Localization of a Locus (GLC1B) for Adult-Onset Primary Open Angle Glaucoma to the 2cen–q13 Region

GENOMICS

36, 142–150 (1996) 0434

ARTICLE NO.

Localization of a Locus (GLC1B) for Adult-Onset Primary Open Angle Glaucoma to the 2cen–q13 Region DILIANA STOILOVA,* ANNE CHILD,† OVIDIU C. TRIFAN,* R. PITTS CRICK,‡ ROGER L. COAKES,§ AND MANSOOR SARFARAZI*,1 *Surgical Research Center, Department of Surgery, University of Connecticut Health Center, Farmington, Connecticut 06030-1110; †Department of Cardiological Sciences, St. George’s Hospital Medical School, London, United Kingdom; and ‡International Glaucoma Association and §Glaucoma Unit, Department of Ophthalmology, King’s College Hospital, London, United Kingdom Received March 26, 1996; accepted June 13, 1996

Primary open angle glaucoma (GLC1) is a common ocular disorder with a characteristic degeneration of the optic nerve and visual field defects that is often associated with an elevated intraocular pressure. The severe but rare juvenile-onset type has previously been mapped to 1q21–q31, and its genetic heterogeneity has been established. Herein, we present a new locus (GLC1B) for one form of GLC1 on chromosome 2cen–q13 with a clinical presentation of low to moderate intraocular pressure, onset in late 40s, and a good response to medical treatment. Two-point and haplotype analyses of affected and unaffected meioses in six families provided maximum linkage information with D2S417, GATA112EO3, D2S113, D2S373, and D2S274 (lod scores ranging from 3.11 to 6.48) within a region of 8.5 cM that is flanked by D2S2161 and D2S2264. Analysis of affected meioses alone revealed no recombination with an additional two markers (D2S2264 and D2S135) in a region of 11.2 cM that is flanked by D2S2161 and D2S176. Analysis of unaffected meioses identified only one healthy 86-year-old male who has inherited the entire affected haplotype and, hence, is a gene carrier for this condition. Eight additional families with similar and/or different clinical presentation did not show any linkage to this region and, therefore, provided evidence for genetic heterogeneity of adultonset primary open angle glaucoma. q 1996 Academic Press, Inc.

INTRODUCTION

Glaucoma is a group of ocular disorders that is generally characterized by a typical degeneration of the optic nerve. This condition is one of the leading causes of blindness worldwide (Thylefors and Negrel, 1994). The insidious type, primary open angle glaucoma (gene symbol GLC1, MIM No. 137760), is the most prevalent 1 To whom correspondence should be addressed. Telephone: (860) 679-3629. Fax: (860) 679-2451. E-mail: [email protected].

0888-7543/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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form in Western societies (Leske, 1983; Thylefors and Negrel, 1994). This disease presents with variable onset usually after the age of 40, normal open anterior chamber angle, and characteristic atrophy of the optic nerve resulting in visual field loss and eventual blindness (Quigley, 1993). Many affected subjects have elevated intraocular pressures (IOP). Although this is no longer an obligatory criterion for diagnosis, it is still regarded as a major risk factor for this condition. Furthermore, about half of confirmed open angle glaucoma patients have a ‘‘screening’’ intraocular pressure within the statistically normal level. Nevertheless, about twothirds of these apparently low-tension glaucoma eyes at some time on follow up have pressures above this level, but the majority of patients with primary open angle glaucoma have intraocular pressure at or below 25 mmHg (Sommer et al., 1991; Hitchings, 1992; Tuck and Crick, 1992; Grosskreutz and Netland, 1994). Those with intraocular pressure constantly below 22 mmHg are referred to as having ‘‘low or normal tension glaucoma (LTG or NTG)’’ or sometimes ‘‘normal pressure glaucoma (NPG).’’ Based on the age of onset and differences in presentation of the phenotype, GLC1 has been arbitrarily divided into two major groups. Juvenile-onset glaucoma (gene symbol GLC1A) usually manifests itself in late childhood and early adulthood (Goldwyn et al., 1970). Typically this form is severe with high intraocular pressure, rapid progression of the condition, and poor response to medical treatment, usually requiring ocular surgery (Ellis, 1948; Johnson et al., 1993). On the other hand, the late-onset form has a milder presentation, gradual development, and slight to moderate elevation of IOP, and medical treatment often yields a satisfactory outcome providing that it is adequately and regularly monitored with appropriate modification of treatment (Vogel et al., 1990; Jay and Murdoch, 1993). As the disease proceeds gradually and painlessly, many individuals may not be aware of being affected until a very late stage when irreversible damage to the optic nerve has occurred. This, together

