Download The Cellular Basis for Different Fetal Hemoglobin

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CONCISE
REPORT
The Cellular
Basis
for
Individuals
By G.J.
Different
With
Dover,
V.T.
Fetal
Two,
Chang,
Hemoglobin
Three,
S.H.
Boyer,
and
G.R.
Fetal hemoglobin
(HbF) levels
vary widely
among
individuals with
sickle
cell anemia
(SS).
Previous
studies
have
suggested
that HbF levels
in SS individuals
with
alphathalassemia
(two or three
functional
alpha-globin
genes)
are lower than HbF levels in SS individuals
with four normal
alpha-globin
genes.
Using
immunocytochemical
techniques,
we studied
F cell production
as measured
by % F
reticulocytes.
the
amount
of HbF per F cell,
and the
preferential
survival
of F cells
versus
non-F
cells
in 51
subjects
with four alpha
genes.
32 subjects
with
three
alpha genes.
and 1 8 subjects
with two alpha genes.
Cornparison
between
alpha-globin
gene groups
was performed
for the total sample
as well as for a subset
of 82 individuals
who
had replicate
samples
and a further
subset
of 39
age-matched
individuals.
%HbF levels
were
6.8. 4.9. and
4.5 percent
for the total
four-,
three-,
and two-alphaglobin-gene
groups.
respectively.
The percentage
of F
A
LPHA-thalassemia
modifies
many
eters
in individuals
who
disease
(55).
SS individuals
genes
counts,
have
with
hematologic
param-
homozygous
only two
have
higher
hemoglobin
levels,
lower
and lower mean corpuscular
hemoglobin
(MCHC)
than SS individuals
with four
tions
genes.’3
In
(HbF)
levels
globin
genes
genes.
patients
the largest
study
to date,3
were lower in 55 individuals
than in SS individuals
with
The reason
has been
In normal
of erythrocytes
for this difference
obscure.
and SS
called
individuals
F cells.4
cells
which
contain
percent
four
in the percent
HbF
HbF,
per
no
independent
variables;
as estimated
by the
detectable
alpha-globin
percentage
F cell
between
genes.6
Now,
uals, we substantiate
the
are lower in 55 individuals
HbF
(non-F
of F reticulocytes,
SS
individuals
in analysis
F cell
production
and
in 55 patients
the percent
with
find that the difference
to the enhanced
survival
with
coexisting
and
of HbF
without
in HbF
of the
per
HbF levels
genes than
F cell
are
alpha-thalassemia.
levels
non-F
the
We
is directly
attributable
cells in 55 individuals
alpha-thalassemia.
MATERIALS
METHODS
All patients
evaluated
attended
the Medical
Research
Council
Sickle Cell Clinic in Kingston,
Jamaica.
Eighty-three
55 individuals
Blood,
Vol 69,
No
1 (January),
1987:
four
selected
pp 34 1-344
& Stratton.
alpha-globin
obtained
clinic
Higgs
from
and
was
clinic
had
no recent
of the
consent
was
Clinical
was
Eighty-two
patients
or crisis.
time
of
had
been
gene
selection.
randomly
as having
not
Alpha-globin
their
were
genes)
randomly
two
Eighteen
selected
from
alpha-globin
genes
population.2
The average
age ( ± SD)
was 20.6 ± 9.5 years,
with no significant
the alpha-globin
gene groups.
Informed
same
clinic
in accordance
University
Investigation
sample
These
identified
obtained
the University
To ensure
siblings
individuals
population
between
Hopkins
alpha-globin
of SS
infection
at the
55
previously
for the entire
age differences
three
study
registry.7
known
gene
in a survey
32 with
a previous
the
not
Inc.
genes,
from
patients
with
Medical
and
the
the
guidelines
School
Clinical
within
a one-year
persons
underwent
such
of
Committee
of
parameters,
interval
from
a second
most
re-examination.
the
Committee
Investigation
of West Indies.
reproducibility
of hematologic
obtained
of both
Joint
subjects.
In addition,
an
age-matched
subset was analyzed
retrospectively
including
13 agematched
individuals
from each alpha-globin
gene number.
