From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
Acquired somatic ATRX mutations in myelodysplastic syndrome associated with
␣ thalassemia (ATMDS) convey a more severe hematologic phenotype than
germline ATRX mutations
David P. Steensma, Douglas R. Higgs, Chris A. Fisher, and Richard J. Gibbons
Acquired somatic mutations in ATRX, an
X-linked gene encoding a chromatinassociated protein, were recently identified in 4 patients with the rare subtype of
myelodysplastic syndrome (MDS) associated with ␣ thalassemia (ATMDS). Here
we describe a series of novel point mutations in ATRX detected in archival DNA
samples from marrow and/or blood of
patients with ATMDS by use of denaturing
high-performance liquid chromatography
(DHPLC), a technique sensitive to low-
level mosaicism. Two of the new mutations result in changes in amino acids
altered in previously described pedigrees
with germ line ATRX mutations (ATR-X
syndrome), but the hematologic abnormalities were much more severe in the
patients with ATMDS than in the corresponding constitutional cases. In one
ATMDS case where DNA samples from
several time points were available, the
proportion of ATRX-mutant subclones
correlated with changes in the amount of
hemoglobin H. This study strengthens
the link between acquired, somatic ATRX
mutations and ATMDS, illustrates how
molecular defects associated with MDS
and other hematologic malignancies
masked by somatic mosaicism may be
detected by DHPLC, and shows that additional factors increase the severity of the
hematologic phenotype of ATRX mutations in ATMDS. (Blood. 2004;103:2019-2026)
© 2004 by The American Society of Hematology
Introduction
Mutations affecting the structure and/or synthesis of hemoglobin
(HbA, ␣2␤2) are most commonly observed in individuals from
tropical and subtropical regions of the world.1-3 Sporadic, inherited
mutations are occasionally found in individual families located
outside of these areas.1 Perturbed patterns of hemoglobin production associated with abnormal erythropoiesis have also been
detected as acquired abnormalities in patients with hematologic
malignancy.4 For example, changes in the normal levels of fetal
hemoglobin (HbF, ␣2␥2) and hemoglobin A2 (HbA2, ␣2␦2) may
occur in acute leukemia and chronic myeloid disorders.5-7 In
addition, there have been a number of reports describing ␣ and ␤
thalassemia as newly acquired traits in the context of hematologic
malignancy.8-11 Acquired ␣ thalassemia due to down-regulation of
␣ globin synthesis is most easily recognized by demonstrating
tetramers of excess ␤ globin chains (␤4, known as hemoglobin H
[HbH]) in the peripheral blood, either by hemoglobin electrophoresis or supravital staining.12 When HbH is detected for the first time
in an individual with a hematologic malignancy, the condition is
often referred to as “acquired HbH disease.”9
Acquired HbH disease has been most commonly reported in
elderly northern European men who develop myelodysplastic
syndrome (MDS) or, less often, other neoplastic myeloid disorders;
this constellation is now referred to as ␣ thalassemia–myelodysplastic syndrome (ATMDS; Mendelian Inheritance in Man [MIM]
catalog n␱. 300448).9 The peripheral blood film in such patients is
characterized by the presence of strikingly hypochromic, micro-
cytic, and anisopoikilocytic red cells (Figure 1A), often intermingled with a variable proportion of apparently normal red cells,
suggesting the coexistence of normal and abnormal hematopoietic
clones. Stratification of red cell precursors in individual ATMDS
cases has revealed a wide distribution of globin chain imbalance,
with ␣/␤ globin chain synthesis ratios ranging from less than 0.1 to
1.0 (normal 0.9-1.2), indicating the presence of normal and
abnormal clones contributing to erythropoiesis. Consistent with
this, in some patients with ATMDS, the proportion of HbH waxes
and wanes during the course of the illness and sometimes
disappears entirely, especially during transformation to overt acute
leukemia. This observation presumably reflects somatic mosaicism
and clonal evolution in MDS.9
The common, inherited forms of ␣ thalassemia result from
deletions of one, 2, 3, or all 4 of the normal ␣ globin genes (normal:
␣␣/␣␣; thalassemic: –␣/␣␣,⫺/␣␣,-␣/-␣,⫺/–␣, or ⫺/⫺) and less
commonly from point mutations in the structural genes.13 In rare
families, deletions of a remote critical regulatory region (HS-40)
have been identified as a cause of ␣ thalassemia.14 In addition,
constitutional mutations in the gene encoding ATRX, an X-linked,
chromatin-associated factor, have been shown to cause a rare
condition (ATR-X syndrome, MIM no. 301040) where mild ␣
thalassemia (usually ⬍ 10% HbH) is found in boys with severe
mental retardation, facial dysmorphism, and urogenital abnormalities.15 In ATMDS, despite the presence of the thalassemic phenotype, extensive mapping and sequence analysis have revealed no
From the Medical Research Council (MRC) Molecular Haematology Unit,
Weatherall Institute of Molecular Medicine, University of Oxford, Headington,
Oxford, United Kingdom; and the Divison of Hematology, Department of
Medicine, Mayo Clinic, Rochester, MN.
the Mayo Foundation, Rochester, MN.
Submitted September 30, 2003; accepted October 24, 2003. Prepublished online as
Blood First Edition Paper, October 30, 2003; DOI 10.1182/blood-2003-09-3360.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported by the Medical Research Council in the United Kingdom (R.J.G.,
C.A.F., D.R.H.). D.P.S. is a Mayo Foundation Research Scholar supported by
BLOOD, 15 MARCH 2004 䡠 VOLUME 103, NUMBER 6
Reprints: David P. Steensma, MRC Molecular Haematology Unit, Weatherall Institute
of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Headington,
Oxford OX3 9DS, United Kingdom; e-mail: [email protected].
