Free
Genetics  |   October 2013
ABCA4 Gene Screening by Next-Generation Sequencing in a British Cohort
Author Affiliations & Notes
  • Kaoru Fujinami
    Laboratory of Visual Physiology, National Institute of Sensory Organs, National Tokyo Medical Center, Tokyo, Japan
  • Jana Zernant
    Department of Ophthalmology, Columbia University, New York, New York
  • Ravinder K. Chana
    UCL Institute of Ophthalmology, London, United Kingdom
    Moorfields Eye Hospital, London, United Kingdom
  • Genevieve A. Wright
    UCL Institute of Ophthalmology, London, United Kingdom
    Moorfields Eye Hospital, London, United Kingdom
  • Kazushige Tsunoda
    Laboratory of Visual Physiology, National Institute of Sensory Organs, National Tokyo Medical Center, Tokyo, Japan
  • Yoko Ozawa
    Department of Ophthalmology, Keio University, School of Medicine, Tokyo, Japan
  • Kazuo Tsubota
    Department of Ophthalmology, Keio University, School of Medicine, Tokyo, Japan
  • Andrew R. Webster
    UCL Institute of Ophthalmology, London, United Kingdom
    Moorfields Eye Hospital, London, United Kingdom
  • Anthony T. Moore
    UCL Institute of Ophthalmology, London, United Kingdom
    Moorfields Eye Hospital, London, United Kingdom
  • Rando Allikmets
    Department of Ophthalmology, Columbia University, New York, New York
    Department of Pathology and Cell Biology, Columbia University, New York, New York
  • Michel Michaelides
    UCL Institute of Ophthalmology, London, United Kingdom
    Moorfields Eye Hospital, London, United Kingdom
  • Correspondence: Rando Allikmets, Columbia University, 630 West 168th Street, New York, NY 10032; rla22@columbia.edu
  • Michel Michaelides, UCL Institute of Ophthalmology, 11-43 Bath Street, London, EC1V 9EL, UK; michel.michaelides@ucl.ac.uk
Investigative Ophthalmology & Visual Science October 2013, Vol.54, 6662-6674. doi:10.1167/iovs.13-12570
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Kaoru Fujinami, Jana Zernant, Ravinder K. Chana, Genevieve A. Wright, Kazushige Tsunoda, Yoko Ozawa, Kazuo Tsubota, Andrew R. Webster, Anthony T. Moore, Rando Allikmets, Michel Michaelides; ABCA4 Gene Screening by Next-Generation Sequencing in a British Cohort. Invest. Ophthalmol. Vis. Sci. 2013;54(10):6662-6674. doi: 10.1167/iovs.13-12570.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose.: We applied a recently reported next-generation sequencing (NGS) strategy for screening the ABCA4 gene in a British cohort with ABCA4-associated disease and report novel mutations.

Methods.: We identified 79 patients with a clinical diagnosis of ABCA4-associated disease who had a single variant identified by the ABCA4 microarray. Comprehensive phenotypic data were obtained, and the NGS strategy was applied to identify the second allele by means of sequencing the entire coding region and adjacent intronic sequences of the ABCA4 gene. Identified variants were confirmed by Sanger sequencing and assessed for pathogenicity by in silico analysis.

Results.: Of the 42 variants detected by prescreening with the microarray, in silico analysis suggested that 34, found in 66 subjects, were disease-causing and 8, found in 13 subjects, were benign variants. We detected 42 variants by NGS, of which 39 were classified as disease-causing. Of these 39 variants, 31 were novel, including 16 missense, 7 splice-site–altering, 4 nonsense, 1 in-frame deletion, and 3 frameshift variants. Two or more disease-causing variants were confirmed in 37 (47%) of 79 patients, one disease-causing variant in 36 (46%) subjects, and no disease-causing variant in 6 (7%) individuals.

Conclusions.: Application of the NGS platform for ABCA4 screening enabled detection of the second disease-associated allele in approximately half of the patients in a British cohort where one mutation had been detected with the arrayed primer extension (APEX) array. The time- and cost-efficient NGS strategy is useful in screening large cohorts, which will be increasingly valuable with the advent of ABCA4-directed therapies.

Introduction
Stargardt disease is the most common form of inherited macular dystrophy and is caused by recessive mutations in the ABCA4 gene. 1,2 Stargardt disease typically presents with central macular atrophy and yellow-white flecks at the posterior pole, primarily at the level of the RPE. 2,3 A highly variable phenotype and progression of Stargardt disease have been documented, and mutations in ABCA4 also have been implicated in cone dystrophy, cone-rod dystrophy, and “retinitis pigmentosa.” 4 12 In this report, we will use the term “ABCA4-associated retinal disease” to refer to the broad range and variability of clinical manifestations of retinopathy due to ABCA4 variants. 
The carrier frequency of likely pathogenic ABCA4 alleles has been reported to be as high as 1:2013,14 and more than 700 ABCA4 variants have been identified so far. 1,2,5 29 The high allelic heterogeneity makes molecular genetic analyses of ABCA4-associated retinal disease very challenging. It has been reported that direct Sanger sequencing of the entire ABCA4 coding region (50 exons) detects between 66% and 80% of disease-causing alleles 13,21 ; however, this approach has significant limitations in large patient cohorts due to the prohibitive time and cost implications. 3,5  
Since the development of the ABCA4 genotyping microarray, using arrayed primer extension (APEX) technology, 14 systematic screening of all known previously reported ABCA4 variants has been available 26,30 ; APEX detects approximately 65% to 75% of all disease-associated alleles. However, by definition, novel variants are not detected by APEX technology, necessitating the use of other methodologies for high-throughput systematic screening of the entire coding region, especially in cases where one or both disease-causing alleles have failed to be identified by the array. 
Zernant et al. recently reported the capability of a next-generation sequencing (NGS) strategy to detect new ABCA4 variants that were not included on the APEX array; all 50 ABCA4 exons of 168 patients were amplified in parallel using an amplicon tagging PCR protocol and NGS was applied to the resulting amplicons. 5 The purpose of this study was to apply this novel NGS strategy for ABCA4 screening in a large, well-characterized British cohort of patients with likely ABCA4-associated phenotypes and report novel disease-causing variants. 
Materials and Methods
Patients
Prescreening with APEX technology was performed in a cohort of 232 patients seen at Moorfields Eye Hospital with a clinical diagnosis of retinopathy compatible with ABCA4-associated retinal disease. Two or more variants were identified in 103 patients, one variant in 79 subjects, and no variants in 50 individuals. The 79 patients with only one ABCA4 allele were recruited for this study. After informed consent was obtained, blood samples were taken from all individuals for NGS of ABCA4. The protocol of the study adhered to the provisions of the Declaration of Helsinki and was approved by the Ethics Committee of Moorfields Eye Hospital. The age at onset was defined as the age at which visual loss was first noted by the patient. The duration of disease was calculated as the difference between age at onset and age at the latest examination. 
Clinical Assessment
A full medical history was obtained and a comprehensive ophthalmologic examination was performed for all patients. Clinical assessment included best-corrected Snellen visual acuity (converted to equivalent logMAR visual acuity), fundus photography, autofluorescence (AF) imaging, spectral domain optical coherence tomography (SD-OCT), and electrophysiologic assessment. 
Color fundus photography was performed with the TRC-50IA Retinal Fundus Camera (Topcon, Tokyo, Japan) and AF images were obtained using either an HRA 2 (excitation wavelength, 488 nm; barrier filter, 500 nm; field of view, 30 × 30°; Heidelberg Engineering, Heidelberg, Germany) 31 or Spectralis with viewing module version 5.1.2.0 (excitation wavelength, 488 nm; barrier filter, 500 nm; fields of view, 30 × 30° and 55 × 55°; Heidelberg Engineering) after pupillary dilation. 32 Patients were classified into one of three AF subtypes based on a recent report in ABCA4-associated retinal disease 31 : type 1—localized low AF signal at the fovea surrounded by a homogeneous background, type 2—localized low AF signal at the macula surrounded by a heterogeneous background, and type 3—multiple areas of low AF signal at the posterior pole with a heterogeneous background. SD-OCT imaging was obtained with the Spectralis with viewing module version 5.1.2.0. 32  
Electrophysiologic assessment included full-field electroretinography (ffERG) and pattern electroretinography (PERG) incorporating the standards of the International Society for Clinical Electrophysiology of Vision (ISCEV). 33,34 All components of the ffERG and PERG were taken into account when classifying patients into one of the three electrophysiologic groups: 4,35 group 1—patients with PERG P50 abnormality with normal ERGs, group 2—subjects with PERG P50 abnormality and additional generalized cone ERG abnormality (assessed with light adapted 30 Hz ERG and light adapted 3.0 ERG), and group 3—individuals with PERG P50 abnormality, and additional generalized cone and rod ERG abnormality (assessed using dark adapted 0.01 dim flash ERG and dark adapted 11.0 bright flash ERG). 
Genetic Screening
Blood samples were collected in EDTA tubes and DNA was extracted with a Nucleon Genomic DNA extraction kit (BACC2; Tepnel Life Sciences, Manchester, UK). Mutation prescreening of ABCA4 was performed with the APEX microarray (ABCR400 chip or ABCR600 chip; Asper Ophthalmics, Tartu, Estonia; available in the public domain at http://www.asperbio.com/genetic-tests/panel-of-genetic-tests/stargardt-disease-cone-rod-dystrophy-abca4) in all probands. 14 We screened 17 patients with ABCR400 (432 mutations on the chip) in 2005, 32 with updated ABCR400 (456 mutations) in 2006, 3 with further updated ABCR400 (480 mutations) in 2007, and 27 with ABCR500 (552 mutations) in 2011. 
All 50 ABCA4 exons and exon-intron boundaries were amplified with tagged PCR primers using an amplicon tagging protocol (Access Array; Fluidigm, South San Francisco, CA; available in the public domain at http://www.fluidigm.com/products/access-array.html) and NGS on the Roche 454 platform (Roche Applied Science, Penzberg, Upper Bavaria, Germany) was performed as reported previously. 5 Sequences of the barcoded samples were analyzed with the NextGENE software for next generation sequence analysis (SoftGenetics, State College, PA), which mapped reads to the reference genome (HG19) and identified all the differences compared to the reference sequence. All the identified variants were confirmed by Sanger sequencing. Segregation analysis was not performed in this study. 
In Silico Molecular Genetic Analysis
All the missense variants identified were analyzed using two software prediction programs: Sorting Intolerant From Tolerant (SIFT; available in the public domain at http://sift.jcvi.org), 36 and PolyPhen2 (available in the public domain at http://genetics.bwh.harvard.edu/pph/index.html). 37 Predicted effects on splicing of all the missense and intronic variants were assessed with the Human Splicing Finder (HSF) program version 2.4.1 (available in the public domain at http://www.umd.be/HSF). The allele frequency of all the variants was estimated by reference to the Exome Variant Server (EVS; NHLBI Exome Sequencing Project, Seattle, WA; available in the public domain at http://snp.gs.washington.edu/EVS). 
All the variants identified were classified into one of three categories based on the bioinformatics prediction protocol described in a previous report, 5 namely disease-causing, possibly disease-causing, and benign. For the purpose of analysis in this study, variants predicted to be possibly disease-causing were included in the total number of variants described as disease-causing variants. The nomenclature of the variants was in the main in keeping with the internationally established guidelines (available in the public domain at http://www.hgvs.org/mutnomen). 38  
Results
Clinical Findings
The clinical findings of the cohort are summarized in Table 1. The study included 40 male (51%) and 39 female (49%) unrelated probands. The median age at onset was 22.0 years, with a median duration of disease of 10.0 years. The median age at the latest examination was 40.0 years, with the median logMAR visual acuities being 1.00. Color fundus photographs were obtained in 75 patients and AF imaging was undertaken in 71 subjects. There were 21 patients (30%) with a type 1 AF pattern, 34 (48%) with type 2, and 16 (22%) with type 3. Of the 70 patients with available electrophysiologic data, 34 subjects (49%) were in ERG group 1 (isolated macular dysfunction), 7 (10%) in ERG group 2 (macular and generalized cone dysfunction), and 29 (41%) in ERG group 3 (macular and generalized cone and rod dysfunction). 
Table 1
 
