ABCMdb

PMID: 23982839

Fujinami K, Zernant J, Chana RK, Wright GA, Tsunoda K, Ozawa Y, Tsubota K, Webster AR, Moore AT, Allikmets R, Michaelides M
ABCA4 gene screening by next-generation sequencing in a British cohort.
Invest Ophthalmol Vis Sci. 2013 Oct 11;54(10):6662-74. doi: 10.1167/iovs.13-12570., [PubMed]

Sentences

No. Mutations Sentence Comment

55 ABCA4 p.Asp2177Asn ABCA4 p.Gly863Ala ABCA4 p.Gly863Ala ABCA4 p.Gly863Ala ABCA4 p.Gly863Ala ABCA4 p.Gly863Ala ABCA4 p.Cys54Tyr ABCA4 p.Cys1490Tyr ABCA4 p.Cys1490Tyr ABCA4 p.Cys75Gly ABCA4 p.Arg602Trp ABCA4 p.Asn965Ser ABCA4 p.Arg653Cys ABCA4 p.Arg653Cys ABCA4 p.Arg1098Cys ABCA4 p.Arg1443His ABCA4 p.Thr1526Met ABCA4 p.Arg1108Cys ABCA4 p.Arg1108Cys ABCA4 p.Arg1108Cys ABCA4 p.Pro1380Leu ABCA4 p.Pro1380Leu ABCA4 p.Pro1380Leu ABCA4 p.Pro1380Leu ABCA4 p.Pro1380Leu ABCA4 p.Pro1380Leu ABCA4 p.Pro1380Leu ABCA4 p.Phe1440Ser ABCA4 p.Arg602Gln ABCA4 p.Arg1705Trp ABCA4 p.Asp576His ABCA4 p.Glu1022Lys ABCA4 p.Glu1022Lys ABCA4 p.Cys641Ser ABCA4 p.Arg1129His ABCA4 p.Arg1129His ABCA4 p.Arg408* ABCA4 p.Gln636* ABCA4 p.Gln636* ABCA4 p.Ile1133Thr ABCA4 p.Tyr856* ABCA4 p.Ser1696Arg ABCA4 p.Asn247Thr ABCA4 p.Gln1412* ABCA4 p.Pro981Leu ABCA4 p.Ala1357Glu ABCA4 p.Gly766Val ABCA4 p.Leu1661* ABCA4 p.Arg1860Trp ABCA4 p.Arg219* ABCA4 p.Arg219* ABCA4 p.Ile1100Asn ABCA4 p.Asp507Tyr ABCA4 p.Ile478Thr ABCA4 p.Ser1545Asn ABCA4 p.Ser1545Asn ABCA4 p.Trp782* 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&#fe;4_&#fe;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 T ABLE 2. Continued 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. Login to comment 56 ABCA4 p.Gly1961Glu ABCA4 p.Gly1961Glu ABCA4 p.Gly1961Glu ABCA4 p.Gly1961Glu ABCA4 p.Gly1961Glu ABCA4 p.Gly1961Glu ABCA4 p.Gly1961Glu ABCA4 p.Arg2107His ABCA4 p.Arg2107His ABCA4 p.Leu2027Phe ABCA4 p.Leu2027Phe ABCA4 p.Leu2027Phe ABCA4 p.Leu2027Phe ABCA4 p.Ile1562Thr ABCA4 p.Arg1108Cys ABCA4 p.Arg1108Cys ABCA4 p.Arg2030Gln ABCA4 p.Thr901Ala ABCA4 p.Leu1201Arg ABCA4 p.Leu1201Arg ABCA4 p.Leu1201Arg ABCA4 p.Leu1201Arg ABCA4 p.Gly991Arg ABCA4 p.Ser1642Arg ABCA4 p.Ile156Val ABCA4 p.Ile156Val ABCA4 p.Thr1572Met ABCA4 p.Thr1428Met ABCA4 p.Gln1513Arg ABCA4 p.Asn1442Lys ABCA4 p.Gln636* ABCA4 p.Arg2149* ABCA4 p.Ser2129Gly ABCA4 p.Gly991Val ABCA4 p.Arg1097* ABCA4 p.Asp1124Tyr ABCA4 p.Leu837Pro ABCA4 p.Lys583Asn ABCA4 p.Lys896Glu ABCA4 p.Ser1518Arg ABCA4 p.Thr2070Arg ABCA4 p.Tyr1652* ABCA4 p.Thr2237Pro 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&#fe;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&#fe;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&#fe;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&#fe;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&#fe;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 Putative novel variants are shown in italics. Login to comment 62 ABCA4 p.Gly863Ala ABCA4 p.Cys54Tyr ABCA4 p.Cys1490Tyr ABCA4 p.Cys75Gly ABCA4 p.Arg602Trp ABCA4 p.Asn965Ser ABCA4 p.Arg653Cys ABCA4 p.Arg1098Cys ABCA4 p.Arg1443His ABCA4 p.Arg1108Cys ABCA4 p.Pro1380Leu ABCA4 p.Thr901Ala ABCA4 p.Leu1201Arg ABCA4 p.Gly991Arg ABCA4 p.Phe1440Ser ABCA4 p.Arg602Gln ABCA4 p.Ile156Val ABCA4 p.Asp576His ABCA4 p.Glu1022Lys ABCA4 p.Cys641Ser ABCA4 p.Thr1428Met ABCA4 p.Arg1129His ABCA4 p.Arg408* ABCA4 p.Gln1513Arg ABCA4 p.Asn1442Lys ABCA4 p.Gln636* ABCA4 p.Asn247Thr ABCA4 p.Gln1412* ABCA4 p.Arg219* 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 (&#fe;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 (&#fe;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&#fe;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 T