|Molecular Vision 2004;
Received 28 July 2004 | Accepted 9 November 2004 | Published 9 November 2004
Absence of myocilin and optineurin mutations in a large Philippine family with juvenile onset primary open angle glaucoma
Dan Yi Wang,1
Bao Jian Fan,1 Oscar Canlas,2
Pancy O. S. Tam,1 Robert Ritch,3,4 Dennis S. C. Lam,1
Dorothy S. P. Fan,1 Chi Pui
(The first two authors contributed equally to this publication)
1Department of Ophthalmology & Visual Sciences, The Chinese University of Hong Kong, Hong Kong, China; 2Jose B. Lingad Memorial Regional Hospital, San Fernando, Philippines; 3Department of Ophthalmology, New York Eye and Ear Infirmary, New York, NY; 4Department of Ophthalmology, New York Medical College, Valhalla, NY
Correspondence to: Professor Chi Pui Pang, Department of Ophthalmology & Visual Sciences, The Chinese University of Hong Kong, Hong Kong Eye Hospital, 147K Argyle Street, Kowloon, Hong Kong, China; Phone: +852 27623169; FAX: +852 27159490; email: firstname.lastname@example.org
Purpose: To analyze the role of the two primary open angle glaucoma (POAG) genes, myocilin (MYOC) and optineurin (OPTN), in a large Philippine family segregating autosomal dominant juvenile onset open angle glaucoma (JOAG).
Methods: The coding sequences of the MYOC and OPTN genes were screened in 27 family members by polymerase chain reaction and direct sequencing. The specific MYOC promoter polymorphism (MYOC.mtl) was identified by restriction endonuclease assay. All of the ABI MD-10 microsatellite markers on chromosomes 1, 2, 3, 7, 8, and 10, which harbor the six known POAG loci, were analyzed for linkage with POAG.
Results: No mutation was identified in this large kindred. Instead, three polymorphisms (-80G->A, -1000G->C, R76K) in MYOC and four polymorphisms (T34T, M98K, R545Q, IVS7+24G->A) in OPTN were found. All markers flanking the six known POAG loci gave LOD scores not more than 1.1. Non-parametric linkage analysis for all these markers resulted in p values more than 0.05.
Conclusions: Both mutation testing and linkage analysis provide strong evidence against MYOC and OPTN being the causative gene in this large family. It indicates that unidentified genes will underlie the occurrence of glaucoma in this family.
Glaucoma consists of a heterogeneous group of progressive neurodegenerative disorders of which together account for one of the leading causes of blindness in virtually every country . Progressive loss of vision can lead to blindness. Primary open angle glaucoma (POAG) is the most common form of glaucoma. There are both sporadic and familial POAG, the latter shows strong evidence of complex inheritance, with variable severity and phenotypic expression. POAG can be arbitrarily subdivided into two different categories depending on the age of disease onset; juvenile onset open angle glaucoma (JOAG) and adult onset open angle glaucoma . In affected families of both groups of POAG, genetic linkage analysis clearly suggests autosomal dominant inheritance with incomplete penetrance . It has also been suggested that adult onset POAG is inherited as a non-Mendelian trait, whereas JOAG exhibits autosomal dominant inheritance [4,5].