Summary of Clinical Features of 79 Patients With ABCA4-Related Retinal Disease
Table 1
 
Summary of Clinical Features of 79 Patients With ABCA4-Related Retinal Disease
Median age of onset, y (range) 22.0 (5–71)
Median age at examination, y (range) 40.0 (15–79)
Median duration of disease, y (range) 10.0 (0–54)
LogMAR visual acuity (range) R 1.00 (−0.08–1.78)
L 1.00 (−0.08–4.00)
AF subtype, n = 71 1 n = 21
2 n = 34
3 n = 16
ERG group, n = 70 1 n = 34
2 n = 7
3 n = 29
Color fundus photographs, AF images, and SD-OCT of three representative cases of “typical” ABCA4-associated retinal disease are shown in Figure 1 (patients 19, 36, and 17), all harboring two disease-causing variants. Six cases with an “atypical” phenotype for ABCA4-associated retinal disease are shown in Figure 2 (patients 74–79), all of them carried only one, likely benign, ABCA4 variant (Table 2). 
Figure 1
 
Color fundus photographs, autofluorescence images, and optical coherence tomography of three representative cases harboring two or more ABCA4 variants with “typical” ABCA4-associated retinal disease (patients 19, 36, and 17). Color fundus photograph of patient 19 shows macular atrophy (A) and AF imaging demonstrates a localized low AF signal at the fovea, with a high signal edge surrounded by a homogeneous background (B). SD-OCT demonstrates marked outer retinal loss at the central macula (C). Patient 36 has macular atrophy surrounded by numerous yellow-white flecks (D) and a localized low AF signal at the macula surrounded by a heterogeneous background, with peripapillary sparing (E). Generalized loss of outer retinal architecture is seen on SD-OCT. Patient 17 has widespread multiple areas of atrophy with patchy pigmentation (G) and multiple areas of low AF signal at the posterior pole with a heterogeneous background (H).
Figure 1
 
Color fundus photographs, autofluorescence images, and optical coherence tomography of three representative cases harboring two or more ABCA4 variants with “typical” ABCA4-associated retinal disease (patients 19, 36, and 17). Color fundus photograph of patient 19 shows macular atrophy (A) and AF imaging demonstrates a localized low AF signal at the fovea, with a high signal edge surrounded by a homogeneous background (B). SD-OCT demonstrates marked outer retinal loss at the central macula (C). Patient 36 has macular atrophy surrounded by numerous yellow-white flecks (D) and a localized low AF signal at the macula surrounded by a heterogeneous background, with peripapillary sparing (E). Generalized loss of outer retinal architecture is seen on SD-OCT. Patient 17 has widespread multiple areas of atrophy with patchy pigmentation (G) and multiple areas of low AF signal at the posterior pole with a heterogeneous background (H).
Figure 2
 
Color fundus photographs and autofluorescence images of six cases harboring a single, likely benign, missense variant with “atypical” clinical features for ABCA4-associated retinal disease (patients 74, 75, 76, 77, 78, and 79). Color photograph of patient 74 shows a geographic atrophy-like appearance (A), which on AF imaging is surrounded by foci of high and low AF signal (B). Patient 75 has evidence of generalized retinal atrophy in addition to marked macular atrophy, with dense pigmentation at the level of the RPE, and bone spicule formation, marked vessel attenuation, and optic disc pallor (C). Patient 76 has extensive macular atrophy extending beyond the arcades, with dense pigmentation at the level of the RPE and slight bone spicule pigmentation in the periphery (D). AF imaging demonstrates a heterogeneous background, but no peripapillary sparing (E). Patient 77 has a large area of macular atrophy extending to the optic disc (F), which on AF imaging is surrounded by an irregular low AF signal with foci of high and low signal, with no peripapillary sparing (G). Patient 78 has multiple widespread areas of atrophy with dense pigmentation at the level of RPE and bone spicule pigmentation in the periphery (H). AF imaging identifies multiple low signal areas with a heterogeneous background and no peripapillary sparing (I). Patient 79 has subtle atrophy confined to the fovea (J). AF imaging demonstrates a localized low AF signal at the fovea surrounded by a homogeneous background (K).
Figure 2
 
Color fundus photographs and autofluorescence images of six cases harboring a single, likely benign, missense variant with “atypical” clinical features for ABCA4-associated retinal disease (patients 74, 75, 76, 77, 78, and 79). Color photograph of patient 74 shows a geographic atrophy-like appearance (A), which on AF imaging is surrounded by foci of high and low AF signal (B). Patient 75 has evidence of generalized retinal atrophy in addition to marked macular atrophy, with dense pigmentation at the level of the RPE, and bone spicule formation, marked vessel attenuation, and optic disc pallor (C). Patient 76 has extensive macular atrophy extending beyond the arcades, with dense pigmentation at the level of the RPE and slight bone spicule pigmentation in the periphery (D). AF imaging demonstrates a heterogeneous background, but no peripapillary sparing (E). Patient 77 has a large area of macular atrophy extending to the optic disc (F), which on AF imaging is surrounded by an irregular low AF signal with foci of high and low signal, with no peripapillary sparing (G). Patient 78 has multiple widespread areas of atrophy with dense pigmentation at the level of RPE and bone spicule pigmentation in the periphery (H). AF imaging identifies multiple low signal areas with a heterogeneous background and no peripapillary sparing (I). Patient 79 has subtle atrophy confined to the fovea (J). AF imaging demonstrates a localized low AF signal at the fovea surrounded by a homogeneous background (K).
Table 2
 
Molecular Genetic Status Identified by NGS in 79 Patients With ABCA4-Related Retinal Disease
Table 2
 
