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Variation of Codons 1961 and 2177 of the Stargardt Disease Gene Is Not Associated With Age-Related Macular Degeneration
Robyn H. Guymer, MD, PhD;
Elise Héon, MD;
Andrew J. Lotery, MD, FRCOphth;
Francis L. Munier, MD;
Daniel F. Schorderet, PhD;
Paul N. Baird, PhD;
Robyn J. McNeil, BSc;
Heidi Haines, MS;
Val C. Sheffield, MD, PhD;
Edwin M. Stone, MD, PhD
Arch Ophthalmol. 2001;119:745-751.
ABSTRACT
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Objectives To investigate the role of 2 specific alleles of the Stargardt disease
gene (ABCA4) in the pathogenesis of age-related macular
degeneration (AMD). Secondary objectives were to investigate differences in
frequency of the G1961E allele in selected ethnic
groups as well as to examine the segregation of both G1961E and D2177N alleles in 5 multiplex families
with AMD.
Methods Five hundred forty-four patients with AMD and 689 controls were ascertained
from 3 continents. Blood samples from 62 normal individuals of Somalian ancestry
were also obtained. Participants were screened for the presence of these ABCA4 alleles with a combination of restriction digestion
and single-strand conformation polymorphism analysis of polymerase chain reaction
amplification products. Detected alleles were confirmed by DNA sequencing.
The number of subjects exhibiting the G1961E or D2177N variants were compared between AMD and control groups
using a 2-tailed Fisher exact test.
Results There was no significant difference (P>.1)
in the frequency of the G1961E and D2177N alleles in patients with AMD (2.2%) vs controls (1.0%). In contrast,
there was a significant difference (P<.001) in
the frequency of the G1961E alleles between normal
individuals of Somali ancestry (11.3%) and normal individuals from other populations
(0.4%). There was no evidence of cosegregation of these alleles and the AMD
phenotype in the 5 multiplex families with AMD examined. These two ABCA4 alleles were slightly more frequent in patients with AMD with
choroidal neovascularization (2.7%) than those without this complication (2.5%).
Conclusions Somali ancestry is more than 100 times more strongly associated with
presence of the G1961E allele than the AMD phenotype.
This study did not find any statistically significant evidence for involvement
of the G1961E or D2177N
alleles of the ABCA4 gene in AMD.
Clinical Relevance The ABCA4 gene is definitively involved in
the pathogenesis of Stargardt disease and some cases of photoreceptor degeneration.
However, it does not seem to be involved in a statistically significant fraction
of AMD cases.
INTRODUCTION
AGE-RELATED macular degeneration (AMD) is the most common cause of severe
visual loss in the developed world.1 There
is a substantial body of evidence that suggests that an important fraction
of AMD has a genetic basis.2-6
However, the high prevalence of this disease, its late onset, and its likely
mechanistic and genetic heterogeneity have all proved to be significant obstacles
to the discovery of the genes involved.
Despite these difficulties, a number of investigators have extensively
pursued AMD predisposition genes because of their potential value in the diagnosis
and treatment of this devastating condition. As a result, several genes responsible
for specific human macular disease phenotypes have been identified.7-13
However, with one possible exception, none of these genes have been found
to be associated with a significant fraction of typical late-onset AMD.
The exception is the ABCA4 gene (formerly ABCR). This gene encodes an adenosine triphosphate–binding
cassette transporter of the retina and was convincingly shown by Allikmets
et al7 to be responsible for autosomal recessive
Stargardt disease. In addition, this gene seems to be capable of causing other
retinal phenotypes, including cone dystrophy and retinitis pigmentosa.14-15 Shortly after the original demonstration
of this gene's role in Stargardt disease, Allikmets and colleagues suggested
that more than 16% of late-onset macular degeneration was also caused by variations
in the ABCA4 gene.7
Subsequent articles16-17 noted
potential difficulties with the statistical methods used in the AMD study
by Allikmets and coauthors,7 while other authors18-20 independently screened
the ABCA4 gene for variations and were unable to
identify any that were significantly more common in patients with AMD than
controls. Other reports21-22 have
offered additional evidence in support of a role for ABCA4 in typical AMD in the form of segregation analysis of ABCA4 sequence variations in families affected with the disease.
