ORIGINAL ARTICLE

Loss of ABCB7 gene: pathogenesis of mitochondrial ironaccumulation in erythroblasts in refractory anemiawith ringed sideroblast with isodicentric (X)(q13)

Kazuya Sato • Yoshihiro Torimoto • Takaaki Hosoki • Katsuya Ikuta • Hiroyuki Takahashi • Masayo Yamamoto •

Satoshi Ito • Naoka Okamura • Kazuhiko Ichiki • Hiroki Tanaka • Motohiro Shindo • Katsuyuki Hirai •

Yusuke Mizukami • Takaaki Otake • Mikihiro Fujiya • Kastunori Sasaki • Yutaka Kohgo

Received: 21 May 2010 / Revised: 8 February 2011 / Accepted: 9 February 2011 / Published online: 8 March 2011

� The Japanese Society of Hematology 2011

Abstract An isodicentric (X)(q13) (idicXq13) is a rare,

acquired chromosomal abnormality originated by deletion

of the long arm from Xq13 (Xq13-qter), and is found in

female patients with hematological disorders involving

increased ringed sideroblasts (RSs), which are character-

ized by mitochondrial iron accumulation around the

erythroblast nucleus. The cause of increased RSs in

idicXq13 patients is not fully understood. Here, we report

the case of a 66-year-old female presenting with refractory

anemia with ringed sideroblasts (RARS), and idicXq13 on

G-banded analysis. We identify the loss of the ABCB7

(ATP-binding cassette subfamily B member-7) gene,

which is located on Xq13 and is involved in mitochondrial

iron transport to the cytosol, by fluorescent in situ

hybridization (FISH) analysis and the decreased expression

level of ABCB7 mRNA in the patient’s bone marrow cells.

Further FISH analyses showed that the ABCB7 gene is lost

only on the active X-chromosome, not on the inactive one.

We suggest that loss of ABCB7 due to deletion of Xq13-

qter at idicXq13 formation may have contributed to the

increased RSs in this patient. These findings suggest that

loss of the ABCB7 gene may be a pathogenetic factor

underlying mitochondrial iron accumulation in RARS

patients with idicXq13.

Keywords RARS � Isodicentric (X)(q13) � ABCB7 �FISH

1 Introduction

An isodicentric (X)(q13) chromosome (idicXq13), which is

a rare chromosomal abnormality originated by deletion of

long arm from Xq13 (Xq13-qter) [1], is found in elderly

female patients with hematological malignant disorders,

such as myelodysplastic syndromes (MDSs), acute myeloid

leukemias (AMLs), and myeloproliferative neoplasms

(MPNs) [1–7]. Interestingly, most reported cases with

idicXq13 are accompanied by increased ringed sideroblasts

(RSs) [1, 3, 5–7], which contain iron granules in the

cytoplasm (that accumulate in the mitochondria) giving a

ringed appearance around the nucleus of the bone marrow

erythroblasts. Especially, refractory anemia with ringed

sideroblasts (RARS), which is one of the MDS subtypes

characterized by more than 15% of RSs in the bone mar-

row, is reported as the most familiar hematological disease

with idicXq13 [1, 5–7].

On the other hand, the ATP-binding cassette subfamily

B member-7 (ABCB7) gene, which is located on the Xq13

[8], encodes a mitochondrial inner membrane transporter

K. Sato (&) � T. Hosoki � K. Ikuta � M. Yamamoto � S. Ito �N. Okamura � K. Ichiki � H. Tanaka � M. Shindo �Y. Mizukami � T. Otake � M. Fujiya � K. Sasaki � Y. Kohgo

Division of Gastroenterology and Hematology/Oncology,

Department of Medicine, Asahikawa Medical University,

Midorigaoka-Higashi 2 jo 1 chome 1-1, Asahikawa,

Hokkaido 078-8510, Japan

e-mail: satofam@asahikawa-med.ac.jp

Y. Torimoto

Oncology Center, Asahikawa Medical University Hospital,

Asahikawa, Hokkaido, Japan

H. Takahashi

Department of Medical Laboratory and Blood Center,

Asahikawa Medical University Hospital, Asahikawa,

Hokkaido, Japan

K. Hirai

Kamifurano Town Hospital, Kamifurano, Hokkaido, Japan

123

Int J Hematol (2011) 93:311–318

DOI 10.1007/s12185-011-0786-y

involved in the transport of iron from mitochondria to

cytosol and in the maturation of cytosolic iron sulfur pro-

teins [8–11]. X-linked sideroblastic anemia with ataxia

(XLSAA), which is a rare inherited Xq13-linked disorder

characterized by anemia with mitochondrial iron accumu-

lation in the bone marrow erythroblasts and spinocerebellar

ataxia [12], is caused by the defects of ABCB7 transporter

[11, 13]. Recently, it is reported that the mRNA expression

level of ABCB7 in the CD34? bone marrow cells (BMCs)

