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Novel Mutation in the TIMP3 Gene Causes Sorsby Fundus Dystrophy
Samuel G. Jacobson, MD, PhD;
Artur V. Cideciyan, PhD;
Jean Bennett, MD, PhD;
Ronald M. Kingsley, MD;
Val C. Sheffield, MD, PhD;
Edwin M. Stone, MD, PhD
Arch Ophthalmol. 2002;120:376-379.
ABSTRACT
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Objective To determine the molecular basis of a retinopathy previously described
as dominant macular subretinal neovascularization with peripheral retinal
degeneration.
Methods The TIMP3 gene was analyzed in family members,
and 4 mutation-positive patients were studied using psychophysics and electroretinography.
Results Cosegregating with disease in the family was a single base pair change
in the TIMP3 gene, altering a conserved tyrosine
to cysteine at amino acid position 172 (Y172C). There was psychophysical and
electroretinographic evidence of rod dysfunction greater than cone dysfunction.
Dark adaptometry showed abnormalities with regional retinal variation in degree.
Conclusions The Y172C mutation in the TIMP3 gene is another
cause of Sorsby fundus dystrophy. The expression of this form of the disease,
as in other C-terminal TIMP3 mutations, is speculated
to be secondary to mutant TIMP-3, causing a decreased turnover of the extracellular
matrix.
Clinical Relevance The molecular clarification of inherited retinal degeneration involving
abnormal extracellular matrix turnover in and around Bruch's membrane should
provide clues to the pathogenesis of not only these particular diseases but
also forms of age-related macular degeneration.
INTRODUCTION
AMONG MANY monogenic retinal degenerative diseases, only Sorsby fundus
dystrophy (SFD) commonly manifests as a hemorrhagic maculopathy secondary
to choroidal neovascularization (CNV).1 This
feature of SFD is interesting because of its clinical similarity to some forms
of age-related macular degeneration (AMD). The hypothesis that there may be
both molecular and clinical similarity to AMD became testable when it was
determined that SFD was caused by mutations in the gene encoding tissue inhibitor
of metalloproteinases-3 (TIMP-3),2 an extracellular
matrix (ECM) protein. However, candidate gene screening with the TIMP3 gene in AMD populations did not reveal a simple genetic relationship
between the rare inherited disease and the common age-related cause of CNV.3-4
The complex pathways leading from the TIMP3
mutation to ECM disturbance and CNV are still being studied.5-6
Results are beginning to reveal a pathogenetic sequence that may explain abnormal
sub-RPE (retinal pigment epithelium) deposition in a number of retinopathies.5-7 Studies to understand
the effects on ECM turnover of mutant TIMP-3 are aided by the knowledge of
disease-causing TIMP3 mutations. Apart from the founder
mutation in codon 181 from the British Isles, only a few other disease-causing TIMP3 gene mutations have been identified.6, 8
More than a decade ago, a family was described as having "dominant macular
subretinal neovascularization with peripheral retinal degeneration."9 Among the diagnostic possibilities considered was
SFD, but the age at disease onset was thought to be too early compared with
other reports in the literature at that time.10-11
We reevaluated the family and report that this retinopathy is a severe form
of SFD caused by a novel mutation in the TIMP3 gene.
PATIENTS AND METHODS
Informed consent was obtained from the study participants for all procedures.
Mutation screening of the TIMP3 gene was performed. Venous blood
samples were collected from 13 family members (generations III and IV in Figure 1A), and DNA was extracted. Techniques
for single-strand polymorphism (SSCP) analysis and direct sequencing of polymerase
chain reaction (PCR) products have been described elsewhere.12
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Figure 1. A, Pedigree of the family in this
study. Filled symbols indicate affected individuals; shaded symbols, individuals
molecularly affected but clinically unaffected to date; open symbols, unaffected
individuals; and slashed symbols, deceased individuals. Patient III-5 is the
proband. B, Single-strand polymorphism analysis of TIMP3 exon
5 polymerase chain reaction products in available family members. Each lane
of this silver nitrate–stained 6% polyacrylamide gel contains the amplified
DNA from the individual whose pedigree number is above it. Individuals with
the mutation (+) have 2 extra bands (arrows), indicating a heterozygous exon
5 mutation. Four individuals (-) lack the extra bands, representing
a homozygous normal sequence in exon 5. C, Fundus photograph of patient III-5
(at age 45 years) showing central retinal scarring and degeneration.
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A subset of 4 patients (III-4, 5, and 6 and IV-14), all positive for
a TIMP3 gene mutation, were assessed with routine ophthalmic
examinations and specialized tests of visual function. Static threshold perimetry
in dark-adapted (500- and 650-nm stimuli) and light-adapted (600-nm stimulus
on a 10 candela/m2 white background) states was performed using
a modified automated perimeter and analyzed for rod and cone threshold elevation.
