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Contract grant sponsor: INSERM; Contract grant sponsor: Association pour la Recherche en Genetique Ophtalmologique; Contract grant sponsor: GREG-genome; Contract grant sponsor: Mutuelle Generale de lEducation Nationale.
*Correspondence to: Henri-Jean Garchon, INSERM U25, Hopital Necker, 161 rue de Se`vres, 75743 Paris, Cedex 15, France.
E-mail: garchon@necker.fr
Received 10 September 1997; Accepted 12 December 1997
KEY WORDS: o p e n - a n g l e g l a u c o m a ;
GLC1A; unaffected carriers;
founder effect
INTRODUCTION
Primary open-angle glaucoma (POAG) is a highly
prevalent optic neuropathy and a leading cause of irreversible blindness in industrialized countries [Leske,
1983]. Its definition associates characteristic cupping
of the optic disc and alteration of the visual field [Quigley, 1993]. Intraocular pressure (IOP) is often elevated
and is a major risk factor [Quigley, 1993]. Prognosis is
strongly dependent on diagnosis at an early stage,
when treatment can prevent irreversible lesions of the
optic nerve [Kass et al., 1989].
Family history is another major risk factor of POAG
[Leske, 1983]. The juvenile-onset form of POAG is often
inherited in an autosomal dominant mode [Johnson et
al., 1993] and a locus, termed GLC1A, has been
mapped to chromosome 1q21-q31 [Sheffield et al.,
1993]. The GLC1A region was progressively restricted
to a 3-cM interval, corresponding to a physical size of 3
Mbases [Belmouden et al., 1997]. Recently, the Trabecular Meshwork Induced Glucocorticoid Response Protein (TIGR) gene was identified as the GLC1A gene
responsible for the disease in several American families and in unrelated POAG patients [Stone et al.,
1997]. Besides its importance for understanding the
pathogenesis of glaucoma, this discovery has major
clinical implications. Direct detection of GLC1A mutations should facilitate the identification of GLC1Alinked families, regardless of their size, and the screening of mutation carriers. This is of particular importance as the risk of developing a severe glaucomatous
optic neuropathy was shown to be greater in GLC1Alinked than in GLC1A-unlinked families [Brezin et al.,
1997].
Initial studies of GLC1A-linked families described a
typical clinical profile with onset before the age of 20,
highly elevated IOP, rapid loss of visual field, con-
439
Fig. 1. Map of the GLC1A region and disease-associated haplotypes. A: Physical map of the GLC1A region integrating data from a YAC contig
[Belmouden et al., 1997] and from a BAC contig (our unpublished data). The markers at the top were positioned approximately in the indicated intervals
by STS-content analysis of BACs. Grouped markers were not dissociated by recombination events or by STS analysis. Polymorphic markers are printed
in boldface. B: Extent of the disease-associated GLC1A haplotypes shared by Families 1, 2, 10, and 19 (top), 27 (middle), and 14 (bottom).
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Brezin et al.
nylon membrane and hybridized with a 17-mer oligonucleotide probe matching either the wild type or the
mutated sequence. Filters were washed to obtain a sequence-specific signal.
RESULTS
Clinical Findings
Six families of unequal size were studied (Fig. 2). The
largest one (Family 1) extended over 9 generations and
included 62 glaucomatous or reportedly blind patients,
of whom 40 were alive. The second largest family (Family 2) comprised 7 generations and 19 glaucomatous
patients, of whom 10 were alive at the time of the
study. The other four families (10, 14, 19, and 27) were
of much smaller size and included 3, 6, 4, and 8 participating patients, respectively. Altogether 205 individuals were contacted and accepted to participate in
this study (132, 31, 15, 9, 6, and 12 for Families 1, 2, 10,
14, 19, and 27 respectively). Most members of these
families were living in Northern France, in the Departments of Nord and Pas-de-Calais for Families 1, 2, and
10, in the Paris area for Family 19, and in Normandy
for Families 14 and 27. Ancestors of four of these families (1, 2, 10, and 19) were identified in a limited area
delineated by Boulogne-sur-mer, Calais, and SaintOmer, in the Pas-de-Calais.
