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Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3582-3592
By
From the Division of Haematology, Department of Cellular and
Molecular Sciences, St George's Hospital Medical School, London, UK.
Improved survival in aplastic anemia (AA) has shown a high incidence
of late clonal marrow disorders. To investigate whether accelerated
senescence of hematopoietic stem cells might underlie the
pathophysiology of myelodysplasia (MDS) or paroxysmal nocturnal hemoglobinuria (PNH) occurring as a late complication of AA, we studied
mean telomere length (TRF) in peripheral blood leukocytes from 79 patients with AA, Fanconi anemia, or PNH in comparison with normal
controls. TRF lengths in the patient group were significantly shorter
for age than normals (P < .0001). Telomere shortening was
apparent in both granulocyte and mononuclear cell fractions, suggesting
loss at the level of the hematopoietic stem cell. In patients with
acquired AA with persistent cytopenias (n = 40), there was
significant correlation between telomere loss and disease duration
(r =
THE PROGNOSIS OF patients with aplastic
anemia (AA) has been greatly improved by treatment with antilymphocyte
globulin (ALG).1-5 However, despite achieving red blood
cell and platelet transfusion independence, many treated patients still
show evidence of impaired hematopoiesis, in the form of persistent
peripheral blood cytopenias and macrocytosis.6-8 A residual
hematopoietic defect can also be inferred from in vitro bone marrow
culture results. Marrows from AA patients who have responded to
treatment may still display a relative deficiency of early progenitor
cells, affecting both total CD34+ cells9-11 and
the CD34+33 With prolonged survival has also come the increasing recognition that
AA patients with autologous marrow recovery are at significant risk of
developing late clonal marrow disorders.8,16-20 The
combined cumulative risk of developing either paroxysmal nocturnal
hemoglobinuria (PNH) or myelodysplastic syndrome (MDS) has been
calculated to be as high as 42% at 15 years after ALG.19
Although an association between AA and PNH has long been
recognised,21,22 to date it has not been possible to
predict which patients are at highest risk of developing secondary
PNH.6-7 By contrast, age has been identified as an
important prognostic factor in the development of secondary MDS, with
the risk of MDS increasing with age as a continuous
variable.23 This led us to hypothesise that accelerated senescence of a reduced population of hematopoietic stem cells could
contribute to the pathogenesis of secondary MDS in AA.
Hematopoietic cells, in common with other somatic
cells,24-30 show evidence of progressive telomere
shortening with age in vivo24,31,32 as well as in
vitro.32-34 Loss of telomeric DNA can therefore be used as
an indicator of hematopoietic aging. In addition, genetic instability
associated with age-related loss of telomeric DNA in hematopoietic stem
cells may be important in the pathogenesis of de novo
MDS,35 which is predominantly a disease of the
elderly.36 We therefore studied an otherwise unselected
group of patients with AA, Fanconi anemia (FA), or PNH, looking at
telomere length in DNA extracted from peripheral blood leukocytes.
We have found evidence for significant telomeric shortening in AA,
affecting both granulocyte and mononuclear cell fractions, and
therefore probably occurring at the level of the hematopoietic stem
cell. Our results suggest that the rate of telomere loss stabilizes
after complete hematological recovery, although progressive telomere
erosion probably continues in those patients who do not fully regain
normal hematopoiesis, with continuing neutropenia, thrombocytopenia,
and macrocytosis. According to the classic model of the role of
telomeres in replicative senescence and
immortalization,37-39 we would predict the subgroup of
patients in whom telomere length was approaching a critical point to be
at highest risk of developing MDS. In keeping with this, the AA
patients in our study with acquired cytogenetic abnormalities were
among those with the shortest telomere lengths. However, there was no
apparent association between telomere loss and the development of
secondary PNH. It is also unlikely that telomere erosion plays a direct
role in the pathogenesis of bone marrow failure in acquired AA; in all
but a small minority of patients in this study, the extent of telomere
loss did not approach that associated with replicative cell senescence
in in vitro cultures of somatic cells.29,30,38,40
Patients
Prevalence of late clonal complications.
Thirteen patients who had been PNH-negative at presentation had
acquired a secondary PNH clone, as shown by a positive Ham test, or by
demonstration of GPI-anchored protein deficiency. Only 1 developed
frank hemolysis, and in 2 cases evidence for PNH was transient, with
expression of GPI-anchored proteins subsequently reverting to normal.
Only 1 patient, an 8-year-old child with FA, had developed overt MDS at
the time of the study, in association with abnormal bone marrow
cytogenetics. However, a further 3 patients with normal cytogenetics at
presentation had developed an abnormal karyotype (2 acquired AA and 1 familial AA). The abnormality was transient in 1 case.
