Download hemoglobin chesterfield (828 leu + arg) produces

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2791
CORRESPONDENCE
HEMOGLOBIN CHESTERFIELD (828 LEU + ARG) PRODUCES THE PHENOTYPE OF
INCLUSION BODY 8 THALASSEMIA
To the Editor:
Several dominant forms of p thalassemia have recently been
identified that result in a thalassemia intermedia phenotype in
individuals who have inherited only a single copy of the abnormal f3
gene.’’6We describe here an individual with severe heterozygous p
thalassemia characterized by large inclusion bodies in the peripheral blood associated with a single-base substitution, CTG -+
CGG, in codon 28 of the p gene. This novel mutation, which is
found in exon 1, leads to the synthesis of an unstable pvariant, p28
Leu + A r g (hemoglobin [Hb] Chesterfield).
The patient was a 34-year-old English woman who presented at
the age of 7 years with abdominal pain and was found to be anemic
and jaundiced with hepatosplenomegaly. She received sporadic
blood transfusions but became transfusion-dependent at the age of
10 and, because of increasing transfusion requirements, underwent
splenectomy 3 years later, followed by a cholecystectomy at the age
of 28. Post-splenectomy, she remained transfusion-dependent
although transfusion requirements were less frequent and she was
started on iron chelation therapy. Recent investigations showed:
Hb 7 to 9 g/dL, MCV 86.6 fL,MCV 30.3 pg, reticulocytes lo%, Hb
A, 4.0%, Hb F 3.4%, and serum ferritin 183 kg/mL. Blood film
examination showed marked erythroblastosis (500 to 800 nucleated
red cells per 100 white blood cells [WBCs]) with mature erythrocytes showing bizarre morphologic changes and basophilic stippling. Numerous inclusion bodies could be demonstrated in peripheral erythrocytes on incubation with methyl violet. No abnormal
Hb was detected on electrophoresis of red cell lysate at pH 8.6, but
separation of in vitro labeled globin chains showed an abnormal
peak (p”) eluting after the normal PA chain (Fig 1A). The total
radioactivity of dPA+ p” was 1.64 while dPAwas 2.47 and P”
accounted for 33% of the total p chains after 60 minutes of
incubation. No abnormal Hb was detected by heat denaturation or
isopropanol tests.
Analysis of seven restriction fragment length polymorphisms
(RFLPs) in the p-globin gene cluster showed: HindII-c -/-,
HindIII-‘yiAy +-/--, HindII-Jrp, 3‘Jrp ++/--, AvaII-P +/+
and BamHI-p +/-. Heterozygosity for five of these sites indicates
the absence of a major rearrangement or deletion. a-globin gene
mapping showed a normal a-genotype (adaa).The P-globin genes
were .enzymatically amplified by the polymerase chain reaction
(PCR) and directly sequenced as previously described.’ Direct
genomic sequencing of 2,100 base pairs (bp) of the p-globin genes,
extending from 307 bp upstream of the Cap site to 320 bp
downstream from the termination site, showed a T
G
( E G CGG) substitution in codon 28 (Fig 1B) of only one of the
p-globin genes; DNA sequence of the other p gene was normal.
The sequence modification abolishes the cleavage site for BstNI in
exon 1 so that BstNI analyses of 355-bp fragment amplified using
primers AP3iAP4 results in only a single 355-bp band if the
mutation is present, but two fragments of 85 bp and 270 bp if the p
allele is normal (Fig 1C). The results confirmed that the patient is
heterozygous for the mutation. Both parents were clinically asymptomatic with normal red blood cell indices and normal levels of Hb
A2 and F. The patient had six siblings, all of whom were clinically
asymtomatic but none were available for investigation. Therefore,
it was likely that the mutation was a de novo genetic event.
Several families with a phenotypically dominant form of p
-+
-+
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CORRESPONDENCE
2792
07
A
0.6
0.5
1
1
0.4
0
Q)
(Y
0.3
P
0
0.2
0.1
!
