Content uploaded by Joanne Elizabeth Sutherland
Author content
All content in this area was uploaded by Joanne Elizabeth Sutherland on Dec 23, 2013
Content may be subject to copyright.
Molecular characterisation of congenital glaucoma
in a consanguineous Canadian community: a step
towards preventing glaucoma related blindness
S Nicole Martin, Joanne Sutherland, Alex V Levin, Robert Klose, Megan Priston,
Elise Héon
Abstract
Glaucoma is a leading cause of irrevers-
ible blindness in Canada. Congenital glau-
coma usually manifests during the first
years of life and is characterised by severe
visual loss and autosomal recessive inher-
itance. Two disease loci, on chromosomes
1p36 and 2p21, have been associated with
various forms of congenital glaucoma. A
branch of a large six generation family
from a consanguineous Amish commu-
nity in south western Ontario was aVected
with congenital glaucoma and was studied
by linkage and mutational analysis to
identify the glaucoma related genetic
defects. Linkage analysis using the MLINK
component of the LINKAGE package (v
5.1) showed evidence of linkage to the 2p21
region (Zmax=3.34, è=0, D2S1348 and
D2S1346). Mutational analysis of the pri-
mary candidate gene, CYP1B1, was done
by direct cycle sequencing, dideoxy fin-
gerprinting analysis, and fragment analy-
sis. Two diVerent disease causing
mutations in exon 3, 1410del13 and
1505G→A, both segregated with the dis-
ease phenotype. The two diVerent combi-
nations of these alleles appeared to result
in a variable expressivity of the pheno-
type. The compound heterozygote ap-
peared to have a milder phenotype when
compared to the homozygotes for the 13
bp deletion. The congenital glaucoma
phenotype for this large inbred Amish
family is the result of mutations in
CYP1B1 (2p21). The molecular infor-
mation derived from this study will be
used to help identify carr iers of the
CYP1B1 mutation in this community and
optimise the management of those at risk
of developing glaucoma.
(J Med Genet 2000;37:422–427)
Keywords: congenital glaucoma; CYP1B1; gene; genetic
counselling
Glaucoma constitutes a leading cause of
irreversible blindness in Canada.
1
Congenital
and infantile glaucoma are associated with
anomalous development of the trabecular
meshwork and anterior chamber angle, which
leads to an increased resistance to aqueous
outflow and raised intraocular pressure (IOP).
2
Clinical characteristics of congenital glaucoma
most often manifest during the first years of life
and include tearing, blepharospasm, and pho-
tophobia. Corneal oedema, Haab striae (breaks
in Descemet membrane), enlarged cornea,
optic nerve cupping, and buphthalmos may
also develop if the pressure is not controlled.
When the diagnosis of congenital glaucoma is
delayed, the resulting visual outcome is often
poor.
34
Even though treatments are available,
earlier detection is needed in order to prevent
or minimise the glaucoma related visual loss.
The primary cause of the disease is
unknown.
25
However, congenital glaucoma is
heritable and the majority of cases are
autosomal recessive.
6
Cases of incomplete pen-
etrance are well documented.
7
The incidence
of congenital glaucoma varies depending on
geographical location: 1:2500 births in the
Middle East, 1:10 000 births in western coun-
tries, and 1:1250 births in the Gypsy popula-
tion of Slovakia.
4 8–10
Congenital glaucoma is genetically heteroge-
neous with two associated loci on chromo-
somes 1p36 and 2p21, respectively.
11 12
Other
forms of early onset glaucoma, mapped to
chromosomes 6p25 and 4q25,
13 14
are usually
associated with other congenital anterior seg-
ment anomalies which only occasionally result
in glaucoma at birth or in infancy and are not
considered as “primary congenital glaucoma”.
CYP1B1, the disease causing gene at the chro-
mosome 2p21 locus, is proposed to be the pre-
dominant congenital glaucoma gene, playing a
role in an estimated 85% of cases.
12
A large, consanguineous Amish family af-
fected with congenital glaucoma, from south
western Ontario, was recruited and studied in
an attempt to identify the disease causing
genetic defect in that community. In addition,
several members of this family were character-
ised with an atypical form of iris hypoplasia
that was studied separately.
