12th Congress of the European Hematology ... - Haematologica

0783
PREVALENCE OF MEMBRANE PROTEIN DEFICIENCIES IN PORTUGUESE PATIENTS WITH
HEREDITARY ELIPTOCYTOSIS AND HEREDITARY SPHEROCYTOSIS
S. Rocha, 1 L. Belo, 1 E.B. Castro, 1 P. Rocha-Pereira, 2 F. Ferreira3, E.
Cleto, 4 J. Barbot, 5 A. Quintanilha, 6 A. Santos-Silva 1
1 Pharmacy Faculty and IBMC Univ. Porto, PORTO; 2 CICS Univ. Beira Interior
and IBMC, COVILHÃ; 3 Hospital S. João, PORTO; 4 Hospital Geral de
Santo António, PORTO; 5 Hospital de Crianças Maria Pia, PORTO; 6 ICBAS
and IBMC Univ. Porto, PORTO, Portugal
The erythrocyte owes its unique shape and mechanical proprieties to
its membrane, which is a complex structure involving interconnections
between transmembrane proteins, the lipid bilayer and cytoskeleton
proteins. Defects in membrane proteins lead to pathologies known as red
cell membrane disorders, being Hereditary Spherocytosis (HS) the most
common. HS and Hereditary Eliptocytosis (HE) are hemolytic anemias
caused by deficiencies in several erythrocyte membrane proteins (spectrin,
ankyrin, band 3 and protein 4.2 for HS; spectrin and protein 4.1 for
HE). The presence of spherocytes in peripheral blood smears, the
increase in osmotic fragility and in reticulocyte count are screening hallmarks
of HS. For HE the presence of eliptocytes in peripheral blood
smears is the major screening hallmark. The aim of our work was to
identify and quantify the membrane protein deficiency underlying HS
and HE, in order to estimate the prevalence of each deficiency in Portuguese
patients. We studied 87 Portuguese patients of the northern
region of Portugal, diagnosed with HS or HE after standard screening
tests. The protein deficiencies that underlie both HS and HE cases were
identified and quantified by standardized electrophoretic erythrocyte
membrane protein analysis. Only 7 patients, out of the 87 patients studied,
presented HE. In HE, 6 patients (85.7%) presented protein 4.1 deficiency
and one patient (14.3%) spectrin deficiency. Considering the HS
cases, we observed band 3 deficiency in 51 (63.7%) patients, ankyrin
deficiency in 20 patients (25.0%), spectrin deficiency in 7 patients (8.8%)
and protein 4.2 deficiency in 2 patients (2.5%). Considering the total of
patients with HE and HS, the prevalence of erythrocyte membrane protein
deficiencies was found to be 58.6% for band 3, 23.0% for ankyrin,
9.2% for spectrin, 6.9% for protein 4.1 and 2.3% for protein 4.2. Our
sample Portuguese population showed band 3 deficiency as the most
prevalent protein deficiency in HS, followed by ankyrin deficiency. The
literature also refers these deficiencies as the most prevalent, although
ankyrin deficiency is the most prevalent in other populations. The prevalence
found for HE in our studied population differs from what is
described by the literature, which states spectrin deficiency as the major
cause. This probably reflects region specificities, and the distribution of
the ethnic groups living in the North of Portugal. A
This study was supported by a PhD grant (SFRH/BD/22442/2005) attributed
to S. Rocha by FCT and FSE.
0784
PYRUVATE KINASE DEFICIENCY: BIOCHEMICAL CHARACTERIZATION OF TWO
MUTATIONS CAUSING INABILITY OF THE ENZYME TO PROPERLY BIND SUBSTRATES
S.M. Morera, 1 L.R. Chiarelli, 1 A. Galizzi, 1 P. Bianchi, 2 E. Fermo, 2
A. Zanella, 2 G. Valentini1 1 2 Università di Pavia, PAVIA; Fondazione IRCCS Ospedale Maggiore,
MILANO, Italy
Background. Pyruvate kinase (PK) deficiency is the most common
enzyme defect of erythrocyte glycolytic pathway causing hereditary
nonspherocytic chronic haemolytic anaemia. PK deficiency is transmitted
as an autosomal recessive trait and 180 mutations associated with the
disorder have been so far reported in the gene encoding the red cell pyruvate
kinase (RPK). The severity of the disorder is highly variable, ranging
from mild to severe anaemia, which can be life-threatening and
require continuous transfusion therapy. RPK catalyzes the conversion of
phosphoenolpyruvate (PEP) to pyruvate with the synthesis of ATP. It is
activated homotropically by PEP and heterotropically by fructose 1,6-bisphosphates
(FBP), and is inhibited by ATP and alanine. The three-dimensional
structure of RPK has been elucidated and ten mutations targeting
distinc regions of RPK structure have been investigated at protein level.