To date, six chromosomal loci; 1q23 (GLC1A), 2cen-q13 (GLC1B), 3q21-q24 (GLC1C), 7q35-q36 (GLC1F), 8q23 (GLC1D), and 10p15-p14 (GLC1E) have been identified for POAG [5-12]. There are two known POAG genes, MYOC in GLC1A and OPTN in GLC1E. When mutated, MYOC causes severe open angle glaucoma, mostly in its juvenile onset form and less commonly the adult onset form. More than 70 MYOC mutations and a few benign polymorphisms have been detected in different population groups [3,13-15]. Most mutations cause an early onset, severe glaucoma, whereas others account for a less typical late onset or even normal tension glaucoma (NTG) [15-17]. Although the exact mechanism is unknown, the MYOC gene has been implicated in causing obstruction of aqueous outflow through the trabecular meshwork, resulting in increased intraocular pressure (IOP) . Defects in the OPTN gene are responsible for moderate to mild forms of late onset glaucoma. It has been suggested that OPTN is specific for NTG . So far, 5 mutations and a few polymorphisms have been reported [12,18]. Ethnic specific OPTN mutation patterns may exist. OPTN mutations were found in 12% of sporadic Caucasian POAG patients . While we identified OPTN mutations in 1.6% of sporadic Chinese POAG patients , no disease causing mutations in OPTN was detected among 148 NTG and 165 POAG patients in the Japanese population . The wild type OPTN protein, operating through the TNF-α pathway, is speculated to play a neuroprotective role in the eye and optic nerve. But when defective, it produces visual loss and optic neuropathy as typically seen in NTG and high tension glaucoma [3,18,20].
Two studies have investigated the role of MYOC and OPTN simultaneously, one on Finnish families  and the other on Barbados glaucoma families of African descent . Except for a few polymorphisms in MYOC and OPTN, no mutation was identified in these families. In this study, we evaluated the role of the MYOC and OPTN genes in a large Philippine family.
One five generation kindred segregating JOAG was recruited from the Philippines (Figure 1). We determined the clinical characteristics of 95 family members. Among them 27 subjects were given a complete ocular examination, and peripheral venous blood was collected and stored at -20 °C for less than 3 months prior to DNA extraction. The clinical status of the other individuals was traced in medical records. JOAG was observed in 22 out of 95 individuals, and 11 of them were male.
Affected individuals were typically diagnosed before the age of 35 years. JOAG was diagnosed as meeting all of the following criteria; (1) exclusion of secondary causes (e.g., trauma, uveitis, or steroid induced glaucoma), (2) anterior chamber angle open (grade III or IV gonioscopy), (3) elevated IOP greater than or equal to 22 mm Hg, (4) characteristic optic disc changes (e.g., vertical cup to disc ratio greater than or equal to 0.7, disk hemorrhage, or thin or notched neuroretinal rim), and (5) characteristic visual field changes with reference to Anderson's criteria for minimal abnormality in glaucoma . Visual acuity was determined using a Snellen eye chart. IOP and visual field were measured by applanation tonometry and Humphrey perimetry with the Glaucoma Hemifield Test, respectively. These criteria also covered glaucoma diagnosis in hospital records or other ophthalmologists' records.
The study was approved by the Ethics Committee for Human Research, the Chinese University of Hong Kong, and followed the tenets of the Declaration of Helsinki. All participants were given an informed consent after explanation of the nature and possible consequences of the study.
Genomic DNA was extracted from 200 μl of blood using a commercial kit (Qiamp Blood Kit; Qiagen, Hilden, Germany). The coding exons and adjacent sequences of MYOC and OPTN were screened for sequence alterations by polymerase chain reaction, as previously reported [14,18], followed by direct sequencing using an ABI 377XL automated DNA sequencer (Applied Biosystems, Foster City, CA). Sequence data were aligned using sequence Navigator analysis software, version 1.0.1 (Applied Biosystems) and compared to the published MYOC (AB006686) and OPTN (AF420371) sequences. For the MYOC.mtl polymorphism, subjects were screened by PCR, followed by restriction endonuclease assay. The PCR products were digested with the restriction enzyme AlwN1 at 37 °C for 4 h prior to polyacrylamide gel electrophoresis.
Statistical analyses were carried out using SAS statistical software version 8.2 (SAS Institute, Cary, NC). Significance of the difference in distribution of each sequence alteration between affected and unaffected subjects was determined by χ2 tests or using Fisher's exact tests.
