Whole Locus Sequencing Identifies a Prevalent Founder Deep Intronic RPGRIP1 Pathologic Variant in the French Leber Congenital Amaurosis Cohort

Abstract

Leber congenital amaurosis (LCA) encompasses the earliest and most severe retinal dystrophies and can occur as a non-syndromic or a syndromic disease. Molecular diagnosis in LCA is of particular importance in clinical decision-making and patient care since it can provide ocular and extraocular prognostics and identify patients eligible to develop gene-specific therapies. Routine high-throughput molecular testing in LCA yields 70%-80% of genetic diagnosis. In this study, we aimed to investigate the non-coding regions of one non-syndromic LCA gene, RPGRIP1, in a series of six families displaying one single disease allele after a gene-panel screening of 722 LCA families which identified 26 biallelic RPGRIP1 families. Using trio-based high-throughput whole locus sequencing (WLS) for second disease alleles, we identified a founder deep intronic mutation (NM_020366.3:c.1468-128T>G) in 3/6 families. We employed Sanger sequencing to search for the pathologic variant in unresolved LCA cases (106/722) and identified three additional families (two homozygous and one compound heterozygous with the NM_020366.3:c.930+77A>G deep intronic change). This makes the c.1468-128T>G the most frequent RPGRIP1 disease allele (8/60, 13%) in our cohort. Studying patient lymphoblasts, we show that the pathologic variant creates a donor splice-site and leads to the insertion of the pseudo-exon in the mRNA, which we were able to hamper using splice-switching antisense oligonucleotides (AONs), paving the way to therapies.

Keywords: Leber congenital amaurosis; RPGRIP1; deep intronic variant; oligotherapy.

Figure 1

Pedigrees of LCA families and segregation analysis of RPGRIP1 pathologic variants. M: c.1468-128T>G; p.?, M1 c.1502-1505insTGTC; p.Ser502Valfs*7, M2 c.2895+1G>T, p?, M3 c.1525C>T, p.Gln509*, M4 c.930+77A>G, p.?, M5 c.3238+1G>A? p.?, M6 c.2437del, p.Ser813Valfs*45; +: wild-type allele. LCA: Leber congenital amaurosis, HL: hearing loss, ID: intellectual disability.

Figure 2

Splicing prediction scores around the RPGRIP1 c.1468-128T>G variant in intron 11, position and sequence of the pseudo-exon amplified from patient mRNA and position of splice-switching antisense oligonucleotides (AONs). (A) Representation of the intron 11 region encompassing the c.1468-128T>G variant and cryptic acceptor and donor splice-sites with splicing score predictions according to the Alamut Software. The pseudo-exon (PE) is framed in red. The sequences of the three AONs designed to target the cryptic acceptor splice site (AON1), one exonic splice enhancer (ESE) sequence in the cryptic exon (AON2) and the cryptic donor splice site (AON3) designed using the ESEFinder 3.0 program are framed in black.(B) Schematic representation of wild-type and mutant RPGRIP1 mRNAs produced from the c.1468-128T>G mutant allele in both homozygous and heterozygous individuals and sequence of the mutant mRNA encompassing the pseudo-exon (PE, framed in red) between exons 11 and 12. (C) RT-PCR from mRNA isolated from controls (C1, C2) and patients carrying the c.1468-128T>G variant in homozygosity (LCA426) and compound heterozygosity (MON035, LCA215) in native condition and following treatment of C1, C2 and LCA215 lymphoblasts with 150 nmoL/L of AON1, AON2, AON3 or AONsense. In native conditions, RT-PCR from mRNA extracted from LCA426 and MON035 fibroblasts and LCA215 lymphoblasts yielded two products, the wildtype product (exon 11–12: 194 bp) and a higher molecular weight fragment (exon 11-PE-12: 312 bp). The controls C1 and C2 only display the wildtype 194-bp product. The treatment of LCA215 lymphoblasts with AON1, AON2, or AON3, but not the negative control sense oligonucleotide (AONsense), resulted in the disappearance of the higher molecular weight product encompassing the aberrant pseudo-exon.