Gln368STOP Myocilin Mutation in Families with Late-Onset Primary Open-Angle

R. Rand Allingbam,1 Janey L Wiggs,2 Monica A. De La Paz,1 Doug Vollrath,5 Deidre A. Tallett,1 Bob Broomer,1 Katherine H. Jones,1 Elizabeth A. Del Bono,2 Jeremy Kern,2 Kara Patterson,2 Jonathan L Haines,4 and Margaret A. Pericak-Vance1

PURPOSE. TO examine families ascertained for late-onset primary open-angle glaucoma (POAG) to determine mutations in the coding for myocilin. METHODS. The diagnosis of late-onset POAG was denned as age at diagnosis more than 35 years, intraocular pressure (IOP) 22 mm Hg or more in both eyes or 19 mm Hg or more while the patient was taking two glaucoma medications, glaucomatous optic neuropathy in both eyes, and visual field loss consistent with optic nerve damage in at least one eye of the proband. Two of three criteria were required in other family members. DNA from all families was screened for polymorphisms in myocilin using single-strand conformation polymorphism analysis. All polymorphisms were se- quenced for mutations.

RESULTS. Eighty-three affected people in 29 families with late-onset POAG were screened for mutations. Three mutations, two novel missense (Thr377Met and Glu352Lys) and one nonsense (Gln368STOP), were identified. The missense mutations did not segregate with the disease phenotype in these families. The nonsense mutation was found in 3 of 29 unrelated families with POAG. All affected family members and 8 of 12 in whom glaucoma was suspected had the Gln368STOP mutation. All people with this mutation had elevated IOP, and 78% had POAG by age 70. CONCLUSIONS. Three mutations were identified in the gene coding for myocilin in families with late-onset POAG. Of these, the Gln368STOP mutation was highly associated with the development of glaucoma. All people with this mutation had glaucoma or elevated IOP by age 70. In the United States, the Gln368STOP mutation in myocilin is strongly associated with the development of late-onset POAG. However, factors in addition to the presence of this mutation seem to play a role in the development of ocular hypertension and glaucoma in these families. (Invest Ophthalmol Vis Sci. 1998;39:2288-2295)

laucoma is a group of disorders characterized by pro- usually consists of mild to moderately elevated IOP, al- gressive excavation of the optic nerve head with asso- though a significant number of patients may not have a Gciated loss of the visual field.Althoug h unnecessary for measured IOP higher than normal.4 Optic nerve damage and diagnosis, elevated intraocular pressure (IOP) is often associ- visual field loss, which may culminate in blindness, occurs ated with glaucoma. Glaucoma is a highly prevalent disorder over many years, usually decades after onset of the disease. and is estimated to be the third most common cause of blind- The mode of inheritance for late-onset POAG is unknown. l ness worldwide. Primary open-angle glaucoma (POAG) is the However, it is thought that POAG is a complex trait that most common form. It is estimated that POAG is responsible results from interaction of multiple in conjunction for blindness in as many as 116,000 people in the United States 23 with environmental influences. and in 3,000,000 worldwide. Juvenile-onset POAG is an uncommon autosomal domi- Late-onset POAG is a bilateral disease that typically nant form of glaucoma. The age of onset is most often before occurs after the fourth decade of life. The clinical course the fourth decade of life and the phenotype is clinically more severe than late-onset POAG. A locus for juvenile-onset POAG was mapped to Iq23 to 25 and confirmed by From the 'Duke University Medical Center, Durham, North Caro- others.5"7 More recently, a candidate gene was localized to 2 lina; New England Medical Center, Boston, Massachusetts; the 'De- chromosome lq23-25, which codes for the described partment of Genetics, Stanford Medical School, Stanford, California; and 4Vanderbilt University, Nashville, Tennessee. as trabecular meshwork-induced glucocorticoid response pro- 8 9 10 Supported in part by Grant EY10886 from the National Eye Institute, tein (TIGR) ' and myocilin. The Organiza- Bethesda, Maryland; the Glaucoma Research Foundation, San Francisco, tion Genome Database Nomenclature Committee has recently California; Research to Prevent Blindness, New York, New York; the adopted the term myocilin. Mutations in myocilin have been American Health Assistance Foundation, Rockville, Maryland; and the Barkhauser Glaucoma Research Fund; Durham, North Carolina. identified in families with juvenile-onset POAG. One mutation Submitted for publication March 19, 1998; revised June 19, 1998; in myocilin (Gln368STOP) has been identified in people with accepted July 2, 1998. late-onset POAG.11 In this article we report on the presence of Proprietary interest category: N. mutations in myocilin in families ascertained for late-onset Reprint requests: R. Rand Allingham, Duke University Eye Center, Box 3802, Erwin Road, Durham, NC 27710. POAG.