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with the late onset and highly variable nature of this phenotype, has complicated the genetic classification of this disorder. One other major risk factor for developing glaucoma is the family history (Shin, 1977; Miller, 1978; Lichter, 1994). Inherited forms of glaucoma have been described as X-linked (Netland et al., 1993), autosomal recessive (Beiguelman and Prado, 1963), autosomal dominant (Crombie and Cullen, 1964; Lee et al., 1985; Bennett et al., 1989), multifactorial (Jay and Peterson, 1970; Lichter, 1994), and glaucomas that are associated with other ocular abnormalities (Netland et al., 1993). Using a single large family with autosomal dominant juvenile-onset GLC1, Sheffield et al. (1993) assigned a locus for this condition (GLC1A) to the 1q21–q31 region. Shortly after that, linkage was confirmed in other pedigrees (Richards et al., 1994; Wiggs et al., 1994; Meyer et al., 1994; Graff et al., 1995). Genetic heterogeneity for this locus has been reported (Graff et al., 1995; Wiggs et al., 1995). In a more recent publication, Morissette et al. (1995) reported linkage to the GLC1A locus in a multigeneration Canadian family segregating for both juvenile and late-onset glaucoma. The authors concluded that the GLC1A gene is responsible for both forms of GLC1 phenotypes. Although it is possible that due to the variable expression of the GLC1A gene the disease manifests itself in a broader range of onset, it does not, however, imply that juvenile and adult-onset GLC1 are caused by the same gene. In fact, there are now considerable data suggesting that the adult-onset type of glaucoma is not linked to the same DNA markers on 1q21–q31 (Seghatoleslami et al., 1994; Wirtz et al., 1995; Richards et al., 1996; Wiggs et al., 1996). Moreover, as there is a considerable delay between the true age of onset as compared to the age of diagnosis at the time of presentation, the numeric onset value reported in each individual member of pedigrees is not reliable, and it is more helpful to report the mean age of onset/detection for each individual pedigree. Therefore, the arbitrary division of the GLC1 families to juvenile and adult-onset that is based merely on the age of onset/detection is not reliable differentiation between these two phenotypes. It would be more accurate to provide a classification that is based on the IOP values reported at the time of presentation, severity of the condition, nature of disease progression, response to medical treatments, and necessity of surgical intervention to control the advancement of the disease. In addition to the GLC1A locus, the chromosomal location of only one other form of glaucoma has thus far been identified. We have recently mapped a new locus (GLC3A) for primary congenital glaucoma (buphthalmos) to the 2p21 region and reported genetic hetereogeneity for this condition (Sarfarazi et al., 1995). More recently, we assigned a second locus (GLC3B) for the primary congenital type to the 1p36 region (Akarsu et al., 1996a), but as yet there are other families that are not caused by either the GLC3A or the GLC3B loci (Akarsu

