Comparison of Fluorescence in Situ Hybridization and Flow Cytometry (Flow-FISH) with Monochrome Multiplex Quantitative Polymerase Chain Reaction (MM-qPCR) for Telomere Length Screening in Adult Patients with Suspected Cryptic Dyskeratosis Congenita (DKC)

Introduction: Dyskeratosis Congenita (DKC) is caused by defective telomere maintenance, mostly due to mutations impacting on the functional activity of telomerase. Classical DKC is characterized by mucocutaneous features and bone marrow failure diagnosed during childhood. In contrast, the cryptic form of DKC often first manifests itself in adulthood and presents clinically with less characteristic manifestations. Correct identification of DKC patients is of utmost importance because of significant implications for the affected patients (treatment, toxicities) and her/his family (donor selection, genetic counselling). Accelerated shortening of telomere length (TL) in peripheral blood leucocytes (PBL) represents the functional read-out and consequently, clinically useful screening parameter of altered telomere function. Consequently, in cases without distinct genetics or clinical presentation, significantly shortened TL is frequently used as a disease-defining marker. TL in PBL of pediatric DKC patients typically is below the first percentile (<1%). However, the difference between the normal range and critically short telomeres decreases with age, thus complicating the diagnosis of DKC in adult patients. Consequently, the TL threshold below 10% of age-adjusted healthy controls is frequently recommended in adults. Currently, fluorescence in situ hybridization and flow cytometry (Flow-FISH), especially of the lymphocyte subpopulation, or monochrome multiplex quantitative polymerase chain reaction (MM-qPCR) are preferred techniques for TL assessment. In our study, we compared MM-qPCR to Flow-FISH regarding their ability to properly identify DKC patients.

Methods and Patients: 105 patients enrolled in the Aachen Telomeropathy Registry from 2015 to 2017 were analysed. Patient inclusion criteria were based on the clinical suspicion of cryptic DKC by the treating physician. Patient`s median age was 47.5 [range 2-88] years. Initial diagnoses were aplastic anemia/paroxysmal nocturnal hemoglobinuria (n=50), unexplained cytopenia (n=20), genetically confirmed DKC mutation/polymorphism or screening of family members (n=14) as well as other disorders (e.g. lung fibrosis, n=21). All patients underwent customized amplicon-based next-generation sequencing analysis (TERT, TERC, DKC1, NOP10, NHP2, USB1, CTC1, RTEL1, TIN2, TCAB1) on a Miseq sequencer. 16/105 patients were identified with a known DKC-related mutation (TERC n=6, TERT n=6, RTEL1 n=3, DKC1 n=1). Median age of the DKC patient cohort was 44.5 (range 21-77) years. Age adaption was performed by using healthy control and TL assessment of PB granulocytes and lymphocytes was carried out in parallel by Flow-FISH and MM-qPCR with all experimental samples assayed in triplicates

Results: We observed that TL measured by MM-qPCR correlates with lymphocyte and granulocyte TL measured by flow-FISH (R²=0.33 and R²=0.25, respectively; both p≤0.001). Assessment of lymphocyte TL by Flow-FISH led to values below the 1% or 10% threshold of normal individuals in 24% (n=26) or 49% (n=51) of the patients, respectively leading to a detection rate of 75% (n=12) or 88% (n=14) of the known DKC patients. In comparison, TL assessment by MM-qPCR resulted in 29% (n=31) or 74% (n=78) of the patients below the 1% or 10% percentile, respectively leading to a detection rate of known DKC cases of 68% (n=11) or 94% (n=15). Thus, the sensitivity of both techniques basically did not differ. When we used the 1% cut off, the specificity of flow-FISH (84%, n=75/89) and MM-qPCR (78%, n=69/89) again did not differ significantly (p=0.74). However, using the 10% threshold, specificity of MM-qPCR (29%, n=26/89) was significantly lower compared to flow-FISH (58%, n=52/89, p=0.02). Finally, the rate of false positive patients using flow-FISH was 13% (n=14) and 19% (n=20) for MM-qPCR for the 1% threshold. Using the 10% threshold, we observed a significantly higher rate of false positive patients using MM-qPCR with 60% (n=63/105) compared to Flow-FISH with 35% (n=37/105, p=0.04).

Conclusions: Depending on the applied threshold, the use of MM-qPCR as a screening and confirmatory method for telomere maintenance disorders was inferior in specificity and showed a significant higher rate of false positive results compared to flow-FISH. Our results suggest the use of flow FISH as the gold standard method of screening especially in adults with suspected cryptic DKC.