Materials and methods
CLINICAL ASSESSMENT
The project was approved by the Toronto Hos-
pital Human Subjects Review Committee and
The Hospital for Sick Children Research Eth-
ics Board. After informed consent, all partici-
pants were questioned on their personal medi-
cal history and a family tree was drawn. All
participants, except for the nuclear family of
the proband, were examined in their home
where no electricity was available. As a result,
no ocular photographs were taken. The
proband and his immediate family had a com-
prehensive eye examination at The Hospital for
Sick Children. The aVected status was defined
by an intraocular pressure greater than 25 mm
Hg in the first years of life and by the presence
J Med Genet 2000;37:422–427422
Vision Science
Research Program,
Toronto Western
Hospital Research
Institute, University
Health Network, 399
Bathurst Street, Room
6-412, Toronto,
Ontario, Canada
M5T 2S8
S N Martin
R Klose
M Priston
E Héon
Department of
Ophthalmology, The
Hospital for Sick
Children, University of
Toronto, Toronto,
Canada
J Sutherland
A V Levin
E Héon
The Research
Institute, The Hospital
for Sick Children,
University of Toronto,
Toronto, Canada
A V Levin
E Héon
Correspondence to:
Dr Héon,
eheon@playfair.utoronto.ca
Revised version received
3 January 2000
Accepted for publication 14
January 2000
of corneal enlargement, scar, or Haab striae or
a history consistent with congenital glaucoma.
As the enlargement of the eye (buphthalmos)
and cornea can only occur in the first few years
of life, people with buphthalmos in their teens
and adulthood are presumed to have a form of
congenital or infantile glaucoma. Eye charts of
older aVected people were reviewed. AVected
subjects and their relatives were examined with
a portable slit lamp and direct ophthalmos-
copy. The charts from their respective eye phy-
sicians were reviewed when available.
GENOTYPING
DNA was prepared from whole blood (10-20
ml) using a non-organic procedure.
15
The
selection of short tandem repeat polymor-
phisms (STRPs) was done using genetic map
information from publications and genome
databases, Généthon, CHLC, and Marshfield.
Primers were obtained from Research Genetics
Inc (screening set 6A) or from ACGT Inc
(Toronto, ON). A fluorescent dye label was
incorporated on the 5' end of one of the prim-
ers and the protocol used for genotyping was
previously described.
16
The genotyping was
done blinded to the aVected status.
LINKAGE ANALYSIS OF CONGENITAL GLAUCOMA
Four STRP markers that mapped to the region
2p21, around CYP1B1, were used and their
telomeric to centromeric order with inter-
marker distances (in cM) were as follows:
D2S1788 - (3) - D2S177 - D2S1348 -
D2S1346. Linkage analysis was performed
with the MLINK component of the LINK-
AGE package (v 5.1). Lod scores were
obtained with the assumption of an autosomal
recessive mode of inheritance, full penetrance,
a disease gene frequency of 0.0001, and equal
allele frequencies.
LINKAGE ANALYSIS OF IRIS HYPOPLASIA
Molecular characterisation of the iris hypopla-
sia variant observed in some family members
was done by linkage analysis using additional
markers covering the three previously reported
loci on chromosomes 4q25, 6p25, and
13q14.
13 17 18
The markers used for each loci
including the intermarker distance (in cM) are
as follows: 4q25 (D4S3240 - D4S2623 - (3) -
D4S406 - (4.5) - D4S2392), 6p25 (D6S1600 -
(1.4) - D6S967 - D6S344 - (6.9) - D6S477),
and 13p14 (D13S1493 - (7.5) - D13S894 -
(1.65) - D13S1253 - (3.8) - D13S263 - (6.3) -
D13S788).
MUTATIONAL ANALYSIS OF CYP1B1
CYP1B1 consists of three exons, only two of
which code for the protein. PCR primers used
for the mutational analysis of exons 2 and 3 of
the gene CYP1B1 were previously
described.
719
Additional exon 3 primers were
designed from the mRNA sequence (Acc No
U56438) (5'catgattcacagaccactgg3'-reverse
and 5'’ccagctcgattcttggacaa3'-forward).
20
SEQUENCING OF CYP1B1
Mutational analysis of CYP1B1 used direct
sequencing from genomic DNA. Methods of
PCR amplification were as previously
described.
