Genome-wide detection of human intronic AG-gain variants located between splicing branchpoints and canonical splice acceptor sites

Research output: Contribution to journalResearch articleContributedpeer-review


  • Peng Zhang - , The Rockefeller University (Author)
  • Matthieu Chaldebas - , The Rockefeller University (Author)
  • Masato Ogishi - , The Rockefeller University (Author)
  • Fahd Al Qureshah - , The Rockefeller University (Author)
  • Khoren Ponsin - , The Rockefeller University (Author)
  • Yi Feng - , The Rockefeller University (Author)
  • Darawan Rinchai - , The Rockefeller University (Author)
  • Baptiste Milisavljevic - , The Rockefeller University (Author)
  • Ji Eun Han - , The Rockefeller University (Author)
  • Marcela Moncada-Vélez - , The Rockefeller University (Author)
  • Sevgi Keles - , Necmettin Erbakan University (Author)
  • Bernd Schröder - , Institute of Physiological Chemistry (Author)
  • Peter D Stenson - , Cardiff University (Author)
  • David N Cooper - , Cardiff University (Author)
  • Aurélie Cobat - , TUD Dresden University of Technology (Author)
  • Bertrand Boisson - , The Rockefeller University (Author)
  • Qian Zhang - , The Rockefeller University, INSERM - Institut national de la santé et de la recherche médicale, Imagine Institute, Université Paris Cité (Author)
  • Stéphanie Boisson-Dupuis - , The Rockefeller University (Author)
  • Laurent Abel - , The Rockefeller University (Author)
  • Jean-Laurent Casanova - , The Rockefeller University (Author)


Human genetic variants that introduce an AG into the intronic region between the branchpoint (BP) and the canonical splice acceptor site (ACC) of protein-coding genes can disrupt pre-mRNA splicing. Using our genome-wide BP database, we delineated the BP-ACC segments of all human introns and found extreme depletion of AG/YAG in the [BP+8, ACC-4] high-risk region. We developed AGAIN as a genome-wide computational approach to systematically and precisely pinpoint intronic AG-gain variants within the BP-ACC regions. AGAIN identified 350 AG-gain variants from the Human Gene Mutation Database, all of which alter splicing and cause disease. Among them, 74% created new acceptor sites, whereas 31% resulted in complete exon skipping. AGAIN also predicts the protein-level products resulting from these two consequences. We performed AGAIN on our exome/genomes database of patients with severe infectious diseases but without known genetic etiology and identified a private homozygous intronic AG-gain variant in the antimycobacterial gene SPPL2A in a patient with mycobacterial disease. AGAIN also predicts a retention of six intronic nucleotides that encode an in-frame stop codon, turning AG-gain into stop-gain. This allele was then confirmed experimentally to lead to loss of function by disrupting splicing. We further showed that AG-gain variants inside the high-risk region led to misspliced products, while those outside the region did not, by two case studies in genes STAT1 and IRF7. We finally evaluated AGAIN on our 14 paired exome-RNAseq samples and found that 82% of AG-gain variants in high-risk regions showed evidence of missplicing. AGAIN is publicly available from and


Original languageEnglish
Pages (from-to)e2314225120
JournalProceedings of the National Academy of Sciences of the United States of America : PNAS
Issue number46
Publication statusPublished - 14 Nov 2023

External IDs

PubMedCentral PMC10655562
Scopus 85176371368


Sustainable Development Goals


  • Humans, RNA Splice Sites, Introns, RNA Splicing, Mutation, Genome