is down-regulated in RARS patients compared with other

subtypes of MDS patients [14, 15]. Moreover, ABCB7-

deficient HeLa cells by RNA silencing of the mitochondrial

ABCB7 transporter caused mitochondrial iron accumula-

tion [10]. These findings suggest that ABCB7 gene is a key

molecule of the pathogenesis of the mitochondrial iron

accumulation in both inherited and acquired sideroblastic

anemias.

The reported idicXq13 breakpoints are not uniform

[7, 16]. Rack et al. [16] demonstrated that those of two

leukemia patients with idicXq13 are at approximately

72.5 Mb on Xq13. Paulsson et al. [7] demonstrated that

those of 11 AML or MDS patients with idicXq13 are

clustering at two regions (70.9 and 72.1 Mb) on Xq13.

They speculated that loss of ABCB7 gene, which is

located at distal region (74.1 Mb) on Xq13, is possibly

occurred by deletion of the Xq13-qter and the gene loss

might contribute to mitochondrial iron accumulation in

cases with idicXq13 [7]. However, these researchers did

not directly demonstrate loss of ABCB7 gene on the

idicXq13. Moreover, it has been reported that there are

some patients with idicXq13 who do not show increased

bone marrow RSs [5], indicating a possibility that idicXq13

breakpoints are not necessarily located at proximal

lesion of ABCB7 gene on Xq13. Therefore, it is worthy to

demonstrate ABCB7 gene loss on the X-chromosome in

cases with increased bone marrow RSs such as RARS

patients with idicXq13. In addition, it is important to know

whether idicXq13 is detected on the active or inactive

X-chromosome (Xa and Xi, respectively), because in

mammalian females most genes on one of the X-chromo-

somes are transcriptionally silenced by gene dosage com-

pensation [17].

To investigate the contribution of loss of ABCB7 gene

to the RS formation in the RARS patient with idicXq13,

we performed fluorescent in situ hybridization (FISH)

analysis for the patient’s BMCs with a bacterial artificial

chromosome (BAC) DNA probe which contains the full-

length sequences to the ABCB7 gene and analyzed the

expression level of ABCB7 mRNA of the BMCs. Fur-

thermore, we determined whether loss of ABCB7 gene

exists on the Xa or Xi by FISH analysis using the ABCB7

gene defect BMCs treated with 5-bromo-2 deoxyuridine

(BrdU).

2 Materials and methods

2.1 Patient profile

A-66-year-old woman was referred to our hospital for

general malaise. A physical examination revealed anemia

of palpebral conjunctiva. A complete blood cell count

showed the following values: white blood cells 2.6 9 109/L,

without an abnormal differential; red blood cells (RBCs)

1760 9 109/L; hemoglobin 5.9 g/dL; hematocrit 19.1%;

mean corpuscular volume 108.5 fL; mean corpuscular

hemoglobin 33.5 pg; mean corpuscular hemoglobin con-

centration 30.9% and platelets 356 9 109/L. The bio-

chemistry profile revealed an elevation of the ferritin level

to 500 ng/mL (normal range 5–120) and Vitamin B12 level

to 1220 pg/mL (normal range 213–914). A bone marrow

aspiration showed hyperplastic marrow with trilinage

dysplasia (Fig. 1a–c) including less than 5% myeloblasts

and more than 60% RSs (Fig. 1d). The patient had a

diagnosis of RARS according to the World Health Orga-

nization classification [18]. At diagnosis, cytogenetic

G-banded analysis on 20 bone marrow metaphases showed

5 metaphases including idicXq13. Her disease status was

intermediate-1 with 1.0 points by International Prognostic

Scoring System of MDS [19]. She was treated with Vita-

min B6 (pyridoxine) and received packed RBC transfu-

sions. Because no improvement was seen for her anemia,

Vitamin D3 (calcitriol), Vitamin K (menatetrenone), and

anabolic steroid (stanozolol) had been administered in

place of pyridoxine. Six months later, G-banded analysis of

BMCs revealed a decrease of idicXq13 to 2 of 20 meta-

phases and she had been free from RBC transfusions

temporarily. However, pancytopenia, especially anemia,

had progressed and she had depended on RBC transfusion.