© 2004 by The American Society of Hematology
2019
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2020
BLOOD, 15 MARCH 2004 䡠 VOLUME 103, NUMBER 6
STEENSMA et al
Figure 1. ATMDS peripheral blood findings. (A) Peripheral blood smear (hematoxylin-eosin; original magnification, ⫻ 400) from ATMDS patient no. 12 (Table 1) demonstrates a typical dimorphic red cell picture with severe
hypochromia, microcytosis, and occasional poorly hemoglobinized “ghost cells.” (B) A hemoglobin H preparation
(brilliant cresyl blue; original magnification, ⫻ 400) from
patient no. 4 at time point 1 reveals many classic “golf ball
cells” (arrow) with intracellular hemoglobin H inclusions.
mutations in the ␣ globin cluster.9 Similarly, we have found no
gross rearrangements in the ATRX gene in ATMDS (D.P.S., D.R.H.,
C.A.F., and R.J.G., unpublished data). However, it was recently
shown that acquired, somatic point mutations in the ATRX gene
(20 ⫹ 1G⬎A in the exon 1–intron 1 canonical splice donor site,
and 236C⬎G; S79X) were present in myeloid but not lymphoid
cells in 2 patients with ATMDS; ATRX mRNA splicing abnormalities were observed in 2 other patients with ATMDS in whom the
causative genomic mutation remains unknown.16
The 2 ATMDS-associated ATRX mutations and 2 ATRX splicing
abnormalities described to date have never been reported in ATR-X
syndrome. One possible reason might be that the ATRX mutations
associated with the much more severe hematopoietic impairment
typical of ATMDS—where the number of HbH-containing erythrocytes usually ranges from 10% to 90% and the ␣/␤ globin chain
synthesis ratio is often less than 0.1—are lethal during development when inherited. However, here we report 8 new ATMDSassociated acquired ATRX mutations and an additional ATRX base
pair change of uncertain significance detected by denaturing
high-performance liquid chromatography (DHPLC). This series
includes one mutation identical to a previously described germ line
ATRX mutation and another mutation that alters the same amino
acid as a constitutional ATRX mutation. In addition, several of the
newly characterized mutations result in the loss of highly conserved domains of the ATRX protein that are also lost in constitutional pedigrees. In each case, the ATMDS hematologic findings
are more severely abnormal than in the previously described cases
where the ATRX mutation was inherited rather than acquired. These
findings expand the spectrum of acquired ATRX mutations and
demonstrate that these are the primary mutations leading to ␣
thalassemia in ATMDS. However, these observations also show
that additional factors in the abnormal erythroid precursors of
patients with ATMDS must contribute to the severe defect in ␣
globin synthesis.
polymerase chain reaction (PCR); reagents included GeneAmp PCR Buffer
II (Applied Biosystems, Foster City, CA), MgCl2 (concentration 1.5 mM to
3 mM depending on the specific amplicon; Applied Biosystems), dNTPs
(200 ␮M; Roche, Mannheim, Germany), forward and reverse primers (list
available on request; final concentration 0.8 ␮M; Invitrogen, Paisley,
United Kingdom), 100 ng template DNA, and a 5:1 ratio of AmpliTaq Gold
DNA polymerase (total 1 U; Applied Biosystems) to Pwo DNA polymerase
(total 0.2 U; Roche). Pwo polymerase was included to provide 3⬘-5⬘
exonuclease activity and thereby increase replication fidelity and improve
DHPLC performance characteristics. Amplicons were designed to cover the
entire 5⬘ untranslated region, protein coding region, canonical splice donor
and splice acceptor sites, and the 3⬘ polyadenylation consensus signal of
ATRX. Total reaction volume was 50 ␮L. Reactions were carried out on a
PTC-200 Peltier Thermocycler (MJ Research, Waltham, MA). Amplification conditions for each amplicon are available on request.
Patients, materials, and methods
Subcloning and sequencing
DNA, polymerase chain reaction for DHPLC, and patients
Subcloning was performed using the pGEM-T Easy Vector System
(Promega; Madison, WI) and XL1-Blue competent cells (Stratagene, La
Jolla, CA) or DH5␣ cells (Invitrogen). Because the Pwo polymerase results
in amplicons with blunt ends, A-tailing was performed to facilitate insertion
into this plasmid vector, which has single 3⬘-T overhangs at either end of the
insertion site. For A-tailing, the PCR product was first purified using either
PEG-NaOAc and 95% ethanol or a QIAquick PCR Purification Kit
(Qiagen, Crawley, United Kingdom), then 2 ␮L purified fragment was
added to 1 ␮L 10 X Taq DNA polymerase reaction buffer with MgCl2
(Roche), dATP (200 ␮M; Roche), Taq DNA polymerase (5 U; Roche), and
6 ␮L dH2O. This mixture was incubated at 70°C for 30 minutes, and 2 ␮L of
the reaction product was used in the ligation reaction. The ligation product
All specimens were collected and analyzed with approval from patients
(parents/legal guardians in the case of minor children) and with permission
of the relevant institutional ethics committees. Hematologic data at the time
of sample acquisition (samples were obtained between 1978 and 2002)
from the 18 adult patients with chronic myeloid disorders analyzed to date
are summarized in Table 1.
Genomic DNA was extracted from heparin-anticoagulated peripheral
blood and/or bone marrow via either a phenol-chloroform method or a
resin-based DNA extraction kit (Nucleon BACC2; Nucleon Biosciences,
Coatbridge, United Kingdom). DNA amplification was performed by
DHPLC
PCR-amplified samples were transferred directly into a skirted 96-well
plate (Thermo Life Sciences, Basingstoke, United Kingdom) and warmed
to 95°C, then cooled to room temperature over approximately 1 hour to
promote heteroduplex formation. A wild-type DNA control was included
for each amplicon. DNA from patients with known or suspected ATRX
constitutional mutations (ie, pure mutant DNA from patients with ATR-X
syndrome) was mixed with equal amounts of wild-type DNA before
heteroduplexing, as was DNA from suspected new cases of ATR-X
syndrome. However, DNA from patients with ATMDS was not mixed, in
view of the tissue DNA admixture in unfractionated blood and marrow
characteristic of chronic myeloid disorders. For DHPLC analysis, we used
the WAVE 3500HT DNA Fragment Analysis System (Transgenomic,
Omaha, NE). Briefly, 5 ␮L of each DNA sample was injected into a
high-throughput DNASep column and eluted over 4 minutes through a 260-nM
photodetector, with concentrations of buffer A (ie, 0.1 M triethylammonium
acetate [TEAA]; Transgenomic) and buffer B (ie, 0.1 M TEAA and 25%
acetonitrile in ultrapure water) adjusted automatically as calculated by the
Navigator software package (Transgenomic). All samples were run at a
minimum of 2 different oven temperatures, including at least one temperature for which the Navigator software predicted each segment of the exonic
component of the amplicon would be under partially denaturing conditions.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 15 MARCH 2004 䡠 VOLUME 103, NUMBER 6
ACQUIRED SOMATIC ATRX MUTATIONS IN ATMDS
2021
Table 1. Hematology parameters and ATRX mutations detected to date in patients with ATMDS
Patient number
(Oxford ATMDS
Registry
database no.)