Molecular Genetic Status Identified by NGS in 79 Patients With ABCA4-Related Retinal Disease
Pt Allele 1 Detected by APEX Allele 2 Detected by NGS Allele 3 Detected by NGS Total N of DC Variants Comments
DNA Change Protein Change/ Effect Pred. Patho. DNA Change Protein Change/ Effect Pred. Patho. DNA Change Protein Change/ Effect Pred. Patho.
1 c.161G>A p.C54Y DC c.2297G>T p.G766V DC 2
2 c.223T>G p.C75G DC c.5088C>G p.S1696R DC 2
3 c.740A>C p.N247T DC c.1433T>C p.I478T B c.2345G>A p.W782* DC 2
4 c.768G>T Splice site DC 1
5 c.1222C>T p.R408* DC c.2568C>A p.Y856* DC 2
6 c.1804C>T p.R602W DC c.859-9T>C Splice site PDC 2
7 c.1805G>A p.R602Q DC c.5113C>T p.R1705W DC 2
8 c.1922G>C p.C641S DC 1
9 c.1957C>T p.R653C DC 1
10 c.1957C>T p.R653C DC 1
11 c.2588G>C p.G863A DC c.655A>T p.R219* DC 2 Allele 2 (p.R219*) was APEX-false-negative
12 c.2588G>C p.G863A DC c.1906C>T p.Q636* DC 2
13 c.2588G>C p.G863A DC c.1906C>T p.Q636* DC 2
14 c.2588G>C p.G863A DC 1
15 c.2588G>C p.G863A DC 1
16 c.2894A>G p.N965S DC c.3322C>T p.R1108C DC 2 Allele 2 (p.R1108C) was APEX-false-negative
17 c.3064G>A p.E1022K DC c.6729+4_+18delAGTTGGCCCTGGGGC Splice site DC 2
18 c.3064G>A p.E1022K DC 1
19 c.3208_3209insGT p.S1071fs DC c.2942C>T p.P981L DC c.6529G>A p.D2177N B 2
20 c.3208_3209insGT p.S1071fs DC c.1519G>T p.D507Y DC 2
21 c.3208_3209insGT p.S1071fs DC c.4634G>A p.S1545N DC 2
22 c.3208_3209insGT p.S1071fs DC 1
23 c.3292C>T p.R1098C DC c.3299T>A p.I1100N DC 2
24 c.3322C>T p.R1108C DC c.4978delC p.L1661* DC 2
25 c.3386G>A p.R1129H DC c.3208_3209insGT p.S1071fs DC c.4634G>A p.S1545N DC 3 Allele 2 (p.S1071fs) was APEX false-negative and allele 1 (p.R1129H) was NGS false-negative
26 c.4139C>T p.P1380L DC c.3191-1G>T Splice site DC 2
27 c.4139C>T p.P1380L DC c.3398T>C p.I1133T PDC 2
28 c.4139C>T p.P1380L DC c.4070C>A p.A1357E DC 2
29 c.4139C>T p.P1380L DC c.4773G>C Splice site DC 2
30 c.4139C>T p.P1380L DC 1
31 c.4139C>T p.P1380L DC 1
32 c.4139C>T p.P1380L DC 1
33 c.4234C>T p.Q1412* DC 1
34 c.4319T>C p.F1440S DC 1
35 c.4328G>A p.R1443H DC c.180delG p.M61fs DC 2
36 c.4469G>A p.C1490Y DC c.1726G>C p.D576H DC 2
37 c.4469G>A p.C1490Y DC 1
38 c.4537_4538insC p.Q1513fs DC c.5578C>T p.R1860W DC 2 Allele 1 (p.Q1513fs) was NGS-false-negative
39 c.4577C>T p.T1526M DC 1
40 c.4926C>G p.S1642R DC c.5041_5055del GTGGTTGCCATCTGC p.V1681_C1685del DC 2
41 c.4956T>G p.Y1652* DC 1
42 c.5018+2T>C Splice site DC 1
43 c.5461-10T>C DC c.6385A>G p.S2129G PDC 2
44 c.5461-10T>C DC 1
45 c.5461-10T>C DC 1
46 c.5461-10T>C DC 1
47 c.5461-10T>C DC 1
48 c.5461-10T>C DC 1
49 c.5461-10T>C DC 1
50 c.5461-10T>C DC 1
51 c.5585-1G>A Splice site DC 1
52 c.5714+5G>A Splice site DC c.6209C>G p.T2070R DC 2
53 c.5882G>A p.G1961E DC c.2686A>G p.K896E B 1
54 c.5882G>A p.G1961E DC c.3050+1G>C Splice site DC 2
55 c.5882G>A p.G1961E DC c.3392delC/3393C>G p.A1131Gfs DC 2
56 c.5882G>A p.G1961E DC c.4539+2T>G Splice site DC 2
57 c.5882G>A p.G1961E DC c.4552A>C p.S1518R DC 2
58 c.5882G>A p.G1961E DC c.5899-2delA Splice site DC 2
59 c.5882G>A p.G1961E DC 1
60 c.6079C>T p.L2027F DC c.1906C>T p.Q636* DC 2
61 c.6079C>T p.L2027F DC c.3322C>T p.R1108C DC 2 Allele 2 (p.R1108C) was APEX-false-negative
62 c.6079C>T p.L2027F DC c.3370G>T p.D1124Y DC 2
63 c.6079C>T p.L2027F DC 1
64 c.6089G>A p.R2030Q DC c.4326C>A p.N1442K DC 2
65 c.6445C>T p.R2149* DC 1
66 c.6709A>C p.T2237P DC c.5899-3_5899-2delTA Splice site DC 2
67 c.2971G>C p.G991R B c.4538A>G p.Q1513R DC 1
68 c.3602T>G p.L1201R B c.1749G>C p.K583N DC 1
69 c.3602T>G p.L1201R B c.1982_1983insG p.A662fs DC 1
70 c.3602T>G p.L1201R B c.2972G>T p.G991V DC 1
71 c.4685T>C p.I1562T B c.3289A>T p.R1097* DC 1
72 c.6320G>A p.R2107H B c.2510T>C p.L837P DC 1
73 c.6320G>A p.R2107H B c.4352+1G>A Splice site DC 1
74 c.2701A>G p.T901A B 0
75 c.3602T>G p.L1201R B 0
76 c.4283C>T p.T1428M B 0
77 c.466A>G p.I156V B 0
78 c.466A>G p.I156V B 0
79 c.4715C>T p.T1572M B 0
Prescreening With APEX Technology
The results of prescreening of ABCA4 in our cohort of 79 patients are summarized in Table 2. We detected 42 variants at the APEX prescreening stage. In silico analysis of these 42 variants suggested that 34 were disease-causing and 8 were considered benign. Therefore, these analyses confirmed at least one disease-causing variant in 66/79 patients, while 13/79 subjects had no disease-causing variants (Tables 2, 3). 
Table 3
 
In Silico Analysis for Previously Reported Variants Identified in 79 Patients With ABCA4-Related Retinal Disease
Table 3
 
In Silico Analysis for Previously Reported Variants Identified in 79 Patients With ABCA4-Related Retinal Disease
Exon/ IVS Nucleotide Substitution Protein Change/ Effect N of Alleles Identified Pt Method Previous Report SIFT Polyphen 2 HSF Matrix Allele Freq. by EVS Reference Comment
APEX NGS Pred. Tol. Index (0–1) Pred. Hum Var Score (0–1) Site Wt CV Mt CV CV % Variation
3 c.161G>A p.C54Y 1 1 Lewis RA, et al.11 Tol. 0.11 PRD 0.994 No change  1/13006 db SNP (rs150774447)
3 c.223T>G p.C75G 1 2 Lewis RA, et al.11 Del. NA POD 0.603 No change ND
5 c.466A>G p.I156V 2 77, 78 Papaioannou M, et al.16 Tol. 0.46 B 0.003 No change 16/13006 db SNP (rs112467008) Benign
6 c.655A>T p.R219* 1 11 Xi Q, et al.27 ND
6 c.740A>C p.N247T 1 3 APEX Del. NA B 0.135 No change ND
6 c.768G>T Splice site 1 4 Klevering BJ, et al.22 Tol. 0.56 NA Don. 70.4 58 Site broken (−17.51) ND
9 c.1222C>T p.R408* 1 5 Webster AR, et al.7 ND
12 c.1726G>C p.D576H 1 36 Downs K, et al.25 POD 0.688 Acc. 68.1 39.1 Site broken (−42.54)  1/13006
13 c.1804C>T p.R602W 1 6 Lewis RA, et al.11 Del. 0.00 B 0.129 No change ND db SNP (rs 6179409)
13 c.1805G>A p.R602Q 1 7 Webster AR, et al.7 Del. 0.04 PRD 0.513 Acc. 48.9 77.9 New site (+59.14)  2/13006 db SNP (rs61749410)
13 c.1906C>T p.Q636* 3 12, 13, 60 Zernant J, et al.5 No change  1/13006 db SNP (rs145961131)
13 c.1922G>C p.C641S 1 8 Stenirri S, et al.24 Del. 0.00 No change ND db SNP (rs61749416)
14 c.1957C>T p.R653C 2 9, 10 Rivera A, et al.17 Del. 0.00 PRD 0.999 No change ND db SNP (rs61749420)
17 c.2588G>C p.G863A/ p.DelG863 5 11, 12, 13, 14, 15 Lewis RA, et al.11/ Maugeri A, et al.29 Del. 0.00 PRD 0.996 No change 68/13006 db SNP (rs76157638)
18 c.2701A>G p.T901A 1 74 APEX Tol. 0.82 B 0.008 23/13006 db SNP (rs139655975) Benign
19 c.2894A>G p.N965S 1 16 Lewis RA, et al.11 Del. 0.03 PRD 0.981 Acc. 53.4 82.3 New site (+54.26) ND db SNP (rs201471607)
20 c.2971G>C p.G991R 1 67 Yatsenko AN, et al.13 Del. 0.02 PRD 0.999 No change 28/13006 db SNP (rs147484266) Benign
22 c.3064G>A p.E1022K 2 17, 18 Webster AR, et al.7 Del. 0.00 PRD 1.000 No change ND db SNP (rs61749459)
22 c.3208_3209insGT p.S1071fs 5 19, 20, 21, 22, 25 APEX ND False-negative in APEX in patient 25
22 c.3292C>T p.R1098C 1 23 Rivera A, et al.17 Del. NA PRD 0.999 No change ND
22 c.3322C>T p.R1108C 3 16, 24, 61 Rozet JM, et al.10 Del. 0.00 PRD 0.986 No change 1/13006 db SNP (rs61750120) False-negative in APEX in patients 16 and 61
23 c.3386G>A p.R1129H 1 25 Zernant J, et al.5 PRD 0.989 No change ND False-negative in NGS in patient 25
24 c.3602T>G p.L1201R 4 72, 73, 74, 79 Lewis RA, et al.11 Tol. 0.37 B 0.052 Don. 61.3 73.7 New site (20.08) 416/13006 db SNP (rs61750126) Benign
28 c.4139C>T p.P1380L 7 30, 31, 32, 33, 34, 35, 36 Lewis RA, et al.11 Del. 0.01 B 0.377 No change 2/13006 db SNP (rs61750130)
28 c.4234C>T p.Q1412* 1 33 Rivera A, et al.17 ND db SNP (rs61750137)
29 c.4283C>T p.T1428M 1 76 APEX Tol. 0.15 B 0.010 No change 2/13006 db SNP (rs1800549) Benign
29 c.4319T>C p.F1440S 1 34 Lewis RA, et al.11 Del. 0.00 POD 0.744 No change ND dbSNP (rs61750141)
29 c.4326C>A p.N1442K 1 64 Zernant J, et al.5 Tol. NA POD 0.374 No change ND
29 c.4328G>A p.R1443H 1 35 Rivera A, et al.17 Del. 0.02 PRD 0.999 No change 1/13006 dbSNP (rs61750142)
IVS29 c.4352+1G>A Splice site 1 73 Zernant J, et al.5 Don. 82.3 55.4 WT site broken (−32.62) ND
30 c.4469G>A p.C1490Y 2 36, 37 Lewis RA, et al.11 Del. 0.00 PRD 0.994 No change ND dbSNP (rs61751402)
30 c.4538A>G p.Q1513R 1 67 Webster AR, et al.7 Tol. NA Benign 0.043 Acc. 91.7 62.8 Site broken (−31.55) ND
30 c.4537_4538insC p.G1513fs 1 38 Briggs CE, et al.19 ND False-negative in NGS in patient 38
31 c.4577C>T p.T1526M 1 39 Lewis RA, et al.11 Del. 0.00 PRD 0.910 No change ND db SNP (rs61750152)
33 c.4685T>C p.I1562T 1 71 Yatsenko, et al.13 Tol. NA PRD 0.783 No change ND Benign
33 c.4715C>T p.T1572M 1 79 Pang CP and Lamm DS23 Del. 0.02 B 0.326 No change ND db SNP (rs185093512) Benign
35 c.4926C>G p.S1642R 1 40 Birch DG, et al.22 Tol. 0.68 B 0.116 No change ND db SNP (rs61753017)
35 c.4956T>G p.Y1652* 1 41 Fumagalli A, et al.16 ND db SNP (rs61750561)
IVS35 c.5018+2T>C Splice site 1 42 APEX Don. 81.2 54.3 WT site broken (−33.07) ND
36 c.5113C>T p.R1705W 1 7 Ernest PJ, et al.26 Del. NA PRD 0.996 Don. 46.5 73.3 No change ND
IVS38 c.5461-10T>C 8 43, 44, 45, 46, 47, 48, 49, 50 Briggs CE, et al.19 No change 3/13006 db SNP (rs1800728)
IVS39 c.5585-1G>A Splice site 1 51 Shroyer NF, et al.21 Acc. 86.3 57.4 WT site broken (−33.53) ND
IVS40 c.5714+5G>A Splice site 1 52 Cremers FP, et al.8 Don. 85.5 73.3 Wild type site broken (−14.23) ND
42 c.5882G>A p.G1961E 7 53, 54, 55, 56, 57, 58, 59 Lewis RA, et al.11 Del. 0.00 PRD 0.998 No change 41/13006 db SNP (rs1800553)
44 c.6079C>T p.L2027F 4 60, 61, 62, 63 Lewis RA, et al.11 Del. 0.00 PRD 1.000 No change 4/13006 db SNP (rs61751408)
44 c.6089G>A p.R2030Q 1 64 Lewis RA, et al.11 Del. 0.00 PRD 0.995 No change 8/13006 db SNP (rs61750641)
46 c.6320G>A p.R2107H 2 72, 73 Fishman GA, et al.15 Del. 0.04 PRD 0.999 No change 91/13006 db SNP (rs62642564) Benign
47 c.6445C>T p.R2149* 1 65 Lewis RA, et al.14 1/13006 db SNP (rs61750654)
48 c.6529G>A p.D2177N 1 19 Rivera A, et al.17 Tol. 0.41 B 0.004 No change 116/13006 db SNP (rs1800555) Benign
48 c.6709A>C p.T2237P 1 66 APEX Del. NA POD 0.719 No change ND
IVS48 c.6729+4_ +18del AGTTGGCCCTGGGGC Splice site 1 17 Littink KW, et al.28 NA ND
Identification of New Variants by NGS
The results of NGS screening in our cohort of 79 patients are summarized in Table 2. We identified 82 variants by NGS in total; 53 missense, 13 splice-site alterations, 10 nonsense, four frameshifts, one in-frame deletion, and one intronic variant of unknown effect (Tables 2, 4). Of a total of 84 different variants identified in this study by APEX and NGS, there were two “NGS false-negative” variants (p.R1129H and p.Q1513fs), which were detected on APEX array, but were not detected by NGS (Table 2, patients 25 and 38). 
Table 4
 