21
Gene specific primers tailed with
M13 universal primer 5'gtaaaacgacggccagt3' or
M13 reverse primer 5'cacaggaaacagctatgac3'
were used. The amplicon was purified using
QIAquick PCR Purification Kit™ (Qiagen,
Mississauga) according to the manufacturer’s
protocol. The column purified amplicon was
then sequenced on a MicroGene Blaster™
automated DNA sequencing unit (Visible
Genetic Inc (VGI), Toronto) using Cy5.5
labelled M13 universal or M13 reverse primers
and the Thermo Sequenase™ Cycle Sequenc-
ing Core Kit (US 79610, VGI) as previously
published.
21
FRAGMENT ANALYSIS OF CYP1B1
Family members were screened for the 13 bp
deletion by fragment analysis as follows. M13
tailed primers were used to amplify a 320 bp
fragment in the 5' region of exon 3. Then 1 µl
of unpurified amplicon, 1.5 mmol/l MgCl
2
,1×
PCR buVer II, 2 mmol/l of each dNTP, and
0.75 pmol of M13 universal primer (CY5.5
labelled) and the unlabelled reverse primer in
the initial amplification were used in a second
PCR reaction. The PCR reaction used the fol-
lowing conditions: 94°C for three minutes,
(94°C for 30 seconds, 51°C for 30 seconds,
70°C for five seconds) × 28 cycles and 70°C for
an eight minute extension. Formamide loading
dye (1.5 µl) was mixed with 1.5 µl of the final
amplified product, denatured for one minute,
and then electrophoresed on a 6% Surefill™
sequencing gel (VGI, Toronto). Two control
size markers were run with each sample.
DIDEOXY FINGERPRINTING ANALYSIS OF CYP1B1
Screening for the 1505G→A missense muta-
tion was done by dideoxythymidine finger-
printing analysis. A cycle sequencing reaction
was performed with only the T termination
mix from Thermo Sequenase™ Cycle Se-
quencing Core Kit and the M13 reverse
primer. The conditions for the cycle sequenc-
ing reaction are the same as described above.
PROTEIN MODELLING
Homology modelling was performed using
Swiss-Model version 2.0. The reference struc-
ture used was P450BM-P which is the best
model for eukaryotic P450s.
19 22
The predicted
structure for CYP1B1 was viewed in the
WEBLAB VIEWER™ PRO (Molecular Simu-
lations Inc, San Diego).
Results
CLINICAL ANALYSIS
Six bilaterally aVected people and 38 unaf-
fected people were recruited. The penetrance
appeared complete and the pedigree (fig 1) was
consistent with autosomal recessive inherit-
ance. However, the severity of the phenotype
appeared to be variable between family mem-
bers. Buphthalmos and increased intraocular
pressure in the first year of life characterised
the aVected status of the sixth generation,
whereas the fourth generation appeared to have
an overall milder course. No specific anterior
Molecular characterisation of congenital glaucoma 423
segment malformation was noted. The clinical
information is summarised in table 1 and
detailed as follows.
VI.1 (4 years old) presented in the first week
of life with corneal oedema and the typical high
iris insertion seen in congenital glaucoma, but
normal corneal diameters (11.25 mm). The
optic nerve could not be seen at presentation.
His glaucoma has been poorly controlled
despite maximum medical and surgical treat-
ments. At 4 years of age he required bilateral
laser cyclophotoablation (destruction of the
ciliary body) and still continues on multiple
topical antiglaucoma medications. His course
has been complicated by a severe cone-rod
retinal dystrophy confirmed by electroretino-
graphy, developmental delay, and failure to
thrive. Karyotype and extensive investigations
to characterise the nature of his non-
ophthalmic findings have been unrevealing.
His current vision is at best hand motion and
felt largely to be because of the retinal
dystrophy. The right optic nerve cup is
approximately 0.3 but is diYcult to assess.
Corneal diameters are now enlarged, 14.0 mm
OD and 14.25 mm OS.
Figure 1 Haplotype analysis and segregation analysis of CYP1B1 mutant alleles. Pedigree with haplotypes for selected markers on chromosome 2p21
markers (marker order: pter, D2S1788, D2S177, D2S1348, D2S1346, cen). Blackened symbols denote subjects aVected with congenital glaucoma while
the asterisk indicates those aVected with iris hypoplasia. The boxed haplotypes segregate with the aVected status. The black boxes indicate the 1410del13
mutant allele, the g rey boxes the 1505G→A mutant allele, and the white boxes indicate a normal allele as determined by mutational analysis. IV.14 showed
a single recombination placing the disease locus centromeric to D2S177. The hatched boxes show the segregation of the 1410del13 mutant allele while the
grey boxes show the segregation of the 1505G→A mutant allele.
