MD-0560ABCA423c.3386G>Tp.Arg1129Leu47c.6410G>Ap.Cys2137Tyr16NPABCR400

MD-0247ABCA423c.3386G>Tp.Arg1129Leu47c.6410G>Ap.Cys2137Tyr12YesABCR400 + dHPLC + HRMAguirre-Lamban et al. 2009 (8); Aguirre-Lamban et al. 2010 (16)

In 20 patients (19 STGD and 1 CRD), 18 different genotypes were identified (Table 1). Except p.Arg187His and p.Tyr954Ser variants, the rest of changes were previously reported as disease-associated allele. 14 The p.Arg187His and p.Tyr954Ser variants were not found in 100 ethnically matched control chromosomes. All the mutations identified in the 20 patients except one were detected by HRM for sensitivity of 95%; however, only 16 variants were identified by dHPLC for sensitivity of 80%.  Table 1. View Table Mutations AnalyzedTable 1. Mutations Analyzed Family Exon Genotype Mutation Detected by dHPLC Mutation Detected by HRM Nucleotide Change Amino Acid Change ARDM-167 5 c.560G>A p.Arg187His No Yes ARDM-257 5 c.560G>A p.Arg187His No Yes ARDM-164 6 c.700C>T p.Gln234X Yes Yes ARDM-135 8 c.1029_1030insT p.Asn344fsX Yes No ARDM-240 15 c.2285C>A p.Ala762Glu Yes Yes ARDM-248 19 c.2861A>C p.Tyr954Ser Yes Yes ARDM-90 — IVS21-2A>T — Yes Yes ARDM-40 27 c.3943C>T p.Gln1315X Yes Yes ARDM-158 30 c.4537delC p.Gln1513fsX1525 Yes Yes ARDM-38 33 c.4739delT p.Leu1580fs Yes Yes ARDM-163 36 c.5172G>T p.Trp1724Cys Not Yes ARDM-197 36 c.5172G>T p.Trp1724Cys Yes Yes ARDM-181 — IVS38+5G>A — Yes Yes ARDM-125 40 — p.KNLFA1876dup Yes Yes ARDM-183 43 c.5929G>A(False −) p.Gly1977Ser(False −) Yes Yes ARDM-146 44 c.6140T>A p.lle2047Asn Yes Yes ARDM-174 — IVS44+2T>A — Yes Yes ARDM-247 47 c.6410G>A p.Cys2137Tyr* Yes Yes ARDM-84 47 c.6410G>A p.Cys2137Tyr† No Yes ARDM-225 48 c.6559C>T p.Gln2187X Yes Yes  Previously unreported mutations are shown in bold. *  Mutation in heterozygous. †  Mutation in homozygous. Homozygous sequence alteration (p.Cys2137Tyr) could be identified from wild-type by HRM analyses (Fig. 1). In contrast, dHPLC did not distinguish any homozygous mutation, except when we mixed it, in a 1:1 proportion, with a previously sequenced wild-type sample at the end of each PCR session and before heteroduplex formation.

In 20 patients (19 STGD and 1 CRD), 18 different genotypes were identified (Table 1). Except p.Arg187His and p.Tyr954Ser variants, the rest of changes were previously reported as disease-associated allele. 14 The p.Arg187His and p.Tyr954Ser variants were not found in 100 ethnically matched control chromosomes. All the mutations identified in the 20 patients except one were detected by HRM for sensitivity of 95%; however, only 16 variants were identified by dHPLC for sensitivity of 80%.  Table 1. View Table Mutations AnalyzedTable 1. Mutations Analyzed Family Exon Genotype Mutation Detected by dHPLC Mutation Detected by HRM Nucleotide Change Amino Acid Change ARDM-167 5 c.560G>A p.Arg187His No Yes ARDM-257 5 c.560G>A p.Arg187His No Yes ARDM-164 6 c.700C>T p.Gln234X Yes Yes ARDM-135 8 c.1029_1030insT p.Asn344fsX Yes No ARDM-240 15 c.2285C>A p.Ala762Glu Yes Yes ARDM-248 19 c.2861A>C p.Tyr954Ser Yes Yes ARDM-90 — IVS21-2A>T — Yes Yes ARDM-40 27 c.3943C>T p.Gln1315X Yes Yes ARDM-158 30 c.4537delC p.Gln1513fsX1525 Yes Yes ARDM-38 33 c.4739delT p.Leu1580fs Yes Yes ARDM-163 36 c.5172G>T p.Trp1724Cys Not Yes ARDM-197 36 c.5172G>T p.Trp1724Cys Yes Yes ARDM-181 — IVS38+5G>A — Yes Yes ARDM-125 40 — p.KNLFA1876dup Yes Yes ARDM-183 43 c.5929G>A(False −) p.Gly1977Ser(False −) Yes Yes ARDM-146 44 c.6140T>A p.lle2047Asn Yes Yes ARDM-174 — IVS44+2T>A — Yes Yes ARDM-247 47 c.6410G>A p.Cys2137Tyr* Yes Yes ARDM-84 47 c.6410G>A p.Cys2137Tyr† No Yes ARDM-225 48 c.6559C>T p.Gln2187X Yes Yes  Previously unreported mutations are shown in bold. *  Mutation in heterozygous. †  Mutation in homozygous. Homozygous sequence alteration (p.Cys2137Tyr) could be identified from wild-type by HRM analyses (Fig. 1). In contrast, dHPLC did not distinguish any homozygous mutation, except when we mixed it, in a 1:1 proportion, with a previously sequenced wild-type sample at the end of each PCR session and before heteroduplex formation.

