclusion in relation to SNP rs9331931 near CLU. Additional genetic studies with large cohorts attaining sufficient power are needed to fully elucidate the role of the CLU and TNF-a genes in the development of PEXS and PEXG.
ACKNOWLEDGMENTS The authors thank all the patients and healthy subjects for participating in this study.
DECLARATION OF INTEREST This study was supported by research grant from ALCON – Aravind Eye Care System, India. The authors have no competing interests to declare.
REFERENCES 1. Ritch R, Schlotzer-Schrehardt U. Exfoliation syndrome. Surv Ophthalmol 2001;45:265–315. 2. Jeng SM, Karger RA, Hodge DO, Burke JP, Johnson DH, Good MS. The risk of glaucoma in pseudoexfoliation syndrome. J Glaucoma 2007;16:117–121. 3. Naumann GO, Schlotzer-Schrehardt U, Kuchle M. Pseudoexfoliation syndrome for the comprehensive ophthalmologist. Intraocular and systemic manifestations. Ophthalmology 1998;105:951–968. 4. Schlotzer-Schrehardt U, Naumann GO. Ocular and systemic pseudoexfoliation syndrome. Am J Ophthalmol 2006;141:921–937. 5. Gottanka J, Flugel-Koch C, Martus P, Johnson DH, LutjenDrecoll E. Correlation of pseudoexfoliative material and optic nerve damage in pseudoexfoliation syndrome. Invest Ophthalmol Vis Sci 1997;38:2435–2446. 6. Schlotzer-Schrehardt U, Naumann GO. Trabecular meshwork in pseudoexfoliation syndrome with and without open-angle glaucoma. A morphometric, ultrastructural study. Invest Ophthalmol Vis Sci 1995;36:1750–1764. 7. Thorleifsson G, Magnusson KP, Sulem P, Walters GB, Gudbjartsson DF, Stefansson H, et al. Common sequence variants in the LOXL1 gene confer susceptibility to exfoliation glaucoma. Science 2007;317:1397–1400. 8. Krumbiegel M, Pasutto F, Schlotzer-Schrehardt U, Uebe S, Zenkel M, Mardin CY, et al. Genome-wide association study with DNA pooling identifies variants at CNTNAP2 associated with pseudoexfoliation syndrome. Eur J Hum Genet 2011;19:186–193. 9. Ringvold A. Epidemiology of the pseudo-exfoliation syndrome. Acta Ophthalmol Scand 1999;77:371–375. 10. Krishnadas R, Nirmalan PK, Ramakrishnan R, Thulasiraj RD, Katz J, Tielsch JM, et al. Pseudoexfoliation in a rural population of southern India: the Aravind Comprehensive Eye Survey. Am J Ophthalmol 2003;135:830–837. 11. Arvind H, Raju P, Paul PG, Baskaran M, Ramesh SV, George RJ, et al. Pseudoexfoliation in South India. Br J Ophthalmol 2003;87:1321–1323. 12. Thomas R, Nirmalan PK, Krishnaiah S. Pseudoexfoliation in southern India: the Andhra Pradesh Eye Disease Study. Invest Ophthalmol Vis Sci 2005;46:1170–1176. 13. Challa P, Schmidt S, Liu Y, Qin X, Vann RR, Gonzalez P, et al. Analysis of LOXL1 polymorphisms in a United States population with pseudoexfoliation glaucoma. Mol Vis 2008;14:146–149. 14. Fan BJ, Pasquale L, Grosskreutz CL, Rhee D, Chen T, DeAngelis MM, et al. DNA sequence variants in the LOXL1 gene are associated with pseudoexfoliation glaucoma in a U.S. clinic-based population with broad ethnic diversity. BMC Med Genet 2008;9:5. 15. Aragon-Martin JA, Ritch R, Liebmann J, O’Brien C, Blaaow K, Mercieca F, et al. Evaluation of LOXL1 gene polymorphisms in exfoliation syndrome and exfoliation glaucoma. Mol Vis 2008;14:533–541. 16. Fingert JH, Alward WL, Kwon YH, Wang K, Streb LM, Sheffield VC, et al. LOXL1 mutations are associated with exfoliation syndrome in patients from the midwestern United States. Am J Ophthalmol 2007;144:974–975. Current Eye Research