I
II
III
IV
V
VI
Family 98-12
**
************
***
*
7
6
2
2
5
2
4
3
7
6
2
2
5
2
4
3
1
4
6
5
5
2
4
3
1
4
6
5
4
6
4
3
1
4
6
5
5
2
4
3
2
5
1
1
3
5
5
4
1
4
6
5
5
2
4
3
4
6
3
6
5
2
4
3
7
6
2
2
5
2
4
3
6
5
2
2
5
2
4
3
6
5
2
2
5
2
4
3
5
2
4
3
5
2
4
3
2
5
1
1
5
2
4
3
2
5
1
1
5
2
4
3
3
5
5
4
5
2
4
3
1
4
6
5
3
5
5
4
7
6
2
2
4
2
1
1
3
5
5
4
6
5
2
2
4
1
2
2
4
2
1
1
22
10
*
*
3
3
2
5
11
Table 1 Clinical characteristics of subjects with congenital glaucoma
Case
Age at
diagnosis
Corneal diameter at diagnosis
OD;OS
IOP at diagnosis
OD;OS
Last vision OD;OS
(age)
Last C/D ratio
(age) OD;OS Treatments
VI.1 <1 wk 11.25 OU (normal) 18; 21 (mm Hg) LP; HM 0.3; N/A Medical treatment, surgery × 3, laser
surgery
VI.2 <1 wk 12 mm; 12.75 (enlarged OS) 22; 49 (mm Hg) Amblyopia OD 0.1 OU Medical treatment, surgery × 3, laser
surgery
CSM OS
IV.7 ∼14 y Enlarged OD Raised OU 20/200; 20/15 (53 y) 0.9; 0.1 1957-73: medical treatment OU
1973: medical and surgical treatment
IV.13 ∼14 y Enlarged OU Not available 20/15 OD teenage None Medical treatment OU/ trauma OS in 1969
IV.14 Birth Enlarged OU Raised OU Enucleated OU Enucleated OU Enucleation at 2 y, traumatic cause?
Buphthalmos OU
IV.15 ∼14 y Buphthalmos OU 37 mm Hg OU Not available Pale nerve OU 1955-67: medical treatment, 1968: medical
and surgical treatment
OD: right eye, OS: left eye, OU: both eyes.
C/D: cup disc ratio of the optic nerve.
IOP: intraocular pressure.
LP: light perception, HM: hand motion.
CSM: central, steady and maintained.
424 Martin, Sutherland, Levin, et al
VI.2 (5 years old) also presented in the first
weeks of life with cloudy, enlarged corneas and
findings consistent with congenital glaucoma.
His condition was also diYcult to control
despite maximum medical and surgical inter-
ventions and laser cyclophotoablation. His
corneal diameters are now 14.25 mm OD and
14.00 mm OS. Optic nerve cuppings are 0.1
OU and pressures are well controlled on medi-
cal therapy.
IV.7 (56 years old) was diagnosed at 14 years
with right eye (OD) buphthalmos and raised
pressures in both eyes (OU). His intraocular
pressures were controlled medically until the
age of 30, when the pressure rose to 36 mmHg
and the vision OD decreased to 20/200.
Surgery of the right eye was required to control
the disease which is currently stabilised with
the addition of medical therapy.
IV.13 (63 years old) had bilateral buphthal-
mos diagnosed in his late teenage years but
retained a good central vision OD with medical
treatment until 1997 when he was last seen. He
suVered traumatic visual loss OS in 1969.
IV.14 (61 years old) was born with glaucoma
and bilaterally enucleated at 2 years of age. It is
unclear if the enucleation was related to
glaucoma or to a severe trauma she suVered in
early childhood.
IV.15 (58 years old) was diagnosed at the age
of 14 years with bilateral buphthalmos, mild
optic nerve pallor, and intraocular pressures of
37 mm Hg in both eyes. He was controlled
medically until 1967 when surgery was re-
quired to control the pressure in both his eyes.
He has remained stable for the last 30 years
with sporadic use of his glaucoma drops.
Twenty people were also aVected with a type
of iris hypoplasia (* in fig 1) that did not segre-
gate with the congenital glaucoma phenotype.
