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Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3428-3435
Unusually Severe Heterozygous  -Thalassemia: Evidence for an
Interacting Gene Affecting Globin Translation
By
P. Joy Ho,
Georgina W. Hall,
Suzanne Watt,
Nicholas C. West,
Jennifer W. Wimperis,
William G. Wood, and
Swee Lay Thein
From the MRC Molecular Haematology Unit, Institute of Molecular
Medicine, John Radcliffe Hospital, Headington, Oxford, UK; Institute of
Haematology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia;
the Department of Haematology, Royal Free Hospital, London, UK; the
Department of Haematology, West Cumberland Hospital, Cumbria, UK; and
the Department of Haematology, Norfolk and Norwich Hospital, Norwich,
UK.
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ABSTRACT |
A common  -thalassemia mutation in Asian populations is the C
 T substitution at position 654 of intron 2, which leads to the activation of two cryptic splicing sites and the incorporation of
73 extra nucleotides into the mutant mRNA. Like most
-thalassemia mutations, it normally exhibits recessive
inheritance. We investigated the unusually severe phenotype in two
heterozygotes for this mutation, father and son, who had thalassemia
intermedia and an apparent dominant mode of inheritance. An increased
level of aberrantly spliced transcript in the reticulocytes of the
probands compared with asymptomatic 654
heterozygotes led us to investigate the production and processing of
654 RNA. We showed that large amounts of the
aberrant 654 transcript were detectable in
erythroblasts from one of the asymptomatic cases. The translation
product of this mRNA was not detectable in vivo, and we were unable to
demonstrate the translation of the mutant mRNA in a cell-free
translation system. Although the reticulocyte  : mRNA
ratios in the two probands were within the range observed in the
asymptomatic heterozygotes, globin chain biosynthesis studies showed
that the probands had considerably greater : chain
imbalance. These results imply that the more severe phenotype may be
due to a second defect, possibly unlinked to the -globin
cluster, that acts at the translational or posttranslational level.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
-THALASSEMIA, ONE OF the most common
single gene disorders, results from the decreased production of
-globin chains. More than 180 mutations affecting almost every known
stage of -globin gene expression result in a reduction
(+) or complete absence (o) of -globin
chain synthesis from the affected allele.1 The excess globin chains that ensue precipitate in the red blood cell
precursors, leading to their premature destruction and ineffective erythropoiesis.2 -Thalassemia usually exhibits Mendelian
recessive inheritance individuals with one allele are clinically
asymptomatic, while the inheritance of two mutant genes produces
disease.
Rarely, -thalassemia can be dominantly inheriteda single mutant
allele results in thalassemia intermedia (TI), with anemia, gross
morphological abnormalities of the erythroid cells, and splenomegaly.3 Unlike the recessive forms characterized by a deficit of normal chains, the pathophysiology of dominantly inherited thalassemias has been attributed to the synthesis of highly
unstable -globin variants, which fail to form functional tetramers
and precipitate intracellularly with the excessive chains, thus
exacerbating ineffective erythropoiesis.4-6 Due to their
instability, demonstration of these -chain variants is often
difficult7,8 or not possible,9,10 but their synthesis is inferred from the presence of mutant mRNA in the peripheral reticulocytes.11
Mutations that cause anomalies of RNA processing represent about one
third of the known -thalassemia alleles, leading to a reduction or
complete inactivation of normal splicing. The C T
mutation in intervening sequence (IVS) 2 position 654 is
commonly found in the Asian population as a cause of recessive
-thalassemia.12,13 Analysis of transfected mutant genes
by S1 nuclease mapping and primer extension14 demonstrated
that the mutation creates a cryptic 5 donor splice site, which
is spliced to the normal 3 acceptor site, while a cryptic
3 acceptor site is activated upstream at IVS 2-579 and spliced
to the normal 5 donor site at the exon 2/intron 2 junction. This
results in the incorporation of 73 extra nucleotides of intron 2 into
the aberrantly spliced mRNA transcript (Fig 1).
