Towards Uncovering the Role of Incomplete Penetrance in Maculopathies through Sequencing of 105 Disease-Associated Genes

Abstract

Inherited macular dystrophies (iMDs) are a group of genetic disorders, which affect the central region of the retina. To investigate the genetic basis of iMDs, we used single-molecule Molecular Inversion Probes to sequence 105 maculopathy-associated genes in 1352 patients diagnosed with iMDs. Within this cohort, 39.8% of patients were considered genetically explained by 460 different variants in 49 distinct genes of which 73 were novel variants, with some affecting splicing. The top five most frequent causative genes were ABCA4 (37.2%), PRPH2 (6.7%), CDHR1 (6.1%), PROM1 (4.3%) and RP1L1 (3.1%). Interestingly, variants with incomplete penetrance were revealed in almost one-third of patients considered solved (28.1%), and therefore, a proportion of patients may not be explained solely by the variants reported. This includes eight previously reported variants with incomplete penetrance in addition to CDHR1:c.783G>A and CNGB3:c.1208G>A. Notably, segregation analysis was not routinely performed for variant phasing-a limitation, which may also impact the overall diagnostic yield. The relatively high proportion of probands without any putative causal variant (60.2%) highlights the need to explore variants with incomplete penetrance, the potential modifiers of disease and the genetic overlap between iMDs and age-related macular degeneration. Our results provide valuable insights into the genetic landscape of iMDs and warrant future exploration to determine the involvement of other maculopathy genes.

Keywords: inherited; macula; maculopathies; penetrance; retinal; sequencing.

Figure 1

Types of variants identified in the 508 individuals, where a genetic diagnosis was considered identified.

Figure 2

Genes involved in the 508 individuals considered very likely or possibly solved following genetic analysis.

Figure 3

ABCA4 splicing assay results in HEK293T cells. (A) Variant c.3329-124G>T in intron 22 was tested in a midigene system using the BA16 construct, which incorporated exons 21–24 of ABCA4. Agarose gel analysis and Sanger sequencing revealed a band of 831 nt corresponding to the wild-type (WT) construct and 940 nt corresponding to 109 nt of intron 22 retention, leading to a frameshift (r.[3328_3329ins3329-109_3329-1,=]; p.[Gly1110Valfs*4,=]). Based on the percentage of aberrant splicing observed (45% of WT RNA remaining), c.3329-124G>T is considered a mild variant. Original images can be found in Supplementary File S1. (B) Variant c.1451A>G in exon 11 was tested in a midigene system using the BA7 construct, which incorporated exons 7–11 of ABCA4. Agarose gel and Sanger sequencing analysis revealed a band of 542 nt corresponding to the WT construct, in addition to a band of 448 nt confirming a loss of 95 of the 198 nt of exon 11, resulting in partial exon skipping. A faint band was also observed at 331 nt, corresponding to exon 10 skipping with partial exon 11 skipping; however, this band contributed to <15% of the total RNA and was thus not considered in the calculations. Based on the percentage of aberrant splicing observed (62% of WT RNA remaining), c.1451A>G is considered a mild variant (r.[=,1357_1451del]; p.[Lys484Arg,Asp453Glyfs*8]) Original images can be found in Supplementary File S1. (C) Variant c.6817-679C>G in intron 49 was tested in a minigene system using the BA39 construct. Agarose gel and Sanger sequencing showed only the WT construct (361 nt; r.(=); p.(=)). Original images can be found in Supplementary File S1.