In these six families, POAG was associated with a
normal iridocorneal angle. Peak IOP was elevated but
varied widely, between 22 and 50 mmHg and above.
Filtration surgery was performed in 93/140 eyes. Age
at diagnosis was also variable and ranged between 10
and 65 years. Figure 3 shows the penetrance curve as a
function of age at diagnosis. Twenty-five, 50, and 75%
of the cases were recognized at the age of 21, 30, and 42
years, respectively. When families were analyzed separately, both juvenile-onset and middle-age onset cases
were seen in Families 1, 2, 14, and 27, whereas POAG
was of the juvenile-onset type in Family 19 (age at
diagnosis ranging between 21 and 28 years) and of the
adult type in Family 10 (40, 40, and 50 years for the 3
patients of this family, respectively).
GLC1A Linkage and Mutation Analysis
Segregation analysis of POAG in Family 1, which
has been almost completely ascertained, was consistent with an autosomal dominant monogenic mode of
inheritance and a high penetrance rate. Fifty-two of
107 children (48.6%) having an affected parent were
glaucomatous. The transmitting parent was the
mother in 17 cases and the father in 16 cases. A similar
analysis could not be performed on Family 2, which has
been characterized only partially, whereas the size of
the other four families was too small to allow firm conclusions.
Linkage of POAG to the GLC1A region was tested in
these families using microsatellite markers spanning
the GLC1A region (Fig. 1). Two-point lod score data for
three of these markers are shown in Table I. These
data strongly support linkage to GLC1A (maximum lod
scores of 24.2, 29.7, and 32.5 for D1S2851, NGA1, and
D1S218, respectively).
Sequencing of the GLC1A gene using PCR-amplified
genomic DNA samples from representative glaucomatous patients belonging to Family 1, 14, and 27 had
identified a C to A transversion at position 1440 [Adam
et al., 1997]. This mutation changed an Asn into a Lys
at residue 480 (N480K). It was also present in Families
2, 10, and 19. A dot-blot assay of PCR-amplified genomic DNA followed by hybridization with oligonucleotide probes matching either the mutated or the wild
type sequence was then set up to facilitate the screening of mutation carriers (Fig. 4). The N480K mutation
was carried by all patients of the six families whereas
it was not detected among 150 unrelated controls.
These findings indicated that the N480K substitution
was responsible for the disease in these pedigrees.
Haplotype Analysis and Founding Effect
To discriminate between a founder effect and a de
novo recurrence, disease-associated haplotypes across
the GLC1A region were determined by typing 26 microsatellite markers covering 8 cM (Fig. 1A). This demonstrated that mutation N480K was associated with a
common haplotype including the 26 markers in families 1, 2, 10, and 19 (P < 1016). Seven of these 26
markers were particularly informative due to the low
frequencies of their disease-associated alleles in the
general population (0.067, 0.072, 0.133, 0.045, 0.0053,
0.121, and 0.033 for NGA1, D1S2851, AFM21, NGA13,
D1S1165, D1S2790, and D1S218, respectively) (P <
108). Part of this extended haplotype was identified in
association with the N480K mutation in Families 14
and 27 (Fig. 1B). It included NGA15 and markers distal
to it in Family 27 (P < 1012) and markers from NGA14
to AFM107yg1 in Family 14 (P < 105).
Two groups of POAG-unrelated patients were then
screened for the presence of the N480K mutation. The
first one included 67 Caucasian patients with POAG
originating from Northern France and who had undergone trabeculectomy before the age of 50. This selection
bias was intended to favor the founder effect associated
with the N480K mutation in Northern France and to
increase the odds of identifying carriers of this mutation. The second group included 56 consecutive POAG
patients who consulted at the Glaucoma Institute of
the Saint-Joseph Hospital in Paris. Only one patient
from the first group was found to carry the N480K
mutation. This patient harbored the extended haplotype of Families 1, 2, 10, and 19, and had at least four
glaucomatous first-degree relatives.
