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Mean Corpuscular Volume of Heterozygotes for p-Thalassemia Correlates
With the Severity of Mutations
By Deborah Rund, Dvora Filon, Nurith Strauss, Eliezer A. Rachmilewitz, and Ariella Oppenheim
The relationship between the degree of microcytosis and the
type of mutation carried by pthalassemia heterozygotes
was investigated. In 113 individuals, 18 different mutations
were identified, correlated with mean corpuscular volume
(MCV) values, and analyzed statistically. Overall, there was a
wide range of MCV (56.3-87.3 fL). In almost all cases, carriers
of f3” mutations had an MCV below 67 fL, whereas all but a
few p’ heterozygotes had MCVs above this cutpoint. Mean
MCV of p” carriers was statistically significantly lower than
those of p’ heterozygotes. The various p’ mutations were
associated with significant differences in mean MCV values.
In contrast, all the f3” (null) mutations had virtually identical
ranges of MCV. The results indicate that degree of reduction
in MCV is directly related to the severity of the mutation.
Deviations, in four cases, were associated with concurrent a
gene rearrangements, whereas in three other cases, the MCV
was not significantly affected by concurrent a rearrangements. The MCV of p-thalassemia heterozygotes is a valuable parameter in planning strategies for rapid identification
of mutations in populations with great mutational diversity.
Its use can be particularly advantageous in the setting of
prenatal diagnosis.The pattern of mean corpuscularhemoglobin (MCH) values was similar to the MCV pattern. However,
our results suggest that MCH may be preferred for carrier
detection in population screening.
0 1992 by The American Society of Hematology.
T
poses great difficulty in performing diagnosis by mutational
analysis.
In the present work we tested the hypothesis that MCV
of heterozygotes is related to the type of mutation they
carry. This hypothesis has two implications: (1) It indicates
that MCV is a phenotypic characteristic related directly to
the genotype, and (2) it suggests that MCV values may
serve as guidelines in prioritizing mutations when testing
heterozygotes for specific mutations. Although only 113
individuals were tested, we found that the MCV values of
individuals were directly related to the type of mutation
they carried.
HE HALLMARKS of P-thalassemia trait are microcytosis and hypochromia, but the mean corpuscular
volume (MCV) of heterozygotes can vary considerably,
from 60 fL or less, to the normal range.’,’ With the advent of
modern techniques of molecular biology, it has become
possible to examine these variations insofar as they relate to
the mutations that are present. A few such studies have
been performed. Rosatelli et a13 reported a relationship
between MCV values and two mutations that led to widely
disparate clinical manifestations, that is, a p” mutation
(nonsense in codon 39) versus one leading to an intermedia
phenotype (-87 C + G). Gonzalez-Redondo et a14noted a
variation in average MCV (and mean corpuscular hemoglobin [MCH]) values studying a wider range of mutations.
Three additional studies failed to show significant differences in MCV in relation to mutation except for isolated
mutations, which were found to be associated with an
unusually high MCV.5-7
At present, over 90 point mutations have been found to
cause P-thalassemia.8The identification of these mutations
has revolutionized prenatal diagnosis in many countries by
facilitating rapid first trimester testing using mutational
analysis. However, this requires prior knowledge of the
mutations present. In homogeneous populations such as
Sardinia, the number of mutations is small, and this is not
problematic. However, in some countries there is a great
mutational diversity. For example, in a small country such
as Israel (population 4.5 million), 20 different mutations
have thus far been discovered. The marked heterogeneity
From the Departments of Hematology and Information Sciences,
Hadassah University Hospital, Jerusalem, Israel.
Submitted December 20, 1990; accepted August 30, I991.
Supported by Grant No. 88-00357 from the United States-Israel
Binational Science Foundation, Jerusalem, and by the Bruno Goldberg Memorial Fund.
Address reprint requests to Deborah Rund, MD, Hematology
Department, Hadassah University Hospital, Jerusalem, Israel, 91 120.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section I734 solely to
indicate this fact.
0 1992 by The American Society of Hematology.
0006-4971I92 I 7901-0024$3.00/0
238
MATERIALS AND METHODS
Subjects were adults and children over 10 years of age who were
referred to Hadassah Medical Center for either genetic counseling
or evaluation of microcytic anemia. They came from many different
ethnic groups: Jews of Kurdish descent or of Mediterranean origin,
Arabs (Moslem or Christian), Druze, and Samaritans.