Statistical analysis
was performed
for the total population,
for subjects
in
which
second
samples
had
been
drawn,
and
for
the
age-matched
group.
From
the
Hopkins
and
of
West
the
August
Supported
by NIH
Medical
Address
reprint
of
more,
MD
Medicine,
Kingston,
The
Human
The
Howard
Biochemical
Council
Johns
Hughes
Genetics,
Laboratories,
Uni-
Jamaica.
accepted
No.
HL
September
28028
15.
and,
1986.
in Jamaica,
by the
requests
to G.J.
Traylor
Dover,
Bldg
Johns
907.
720
Hopkins
Rutland
University
Aye,
Balti-
21205.
“advertisement”
J 987
Research
Medicine,
payment.
(#{231}
for
Medical
Grant
and
Medicine;
Council.
publication
indicate
of
6, 1986,’
Research
School
Pediatrics
Laboratory
Indies,
Submitted
The
of
School
lnstitute,
Baltimore;
versity
Department
University
Medical
charge
AND
D.R.
two or
of 101 SS individ-
finding
of others3
that
with two alpha-globin
and
or the
with
in those with four alpha-globin
genes.
Furthermore,
in this
report,
we analyze
in detail
the three cellular
variables
that
determine
HbF
levels
in order
to determine
the basis
for
different
levels of HbF in these individuals.
We conclude
that
same
with
number
cells).5
In a previous
study
utilizing
a small
number
of SS
individuals
(10 to 15 subjects
per group)
we saw no difference
by Grune
two-alpha-globin
HbF is confined
to a subset
HbF levels in patients
with SS
Genes
S. Antonarakis,
1987
transfused
percentage
of F reticulocytes;
second,
the amount
of HbF per
F cell; and third,
the preferential
survival
of F cells versus
red
a
(51
SS
Cell
percentage
HbF per F cell. and the enrich(% F cell/%
F reticulocytes)
did not change
significantly
with
alpha-globin
gene
number.
Moreover,
no correlation
existed
between
alpha-globin
gene number
and the absolute
number
of F cells
in any group
studied. However.
there
was
a strong
inverse
correlation
(r = -0.407.
P = .0001 1 between
non-F cell levels
(1 .7 ± 2.
2.2 ± 5, 3.0 ± 1 .0 x 1012/1)
and
decreasing
alpha-globin
gene
number.
These
data suggest
that falling
HbF levels
among
SS individuals
with
lessened
numbers
of alphaglobin
genes
reflect
prolonged
survival
of non-F cells and
are not due to intrinsic
differences
in F cell production
or in
the amount
of HbF per F cell. The improved
survival
of
non-F cells in SS alpha-thalassemia
is presumed
to be due
to the lower MCHC observed
in such individuals.
reticulocyte
concentraalpha-globin
among
Sickle
reticulocytes.
ment
ratio
were
levels
Among
Alpha-Globin
Serjeant,
Johns
disease
is the summation
of three
first, the number
of F cells produced,
those
Four
sickle
cell
alpha-globin
fetal
hemoglobin
with two alphafour alpha-globin
in HbF
Levels
costs
ofthis
article
This
article
must
in
this
accordance
with
were
defrayed
therefore
18
U.S.C.
be
in part
hereby
§1734
by page
marked
solely
to
fact.
by Grune
& Stratton,
Inc.
0006-4971/87/6901-0063$03.00/0
341
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
342
DOVER
All samples
of blood were obtained
from venous
blood collected
in
EDTA.
Peripheral
blood
reticulocyte
counts
were obtained
using
conventional
new methylene
blue staining,
and red cell indices were
performed
using an electronic
cell counter
(Coulter
ZBI 6, Coulter
Electronics,
Hialeah,
FL). Alpha-globin
gene number
was determined
or redetermined,
in cases
of two-alpha-globin
by DNA restriction
endonuclease
from peripheral
blood leukocytes.8
assayed
by radial
either
cells
with
a monoclonal
levels
was
based
used
to
the
test
normally
for
of
(%
was
used.
tested
mean
equality
distributed
formation
denaturation
were
observed
counts
Bartlett’s
For
values
% reticulocytes),
degree
of
test
that
linear
correlation
number.