While most heterozygotes for the IVS 2-654 mutation have a typical
thalassemia trait phenotype, two cases, one Chinese and one Japanese,
have been previously reported to have TI,15,16 but the
cause of their unusually severe phenotype was not elucidated. We have
studied two Chinese individuals, father and son, who manifest the
phenotype of TI despite having inherited only a single copy of the
mutant allele. To understand this phenotypic difference, we have
compared these two cases with typical asymptomatic IVS 2-654 heterozygotes. Differences were observed in mRNA processing and in the
severity of globin chain imbalance, implying a second defect
interacting with the thalassemia allele to produce the thalassemia
intermedia condition.
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MATERIALS AND METHODS |
Blood Samples and Hematological Studies
Blood samples were collected in EDTA and full blood profiles obtained
using an automated cell counter. Hemoglobin A2
(HbA2) and HbF levels were measured using
standard techniques. Fresh blood was collected in heparin and kept on
ice for globin chain biosynthesis studies, performed by
3H-leucine labeling and carboxymethyl-cellulose column
chromatography at pH 6.4, as previously described.17
Chromatography was also performed at pH 6.0 to detect a more
negatively-charged -globin variant. In time-course experiments,
samples were removed at 5, 10, 15, and 60 minutes from the start of
incubation and a pulse-chase was performed by incubating the cells for
5 minutes with 3H leucine followed by 60 minutes incubation
in excess unlabeled leucine. Informed consent was obtained in all cases
before the collection of blood samples.
Culture of CD34+ Erythroid Progenitors From
Peripheral Blood
Fresh blood was collected in acid citrate dextrose (ACD), diluted 1:2
with phosphate-buffered saline (PBS; calcium- and magnesium-free) containing 0.6% ACD and fractionated on Ficoll-Hypaque (1.077 g/mL;
Sigma, St Louis, MO) at 1,600 rpm for 40 minutes at room temperature. The light density cells were washed in PBS-ACD by centrifugation at 900 rpm for 15 minutes to remove contaminating platelets. The resulting nucleated cells were washed three times in 2 mmol/L sodium phosphate buffer and 0.145 mol/L NaCl containing 0.5%
bovine serum albumin and 2 mmol/L EDTA or 0.6% ACD. CD34+
cells were isolated using the Mini-MACS CD34 stem cells isolation kit
from Miltenyi Biotech (Bergisch Gladbach, Germany) according to the
manufacturer's instructions. The CD34+ cells were selected
twice by passage through magnetic bead separation columns, allowing
recoveries of 0.01% to 0.05% of the original leukocytes. When stained
with phycoerythrin-conjugated CD34 or isotype matched IgG1
monoclonal antibodies and analyzed by fluorescence-activated cell
sorting (FACS), 80% to 85% CD34+ cells within the
nucleated fraction was obtained. These cells were cultured in Falcon
3047 tissue culture plates in erythroid-specific serum-free medium
containing optimal concentrations of interleukin-3 (IL-3), IL-6, Steel
Factor, and erythropoietin, at 37°C in 4.5% CO2 and
5% O2 gas mixture. The cells were harvested at days 9 to
10. The number of nucleated cells was determined, and their phenotypes
were assessed with antiglycophorin A and antiglycophorin C
(IgG1 isotypes) using FACS and single color
immunofluorescence of cytospins.18,19 Anti-CD3 was used as
a negative control. All antibodies were purchased from Dakopatts
(Copenhagen, Denmark).
DNA Analysis
DNA was extracted from peripheral blood leukocytes using standard
techniques. haplotypes were derived from seven restriction fragment
length polymorphisms (RFLPs) in the -globin gene cluster; HindII- , HindIII-G ,
HindIII-A, HindII- ,
HindII-3, AvaII-, and
BamHI-.20 The RFLPs were analyzed by restriction
enzyme digestion and Southern blot hybridization.21
BamHI- and BglII-digested DNA was hybridized with and  globin gene probes, respectively, to determine the number of
globin genes.21
The -globin genes were amplified by the polymerase chain reaction
(PCR); single-stranded template was prepared and directly sequenced as
described.21,22
RNA Analysis
Total RNA was extracted from fresh blood and cultured erythroid
progenitors by standard methods.23
Reverse transcription (RT)-PCR.
