Blood, 1 December 2002, Vol. 100, No. 12, pp. 4236-4238
BRIEF REPORT
Absent phenotypic expression of X-linked sideroblastic anemia in
one of 2 brothers with a novel ALAS2 mutation
Mario Cazzola,
Alison May,
Gaetano Bergamaschi,
Paola Cerani,
Sara Ferrillo, and
David F. Bishop
From the Department of Hematology and the Department of
Internal Medicine and Medical Therapy, University of Pavia Medical
School, and Istituto 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.
 |
Abstract |
X-linked sideroblastic anemia (XLSA) is caused by mutations in the
erythroid-specific 5-aminolevulinic acid synthase (ALAS2) gene. Hemizygous males have microcytic anemia and iron overload. A
38-year-old male presented with this phenotype (hemoglobin
[Hb] 7.6 g/dL, mean corpuscular volume [MCV] 64 fL, serum ferritin 859 µg/L), and molecular analysis of ALAS2 showed a
mutation 1731G>A predicting an Arg560His amino acid change. A
36-year-old brother was hemizygous for this mutation and expressed the
mutated ALAS2 mRNA in his reticulocytes, but showed almost
no phenotypic expression. All 5 heterozygous females from this family,
including the 3 daughters of the nonanemic hemizygous male, showed
marginally increased red-cell distribution width (RDW). Although
variable penetrance for XLSA in males has been previously described,
this is the first report showing that phenotypic expression can be
absent in hemizygous males. This observation is relevant to genetic
counseling, emphasizing the importance of gene-based diagnosis.
(Blood. 2002;100:4236-4238)
© 2002 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.1,2
Affected males usually present in the first 2 decades of life with
symptoms of anemia, and in middle age with manifestations of secondary
iron overload. Phenotypic expression of XLSA varies considerably in
males, and is partly related to the type of ALAS2 mutation,
with more than 25 mutations described so far in more than 30 kindreds.3,4 In addition, modifying genes such as those
for genetic hemochromatosis may significantly exacerbate XLSA in
hemizygous males.3 On the other hand, genetic and acquired
factors responsible for skewed X-chromosome inactivation may lead to
late-onset XLSA in heterozygous females.5,6
Whereas the existence of modifying genes capable of worsening XLSA is
well established, little information is available on genetic factors
that may attenuate or suppress its phenotypic expression.1
By studying a family with a new ALAS2 mutation, we provide
evidence that phenotypic expression of XLSA may be absent in hemizygous males.
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Study design |
The proband (subject II-2 in Figure
1A) was found to have microcytic anemia
(hemoglobin [Hb] level 7.6 g/dL; mean corpuscular volume [MCV] 64 fL) with high red-cell distribution width (RDW) (30.9%) at the age of
38. Serum ferritin was raised, transferrin was 82% saturated, and 30%
of the patient's bone marrow erythroblasts were ring sideroblasts. A
diagnosis of XLSA was made. On treatment with 300 mg/d pyridoxine,
hemoglobin level averaged around 10 g/dL. Iron chelation therapy
with deferoxamine (DFO) normalized serum ferritin.
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| Figure 1.
Most relevant clinical and molecular findings in
the family studied.
(A) Pedigree of the family studied. Circles denote female family
members; squares, male family members; and diamonds, additional members
of either sex (the number of additional members is shown in the
diamonds). Symbols with diagonal lines indicate deceased members.
Filled squares indicate hemizygous males; half-filled circles indicate
heterozygous women. (B) Red cell volume histograms obtained with a
Bayer Technicon H3 in 3 family members. The vertical tick marks
correspond to 10 fL increments. Subject II-7 shows a typical normal
picture. Subject II-4 is a typical heterozygous woman with a small
proportion of microcytic red cells (tail on the left side of the
histogram). The nonanemic hemizygous brother (subject II-3) shows a red
cell volume histogram that is almost normal. (C) Dideoxy dye terminator
sequence analysis of polymerase chain reaction-amplified, exon 11 genomic DNA from a healthy individual (NL), the proband (II-2),
and his brother (II-3). (D) Sequence analysis of reverse transcribed
ALAS2 mRNA expressed in reticulocytes from the anemic (II-2)
and nonanemic (II-3) hemizygous brothers.
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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. Routine hematologic
measurements were obtained using a Bayer Technicon (Milan, Italy) H3
automatic cell analyzer. For evaluation of potential thalassemia
defects, standard procedures were used.7 Molecular analyses of the HFE and ALAS2 genes were performed as
previously described.3 The expression of mutant
ALAS2 messenger RNA (mRNA) was evaluated in peripheral blood
reticulocytes from the proband and his brother, as previously
described.6,8
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Results and discussion |
The pedigree of the proband's family is shown in Figure 1A, while
hematologic and iron status data for the family members are reported in
Table 1.
