|Molecular Vision 2006;
Received 3 April 2006 | Accepted 29 September 2006 | Published 26 October 2006
The association of single nucleotide polymorphisms in the MMP-9 genes with susceptibility to acute primary angle closure glaucoma in Taiwanese patients
I-Jong Wang,1 Ting-Hsuan
Chiang,1 Yung-Feng Shih,1
Shao-Chun Lu,2 Luke Long-Kuang Lin,1 Jui-Wen Shieh,3
Tsing-Hong Wang,1 John R. Samples,4 Por-Tying Hung1,3
1Department of Ophthalmology and 2Department of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan; 3Department of Ophthalmology, Mackay Memorial Hospital, Taipei, Taiwan; 4Casey Eye Institute, Oregon Health & Science University, Portland, OR
Correspondence to: Yung-Feng Shih, M.D., Department of Ophthalmology, National Taiwan University Hospital, 7, Chung-Shan S. Rd., Taipei, Taiwan; Phone: 886-2-23123456; FAX: 886-2-23412875; email: email@example.com
Purpose: To study the relationships between single nucleotide polymorphisms (SNPs) of extracellular matrix, matrix metalloproteases (MMPs), tissue inhibitors of MMPs, and other glaucoma-associated genes and acute primary angle closure glaucoma (PACG).
Methods: We extracted DNA samples from 78 adult patients with acute PACG and 86 control subjects to study the relationships between these specific genes and acute PACG. Genotyping was performed for 35 genes by the GenomeLab SNPstream genotyping system after PCR amplification of chromosomal DNA. The association between these genetic polymorphisms and risk of primary PACG was estimated by χ2 and logistic regression.
Results: The genotyping success rate was 99%. Genotyping for the MMP9 site (rs2664538) was significantly different between the two groups (p=0.000001) and the odds ratio was 2.586 (95% CI: 1.715-3.898, p<0.00001). However, there were no associations of SNPs to other genes in patients with acute PACG.
Conclusions: Our results reveal that SNP rs2664538, which is located at the MMP9 gene, is likely to be associated with acute PACG.
Glaucoma is a group of diseases causing optic neuropathy and it is characterized by optic disc cupping and loss of visual field. Glaucoma is the second leading cause of blindness worldwide, estimated to affect about 70 million people, with 6.7 million of these being bilaterally blind . Categorized according to the anatomy of the anterior chamber angle, there are two main forms of glaucoma: primary open-angle (POAG) and primary angle-closure (PACG) glaucoma. Acute PACG is more frequent in middle aged women and is a major form of glaucoma in Asians [2,3], compared with POAG, which is the predominant disease among whites and Africans [4,5]. Angle closure is responsible for most bilateral glaucoma-caused blindness in Singapore, China, and India, and it has been estimated that PACG blinds more people than POAG worldwide [6-8].
Angle closure may be acute, subacute, or chronic . An episode of acute angle closure with a painful red eye and a fixed dilated pupil is a defining event. It is well known that PACG is associated with certain biometric ocular features such as shallow anterior chamber [10,11], increased thickness of the lens , and short axial length . These patients usually have a hyperopic refractive error . The mechanisms involved in the pathophysiology and development of PACG are complicated and involve the anatomy of the angle, and the spatial and anatomical relationships between the lens and the anatomy of the angle, the iris, and the lens . Most mechanisms for PACG impute increasing lens thickness during aging in a relatively small eye and a shallow anterior chamber. Such a hyperopic eye is in contrast to high myopia, in which a thin sclera, particularly at the posterior pole of the eye, is an important feature . Acute PACG has a relatively short axial length. Human sclera undergoes active remodeling during the development of myopia. Biochemical assays from highly myopic eyes show markedly reduced amounts of biochemical markers for collagen and glycosaminoglycans, when compared with the similar sclera of emmetropic eyes . This remodeling process occurs in concert with an increase in the production and enhanced activation of collagen degrading enzymes, particularly matrix metalloproteases (MMPs) . The net effect of these changes is a loss of scleral tissue at the posterior pole of the eye. Therefore, it is of interest to study the scleral remodeling and development in acute PACG. In both the small hyperopic eye and long myopic eye, extracellular matrix remodeling is likely an important determinant.