Thereafter (71 months after diagnosis), the idicXq13 in

BMCs had increased to 6 of 20 metaphases by G-banded

analysis (Fig. 2). She died from pneumonia without

recovery of her pancytopenia 9 years after diagnosis.

2.2 FISH probes

To detect ABCB7 gene by FISH analysis, a probe from

a BAC DNA (PR11-79C1, Invitrogen, Carlsbad, CA)

containing full-length sequences of ABCB7 gene was

constructed (Fig. 3a) and used by labeling with Rhoda-

mine. To detect X-chromosome, X-centromere-specific

probe CEP X SpectrumGreen (VYSIS, Princeton, NJ)

was used.

To identify ABCB7 gene defect on Xi, ABCB7-BAC

DNA probe labeled by Cy3 (Chromosome Science Labo

Inc., Sapporo, Japan) and X-centromere-specific probe

pBamX7 labeled by Cy5 (Oncor, Gaithersburg, MD) were

used. For XIST-FISH analysis, digoxigenin-labeled XIST

312 K. Sato et al.

123

DNA probe (Oncor) containing specific sequences to the

XIST gene [20] was used.

2.3 FISH analysis for ABCB7 and XIST gene

To obtain metaphase chromosomes (for ABCB7), the BMCs

of the patient were treated with colcemid (N-deacetyl-N-

methylcolchicine, NAKARAI TESQUE Inc., Kyoto, Japan)

for 30 min and were incubated for 48–72 h. The metaphase

(for ABCB7) or interphase (for XIST) BMCs were treated with

hypotonic buffer (0.075 M KCl). The chromosomal DNA of

the cells was denatured by 70% formamide at 70�C for 2 min

to fix to the slide glasses. Appropriate probe mixtures were

placed at 75�C and were added to each glass. The DNA fixed

glasses were hybridized overnight at 37�C in a humidified

chamber, and then applied with (for XIST) or without

74.0M

a b

X chromosome

q13 74.1M

74.2M

FISH probe

ABCB7 gene

PR11-79C1

(BAC clone)

X

idicXq13

normal X

Fig. 3 FISH analysis for ABCB7 gene on X-chromosome. a BAC

DNA (clone: PR11-79C1) FISH probe which contains the full-length

sequences to the ABCB7 gene. b Loss of ABCB7 gene in the bone

marrow metaphase of the idicXq13 patient. FISH analysis was

performed with a mixture of the BAC DNA probe and the

X-centromere probe. Normal X-chromosome shows a green signal

(X-centromere) and two red signals (ABCB7). IdicXq13 shows two

X-centromere green signals. ABCB7 gene-deleted idicXq13 was

detected in 34% metaphases (126 of 360 analyzed cells) and all of

them lost ABCB7 signals hemizygously. These analyses were

performed using the same sample of G-banded analysis in Fig. 2

Fig. 1 Bone marrow smears of

the patient at diagnosis.

a Myeloid dysplasias,

b erythroid dysplasias, c a

micromegakaryocyte, d ringed

sideroblasts in the bone marrow

smear. a–c May-Giemsa

staining (91000), d iron

staining (91000)

Fig. 2 G-banded analysis of the bone marrow cells. G-banded

chromosomal analysis showed abnormal karyotype of 46,X,idicXq13

(an arrow) in 6 and normal female karyotype of 46,XX in 14 of 20

analyzed metaphases

Loss of ABCB7 and idicXq13 313

123

(for ABCB7) fluorescein isothiocyanate (FITC)-labeled anti-

digoxigenin and incubated at 37�C. The chromosomes were

counterstained with 40-6-diamidine-2-phenylindole dihydro-

chloride (for ABCB7) or propidium iodide (for XIST). The

cells were viewed with a fluorescent microscope. All the FISH

analyses were performed using the same sample of G-banded

analysis in Fig. 2 (71 months after diagnosis), in which the

idicXq13 chromosome was detected in 6 of 20 metaphases.

