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Accelerated telomere shortening in young recipients of allogeneic bone-marrow transplants
EARLY REPORT
Early Report
Accelerated telomere shortening in young recipients of allogeneic bone-marrow transplants
Robert F Wynn, Michael A Cross, Claire Hatton, Andrew M Will, Linda S Lashford, T Michael Dexter, Nydia G Testa
Summary Background The establishment of donor-derived haemopoiesis in the recipients of allogeneic bone-marrow transplants (BMT) involves extensive proliferation of haemopoietic stem cells. The biological consequences of this replicative stress are ill defined, but any “ageing” effect would carry the risk of an increased frequency of clonal disorders during later life. We compared blood-cell mean telomere lengths in donor/recipient pairs. Methods Mean telomere length was calculated by in-gel hybridisation to leucocyte DNA from 56 normal individuals aged 0–96 years, and from 14 consecutive BMT recipients (aged 2–14 years) plus their respective donors (aged 2–46 years). Engraftment was confirmed by variable numbers of tandem repeats (VNTR) or gender analysis. Findings On average, blood-cell telomeres of transplant recipients were 0·4 kb (95% CI ⫺0·2 to ⫺0·6) shorter than those of their respective donors. This degree of telomere loss is equivalent to a median of 15 years’ (range 0–40) ageing in the healthy controls. Interpretation The kinetics of haemopoietic engraftment impose replicative stress on the haemopoietic stem cells, resulting in a pronounced ageing effect, which may be sufficient to accelerate the onset of clonal haemopoietic disorders usually associated with later life. Monitoring of haemopoietic status in BMT recipients as time since BMT increases will be important. Assessment of transplant protocols under development in terms of their effects on telomere shortening is also indicated.
Lancet 1998; 351: 178–81 See Commentary page XXX
Cancer Research Campaign Department of Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust (R F Wynn MRCP, M A Cross PhD, L S Lashford PhD, T M Dexter PhD, N G Testa PhD); and Willink Biochemical Genetics Unit (C Hatton BSc) and Department of Paediatric Haematology and Oncology, Royal Manchester Children’s Hospital (R Wynn, A M Will MRCP, L S Lashford), Manchester, UK Correspondence to: Dr Robert F Wynn, CRC Department of Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, UK
178
Introduction The widespread use of intensive cytotoxic treatment and bone-marrow transplantation (BMT) since the 1970s has greatly increased life expectancy for many cancer patients. However, the longer survival has revealed a high frequency of secondary neoplasms in this group compared with age-matched controls. 1,2 DNA damage accumulating during treatment with mutagenic agents, such as etoposide and alkylating agents, may be one of the main factors contributing to the genesis of secondary malignant disorders in patients undergoing autologous BMT. However, there is also some evidence of an unusually high frequency of secondary leukaemias in the recipients of allogeneic (normal donor) BMT, which suggests the involvement of factors other than therapeutic history and conditioning. 2,3 Since the frequency of clonal disorders of the blood in the general population increases greatly with age, 4 one possibility is that the reconstitution of donor-derived haemopoiesis in the recipient is associated with accelerated “ageing” of the progeny of the transplanted cells. Although the molecular basis of ageing is not clear, reductions in telomere length are known to play an important part. For example, progressive telomeric shortening in explanted primary fibroblasts leads ultimately to replicative senescence. 5 In the blood, as in other replenishing tissues, the mean telomere length of both early progenitors6 and mature cells7–9 decreases with age. This finding suggests either that the mechanisms for maintenance of telomeres in the stem and progenitor cells become less effective, or that the replicative stresses imposed on individual stem cells increase throughout life. Rates of telomere length reduction do depend on replicative stresses in at least some tissues, since the reduction in mean telomere length in vascular endothelial cells from areas of turbulent flow is greater than that in cells from areas of linear flow.10 These observations suggest that the replicative stresses imposed on a reconstituting haemopoietic stem cell might result in detectable telomere shortening in BMT patients. Furthermore, telomere shortening is associated not only with replicative senescence5 but also with chromosomal instability.11,12 Thus, it may not simply reflect ageing, but may also contribute directly to the incidence of clonal disorders. For these reasons we set out to establish whether allogeneic BMT and reconstitution is associated with a significant loss of telomere length, particularly in young recipients with a lifetime of haemopoietic demand before them.
