Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 368-369
CORRESPONDENCE
-Spectrin Promissão: A Translation Initiation Codon
Mutation of the -Spectrin Gene (ATG 
GTG) Associated With
Hereditary Spherocytosis and Spectrin Deficiency in a Brazilian Family
 |
LETTER |
To the Editor:
Hereditary spherocytosis (HS) is a common inherited anemia
characterized by the presence of spheroidal red cells and increased
osmotic fragility of erythrocytes.1 This disorder is
heterogeneous in terms of its clinical presentation, molecular basis,
and inheritance.2 HS mutations have been ascribed to
several genes,1 including the 
-spectrin gene. So far 13
-spectrin mutations have been described associated with
HS.3-6
We have studied a Brazilian family with HS diagnosed in eight
subjects from two generations and inherited in an autosomal dominant
fashion (Fig 1). The propositus was a
28-year-old black man, who presented compensated hemolytic disease with
splenomegaly, hyperbilirubinemia, increased osmotic fragility, and a
regular number of spherocytes and acanthocytes in the blood smear (Fig
1). His recent hematological profile was: hemoglobin (Hb) 15.0 g/dL,
red blood cell 4.49 × 1012/L, mean corpuscular volume 88
fL, mean corpuscular hemoglobin concentration 38.0 g/dL, reticulocyte
count 530 × 109/L (11.8%). His mother, uncle, and two
cousins were splenectomized. Densitometric scans of Coomassie
blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) of the propositus membrane proteins showed an 18% reduction
in spectrin content (Fig 1). This pointed to the -spectrin gene as
the most likely candidate for bearing the primary defect. Therefore, we
started screening for mutations in the -spectrin gene. This was
performed through the amplification of the individual exons of the
-spectrin gene with intronic primers. The amplification products
were submitted to nonradioactive single-strand conformation
polymorphism (SSCP) technique in a PhastSystem apparatus (Pharmacia,
Uppsalla, Sweden) to detect sequence abnormalities. The DNA
amplification products of exon 2 of the patient and his mother showed
an identical band shift in two independent experiments (Fig
2). No such band pattern was observed in 2
independent controls, nor was it observed in 12 other HS patients with
spectrin deficiency and acanthocytes in the blood smear, suggesting
that this patient bore a unique or at least a rare sequence alteration
in this region of the gene. Sequencing revealed an heterozygous
A 
G nucleotide substitution at the translation initiation codon
of the -spectrin gene (ATG GTG) (Fig
3). This is the first report of an
initiation codon mutation in this gene, but such mutations have already
been reported for other genes, including the -globin
gene.7 In this case, the mutations led to
0-thalassemia phenotypes, suggesting that alternative
triplets for ATG are nonfunctional as initiation codons for this gene.
Therefore, we suggest that in our case translation from the mutated
allele is also impaired. Based on this supposition, the ATG GTG
mutation could be held responsible for the reduced spectrin content
observed in the patient. The propositus would have only one
functional allele and, as -spectrin quantities are considered
limiting for membrane assembly, this could account for the picture of
spherocytosis observed in this patient.
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| Fig 1.
(Left) 3.5% to 17% exponential gradient
SDS-polyacrylamide gel of total membrane proteins stained with
Coomassie blue, showing a reduction of spectrin content in the patient
(lane 2) compared with the control (lane 1). (Upper right) Blood smear
of the propositus showing regular numbers of spherocytes and
acanthocytes. (Lower right) Family pedigree showing all affected
members from two generations. Splenectomized individuals are indicated
by asterix and the proband is indicated by an arrow.
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| Fig 2.
Nonradioactive SSCP of exon 2 showing a band shift in the
amplification products of the patient (lane 1) and his mother (lane 2),
but absent in a control (lane 3).
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| Fig 3.
Direct sequencing of the amplified DNA from a control (B)
and the patient (A), showing an A G substitution in the latter. The
initiation codon is indicated by bold letters.
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| |
ACKNOWLEDGMENT |
This work was supported by the Conselho Nacional de Desenvolvimento
Cientifico e Tecnológico (CNPq) and Fundação de
Amparo à Pesquisa do Estado de São Paulo
(Fapesp).
Daniela S. Bassères
Dilmara L. Vicentim
Fernando F. Costa
Sara T.O. Saad
Hemocentro
Universidade Estadual de Campinas,
Brazil
Hani Hassoun
Division of Hematology/Oncology
St
Elizabeth's Medical Center, Boston, MA
| |
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