A total of 1 µg total RNA was reverse transcribed into cDNA using an
oligo(dT)15 primer and avian myeloblastosis virus
(AMV) reverse transcriptase in a buffer containing
ribonuclease inhibitor 20 U, 1 mmol/L each of
deoxyguanosine triphosphate (dGTP), deoxyadenosine triphosphate
(dATP), deoxythymidine triphosphate (dTTP), and deoxycytidine triphosphate (dCTP), 5 mmol/L MgCl2, 10 mmol/L
Tris-HCl pH 8.8, 50 mmol/L KCl, and 0.1% Triton X-180 in a 20-µL
volume, by incubation at 42°C for 15 minutes followed by
inactivation of the reverse transcriptase at 99°C for 5 minutes. All reagents were supplied by Promega, Southampton, UK.
-Globin cDNA comprising exons 3 and 2, was amplified by PCR in a
100-µL volume containing 0.2 mmol/L each of deoxynucleoside triphosphates (dNTPs), 50 mmol/L KCl, 10 mmol/L Tris-HCl
pH 8.3, 2.0 mmol/L MgCl2, 2.5 U Taq polymerase, and
10 pmol of primers AP1 and AP2 (see Fig 1). After initial denaturation
at 94°C for 4 minutes, 25 cycles of 94°C for 1 minute, 52°C
for 1 minute, and 72°C for 30 seconds were applied. As shown in Fig
1, the normal and aberrantly spliced transcripts would yield cDNA
fragments of 240 bp and 313 bp, respectively. The RT-PCR products were
electrophoresed in 2% ethidium bromide-stained agarose gels,
transfered to positively-charged nylon membranes (Biodyne B; Pall
Biodyne, Portsmouth, UK), and hybridized with an oligonucleotide probe
(OP, Fig 1) complementary to a sequence common to the normal and
aberrantly spliced transcripts. The oligoprobe was 3
end-labeled with 32P-dCTP using calf thymus
deoxynucletide terminal transferase (Boehringer, Mannheim,
Germany).
To quantitate the relative amounts of normal and aberrantly-spliced
-globin cDNA, AP2 was 5 end-labeled with
32P-dCTP using T4 polynucleotide kinase (Amersham
International, Amersham, UK). Using 10 pmol of radio-labelled AP2
(diluted 1:9 with unlabelled AP2), 10 pmol of AP3 and the conditions as
described for AP1/AP2 PCR (see above), a variable number (18, 20, 22, 25, and 28) of PCR cycles was applied. The normal and
aberrantly-spliced transcripts yield cDNA fragments of 430 and 503 bp,
respectively (see Fig 1). The RT-PCR products were resolved on a
denaturing 6% polyacrylamide gel. Radioactivity was measured by
phosphorimager analysis (Molecular Dynamics, Sunnyvale,
CA).
Ribonuclease (RNase) protection assays.
RNase protection assays were performed as described by Zinn et
al.24 A total of 0.5 to 1 µg total RNA from peripheral
reticulocytes and erythroid progenitors was hybridized with
106 cpm of each riboprobe in probe excess. The protected
fragments were electrophoresed on 8% polyacrylamide 8M Urea gels.
Radioactivity in protected fragments was measured by phosphorimager
analysis (Molecular Dynamics) and corrected to account for the number
of labeled G residues in the protected fragments.
A probe specific for the detection of the aberrantly spliced
654 transcript was generated by RT-PCR of RNA from AH, a
hemizygote, using primers AP4 and AP2 (see Fig 1). The fragment was
cloned into PCR2 TA vector (Invitrogen BV, Leek, The
Netherlands). The protected fragment of 189 bp includes 60 of the 73-bp
IVS 2 insert as well as exon 3.
In Vitro Cell-Free Translation of RNA
Total RNA extracted from peripheral reticulocytes and nucleated
erythroid cells derived from cultured CD34+ progenitors
were subjected to in vitro cell-free translation in rabbit reticulocyte
lysate. The "rabbit reticulolysate translation kit" (Amersham
International) was used, containing reticulolysate prepared from New
Zealand white rabbits and treated with micrococcal nuclease to destroy
endogenous mRNA. Total RNA (2.5 to 10 µg) was combined with 10 U
RNase inhibitor, 2 µL [35S]Methionine (translational
grade), 10 µL reticulocyte lysate in a translation mixture containing
100 mmol/L potassium acetate (KOAc), 0.5 mmol/L magnesium acetate
(Mg[OAc]2), 2 mmol/L dithiothreitol (DTT),
20 mmol/L HEPES pH 7.6, 8 mmol/L creatine phosphate, and 25 mmol/L each
of 19 amino acids (except methionine), in a reaction volume of 25 µL.
