Abstract

Adult onset primary open angle glaucoma is a leading cause of blindness throughout the world. The disease results in an apoptotic death of retinal ganglion cells that is usually associated with an elevation of intraocular pressure. Familial aggregation of the disorder provides evidence for strong genetic influences that are likely to be the result of multiple susceptibility genes. A two-stage genome scan to identify the genomic locations of glaucoma susceptibility genes was performed using an initial pedigree set of 113 affected sibpairs and a second pedigree set of 69 affected sibpairs. Linkage analysis was performed using both model-dependent (lod score) and model-independent affected relative pair and sibpair methods. Twenty-five regions identified by the initial scan were further investigated using the second pedigree set. In the combined data analysis, regions located on chromosomes 2, 6, 9, 11, 14, 17 and 19 continued to produce model-dependent lod scores and/or an MLS >1.0, while five regions (2, 14, 17p, 17q and 19) produced an MLS >2.0. Multipoint analysis using ASPEX also showed significant results on chromosomes 2, 14, 17p, 17q and 19. These results are an important step towards the identification of genes responsible for the genetic susceptibility to this blinding condition.

Received 17 December 1999; Revised and Accepted 14 February 2000.

INTRODUCTION

Glaucoma is a term used to describe a group of disorders that have in common a characteristic degeneration of the optic nerve that is usually associated with elevated intraocular pressure. In most instances, the elevation of intraocular pressure results from impaired drainage of aqueous humor (produced by the ciliary body) through the trabecular meshwork outflow pathways (1). The elevation of intraocular pressure is correlated with the onset of an apoptotic death of retinal ganglion cells resulting in optic nerve degeneration and irreversible loss of sight. Collectively, glaucoma is the third most prevalent cause of visual impairment and blindness among white Americans and is the leading cause of blindness among black Americans (2). Of the different forms of glaucoma, primary open angle glaucoma (POAG) of adult onset is the most common, representing approximately half of all cases of the disease (3).

A family history has long been recognized as a major risk factor for adult onset POAG, suggesting that specific genetic defects are likely to contribute to the pathogenesis of the disease. The prevalence of POAG in first degree relatives of affected patients has been documented to be as high as 7–10 times that of the general population (47). A high concordance of glaucoma between monozygotic twins has been observed, consistent with a significant genetic predisposition (810). Although the heritability of POAG is high, a simple mode of inheritance or a single underlying susceptibility gene is not likely and cannot be assumed in genetic studies designed to identify POAG loci.

The notion that multiple genes may be involved in the pathogenesis of POAG has been supported by results of recent investigations. Five genomic regions [(2cen-q13 (GLC1B), (11); 3q21-q24 (GLC1C), (12); 8q23 (GLC1D) (13); 10p15-p14 (GLC1E) (14); and 7q35 (GLC1F) (15)] have been shown to segregate with POAG in unconfirmed studies of a small number of independent families. Although relevant genes in these regions have not yet been cloned, and their contributions to the general POAG population have not yet been evaluated, these results do suggest that more than one gene may play a role in the disease. Sequence abnormalities in one gene primarily responsible for juvenile onset POAG [TIGR/Myocilin, 1q23 (GLC1A), (16)] are associated with POAG in a small percentage (3–5%) of affected individuals (1719). The TIGR/Myocilin protein appears to be expressed in multiple ocular tissues, including the trabecular meshwork (2022). Presumably, alterations of this protein cause abnormal egress of aqueous humor through the trabecular pathways leading to an increase in intraocular pressure. Genetic abnormalities that may result in retinal ganglion cell death have not yet been discovered.

We have completed a genome screen using a total of 113 affected sibpairs with POAG. We have genotyped 445 markers with a 7 cM grid and analyzed the results using a multi-analytical strategy consisting of model-dependent lod score analysis and model-independent affected relative pair and affected sibpair approaches. Regions with positive results were followed up with additional markers and a second set of 69 affected sibpairs.