A single point mutation in exon 11 of the ALAS2 gene was
found in the proband (Table 1 and Figure 1C), after all exons,
intron-exon boundaries, and the 5' and 3' flanking regions were
sequenced in both directions.3 The only mutation found was
a transition from G to A at nucleotide 1731 that predicts an amino acid
change of a conserved arginine to the more compact histidine at
position 560 (Arg560His). There were 2 males (including the proband)
who were hemizygous, and 5 females, including the 3 obligate
heterozygote daughters of the nonanemic hemizygous male (II-3), who
were heterozygous for this mutation (Table 1). These results were
independently obtained in 2 different laboratories and the mutation was
confirmed in a third. In addition, sequence analysis of cDNA derived
from reticulocyte RNA revealed that both the proband and his brother expressed mRNA from the mutated ALAS2 allele in erythroid
cells (Figure 1D). The 2 brothers' DNA samples that were used for
sequencing were shown to be nonidentical and derived from the same
parents by genotyping of informative short tandem repeat polymorphisms at 15 loci (data not shown).
In order to demonstrate that the above mutation was not a polymorphism,
we studied 100 DNA samples from unrelated females, none of whom had
this change. This indicates that the transition from G to A at
nucleotide 1731 is not a polymorphism, since it was found in less than
1% of the population. In addition, significant difference was found
between the mean RDW values (a phenotypic marker of XLSA3)
for 5 healthy and 5 heterozygous females carrying the 1731G>A
mutation. As detailed in Table 1 (footnote on RDW), these latter had
markedly higher RDW values (F = 50, P = .0001), indicating that all of them had the typical hematologic phenotype of
female carriers.
By contrast, the 2 hemizygous brothers had markedly different
hematologic phenotypes. The proband (II-2) showed a classical microcytic anemia with high RDW and secondary iron overload. In comparison, his brother (II-3) had normal red cell counts and no
evidence of iron overload. However, he showed marginally low MCV and
marginally elevated RDW on some occasions. A feature of XLSA is the
presence of microcytic red cells. Figure 1B shows red cell volume
histograms of 3 representative family members: the nonanemic hemizygous
brother shows an almost normal pattern.
Although unlikely, we could not exclude that the mutant
ALAS2 in the nonanemic healthy brother was silenced
through a mechanism of intercistronic suppression.9 In
order to establish whether other inherited disorders could explain the
different phenotypes, additional studies were performed. Molecular
analysis of HFE excluded the Cys282Tyr or His63Asp
mutations, while investigations for beta or alpha thalassemia defects
revealed no imbalance in globin chain synthesis in both brothers. No
potentially causative nongenetic factors were identified.
Variable penetrance of pyridoxine-responsive XLSA has been previously
reported. In a family with the mutation 1215C>G predicting an
Ser388Thr amino acid change,10 3 hemizygous males were
studied. Their hemoglobin levels at clinical onset ranged from 5.0 g/dL to 14.4 g/dL, but all of them showed microcytosis (60-73 fL). There
were 4 hemizygous males who were studied in a family with the mutation
923G>A predicting a Gly291Ser amino acid change,11 and
variable hemoglobin levels at clinical onset were found. All patients,
however, were anemic (hemoglobin from 9.2 g/dL to 12.5 g/dL) and showed
reduced MCV values (60-74 fL). Therefore, all these patients clearly
had the XLSA phenotype, and modifying genes or acquired factors capable
of worsening or ameliorating XLSA were likely playing a role in
various individuals.
The present family is unique because the severe hematologic phenotype
of the proband was almost completely absent in his brother. The fact
that all 3 of obligate heterozygote daughters of this nonanemic
hemizygote had mild phenotypes (marginally elevated RDW), argues
against worsening factors in these family members, and would be
consistent with the presence of a silencing factor(s) in the nearly
normal hemizygote, as in the case of complete suppression of congenital
dyserythropoietic anemia type II in a homozygous male.12
On the other hand, different genetic backgrounds may be responsible for
the expression of XLSA in the proband, similar to the variable clinical
expression in heterozygous gene carriers for mutations in the autosomal
dominant porphyrias.13
Hemizygous males with ALAS2 mutations and absent XLSA
phenotype may be less rare than thought. In fact, these individuals are
likely to go unnoticed, since only males with abnormal phenotype are
usually studied in families with X-linked inherited disorders. This
emphasizes the importance of gene-based diagnosis in families with
XLSA. Identification of the ALAS2 mutation in the subject II-3 has led to the recognition of a heterozygous state in his 3 daughters, who will now benefit from genetic counseling. In the future,
they will be advised that their offspring might be at risk for anemia
and iron overload14 but could benefit from a simple oral
administration of pyridoxine that might prevent development of XLSA. We
believe that all at-risk individuals in families with XLSA who request
testing should have their DNA examined for ALAS2 mutations, regardless
of normal hematologic findings, sex, and age.
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Acknowledgments |
The authors thank Joyce Hoy, Barrie Francis, and Maria Ramirez for
technical help with DNA sequencing and genotype analyses.
| |
Footnotes |
Submitted March 5, 2002; accepted July 9, 2002.
Prepublished online as
Blood First Edition Paper, August 8, 2002; DOI
10.1182/blood-2002-03-0685.
Supported by grants from MIUR, Rome, Italy, from AIRC, Milan, Italy,
from IRCCS Policlinico S. Matteo and from Fondazione Ferrata Storti,
Pavia, Italy (M.C.); grants R01 DK40895 and R01 DK26824 from the
National Institutes of Health, Bethesda, MD, and grant 584 from the
March of Dimes Birth Defects Foundation (D.F.B.).
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:
mario.cazzola{at}unipv.it.
| |
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Abstract
Introduction