2.4 Identification of Xi in ABCB7 gene defect BMCs

To identify the Xi in ABCB7 defect cells, we followed the

methods to identify the Xi, which is more chromogenic than

the Xa, as previously described [21]. In brief, 48-h-cultured

BMCs were pre-incubated with BrdU for 5.5 h before

induction of metaphase. Afterward, G-banded metaphase

chromosomes were stained with Hoechst 33258 and processed

by ultraviolet light. Then ABCB7-BAC and X-centromere-

specific probes were applied. Induction of metaphases, sample

denaturing or -fixation, probe hybridization, and detection of

the genes were performed by the same methods as described in

Sect. 2.3. These analyses were performed using the same BMC

sample of G-banded analysis in Fig. 2.

2.5 Quantitative RT-PCR for ABCB7 gene

Total RNA was isolated from the BMCs of the present

RARS patient with idicXq13, patients without idicXq13,

refractory anemia with excess blasts (RAEB), and non-

Hodgkin lymphoma (NHL) patient with normal bone

marrow as a control. Quantitative RT-PCR (qRT-PCR) was

performed in reaction mix containing TaqMan Universal

PCR Master Mix No AmpErase UNG (Applied Biosystems),

specific human ABCB7 primers, and probe (pre-validated

Taqman gene expression assay, Applied Biosystems), and

150 ng of cDNA. All reactions were multiplexed with the

housekeeping gene 18S, provided as a pre-optimized

control probe (Applied Biosystems) enabling data to be

expressed as delta threshold (DCt) values (DCt = Ct of

18s subtracted from Ct of gene of interest). All mea-

surements were performed in triplicate and relative

ABCB7 mRNA expression was described as fold expres-

sion over the average of ABCB7 mRNA expression in the

BMCs in the NHL patient.

3 Results

3.1 FISH analysis for ABCB7 gene on X-chromosome

To clarify loss of ABCB7 in BMCs of the patient with

idicXq13, we firstly performed metaphase FISH analysis

for the BMCs of the patient using BAC DNA probe con-

taining full-length DNA sequences of the ABCB7 gene. As

shown in Fig. 3b, normal X-chromosome shows a green

signal (X-centromere) and two red signals (ABCB7).

Meanwhile, on the idicXq13, which shows two X-centro-

mere green signals, loss of ABCB7 red signals was con-

firmed. IdicXq13 was detected in 126 of 360 analyzed

metaphases (34%) and all of them lost ABCB7 signals

hemizygously.

3.2 ABCB7 expression levels in the BMCs

in the patients with or without idicXq13

To confirm the level of ABCB7 transcript, we determined

ABCB7 mRNA level in the present case (sample #2a and

2b) and those in the patients without idicXq13 (#1, NHL;

#3, RAEB). The characteristics of the three patients are

shown in Fig. 4a. As shown in Fig. 4b, ABCB7 expression

ratio in the BMCs in the present case, whose RSs were 50%

(#2a) and 63% (#2b), respectively, was significantly lower

than those in the NHL patient (*p \ 0.05, #2a vs. #1;

**p \ 0.01, #2b vs. #1; t test) or the RAEB patient

(**p \ 0.01, #2a vs. #3; ***p \ 0.0001, #2b vs. #3; t test).

In addition, the expression of ABCB7 mRNA tended to be

negatively correlated with number of idicXq13 metaphases

(Fig. 4a, b). These findings indicate that the decreased

transcript level of ABCB7 originated from idicXq13 for-

mation would contribute to the pathogenesis of mitochon-

drial iron accumulation of the erythroblasts in the present

RARS patient.

3.3 Identification of Xi in ABCB7 defect cells

In mammalian females including human, it is known that

most genes on either X-chromosome are transcriptionally

silenced as a result of the X-chromosome inactivation [16].

To confirm whether loss of ABCB7 gene on the idicXq13 is

involved with Xi or Xa, we further performed FISH ana-

lysis using the same BAC DNA probe for the BMCs added

by BrdU at middle S-phase to identify Xi. Normal

X-chromosome has a red signal (X-centromere) and yellow

signals (ABCB7) (Fig. 5a, b), and idicXq13 has only two

red signals (Fig. 5b). As shown in Fig. 5a, in normal

karyotype BMCs (81 of 100 analyzed metaphases), Xi is

obviously identified as a more chromogenic chromosome

than Xa, because Xi starts DNA synthesis at late S-phase so

that the level of BrdU intake is high. Interestingly, idi-

cXq13, which was detected 19 of 100 analyzed metapha-

ses, as well as normal X-chromosome was Xa in all the

ABCB7 defect BMCs (Fig. 5b). The result indicates that

both X-chromosomes are activated in the ABCB7 defect

BMCs.