The present study had 3 objectives. (1) The primary purpose was to try
to clarify the role of ABCA4 in AMD by screening
a large cohort of patients with AMD and ethnically matched controls for the
presence of the 2 sequence variations (G1961E and D2177N) that were most highly associated with AMD in the
study by Allikmets et al.7 (2) A secondary
purpose was to investigate the variation in allele frequency of G1961E among selected ethnic groups. (3) The final purpose was to examine
the segregation of the G1961E and D2177N alleles in 5 multiplex families with AMD from Australia.
PATIENTS, MATERIALS, AND METHODS
PATIENT ASCERTAINMENT
Informed consent was obtained from all study patients or their legal
guardians. Five hundred forty-four patients with a clinical diagnosis of AMD
were ascertained from the United States (304), Australia (201), and Switzerland
(39). Patients were classified as having AMD based on an established international
classification system.23 Clinical information
about the patients from the United States and Switzerland was obtained by
a retrospective analysis of medical records and fundus photographs, while
the Australian patients with AMD were each examined by a small team of ophthalmologists
as part of the Australian AMD inheritance study. The average age of patients
with AMD ascertained in the United States, Australia, and Switzerland was
75, 78, and 79 years, respectively. All were aged 55 years or older. Of the
235 Iowans with AMD, 110 had definite evidence of choroidal neovascularization
(CNV) while an equal number were free of this complication. The presence or
absence of CNV could not be determined with certainty in the remaining 15.
Of the 201 Australian patients with AMD, 99 had definite evidence of CNV,
100 were free of this complication, and 2 had an unknown status. Of the 39
Swiss patients with AMD, 13 had definite CNV and 26 did not. Six hundred eighty-nine
control subjects with no history of AMD were ascertained from the United States
(408), Australia (187), and Switzerland (94). The minimum age of the control
subjects from Switzerland was 25 years; Australia, 51 years; and United States,
40 years.
In addition, blood samples were obtained from 42 unrelated individuals
born in Somalia but living in Canada as well as 20 unrelated individuals born
in Somalia but living in Australia. All of these individuals had normal vision
and had no personal or familial history of any form of macular degeneration,
including Stargardt disease.
MOLECULAR ANALYSIS
The DNA was extracted from venous blood as previously described.24 Exons 42 and 48 (containing codons 1961 and 2177
respectively) of the ABCA4 gene were amplified using
previously reported oligonucleotide primers.7, 25
The G1961E variant was detected using a Taq 1 restriction
digestion as follows. The 8.35 µL PCR product was mixed with 1.0 µL
10X Taq 1 buffer, 1 µL 10X bovine specific albumin (BSA), and 4 U of
Taq 1 and then incubated at 65°C for 2 hours. Five microliters of the
digested product were electrophoresed on 6% polyacrylamide, 5% glycerol nondenaturing
gels, and stained with silver nitrate using a standard protocol.26
The G1961E allele was recognized by the appearance
of 2 new fragments 135 base pair (bp) and 77 bp in size and confirmed by automated
DNA sequencing using dye-terminator chemistry and an ABI 377 sequencer. The D2177N allele was detected by single-strand conformation
polymorphism analysis. Amplimers were denatured at 95°C for 3 minutes,
electrophoresed on 6% polyacrylamide, 5% glycerol nondenaturing gels, and
stained with silver nitrate as mentioned earlier in this article.26 Samples exhibiting band shifts were sequenced as
described earlier. All samples were screened in a single laboratory, and all
gels were examined by a minimum of 2 experienced investigators. In addition,
the samples from the Australian patients were independently screened in a
second laboratory with identical results.