Patient age in
years, sex, clinical
diagnosis
1 (O51)
60, M, MDS (RA)
4
0
NA
69.8
ND; ⫺14G⬎A (5⬘UTR) polymorphism
2 (O65)
70, M, CMD NOS
16
5.2
NA
62
20⫹1G⬎A (exon 1–intron 1 consensus splice
3 (O45)
60, M, MDS (RA)
35
8.7
0.11
67
236C⬎G, S79X16
% HbHcontaining
cells by BCB
% HbH by
electrophoresis
␣/␤ globin
synthesis
ratio
MCV, fL
ATRX mutation cDNA; predicted protein
abnormality if applicable (description)
donor site16)
4 (O27)
76, M, CMD NOS
50, ⬍ 1†
5 (O46)
70, M, MDS (RA)
48
25, ⬍ 1†
8.4
6 (O54)
83, M, MDS (RAEB)
75
37.6
0.09
69.3
794G⬎A; C265Y
7 (O58)
73, M, MDS (RA)
16*
0*
0.58*
82*
2692G⬎C; D898H‡
8 (O53)
76, M, MDS (RAEB)
25
NA
68.4
Splicing abnormality: exclusion of exon 16,
NA
0.09, 1.02†
0.09
65, 78†
576G⬎C; L192F
80.4
718T⬎G; C240G
inclusion of segment from intron 16 (see
Gibbons et al16 for details)
9 (O61)
67, M, MDS (RAEB)
60*
15*
0.49*
66*
Splicing abnormality: inclusion of segment from
10 (O29)
86, M, MDS
14*
10*
0.29*
82*
5567-3C⬎G (intron 23–exon 24 consensus splice
11 (O50)
79, M, MDS (RA)
10*
8*
NA
82.6*
6083G⬎C; R2028P
12 (O42)
71, M, MDS (RA)
40
16.9
0.09
59.8
6884_6885insT; 2293Tfs2319X
13 (O49)
60, M, MDS (RA)
25
13
0.07
69.4
7014_7017del; N2340fsX2341
14 (O26)
61, M, MMM
0.08*
92*
7049delT; E2351fsX2353
15 (O41)
87, F, ET
0.62
65
ND
16 (O43)
68, M, MDS (RA)
6.6*
0*
0.60*
88.6*
ND
17 (O69)
69, M, MDS (RA)
0.5
0
NA
72
ND
18 (O68)
68, M, MDS NOS
0.1
0
NA
72.9
ND
intron 18 (see Gibbons et al16 for details)
(?RARS)
acceptor site)
5*
20
0*
NA
Patients no. 2, 3, 8, and 9 were described in Gibbons et al16; all other ATRX mutations are newly described here. The precise gDNA mutation responsible for the splicing
abnormality detected in cDNA from patients no. 8 and no. 9 is currently unknown.
HbH indicates hemoglobin H; MDS, myelodysplastic syndrome; BCB, brilliant cresyl blue supravital stain; MCV, mean corpuscular volume (normal, 81-99 fL); NA, data not
available or assay not performed; ND, no pathologic mutation detected; cDNA, complementary deoxyribonucleic acid; ET, essential thrombocythemia; MMM, myelofibrosis
with myeloid metaplasia; CMD NOS, chronic myeloid disorder not otherwise specific; MDS NOS, myelodysplastic syndrome not otherwise specified; RA, refractory anemia;
RAEB, refractory anemia with excess blasts; RARS, refractory anemia with ringed sideroblasts (French-American-British Co-operative Group Classification was used to
describe MDS subtypes, as most predated the recent reclassification by the World Health Organization); and UTR, untranslated region. ? indicates diagnostic uncertainty.
*Patient had been transfused with packed red cells prior to obtaining sample, and HbH level, MCV, and globin synthesis ratio may reflect admixture of patient and donor
cells.
†Samples were obtained at 2 time points for patient no. 4 (see “Sensitivity of DHPLC for detection of mutations in the ATRX gene”).
‡Base pair change of uncertain significance, possible polymorphism (see “Comparison of acquired and germ line ATRX mutations”).
was transformed into competent cells according to a heat-shock protocol
recommended by the manufacturer. After transformation, cells were grown
overnight at 37°C in 5% CO2 on duplicate LB/Ampicillin/IPTG/X-Gal
(Sigma and Bio101, Carlsbad, CA) plates; white colonies were harvested
and grown in 3 mL SOC medium at 37°C in 5% CO2 for 18 hours. Plasmid
DNA was extracted using QIAprep Spin Miniprep Kit (Qiagen). The
presence of inserts of appropriate length in the extracted plasmids was
confirmed by digesting at 37°C for 1 hour using EcoRI restriction
endonuclease (Roche) in SuRE/Cut Buffer (Roche); digests were electrophoresed through a 1% agarose gel stained with ethidium bromide and
visualized with a phosphoimager.
Sequencing of subclones bearing inserts of the proper length was
performed using the ABI PRISM BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and the ABI PRISM 3100 Genetic Analyzer
(Applied Biosystems). The generated sequence data were analyzed using
the Sequencher v4.1.2 software package (Gene Codes, Ann Arbor, MI).
Mutations and polymorphisms are described here using the nomenclature
and numbering scheme suggested by den Dunnen and Antonarakis.17
Although all the ATMDS mutations in this study were detected at the
genomic DNA (gDNA) level, they are described in terms of cDNA for the
sake of clarity because of the lack of a consensus numbering system for
ATRX gDNA.
Hemoglobin H and globin chain analysis
Fresh peripheral blood was incubated for 4 hours with 1% brilliant cresyl
blue (Merck, Poole, United Kingdom) in 0.9% NaCl, smeared on a glass
slide and examined for intraerythrocytic hemoglobin H inclusions.
Hemoglobin subtype analysis was performed on an automated highperformance liquid chromatography unit (Variant Hemoglobin Testing
System; Bio-Rad, Hercules, CA) using a protocol recommended by the
manufacturer.
Measurement of globin chain synthesis was performed according to the
method of Weatherall et al.18,19 Briefly, an enriched reticulocyte preparation
derived from fresh heparinized marrow was resuspended in 2.5% fetal calf
serum and RPMI 1640 without leucine (Gibco BRL, Grand Island, NY),
incubated for 1 hour at 37°C with 50 ␮Ci (1.85 MBq) [3H]leucine
(Amersham) and ferrous ammonium sulfate (Sigma), then cooled and
washed to eliminate unincorporated radiolabeled amino acid. Cells were
then lysed with distilled water. Globin chains were precipitated with 2%
HCl in acetone with trace amounts of ␤-mercaptoethanol (Sigma) and
separated by Whatman CM-23 cellulose column chromatography using a
urea-phosphate buffer gradient at pH 6.4 in the presence of dithiothreitol
(Sigma). Absorbance of collected eluted fractions was measured at 280 nM
on a spectrophotometer, and aliquot radioactivity was quantified on a
scintillation counter.