POAG is genetically heterogeneteous . To date more than 15 loci and seven glaucoma-causing genes have been identified . Among them, two genes have been identified as harboring mutations causing POAG; MYOC encoding myocilin and OPTN encoding optineurin [21-23]. Since the suggestion that transmission was via a single, dominant gene in 1953, there has been a paucity of research into the genetic basis of PACG . Despite the observation that PACG frequently runs in families, no genetic locus has yet been discovered for PACG. Nanophthalmos, an autosomal dominant disorder, has been known a significant association with PACG and two chromosomal loci have been identified which are specifically linked to nanophthalmos . However, nanophthalmos is very different from primary angle closure from a clinical perspective.
Two recent studies in Canadian reported the presence of MYOC mutations in a few individuals with PACG [25,26]. Of 17 subjects with PACG who were studied, two MYOC mutations were described, one each with Pro481Leu and Gln368STOP . A patient with mixed POAG-PACG via a Gly399Val mutation has also been reported . The importance of this finding remains to be elucidated. These reports provide suggest that some PACG patients may carry similar genes to POAG.
The analysis of complex human diseases has been spurred by the number of genomic sequence variants discovered in the course of sequencing the human genome . Single-nucleotide polymorphisms (SNPs), common variations among the DNA of individuals, are being assembled into large SNP databases that promise to elucidate the genetic basis of many diseases and drug responses . For example, Aung et al.  found that eight sequence variants in the myocilin gene make it a probable candidate gene for PACG. Therefore, it is of interest to examine differences of SNPs in the extracellular matrix (ECM), MMPs, tissue inhibitors of MMPs (TIMPs), and other glaucoma-associated gene(s) which may regulate the growth of scleral coats in acute PACG patients.
We report a case-controlled study in which an attempt was made to identify these SNPs and their association with acute PACG. We found that SNP rs17576 of the MMP9 gene located at chromosome 20q11.2-q13 by using a multiplex PCR-oligonucleotide extension assay with a SNP genotyping platform, is strongly associated with acute PACG.
Unrelated Taiwanese subjects with acute PACG and unrelated control subjects with normal eyes were recruited at the National Taiwan University and Mackay Memorial Hospitals. All participants had similar social backgrounds and were from the local ethnic Han Chinese population, with no ethnic subdivision. Informed consent was obtained from all subjects. This project had the approval of the Institutional Review Board (IRB)/Ethics Committees of both hospitals and was carried out in accordance with the World Medical Association's Declaration of Helsinki.
Patients were eligible for study participation if their acute PACG met the following diagnostic criteria: (1) the presence of at least two symptoms: eye pain, headache, blurred vision, and vomiting; (2) the presence of the following signs: conjunctival congestion, a mid-dilated unreactive pupil, and corneal edema; (3) a greater than or equal 270 ° of closure of the chamber angle found on gonioscopic examination; and, (4) IOP>40 mm Hg using Goldmann applanation tonometry measured with the Perkins hand-held device. All of these criteria were in compliance with the ISGEO classification of angle closure glaucoma by Foster et al. . In contrast, none of the control subjects had any of the aforementioned symptoms and signs. Every participant received a complete ocular examination which included retinoscopy, slit-lamp evaluation of the anterior segment, measurement of intraocular pressure, axial-length measurements (Sonomed Ultrasound A-1500, Warszawa, Poland), keratometry measurements, and a fundus examination, with special notation as to the health and degree of cupping of the optic nerve head.
