The Natural History of Stargardt Disease with Specific Sequence Mutation in the ABCA4 Gene

Abstract

Purpose.: To determine longitudinal changes in fundus appearance and visual function in patients with Stargardt with at least one allelic mutation (Gly1961Glu) in the ABCA4 gene.

Methods.: Sixteen patients with a diagnosis of Stargardt disease and a Gly1961Glu mutation were enrolled. All patients underwent a complete ocular examination including best corrected visual acuity, Goldmann visual field (GVF), and full-field ERG examinations. The percentage of patients who showed at least a doubling in the log of the minimum angle of visual resolution (logMAR) between their initial and most recent visits was determined, as was the percentage of patients who showed a doubling in the size of the central scotoma over this duration.

Results.: Nine patients had at least a doubling of the logMAR visual acuity in their right eyes and 10 patients in their left eyes, over a mean follow-up (FU) period of 18.6 years. Of 15 patients, 46.7% had equal to or more than a doubling of the central scotoma area in response to a II2e test stimulus in the right eye and 60.0% in the left eyes. Among 10 patients whose ERGs were initially normal for rod and cone responses, 8 remained normal at their most recent FU visit.

Conclusions.: In these patients with Stargardt disease and a Gly1961Glu mutation, most showed a clinical phenotype characterized by fundus changes localized to the foveal and parafoveal regions, normal ERG amplitudes, absence of a silent or masked choroid, and a mean age at initial presentation in the third decade.

Stargardt disease is a recessively inherited macular dystrophy with an estimated prevalence of between 1 in 8,000 to 10,000. ¹ The disorder is typically characterized by impairment of central vision within the first 10 to 20 years of life that often progresses to the level of legal blindness. ^2,3 The peripheral vision is usually preserved. ^4,5 There is a characteristic beaten-bronze appearance of the macula, with small yellow-white flecks scattered within the posterior pole and, to a lesser extent, in the midperipheral retina. In the majority of affected patients, fluorescein angiography (FA) shows a masking of the fluorescence from the choroidal circulation (dark choroid) suggesting a diffuse abnormality at the level of the retinal pigment epithelium (RPE). ²  

In 1994, Stargardt disease was mapped to the long arm of chromosome 1 ⁶ and was later shown to be caused by mutations in the ABCA4 (formerly ABCR) gene. ⁷ The ABCA4 gene encodes an ATP-binding cassette (ABC) transport protein located in the disc membranes of rod and cone outer segments. The ABCA4 protein is involved in the transport of all-trans-retinal conjugates across the disc membranes. In the absence of a functional ABCA4 protein due to mutations in the gene, vitamin A aldehyde forms bisretinoid products that are deposited in retinal pigment epithelial (RPE) cells during the process of disc shedding and phagocytosis. Consequently, abnormally high levels of lipofuscin pigments, such as A2E and the all-trans-retinal dimer phosphatidylethanolamine, accumulate in the RPE, triggering RPE-cell death and causing secondary photoreceptor degeneration. Mutations in the ABCA4 gene are responsible not only for Stargardt disease (STGD1), ⁷ but also have been implicated in autosomal recessive forms of cone–rod dystrophy (arCRD) and retinitis pigmentosa (arRP). ^8–11  

Clinical observations have confirmed the presence of a wide spectrum of retinal phenotypes in patients with Stargardt with sequence mutations in the ABCA4 gene. In a previous study by Fishman and et al., ¹² an association was observed between a certain ABCA4 genotype, in which the amino acid glutamic acid is substituted for glycine (Gly1961Glu), and a Stargardt disease phenotype. The phenotype was classified as stage I, which is typically characterized by a localized atrophic-appearing foveal lesion surrounded by parafoveal or perifoveal yellowish-white lesions (flecks). In stage II, the retinal flecks appear throughout the posterior pole, anterior to the vascular arcades and/or nasal to the optic disc, while in stage III, the flecks are almost entirely reabsorbed. In stage IV, there is extensive atrophy of the RPE and choroid. ¹² Eleven of the patients with Gly1961Glu mutations cited in Fishman et al. ¹² were also included in the current natural history study. 