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CLU and TNF-a Gene Variants in Pseudoexfoliation 17. Pasutto F, Krumbiegel M, Mardin CY, Paoli D, Lammer R, Weber BH, et al. Association of LOXL1 common sequence variants in German and Italian patients with pseudoexfoliation syndrome and pseudoexfoliation glaucoma. Invest Ophthalmol Vis Sci 2008;49:1459–1463. 18. Hewitt AW, Sharma S, Burdon KP, Wang JJ, Baird PN, Dimasi DP, et al. Ancestral LOXL1 variants are associated with pseudoexfoliation in Caucasian Australians but with markedly lower penetrance than in Nordic people. Hum Mol Genet 2008;17:710–716. 19. Mossbock G, Renner W, Faschinger C, Schmut O, Wedrich A, Weger M. Lysyl oxidase-like protein 1 (LOXL1) gene polymorphisms and exfoliation glaucoma in a Central European population. Mol Vis 2008;14:857–861. 20. Malukiewicz G, Lesiewska-Junk H, Linkowska K, Mielnik M, Grzybowski T, Sulima N. Analysis of LOXL1 single nucleotide polymorphisms in Polish population with pseudoexfoliation syndrome. Acta Ophthalmol 2011;89: e64–e66. 21. Fuse N, Miyazawa A, Nakazawa T, Mengkegale M, Otomo T, Nishida K. Evaluation of LOXL1 polymorphisms in eyes with exfoliation glaucoma in Japanese. Mol Vis 2008;14: 1338–1343. 22. Ozaki M, Lee KY, Vithana EN, Yong VH, Thalamuthu A, Mizoguchi T, et al. Association of LOXL1 gene polymorphisms with pseudoexfoliation in the Japanese. Invest Ophthalmol Vis Sci 2008;49:3976–3980. 23. Tanito M, Minami M, Akahori M, Kaidzu S, Takai Y, Ohira A, et al. LOXL1 variants in elderly Japanese patients with exfoliation syndrome/glaucoma, primary open-angle glaucoma, normal tension glaucoma, and cataract. Mol Vis 2008;14:1898–1905. 24. Chen L, Jia L, Wang N, Tang G, Zhang C, Fan S, et al. Evaluation of LOXL1 polymorphisms in exfoliation syndrome in a Chinese population. Mol Vis 2009;15:2349–2357. 25. Sagong M, Gu BY, Cha SC. Association of lysyl oxidase-like 1 gene polymorphisms with exfoliation syndrome in Koreans. Mol Vis 2011;17:2808–2817. 26. Dubey SK, Hejtmancik JF, Krishnadas SR, Sharmila R, Haripriya A, Sundaresan P. Lysyl oxidase-like 1 gene in the reversal of promoter risk allele in pseudoexfoliation syndrome. JAMA Ophthalmol 2014;132:949–955. 27. Williams SE, Whigham BT, Liu Y, Carmichael TR, Qin X, Schmidt S, et al. Major LOXL1 risk allele is reversed in exfoliation glaucoma in a black South African population. Mol Vis 2010;16:705–712. 28. Kim S, Kim Y. Variations in LOXL1 associated with exfoliation glaucoma do not affect amine oxidase activity. Mol Vis 2012;18:265–270. 29. Burdon KP, Sharma S, Hewitt AW, McMellon AE, Wang JJ, Mackey DA, et al. Genetic analysis of the clusterin gene in pseudoexfoliation syndrome. Mol Vis 2008;14:1727–1736. 30. Krumbiegel M, Pasutto F, Mardin CY, Weisschuh N, Paoli D, Gramer E, et al. Exploring functional candidate genes for genetic association in german patients with pseudoexfoliation syndrome and pseudoexfoliation glaucoma. Invest Ophthalmol Vis Sci 2009;50:2796–2801. 31. Razeghinejad MR, Rahat F, Kamali-Sarvestani E. Association of TNFA -308 G/A and TNFRI +36 A/G gene polymorphisms with glaucoma. Ophthalmic Res 2009;42:118–124. 32. Khan MI, Micheal S, Rana N, Akhtar F, den Hollander AI, Ahmed A, et al. Association of tumor necrosis factor alpha gene polymorphism G-308A with pseudoexfoliative glaucoma in the Pakistani population. Mol Vis 2009;15: 2861–2867.