A total of 1 µg "RNA B" transcript was translated in parallel
as a control. The reactions were incubated at 30°C for 60 minutes
and placed on ice. The products were resolved in 15% sodium dodecyl
sulfate (SDS)-polyacrylamide25 and acid triton urea26 gels, treated with a fluorographic agent and
autoradiographed.
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RESULTS |
Hematological Data
The probands studied were father and son from a Chinese family. The
father (I-1) was a 58-year-old man who suffered from chronic ill health
and jaundice since childhood, requiring intermittent transfusions for
20 years. He had gross hepatosplenomegaly with hypochromic microcytic
anemia (Hb 6.7 g/dL, mean corpuscular volume [MCV] 63.6 fL, mean corpuscular hemoglobin [MCH] 19.0 pg) and reticulocytosis of 9%. Blood film examination showed nucleated red
blood cells, target cells, tear drop poikilocytes, and
gross basophilic stippling. Both HbA2 (5.5%) and HbF
(4.5%) were raised. The 51Chromium red blood cell
half-life was reduced at 12 days. Hereditary enzyme deficiencies
including glucose-6-phosphate dehydrogenase (G6PD), 6PGD, pyruvate
kinase, hexokinase, and glucose phosphate isomerase were excluded.
Serum ferritin was markedly raised at 4,900 µg/L. At the age of 58, he suffered an episode of cardiovascular collapse with atrial
fibrillation and hypoglycemia, from which he did not recover. As I-1
was adopted, little was known of his family history, but his father and
brother were said to have died of thalassemia at the age of 35 and
infancy, respectively. I-1 married an Irish woman (I-2) who was
hematologically normal. They had three sons, of whom one (II-3) had the
same blood profile as his father (Hb 8.6 g/dL, MCV 68.8 fL,
MCH 20.8 pg), with increased HbA2 (4.2%) and HbF (9.5%)
levels, and reticulocytosis of 8%. He had mild splenomegaly and
evidence of iron overload (serum ferritin 250 µg/L) despite the
absence of blood transfusions. His two siblings were clinically and
hematologically normal with serum ferritin levels within the normal
range (26.1 and 39.3 µg/L, normal range, 14 to 150 µg/L). The
relevant hematologic results of the family are shown in
Table 1. In view of the markedly elevated serum ferritin in I-1, the family was screened for the Cys282Tyr and
His63Asp mutations in the HFE gene for a possible interaction with hereditary hemochromatosis.27 The mutations were
detected by restriction enzyme analysis of specifically amplified
DNA,28 but neither of the probands carried the Cys282Tyr or
the His63Asp mutation.
Six control subjects heterozygous for IVS 2-654 C T
mutation with asymptomatic thalassemia trait were examined, together with one homozygote and one compound heterozygote for IVS
2-654/Chinese (A )0 thalassemia. Relevant
hematologic data are shown in Table 1.
DNA Analysis
The -globin genes from both probands were sequenced from position
 710 5 to the cap site to 240 bp downstream of the
termination site. The IVS 2-654 C T mutation was found
in both individuals (associated with haplotype I, +----++),
together with a normal A allele. No other mutational or
deletional defects were found. Gene mapping showed a normal number of
globin genes.
The IVS 2-654 mutation in the other patients was determined by
a combination of allele-specific priming and direct sequence analysis
of specifically amplified -globin genes.
Globin Chain Biosynthesis
Globin chain biosynthesis studies (Table 1) in the two probands showed
considerably greater globin chain imbalance (/non- ratios of 3.6 and 3.8) than in the asymptomatic IVS 2-654 cases studied (1.7 and
1.8) or in other -thalassemia traits (1.8 to 2.3). No abnormal
globin chains were observed on chromatography at pH 6.4 or 6.0. A time
course experiment on the blood of L II-3 did not show any evidence of
globin chain instability, nor did the /non- ratio change with a
pulse-chase experiment (data not shown).
