The genetics of WALLACE L.M. ALWARD open-angle glaucoma: the story of GLC1A and myocilin Abstract There are three main approaches to identifying a disease-causing gene (Table 1). A linkage analysis study was performed on a The candidate gene approach is useful when single large family with juvenile-onset there is a known gene whose function makes it a primary open-angle glaucoma (POAG). This strong suspect. For example rhodopsin was a led to the recognition that there was a region very reasonable candidate for retinitis of chromosome lq that harboured a gene for pigmentosa and proved to be the disease­ juvenile-onset POAG. This chromosomal site causing gene in some cases.s Unfortunately was called GLCIA. It was discovered that a there are too many potential candidate genes for gene that produces the protein myocilin POAG, including all the genes involved in the resides within this interval and that mutations development, structure and function of the in myocilin caused most cases of autosomal trabecular meshwork and optic nerve head. dominant juvenile-onset POAG. More A second approach is to identify patients importantly myocilin mutations also cause up who manifest the disease of interest and also to 4.6% of cases of adult-onset POAG. The have a chromosomal deletion or translocation; prevalence of myocilin mutations is similar the break in the chromosome might be in or regardless of race or geographic location. near the disease-causing gene. This technique There are widely variable glaucoma phenotypes depending on the specific has proved helpful for a variety of mutation in myocilin. Myocilin is expressed in developmental glaucomas. Recently Nishimura 6 multiple tissues throughout the eye and in et a/. discovered the forkhead transcription many other organs. In the trabecular factor gene FKHL7 by studying the breakpoints meshwork the production of myocilin can be in chromosome 6 of two children with primary induced by the application of topical congenital glaucoma and chromosomal corticosteroids. The exact function of myocilin trans locations. Mutations in this gene have been in health and disease remains a mystery. demonstrated to cause a variety of anterior segment abnormalities including Axenfeld anomaly and Rieger anomaly.6,7 Key words Genetics, Glaucoma, GLC1A, In the absence of any other clues as to the Myocilin, TIGR location and nature of the gene causing a disease, the search typically begins by performing linkage analysis studies on large families affected with the disease. In this Glaucoma is the leading cause of irreversible technique there is a search for co-segregation blindness in the world. It is estimated that between a disease phenotype and polymorphic approximately 67 million people worldwide are genetic markers. Unfortunately, POAG is not an affected with glaucoma and, of these, 6.7 million ideal disease for linkage studies. Linkage are bilaterally blind.l In Western countries, analysis requires large numbers of living primary open-glaucoma (POAG) is by far the affected individuals. Because POAG is a late­ most common form of glaucoma? The four onset disease, the parents of affected most important risk factors for the development individuals are almost always deceased, as are Wallace L.M. Alward, MD � Depa rtment of of POAG include advanced age, black race, many of the siblings. Additionally, the children Ophthalmology elevated intraocular pressure (lOP) and positive University of Iowa College family history? The risk of developing Table 1. Major techniques used in searching for of Medicine 200 glaucoma for individuals with a history of disease-causing genes Hawkins Drive glaucoma among first-degree relatives is up to Iowa City Candidate gene approach IA 52242-1091, USA 8 times higher than the risk for the general Utilising clues from chromosomal deletions and Tel: +1 (319) 356 3938 population.2-4 This heritable nature of glaucoma translocations Fax: +1 (319) 353 7699 Linkage analysis permits the use of molecular genetic techniques e-mail: Combination of the above techniques to study this important blinding disease. [email protected] Eye (2000) 14, 429-436 © 2000 Royal College of Ophthalmologists 429 II 4 III IV V VI Fig. 1. Pedigree of the proband's family. Individuals with glaucoma are indicated with filled symbols. Those individuals indicated with half-filled symbols have ocular hypertension and are known to have the disease-causing mutation because they have affected children. Reproduced with 1.l permission from Johnson et al. are often too young to manifest signs of the disease. The proband and his family Therefore, obtaining large numbers of affected As with all genetics studies, this story begins with a individuals in a single extended family is very difficult. patient. The proband presented to the University of Iowa