IOVS Papers in Press. Published on June 10, 2014 as Manuscript iovs.14-13991 1
1
Association of LOXL1 polymorphisms with pseudoexfoliation, glaucoma,
2
intraocular pressure, and systemic diseases in a Greek population. The
3
Thessaloniki Eye Study.
4 5
Anastasopoulos E1, Coleman AL2, Wilson MR3, Sinsheimer JS4, Yu F2,
6
Katafigiotis S5, Founti P1, Salonikiou A1, Pappas T1, Koskosas A1, Katopodi T5,
7
Lambropoulos A5, Topouzis F1
8 9 10 11 12 13 14 15
1. Department of Ophthalmology, School of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece 2. Center for Eye Epidemiology, Jules Stein Eye Institute, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 3. School of Medicine, Wayne State University, Detroit, MI 4. Department of Human Genetics, Department of Biomathematics, David
16
Geffen School of Medicine at UCLA and Department of Biostatistics, UCLA
17
School of Public Health.
18 19
5. Laboratory of General Biology, School of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece
20 21
Funding:
22
1. Co-funded by the European Union (European Social Fund) and Greek national
23
funds under the Act "Aristia" of the Operational Program "Education and Lifelong
24
Learning".
25
2. NIH grand GM053275
26
Copyright 2014 by The Association for Research in Vision and Ophthalmology, Inc.
2
1
Corresponding Author:
2
FotisTopouzis, MD, PhD
3
Aristotle University of Thessaloniki
4
A’ Department of Ophthalmology
5
A.H.E.PA. Hospital St.Kiriakidi 1 54636
6
Thessaloniki, Greece
7
Tel. Number: +302310994920
8
Fax Number: +302310839497 e-mail: [email protected]
9 10
3
1
Abstract
2
Purpose: To investigate the association of the two SNPs in the LOXL1 gene with
3
pseudoexfoliation syndrome (PEX), pseudoexfoliative glaucoma (PEXG) and primary
4
open-angle glaucoma (POAG) in a Greek population-based setting from
5
Thessaloniki Eye study.
6
Methods: A total of 233 subjects with successful DNA extraction, PCR amplification
7
and genotyping were included in the genetic analysis of G153D and R141L SNPs of
8
LOXL1 gene and classified into four groups: controls (n= 93), subjects with PEX
9
(n=40), POAG (n=66) and PEXG (n=34). Multinomial logistic regression was used to
10
test their association with LOXL1 SNPs with adjustment for covariates. The
11
association of LOXL1 with IOP (in untreated subjects) and with systemic diseases
12
was explored.
13
Results: Both LOXL1 SNPs were present in high frequencies in controls and cases.
14
The G153D was strongly associated with both PEX (OR=23.2, p=0.003 for allele G)
15
and PEXG (OR =24.75, p=0.003 for allele G) and was not associated with POAG
16
(p=0.451). In contrast, the R141L was not associated with PEX (p=0.81), PEXG
17
(p=0.063) or POAG (p=0.113). No association of the G153D with either IOP or
18
systemic diseases was found.
19
Conclusions: In Thessaloniki Eye study, the G153D SNP of LOXL1 gene was
20
strongly associated with both PEX and PEXG, whereas the R141L was not
21
associated. No association of the LOXL1 with IOP or with systemic diseases was
22
found. These findings further support the hypothesis that LOXL1 gene contributes to
4
1
onset of PEXG through PEX. LOXL1 gene variants do not help to identify those with
2
PEX at increased risk for glaucoma development.
5
1 2
Introduction Pseudoexfoliation syndrome (PEX) is an age-related disorder of the
3
extracellular matrix characterized by production and progressive accumulation of
4
small, white deposits of a fibrillar material in various intraocular and extraocular
5
tissues. PEX occurs worldwide. However, the reported prevalence estimates vary
6
considerably.1
7
PEX is the most common identifiable cause of secondary glaucoma named
8
pseudoexfoliative glaucoma (PEXG).2 PEXG usually presents with higher IOP than
9
the primary open-angle glaucoma (POAG) and its prevalence is more IOP-
10 11
dependent than POAG.3 Only few large-population-based studies have specifically investigated PEX
12
and PEXG prevalence or incidence.4-6 Thessaloniki Eye Study (TES) was designed
13
to address the prevalence of the major eye diseases and disorders, including PEX,
14
in the Greek population of Thessaloniki. The reported prevalence of PEX in subjects
15
over 60 years of age was 11.9%.4 Among those with pseudoexfoliation, only the 15%
16
presented with glaucoma.4 In the absence of glaucoma, subjects with PEX
17
compared to those without PEX, presented with similar clinical characteristics except
18
that they have higher IOP, only by 0.6 mmHg.7The reasons why some subjects
19
develop PEX currently remain unclear. In addition it remains unclear why a
20
proportion of subjects with PEX develop glaucoma while the majority of those with
21
PEX do not get glaucoma.