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et al., 1996b). Therefore, the data published so far indicate that at minimum there are two loci for juvenileonset GLC1 and an additional three loci for the congenital type of glaucoma. Herein we present clinical, linkage, and haplotype data from a study of GLC1 families and report the assignment of a new locus for one form of adult-onset primary open angle glaucoma to the 2cen–q13 region. We also provide evidence for the existence of at least one more locus for adult-onset GLC1 on a different part of the genome. Therefore, there is now evidence for the existence of at least seven different loci for these three types of glaucoma. Many more loci are expected to exist in the genome, especially for adult-onset GLC1. MATERIALS AND METHODS Family panel. Our family panel either has been ascertained through the glaucoma registries of King’s College Hospital London (KCH) or the database of the International Glaucoma Association (IGA) or has been referred to us by other ophthalmologists. The two registries have been in operation at the glaucoma unit of the KCH since 1969 (Crick, 1974; Crick and Blackmore, 1979). The KCH/IGA computer databases contain information on visual acuity, tonometry, gonioscopy, visual fields, and many other data that are standardized and recorded numerically. The visual fields in this database are recorded as a parabolic projection by a modification of the Friedmann Visual Field Analyzer. Of 1786 GLC1 entries in the database, 1021 are registered to have chronic simple glaucoma. Family history is recorded for a total of 559 people. However, the actual number of families will be smaller because the sample includes members of the same family found as the result of screening for hereditary tendency. One of the unique features of this database is the fact that annually the affected members of the families have been monitored for the past 25 years at KCH by the same ophthalmologists (R.P.C. and R.L.C.). Additionally, the unaffected members of these families are aware of their genetic risk; therefore, they are regularly being examined by an eye specialist. The KCH/IGA registries have been subjected to a variety of analyses providing clinical data for publication of over 30 papers in the peer reviewed journals (Crick, 1979; Crick and Daubs, 1980; Crick et al., 1983, 1989; Vogel et al., 1990). Of the 89 families collected so far, 61 were ascertained through this source alone. The remaining families were either referred to us by other ophthalmologists or have responded to the invitation to participate in the study given at the several Annual General Meetings of the IGA and in the subsequent newsletters sent to 12,500 IGA members. The ophthalmologist responsible for the primary care of each of the affected member in these pedigrees was contacted to ensure accurate diagnosis of the affected members utilizing the same diagnostic criteria as is currently being used for GLC1 at King’s College Hospital. Detailed clinical information on the age of onset/diagnosis, intraocular pressure before treatment, stages of visual field loss, status of the optic nerve damage, type of current and past eye surgery, current eye medication, type and primary/secondary nature of glaucoma, and other associated clinical findings of each individual member of our GLC1 pedigrees has also been obtained. Ascertainment of another 47 families from Gambia and the US is currently in progress. For the purpose of positional mapping, we selected a group of 17 pedigrees as our initial screening panel, that met the following criteria: (1) All affected members had bilateral primary open angle glaucoma and were diagnosed in early to late adulthood without any other associated abnormalities; (2) The phenotype transmitted as an autosomal dominant trait in their respective pedigrees; (3) Kindreds consisted of at least 2–3 affected offspring and one living parent; (4) All families are Caucasian and ascertained from the UK (except one). This panel consists of a total of 203 subjects (90 affected), providing

