high-dose cytarabine are administered. J Clin Oncol 1999; 17:
3 Grimwade D, Walker H, Oliver F, Wheatley K, Harrison C,
Harrison G et al. The importance of diagnostic cytogenetics on
outcome in AML: analysis of 1612 patients entered into the MRC
AML 10 trial. The Medical Research Council Adult and Children’s
Leukaemia Working Parties. Blood 1998; 92: 2322–2333.
4 Marcucci G, Mrozek K, Ruppert AS, Maharry K, Kolitz JE, Moore JO
et al. Prognostic factors and outcome of core binding factor acute
myeloid leukemia patients with t(8;21) differ from those of patients
with inv(16): a Cancer and Leukemia Group B study. J Clin Oncol
2005; 23: 5705–5717.
5 Nishii K, Usui E, Katayama N, F Lorenzo V, Nakase K, Kobayashi T
et al. Characteristics of t(8;21) acute myeloid leukemia (AML) with
additional chromosomal abnormality: concomitant trisomy 4 may
constitute a distinctive subtype of t(8;21) AML. Leukemia 2003; 17:
6 Nguyen S, Leblanc T, Fenaux P, Witz F, Blaise D, Pigneux A et al. A
white blood cell index as the main prognostic factor in t(8;21) acute
myeloid leukemia (AML): a survey of 161 cases from the French
AML Intergroup. Blood 2002; 99: 3517–3523.
7 Schlenk RF, Benner A, Krauter J, Buchner T, Sauerland C, Ehninger
G et al. Individual patient data-based meta-analysis of patients aged
16 to 60 years with core binding factor acute myeloid leukemia: a
survey of the German Acute Myeloid Leukemia Intergroup. J Clin
Oncol 2004; 22: 3741–3750.
8 Appelbaum FR, Kopecky KJ, Tallman MS, Slovak ML, Gundacker
HM, Kim HT et al. The clinical spectrum of adult acute myeloid
leukaemia associated with core binding factor translocations.
Br J Haematol 2006; 135: 165–173.
Center, Nagoya; Toyohashi Municipal Hospital, Toyohashi;
Hospital, Toyoake; Komaki City Hospital, Komaki; Yokkaichi
Central Hospital, Yamanashi; Okazaki City Hospital, Okazaki,
Congenital transfusion-dependent anemia and thrombocytopenia with myelodysplasia
due to a recurrent GATA1G208Rgermline mutation
Leukemia (2008) 22, 432–434; doi:10.1038/sj.leu.2404904;
published online 23 August 2007
The X-linked gene GATA1 encodes a 414-amino-acid hemato-
poietic transcription factor that controls erythroid and mega-
karyocytic differentiation. Virtually all cases of transient
myeloproliferative disease and acute megakaryoblastic leuke-
mia in children with Down syndrome harbor somatic GATA1
mutations typically affecting exon 2 and leading to expression of
the short isoform, GATA-1s, which lacks the transcriptional
activation domain. Moreover, germline missense mutations in
exon 4 of GATA1 that predict alterations of amino acids Val205,
Gly208, Arg216 or Asp218 of the N-terminal zinc-finger domain
(residues 204–228) have been reported in nine families.1–9One
additional family has been found to harbor a germline mutation,
GATA1 c.322G4C, which leads to expression of GATA-1s.10
Consistent with X-linked inheritance and full penetrance,
germline GATA1 mutations disrupt hematopoiesis in males
who harbor a hemizygous mutant GATA1 allele. In contrast,
female heterozygous carriers have no or minor hematopoietic
defects such as mild chronic thrombocytopenia.1–10
spectrum of abnormalities caused by different GATA1 mutations
probably depends on the function of the predicted mutant
protein such as the ability to associate with cofactor FOG-1.9
Hematologic abnormalities include dyserythropoietic anemia
and thrombocythemia (V205M, G208R, D218Y and GATA-
congenital erythropoietic porphyria (R216W),7
and gray platelet syndrome (R216Q).6Splenomegaly is noted
in some cases5,7and is likely to be due to ineffective and
consecutive extramedullary hematopoiesis. To date, only the
GATA1R216Qmutation has been identified in more than one
family,4–6hampering phenotype–genotype correlation.
We identified a second family with a GATA1G208Rmutation.