The purpose of the present study was to determine longitudinal changes in visual acuity, fundus morphologic features, central scotoma size, and ERG amplitudes in patients with Stargardt with at least one allelic mutation (Gly1961Glu) in the ABCA4 gene. 

Materials and Methods

Patient Ascertainment

One of the authors (GAF) referred 16 patients with a diagnosis of Stargardt macular dystrophy and a specific ABCA4 sequence mutation (Gly1961Glu) on at least one allele for the present study. The inclusion criteria included those patients with a decrease in central vision, yellowish-white retinal flecks, a minimum of three visits, and at least 6 years of follow-up. The study conformed to the tenets of the Declaration of Helsinki and was approved by a University of Illinois Institutional Review Board. Informed consent was obtained from all participants. 

All patients underwent a complete ocular examination including best corrected visual acuity, using either a Snellen projection chart or the Early Treatment Diabetic Retinopathy Study (ETDRS) chart (The Light House, Long Island City, NY), slit lamp biomicroscopy, ocular pressure measurement by applanation tonometry, and dilated fundus examination with direct and indirect ophthalmoscopy. Fundus color photography was obtained for documentation of retinal changes. These photographs and findings on clinical fundus examination were used to classify patients into one of the four stages described in the introduction and published elsewhere. ¹² A fluorescein angiogram was obtained in 12 of the 16 patients. 

Visual Field Examination

Visual field examination was performed by Goldmann kinetic perimetry (model 940; Haag-Streit AG, Köniz, Switzerland), using II2e, II4e, III4e, and V4e test targets. Not all these stimulus targets were used for testing on each patient. Corrective trial lenses commensurate to refractive status and age of the patient were used only for fields within a radius of 30° or less and only with the II2e target. To determine changes in the size of a central scotoma among the studied patients over time, the area of the scotoma was determined by planimetry with the use of a digitizing tablet (SummaSketch III; Summagraphics, Scottsdale, AZ) and computer software. The central scotoma area was measured and expressed in square inches. Since a change in visual field area is best described by an exponential function, the central scotoma area data were transformed to natural log units. We determined how many patients showed at least a doubling in the area of the central scotoma (change of 0.3 log units or more) over the duration of the follow-up. The criterion for a doubling in size of the scotoma was used because this degree of change was likely to be of clinical significance. 

Electroretinogram Examination

A full-field electroretinogram (ERG) was obtained monocularly by either of two procedures previously described with the use of a unipolar Burian-Allen contact lens electrode. ^13,14 One eye was tested after dilating with 2.5% phenylephrine and 1% tropicamide. The ERG responses were obtained according to the International Society for Clinical Electrophysiological of Vision (ISCEV) guidelines, which included a dark-adapted, rod-isolated response; a dark-adapted, rod-dominant response; a 32-Hz flicker response; and a light-adapted, single flash response. Parameters included amplitudes and implicit times for each of the major waveform components. ¹⁵ All results were compared with 90% tolerance limits or an appropriate range for a visually normal population. 

Molecular Analysis

Blood samples were obtained from all patients, and the DNA was extracted from venous blood, as previously described. ¹⁶ Two mutation screening techniques were used to detect mutations in the ABCA4 gene. The first was a single-strand conformation polymorphism analysis (SSCP) followed by automated direct DNA sequencing. ¹² The second was the ABCA4 genotyping microarray (gene chip), followed by confirmation of the detected mutations by direct sequencing. ¹⁷ Exon numbers are reported according to Azarian et al. ¹⁸ and Gerber et al. ¹⁹  

Statistical Analysis

Qualitative variables such as sex and race were described as frequencies. For a comparison of visual acuity between initial and most recent visits, a nonparametric Mann-Whitney rank sum test was used. The level of significance was considered to be P < 0.05. For calculating average values for years of follow-up, intervals between ERG recordings, and ages at initial and most recent visits, we calculated the mean and standard deviations and provide the medians as well as the ranges. 

Results

Of the 16 patients with Gly1961Glu mutations, 6 (37.5%) had the Gly1961Glu mutation on one allele (heterozygous), whereas 1 (6.2%) was homozygous for the mutation and 9 (56.2%) had a compound heterozygous mutation (Table 1). 