The absence of iris colarette and a variable
degree of thinned anterior leaflet of the iris
from the pupillary border to the iris base with
the iris base being the thinnest characterised
this phenotype. The stromal strands had a tight
appearance and the pigment epithelium of the
iris could be seen through the strands to a vari-
able degree. These people were otherwise nor-
mal; no transillumination defects were ob-
served, nor were the classical features of Rieger
syndrome.
23
The iris hypoplasia appeared to
have an autosomal dominant mode of inherit-
ance.
LINKAGE ANALYSIS OF CONGENITAL GLAUCOMA
A subset of the branch of the family was geno-
typed and studied by linkage analysis (fig 1).
This included five aVected subjects and 14
unaVected subjects. AVected subject VI.2 was
recruited after linkage analysis was completed.
The first candidate locus was the CYP1B1
locus on chromosome 2p21 because of its sug-
gested role in congenital glaucoma. Two point
linkage data for the four STRP markers are
summarised in table 2. Evidence for linkage to
the locus at 2p21 was obtained with markers
D2S1348 and D2S1346 (Zmax=3.34,
èmax=0).
Haplotype analysis showed that the aVected
haplotypes segregated perfectly with the con-
genital glaucoma phenotype in this family (fig
1). A recombination event observed in aVected
subject IV.14 places the candidate gene
CYP1B1 centromeric to D2S177. This recom-
bination event also clarifies the order of the two
previously non-recombinant markers, D2S177
and D2S1346 (fig 1).
LINKAGE ANALYSIS OF IRIS HYPOPLASIA
Two point linkage data for the iris hypoplasia
phenotype at loci 4q25, 6p25, and 13q14 failed
to identify linkage to these loci. Lod score
values lower then −2 were obtained for several
markers at each locus (data not shown).
MUTATIONAL ANALYSIS OF CYP1B1
CYP1B1 was the primary candidate gene to
study. The two coding exons were directly
sequenced in one aVected person (IV.7, fig 1)
and two mutations within exon 3 were
identified (data not shown). One mutation is a
13 bp deletion of nucleotides 1410-1422
(1410del13), which creates a frameshift pre-
mature stop codon, and truncation of the pro-
tein (Acc No U56438).
20
The other mutation
was a missense mutation, 1505G→A, substi-
tuting the highly conserved amino acid
glutamic acid for lysine (Glu387Lys, Acc No
3913312 ). These mutations were previously
reported separately, as was the screening of a
randomly selected control population. Neither
mutation was present in 200 control
chromosomes.
12 19
Screening of the other 43 family members
using fragment analysis and dideoxythymidine
fingerprinting confirmed that both mutations
cosegregated with the disease phenotype (fig
1). Segregation of the mutant alleles was
consistent with an autosomal recessive mode of
inheritance. Mutational analysis identified four
aVected subjects who were compound hetero-
zygotes, two aVected subjects who were homo-
zygous for the 13 bp deletion, and 19 subjects
who were carriers of either mutation (fig 1).
The congenital glaucoma phenotypes associ-
ated with the diVerent combination of muta-
tions showed variable degrees of severity.
Homozygotes for the 1410del13 appeared to
have a more severe phenotype than those who
were compound heterozygotes for the two
described mutations. Unfortunately, there is
missing clinical information for IV.14, leaving
unclear the severity of her disease.
No correlations were observed between the
CYP1B1 genotypes and the iris hypoplasia
phenotype confirming that the iris hypoplasia
observed in this family is unrelated to the con-
genital glaucoma. There was no evidence of
linkage of the iris hypoplasia phenotype to the
previously documented loci (data not shown),
Table 2 Two point linkage data
Marker
IMD
(cM)
Lod score at è =
è max Zmax0.00 0.05 0.10 0.20 0.30 0.40
D2S1788 −∞ 1.74 1.71 1.34 0.84 0.35 0.066 1.76
D2S177 3 −∞ 1.52 1.51 1.19 0.74 0.30 0.071 1.54
D2S1346 0 3.34 3.0 2.64 1.91 1.16 0.44 0.000 3.34
D2S1348 0 3.34 3.0 2.64 1.91 1.16 0.44 0.000 3.34
IMD: intermarker distance.
Molecular characterisation of congenital glaucoma 425
supporting the genetic heterogeneity of this
condition and suggesting the possibility of an
additional iris hypoplasia locus. No person
with iris hypoplasia had congenital glaucoma
with the exception of IV.7 and IV.13.