STATISTICAL ANALYSIS
The proportion of subjects showing the G1961E
or D2177N variant were compared between AMD and control
groups using the Fisher exact test (2-tailed). This analysis was calculated
for the sample group as a whole and for each individual subgroup. Correction
for multiple measurements and the power calculation were performed as described
elsewhere.27-28
RESULTS
The distributions of G1961E and D2177N sequence variations in the various AMD and control populations
are summarized in Table 1. Overall,
544 patients with AMD and 689 ethnically matched population controls were
studied. Five instances of the G1961E variation and
7 instances of the D2177N change were observed among
the patients with AMD, while 3 instances of G1961E
and 4 of D2177N were observed among the controls.
These changes were heterozygous in all 12 cases. A Fisher exact test revealed
the differences in allele frequency between these groups to be insignificant
whether the alleles were considered together or separately (P>.10 in all cases) even without correction for multiple measurements.27 Collectively, the G1961E
and D2177N sequence changes were found in 2.2% of
the 544 patients with AMD in this study, ranging from 0.99% in patients from
the United States to 5.1% in patients from Switzerland. This variation suggested
that ethnic differences in ABCA4 allele frequencies
might exist to a degree that could affect the interpretation of a study if
they were not taken into account.
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Table 1. G1961E Association With Age-Related
Macular Degeneration*
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The latter idea was strengthened by a chance observation made during
our study of a large group of probands with Stargardt disease (Andrew R. Webster,
MD, FRCOphth, E.H., A.J.L., et al, unpublished data, February 2000). Specifically,
Webster and co-workers found that only 5 of 386 Stargardt probands were homozygous
for a likely disease-causing mutation in the ABCA4
gene. Two of these 5 individuals were homozygous for G1961E, and both of them were patients with Somali ancestry. The fact that
these were the only 2 Stargardt probands in the entire cohort who were known
to have Somali ancestry suggested that the G1961E
allele frequency might be much higher in individuals from Somalia than those
from other ethnic backgrounds. To test this hypothesis, 62 unrelated normal
individuals with Somali ancestry were screened for the G1961E change, and 7 were found to be heterozygous for this variant
(11.2%). Table 2 gives the result
of sequential comparisons (Fisher exact test) of the frequency of the G1961E change in normal individuals in Somalia and normal
individuals from our other study populations. The frequency of the G1961E change is significantly greater in normal individuals of Somali
ancestry than in normal individuals from other ethnic backgrounds. Table 3 gives a similar set of sequential
comparisons between the frequency of the G1961E change
in normal individuals from Somalia and the frequency of the change in patients
with AMD in our study. The frequency of the G1961E
change is significantly greater in unaffected individuals from Somalia than
in the entire cohort of patients with AMD in this study (P<.001).
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Table 2. G1961E Association With Ethnicity
(Somali Controls vs Other Controls)*
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Table 3. G1961E Association With Ethnicity
(Somali Controls vs Patients With AMD)*
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Five of the 7 AMD probands from Australia who harbored a G1961E or D2177N change had family members
who also carried the clinical diagnosis of AMD. Clinicians who were masked
to the molecular status of these individuals ascertained all living siblings
of the affected individuals and diagnosed them as affected (Figure 1A) or unaffected (Figure
1B) with AMD. Figure 2
shows the results of these examinations as well as the associated molecular
findings. Of the 15 family members diagnosed with AMD, 8 harbored a G1961E or D2177N change. Similarly,
of the 5 siblings who were clinically unaffected, 3 had the ABCA4 sequence variation that was present in the proband of their family.
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Figure 1. A, Fundus photograph of the right
eye of a 71-year-old woman (family 1, patient 3 in Figure 2) with age-related
macular degeneration (AMD). Her visual acuity was 20/200 OD. Although 5 of
her 6 siblings harbored the G1961E allele, she did
not. B, Fundus photograph of the left eye of a 66-year-old woman (family 1,
patient 5 in Figure 2). Although this woman did harbor the G1961E allele, the clinicians who examined her felt that she was unaffected
by AMD.
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Figure 2. Pedigree drawings of 5 Australian
families with age-related macular degeneration (AMD) and ABCA4 sequence variations. Closed symbols indicate the diagnosis of
AMD by a clinician who was masked to the molecular status of the individual.