Investigative Ophthalmology & Visual Science, November 1998, Vol. 39, No. 12 2288 Copyright © Association for Research in Vision and Ophthalmology

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12 METHODS tion polymorphism (SSCP) analysis. Individual samples sub- ject to SSCP analysis were amplified by polymerase chain re- Family Collection and Ascertainment action using DNA extracted from whole blood. The following primer pairs were used to amplify overlapping segments of the Families were ascertained through the Departments of Oph- coding sequence of the gene: thalmology at the Duke University Medical Center in Durham, North Carolina, and the Tufts New England Medical Center in cDNA Sequence Primer pairs (5' to 3'): Boston, Massachusetts. Some family members were ascertained by referring ophthalmologists. Predominately, families were -36 to 402 (F) AGAGCTTTCCAGAGGAAGCC identified from the southeastern United States and New En- (R) TTGGGTTTCCAGCTGGTCCC gland. Informed consent was obtained from all participating 300 to intron 1 (F) CAATTGACCTTGGACCAGGC family members. This study was approved by the Duke Univer- (R) TCATCTGTCTTGTGCTAGCT sity and New England Medical Center Investigational Review Intron 1 to 701 (F) CAGCCTATTTAAATGTCATC Boards. All procedures used in the execution of this study (R) CCAGATGGGCTCTCCTTCAA adhered to the tenets of the Declaration of Helsinki. All family members were personally examined or had their 604 to intron 2 (F) TTTCTACGTGGATTTGGAC medical records reviewed QLW or RRA). Examiners were (R) CTGAACACAGCACTAGACAT masked to family members' mutation status. Ascertainment for Intron 2 to 1005 (F) CTTCCGCATGATCATTGTCT proband, affected (late-onset POAG), suspected glaucoma, and (R) ATAGAGGCTCCCCGAGTACA unaffected was defined as follows. All those affected had age at 911 to 1364 (F) CAGCCAGTTTATGCAGGGCT diagnosis of more than 35. Proband had IOP measured by (R) TCATAAGCAAAGTTGACGGT applanation tonometry in both eyes of 22 mm Hg or more or 19 mm Hg or more while taking two glaucoma medications, 1285 to 1626 (F) GCCTTCATCATCTGTGGCAC glaucomatous optic neuropathy in both eyes, and visual field (R) AAAGCAGTCAAAGCTGCCTG loss consistent with optic nerve damage in at least one eye. To be considered glaucomatous optic neuropathy, documented One hundred nanograms genomic DNA was amplified 7 progression of optic nerve cupping or two of the following using standard conditions. PCR products were screened for were required: focal thinning of the neuroretinal rim, vertical sequence alterations using SSCP. Any abnormality identified by cup-to-disc ratio more than 0.7 in both eyes, optic cup asym- SSCP was sequenced. For sequencing, the PCR products were metry more than 0.2, or presence of an optic disc hemorrhage. amplified using a PCR purification kit (Quiagen QLAquick, To be considered affected (other than proband) required two Chatsworth, CA). Aliquots of 300 ng were sequenced using a of the three ocular criteria just described. Those with sus- dideoxy sequencing kit (Amersham Life Science, Arlington pected glaucoma had to have IOP of 22 mm Hg or more or Heights, IL). optic nerves that seemed glaucomatous to the examiner. Visual All polymorphisms identified with SSCP were sequenced. field evaluations were performed on all family members who All members of from families identified with at least one person had elevated IOP or optic nerves that raised suspicion of with a myocilin mutation were sequenced, regardless of af- glaucoma. Unaffected people had IOP in the normal range fected status. (<22 mm Hg) and normal appearing optic nerves. People with a history of incisional surgery (e.g., cataract extraction) before Statistical Analysis documentation of glaucoma status, a history of penetrating or The two-tailed Student's f-test was used for statistical analysis. blunt ocular trauma, or any secondary form of glaucoma were A value of P < 0.05 was considered significant. not included in the initial screen for mutations in myocilin. Families with any member identified with pseudoexfoliation or pigment dispersion syndrome were excluded from analysis. All RESULTS families had at least two siblings affected by POAG. Age at diagnosis, race, sex, visual acuity, applanation tonometry, slit Eighty-three affected people in 29 families in which POAG was lamp examination, gonioscopy, fundus examination, visual identified (2 African-American and 27 white) were screened for fields, refraction, and available ophthalmic medical and surgi- mutations in myocilin. One hundred four unrelated white peo- cal history were obtained on all family members. ple with age-related macular degeneration (n = 88) and their spouses (n = 16) with no personal or family history of glau- All available members of families identified with the myo- coma were used as control subjects. The mean age of control cilin Gln368STOP mutation were examined and sampled. Af- subjects was 72.8 years. fected status was classified according to the described criteria Three mutations were identified in five families. One fam- with the exception that people with previous ocular surgery ily (no. 5039) with mutations in myocilin was of African- were included in the final analysis. DNA was obtained from American ancestry, the other families were of white descent. people with age-related macular degeneration without per- All mutations were located in the third codon of myocilin. Two sonal or family history of glaucoma to be used as control missense mutations were found, one in each of two families. subjects. One mutation changes glutamate to lysine at 352 (Glu352Lys) and the other changes threonine to methionine at Single-Strand Conformation Polymorphism and amino acid 377 (Thr377Mef). A nonsense mutation, Sequencing Analyses Gln368STOP, was identified in three families. A wobble muta- DNA from all family members and control subjects was tion, Tyr347Tyr, was identified in six families that did not screened for myocilin mutations using single-strand conforma- segregate with the disease phenotype and eight control sub-