MCV and MCH were determined at Hadassah Hospital using an
automated Coulter S Plus IV analyzer. Hemoglobin A2(HbA,) was
determined spectrophotometrically after electrophoretic separation on cellulose acetate. DNA was isolated from peripheral blood
leukocytes using standard procedures? DNA samples were amplified by the polymerase chain reaction (PCR).“’ Mutations were
determined by screening the amplified DNA using radioactive
oligonucleotide probes.’’ Some of the mutations were identified by
restriction enzyme digestion of the amplified DNA fragments.”
p-globin gene haplotype analysis was performed, as described by
Orkin et al,” using seven polymorphic restriction sites throughout
the p-globin gene cluster (HincII E; Hind111 y, two sites; HincII $p,
two sites;Ava I1 p, and BamHI, 3’ to p).
The number of a-globin genes was determined by BamHI
dige~ti0n.I~
Statistical analysis was performed as follows. A two-sample t test
was used to compare the mean values of MCV between p” and p’
groups. Testing the equality of MCV means among the different
mutations was performed using analysis of variance. Methods of
discriminant analysis were applied to estimate functions that
discriminate between p” and p+, and, similarly, among the mutations. Prior probabilities for each group were assumed equal to
their relative frequencies in the sample. Fisher’s linear discriminant functions were used to estimate cut points of MCV that
distinguish between p” and p+, and, in a separate analysis, among
the different mutations, given MCV and ethnic origin. SPSSiPC
Blood, Vol79, No 1 (January I), 1992: pp 238-243
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GENOTYPE AND MCV OF P-THALASSEMIA CARRIERS
239
(SPSS, Inc, Chicago, IL) software was used to perform the analysis,
at the Hadassah Computer Center.
-101 C-T
Poly(A1 6
RESULTS
-28 A-C
A Poly (A)
Individuals with p” mutations have a lower MCVthan those
with p’. A family study (Fig l), which antedated the
technique of PCR, illustrates the observations that led us to
the present studies. A Kurdish Jewish couple (both carriers
of P-thalassemia) sought prenatal diagnosis for their sixth
pregnancy. They had one unaffected child and three children who were heterozygotes. A previous pregnancy had
been terminated after prenatal diagnosis by fetal blood
sampling. Routine hematologic tests and haplotype analysis
were performed in an attempt to identify a nonthalassemic
(A/A) sibling, and to facilitate DNA-based prenatal diagnosis by restriction fragment length polymorphism (RFLP).
The analysis suggested that the parents carried two different mutations, with a high MCV phenotype (mother)
and a lower MCV phenotype (father), which segregated in
the children (Fig 1).
We therefore commenced studies of all thalassemia
carriers who were available. Thus far, 113 P-thalassemia
heterozygotes, who were found to carry any of 18 different
point mutations, were examined. Nine of these were mutations leading to a p” phenotype and nine to a P’ phenotype.
Sixty-three individualswere carriers of 6’ mutations (55.8%)
and 50 of p” mutations (44.2%). These individuals represent a number of different ethnic groups: Arabs (54.9%),
Kurdish Jews (30.1%), Druze (10.6%), and others, which
include Samaritans and Jews of other Mediterranean countries (4.4%). Carriership of p” and P’ mutations was
approximately equal for individuals from the major ethnic
groups (Arab and Kurdish Jewish).
Figure 2 is a scattergram of the MCV of the subjects,
2
1
I
MCV76.5
MCV 73.8
II/vlI
Age13
MCV 72.5
II/vII
Age11
MCV 82.8
1/11
I
MCV 69
MCV 57
I/I
Aw3
I / VI1
Fig 1. Pedigree of S. family. Carriers of @-thalassemiaare designated as follows: vertically striped symbols, high MCV; horizontally
striped symbols, low MCV. Roman numerals under the symbols
indicate Mediterranean haplotypes as designated by Orkin et al.”