A plus indicates the presence of a heterozygousABCA4
sequence variation that was present in the family's proband (G1961E for family 1, and D2177N for families
2-5). Fundus photographs of 2 members of family 1 are shown in Figure 1, while
photographs of 4 members of family 2 are shown in Figure 3. The age (y) of
each individual is shown beneath their pedigree symbol, while the proband
of each family is identified with an arrow.
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Of the 544 patients with AMD in this study, we had clinical information
sufficient to evaluate the presence of CNV in 458. Of this group, 222 (48%)
had definite evidence of CNV (Figure 3), while 236 were free of this complication. The G1961E
or D2177N sequence variations were found in 6 of
the patients (2.7%) with CNV and 6 of the patients (2.5%) who were free from
this complication.
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Figure 3. A, Fundus photograph of the right
eye of an 81-year-old woman (family 2, patient 1 in Figure 2) with age-related
macular degeneration (AMD) and choroidal neovascularization (CNV). Her visual
acuity in this eye was hand motions. She was found to harbor the D2177N allele. B, Fundus photograph of the left eye of an 80-year-old
woman (family 2, patient 2 in Figure 2) with AMD and CNV. Her visual acuity
in this eye was hand motions. She was found to harbor the D2177N allele. C, Fundus photograph of the left eye of a 66-year-old
man (family 2, patient 3 in Figure 2) with AMD and CNV. His visual acuity
was 20/100 OS. Although 2 of his 3 relatives harbored the D2177N allele, he did not. D, Fundus photograph of the right eye of
a 78-year-old woman (family 2, patient 4 in Figure 2) with AMD. Her visual
acuity was 20/100 OD. Although 2 of her 3 relatives harbored the D2177N allele, she did not.
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COMMENT
Perhaps the greatest challenge in interpreting the clinical significance
of sequence variations in the ABCA4 gene is the relatively
high frequency of these variations in both patient and control populations.
For example, when Stone et al18 initially examined
a cohort of patients with Stargardt disease, patients with AMD, and controls,
they found more than 1100 instances of 140 different sequence variations.
In principle, any of these sequence variations could be disease causing while
in fact, many of these changes probably represent non–disease-causing
polymorphisms. Before screening a population of patients for potential disease-causing
variations in a gene, it is difficult to know what types of variations are
likely to be found. However, if one uses the distribution of different alleles
between patients and controls as the criterion for determining the pathogenicity
of those alleles, it is statistically invalid to use the same set of data
to argue that the gene is involved in the pathogenesis the disease (ie, based
on the skewed distribution of "pathogenic" alleles). It would be valid to
use the first set of data as a pilot experiment to identify the characteristics
of likely disease-causing mutations and then to sample a new population to
see whether the hypothesis generated from the first experiment can be supported
by the second. Therefore, in this study, we focused on the 2 sequence variations
(G1961E and D2177N) that
were most plausibly associated with AMD in the study by Allikmets et al.7 In that study, Allikmets and colleagues found evidence
that one or the other of those 2 sequence variations were found in approximately
8% of patients with AMD (one-half of the total ABCA4
association they observed). Although these 2 sequence variations were statistically
associated with AMD when considered by themselves,7
there was no statistical association with AMD if all of the observed missense
variations were included in the analysis, or alternatively, if all of the
various subgroup analyses were subjected to a statistical correction for multiple
measurements.16 In this study, we failed to
find any statistically significant association between AMD and the presence
of the G1961E and D2177N ABCA4 sequence variations. We found these 2 ABCA4
variations in 12 (2.2%) of 544 patients with AMD. This was similar to the
frequency we had observed in patients with AMD in our previous study18 (3 [1.6%] of 182) but more than 3-fold lower than
that observed by Allikmets and coworkers (13 [7.8%] 167).7
With the sample size that we used, our study had a power of greater than 95%
( = .05) to detect the difference in frequency of these alleles between
patients with AMD and controls who had been previously reported (7.8% vs 0.5%).7 However, it should be emphasized that the control
individuals in the present study were not examined for the presence of AMD
and that many of them were not old enough to manifest the signs of the disease
even if they had been examined. As a result, a portion of these control subjects
would be expected to harbor the same AMD-causing variations that were present
in an AMD patient cohort (thereby lessening the difference between the 2 groups).