Through a meta-analysis of published literature38,39, we also found that individuals with inherited retinal disease caused by recessive ABCA4 deficiency (Stargardt disease or cone rod dystrophy) are likely to have ASO-amenable variants in similar proportions (Supplementary Note 7 and Supplementary Tables 15 and 16), leading us to suggest that around 15% is a reasonable first estimate for other recessive genetic conditions.

MD-1146 STGD1 14 c.2023G>A p.(Val675Ile) 47 c.6410G>A p.(Cys2137Tyr) Yes 46 - - 46y Normal 1/1 This study

MD-0740 STGD1 47 c.6410G>A p.(Cys2137Tyr) IVS28 c.4253+43G>A p.[=, Ile1377Hisfs*3] - 61 No - 62y - 0.8/0.16 This study

MD-0560 STGD1 23 c.3386G>T p.(Arg1129Leu) 47 c.6410G>A p.(Cys2137Tyr) Yes 16 20 - 53y Normal 0.1/0.1 Riveiro-Alvarez et al.,2013

MD-1001 STGD1 44 c.6089G>A p.(Arg2030Gln) 47 c.6410G>A p.(Cys2137Tyr) - - - - - - - This study

MD-0247 STGD1 23 c.3386G>T p.(Arg1129Leu) 47 c.6410G>A p.(Cys2137Tyr) Yes 12 12 - 22y Cone-pattern 0.05/0.1 Riveiro-Alvarez et al.,2013

47 c.6410G>A p.(Cys2137Tyr) Aguirre-Lamban (2008) Hum Genet 123 547 8 missens

MD-0723 STGD1 23 c.3386G>T p.(Arg1129Leu) 47 c.6410G>A p.(Cys2137Tyr) - 13 13 Yes 15y Cone-pattern 0.2/0.2 This study

MD-0084 STGD1 47 c.6410G>A p.(Cys2137Tyr) 47 c.6410G>A p.(Cys2137Tyr) Yes 7 7 - 23y - <0.05/<0.05 Riveiro-Alvarez et al.,2013

p.Cys2137Tyr (c.6410G>A)0.63±0.090.45±0.03 0.32±0.120Severe 17(Riveiro-Alvarez et al., 2013)

93RECRDrecessive ABCA4 c.3988G>Tp.E1330XHet[a]c.6410G>Ap.C2137YHet[a]

Screening of reported pathogenic variants in ABCA4 for Stargardt (STGD) The disease prevalence of STGD is estimated as 1 in 10000 individuals4. It has been estimated that about 70% of STGD patients carry variants in ABCA45. Therefore, this represents the scenario of a recessive disease with a relatively homogeneous genetic cause. We screened 945 reported pathogenic variants in ABCA4 genes collected in HGMD. Among them, 11 variants are likely benign, as their population AF in is higher than 0.7% (1/20000‾‾‾‾‾‾‾‾√)<math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline" id="M11"><mrow><mrow><mo>(</mo><mrow><msqrt><mrow><mn>1</mn><mo>/</mo><mn>20000</mn></mrow></msqrt></mrow><mo>)</mo></mrow></mrow></math>, the cutoff based on STGD disease prevalence, therefore were excluded from further analysis. The remaining 934 variants were subjected to our test model. As a result, 26 variants with the AF in the range of 0.46% to 0.03% were identified as likely benign (Binomial test1, Bonferroni correction p-value ≤ 0.05/934 and test2 Bonferroni correction p-value > 0.05/934) (Figure 3A).

Supplementary TableS5

Supplementary TableS10

Interestingly, we identified one 30-year-old patient (ARDM-247), double heterozygous for the p.Arg1129Leu and p.Cys2137Tyr alleles, who presented a CRD phenotype. This p.Cys2137Tyr change was located more towards the amino terminus. Moreover, in other study, the results showed that the changes located in this zone appear to result in altered processing of the protein and to be associated with an earlier onset of disease.16 The p.Cys2137Tyr change in combination with the p.Arg1129Leu allele produced a CRD phenotype. Therefore, we speculate that the novel p.Cys2137Tyr variant could be a severe allele which is modifying the patient’s phenotype.

Microarray-based mutation analysis of the ABCA4 gene in Spanish patients with Stargardt disease: evidence of a prevalent mutated allele