Discussion
Linkage and haplotype analysis identified the
disease causing locus on chromosome 2p21
with CYP1B1 being the disease causing gene
for the glaucoma segregating in this Amish
family. CYP1B1 encodes for a cytochrome
P450 enzyme that belongs to the multigene
superfamily of monomeric mixed function
mono-oxygenases responsible for phase 1
metabolism of numerous structurally diverse
substrates. It is postulated that the oxygenation
of a CYP1B1 substrate, still unknown in the
eye, would allow the proper functioning of sig-
nal transduction pathways involved in eye
growth and diVerentiation.
112
By directly sequencing CYP1B1 in this family,
two mutations in exon 3 were identified. The
1410del13 mutation introduces a stop codon
203 bp downstream of the deletion. The trunca-
tion of the protein (amino acids 422-453) elimi-
nates the carboxy-terminus end, which includes
the essential haem domain. This may have a
deleterious eVect on the function of the protein
(data not shown).
22–25
This mutation is suggested
to create a functional null allele.
6
The other
mutation altered amino acid 387 from glutamic
acid to lysine. Glu387 is located in helix K,
which is one of the highly conserved core struc-
tural elements.
19
The core structural elements
are suspected to be involved in proper folding of
the protein and in active haem binding.
19 22
Glu387 is also conserved across all documented
species and diVerent P450 enzymes and is one of
three absolutely conserved residues identified in
P450s.
22
This conservation of the amino acid
supports its importance and suggests that this
mutation is likely to aVect the proper function of
the protein.
The phenotypes between the two aVected
generations show some diVerences in severity.
Children who were homozygous for the
deletion appeared to be more severely aVected
than those in generation IV who were com-
pound heterozygotes, with perhaps the excep-
tion of IV.14. This is not surprising since an
important part of this protein is eliminated by
the 1410del13 mutation. This homozygous
deletion was previously reported but no
phenotype was described,
12
whereas a case
homozygous for the 1505G→A mutation was
previously reported with a severe phenotype.
8
One could speculate that any homozygous
impairment of the haem binding domain could
lead to a severe phenotype. This combination
of the mutations described has not been previ-
ously reported and this is the first report of the
respective genotype-phenotype correlation. As
more mutations are identified, the definition of
phenotype-genotype correlation may help to
improve the management.
All 44 members of the Amish family were
screened for both mutations and the carriers of
the disease were identified along with couples
whose future children may be at risk of devel-
oping glaucoma. Identifying two mutations
suggests that there are two distinct founders
for the glaucoma segregating in this family.
The mutant allele containing the 13 bp
deletion is found in both branches of this fam-
ily increasing the risk of the development of
congenital glaucoma in future children consid-
ering the high degree of “intracommunity”
relationships (not all illustrated in the pedigree
shown). The information has been returned to
the community to improve genetic counselling
and management. The opportunity has also
been given to other members of the Amish
community to assess their carrier status and to
proceed with early screening of their children
at risk. Although genetic counselling may be
less likely to alter mating patterns of this highly
consanguineous population, early identifica-
tion of at risk couples will hopefully improve
the management and outcome of oVspring and
provide an opportunity to prevent or minimise
glaucoma related visual loss.
Data access: Généthon: www.genethon.fr/, Marshfield:
www.marshmed.org/genetics/, CHLC: www.chlc.org/, Molecu-
lar Simulation Inc: www.msi.com, SWISS-MODEL version
2.0: www.expasy.ch/swissmod/SWISS-MODEL. html, Gen-
bank: www.ncbi.nlm.nih.gov/Web/Search/index.html. The au-
thors are grateful to Gail Billingsley MSc for her constructive
discussions, to the families for their enthusiastic participation, to
Dr J R Walker who kindly provided medical records, and to Dr
V Siu, Dr D Williams-Lyn and Ms E Perruzza for their support
in the organisation of this project. This work was funded by the
Glaucoma Research Society of Canada, the Weston Founda-
tion, PSI grant No 97-02, Fight For Sight, and the Canadian
Genetic Disease Network.
1 Friedman J, Walter M. Glaucoma genetics, present and
future. Clin Genet 1999;55:71-9.
2 DeLuise V, Anderson D. Primary infantile glaucoma
(congenital glaucoma). Surv Ophthalmol 1983;28:1-19.
3 ShaVer R. Genetics and the congenital glaucoma. Trans Am
Acad Ophthalmol Otol 1965;69:253-68.