The severe clinical course and unusually severe chain imbalance in
these two heterozygotes for the IVS 2-654 allele suggest that an
additional defect is present in these cases. An interacting erythrocyte
membrane abnormality would not explain the unusually imbalanced globin
chain biosynthesis ratios. The limited size of the pedigree precludes
the determination as to whether this is a cis or
trans-effect. Therefore, we undertook RNA analysis to try to
identify that part of the globin gene regulatory pathway, which was
likely to be affected.
RNA Analysis In Vivo
cDNA encompassing exons 3 and 2 obtained by RT-PCR (primers AP1 and
AP2) from peripheral blood RNA in normal subjects produces a band of
240 bp (Fig 1). In proband LI-1, a larger
313-bp fragment, representing the abnormally spliced cDNA, is also
seen (Fig 2A, lane 2). This band is only
just visible in the control heterozygotes (lanes 4 and 5) and absent in
the normal individuals (lanes 1 and 3). Only the aberrantly spliced
cDNA is seen in the IVS 2-654 homozygote (Fig 2A, lane 6). The
abnormal fragments were confirmed by Southern blot hybridization using
an internal oligoprobe, OP (Figs 1 and 2A, right).
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| Fig 1.
Representation of the genomic and cDNA structure of the
normal allele (A) and the mutant allele (B) showing the
products obtained by RT-PCR using the primers AP1/AP2 and AP3/AP2. OP
(5-GTCTGTGTGTGCTGGCCCATCA-3) is an oligonucleotide probe
used to hybridize Southern-blotted cDNA fragments. Sequences of the
primers are AP1: 5-CTGAGGAGAAGTCTGCCGTT-3, AP2:
5-GCTTAGTGATACTTG TGGGCC-3, and AP3:
5-TGAGGAGAAGTCTCGCGTTAC-3. A 654 riboprobe
for detecting the normal and aberrantly spliced transcripts was
generated by RT-PCR from a hemizygote for the IVS 2-654 mutation,
using primers AP4 (5-CAATGTATCATGCCTCTTTGCAC-3) and
AP2.
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| Fig 2.
(A) RT-PCR products of proband LI-1 and controls using
primers AP1 and AP2 (see Fig 1). Ethidium bromide-stained 2% agarose
gel is on the left and autoradiograph of Southern blot of the gel,
hybridized with oligoprobe OP, is shown on the right. The lanes are
represented by M:  X174 RF DNA-HaeIII; 1 and 3: normal
controls; 2: LI-1 (proband); 4 and 5: CI-1 and CI-2 (asymptomatic
heterozygotes); and 6: CII-1 (homozygote for the IVS 2-654 mutation).
(B) Radioactive RT-PCR of mRNA of probands and controls using
primers AP3 and radiolabelled AP2 after 20 cycles of amplification. The
mutant (654) and normal (N) cDNAs are 503 and 430 bp,
respectively. The lanes are represented by M: pBR322 DNA-Msp I
marker; 1: Proband LI-1; 2: Proband LII-3; 3: CII-1 (homozygote for IVS 2-654); lanes 4 to 9: asymptomatic heterozygote controls; 10: AH
( IVS 2-654/(A)0 thalassemia); 11: normal
control.
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Quantitative RT-PCR was performed using radiolabeled AP2 and AP3
probes, as shown in Fig 2B. A variable number of PCR cycles (18, 20, 22, 25, and 28) were applied to ensure that the samples analyzed were
in the linear phase of the PCR. Ratios of radioactivity of the aberrant
(503 bp) to normal (430 bp) cDNA were highly reproducible and
substantially greater in the probands (Fig 2B, lanes 1 and 2), ranging
from 4% to 11%, than in the asymptomatic heterozygotes (Fig 2B, lanes
4 to 9), 0.2% to 1%. The mean values were 6.7% and 0.6% in the
probands and control heterozygotes, respectively, representing an
approximately 10-fold increase in the proportion of the abnormally
spliced mRNA. In the homozygote (C II-1, Fig 2B, lane 3) and the
hemizygote (AH, Fig 2B, lane 10), the vast majority of cDNA was
abnormally spliced, as expected. These results were confirmed by RNase
protection assay, using the 654 riboprobe. The
abnormally spliced transcript was estimated to be 3.1% to 3.2% of
normal in the probands, substantially greater than the 0% to 0.5% in
the asymptomatic carriers (Fig 3A).
