Blood, 1 September 2002, Vol. 100, No. 5, pp. 1913-1914
BRIEF REPORT
Homozygosity for nondeletion 
-
0 thalassemia
resulting in a silent clinical phenotype
Renzo Galanello,
Susanna Barella,
Stefania Satta,
Liliana Maccioni,
Carlo Pintor, and
Antonio Cao
From the Ospedale Regionale Microcitemie; and
Dipartimento di Scienze Biomediche e Biotecnologie and Dipartimento di
Scienze Pediatriche, Università di Cagliari, Cagliari, Italy.
 |
Abstract |
The clinical phenotype of homozygous 
thalassemia varies in
severity from the mild thalassemia intermedia to the severe thalassemia major. This variability depends largely on the molecular heterogeneity of thalassemia defects. We report the first case of a homozygous state for nondeletion Sardinian 
-0 thalassemia, which
resulted in a symptomless clinical phenotype with a peculiar hemoglobin
(Hb) pattern (99.8% Hb F and 0.2% Hb A2). The molecular
defect was characterized by the presence of 2 nucleotide substitutions:
196C>T in the promoter of the A
-globin gene and 39C>T
nonsense mutation. The absence of typical thalassemia clinical
findings was due to the high Hb F output, which compensated for the
absence of chains. The near absence of Hb A2 may have resulted from either alterations in the globin gene transcriptional complex with preferential activation of -globin genes and
suppression of -globin genes or preferential survival of red blood
cells with the highest Hb F content and low Hb A2 level.
(Blood. 2002;100:1913-1914)
© 2002 by The American Society of Hematology.
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Introduction |
Thalassemia intermedia is a clinical definition for
a heterogeneous group of conditions ranging in severity from the thalassemia carrier state to the transfusion-dependent thalassemia
major phenotype. Characteristic of these mild clinical forms is the
absence of an absolute requirement for regular transfusions for
survival. The remarkable variability in clinical severity of
thalassemia intermedia results largely from the genetic heterogeneity
of thalassemias. Progress in molecular biology has allowed definition
of globin gene defects and partial elucidation of the relation between
phenotype and genotype.1 The defined molecular mechanisms
for thalassemia intermedia have as a common hallmark a reduction in the
imbalance of synthesis of 
and non- globins. One of these
mechanisms is an increase in production of -globin chains sufficient
to reduce the /non- imbalance, which results from the
coinheritance of genetic determinants such as - thalassemia or
hereditary persistence fetal hemoglobin (HPFH).
- Thalassemia is usually caused by large deletions of
variable extent in the -globin cluster.2 One form of
nondeletion -0 thalassemia has been described; this
disorder results from the presence in cis in the
-globin cluster of 2 different nucleotide substitutions, one in the
promoter of the A gene (196C>T) and the other in the -globin
gene (the 0 39C>T nonsense mutation).3
Carriage of nondeletion -0 thalassemia is
characterized by high levels of hemoglobin F (Hb F; range, 10%-20%)
containing mainly A chains and normal levels of Hb
A2.4 Nondeletion -0
thalassemia is a rare form of - thalassemia in the Sardinian population. Compound heterozygosity for -0
thalassemia and the common 0 39 nonsense mutation
results in the clinical phenotype of thalassemia intermedia.5 Here, we report the first case of a
homozygous state for nondeletion -0 thalassemia,
which produced a symptomless clinical phenotype with a peculiar Hb pattern.
