Telomere variant sequences encode the genetic blueprint for allele-specific telomere length

The ends of human chromosomes are capped by specialized nucleoprotein structures known as telomeres, which are essential for genome stability. Recent advances in long-read sequencing have enabled allele-specific telomere length measurements at nucleotide resolution, uncovering extreme heterogeneity in telomere length between alleles that is determined at birth and gradually shortens with age. However, the mechanisms underlying allele-specific telomere maintenance and its stability remain poorly understood. Here, we developed a high-resolution workflow combining PacBio and Nanopore long-read sequencing platforms to map allele-specific telomere length in clinical patient samples as well as telomerase-positive cancer cell lines. By tracing allele-specific telomeric sequence in family members across multiple generations, we show that telomeric variant sequences (TVSs) interspersed throughout the canonical repeat region are heritable (with mean similarity score > 0.95), allele-specific, and account for the extreme heterogeneity of telomere length between alleles. Targeted deletion of allele-specific TVSs using CRISPR-Cas9 resets telomere length, further confirming their causal role in the control of allele-specific telomere maintenance. Continuous cell proliferation likely drives the slow but stochastic evolution of allele-specific TVSs distribution, resulting in asymmetry in telomere inheritance from father and mother (p-value < 0.045). These results illustrate how the telomere inheritance may predict allele-specific vulnerability in telomere protection, providing a new paradigm for personalized telomere profiling in aging-related diseases.