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Family: 5055 2f rp T000 T001

113 -a 0114 6" o 9056 57 9058 9062 9054 63 yr 63 yr 58 yr 49 yr 6Y6 666 8086 8088 8090 8056 8058 8059 8060 8069 8071 42 yr 42 yr 39 yr

^O 0001 0110 0101 0103 0100 0104 0108 0109 53 yr 82 yr 72 yr 73 yr 61 yr

| ** 6- r-o 9002 9004 9005 9007 9013 9014 9016 9019 9023 9078 9077 9076 9079 9070 9071 9074 9020 9080 55 yr 49 yr 43 yr 58 yr 55 yr 55 yr 50 yr 41 yr 59 yr 60 yr 59 yr 64 yr 67 yr 61 yr 34 yr 51 yr 6+ i+ r 6 8134 8136 8138 8139 8131 8132 8114 8116 8141 8021 8030 8027 27 yr 35 yr 23 yr 31 yr 28 yr 47 yr 45 yr 40 yr 37 yr 33 yr 29 yr

Symbol definitions • 0 unaffected • • POAG Family: 125 B © Suspect Family: 5052 Mutation present 4- T000 i001 — Mutation absent

0001 0100 0101 0102 56 yr 92 yr 0001 0100 0102 0105 75 yr 73 yr 73 yr 41 yr + + L + £1> 9000 9002 9003 9005 9006 42 yr 39 yr 35 yr 41 yr 36 yr

FIGURE 1. Pedigrees of families with late-onset primary open-angle glaucoma in which the Gln368STOP myocilin mutation was identified. Age at last examination is shown.

jects. No other mutations were identified in the control pop- The Gln368STOP mutation consisted of a CAG to TAG bp ulation. The missense mutations occurred in small families and change at codon 368 in all 3 families. Pedigrees of these did not segregate with the disease phenotype. These mutations families are depicted in Figure 1. In family 5052 there were will be discussed in more detail elsewhere (Wiggs et al., un- three members with the Gln368STOP mutation, all of whom published results). were affected with POAG. The fourth family member had