Table 1.

 

Genetic Mutations in the ABCA4 Gene in the Patients

Patient No.	Genetic Mutation Allele 1	Genetic Mutation Allele 2	Comments
1	gly1961glu exon 42		Heterozygous
2	gly1961glu exon 42	ala1038val exon 21 and leu541pro exon 12	Compound heterozygous
3	gly1961glu exon 42		Heterozygous
4	gly1961glu exon 42	arg2077trp exon 45	Compound heterozygous
5	gly1961glu exon 42	gly65glu exon 3	Compound heterozygous
6	gly1961glu exon 42	leu1201arg exon 24	Compound heterozygous
7	gly1961glu exon 42		Heterozygous
8	gly1961glu exon 42		Heterozygous
9	gly1961glu exon 42	del Co 1620–1622 exon 35	Compound heterozygous
10	gly1961glu exon 42		Heterozygous
11	gly1961glu exon 42	1bp del(T) Co 36 which creates stop at Co 38	Compound heterozygous
12	gly1961glu exon 42	IVS38-10 T>C	Compound heterozygous
13	gly1961glu exon 42		Heterozygous
14	gly1961glu exon 42	pro1380leu	Compound heterozygous
15	gly1961glu exon 42	pro1380leu	Compound heterozygous
16	gly1961glu exon 42	gly1961glu exon 42	Homozygous

There were 12 (75.0%) females and 4 (25.0%) males. In the studied cohort, there were 14 (87.5%) Caucasians, 1 (6.2%) African American, and 1 (6.2%) Asian patient (Table 2). The mean age of the patients at the initial visit was 27.6 ± 8.05 (SD) (median, 27.3; range, 13–43 years). The mean age at the most recent follow-up (FU) visit was 46.3 of ± 12.2 (median, 46.5; range: 24–74 years). The average number of the visits was 10.3 ± 6.9 with a range between 3 and 27 visits. The overall mean FU period was 18.6 ± 8.7 years (median, 15.5; range: 6–34 years). Three (18.8%) patients were followed up from 6 to 10 years, four (25.0%) from 11 to 15 years, and nine (56.2%) for more than 15 years (Tables 2, 3). 

Table 2.

 

Demographics of the Study Population

Patient No.	Sex	Race	Age at Initial Visit (y)	Age at Most Recent Visit (y)	Visits (n)	Follow-up Period (y)
1	F	C	30	41	3	12
2	M	C	27	42	6	15
3	F	C	25	57	9	33
4	M	C	36	49	4	12
5	F	C	24	39	16	15
6	F	AA	38	47	4	8
7	M	C	23	57	6	34
8	F	C	32	55	23	23
9	F	C	43	74	15	32
10	M	C	13	24	4	10
11	F	C	18	43	27	25
12	F	C	30	46	11	16
13	F	C	34	59	15	25
14	F	C	15	31	7	15
15	F	C	31	48	10	16
16	F	AS	22	29	5	6
			Mean 27.6	Mean 46.3	Mean 10.3	Mean 18.6
			SD8.05	SD12.2	SD6.9	SD8.7
			Range 13–43	Range 24–74	Range 3–27	Range 6–34

 C, Caucasian (n = 14 [87.5%]); AA, African American (n = 1 [6.25%]); AS, Asian (Indian) (n = 1 [6.25%]).

Table 3.

 

Follow-up Period in Stargardt Macular Dystrophy with Gly1961Glu Mutations

Follow-up Period (y)	Patients, n (%)
6–10	3 (18.8)
11–15	4 (25.0)
>15	9 (56.2)
Total	16 (100)

The number of patients with various stages of Stargardt dystrophy ²⁰ at the time of the initial presentation was as follows: 13 (81.2%) patients showed a stage I phenotype, 2 (12.5%) a stage II, and 1 (6.2%) a stage IV. At the most recent FU visit, among the 13 patients who were initially at stage I, 10 (76.9%) remained at stage I, whereas 3 (23.1%) had progression to stage II. Of the two patients who were at stage II initially, one remained in stage II, and one had progression to stage III. One patient remained in stage IV (Table 4). 