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33. Wilson MR, Easterbrook-Smith SB. Clusterin is a secreted mammalian chaperone. Trends Biochem Sci 2000;25:95–98. 34. Zenkel M, Kruse FE, Junemann AG, Naumann GO, Schlotzer-Schrehardt U. Clusterin deficiency in eyes with pseudoexfoliation syndrome may be implicated in the aggregation and deposition of pseudoexfoliative material. Invest Ophthalmol Vis Sci 2006;47:1982–1990. 35. Ovodenko B, Rostagno A, Neubert TA, Shetty V, Thomas S, Yang A, et al. Proteomic analysis of exfoliation deposits. Invest Ophthalmol Vis Sci 2007;48:1447–1457. 36. Zhang S, Zhang D, Jiang Y, Wu L, Shang H, Liu J, et al. CLU rs2279590 polymorphism contributes to Alzheimer’s disease susceptibility in Caucasian and Asian populations. J Neural Transm 2014;122:433–439. 37. Wilson AG, Symons JA, McDowell TL, McDevitt HO, Duff GW. Effects of a polymorphism in the human tumor necrosis factor alpha promoter on transcriptional activation. Proc Natl Acad Sci U S A 1997;94:3195–3199. 38. Meijer MJ, Mieremet-Ooms MA, van Hogezand RA, Lamers CB, Hommes DW, Verspaget HW. Role of matrix metalloproteinase, tissue inhibitor of metalloproteinase and tumor necrosis factor-alpha single nucleotide gene polymorphisms in inflammatory bowel disease. World J Gastroenterol 2007;13:2960–2966. 39. Schlotzer-Schrehardt U, Lommatzsch J, Kuchle M, Konstas AG, Naumann GO. Matrix metalloproteinases and their inhibitors in aqueous humor of patients with pseudoexfoliation syndrome/glaucoma and primary open-angle glaucoma. Invest Ophthalmol Vis Sci 2003;44:1117–1125. 40. Maatta M, Tervahartiala T, Harju M, Airaksinen J, AutioHarmainen H, Sorsa T. Matrix metalloproteinases and their tissue inhibitors in aqueous humor of patients with primary open-angle glaucoma, exfoliation syndrome, and exfoliation glaucoma. J Glaucoma 2005;14:64–69. 41. Tekeli O, Turacli ME, Egin Y, Akar N, Elhan AH. Tumor necrosis factor alpha-308 gene polymorphism and pseudoexfoliation glaucoma. Mol Vis 2008;14:1815–1818. 42. Mossbock G, Renner W, El-Shabrawi Y, Faschinger C, Schmut O, Wedrich A, et al. TNF-alpha -308 G4A and -238 G4A polymorphisms are not major risk factors in Caucasian patients with exfoliation glaucoma. Mol Vis 2009;15:518–522. 43. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16:1215. 44. Purcell S, Cherny SS, Sham PC. Genetic Power Calculator: design of linkage and association genetic mapping studies of complex traits. Bioinformatics 2003;19:149–150. 45. Harold D, Abraham R, Hollingworth P, Sims R, Gerrish A, Hamshere ML, et al. Genome-wide association study identifies variants at CLU and PICALM associated with Alzheimer’s disease. Nat Genet 2009;41:1088–1093. 46. Xing YY, Yu JT, Cui WZ, Zhong XL, Wu ZC, Zhang Q, et al. Blood clusterin levels, rs9331888 polymorphism, and the risk of Alzheimer’s disease. J Alzheimers Dis 2012;29: 515–519. 47. Kaarniranta K, Salminen A, Haapasalo A, Soininen H, Hiltunen M. Age-related macular degeneration (AMD): Alzheimer’s disease in the eye? J Alzheimers Dis 2011;24: 615–631. 48. Jun G, Moncaster JA, Koutras C, Seshadri S, Buros J, McKee AC, et al. delta-Catenin is genetically and biologically associated with cortical cataract and future Alzheimerrelated structural and functional brain changes. PLoS One 2012;7:e43728.
ORIGINAL ARTICLE
Evaluation of Genetic Polymorphisms in Clusterin and Tumor Necrosis Factor-Alpha Genes in South Indian Individuals with Pseudoexfoliation Syndrome
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Sushil K. Dubey1, James F. Hejtmancik2, Subbaiah R. Krishnadas3, Rajendrababu Sharmila3, Aravind Haripriya4 and Periasamy Sundaresan1 1
Department of Genetics, Dr. G. Venkataswamy Eye Research Institute, Aravind Medical Research Foundation, Madurai, Tamil Nadu, India, 2Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health, Rockville, MD, USA, 3Glaucoma Clinic, Aravind Eye Hospital, Madurai, Tamil Nadu, India, and 4Intraocular Lens and Cataract Clinic, Aravind Eye Hospital, Madurai, Tamil Nadu, India
ABSTRACT Purpose: The aim of this study was to explore the potential association of genetic variants across clusterin (CLU) and tumor necrosis factor-alpha (TNF- ) genes in South Indian individuals with pseudoexfoliation syndrome (PEXS) and pseudoexfoliation glaucoma (PEXG). Materials and Methods: A total of 523 individuals including 299 unrelated cases (150 PEXS and 149 PEXG) and 224 age- and ethnically-matched healthy controls were recruited for genetic analysis. Six single-nucleotide polymorphisms (SNPs) including, five CLU SNPs (rs11136000, rs2279590, rs9331888, rs9331931, rs3087554) and one promoter SNP (rs1800629) of TNF- were genotyped in all study subjects. Genotyping of CLU SNPs were performed using the TaqMan allelic discrimination assay while TNF- SNP was genotyped using polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis. Association analysis was performed by determining the distributions of genotype and allele frequencies, Hardy–Weinberg