Results
Following the identification of ATRX mutations or splicing abnormalities in 4 patients with ATMDS as described,16 further investigation was hampered by the rarity of the syndrome, the mixed
cellularity of archival samples, and the admixture of neoplastic
clonal and residual normal polyclonal progenitors that is characteristic of MDS. To overcome the latter 2 limitations, we developed a
screening protocol to search for mutations in the relatively large
(10.1 kb mRNA) ATRX gene using DHPLC.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2022
STEENSMA et al
BLOOD, 15 MARCH 2004 䡠 VOLUME 103, NUMBER 6
Figure 2. Sensitivity of DHPLC for mosaic mutation detection. DHPLC
tracing of the following mixtures of abnormal (purified neutrophil) DNA from
ATMDS patient no. 2 with a 20 ⫹ 1G⬎A ATRX mutation and normal,
wild-type ATRX DNA: 10% mutation-enriched DNA (from the patient’s
purified peripheral blood granulocytes) with 90% wild-type (cyan), 5%
mutant DNA with 95% wild-type (green), 2.5% mutant DNA with 97.5%
wild-type (red), and 100% wild-type DNA (purple). The left peak or
“shoulder” represents DNA heteroduplexes, the larger central peak represents homoduplexes. Even 2.5% abnormal DNA is clearly differentiated
from the pure wild-type DNA. If the peripheral blood granulocytes from the
patient with ATMDS included the progeny of any residual normal clones
(ie, if the granulocyte DNA was not 100% ATRX mutant), the actual mutant
DNA would be even less than 2.5% and the sensitivity correspondingly
greater.
Sensitivity of DHPLC for detection of mutations
in the ATRX gene
To test the sensitivity of DHPLC for ATRX mutation detection in
the setting of somatic mosaicism, DNA derived from the purified
peripheral blood neutrophils of a previously described16 patient
with ATMDS with an ATRX mutation in the canonical splice donor
site at the exon 1–intron 1 boundary (20 ⫹ 1G⬎A) was mixed with
wild-type DNA in varying proportions. As expected, with either
100% mutant DNA (derived from the patient’s purified peripheral
blood granulocytes) or 100% wild-type DNA alone, no heteroduplex formation was detected, and DHPLC analysis of the amplicon
containing exon 1 revealed only a single narrow homoduplex peak.
However, for all of the mixtures tested (containing 80%, 60%,
40%, 20%, 10%, 5%, and 2.5% mutant granulocyte DNA) either a
distinct second heteroduplex peak or an abnormal “shoulder” on
the homoduplex peak was apparent (Figure 2).
To assess the ability of DHPLC to detect a wide range of
pathogenic ATRX mutations, we screened archival DNA samples
from boys with ATR-X syndrome that included 49 unique constitutional ATRX mutations involving 17 of the 35 exons (list available
on request). DHPLC clearly resolved 48 of the 49 mutations (98%),
including examples of substitutions, insertions, and deletions. The
only mutation that the WAVE system missed was a 580G⬎A in
exon 8; the available archival DNA from this pedigree was partially
degraded and amplified poorly, and the DHPLC system was able to
detect several other single base-pair changes in this region,
including 576G⬎C and 599G⬎C. Subsequently, screening of new
blood samples from undiagnosed boys with clinical features of
constitutional ATR-X syndrome using our DHPLC conditions
revealed 5 new germ line ATRX mutations (data not shown)
including 3 in exons without previously described mutations,
expanding the number of validated amplicons to 20.
amount of HbH remained stable over time or an archival DNA
sample was only available from one time point. DHPLC analysis of
an amplicon containing ATRX exon 8 from patient no. 4 showed a
second peak suggestive of a heteroduplex (Figure 3A). This peak
was not seen in the wild-type control or any other ATMDS samples
screened at the same time. The size of the peak was greater in a
sample of bone marrow obtained when patient no. 4 had 25% HbH
by electrophoresis and 50% HbH-containing cells by supravital
staining compared with a subsequent marrow sample when he had
Characterization of ATRX mutations in patients with ATMDS
Since the DHPLC method appeared sensitive and able to detect a
wide range of mutations, we next analyzed unfractionated archival
DNA samples from the 14 patients with myeloid disorders and
hypochromic red cell indices summarized in Table 1. This screen
detected the 9 novel ATRX mutations listed in Table 1 as well as a
new polymorphism (-14G/A).
Because MDS is a clonal disorder and previous findings had
suggested that the hematologic features of ATMDS might vary in a
manner reflecting this clonality, it was of interest to compare the
markers of ␣ thalassemia with the proportion of the abnormal
clone. For ATMDS patient no. 4, archival DNA samples from 2
different time points when the patient had very different amounts of
HbH were available for analysis. In all the other patients, either the
Figure 3. 576G>C ATRX mutation in a patient with ATMDS. (A) DHPLC tracing of
the exon 8 amplicon from patient no. 4. The lighter and darker purple tracings
represent unfractionated peripheral blood and bone marrow from a sample obtained
when the patient had 50% erythrocytes with HbH inclusions by brilliant cresyl blue
(BCB) staining and 25% HbH on electrophoresis. The brick red tracing represents
unfractionated marrow from a time point 6 months later when the amount of HbH by
electrophoresis had fallen to less than 1% and only rare HbH inclusions were
detectable on supravital staining. The dark green tracing is a wild-type control. (B) Big
Dye–generated sequence of 2 subclones (top 2 lines) and unfractionated marrow
DNA (bottom line) from patient no. 4, exon 8 (coding strand 5⬘ to 3⬘, running left to
right). The top line is sequence of a subclone with normal DNA (ie, 576G), the middle
line is from one of the subclones demonstrating the 576G⬎C mutation (arrowhead),
and the bottom line represents DNA from unfractionated marrow where a contribution
from cytidine (blue, with arrow) can be seen as a minor peak under the primary
fluorochrome representing guanidine (black).
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 15 MARCH 2004 䡠 VOLUME 103, NUMBER 6
less than 1% HbH-containing cells and undetectable HbH on
electrophoresis (Figure 3A). Subcloning and sequencing of this
amplicon revealed 8 of 23 (35%) of the clones with a 576G⬎C;
L192F substitution (Figure 3B) when patient no. 4 had 25% HbH
by electrophoresis, and only 3 of 40 (7.5%) clones when he had less
than 1% HbH, demonstrating a correlation between the proportion
of ATRX mutation–containing clones and the severity of the
thalassemic phenotype. The L192F mutation alters an amino acid
in a loop-helix-loop motif in one of the critical zinc-finger
DNA-binding domains of the ATRX protein.20
In 2 additional patients with ATMDS from whom only one
DNA sample was available, the proportion of subclones with an
ATRX mutation also roughly correlated with the amount of HbH.