DNA extraction, amplification, and mutation screening
Total genomic DNA was extracted from 10 to 15 ml of venous blood from each participant after informed consent was obtained. DNA was purified from lymphocyte pellets according to standard procedures using a Puregene kit (Gentra Systems, Minneapolis, MN) or the phenol-chloroform extraction method . Sixty-seven SNPs in 35 different genes were identified using GenBank along with a database of Japanese single nucleotide polymorphisms (JSNP; Table 1). All SNPs are located in the exons of these genes. Multiplex PCR and SNP analyses were performed with the GenomeLab SNPstream genotyping platform (Beckman Coulter Inc. Fullerton, CA) and its accompanying SNPstream software suite. The primers for the multiplex PCR and single base extension primers were optimally designed by Web-based software provided at Beckman Coulter Inc. PCR primers were designed to amplify a short stretch of DNA (90 bp) that encompasses the SNP of interest (Table 1). The tagged extension primers (Table 1) used to identify the SNPs were designed in two parts. The 5' portion of the probe is complementary to one of 12 unique single stranded DNA oligonucleotides that are microarrayed at a specific location within each well of a 384-well microplate. The 3' portion of the probe is complementary and precisely adjacent to the SNP, which enables detection of the presence of either or both nucleotides of the SNP through the incorporation of a fluorescent-labeled terminating nucleotide. Twelve-plex PCR reactions were performed in 384-well plates (MJS BioLynx, Brockville, ON) in a 5 μl volume using 6 ng of DNA, 75 μM dNTPs, 0.5 U of AmpliTaq Gold (Perkin-Elmer, Wellesley, MA), and the 24 PCR primers at a concentration of 50 nM each in 1 X PCR buffer. Thermal cycling was performed in GeneAmp PCR system 9700 thermal cyclers (Applied Biosystems, Foster City, CA) using the following program: initial denaturation at 95 °C for 5 min followed by 40 cycles of 95 °C for 30 s, 50-55 °C for 55 s, 72 °C for 30 s. After the last cycle, the reaction was held at 72 °C for 7 min. Following PCR, plates were centrifuged briefly and 3 μl of a mixture containing 0.67 U Exonuclease I (Amersham Pharmacia, Buckinghamshire, UK) and 0.33 U shrimp alkaline phosphatase (Amersham Pharmacia) were added to each well. The plates were sealed and incubated for 30 min at 37 °C and at 95 °C for 10 min. The tagged extension primers were extended with single TAMRA- or bodipy-fluorescein-labeled nucleotide terminator reactions and then spatially resolved by hybridization to the complementary oligonucleotides arrayed on the 384-well microplates (SNPware Tag array). The Tag array plates were imaged with a two-laser, two-color charged couple device-based imager (GenomeLab SNPstream array imager). The 12 individual SNPs were identified by their position and fluorescent color in each well according to the position of the tagged oligonucleotides. Sample genotype data were generated on the basis of the relative fluorescent intensities for each SNP and computer processed for graphical review.
Direct DNA Sequencing
The forward primer (TTC TCC CCC TTT CCC ACA) and the reverse primer (AGA TGA ATG GAA ACT GGC AG) were designed to amplify the sequence of the identified SNP which includes rs2664538. PCR was performed on 50 ng of genomic DNA with a GeneAmp PCR system 9700 thermocycler (Applied Biosystems). The Touchdown PCR cycling condition was performed as follows: initial preheating step for 11 min at 95 °C to achieve a hot start, followed by 12 cycles of 94 °C for 20 s, 68 °C for 20 s, and 72 °C for 45 s, the annealing temperature decreased 0.5 °C per cycle until 62 °C; followed by 30 cycles of 94 °C for 20 s, 62 °C for 20 s, and 72 °C for 45 s; with a final elongation at 72 °C for 10 min. Amplified PCR products were separated by agarose gel electrophoresis and visualized by staining with ethidium bromide. They were then purified in purification columns (QIAquick; Qiagen, Valencia, CA) and sequenced using dye terminator chemistry (BigDye Terminator ver. 3.1 on a model 3100 Genetic Analyzer; Applied Biosystems, Foster City, CA). Sequences were trimmed for quality and aligned using BioEdit software (version 5.0.6). Normal and affected individual DNA sequences were aligned to the known reference genomic sequence (GenBank NT_011362), available via the National Center for Biotechnology Information (NCBI) database and compared for sequence variation.
To examine the association of the SNP, we used the χ2 test to compare the alteration of genotypes between patients and controls. The χ2 test evaluates the difference between the observed genotype frequency and the expected frequency under the null hypothesis of no association. However, when the expected frequency is small, the Fisher exact test is conducted instead. Bonferroni correction was applied for the multiple tests. Next, all detected SNPs were assessed for Hardy-Weinberg disequilibrium using the χ2 test . We conducted logistic regression analysis with a stepwise approach . The dependent variable was the disease status (patient, 1; control subject, 0), and the independent variables were values of SNP (homozygote, 2; heterozygote, 1; wild type, 0). The covariates of interactions between SNP were also included in the model. The final optimal model contains the statistically significant variables (SNP) that were selected by the stepwise procedure. These statistical analyses were performed using SPSS software (ver. 10.1; SPSS Science, Chicago, IL).