22
Further pseudoexfoliative material has been also identified by electron
23
microscopy in visceral organs such as lung, heart, liver, kidney, gall bladder and
24
meninges. This widespread distribution led to the hypothesis of possible association
25
of PEX with systemic cardiovascular or cerebrovascular diseases. Although there is
6
1
some evidence in favor of this hypothesis, other studies did not confirm this
2
association and thus, this hypothesis remains controversial.7-10 LOXL1 gene has been identified as a potential genetic risk factor for
3 4
both PEX and PEXG since 2007 when a genome-wide association study on
5
pseudoexfoliative individuals from Iceland and Sweden was performed.11 This study
6
demonstrated a strong association (>99% population attributable risk) of PEX and
7
PEXG conferred by three SNPs in the LOXL1 gene on chromosome 15q24.1. Two
8
SNPs were identified in the first coding exon (rs3825942 or G153D and rs1048661
9
or R141L) and one within the first intron of this gene (rs2165241). Although the
10
rs2165241 was more significantly associated with PEXG than the two exonic SNPs,
11
this association was not statistically significant after adjusting the results for the two
12
other SNPs.11 Several studies of different ethnic populations have reported
13
associations of LOXL1 with PEX and PEXG. Many of them did not differentiate
14
between PEX and PEXG. Recently, two clinical-based studies in Greece examined
15
the association of LOXL1 SNPs with PEX and PEXG and reported conflicting
16
results.12,13
17
The discovery of common sequence variants in the LOXL1 gene that confer
18
susceptibility to PEXG, primarily through PEX syndrome, has shed some light in the
19
pathogenesis of the disease.14 LOXL1 is one of the five enzymes in the family of lysyl
20
oxidases, which are copper-dependent monoamino oxidases secreted by fibrogenic
21
cells including fibroblasts and smooth muscle cells. These enzymes are involved in
22
the covalent cross-linking of collagen and elastin polymers in extracellular matrix
23
formation. LOXL1 is necessary for tropoelastin cross-linking and elastic fiber
24
formation, maintenance, and remodeling.14
7
1
The purpose of the present study is to explore the association of two non-
2
synonymous LOXL1 SNPs (G153D and R141L) with PEX, PEXG and primary open-
3
angle glaucoma (POAG) in a population-based setting using a well-defined,
4
ethnically homogenous population in Thessaloniki, Greece. In addition, in previous
5
studies no information is provided on the potential association of LOXL1 with IOP or
6
with systemic diseases. This study aims to identify whether the two LOXL1 SNPs
7
were associated with differences in IOP or with the self-reported history of systemic
8
diseases (diabetes mellitus, cardiovascular disease or cardiovascular surgery) in
9
subjects with PEX, PEXG, POAG and controls.
10 11 12
Methods The Thessaloniki Eye Study is a cross-sectional, population-based study of
13
chronic eye diseases in the Greek population of Thessaloniki. Thessaloniki is the
14
major urban centre in Northern Greece and the second largest city in Greece, after
15
Athens. This urban area has a stable and homogenous population with 97.7% of the
16
population identified as being of Greek ethnicity.The study was approved by the
17
Aristotle University Medical School, Ethics Committee and the Institutional Review
18
Board of the University of California, Los Angeles. All study procedures adhered to
19
the principles outlined in the Declaration of Helsinki for research involving human
20
subjects and all participants gave written informed consent prior to their participation.