Numbers and Types of Variants Detected by APEX Technology and NGS
Table 4
 
Numbers and Types of Variants Detected by APEX Technology and NGS
Total Null Non-Null Benign Missense
Splice-Site Altering Nonsense Frameshift Unknown In-Frame Deletion Disease-Causing Missense
Variants detected by prescreening with APEX 42 4 4 2 1 0 23 8
New variants detected by NGS alone 42 (33) 9 (7) 6 (4) 3 (3) 0 (0) 1 (1) 20 (16) 3 (2)
Gross total 84 (33) 13 (7) 10 (4) 5 (3) 1 (0) 1 (1) 43 (16) 11 (2)
A total of 42 additional variants, which were not detected by APEX, were identified in 45 (57%) patients screened by NGS (Tables 2, 4). Three variants (p.R219*, p.R1108C, and p.S1071fs) found by NGS, were not identified by APEX at the prescreening stage despite being represented on the array (“APEX false-negative”; Table 2, patients 11, 16, 25, and 61). Three (3/45) subjects had two new variants and 42 (42/45) individuals had one new variant (Table 2). Of the 42 new variants detected by NGS, there were 23 missense, 9 splice-site alterations, 6 nonsense, 3 frameshifts, and 1 in-frame deletion (Table 4). 
Of the 42 new variants identified by NGS, 33 (79%) were novel, including 18 missense, 7 splice-site alterations, 4 nonsense, 3 frameshifts, and 1 in-frame deletion (Tables 2, 4, 5). Seven variants identified only by NGS already were known, but not yet added to the ABCA4 array by the time of the prescreening of those samples. 
Table 5
 
In Silico Molecular Genetic Analysis for Novel ABCA4 Variants Identified by NGS
Table 5
 
In Silico Molecular Genetic Analysis for Novel ABCA4 Variants Identified by NGS
Exon/ IVS DNA Change Protein Change/ Effect N of Alleles Identified Pt SIFT Polyphen2 HSF Matrix Allele Freq. by EVS Reference Comments
Pred. Tol. Index (0–1) Pred. Hum Var Score (0–1) Site Wt CV Mt CV CV % Variation
3 c.180delG p.M61fs 1 35 ND
IVS7 c.859-9T>C Splice site 1 5 Acc. 78.18 76.99 Possibly site broken (−1.52) ND Possibly disease-causing
11 c.1433T>C p.I478T 1 1 Tol. B 0.007 No change ND Benign
11 c.1519G>T p.D507Y 1 20 Del. 0.01 POD 0.641 No change 1/13006 dbSNP (rs148234178)
12 c.1749G>C p.K583N 1 68 Del. 0.04 POD 0.893 Acc. 66.17 37.22 Site broken (−43.75) 1/13006 dbSNP (rs145265791)
14 c.1982_ 1983insG p.A662fs 1 69 ND
15 c.2297G>T p.G766V 1 1 Tol. NA POD 0.557 Don. 69.18 42.34 Site broken (−38.79) ND
15 c.2345G>A p.W782* 1 3 ND
16 c.2510T>C p.L837P 1 72 Tol. NA POD 0.905 No change ND
16 c.2568C>A p.Y856* 1 5 ND
18 c.2686A>G p.K896E 1 53 Tol. NA B 0.002 ND Benign
20 c.2942C>T p.P981L 1 19 Del. 0.00 POD 0.813 No change 1/13006 dbSNP (rs147826775)
20 c.2972G>T p.G991V 1 70 Del. NA PRD 0.998 Donor 64.62 91.45 New site (41.53) ND
IVS20 c.3050+1G>C Splice site 1 54 Acc. 86.43 57.49 Site broken (−33.49) ND
IVS21 c.3191-1G>T Splice site 1 26 Acc. 94.38 65.44 WT site broken (−30.67) ND
22 c.3289A>T p.R1097* 1 71 ND
22 c.3299T>A p.I1100N 1 23 Del. NA PRD 0.986 No change ND
23 c.3370G>T p.D1124Y 1 62 Del. NA PRD 0.998 No change ND
23 c.3392delC/ 3393C>G p.A1131Gfs 1 55 ND
23 c.3398T>C p.I1133T 1 27 Del. NA B 0.100 No change ND Possibly disease-causing
27 c.4070C>A p.A1357E 1 28 Del. NA PRD 0.94 Acc. 40.92 69.86 New site (+70.74) ND
IVS30 c.4539+2T>G Splice site 1 56 Don. 79.18 52.35 WT site broken (−33.89) ND
31 c.4552A>C p.S1518R 1 57 Del. NA POD 0.871 Acc. 76.3 47.36 Site broken (−37.94) ND
31 c.4634G>A p.S1545N 2 21, 25 Tol. NA B 0.253 Acc. 80.04 51.1 Site broken (−36.16) ND
33 c.4773G>C Splice site 1 29 Don. 84.58 73.57 Site broken (−13.02) ND
35 c.4978delC p.L1661* 1 24 ND
36 c.5041_5055del GTGGTTGCCATCTGC p.V1681_C1685del 1 40 NA ND db SNP (rs62646872)
36 c.5088C>G p.S1696R 1 10 Tol. NA PRD 0.780 Don. 59.34 86.17 New site (45.23) ND
39 c.5578C>T p.R1860W 1 38 Del. 0.02 B 0.025 No change ND db SNP (rs200849015)
IVS42 c.5899-3_ 5899-2delTA Splice site 1 66 NA ND
IVS42 c.5899-2delA Splice site 1 58 Acc. 82.1 28.26 WT site broken (−65.58) ND
45 c.6209C>G p.T2070R 1 52 Tol. NA PRD 0.996 Acc. 57.41 86.36 New site (50.42) ND
46 c.6385A>G p.S2129G 1 43 Del. NA B 0.001 ND Possibly disease-causing
In Silico Molecular Genetic Analysis for New Variants Identified by NGS
In silico analysis of the 42 variants identified by NGS, including the 9 previously reported variants and the 33 novel variants, are shown in Tables 3 and 5, respectively. 
Of the 9 previously reported variants that were detected by NGS, there were 4 null variants (2 nonsense and 2 splice-site alterations); 4 disease-causing missense variants, with deleterious or damaged protein function predicted by SIFT and Polyphen2; and one benign missense variant (p.D2177N, Table 3). 
Deleterious or damaged protein function was predicted by SIFT and Polyphen2 in 16 of 18 novel missense variants (Table 5). Two variants (p.I478T and p.K896E) were predicted to be tolerated and benign. The predicted effects on splicing of these 16 missense variants, one variant resulting in a nucleotide substitution at the end of exon 33 (c.4773G>C), and five intronic variants, were assessed using the HSF program. Altered splicing was suggested for 7 of the missense variants, the (c.4773G>C) variant, and all 5 intronic variants (Table 5). The allele frequencies for the 33 novel variants were, at most, 1 in 13006, suggesting that these are all very rare. Overall, 31 of the 33 novel variants were considered disease-causing, except for only the two missense variants, p.I478T and p.K896E (Table 5). 
Disease-Causing Variants
A total of 73 (31 novel and 42 previously identified) disease-causing variants was identified in this cohort of 79 patients (Table 4). The distribution of the number of alleles in the cohort is summarized in Table 6. One patient (1%) harbored three disease-causing variants, 36 (46%) had two disease-causing variants, 36 (46%) had one disease-causing variant, and six patients (7%) remained with no disease-causing variant identified (Table 6). 
Table 6
 
Distribution of 79 Patients With ABCA4-Related Retinal Disease Based on Number of Identified Disease-Causing Variants
Table 6
 