equilibrium, and chi-square p values and odds ratios as implemented in the Golden Helix SNP & Variation Suite (SVS). Results: Five CLU SNPs did not show any significant differences in allele frequencies between patients and control subjects (rs3087554, p = 0.919, OR = 1.01, 95% CI: 0.77–1.33; rs2279590, p = 0.432, OR = 1.12, 95% CI: 0.84–1.51; rs9331931, p = 0.310, OR = 1.24, 95% CI: 0.81–1.89; rs11136000, p = 0.072, OR = 1.31, 95% CI: 0.97–1.76; rs9331888, p = 0.911, OR = 1.01, 95% CI: 0.78–1.31). The investigation of TNF- SNP established a significant association with PEXS and PEXG (p = 0.042, OR = 0.61, 95% CI: 0.38–0.99). However, this association did not remain significant after Bonferroni correction. Conclusions: Our data suggest that genetic variants in CLU and TNF-a genes do not play a major role in the development of PEXS and PEXG in the South Indian population. Keywords: CLU, pseudoexfoliation glaucoma, pseudoexfoliation syndrome, SNP, TNF-
INTRODUCTION
pathologic deposition of abnormal microfibrillar aggregates on both intra- and extra-ocular tissues. In the eye, these materials are primarily seen on tissues lining the aqueous-bathed surfaces of anterior segment.1 The origin of these abnormal microfibrils is unclear but they are believed to be multifocally
Pseudoexfoliation syndrome (PEXS) is an age-related systemic disorder of the extracellular matrix and the most common identifiable cause of secondary openangle glaucoma.1,2 PEXS is characterized by the
Received 7 July 2014; revised 4 December 2014; accepted 8 December 2014; published online 2 April 2015 Correspondence: Periasamy Sundaresan, Department of Genetics, Dr. G. Venkataswamy Eye Research Institute, Aravind Medical Research Foundation, No 1, Anna Nagar, Madurai, Tamil Nadu, India. E-mail: [email protected]
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produced by various ocular tissues including the corneal endothelium, iris, non-pigmented ciliary epithelium, lens epithelium and trabecular meshwork.3 PEXS-associated glaucoma (pseudoexfoliation glaucoma, PEXG) is characterized by rapid progression, high resistance to medical therapy and poorer prognosis than the more common primary open-angle glaucoma.4 Progressive accumulation of pseudoexfoliation (PEX) material on the trabecular meshwork presumably causes obstruction of aqueous out flow and correlates with elevated intraocular pressure (IOP) and severity of optic nerve damage.5,6 Hence, PEXG represents PEXS accompanied by outflow obstruction causing elevated intra-ocular pressure. Approximately 60% of the eyes with PEXS develop PEXG within 15 years of diagnosis.2 Thus, while there might be some additional components (genetic or environmental) to PEXG, certainly every PEXG patient can be counted as having PEXS. Genetic studies also supported that both PEXS and PEXG have overlapping, if not necessarily identical, genetic risk factors.7,8 The average worldwide prevalence rate of PEXS is 10–20% of the general population over 60 years of age.9 Population-based studies in South India have reported the overall prevalence of 3–6% among subjects older than 40 years.10–12 Thorleifsson et al.7 performed a genome-wide association study in Icelandic and Swedish populations and identified a strong association between PEXS and three common single-nucleotide polymorphisms (SNPs) in lysyl oxidase-like 1 (LOXL1; OMIM: 153456) gene. Subsequently, these findings were replicated in multiple populations globally, which established LOXL1 as a major genetic risk factor for PEXS and PEXG.13–27 However, the risk associated alleles of these LOXL1 SNPs are common and relatively more prevalent – up to 88% – among the general populations. In some populations, while the frequency of risk allele is high, the disease penetrance is low.7,16,18 Moreover, recent studies demonstrated the reversal of LOXL1 risk allele in some populations21–27 as well as lack of impact of two non-synonymous variants (R141L and G153D) on amine oxidase activity of LOXL1 protein.28 Taken together, these data suggest that common SNPs in LOXL1 are not the causative variants and additional as yet unrevealed genetic and environmental factors might be involved in the development of PEXS and PEXG. Previous studies in some populations have reported association of SNP allele in the clusterin (CLU) and tumor necrosis factor-alpha (TNF- ) genes with PEXS and/or PEXG.29–32 CLU is an extracellular multifunctional protein that primarily acts as a molecular chaperone, preventing the precipitation and aggregation of misfolded extracellular proteins.33 Lower levels of CLU protein in aqueous humor as well as down regulation of CLU mRNA in many