21
Details about the recruitment process in the Thessaloniki Eye Study have
22
been previously described.4 In brief, 5000 subjects 60 years of age or older were
23
randomly selected in February 1999 from approximately 321,000 persons registered
8
1
in the municipality registers of the city of Thessaloniki. Subjects who agreed to
2
participate were invited to the Thessaloniki Eye Study centre (Laboratory of
3
Research and Clinical Applications in Ophthalmology at the Aristotle University of
4
Thessaloniki) for an extensive ophthalmologic examination. Clinic-visit examination
5
included measurement of visual acuity using Early Treatment Diabetic Retinopathy
6
Study (ETDRS) charts, Humphrey automated perimetry, Goldmann applanation
7
tonometry (three readings from each eye), gonioscopy, slit-lamp examination (before
8
and after pupil dilation) and fundus biomicroscopy. Details of observation procedures
9
were described elsewhere.4
10
A home-visit eye examination was organized, proposed and arranged for
11
persons unable to visit the study examination centre due to major disability or illness.
12
Home-visit examination included ETDRS visual acuity measurement, Perkins
13
applanation tonometry, slit-lamp examination, and dilated fundus examination.
14
Prior to clinical examination, a questionnaire was administered including
15
demographic data, ophthalmologic and medical history. In particular, subjects were
16
queried whether they had ever been diagnosed with hypertension, diabetes mellitus
17
(treated or not with insulin), any cardiovascular disease, heart attack or they had
18
undergone coronary artery bypass or vascular surgery.
19
Definitions
20
Particular attention was given to identify the presence of PEX. PEX was
21
defined by the presence of pseudoexfoliative material either at the pupil margin or on
22
the lens capsule. Prior to pupil dilation a detailed high magnification slit-lamp
23
assessment of the pupil margin was performed. After pupil dilation, the anterior lens
9
1
surface from each eye was scanned from left to right using a narrow slit-lamp beam
2
and then was examined using a vertical broad slit-lamp beam, looking specifically for
3
early signs of PEX, including pregranular radial lines, as well as established granular
4
deposits. The location of PEX either on the iris, the lens or both was recorded. The
5
PEX detection and the exact location required consensus agreement between at
6
least two of the three ophthalmologist graders. Dilation was conducted in all patients
7
and even in those with narrow-occludable angle, laser peripheral iridotomy was
8
performed and they were examined later under dilation.
9
Glaucoma was defined according to specific criteria.4 Subjects were classified
10
as having primary open-angle glaucoma (POAG) if they had glaucoma and open,
11
normal-appearing anterior chamber angle and absence of other secondary causes of
12
glaucoma in either eye. Subjects were classified as having pseudoexfoliative
13
glaucoma (PEXG) if they had glaucoma and presence of pseudoexfoliation in either
14
eye. A consensus agreement between at least two of the three ophthalmologists was
15
required to assign the diagnosis of glaucoma. Three ophthalmologists were
16
responsible for the ophthalmic examination. At least two of the three examined each
17
patient and all diagnoses were in consensus agreement.
18
Among the 3,617 subjects who were deemed eligible, 2,554 participated in
19
the study (participation rate 71%). Of those participating, 2261 (89%) had the clinic-
20
visit examination and 293 (11%) had the home-visit examination.
21
A blood sample was taken from all subjects with POAG and PEXG and in a
22
consecutive subset of participants without glaucoma. Written consent was obtained
23
before the blood draw. A total of 233 subjects who gave blood sampleswere included
24
in this analysis. Among them, 93 had neither PEX nor glaucoma in both eyes
10
1
(controls), 40 had pseudoexfoliation and no glaucoma in either eye (PEX), 66were
2
diagnosed with POAG and 34 with PEXG.
3
Peripheral blood samples were collected from each subject and DNA was
4
extracted using QIAamp DNA Mini Kit (Qiagen). DNA samples were amplified by
5
PCR as previously described using the sense: 5’ GCAGGTGTACAGCTTGCTCA 3’
6
and antisense 5’ ACACGAAACCCTGGTCGTAG 3’ primers.15 PCR reactions were
7
performed in a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems Inc.
8
[ABI], Foster City, CA).PCR products were sequenced using the same primers as in
9
PCR amplification in a 310 DNA Analyzer (Applied Biosystems Inc. Foster City, CA),
10
using manufacturer’s protocol. Analysis of individual sequences was carried out
11
using the AB DNA sequencing analysis software v5.2.