Table 4.

 

The Stages of Stargardt Dystrophy and History of Nyctalopia among the Study Population

Patient No.	Stage at Initial Visit	Stage at Most Recent Visit	Hx of Nyctalopia at Initial Visit	Hx of Nyctalopia at Most Recent Visit
1	1	1	−	−
2	1	1	+	−
3	2	2	−	−
4	1	1	+	+
5	1	1	−	−
6	1	1	+	−
7	1	1	−	−
8	1	1	−	−
9	4	4	−	−
10	1	2	−	−
11	1	2	−	−
12	2	3	−	−
13	1	2	−	−
14	1	1	+	−
15	1	1	+	+
16	1	1	−	−

 Hx, history; +, present; −, absent.

We determined that five (31.2%) patients had a history of nyctalopia at the initial visit, whereas six (37.5%) had a history of photoaversion. 

Using a nonparametric Mann-Whitney rank sum test, we determined that the median logMAR visual acuity (VA) at the initial visit in the right eye was 0.87 (range, 0.10–1.40), whereas the median logMAR VA at the most recent FU visit was 1.00 (range, 0.18–2.80; P = 0.325). In the left eye, the initial median logMAR VA was 0.65 (range, 0.18–1.90), whereas the median logMAR VA at the most recent FU visit was 1.00 (range, 0.40–2.80; P = 0.074; Table 5). 

Table 5.

 

VA Changes over Follow-up Periods

Patient No.	Initial VA LogMAR OD	Most Recent VA LogMAR	Initial VA Snellen OD	Most Recent VA Snellen OD	LogMAR Difference* OD	Initial VA LogMAR OS	Most Recent VA LogMAR OS	Initial VA Snellen OS	Most Recent VA Snellen OS	LogMAR Difference* OS	Follow-up (y)
1	0.1	0.54	20/25−1	20/70−2	−0.44	0.18	1	20/30−2	20/200+1	−0.82	12
2	1.3	1.0	20/400	20/400	0	0.18	0.98	20/30+2	20/400	−1.12	15
3	1.18	1.3	20/300	20/400	−0.12	1.18	1.0	20/300	20/200	−0.18	33
4	0.1	1.3	20/25−2	20/400	−1.2	1	1.3	20/200	20/400	−0.3	12
5	1.0	0.7	20/200	20/100−1	0.3	0.6	0.7	20/80+3	20/100−1	−0.1	14
6	0.1	0.18	20/25+2	20/30−2	−0.08	1	1	20/200	20/200	0.00	8
7	1.18	1.0	20/300	20/200	0.18	0.4	1	20/50	20/200	−0.6	34
8	1.0	1.0	20/200−1	20/200	0.00	0.48	1	20/60+2	20/200	−0.52	23
9	1.4	2.8	8/200	LP	−1.4	1.9	2.8	5/400	LP	−0.9	32
10	1.0	1.0	20/200	20/200	0.00	1	1	20/200	20/200	0.00	10
11	0.40	0.48	20/50	20/60	−0.08	0.48	0.70	20/60+1	20/100−2	−0.22	25
12	1.0	1.3	20/200	20/400	−0.3	1	1.3	20/200	20/400	−0.3	16
13	1.0	0.70	20/200	20/100−2	0.3	1.0	0.70	20/200	20/100−2	0.3	25
14	0.54	1.0	20/70+2	20/200	−0.46	0.70	1.0	20/100+2	20/200+1	−0.3	15
15	0.10	0.90	20/25−1	20/200	−0.8	0.18	0.92	20/30	20/200	−0.74	16
16	0.40	1.00	20/50+2	20/200+1	−0.6	0.18	0.40	20/30	20/50	−0.22	6

*  LogMAR difference is the difference in VA in LogMAR between most recent and initial visits.

Using a doubling of the log of the minimum angle of visual resolution (logMAR) as a criterion to determine the visual acuity changes over the FU period (doubling of the visual angle is equal to a change of 0.3 logMAR units, which is a 3-line difference on an ETDRS chart), we found that in seven patients (43.8%) the logMAR difference was less than double, whereas in nine (56.2%) it was equal to or more than a doubling of their visual angle of resolution in the right eye. For the left eye, in six (37.5%) patients, the logMAR difference was less than a doubling of the minimum angle of visual resolution (Table 6). 