Note that the maternal thalassemic allele is linked t o Mediterranean
haplotype VII, whereas the paternal mutant allele is linked t o haplotype I (identical t o the haplotype of the normal maternal chromosome). Boldface Roman numerals refer t o generation number. In
subsequent analyses, the father (1-2) and child 11-5 were found t o have
lVSl nt 110, whereas the mother (1-1) and children 11-1 and 11-2 carry
the poly(A) point mutation. The MCV of child 11-5 was omitted from
the analysis presented in Fig 2 because of his age (3 years). His young
age accounts for the discrepancy seen between MCV values of father
and son. The sixth pregnancy was electively terminated after diagnosis of an affected fetus using haplotype analysis.
IVSl 6
I V S l -I
IVSl 5
I V S l I IO
I V S 2 745
IVS2 I
IVSl I
N39
8888
N37
FS 10617
FS 44
o~
0
p
oo
0
FS 3617
0
o”0
0
FS 8
FS 5
0
00
0
1
1
1
0
1
1
1
60
1
1
1
1
1
65
1
4
~
~
1
70
~
~
1
75
1
1
1
80
I
l I II
I I I II
II I ~
85
MCV ( f l )
Fig 2. Relationship of MCV t o mutation in heterozygotes for
@-thalassemia.Each circle represents an individual carrying any of the
mutations; (01,
18 mutations, as designated at the left. (01,
mutations. Diagonal lines represent individuals tested for deletional
a-thalassemia and found t o be normal. Additional symbols indicate
a-globin gene rearrangements found in the specific individuals. (0).
-&’loa; (0).deletion of 2 a genes in trans; (A), cmd“37/aa;(0).
-/au.
The mutations analyzed are designated on the left. N,
nonsense; FS, frameshift. The mutations are, from top t o bottom:
- 101 C + T; poly(A) nt 6 (AATAAA -+ AATAAG); -28 A + C; poly(A)
deletion (AATAAA + A-----); lVSl nt 6 (T C); lVSl -1 (G C); lVS1
nt 5 (G C); l V S l n t 110 (G A); IVS2 nt 745 (C -+ G); IVS2 n t 1
nonsense 39 (C-+T); nonsense 37
(G+A); lVSl n t 1 (G-A);
(G +A); frameshift 106/107 (+G); frameshift 44 ( 4 ) ; frameshift
36/37 (-T); frameshift 8 (-AA); and frameshift 5 (-CT).
-
- -
-
grouped according to the mutations that they carry. The
mean MCV value for p” mutations was 63.1 (SD = 3.4),
whereas for P’ mutations it was 69.3 (SD = 5.6). The
difference between the mean values of MCV of the two
groups was statistically significant (P < .mol).
Could MCV have a predictive value for a p” mutation
versus P’? This question was answered positively by discriminant analysis, which yielded a significant discriminant
function for p” versus P’ that includes MCV (P < .0001).
This means that MCV can be used to predict whether a
heterozygote carries a p” or a P’ mutation. Estimating
Fisher’s linear discriminant functions, we arrived at the
classification rule that if the MCV is greater than 66.96, the
mutation is P+, and if the MCV is lower, it is p” (arrow, Fig
2). Using this cutpoint, we obtained the results presented in
Table 1, with 81.4% of individuals correctly classified as P’
or p”. The major source of erroneous predictions was the
MCV of individuals with the p’ mutation IVSl nt 110, half
(11 of 21) of whom had MCV values below the p”/P’
boundary (Fig 2). Another source of error was the low
MCV of two individuals with the IVS2 nt 745 mutation.
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RUND ET AL
240
Table 1. Use of MCV in PredictingWhether Heterozygotes Carry
or p’ Mutations: Classification Results
Type of
Mutation Predicted
p”
P‘
No. of
Individuals
Po
P‘
60 (95.2%)
18 (36.0%)
Total
78
3 (4.8%)
32 (64.0%)
35
63
50
113
Type of
Mutation Found
The MCV value used to predict the type of mutation (p” or
66.96 fL. Correctlv classified cases: 92/113 = 81.4%.
p’) was
Different p’ mutations result in different MCV values.
There was a wide range of MCV values (56 to 87 fL), and
their distribution (Fig 2) is nonrandom. We asked whether
the different mutations have MCV values that are distinguishable from each other in a statistically significant
fashion.
We tested this hypothesis using analysis of variance. All
of the groups (a set of individuals carrying a given mutation) carrying p” mutations were found to have no statistically significant intermutational variation in MCV (P = .86).