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elevated IOP but did not have the mutation. In family 125, five mutations (Thr377Met and Glu352Lys) were found in one members had the Gln368STOP mutation: Two were affected, family each. Of note, neither mutation segregated with the and two had elevated IOP. There was another family member POAG phenotype in these families. These mutations may with elevated IOP who did not have the Gln368STOP muta- represent rare polymorphisms, although they were not tion. In family 5055 there were three affected people, all of found in the control population nor have they been de- whom had the Gln368STOP mutation. There were 15 addi- scribed elsewhere. It is possible that factors in addition to tional people with the Gln368STOP mutation, 6 of whom had these mutations may be required to produce the POAG elevated IOP. Two members of family 5055 without the phenotype in these families. The third mutation was a non- Gln368STOP mutation had suspected glaucoma, one with mar- sense mutation (Gln368STOP) and was found in three fam- ginally elevated IOP and another with optic nerve cupping ilies. The missense mutations found in these families will be suggestive of glaucoma. described in more detail elsewhere (Wiggs et al., unpub- Clinical information on people in the three families with lished). The primary focus of this article is on families with the Gln368STOP mutation is presented in Table 1. Forty-eight the Gln368STOP mutation. people were screened. Of these, 26 family members had the In all families in which the Gln368STOP mutation was Gln368STOP mutation. There were 8 affected people, 8 with identified, all affected people with POAG had the mutation. All suspected glaucoma, and 10 unaffected people with the muta- family members older than 70 years with the Gln368STOP tion. The remaining 22 people did not have the mutation. mutation had elevated IOP, and 78% of these had glaucoma. All affected people demonstrated elevated IOP, normal The association between mutation status and classification as a angles, open angles on gonioscopy, characteristic glaucoma- glaucoma suspect was less clear. Four of 12 of of those with tous optic nerve damage, and associated visual field loss (Figs. suspected glaucoma and 3 of 11 with ocular hypertension did 2, 3, 4). The Gln368STOP mutation was present in all affected not have the Gln368STOP mutation, although those with sus- people. Five of eight of those affected had moderate to ad- pected glaucoma who had the Gln368STOP mutation had a vanced optic nerve damage and visual field loss and five re- higher average IOP than those without the mutation. In addi- quired laser or incisional surgery in addition to medical man- tion, there was significant overlap in the age at diagnosis of agement to control their glaucoma. affected people, people with suspected glaucoma, and the ages Glaucoma was suspected in twelve family members. of unaffected people with the Gln368STOP mutation in these Eleven of the 12 had elevated IOP in one or both eyes. Eight of families. Therefore, it seems that there are important genetic 12 had the Gln368STOP mutation. The one (family no. 5055, and/or environmental factors in these families that play an individual no. 9036) who was classified on the basis of optic important role in the pathogenesis of ocular hypertension and nerve cupping suggestive of glaucoma had normal IOP and did glaucoma. not have the Gln368STOP mutation. All had normal visual These data suggest that the Gln368STOP mutation in myo- fields. There were 28 unaffected family members of whom 10 cilin, a novel protein, is associated with the development of had the Gln368STOP mutation and 18 did not. glaucoma in families with late-onset POAG. Little is known Mean IOP and age at diagnosis are summarized in Table 2. regarding the function of myocilin. The deduced amino acid The mean IOP of those with POAG was 311 mm Hg. The mean sequence of myocilin shares significant homology with non- IOP in all with suspected glaucoma was 24.2 mm Hg. The muscle at the N terminus and neural olfactomedin at mean IOP in those with the mutation was 25.8 mm Hg and in the C terminus.1013 Evidence to date suggests that myocilin those without the mutation was 21.1 mm Hg. The difference in may be a membrane-associated cytoskeletal protein, an extra- IOP between these two groups was significant (P = 0.05). The cellular protein, or both.910 mean IOP in the unaffected group in people with and without The gene for myocilin has three .1013 To date, all the mutation was 16.8 mm Hg and 16.2 mm Hg, respectively, mutations associated with the open-angle glaucoma phenotype and did not differ significantly (P = 0.25). The mean age at are located in the third or that region coding for the diagnosis of those with POAG was 61.8 years (range, 41-75 olfactomedinlike domain.111415 All these are missense muta- years). The mean age at diagnosis of all with suspected glau- tions, which produce amino acid substitutions in the protein, coma was 53-8 years (range, 39-73 years). Mean age at diag- except for one nonsense mutation, Gln368STOP. This muta- nosis of those with suspected glaucoma who had the mutation tion codes for premature truncation of 135 amino acids of the was 49.5 years (range, 23-72 years) and in those without the C terminus of the myocilin protein, or approximately half of mutation was 62.5 years (range, 41-73 years), which was not the olfactomedinlike domain. significantly different (P = 0.20). The mean age at the time of Excluding the Thr377Met and Glu352Lys mutations de- examination for unaffected people was 47.1 years (range, scribed that do not segregate with the disease phenotype, the 28-91 years). Of those with the Gln368STOP mutation who mean age at glaucoma diagnosis with missense mutations is were older than 70 years at the time of their last examination, under age 35 (Table 3). In contrast, the age at diagnosis for the 100% (9/9) had elevated IOP, and 78% (7/9) were classified as Gln368STOP mutation occurs at least 3 decades after these affected. missense mutations. The reason for this striking difference in the age at diag- nosis of POAG between missense mutations and the DISCUSSION Gln368STOP mutation is unclear. Although studies are needed to answer many of these questions, it is Mutations in the myocilin gene have been identified that are reasonable to speculate that this observation is attributable to associated with the development of POAG, usually of juve- differences in expression or function between with nile onset.11 We have identified three mutations in myocilin amino acid substitutions and those that are prematurely trun- in families screened for late-onset POAG. Two missense cated. Missense and nonsense mutations alter the tertiary struc-