Table 6.

 

The Difference in Doubling of logMAR VA in the Patients

	Patients, n (%)	Age (y) at Most Recent Visit		Visits (n)		Follow-up Period (y)
		Mean ± SD	Range	Mean ± SD	Range	Mean ± SD	Range
Right eye
<Doubling of VA (<0.3)	7 (43.8)	42.10 ± 11.8	24–57	9.59 ± 8.9	4–27	21.14 ± 9.1	8–34
≥Doubling of the VA (≥0.3)	9 (56.2)	42.00 ± 13.9	29–74	9.11 ± 5.1	3–16	15.66 ± 7.7	6–32
Left eye
<Doubling of VA (<0.3)	6 (37.5)	35.86 ± 12.0	24–57	10.59 ± 9.2	4–27	16.17 ± 4.2	6–33
≥Doubling of VA (≥0.3)	10 (62.5)	46.71 ± 11.8	31–74	9.65 ± 6.2	3–23	20.0 ± 14.9	1–34

At the most recent visit, all of the 16 patients had an atrophic-appearing lesion (that included geographic atrophy or a bull's eye–appearing macular lesion) in the macula of both eyes compared with 15 (93.8%) patients who had an atrophic macular lesion at the time of presentation. Among the 16 patients at their most recent visit, 4 (25.0%) had a bull's eye maculopathy in at least one eye. At their initial visit, eight (50.0%) patients showed a bull's eye-appearing macular lesion in each eye. Of the 12 patients who had fluorescein angiographic studies, 11 did not show a masking of the background choroidal fluorescence (dark choroid). 

Goldmann visual field testing at the most recent visit showed that in the 16 patients, 30 (93.8%) eyes of 15 of them had a central scotoma in response to the II2e stimulus, 30 eyes of 15 had a central scotoma in response to the II4e stimulus, and 8 (25.0%) eyes of 4 patients showed a central scotoma in response to the III4e stimulus. 

Using a doubling of the log difference of the central scotoma area as a criterion to determine the changes in the central scotoma size over time, for the right eyes we found that 7 (46.7%) patients had equal to or more than a doubling of the log unit difference in response to a II2e stimulus over a mean of 18.4 years (median, 17.2; range, 10–25), 10 (66.7%) in response to a II4e stimulus over a mean of 16.4 years (median, 15; range, 8–25), and 2 (50.0%) in response to a III4e stimulus over a mean of 20 years (median, 20; range,15–25). For the left eyes, 9 (60.0%) patients showed an equal or more than a doubling of the log unit difference in response to a II2e stimulus over a mean of 16.3 years (median, 15.5; range, 6–33), 10 (66.7%) in response to a II4e stimulus over a mean of 16.0 years (median, 15; range, 6 to 25 years), and 2 (50.0%) in response to a III4e stimulus over a mean of 15.5 years (median, 15.5; range, 6 to 25 years; Table 7). 

Table 7.

 

Number and Percentage with at Least a Doubling of the Area of the Central Scotoma (≥0.3 Log Units)

Target Size	Central Scotoma at Most Recent Visit		OD	OS	Prevalence (%) of Central Scotoma at Most Recent Visit
	Eyes (n)	Patients (n)	Patients, n (%)	Patients, n (%)
II2e	30	15	7 (46.7)	9 (60.0)	93.8
II4e	30	15	10 (66.7)	10 (66.7)	93.8
III4e	8	4	2 (50.0)	2 (50.0)	25.0

 Data were obtained at the most recent visit by kinetic perimetry, and the number and percentage of doubling of the log unit difference was recorded among the study population in response to different target size stimuli during a follow-up period.