In total, 78 patients (20 males and 58 females) with PACG and 86 healthy subjects (30 males and 56 frmales) were enrolled in this study. The mean age in the PACG patients and controls were 68.01±8.67 and 64.83±7.12 years, respectively. The mean axial lengths of the eyes were 23.37±0.75 mm (range from 21.57 mm to 25.21 mm) for right eyes and 23.32±0.76 mm (range from 21.35 mm to 24.63 mm) for left eyes of the control group. The mean axial length was 22.57±1.07 mm (range from 20.58 mm to 26.03 mm) for the eye which underwent PACG sugery and 22.52±1.02 mm (range from 20.63 mm to 25.92 mm) for the fellow eye in PACG subjects (Table 2). Gender difference in both groups was tested with the χ2 test, and the p-value was 0.111. Age difference in both groups was tested with the t-test, and the p-value was 0.231. Axial length difference between the attacked eye and their fellow eye was tested with the paired t-test, and the p-value was 0.692. Axial length difference of eyes with PACG in acute PACG groups and mean axial lengths of control subjects was tested with the t-test, and the p-value was <0.0001. The axial lengths AA genotype (n=44, 22.49±1.01 mm) were less than those of GG (n=17, 22.65±1.33 mm), GA (n=17, 22.63±1.24 mm), however, there was no statistical difference between the groups (one-way ANOVA, p=0.084). Not only does this indicate that the groups are homogeneous but it also clearly demonstrates that there are significant differences in axial length between cases and controls.
The genotyping success rate was 99%. There were no associations of these genotypes with acute PACG except for the MMP9 gene, rs2664538 (Table 3). This was a transition of A to G at nucleotide 855 in exon 7 and was associated with the substitution of glutamine for arginine at codon 279. This was confirmed by direct DNA sequencing. The genotyping for the MMP9 (rs2664538) was significantly different between the two groups (p=0.000001), and the odds ratio was 6.20 (95% CI: 2.52-15.223, p<0.00001). After Bonferroni correction by comparing each p value with the adjusted significance level, α/67=0.05/67=0.00075, it is still significant. The distributions of MMP9 genotypes among PACG patients (Arg/Arg, 75%; Arg/Gln, 19%; Gln/Gln, 6%) were different from those of the control subjects (Arg/Arg, 5%; Arg/Gln, 28%; Gln/Gln, 67%). All detected SNPs were assessed for Hardy-Weinberg disequilibrium using the χ2 test (Table 4).
There was one homozygous genotype found in 46 of 67 SNPs. These SNPs were reexamined for population diversity with reference to the GenBank web site, especially for Asian population, such as HapMap-JPT and HapMap-HCB. One homozygous genotype in all ethnic populations was found in 14 SNPs showed an extreme imbalance in the distribution of genotypes and could possibly explain why only one homozygous genotype was found in them: No population diversity data was found for another 26 SNPs. Six SNPs, rs3138289 (genotype C:T=0.011:0.989 in HapMap-HCB, C:T=0.012:0.988 in HapMap-JCP), rs12721427 (genotype G:A=0.989:0.011 in HapMap-HCB, G:A=0.978:0.022 in HapMap-JCP), rs1800440 (genotype G:A=0.011:0.989 in HapMap-HCB, G:A=0:1 in HapMap-JCP), rs363830 (genotype G:A=0.967:0.033 in HapMap-HCB, no available data from HapMap-JCP), rs17883861 (genotype G:A=0.989:0.011 in HapMap-HCB, G:A=1:0 in HapMap-JCP), and rs1042704 (genotype G:A=0.977:0.023 in HapMap-HCB, G:A=1:0 in HapMap-JCP).