decrease
This decrease
reticulocyte
levels
bers,
the
negatively
not
is associated
to fall
between
gene.
percentage
of reticulocytes;
alpha-globin
data for the overall
were
found
between
The
and F enrichment
ratios,
F reticuof HbF per F cell. The remaining
were highly
correlated
with alpha-
bin
and
levels
alpha-globin
red
gene
all decreased
gene
cell
with
F
num-
gene
decreasing
alpha-globin
The
non-F
upshot
cells
=
P
is that
survival
F cell
num-
per liter
was
with decreas-
.0001)
alpha-thalassemia
of
non-F
number.
number
significantly
In contrast,
increased
1 . Hematologic
Features
previous
investigators,2”2
differences,
percentage
decreasing
of these,
different
and
Varying
cells.
The
2 is
had
was
to analyze
the
levels among
individuals
numbers
of alpha-globin
we
found
we considered
a
the
of HbF
levels
found
of Alpha-Globin
inverse
and the number
the origins
of
that
HbF
is discon-
in these
individuals.5
that contribute
to the
in peripheral
blood.
ie, the percentage
of F reticulocytes,
among
the three
alpha-globin
groups
Num bers
of
SS disease
Like some
significant
fact
red cells
variables
origins
with
genes.
Gen e (Mean
±
The
first
was not
studied
(Ta-
SD)
Genes
Alpha-Globin
4
N-51
in HbF
different
tinuously
distributed
among
We analyzed
the three major
with
in 55 In dividuals
paper
differences
who have
these
hemoglo-
with
of this
intent
correlation
between
the percentages
of HbF
of alpha-globin
genes.
In order
to determine
numbers.
Table
alpha-globin
outcome
is the same when analysis
like that in Table
applied
to either
mean
values
from
82 subjects
who
repeat
tests or to 39 age-matched
subjects.
globin
gene groups:
Picograms
of HbF per F cell, percentage
of F cells,
percentage
of HbF,
MCHC,
mean
corpuscular
volume
(MCV),
mean corpuscular
hemoglobin
(MCH),
and
decreasing
of
DISCUSSION
the hematologic
correlations
alpha-globin
gene number
locytes,
or the percentage
hematologic
parameters
percentage
so. Although
absolute
with alpha-globin
gene
prolonged
RESULTS
1 summarizes
No
significant
as the
number
of
(r = -0.407,
with
the
of
to
is due to the significant
decrease
in
percentages
noted
in Table
1 . Non-F
seem
absolute
correlated
ing alpha-globin
trans-
alpha-globin
gene numbers
and hematologic
parameters
were
assessed by linear correlation
coefficient
(r). Where the distributions
were nonnormal,
or the variances
were unequal, the Spearman-Rank
correlation
test was used.
Table
group.
Although
numbers
according
AL
not correlated
with alpha-globin
numbers
of F reticulocytes
(Ta-
genes
but not significantly
numbers
were not correlated
was
were
logarithmic
(Table
1) are
the absolute
reticulocyte
red
of HbF
by a chi-square
variance.
gene
reticulocytes
gene number,
ber falls.
the total
were
of fixed
alpha-globin
ble 2) significantly
techniques.
for normality
and
variance.