Characterization of mutant proteins may serve as a valuable tool to assist
with diagnosis and genetic counseling. Aims. To improve knowledge on
molecular basis of the haemolytic anaemia caused by PK deficiency two
additional mutant forms of RPK have been subjected to biochemical
characterization. The variants investigated Gly159Val (c.475G>T) and
Arg163Cys (c.487C>T) have been described in two young PK-deficient
patients affected by severe haemolytic anaemia. 1,2 Methods. The DNA
bearing the desired mutations were obtained from the cloned wild-type
cDNA using standard methods of site-directed mutagenesis. The mutant
enzymes were expressed in E.coli DH5α and purified to homogeneity
following the procedure developed for recombinant wild-type protein. 3
Results. Arg163Cys RPK showed a drastic reduction of the catalytic efficiency
expecially versus ADP (3 orders of magnitude) due to the
increased Km value (42-fold higher). Gly159Val had kinetic properties
altered either vs ADP or PEP (Km values, 8- and 4-fold higher; catalytic
efficiency, about 15-fold reduced). Moreover, both mutant enzymes did
not gain the fully active conformation in the presence of 1mM FBP (nH
1.3 and 1.5 for Arg163Cys and Gly159Val, respectively, vs 1.05 of the
wild-type enzyme) and resulted quite insensitive to ATP inhibition (IC50
at least 20-fold higher). Conclusions. The mutation c.487C>T affects an
arginine essential for ADP binding, and thus prevents the enzyme from
accomplishing his function. The mutation c.475G>T affects a functionally
crucial residue involved in the allosteric transition, thus impairing the
substrates binding site to adopt the appropriate geometry required for
the catalytic cycle. The altered properties displayed by the mutant forms
account for the reduced activity observed in RBCs of patients affected
by this pathology.
This work was supported by grants from University of Pavia (FAR).
References
12 th Congress of the European Hematology Association
1. Neubauer et al. Blood 1991;77:1871-5.
2. Demina et al. Blood 1998;92:647-52.
3. Valentini et al. J Biol Chem 2002;277:23807-14
0785
IDENTIFICATION AND CHARACTERIZATION OF THE FIRST MUTATION IN THE RED BLOOD
CELL SPECIFIC HEXOKINASE PROMOTER IN A PATIENT WITH MILD HEXOKINASE
DEFICIENCY
K. de Vooght, W.W. Van Solinge, A.C. Van Wesel, S. Kersting,
G. Rijksen, R. Van Wijk
University Medical Center Utrecht, UTRECHT, Netherlands
Background. Hexokinase (HK) is one of the key enzymes of glycolysis
and catalyzes the phosphorylation of glucose to glucose-6-phosphate.
HK-I, that is encoded by the HK-I gene (HK1), is the predominant
isozyme in a variety of tissues including brain, kidney, erythrocytes, and
platelets. Red blood cell hexokinase (HK-R) is a HK isozyme that is transcribed
from HK1 by use of an erythroid-specific promoter. HK-R is
mainly expressed in reticulocytes and young erythrocytes and its half life
is much shorter than that of HK-I. Thus HK-I replaces HK-R as the erythrocyte
matures. Hexokinase deficiency is a very rare cause of hereditary
non-spherocytic haemolytic anaemia. To date less than 20 cases
have been described. Aims. The aim of this study was to investigate the
molecular basis for HK deficiency in a patient with chronic haemolysis.
Methods. HK activity, and activity of the red blood cell age-related
enzymes, was determined according to standardized procedures. Functional
studies were performed using transient transfection of HK promoter
constructs in human K562 erythroleukemia cells. DNA-protein interaction
at the promoter of HK was studied using Electrophoretic Mobility
Shift Assay’s (EMSA) with nuclear extracts from K562 cells. DNA
analysis and RT-PCR were performed according to standardized procedures.
Results. In the patient, HK activity was 61% of the mean reference
value, whereas PK and G6PD activities were normal. Consequently, we
interpreted the HK activity as too low. By DNA sequence analysis we
identified on the paternal allele in the erythroid-specific promoter of
HKI two novel mutations in cis: -373A>C and -193A>G (relatively to the
start codon). These mutations were absent in a Caucasian reference population.
Transfection of promoter constructs into K562 cells showed that
the most upstream 373A>C mutation was nonfunctional. In contrast, the
193A>G mutation reduced promoter activity by 92%. EMSA using K562
nuclear extracts indicated binding of a trans-acting factor. On the maternal
allele we identified a novel mutation in exon 3 (c.278G>A). Three
PCR products were amplified from the patient’s mRNA by HK-R specific
RT-PCR: one normal PCR product, one lacking exon 3 and one alternatively
processed transcript. These results suggested that the exon 3
mutation compromises normal pre-mRNA processing. Summary and conclusions.
We investigated the molecular basis for HK deficiency in a
patient with hexokinase deficiency. The -193A>G mutation was shown
to cause a dramatic decrease in promoter activity in vitro, and is therefore
the first mutation known to cause HK-R specific HK deficiency.
Furthermore, the c.278G>A exon 3 mutation on the second allele was
haematologica/the hematology journal | 2007; 92(s1) | 293