Correlation of Telomere Length in Blood, Buccal Cells, and Fibroblasts From Patients with Inherited Bone Marrow Failure Syndromes.

Patients with dyskeratosis congenita (DC), an inherited bone marrow failure syndrome (IBMFS), have defects in telomere biology. Measurement of telomere length (TL) by flow fluorescence in situ hybridization (FISH) in white blood cell (WBC) subsets is a very important diagnostic tool in DC. Quantitative PCR (Q-PCR) is a high-throughput method of TL measurement that is currently used in large epidemiologic studies. It measures telomere length as a ratio of TTAGGG repeat copy number to a single copy gene number (T/S), and is often used on DNA derived from blood or buccal cells. There are limited data on tissue-specific correlations of TL and on the comparability of studies using different methods. In order to better understand tissue TL variability, we evaluated intra-individual correlations of DNA extracted from frozen blood, fibroblasts, and buccal cells by Q-PCR; and flow-FISH TL in WBC subsets.

We studied 21 patients: 5 Diamond-Blackfan Anemia (DBA), 6 DC, 5 Fanconi anemia (FA), and 4 Shwachman-Diamond Syndrome (SDS), enrolled in the National Cancer Institute's IBMFS study who contributed blood, buccal cells, and fibroblasts, and also had WBC flow-FISH TL measured. Genomic DNA was extracted from either whole blood (n=4) or the WBC pellet remaining after ficoll separation (n=17, primarily granulocytes) by manual Gentra Puregene. DNA was isolated from fibroblasts and buccal cells by phenol-chloroform extraction. TL was measured by Q-PCR of DNA from the three tissue types, and by flow-FISH in WBC subsets (lymphocytes and cell types matched to the types of WBC used in DNA extraction). We used the Wilcoxon signed-rank test to compare the median ranks of paired TL, and Spearman rank correlation coefficient to measure the strength of the associations between these measurements.

TL in patients with DC was significantly (p<0.01) shorter than in patients with other IBMFS in all Q-PCR measurements of DNA from blood, fibroblasts, buccal cells; and flow-FISH WBC lymphocytes and matched cell types of WBC used in DNA extraction (86% were from granulocytes). Across all disorders, the median Q-PCR TL was longer in fibroblast and buccal cell DNA when compared with peripheral blood DNA (overall T/S ratio= 1.42 and 1.16 vs. 1.05, p=0.0002, 0.002, respectively). Although the absolute values varied, we observed in all IBMFS overall statistically significant (p≤0.001) intra-individual correlations in TL measured by Q-PCR in blood and fibroblast (r=0.67), blood and buccal cells (r=0.77), as well as fibroblast and buccal cells (r=0.67). When stratifying by disease subtype, statistically significant (p<0.05) correlations of Q-PCR in blood and buccal cells (r=0.9), and fibroblast and buccal cells (r=0.9) were observed in DC, and Q-PCR in blood and fibroblasts (r=1.0) in SDS. None of the Q-PCR tissue correlations reached statistical significance in FA or DBA. In addition, overall statistically significant (p≤0.01) correlations between TL in flow-FISH WBC subsets and Q-PCR in blood, fibroblast and buccal cells were observed. The correlations were driven mainly by the correlations with Q-PCR in blood and buccal cells in DC patients and with Q-PCR in fibroblasts in SDS patients.

Overall Q-PCR TL was correlated between blood, buccal cells and fibroblasts and in comparison with flow-FISH TL, but there was some variability within different IBMFS. The poor correlation between tissues in FA and DBA might reflect blood-specific TL attrition in response to bone marrow stress. Most important, the correlation profile observed in DC suggests that the heritability of TL in DC is tissue independent. Blood or tissue Q-PCR TL appear to be useful in identifying individuals with DC who are unable to provide a fresh blood sample (e.g. epidemiologic studies or patients after bone marrow transplantation), although larger studies are needed to confirm or refute these findings. When possible, flow-FISH TL in subsets of leukocytes remains the modality of choice for the diagnosis of DC.

Lansdorp:Repeat Diagnostics Inc.: Equity Ownership.