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| Fig 3.
(A) RNase protection assay of reticulocyte RNA, using the
654 riboprobe. Protected fragments of normal and
aberrant transcripts are 129 bp and 189 bp, respectively. The lanes are
represented by M: Marker (pBR322 DNA-Msp I); 1: Proband LI-1;
2: Proband LII-3; lanes 3 to 8 are the asymptomatic heterozygotes; N:
normal; 9: CII-1 ( IVS 2-654 homozygote) and 10: AH ( IVS
2-654/(A)0 thalassemia). (B) RNase
protection assay of RNA extracted from erythroblasts, cultured from
CD34+ peripheral progenitors, using the
654 probe. Protected fragments of the normal
(N) and aberrantly spliced (654)
transcripts, at 129 and 189 bp, are indicated. The lanes are
represented by M, pBR322 DNA-Msp I; 1, RH (5 µg RNA); 2, RH (1 µg
RNA); 3, normal control.
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This 10-fold increase in the proportion of abnormally spliced mRNA in
the probands suggested that the difference between them and the typical
heterozygotes might lie in the RNA processing pathway. Most of this
process takes place in nucleated erythroblasts, but bone marrow samples
were unavailable and further blood samples from either of the probands
were not accessible. It was hoped that analysis of erythroblasts from
an asymptomatic heterozygote may shed some light on any abnormality in
the RNA processing pathway. Therefore, CD34+ progenitor
cells from the peripheral blood of RH (an asymptomatic heterozygote)
were cultured under conditions for erythroid differentiation. Analysis
of the RNA from the these erythroblasts showed high levels of the
aberrantly spliced transcript,  30% of the normal (Fig 3B). This is
significantly higher than the 1% detected in peripheral reticulocytes,
suggesting that even in the asymptomatic patients there is substantial
transcription and processing of this message in erythroblasts, but that
it is less stable than the normal mRNA during erythroid cell
maturation.
Ratios of : reticulocyte mRNA were measured in the two probands
and two asymptomatic heterozygotes (RH and ST) using a H
riboprobe that protects both the normal and aberrant mRNAs
(Fig 4). The : mRNA ratios were also
assessed in erythroid progenitor RNA from RH and a normal control. No
clear cut difference in the ratio was seen in reticulocyte mRNA from
the probands and the asymptomatic patients. We have observed a similar
unexplained broad range of ratios in normal individuals and other
thalassemic patients (see legend to Fig 4). The : ratio of 1.9 in
the erythroblast RNA of RH is substantially lower than the ratio of 5.5 in reticulocyte RNA, confirming the greater amount of total mRNA in
the former.
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| Fig 4.
RNase protection assays to assess the : mRNA ratio
in CD34+ nucleated erythroblasts and reticulocytes of
heterozygotes of the IVS 2-654 C-T mutation. RNA was extracted from
the nucleated erythroblasts of one asymptomatic heterozygote (RH, lane
1) and one normal subject (lane 2). This was compared with reticulocyte
RNA of RH (lane 3), the probands LI-1 (lane 4) and LII-3 (lane 5), and
another asymptomatic heterozygote control (ST, lane 6). Protected
fragments of and mRNA, at 97 and 135 bp, are indicated. M
represents size marker pBR322 DNA-Msp I. The : mRNA
ratios in lanes 1 to 6 were 1.9, 2.1, 5.5, 5.4, 4.1, and 3.5, respectively. For comparison, : mRNA ratios in controls were: 1.8 to 2.7 (normals, n = 11); 3.3 to 5.8 (-thalassemia traits, n = 15), and 4.9, 5.1, and 6.4 (thalassemia intermedias).