Patient no. 11 had 10% HbH-containing cells and 8% HbH by
electrophoresis, and after subcloning, 10% of clones were found to
harbor a 6083G⬎C; R2028P mutation. Patient no. 12 had 40%
HbH-containing cells and 16.9% HbH by electrophoresis; 30% of
clones contained the insertion 6884_6885insT; 2293Tfs2319X
(Figure 4A). The latter mutation creates a new Hinf I restriction
site, and Hinf I (New England Biolabs) digestion confirmed the
presence of the mutation in both a mutant subclone and unfractionated marrow-derived DNA from patient no. 12 (Figure 4B).
Comparison of acquired and germ line ATRX mutations
A 576G⬎C; L192F mutation identical to that observed in ATMDS
patient no. 4 was previously detected in a boy with the constitutional ATR-X syndrome who had 0.1% HbH-containing erythrocytes. While this strongly supports the pathologic significance of
the acquired mutation in the patient with ATMDS, it is not clear
why the patient with ATMDS had a much more severe hematologic
phenotype than the patient with ATR-X.
An acquired ATRX mutation resulting in an amino acid change
similar to that previously found in an ATR-X pedigree was found in
ATMDS patient no. 5. This patient with ATMDS had a 718T⬎G;
C240G mutation and 48% HbH-containing red cells, while the
ATR-X pedigree proband had 719G⬎T; C240F and only 6%
HbH-containing cells. Both mutations result in a loss of the same
ACQUIRED SOMATIC ATRX MUTATIONS IN ATMDS
2023
cysteine residue in the highly conserved plant homeodomain
(PHD) finger, a zinc-finger motif that has been identified in many
proteins, some of which appear to be involved in chromatinmediated transcriptional regulation.21 The consensus PHD motif
and adjacent cysteine-rich domain (protein residues 220 to 268) is
the most common site of germ line ATRX mutations associated with
ATR-X syndrome (Figure 5A).20 Loss of C240 may result in
similar loss of a disulfide bond and unfolding of the PHD motif
with both germ line and acquired mutations, but again the severity
of the red cell abnormality was much greater in the patient with
ATMDS. A 794G⬎A; C265Y mutation was found in ATMDS patient
no. 6; although this also results in a loss of a cysteine residue in the PHD
finger and may lead to protein unfolding, no directly comparable germ
line ATRX mutations have yet been described.
Three of the new ATRX mutations (6884_6885insT;
2293Tfs2319X, 7014_7017del; N2340fsX2341, and 7049delT;
E2351fsX2353 in patient nos. 12-14, respectively) result in a
frame-shift and generation of a premature stop codon near the
C-terminus of ATRX. This is predicted to result in the loss of 2
highly conserved ATRX domains: the P-box (protein residues
2385-2401), an element conserved among SNF2-like family members involved in transcriptional regulation, and the Q-box, a stretch
of glutamine residues (protein 2415-2425) representing a potential
protein interaction domain.22 Several germ line ATRX mutations
resulting in the loss of these domains have been described (Figure
5A).23 In contrast to patients with ATMDS, all of the affected
individuals in those ATR-X pedigrees have only rare HbHcontaining cells.
ATMDS patient no. 10 had a 5567-3C⬎G ATRX mutation in the
consensus splice acceptor site 5⬘ to exon 24 (in intron 23), and
patient no. 11 had a 6083G⬎C; R2028P mutation in exon 27—both
located in the region between the third and fourth of the 7 highly
conserved helicase domains of ATRX. Archival mRNA was not
available from patient no. 10 (deceased) with 5567-3C⬎G in order
to confirm the presumed splicing abnormality. Although mutations
in the region of the ATRX helicase domains are relatively common
in germ line ATR-X patients (Figure 5), the associated hematologic
Figure 4. 6884_6885 insT ATRX mutation in a patient with ATMDS. (A) Sequences from patient no. 12, exon 33 (5⬘ to 3⬘, running left to right). The top line is a sequence from
a normal subclone, the middle line represents one of the 10% of subclones bearing the thymidine insertion 6884_6885insT (arrowhead) that results in a frame shift, and the
lower line is unfractionated marrow DNA, where the inserted thymidine (red, with arrow) can be seen as a secondary peak under the 3⬘ cytidine (blue); this is followed by a
series of secondary peaks (denoted in red letters under primary base calls) as a result of the frame shift. (B) Hinf I digest from patient no. 12. The predicted normal exon 33
amplicon digestion fragments are 218-bp and 21-bp long. The T insertion at 6884_6885insT expands the amplicon from 239 bp to 240 bp and creates a new restriction site,
leading to 3 digestion fragments of lengths 115 bp, 104 bp, and 21 bp. A 2-log DNA ladder (New England Biolabs) is at far left. Lane 2 is a digest of a subclone containing
6884_6885insT, and has the predicted 115-bp and 104-bp fragments. Lane 3 is a digest of a subclone from patient no. 12 where sequencing did not show the mutation, and only
the 218-bp fragment can be seen, similar to lane 4, a wild-type control with only the 218-bp fragment. Lane 1 is a digest of amplified unfractionated marrow DNA from patient no.
12, and contains both the normal 218-bp fragment as well as faint bands corresponding to the 115-bp and 104-bp fragments from the minor population of mutant DNA.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2024
STEENSMA et al
BLOOD, 15 MARCH 2004 䡠 VOLUME 103, NUMBER 6
Figure 5. Acquired versus constitutional ATRX mutations. (A) Comparison of the spectrum of ATRX mutations described in boys with ATR-X syndrome with those found in
ATMDS. The ATRX gene (top, with introns not to scale) is aligned with ATRX protein (bottom) to allow comparison of mutation site with functionally important protein domains
such as the ADD (ie, ATRX, DNMT3, DNMT3L) zinc-finger domain (ADD includes a C2-C2 type of zinc finger and the closely located PHD motif), helicase domains, P-box, and
Q-box. ATR-X–associated mutations are annotated above the schematic representation of the ATRX gene at the mutation loci, while mutations associated with ATMDS are
denoted below. F represents mutations predicted to cause protein truncation (ie, frame shifts and nonsense mutations) and null mutations, whereas E represents amino acid
changes, including in-frame insertions and deletions. Unfilled boxes denote the newly described -14G⬎A polymorphism and 2692G⬎C; D898H base-pair change of uncertain
significance as described in the text. (B) Comparison of the fraction of erythrocytes containing HbH inclusions in 118 patients with ATR-X syndrome with the fraction detected in
the 18 patients with ATMDS analyzed in this study. Transfused and untransfused patients with ATMDS are tallied together; the possibility of dilution by donor erythrocytes
contributing to artifactually decreased HbH-containing cells in the former group should be recognized.
phenotypes in these pedigrees are quite variable, and the 2 mutations
detected in the patients with ATMDS have not been described to
date in ATR-X pedigrees.