Dark adaptometry was tested with 500- and 650-nm stimuli after a retinal exposure
of 7.8 log scotopic-troland-seconds, estimated to bleach about 99% of the
rhodopsin present, and recovery was measured until prebleach baseline dark-adapted
(>3-hour) thresholds were attained. Details of these procedures have been
published previously.13-15
For the patients with central scotomas, bleaching and testing were performed
using infrared visualization of the fundus with a modified fundus photoperimeter.
Electroretinogram (ERG) photoresponses were evoked in the dark-adapted state
with high-energy blue (2.3-4.6 log scotopic-troland-seconds) and red (1.4-3.6
log photopic-troland-seconds) stimuli and in the light-adapted (3.2 log troland
white background) state with red (2.2-4.1 log photopic-troland-seconds) stimuli.
A model of phototransduction activation consisting of the sum of rod and cone
components was used to quantify the leading edges of dark-adapted waveforms;
a model of cone phototransduction was used to quantify the leading edges of
light-adapted waveforms. Details of recording and analysis methods have been
published previously.16-17
RESULTS
The PCR product from exon 5 of the TIMP3 gene
amplified from the proband (patient III-5) migrated in an aberrant electrophoretic
pattern compared with normal control DNA samples run in parallel during SSCP
analysis. Direct sequencing of the PCR product revealed a single base pair
change, altering a conserved tyrosine to cysteine at amino acid position 172.
This Y172C TIMP3 gene mutation cosegregates with
the disease in generation III, which has the only living clinically affected
members at this time; there were both mutation-positive and mutation-negative
members in generation IV (Figure 1B).
The fundus photograph of patient III-5 at age 45 years (Figure 1C) is representative of the central retinal scarring from
hemorrhagic macular degeneration found in affected members in the fourth and
fifth decades of life. Fundus appearance and fluorescein angiography in patients
III-3, 4, 6, and 7 documenting RPE abnormalities in the macula and midperipheral
retina were previously reported.9 Five of the
6 affected patients had an acute loss of central vision in one eye between
ages 26 and 29 years, with the second eye losing vision within 2 years of
the first event. The affected half brother (patient III-1) lost central vision
between ages 35 and 36 years. In generation IV, 4 younger asymptomatic family
members (ages 7-21 years) carry the mutation and are at risk for developing
the disease (Figure 1A).
Visual function studies in a subset of heterozygotes for the Y172C TIMP3 mutation are shown in Figure 2. Rod- and cone-mediated thresholds throughout the field
of vision are shown for a 39-year-old patient (III-6) with a large central
scotoma. Throughout the peripheral visual field were significant rod threshold
elevations (mean, 2.0 log units); beyond the central scotoma, most cone thresholds
were within normal limits. Patients III-3 and III-5 also showed more rod than
cone threshold elevations in the peripheral visual field beyond their central
scotomas. An asymptomatic 20-year-old heterozygote (patient IV-14) had normal
results on eye examination and visual function tests.
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Figure 2. Functional phenotype of heterozygotes
with the Y172C TIMP3 mutation. A, Static threshold dark- and
light-adapted perimetry in a patient representing a late stage of the disease.
Elevation of rod and cone thresholds across the visual field are shown as
gray-scale maps, with 16 levels representing 0 to 3 log units of threshold
elevation. Black squares indicate no detection of stimuli. T, N, I, and S
indicate temporal, nasal, inferior, and superior visual fields, respectively.
B, Rod- and cone-isolated photoresponses (symbols connected by thin black
lines) fitted with a model of phototransduction activation (thicker gray lines).
Photoresponses evoked with blue 4.6 log scotopic-troland-second (ROD) and
red 4.1 log photopic-troland-second (CONE) flashes are shown for each patient.
Arrows on the y-axis denote the lower limit (mean - 2 SD)
of normal maximum amplitude. C and D, Dark adaptation with 500-nm stimulus
after a full bleach exposure in patients (symbols and thin lines) compared
with the normal range (gray, thicker lines). Prebleach thresholds are shown
preceding time zero; numbers represent test location in degrees in the T and
N visual fields.
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Rod and cone ERG photoresponses suggest that the abnormalities on psychophysical
testing have a photoreceptor basis (Figure
2B). Patient IV-14 had normal rod and cone responses, whereas 3
older patients (patients III-3, 5, and 6) had rod maximum amplitudes reduced
to approximately half of the mean normal value and normal or slightly reduced
cone maximum amplitudes. Rod photoresponse sensitivities were within normal
limits in 2 patients (III-6 and IV-14), whereas 2 other heterozygotes (patients
III-3 and 5) had sensitivity losses of about 0.4 log units. Cone photoresponse
sensitivities were within normal limits for all 4 patients.