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In Vitro Cell-Free Translation of mRNA Transcript
As aberrant splicing of the IVS 2-654 pre-mRNA results in
frameshift and premature termination at codon 121, the predicted -globin chain would be truncated by 26 residues. Taking the average molecular weight of an amino acid to be approximately 110 kD, the globin variant would be reduced from 16.5 kD to
approximately 13.6 kD. The predicted chain is also likely to be highly
unstablethe carboxy terminus is abnormal, with the net loss of 20 hydrophobic amino acids and the associated stabilizing interactions,
while the 11 contact would be greatly
affected, as all but one of the contact points in helix G are disrupted
and all those in Helix H are lost. As no abnormal protein was
detectable in the patient's red blood cells, in vitro cell-free
translation was performed.
Total reticulocyte RNA from proband I-1, a normal control, and an
asymptomatic heterozygote (ST) were subjected to in vitro translation
in rabbit reticulocyte lysate. In the SDS-PAGE gel, a 16-kD band was
identified in LI-1, the normal control, and the asymptomatic
heterozygote. An identical pattern was obtained in all cases with no
trace of a 13.6-kD band in the samples from the proband or the
asymptomatic heterozygote. Total RNA from cultured erythroblasts of RH
were also subjected to in vitro translation, as the proportion of
aberrant mRNA was much higher in these samples. Again, no lower
molecular weight (MW) band was detectable. On acid triton
urea electrophoresis, the translated , , G, and
A globin chains were identifiable, but again no
extraneous band could be seen (results not shown). In both the proband
LI-1 and the asymptomatic heterozygotes (ST), a reduction of compared with chains was demonstrated, the reduction being more
severe in LI-1, confirming the results of the globin chain biosynthesis studies.
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DISCUSSION |
The clinical course and the hematological findings in the two probands
in this study clearly demonstrated that both father and son have
thalassemia intermedia. Analysis of the -globin genes showed a
single IVS 2-654 C T mutation, together with a normal
-globin gene. This apparent dominant pattern of inheritance in
heterozygous -thalassemia has been well documented in cases with
premature stop codons in the third exon or point mutations leading to
highly unstable, abnormal globin chains.3 The necessity to
remove both the truncated or abnormal chains, as well as the excess
chains that ensue, is believed to overload the proteolytic mechanism of the erythroid precursors resulting in sufficient red
blood cell damage to produce the ineffective erythropoiesis characteristic of thalassemia intermedia.
The IVS 2-654 C T mutation results in an abnormally
spliced mRNA that, if translated, would lead to a -globin chain
truncated at codon 121 that would be predicted to be highly unstable.
However, the majority of cases that have been described have the
phenotype of asymptomatic -thalassemia trait and not the pattern of
dominant -thalassemia. In addition to our two cases, two other
Oriental IVS 2-654 heterozygotes have been described with
thalassemia intermedia.15,16 No other -globin gene
abnormality was detectable in these patients and family studies were
uninformative. It was not clear, therefore, whether these and our cases
represented a "dominant" IVS 2-654 allele or the interaction
of a second defect. Hence, we examined globin gene transcription, RNA
processing, and globin chain synthesis of the IVS 2-654 allele in
the probands and in the asymptomatic carriers.
Large amounts of aberrantly spliced mRNA from the IVS 2-654 allele
were detectable in early erythroblasts in one asymptomatic case. If
large amounts of the aberrant mRNA were to accumulate and be
translated, the abnormal globin chains produced should be highly
unstable and should result in most IVS 2-654 heterozygotes having
thalassemia intermedia, yet this is not the case. The answer to this
paradox may lie in the fact that no abnormal protein was detectable in
vivo, and we were unable to demonstrate the translation of the
aberrantly spliced 654 mRNA in a cell-free translation
system. It may be, therefore, that little, if any, of this abnormal
mRNA is translated and further investigation of this aspect seems
warranted.