Distribution of 79 Patients With ABCA4-Related Retinal Disease Based on Number of Identified Disease-Causing Variants
Comprehensive Screening With APEX and NGS
No Disease-Causing Variant 1 Disease-Causing Variant 2 Disease-Causing Variants 3 Disease-Causing Variants
Prescreening with APEX 1 disease causing-variant, n = 66 29 36 1
No disease-causing variants, n = 13 6 7
Total, n = 79 6 36 36 1
Discussion
Our study reports the molecular genetic findings using a PCR-enrichment–based NGS strategy in a large well-characterized British cohort with a clinical diagnosis of ABCA4-associated retinal disease. The NGS revealed two or more disease-causing variants in 37 (47%) of 79 patients, in whom only one variant was detected in prescreening with APEX array technology. 
Of the 66 subjects with one disease-causing allele identified previously by APEX, the second disease-causing allele was identified in 37 individuals (56%). In keeping with our findings, Zernant et al. reported that the same NGS strategy identified the second disease-causing allele in 48% of their cohort who also only had one allele found previously with APEX. 5 These findings suggest that many disease-associated mutations in the ABCA4 gene are very rare and yet unknown, supporting the validity of the PCR-enrichment–based NGS method either as the screening method of choice, or as an additional screening method for patients in whom APEX does not reveal two variants. Of note, the NGS method is cost- and time-efficient at this time only for large (at least 96 samples) cohorts. 
Of the 13 patients with initially no disease-causing variant found by APEX, one disease-causing ABCA4 allele was identified in seven subjects (54%) by NGS, with six remaining with no likely disease-causing allele (46%). Further screening with NGS, including screening all intronic regions, and upstream and downstream control regions of the ABCA4 gene, as well as other candidate genes, in larger well-characterized cohorts will be needed to identify fully all pathogenic alleles in these patients. It recently has been proposed that intronic and synonymous variants may account for a significant proportion of the remaining disease-causing variants not identified with exomic NGS. 5,39  
There were two “NGS false negative” variants (p.R1129H and p.Q1513fs) and three “APEX false negative” variants (p.R219*, p.R1108C, and p.S1071fs) in our cohort. The missense variant p.R1129H was not detected by NGS, most likely due to allele-specific amplification. The frameshift variant c.4537_4538insC, p.Q1513fs, is located in a homopolymer of seven C-nucleotides, where an insertion of another C nucleotide presents a challenge to identify by the Roche 454 sequencing platform. The “APEX false negative” variants were caused by technical issues with the specific array, ABCR400 (432 mutations on the chip) in 2005. Two of those variants, p.R1108C, and p.S1071fs, were detected by APEX in other patients (Table 2). Nevertheless, these findings suggest that combined APEX/NGS analysis may be worthy of consideration for comprehensive mutation detection. 
In silico molecular genetic analysis was performed for all 84 variants identified in our cohort, with 73 of these determined to be likely disease-causing. Review of the clinical findings of the six patients harboring only one, likely benign, missense variant, revealed that they had a less typical (“atypical”) phenotype for ABCA4-associated retinal disease, including an absence of flecks in all patients, significant peripheral retinal bone spicule pigmentation in three subjects, geographic-like atrophy in one individual, a subtle atrophic change confined to the fovea in one patient, and a lack of peripapillary sparing on AF imaging in three subjects. In this study, patients with bilateral macular atrophy, with or without surrounding flecks, potentially were included as having Stargardt disease, with other less typical (“atypical”) findings also having been reported in ABCA4-associated retinal disease. 18,40,41 However, other genes associated with autosomal recessive macular dystrophy, autosomal recessive cone-rod dystrophy, and autosomal recessive retinitis pigmentosa also should be considered for these patients with no identified likely disease-causing ABCA4 alleles, in addition to the possibility of missed ABCA4 alleles due to the inherent limitations of the molecular testing approach. 
In our cohort, in keeping with previous reports, there was one well-known intronic variant (c.5461-10T>C) of equivocal pathogenicity following detailed in silico analysis, 4,5,16 ; highlighting the need for an effective assay to determine functionally the effects of ABCA4 variants, or alternatively to consider investigating mRNA expression. The allele, c.5461-10T>C, was the most common in our cohort, with 8/79 patients (10%) harboring this variant. Interestingly, the second allele was not identified in six of these patients. However, co-segregation of c.5461-10T>C with the disease has been documented in several studies, thereby strongly suggesting its disease-causation. 4  
In summary, we have demonstrated the validity and utility of ABCA4 mutation screening with an NGS-based protocol in a large British cohort, with successful identification of the new disease-causing alleles in approximately half of the cases harboring one allele detected by prescreening with APEX technology. The identification of both disease-causing alleles will improve the accuracy of diagnosis and the counselling of patients, and also will assist in more effective patient selection of genetically confirmed participants for current and future clinical trials for ABCA4-associated retinal disease. 
Acknowledgments
The authors thank the patients who kindly agreed to take part in this study and colleagues who referred individuals to us at Moorfields Eye Hospital, and those who contributed to the assembly of the ABCA4 panel, particularly Naushin Waseem, Bev Scott, and Sophie Devery. The authors also thank Graham E. Holder, Anthony G. Robson, and Magella M. Neveu for interpretation of electrophysiologic data, and Yozo Miyake, Arundhati Dev Borman, Rajarshi Mukherjee, Eva Lenassi, Panagiotis I. Sergouniotis, and Aman Chandra for their insightful comments. 
Supported by grants from the National Institute for Health Research Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology; Foundation Fighting Blindness (USA); Fight For Sight; Moorfields Eye Hospital Special Trustees; Macular Disease Society; National Eye Institute/NIH Grants EY021163, EY019861, and EY019007 (Core Support for Vision Research); unrestricted funds from Research to Prevent Blindness (New York, NY) to the Department of Ophthalmology; Columbia University; Suzuken Memorial Foundation; Mitsukoshi Health and Welfare Foundation; Daiwa Anglo-Japanese Foundation; and Grant-in-Aid for Young Scientists (B) of the Ministry of Education, Culture, Sports, Science, and Technology (Japan); and by a Foundation Fighting Blindness Career Development Award (MM). The authors alone are responsible for the content and writing of the paper. 
Disclosure: K. Fujinami, None; J. Zernant, None; R.K. Chana, None; G.A. Wright, None; K. Tsunoda, None; Y. Ozawa, None; K. Tsubota, None; A.R. Webster, None; A.T. Moore, None; R. Allikmets, None; M. Michaelides, None 
References
Allikmets R Singh N Sun H A photoreceptor cell-specific ATP-binding transporter gene (ABCR) is mutated in recessive Stargardt macular dystrophy. Nat Genet . 1997; 15: 236– 246. [CrossRef] [PubMed]
Michaelides M Chen LL Brantley MA Jr ABCA4 mutations and discordant ABCA4 alleles in patients and siblings with bull's-eye maculopathy. Br J Ophthalmol . 2007; 91: 1650– 1655. [CrossRef] [PubMed]
Burke TR Tsang SH. Allelic and phenotypic heterogeneity in ABCA4 mutations. Ophthalmic Genet . 2011; 32: 165– 174. [CrossRef] [PubMed]
Fujinami K Lois N Davidson AE A longitudinal study of Stargardt disease: clinical and electrophysiological assessment, progression and genotype correlations. Am J Ophthalmol . 2013; 155: 1075– 1088. [CrossRef] [PubMed]
Zernant J Schubert C Im KM Analysis of the ABCA4 gene by next-generation sequencing. Invest Ophthalmol Vis Sci . 2011; 52: 8479– 8487. [CrossRef] [PubMed]
Fujinami K Akahori M Fukui M Tsunoda K Iwata T Miyake Y. Stargardt disease with preserved central vision: identification of a putative novel mutation in ATP-binding cassette transporter gene. Acta Ophthalmol . 2011; 89: 297– 298. [CrossRef]
Webster AR Heon E Lotery AJ An analysis of allelic variation in the ABCA4 gene. Invest Ophthalmol Vis Sci . 2001; 42: 1179– 1189. [PubMed]
Cremers FP van de Pol DJ van Driel M Autosomal recessive retinitis pigmentosa and cone-rod dystrophy caused by splice site mutations in the Stargardt's disease gene ABCR. Hum Mol Genet . 1998; 7: 355– 362. [CrossRef] [PubMed]
Martinez-Mir A Paloma E Allikmets R Retinitis pigmentosa caused by a homozygous mutation in the Stargardt disease gene ABCR. Nat Genet . 1998; 18: 11– 12. [CrossRef] [PubMed]
Rozet JM Gerber S Souied E Spectrum of ABCR gene mutations in autosomal recessive macular dystrophies. Eur J Hum Genet . 1998; 6: 291– 295. [CrossRef] [PubMed]
Lewis RA Shroyer NF Singh N Genotype/phenotype analysis of a photoreceptor-specific ATP-binding cassette transporter gene, ABCR, in Stargardt disease. Am J Hum Genet . 1999; 64: 422– 434. [CrossRef] [PubMed]
Rozet JM Gerber S Ghazi I Mutations of the retinal specific ATP binding transporter gene (ABCR) in a single family segregating both autosomal recessive retinitis pigmentosa RP19 and Stargardt disease: evidence of clinical heterogeneity at this locus. J Med Genet . 1999; 36: 447– 451. [PubMed]
Yatsenko AN Shroyer NF Lewis RA Lupski JR. Late-onset Stargardt disease is associated with missense mutations that map outside known functional regions of ABCR (ABCA4). Hum Genet . 2001; 108: 346– 355. [CrossRef] [PubMed]
Jaakson K Zernant J Kulm M Genotyping microarray (gene chip) for the ABCR (ABCA4) gene. Hum Mutat . 2003; 22: 395– 403. [CrossRef] [PubMed]
Fishman GA Stone EM Grover S Derlacki DJ Haines HL Hockey RR. Variation of clinical expression in patients with Stargardt dystrophy and sequence variations in the ABCR gene. Arch Ophthalmol . 1999; 117: 504– 510. [CrossRef] [PubMed]
Papaioannou M Ocaka L Bessant D An analysis of ABCR mutations in British patients with recessive retinal dystrophies. Invest Ophthalmol Vis Sci . 2000; 41: 16– 19. [PubMed]
Rivera A White K Stohr H A comprehensive survey of sequence variation in the ABCA4 (ABCR) gene in Stargardt disease and age-related macular degeneration. Am J Hum Genet . 2000; 67: 800– 813. [CrossRef] [PubMed]
Birch DG Peters AY Locke KL Spencer R Megarity CF Travis GH. Visual function in patients with cone-rod dystrophy (CRD) associated with mutations in the ABCA4(ABCR) gene. Exp Eye Res . 2001; 73: 877– 886. [CrossRef] [PubMed]
Briggs CE Rucinski D Rosenfeld PJ Hirose T Berson EL Dryja TP. Mutations in ABCR (ABCA4) in patients with Stargardt macular degeneration or cone-rod degeneration. Invest Ophthalmol Vis Sci . 2001; 42: 2229– 2236. [PubMed]
Fumagalli A Ferrari M Soriani N Mutational scanning of the ABCR gene with double-gradient denaturing-gradient gel electrophoresis (DG-DGGE) in Italian Stargardt disease patients. Hum Genet . 2001; 109: 326– 338. [CrossRef] [PubMed]
Shroyer NF Lewis RA Yatsenko AN Wensel TG Lupski JR. Cosegregation and functional analysis of mutant ABCR (ABCA4) alleles in families that manifest both Stargardt disease and age-related macular degeneration. Hum Mol Genet . 2001; 10: 2671– 2678. [CrossRef] [PubMed]
Klevering BJ Blankenagel A Maugeri A Cremers FP Hoyng CB Rohrschneider K. Phenotypic spectrum of autosomal recessive cone-rod dystrophies caused by mutations in the ABCA4 (ABCR) gene. Invest Ophthalmol Vis Sci . 2002; 43: 1980– 1985. [PubMed]
Pang CP Lam DS. Differential occurrence of mutations causative of eye diseases in the Chinese population. Hum Mutat . 2002; 19: 189– 208. [CrossRef] [PubMed]
Stenirri S Fermo I Battistella S Denaturing HPLC profiling of the ABCA4 gene for reliable detection of allelic variations. Clin Chem . 2004; 50: 1336– 1343. [CrossRef] [PubMed]
Downs K Zacks DN Caruso R Molecular testing for hereditary retinal disease as part of clinical care. Arch Ophthalmol . 2007; 125: 252– 258. [CrossRef] [PubMed]
Ernest PJ Boon CJ Klevering BJ Hoefsloot LH Hoyng CB. Outcome of ABCA4 microarray screening in routine clinical practice. Mol Vis . 2009; 15: 2841– 2847. [PubMed]
Xi Q Li L Traboulsi EI Wang QK. Novel ABCA4 compound heterozygous mutations cause severe progressive autosomal recessive cone-rod dystrophy presenting as Stargardt disease. Mol Vis . 2009; 15: 638– 645. [PubMed]
Littink KW Koenekoop RK van den Born LI Homozygosity mapping in patients with cone-rod dystrophy: novel mutations and clinical characterizations. Invest Ophthalmol Vis Sci . 2010; 51: 5943– 5951. [CrossRef] [PubMed]
Maugeri A van Driel MA van de Pol DJ The 2588G-->C mutation in the ABCR gene is a mild frequent founder mutation in the Western European population and allows the classification of ABCR mutations in patients with Stargardt disease. Am J Hum Genet . 1999; 64: 1024– 1035. [CrossRef] [PubMed]
Klevering BJ Yzer S Rohrschneider K Microarray-based mutation analysis of the ABCA4 (ABCR) gene in autosomal recessive cone-rod dystrophy and retinitis pigmentosa. Eur J Hum Genet . 2004; 12: 1024– 1032. [CrossRef] [PubMed]
Fujinami K Sergouniotis PI Davidson AE The clinical effect of homozygous ABCA4 alleles in 18 patients [published online ahead of print June 11, 2013]. Ophthalmology . doi:10.1016/j.ophtha.2013.04.016 .
Sergouniotis PI Davidson AE Lenassi E Devery SR Moore AT Webster AR. Retinal structure, function, and molecular pathologic features in gyrate atrophy. Ophthalmology . 2012; 119: 596– 605. [CrossRef] [PubMed]
Bach M Brigell MG Hawlina M ISCEV standard for clinical pattern electroretinography (PERG): 2012 update. Doc Ophthalmol . 2013; 126: 1– 7. [CrossRef] [PubMed]
Marmor MF Fulton AB Holder GE Miyake Y Brigell M Bach M. ISCEV standard for full-field clinical electroretinography (2008 update). Doc Ophthalmol . 2009; 118: 69– 77. [CrossRef] [PubMed]
Lois N Holder GE Bunce C Fitzke FW Bird AC. Phenotypic subtypes of Stargardt macular dystrophy-fundus flavimaculatus. Arch Ophthalmol . 2001; 119: 359– 369. [CrossRef] [PubMed]
Ng PC Henikoff S. SIFT: predicting amino acid changes that affect protein function. Nucleic Acids Res . 2003; 31: 3812– 3814. [CrossRef] [PubMed]
Adzhubei IA Schmidt S Peshkin L A method and server for predicting damaging missense mutations. Nat Methods . 2010; 7: 248– 249. [CrossRef] [PubMed]
den Dunnen JT Antonarakis SE. Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion. Hum Mut . 2000; 15: 7– 12. [CrossRef] [PubMed]
Braun TA Mullins RF Wagner AH Non-exomic and synonymous variants in ABCA4 are an important cause of Stargardt disease. Hum Mol Genet. In press.
Burke TR Allikmets R Smith RT Gouras P Tsang SH. Loss of peripapillary sparing in non-group I Stargardt disease. Exp Eye Res . 2010; 91: 592– 600. [CrossRef] [PubMed]
Fritsche LG Fleckenstein M Fiebig BS A subgroup of age-related macular degeneration is associated with mono-allelic sequence variants in the ABCA4 gene. Invest Ophthalmol Vis Sci . 2012; 53: 2112– 2118. [CrossRef] [PubMed]
Figure 1
 