ABLE 3. Continued 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. Login to comment 63 ABCA4 p.Asp2177Asn ABCA4 p.Arg2107His ABCA4 p.Leu2027Phe ABCA4 p.Ile1562Thr ABCA4 p.Thr1526Met ABCA4 p.Arg2030Gln ABCA4 p.Ser1642Arg ABCA4 p.Arg1705Trp ABCA4 p.Thr1572Met ABCA4 p.Arg2149* ABCA4 p.Tyr1652* Hum Var Score (0-1) Site Wt CV Mt CV CV % Variation 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 DS 23 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&#fe;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&#fe;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&#fe;4_ &#fe;18del AGTTGGCCCTGGGGC Splice site 1 17 [ Littink KW, et al. 28 NA ND Splice-site alteration (described as splice site) includes the change expected to affect splicing, for example, when the splice donor or splice acceptor site is changed, and the change that might affect splicing, for example, changes close to the splice donor or splice acceptor site, or in the first or last nucleotide of an exon. SIFT (version 4.0.4) results are reported to be tolerant if tolerance index is ߥ0.05 or deleterious if tolerance index is <0.05. Login to comment 97 ABCA4 p.Arg1129His 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). Login to comment 100 ABCA4 p.Arg1129His Two variants were detected only on APEX array, but not identified by NGS; one frameshift variant (p.Q1513fs) and one disease-causing missense variant (p.R1129H). Login to comment 103 ABCA4 p.Ile1133Thr ABCA4 p.Tyr856* ABCA4 p.Pro981Leu ABCA4 p.Ala1357Glu ABCA4 p.Gly766Val ABCA4 p.Ile1100Asn ABCA4 p.Asp507Tyr ABCA4 p.Ile478Thr ABCA4 p.Ser1545Asn ABCA4 p.Trp782* ABCA4 p.Gly991Val ABCA4 p.Arg1097* ABCA4 p.Asp1124Tyr ABCA4 p.Leu837Pro ABCA4 p.Lys583Asn ABCA4 p.Lys896Glu ABCA4 p.Ser1518Arg 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&#fe;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 (&#fe;70.74) ND IVS30 c.4539&#fe;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 5. Continued 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. Login to comment 104 ABCA4 p.Ser1696Arg ABCA4 p.Leu1661* ABCA4 p.Arg1860Trp ABCA4 p.Ser2129Gly ABCA4 p.Thr2070Arg Hum Var Score (0-1) Site Wt CV Mt CV CV % Variation 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 Splice-site alteration (described as splice site) includes the change expected to affect splicing, for example, when the splice donor or splice acceptor site is changed, and the change that might affect splicing, for example, changes close to the splice donor or splice acceptor site, or in the first or last nucleotide of an exon. SIFT (version 4.0.4) results are reported to be tolerant if tolerance index is ߥ0.05 or deleterious if tolerance index is <0.05. Login to comment 109 ABCA4 p.Arg1108Cys ABCA4 p.Arg219* 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). Login to comment 115 ABCA4 p.Asp2177Asn 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). Login to comment 117 ABCA4 p.Ile478Thr ABCA4 p.Lys896Glu Two variants (p.I478T and p.K896E) were predicted to be tolerated and benign. Login to comment 121 ABCA4 p.Ile478Thr ABCA4 p.Lys896Glu 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). Login to comment 132 ABCA4 p.Arg1108Cys ABCA4 p.Arg1129His ABCA4 p.Arg219* 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. Login to comment 133 ABCA4 p.Arg1129His The missense variant p.R1129H was not detected by NGS, most likely due to allele-specific amplification. Login to comment 136 ABCA4 p.Arg1108Cys Two of those variants, p.R1108C, and p.S1071fs, were detected by APEX in other patients (Table 2). Login to comment