A unified nanopore-based approach for direct telomere sequencing and genetic diagnosis in inherited bone marrow failure syndromes

Abstract Introduction: Telomeres are repetitive sequences located at the ends of chromosomes. Impaired telomere maintenance in telomere biology disorders, including dyskeratosis congenita (DC), is caused by pathogenic alterations in several genes, such as DKC1, TINF2, TERC, and TERT, resulting in bone marrow failure and various systemic manifestations. Therefore, telomere length measurement is useful for the diagnosis of inherited bone marrow failure syndromes (IBMFSs), along with genetic testing. However, telomere length measurement and genetic analysis are performed through separate assays: telomere length is typically evaluated using flow cytometry with fluorescent in situ hybridization method or quantitative PCR, whereas genetic abnormalities are generally detected through next-generation sequencing approaches. Herein, we established an analytical pipeline for diagnosis of IBMFSs through simultaneous direct telomere sequencing and genetic analysis by targeted adaptive sampling long-read sequencing (TAS-LRS), a computational targeted sequencing technique using a nanopore sequencer. Methods: Ten peripheral blood or bone marrow samples were analyzed: two patients with DC, one with Fanconi anemia, six with idiopathic aplastic anemia, and one healthy participant (age, 1.4–33.5 years). DNA was mildly fragmented, targeting an N50 of ~15,000 bp. Our TAS-LRS pipeline consisted of two modules: direct telomere sequencing and genomic diagnosis of IBMFSs. The direct telomere sequencing module enriched telomeric/subtelomeric regions, then identified reads that were aligned to the subtelomeric regions of the T2T-CHM13 and also contained the canonical telomere repeat (TTAGGG) or telomere variant repeats (TVRs; TGAGGG, TCAGGG, and TTGGGG) in more than 80% of the contiguous sequence extending from the subtelomeric regions. The telomere lengths of these reads were calculated. The genomic diagnosis module enriched 188 genes associated with IBMFSs, and single-nucleotide variants, small insertions/deletions, structural variations, and copy number variations were identified. TAS-LRS was performed using a GridION sequencer and R10.4 flowcells (Oxford Nanopore Technologies, OX, UK). Results: Mean depth of on-target regions was 17.1× in median (10.6×–25.9×). N50 of on-target regions was 14,832 bp in median (13,328–18,132 bp) with no statistical significance between those with and without DC (average, 15,230 bp vs. 15,194 bp, P = 0.96). The direct telomere sequencing module revealed that estimated telomere length was significantly lower in samples with DC than those without DC (average, 3,856 vs. 6,249 bp, P < 1×10-15). Regarding telomere composition, the DC group exhibited a significantly lower proportion of canonical telomere repeats (average, 82.9% vs. 87.8%, P < 1×10-15) and a significantly higher proportion of TVRs (average, 8.2% vs. 6.1%, P = 1.0×10-6) compared to the non-DC group. These observations were consistent with the known localization of TVRs predominantly in the proximal regions of telomeres and the progressive shortening of telomeres from the distal ends. In the eight samples without DC, telomere length demonstrated an age-associated shortening trend, with a mild negative correlation (r = -0.59). The genomic diagnosis module identified a pathogenic variant of TINF2 in a patient with DC. Also, compound heterozygous variants of FANCG were detected in the patient with Fanconi anemia: a nonsense variant and a complex structural variation resulting in complete deletion of FANCG.Conclusions: This study suggested the validity of telomere length measurement and the capability to detect telomere shortening in patients with DC using the direct telomere sequencing module of our TAS-LRS pipeline. Furthermore, the genomic diagnosis module successfully identified pathogenic variants associated with IBMFSs, with the advantage of long-read sequencing particularly evident in detecting complex structural variations. Collectively, this study demonstrated the utility of TAS-LRS as a comprehensive diagnostic approach for IBMFS through simultaneous direct telomere sequencing and genetic analysis.