THE LANCET • Vol 351 • January 17, 1998
EARLY REPORT
gel since there is substantial variation in results between gels. In addition, duplicate or triplicate samples of eight pairs were repeated on separate gels with consistent results. Three lanes of each gel were loaded with a radiolabelled 3 kb DNA ladder. The gel was dried stringently in a vacuum drier without heat for 1 h, and then at 65°C for 45 min. The dried gel was denatured (1·5 mol/L sodium chloride, 0·5 mol/L sodium hydroxide) for 20 min then neutralised (1 mol/L Tris-HCl, 1·5 mol/L sodium chloride) for 15 min, and rinsed briefly in water. The gel was hybridised to a phosphorus-32-labelled 5'-(CCCTAA)3 telomeric probe in 500 mmol/L Na2HPO4, 1% bovine serum albumin, 7% sodium dodecyl sulphate, and 10 mmol/L edetic acid overnight at 37°C. After three highstringency washes in 0·1⫻SSC (15 mmol/L sodium chloride, 1·5 mmol/L sodium citrate) at room temperature, the gel was exposed to a PhosphorImager plate (Molecular Dynamics, Amersham, UK). The position of the size markers was used to calculate the distances migrated in the gel by fragments of known molecular weight. The lanes occupied by the DNA samples were divided into 30 regions of equal size between 3 kb and 18 kb by means of ImageQuant software (Molecular Dynamics) and the molecular weight of each of these regions calculated from the inverse logarithmic relation between molecular weight and distance migrated and the known position of the size markers. The telomeric DNA smear was quantified and the signal recorded for each of these regions (optical density). The mean telomere length was then calculated as the sum of (optical density⫻molecular weight) divided by the sum of optical densities.13 Use of another equation that takes account of increased probe binding at higher molecular weights (telomere restriction fragment=sum of optical densities divided by the sum of [optical density⫼molecular weight]) changed absolute telomere length slightly but made no difference to the relative differences in length between samples. The same sample when run in different lanes of the same gel yielded the same mean telomere length, although there is substantial variation in the mean telomere length of the same sample run on different gels. Variable number of tandem repeats (VNTR) analysis was done by PCR amplification of three VNTR loci—Apo-B,14 DlS80,15 and COL2Al.16 Reactions were carried out in 0·5 mL Eppendorf tubes with 100 µmol/L of each dNTP, 1⫻Supertaq buffer, 0·2 U Supertaq (HT Biotechnology, Cambridge, UK), 40 pmoles/tube primers, and 0·1 µg DNA template in a total volume of 50 µL. The reactions were overlaid with paraffin oil and the tubes were placed in a Hybaid (Teddington, Middlesex, UK) Omnigene Thermocycler for amplification by modifications of temperature cycling conditions previously described;14–16 Apo-B, DlS80, and COL2Al annealing
12
Mean telomere length (kb)
11 10 9 8 7 6 5 0
20
40 60 Age (years)
80
100
Figure 1: Mean telomere length versus age in normal individuals
Methods Blood was taken from 14 consecutive young (aged 2–14 years) recipients of BMT who attended a follow-up clinic after engraftment. Blood was also taken from their family donors at the same time, according to a protocol approved by the local research ethics committee. After red-cell lysis, the white-cell pellet was resuspended and digested in 10 mmol/L Tris-HCl (pH 8·0), 1 mmol/L edetic acid, 100 mmol/L sodium chloride, 1% sodium dodecyl sulphate, and 0·1 mg/mL proteinase K at 37°C overnight or at 55°C for 2 h. Unfractionated leucocytes were used because of the small volume of many of the samples and because other investigators have shown that T and B lymphocytes and neutrophils have similar telomere lengths and show similar changes with ageing.9 After one extraction with phenol, one with phenol/chloroform (1/1), and one with chloroform, DNA was precipitated, washed, and resuspended in 10 mmol/L Tris-HCl and 1 mmol/L edetic acid. The concentration of genomic DNA was measured spectrophotometrically and its integrity by agarose-gel electrophoresis. 15 µg genomic DNA was digested to completion with 30 U Rsa1 and Hinf1. 5 µg samples (measured by fluorimetry) were loaded onto 0·5% agarose gels and separated by electrophoresis at 60 V overnight. Digests from donor/recipient pairs were run on adjacent lanes of the same Transplant reference number 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Recipient*