If one assumes that one third of the population is at risk for the degree
of AMD manifested by the patients in this study, one would predict a 3-fold
greater frequency of a true disease-causing mutation in the AMD cohort than
in an unexamined and/or young control population. The sample size of this
study was also large enough to result in a 95% power ( = .05) to detect
such a 3-fold difference if the frequency in the AMD cohort was as large as
had been previously suggested (ie, 7.8% vs 2.6%).7
An interesting observation in the study by Allikmets and coauthors7 was that the G1961E and D2177N sequence variations were more commonly found in
patients who had not experienced CNV. Specifically, only 1 of their 33 patients
with ABCA4 sequence variations had experienced that
complication. In the present study, we found no evidence to support the idea
that ABCA4 sequence variations are more likely to
be present in patients with AMD who do not have CNV. Of the 12 patients with
AMD in this study who harbor G1961E or D2177N sequence variations, 6 had CNV.
One of the difficulties of studying disease-associated allelic variation
in ethnically diverse human populations is that some of the variation that
is observed may be secondary to unrecognized variations in ethnicity instead
of variations in the disease state. High frequencies of certain alleles can
be present in certain ethnic groups because of geographic or cultural barriers
that existed hundreds of years earlier. It often requires a chance observation
to discover a population in which a specific allele is enriched or depleted
because the number of different ancestral ethnic groups worldwide is so great.
For example, in this study, we found that the frequency of the G1961E allele was significantly higher in patients of Somali ancestry
than in control populations from the United States, Australia, and Switzerland
(Table 2) or AMD populations in
the United States and Australia (Table 3). Although further work needs to be performed to carefully characterize
the phenotype of G1961E heterozygotes with Somali
ancestry, the point to be made in the present context is that Somali ancestry
is more than 100 times more highly correlated to the presence of the G1961E sequence variation than the presence of AMD.
A common strategy for inferring the pathogenicity of sequence variations
in human populations is to demonstrate a cosegregation of the variation and
the phenotype in a large number of individuals. This is the basis of the familiar
lod score method for chromosomal localization of disease genes. Some authors
have sought to investigate the possible role of ABCA4
sequence variations in macular degeneration by examining the cosegregation
of the AMD phenotype with certain ABCA4 alleles in
patients with familial AMD.21-22,29
These studies have taken 2 forms. In the first, the grandparents of patients
with Stargardt disease are studied to determine whether the 2 grandparents
who harbor one of the ABCA4 alleles present in the
affected child also manifest AMD.21-22
In the second, siblings of an AMD proband with a variant ABCA4 allele are examined for the cosegregation of the AMD phenotype
and the ABCA4 allele.29
For this type of experiment to be valid, it is necessary for the clinicians
making the phenotypic judgments to be masked to the genotypic information
and for all of the relatives of the proband to be equally ascertained. In
addition, an adequate number of individuals need to be examined to allow the
possibility of observing a statistically significant association. None of
the studies currently in the literature have demonstrated a statistically
significant association of ABCA4 alleles with an
AMD phenotype in this type of experiment.
In the present study, we examined all of the siblings of 5 families
affected with AMD who were also found to harbor a G1961E or D2177N ABCA4 allele. In all cases, we
observed at least one individual with the AMD phenotype who did not harbor
the proband's ABCA4 allele, and in 2 of the families,
there were also unaffected siblings who did harbor the proband's allele (Figure 2). These findings by themselves do
not demonstrate that ABCA4 alleles lack a phenotype
in the heterozygous state. That is, if these alleles represent only a small
fraction of all patients with AMD, one would expect to find many examples
in which some other genetic cause of AMD was present within a nuclear family.