12
Statistical analysis
13
All statistical analyses were performed with statistical software package Stata
14
version 10.0 (Stata corporation, 4905 Lakeway Drive, College Station, Texas 77845
15
USA). Fisher’s exact test was used for the genotypic comparison using as a) control
16
group: subjects without PEX, PEXG, POAG, b) cases 1: subjects with POAG, c)
17
cases 2: subjects with PEX, d) cases 3: subjects with PEXG. Multinomial logistic
18
regression analysis was conducted in order to examine the association between the
19
two LOXL1 polymorphisms and POAG, PEX or PEXG, adjusted for the following
20
factors: age, gender, history of diabetes, history of cardiovascular disease, history of
21
cardiovascular surgery and the higher IOP for the two eyes (possible confounding
22
factors for glaucoma, as identified in a previous report of Thessaloniki Eye study).16.
23
Logistic regression analysis adjusted for age was also conducted in order to examine
24
the association of the two LOXL1 SNPs with IOP in controls and cases not receiving
11
1
IOP-lowering treatment. The association of the LOXL1 SNPs with the history of the
2
aforementioned systemic diseases was also examined with regression analysis
3
adjusted for age and gender. Hardy-Weinberg equilibrium (HWE) testing was
4
performed using Mendel version 13.0.17,18 Linkage Disequilibrium and haplotype
5
analysis was also performed using Mendel version 13.0.19 Odds Ratios (ORs) with
6
95% Confidence Intervals (CIs) were calculated as measures of each strength of the
7
allele’s association with POAG, PEX, PEXG. P-values were not adjusted for multiple
8
testing.
9
Results
10
Successful DNA extraction, PCR amplification and genotyping were carried
11
out for all of the 233 enrolled subjects. The general characteristics of the participants
12
(mean age, gender, IOP, history of diabetes, insulin use, cardiovascular disease or
13
cardiovascular surgery) in the four groups are presented in Table 1.
14
The allelic and genotypic distribution of the G153D (rs3825942) and
15
R141L (rs1048661) polymorphisms in controls, POAG, PEX and PEXG groups are
16
presented in Table 2. All frequencies were in Hardy Weinberg Equilibrium using a
17
heterozygote-homozygote test (p=0.486 for R141L, p=0.603 for G143D).18 With
18
regards to the G153D polymorphism, there was a statistically significant difference in
19
subjects with PEX and PEXG compared to controls using either the log-additive
20
allelic model (G vs. A allele, p
1
Association of LOXL1 polymorphisms with pseudoexfoliation, glaucoma,
2
intraocular pressure, and systemic diseases in a Greek population. The
3
Thessaloniki Eye Study.
4 5
Anastasopoulos E1, Coleman AL2, Wilson MR3, Sinsheimer JS4, Yu F2,
6
Katafigiotis S5, Founti P1, Salonikiou A1, Pappas T1, Koskosas A1, Katopodi T5,
7
Lambropoulos A5, Topouzis F1
8 9 10 11 12 13 14 15
1. Department of Ophthalmology, School of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece 2. Center for Eye Epidemiology, Jules Stein Eye Institute, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 3. School of Medicine, Wayne State University, Detroit, MI 4. Department of Human Genetics, Department of Biomathematics, David
16
Geffen School of Medicine at UCLA and Department of Biostatistics, UCLA
17
School of Public Health.
18 19
5. Laboratory of General Biology, School of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece
20 21
Funding:
22
1. Co-funded by the European Union (European Social Fund) and Greek national
23
funds under the Act "Aristia" of the Operational Program "Education and Lifelong
24
Learning".
25
2. NIH grand GM053275
26
Copyright 2014 by The Association for Research in Vision and Ophthalmology, Inc.
2
1
Corresponding Author:
2
FotisTopouzis, MD, PhD
3
Aristotle University of Thessaloniki
4
A’ Department of Ophthalmology
5
A.H.E.PA. Hospital St.Kiriakidi 1 54636
6
Thessaloniki, Greece
7
Tel. Number: +302310994920
8
Fax Number: +302310839497 e-mail: [email protected]
9 10
3
1
Abstract
2
Purpose: To investigate the association of the two SNPs in the LOXL1 gene with
3
pseudoexfoliation syndrome (PEX), pseudoexfoliative glaucoma (PEXG) and primary
4
open-angle glaucoma (POAG) in a Greek population-based setting from
5
Thessaloniki Eye study.