Microsatellite markers of ABI PRISM Linkage Mapping Set MD-10 (Applied Biosystems) on chromosomes 1, 2, 3, 7, 8, and 10 were genotyped on 27 participants. The Genescan and Genotyper software packages (Applied Biosystem) were used to call genotypes. The GenoPedigree and GenBase software packages (Applied Biosystem) were used to draw pedigree and to export data for linkage analysis. Prior to performance of linkage analysis, we eliminated of all Mendelian inconsistencies in the pedigree data using the PedCheck program . The JOAG gene frequency was set as 0.0001. An autosomal dominant mode of inheritance was used with one liability class with penetrance values 0, 1, 1, respectively. Two point analyses were performed with the MLINK and ILINK programs from the FASTLINK version 4.1P software package . Nonparametric linkage analyses were carried out with Simwalk2 version 2.83 .
Segregation analysis showed autosomal dominant inheritance in this family. We screened a total of 27 subjects (9 with JOAG) for sequence alterations in the coding regions of MYOC and OPTN. No disease causing mutations were identified in MYOC in this family. Instead, three neutral polymorphisms, -1000C->G, -83G->A, and R76K, were found (Figure 1). The most common polymorphism was R76K, detected in 33% (9/27) of subjects. No significant difference was found in all polymorphisms between affected and unaffected individuals (p>0.05; Table 1).
For OPTN also, no mutation was identified. However, 4 known polymorphisms, T34T, M98K, R545Q, and IVS7+24G->A were found (Figure 1). The most common polymorphism was T34T identified in 40% (11/27) of study subjects. There was also no significant difference in all polymorphisms between affected and unaffected individuals (p>0.05; Table 1). Linkage analysis of markers flanking all six POAG loci showed no linkage with this family. For each of these six chromosomes, the highest two point LOD scores were detected in D1S484 (Zmax=0.89 at θ=0.0), D2S2211 (Zmax=1.07 at θ=0.05), D3S1278 (Zmax=0.74 at θ=0.0), D8S258 (Zmax=0.53 at θ=0.20), D10S548 (Zmax=0.68 at θ=0.0), and D7S517 (Zmax=1.06 at θ=0.0), respectively (Figure 2). Non-parametric linkage analysis resulted in p values more than 0.05 for all markers on these chromosomes.
In this study on a large five generation family, which segregates JOAG in an autosomal dominant fashion, no disease causing mutations were identified in MYOC and OPTN. Only three known MYOC polymorphisms (-1000C->G, -83G->A, and R76K) and four known OPTN polymorphisms (T34T, M98K, R545Q, and IVS7+24G->A) were found. These sequence alterations were not classified as causative mutations because of similar frequencies in JOAG patients and the unaffected family members.
In the MYOC gene, the most common polymorphism R76K was not associated with glaucoma in this family. R76K has no critical effect. It is located outside the olfactomedin-like region and the leucine zipper region (codons 117-166) which has been implicated as another potential site for MYOC dimerization and oligomerization . However, R76K and -83G->A are in linkage disequilibrium in Chinese, Japanese, and Indian populations [3,14,15]. In contrast, evidence for linkage disequilibrium is not obvious in Caucasians and African-Americans [13,16]. In the present study of Philippine subjects, the linkage disequilibrium of these two polymorphisms is obvious, consistent with other populations in Asia.
Polymorphism -1000C->G in the MYOC gene, also designated as MYOC.mt1, did not segregate with the disease phenotype in the present study. In one report, it was associated with increased IOP and greater visual field damage, especially in women . Time to event analysis showed that MYOC.mt1 accelerated deterioration of both optic disc and visual field . However, the association of this specific polymorphism with the severity of POAG is still controversial. One reported study showed no significant difference of the distribution of MYOC.mtl in any measure of disease severity from 393 POAG patients . Our recent study on the MYOC promoter in unrelated individuals also indicated that MYOC.mt1 is not associated with the risk and severity of POAG .