anterior segment tissues has been detected in eyes with the PEX syndrome.34 CLU is also a major component of PEX material.35 Burdon et al.29 reported a nominal association between rs3087554 of CLU and PEXS in Australian Blue Mountains Eye Study cohort. Subsequently, Krumbiegel et al.30 genotyped five SNPs of CLU in PEXS, PEXG and controls from Europeans and reported that allelic variant of rs2279590 is a risk factor for the development of PEXS in German patients but not in Italian patients. SNP rs3087554 alleles, which showed association with PEXS in Australians, were not associated with PEXS in either German or Italian populations. Interestingly, rs2279590 has shown association with Alzheimer disease in Caucasian and Asian populations.36 Therefore, it would be of interest to know whether CLU SNPs associated with Alzheimer’s are also associated with PEX. Studies associating CLU gene with PEXS and PEXG have not been conducted previously in Asian populations. A functional SNP (rs1800629) at position –308 in the promoter region of TNF- gene, characterized by G to A substitution, is associated with higher TNF-a production.37 TNF-a, a pleiotropic proinflammatory cytokine, regulates the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs).38 Both MMPs and TIMPs are required for proper turnover of extracellular matrix components.38 Deposition of abnormal extracellular matrix components in PEXS may be due to reduced turnover of matrix components. Interestingly, decreased level of MMP-2 and increased level of both TIMP-1 and TIMP-2 have been detected in the aqueous humor of the PEX patients, suggesting the involvement of TNF-a in PEXS and PEXG.39,40 Association studies of rs1800629 with PEXG have been done in few populations which showed contradictory results.31,32,41,42 Therefore, further replication studies in different populations are essential for a comprehensive understanding of the role of rs1800629 in PEX pathogenesis. Both CLU and TNF- genes have not been characterized in PEX patients of South Indian ethnic background. The purpose of this study was to evaluate the possible association of genetic variants in CLU and TNF- genes with PEXS and PEXG in a South Indian population.
MATERIALS AND METHODS Study Population The study protocol was approved by the Institutional Review Board of Aravind Eye Hospital and was carried out in accordance with the tenets of the Declaration of Helsinki. Informed consent was obtained from all study participants after explaining the nature of the study. 299 unrelated cases (PEXS, Current Eye Research
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CLU and TNF-a Gene Variants in Pseudoexfoliation 150; PEXG, 149) and 224 age- and ethnicity-matched controls were enrolled for this study. All participants underwent a complete ophthalmic examination including visual acuity, slit lamp biomicroscopy, tonometric assessment of IOP, gonioscopy and retinal examination. PEXS cases were diagnosed as those with clinical evidence of PEX material on pupillary margin and/or anterior lens capsule, IOP 519 mm Hg and no glaucomatous optic atrophy and visual field defects. PEXG were defined as the presence of PEX material, open iridocorneal angles, IOP 22 mm Hg in either eye, typical glaucomatous optic disc changes (defined as a thinning or notching of neuroretinal rim, vertical cup-to-disc ratio 40.7 or cup-to-disc ratio asymmetry 0.3 between both eyes) and typical visual field defects in computed perimetry. Control subjects had no evidence of PEX material, IOP 519 mm Hg, no glaucomatous changes in optic disc (no thinning or notching of neuroretinal rim, cup-to-disc ratio range was 0.3–0.5 or cup-to-disc ratio asymmetry 50.2 between both eyes), normal visual fields and had no medical or family history of glaucoma.
Genotyping Venous blood samples (5 ml) were collected from all participants and total genomic DNA was isolated by salt precipitation method.43 Five CLU SNPs (rs11136000, rs2279590, rs9331888, rs9331931, rs3087554) were selected for this study as these SNPs are in a large loose LD block that covers most of CLU gene. The same SNPs were accessed for association with PEXS and PEXG in a previous study.30 Promoter SNP of TNF- , rs1800629, was selected based on its association with PEXG in some populations.31,32 CLU SNPs were genotyped using pre-developed TaqMan SNP genotyping assays (Applied Biosystems, Foster City, CA). Assays were performed according to manufacturer’s instructions and each 5 ml reaction mixture was prepared in a 384well plate, containing 2.5 ml TaqMan universal polymerase chain reaction (PCR) master mix (2), 0.125 ml of SNP genotyping assay (40), 2 ml of DNA (20 ng/ ml) and 0.375 ml MilliQ water. Reactions were amplified using standard thermal cycling conditions and analyzed on an ABI 7900 HT Fast real-time PCR system (Applied Biosystems, Foster City, CA). SNP rs1800629 of TNF- were genotyped by PCR-based restriction fragment length polymorphism (RFLP) approach as described earlier42 with certain modifications in PCR parameter and RFLP. PCR, using gradient thermo cycler (ASTEC, Fukuoka, Japan), was performed in total volume of 20 ml, containing 1 PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, and 0.001% gelatin), 100 ng of genomic DNA, 0.5 pmol of each primer, 200 mM of dNTPs (Medox !