6
Methods: A total of 233 subjects with successful DNA extraction, PCR amplification
7
and genotyping were included in the genetic analysis of G153D and R141L SNPs of
8
LOXL1 gene and classified into four groups: controls (n= 93), subjects with PEX
9
(n=40), POAG (n=66) and PEXG (n=34). Multinomial logistic regression was used to
10
test their association with LOXL1 SNPs with adjustment for covariates. The
11
association of LOXL1 with IOP (in untreated subjects) and with systemic diseases
12
was explored.
13
Results: Both LOXL1 SNPs were present in high frequencies in controls and cases.
14
The G153D was strongly associated with both PEX (OR=23.2, p=0.003 for allele G)
15
and PEXG (OR =24.75, p=0.003 for allele G) and was not associated with POAG
16
(p=0.451). In contrast, the R141L was not associated with PEX (p=0.81), PEXG
17
(p=0.063) or POAG (p=0.113). No association of the G153D with either IOP or
18
systemic diseases was found.
19
Conclusions: In Thessaloniki Eye study, the G153D SNP of LOXL1 gene was
20
strongly associated with both PEX and PEXG, whereas the R141L was not
21
associated. No association of the LOXL1 with IOP or with systemic diseases was
22
found. These findings further support the hypothesis that LOXL1 gene contributes to
4
1
onset of PEXG through PEX. LOXL1 gene variants do not help to identify those with
2
PEX at increased risk for glaucoma development.
5
1 2
Introduction Pseudoexfoliation syndrome (PEX) is an age-related disorder of the
3
extracellular matrix characterized by production and progressive accumulation of
4
small, white deposits of a fibrillar material in various intraocular and extraocular
5
tissues. PEX occurs worldwide. However, the reported prevalence estimates vary
6
considerably.1
7
PEX is the most common identifiable cause of secondary glaucoma named
8
pseudoexfoliative glaucoma (PEXG).2 PEXG usually presents with higher IOP than
9
the primary open-angle glaucoma (POAG) and its prevalence is more IOP-
10 11
dependent than POAG.3 Only few large-population-based studies have specifically investigated PEX
12
and PEXG prevalence or incidence.4-6 Thessaloniki Eye Study (TES) was designed
13
to address the prevalence of the major eye diseases and disorders, including PEX,
14
in the Greek population of Thessaloniki. The reported prevalence of PEX in subjects
15
over 60 years of age was 11.9%.4 Among those with pseudoexfoliation, only the 15%
16
presented with glaucoma.4 In the absence of glaucoma, subjects with PEX
17
compared to those without PEX, presented with similar clinical characteristics except
18
that they have higher IOP, only by 0.6 mmHg.7The reasons why some subjects
19
develop PEX currently remain unclear. In addition it remains unclear why a
20
proportion of subjects with PEX develop glaucoma while the majority of those with
21
PEX do not get glaucoma.
22
Further pseudoexfoliative material has been also identified by electron
23
microscopy in visceral organs such as lung, heart, liver, kidney, gall bladder and
24
meninges. This widespread distribution led to the hypothesis of possible association
25
of PEX with systemic cardiovascular or cerebrovascular diseases. Although there is
6
1
some evidence in favor of this hypothesis, other studies did not confirm this
2
association and thus, this hypothesis remains controversial.7-10 LOXL1 gene has been identified as a potential genetic risk factor for
3 4
both PEX and PEXG since 2007 when a genome-wide association study on
5
pseudoexfoliative individuals from Iceland and Sweden was performed.11 This study
6
demonstrated a strong association (>99% population attributable risk) of PEX and
7
PEXG conferred by three SNPs in the LOXL1 gene on chromosome 15q24.1. Two
8
SNPs were identified in the first coding exon (rs3825942 or G153D and rs1048661
9
or R141L) and one within the first intron of this gene (rs2165241). Although the
10
rs2165241 was more significantly associated with PEXG than the two exonic SNPs,
11
this association was not statistically significant after adjusting the results for the two
12
other SNPs.11 Several studies of different ethnic populations have reported
13
associations of LOXL1 with PEX and PEXG. Many of them did not differentiate
14
between PEX and PEXG. Recently, two clinical-based studies in Greece examined
15
the association of LOXL1 SNPs with PEX and PEXG and reported conflicting
16
results.12,13
17
The discovery of common sequence variants in the LOXL1 gene that confer
18
susceptibility to PEXG, primarily through PEX syndrome, has shed some light in the
19
pathogenesis of the disease.14 LOXL1 is one of the five enzymes in the family of lysyl
20
oxidases, which are copper-dependent monoamino oxidases secreted by fibrogenic
21
cells including fibroblasts and smooth muscle cells. These enzymes are involved in
22
the covalent cross-linking of collagen and elastin polymers in extracellular matrix
23
formation. LOXL1 is necessary for tropoelastin cross-linking and elastic fiber
24
formation, maintenance, and remodeling.14
7
1
The purpose of the present study is to explore the association of two non-
2
synonymous LOXL1 SNPs (G153D and R141L) with PEX, PEXG and primary open-
3
angle glaucoma (POAG) in a population-based setting using a well-defined,
4
ethnically homogenous population in Thessaloniki, Greece. In addition, in previous
5
studies no information is provided on the potential association of LOXL1 with IOP or
6
with systemic diseases. This study aims to identify whether the two LOXL1 SNPs
7
were associated with differences in IOP or with the self-reported history of systemic
8
diseases (diabetes mellitus, cardiovascular disease or cardiovascular surgery) in
9
subjects with PEX, PEXG, POAG and controls.