For the OPTN gene, the most common polymorphism was T34T (c.412G->A). This does not alter the amino acid sequence and has been reported as a synonymous codon change and neutral polymorphism [12,18]. We also found no segregation of M98K and R545Q with JOAG in this family, although they had been reported as mutation or risk associated genetic factor for glaucoma [12,31]. Since M98K is located within a putative bZIP domain and is conserved in macaques, it may represent a risk associated factor or a dominant susceptibility allele. M98K also did not segregate with POAG in a study on Caucasian families , but it could be a common polymorphism in Chinese and Japanese [18,20]. R545Q is not part of a known protein domain, it is situated near the only zinc finger motif within optineurin. This motif is normally seen in transcription factors. The R545Q variant appears to be a neutral polymorphism that is much more prevalent among Asians than Caucasians .
In the present study, MYOC IVS7+24G->A did not show a significant association with JOAG. In our previous report, the mutation was associated with an increased cup to disc ratio and was potentially related to disruption of optic nerve . However, before it can be concluded as a glaucoma causing mutation, it still has to be affirmatively linked with glaucoma through segregation in families, sequence analysis of relevant cDNA regions, assessment of the level of the mature transcript, or expression studies.
To date, the genes that underlie two of the six named POAG loci have been identified [6-11]; MYOC is the gene for GLC1A and OPTN is the gene for GLC1E, whereas genes underlying the other reported loci have not yet been identified. In families with mixed onset POAG containing both JOAG and adult onset open angle glaucoma, the MYOC mutation prevalence is 31% . MYOC mutations have also been identified in 2-4% of unrelated individuals with POAG in various populations [3,6,14,15]. Meanwhile, OPTN mutations had been found in 16.7% of the hereditary form of NTG  and in about 1-2% of sporadic POAG patients [3,18,20]. All these findings show that MYOC and OPTN mutations only account for a minority of POAG.
Apart from the lack of MYOC and OPTN mutations, our linkage analysis also showed no evidence for linkage to any of six known POAG loci. All markers flanking these POAG loci gave LOD scores of not more than 1.1, and p values more than 0.05 were obtained by non-parametric linkage analysis for all these markers. Furthermore, no MYOC and OPTN mutations had been found in studies on Finnish and Barbados POAG families [21,22]. Linkage analysis was also carried out in the Barbados study, in which glaucoma did not show linkage with markers near the MYOC and OPTN regions. Although both our study and the above studies did not exclude a role for MYOC and OPTN in POAG, they did not support a major role for any of them as a cause of POAG. Further studies with familial and sporadic glaucoma patients are needed to detect susceptibility genes for POAG in the future. A genome wide linkage analysis in this family to discover the disease loci for JOAG is in progress.
We are grateful to the family that participated in this study. The work described in this paper was partially supported by a block grant from the Chinese University of Hong Kong and by the Mrs. Annie Wong Eye Foundation, Hong Kong.
1. Quigley HA. Number of people with glaucoma worldwide. Br J Ophthalmol 1996; 80:389-93.
2. Ritch R, Shields MB, Krupin T. Classification and mechanisms of the glaucomas. In: Ritch R, Shields MB, Krupin T, editors. The Glaucomas. Vol. II. Mosby: St. Louis; 1996. p. 717-25.
3. Ray K, Mukhopadhyay A, Acharya M. Recent advances in molecular genetics of glaucoma. Mol Cell Biochem 2003; 253:223-31.
4. Wiggs JL, Allingham RR, Vollrath D, Jones KH, De La Paz M, Kern J, Patterson K, Babb VL, Del Bono EA, Broomer BW, Pericak-Vance MA, Haines JL. Prevalence of mutations in TIGR/Myocilin in patients with adult and juvenile primary open-angle glaucoma. Am J Hum Genet 1998; 63:1549-52.
5. Stone EM, Fingert JH, Alward WL, Nguyen TD, Polansky JR, Sunden SL, Nishimura D, Clark AF, Nystuen A, Nichols BE, Mackey DA, Ritch R, Kalenak JW, Craven ER, Sheffield VC. Identification of a gene that causes primary open angle glaucoma. Science 1997; 275:668-70.
6. Sheffield VC, Stone EM, Alward WL, Drack AV, Johnson AT, Streb LM, Nichols BE. Genetic linkage of familial open angle glaucoma to chromosome 1q21-q31. Nat Genet 1993; 4:47-50.