4 Francois J. Congenital glaucoma and its inheritance.
Ophthalmologica 1980;181:61-73.
5 Jerndal T. Congenital glaucoma due to dominant goniodys-
genesis. A new concept of the heredity of glaucoma. Am J
Hum Genet 1983;35:645-51.
6 Sarfarazi M. Recent advances in molecular genetics of glau-
comas. Hum Mol Genet 1997;6:1667-77.
7 Bejjani B, Lewis R, Tomey K, et al. Mutations in CYP1B1,
the gene for cytochrome P450B1, are the predominant
cause of primary congenital glaucoma in Saudi Arabia. Am
J Hum Genet 1998;62:325-33.
8 Plasilova M, Ferakova E, Kadasi L, et al. Linkage of
autosomal recessive primary congenital glaucoma to the
GLC3A locus in Roms (gypsies) from Slovakia. Hum Hered
1998;48:30-3.
9 Gencik A, Gencikova A, Ferak V. Population genetical
aspects of primary congenital glaucoma. I. Incidence,
prevalence, gene frequency, and age of onset. Hum Genet
1982;61:193-7.
10 Jaafar M. Care of the infantile glaucoma patient. In:
Reinecke RD, ed. Ophthalmology annual 1988. New York:
Raven Press, 1988:15.
11 Akarsu A, Turacli M, Aktan S, et al. A second locus
(GLC3B) for primary congenital glaucoma (buphthalmos)
maps to the 1p36 region. Hum Mol Genet 1996;5:1199-
203.
12 Stoilov I, An A, Sarfarazi M. Identification of three truncat-
ing mutations in cytochrome P4501B1 (CYP1B1)asthe
principal cause of primary congenial glaucoma (buphthal-
mos) in families linked to the GLC3A locus on
chromosome 2p21. Hum Mol Genet 1997;6:641-7.
13 Semina E, Reiter R, Leysens N, et al. Cloning and
characterization of a novel bicoid-related homeobox
transcription factor gene, REIG, involved in Rieger
syndrome. Nat Genet 1996;14:392-9.
14 Nishimura D, Swiderski R, Alward W, et al. The forkhead
transcription factor gene FKHL7 is responsible for
glaucoma phenotypes which map to 6p25. Nat Genet 1998;
19:140-7.
15 Miller S, Dykes D, Polesky H. A simple salting out
procedure for extracting DNA from human nucleated cells.
Nucleic Acids Res 1988;16:1215.
16 Heon E, Liu S, Billingsley G, et al. Gene localization for
aculeiform cataract, on chromosome 2q33-35. Am J Hum
Genet 1998;63:921-6.
17 Mears A, Farideh M, Gould D, Pearce W, Walter M. Auto-
somal dominant iridogoniodysgenesis anomaly maps to
6p25. Am J Hum Genet 1996;59:1321-7.
426 Martin, Sutherland, Levin, et al
18 Phillips J, Del Bono E, Haines J, et al. A second locus for
Rieger syndrome maps to chromosome 13q14. Am J Hum
Genet 1996;59:613-19.
19 Stoilov I, Akarsu A, Alozie I, et al. Sequence analysis and
homology modeling suggest that primary congenital
glaucoma on 2p21 results from mutations disrupting either
the hinge region or the conserved core structures of
cytochrome P4501B1. Am J Hum Genet 1998;62:573-84.
20 Sutter T, Tang Y, Hayes C, et al. Complete cDNA sequence
of a human dioxin-inducible mRNA identifies a new gene
subfamily of cytochrome P450 that maps to chromosome
2. J Biol Chem 1994;269:13092-9.
21 Heon E, Priston M, Schorderet D, et al. The gamma crystal-
lins and human cataracts: a puzzle made clearer. Am J Hum
Genet 1999;65:1261-7.
22 Graham-Lorence S, Peterson J. Structural alignments of
P450s and extrapolations to the unknown. Methods
Enzymol 1996;272:315-27.
23 Shields M, Buckley E, Klintworth G, Thresher R. Axenfeld-
Rieger syndrome. A spectrum of developmental disorders.
Surv Ophthalmol 1985;29:387-409.
24 Guengerich F. Mini review. Cytochrome P-450. Comp
Biochem Physiol 1988;89C:1-4.
25 Gonzalez F. The molecular biology of cytochrome P450s.
Pharmacol Rev 1989;40:443-288.
Molecular characterisation of congenital glaucoma 427