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a total of 96 informative meioses, 48 of whom are affected. Twelve families have a three or more generation structure. The statuses of both affected and normal members of these families have been confirmed with their respective ophthalmologists using the following uniform diagnostic criteria: (1) intraocular pressure equal or more than 22 mmHg; (2) characteristic appearance of the optic disc; and (3) characteristic loss of visual fields. Subjects with confirmed lowtension glaucoma were also classified as affected. Members that met only one diagnostic criterion were grouped as suspects and were not included in our initial genome-wide search and linkage analysis. However, when the family of such individuals showed any indication of linkage, subsequently they were genotyped for the DNA markers, and their clinical status was coded as unknown for the purpose of linkage analysis. DNA study. Blood (10–20 ml) was collected by venipuncture from 155 willing members of these families, and DNA was extracted as described elsewhere (Richards, 1994). We selected a group of highly polymorphic short tandem repeat polymorphisms (STRP) with a firm genetic linkage map location and used multiplex polymerase chain reaction for amplification. The information on primer sequences, band sizes, and number of alleles was obtained from the Genome Data Base (GDB; Fasman et al., 1994), Ge´ne´thon (Gyapay et al., 1994; Dib et al., 1996), Utah Marker Development Group (1995), and Cooperative Human Linkage Center (CHLC; Murray et al., 1994; Sheffield et al., 1995; Gastier et al., 1995). Amplifications were performed in a 25-ml reaction volume containing 100 ng genomic DNA, 50 pM each primer, 200 mM each dNTP, 50 mM KCl, 10 mM Tris (pH 8.4), 1.5 mM MgCl2 , 0.01% gelatin, and 0.5 U Taq polymerase (AmpliTaq, Perkin–Elmer). Amplifications were carried out in a Gene Amp 9600 thermocycler (Perkin–Elmer) under the following conditions: initial denaturation for 3 min at 947C, 30–35 cycles of 5s denaturation at 947C and 30-s annealing at 55–607C, followed by a final extension at 727C for 3 min. The amplified products were separated by electrophoresis on 7% denaturing acrylamide gels, visualized by silver staining (Bassam and Caetano-Annoles, 1993), photographed, and subsequently genotyped. Linkage analysis. The data obtained from the genotyping of STRPs were entered into a dedicated computer program (DMS; unpublished), checked for data errors and inconsistencies, and prepared for the LINKAGE program (Lathrop and Lalouel, 1984; Schaffer et al., 1994). Two-point linkage was calculated with the MLINK module of the LINKAGE package. All of the calculations were made under the assumption of an autosomal dominant trait. Due to the late onset of this disease and lack of reliable penetrance ratios and to eliminate the effect of possible incomplete penetrance, the lod score calculations were repeated for the affected meioses only. The heterogeneity tests were carried out with the HOMOG program (Ott, 1983).

RESULTS

Exclusion Mapping We have used 11 microsatellite markers from the 1q21–q31 region to test all the families in our initial screening panel for possible linkage to the GLC1A locus. The obtained lod scores were significantly negative for DNA markers from this region. For example, the exclusion area for markers D1S210 and D1S452 is about 12 cM (lod score of 03.46). Construction and inspection of haplotypes from this region also confirmed that the GLC1 phenotype in these families is not linked to the juvenile-onset glaucoma locus at the 1q region (Seghatoleslami et al., 1994, 1995; Stoilova et al., 1995). Linkage Analysis After genotyping a total of 215 STRP markers and excluding a total of 25 candidate gene regions (includ-

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ing 17 intragenic markers), an allele from D2S436 was found to segregate with this phenotype in six of our families. Additional STRPs from either side of the chromosome 2 centromere were used to saturate the region. A significant lod score of over 3.0 was obtained for a total of 12 STRP markers (Table 1). To account for possible bias due to the existence of incomplete penetrance and through the contribution of normal subjects who may be asymptomatic gene carriers, lod score calculations were repeated for the affected meioses only. As shown in Table 1 and as expected, the lod score values were reduced, but they are still statistically significant (i.e., lod scores ú3.0). Two-point linkage of each individual pedigree from the initial screening panel indicated that eight families are not linked to this region of chromosome 2. The results from three other families were inconclusive, and they are currently under investigation. Therefore, we designated the locus on this region of chromosome 2 GLC1B, and further analysis was performed only for those families that showed linkage to this region of chromosome 2. As there is no accurate cytogenetic mapping for the STRP markers used in this study, the exact site of the GLC1B locus in relation to the centromere remains to be determined. However, two of the STRP markers in this region have been mapped to 2p13–q21 (i.e., D2S113) and p13–q13 (i.e., D2S160). The maximum linkage information obtained in this study is with D2S113, a marker tightly linked to D2S25 on 2q11.1. We also observed affected recombination with D2S1892, a marker tightly linked to D2S160. Therefore, it is likely that the GLC1B locus is on the long arm and is located in the region 2cen–q13. Clinical Observation of the Linked Families A brief description of the clinical findings in the living affected individuals of the linked families is listed in Table 2. The phenotype of glaucoma patients presented here is generally homogeneous and very distinct from that reported to be linked to the GLC1A locus on 1q. The mean age of onset/diagnosis in this group of families was 47 years. The intraocular pressure was slightly elevated but in about half of the patients it was within a statistically normal range. Fifty percent of the affected subjects had intraocular pressures of õ22 mmHg, and the remaining had moderate pressures between 22 and 30 mmHg. Only one subject was identified in this group of families who had pressure of ú30 mmHg. In 54% of the affected subjects, medical treatment yielded a satisfactory result that required no surgery to control the course of glaucoma. The GLC1B families that are linked to this region of chromosome 2 may include a group of specific kindreds that are sometimes referred to as ‘‘low or normal tension glaucoma.’’ However, as stated above, these families have a combination of both normal and moderate tension group of patients in each distinct kindred. As presented