Donor
Age (years)
Sex
Age (years)
Sex
14 10 11 12 5 7 7 10 11 4 2 10 11 4
M M M M F M M M F M F F M M
12 5 7 11 1·5 31 5 8 8 2 9 9 9 46
F F F F F F F F F F M M F M
Relation to recipient
Sister Sister Sister Sister Sister Mother Sister Sister Sister Sister Brother‡ Brother‡ Sister Grandfather
Recipient
Mean telomere length (kb) Donor
Difference in telomere length between donor and recipient (kb)
Time since BMT (months)
Degree of chimerism
9·1 9·0 8·4 8·0 9·4 7·5 8·5 7·9 9·0 8·9 8·3 8·2 8·5 8·1
10·1 9·4 8·6 8·5 10·3 7·5 8·6 8·0 9·3 9·7 8·5 8·5 8·6 7·8
⫺1·0 ⫺0·4 ⫺0·2 ⫺0·5 ⫺0·9 ⫺0 ⫺0·1 ⫺0·1 ⫺0·3 ⫺0·8 ⫺0·2 ⫺0·3 ⫺0·1 0·3
54 17 11 27 5 76 5 6 11 10 4 82 40 42
Complete† Complete† Complete† Complete Complete† Complete† Complete Complete† Complete† Complete Complete Complete† Complete† Mixed§†
*Diagnosis: aplasia patient 1; acute lymphocytic leukaemia patients 2, 7, 9, and 10; myelodysplastic syndrome patient 3; acute myelogenous leukaemia patients 4 and 5; Hurler’s syndrome patient 6; Diamond-Blackfan anaemia patient 8; thalassaemia patients 11 and 12; osteopetrosis patient 14. †Engraftment established by VNTR; otherwise by cytogenetics. ‡Same brother donor to two thalassaemic sisters. §50/50 donor recipient mixed chimera.
Donor and recipient characteristics
THE LANCET • Vol 351 • January 17, 1998
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EARLY REPORT
Figure 2: Example of in-gel hybridisation to measure mean telomere length M=3 kb DNA ladder (sizes in kb indicated on right); D=donor; R=recipient (numbers correspond to transplant reference numbers shown in table). R 11 and R12 received marrow from a single donor, assayed twice as D 11 and D12. CB=cord blood. (Lane AA is a sample from a patient with aplastic anaemia who is not included in this study.)
temperatures were 53°C, 67°C, and 58°C, respectively. Electrophoresis of the amplified DNA was done on 1·5% agarose gels, and the amplification products were visualised directly after staining with ethidium bromide.
Results Peripheral-blood samples were obtained from 14 consecutive attenders at the clinic, who had received allogeneic BMT 4–82 months previously (median 11 months) from family members (table). Samples were taken at the same time from their respective donors. VNTR analysis was informative for ten recipients and confirmed complete engraftment in nine cases. Recipient 14 showed a chimeric mixture of donor and recipient cells and was excluded from further analysis. For the four cases in which VNTR analysis was uninformative, confirmation of complete engraftment by gender analysis was possible. Blood samples were also collected from 56 normal individuals ranging in age from newborn infants (cord blood) to 96 years. These individuals had no abnormality of their blood counts and were attending hospital for elective surgery or an acute illness. Mean telomere length in these samples showed a net decrease with age of 27 bp per year, on the assumption of a constant rate of loss throughout life (figure 1). Our observations accord closely with published data7–9 both in terms of the rate of telomere loss and the greater degree of variability in samples taken from younger individuals.9 Measurement of telomere lengths in the transplant pairs revealed a significant net decrease in telomere length in the recipients compared with their respective donors (two-tailed paired t test, p<0·002). On average, the blood-cell telomeres of the recipients were 0·4 kb (95% CI ⫺0·2 to ⫺0·6) shorter than those of their respective donors (table, figure 2).