10 11 12
Methods The Thessaloniki Eye Study is a cross-sectional, population-based study of
13
chronic eye diseases in the Greek population of Thessaloniki. Thessaloniki is the
14
major urban centre in Northern Greece and the second largest city in Greece, after
15
Athens. This urban area has a stable and homogenous population with 97.7% of the
16
population identified as being of Greek ethnicity.The study was approved by the
17
Aristotle University Medical School, Ethics Committee and the Institutional Review
18
Board of the University of California, Los Angeles. All study procedures adhered to
19
the principles outlined in the Declaration of Helsinki for research involving human
20
subjects and all participants gave written informed consent prior to their participation.
21
Details about the recruitment process in the Thessaloniki Eye Study have
22
been previously described.4 In brief, 5000 subjects 60 years of age or older were
23
randomly selected in February 1999 from approximately 321,000 persons registered
8
1
in the municipality registers of the city of Thessaloniki. Subjects who agreed to
2
participate were invited to the Thessaloniki Eye Study centre (Laboratory of
3
Research and Clinical Applications in Ophthalmology at the Aristotle University of
4
Thessaloniki) for an extensive ophthalmologic examination. Clinic-visit examination
5
included measurement of visual acuity using Early Treatment Diabetic Retinopathy
6
Study (ETDRS) charts, Humphrey automated perimetry, Goldmann applanation
7
tonometry (three readings from each eye), gonioscopy, slit-lamp examination (before
8
and after pupil dilation) and fundus biomicroscopy. Details of observation procedures
9
were described elsewhere.4
10
A home-visit eye examination was organized, proposed and arranged for
11
persons unable to visit the study examination centre due to major disability or illness.
12
Home-visit examination included ETDRS visual acuity measurement, Perkins
13
applanation tonometry, slit-lamp examination, and dilated fundus examination.
14
Prior to clinical examination, a questionnaire was administered including
15
demographic data, ophthalmologic and medical history. In particular, subjects were
16
queried whether they had ever been diagnosed with hypertension, diabetes mellitus
17
(treated or not with insulin), any cardiovascular disease, heart attack or they had
18
undergone coronary artery bypass or vascular surgery.
19
Definitions
20
Particular attention was given to identify the presence of PEX. PEX was
21
defined by the presence of pseudoexfoliative material either at the pupil margin or on
22
the lens capsule. Prior to pupil dilation a detailed high magnification slit-lamp
23
assessment of the pupil margin was performed. After pupil dilation, the anterior lens
9
1
surface from each eye was scanned from left to right using a narrow slit-lamp beam
2
and then was examined using a vertical broad slit-lamp beam, looking specifically for
3
early signs of PEX, including pregranular radial lines, as well as established granular
4
deposits. The location of PEX either on the iris, the lens or both was recorded. The
5
PEX detection and the exact location required consensus agreement between at
6
least two of the three ophthalmologist graders. Dilation was conducted in all patients
7
and even in those with narrow-occludable angle, laser peripheral iridotomy was
8
performed and they were examined later under dilation.