The index patient was a male neonate born at term to healthy
European non-consanguineous parents. Family history was
unremarkable and the mother previously gave birth to a healthy
boy. At birth, petechiae and ecchymoses on skin and mucosa as
well as enlargement of liver and spleen were noted. Hemoglobin
measured 8.9g/dl, leukocytes 54900/ml and thrombocytes
54000/ml. Repeated platelet and packed red blood cell transfu-
sions were administered. A bone marrow smear revealed
dyserythropoiesis (Figure 1) and dysmegakaryopoiesis but no
increase in blasts. A liver biopsy taken at the age of 16 days
revealed siderosis, cholestasis and extramedullary hematopoi-
esis. Mutation analysis with published methods,1,10uncovered a
hemizygous G to A transition at nucleotide position c.622 in
exon 4 of GATA1 predicting a p.G208R change in the highly
conserved N-terminal zinc-finger domain of GATA-1. The
patient inherited this allele from his heterozygous mother
(Figure 1), who had a hemoglobin level of 11.3g/dl, mean
erythrocyte volume 81fl, leukocytes 12700/ml and thrombocytes
172000/ml. At the time of this report, the patient was 6 months of
age and was in stable condition requiring platelet transfusions
every week and red packed cell transfusions every second to
third week. To obtain the option of hematopoietic stem cells
transplantation (HSCT) a donor search has been initiated. The
same mutation, GATA1G208Rhas been described in another
individual with dyserythropoietic anemia and thrombocytopenia
who was found to have anemia and thrombocytopenia at birth
requiring transfusions.3This patient received his last red packed
cell transfusion at 5 years of age and the frequency of mucosal
and severe bleeding decreased in adulthood. At the age of 17
years he had a hemoglobin level of 9.6g/dl, mean erythrocyte
volume 103fl and thrombocytes 12000/ml.3Notably, this
patient’s mother was mildly thrombocytopenic with platelets
measuring 140000/ml.3The similar clinical presentation with
transfusion-dependent cytopenia at birth underscores the notion
Letters to the Editor
of a genotype–phenotype relationship of different GATA1
defects. However, in addition to dyserythropoietic anemia and
thrombocytopenia, our patient presented with marked organo-
megaly. Although bleeding complications in patients with
germline GATA1 mutations may decrease with age,3this
disorder may lead to early death.2,9Notably, in one family with
a GATA1D218Yallele, six affected boys died before the age of 2
years.9In cases of GATA1 defects with significant thrombo-
cytopenia and severe bleeding diathesis, HSCT may be warranted.
Indeed, related or unrelated HSCT has been performed in a small
number of cases.1,7,10In conclusion, we describe the second
family with a GATA1G208Rallele causing congenital dyserythro-
poietic anemia and thrombocytopenia. Additionally, the affected
male showed marked organomegaly. Besides GATA1G216Q, the
GATA1G208Rallele is the only mutation that has been identified in
more than one family. The identification of a larger number of
families with these rare mutations that lead to a spectrum of mild
to severe hematologic defects will help to perform phenotype–
genotype correlations that eventually will facilitate treatment
decisions and patient care.
We thank Cornelia Klein for technical assistance.
CP Kratz1, CM Niemeyer1, A Karow1, M Volz-Fleckenstein2,
A Schmitt-Gra ¨ff3and B Strahm1
1Department of Pediatrics and Adolescent Medicine, Pediatric
Hematology/Oncology, University of Freiburg, Freiburg,
2Krankenhaus Barmherzige Bru ¨der Regensburg, Klinik St
Hedwig/Pa ¨diatrische Onkologie und Ha ¨matologie,
Regensburg, Germany and
3Department of Pathology, University of Freiburg, Freiburg,
1 Nichols KE, Crispino JD, Poncz M, White JG, Orkin SH, Maris JM
et al. Familial dyserythropoietic anaemia and thrombocytopenia
due to an inherited mutation in GATA1. Nat Genet 2000; 24:
2 Mehaffey MG, Newton AL, Gandhi MJ, Crossley M, Drachman JG.
X-linked thrombocytopenia caused by a novel mutation of
GATA-1. Blood 2001; 98: 2681–2688.
3 Del Vecchio GC, Giordani L, De Santis A, De Mattia D.
Dyserythropoietic anemia and thrombocytopenia due to a novel
mutation in GATA-1. Acta Haematol 2005; 114: 113–116.
4 Yu C, Niakan KK, Matsushita M, Stamatoyannopoulos G, Orkin
SH, Raskind WH. X-linked thrombocytopenia with thalassemia
from a mutation in the amino finger of GATA-1 affecting
DNA binding rather than FOG-1 interaction. Blood 2002; 100:
5 Balduini CL, Pecci A, Loffredo G, Izzo P, Noris P, Grosso M et al.
Effects of the R216Q mutation of GATA-1 on erythropoiesis and
megakaryocytopoiesis. Thromb Haemost 2004; 91: 129–140.
6 Tubman VN, Levine JE, Campagna DR, Monahan-Earley R,
Dvorak AM, Neufeld EJ et al. X-linked gray platelet syndrome
due to a GATA1 Arg216Gln mutation. Blood 2007; 109:
7 Phillips JD, Steensma DP, Pulsipher MA, Spangrude GJ, Kushner JP.
Congenital erythropoietic porphyria due to a mutation in GATA1:
the first trans-acting mutation causative for a human porphyria.
Blood 2007; 109: 2618–2621.
