|Molecular Vision 2007;
Received 16 August 2007 | Accepted 5 October 2007 | Published 9 October 2007
Association between primary open-angle glaucoma and WDR36 DNA sequence variants in Japanese
Department of Ophthalmology, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Miyagi, Japan
Correspondence to: N. Fuse, Department of Ophthalmology, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8574, Japan; Phone: 81-22-717-7294; FAX: 81-22-717-7298; email: firstname.lastname@example.org
Purpose: To determine whether mutations in the WD repeat domain 36 gene (WDR36) are associated with primary open-angle glaucoma (POAG) in Japanese. Subjects with high tension glaucoma (HTG) and normal tension glaucoma (NTG) were analyzed separately.
Methods: One hundred and thirty-six unrelated Japanese patients with HTG and 103 unrelated patients with NTG were studied. Genomic DNA was extracted from peripheral blood leukocytes, and all 23 exons were amplified by polymerase chain reaction (PCR) and directly sequenced bidirectionally.
Results: Twenty sequence alterations were identified: 10 have already been reported (p.I264V, c.1494+90C>T, c.1494+143A>G, c.1609+89G>A, c.1775+89C>A, c.1965-30A>G, p.V714V, c.2170+217C>T, p.V727V, and c.2518+60G>C) and 10 were novel (p.D179D, p.Q270Q, p.M283R, c.898+63C>G, c.1074+20C>T, p.G459G, c.1884+26C>G, p.S664L, p.S664S, and p.P744P). One nonsynonymous amino acid change in exon 17, p.S664L, was identified in a patient with HTG. The frequency of the p.I264V variant was significantly higher in the HTG group than in the control group (p=0.01), but the frequency in the NTG group was not significantly different from the control group (p=0.12). The frequency of the c.1965-30A>G variant was also significantly higher in the HTG group than in the control group (p=0.03), but the frequency in the NTG group was not significantly different from the control group (p=0.06).
Conclusions: One nonsynonymous variant, p.S664L, and the association of the allelic variants (p.I264V and c.1965-30A>G) in WDR36 and their prevalence in unrelated Japanese patients with HTG suggest that they are probably involved in the pathogenesis of HTG.
Glaucoma is a complex, heterogeneous disease characterized by a progressive degeneration of the optic nerve axons and is the second highest cause of blindness worldwide, affecting approximately 70 million people . Because of the loss of the optic nerve axons, visual field defects develop. Primary open-angle glaucoma (POAG), the most common type of glaucoma, is associated with an elevated intraocular pressure (IOP)  and includes subgroups called high tension glaucoma (HTG) and low-tension glaucoma (LTG) [3,4] or normal tension glaucoma (NTG) . The prevalence of NTG is higher among the Japanese than among Caucasians [6,7].
Although the precise molecular basis of POAG is not known, it is probably a genetically heterogeneous disorder caused by the interaction of multiple genes and environmental factors [8,9]. POAG is more likely a complex or multifactorial disease, and numerous genes can independently cause the disease but do not necessarily implicate the environment in modulating the disease.
Several genetic loci that contribute to the susceptibility of eyes to POAG have been identified. To date, at least 14 loci from GLC1A to GLC1N have been linked to POAG, and three genes have been identified: the myocilin gene (MYOC), the optineurin gene (OPTN), and the WD repeat domain 36 gene (WDR36). MYOC is mutated in juvenile-onset primary open-angle glaucoma , and MYOC mutations account for approximately 2%-4% of POAG cases [11,12]. However, the percentage of MYOC mutations might be as high as 22.2%  to 36%  in families with juvenile hereditary POAG. OPTN is mutated in 16.7% of families with autosomal dominant, adult-onset POAG, including some with NTG . However, one study reported that OPTN variants do not predispose the subject to POAG , and another study reported a lack of association between OPTN and POAG/NTG .
WDR36 is the POAG gene at the GLC1G locus and is composed of 23 exons  that encodes a 951 amino acid protein with multiple G beta winged domain 40 (WD40) repeats. Four mutations (p.N355S, p.A449T, p.R529Q, and p.D658G) were identified in 17 (5.02%-6.92%) unrelated POAG subjects, 11 with HTG, and six with NTG. However, the magnitude of its role and association with glaucoma remains controversial, and the main single nucleotide polymorphism (SNP; p.D658G), which did segregate with the original families linked to the 5q region, was later identified as a neutral variant in an Australian population . Furthermore, the findings in other studies indicated that WDR36 variants may be only a rare cause of NTG in a German population  or no causal association with POAG .