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TABLE 1 Two-Point Lod Scores between the GLC1B Locus and 16 DNA Markers on Chromosome 2 All meioses

u

D2S428 D2S2161 D2S417 GATA112E03 D2S113 D2S373 D2S274 D2S2264 D2S135 D2S176 D2S1897 D2S436 D2S1890 D2S1784 D2S340 D2S1892

Affected meioses only

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

Zmax

umax

Zmax

umax

0` 0` 3.11 4.88 6.48 5.36 5.86 0` 0` 0` 0` 0` 0` 0` 0` 0`

3.95 3.61 2.69 4.39 5.81 4.78 5.26 5.12 2.98 2.07 4.16 3.77 4.03 0.77 2.38 3.85

3.68 3.55 2.28 3.87 5.12 4.17 4.63 4.69 2.78 2.04 3.87 3.49 3.71 1.09 2.47 3.75

3.26 3.22 1.90 3.32 4.41 3.55 3.98 4.13 2.44 1.86 3.44 3.07 3.25 1.14 2.29 3.38

2.77 2.77 1.53 2.76 3.67 2.92 3.32 3.50 2.05 1.62 2.95 2.59 2.71 1.07 2.01 2.89

2.24 2.25 1.18 2.19 2.92 2.28 2.65 2.82 1.63 1.35 2.42 2.08 2.15 0.94 1.67 2.34

1.68 1.70 0.85 1.63 2.16 1.66 1.98 2.13 1.21 1.07 1.88 1.57 1.58 0.76 1.30 1.76

1.13 1.15 0.55 1.11 1.45 1.09 1.35 1.46 0.81 0.78 1.34 1.07 1.03 0.55 0.93 1.20

0.62 0.65 0.30 0.64 0.81 0.59 0.78 0.84 0.47 0.49 0.84 0.63 0.56 0.34 0.57 0.69

0.23 0.25 0.11 0.26 0.31 0.22 0.33 0.34 0.20 0.23 0.38 0.27 0.21 0.15 0.25 0.28

3.96 3.64 3.11 4.88 6.48 5.36 5.86 5.18 2.98 2.09 4.18 3.78 4.05 1.14 2.48 3.87

0.04 0.07 0.00 0.00 0.00 0.00 0.00 0.03 0.04 0.06 0.04 0.04 0.04 0.14 0.08 0.06

0.46 0.51 1.01 2.57 2.84 3.40 2.28 3.11 2.35 0.72 1.60 1.23 2.59 1.14 2.29 1.22

0.10 0.14 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.10 0.06 0.07 0.00 0.07 0.00 0.11

Note. Zmax , maximum lod score; umax , maximum recombination fraction.