the haemopoietic cells of young patients who have received allogeneic BMT. Comparison both with published data7–9 and with our own confirmatory observations shows that the average reduction is equivalent to roughly 15 years’ ageing (with the assumption of a constant rate of loss in the normal population), but it approaches 40 years in some patients. If telomere shortening does reflect ageing in the haemopoietic system, this degree of loss from young transplant patients might well affect the frequency and age of onset of clonal disorders during later life. Of particular relevance are disorders such as myelodysplasia, the frequency of which increases 100 fold between the fourth and seventh decades of life. 4 A definitive conclusion about the relevance of the telomere shortening reported here to the genesis of late effects will be possible only after monitoring for secondary effects over a long period of large enough numbers of patients with known telomere length reductions at transplantation. The variability in the degree of telomere loss associated with allogeneic transplantation may be encouraging, since it suggests that there may be scope for interventions to decrease the effect. To this end, identification of the stage at which the telomere loss occurs will be interesting. If the decrease in telomere length results purely from increased replicative demand on engrafted stem cells that are essentially normal, use of a larger number of repopulating cells should be beneficial. Our results support the observation of telomeric shortening during ex-vivo stem-cell expansion despite the up-regulation of telomerase.17 These findings raise the question of whether current procedures used to harvest, manipulate, and transplant haemopoietic cells, as well as more innovative techniques such as ex-vivo expansion, are the best to maintain reproductive integrity of the repopulating cells.
Discussion
Contributors
Our results indicate accelerated telomeric reduction in
Robert Wynn carried out the laboratory work for telomere length
180
THE LANCET • Vol 351 • January 17, 1998
EARLY REPORT calculation and did the data analysis. Michael Cross supervised the laboratory work for telomere length calculation and reviewed the raw data and its analysis. Claire Hatton carried out the VNTR data analysis. Andrew Will prepared and stored pretransplant DNA from patients and donors used to establish informative post-transplant VNTR loci, took samples, and critically assessed the clinical importance of the data. Linda Lashford was involved in design of the study, data interpretation, and discussion. Michael Dexter was responsible for establishment of the study and critical evaluation. Nydia Testa was responsible for study design and evaluation of results.
Acknowledgments We thank O B Eden and Mike Hawkins for helpful discussion, S N Jowett for assistance in the provision of normal donor blood, and R F Stevens for permission to study some of his patients. RFW is the Margaret Dobson Trust senior registrar in paediatric haematology; TMD is a CRC Gibb research fellow. This work was supported by grants from the Margaret Dobson Trust and the Cancer Research Campaign of Great Britain.
7
8
9
10
11 12 13
References 1 2
3 4 5 6
Curtis RE, Rowlings PA, Deeg HJ, et al. Solid cancers after bone marrow transplantation. N Engl J Med 1997; 336: 897–905. Witherspoon RP, Fisher LID, Schoch G, et al. Secondary cancers after bone marrow transplantation for leukemia or aplastic anemia. N Engl J Med 1989; 321: 784–89. Brown SA, Bashey A, Schey SA. Donor cell leukaemia—an unresolved problem. Eur J Haematol 1995; 54: 98–99. Oscier DG. Myelodysplastic syndromes. Baillieres Clin Haematol 1987; 1: 389–426. Allsopp RC, Harley CB. Evidence for a critical telomere length in senescent human fibroblasts. Exp Cell Res 1995; 219: 130–36. Vaziri H, Dragowska W, Allsopp RC, Thomas TE, Harley CB, Lansdorp PM. Evidence for a mitotic clock in human hematopoietic
