Blood, 15 December 2000, Vol. 96, No. 13, pp. 4363-4365
BRIEF REPORT
Familial-skewed X-chromosome inactivation as a predisposing
factor for late-onset X-linked sideroblastic anemia in
carrier females
Mario Cazzola,
Alison May,
Gaetano Bergamaschi,
Paola Cerani,
Vittorio Rosti, and
David F. Bishop
From the Department of Hematology and the
Department of Internal Medicine and Medical Therapy, University of
Pavia Medical School, and Instituto di Ricovero e Cura a Carattere
Scientifico (IRCCS) Policlinico S. Matteo, Pavia, Italy; the Department
of Haematology, University of Wales College of Medicine, Cardiff,
Wales; and the Department of Human Genetics, Mount Sinai School of
Medicine, New York, NY.
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Abstract |
X-linked sideroblastic anemia (XLSA) is caused by mutations in the
erythroid-specific 5-aminolevulinic acid synthase
(ALAS2) gene. An elderly woman who
presented with an acquired sideroblastic anemia is studied. Molecular
analysis revealed that she was heterozygous for a missense mutation in
the ALAS2 gene, but she expressed only the mutated gene in
reticulocytes. Her 2 daughters and a granddaughter were heterozygous
for this mutation, had normal hemoglobin levels, and expressed the
normal ALAS2 gene in reticulocytes. A grandson with a
previous diagnosis of thalassemia intermedia was found to be hemizygous
for the ALAS2 mutation. Treatment with pyridoxine completely corrected the anemia both in the proband and her grandson. All women who were analyzed in this family showed skewed X-chromosome inactivation in leukocytes, which indicated a hereditary condition associated with unbalanced lyonization. Because the preferentially active X chromosome carried the mutant ALAS2 allele,
acquired skewing in the elderly likely worsened the genetic condition
and abolished the normal ALAS2 allele expression in the proband.
(Blood. 2000;96:4363-4365)
© 2000 by The American Society of Hematology.
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Introduction |
X-linked sideroblastic anemia (XLSA) is caused by
mutations (primarily missense) in the erythroid-specific
5-aminolevulinic acid synthase (ALAS2) gene.1
Affected hemizygous males may present in the first 2 decades of life
with symptoms of anemia or in middle age with manifestations of
secondary iron overload.2 The majority of heterozygous
females have no clinical signs because immature red blood cells (RBCs)
expressing the normal ALAS2 are sufficient to sustain a
normal level of RBC production. As in any X-linked disorder, however,
the clinical phenotype of female carriers may be influenced by
the pattern of X-chromosome inactivation (or
lyonization).3 Different genetic mechanisms may lead to a
skewed pattern of X-chromosome inactivation in
females.4,5 In addition, recent studies have
shown that skewed lyonization can also be an acquired pattern in
hematopoietic cells.6-9
Late-onset XLSA has been already described.10 By studying
the molecular basis for late-onset XLSA in an elderly woman, we report
here a potential mechanism involving late-onset X-linked disorders in
female obligate carriers of X-linked hematopoietic disorders.
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Study design |
The proband is a 72-year-old female of Italian ancestry. This
woman was an active farm worker until her forties. At the age of 36 her
hemoglobin (Hb) level was normal (Table
1). She later presented at the age of 64 with breathlessness and fatigue and was found to have severe microcytic
anemia, with a Hb level of 52 g/L (5.2 g/dL; reference range,
120-160 g/L [12-16 g/dL] and a mean cell volume (MCV) of 74 fL
(reference range, 83-97 fL). A presumptive diagnosis of
myelodysplastic syndrome (refractory anemia with ringed sideroblasts)
was made, and a transfusion therapy (3-4 units per month) was started.
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Table 1.
Hematological data, ALAS2 exon 9 sequence (from 1230 to
1240 nucleotide), and results of X-chromosome inactivation studies
using HUMARA assay in the family members
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When we saw the proband at the age of 71 years, her serum
ferritin level was 3954 µg/L (female reference range, 12-200 µg/L) and transferrin saturation was 96% (female reference range, 15% to
45%), which indicated severe iron overload. She was treated with 4000 U/d subcutaneous (sc) recombinant human erythropoietin for 5 days a
week and 300 mg/d oral pyridoxine. Four weeks later, her Hb level had
increased from 77 to 176 g/L (7.7 to 17.6 g/dL); administration of
erythropoietin was immediately discontinued, while oral pyridoxine was
maintained. Hb levels stabilized between 110 and 120 g/L (11 and 12 g/dL) with no transfusion requirement, and MCV values ranged from 80-85 fL. Iron chelation therapy with sc deferoxamine was started.