TABLE 2 Brief Description of Clinical Findings in Six GLC1B Linked Families

Sex

Age of diagnosis

Highest IOP (mm Hg)

5-II-1 5-II-3 5-III-1 5-III-2

F M F F

43 55 45 43

11/11 Normal 13/12 11/16

29-I-1 29-II-1 29-II-2

F M F

64 57 45

38-I-1 38-II-1 38-II-4

F F F

39-II-4 39-II-6 39-II-8 39-III-2 39-III-6 39-IV-1 39-IV-2

Case

Stage of OND at diagnosis

VFL (Yes/no)

Treatment

Late Late Early Early

Yes Yes Yes Yes

Nil Timolol Maleate Timolol Maleate Timolol Maleate

23/23 25/27 21/21

Early Medium Early

Yes Yes Yes

Trabeculectomy Trabeculectomy Trabeculectomy

58 45 40

28/28 23/24 23/23

Medium Early Early

Yes Yes Yes

Trabeculectomy Timolol Maleate Trabeculectomy

M F M F F F F

54 66 44 36 45 25 20

18/18 20/20 23/25 18/18 23/22 24/26 23/25

Late Medium Early Late Early Medium Early

Yes Yes Yes Yes Yes Yes Yes

Nil Timolol Maleate Timolol Maleate Trabeculectomy Betaxolol hydrochloride Trabeculectomy Trabeculectomy

54-I-2 54-II-1 54-II-2

M M M

68 48 45

21/21 30/28 29/28

Early Early Early

Yes No Yes

Trabeculectomy Timolol Maleate Timolol Maleate

86-III-1 86-III-3 86-III-4 86-III-5

M F F F

63 40 45 38

18/20 19/19 34/31 18/18

Early Medium Medium Medium

Yes Yes Yes Yes

Betaxolol hydrochloride Laser trabeculoplasty Iridectomy Betaxolol hydrochloride

Note. IOP, intraocular pressure; OND, optic nerve damage; VFL, visual field loss.

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here, this type of GLC1 families also showed genetic heterogeneity at the GLC1B locus. Other families with a similar phenotype but with an earlier age of onset have been described and catalogued as autosomal dominant low-tension glaucoma (Bennett et al., 1989). As this type of GLC1 may account for about 20% of confirmed cases (Sommer et al., 1991; Hitchings, 1992; Tuck and Crick, 1992; Grosskreutz and Netland, 1994), the GLC1B locus described here may also potentially be a major locus for this inherited form of adult-onset primary open angle glaucoma. Test for Genetic Homogeneity The two-point linkage obtained in the linked and unlinked families was used to test for genetic homogeneity using an admixture test that has previously been described (Ott, 1983). To maximize the use of linkage information, we first manually haplotyped D2S417 with GATA112EO3 and D2S1897 with D2S436. Their combined lod scores were calculated with the MLINK module of the LINKAGE program and subsequently used in the HOMOG program to test for genetic homogeneity. Statistically significant evidence for the presence of heterogeneity was obtained with markers D2S1897/D2S436 (P Å 0.0033; likelihood test ratio L(r) Å 40.04) but not with the D2S417/GATA112EO3 haplotypes (P Å 0.1419; L(r) Å 1.78) as previously anticipated by the haplotype analysis.

loss or any detectable damage to the optic nerves. This individual does not have any offspring and, therefore, his status as an asymptomatic gene carrier remains unknown. In the same family we detected an unaffected individual (III-5) who carries the normal portion of the chromosome for D2S135 and all the markers below it, but is recombinant for D2S113 and all the markers above it. His affected parent (II-8) is homozygote for the marker D2S373 and, therefore, the precise position of the crossover remains unknown. Although this individual is 52 years old, which is well past the earliest and the mean age of onset/diagnosis for the affected members of his family, he may still develop glaucoma in the future. On other hand, he may be another case of an asymptomatic gene carrier. For the purpose of linkage analyses the statuses of these individuals were coded as unknown. DISCUSSION