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Biotech India Pvt. Ltd, Chennai, India) and 1 U of Taq DNA polymerase (Sigma, Saint Louis, MO). Thermal cycling conditions were 5 min at 95 C, followed by 36 cycles (30 s at 95 C, 30 s for primer annealing at 62 C and 45 s at 72 C) and a final extension for 7 min at 72 C. PCR amplicon (13 ml) was restriction digested at 37 C overnight with 10 U of Bsp19I restriction enzyme (Fermentas, India) and electrophoresed in a 3% (wt/ vol) agarose gels. Amplicons carrying the A allele remained uncut (212 bp), while amplicons carrying the G allele were digested into two fragments of 192 bp and 20 bp.
Statistical Analysis The observed genotype frequencies for each SNP were tested for Hardy–Weinberg equilibrium (HWE) in both cases and controls and the difference between the observed and expected frequencies was tested for significance using Fisher exact test. 2 and Fisher exact tests were used to examine the genotypic and allelic associations of all the SNPs, as implemented in the Golden Helix SVS software suite 7 (Golden Helix, Bozeman, MT). Call rates, odds ratios (ORs), and confidence intervals (CIs) were also calculated using the same program. A p50.05 was considered statistically significant in hypothesis testing and 95% CIs were used to describe the estimation of unknown parameters. p Values were corrected for multiple testing using a Bonferroni correction. Power was estimated as described by Purcell et al.44 assuming a high-risk allele frequency and prevalence of 0.15, genotype relative risks for both heterozygotes and homozygous risk alleles of 2, a significant p at 0.05, and D’ = 1. Logistic regression analysis was done after adjusting for age and sex to assess the association of CLU and TNF- SNPs with PEXS and PEXG.
RESULTS The present study group comprised 299 PEX patients and 224 age- and ethnicity- matched healthy control subjects. The mean age was 65.0 ± 7.0 years (range, 45– 80) in PEXS cases, 67.0 ± 7.6 years (range, 46–95) in PEXG cases and 66.0 ± 6.2 years (range, 55–92) in controls. There were no significant differences found among the means of these three groups (p = 0.134, PEXS versus control; p = 0.188, PEXG versus control). The observed genotype frequencies of CLU and TNF- SNPs were in HWE in both cases and control groups. The distribution of genotype and allele frequencies of PEX cases and controls are shown in Table 1. Statistical analysis was performed to establish the association of these SNPs with PEXS and PEXG after adjusting for age and sex (Table 2). After controlling for the effects of age and sex, logistic
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TABLE 1 Distribution of genotype and allele frequencies of CLU and TNF- SNPs in PEXG, PEXS and controls in a South Indian cohort. SNP (gene) rs3087554 (CLU)
Genotype frequency (%) Study subjects (N) PEXG (149) PEXS (150) PEXG + PEXS (299) Control (222)
rs2279590 (CLU)
68 59 127 100
(45.6) (39.3) (42.5) (45.0)
TC 66 75 141 93
CC PEXG (147) PEXS (150) PEXG + PEXS (297) Control (224)
rs9331931 (CLU) Curr Eye Res Downloaded from informahealthcare.com by University of Victoria on 04/25/15 For personal use only.
TT
81 90 171 122
(55.1) (60.0) (57.6) (54.5)
rs11136000 (CLU)
119 111 230 183
(79.9) (74.0) (76.9) (82.1)
57 51 108 86
rs9331888 (CLU)
82 90 172 115
(55.0) (60.0) (57.5) (51.3)
29 38 67 36
rs1800629 (TNF-a)
49 42 91 64
(32.9) (28.0) (30.4) (28.6)
57 51 108 93
127 128 255 172
(87.0) (88.3) (87.6) (81.9)
9(6.1) 9 (6.0) 18(6.0) 16(7.1)
(19.4) (25.3) (22.4) (16.1)
(38.3) (34.0) (36.1) (41.5)
71 77 148 113
(47.6) (51.3) (49.5) (50.4)
GA 17 16 33 35
(11.6) (11.0) (11.4) (16.7)
202 193 395 293
1(0.7) 1 (0.7) 2 (0.7) 4 (1.8)
219 231 450 330
267 260 527 402
(19.5) (20.7) (20.1) (21.0)
221 231 452 323
169 161 330 241
(74.5) (77.0) (75.8) (73.7)
75 69 144 118
(25.5) (23.0) (24.2) (26.3) C
(89.6) (86.7) (88.1) (90.1)
31 40 71 44
(10.4) (13.3) (11.9) (9.9) T
(74.2) (77.0) (75.6) (72.1)
77 69 146 125
(25.8) (23.0) (24.4) (27.9) G
(56.7) (53.7) (55.2) (53.8)
129 139 268 207
G 271 272 543 379
(32.2) (35.7) (33.9) (34.0) T
C
AA 2 (1.4) 1 (0.7) 3(1.0) 3 (1.4)
96 107 203 151
C
GG 29 31 60 47
(67.8) (64.3) (66.1) (66.0)
G
TT 10(6.7) 9 (6.0) 19(6.4) 16(7.2)
C
C
CC
CG
GG PEXG (146) PEXS (145) PEXG + PEXS (291) Control (210)
(38.8) (34.0) (36.4) (38.4)
(10.1) (10.7) (10.4) (13.1)
T
TT
CT
CC PEXG (149) PEXS (150) PEXG + PEXS (299) Control (224)
15 16 31 29
GC
CC PEXG (149) PEXS (150) PEXG + PEXS (299) Control (224)
(44.3) (50.0) (47.1) (41.9)
CC
CT
GG PEXG (149) PEXS (150) PEXG + PEXS (299) Control (223)
Allele frequency (%)
(43.3) (46.3) (44.8) (46.2) A
(92.8) (93.8) (93.3) (90.2)
21 18 39 41
(7.2) (6.2) (6.7) (9.8)
PEXG, pseudoexfoliation glaucoma; PEXS, pseudoexfoliation syndrome.