A large decrease in the amount of the abnormally spliced
654 mRNA was observed during the maturation of
erythroblasts to reticulocytes suggesting instability of this mRNA,
which was also demonstrated in a functional expression system we have
devised using murine erythroleukemia (MEL) cells
(manuscript in preparation). Interestingly, the
proportion of the aberrant mRNA was 10-fold higher in the peripheral
blood of the probands than in their asymptomatic counterparts, one of
the few detectable differences between the two. This difference could
indicate that proteins involved in maintaining RNA stability or
responsible for its destruction differ in the affected and unaffected
carriers. However, the higher levels of 654 mRNA in the
thalassemia intermedia cases could simply reflect the generally younger
population of red blood cells in the circulation in these cases, as
shown by the presence of nucleated red blood cells and the higher
reticulocyte counts. As the proportion of this mRNA decreases with
erythroid maturation, younger cells should contain higher amounts.
The major difference observed between the two groups of patients was in
the /non- globin chain synthesis ratios. The ratios in the
thalassemia intermedia cases were not only higher than in the
asymptomatic cases, but were much higher than usually found in
-thalassemia heterozygotes and similar to that observed in the IVS 2-654 homozygote (C II-1) with thalassemia major. This result
implies that either there is reduced product from the apparently normal
-globin gene or that there is increased production of globin.
The difference in /non- ratios was not observed at the mRNA
level, where the range in the two probands was 4.1 to 5.4, within the
range for the asymptomatic patients, 3.5 to 5.5. This leads to the
conclusion, therefore, that the difference between the two groups lies
at or below the level of translation.
Little is known about the differential translation of and mRNAs; the level of mRNA is higher than mRNA in normal red blood cells, but equal amounts of the respective globin chains are
produced. Whether the reduced rate of translation of the mRNA is
solely due to a difference in the structure of the RNA itself or
whether it involves alterations in the binding of proteins other than
the ribosomes has not been established. Certainly there are differences
in the proteins that bind to the 3 untranslated regions of the
and mRNAs.29-31 A mutation in a protein that differentially affects the translation of and mRNAs could result in either increased chain production or decreased -chain production. Such an effect could be quite small and would not produce a
readily detectable effect in normal individuals, but when the cell is
already compromised by the presence of a -thalassemia gene, the
effect of the mutation may convert a phenotype of thalassemia trait
into thalassemia intermedia.
Alternatively, we cannot exclude the possibility that there may be
differences in the proteolytic capacities of the erythroid cells from
the probands and the asymptomatic cases. In asymptomatic -thalassemia heterozygotes, most of the excess chains do not accumulate within the cells and are presumably degraded by proteolytic mechanisms, particularly in the marrow erythroblasts.32
Degradation of newly synthesized chains is seen in the bone marrow
erythroblasts, but not in peripheral blood reticulocytes. Therefore,
any putative difference between the probands and the asymptomatic cases
might not be detectable in a peripheral blood incubation. If there is a
second defect in proteolysis, it would not be expected to affect globin
production in normal individuals and would be "silent."
If either of the interpretations offered above is correct, one would
predict that such a gene(s) affecting globin production would be found
in other situations. Moreover, the putative gene(s) need not be linked
to the -globin locus. We have previously documented a family with a
-thalassemia phenotype that was unlinked to the -globin
cluster,21 and other such cases have been
reported.33,34 Furthermore, heterozygotes for the codon
39 thalassemia allele with the phenotype of thalassemia intermedia have
recently been reported in two large families from
Sardinia.35 Family studies in these cases demonstrated
unequivocally that the postulated second defect was not linked to the
-globin cluster. Identification of such defects may be important for
a fuller understanding of globin chain production, as well as in
understanding genotype-phenotype relationships in the thalassemias.
| |
FOOTNOTES |
Submitted March 25, 1998;
accepted June 23, 1998.
Supported by the MRC, UK. P.J.H. was a Nuffield Dominions Fellow and
G.W.H. an MRC Training Fellow.
Address reprint requests to Swee Lay Thein, MD, MRC
Molecular Haematology Unit, Institute of Molecular Medicine, John
Radcliffe Hospital, Headington, Oxford, OX3 9DS, UK; e-mail:
swee.thein{at}imm.ox.ac.uk.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Liz Rose and Milly Graver for preparation of the manuscript,
Prof Sir D.J. Weatherall for his continuing encouragement and support,
the L family for their cooperation, Dr F Morlé for advice on the
reticulocyte lysate translation, and Jackie Sloane-Stanley for her
technical expertise.
| |
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Abstract
Introduction
Abstract