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14
15
16
17
stem cells: loss of telomeric DNA with age. Proc Natl Acad Sci USA 1994; 91: 9857–60. Hastie ND, Dempster M, Dunlop MG, Thompson AM, Green DK, Allshire RC. Telomere reduction in human colorectal carcinoma and with ageing. Nature 1990; 346: 866–68. Slagboom PE, Droog S, Boomsma DI. Genetic determination of telomere size in humans: a twin study of three age groups. Am J Hum Genet 1994; 55: 876–82. Vaziri H, Schachter F, Uchida I, et al. Loss of telomeric DNA during aging of normal and trisomy 21 human lymphocytes. Am J Hum Genet 1993; 52: 661–67. Chang E, Harley CB. Telomere length and replicative aging in human vascular tissues. Proc Natl Acad Sci USA 1995; 92: 11 190–94. Lundblad V, Szostak JW. A mutant with a defect in telomere elongation leads to senescence in yeast. Cell 1989; 57: 633–43. Metcalfe JA, Parkhill J, Campbell L, et al. Accelerated telomere shortening in ataxia telangiectasia. Nat Genet 1996; 13: 350–53. Kruk PA, Rampino NJ, Bohr VA. DNA damage and repair in telomeres: relation to aging. Proc Natl Acad Sci USA 1995; 92: 258–62. Boerwinkle E, Xiong WJ, Fourest E, Chan L. Rapid typing of tandemly repeated hypervariable loci by the polymerase chain reaction: application to the apolipoprotein B 3' hypervariable region. Proc Natl Acad Sci USA 1989; 86: 212–16. Kasai K, Nakamura Y, White R. Amplification of a variable number of tandem repeats (VNTR) locus (pMCT118) by the polymerase chain reaction (PCR) and its application to forensic science. J Forensic Sci 1990; 35: 1196–200. Priestley L, Kumar D, Sykes B. Amplification of the COL2A1 3' variable region used for segregation analysis in a family with the Stickler syndrome. Hum Genet 1990; 85: 525–26. Engelhardt M, Kumar R, Ablanell J, Pettengell R, Wei Han, Moore MAS. Telomerase regulation, cell cycle and telomere stability in primitive haemopoietic cells. Blood 1997; 90: 182–93.
181
Early Report
Accelerated telomere shortening in young recipients of allogeneic bone-marrow transplants
Robert F Wynn, Michael A Cross, Claire Hatton, Andrew M Will, Linda S Lashford, T Michael Dexter, Nydia G Testa
Summary Background The establishment of donor-derived haemopoiesis in the recipients of allogeneic bone-marrow transplants (BMT) involves extensive proliferation of haemopoietic stem cells. The biological consequences of this replicative stress are ill defined, but any “ageing” effect would carry the risk of an increased frequency of clonal disorders during later life. We compared blood-cell mean telomere lengths in donor/recipient pairs. Methods Mean telomere length was calculated by in-gel hybridisation to leucocyte DNA from 56 normal individuals aged 0–96 years, and from 14 consecutive BMT recipients (aged 2–14 years) plus their respective donors (aged 2–46 years). Engraftment was confirmed by variable numbers of tandem repeats (VNTR) or gender analysis. Findings On average, blood-cell telomeres of transplant recipients were 0·4 kb (95% CI ⫺0·2 to ⫺0·6) shorter than those of their respective donors. This degree of telomere loss is equivalent to a median of 15 years’ (range 0–40) ageing in the healthy controls. Interpretation The kinetics of haemopoietic engraftment impose replicative stress on the haemopoietic stem cells, resulting in a pronounced ageing effect, which may be sufficient to accelerate the onset of clonal haemopoietic disorders usually associated with later life. Monitoring of haemopoietic status in BMT recipients as time since BMT increases will be important. Assessment of transplant protocols under development in terms of their effects on telomere shortening is also indicated.