Normal Controls
Leukocyte Separation and DNA Extraction
Mean Terminal Restriction Fragment Length (TRF) Measurement Five-microgram aliquots of genomic DNA were digested to completion with RsaI (Promega) and resolved on 0.7% agarose gels, alongside 35S-labeled DNA size markers (Amersham UK, Little Chalfont, UK). Completeness of digestion was confirmed by ethidium bromide staining. Gels were blotted onto Hybond N+ filters (Amersham) and alkali-fixed according to the manufacturer's instructions. Filters were prehybridized in 5× SSC/4× Denhardt's/0.5% sodium dodecyl sulfate (SDS)/100 mg/mL herring sperm DNA at 48°C and then hybridized with 32P end-labeled (TTAGGG)4
telomere repeat probe in 5× SSC overnight at 48°C. Filters
were rinsed in 4× SSC at room temperature and then washed twice
for 10 minutes in 4× SSC/0.1% SDS at 48°C. Filters were
exposed to x-ray film (Hyperfilm MP; Amersham) for 24 to 72 hours. The
mean TRF was determined by densitometric analysis of autoradiographs
using ImageQuant software (Molecular Dynamics, Sunnyvale,
CA).25 Analysis was performed without
knowledge of the identity of the samples, and normals were included on
each gel to minimize the risk of bias and to confirm that results were comparable between different experiments, because there was usually insufficient sample to allow replicate TRF measurement.
Statistical Analysis Linear regression analysis was performed using Fig.P software (Biosoft, Cambridge, UK). The Mann-Whitney U test was used in the comparison of telomere loss and MCV between subgroups, and the Wilcoxon matched-pairs test was used to compare results from different cell fractions (Stat-100 software; Biosoft).
Telomere Shortening With Age in Normals and Patients As expected, there was progressive shortening of total leukocyte TRF with age in normals (Fig 1), equivalent to a loss of 36 bp per year (r = .838, P < .0001), consistent
with the rate of loss reported by others.24,31 TRF lengths
in the patient group, although also related to age, were significantly
shorter than normals (Fig 2), with 27 of 79 (34%) below
the normal 95% confidence interval for age (P < .0001).
Results from different subgroups of patients were then analyzed
separately to identify any factors that might predispose to telomere
loss in AA. TRF loss was expressed as the residual value between the
observed TRF and the regression estimate of TRF for age in normals
(TRFO-E), representing the difference in kilobases between
the measured TRF and the age-appropriate point on the normal regression
line (Fig 2). This facilitated comparison between normals and subgroups of patients of different ages by allowing for the normal decline of TRF
length with age.
Acquired AA This category excluded patients with FA or familial AA or those with de novo PNH without evidence for AA at presentation. Five patients who had successful allografts for AA were also considered separately, because such patients do not appear to be at the same risk of late clonal complications.17,23 However, this analysis did include 3 patients in whom allogeneic bone marrow transplantation (BMT) had resulted in graft rejection followed by autologous marrow recovery, as demonstrated by analysis of DNA polymorphisms (data not shown). Patients for whom hematological and clinical data were incomplete were also excluded from this analysis. There was a tight scatter of TRFO-E for normals (n = 60; mean, 0.069 kb; Fig 3). The TRFO-E values for the acquired aplastic anemia group showed a wider scatter, but were significantly lower than normals (n = 60; mean,0.780 kb; P < .0001). There was no apparent correlation between TRFO-E
and age, whether at presentation or at time of testing, or with severity at presentation and treatment received (data not shown).
Hematological status at time of TRF measurement.
Patients with acquired AA were subdivided into those who had achieved
complete remission (recovered AA; n = 20) and those who still had
active AA, as shown by persistent cytopenia (active AA; n = 40). In this study, complete remission was defined by transfusion
independence, with neutrophils greater than 1.8 × 109/L and platelets greater than 100 × 109/L.41 The recovered AA and active AA groups
differed significantly with respect to MCV, endorsing the selection of
these criteria. Median MCV for patients with active AA who were not red
blood cell transfusion-dependent was 108 fL (mean, 106.5 fL; n = 25), whereas the median MCV for the recovered AA group was 96 fL (mean, 95.5 fL; n = 20; P < .01). The mean TRFO-E was
equivalent for both groups ( Evidence for progressive TRF loss.
TRFO-E was plotted against time since diagnosis to
investigate at what stage TRF loss had occurred during the clinical
course. There was some evidence for progressive loss of TRF length when the whole acquired AA group was included (r =
TRF Loss After BMT Eight patients in the total group had been treated by BMT 4 to 174 months before this study. Three had failed to engraft and subsequently showed evidence of autologous marrow recovery. These are included in the acquired AA group considered above. Five patients had successfully engrafted with donor marrow after matched sibling donor BMT. For these 5 patients, the mean TRFO-E calculated on the basis of recipient age was1.07 kb (range, 2.28 to 0.14 kb) and
1.11 kb on the basis of donor age. There was no apparent
correlation with time since BMT, suggesting stable loss, but the
numbers are small.
TRF Loss and PNH PNH at presentation.