HbF,
The
F cell
or by analysis
antibody.’#{176}Percentage
by alkaline
distributions
on
procedure
anti-HbF
determined
Statistical
test,
reactions,’
above
subjects,
analysis
utilizing
DNA obtained
Percentage
of F reticulocytes
was
immunoprecipitate
by the
performed
gene
Table 2 provides
a comparison
of the absolute
F cells and non-F cells in SS patients
partitioned
ET
3
N-32
Statistics
2
N-18
ru
Il
Fenrichmentratio’
1.75
±
.89
1.58
±
.90
1.47
±
.55
.130
NS
Freticulocyte(per-
20.9
±
10.5
19.7
±
11.3
16.5
±
7.9
.149
NS
34.4
±
17.0
26.5
±
14.6
24.0
±
15.3
.262
.01
±
1.8
5.5
±
2.7
.403
.0001
±
6.2
23.2
±
12.2
.167
±
.28
±
.24
.242
cent)
Fcell(percent)
7.1
HbF/FcelI(pg)t
22.2
HbF/Fcell(%)
Hemoglobin
F
(per-
.89(6.8)
±
2.2
6.1
±
6.4
21.9
±
.28
.77(4.9)
.73(4.3)
NS
.02
cent)
Hemoglobin
MCHC
8.2
(g/dL)
Redcells(x
10’2/L)
MCV(fl)
MCH
(pg)
Reticulocytes
(per-
±
1.0
8.4
±
1.4
9.2
±
1.6
34.1
±
4.5
33.3
±
2.6
32.1
±
1.7
2.63
±
.39
3.04
±
.60
3.91
±
.82
91.6
±
6.9
82.5
±
6.8
72.7
±
5.5
31.6
±
2.9
28.0
±
3.0
23.7
±
±
.21
±
.19
±
1.03(9.6)
.96(8.1)
.83(5.8)
-.241
.02
.304
.005
-.627
.001
.725
.0001
2.1
.716
.0001
.19
.316
.002
cent)
F enrichment
ratio
HbF/F
celI(%)
-
§These
values
were
Figures
Hr (>0.05).
calculated
(pg) of HbF/F
fPicowams
in parentheses
regression
as the (F cell percent)/(F
cell
[pgHbF/F
(MCH
cell/MCH]
calculated
x %HbF)/F
x
100.
after logarithmic
represent
coefficient;
-
means
P
-
Note
MCH
percent).
of F cell and non-F
transformation.
reexpressed
probability
reticulocyte
cell%.
in original
of hematologic
For hemoglobin
cells are the same.’#{176}
F values,
log(%HbF
+
1 ); for
gene
number
reticulocytes,
log(%reticulocytes
+ 1).
units.
variable
correlated
with
alpha-globin
(see
Methods);
NS
-
not
significant
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HbF
LEVELS
IN
2.
Table
SS
DISEASE-ALPHA
Absolute
Number
343
THALASSEMIA
of F Cells
and Non-F
Cells
in 55 Individuals
and Varying
Alpha-Globin
4
F reticulocytes’
(x
Fcells(x
10’2/L)
reticulocytes
Non-FceIIs(x
(x
of F or non-F
reticulocytes
number
of F or non-F
cells
ble 1 ). However,
the
decreased
significantly
the
ber,
level
percentage
significant
(Table
is due
number
decrease
I ). These
in
themselves,
represent
While
on
there
F cell,
among
in the
actual
The
third
would
not
the
gene
of F cells.
of HbF
that
.13
.21
±
.09
.20
±
.12
.106
±
.17
±
.49
2.95
±
1.0
F cells
versus
the
ratios
contribute
to it. Consequently,
between
in
in the
second
of HbF
there
per
was
a
developed
of this
measured
ratios
such
ratios
1) was
was
not
analysis
of
by known
variation
and F cells, that
such estimates
are less precise
numbers
of non-F
cells which
variable.
Here,
a highly
in the number
of non-F
cells
of
individuals
with
decreasing
number
was observed
(Table
2). Since
did not change
between
groups,
the
of non-F
concentration
groups
in the
cells
indicate
that
two-
non-F
and
cells
threesurvive
reticulocytes).
with
come
MCHC
of a lessened
(Table
l).136
is highly
dependent
cell.’4 Recently,
HbS polymer
accounted
levels
leads
proportion
to a decreased
less dense
should
between
is that
the
of the
55
in all
irreversibly
A decrease
in the
with
of non-F
cells
have
MCHC
genes
two
and
three
in 55
in HbF
of alpha-globin
numbers
to differences
The
survival
of
this
to non-F
to
in 55
that
than
are
55
individunot
due
per
are
relatively
prolonged
55
individuals
consis-
of HbF
genes
the
We speculate
that previously
among SS alpha-thalassemic
prolonged
among
in alpha-thalassemic
cells
near
respects,
our
cells
in 55
or percentage
is due
cells
non-F
compared
genes.
MCHC
difference
are
expect
longer
genes
levels
in F cell production
F cell.
of
individuals
that
alpha-globin
als
different
has two
shorter
proportion
one would
would last
tent with previous
reports.
In summary,
differences
with
cells6
sickled
37 g/dL)
exhibits
>
and
do when MCHC
is elevated.