regression analysis revealed that none of the CLU SNPs were associated with PEXS and PEXG (rs3087554, p = 0.919, OR = 1.01; rs2279590, p = 0.432, OR = 1.12; rs9331931, p = 0.310, OR = 1.24; rs11136000, p = 0.072, OR = 1.31; rs9331888, p = 0.911, OR = 1.01). However, rs1800629 of TNF- showed a marginal association with PEXS and PEXG after controlling for age and sex (p = 0.042, OR = 0.61, 95% CI: 0.38–0.99; Table 2), which did not remain statistically significant after Bonferroni correction (data not shown). Association tests of specific inheritance models also gave similar results as no significant association was seen under a dominant or recessive model (Table 2). While analysis of the above SNPs did not provide any support for association of CLU and TNF- with either PEXS or PEXG, the study did not have sufficient power to exclude lower levels of association. Assuming an OR = 2 with an allelic association model, the sample set used for this study was predicted to
have about 80% power to detect association with rs1800629, 73% power with rs3087554, 87% with rs2279590 and rs11136000, 92% power with rs11136000, and 54% power with rs9331888. Similarly, the 95% CIs for the ORs range from 0.38 to 0.99 for rs1800629 to 0.81–1.89 for rs9331931 (Table 2), suggesting that any association that might exist would lie within these bounds.
DISCUSSION Here we have examined possible association between SNPs in the CLU and TNF- genes with PEXS and PEXG in a South Indian population. In contrast to previous reports28–31 in other populations, the present study demonstrates that CLU and TNF- genes are not the major risk factors for either PEXS or PEXG in the South Indian population. While the relatively Current Eye Research
CLU and TNF-a Gene Variants in Pseudoexfoliation
5
TABLE 2 Association of CLU and TNF- SNPs with PEXG + PEXS after controlling for the effects of age and sex and analysis under specific models. SNP rs3087554
rs2279590
rs9331931
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rs11136000
rs9331888
rs1800629
Minor allele
Cases (PEXG + PEXS)
Controls
P_Age & sex*
OR (95% CI)*
Model
p Valuey
OR (95% CI)
C
33.9%
34.0%
0.919
1.01 (0.77–1.33)
Genotypic Dominant Recessive
0.410 0.558 0.340
0.90 (0.63–1.28) 0.77 (0.45–1.32)
Genotypic Dominant Recessive
0.744 0.478 0.620
1.13 (0.80–1.61) 0.84 (0.42–1.68)
Genotypic Dominant Recessive
0.112 0.152 0.233
0.73 (0.47–1.12) 0.37 (0.07–2.03)
Genotypic Dominant Recessive
0.371 0.159 0.721
1.28 (0.91–1.82) 0.88 (0.44–1.76)
Genotypic Dominant Recessive
0.893 0.644 0.797
1.09 (0.75–1.60) 0.94 (0.61–1.45)
Genotypic Dominant Recessive
0.203 0.074 0.686
1.56 (0.95–2.57) 0.72 (0.14–3.60)
T
C
T
G
A
24.2%
11.9%
24.4%
44.8%
6.7%
26.3%
9.9%
27.9%
46.2%
9.8%
0.432
0.310
0.072
0.911
0.042
1.12 (0.84–1.51)
1.24 (0.81–1.89)
1.31 (0.97–1.76)
1.01 (0.78–1.31)
0.61 (0.38–0.99)
OR, odds ratio; PEXG, pseudoexfoliation glaucoma; PEXS, pseudoexfoliation syndrome; SNP, single-nucleotide polymorphism. The asterisk that p values and odds ratios (ORs) with 95% confidence intervals (CI) were obtained from logistic regression analysis of SNPs with PEXG + PEXS after controlling for the effects of age and sex. yChi-square test was used to calculate genotypic p value, dominant and recessive model p values.