9
Glaucoma was defined according to specific criteria.4 Subjects were classified
10
as having primary open-angle glaucoma (POAG) if they had glaucoma and open,
11
normal-appearing anterior chamber angle and absence of other secondary causes of
12
glaucoma in either eye. Subjects were classified as having pseudoexfoliative
13
glaucoma (PEXG) if they had glaucoma and presence of pseudoexfoliation in either
14
eye. A consensus agreement between at least two of the three ophthalmologists was
15
required to assign the diagnosis of glaucoma. Three ophthalmologists were
16
responsible for the ophthalmic examination. At least two of the three examined each
17
patient and all diagnoses were in consensus agreement.
18
Among the 3,617 subjects who were deemed eligible, 2,554 participated in
19
the study (participation rate 71%). Of those participating, 2261 (89%) had the clinic-
20
visit examination and 293 (11%) had the home-visit examination.
21
A blood sample was taken from all subjects with POAG and PEXG and in a
22
consecutive subset of participants without glaucoma. Written consent was obtained
23
before the blood draw. A total of 233 subjects who gave blood sampleswere included
24
in this analysis. Among them, 93 had neither PEX nor glaucoma in both eyes
10
1
(controls), 40 had pseudoexfoliation and no glaucoma in either eye (PEX), 66were
2
diagnosed with POAG and 34 with PEXG.
3
Peripheral blood samples were collected from each subject and DNA was
4
extracted using QIAamp DNA Mini Kit (Qiagen). DNA samples were amplified by
5
PCR as previously described using the sense: 5’ GCAGGTGTACAGCTTGCTCA 3’
6
and antisense 5’ ACACGAAACCCTGGTCGTAG 3’ primers.15 PCR reactions were
7
performed in a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems Inc.
8
[ABI], Foster City, CA).PCR products were sequenced using the same primers as in
9
PCR amplification in a 310 DNA Analyzer (Applied Biosystems Inc. Foster City, CA),
10
using manufacturer’s protocol. Analysis of individual sequences was carried out
11
using the AB DNA sequencing analysis software v5.2.
12
Statistical analysis
13
All statistical analyses were performed with statistical software package Stata
14
version 10.0 (Stata corporation, 4905 Lakeway Drive, College Station, Texas 77845
15
USA). Fisher’s exact test was used for the genotypic comparison using as a) control
16
group: subjects without PEX, PEXG, POAG, b) cases 1: subjects with POAG, c)
17
cases 2: subjects with PEX, d) cases 3: subjects with PEXG. Multinomial logistic
18
regression analysis was conducted in order to examine the association between the
19
two LOXL1 polymorphisms and POAG, PEX or PEXG, adjusted for the following
20
factors: age, gender, history of diabetes, history of cardiovascular disease, history of
21
cardiovascular surgery and the higher IOP for the two eyes (possible confounding
22
factors for glaucoma, as identified in a previous report of Thessaloniki Eye study).16.
23
Logistic regression analysis adjusted for age was also conducted in order to examine
24
the association of the two LOXL1 SNPs with IOP in controls and cases not receiving
11
1
IOP-lowering treatment. The association of the LOXL1 SNPs with the history of the
2
aforementioned systemic diseases was also examined with regression analysis
3
adjusted for age and gender. Hardy-Weinberg equilibrium (HWE) testing was
4
performed using Mendel version 13.0.17,18 Linkage Disequilibrium and haplotype
5
analysis was also performed using Mendel version 13.0.19 Odds Ratios (ORs) with
6
95% Confidence Intervals (CIs) were calculated as measures of each strength of the
7
allele’s association with POAG, PEX, PEXG. P-values were not adjusted for multiple
8
testing.
9
Results
10
Successful DNA extraction, PCR amplification and genotyping were carried
11
out for all of the 233 enrolled subjects. The general characteristics of the participants
12
(mean age, gender, IOP, history of diabetes, insulin use, cardiovascular disease or
13
cardiovascular surgery) in the four groups are presented in Table 1.
14
The allelic and genotypic distribution of the G153D (rs3825942) and
15
R141L (rs1048661) polymorphisms in controls, POAG, PEX and PEXG groups are
16
presented in Table 2. All frequencies were in Hardy Weinberg Equilibrium using a
17
heterozygote-homozygote test (p=0.486 for R141L, p=0.603 for G143D).18 With
18
regards to the G153D polymorphism, there was a statistically significant difference in
19
subjects with PEX and PEXG compared to controls using either the log-additive
20
allelic model (G vs. A allele, p