7. Stoilova D, Child A, Trifan OC, Crick RP, Coakes RL, Sarfarazi M. Localization of a locus (GLC1B) for adult-onset primary open angle glaucoma to the 2cen-q13 region. Genomics 1996; 36:142-50.
8. Wirtz MK, Samples JR, Kramer PL, Rust K, Topinka JR, Yount J, Koler RD, Acott TS. Mapping a gene for adult-onset primary open-angle glaucoma to chromosome 3q. Am J Hum Genet 1997; 60:296-304.
9. Trifan OC, Traboulsi EI, Stoilova D, Alozie I, Nguyen R, Raja S, Sarfarazi M. A third locus (GLC1D) for adult-onset primary open-angle glaucoma maps to the 8q23 region. Am J Ophthalmol 1998; 126:17-28.
10. Wirtz MK, Samples JR, Rust K, Lie J, Nordling L, Schilling K, Acott TS, Kramer PL. GLC1F, a new primary open-angle glaucoma locus, maps to 7q35-q36. Arch Ophthalmol 1999; 117:237-41.
11. Sarfarazi M, Child A, Stoilova D, Brice G, Desai T, Trifan OC, Poinoosawmy D, Crick RP. Localization of the fourth locus (GLC1E) for adult-onset primary open-angle glaucoma to the 10p15-p14 region. Am J Hum Genet 1998; 62:641-52.
12. Rezaie T, Child A, Hitchings R, Brice G, Miller L, Coca-Prados M, Heon E, Krupin T, Ritch R, Kreutzer D, Crick RP, Sarfarazi M. Adult-onset primary open-angle glaucoma caused by mutations in optineurin. Science 2002; 295:1077-9.
13. Fingert JH, Heon E, Liebmann JM, Yamamoto T, Craig JE, Rait J, Kawase K, Hoh ST, Buys YM, Dickinson J, Hockey RR, Williams-Lyn D, Trope G, Kitazawa Y, Ritch R, Mackey DA, Alward WL, Sheffield VC, Stone EM. Analysis of myocilin mutations in 1703 glaucoma patients from five different populations. Hum Mol Genet 1999; 8:899-905.
14. Lam DS, Leung YF, Chua JK, Baum L, Fan DS, Choy KW, Pang CP. Truncations in the TIGR gene in individuals with and without primary open-angle glaucoma. Invest Ophthalmol Vis Sci 2000; 41:1386-91.
15. Mabuchi F, Yamagata Z, Kashiwagi K, Tang S, Iijima H, Tsukahara S. Analysis of myocilin gene mutations in Japanese patients with normal tension glaucoma and primary open-angle glaucoma. Clin Genet 2001; 59:263-8.
16. Alward WL, Fingert JH, Coote MA, Johnson AT, Lerner SF, Junqua D, Durcan FJ, McCartney PJ, Mackey DA, Sheffield VC, Stone EM. Clinical features associated with mutations in the chromosome 1 open-angle glaucoma gene (GLC1A). N Engl J Med 1998; 338:1022-7.
17. Mardin CY, Velten I, Ozbey S, Rautenstrauss B, Michels-Rautenstrauss K. A GLC1A gene Gln368Stop mutation in a patient with normal-tension open-angle glaucoma. J Glaucoma 1999; 8:154-6.
18. Leung YF, Fan BJ, Lam DS, Lee WS, Tam PO, Chua JK, Tham CC, Lai JS, Fan DS, Pang CP. Different optineurin mutation pattern in primary open-angle glaucoma. Invest Ophthalmol Vis Sci 2003; 44:3880-4.
19. Tang S, Toda Y, Kashiwagi K, Mabuchi F, Iijima H, Tsukahara S, Yamagata Z. The association between Japanese primary open-angle glaucoma and normal tension glaucoma patients and the optineurin gene. Hum Genet 2003; 113:276-9.