Fraction number
TGCAGAT C
IG
-
B
27 A la Ala
28 Leu Arg*
29 GlY
\A
thalassemia have now heen reported. Unlike typical B thalassemia,
which is prevalent in walaria-endemic populations. dominant B
thalasemias are rare with a wider geographical distribution. At the
molecular level they fall into three groups: those resulting in a
highly unstable B chain as a result of a single base substitution,'"
thcm with a truncated f3 chain due a premature termination codon
as a result of a base suhstitution.'." and those with an abnormal f3
globin that is elongated with an altered carhoxy-terminal end as a
result of a frameshift mutation."." These mutations result in the
production of unstable B chains that are not capable of forming
viahle tetramers with a chains and, thus. are not only nonfunctional. hut prove to he an additional burden to the cell's
proteolytic machinery.
The single base suhstitution C G -+ e G in d o n 28. results in
BWLeu Arg. Although there i s an increase i n the net positive
charge of the I l b molecule, no abnormal Hb could he detected by
-
5 . 1 . (A) Chromatography of
the I a M e d globin chaim. The
peripheral blood takul0cyt.r of
the patient were incubated inthe
presence of 'H-leucine for 60 minutes at 37C. The labeled globin
chains were analyzed by CMcellulo8echromatography. r e p
resents the abnormal peak corresponding t o the expected
position of the abnormal p chain.
( 8 )DNA sequence of part of the
p-globin gene in exon 1. Eruymatically amplified p-globin genes
were purified and sequenced directly using the dideoxynucleotide chain termination method.'
The sequence reaction was
loaded in the order TGCA for the
first four lanes and repeated in
the order GATC for the second
four lanes. The PCR rimultanbourly amplifies the wild-type
and the p-thalasuemia alleles so
that both alleles are sequenced.
In this case. if both alleles had
been normal, only a T should be
present in the second position of
codon 28. The presence of a G as
well as a T in this position (orrowed) indicates a G substthction in the mutant allele, altering
Leu for Arg (*I.
electrophoresis. CM-cellulosc chromatography demonstrated an
abnormal peak in the position expected for the abnormal f3 chain
but with no detectable corresponding protein peak, suggesting the
production of a Bthain variant that is highly unstable and rapidly
degraded after synthesis. The clinical severity of unstable Hb
variants presumahly depends on the degree of instahility of the
abnormal chain. The most severely unstable variants produce the
picture of dominant B thalassemia with a clinical picture of marked
ineffective erythropoiesis and thalassemia intermedia. as seen in
the current case. Those with a lesser degree of instahility that are
still capable of forming viable tetramers may give rise to less
inefTective erythropoiesis and a more typical picture of hemolytic
anemia. Two other p variants due to amino acid substitutions
involving g2S Leu have been descrihed. H b Genova (g28
Leu + Pro)' is an unstable Hb producing a phenotype of severe
congenital hemolytic anemia associated with Heinz b d i e s in the
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2793
CORRESPONDENCE
erythrocytes. I t appears that the replacement of 828 leucine by a
proline residue, although causing a profound distortion o f the B
helix, seems less destructive than the replacement 828 Leu to Arg,
because 828 Pro is stable enough to allow the formation of viable
tetramers (Hb Genova). which then fall apart in the peripheral
circulation, causing a hemolytic anemia. Hb St Louis (828
Leu +Gin)" is also an unstable Hb but produces a distinctly
different clinical phenotype of severe hemolytic anemia associated
with methemoglobinemia. In this case, 828 (BIO) glutamine and
the distal histidine (histidine E7) swing towards each other.
stablizing a water molecule in the normally hydrophobic heme
pocket. which results in thermal instability and the high rate of
methemoglobin formation. These findings illustrate that the nature
of the amino acid substitution as well as its position in the globin
chain i s a critical determinant of the resulting phenotype.
ACKNOWLEDGMENT
We thank Liz Rose and Linda Roberts for preparation of the
manuscript, Drs Max Perutz, Bill Wood, and John Clegg for helpful
criticisms. and Prof Sir D.J. Weatherall for encouragement and
support.