RESULTS

Family recruitment

The first stage of the analysis included 41 multiplex families comprising 126 affected individuals and 113 affected sibling pairs. The majority of families consisted of two, three or four affected individuals in a single generation. Five larger families were included in this pedigree set: three families with five, one family with six and one family with nine affected individuals. Affected individuals were present in two generations in seven pedigrees. All of the families are of North American origin and represent a variety of ethnic backgrounds, but are mainly Caucasian. The age range of affected individuals when entered into the study was 46–95 years. The average age of diagnosis was 57.6 years with a range of 33–84 years. The average highest intraocular pressure was 27 mm Hg, with a range of 19–58. All patients entered into the study had evidence of optic nerve degeneration judged by visual field abnormalities and visual inspection of the optic nerve. The second group of 69 affected sibpairs included 33 multiplex families and 81 affected individuals. The majority of families in the second pedigree set also consisted of two, three or four affected individuals in a single generation. One larger family consisting of seven affected individuals was included in this pedigree set, and four pedigrees had affected individuals present in two generations. These families are also of North American origin, representing a variety of ethnic backgrounds and are also mainly Caucasian. This second group of pedigrees was ascertained for the study using the same set of affected criteria as the initial group of pedigrees. In the second set, the average age of diagnosis was 58 years with a range of 33–84 years. The average highest intraocular pressure was 27 mm Hg, with a range of 19–58 (Table 1). All of the individuals in the second set of families also had evidence of optic nerve degeneration.

Initial genome screen

Our data set contains a variety of pedigree structures, with some pedigrees having as many as nine affected individuals. Thus, genomic regions demonstrating nominal evidence for POAG linkage were initially identified using three methods of analysis. The first is model-dependent lod score analysis using the VITESSE computer program (23). For this analysis, we used an affected-only autosomal dominant model and an affected-only autosomal recessive model. The second method is a model-free affected relative pair approach using the GENEHUNTER computer program (24). This analysis eliminates the possibility of model mis-specification and allows the inclusion of all affected relatives. The third method is a model-free sibpair approach using ASPEX (25).

For the initial genome screen, 445 markers resulting in a 7 cM grid were tested using all three of the above methods. A region was identified as ‘promising’ if: (i) the model-dependent lod score was >1.0; (ii) a GENEHUNTER NPL was >1.0; or (iii) the sibpair maximum lod score (MLS) was >1.0. These criteria were chosen not to declare linkage, but to identify as many true-positive results as possible, accepting the fact that we will also have many false-positive results. Twenty-five regions showed promising results for at least one method, 17 for at least two methods and five for all three methods. Seven regions produced values >2.0 using one of the three methods (Table 2).

For the second stage of the analysis, a second set of 69 affected sibling pairs was genotyped using these same markers and an additional 68 markers located in the regions of interest. The clinical features of the second set of 69 affected sibling pairs were similar to those of the first set of 113 sibling pairs (Table 1). Because of the relatively small number of pedigrees in the second data set, the data were analyzed together rather than treating the second group of pedigrees as a ‘replication’ data set. In the combined data set, regions on chromosomes 2, 6, 9, 11, 14, 17 and 19 continued to show positive results (Table 3).

Sibpair multipoint analysis was performed using ASPEX for the entire set of markers for pedigree set I and for the regions selected for follow-up in the combined pedigree set of 182 affected sibpairs. The results shown in Figure 1 indicate significant results for chromosomes 2, 4, 11, 14, 17 and 19. The addition of the second pedigree set increased the multipoint lod scores for the regions on chromosomes 2, 14 and 17. The multipoint curves for the combined pedigree set for chromosomes 11 and 19 are similar to the curve for the initial data set. These results probably reflect the underlying genetic heterogeneity of the combined pedigree set. Multipoint analysis using a single independent sibpair from each pedigree was also performed (data not shown). All the graphs using a single sibpair were similar to those obtained with all possible sibpairs, with the one exception of chromosome 10. The peak MLS for chromosome 10 with independent sibpairs was 1.37, while with all possible sibpairs a peak value of 0.62 was obtained.

