To investigate the frequency of deep intronic variants in the
ABCA4 locus in our patient cohort, we sequenced genomic regions encompassing putative alternative exons in intron 30 and 36, namely, exon 30.1, exon 36.01, and exon 36.1. These regions were previously identified by RNA sequencing of retinal tissue (
Supplementary Fig. S2).
24,37,38 Ten different sequence variants were found in 236 patients, of which four (c.4539+2028C>T, c.4540-2036C>A, c.5196+1013A>G, c.5196+1056A>G) were defined as rare variants with a MAF ≤ 0.005 in the 1000 Genomes catalog, phase 3 (
Table 3;
Supplementary Fig. S2). These changes were each identified once in the 472 alleles analyzed. Among these, c.4539+2028C>T and c.5196+1056A>G have been described previously by Braun et al. (2013)
24 as V5 and V3, respectively, and were later also found in another cohort of STGD1 patients.
20 Deep intronic variants c.4540-2036C>A and c.5196+1013A>G are reported for the first time. The c.5196+1056A>G (V3) variant is predicted to strengthen a cryptic donor splice site. Reverse transcriptase–PCR analysis using keratinocyte mRNA of patients carrying the V3 variant revealed the inclusion of a 177-bp segment into the
ABCA4 transcript (termed “alternative exon 36.01”), thus providing experimental evidence for a loss-of-function effect of this variation.
24 Patient 230 in our cohort carried the V3 variant and a second variant (c.2588G>C) annotated as pathogenic. This suggests that the V3 mutation is indeed the second disease-associated allele in our patient. A novel sequence change, c.5196+1013A>G, was found in patient 79 in combination with c.1654G>A, a likely pathogenic missense mutation [p.(V552I)]. Similar to the V3 variant, c.5196+1013A>G is predicted to strengthen a cryptic donor splice site and thus may also result in deleterious effects upon splicing. Based on bioinformatics, variants c.4539+2028C>T (V5) and c.4540-2036C>A are not predicted to affect splicing. The c.4539+2028C>T (V5) sequence change was identified in patient 42 with no other probable mutant
ABCA4 allele; variant c.4540-2036C>A was detected in patient 159 in addition to pathogenic mutation c.[1622T>C(;)3113C>T] and likely pathogenic mutation c.3261A>C. A disease association of these intronic variants in our patients is thus unclear. Six previously reported intronic sequence changes (c.4539+2001G>A, c.4539+2064C>T, c.5196+1136C>A, c.5196+1137G>A, c.5196+1159G>A, c.5196+1216C>A) were not found in our patient cohort (
Table 3).