Comparison of flow‐FISH and MM–qPCR telomere length assessment techniques for the screening of telomeropathies

Abstract

Assessment of telomere length (TL) in peripheral blood leukocytes is part of the diagnostic algorithm applied to patients with acquired bone marrow failure syndromes (BMFSs) and dyskeratosis congenita (DKC). Monochrome multiplex-quantitative polymerase chain reaction (MM-qPCR) and fluorescence in situ hybridization (flow-FISH) are methodologies available for TL screening. Dependent on TL expressed in relation to percentiles of healthy controls, further genetic testing for inherited mutations in telomere maintenance genes is recommended. However, the correct threshold to trigger this genetic workup is still under debate. Here, we prospectively compared MM-qPCR and flow-FISH regarding their capacity for accurate identification of DKC patients. All patients (n = 105) underwent genetic testing by next-generation sequencing and in 16 patients, mutations in DKC-relevant genes were identified. Whole leukocyte TL of patients measured by MM-qPCR was found to be moderately correlated with lymphocyte TL measured by flow-FISH (r 2 = 0.34; P < 0.0001). The sensitivity of both methods was high, but the specificity of MM-qPCR (29%) was significantly lower compared with flow-FISH (58%). These results suggest that MM-qPCR of peripheral blood cells is inferior to flow-FISH for clinical routine screening for suspected DKC in adult patients with BMFS due to lower specificity and a higher rate of false-positive results.

Comparable Effects of the Androgen Derivatives Danazol, Oxymetholone and Nandrolone on Telomerase Activity in Human Primary Hematopoietic Cells from Patients with Dyskeratosis Congenita

“…TL qPCR was performed using the Absolute Human Telomere Length Quantification qPCR Assay Kit (ScienCell, Carlsbad, CA, USA) and FastStart Essential DNA Green Master (Roche, Basel, Switzerland). Leukocytes from healthy subjects ( n = 104) were used for age adaptation of TL, which is given in T/S ratios [ 26 ]. A T/S ratio is calculated by dividing the number of copies of the telomere template (T) by the single copy reference (SCR) template (S) which is an amplified 100 bp-long region on human chromosome 17.…”

“… Telomere length of four patients analyzed in comparison to telomere length distributions derived from healthy individuals. Telomere length was assessed with flow-FISH as previously described [ 24 , 25 , 26 ]. ( A ) Lymphocyte telomere length ( B ) Granulocyte telomere length.…”

PRDM8 reveals aberrant DNA methylation in aging syndromes and is relevant for hematopoietic and neuronal differentiation

“…All DKC patients revealed lymphocyte telomere length below 1% percentile and diagnosis was complemented by clinical and genotypic characteristics [ 12 , 58 ]. All AA and DKC samples were screened by NGS using a self-customized gene panel containing the entire coding sequence of genes that are known to play a relevant role in DKC ( CTC1 , DKC1 , NHP2 , NOP10 , RTEL1 , TERC , TERT , TCAB1 , USB1 , and exon 6 of TINF2 , which is a known hot spot region) [ 12 ]. Library preparation and sequencing were performed with the TruSeq Custom Amplicon Kit and the MiSeq Reagent Kit v2 (all from Illumina) using a MiSeq Illumina platform.…”

“…Of note, in acquired AA, telomere length in the granulocyte compartment can also show substantial attrition and was shown to reflect the degree of autoimmune-mediated damage to the hematopoietic stem cell compartment [ 11 ]. This may hamper correct diagnosis of DKC [ 12 ], which is of utmost importance since DKC, in contrast to acquired AA, is not responding to immunosuppressive therapy and requires optimized conditioning protocols in case of allogeneic stem cell transplantation [ 13 ].…”

Molecular Mechanisms Contributing to Mesenchymal Stromal Cell Aging

“…Southern blotting [170] Flow FISH [194] Real-time PCR [194] STELA [195] Telomere attrition is directly correlated to replicative senescence, but it also occurs after exposure to oxidative damage. The subpopulation heterogeneity must be taken into account and may be addressed with emerging techniques such as STELA, detecting individual telomere IHC, IF, ELISA [192], HPLC [192] [190] Senescent cells at low density express a lysosomal β-galactosidase active at pH 6.0, detectable either with activity assays or with a specific antibody.…”