The development of an accurate diagnostic test for pre-symptomatic individuals at risk for glaucoma is urgently needed. Thus, the screening of the glaucoma causal gene may contribute to the identification of pre-symptomatic cases in the general population. The purpose of this study was to determine whether mutations in WDR36 are associated with HTG and NTG in the Japanese.
One hundred and thirty-six unrelated Japanese patients with HTG (73 men and 63 women; mean age 58.6±12.4 years) and 103 unrelated Japanese patients with NTG (53 men and 50 women; mean age 61.8±11.7 years), who were examined at our Ophthalmic clinic at the Tohoku University Hospital, Sendai, Japan, were studied. The purpose and procedures of the study were explained to all patients, and informed consent was obtained. This study was approved by the Tohoku University Institutional Review Board, and the procedures used conformed to the tenets of the Declaration of Helsinki. Routine ophthalmic examinations were performed on all patients.
The criteria for classifying a patient as having HTG were: (1) An applanation IOP 22 mmHg or more in each eye; (2) glaucomatous cupping in each eye including a cup-to-disc ratio >0.7; (3) visual field defects determined by Goldmann perimetry and/or Humphrey field analyzer consistent with glaucomatous cupping in at least one eye; and (4) an open anterior chamber angle. Patients with glaucoma of secondary causes, e.g. trauma, uveitis, or steroid-induced, were excluded. The criteria for NTG were: (1) Untreated peak IOP measured with a Goldmann applanation tonometer was <22 mmHg in both eyes at all examinations, including the three baseline measurements and those during the diurnal test (every 2 or 3 h from 6 AM to 12 PM) with or without medication. In addition, the IOP was consistently <22 mmHg throughout the follow-up period. The other criteria were the same as (2), (3), and (4) of the HTG group. The central corneal thickness (CCT)was measured with a ultrasound CCT measurement apparatus (SP-3000; TOMEY, Nagoya, Japan). Intraocular pressure was measured three times with a Goldmann applanation tonometer under topical anesthesia, and the median value was used for analysis.
The mean IOP at diagnosis was 27.2±5.1 mmHg in the 136 patients with HTG and 16.3±3.5 mmHg in the 103 patients with NTG. To make an exact clinical definition of the HTG and NTG for each patient with OAG based on IOP, the central corneal thickness (CCT) should be considered. The CCT was measured with a pachymeter in all OAG patients who had borderline IOP, i.e. 21-22 mmHg. The following formula was used to calculate the corrected IOP; corrected IOP=IOP reading-0.012 (CCT (μm)-520) .
Control subjects (62 men and 56 women; mean age 68.0±7.7 years) had the following characteristics: An IOP less than 22 mmHg, normal optic discs, and no family history of glaucoma. To decrease the chance of enrolling individuals with pre-symptomatic glaucoma, we limited this group to individuals older than 60 years.
Sample preparation and mutation screening
Genomic DNA was extracted from peripheral blood leukocytes and purified by the Qiagen QIAamp Blood Kit (Qiagen, Valencia, CA). All 23 exons that code for a 951 amino acid protein were amplified by polymerase chain reaction (PCR) using 0.5 μM of the 23 pairs of intronic primers in an amplification mixture (25 μl) containing 0.2 mM dNTPs and 0.5 U Ex Taq polymerase (Takara, Shiga, Japan) with 30 ng template DNA. The oligonucleotides for amplification and sequencing were selected using Primer3 software, (provided in the public domain by the Massachusetts Institute of Technology, Cambridge, MA).
PCR fragments were purified by ExoSAP-IT (USB, Cleveland, OH), and sequenced with the BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Foster City, CA) on an automated DNA sequencer (ABI PRISMTM 3100 Genetic Analyzer, Perkin-Elmer). All HTG and NTG subjects and controls were fully sequenced. All sequencing results were confirmed by sequencing the opposite strand. To describe the variations in the DNA sequence, we used a website and read the editorial by den Dunnen [23,24]. The nomenclature of the mutations was based on GenBank NM_139281.