Color fundus photographs, autofluorescence images, and optical coherence tomography of three representative cases harboring two or more ABCA4 variants with “typical” ABCA4-associated retinal disease (patients 19, 36, and 17). Color fundus photograph of patient 19 shows macular atrophy (A) and AF imaging demonstrates a localized low AF signal at the fovea, with a high signal edge surrounded by a homogeneous background (B). SD-OCT demonstrates marked outer retinal loss at the central macula (C). Patient 36 has macular atrophy surrounded by numerous yellow-white flecks (D) and a localized low AF signal at the macula surrounded by a heterogeneous background, with peripapillary sparing (E). Generalized loss of outer retinal architecture is seen on SD-OCT. Patient 17 has widespread multiple areas of atrophy with patchy pigmentation (G) and multiple areas of low AF signal at the posterior pole with a heterogeneous background (H).
Figure 1
 
Color fundus photographs, autofluorescence images, and optical coherence tomography of three representative cases harboring two or more ABCA4 variants with “typical” ABCA4-associated retinal disease (patients 19, 36, and 17). Color fundus photograph of patient 19 shows macular atrophy (A) and AF imaging demonstrates a localized low AF signal at the fovea, with a high signal edge surrounded by a homogeneous background (B). SD-OCT demonstrates marked outer retinal loss at the central macula (C). Patient 36 has macular atrophy surrounded by numerous yellow-white flecks (D) and a localized low AF signal at the macula surrounded by a heterogeneous background, with peripapillary sparing (E). Generalized loss of outer retinal architecture is seen on SD-OCT. Patient 17 has widespread multiple areas of atrophy with patchy pigmentation (G) and multiple areas of low AF signal at the posterior pole with a heterogeneous background (H).
Figure 2
 
Color fundus photographs and autofluorescence images of six cases harboring a single, likely benign, missense variant with “atypical” clinical features for ABCA4-associated retinal disease (patients 74, 75, 76, 77, 78, and 79). Color photograph of patient 74 shows a geographic atrophy-like appearance (A), which on AF imaging is surrounded by foci of high and low AF signal (B). Patient 75 has evidence of generalized retinal atrophy in addition to marked macular atrophy, with dense pigmentation at the level of the RPE, and bone spicule formation, marked vessel attenuation, and optic disc pallor (C). Patient 76 has extensive macular atrophy extending beyond the arcades, with dense pigmentation at the level of the RPE and slight bone spicule pigmentation in the periphery (D). AF imaging demonstrates a heterogeneous background, but no peripapillary sparing (E). Patient 77 has a large area of macular atrophy extending to the optic disc (F), which on AF imaging is surrounded by an irregular low AF signal with foci of high and low signal, with no peripapillary sparing (G). Patient 78 has multiple widespread areas of atrophy with dense pigmentation at the level of RPE and bone spicule pigmentation in the periphery (H). AF imaging identifies multiple low signal areas with a heterogeneous background and no peripapillary sparing (I). Patient 79 has subtle atrophy confined to the fovea (J). AF imaging demonstrates a localized low AF signal at the fovea surrounded by a homogeneous background (K).
Figure 2
 
Color fundus photographs and autofluorescence images of six cases harboring a single, likely benign, missense variant with “atypical” clinical features for ABCA4-associated retinal disease (patients 74, 75, 76, 77, 78, and 79). Color photograph of patient 74 shows a geographic atrophy-like appearance (A), which on AF imaging is surrounded by foci of high and low AF signal (B). Patient 75 has evidence of generalized retinal atrophy in addition to marked macular atrophy, with dense pigmentation at the level of the RPE, and bone spicule formation, marked vessel attenuation, and optic disc pallor (C). Patient 76 has extensive macular atrophy extending beyond the arcades, with dense pigmentation at the level of the RPE and slight bone spicule pigmentation in the periphery (D). AF imaging demonstrates a heterogeneous background, but no peripapillary sparing (E). Patient 77 has a large area of macular atrophy extending to the optic disc (F), which on AF imaging is surrounded by an irregular low AF signal with foci of high and low signal, with no peripapillary sparing (G). Patient 78 has multiple widespread areas of atrophy with dense pigmentation at the level of RPE and bone spicule pigmentation in the periphery (H). AF imaging identifies multiple low signal areas with a heterogeneous background and no peripapillary sparing (I). Patient 79 has subtle atrophy confined to the fovea (J). AF imaging demonstrates a localized low AF signal at the fovea surrounded by a homogeneous background (K).
Table 1
 
Summary of Clinical Features of 79 Patients With ABCA4-Related Retinal Disease
Table 1
 
Summary of Clinical Features of 79 Patients With ABCA4-Related Retinal Disease
Median age of onset, y (range) 22.0 (5–71)
Median age at examination, y (range) 40.0 (15–79)
Median duration of disease, y (range) 10.0 (0–54)
LogMAR visual acuity (range) R 1.00 (−0.08–1.78)
L 1.00 (−0.08–4.00)
AF subtype, n = 71 1 n = 21
2 n = 34
3 n = 16
ERG group, n = 70 1 n = 34
2 n = 7
3 n = 29
Table 2
 