The sclera is a typical fibrous connective tissue consisting primarily of collagen. In mammals, collagen accounts for as much as 90% of the scleral dry weight and the vast majority of this collagen (as much as 99%) is type I collagen . However, low levels of other fibrillar collagen subtypes, including type III and V, have been reported in the mammalian sclera. It is possible to attribute specific functions to each of these subtypes . Other reported collagen subtypes of the sclera include types VI , and XII , both of which are considered fibril-associated collagens, and the nonfibril forming collagen types VIII  and XIII . In our study, we could not identify any associations of collagen 1A2 (COL1A2), collagen 2A1 (COL2A1), and fibrillin-1 with acute PACG. These SNPs were selectively chosen from the primer program. They did not cover the whole SNPs of these genes; however, they were located in the exons. In the present studies, we did not find any association of acute PACG with SNPs of these collagen-associated genes. SNPs at rs2229806 (C->T, Val->Ala), rs2857400 (C->T, Gln->Glu), rs6946698 (G->A, Asn->Asp) and rs12721427 (G- A, Ile->Val) are nonsynonymous. Although they will cause substitution of an amino acid, they were not associated with an alteration in protein function. The SNP rs2070739 (C->T, Ser->Gly) will change protein function, but it was not different between the acute PACG group and the control group, which may mean that this SNP in COL2A1 is not associated with scleral changes of eyeball size.. There is only one homozygous genotype for rs2229806, rs2857400, rs6946698, and rs12721427. Yet rs17122498, rs2384472, rs384444, and rs1793948 are synonymous, whereas rs3803182 is located in the 3'-untranslated region of the COL2A1 gene.
Proteoglycans are also a major component of the scleral extracellular matrix (ECM). The mammalian sclera is rich in hyaluronan, a unique, nonsulphated glycosaminoglycan that does not associate with a core protein of its own. Sclera also contains large amounts of dermatan and chondroitin sulphate-based proteoglycans , particularly the small proteoglycans, decorin and biglycan [40,41]. These small proteoglycans play an important role in regulating collagen fibril assembly and interaction . In addition, larger proteoglycans such as aggrecan are also present in the scleral ECM. Aggrecan is likely to be important in the regulation of scleral hydration . Furthermore, a recent study of the sclera in mice deficient in lumican, a small keratan sulphate based proteoglycan in the sclera, demonstrated the importance of this proteoglycan in the formation and organization of collagen fibrils in the sclera . We also could not find any associations of keratocan, decorin, and DSPG3 with acute PACG. The amino acid changes from the SNPs of these three proteoglycan genes are also not linked to the anatomic changes of acute PACG.
The sclera is mediated by a number of protease enzymes, the most extensively studied of these being the MMP family, which currently numbers at least 26 related enzymes . Gelatinase (MMP-2 and MMP-9) and stromelysin (MMP-3) are present in the sclera and are presumed to be involved in scleral remodeling during growth and development, since these enzymes are all known to be involved in the breakdown of collagen [46,47]. At least two of the four natural regulators of MMPs, the TIMPs, are also present in the sclera. This is expected given the close relationship between these enzymes and their inhibitors, with reports of TIMP-1 and TIMP-2 in mammalian species [18,48]. We tested the associations of MMPs-1, 2, 7, 9, 11, 13, 14, 16, and 19, MMPL1 (matrix metalloprotease-like 1), and TIMPs1 and 3 with acute PACG. Only MMP9 (rs2664538) showed a statistical significance for acute PACG. It is a transition of A to G at nucleotide 855 in exon 6 and is associated with the substitution of glutamine for arginine at codon 279.
MMP-9 is a secreted multidomain enzyme that is important for the remodeling of the ECM and the migration of normal and tumor cells. It cleaves denatured collagens (gelatins) and type IV collagen present in basement membranes. Increased expression of MMP-9 has been found in vivo in scleritis tissue and in vitro in cultured human scleral fibroblasts . It also can be found in other organ remodeling, such as in lung . Activation of MMP-9 via neuronal nitric oxide synthase contributes to N-methyl-D-aspartate (NMDA) induced retinal ganglion cell death . MMP-9 is also associated with leaking glaucoma filtering blebs , and normal tension glaucoma . In the current study, we found that MMP-9 is associated with acute PACG. Liu et al.  have found that decreased mRNA transcripts of MMP-2, MMP-9, TIMP-1, and TIMP-2 can be found in the Tenon's capsule in PACG and decreased MMP-2 and TIMP-1 can be found in POAG. They postulated that these alterations are due to the long-term use of antiglaucomatous agents. MMP-2, mainly in proform, is elevated and is the predominant gelatinase in aqueous humor in cataract and POAG [55,56]. This fits well with the apparent nature of gelatinases, of which MMP-2 is mainly constitutively expressed, whereas MMP-9 is highly inducible. Trabecular meshwork (TM) in POAG contains decreased amounts of hyaluronan and its binding receptor CD44H [57,58], which may serve as an anchoring site for MMP-9 . Therefore, MMP-9 is not upregulated in POAG.