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TABLE 1. Genotype and Clinical Data of Study Participants in Families with Gln368STOP Myocilin Mutation Visual Acuity IOP Vertical Family/Individual Gln368STOP Age at Highest Cup-to-Disc Number Mutation Diagnosis Sex OD OS (mm Hg) Ratio Surgery Affected with POAG 5055/0001 53 M 20/30 20/40 31/32 0.7/0.7 LTP 5055/0108 61 F LP NLP 29/32 1.0/1.0 CE; TRAB 5055/9056 63 F 20/20 20/20 20/40 0.6/0.7 5052/0001 75 F 20/30 20/50 28/24 0.9/0.9 LTP; RS 5052/0102 73 M CF 20/40 30/24 1.0/0.9 LTP; TRAB; CE 5052/0105 41 M 20/30 20/25 30/30 0.7/0.7 CE 125/0001 56 M 20/20 20/25 34/38 1.0/1.0 LTP; TRAB; CE 125/0101 72 F 20/20 20/20 25/25 0.7/0.7 Possible POAG 5055/0100 61 F 20/25 20/25 24/24 0.3/0.5 5055/8138 23 M 20/15 20/15 23/20 0.1/0.1 5055/9076 59 F 20/20 20/20 30/32 0.7/0.5 5055/9023 41 F 20/20 20/15 23/20 0.2/0.2 5055/0101 72 F 20/300 20/40 30/34 0.5/0.3 5055/9078 59 F 20/20 20/20 27/25 0.3/0.4 125/9002 39 F 20/15 20/15 24/27 Tilted 125/9000 42 M 20/20 20/25 24/25 0.3/0.3 5055/9036 72 M 20/50 20/30 18/19 0.6/0.6 CE 5055/9042 64 F 20/25 20/25 22/19 0.3/0.25 125/9005 41 M 20/25 20/25 26/19 0.1/0.1 5052/0100 73 M 20/30 20/25 23/23 0.1/0.1 Unaffected by POAG 5055/8136 35 M 20/20 20/15 15/16 0.4/0.3 5055/9062 58 F 20/20 20/20 18/18 0.5/0.5 5055/9007 59 F 20/20 20/25 19/19 0.3/0.3 5055/9005 43 F 20/20 20/20 18/17 0.2/0.3 5055/9016 55 M 20/40 20/25 13/14 0.2/0.2 5055/9019 50 M 20/20 20/20 18/18 0.25/0.25 RL 5055/9070 67 F 20/20 20/25 17/18 0.2/0.2 5055/9054 49 F 20/15 20/20 14/14 0.4/0.3 5055/8134 38 F 20/20 20/20 17/18 0.1/0.1 125/9006 36 F 20/25 20/40 17/17 0.3/0.3 5055/9058 63 F 20/40 20/25 18/18 0.3/0.3 5055/9004 49 F 20/25 20/25 20/18 0.3/0.3 5055/8139 31 F 20/20 20/20 16/16 0.2/0.2 5055/8131 28 F 20/15 20/15 16/17 0.2/0.2 5055/8114 47 M 20/30 20/20 16/11 0.3/0.3 5055/8116 45 F 20/15 20/15 16/17 0.2/0.2 5055/8141 40 M 20/20 20/20 13/13 0.2/0.1 5055/8021 37 F 20/15 20/15 18/18 0.1/0.1 5055/8030 33 F 20/15 20/15 15/15 0.2/0.2 5055/8027 29 M 20/20 20/30 18/16 0.2/0.1 5055/9044 62 F 20/25 20/100 17/21 0.2/0.2 5055/8088 42 F 20/20 20/20 15/16 0.3/0.3 5055/8056 42 F 20/15 20/15 12/12 0.3/0.3 5055/8069 39 F 20/15 20/15 15/15 0.3/0.3 5055/9002 55 F 20/25 20/50 18/19 0.1/0.1 5055/9074 61 F 20/25 20/25 16/16 0.25/0.25 5055/9020 34 M 20/30 20/30 15/15 0.2/0.2 5055/115 91 M 20/30 20/100 12/14 0.3/0.3 CE