That is, all carriers of p” mutations behave as a single group
with regard to MCV. In contrast, the different @’ mutations
showed statistically significant differences in mean MCV
(P < .0001) between groups.
We wished to test how useful these data would be in
predicting mutations among the p’ individuals, as an aid in
mutational screening of heterozygotes. This was performed
using discriminant analysis in a similar way to the analysis of
p” versus p’ mutations. The results showed that even
though the present data are limited, ranges of expected
MCV values could be delineated for the different mutations. For certain mutations there was an overlap in the
MCV ranges. That is, an individual with a certain MCV
value would be expected to have any of several mutations.
It is a common practice in mutational screening to use
ethnic origin as a guideline. Therefore, we also tested the
effect of introducing ethnic origin as an additional variable.
This resulted in a better ability to discriminate among the
various mutations on the basis of MCV. This is primarily
because the ethnic background helped pinpoint the mutation; some of the p’ mutations (three of nine) are ethnic
group specific. These results, and a more expanded statistical treatment of the data, will be published elsewhere.
MCV and ethnic background. Because specific mutations are characteristic of different ethnic groups, the
different MCVs could reflect the ethnic background rather
than the type of mutation. However, the findings thus far
also raised the interesting possibility that the reduction in
MCV directly results from the severity of the mutation. We
therefore wanted to determine whether MCV is influenced
by ethnic background for any given mutation. The availability of mutations that are widespread across different ethnic
groups enabled us to do the analysis.
Regression analysis was performed on the carriers of the
p’ mutations only, because p” mutations were homogeneous and thus were not expected to be informative. To test
this hypothesis directly and eliminate a factor that could
introduce bias, individuals with known a-globin gene rearrangements were excluded. Using this analysis, the only
factor of significance in determining MCV was found to be
the severity of mutation (P < .OO01) and the ethnic origin
had no additional significant contributory effect (P = .46).
Confounding factors. Because concomitant presence of
a-thalassemia may affect the MCV of heterozygotes,l’Z
analysis of a-globin gene number was performed in 32 of
the individuals. Because we wanted to find whether a-thalassemia affected the MCV distribution, the individuals were
selected in order to increase the likelihood of identifying
those with concomitant a-globin rearrangements. Most of
the individuals selected for analysis had an MCV that was
outside, or at the extremes of, the range of MCV seen in
others with the same mutation. These individuals are
designated (by a diagonal line) in Fig 2.
Twenty-five of the 32 individuals examined had a normal
a-globin gene pattern. Of the remaining seven, three had
one a gene deleted ( -a3’/aa);in a fourth, two a genes were
deleted in trans; in the fifth and sixth, a triplicated Q gene
and in the seventh, an a-globin
was present (aaaantJ7/aa);
quadruplication was found (aaaa/aa).
The high percentage of individuals found to have a-globin gene rearrangements (7 of 32, 21.8%) is most likely due to the biased
sampling, as already explained, because a-thalassemia is
not known to be highly prevalent in 1~rael.l~
In evaluating the effect of concurrent p- and a-thalassemia on the MCV of the heterozygotes tested, we found that
in two of these individuals, a-globin gene deletion(s)
appeared to contribute to the unexpectedly high MCV
(inverted triangle and diamond, Fig 2). In two other
individuals, the triplicated a-globin locus may have contributed to an unexpectedly low MCV (triangles, Fig 2). In
three individuals the MCV stayed within the range despite
a rearrangement. Overall, statistical analysis for the values
described (Table 2) were only slightly changed if carriers
doubly heterozygous for a- as well as P-thalassemia were
omitted from the analysis. This is remarkable considering
that four of the seven data points omitted were MCVvalues
that deviated considerably from the mean.
Relationship of mutations to MCH. The MCH data for
106 of these same individuals and mutations were also
evaluated (MCH data were unavailable for seven individuals) (Fig 3). Overall, we found that the visual spread of the
points was similar to the MCV data. There was a wide range
of MCH, 17-28.2 pg/cell, yet the point values were distributed in a nonrandom fashion. Carriers of p” mutations had
a mean MCH of 19.7 (SD = 1.26), whereas heterozygotes
for @’ mutations had a mean MCH of 21.8 (SD = 2.03).