Primary open angle glaucoma is a group of common ocular disorders presenting with a characteristic degeneration of the optic nerve and visual field loss and often associated with an elevated intraocular pressure. A severe but relatively uncommon form of this condition known as the juvenile-onset type (i.e., GLC1A) has already been mapped to the 1q21–q31 region (Sheffield et al., 1993; Richards et al., 1994; Meyer et al., 1994; Wiggs et al., 1994), and genetic heterogeneity has been reported for it (Graff et al., 1995; Wiggs et al., 1995). Haplotype Analysis However, the chromosomal location of the most prevaWe used a total of 16 polymorphic markers to estab- lent form of this condition with a later age of onset has lish the smallest region that cosegregates with the remained unknown. We used a combination of candiGLC1B phenotype in our pedigrees. The information on date gene/region and a general positional mapping the marker positions was obtained from the Ge´ne´thon, strategy and identified a locus (GLC1B) for one form Utah, and CHLC maps. Haplotypes were created based of later onset primary open angle glaucoma on chromoon the assumption of a minimum number of recombina- some 2. Six families providing a total of 31 informative tion events (Fig. 1). A summary of the observed recom- meioses (16 affected) were linked to a group of STRP binations in the affected members of these families is markers that are located on the 2p11–2q13 region. The presented in Fig. 2. By constructing the haplotypes, we highest lod score was obtained with the D2S113 detected four critical crossovers in the affected individ- marker (Z Å 6.48). Considering the recombination uals. The recombinations in families 39 (Fig. 1, person events only in the affected subjects places the GLC1B IV-2) and 86 (III-3; not shown) placed the disorder be- locus on an 11.2-cM region that is flanked by the low the D2S2161 marker, while a second cross over in D2S2161 and D2S176 markers. Based on the cytogefamily 39 (Fig. 1, person III-6) positioned the GLC1B netic localization of the markers studied, it is likely locus above the D2S176 marker and within an esti- that the GLC1B locus is located in the region 2cen– q13. The clinical observation in the linked families remated region of about 11.2 cM. vealed a phenotype very distinct from that of GLC1A. The disorder is less severe, appears later in life, and Gene Carriers responds better to medical treatment than the GLC1A Two-point linkage and inspection of haplotype data phenotype. When compared with the phenotype of the identified only one normal individual who has inher- kindreds that are not linked to this region of chromoited the entire affected chromosome from his affected some 2, only one major difference was noted: the prevaparent. This subject is an 86-year-old male (Fig 1, fam- lence of glaucoma patients with normal intraocular ily 39, person II-3) whose normal clinical status has pressure in the linked families. Therefore, the region been confirmed by an recent complete ophthalmologi- reported here may be a locus for cases of primary open cal examination. His highest recorded IOP is 16 angle glaucoma that include those with moderately mmHg in both eyes with no evidence of visual field raised intraocular pressures and those with so-called

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FIG. 1. Example pedigree segregating for the GLC1B locus. Genotypic data for eight markers from the 2p11–q13 region are shown below each family member. The solid bars represent the inherited affected chromosome, the open bars the normal chromosome, and the thin black line the uninformative portion of the chromosome.

‘‘low/normal tension glaucoma,’’ as half of the affected patients in the linked families have intraocular pressure within the normal range. This is what might be expected to result from the interaction of the two continuous variables as encountered above. However, such a conclusion remains to be determined in the future after other kindreds with similar and different phenotypes are tested for linkage to the locus identified in this study. We observed only one normal individual who has inherited the entire affected chromosome from his affected parent. As this individual does not have any affected offspring and, therefore, there is no proof of a skipped generation, his clinical status as an asymptomatic gene carrier remains to be determined when the causative mutation in his family has been identified. Furthermore, he may well be a gene carrier for the GLC1B locus, as incomplete penetrance is known to exist in this group of conditions; it has already been reported for the juvenile-onset form of GLC1 (Johnson et al., 1993; Sheffield et al., 1993). The region containing this new GLC1B gene on 2cen–q13 is part of a large DNA segment that contains a number of functional genes. There are also at least six overlapping contigs within the GLC1B-flanking markers that collectively encompass at least 34 STRPs, 18 random DNA markers (STS), 17 expressed sequences (EST), and 177 different overlapping yeast ar-

