Germline mutations in POT1 are linked to familial cancer predisposition, and somatic POT1 mutations occur recurrently in tumors. These mutations promote oncogenesis by enabling aberrant telomere elongation. For inherited POT1 mutations, a critical question is the extent to which elongated telomeres are transmitted to the next generation from the POT1 carrier parent and whether the inherited hyper-elongated telomeres elevate cancer risk. Using a nanopore sequencing approach that provides haplotype-specific telomere length measurements, we examined telomere inheritance in families harboring POT1 mutations. We found that individuals preferentially inherit their longest telomeres from the carrier parent, consistent with extensive telomere elongation in the carrier germline, whereas their comparatively short telomeres originate from the non-carrier parent. Analysis of carrier and non-carrier siblings showed that both sets of parental telomeres are longer in POT1 carriers, yet the shortest non-carrier-derived telomeres undergo disproportionately greater elongation than those inherited from the carrier parent. This identifies a mechanism of genetic anticipation in which the inheritance of long telomeres from one parent drives excessive extension of shorter telomeres. These findings demonstrate that telomere length inherited from both parents jointly defines the telomere-based tumor suppressor mechanism.

Human telomeres play critical roles in protecting chromosome ends and preserving genomic integrity. Telomerase, essential for maintaining telomere length and cellular replicative capacity, is only expressed in a small subset of human cells: stem and progenitor populations. Conversely, most somatic cells' telomeres shorten with each cell division; this shortening provides a potent tumor suppressor mechanism. Thus, telomerase regulation shapes not only cellular life span and differentiation, but also the regenerative capacity and long-term integrity of tissues. Here, we review the current understanding of telomere length control and telomerase regulation in humans, from molecular interactions at chromosome ends to the tissue-specific variation of telomere length dynamics, drawing insight from pluripotent and adult stem cell populations, as well as telomerase dysregulation in cancer and telomere biology disorders.

Mutations in the shelterin protein POT1 are associated with diverse cancers and thought to drive carcinogenesis by impairing POT1's suppression of aberrant telomere elongation. To classify clinical variants of uncertain significance (VUSs) and identify cancer-driving loss-of-function mutations, we developed a locally haploid human stem cell system to evaluate >1900 POT1 mutations, including >600 VUSs. Unexpectedly, many validated familial cancer-associated POT1 (caPOT1) mutations are haplosufficient for cellular viability, indicating that some pathogenic alleles do not act through a loss-of-function mechanism. Instead, POT1's DNA damage response suppression and telomere length control are genetically separable. ATR inhibition enables isolation of frameshift mutants, demonstrating that the only essential function of POT1 is to repress ATR. Furthermore, comparison of caPOT1 and frameshift alleles reveals a class of caPOT1 mutations that elongate telomeres more rapidly than full loss-of-function alleles. This telomere length-promoting activity is independent from POT1's role in overhang sequestration and fill-in synthesis.

This protocol describes MULTI-Seq barcoding of hESCs and library preparation, it is based on McGinnis et. al. 2019. PMID: 31209384 and the 10x Genomics user guide “Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 with Feature Barcoding technology for CRISPR Screening” Protocol overview A. Oligonucleotides B. Sequencing Initial notes A list of reagents and relevant vendor information can be found in the table listed under the materials tab.