| |
Study design |
An 18-month-old girl was admitted to the hospital because of
high fever. Venous blood was drawn for hematologic and molecular analysis. Red blood cell (RBC) indices were determined by using a
Coulter STKS device (Beckman Coulter, Milan, Italy). Qualitative and
quantitative Hb analysis was done with high-pressure liquid chromatography (Variant; Bio-Rad, Milan, Italy)6 and
globin-chain analysis with reversed-phase high-performance liquid
chromatography (Gold System; Beckman Coulter). Analysis of globin-chain
synthesis was carried out according to the Clegg
method.7
Serum levels of erythropoietin (Epo) and transferrin receptor (TfR)
were determined by enzyme immunoassays (Immulite Epo; DPC, Los Angeles,
CA, and Ramco Laboratories, Houston, TX, respectively). DNA was
extracted from peripheral blood (PB) leukocytes, and cluster mutations
in the -globin gene were defined by sequencing DNA amplified by
polymerase chain reaction.8
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Results and discussion |
Analysis of RBC indices and Hb level performed as part of a
routine hematologic evaluation in our patient showed a normal Hb level
(125 g/L), microcytosis (mean corpuscular volume [MCV], 61.6 fL), and an Hb pattern indicative of homozygous 0
thalassemia with only Hb F and a near absence of Hb A2 (Hb
F, 99.7%; and HbA2, 0.3%; Figure
1). Hb F was composed of 72% A and
28% G chains (ratio of G to A, 0.38). Analysis of
globin-chain synthesis in PB reticulocytes showed an -to- ratio
of 1.9, which is in the range of that of carriers of thalassemia. A
PB smear revealed only mild morphologic changes in RBCs (hypochromia
and anisopoikilocytosis) and absence of nucleated RBCs. Clinical
examinations at presentation and follow-up (6 years) showed no
enlargement of the liver or spleen, growth within the normal range
(10th percentile for both weight and height), and absence of
thalassemia-associated bone changes.
DNA analysis of the -globin gene cluster in the patient revealed
homozygosity for the codon 39 nonsense (C>T) mutation and the
196C>T substitution in the promoter of the A-globin gene. These
mutations are typical of nondeletion Sardinian -0
thalassemia.3,9 The patient's mother was found to have
the hematologic characteristics of carriers of this genetic determinant (MCV, 73 fL; Hb A2, 2.8%; and Hb F, 18.8%), and DNA
analysis confirmed the presence of the heterozygous state for the
mutations observed in the patient (Figure 1). The patient's
father was not available for testing. Repeated clinical and hematologic
evaluations of the patient during follow-up (up to 6 years) did not
show any clinical abnormalities. In particular, Hb levels remained
within the expected range for the patient's age, and erythropoiesis
was not quantitatively increased as indicated by normal reticulocyte numbers and normal serum levels of Epo and TfR (Figure 1). The total
bilirubin level was not increased (8.5 µM/L).
Compound heterozygotes for nondeletion Sardinian -0
thalassemia and the 0 39 nonsense mutation have the
classic clinical phenotype of thalassemia intermedia characterized by
moderate to severe anemia, hepatosplenomegaly, and mild jaundice
because the increased production of Hb F associated with the -
thalassemia determinant partly compensates for the absence of chains.5,9 The homozygous state for nondeletion -0 thalassemia in our patient resulted in a marked
increase in Hb F that mostly compensated for the absence of chains.
The increased number of RBCs contributed to achievement of normal Hb
levels. The imbalance in the ratio of to , similar to that in
heterozygous thalassemia, explains the reduction in MCV and mean
corpuscular Hb. Heterozygotes for Sardinian nondeletion -
thalassemia have a high proportion of A-globin chains (range,
80%-93%) resulting from overexpression of the A gene associated
with the absence of -globin synthesis due to the presence of the
0 39 nonsense mutation. A chains were also prevalent
in our homozygous patient, who had 72% A-globin chains. The higher
G content compared with that in the heterozygous state may have been
due to mild stress erythropoiesis, which was also reflected in the
increased RBC production.
Patients who are heterozygous for nondeletion - thalassemia have
reduced Hb A2 levels (0.6 pg/cell compared with 0.75 pg/cell in healthy subjects and 1.1 pg/cell in carriers of thalassemia).10 Interestingly, our patient with
nondeletion homozygous -0 thalassemia had almost no
Hb A2 (0.3%). We previously reported that the -globin
gene sequence in a -0 thalassemia chromosome from
position 360 to the Cap site to 343 nucleotides 3' to the termination
codon was entirely normal.10 Therefore, the reduced
expression of the -globin gene of the Sardinian - thalassemia
chromosome may result from the suppressive effect of the in
cis A 196C>T mutation. This suppressive in cis effect has already been reported for similar mutations,
such as the 202 G HPFH, and may be explained by assuming a
reciprocal and coordinated regulation of globin gene expression through
the interaction of the locus control region and the individual
genes.11,12 However, the very low Hb A2 level
could also be related to the preferential survival of RBCs with the
highest Hb F content, since it is well known that Hb A2
levels have a strong negative correlation with Hb F
levels.13
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Acknowledgments |
We thank Valeria Siccardo and Franca Rosa Demartis for editorial assistance.