Altogether, these findings indicated that the 71 patients from Families 1, 2, 10, 14, 19, and 27 and the
patient identified by screening were all linked by a
founding effect and had inherited their mutation from
a common ancestor.
Variable Expressivity of the GLC1A Gene
The set of 71 patients described above provides a
unique opportunity for analyzing the variability of the
phenotype determined by the N480K mutation. The
age at diagnosis is one parameter of this variability. In
light of the mutation typing data, cumulated penetrance rates of the disease gene as a function of the
age of patients at diagnosis or of the age of unaffected
Fig. 2. Family trees. Filled and crossed symbols indicate POAG patients and unaffected individuals under 18 years of age, respectively. Among living subjects, those who were genotyped are marked
by a slanted T.
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Brezin et al.
carriers at the time of the study could then be reevaluated as follows: 25% at 19 years, 50% at 26 years, 75%
at 32 years, and >95% at 57 years.
Whereas juvenile-onset is usually associated with a
severe course of the disease, a later age at diagnosis
may be the result of either a late diagnosis or a late
onset of disease. The following three selected cases examplify three typical clinical situations encountered
among carriers of the N480K mutation.
Juvenile POAG. The daughter of a severely affected woman from Family 14 was first diagnosed with
POAG when she was 21 years old. Her peak IOP was
then 28 mmHg OU. Treatment was initiated by a topical beta-blocker alone, then in combination with pilocarpine. Filtrating surgery was perfomed on both eyes
1 year later. Now, at the age of 23, her current IOP is
OD: 15 mmHg, OS: 12 mmHg. Her optic discs are excavated, with a cup/disk ratio of 0.8. Her visual fields
show severe defects (Fig. 5A).
Middle-age POAG. A 52-year-old woman from
Family 1 with previously normal ophthalmic examinations was genotyped. She carried the diseaseassociated haplotype of her family and the N480K
GLC1A mutation. Recently, her IOP was measured at
TABLE I. Two-Point Lod Scores for OAG and GLC1A-Linked Microsatellite Markers
Recombination fraction
Marker
Family
D1S2851
1
2
10
19
14
27
NGA1
1
2
10
19
14
27
D1S218
1
2
10
19
14
27
0.0
0.01
0.05
0.1
0.2
0.3
0.4
Zmax
Theta (%)
23.84
14.93
5.05
1.19
0.47
1.34
0.86
29.36
18.62
6.00
1.21
0.81
1.26
1.46
27.57
16.16
6.33
1.25
0.85
1.34
1.64
24.21
15.50
4.94
1.17
0.45
1.31
0.84
29.65
19.06
5.94
1.18
0.80
1.24
1.43
32.49
21.28
6.24
1.22
0.83
1.32
1.60
22.91
14.99
4.51
1.05
0.40
1.20
0.76
28.17
18.36
5.59
1.06
0.73
1.13
1.30
30.99
20.59
5.86
1.10
0.77
1.20
1.47
20.54
13.67
3.94
0.90
0.34
1.05
0.64
25.50
16.79
5.04
0.91
0.64
0.98
1.14
28.19
18.90
5.32
0.95
0.68
1.05
1.29
15.20
10.42
2.78
0.61
0.23
0.74
0.42
19.31
12.99
3.75
0.61
0.47
0.69
0.80
21.64
14.74
4.08
0.64
0.50
0.75
0.93
9.54
6.78
1.63
0.34
0.13
0.44
0.22
12.54
8.68
2.34
0.33
0.30
0.42
0.47
14.35
9.96
2.69
0.36
0.32
0.46
0.56
4.21
3.10
0.66
0.14
0.06
0.19
0.06
5.74
4.12
1.00
0.12
0.14
0.18
0.18
6.80
4.83
1.26
0.14
0.15
0.20
0.22
24.2
0.8
29.7
0.7
32.5
0.9
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Fig. 4. N480K mutation detection by dot-blot hybridization with indicated sequence-specific oligonucleotide probes of PCR-amplified genomic
DNA from a noncarrier (lane 1) and a carrier (lane 2), verified by nucleotide sequencing. Lane 3: no DNA.
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