The ancestral allele of rs2664538 is not available from GeneBank, but it is a nonconservative amino acid substitutions occurring at residues that are identical in human and mouse. It is located in the second internal repeat of fibronectin Type II domain (FN2; total 3 FN2 internal repeat). FN2 is one of three types of internal repeats that combine to form larger domains within fibronectin. Fibronectin, a plasma protein that binds cell surfaces and various compounds including collagen, fibrin, heparin, DNA, and actin, usually exists as a dimer in plasma and as an insoluble multimer in extracellular matrices. The FN2 of MMP-9 was found to have an affinity for denatured collagen, suggesting that these domains may be responsible for the collagen-affinity of MMP-9 . Therefore, the SNP at this codon may affect the enzymatic activity of MMP-9 through the affinity to its substrate in the scleral coat and thus affect the eyeball size. We cannot exclude the possibility that the identified rs2664538 SNP is not directly involved in the disease, since there are no axial length differences between these genotypes and there could be a linkage disequilibrium with another causative locus.
Latent MMP-2 and upregulated TIMP-2 are found in the aqueous humor of POAG. Presently, we have used SNPs to study the association of MMP-9 with acute PACG. We postulate that the activity of MMP-9 may be downregulated in acute PACG and the relatively short axial length in acute PACG is possibly due to an alteration in the activity of MMP-9 in the remodeling of ECM during ocular growth and development. The possible mechanism is not known at the present time because the substrates of MMP-9 are gelatin and type IV collagen, which are not the predominant collagen fibrils in the sclera. Therefore, we need to perform additional studies in acute PACG eyes to confirm the role of MMP-9.
We also screened subjects for CYP1B1 (which may be associated with primary congenital glaucoma), MYOC (which may be associated with juvenile POAG), and OPTN (which may be associated with POAG/NTG) [21,61,62]. However, there was no association of those of SNPs in these genes with acute PACG. Aung et al.  reported that a combination of two polymorphic markers in the gene known to cause dominant optic atrophy (OPA1) are associated with NTG. We did not find any association of OPA1 with acute PACG.
For congenital glaucoma, we also screened several SNPs for known genes. Mutations in the human PAX3 gene have been described in patients with Waardenburg syndrome , displaying abnormalities in eye and nose formation, deafness, and pigmentation disturbances. Myotubularin-related 13 genes (MTMR13), a member of the MTMR protein family includes proteins with a phosphoinositide phosphatase activity, as well as proteins in which key catalytic residues are missing, and thus are called "pseudophosphatases." Mutations in MTMR13 also cause developmental anomalies of both the peripheral nerves and the trabecular meshwork, which permits the outflow of the aqueous humor . β2-Aadrenergic receptor polymorphism has been shown to be susceptible to primary congenital and primary open angle glaucoma . Four soluble human 3α-hydroxysteroid dehydrogenase (HSD) isoforms exist that are aldo-keto reductase (AKR) superfamily members. They share 86% sequence identity and correspond to AKR1C1 (20α[3α]-HSD); AKR1C2 (type 3 3α-HSD and bile-acid binding protein); AKR1C3 (type 2 3α-HSD and type 5 17β-HSD); and AKR1C4 (type 1 3α-HSD) . Agapova, et al.  found that glaucomatous optic nerve head astrocytes expressed higher levels of AKR1C1, AKR1C2, and AKR1C3 mRNA than normal astrocytes, with significant differential increase of AKR1C2 expression, and exhibited higher enzymatic activity forming 3α-androstanediol, a well-recognized neurosteroid compound. Normal astrocytes exposed to elevated hydrostatic pressure selectively increased AKR1C2 expression. The authors suggested that increased expression of 3alpha-HSDs in glaucomatous optic nerve head astrocytes might be a possible role for neurosteroids in the pathophysiology of glaucoma. However, our present observations do not confirm the relationship of our SNPs with acute PACG.
In conclusion, our results demonstrate that SNP (rs2664538) may be associated with acute PACG. Elucidation of the exact mechanism will be the subject of further study.
This study was supported by NRPGM, Microarray and SNP Core Facility for Genomic Medicine, NSC 93-2811-B-002-018, 94-3112-B-002-031-Y, 95-3112-B-002-022.
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