N terminal zinc finger
GATA1 mutation, c.622G4A, is detected in the patient’s mother. The patient harbors a hemizygous mutant allele (a). This alteration in exon 4
predicts a G208R change in the N-terminal zinc finger (N) of the protein (TD: transcriptional activation domain; C: C-terminal zinc finger). This
mutation locates near residues that are altered by other known germline mutations (red) (b). A bone marrow slide shows severe dyserythropoietic
changes (arrows) (c).
The recurrent GATA1G208Rmutation causes congenital dyserythropoietic anemia and thrombocytopenia. A heterozygous germline
Letters to the Editor
8 Freson K, Devriendt K, Matthijs G, Van Hoof A, De Vos R, Thys C
et al. Platelet characteristics in patients with X-linked macro-
thrombocytopenia because of a novel GATA1 mutation. Blood
2001; 98: 85–92.
9 Freson K, Matthijs G, Thys C, Marien P, Hoylaerts MF, Vermylen J
et al. Different substitutions at residue D218 of the X-linked
transcription factor GATA1 lead to altered clinical severity of
with variable skewed X inactivation. Hum Mol Genet 2002; 11:
10 Hollanda LM, Lima CS, Cunha AF, Albuquerque DM, Vassallo J,
Ozelo MC et al. An inherited mutation leading to production of
only the short isoform of GATA-1 is associated with impaired
erythropoiesis. Nat Genet 2006; 38: 807–812.
and anemiaand areassociated
A simple FISH assay for the detection of 3q26 rearrangements in myeloid malignancy
Leukemia (2008) 22, 434–437; doi:10.1038/sj.leu.2404906;
published online 13 September 2007
In myeloid malignancy, a number of recurrent and sporadic
rearrangements of 3q26 are associated with transcriptional
activation of EVI1 and/or EVI1 chimaeric genes.1Recurrent
rearrangements include the inv(3)(q21q26) and its variants
t(3;3)(q21;q26) and ins(3;3)(q26;q21q26), as well as transloca-
tions involving other chromosomes such as the t(3;21)(q26;q22).
In general, 3q26 rearrangements are associated with a poor
Given the unfavourable outcome of 3q26 rearrangements,
methods for establishing their presence at diagnosis and during
treatment or disease progression are highly desirable, particu-
larly when chromosome banding analysis is hampered by low
quality or insufficient metaphase cells. To date, however, the
wide 3q26 breakpoint region has precluded development of a
single fluorescent in situ hybridization (FISH) probe sensitive
enough to monitor residual disease. We describe a novel dual-
colour FISH probe designed to span the entire 3q26 breakpoint
region in a single hybridization, which allowed successful
detection and quantification of the level of leukaemic cells in 11
patients with 3q26 rearrangements. Physical mapping data
obtained with this probe further support the notion of a degree of
rearrangement-specific breakpoint clustering within cytogenetic
subgroups of 3q26 abnormality.
The dual-colour EVI1 FISH probe was designed to comprise
two differentially labelled DNA contigs flanking the common
3q26 breakpoint region (Figure 1). Expected signal patterns from
differentially labelled contigs specific for the 3q26 locus (Kreatech Biotechnologies, Amsterdam, The Netherlands). The most centromeric contig,
labelled in green fluorochrome, hybridizes to a region extending 460kb from the centromeric (30) end of the EVI1 gene. The second, most
telomeric contig, labelled with a red fluorochrome, is specific for a region beginning approximately 500kb 30of EVI1 and extends 370kb in a
telomeric direction. The distance between the hybridization regions of the two contigs is 530kb. The breakpoint region associated with 3q26
abnormalities is indicated. The lower portion of the figure shows the principle of the EVI1 break-apart FISH assay with the expected normal and
abnormal hybridization patterns. For simplicity, an inv(3)(q21q26) is depicted in the scheme, however, the principles of this approach apply to all
3q26 rearrangements. (i) Expected hybridization pattern on normal chromosome 3 and corresponding interphase nuclei. Two red-green fusion
signals are produced (2F). (ii) Expected pattern on metaphase chromosomes and in interphases with a 3q26 rearrangement involving a breakpoint
mapping 30of EVI1, within the hybridization region of the green probe component (indicated by the first square bracket). Interphase cells show a
pattern of one green and two fusion signals (1G2F). (iii) Hybridization pattern expected in cells with a more centromeric 3q26 breakpoint, up to
500kb 50of EVI1 between the hybridization regions of the two probe contigs (middle square bracket). Interphase cells show one red, one green and
one fusion signal (1R1G1F). (iv) Pattern expected in cells with a 3q26 breakpoint more than 500kb 50of EVI1 (third square bracket), resulting in a
split red signal and an interphase pattern of one red and two fusion signals (1G2F).
Structure and principle of the EVI1 break-apart fluorescent in situ hybridization (FISH) probe system. The probe is composed of two
Letters to the Editor