Z-DNA-associated genomic instability in the human pangenome.

Z-DNA is a non-canonical, left-handed nucleic acid conformation implicated in gene regulation and genome instability. Long-read sequencing and telomere-to-telomere genome assemblies now enable analysis of repetitive regions that were absent or incompletely represented in earlier human references. Leveraging T2T-CHM13 and 464 haplotype-resolved Human Pangenome Reference Consortium assemblies, we mapped predicted Z-DNA-forming sequences and characterized their genomic and mutational landscape. Z-DNA density was broadly similar across human haplotypes and superpopulations but differed substantially between T2T-CHM13 and GRCh38 in several repetitive genomic compartments, including acrocentric loci. Across more than 52 million HPRC variants, predicted Z-DNA-forming loci showed elevated mutation density relative to length-, GC-, and CpG/GpC-matched controls across most mutation classes, with the strongest associations observed for small and intermediate insertions, deletions, and complex insertion- and deletion-like events. Enrichment was concentrated near predicted B-Z junctions and varied by genomic context, with the strongest effects in genic and euchromatic compartments. Similar mutation-class associations were observed in more than 250,000 de novo mutations. Together, these analyses define the mutation-class, spatial, and genomic-context landscape associated with predicted Z-DNA-forming sequences across the human pangenome.