Of the 16 patients, full-field electroretinogram (ERG) recordings were obtained in 15 (93.8%) at their initial visit, whereas 12 (75.0%) had ERG measurements at their most recent visit. The mean interval between ERG recordings was 15.9 ± 7.9 years (median, 15.0; range, 6–34). The initial ERG responses among the cohort were as follows: 10 (66.7%) patients had normal cone and rod responses whereas only 3 (20.0%) had abnormal cone and rod responses. One (6.7%) patient had normal rod function and abnormal cone function and one (6.7%) had normal cone and abnormal rod function. Among the 12 patients who had ERG testing at their most recent FU visit, 8 (66.7%) had normal rod and cone responses, whereas only 3 (25.0%) had abnormal cone and rod responses and 1 (8.3%) had normal rod and abnormal cone function. 

Among the 10 patients whose ERGs were initially normal for both rod and cone responses, 8 (80.0%) remained normal at their most recent FU visit, which ranged from 8 to 34 years, with a mean of 16.9 years. One (10.0%) patient refused to have an ERG obtained at the most recent visit, and one had an abnormal ERG at the most recent visit in the form of a 50% reduction in rod function and 33% reduction in cone amplitudes when compared with the normal initial ERG measured 15 years previously. 

Discussion

The present study, to our knowledge, is the first to demonstrate the natural history of structural and functional changes observed in the course of disease in patients with Stargardt disease and a Gly1961Glu sequence mutation in the ABCA4 gene. 

If our 16 study patients, 10 (62.5%) remained at stage I ²⁰ at their most recent visit, with atrophic lesions at the macula surrounded by a ring of yellowish white perifoveal or parafoveal flecks over a mean FU period of 18.6 ± 8.7 years (range, 6–34; median, 15.5). 

In our cohort of patients with Stargardt disease, 14 (88.0%) were Caucasian of different ancestries (Eastern European, British, German, and Irish). In a previous report, Guymer et al. ²¹ described that there was a significant difference in the frequency of the G1y1961Glu allele between visually normal individuals of Somali ancestry (11.3%) and visually normal individuals from a United States population (0.4%).This finding implies a potential for a higher prevalence of individuals who are homozygous for the Gly1961Glu mutation in the Somali population. 

In our group of patients with Stargardt disease and at least one Gly1961Glu mutation, visual acuity was eventually reduced to a level of 20/200 to 20/400. Similarly, other reports have described progressive loss of visual acuity in patients with Stargardt to a level of 20/200. ^2,4 Our data showed that there was at least a doubling in logMAR in the right eyes of 9 (56.2%) patients and in the left eyes of 10 (62.5%) patients over a period of 6 to 34 years, with a mean of 18.3 years (median, 15.5). The data also showed that on Goldmann kinetic perimetry testing, 46.7% of patients had equal to or more than a doubling of central scotoma area in response to a II2e stimulus in the right eye over a mean duration of 18.4 years, whereas 60.0% showed the doubling over a mean duration of 16.3 years in the left eye. 

We observed that, not surprisingly, the atrophic-appearing macular lesions in our cohort of patients became more evident over time. Of note, except for one patient, there was no evidence of a dark choroid on fluorescein angiography. 

An ERG obtained in 12 patients at their most recent visit showed that in 66.7% of them, normal rod and cone amplitudes and implicit times were found. This finding is also consistent with observations of Fishman et al., ¹² who showed that 90.0% (9/10) of patients with Stargardt with a Gly1961Glu mutation showed normal rod and cone ERG responses. 

In our patients with Stargardt disease and a Gly1961Glu mutation, a high percentage initially presented and then maintained stage I disease; did not manifest a dark choroid; and as a group, maintained normal ERG cone and rod amplitudes. Only one patient showed visual acuity of less than 20/400 in her better seeing eye at the most recent visit. 

Comprehensive studies that correlate genotype data with phenotypic findings, including natural history for visual loss, will play a useful, if not important, role in patient selection in future therapeutic clinical trials, as well as in patient counseling in those with Stargardt macular dystrophy. 

Footnotes

 Supported by funds from the Foundation Fighting Blindness, Owings Mills, Maryland; Grant Healthcare Foundation, Lake Forest, Illinois; a National Institutes of Health Core Grant EY01792; and an unrestricted departmental grant from Research to Prevent Blindness.

Footnotes

 Disclosure: M.A. Genead, None; G.A. Fishman, None; E.M. Stone, None; R. Allikmets, None

Footnotes

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.