POAG, primary open-angle glaucoma; IOP, intraocular pressure; LTP, laser trabeculoplasty; CE, cataract extraction; TRAB, trabeculectomy; RS, retinal surgery; RL, retinal laser; LP, light perception; NLP, no light perception; CF, counts fingers.

ture of proteins, which in turn can alter their function, half-life, rapidly eliminated by the cell.17 19 Therefore, the protein coded and interaction with other proteins.16 Altered forms of myoci- for by the Gln368STOP mutation may be eliminated more rapidly, lin may compete with or bind normal myocilin produced by producing a relative deficiency of myocilin. Abnormal myocilin the normal homologue or interact with other cellular proteins. produced by missense mutations, particularly if these have a Truncated proteins, because of greater alterations in structure, longer half-life, may compete more effectively with the wild-type are more likely to be recognized as abnormal and are often protein, causing greater cellular dysfunction.

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FIGURE 2. Gonioscopic appearance in a person affected with primary open-angle glaucoma with the Gln368STOP myocilin mutation. The filtering angle is normal in appearance.

Regardless, these data suggest that chronic reduction of or 10 years more than that of those with suspected glau- interference with normal myocilin function produced by these coma. Additionally, all with suspected disease who had mutations is associated with increased risk of IOP elevation and this mutation and those affected by glaucoma had elevated glaucoma. It is well known that the administration of exoge- IOP. It appears that elevated IOP precedes optic nerve nous glucocorticoids is also associated with increased risk of damage by many years in people with this mutation in elevated IOP and glaucoma, particularly in people with POAG myocilin. It is impossible to determine the precise risk of and their close relatives.20"23 Of interest, glucocorticoids in- development of elevated IOP or glaucoma from this small crease production of myocilin in human trabecular cells.9 Al- number of families; however, it is important to note that though the time course in steroid-induced IOP elevation is fully 60% of those family members with the Gln368STOP measured in weeks to months versus decades in people with mutation who are aged more than 35 years had elevated IOP mutations in myocilin, it is interesting that abnormal myocilin or glaucoma. Of additional interest, the three people with- and excess levels of myocilin are both associated with in- out this mutation have elevated IOP. It is clear that prospec- creased IOP and glaucoma. tive longitudinal studies are needed to define further the In family members with the Gln368STOP mutation, the risks and appropriate methods for observing people with age of patients at diagnosis of POAG is approximately these mutations.

FIGURE 3- Optic disc photographs of eye in Figure 2 affected with primary open-angle glaucoma. Saucerization and glaucomatous cupping of both optic nerves are present. Drusen are also observed.

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RIGHT 18 18 •3 (3) HE 33 9 B FXXHTXGN LOSSES 8/13 rib 6) (14) 8 12 FfiLSE PUS ERRORS ft'll & 11 13 12 14 Fft.Sc !£C ERRORS 8/7 <9 <9 9 8 28 16 14. 18 9. 19 (8 14 QUESTIONS fiSKED 229 (I?) 19 23 22 18 14 TEST Til* 37U4 (i?)» & 19 12 & l7 " ffflVN 15 19 ;4 14 .4) (11)

LEFT 14 It RCE 33 17 FICTION LOSSES 0/t3 (35 (IS) (ff> & 14 9 18 14 24 FH.SE POS ERRCRS 8/3 (13) & " FitSE f€G ERRORS 8^ 24 23 , 19 1? 13 QUEST IOKS; ASKED 221 24 23 17 14 I) TEST TIK 87:82 13 2« J 22 ( (25) (28) 21 " 23 H & 16 19 21 17

FIGURE 4. Visual fields of eyes in Figure 2 affected with primary open-angle glaucoma. Generalized depression and peripheral constriction are present in both eyes. Superior visual field loss is present in the right eye.