Often a combination of the techniques mentioned Department of Ophthalmology in 1986 requesting above is used to find a disease-causing gene. Murray glaucoma filtration surgery. He was 28 years old and had et ai.8 found that a gene on the long arm of chromosome 4 been aware that he had glaucoma since age 16. At the could cause Rieger syndrome by performing linkage time we first examined him he was using timolol analysis on relatively small families. They were able to maleate, dipivefrine HCl and pilocarpine gel. His lOPs were 24 mmHg in the right eye and 25 mmHg in the left. use small families because they had narrowed the region He supported the request for filtration surgery by of search by targeting a small portion of the genome presenting a simple pedigree that he had constructed. using clues from the coexistence of Rieger syndrome and Half his relatives were affected by a form of glaucoma translocations and deletions in chromosome 4q.8 that had a very early age of onset, was resistant to This article will discuss the story behind the discovery medical therapy, was resistant to laser trabeculoplasty, of the first linkage for POAG (GLCIA) and ultimately the and generally did well after filtration surgery. He gene at this locus (myocilin). Mutations in myocilin have ultimately had bilateral trabeculectomies with resultant been demonstrated to be capable of causing juvenile­ excellent control of his lOP and stability of his optic onset POAG,9 adult-onset POAG9 and normal tension nerves and visual fields. In 1993, Johnson described this family in detailY The glaucoma.lO The discovery was made through the use of family was of German ancestry. Of 59 individuals in five linkage analysisll followed by evaluation of candidate generations who were at risk for the disease, 30 were genes in the linked interva1.9 This approach has been affected (Fig. 1). They had a POAG with a very early age 1 called the positional candidate approach. 2 Because most of onset (mean 18 years) and very high lOPs (mean of the papers about this discovery have appeared in the 45 mmHg). A sampling of the age of onset and lOPs of basic science, genetic and general medical literature it is some of the more dramatic cases is shown in Table 2. useful to review them for an ophthalmology audience. Eighteen (82%) of 22 living affected family members had Table 2. Examples of the early age of onset and dramatically high lOP found in members of this pedigree Peak lOP (mmHg) Pedigree no. Age at diagnosis (years) Right eye Left eye IV-9 17 54 54 IV-ll 16 66 58 V-2 18 62 63 V-3 27 59 53 V-ll 22 57 56 V-24 16 52 56 V-27 16 52 56 V-29 8 50 43 l3 Adapted with permission from Johnson et al. These are some of the most striking examples from this pedigree. 430 undergone filtering surgery. Affected members of the eM 0 015194 family were usually myopic (mean refractive error I 1 I -4.06 D) but otherwise had normal ocular anatomy. 3 I I It was felt that juvenile-onset POAG could serve as a I 21 I 015196 model for the adult-onset disease because it shared the I 13.3 I 5 clinical phenotype, except for the following differences: a 13 2 I . 13.1 12 I very early age of onset, a striking autosomal dominant 11 I 11 I inheritance pattern with high penetrance, and very high 12 I IOPs.13 Otherwise, the diseases were identical; the 21 1 21 .2 . iridocorneal angle was open and the trabecular 2�� 10 23 meshwork was normal in appearance. 24 25 I 31 015218 \ \ 15 32.1 \ 015212 Linkage analysis 32.2 \ 32.3 \ 015215 41 \ DNA samples were obtained from 22 affected 42 1 \ 42.2 42.3. \ individuals and tested for linkage with 90 short tandem 43 44 �o repeat polymorphism (STRP) markers scattered across \ the entire human genome. These markers, also called \ \ 015240 microsatellites, are areas of normal variability in the 21 015191 DNA sequence. There are thousands of these markers Fig. 2. Location of the disease-causing gene on chromosome 1. The located on all chromosomes. These portions of the DNA heavy vertical bar to the right of the chromosome drawing denotes the can be amplified by polymerase chain reaction (PCR) and interval q21-q31, which contains the gene. Reproduced with permis­ 11 assayed to see whether one of the possible variations in sion from Sheffield et al. DNA sequence (alleles) segregates with the affected phenotype. Trabecular meshwork inducible glucocorticoid In this family there was linkage between the affected receptor protein status and markers on the long arm of chromosome 1 During the years that these family studies were in (lq21-q31) (Fig. 2).11 After this linkage was reported it progress Drs Nguyen and Polansky were performing an was confirmed in populations in the United States14,15 independent and seemingly unrelated series of and throughout the world.16-18 The majority of large experiments.