Three patients had hemolytic PNH at presentation, without evidence for
AA. TRFO-E ranged from Secondary PNH.
Thirteen patients within the study had become PNH-positive during the
course of their illness, having been negative at presentation. In all
but 1 case this represented the finding of a deficiency in GPI-anchored
proteins on FACS analysis, rather than hemolytic PNH. The median
interval between diagnosis and development of PNH was 34 months. Seven
patients with secondary PNH were still cytopenic at the time of TRF
measurement, whereas 6 had fully recovered blood counts at the time of
study. All 6 had achieved normal counts before the PNH clone was
detectable, including 1 patient who had autologous bone marrow recovery
after a failed allogeneic BMT. PNH did not develop in any of the 5 patients who had successfully engrafted after BMT. There was no
apparent association between the development of PNH positivity and age
or severity at diagnosis or previous therapy (data not shown). The 13 patients with late onset PNH are included in the recovered AA and
active AA subgroups analyzed above, but were also analyzed separately to determine whether TRF loss might be influenced by the development of
a PNH clone. AA patients with secondary PNH showed a similar pattern of
telomere loss to the whole acquired AA group. TRFO-E for
the 6 with recovered AA and secondary PNH ranged from FA The study included 6 patients with FA from 4 families, including 2 sibling pairs. Both sibling pairs showed telomere loss that was more pronounced in the older child, consistent with progressive TRF loss with age. One pair had TRFO-E values of1.50 and
1.21 kb aged 15 and 5 years, respectively, and the other had
TRFO-E of 2.24 and 1.95 kb aged 6 and 4 years, respectively. However, the other 2 FA patients had TRF values
within the normal range. TRFO-E was 0.55 kb in a girl 8 years of age and 0.18 kb in another 18 years of age, despite both
having progressed to end-stage bone marrow failure and the 8-year-old
having subsequently developed MDS.
Non-Fanconi Familial AA Significant telomere loss was apparent in 2 patients who gave a history of AA in a first-degree relative, but who tested negative for FA on induced breakage studies. One had TRFO-E of4.13 kb
(15 years of age) and the other had TRFO-E of 3.11
kb (36 years of age).
TRF Loss and Risk of MDS An association between TRF loss and MDS in AA is suggested by the finding that, of the 5 patients in this study with TRF length less than 5.0 kb, 3 (2 with acquired AA and 1 with familial AA) had acquired an abnormal karyotype, although none had developed morphological evidence of MDS at the time of testing. The cytogenetic abnormality was transient in 1 patient and was not subsequently detected. The 2 patients with acquired, nonfamilial AA were both in the group with residual cytopenia (active AA), in which there was evidence for progressive telomere loss. Serial cytogenetic data were not available for every patient, reflecting the difficulties inherent in marrow chromosome studies in the presence of profound marrow hypoplasia. However, cytogenetic abnormalities were detected in a further 2 cases, neither of whom had excessive telomere loss (Table 2). One patient had pericentric inv(9), detected at presentation, which probably represents a normal polymorphic variant, seen in up to 1% of white caucasians.42 The other patient, who had underlying FA, did have frank MDS, with a hypercellular marrow and trilineage dysplasia, but no excess of blasts.
TRF Loss at the Level of the Hematopoietic Stem Cell The above-reported data refer to TRF length measurement in total leukocyte DNA, extracted from blood samples containing differing proportions of granulocytes and lymphocytes. In some cases, the granulocytes represented less than 10% of the samples studied. TRF loss is therefore likely to have occurred in both myeloid and lymphoid cells, consistent with loss at the level of the hematopoietic stem cell. To investigate this further, DNA was extracted from peripheral blood mononuclear cells (MNC) and granulocytes separated on density gradients. In normals (n = 6), there was no significant difference between the TRF lengths (P = .345) measured in granulocyte and mononuclear cell fractions (Fig 5), consistent with previous reports.43
We have found evidence for significant telomere shortening in a wide
cross-section of patients with AA of varying disease severity and
duration. Moreover, we have shown that there is evidence for
progressive telomere erosion in patients with ongoing disease. Our
results suggest that a proportion of the excessive telomere loss occurs
at the level of the hematopoietic stem cell, in that there was evidence
for telomere shortening affecting both granulocyte and mononuclear cell
fractions, although it was more pronounced in granulocytes. This
discrepancy between the different cell fractions could be in part
attributable to the longer half life of lymphocytes or to differing
regulation of telomere length between cell
types.
Submitted January 21, 1998;
accepted March 3, 1998.
The authors thank Drs Rose Ann Padua and Tim Rutherford for helpful
discussion.
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Abstract
Introduction
Abstract
subset.
) show a progressive shortening of TRF with age. The regression line
for normals is shown as a solid line, and the 95% confidence limits
are shown as broken lines (r = 
) TRF
values for patients (n = 79).
; n = 20), mean TRFO-E = 