314 K. Sato et al.

123

3.4 FISH analysis for XIST gene on X-chromosome

XIST gene, which induces the inactivation of one of the

X-chromosomes, is reported to exist on 0.7–2.2 Mb distal

region of the breakpoints in the idicXq13 [22] and only 1 Mb

proximal region of ABCB7 gene on the X-chromosome [7].

From the result of Fig. 4b, XIST gene is possibly lost on the

idicXq13 because we did not detect any Xis on ABCB7 defect

BMCs. We therefore investigated whether XIST gene on the

idicXq13 in the patient’s BMCs is lost. We firstly tried to

perform metaphase FISH analysis. However, we could not

get enough metaphases so that interphase FISH analysis was

substituted. As a result, single XIST signal was detected in

19% of interphase BMCs (Fig. 6) and double signals in 81%

(data not shown), indicating that 19% of analyzed BMCs of

the patient would hemizygously lost XIST gene. From FISH

analysis results that the percentage (19%) of ABCB7 defect

Xa in the patient’s BMCs and that (19%) of XIST defect

BMCs are equal, it is speculated that loss of both XIST and

ABCB7 gene would occur simultaneously at the formation of

the idicXq13 and active X-chromosome.

4 Discussion

In the present study, we described a female RARS patient

with idicXq13 and demonstrated loss of ABCB7 gene by

FISH analysis using a probe of BAC DNA for ABCB7 gene

and the decreased expression level of ABCB7 mRNA in the

patient’s BMCs. We further showed that the ABCB7 gene

loss was definitely detected on the Xa. These findings

indicate that acquired loss of ABCB7 gene by the deletion

of Xq13-qter at the idicXq13 formation would contribute

to a mitochondrial iron accumulation in the patient’s

erythroblasts.

From the FISH analyses, we demonstrated that ABCB7

defect in the idicXq13-BMCs was detected on the Xa and

the BMCs had an another Xa (a normal X-chromosome).

Furthermore, we indicated the possibility that an XIST

gene, which induces an X-chromosome inactivation, would

also be lost in the idicXq13-BMCs. These findings are

compatible with two previous reports [6, 7] describing

patients with active idicXq13. Though it is still a matter of

speculation, loss of a XIST by the formation of idicXq13

b

* *

*

p < 0.01

p < 0.05

a b

* * * p < 0.0001

* * *1.4

* * * * *

1

1.2

0.6

0.8

AB

CB

7 e

xpre

ssio

n r

atio

0

0.2

0.4

sample #1 2a 2b 3

Fig. 4 Expressions of ABCB7 in BMCs in the patients with or

without idicXq13. qRT-PCR for ABCB7 gene was performed using

cDNA from bone marrow cells (BMCs) of the present RARS patient

with idicXq13 or patients without idicXq13. a The characteristics of

the three patients used in this analysis. Chromosomal status was

determined by G-banded analysis. Ringed sideroblasts (RSs) were

defined as the erythroblasts which have both characteristics of more

than 5 iron granules stained by Prussian blue and a ringed appearance

around at least 30% of the nucleus [27]. b ABCB7 mRNA expression

levels in the BMCs in the patients with or without idicXq13. The

mRNA expression levels were standardized by 18S rRNA. Relative

ABCB7 mRNA expression levels are shown as fold expression over

the average of ABCB7 mRNA expression in the BMCs in the NHL

patient as a control (sample #1). Sample 1, 2a, 2b, 3 correspond to the

sample #1, 2a, 2b, 3 in a, respectively. ABCB7 expression ratio in the

BMCs in the present case, whose RSs were 50% (#2a) and 63% (#2b),

respectively, was significantly lower than those in the NHL patient

(*p \ 0.05, #2a vs. #1; **p \ 0.01, #2b vs. #1; t test) or the RAEB

patient (**p \ 0.01, #2a vs. #3; ***p \ 0.0001, #2b vs. #3; t test).