DISCUSSION

In a genome screen of 113 sibpairs affected by adult onset glaucoma, we initially identified twenty-five genomic regions that showed interesting results, with seven regions producing a lod score ≥ 2.0 using either model-dependent or -independent methods. When a second set of 69 affected sibpairs was included in the analysis, five regions (chromosomes 2, 14, 17p, 17q and 19) continued to produce lod scores > 2.0 and three regions produced lod scores between 1.0 and 2.0 with one of the analytical methods. Sibpair multipoint analysis also showed interesting results for the regions on chromosomes 2, 14, 17 and 19.

Of the genomic regions corresponding to previously identified glaucoma loci, we only observed significant results with markers located near the GLC1B locus on chromosome 2 (D2S441 and D2S2232). Modest results were obtained for markers D8S534 and D10S1208 located ~30–50 cM from the peak markers defining GLC1D (8q23) and GLC1E (10p14-p15), respectively. An MLS of 0.78 was obtained for marker D1S397 located ~10 cM from GLC1A (TIGR/Myocilin). The other analyses for marker D1S397 were negative (data not shown). Negative results were also obtained for markers located near the GLC1C locus (3q25) and the GLC1F locus (7q35) (data not shown). These results suggest that the glaucoma genes localized to these regions do not significantly contribute to the disease affecting our study population.

This is the first genome screen to be completed for adult onset glaucoma. The results of this study have revealed several interesting loci including regions on chromosomes 2, 14, 17 and 19. Follow-up studies to refine the genetic intervals and to assess possible candidate genes located in these regions are currently underway. The results of these studies will help identify genes that contribute to a susceptibility to this blinding condition.

MATERIALS AND METHODS

Families

The families used for this study were collected from the New England area and from the Southeast area of the United States, and represent a wide variety of ethnic backgrounds. The families are mainly Caucasian (88%). At least two members of each family were affected by POAG. The family size ranged from nine affected individuals to a single affected sibpair, with the majority of families consisting of two, three or four affected individuals in a single generation (see Results). All patients and appropriate family members had a complete ocular examination including visual acuity, refraction, tonometry, slit beam evaluation, gonioscopy and funduscopic evaluation. Optic nerve evaluation (usually including photography) and visual field testing were done on all affected individuals. The following criteria was used to assign affected status: intraocular pressure >22 mm Hg in both eyes on two occasions or intraocular pressure >19 mm Hg in both eyes on treatment with two or more glaucoma medications; evidence of optic nerve damage in both eyes; visual field defects consistent with optic nerve damage and characteristic for glaucoma in at least one eye, and onset before age 35.

Genotyping

DNA samples were prepared from lymphocyte pellets using standard techniques. Genotyping was performed in one of two ways. One method used fluorescent-tagged oligonucleotides for PCR amplification of microsatellite repeat markers. The fluorescent DNA fragments were detected by the Hitachi FMBIO II laser scanner and alleles were recorded as base pair lengths. Alternatively, genotyping was performed using PCR incorporation of 32P-radiolabeled nucleotides. Amplification products were electrophoresed on a 6% polyacrylamide gel. Alleles were scored manually as base pair lengths after exposure of the gel to X-ray film. The PCR conditions have been published previously (25,16).

Markers

A total of 445 microsatellite markers obtained from Genethon and CHLC were used for the initial genome screen. The average distance between markers was 7 cM and the average heterozygosity was 81%.

