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Contribution and therapeutic implications of retroelement insertions in ataxia telangiectasia - PubMed

  • ️Sun Jan 01 2023

Contribution and therapeutic implications of retroelement insertions in ataxia telangiectasia

Boxun Zhao et al. Am J Hum Genet. 2023.

Abstract

Certain classes of genetic variation still escape detection in clinical sequencing analysis. One such class is retroelement insertion, which has been reported as a cause of Mendelian diseases and may offer unique therapeutic implications. Here, we conducted retroelement profiling on whole-genome sequencing data from a cohort of 237 individuals with ataxia telangiectasia (A-T). We found 15 individuals carrying retroelement insertions in ATM, all but one of which integrated in noncoding regions. Systematic functional characterization via RNA sequencing, RT-PCR, and/or minigene splicing assays showed that 12 out of 14 intronic insertions led or contributed to ATM loss of function by exon skipping or activating cryptic splice sites. We also present proof-of-concept antisense oligonucleotides that suppress cryptic exonization caused by a deep intronic retroelement insertion. These results provide an initial systematic estimate of the contribution of retroelements to the genetic architecture of recessive Mendelian disorders as ∼2.1%-5.5%. Our study highlights the importance of retroelement insertions as causal variants and therapeutic targets in genetic diseases.

Keywords: Mendelian disease; antisense oligonucleotides; ataxia telangiectasia; cryptic exon; exon skipping; genetic diagnosis; retroelement; splicing; transposable element; whole-genome sequencing.

Copyright © 2023 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

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Conflict of interest statement

Declaration of interests T.W.Y. received research funding from ATCP and EveryOne Medicines; has served as a scientific consultant to Biomarin, GeneTx, Alnylam, and Servier Pharmaceuticals; and is a volunteer scientific advisor to several nonprofit rare disease foundations. E.A.L. received research funding from ATCP and has served on the scientific advisory board to Genome Insight.

Figures

Figure 1
Figure 1

Retroelement insertions detected in ATM in individuals with A-T (A–E) The schematic diagrams and corresponding Integrative Genomics Viewer (IGV) screenshots of WGS data illustrate four Alu insertions and one DUSP16 pseudogene insertion in ATM: (A) Alu #1 in exon 50, (B) Alu #2 in intron 54, (C) Alu #3 in intron 5, (D) Alu #4 in intron 32, and (E) processed DUSP16 mRNA insertion in intron 54. Each IGV image shows two different types of reads supporting a non-reference retroelement insertion: (1) soft-clipped reads “clipped” at insertion breakpoints, with colored bases indicating mismatches to the reference genome, and (2) discordant reads near the breakpoints whose mate reads map to retroelement sequences interspersed in the genome (blue and yellow reads colored by mapping orientation). It also shows genomic hallmarks of TPRT-mediated retrotransposition: (1) poly(A) tails (consecutive T bases in red at an insertion breakpoint), (2) increased read coverage between the two breakpoints (red dashed lines) indicating target site duplication (TSD), and (3) L1 endonuclease cleavage site (blue underline). (F) Characterization of processed DUSP16 pseudogene and the corresponding source gene locus. Features from the IGV screenshot of the source DUSP16 locus enable the reconstruction of a 5′ capped (red base G) DUSP16 mature mRNA with a partially truncated 3′ UTR due to alternative polyadenylation. The coverage track shows increased depth only in exons (red dashed lines), not in introns, and split reads across adjacent exons (red lines) reflect the integration of cDNA derived from processed mRNA after splicing. Loci with sequence variations relative to Alu consensus or DUSP16 source gene sequences are marked by red triangles on the schematic diagrams.

Figure 2
Figure 2

Exon skipping and cryptic exonization caused by Alu insertions and DUSP16 pseudogene insertion, respectively, and ASOs correcting the aberrant exonization (A) Intronic antisense Alu insertions proximal (all <50 bp) to the exon-intron boundaries are predicted to cause exon skipping. (B) Splice analysis of the deep intronic DUSP16 pseudogene insertion predicts strong splice donor and acceptor sites (blue and yellow lollipops) within the insertion leading to pseudoexon inclusion (red underline) with a premature termination codon (red stop sign). (C) Minigene constructs for Alu allele, reference allele, and DUSP16 insertion alleles are shown. (D and E) RT-PCR readouts of minigene splicing assays confirm (D) exon skipping caused by all three Alu insertions (Alu #2, #3, and #4) and (E) pseudoexon inclusion by DUSP16 pseudogene insertion. Minigene assays demonstrate that Alu insertions #2, #3, and #4 weaken exon definition. The observation of some exon skipping in reference constructs also suggests that these ATM exons, at least in isolation in a minigene context, may be weakly defined in the first place. (F) IGV sashimi plot of RNA-seq data from six A-T individuals with Alu #2 and two control individuals confirm that reads skipping exon 54 are observed only in the six A-T individuals with Alu #2, not in controls. The plot shows exon-exon junctions that are specifically linked to exon 53 (depicted as a blue empty box). (G and H) Direct RT-PCR of blood RNA from A-T individuals with Alu #2 (G) and Alu #3 (H) and unrelated healthy controls confirms exon skipping. (I) Nine 22-mer ASOs were designed to cover cryptic splice donor, acceptor, and multiple splice enhancer sites within the exonized region. RT-PCR readouts of minigene and ASO co-transfection show the efficacy of multiple ASOs to correct pseudoexon inclusion. Null, no transfection; scramble, a non-targeting ASO as a negative control. PCR amplicon bands with normal and aberrant splicing were indicated by green and red arrows, respectively.

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