Molecular Genetic Status Identified by NGS in 79 Patients With ABCA4-Related Retinal Disease
Table 2
 
Molecular Genetic Status Identified by NGS in 79 Patients With ABCA4-Related Retinal Disease
Pt Allele 1 Detected by APEX Allele 2 Detected by NGS Allele 3 Detected by NGS Total N of DC Variants Comments
DNA Change Protein Change/ Effect Pred. Patho. DNA Change Protein Change/ Effect Pred. Patho. DNA Change Protein Change/ Effect Pred. Patho.
1 c.161G>A p.C54Y DC c.2297G>T p.G766V DC 2
2 c.223T>G p.C75G DC c.5088C>G p.S1696R DC 2
3 c.740A>C p.N247T DC c.1433T>C p.I478T B c.2345G>A p.W782* DC 2
4 c.768G>T Splice site DC 1
5 c.1222C>T p.R408* DC c.2568C>A p.Y856* DC 2
6 c.1804C>T p.R602W DC c.859-9T>C Splice site PDC 2
7 c.1805G>A p.R602Q DC c.5113C>T p.R1705W DC 2
8 c.1922G>C p.C641S DC 1
9 c.1957C>T p.R653C DC 1
10 c.1957C>T p.R653C DC 1
11 c.2588G>C p.G863A DC c.655A>T p.R219* DC 2 Allele 2 (p.R219*) was APEX-false-negative
12 c.2588G>C p.G863A DC c.1906C>T p.Q636* DC 2
13 c.2588G>C p.G863A DC c.1906C>T p.Q636* DC 2
14 c.2588G>C p.G863A DC 1
15 c.2588G>C p.G863A DC 1
16 c.2894A>G p.N965S DC c.3322C>T p.R1108C DC 2 Allele 2 (p.R1108C) was APEX-false-negative
17 c.3064G>A p.E1022K DC c.6729+4_+18delAGTTGGCCCTGGGGC Splice site DC 2
18 c.3064G>A p.E1022K DC 1
19 c.3208_3209insGT p.S1071fs DC c.2942C>T p.P981L DC c.6529G>A p.D2177N B 2
20 c.3208_3209insGT p.S1071fs DC c.1519G>T p.D507Y DC 2
21 c.3208_3209insGT p.S1071fs DC c.4634G>A p.S1545N DC 2
22 c.3208_3209insGT p.S1071fs DC 1
23 c.3292C>T p.R1098C DC c.3299T>A p.I1100N DC 2
24 c.3322C>T p.R1108C DC c.4978delC p.L1661* DC 2
25 c.3386G>A p.R1129H DC c.3208_3209insGT p.S1071fs DC c.4634G>A p.S1545N DC 3 Allele 2 (p.S1071fs) was APEX false-negative and allele 1 (p.R1129H) was NGS false-negative
26 c.4139C>T p.P1380L DC c.3191-1G>T Splice site DC 2
27 c.4139C>T p.P1380L DC c.3398T>C p.I1133T PDC 2
28 c.4139C>T p.P1380L DC c.4070C>A p.A1357E DC 2
29 c.4139C>T p.P1380L DC c.4773G>C Splice site DC 2
30 c.4139C>T p.P1380L DC 1
31 c.4139C>T p.P1380L DC 1
32 c.4139C>T p.P1380L DC 1
33 c.4234C>T p.Q1412* DC 1
34 c.4319T>C p.F1440S DC 1
35 c.4328G>A p.R1443H DC c.180delG p.M61fs DC 2
36 c.4469G>A p.C1490Y DC c.1726G>C p.D576H DC 2
37 c.4469G>A p.C1490Y DC 1
38 c.4537_4538insC p.Q1513fs DC c.5578C>T p.R1860W DC 2 Allele 1 (p.Q1513fs) was NGS-false-negative
39 c.4577C>T p.T1526M DC 1
40 c.4926C>G p.S1642R DC c.5041_5055del GTGGTTGCCATCTGC p.V1681_C1685del DC 2
41 c.4956T>G p.Y1652* DC 1
42 c.5018+2T>C Splice site DC 1
43 c.5461-10T>C DC c.6385A>G p.S2129G PDC 2
44 c.5461-10T>C DC 1
45 c.5461-10T>C DC 1
46 c.5461-10T>C DC 1
47 c.5461-10T>C DC 1
48 c.5461-10T>C DC 1
49 c.5461-10T>C DC 1
50 c.5461-10T>C DC 1
51 c.5585-1G>A Splice site DC 1
52 c.5714+5G>A Splice site DC c.6209C>G p.T2070R DC 2
53 c.5882G>A p.G1961E DC c.2686A>G p.K896E B 1
54 c.5882G>A p.G1961E DC c.3050+1G>C Splice site DC 2
55 c.5882G>A p.G1961E DC c.3392delC/3393C>G p.A1131Gfs DC 2
56 c.5882G>A p.G1961E DC c.4539+2T>G Splice site DC 2
57 c.5882G>A p.G1961E DC c.4552A>C p.S1518R DC 2
58 c.5882G>A p.G1961E DC c.5899-2delA Splice site DC 2
59 c.5882G>A p.G1961E DC 1
60 c.6079C>T p.L2027F DC c.1906C>T p.Q636* DC 2
61 c.6079C>T p.L2027F DC c.3322C>T p.R1108C DC 2 Allele 2 (p.R1108C) was APEX-false-negative
62 c.6079C>T p.L2027F DC c.3370G>T p.D1124Y DC 2
63 c.6079C>T p.L2027F DC 1
64 c.6089G>A p.R2030Q DC c.4326C>A p.N1442K DC 2
65 c.6445C>T p.R2149* DC 1
66 c.6709A>C p.T2237P DC c.5899-3_5899-2delTA Splice site DC 2
67 c.2971G>C p.G991R B c.4538A>G p.Q1513R DC 1
68 c.3602T>G p.L1201R B c.1749G>C p.K583N DC 1
69 c.3602T>G p.L1201R B c.1982_1983insG p.A662fs DC 1
70 c.3602T>G p.L1201R B c.2972G>T p.G991V DC 1
71 c.4685T>C p.I1562T B c.3289A>T p.R1097* DC 1
72 c.6320G>A p.R2107H B c.2510T>C p.L837P DC 1
73 c.6320G>A p.R2107H B c.4352+1G>A Splice site DC 1
74 c.2701A>G p.T901A B 0
75 c.3602T>G p.L1201R B 0
76 c.4283C>T p.T1428M B 0
77 c.466A>G p.I156V B 0
78 c.466A>G p.I156V B 0
79 c.4715C>T p.T1572M B 0
Table 3
 
In Silico Analysis for Previously Reported Variants Identified in 79 Patients With ABCA4-Related Retinal Disease
Table 3
 