Lancet 1998; 351: 178–81 See Commentary page XXX
Cancer Research Campaign Department of Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust (R F Wynn MRCP, M A Cross PhD, L S Lashford PhD, T M Dexter PhD, N G Testa PhD); and Willink Biochemical Genetics Unit (C Hatton BSc) and Department of Paediatric Haematology and Oncology, Royal Manchester Children’s Hospital (R Wynn, A M Will MRCP, L S Lashford), Manchester, UK Correspondence to: Dr Robert F Wynn, CRC Department of Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, UK
178
Introduction The widespread use of intensive cytotoxic treatment and bone-marrow transplantation (BMT) since the 1970s has greatly increased life expectancy for many cancer patients. However, the longer survival has revealed a high frequency of secondary neoplasms in this group compared with age-matched controls. 1,2 DNA damage accumulating during treatment with mutagenic agents, such as etoposide and alkylating agents, may be one of the main factors contributing to the genesis of secondary malignant disorders in patients undergoing autologous BMT. However, there is also some evidence of an unusually high frequency of secondary leukaemias in the recipients of allogeneic (normal donor) BMT, which suggests the involvement of factors other than therapeutic history and conditioning. 2,3 Since the frequency of clonal disorders of the blood in the general population increases greatly with age, 4 one possibility is that the reconstitution of donor-derived haemopoiesis in the recipient is associated with accelerated “ageing” of the progeny of the transplanted cells. Although the molecular basis of ageing is not clear, reductions in telomere length are known to play an important part. For example, progressive telomeric shortening in explanted primary fibroblasts leads ultimately to replicative senescence. 5 In the blood, as in other replenishing tissues, the mean telomere length of both early progenitors6 and mature cells7–9 decreases with age. This finding suggests either that the mechanisms for maintenance of telomeres in the stem and progenitor cells become less effective, or that the replicative stresses imposed on individual stem cells increase throughout life. Rates of telomere length reduction do depend on replicative stresses in at least some tissues, since the reduction in mean telomere length in vascular endothelial cells from areas of turbulent flow is greater than that in cells from areas of linear flow.10 These observations suggest that the replicative stresses imposed on a reconstituting haemopoietic stem cell might result in detectable telomere shortening in BMT patients. Furthermore, telomere shortening is associated not only with replicative senescence5 but also with chromosomal instability.11,12 Thus, it may not simply reflect ageing, but may also contribute directly to the incidence of clonal disorders. For these reasons we set out to establish whether allogeneic BMT and reconstitution is associated with a significant loss of telomere length, particularly in young recipients with a lifetime of haemopoietic demand before them.
THE LANCET • Vol 351 • January 17, 1998
EARLY REPORT
gel since there is substantial variation in results between gels. In addition, duplicate or triplicate samples of eight pairs were repeated on separate gels with consistent results. Three lanes of each gel were loaded with a radiolabelled 3 kb DNA ladder. The gel was dried stringently in a vacuum drier without heat for 1 h, and then at 65°C for 45 min. The dried gel was denatured (1·5 mol/L sodium chloride, 0·5 mol/L sodium hydroxide) for 20 min then neutralised (1 mol/L Tris-HCl, 1·5 mol/L sodium chloride) for 15 min, and rinsed briefly in water. The gel was hybridised to a phosphorus-32-labelled 5'-(CCCTAA)3 telomeric probe in 500 mmol/L Na2HPO4, 1% bovine serum albumin, 7% sodium dodecyl sulphate, and 10 mmol/L edetic acid overnight at 37°C. After three highstringency washes in 0·1⫻SSC (15 mmol/L sodium chloride, 1·5 mmol/L sodium citrate) at room temperature, the gel was exposed to a PhosphorImager plate (Molecular Dynamics, Amersham, UK). The position of the size markers was used to calculate the distances migrated in the gel by fragments of known molecular weight. The lanes occupied by the DNA samples were divided into 30 regions of equal size between 3 kb and 18 kb by means of ImageQuant software (Molecular Dynamics) and the molecular weight of each of these regions calculated from the inverse logarithmic relation between molecular weight and distance migrated and the known position of the size markers. The telomeric DNA smear was quantified and the signal recorded for each of these regions (optical density). The mean telomere length was then calculated as the sum of (optical