The functional consequence (if any) of the remaining 2 newly
described ATRX base-pair changes, in patients no. 1 and no. 7,
respectively, is uncertain. Unlike the other 8 patients with ATMDS
with novel ATRX mutations described here, in these cases no
mosaicism was apparent on sequencing and/or subcloning. In
addition, these single base-pair ATRX changes were also present in
Epstein-Barr virus (EBV)–transformed lymphoblastoid cell lines
derived from the patients. The -14G⬎A change in patient no. 1 is
located in a conserved area of the 5⬘ untranslated region of the
ATRX gene, but we were able to obtain gDNA from this deceased
patient’s daughter (with her informed consent) and found that she is
heterozygous for -14G and -14A, demonstrating that this base-pair
change is a germ line polymorphism. In patient no. 7, the
2692G⬎C; D898H mutation is in the middle of exon 10, 3⬘ to the
PHD motif; this region of ATRX is not highly conserved in the
mouse.22 Unfortunately, patient no. 7 is also dead, and no other
genetic material is availabe to help resolve the mutation versus
polymorphism issue (eg, DNA from buccal smears or fibroblast cell
lines derived from the patient or from a surviving daughter).
Neither -14G⬎A nor the 2692G⬎C; D898H variant have been
detected in previous studies of pedigrees with ATR-X syndrome,
patients with ATMDS, or healthy controls.
Patients in whom no ATRX mutation was detected
No pathologic mutations in ATRX were found in patients no. 15
through no. 18 or patient no. 1 with chronic myeloid disorders,
HbH inclusions, and hypochromic, microcytic red cells. These
patients were atypical of ATMDS in several respects. In patients no.
1 and no. 16 through no. 18, the amount of HbH was so small that it
was not detectable by electrophoresis. Patients no. 17 and no. 18
had only very rare (0.01% and 0.3%) HbH-containing erythrocytes
on supravital staining. Patient no. 15 was the only female patient
with ATMDS studied; approximately 90% of patients with ATMDS
described have been men.9
Discussion
The data presented here add further support to recent observations16
showing that point mutations in the ATRX gene at Xq13.3 are
strongly associated with the rare ATMDS subtype and are responsible for the associated ␣ thalassemia seen in such cases. However, these data also raise several new questions regarding the
mechanism by which ␣ globin expression is down-regulated in
this condition.
The normal role of ATRX protein in the cell is not known, but it
is a member of the SWI/SNF group of proteins that are thought to
influence a wide range of nuclear processes (eg transcription,
replication, recombination) via effects on chromatin.23,24 ATRX is
found in the nucleus where it localizes to pericentromeric heterochromatin during interphase and mitosis.25,26 Consistent with this,
ATRX has been shown to interact with the major heterochromatinassociated protein HP1.25 A proportion of ATRX is also found in
promyelocytic leukemia (PML) bodies,27 which contain a variety
of nuclear factors that influence apoptosis, regulate gene expression, and control progression through the cell cycle. These bodies
are characteristically disrupted in AML subtype M3.28 ATRX has
also recently been shown to form a stable complex with Daxx, a
protein that is found in PML bodies and has been implicated in the
regulation of apoptosis.27
Perhaps the best guide to the role played by ATRX in the cell
comes from analysis of the consequences of germ line ATRX
mutations in the inherited ATR-X syndrome. Such mutations are
associated with widespread changes in the patterns of DNA
methylation in the genome together with alterations in gene
expression.24 At present, the best documented of these changes in
gene expression is that which occurs at the ␣ globin cluster, where
ATRX mutations are consistently associated with down-regulation
of gene expression, resulting in mild ␣ thalassemia. As shown here
and in our previous report,16 acquired ATRX mutations in the
ATMDS syndrome may be associated with a relatively severe
degree of ␣ thalassemia. At present it is not yet clear how ATRX
contributes to normal ␣ globin gene expression, or at what stage(s)
of development and red cell differentiation ATRX is involved.
The findings presented here raise a new issue in our understanding of the pathophysiology of ATMDS. The dramatically reduced
␣/␤ globin synthesis ratios, the consequent hematologic changes,
and the relatively high levels of HbH in ATMDS cases with
acquired ATRX mutations demonstrate that the associated ␣
thalassemia is, in general, more severe in ATMDS than in ATR-X
syndrome. Individuals with ATMDS have been described in whom
up to 90% of the peripheral blood erythrocytes contain HbH
inclusions,9 whereas patients with constitutional ATRX mutations
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 15 MARCH 2004 䡠 VOLUME 103, NUMBER 6
ACQUIRED SOMATIC ATRX MUTATIONS IN ATMDS
have much lower levels of HbH (90% have 10% HbH inclusions or
less, while 53% have ⱕ 1% HbH inclusions) and some have no
HbH inclusions (Figure 5B).29 One explanation may be that the
spectrum of acquired somatic ATRX mutations in ATMDS may
include severe, null mutations that are not seen in patients with the
inherited ATR-X syndrome because such mutations may be lethal
early in development.16,30 However, in this study we found a patient
with severe hypochromic microcytic anemia and up to 50% of red
cells containing HbH inclusions who has acquired the same
mutation (576G⬎C; L192F) previously identified in a patient with
ATR-X syndrome who had minimal red cell changes and only very
rare HbH inclusions. We also found an ATRX mutation (718T⬎G;
C240G) in another patient with ATMDS whose mutation affects the
same amino acid as a congenital ATR-X case, as well as a series of
ATRX mutations that may have similar functional consequences as
previously described constitutional cases. In each instance the
ATMDS hematologic phenotype was more severe than in the
corresponding ATR-X cases. Although it seems almost certain that
the ATRX mutation is responsible for the ␣ thalassemia in both
groups, the degree to which ␣ globin expression is down-regulated
must depend on other interacting molecular and/or cellular factors.
This might have been expected, since we have previously noted
that in ATR-X syndrome, the number of HbH inclusions may vary
over several orders of magnitude in different pedigrees with the
same ATRX mutation, and there is also some variation within
affected families. It will be important to understand which factors
are modifying the effects of mutations in ATRX.