Results in b show a representative experiment in triplicate. Data

represent mean ± SD. RARS indicates refractory anemia with ringed

sideroblasts; RAEB refractory anemia with excess blasts, NHL non-

Hodgkin lymphoma, RSs ringed sideroblasts, F female, M male,

idicXq13 isodicentric (X)(q13)

Loss of ABCB7 and idicXq13 315

123

might contribute to the occurrence of two Xas in the

idicXq13-BMCs.

We assume mitochondrial iron accumulation in this

patient’s erythroblasts as follows: by the formation of two

Xas in the idicXq13-BMCs, total gene dosage of the entire

X-chromosomes would be increased; therefore, chromo-

some-wide transcription levels are possibly decreased

according to the mechanism of gene dosage compensation

[23], then the total expression level of ABCB7 protein

might be decreased; in consequence, total mitochondrial

ABCB7 transporters would be decreased, and then reduc-

tion of the iron transport from the mitochondria to the

cytosol causes the iron accumulation in the mitochondria of

the erythroblasts.

Some gene mutations as pathogenesis, such as ALAS2

(d-aminolevulinate synthase-2) [24] in XLSA, ABCB7 [11,

13] in XLSA/A, and PUS1 (pseudouridine synthase-1) in

mitochondrial myopathy and sideroblastic anemia (MLA-

SA) [25], have been identified for inherited sideroblastic

anemias. However, molecular pathogenesis of acquired

sideroblastic anemias including RARS is not fully eluci-

dated. Most recently, a few findings concerning this issue

have been reported [14, 15]. Boultwood et al. [15] analyzed

122 MDS patients and demonstrated that mRNA expres-

sion levels of ABCB7 in RARS patients not only in the

CD34? BMCs, but also in cultured erythroblasts were

significantly lower than those in other subtypes of MDS

patients. Interestingly, they also indicated the relationship

between increased rate of bone marrow RSs and decreased

expression levels of ABCB7 [15]. These findings support

our results that ABCB7 mRNA levels in the BMCs in the

Fig. 5 Identification of inactive X-chromosome (Xi) on the ABCB7gene defect cells. Metaphase FISH analysis in the patient’s bone

marrow cells (BMCs) with idicXq13 was performed with a mixture of

the BAC DNA (PR11-79C1) probe, and the X-centromere probe after

treatment the bone marrow cells with the BrdU. Normal X-chromo-

some shows a red signal (X-centromere) and yellow signals (ABCB7)

(a, b), and idicXq13 shows only two red ABCB7 signals (b).

a Identification of Xi in the normal BMCs. An Xi is shown more

chromogenic than an active X-chromosome (Xa). b State of activation

of X-chromosomes on the ABCB7 gene defect BMCs. Both normal

X-chromosome and idicXq13, which shows ABCB7 gene defect, are

not chromogenic Xas. These analyses were performed using the same

sample of G-banded analysis in Fig. 2

Fig. 6 FISH analysis for XIST gene on X-chromosome. Interphase

FISH analysis for the patient with idicXq13 was performed with

digoxigenin-labeled XIST DNA probe, which contains specific

sequences to the XIST gene. A green signal (an arrow) shows XISTgene. This analysis was performed using the same sample of

G-banded analysis in Fig. 2

316 K. Sato et al.

123

RARS patient with idicXq13, who had more RSs, were

lower than those in the patients without idicXq13, who had

fewer RSs. On the other hand, Pondarre et al. [11] dem-

onstrated that an emergence of the siderocytic reticulocytes

in the peripheral blood and severe pancytopenia accom-

panied by markedly hypocellular marrow were revealed in

the ABCB7 conditional knockout mice, indicating ABCB7

is essential not only for erythropoiesis but also for total

hematopoiesis. These basic and clinical findings not only

support our hypothesis that loss of ABCB7 gene would be

responsible for the increased RSs in the RARS patient with

idicXq13, but also can be a part of explanations of our

patient’s progressed pancytopenia with an increase of idi-

cXq13 metaphases.

