Distinct genome wide DNA methylation signatures due to pathogenic germline variants in DKC1 , TERC , and TINF2

Germline pathogenic variants (GPVs) in DKC1 , TERC , or TINF2 lead to defective telomere maintenance and cause the cancer-prone telomere biology disorders (TBDs). DNA methylation (DNAm) can contribute to disease expressivity and penetrance in TBDs; however, epigenetic signatures across patients with TBDs remain undefined. As telomere shortening is a hallmark of aging, TBDs also offer a framework to study its link to epigenetic aging. We sought to identify DNAm alterations and evaluate epigenetic aging in patients with TBDs. Genome-wide DNAm profiling was performed using the Illumina Infinium Methylation EPICv2 array on blood samples from individuals with heterozygous TERC (n=15) or TINF2 (n=13) GPVs, hemizygous DKC1 males (n=7), and 41 controls participating in our IRB-approved study (NCT00027274). Differentially methylated probe (DMP) analysis was conducted using limma , adjusting for age, sex, DNAm-derived cell type composition, and family structure (FDR < 0.05 and |Δbeta| ≥ 0.20); group differences were evaluated with mixed-effects regression models. Differentially methylated regions (DMRs) were identified with DMRcate (HMFDR < 0.05; |mean Δbeta| ≥ 0.10). Functional enrichment of DMP-associated genes was assessed via KEGG pathway analysis using missMethyl . Epigenetic aging was estimated using the DunedinPACE algorithm. We identified 499, 167, and 164 DMPs in TINF2 , DKC1 , and TERC , respectively. Hypermethylation was mainly found in OpenSea regions (59-79%), except for TERC , which showed enrichment in CpG islands (45%). Hypomethylated probes were largely OpenSea (≥90%) across all cohorts. Genomic mapping showed enrichment of hypermethylation on chromosome X in TERC carriers, whereas TINF2 and DKC1 showed widespread autosomal changes. We detected 403 DMRs in TINF2 , 158 in DKC1 , and 119 in TERC , predominantly within promoter-proximal (21-30%) and intronic (27-38%) segments. Promoter hypermethylation was most common in TERC and DKC1 , whereas TINF2 exhibited intronic hypermethylation (36%). Nine genes with promoter hypermethylation and five with hypomethylation were recurrently detected across all patients. Pathway analysis showed enrichment in hematopoietic lineage and calcium signaling, with gene-specific trends in immune ( DKC1 ), stem cell ( TERC ), and growth factor ( TINF2 ) pathways. Epigenetic aging was accelerated in all patients, most prominently in DKC1 (beta=0.225, p<0.001) and TINF2 (beta=0.190, p<0.001). We detected distinct yet convergent DNAm signatures across individuals with TBDs. Individuals with GPVs in TINF2 exhibited the most extensive methylation remodeling. All patients demonstrated accelerated epigenetic aging. These findings suggest that DNAm alterations accompany telomere dysfunction in TBDs and underscore the need for integrated multi-omic and longitudinal studies to clarify its mechanistic basis, tissue specificity, and clinical implications.