Statistical analysis

Three methods of analysis were used. Two-point lod scores were calculated using the VITESSE (23) computer program. Both autosomal dominant and autosomal recessive models with a low penetrance, ‘affected-only’ penetrance, function were used. Such a model allows us to incorporate genotype information on all individuals, but phenotype information only on affected individuals. The autosomal dominant model used a disease allele frequency of 0.0010 and the autosomal recessive model used a disease allele frequency of 0.0100. Multipoint lod score analysis was not possible due to the large size of some pedigrees. Marker allele frequencies were calculated from all independent chromosomes in the pedigrees, and when compared against known population frequencies no significant differences were observed. Affected relative pair analysis was performed using the NPL (all) statistic in the GENEHUNTER program. This analysis allows the use of all affected relatives in a pedigree without having to assume a specific genetic model. Scores are reported from whole-chromosome multipoint analysis. Affected sibpair analysis was performed using ASPEX (24), using a whole-chromosome multipoint analysis. The all-pairs option was used. Choosing just independent sibpairs from each family did not significantly alter the results.

Error analysis for the genotypes was performed using three different procedures. First, Hardy–Weinberg calculations were performed for each marker analyzed. An excess of homozygosity, consistent with genotyping error, was found for 11 markers (2.5%). These genotypes were either repeated with satisfactory results, or the markers were not used for the analysis. Secondly, haplotypes were constructed in each family in regions showing the strongest evidence of linkage. Excess recombination (double or triple recombinants) was not observed. Thirdly, to check for errors between the two laboratories performing genotyping, we randomly selected 20 markers for genotyping in each laboratory using independent methods. The results between laboratories were highly consistent.

ACKNOWLEDGEMENTS

We thank the families for their participation and the following clinicians for referring appropriate individuals for the study: Drs Joel Schuman, Cynthia Mattox, Evan Dreyer, Cynthia Grosskreutz, Lou Pasquale, Tom Hutchinson, Bob Bellows, Brad Shingleton, Ramesh Tripathi, Bob Allen, David Epstein, Paul Lee, Leon Herndon, Andrew J. Michael, Harold E. Shaw Jr, Dorothy M. Bell, Michael W. Brennan, David L. Smith, Sandra M. Johnson and Martin Wand. We also acknowledge support from NEI (EY10886), the Glaucoma Research Foundation, the Massachusetts Lions Fund and the Barkhouser Glaucoma Research Fund.

+

To whom correspondence should be addressed. Tel: +1 617 636 5484; Fax: +1 617 636 6126; Email: jwiggs@lifespan.org

Figure 1. Multipoint lod scores. The graphs for the initial pedigree set (113 sibpairs) are shown as continuous lines. The graphs for the combined pedigree set (182 sibpairs) are shown as dashed lines.

Figure 1. Multipoint lod scores. The graphs for the initial pedigree set (113 sibpairs) are shown as continuous lines. The graphs for the combined pedigree set (182 sibpairs) are shown as dashed lines.

Table 1.

Clinical characteristics of pedigree sets I and II

 Set I Set II 
Families 41 33 
Affected individuals 126 81 
Affected sibpairs 113 69 
 Mean (range) Mean (range) 
Age when entered into study 72.2 (46–95) 69.4 (42–86) 
Age at onset of glaucoma 57.6 (33–84) 58.6 (33–84) 
Highest intraocular pressure (mm Hg) 27.0 (19–58) 27.0 (19–58) 
 Set I Set II 
Families 41 33 
Affected individuals 126 81 
Affected sibpairs 113 69 
 Mean (range) Mean (range) 
Age when entered into study 72.2 (46–95) 69.4 (42–86) 
Age at onset of glaucoma 57.6 (33–84) 58.6 (33–84) 
Highest intraocular pressure (mm Hg) 27.0 (19–58) 27.0 (19–58) 
Table 2.