Differences in the genotype frequencies among the cases and controls were tested by Fisher's exact test depending on cell counts. Odds ratios (approximating to relative risk) were calculated as a measure of the association between the WDR36 genotype and the phenotype of HTG/NTG with the effects of the mutant allele assumed to be dominant (wild/wild versus wild/mutant and mutant/mutant combined). For each odds ratio, the p-values and 95% confidence intervals were calculated. The clinical characteristics of the subjects who were homozygotes but not-at-risk genotype, heterozygote genotype, and the homozygote genotype were examined. The clinical characteristics were: age at diagnosis, initial IOP, and global indices of the visual field (mean deviation [MD]) of the Humphrey field analyzer.
The inferred haplotypes, quantified between all pairs of biallelic loci, were estimated using the SNPAlyze program version 4.0 (Dynacom, Yokohama, Japan). Additionally, a permutation test was performed to test the deviations of allelic frequencies of SNPs and haplotypes . The significance of association was determined by contingency table analysis using χ2 or Fisher's exact test. The Hardy-Weinberg (HW) equilibrium was analyzed using gene frequencies obtained by simple gene counting and the χ2 test with Yates' correction for comparing observed and expected values.
WDR36 variants detected in subjects
After direct sequencing, 20 variants were identified: three were nonsynonymous changes, seven were synonymous codon changes, and 10 were changes in the noncoding sequences (Table 1). Ten have already been reported (p.I264V, c.1494+90C>T, c.1494+143A>G, c.1609+89G>A, c.1775+89C>A, c.1965-30A>G, p.V714V, c.2170+217C>T, p.V727V, and c.2518+60G>C), and 10 were novel (p.D179D, p.Q270Q, p.M283R, c.898+63C>G, c.1074+20C>T, p.G459G, c.1884+26C>G, p.S664L, p.S664S, and p.P744P). The frequency of the genotypes of WDR36 in patients with HTG/NTG and control subjects is shown in Table 1.
Distribution of WDR36 variants in high tension glaucoma and normal tension glaucoma patients and control subjects
p.S664L, identified in exon 17, was a heterozygous mutation (C>T) in the second nucleotide, which changed serine to leucine. We did not find the p.S664L alteration in 118 ethnically-matched controls. We could not detect the previously identified sequence alterations (p.N355S, p.A449T, p.R529Q, and p.D658G ) in our glaucomatous patients. p.M283R, identified in exon 7, was a heterozygous mutation (T>G) in the second nucleotide, which changed methionine to arginine. We found the p.M283R alteration in five controls and in only one NTG subject.
The previously reported nonsynonymous SNP (p.I264V) was a common sequence variant that was found evenly distributed between HTG probands and control subjects. The frequency of the p.I264V variant was significantly higher in the HTG group than in the control group (55.1%versus 39.0%; p=0.01; Fisher two-tailed exact test) but not in the NTG group (28.2% versus 39.0%; p=0.12; Fisher two-tailed exact test) for the dominant effect of the I264V allele.
Among the noncoding sequences, the intronic c.1074+20C>T variant was found in only HTG and NTG subjects (p=0.03 and p=0.02, respectively), but the percentage of the mutated allele was less than 10% (Table 1). We also found c.898+63C>G in one HTG (p=1.0) and c.1884+26C>G in two HTG (p=0.50) individuals, but we could not determine whether those would be a glaucoma-causing alteration because they were minor SNPs.
The frequency of the c.1965-30A>G variant was higher in the HTG group than in the control group (53.7% versus 39.0%; p=0.03) but not in the NTG group (26.2% versus 39.0%; p=0.06; Fisher two-tailed exact test for the dominant effect of the WDR36 G allele; Table 2). In addition, the frequency of the c.1609+89G>A and c.1775+89C>A variants was higher in the HTG group than in the control group (p=0.01 and p=0.04, respectively; Table 1). All polymorphisms adhered to the HW expectations (p>0.05).
Association of WDR36 haplotype blocks with high tension glaucoma
In the studied SNPs, four SNPs were significantly associated (permutation p<0.05). The haplotype-based associations were tested with a 1,000 iterated permutation test. Three major haplotypes; A-C-A-A, G-T-G-G, and A-T-G-A (each frequency >5%) and one minor haplotype, G-T-G-A, were found in the subjects (Table 3.). One haplotype, G-T-G-G, was over-represented in HTG subjects, showing a highly significant difference in frequency between the HTG and control group (p=0.006, permutation p=0.005), but the same G-T-G-G haplotype was less-represented in NTG subjects (p=0.033, permutation p=0.036).