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tificial chromosome (YAC) clones (Hudson et al., 1995). The region is estimated to be around 8–17 cM. Based on the radiation hybrid (RH) map, this region is approximately 147 cR. The current estimate of the RH map is 3.7 cR/Mb for the entire human genome; therefore, the GLC1B candidate region may contain around 40 Mb of DNA. However, this estimate may not be accurate, as both retention rates in RH mapping and recombination rates in linkage mapping are significantly different for the centromeric region of most of the chromosomes. A partial list of candidate genes in this region of chromosome 2 includes RALB (ras-like protein b; Hsieh et al., 1990), ADRA2B (a-2b-adrenergic receptor; Lomasney et al., 1990), and PAX8 (paired box homeotic gene-8; Stapleton et al., 1993). Other genes loosely mapped to this region include IL1A (interleukin 1a; Webb et al., 1986), IL1B (interleukin 1b; Webb et al., 1986), their receptors IL1RA and IL1RB (Copeland et al.,1991), NCL (nucleolin; Srivastava et al., 1990), CD8 antigen (Bowcock et al., 1986) and EN1 (engrailed-1; Kohler et al., 1993), among others. Therefore, establishing the boundaries of a region containing the GLC1B gene would be crucial before any attempt could be made to search for mutations in the known or unknown genes in the region. To achieve this, we are currently using a combination of newly released Ge´ne´thon CA-repeat markers to saturate the region and the ‘‘GeneBridge 4

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FIG. 2. Map order of STRPs from the 2p11–q13 region: on the right side of the map, the Ge´ne´thon markers and their genetic distances are from Dib et al. (1996); on the left side are the CHLC and Utah markers. The 16 markers used in our study are underlined. Observed recombinations in four affected individuals are shown. The inherited affected chromosome is represented by a solid black bar, the normal chromosome with a hatched bar, and the uninformative portion of the chromosome by a thin line.

Radiation Hybrid Panel’’ to identify potential genes that map within the GLC1B candidate region. We also intend to identify the minimum sized contigs (i.e., YAC and BAC clones) that are expected to cover the region containing the GLC1B gene. Future plans include expanding this initial mapping to other GLC1 families with similar or distinct phenotype presentation.

locus (GLC3B) for primary congenital glaucoma (Buphthalmos) maps to the 1p36 region. Hum. Mol. Genet., in press. Akarsu, A. N., Turacli, M. E., Aktan, S. G., Hossain, A., BarsoumHomsy, M., Chevrette, L., Sayli, B. S., and Sarfarazi, M. (1996b). Exclusion of primary congenital glaucoma from two candidate regions of chromosome arm 6p and chromosome 11. Am. J. Med. Genet. 61: 290–292. [Erratum 62: 102]. Bassam, B. S., and Caetano-Annoles, G. (1993). Silver staining of DNA in polyacrylamide gels. Protocols Biotechnol. 42: 181–188.

ACKNOWLEDGMENTS

Beiguelman, B., and Prado, D. (1963). Recessive juvenile glaucoma. J. Genet. Hum. 12: 53–54.

We thank Sarah Child, Ursula Kalichevsky, and Glen Brice for assistance in compilation of the clinical database and the families and their ophthalmologists and opticians for participating in this study. A.C. and M.S. gratefully acknowledge the continuous financial support of the International Glaucoma Association (IGA-G249) over the past few years. This work was also supported by grants from the National Eye Institute (EY-09947) and the University of Connecticut General Clinical Research Center (M01-RR-06192) to M.S.

Bennett, S. R., Alward, W. L., and Folberg, R. (1989). An autosomal dominant form of low tension glaucoma. Am. J. Ophthalmol. 108: 238–244.

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