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Footnotes |
Submitted August 15, 2001; accepted April 1, 2002.
Supported by grant LR 11 del 30.4.90 from Progetti di Ricerca
Scientifica Locale, Cofin 99, MURST, Italy.
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: Renzo Galanello, Ospedale Regionale Microcitemie,
Via Jenner SN 09121, Cagliari, Italy; e-mail:
renzo.galanello{at}mcweb.unica.it.
| |
References |
1.
Galanello R, Cao A.
Relationship between genotype and phenotype. Thalassemia intermedia.
Ann N Y Acad Sci.
1998;850:325-333[CrossRef][Medline]
[Order article via Infotrieve].
2.
Stamatoyannopoulos G, Grosveld F.
Hemoglobin switching. In:
Stamatoyannopoulos G,Majerus PW,Perlmutter RM,Varmus HE, eds.
The Molecular Basis of Blood Diseases. 3rd ed. Philadelphia, PA: WB Saunders; 2001:135-182.
3.
Pirastu M, Kan YW, Galanello R, Cao A.
Multiple mutations produce 0 thalassemia in Sardinia.
Science.
1984;223:929-930[Abstract/Free Full Text].
4.
Cao A, Melis MA, Galanello R, et al.
F-Thalassaemia in Sardinia.
J Med Genet.
1982;19:184-192[Abstract/Free Full Text].
5.
Galanello R, Dessi E, Melis MA, et al.
Molecular analysis of zero-thalassemia intermedia in Sardinia.
Blood.
1989;74:823-827[Abstract/Free Full Text].
6.
Galanello R, Barella S, Gasperini D, et al.
Evaluation of a new automatic HPLC analyser for thalassemia and hemoglobin variants screening.
J Automatic Chem.
1995;17:73-76.
7.
Clegg JB.
Hemoglobin synthesis. In:
Weatherall DJ, ed.
The Thalassemias. Methods in Hematology. Vol 6. Edinburgh, United Kingdom: Churchill Livingstone; 1983:54-73.
8.
Ansubel FM,Brent R,Kingston SE,,et al
eds.
Short Protocols in Molecular Biology: a Compendium of Methods from Current Protocols in Molecular Biology. New York, NY: Greene Publishing/Wiley Interscience; 1989.
9.
Ottolenghi S, Giglioni B, Pulzini A, et al.
Sardinian zero-thalassemia: a further example of a C to T substitution at position 196 of the A globin gene promoter.
Blood.
1987;69:1058-1061[Abstract/Free Full Text].
10.
Loudianos G, Moi P, Lavinha J, Galanello R, Cao A, Pirastu M.
Normal -globin gene sequences in Sardinian nondeletional -thalassemia.
Hemoglobin.
1992;16:503-509[Medline]
[Order article via Infotrieve].
11.
Collins FS, Stoeckert CJ Jr, Serjeant GR, Forget BG, Weissman SM.
G gamma beta+ hereditary persistence of fetal hemoglobin: cosmid cloning and identification of a specific mutation 5' to the G gamma gene.
Proc Natl Acad Sci U S A.
1984;81:4894-4898[Abstract/Free Full Text].
12.
Gilman JG, Mishima N, Wen XJ, Kutlar F, Huisman THJ.
Upstream promoter mutation associated with a modest elevation of fetal hemoglobin expression in human adults.
Blood.
1988;72:78-81[Abstract/Free Full Text].
13.
Weatherall DJ, Clegg JB.
Hereditary persistence of fetal hemoglobin The Thalassaemia Syndromes. 4th ed. Oxford, United Kingdom: Blackwell Science; 2001:450-455.
Top
Abstract
Introduction