small size of our study does not allow exclusion of low levels of association with these markers, these results strongly suggest that if such association exists, it is relatively weak or perhaps impacts only a small part of the population. This is in strong contrast to our previous results with LOXL1-related SNPs, which showed association of some SNPs with ORs as high as 15, and strong haplotypic association with an OR as low as 0.1.26 Indeed, a number of genetic association studies in multiple populations have convincingly shown that LOXL1-related polymorphisms are a major risk factor for the development of PEXS and PEXG.13–27 LOXL1 SNPs hold promise as potential markers for identification of individuals predisposed to this condition. However, several lines of evidence, including reversal of LOXL1 risk alleles in some populations,21–27 the high prevalence of LOXL1 risk alleles in general population, and reduced disease penetrance in some populations regardless of the high frequency of risk alleles,7,16,18 all suggest that LOXL1 variants themselves are not biologically causative. Other genetic and/or environmental factors are likely to contribute to the development of PEXS and PEXG. Genetic studies in some populations have reported association of SNPs tagging PEXS with CLU and TNF- . However, the strength of association of these markers was not as strong as that seen with the LOXL1 gene, suggesting that the CLU and TNF- gene variants are not major contributors to the development of PEXS. !
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Furthermore, some studies did not show association of these genes with PEXS. Therefore, independent replication studies are required to precisely evaluate the role of CLU and TNF- gene variants in PEX pathogenesis. CLU has shown the association with PEXS in Australian and German populations but not in an Italian cohort.29,30 A significant study by Ovodenko et al.35 identified CLU as a prominent component of PEX material. Zenkel et al.34 demonstrated a reduced expression of CLU in the anterior segment tissues and aqueous humor of PEX-affected eyes, which has been suggested to promote the aggregation and deposition of pseudoexfoliative material in the extracellular space. These findings strongly suggest a role for CLU in the development of PEXS. In this study, we evaluated the hypothesis that common genetic variants in CLU might be associated with the PEXS in a South Indian population. In the present study, genotypic and allelic analyses showed that common variants in CLU do not contribute significantly to the risk of PEXS. Additionally, logistic regression failed to identify significant association between CLU SNPs and PEXS and PEXG after controlling for the effects of age and/or sex as well as correcting for multiple testing (Table 2). Therefore, these data indicate that common variants in the CLU gene do not represent a strong genetic contributor in the development of PEXS and PEXG in a South Indian population.
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6
S. K. Dubey et al.
Previous studies36,45,46 have revealed association of rs11136000, rs2279590, and rs9331888 of CLU with Alzheimer disease. SNP rs2279590 has shown association with both PEX and Alzheimer disease. Aging is the principal risk factor for PEX, Alzheimer’s disease, age-related macular degeneration and age-related cataract. Alzheimer-like changes and SNPs associated with Alzheimer disease have been reported for other age-related eye disorders including age-related macular degeneration and age-related cataract.47,48 However, our study did not find association of any of these SNPs with PEX. An increased or decreased level of TNF-a has been shown to affect the activity and synthesis of MMPs and TIMPs, which play a key role in accumulation of abnormal extracellular PEX material.38,39 Genetic evaluation between PEX syndrome and rs1800629, a SNP associated with elevated TNF levels,37 showed conflicting outcomes in different populations. SNP rs1800629 has been found to be significantly associated with PEXG in Pakistani and Iranian individuals whereas in a European population of Caucasian descent this SNP did not show any association with PEXG.31,32,42 In another study conducted in Turkish population, this variant played a role as a possible protective factor against PEXG.41 In the present study, no significant associations were detected either in the genotype distribution or in allelic frequency of the TNF- –308 G4A polymorphism. Although, logistic regression analysis after controlling for age and sex identified a marginal association of rs1800629 with PEXS and PEXG (Table 2), this association disappeared after Bonferroni correction for multiple testing. Our results are in contradiction with two previous Asian studies, which have shown significant association of the SNP rs1800629 with PEXG.31,32 Possible explanations for these conflicting results may be due to different genetic makeup and varying genotype distributions among different ethnic groups. In conclusion, polymorphisms in CLU and TNF- genes are not major risk factors for the pathogenesis of PEXS and PEXG in the South Indian population. However, low levels of association of these SNPs with PEX cannot be ruled out, nor can the possibility that other, as yet unidentified functional variants in these genes are linked in the development of PEXS and PEXG. These factors make it challenging to draw a firm conclusion in relation to SNP rs9331931 near CLU. Additional genetic studies with large cohorts attaining sufficient power are needed to fully elucidate the role of the CLU and TNF-a genes in the development of PEXS and PEXG.
ACKNOWLEDGMENTS The authors thank all the patients and healthy subjects for participating in this study.
DECLARATION OF INTEREST This study was supported by research grant from ALCON – Aravind Eye Care System, India. The authors have no competing interests to declare.
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