Based on both RBC microcytosis and pyridoxine
responsiveness, we hypothesized that this patient had late-onset
X-linked sideroblastic anemia.10 A careful review
of family history revealed that an anemic grandson had been
presumptively diagnosed with thalassemia intermedia. This 14-year-old
boy was given pyridoxine and folic acid, although there was no evidence
of folate deficiency, and his Hb level normalized, while MCV values
remained low (Table 1). He was still following his daily course of 300 mg pyridoxine when we saw him at the age of 29.
The procedures followed were in accordance with the ethical
standards of the institutional committee on human experimentation and
with the Helsinki Declaration of 1975, as revised in 1983. Molecular
analysis of the ALAS2 gene was performed as previously described.2 Analysis of X-chromosome inactivation was
performed as previously described in detail, with minor
modifications.8 Relative expression of mutant and
wild-type ALAS2 messenger RNAs (mRNAs) was evaluated in peripheral
blood reticulocytes from the proband and her relatives.11
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Results and discussion |
The pedigree of the proband's family is shown in Figure
1A. A single-point mutation in exon 9 of
the ALAS2 gene was found in 5 family members (Table 1). This
is a transition from G to A at nucleotide 1236 that predicts an amino
acid change of cysteine to tyrosine at position 395 (TGT
TAT; C395Y).
Position 395 is very close to the lysine at position 391, which forms
the Schiff base with the PLP aldehyde group. The point mutation might
therefore interfere with binding of PLP or with the catalytic reaction
itself. All 4 women studied, including the proband, were heterozygous for this exon 9 mutation (Table 1). The grandson had the mutation and
therefore was a typical hemizygote. Studies on 200 normal alleles
indicated that the transition from G to A at the 1236 nucleotide is not
a polymorphism.
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| Figure 1.
Molecular analysis of ALAS2 and clonal analysis of
hematopoiesis.
(A) Pedigree of the family. Circles denote female family members;
squares, male family members; and symbols with diagonal lines, deceased
members. The proband is denoted by an arrow. All women were
heterozygotes for the ALAS2 mutation; the only available
male was hemizygote. (B) Clonal analysis of hematopoiesis using HUMARA
assay on DNA from peripheral blood leukocytes. The  and + signs indicate sample aliquots undigested () or digested (+) with the
methylation-sensitive restriction endonuclease HpaII.
HUMARA alleles (indicated by arrow) are represented by the
lower band in the proband's sample (I-2, homozygous woman), and by the
2 lower bands in the other samples (heterozygous women). Upper bands
probably derive from intrastrand secondary structures due to the high
G + C content. In HpaII digested samples from all the
heterozygous women, the lower allele (paternally derived) is amplified
more than the upper allele (maternally derived) compared with
undigested samples. This indicates that the maternally derived
HUMARA allele (carrying the mutant ALAS2) was
less methylated (more active) and more digested by HpaII
than the paternally derived allele. In conclusion, unbalanced
X-chromosome inactivation, leading to prevalent inactivation of the
paternally derived chromosome (carrying the normal ALAS2
allele), occurred in hematopoietic cells from these women. (C)
Wild-type and mutant ALAS2 mRNA expression in reticulocytes from 4 family members. RNA was isolated from peripheral blood reticulocytes
and reverse transcribed into cDNA; arrows indicate nucleotide 1236 of
ALAS2 cDNA. III-1 (hemizygous grandson): As expected, only mutant cDNA
(carrying A [adenine] at position 1236) was amplified. I-2 (proband):
Only mutant cDNA was amplified, indicating that most of the
reticulocyte ALAS2 mRNA derived from the mutant allele. II-2 (elder
daughter): Only wild-type cDNA (carrying [guanine] at position 1236)
was amplified, indicating that most of the reticulocyte ALAS2 mRNA
derived from the wild-type allele. III-2 (granddaughter): Both
wild-type and mutant cDNA were amplified. (N indicates the ambiguity
resulting from overlapping signals corresponding to both guanine
and adenine.)