Thus,
in these
indicating
prolonged
survival
of non-F
individuals
varia-
syndromes.’5
MCHC
normal.6
Consequently,
in this setting,
non-F cells with near-normal
MCHC
they
data
red
lead to a relative
decrease
in the
survival
and non-F
cell survival.
majority
alpha-globin
polymerization
within
the
80%
of dense
cells.6
therefore
F cell
out-
alpha-thalasse-
different
dense cell fraction
(MCHC
it contains
very few F cells
than
with
indirect
that intracellular
basis of MCHC
and
to decreased
Alpha-thalassemia
two
with
for at least
among
and
fact
it is the
Brittenham
demonstrated
levels,
calculated
on the
concentration,
The
in comparigenes.
individuals
Sickle
hemoglobin
(HbS)
upon HbS concentration
in hemoglobin
with
that
associated
HbS
cells.6’6
alpha-thalassemia
suppose
tion
The
(%
NS
.0001
with four alpha-globin
cells last longer
in 55
We
dense cells
difference
peripheral
change
with
individuals
alpha-thalassemia?
survival
in the
NS
-.407
of F or non-F
in SS
hallmarks:
is the difference
survival
was
of enrichment
F reticulocytes
groups
alpha-globin-gene
to
(Ta-
.023
cells).
mia
and
an
cells
difficulty
of absolute
HbF-centered
correlation
that
have
levels
it is perturbable
is that
2).
in
F reticulocytes,
of all HbF-bearing
no change
x (percentage
longer
being
production
.043
son to 55 individuals
Why
do non-F
F cell.
HbF
non-F
The
of the two estimates,
alpha-globin
gene
non-F
reticulocytes
percent)
of F cells or non-F
a decline
in enrichment
gene
number
(Table
in each
than the measure
utilize
only
one
significant
inverse
per
magnitude
significant.
enrichment
x (reticulocyte
categories,
in preferential
by comparison
However,
statistically
(RBC)
expected
Thus
affects
F cells/%
F reticulocytes),
with decreasing
alpha-globin
higher
±
ie, picograms
alpha-globin
blood.
This difference
two ways. As assessed
liter
.23
differences
production,
percentage
of
be
significant
HbF
variable
in survival
per
.129
1), we presume
HbF
levels,
since
only a few percent
fall in the MCH
observed.
.57
to a concomitant
reticulocyte
were
affecting
parallel
.231
±
x (percentage
overall
changes
effect
variable
.030
.98
individuals
absolute
appreciable
cells.
±
.57
individuals
(Table
between-group
differences
of F reticulocytes
the fall in their
ble
.035
±
in two-alpha-globin-gene
of that in four-alpha-globin-gene
In the absence
of significant
the
±
.84
absolute
number
of F reticulocytes
with falling
alpha-globin
gene num-
77%
P
.50
2.20
SD)
±
r
.032
=
(RBC)
=
2
±
1.70
number
I oci (Me an
±
10’2/L)t
tAbsolute
Gene
Statistics
3
.051
obin
.93
10’2/L)
‘Absolute
of Alpha-GI
.056
10’2/L)t
Non-F
Numbers
Genes
individuals
with
four
demonstrated
individuals
alpha
declines
account
in
for
survival.
REFERENCES
1. Embury
SH, Dozy AM, Miller i, Davis iR, Kleman
KM.
Preisler H, Vichinsky
E, Lande WN, Lubin BH, Kan YW, Mentzer
WC: Concurrent
sickle-cell
anemia
and alpha-thalassemia.
Effect
on severity of anemia.
N Engl i Med 306:270,
1982
2.
Higgs
Hayes
Ri,
Serjeant
DR,
Aldridge
Grandison
BE,
Y,
Lowrie
GR: The interaction
sickle-cell
disease.
N EngI
Lamb
i, Clegg
Y,
Mason
of alpha-thalassemia
i Med
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1987 69: 341-344
The cellular basis for different fetal hemoglobin levels among sickle cell
individuals with two, three, and four alpha-globin genes
GJ Dover, VT Chang, SH Boyer, GR Serjeant, S Antonarakis and DR Higgs
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