density⫻molecular weight) divided by the sum of optical densities.13 Use of another equation that takes account of increased probe binding at higher molecular weights (telomere restriction fragment=sum of optical densities divided by the sum of [optical density⫼molecular weight]) changed absolute telomere length slightly but made no difference to the relative differences in length between samples. The same sample when run in different lanes of the same gel yielded the same mean telomere length, although there is substantial variation in the mean telomere length of the same sample run on different gels. Variable number of tandem repeats (VNTR) analysis was done by PCR amplification of three VNTR loci—Apo-B,14 DlS80,15 and COL2Al.16 Reactions were carried out in 0·5 mL Eppendorf tubes with 100 µmol/L of each dNTP, 1⫻Supertaq buffer, 0·2 U Supertaq (HT Biotechnology, Cambridge, UK), 40 pmoles/tube primers, and 0·1 µg DNA template in a total volume of 50 µL. The reactions were overlaid with paraffin oil and the tubes were placed in a Hybaid (Teddington, Middlesex, UK) Omnigene Thermocycler for amplification by modifications of temperature cycling conditions previously described;14–16 Apo-B, DlS80, and COL2Al annealing
12
Mean telomere length (kb)
11 10 9 8 7 6 5 0
20
40 60 Age (years)
80
100
Figure 1: Mean telomere length versus age in normal individuals
Methods Blood was taken from 14 consecutive young (aged 2–14 years) recipients of BMT who attended a follow-up clinic after engraftment. Blood was also taken from their family donors at the same time, according to a protocol approved by the local research ethics committee. After red-cell lysis, the white-cell pellet was resuspended and digested in 10 mmol/L Tris-HCl (pH 8·0), 1 mmol/L edetic acid, 100 mmol/L sodium chloride, 1% sodium dodecyl sulphate, and 0·1 mg/mL proteinase K at 37°C overnight or at 55°C for 2 h. Unfractionated leucocytes were used because of the small volume of many of the samples and because other investigators have shown that T and B lymphocytes and neutrophils have similar telomere lengths and show similar changes with ageing.9 After one extraction with phenol, one with phenol/chloroform (1/1), and one with chloroform, DNA was precipitated, washed, and resuspended in 10 mmol/L Tris-HCl and 1 mmol/L edetic acid. The concentration of genomic DNA was measured spectrophotometrically and its integrity by agarose-gel electrophoresis. 15 µg genomic DNA was digested to completion with 30 U Rsa1 and Hinf1. 5 µg samples (measured by fluorimetry) were loaded onto 0·5% agarose gels and separated by electrophoresis at 60 V overnight. Digests from donor/recipient pairs were run on adjacent lanes of the same Transplant reference number 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Recipient*
Donor
Age (years)
Sex
Age (years)
Sex
14 10 11 12 5 7 7 10 11 4 2 10 11 4
M M M M F M M M F M F F M M
12 5 7 11 1·5 31 5 8 8 2 9 9 9 46
F F F F F F F F F F M M F M
Relation to recipient
Sister Sister Sister Sister Sister Mother Sister Sister Sister Sister Brother‡ Brother‡ Sister Grandfather
Recipient
Mean telomere length (kb) Donor
Difference in telomere length between donor and recipient (kb)
Time since BMT (months)
Degree of chimerism
9·1 9·0 8·4 8·0 9·4 7·5 8·5 7·9 9·0 8·9 8·3 8·2 8·5 8·1
10·1 9·4 8·6 8·5 10·3 7·5 8·6 8·0 9·3 9·7 8·5 8·5 8·6 7·8
⫺1·0 ⫺0·4 ⫺0·2 ⫺0·5 ⫺0·9 ⫺0 ⫺0·1 ⫺0·1 ⫺0·3 ⫺0·8 ⫺0·2 ⫺0·3 ⫺0·1 0·3
54 17 11 27 5 76 5 6 11 10 4 82 40 42
Complete† Complete† Complete† Complete Complete† Complete† Complete Complete† Complete† Complete Complete Complete† Complete† Mixed§†
*Diagnosis: aplasia patient 1; acute lymphocytic leukaemia patients 2, 7, 9, and 10; myelodysplastic syndrome patient 3; acute myelogenous leukaemia patients 4 and 5; Hurler’s syndrome patient 6; Diamond-Blackfan anaemia patient 8; thalassaemia patients 11 and 12; osteopetrosis patient 14. †Engraftment established by VNTR; otherwise by cytogenetics. ‡Same brother donor to two thalassaemic sisters. §50/50 donor recipient mixed chimera.
Donor and recipient characteristics
THE LANCET • Vol 351 • January 17, 1998
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EARLY REPORT
Figure 2: Example of in-gel hybridisation to measure mean telomere length M=3 kb DNA ladder (sizes in kb indicated on right); D=donor; R=recipient (numbers correspond to transplant reference numbers shown in table). R 11 and R12 received marrow from a single donor, assayed twice as D 11 and D12. CB=cord blood. (Lane AA is a sample from a patient with aplastic anaemia who is not included in this study.)
temperatures were 53°C, 67°C, and 58°C, respectively. Electrophoresis of the amplified DNA was done on 1·5% agarose gels, and the amplification products were visualised directly after staining with ethidium bromide.