A mechanism of idicXq13 formation is thought as fol-

lows: firstly, DNA-break Xq13 is occurred during S-phase

and then the broken sister chromatids are fused at Xq13;

finally, normal centromere separation of the fused chro-

mosome produces idicXq13 [1]. Importantly, as a result of

idicX formation, certain tumor suppressor gene as well as

ABCB7 on the Xq13-qter would be lost. Interestingly, all

the reported cases with idicXq13 including ours were

hematopoietic stem cell malignancies, such as MDSs,

AMLs, and MPNs, though approximately 40 cases have

been reported to date [1–7]. It is noteworthy that tumor

suppressor gene RPL10 encoded at the Xq28, which is

located on the Xq13-qter, has been reported down-regu-

lated gene expression levels in some human hepatocellular

carcinoma cell lines [26]. Though loss of the gene was not

investigated in our patient, such gene loss might contribute

to the oncogenesis of the hematological malignancies with

idicXq13.

Although metaphases carrying idicXq13 are minor

population in this patient, 60% of erythroblasts were sid-

eroblasts. In the present study, the ratio of metaphases with

idicXq13, which was almost the same ratio of ABCB7

defect metaphases, was determined by G-banded analysis

from a proportion in the whole bone marrow mononuclear

cells (MNCs) including erythroid precursors. Though it is

still a matter of speculation, the MNCs in which idicXq13

formation occurred may tend to differentiate into the ery-

throblasts. As a result, the proportion of RS would be more

than that of metaphases carrying idicXq13 by G-banded

analysis.

Inherited sideroblastic anemia XLSA/A, which caused

by a single missense mutation of ABCB7, exhibits micro-

cytic anemia [11]. As a result of mitochondrial iron accu-

mulation due to impairment of iron transport from

mitochondria to cytosol, defective heme synthesis caused

by cytosolic iron shortage occurs then the erythrocyte

volume would be reduced [10]. On the other hand, cases of

RARS with idicXq13, including the present case, have

been reported to exhibit macrocytic anemia [1, 3]. In this

patient with deletion of Xq13-qter by idicXq13 formation,

the dyserythropoiesis cannot be explained only by the

deletion of single ABCB7 gene. In addition to this gene

loss, other genetic or epigenetic alterations involved in

erythroblast maturation may have occurred like in other

MDS patients. As a result, macrocytic erythrocytes in this

patient may have been predominantly generated.

In conclusions, we demonstrated the loss of ABCB7

gene by FISH analyses and the decreased expression level

of ABCB7 mRNA in the BMCs in a RARS patient with

idicXq13 and loss of the gene perhaps contributes to the

pathogenesis of mitochondrial iron accumulation of the

erythroblasts in this patient. Although further investiga-

tions are needed for the deletion status of ABCB7 in the

BMCs of the patients with increased RSs, loss of ABCB7

gene can be one of the mechanisms of RS formation in

RARS.

References

1. Dewald GW, Pierre RV, Phyliky RL. Three patients with struc-

turally abnormal X chromosomes, each with Xq13 breakpoints

and a history of idiopathic acquired sideroblastic anemia. Blood.

1982;59:100–5.

2. Petit P, Fryns JP, Masure R, Berghe VH. Isodicentric (X)(q13): a

new characteristic chromosomal anomaly in myeloproliferative

syndrome? Cancer Genet Cytogenet. 1982;7:339–47.

3. Morgan RJ, Milligan DW, Williams J. Isodicentric X chromo-

some in a patient with myelodysplastic syndrome. Cancer Genet

Cytogenet. 1987;27:215–8.

4. Mackinnon WB, Michael PM, Webber LM, Garson OM. Isodi-

centric X chromosome involving the Xq13 breakpoint in mye-

lodysplasia and acute nonlymphocytic leukemia. Cancer Genet

Cytogenet. 1988;30:43–52.

5. Dewald GW, Brecher M, Travis LB, Stupca PJ. Twenty-six

patients with hematologic disorders and Xq13 anomalies asso-

ciated with pathologic ringed sideroblasts. Cancer Genet Cyto-

genet. 1989;42:173–85.

6. Judith D, Lucinne M, Arnold C, et al. Isodicentric (X)(q13) in

haematological malignancies: presentation of five new cases,

application of fluorescence in situ hybridization (FISH) and

review of the literature. Br J Haematol. 1995;91:885–91.

7. Paulsson K, Haferlach C, Fonatsch C, et al., on behalf of the

MDS Foundation. The idic(X)(q13) in myeloid malignancies:

breakpoint clustering in segmental duplications and association

with TET2 mutation. Hum Mol Genet. 2010. [Epub ahead of

print].