Regions showing initial evidence for POAG linkage

 Two-point LOD scores    Sibpair NPL Map**  
 Dominant model  Recessive model  MLS    
Marker Lod  Theta Lod Theta     
D2S391 1.05 0.15 1.44 0.20 1.16 0.42 74 cM  
D2S441 2.11 0.15 0.95 0.20 0.92 0.23 90 cM  
D2S2232 0.33 0.30 1.44 0.20 1.14 0.83 107 cM  
D2S2968 1.35 0.15 1.90 0.20 1.27 0.66 251 cM  
D4S2397 1.28 0.15 1.11 0.20 1.17 2.05 42 cM  
D4S3248 1.01 0.15 NEG  0.50 1.31 72 cM  
D4S400 1.24 0.15 2.65 0.15 1.67 0.49 88 cM  
D4S1554 1.31 0.15 NEG  1.35 NEG 192 cM  
D5S644 0.71 0.20 1.26 0.30 1.27 NEG 104 cM  
D5S422 1.62 0.15 1.22 0.20 1.08 0.41 164 cM  
D6S287 1.05 0.15 1.12 0.20 0.73 NEG 122 cM  
D8S136 1.49 0.15 NEG  0.01 1.02 44 cM  
D8S534 1.66 0.15 NEG  0.55 NEG 154 cM  
D9S281 1.49 0.20 1.05 0.20 1.29 0.59 15 cM  
D10S1208 0.56 0.20 1.02 0.20 0.02 NEG 63 cM  
D11S4131 0.52 0.30 1.04 0.30 1.08 0.56 138 cM  
D12S373 1.36 0.10 NEG  0.01 1.29 36 cM  
D14S72 2.07 0.10 1.16 0.20 1.18 2.42 9 cM  
D14S742 2.75 0.05 3.03 0.15 3.52 2.27 12 cM  
D14S283 1.41 0.15 1.41 0.20 0.88 2.16 14 cM  
D14S608 0.68 0.20 1.21 0.20 0.83 0.52 28 cM  
D14S1279 1.01 0.15 NEG  NEG NEG 87 cM  
D15S165 1.27 0.15 0.70 0.30 0.61 0.28 20 cM  
D15S87 0.96 0.20 2.00 0.20 0.93 0.42 116 cM  
D17S926 2.11 0.10 2.23 0.15 2.42 1.40 1 cM  
D17S801 1.92 0.15 1.86 0.20 1.40 2.13 103 cM  
D19S414 2.27 0.10 3.11 0.15 2.66 1.40 53 cM  
 Two-point LOD scores    Sibpair NPL Map**  
 Dominant model  Recessive model  MLS    
Marker Lod  Theta Lod Theta     
D2S391 1.05 0.15 1.44 0.20 1.16 0.42 74 cM  
D2S441 2.11 0.15 0.95 0.20 0.92 0.23 90 cM  
D2S2232 0.33 0.30 1.44 0.20 1.14 0.83 107 cM  
D2S2968 1.35 0.15 1.90 0.20 1.27 0.66 251 cM  
D4S2397 1.28 0.15 1.11 0.20 1.17 2.05 42 cM  
D4S3248 1.01 0.15 NEG  0.50 1.31 72 cM  
D4S400 1.24 0.15 2.65 0.15 1.67 0.49 88 cM  
D4S1554 1.31 0.15 NEG  1.35 NEG 192 cM  
D5S644 0.71 0.20 1.26 0.30 1.27 NEG 104 cM  
D5S422 1.62 0.15 1.22 0.20 1.08 0.41 164 cM  
D6S287 1.05 0.15 1.12 0.20 0.73 NEG 122 cM  
D8S136 1.49 0.15 NEG  0.01 1.02 44 cM  
D8S534 1.66 0.15 NEG  0.55 NEG 154 cM  
D9S281 1.49 0.20 1.05 0.20 1.29 0.59 15 cM  
D10S1208 0.56 0.20 1.02 0.20 0.02 NEG 63 cM  
D11S4131 0.52 0.30 1.04 0.30 1.08 0.56 138 cM  
D12S373 1.36 0.10 NEG  0.01 1.29 36 cM  
D14S72 2.07 0.10 1.16 0.20 1.18 2.42 9 cM  
D14S742 2.75 0.05 3.03 0.15 3.52 2.27 12 cM  
D14S283 1.41 0.15 1.41 0.20 0.88 2.16 14 cM  
D14S608 0.68 0.20 1.21 0.20 0.83 0.52 28 cM  
D14S1279 1.01 0.15 NEG  NEG NEG 87 cM  
D15S165 1.27 0.15 0.70 0.30 0.61 0.28 20 cM  
D15S87 0.96 0.20 2.00 0.20 0.93 0.42 116 cM  
D17S926 2.11 0.10 2.23 0.15 2.42 1.40 1 cM  
D17S801 1.92 0.15 1.86 0.20 1.40 2.13 103 cM  
D19S414 2.27 0.10 3.11 0.15 2.66 1.40 53 cM  