TABLE 2. Age at Diagnosis, Mutation Status, Sex, and We identified the Gln368STOP mutation in more than Mean IOP of Individuals with the Gln368STOP 10% (3/29) of probands and none of 104 control subjects. This 11 Mutation of Myocilin association was highly significant (P ~ 0.0002). Stone et al. found the Gln368STOP mutation in 2.6% (6/227) of probands. Sex Mean IOP Because their sample included many families with juvenile- Number Age* (M/F) (mm Hg) onset glaucoma this figure may underestimate the percentage POAGt 8 61.8 4/4 31.1 Suspected Glaucoma 12 53.8 5/7 24.2 Gln368STOP TABLE 3. Mean Age of Diagnosis for TIGR and mutation + 8 49.5 2/6 25.8 MYOC Mutations Gln368STOP mutation - 4 62.5 3/1 21.1 Mutation Mean Age at Unaffected by POAG 28 47.1 8/20 16.4 (Type) Diagnosis (y) Reference Gln368STOP mutation + 10 49 3/7 16.8 Gln368STOP 62-64 Current study, 11 Gln368STOP Pro370Leu 10-11 15, 14 mutation - 18 46 5/13 16.2 Ile477Ser 33 15 Asn480Lys 30-35 15 POAG, primary open-angle glaucoma; IOP, intraocular pres- Ile499Phe 31 15 sure. Gly246Arg 20 15 • Values are means at time of diagnosis for those with POAG and Tyr437His 22 11 those with suspected POAG and at time of last examination for those Gly364Val 33 11 who were unaffected. Lys423Glu 15, 24 t All Gln368STOP mutation +. 30

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that would have been found in families with late-onset POAG. 9- Polansky JR, Fauss DJ, Chen P, et al. Cellular pharmacology and Therefore, the actual prevalence of the Gln368STOP mutation molecular biology of the trabecular meshwork inducible glucocor- in families with late-onset POAG may be greater than previ- ticoid response gene product. Ophthalmologica. 1997;211:126- 139. ously thought. If true, this would have significant implications 10. Kubota R, Noda S, Wang Y, et al. A novel myosin-like protein regarding population screening for mutations and the ap- (myocilin) expressed in the connecting cilium of the proach to medical intervention. photoreceptor: molecular cloning, tissue expression, and chromo- In summary, we have identified three mutations in the somal mapping. Genomics. 1997;4l:360-369. gene coding for myocilin in families with late-onset POAG. Of 11. Stone EM, FingertJH, Alward WLM, et al. Identification of a gene these, the Gln368STOP mutation is highly associated with the that causes primary open angle glaucoma. Science. 1997;275:668- development of the disease phenotype. Glaucoma developed 670. or was suspected by age 70 in all people with this mutation. 12. Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T. Detection of polymorphisms of human DNA by gel electrophoresis as single- However, factors in addition to the presence of this mutation strand conformation polymorphisms. Proc Natl Acad Sci USA. appear to play a role in the development of ocular hyperten- 1989;86:2766-2770. sion and glaucoma in these families. 13- Ortego J, Escribano J, Coca-Prados M. Cloning and characteriza- tion of subtracted cDNAs from a human ciliary body library en- Acknowledgments coding TIGR, a protein involved in juvenile open angle glaucoma with homology to myosin and olfactomedin. FEBS Lett. 1997;4l3: The authors thank the families who enthusiastically participated in this 349-353- project; David L. Epstein, W. Daniel Stamer, and Brian S. McKay for 14. Suzuki Y, Shirato S, Taniguchi F, Ohara K, Nishimaki K, Ohta S. their critical review of the manuscript; and the Center for Human Mutations in the TIGR gene in familial open-angle glaucoma in Genetics of the Duke University Medical Center for permitting the use Japan. Am J Hum Genet. 1997:1202-1204. of resources and staff. 15. Adam MF, Belmouden A, Binisti P, et al. Recurrent mutations in a single exon encoding the evolutionary conserved olfactomedin- References homology domain of TIGR in familial open-angle glaucoma. Hum Mol Genet. 1997;6:2091-2097. 1. Thylefors B, Negrel AD, Pararajasegaram R, Dadzie KY. Global data on blindness. Bull World Health Org. 1995;73:115. 16. Creighton TE. Proteins. New York: WH Freeman, 1984. 2. The International Bank for Reconstruction and Development. The 17. 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