Deep intronic founder mutations identified in the ERCC4/ XPF gene are potential therapeutic targets for a high-frequency form of xeroderma pigmentosum

Abstract

Xeroderma pigmentosum (XP) is a genodermatosis defined by cutaneous photosensitivity with an increased risk of skin tumors because of DNA repair deficiency. The worldwide prevalence of XP is ~1 to 4 in million, with higher incidence in some countries and regions including Japan (1 in 22,000) and North Africa due to founder mutations and a high degree of consanguinity. Among XP, the complementation group F (XP-F), is a rare form (1% of worldwide XP); however, this is underdiagnosed, because the ERCC4/XPF gene is essential for fetal development and most of previously reported ERCC4/XPF pathogenic variants are hypomorphs causing relatively mild phenotypes. From the largest Japanese XP cohort study, we report 17 XP-F cases bearing two pathogenic variants, both identified in deep intronic regions of the ERCC4/XPF gene. The first variant, located in intron 1, is a Japanese founder mutation, which additionally accounts for ~10% of the entire Japanese XP cases (MAF = 0.00196), causing an aberrant pre-mRNA splicing due to a miss-binding of U1snRNA. The second mutation located in intron eight induces an alternative polyadenylation. Both mutations cause a reduction of the ERCC4/XPF gene expression, resulting in XP clinical manifestations. Most cases developed early-onset skin cancers, indicating that these variants need critical attention. We further demonstrate that antisense oligonucleotides designed for the mutations can restore the XPF protein expression and DNA repair capacity in the patients' cells. Collectively, these pathogenic variants can be potential therapeutic targets for XP.

Keywords: DNA repair; artificial antisense oligonucleotides (ASOs); nucleotide excision repair (NER); oligonucleotide therapeutics; xeroderma pigmentosum.

Fig. 1.

Japanese XP-F cases. (A) Photos of representative cases. XP165KO, age 45 (first and second panels); XP133KO, age 24 (third panel); XP97NG, age 71. (B) Lentivirus-based complementation assay. Exclusive rescue of the UDS deficiency by the infection of recombinant lentivirus expressing the wild-type ERCC4/XPF cDNA in XP43NG cells (filled bars, 20 J/m² UVC; open bars, without UV). (C) Lentivirus-based complementation assay. XP2YO was also assigned to the XP-F complementation group. UDS was normalized to activity in nonirradiated cells. Error bars: SD of means of at least quintuplicate wells (B and C, representative data). See also SI Appendix, Fig. S1.

Fig. 2.

Locations of the ERCC4/XPF intron variants identified in the Japanese XP-F cases. (A) Estimated structures of pre-mRNA products resulting from the ERCC4/XPF intron variants. Cryptic intron fragments identified in the patients’ mRNA are shown in blue lines. (B) cDNA sequences of the ERCC4/XPF exons 1 to 2 boundary in 48BR (normal) and XP43NG (XP-F). The 5′ cryptic intron 1 fragment is shown in blue letters. (C) A cDNA sequence of the ERCC4/XPF exon8–intron9 boundary in XP2YO (XP-F). The cryptic intron 8 fragment is shown in blue letters. Arrowhead indicates the variant position. (D) Digital quantitative PCR (digital qPCR) detected the reduction of ERCC4/XPF expression and the aberrant splicing product of intron 1 in XP43NG (filled bars, PCR amplifying the ERCC4/XPF exons 1 to 2 boundary; gray bars, PCR amplifying the 5′ cryptic fragment of ERCC4/XPF intron 1; open bars, a control PCR product of the TBP gene). (E) Digital qPCR detected the reduction of ERCC4/XPF expression in XP3YO (XP-F) (filled bars, PCR amplifying the ERCC4/XPF exons 1 to 2 boundary; hatched bars, exons 7 to 8 boundary; gray bars, exons 8 to 9 boundary; open bars, TBP). (F) Immunoblotting of the XPF protein. wild type and ΔERCC4/XPF, wild type and ERCC4/XPF-deficient HeLa cells; 48BR, normal; XP24BR, XP-F control; [XP136KO, XP37NG, XP43NG, XP101OS, XP97NG, XP165KO, XP103NG, XP4NG, XP48NG, XP90NG, XP95NG, XP18NG, XP133KO, XP23OS, XP96NG, XP2YOSV40, and XP3YO], the Japanese XP-F cases. b-actin (ACTB) as a loading control. XPF-upper bands represent the stop-loss product, p.*917Rext*83. The PCR primers are listed in SI Appendix, Fig. S2 and Table S2.

Fig. 3.

Aberrant pre-mRNA splicing and alternative polyadenylation events of ERCC4/XPF in the cases. (A) Schematic representation of the minigene assay. GFP-ERCC4/XPF partial gene fusion vectors with or without the intron 1 variant, their corresponding transcripts, and the size of PCR amplified cDNAs are shown. Int1Wt, a wild-type vector with the wild-type intron 1 sequence amplified from normal 48BR cells; Int1Mut, a mutant vector with the intron 1 variant amplified from XP43NG; Int1Rev, a vector with a wild-type revertant of the intron 1 variant generated from Int1Mut. (B) The GFP-ERCC4/XPF minigene cDNA products were resolved on agarose-gel electrophoresis. RT-PCR was performed using the primers shown in A. Int1Wt and Int1Rev gave the same sized band, while Int1Mut gave a 192bp longer product. 18SrRNA as a control. (C) Direct Sanger sequencing of the cDNA products. Int1Mut gave a fragment with the cryptic intron 1 insertion. (D) Minigene vectors with or without the intron 8 variant and the corresponding transcripts are shown. Int8Wt, a wild-type vector with the wild-type intron 8 sequence; Int8Mut, a mutant vector with the intron 8 variant amplified from XP3YO; Int8Rev, a wild-type revertant of the intron 8 variant generated from Int8Mut. (E) Minigene cDNA products were resolved on a capillary-gel electrophoresis system, MultiNa (SHIMADZU). ex9 denotes the PCR products that spans exons 8 to 9, being amplified from the normal transcript, while oligodT represents the PCR products amplified from both normal- and 3′ truncated-mRNAs. (F and G) Direct Sanger cDNA sequencing of the exons 8 to 9 boundary (F) and the transcription termination site (G). Int8Mut gave a fragment with the cryptic intron 8 insertion. The PCR primers are listed in SI Appendix, Fig. S3 and Table S2.

Fig. 4.

Recovery of DNA repair activity by antisense nucleotide treatments. (A) Schematic representation of the artificial antisense oligonucleotides (ASOs) designed for the intron 1 and the intron 8 variants. (B and C) Recovery of the XPF protein expression by the ASO treatments. (B) XP43NG, bearing the intron 1 variant, was treated with the LNA- as well as with the morpholino-modified oligonucleotides shown in A. (C) XP3YO, with the intron 8 variant, was treated with the ASOs shown in A. (D and E) Recovery of DNA repair activity by the ASO treatments. The reduced UDS was restored by the ASOs designed for the intron 1 variant in XP43NG cells (D) and for the intron 8 variant in XP3YO cells (E) (filled bars, 20 J/m² UVC; open bars, without UV). Red bars indicate appropriate recoveries of ERCC4/XPF expression and DNA repair activity by the ASO treatments. The ASOs are listed in SI Appendix, Fig. S4 and Tables S3 and S4.