8. Shimada Y, Okuno S, Kawai A, et al. Cloning and chromosomal

mapping of a novel ABC transporter gene (hABC7), a candidate

for X-linked sideroblastic anemia with spinocerebellar ataxia.

J Hum Genet. 1998;43:115–22.

9. Bekri S, Kispal G, Lange H, et al. Human ABC7 transporter: gene

structure and mutation causing X-linked sideroblastic anemia

with ataxia with disruption of cytosolic iron-sulfur protein mat-

uration. Blood. 2000;96:3256–64.

10. Cavadini P, Biasiotto G, Poli M, et al. RNA silencing of the

mitochondrial ABCB7 transporter in HeLa cells causes an iron-

deficient phenotype with mitochondrial iron overload. Blood.

2007;109:3552–9.

Loss of ABCB7 and idicXq13 317

123

11. Pondarre C, Campagna DR, Antiochos B, et al. Abcb7, the gene

responsible for X-linked sideroblastic anemia with ataxia, is

essential for hematopoiesis. Blood. 2007;109:3567–9.

12. Raskind WH, Wijsman E, Pagon RA, et al. X-linked sideroblastic

anemia and ataxia: linkage to phosphoglycerate kinase at Xq13.

Am J Hum Genet. 1991;48:335–41.

13. Allikmets R, Raskind WH, Hutchinson A, et al. Mutation of a

putative mitochondrial iron transporter gene (ABC7) in X-linked

sideroblastic anemia and ataxia (XLSA/A). Hum Mol Genet.

1999;8:743–9.

14. Malcovati L, Della Porta MG, Pietra D, et al. Molecular and clinical

features of refractory anemia with ringed sideroblasts associated

with marked thrombocytosis. Blood. 2009;114:3538–45.

15. Boultwood J, Pellagatti A, Nikpour M, et al. The role of the iron

transporter ABCB7 in refractory anemia with ring sideroblasts.

PLoS One. 2008;3:e1970.

16. Rack KA, Chelly J, Gibbons RJ, et al. Absence of the XIST gene

from late-replicating isodicentric X chromosomes in leukaemia.

Hum Mol Genet. 1994;3:1053–9.

17. Heard E. Recent advances in X-chromosome inactivation. Curr

Opin Cell Biol. 2004;16:247–55.

18. Harris NL, Jaffe ES, Diebold J, et al. The World Health Orga-

nization classification of neoplastic diseases of the hematopoietic

and lymphoid tissues. Report of the Clinical Advisory Committee

meeting, Airlie House, Virginia, November, 1997. Ann Oncol.

1999;10:1419–32.

19. Greenberg P, Cox C, LeBeau MM, et al. International scoring

system for evaluating prognosis in myelodysplastic syndromes.

Blood. 1997;89:2079–88.

20. Willard HF, Breg WR. Human X chromosomes: synchrony of

DNA replication in diploid and triploid fibroblasts with multiple

active or inactive X chromosomes. Somatic Cell Genet. 1980;6:

187–98.

21. Brown CJ, Ballabio A, Rupert JL, Ronald G, et al. A gene from the

region of the human X inactivation centre is expressed exclusively

from the inactive X chromosome. Nature. 1991;349:38–44.

22. Payer B, Lee JT. X chromosome dosage compensation: how

mammals keep the balance. Annu Rev Genet. 2008;42:733–72.

23. Lucchesi JC, Kelly WG, Panning B. Chromatin remodeling in

dosage compensation. Annu Rev Genet. 2005;39:615–51.

24. Fleming MD. The genetics of inherited sideroblastic anemias.

Semin Hematol. 2002;39:270–81.

25. Casas K, Bykhovskaya Y, Mengesha E, et al. Gene responsible for

mitochondrial myopathy and sideroblastic anemia (MSA) maps to

chromosome 12q24.33. Am J Med Genet A. 2004;127:44–9.

26. Yoon SY, Kim JM, Oh JH, et al. Gene expression profiling of

human HBV- and/or HCV-associated hepatocellular carcinoma

cells using expressed sequence tags. Int J Oncol. 2006;29:315–27.

27. Greenberg PL, Young NS, Gattermann N. Myelodysplastic syn-

dromes. Hematology Am Soc Hematol Educ Program. 2002;1:

136–61.

318 K. Sato et al.

123