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Analysis of DNA from peripheral blood leukocytes for
polymorphisms at 2 X chromosome loci showed a status of homozygosity for all the loci examined: human androgen receptor (HUMARA),
phosphoglycerate kinase (PGK), and DXS255 (probe M27
) (Figure
1B, HUMARA assay). This prevented clonal analysis of hematopoiesis to
be carried out in the proband. The initial screening at the HUMARA
locus revealed that the proband's daughters and granddaughter were
heterozygous (Figure 1B). All 3 women had skewed X-chromosome
inactivation because their cleavage ratios between alleles ranged from
3.2 to 4.0 (Table 1), and the preferentially active X chromosome carried the mutant ALAS2 allele.
Sequence analysis of complementary DNA (cDNA) derived from reticulocyte
RNA (Figure 1C) revealed that the proband and her grandson (both under
pyridoxine treatment) expressed only the mutated ALAS2
allele, with the G to A transition at nucleotide 1236. On the contrary,
the elder heterozygous daughter expressed exclusively the wild-type
allele, and the granddaughter expressed both the wild-type and mutated
alleles. No female in the family exhibited the cytosine to guanine
mutation in the XIST minimal promoter found by Plenge et
al.5 This does not exclude the possibility of a different
mutation in the same gene involving XIST underexpression and
preferential inactivation of the X chromosome carrying such mutation.
This case is interesting not only because the patient is a female and
XLSA, as all the X-linked recessive diseases, normally affects
hemizygous males, but also because the anemic condition was acquired in
spite of the fact that the ALAS2 defect was present from
birth. In addition, skewed lyonization in leukocytes with preferential
inactivation of the X chromosome carrying the normal ALAS2
allele was found in all women examined, which clearly indicates that
congenital skewing was also present in this family.12 The available evidence suggests that late in this woman's life, an additional event following inheritance of the ALAS2 mutation
and congenital skewing led to dominance of hematopoietic cells
expressing the X chromosome with the mutant gene.
The most likely explanation of the above findings is that the
proband, despite a markedly unbalanced X-chromosome inactivation in her
hematopoietic cells, was able to produce normal amounts of RBCs for the
first 6 decades of her life, as do her daughters and granddaughter. In
the seventh decade she developed acquired skewing, as do approximately
one-third of elderly women.13 She unfortunately further
inactivated the parental X chromosome carrying the normal
ALAS2 gene, and when nearly all RBC precursors
expressed the mutant gene, she became severely anemic.
A search of the literature reveals that at least 8 cases of late-onset
pyridoxine-responsive sideroblastic anemia have been described
(including the present case),14-17 and 7 of these patients are women. Interestingly, Aivado et al17 have recently
described a family from Germany with a proven ALAS2 mutation
present in the mother and 2 of her 3 daughters. Excessively skewed
lyonization was found in all women, and 2 of them developed microcytic
anemia. Therefore, a combination of congenital and acquired skewing was likely present also in this family, although age-dependency was less clear.
Clinicians should be alerted to the possibility that an elderly woman
may present with manifestations of X-linked hematopoietic disorders. In
addition to XLSA, these may include glucose-6-phosphate dehydrogenase
deficiency,18 X-linked agammaglobulinemia or
hyper-immunoglobulin (Ig) M (hyper-IgM) syndrome,19 severe
combined immunodeficiency,20 and chronic granulomatous
disease.21,22
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Acknowledgments |
We thank Joyce Hoy and Barrie Francis, Department of Medical
Biochemistry, University of Wales College of Medicine, Cardiff, Wales,
for their help with the ABI 377 electrophoresis and analysis.
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Footnotes |
Supported by a grant from MURST, Rome, Italy; a grant from the IRCCS
Policlinico S. Matteo and a grant (M.C.) from the Fondazione Ferrata
Storti, Pavia, Italy; grant R01 DK40895 (D.F.B.) from the National
Institutes of Health, Bethesda, MD; and grant 584 (D.F.B.)
from the March of Dimes Birth Defects Foundation.
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.
Reprints: Mario Cazzola, Division of Hematology, IRCCS
Policlinico S. Matteo, 27100 Pavia, Italy; e-mail: m.cazzola{at}iol.it.
| |
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Abstract
Introduction