To date, all 12 of the patients with ATMDS in whom we have
identified clearly pathogenic ATRX mutations or ATRX splicing
abnormalities have the typical features of this rare syndrome with
hypochromic, microcytic anisopoikilocytic red cells associated
with substantial amounts of HbH and significantly reduced ␣/␤
globin chain synthesis ratios. The only ATMDS patients with an
␣/␤ globin chain synthesis ratio more than 0.2 in whom we found
an ATRX mutation were those who had received red cell transfusions prior to study, which likely affected their red cell indices and
globin chain synthesis ratio.
The red cell indices of patients with ATMDS in general are quite
distinct from the majority of patients with MDS, who typically
have normocytic or macrocytic anemia.31 We have observed other
patients with MDS and similar hypochromic microcytic red cell
morphology but with only rare or no HbH inclusions; to date, no
mutations in the ATRX gene have been detected in such individuals.
It is not clear whether this is a result of insensitivity of our current
detection techniques to very small subpopulations of cells with an
ATRX mutation, or whether a different molecular mechanism might
be responsible.
It seems unlikely that ATRX mutations play a key role in the
development of MDS. Patients with the inherited ATR-X syndrome
do not show any evidence of genome instability and have no
2025
increased incidence of MDS or hematologic malignancy.29 Furthermore, if ATRX mutations were involved in the origin or evolution
of MDS, ␣ thalassemia should be encountered more frequently in
such patients. It seems likely that mutations in ATRX have come to
attention in MDS only because the resulting ␣ thalassemia has an
easily detectable and dramatic red cell phenotype.
Before we studied this series of patients with ATMDS with
DHPLC, the ATRX gene had been sequenced in a subset of the
patients without detection of additional mutations. Several possibilities might account for difficulty in finding ATRX mutations in
ATMDS. Mutations in MDS are often lineage restricted.32 This also
appears to be true in ATMDS, where the mutations described above
(with the exception of the one that may represent a polymorphism)
were found in DNA isolated from myeloid cells but not DNA from
lymphoblastoid cell lines derived from the patients, where such cell
lines were available. Although malignant stem cell transformation
has been described in MDS, the usual cell of origin is thought to be
a committed myeloid progenitor and lymphocytes are often not
involved in the neoplastic clone.33-35 In addition, ATRX analysis
could be confounded by somatic mosaicism in ATMDS. These
issues are pertinent to genetic and pathophysiologic investigations
in all types of MDS, and therefore devising a strategy to identify
ATRX mutations in unfractionated samples of DNA from patients
with ATMDS provides a good model for developing general
approaches for mutation analysis in MDS and related hematologic
malignancies.
Direct sequencing can detect point mutations in mixtures of
genomic DNA containing 20% to 30% mutant DNA.36 Denaturing
gradient gel electrophoresis (DGGE) is more sensitive than sequencing (DGGE can detect ⬍ 5% mutant DNA) but is cumbersome and
only a few samples can be analyzed at a time.37 Recently, DHPLC
has proven to be a powerful, sensitive, high-throughput technique
for mutation detection38 that may be particularly useful in the
setting of germ line and somatic mosaicism.39-41 DHPLC is
therefore an attractive technique for mutational analysis of neoplastic conditions such as MDS, including the ATMDS subtype, where
the abnormal cells are not easy to separate from normal hemopoietic cells. This is also relevant to archival DNA obtained from
unfractionated blood or marrow specimens.
Somatic mosaicism has likely contributed to the difficulty many
investigators have faced in describing specific molecular lesions
associated with various MDS phenotypes and cytogenetic patterns—
compare, for example, the many specific mutations that have been
recognized in acute leukemia42 with the relatively small number
fully characterized in MDS.43 The present study demonstrates
that DHPLC can be a powerful tool for detecting mutations in
the setting of mosaicism in MDS. Therefore, in addition to
confirming the importance of ATRX in ATMDS, our findings
have important implications for identifying other genes that may
be mutated in MDS.
References
1. Weatherall DJ, Clegg JB. The Thalassaemic Syndromes. 4th ed. Malden, MA: Blackwell Science;
2001.
2. Bernini LF, Harteveld CL. Alpha-thalassaemia. Baillieres Clin Haematol. 1998;11:
53-90.
3. Olivieri NF. The beta-thalassemias. N Engl J Med.
1999;341:99-109.
4. Bradley TB, Ranney HM. Acquired disorders of
hemoglobin. Prog Hematol. 1973;8:77-98.
5. Wood WG. Increased HbF in adult life. Baillieres
Clin Haematol. 1993;6:177-213.
6. Rochette J, Craig JE, Thein SL. Fetal hemoglobin
levels in adults. Blood Rev. 1994;8:213-224.
beta thalassaemia trait in MDS. Br J Haematol.
1991;79:116-117.
7. Aksoy M, Erdem S. Decrease in the concentration of haemoglobin A2 during erythroleukaemia.
Nature. 1967;213:522-523.
11. Markham RE, Butler F, Goh K, Rowley PT. Erythroleukemia manifesting delta beta-thalassemia.
Hemoglobin. 1983;7:71-78.
8. Weatherall DJ, Old J, Longley J, et al. Acquired haemoglobin H disease in leukemia: pathophysiology and
molecular basis. Br J Haematol. 1978;38:305-322.
12. Chui DH, Fucharoen S, Chan V. Hemoglobin H
disease: not necessarily a benign disorder. Blood.
2003;101:791-800.
9. Higgs DR, Wood WG, Barton C, Weatherall
DJ. Clinical features and molecular analysis of
acquired hemoglobin H disease. Am J Med.
1983;75:181-191.
13. Higgs DR, Vickers MA, Wilkie AO, Pretorius IM,
Jarman AP, Weatherall DJ. A review of the
molecular genetics of the human alpha-globin
gene cluster. Blood. 1989;73:1081-1104.
10. Hoyle C, Kaeda J, Leslie J, Luzzatto L. Acquired
14. Viprakasit V, Kidd AM, Ayyub H, Horsley S,
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2026
BLOOD, 15 MARCH 2004 䡠 VOLUME 103, NUMBER 6
STEENSMA et al
Hughes J, Higgs DR. De novo deletion within the
telomeric region flanking the human alpha globin
locus as a cause of alpha thalassaemia. Br J
Haematol. 2003;120:867-875.
24. Gibbons RJ, McDowell TL, Raman S, et al. Mutations in ATRX, encoding a SWI/SNF-like protein,
cause diverse changes in the pattern of DNA
methylation. Nat Genet. 2000;24:368-371.
15. Gibbons RJ, Picketts DJ, Villard L, Higgs DR. Mutations in a putative global transcriptional regulator cause X-linked mental retardation with alphathalassemia (ATR-X syndrome). Cell. 1995;80:
837-845.