These differences were statistically significant. Further
statistical analysis resulted in a cutpoint of 20.94 to discriminate between carriers of p” and p’ mutations. However,
Table 2. Effect of Concurrent u Gene Rearrangement on Mean MCV
of p-Thalassemia Heterozygotes
Mean MCV p”
Mean MCV p’
Cutpoint for p” or p’
No. of individuals tested
a-Thalassemia
Individuals
Included
wThalassemia
Individuals
Not Included
63.1
69.3
66.96
113
62.9
69.3
66.74
106
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GENOTYPE AND MCV OF P-THALASSEMIA CARRIERS
Fig 3, Relationship of MCH to tVpe of mutation. Each circle
represents an individual carrying any of the 18 mutations as designated at the left. (0).B” mutations; (01,p’ mutations. Diagonal lines
and additional symbols are as designated in Fig 2. The mutations are
identicalto those in Fig 2. N, nonsense; FS, frameshift.
the MCH is less sensitive to the difference between p” and
severe p+mutations. Almost all of the individuals (21 of 22)
with the p’ mutation intervening sequence (IVS) nt 110
had MCH below the 20.94 cutpoint. similarly, SOme beterozygotes for Ivsl nt 5 and lvsl nt -1 would have been
misinterpreted as p” carriers if MCH had been used as a
guideline for mutational screening.
ffbA, levels did not c o ~ e l ~ with
t e spec@ mutations.
HbA, levels are a parameter less readily available than
MCV. These data were available on 77 of the individuals.
There were significant differences ( p = .0008) in HbA,
levels for p” (mean = 5-13, SD = 937) versus p+ carriers
(mean = 4.28, SD = 1.193). However, the difference was
less significant than for the analogous data for MCV and
MCH, with a considerable overlap in values (data not
shown). We tested for differences among mutations within
groups of p” and p’ heterozygotes separately. The analysis
showed no significant differences for the p” mutations as
well as for the p’ group (p = .46). This means that, in
contrast to MCV, HbA, cannot be used to predict type of p’
mutation. Others have also found heterogeneity in HbA,
levels of different thalassemia mutation^.^.'^
DISCUSSION
As has been noted by Weatherall and Clegg,’ it would be
useful to differentiate between p” and p’ thalassemia in
heterozygous carriers. Attempts to do this have been made
in various populations (Jamaican blacks, Thais, and Greeks)
241
before the advent of molecular techniques. In blacks, there
was a significant difference in the average MCV and MCH
of p” versus p’ carriers. However, in the other groups
studied, such a distinction was not found. Weatherall and
Clegg concluded that the heterozygous state for p” thalassemia and the more severe form of p’ thalassemia were
indistinguishable by the techniques that were then available.
Our results confirm and extend these findings. We found
that the p” mutations were homogeneous, as would be
expected due to their null phenotype. This homogeneity
was supported by statistical analysis. However, the use of
molecular techniques and precise identification of the
various p’ mutations showed that not only were they
distinguished from p” mutations, but the various p’ mutations were associated with different MCV values. In general, it would appear that internal IVS mutations (activation of cryptic splice sites) cause lower MCV than mutations
in consensus sequences of splice signals. Transcriptional
mutations and polyadenylation signal mutations are milder.
Moreover, it is of note that a mutation in the TATA box
lowers the MCV more than one in the CACACCC upstream element. A deletion of 5 bp in the poly(A) signal
sequence is more severe than a point mutation in the signal.
Other investigators have reported MCV values for heterozygotes that are consistent with our findings. For example, in U.S. blacks, heterozygotes for a different TATA box
mutation, -29 A + G, had MCV 70-72.8 fL,4 almost
identical to our findings for -28 A --* C. The - 101 c --* T
mutation in Turkey was associated with a normal to high
MCV” and a normal MCV in Italians,’* as we found. In
addition, three other point mutations in the poly(A) signal
were repofled to lead to MCVs in the Same range (71-78
fL)””)as found for heterozygotes for the PolY(A) mutations
in our region. This suggests that the nature of the mutation
is a crucial determinant of the MCV even in disparate
Populations.
The clustering of MCV values for each mutation is
Striking in View Of the many factors known to influence the
volume of the erythrocyte. Some of the factors that lower
the MCV are those that decrease Heme production, such as
iron deficiency and lead poisoning. Other factors can raise
the MCV, for example, ethanol abuse and folate deficiency.