Evolutionary conservation of WDR36 variants in humans and four other species
Two nonsynonymous SNPs, p.M283R and p.S664L, which were not previously reported, were found in our population. We showed amino acid alignment and evolutionary conservation of p.I264V, p.M283R, and p.S664L of WDR36 variants. WDR36 orthologs were evolutionarily conserved in humans, chimpanzees, rats, mice, and dogs. The p.I264V, p.M283R in exon 7, and the p.S664L in exon 17 are located in the G-beta WD40 repeat (Figure 1) and in the Cytochrome cd1-nitrite reductase-like COOH-terminal haem d1 (cyt cd1) domain.
Characteristics of patient with p.I264V, c.1965-30A>G, and p.S664L
Homozygote genotypes for the p.I264V change and the c.1965-30A>G change had modest significance in the HTG group. Between the subjects having homozygote not-at-risk genotype, heterozygote genotype, and the homozygote genotype, the mean deviation (MD) of the visual field was statistically different. The MDs of subjects having the homozygote genotype were worse than those of heterozygote genotype and homozygote not-at-risk genotype (p<0.001; Table 4).
The patient with the p.S664L mutation was a 70-year-old woman diagnosed with HTG at age 51 years. She had no family history of glaucoma, and her IOPs were between 16 and 23 mmHg under the control of eye drops. Visual field (Humphrey Field Analyzer) MDs were Rt -9.68 dB, and Lt, -4.52 dB.
We found 20 sequence changes; 10 sequence changes (p.I264V, c.1494+90C>T, c.1494+143A>G, c.1609+89G>A, c.1775+89C>, c.1965-30A>G, p.V714V, c.2170+217C>T, p.V727V, and c.2518+60G>C), which were previously identified [18,26,27], and 10 were novel (p.D179D, p.Q270Q, p.M283R, c.898+63C>G, c.1074+20C>T, p.G459G, c.1884+26C>G, p.S664L, p.S664S, and p.P744P). Other groups have reported on the distribution of WDR36 sequence variants in a cohort of patients with HTG in the United States , and they found 32 WDR36 sequence variants including six novel nonsynonymous SNPs, four synonymous SNPs, and nine intronic changes. Interestingly, 10 novel sequence changes that we found were not found in the other studies. One nonsynonymous SNP, p.I264V, was a common sequence variant and was found evenly distributed between HTG probands and control subjects. Our data demonstrated substantial differences and ethnic variations in the frequencies of SNPs in WDR36 between Caucasian and Japanese populations.
p.M283R is a nonsynonymous amino acid change and was found in patients and controls (Table 1). The incidence of the p.M283R was higher in the controls, but more statistical analyses will be required to determine that p.M283R would be protective against glaucoma.
Among our sequence variants, p.I264V and c.1965-30A>G had a significantly higher frequency in patients with HTG than in control subjects (Table 2). p.I264V is conserved in four species and is located in the second G-beta WD40 repeat and the Cytochrome cd1-nitrite reductase-like, COOH-terminal haem d1 (cyt cd1) domain. Thus, p.I264V might be an important part of the WDR36 domain. Although c.1965-30A>G is an intronic alteration, it has the possibility of altering the expression of the gene or affect the RNA stability or protein function. Its mRNA level and RNA secondary structure are still to be determined [28,29]. p.I264V and c.1965-30A>G could be glaucoma-associated (risk-associated) or dominant susceptibility alleles.
Among the sequence changes, p.S664L has a nonsynonymous amino acid change and is also likely to be disease-causing. It accounted for 0.7% (1/136) of the sporadic Japanese patients with HTG in this study. p.S664L in exon 17 is located in the eighth G-beta WD40 repeat and also maps to the Cytochrome cd1-nitrite reductase-like, COOH-terminal haem d1 (cyt cd1) domain (Figure 1). So it may have a dominant-negative effect or cause haploinsufficiency. All of the sequence changes, their protein level, and their functions should be assessed in more detail before it can be concluded that they are glaucoma-causing alterations. We could not recruit the family members of mutation carrying probands, but from the medical history, there was no affected member in their families.