In Silico Analysis for Previously Reported Variants Identified in 79 Patients With ABCA4-Related Retinal Disease
Exon/ IVS Nucleotide Substitution Protein Change/ Effect N of Alleles Identified Pt Method Previous Report SIFT Polyphen 2 HSF Matrix Allele Freq. by EVS Reference Comment
APEX NGS Pred. Tol. Index (0–1) Pred. Hum Var Score (0–1) Site Wt CV Mt CV CV % Variation
3 c.161G>A p.C54Y 1 1 Lewis RA, et al.11 Tol. 0.11 PRD 0.994 No change  1/13006 db SNP (rs150774447)
3 c.223T>G p.C75G 1 2 Lewis RA, et al.11 Del. NA POD 0.603 No change ND
5 c.466A>G p.I156V 2 77, 78 Papaioannou M, et al.16 Tol. 0.46 B 0.003 No change 16/13006 db SNP (rs112467008) Benign
6 c.655A>T p.R219* 1 11 Xi Q, et al.27 ND
6 c.740A>C p.N247T 1 3 APEX Del. NA B 0.135 No change ND
6 c.768G>T Splice site 1 4 Klevering BJ, et al.22 Tol. 0.56 NA Don. 70.4 58 Site broken (−17.51) ND
9 c.1222C>T p.R408* 1 5 Webster AR, et al.7 ND
12 c.1726G>C p.D576H 1 36 Downs K, et al.25 POD 0.688 Acc. 68.1 39.1 Site broken (−42.54)  1/13006
13 c.1804C>T p.R602W 1 6 Lewis RA, et al.11 Del. 0.00 B 0.129 No change ND db SNP (rs 6179409)
13 c.1805G>A p.R602Q 1 7 Webster AR, et al.7 Del. 0.04 PRD 0.513 Acc. 48.9 77.9 New site (+59.14)  2/13006 db SNP (rs61749410)
13 c.1906C>T p.Q636* 3 12, 13, 60 Zernant J, et al.5 No change  1/13006 db SNP (rs145961131)
13 c.1922G>C p.C641S 1 8 Stenirri S, et al.24 Del. 0.00 No change ND db SNP (rs61749416)
14 c.1957C>T p.R653C 2 9, 10 Rivera A, et al.17 Del. 0.00 PRD 0.999 No change ND db SNP (rs61749420)
17 c.2588G>C p.G863A/ p.DelG863 5 11, 12, 13, 14, 15 Lewis RA, et al.11/ Maugeri A, et al.29 Del. 0.00 PRD 0.996 No change 68/13006 db SNP (rs76157638)
18 c.2701A>G p.T901A 1 74 APEX Tol. 0.82 B 0.008 23/13006 db SNP (rs139655975) Benign
19 c.2894A>G p.N965S 1 16 Lewis RA, et al.11 Del. 0.03 PRD 0.981 Acc. 53.4 82.3 New site (+54.26) ND db SNP (rs201471607)
20 c.2971G>C p.G991R 1 67 Yatsenko AN, et al.13 Del. 0.02 PRD 0.999 No change 28/13006 db SNP (rs147484266) Benign
22 c.3064G>A p.E1022K 2 17, 18 Webster AR, et al.7 Del. 0.00 PRD 1.000 No change ND db SNP (rs61749459)
22 c.3208_3209insGT p.S1071fs 5 19, 20, 21, 22, 25 APEX ND False-negative in APEX in patient 25
22 c.3292C>T p.R1098C 1 23 Rivera A, et al.17 Del. NA PRD 0.999 No change ND
22 c.3322C>T p.R1108C 3 16, 24, 61 Rozet JM, et al.10 Del. 0.00 PRD 0.986 No change 1/13006 db SNP (rs61750120) False-negative in APEX in patients 16 and 61
23 c.3386G>A p.R1129H 1 25 Zernant J, et al.5 PRD 0.989 No change ND False-negative in NGS in patient 25
24 c.3602T>G p.L1201R 4 72, 73, 74, 79 Lewis RA, et al.11 Tol. 0.37 B 0.052 Don. 61.3 73.7 New site (20.08) 416/13006 db SNP (rs61750126) Benign
28 c.4139C>T p.P1380L 7 30, 31, 32, 33, 34, 35, 36 Lewis RA, et al.11 Del. 0.01 B 0.377 No change 2/13006 db SNP (rs61750130)
28 c.4234C>T p.Q1412* 1 33 Rivera A, et al.17 ND db SNP (rs61750137)
29 c.4283C>T p.T1428M 1 76 APEX Tol. 0.15 B 0.010 No change 2/13006 db SNP (rs1800549) Benign
29 c.4319T>C p.F1440S 1 34 Lewis RA, et al.11 Del. 0.00 POD 0.744 No change ND dbSNP (rs61750141)
29 c.4326C>A p.N1442K 1 64 Zernant J, et al.5 Tol. NA POD 0.374 No change ND
29 c.4328G>A p.R1443H 1 35 Rivera A, et al.17 Del. 0.02 PRD 0.999 No change 1/13006 dbSNP (rs61750142)
IVS29 c.4352+1G>A Splice site 1 73 Zernant J, et al.5 Don. 82.3 55.4 WT site broken (−32.62) ND
30 c.4469G>A p.C1490Y 2 36, 37 Lewis RA, et al.11 Del. 0.00 PRD 0.994 No change ND dbSNP (rs61751402)
30 c.4538A>G p.Q1513R 1 67 Webster AR, et al.7 Tol. NA Benign 0.043 Acc. 91.7 62.8 Site broken (−31.55) ND
30 c.4537_4538insC p.G1513fs 1 38 Briggs CE, et al.19 ND False-negative in NGS in patient 38
31 c.4577C>T p.T1526M 1 39 Lewis RA, et al.11 Del. 0.00 PRD 0.910 No change ND db SNP (rs61750152)
33 c.4685T>C p.I1562T 1 71 Yatsenko, et al.13 Tol. NA PRD 0.783 No change ND Benign
33 c.4715C>T p.T1572M 1 79 Pang CP and Lamm DS23 Del. 0.02 B 0.326 No change ND db SNP (rs185093512) Benign
35 c.4926C>G p.S1642R 1 40 Birch DG, et al.22 Tol. 0.68 B 0.116 No change ND db SNP (rs61753017)
35 c.4956T>G p.Y1652* 1 41 Fumagalli A, et al.16 ND db SNP (rs61750561)
IVS35 c.5018+2T>C Splice site 1 42 APEX Don. 81.2 54.3 WT site broken (−33.07) ND
36 c.5113C>T p.R1705W 1 7 Ernest PJ, et al.26 Del. NA PRD 0.996 Don. 46.5 73.3 No change ND
IVS38 c.5461-10T>C 8 43, 44, 45, 46, 47, 48, 49, 50 Briggs CE, et al.19 No change 3/13006 db SNP (rs1800728)
IVS39 c.5585-1G>A Splice site 1 51 Shroyer NF, et al.21 Acc. 86.3 57.4 WT site broken (−33.53) ND
IVS40 c.5714+5G>A Splice site 1 52 Cremers FP, et al.8 Don. 85.5 73.3 Wild type site broken (−14.23) ND
42 c.5882G>A p.G1961E 7 53, 54, 55, 56, 57, 58, 59 Lewis RA, et al.11 Del. 0.00 PRD 0.998 No change 41/13006 db SNP (rs1800553)
44 c.6079C>T p.L2027F 4 60, 61, 62, 63 Lewis RA, et al.11 Del. 0.00 PRD 1.000 No change 4/13006 db SNP (rs61751408)
44 c.6089G>A p.R2030Q 1 64 Lewis RA, et al.11 Del. 0.00 PRD 0.995 No change 8/13006 db SNP (rs61750641)
46 c.6320G>A p.R2107H 2 72, 73 Fishman GA, et al.15 Del. 0.04 PRD 0.999 No change 91/13006 db SNP (rs62642564) Benign
47 c.6445C>T p.R2149* 1 65 Lewis RA, et al.14 1/13006 db SNP (rs61750654)
48 c.6529G>A p.D2177N 1 19 Rivera A, et al.17 Tol. 0.41 B 0.004 No change 116/13006 db SNP (rs1800555) Benign
48 c.6709A>C p.T2237P 1 66 APEX Del. NA POD 0.719 No change ND
IVS48 c.6729+4_ +18del AGTTGGCCCTGGGGC Splice site 1 17 Littink KW, et al.28 NA ND
Table 4
 
Numbers and Types of Variants Detected by APEX Technology and NGS
Table 4
 
Numbers and Types of Variants Detected by APEX Technology and NGS
Total Null Non-Null Benign Missense
Splice-Site Altering Nonsense Frameshift Unknown In-Frame Deletion Disease-Causing Missense
Variants detected by prescreening with APEX 42 4 4 2 1 0 23 8
New variants detected by NGS alone 42 (33) 9 (7) 6 (4) 3 (3) 0 (0) 1 (1) 20 (16) 3 (2)
Gross total 84 (33) 13 (7) 10 (4) 5 (3) 1 (0) 1 (1) 43 (16) 11 (2)
Table 5
 
In Silico Molecular Genetic Analysis for Novel ABCA4 Variants Identified by NGS
Table 5
 
In Silico Molecular Genetic Analysis for Novel ABCA4 Variants Identified by NGS
Exon/ IVS DNA Change Protein Change/ Effect N of Alleles Identified Pt SIFT Polyphen2 HSF Matrix Allele Freq. by EVS Reference Comments
Pred. Tol. Index (0–1) Pred. Hum Var Score (0–1) Site Wt CV Mt CV CV % Variation
3 c.180delG p.M61fs 1 35 ND
IVS7 c.859-9T>C Splice site 1 5 Acc. 78.18 76.99 Possibly site broken (−1.52) ND Possibly disease-causing
11 c.1433T>C p.I478T 1 1 Tol. B 0.007 No change ND Benign
11 c.1519G>T p.D507Y 1 20 Del. 0.01 POD 0.641 No change 1/13006 dbSNP (rs148234178)
12 c.1749G>C p.K583N 1 68 Del. 0.04 POD 0.893 Acc. 66.17 37.22 Site broken (−43.75) 1/13006 dbSNP (rs145265791)
14 c.1982_ 1983insG p.A662fs 1 69 ND
15 c.2297G>T p.G766V 1 1 Tol. NA POD 0.557 Don. 69.18 42.34 Site broken (−38.79) ND
15 c.2345G>A p.W782* 1 3 ND
16 c.2510T>C p.L837P 1 72 Tol. NA POD 0.905 No change ND
16 c.2568C>A p.Y856* 1 5 ND
18 c.2686A>G p.K896E 1 53 Tol. NA B 0.002 ND Benign
20 c.2942C>T p.P981L 1 19 Del. 0.00 POD 0.813 No change 1/13006 dbSNP (rs147826775)
20 c.2972G>T p.G991V 1 70 Del. NA PRD 0.998 Donor 64.62 91.45 New site (41.53) ND
IVS20 c.3050+1G>C Splice site 1 54 Acc. 86.43 57.49 Site broken (−33.49) ND
IVS21 c.3191-1G>T Splice site 1 26 Acc. 94.38 65.44 WT site broken (−30.67) ND
22 c.3289A>T p.R1097* 1 71 ND
22 c.3299T>A p.I1100N 1 23 Del. NA PRD 0.986 No change ND
23 c.3370G>T p.D1124Y 1 62 Del. NA PRD 0.998 No change ND
23 c.3392delC/ 3393C>G p.A1131Gfs 1 55 ND
23 c.3398T>C p.I1133T 1 27 Del. NA B 0.100 No change ND Possibly disease-causing
27 c.4070C>A p.A1357E 1 28 Del. NA PRD 0.94 Acc. 40.92 69.86 New site (+70.74) ND
IVS30 c.4539+2T>G Splice site 1 56 Don. 79.18 52.35 WT site broken (−33.89) ND
31 c.4552A>C p.S1518R 1 57 Del. NA POD 0.871 Acc. 76.3 47.36 Site broken (−37.94) ND
31 c.4634G>A p.S1545N 2 21, 25 Tol. NA B 0.253 Acc. 80.04 51.1 Site broken (−36.16) ND
33 c.4773G>C Splice site 1 29 Don. 84.58 73.57 Site broken (−13.02) ND
35 c.4978delC p.L1661* 1 24 ND
36 c.5041_5055del GTGGTTGCCATCTGC p.V1681_C1685del 1 40 NA ND db SNP (rs62646872)
36 c.5088C>G p.S1696R 1 10 Tol. NA PRD 0.780 Don. 59.34 86.17 New site (45.23) ND
39 c.5578C>T p.R1860W 1 38 Del. 0.02 B 0.025 No change ND db SNP (rs200849015)
IVS42 c.5899-3_ 5899-2delTA Splice site 1 66 NA ND
IVS42 c.5899-2delA Splice site 1 58 Acc. 82.1 28.26 WT site broken (−65.58) ND
45 c.6209C>G p.T2070R 1 52 Tol. NA PRD 0.996 Acc. 57.41 86.36 New site (50.42) ND
46 c.6385A>G p.S2129G 1 43 Del. NA B 0.001 ND Possibly disease-causing
Table 6
 
Distribution of 79 Patients With ABCA4-Related Retinal Disease Based on Number of Identified Disease-Causing Variants
Table 6
 
Distribution of 79 Patients With ABCA4-Related Retinal Disease Based on Number of Identified Disease-Causing Variants
Comprehensive Screening With APEX and NGS
No Disease-Causing Variant 1 Disease-Causing Variant 2 Disease-Causing Variants 3 Disease-Causing Variants
Prescreening with APEX 1 disease causing-variant, n = 66 29 36 1
No disease-causing variants, n = 13 6 7
Total, n = 79 6 36 36 1
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×