Dark adaptation was tested at several different retinal loci in the
4 heterozygotes; representative functions obtained at  30° eccentric
to the anatomical fovea are shown (Figure
2C and D). In patients IV-14 and III-6, dark-adaptation functions
at 3 different loci were normal; in patient III-5, dark adaptation was borderline
normal at 4 loci; and in patient III-3, it ranged from normal to abnormal
at 4 loci tested. The abnormality, when present, consisted mainly of threshold
elevation (Figure 2D).
COMMENT
Following the first association between a TIMP3
mutation and SFD,2 several reports of other TIMP3 mutations in SFD have appeared.6, 8
Almost all mutations documented in SFD to date, including Y172C, would be
expected to alter residues in the C-terminal domain of the molecule. There
is still no biological explanation of how mutation in an inhibitor of matrix
metalloproteinase may be leading to decreased ECM turnover and extreme thickening
of Bruch's membrane, the histopathological hallmark of SFD.18-19
Recent in vitro studies of 4 SFD mutations suggest that the pathogenesis is
probably not caused by haploinsufficiency but by persistent increased function.
Evidence has appeared that mutant TIMP-3 may be accumulating as a dimer and
functionally contributing to a slowed turnover of the ECM.6
Immunocytochemical studies in a donor retina with SFD previously demonstrated
an accumulation of TIMP-3 in Bruch's membrane.19
Recently, the need for a greater understanding of ECM biology to interpret
disease was further emphasized when another counterintuitive observation was
made in patients with multicentric osteolysis and arthritis syndrome, disorders
characterized by an enhanced ECM breakdown. Causative mutations were found
in the gene encoding matrix metalloproteinase-2,20
suggesting that like SFD this was another ECM disorder with a complex mechanism.21
Considering the onset ages reported for hemorrhagic macular degeneration
in SFD families with other causative mutations,8
the family with the Y172C TIMP3 mutation is among
those with an earlier onset of macular disease. The clinical manifestations
in SFD are all likely to be secondary to the slowly progressive thickening
of Bruch's membrane from the ECM imbalance discussed previously. Until the
disease threshold is reached, the retinas of these patients are clinically
and functionally normal. Choroidal neovascularization could be secondary to
a hypoxic stimulus from the retina, which becomes separated by a critical
distance from its choriocapillaris blood supply by ECM buildup. The photoreceptor
abnormalities leading to extramacular vision loss may be secondary to RPE
dysfunction.14 The finding of increased TIMP-3
content of Bruch's membrane with normal aging and in AMD7, 22
raises the suspicion that this molecule may be a marker for pathological processes
that lead to a disturbed ECM turnover. Abnormal binding and activity of TIMP-3
may be one of the aberrant pathways leading to many diseases of Bruch's membrane.
AUTHOR INFORMATION
Submitted for publication July 17, 2001; final revision received October
25, 2001; accepted November 16, 2001.
This study was supported in part by grants EY-05627, EY-13203, and EY-12156
from the National Institutes of Health, Bethesda, Md; the Macular Disease
Foundation, Virginia Beach, Va; the Macula Vision Research Foundation, West
Conshohocken, Pa; Foundation Fighting Blindness Inc, Owings Mills, Md; and
the F. M. Kirby Foundation, Morristown, NJ.
Dr Jacobson is a Research to Prevent Blindness (New York, NY) Senior
Scientific Awardee, Drs Bennett and Cideciyan are Research to Prevent Blindness
Special Scholars, and Dr Sheffield is an associate investigator of the Howard
Hughes Medical Institute, Iowa City, Iowa.
We are grateful to M. Benegas, D. Hanna, J. Emmons, L. Gardner, Y. Huang,
D. Marks, J. Huang, and C. Taylor for help with the conduct of this study.
Corresponding author and reprints: Samuel G. Jacobson, MD, PhD, Scheie
Eye Institute, 51 N 39th St, Philadelphia, PA 19104 (e-mail: jacobsos{at}mail.med.upenn.edu).
From the Department of Ophthalmology, Scheie Eye Institute, University
of Pennsylvania, Philadelphia (Drs Jacobson, Cideciyan, and Bennett); Department
of Ophthalmology, University of Oklahoma Health Sciences Center, Dean A. McGee
Eye Institute, Oklahoma City (Dr Kingsley); and Departments of Ophthalmology
(Dr Stone) and Pediatrics and the Howard Hughes Medical Institute (Dr Sheffield),
University of Iowa College of Medicine, Iowa City.
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ABSTRACT
SECTION EDITOR: EDWIN M. STONE, MD, PHD
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