The latter are rare in Israel. The incidence of concurrent
iron deficiency in Israeli f3-thalassemia carriers was rePorted to be low (4%) and confined, in the adult population, to women of childbearing age.” Because ours was
Primarily a retrospective analysis, iron deficiency was not
tested. Young adult males, in the age range of most of the
heterozygotes we studied, were not iron deficient,” as was
also reported for other Middle Eastern populations.2’
One of the confounding factors affecting MCV values is
concurrent a-thalassemia or a-globin gene multiplicity.
Because for every mutation there is a range of MCV values,
it is expected that in some individuals the MCV will remain
within the range in spite of coexistent a-globin gene
abnormalities. Indeed, that was found for three individuals
in our present study (Fig 2). In four other individuals,
a-gene rearrangements appeared to be responsible for
deviations in MCV. The reasons for MCV values that
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RUND ET AL
242
departed from the expected range in several other individuals remains unexplained. These may be due to nondeletional a-thalassemia, which was not excluded in the present
study (or to any of the other possible confounding factors
already mentioned).
Despite the many variables affecting MCV, this parameter is extremely useful in planning strategies for the rapid
characterization of mutations. We found that the MCV is a
reliable indicator in planning how to prioritize which
mutations are to be sought. In Israel, ethnic group identification is also a valuable consideration, as is the precise
geographic origin of the family and its forbears, because
some ethnic groups have remained relatively inbred for
extended periods.= Obviously, expediting screening for
multiple mutations is crucial under the time constraints
imposed by an ongoing pregnancy. Furthermore, cost and
use of technician time are thereby minimized.
The MCH values were generally distributed similarly to
MCV values (Fig 3), but we found the MCH to be a less
sensitive discriminator between the p" and some of the
more severe P' forms of thalassemia trait. However, for the
purposes of carrier detection, MCH seemed superior to
MCV. Using accepted normal values (MCV 84 & 7 fL,
MCH 29 3 pg/cell) six P-thalassemia heterozygotes would
not have been identified as carriers using MCV as a
screening criterion. Only three of these would have been
missed using MCH (Figs 2 and 3). This supports the
conclusion of Modell and Berdoukas: who suggested that
the MCH is more useful for population screening.
Our results suggest that the MCV is not affected by the
general genetic background or by the haplotype of the
p-globin gene cluster. Mutations that are found in different
ethnic groups, and are associated with different P-globin
haplotypes (unpublished results) do not seem to result in
markedly different MCV values. It will be interesting to
reexamine this point as additional data accumulate from
other populations. In homozygous P-thalassemia patients,
disease severity may vary according to the haplotypic
background of the mutation, generally in conjunction with
variations in Hb F prod~ction.5"~~
There is considerable evidence that the nature of the
P-thalassemia mutation directly affects the level of P-globin
chain production. The direct relationship to the hemoglobin content of the erythrocyte (MCH) implies, perhaps not
surprisingly, that in P-thalassemia heterozygotes the level
of P-chains is a major limiting factor in hemoglobin production. However, the direct genotype-phenotype relationship
between mutations and MCV is intriguing, We would like
to speculate that the relative excess of a-chains plays an
important role. It is known that precipitated excess a-chains
have a profound effect on the structure of the erythrocyte
cyto~keleton?~
One might predict that MCV (moreso than
MCH) would be sensitive to a-gene deletions, because
these would ameliorate the chain imbalance. Indeed, the
three individuals with a-gene deletions for whom both
MCV and MCH values were available show a shift in their
MCV to higher values compared with other individuals
carrying the same mutation. The MCH value for only one of
these individuals (with two a-genes deleted, carrying IVS2
nt 1) was elevated, but to a lesser degree than the MCV
(compare Figs 2 and 3). The precise mechanism by which
the level of chain synthesis lowers the volume of the
erythrocyte remains to be elucidated.
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GENOTYPE AND MCV OF B-THALASSEMIA CARRIERS
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Mean corpuscular volume of heterozygotes for beta-thalassemia
correlates with the severity of mutations
D Rund, D Filon, N Strauss, EA Rachmilewitz and A Oppenheim
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