2008/02/21 by Michael Kirwan, Tom Vulliamy, Richard Beswick +3 · 3 citations
Medicine · Biochemistry, Genetics and Molecular Biology · #Telomeres, Telomerase, and Senescence #Nuclear Structure and Function #DNA Repair Mechanisms
paper · pdf · doi:10.1111/j.1365-2141.2008.06991.x
Dyskeratosis congenita (DC) is an inherited multi-system disorder classically characterized by a triad of abnormal skin pigmentation, nail dystrophy and leucoplakia. Bone marrow (BM) failure is the principal cause of mortality and would have developed in the majority of patients by 30 years of age. DC has been shown to be linked to mutations in components of the telomerase complex including the nucleolar protein dyskerin, the RNA component (TERC) and the reverse transcriptase component (TERT) (Marrone and Dokal 2006). While reduced BM haematopoiesis and a reduction in the numbers of haematopoietic progenitors and their ability to expand have already been reported in several cases of DC (Colvin, et al 1984, Friedland, et al 1985, Marley, et al 1999, Marsh, et al 1992, Yamaguchi, et al 2005), to date no comparison of progenitors has been made between the different genetic subtypes. To this end, peripheral blood (PB) was obtained from nine patients with DC (seven males, two females; mean age 24 years) of different subtypes and eight healthy volunteers (seven males, one female; mean age 39 years) attending the Royal London Hospital with informed, written consent and approval from Research Ethics Committee (Table I). Within 6 h of venesection, mononuclear cells (MNCs) were separated using Lymphoprep, washed twice with phosphate-buffered saline, resuspended at 1·5 × 106 cells/ml in Iscove’s modified Dulbecco’s medium + 2% fetal calf serum and incubated in tissue culture plates for 2 h at 37°C in 5% CO2 humidified atmosphere to remove plastic-adherent cells. Non-adherent cells were plated at 105 MNCs/1·1 ml in methylcellulose-containing medium (StemCell Technologies 0435, London, UK) containing stem cell factor, granulocyte-macrophage colony-stimulating factor, interleukin-3, interleukin-6, granulocyte colony-stimulating-factor, erythropoietin and serum in 35-mm Petri dishes and incubated at 37°C in a 5% CO2 humidified atmosphere for 12 days. Progenitor colonies were identified and enumerated by light microscopy using visual identification guidelines set out by StemCell Technologies. For all statistical analyses, P-values <0·05 were considered to be significant. Overall, numbers of myeloid and erythroid progenitors were reduced in all DC patients (Fig 1A). However, the genotype of the patient correlated with the extent of reduction with DKC1 mutations resulting in approximately fivefold (P < 0·01), fourfold (P = 0·07) and twofold (P = 0·07) reductions in myeloid (granulocyte/macrophage colony-forming units; CFU-GM), late erythroid CFUs and early erythroid (erythroid burst-forming units; BFU-E) colonies respectively, while TERC mutations resulted in the production of almost no countable colonies. Only the CFU-GM colony counts for DKC1 mutant samples reached a statistically significant difference (P < 0·05) using a Mann–Whitney U-test, perhaps because of the small numbers. All P-values for TERC mutant samples were <0·05. The only TERT mutant sample available for analysis gave a count intermediate between that of the DKC1 and TERC mutants. Progenitor colony counts from the peripheral blood of patients with dyskeratosis congenita (DC) and healthy controls: A) Average granulocyte/macrophage colony-forming units (CFU-GM), erythroid-colony forming units (CFU-E) and erythroid burst-forming units (BFU-E) progenitor colony counts from 5 × 105 mononuclear cells from peripheral blood of normal, wild-type controls and DC patients with DKC1, TERC or TERT mutations. B) Percentage of primary CFU-GM colonies which when transferred to fresh medium gave rise to secondary colonies. C) The average number of secondary colonies produced from each primary CFU-GM colony when transferred to fresh medium. Dyskeratosis congenita is such a rare disorder that large numbers of patient samples will always be difficult to assemble (particularly where it is important to set up cultures within 12 h of venesection) and as such, the small numbers here prevent the drawing of rigorous conclusions. However, given that the patients were not selected on any other basis than they presented consecutively at the clinic, the trend is unlikely to be an experimental or statistical artefact. There is an apparent link between the critically low progenitor colonies from the PB of patients with TERC mutations and the disease phenotype – DC patients with TERC mutations tend to present initially with aplastic anaemia (AA). BM progenitors from AA patients have been shown to be particularly deficient in proliferation and potential for expansion (Martinez-Jaramillo, et al 2002) and in this context the link between progenitor count and clinical presentation is logical enough. However, the findings were surprising as the X-linked form of DC is generally considered to be more severe. Taking our cohort into consideration, though the patients with DKC1 mutations had relatively mild haematological deficiencies compared with those with TERC mutations, their mucocutaneous abnormalities were more severe. This dissociation might suggest that a telomerase defect is primarily responsible for the haematological aspects of the disorder while other functions associated with dyskerin (e.g. ribosome biogenesis) might play a greater role in development of mucocutaneous abnormalities. Looking more closely at the DKC1 mutant samples of the three progenitor colony types assayed here, the BFU-E colonies were the least reduced. As this colony type represents a more primitive erythroid lineage, this is consistent with the hypothesis that much of the DC phenotype is because of premature death or senescence of haematopoietic cells. More primitive cells might therefore have greater potential to expand before reaching a critical check-point such as critically short telomeres. To determine the ability of progenitor cells to self-renew, replica dishes were set up on the day of the initial colony count for colony replating assays. After 7 days incubation, primary myeloid colonies were picked and transferred to methylcellulose-containing medium in 96-well plates. Plates were incubated as above for a further 7 days before enumeration of secondary colonies. Interestingly, the proportion of primary colonies giving rise to secondary colonies was broadly similar for DKC1 mutations and wild-type controls (Fig 1B) as were the average numbers of secondary colonies produced by each primary colony (Fig 1C). For the single sample with a TERT mutation, both parameters were slightly below those for wild-type and DKC1 mutant samples. As TERC mutants produced few or no colonies, it was not possible to perform a reliable replating assay. The similar results between wild-type and DKC1 mutations are intriguing, suggesting that the potential for renewal of individual progenitors is not significantly reduced, only that there are either fewer progenitors present in the PB of DC patients or that fewer of those present are capable of initial expansion. This report highlights another differentiating factor among the various subtypes of DC, demonstrating a new link between genotype and phenotype in the disease and has implications for the diagnosis and treatment of DC patients dependent upon the causative mutation in each case. This work was supported by the Medical Research Council and The Wellcome Trust.