20. Alward WL, Kwon YH, Kawase K, Craig JE, Hayreh SS, Johnson AT, Khanna CL, Yamamoto T, Mackey DA, Roos BR, Affatigato LM, Sheffield VC, Stone EM. Evaluation of optineurin sequence variations in 1,048 patients with open-angle glaucoma. Am J Ophthalmol 2003; 136:904-10.
21. Forsman E, Lemmela S, Varilo T, Kristo P, Forsius H, Sankila EM, Jarvela I. The role of TIGR and OPTN in Finnish glaucoma families: a clinical and molecular genetic study. Mol Vis 2003; 9:217-22 <http://www.molvis.org/molvis/v9/a32/>.
22. Nemesure B, Jiao X, He Q, Leske MC, Wu SY, Hennis A, Mendell N, Redman J, Garchon HJ, Agarwala R, Schaffer AA, Hejtmancik F, Barbados Family Study Group. A genome-wide scan for primary open-angle glaucoma (POAG): the Barbados Family Study of Open-Angle Glaucoma. Hum Genet 2003; 112:600-9.
23. Anderson DR. Automated static perimetry. St. Louis: Mosby-Year Book; 1992: p 12.
24. O'Connell JR, Weeks DE. PedCheck: a program for identification of genotype incompatibilities in linkage analysis. Am J Hum Genet 1998; 63:259-66.
25. Cottingham RW Jr, Idury RM, Schaffer AA. Faster sequential genetic linkage computations. Am J Hum Genet 1993; 53:252-63.
26. Sobel E, Lange K. Descent graphs in pedigree analysis: applications to haplotyping, location scores, and marker-sharing statistics. Am J Hum Genet 1996; 58:1323-37.
27. Colomb E, Nguyen TD, Bechetoille A, Dascotte JC, Valtot F, Brezin AP, Berkani M, Copin B, Gomez L, Polansky JR, Garchon HJ. Association of a single nucleotide polymorphism in the TIGR/MYOCILIN gene promoter with the severity of primary open-angle glaucoma. Clin Genet 2001; 60:220-5.
28. Polansky JR, Juster RP, Spaeth GL. Association of the myocilin mt.1 promoter variant with the worsening of glaucomatous disease over time. Clin Genet 2003; 64:18-27.
29. Alward WL, Kwon YH, Khanna CL, Johnson AT, Hayreh SS, Zimmerman MB, Narkiewicz J, Andorf JL, Moore PA, Fingert JH, Sheffield VC, Stone EM. Variations in the myocilin gene in patients with open-angle glaucoma. Arch Ophthalmol 2002; 120:1189-97.
30. Fan BJ, Leung YF, Pang CP, Fan DS, Wang DY, Tong WC, Tam PO, Chua JK, Lau TC, Lam DS. Polymorphisms in the myocilin promoter unrelated to the risk and severity of primary open-angle glaucoma. J Glaucoma 2004; 13:377-84.
31. Aung T, Ebenezer ND, Brice G, Child AH, Prescott Q, Lehmann OJ, Hitchings RA, Bhattacharya SS. Prevalence of optineurin sequence variants in adult primary open angle glaucoma: implications for diagnostic testing. J Med Genet 2003; 40:e101.
32. Wiggs JL, Auguste J, Allingham RR, Flor JD, Pericak-Vance MA, Rogers K, LaRocque KR, Graham FL, Broomer B, Del Bono E, Haines JL, Hauser M. Lack of association of mutations in optineurin with disease in patients with adult-onset primary open-angle glaucoma. Arch Ophthalmol 2003; 121:1181-3.
33. Shimizu S, Lichter PR, Johnson AT, Zhou Z, Higashi M, Gottfredsdottir M, Othman M, Moroi SE, Rozsa FW, Schertzer RM, Clarke MS, Schwartz AL, Downs CA, Vollrath D, Richards JE. Age-dependent prevalence of mutations at the GLC1A locus in primary open-angle glaucoma. Am J Ophthalmol 2000; 130:165-77.