p.I264V and c.1965-30A>G were associated with HTG, and haplotype analysis showed that the second major G-T-G-G haplotype represents individuals statistically susceptible to HTG. However, the haplotype statistically appears to reduce the susceptibility to NTG. It remains to be determined whether WDR36 is associated with the pathogenesis of NTG. Interestingly, the homozygote genotypes for the p.I264V change and the c.1965-30A>G change have modest significance in the HTG group. We showed one of the phenotypes (mean deviation of Humphrey Field Analyzer of the patient harboring p.I264V, c.1965-30A>G) of the individuals carrying the homozygote, heterozygote, and the homozygote not-at-risk genotype (Table 4). There was no difference between heterozygote and the homozygote not-at-risk genotype, but the individuals carrying the homozygote had worse visual field scores; mean deviation (MD; p<0.001). So, it might be associated with the phenotype of the HTG.
To gain insight into what role WDR36 plays in the development of glaucoma, it is necessary to study its regulation by interleukin 2 (IL2) and its relationship with T-cell activation. WDR36 is expressed in lens, iris, sclera, ciliary muscles, ciliary body, trabecular meshwork, retina, and optic nerve. It still has not been determined whether the expression of WDR36 is involved in the mechanisms regulating aqueous humor outflow or whether it is involved in the neurodegeneration of the ganglion cell and optic nerve. Skarie JM et al. showed that loss of WDR36 function in the zebrafish resulted in an ocular phenotype during development. Histological analysis revealed a decrease in the size of the retinal marginal zone and a thickened lens epithelium in analysis of WDR36 morphants . Therefore, the missense variants in WDR36 might be related with the development of the ganglion cell, optic nerve, and probably normal outflow pathway.
To date, three genes have been identified as causing POAG: MYOC, OPTN, and WDR36. WDR36 was originally reported in 65% of the HTG subjects with this mutation, and 35% of the subjects with this mutation had NTG, but there was a relatively weak statistical association for this gene . The magnitude of its role and association with glaucoma remains controversial, and the main SNP (p.D658G), which did segregate with the original families linked to the 5q region, was later identified as a neutral variant in an Australian population . A large family linked to this region has not been found to have a coding mutation . Furthermore, the findings in two other studies indicated that WDR36 variants may only be rare causes of normal tension glaucoma in a German population  or have no causal association with POAG .
Recent studies have revealed that patients with ocular hypertension had thicker than normal central corneas [7,22,31], and those with normal tension glaucoma had thinner than normal central corneas . Thus, an accurate measurement of the central corneal thickness (CCT) is important not only for patient care but also for clinical studies by using a more reliable classification of subjects . In a Japanese population, the epidemiological study of glaucoma showed no significant difference in CCT among OAG patients with IOPs >22 mmHg (523±35 μm), OAG with IOP <22 mmHg (518±29 μm), and subjects without glaucoma (520±32 m) . Moreover, the IOP measured with the Goldmann applanation tonometer was positively correlated with the CCT and the corrected IOP=IOP reading-0.012 (CCT (μm)-520) . This formula indicates that a 1 mmHg change in IOP would need a 83.3 μm change in the CCT in the Japanese population. In our subjects with borderline IOP around 21 mmHg (n=5) to 22 mmHg (n=8), their diagnosis was not altered by correcting for the CCT. The adjustment of the IOP for the CCT (1 mmHg/83.3 μm CCT) would be small in our study, and the CCT would most likely not significantly influence our statistical analysis of association in this study.
In conclusion, we have identified WDR36 SNPs as a genetic susceptibility allele for HTG in the Japanese populations. However, these results suggest that WDR36 is not a major contributing factor to POAG in the Japanese population, and additional populations should be carefully assessed for WDR36 variant. Further investigations on the structure and function of the WDR36 protein would be helpful in understanding the pathogenesis of POAG and whether the WDR36 protein might have a role in either glaucoma susceptibility or protection.
AcknowledgementsThis study was supported in part by a Grant-In-Aid for Scientific Research from the Ministry of Education, Science, and Culture of the Japanese Government (NF; C-18591905), Tokyo, Japan. This material was presented in part at the Association for Research in Vision and Ophthalmology annual meetings in 2006 in Fort Lauderdale, Florida. The authors thank Dr. Makoto Tamai for encouraging us to undertake the investigation. We thank Dr. D.I. Hamasaki for editing the manuscript.
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