Results Peripheral-blood samples were obtained from 14 consecutive attenders at the clinic, who had received allogeneic BMT 4–82 months previously (median 11 months) from family members (table). Samples were taken at the same time from their respective donors. VNTR analysis was informative for ten recipients and confirmed complete engraftment in nine cases. Recipient 14 showed a chimeric mixture of donor and recipient cells and was excluded from further analysis. For the four cases in which VNTR analysis was uninformative, confirmation of complete engraftment by gender analysis was possible. Blood samples were also collected from 56 normal individuals ranging in age from newborn infants (cord blood) to 96 years. These individuals had no abnormality of their blood counts and were attending hospital for elective surgery or an acute illness. Mean telomere length in these samples showed a net decrease with age of 27 bp per year, on the assumption of a constant rate of loss throughout life (figure 1). Our observations accord closely with published data7–9 both in terms of the rate of telomere loss and the greater degree of variability in samples taken from younger individuals.9 Measurement of telomere lengths in the transplant pairs revealed a significant net decrease in telomere length in the recipients compared with their respective donors (two-tailed paired t test, p<0·002). On average, the blood-cell telomeres of the recipients were 0·4 kb (95% CI ⫺0·2 to ⫺0·6) shorter than those of their respective donors (table, figure 2).
the haemopoietic cells of young patients who have received allogeneic BMT. Comparison both with published data7–9 and with our own confirmatory observations shows that the average reduction is equivalent to roughly 15 years’ ageing (with the assumption of a constant rate of loss in the normal population), but it approaches 40 years in some patients. If telomere shortening does reflect ageing in the haemopoietic system, this degree of loss from young transplant patients might well affect the frequency and age of onset of clonal disorders during later life. Of particular relevance are disorders such as myelodysplasia, the frequency of which increases 100 fold between the fourth and seventh decades of life. 4 A definitive conclusion about the relevance of the telomere shortening reported here to the genesis of late effects will be possible only after monitoring for secondary effects over a long period of large enough numbers of patients with known telomere length reductions at transplantation. The variability in the degree of telomere loss associated with allogeneic transplantation may be encouraging, since it suggests that there may be scope for interventions to decrease the effect. To this end, identification of the stage at which the telomere loss occurs will be interesting. If the decrease in telomere length results purely from increased replicative demand on engrafted stem cells that are essentially normal, use of a larger number of repopulating cells should be beneficial. Our results support the observation of telomeric shortening during ex-vivo stem-cell expansion despite the up-regulation of telomerase.17 These findings raise the question of whether current procedures used to harvest, manipulate, and transplant haemopoietic cells, as well as more innovative techniques such as ex-vivo expansion, are the best to maintain reproductive integrity of the repopulating cells.
Discussion
Contributors
Our results indicate accelerated telomeric reduction in
Robert Wynn carried out the laboratory work for telomere length
180
THE LANCET • Vol 351 • January 17, 1998
EARLY REPORT calculation and did the data analysis. Michael Cross supervised the laboratory work for telomere length calculation and reviewed the raw data and its analysis. Claire Hatton carried out the VNTR data analysis. Andrew Will prepared and stored pretransplant DNA from patients and donors used to establish informative post-transplant VNTR loci, took samples, and critically assessed the clinical importance of the data. Linda Lashford was involved in design of the study, data interpretation, and discussion. Michael Dexter was responsible for establishment of the study and critical evaluation. Nydia Testa was responsible for study design and evaluation of results.
Acknowledgments We thank O B Eden and Mike Hawkins for helpful discussion, S N Jowett for assistance in the provision of normal donor blood, and R F Stevens for permission to study some of his patients. RFW is the Margaret Dobson Trust senior registrar in paediatric haematology; TMD is a CRC Gibb research fellow. This work was supported by grants from the Margaret Dobson Trust and the Cancer Research Campaign of Great Britain.
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