Z-DNA-induced genomic instability in the human pangenome

Z-DNA is a non-canonical, left-handed nucleic acid conformation with roles in gene regulation, genomic variation, and genome instability. Recent advances in long-read sequencing enable systematic interrogation of Z-DNA in repetitive and previously unresolved parts of the human genome. Leveraging the complete telomere-to-telomere reference human genome and 464 haplotype-resolved genome assemblies from individuals of multiple ethnicities, we systematically mapped Z-DNA-forming sequences and investigated their genomic and mutational landscape. We show that Z-DNA density is highly constrained across human haplotypes and superpopulations, with substantial enrichment in repetitive regions absent or unresolved in GRCh38, including centromeric, pericentromeric, and acrocentric loci. By analyzing more than 52 million variants across multiple mutation categories in the human pangenome, we show that Z-DNA loci are strongly enriched for mutational events not explained by sequence composition alone. The strongest associations were observed for small insertions, deletions, and complex structural variants. Finally, we replicated our findings using more than 250,000 de novo mutations. Together, these findings establish Z-DNA as a functional element of the human genome, with important implications for genome instability and human disease.