25. McDowell TL, Gibbons RJ, Sutherland H, et al.
Localization of a putative transcriptional regulator
(ATRX) at pericentromeric heterochromatin and
the short arms of acrocentric chromosomes. Proc
Natl Acad Sci U S A. 1999;96:13983-13988.
16. Gibbons RJ, Pellagatti A, Garrick D, et al. Identification of acquired somatic mutations in the gene
encoding chromatin-remodeling factor ATRX in
the alpha-thalassemia myelodysplasia syndrome
(ATMDS). Nat Genet. 2003;34:446-449.
26. Berube NG, Smeenk CA, Picketts DJ. Cell cycledependent phosphorylation of the ATRX protein
correlates with changes in nuclear matrix and
chromatin association. Hum Mol Genet. 2000;9:
539-547.
17. den Dunnen JT, Antonarakis SE. Nomenclature
for the description of human sequence variations.
Hum Genet. 2001;109:121-124.
27. Xue Y, Gibbons R, Yan Z, et al. The ATRX syndrome protein forms a chromatin-remodeling
complex with Daxx and localizes in promyelocytic
leukemia nuclear bodies. Proc Natl Acad Sci
U S A. 2003;100:10635-10640.
18. Weatherall DJ, Clegg JB, Naughton MA. Globin
synthesis in thalassaemia: an in vitro study.
Nature. 1965;208:1061-1065.
19. Clegg JB, Weatherall DJ, Na-Nakorn S, Wasi P.
Haemoglobin synthesis in beta-thalassaemia.
Nature. 1968;220:664-668.
20. Gibbons RJ, Bachoo S, Picketts DJ, et al. Mutations in transcriptional regulator ATRX establish
the functional significance of a PHD-like domain.
Nat Genet. 1997;17:146-148.
21. Aasland R, Gibson TJ, Stewart AF. The PHD finger: implications for chromatin-mediated transcriptional regulation. Trends Biochem Sci. 1995;
20:56-59.
22. Picketts DJ, Tastan AO, Higgs DR, Gibbons
RJ. Comparison of the human and murine ATRX
gene identifies highly conserved, functionally important domains. Mamm Genome. 1998;9:400403.
23. Picketts DJ, Higgs DR, Bachoo S, Blake DJ,
Quarrell OW, Gibbons RJ. ATRX encodes a novel
member of the SNF2 family of proteins: mutations
point to a common mechanism underlying the
ATR-X syndrome. Hum Mol Genet. 1996;5:18991907.
28. Eskiw CH, Bazett-Jones DP. The promyelocytic
leukemia nuclear body: sites of activity? Biochem
Cell Biol. 2002;80:301-310.
29. Gibbons RJ, Brueton L, Buckle VJ, et al. Clinical
and hematologic aspects of the X-linked alphathalassemia/mental retardation syndrome (ATRX). Am J Med Genet. 1995;55:288-299.
30. Gibbons RJ, Garrick D, Sharpe J, et al. Disruption
of ATRX in mouse by conditional knockout [abstract]. Am Soc Hum Genet. Baltimore, MD;
2002:116.
31. List AF, Doll DC. The myelodysplastic syndromes.
In: Lee GR, ed. Wintrobe’s Clinical Hematology.
10th ed. Baltimore, MD: Lippincott, Williams &
Wilkins; 1999:2320-2341.
32. Kibbelaar RE, van Kamp H, Dreef EJ, et al. Combined immunophenotyping and DNA in situ hybridization to study lineage involvement in patients with myelodysplastic syndromes. Blood.
1992;79:1823-1828.
33. van Kamp H, Fibbe WE, Jansen RP, et al. Clonal
involvement of granulocytes and monocytes, but
not of T and B lymphocytes and natural killer cells
in patients with myelodysplasia: analysis by X-
linked restriction fragment length polymorphisms
and polymerase chain reaction of the phosphoglycerate kinase gene. Blood. 1992;80:17741780.
34. Asano H, Ohashi H, Ichihara M, et al. Evidence
for nonclonal hematopoietic progenitor cell populations in bone marrow of patients with myelodysplastic syndromes. Blood. 1994;84:588-594.
35. Boultwood J, Wainscoat JS. Clonality in the myelodysplastic syndromes. Int J Hematol. 2001;73:
411-415.
36. Levin BC, Tully LA, Hancock DK, Schwarz FP,
Lee MS. A new NIST mitochondrial DNA interactive website. MitoMatters. 2002;1:5-6.
37. Borgione E, Sturnio M, Spalletta A, et al. Mutational analysis of the ATRX gene by DGGE: a
powerful diagnostic approach for the ATRX syndrome. Hum Mutat. 2003;21:529-534.
38. Liu W, Smith DI, Rechtzigel KJ, Thibodeau SN,
James CD. Denaturing high performance liquid
chromatography (DHPLC) used in the detection
of germline and somatic mutations. Nucleic Acids
Res. 1998;26:1396-1400.
39. Emmerson P, Maynard J, Jones S, Butler R,
Sampson JR, Cheadle JP. Characterizing mutations in samples with low-level mosaicism by collection and analysis of DHPLC fractionated heteroduplexes. Hum Mutat. 2003;21:112-115.
40. Jones AC, Sampson JR, Cheadle JP. Low level
mosaicism detectable by DHPLC but not by direct
sequencing. Hum Mutat. 2001;17:233-234.
41. van Den Bosch BJ, de Coo RF, Scholte HR, et al.
Mutation analysis of the entire mitochondrial genome using denaturing high performance liquid
chromatography. Nucleic Acids Res. 2000;28:
E89.
42. Giles FJ, Keating A, Goldstone AH, Avivi I, Willman CL, Kantarjian HM. Acute myeloid leukemia.
American Society of Hematology Education
Book. 2002:73-110.
43. Fenaux P. Chromosome and molecular abnormalities in myelodysplastic syndromes. Int J Hematol. 2001;73:429-437.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2004 103: 2019-2026
doi:10.1182/blood-2003-09-3360 originally published online
October 30, 2003
Acquired somatic ATRX mutations in myelodysplastic syndrome
associated with α thalassemia (ATMDS) convey a more severe
hematologic phenotype than germline ATRX mutations
David P. Steensma, Douglas R. Higgs, Chris A. Fisher and Richard J. Gibbons
Updated information and services can be found at:
http://www.bloodjournal.org/content/103/6/2019.full.html
Articles on similar topics can be found in the following Blood collections
Clinical Trials and Observations (4563 articles)
Neoplasia (4182 articles)
Red Cells (1159 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society
of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.