Similarly, the absence of the phenotype in individuals who carry a certain
allele can be explained by the incomplete penetrance that is a common feature
of all late-onset dominant diseases. However, these 5 families do illustrate
the difficulty that one encounters trying to establish a statistically significant
association between certain ABCA4 alleles and a common
phenotype like AMD.
The data in this article notwithstanding, there is no doubt that variations
in the ABCA4 gene cause human retinal disease. The
data supporting the involvement of this gene in autosomal recessive Stargardt
disease are extensive,7, 21, 30-32
and the data for its role in other recessive retinal degenerations are also
strong.14-15,30 However,
much remains to be learned about the phenotypic effect of specific alleles.
The fact that 11% of people of Somali ancestry are heterozygous for the G1961E allele would predict that more than 1% of that population
would be homozygous. The fact that Stargardt disease is not known to be 100
times more prevalent in this population than in the United States, Switzerland,
or Australia suggests that G1961E does not frequently
cause disease in the homozygous state. It raises the testable hypothesis that G1961E is more likely to cause disease in the compound
heterozygous state than in the homozygous state. Thus at the present time,
it seems that we do not fully understand the pathogenic role of the G1961E allele even in patients with Stargardt disease (with
which the allele has been significantly associated in multiple studies). It
therefore seems premature to suggest a pathogenic role for this allele in
a much more common and more genetically heterogeneous disease (AMD).
Given the extreme allelic diversity of the ABCA4
gene in the human population, the most straightforward way to investigate
the possibility of its role in dominantly inherited (ie, heterozygous) late-onset
disease may be with the use of transgenic animal models and/or in vitro systems.33-34
AUTHOR INFORMATION
Accepted for publication August 12, 2000.
This study was supported in part by the National Institutes of Health,
Bethesda, Md, grant EY10539; the Foundation Fighting Blindness, Hunt Valley,
Md; the Carver Charitable Trust, Muscatine, Iowa; the Ronald McDonald House
Charities, Oak Brook, Ill; the Grousbeck Family Foundation, Stanford, Calif;
the Ruth and Milton Steinbach Foundation, New York, NY; the Royal Victorian
Institute for the Blind, Melbourne, Victoria, Australia; the Swiss National
Science Foundation, Bern, Switzerland, grant 32-053750.98; the Fondation Telethon
Action Suisse, Aubonne, Switzerland; and an unrestricted grant from Research
to Prevent Blindness Inc, New York, NY. Dr Lotery is the recipient of a Research
to Prevent Blindness Career Development Award.
The authors would like to thank the patients for their enthusiastic
participation, and especially the Somalian communities in Toronto and Melbourne.
The authors are indebted to Alex Levin, MD, for facilitating their contact
with the Somalian community in Toronto; to Penelope Allen, MD, Melinda Cain,
and Catherine McCarty, MD, for their help in recruiting patients in Australia;
and to Andrew Webster, MD, FRCOphth, for his help with the statistical analyses.
The authors thank Luan Streb, Kim Vandenburgh, Robin Hockey, Gretel Beck,
and Chris Taylor for their excellent technical assistance.
Corresponding author and reprints: Edwin M. Stone, MD, PhD, Department
of Ophthalmology, University of Iowa College of Medicine, Iowa City, IA 52242
(e-mail: Edwin-Stone{at}uiowa.edu).
From the Centre for Eye Research Australia (Drs Guymer and Baird and
Ms McNeil), University of Melbourne, and Royal Victorian Eye and Ear Hospital
(Dr Guymer), Melbourne, Victoria; Department of Ophthalmology (Dr Héon),
University of Toronto, Toronto, Canada; Vision Science Research Program (Dr
Héon), University Health Network, Toronto; Departments of Ophthalmology
(Drs Lotery and Stone and Ms Haines) and Pediatrics (Dr Sheffield), University
of Iowa College of Medicine, Iowa City; Hôpital Jules Gonin (Dr Munier),
Lausanne, Switzerland; Division de Génétique Médicale
(Drs Munier and Schorderet), Centre Hospitalier Universitaire Vaudois, Lausanne;
and the Howard Hughes Medical Institute (Dr Sheffield), Chevy Chase, Md.
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