S.L. THElN
S. BEST
J. SHARPE
MRC Molecular Haematology Unit
Institute of Molecular Medicine
John Radclife Hospital
Headington. Oxford
B. P A U L
Fig. 1. (cant'd) (C) IdentHkatlon of the CTG -L CGG mutation in
codon 28 of the p-globin gene by BstN1 rertriction analysis of
AP3/AP3 amplified DNA. BstNl cuts the 355 bp resulting in two
fragments of 85 and 270 bp; presence of the mutation removes the
BstNl cleavage site. The sequences of the primers are 5'
3': AP3:
ATGGTGCACCTG ACTCCTGAGG. AP4-GCCATCACTAAAGGCACCGAGC. Lanes a represent amplified DNA cleaved with BstNI, and
lanes b, the corresponding un-cleaved DNA; 1, uncharacterized
p-thalassaemia heterozygote; 2, mother of the patient; 3, patient; 4,
normal individual. The lanes M represent d q Hae 111 size markers.
Presence of the three bands of size 355,270. and 85 bp in lane a, no. 3,
after digestion with BstNl indicates that the patient is heterozygous
for the T G mutation in codon 28.
-
D.J. CLARK
kpanment of Haematology
Chesterjieldand North Derbyshire Royal Hospital
Calow, Chesterjield
M.J. BROWN
Depanment of Haematology
Nonhem General Hospital
Shefield, UK
-.
REFERENCES
1. Thein SL, Hesketh C, Taylor P, Temperley IJ, Hutchinson
RM, Old JM, Wood WG, Clegg JB. Weatherall DJ: Molecular
basis for dominantly inherited inclusion body 8-thalassemia. Proc
Natl Acad Sci USA 873924,1990
2. Fucharoen S , Kobayashi Y, Fucharoen G, Ohba Y, Miyazono
K, Fukumaki Y, Takaku F A single nucleotide deletion in codon
123 of the f3-globin gene causes an inclusion body f3-thalassaemia
Br J Haematol75:393,
trait: A novel elongated globin chain pWab".
1990
3. Fei YJ, Stoming TA, Kutlar A, Huisman THJ. Stamatoyannopoulos G: One form of inclusion body 8-thalassemia i s due to a
GAA + TAA mutation at codon 121of the 8 chain. Blood 73:1075,
1989
4. Kazazian HH Jr, Dowling CE, H u w i t z RL, Coleman M,
Adams JG 111: Thalassemia mutations in exon 3 of the &globin
gene often cause a dominant form of thalassemia and show no
predilection for malarial-endemic regions of the world. Am J Hum
Genet 29950, 1989 (suppl)
5. Beris P, Miescher PA, Diaz-Chico JC, Han I-S, Kutlar A, H u
H, Wilson JB, Huisman THJ: Inclusion body 8-thalassemia trait in
a Swiss family i s caused by an abnormal hemoglobin (Geneva) with
an altered and extended 8 chain carboxy-terminus due to a
modification in codon 81 14. Blood 72:801. 1988
6. Kobayashi Y, Fukumaki Y, Komatsu N. Ohba Y. Miyaji T,
Miura Y: A novel globin structural mutant, Showa-Yakushiji (f31 IO
Leu-Pro) causing a 8-thalassemia phenotype. Blood 7031688, 1987
7. Sansone G . Carrel1 RW, Lehmann H: Haemoglobin Genova:
828 (BIO) Leucine 4 Proline. Nature 214:877, 1967
8. Thillet J, Cohen-Sola1 M, Seligmann M, Rosa J: Functional
and physicochemical studies of hemoglobin St. Louis 828 (BIO)
Leu -t Gln. A variant with ferric 8 heme iron. J Clin Invest
58:1098,1976
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1991 77: 2791-2793
Hemoglobin Chesterfield (beta 28 Leu----Arg) produces the phenotype
of inclusion body beta thalassemia [letter]
SL Thein, S Best, J Sharpe, B Paul, DJ Clark and MJ Brown
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