The lod score listed is the maximum lod score obtained for the analysis, and was obtained at the value of theta shown.

**The map location indicates the position of the marker in centimorgans from the top of the relevant chromosome based on the Marshfield genetic maps (www.marshmed.org ).

NEG = negative.

Table 3.

Regions showing evidence for POAG linkage (pedigree sets I and II)

 Two-point LOD scores    Sibpair   
 Dominant model  Recessive model  MLS   
Marker Lod Theta Lod Theta    
D2S441 3.50 0.10 3.05 0.20 2.67   
D2S2232 0.25 0.30 0.84 0.20 0.83   
D6S287 1.6 0.15 1.2 0.30 0.80   
D9S1850 1.1 0.15 0.82 0.30 0.65   
D11S1307 1.19 0.20 1.90 0.20 1.66   
D14S122 2.49 0.15 1.26 0.30 1.41   
D14S72 2.12 0.15 1.27 0.30 1.16   
D14S742 3.99 0.05 3.54 0.20 3.68   
D14S283 1.12 0.20 1.21 0.30 0.84   
D17S926 3.61 0.10 3.62 0.20 2.73   
D17S801 2.47 0.15 2.40 0.20 2.45   
D19S414 0.64 0.20 0.67 0.30 0.58   
D19S408 2.45 0.10 1.74 0.20 2.17   
D19S412 0.25 0.30 0.12 0.30 0.13   
D19S246 1.98 0.20 0.76 0.30 0.75   
 Two-point LOD scores    Sibpair   
 Dominant model  Recessive model  MLS   
Marker Lod Theta Lod Theta    
D2S441 3.50 0.10 3.05 0.20 2.67   
D2S2232 0.25 0.30 0.84 0.20 0.83   
D6S287 1.6 0.15 1.2 0.30 0.80   
D9S1850 1.1 0.15 0.82 0.30 0.65   
D11S1307 1.19 0.20 1.90 0.20 1.66   
D14S122 2.49 0.15 1.26 0.30 1.41   
D14S72 2.12 0.15 1.27 0.30 1.16   
D14S742 3.99 0.05 3.54 0.20 3.68   
D14S283 1.12 0.20 1.21 0.30 0.84   
D17S926 3.61 0.10 3.62 0.20 2.73   
D17S801 2.47 0.15 2.40 0.20 2.45   
D19S414 0.64 0.20 0.67 0.30 0.58   
D19S408 2.45 0.10 1.74 0.20 2.17   
D19S412 0.25 0.30 0.12 0.30 0.13   
D19S246 1.98 0.20 0.76 0.30 0.75   

The lod score listed is the MLS obtained for the analysis, and was obtained at the value of theta shown. Several additional markers not part of the first stage of the genome screen (Table 2) were included in this analysis: D14S122, D19S408, D19S412 and D19S246. D2S441 and D2S2232 are located within a 30 cM interval and are in the vicinity of markers that define a previously identified glaucoma locus (GLC1B). Markers D14S122, D14S72, D14S742 and D14S283 are located within a 12 cM interval, and markers D19S414, D19S408, D19S412 and D19S246 are located within an 18 cM interval. Markers D17S926 and D17S801 are separated by > 50 cM.

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