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Hereditary
Poikilocytic
Anemia
Associated
Alpha
By Tern
This
report
describes
structural
and
A. larocci,
a black
defects
of alpha
in combination.
The
hemolytic
anemia
hereditary
pyropoikilocytosis
cytosis.
is a compound
alpha”5
and
spectrin
sibling.
while
the
father
tent
was
the
were
normal
H
EREDITARY
HPP
severe
and
those
who
had
found
The
in an
alpha#{176}
hemolytic
extreme
poikilocytic
for thermal
red
cell
have
also
alpha
submembranous
and visco-elastic
but
patient
cy, a finding
in
morpholoof red
fragility.’4
Nearly
in homozygous
Structural
and quantitative
a major
component
of the
skeleton
responsible
for the shape
of red cells, form the molecular
spectrin,
protein
properties
for the hematologic
findings
HE.5’6
in HPP2’4
and
some
cases
biochemically
distinct
report here a family
in which
two
alpha
spectrin
defects
were identified.
The
spectrin
lesion
of homozygous
maternal
We
was
associated
with
hereditary
but no abnormalities
of red cell morphology
the paternal
defect.
A male
offspring
who
the maternal
and paternal
spectrin
abnormali-
elliptocytosis,
accompanied
inherited
both
ties exhibited
poikilocytosis
and severe hemolytic
anemia
during
infancy.
Although
the anemia
improved
by the age of
1 year,
hemolysis
continued
and
red cell
morphology
remained
poikilocytic
over the first
course
compatible
with that of HPP
findings
in this
child,
the first
4 years of life,
or homozygous
reported
example
heterozygosity
for the spectnin
alpha”65
tions,
suggested
a cumulative
effect
membrane
skeletal
function
and clinical
From
the
Departments
University
of California,
tal ofOakland
Research
Submitted
May
Supported
by
(AM32094,
DK26263.
Address
2108,
reprint
Bldg
San Francisco,
indicate
©
1988
the Children’s
accepted
from
and
January
National
Medicine,
Hospi-
4. 1988.
Institutes
of
Health
MD,
Room
HD06633).
to William
Francisco
This
article
General
in accordance
article
must
with
this fact.
by Grune
& Stratton,
0006-4971/88/7105-0027$3.00/0
1390
the
costs ofthis
payment.
“advertisement”
Laboratory
and
C. Mentzer,
Hospital,
Jr.
1001
Potrero
Ave.
CA 94110.
The publication
charge
and
requests
20, San
of compound
and alphah/5a
mutaof these
defects
on
severity.
of Pediatrics
14. 1987;
grants
a clinical
HE. The
San Francisco;
Institute,
CA.
Inc.
were defrayed
therefore
18 U.S.C.
in part
be hereby
section
a 1988
by page
marked
1 734 solely
to
absent
members.
classic
two
alpha
hemolytic
greater
of the
in the
HPP
and
family
in the
members.
defects
milder
clinical
presence
was
proposi-
ektacytome-
spectrin
anemia
changes
Mechanical
by
in other
at
and
dimers
in the
measured
in both
The
minimal
sevenfold
moderately
in HPP.
only
of spectrin
family
ghosts.
of
by Grune
but
HPP.
other
be a consequence
can
than
severity
of spectrin
of
that
HPP
deficien-
propositus.
& Stratton, Inc.
MATERIALS
is a
characterized
reported
co-inheritance
found
cells
severely
in a poikilocytic
may
and
percentage
only
result
usually
red
The
in
Thus,
defects
well.
Prolonged
incubation
budding,
fragmentation,
in classic
threefold
reduced
(HE).56
elliptocytosis
of
been
of Two
C. Mentzer
HPP
of erythrocyte
was
(HPP)
mechanical
and
stability
propositus.
in
defect
erythrocyte
fragmentation
tenfold
try.
con-
unrelated
anemia
cells.
and
functional
spectrin
of classic
The
present
spectrin
spectrin
two
tus,
by
mother
was
cell
members.
the
spectrin
fragments.
PYROPOIKILOCYTOSIS
findings
hereditary
basis
with
the
and William
deficient
as
in striking
in propositus
increased
ellipto-
in his
red
of these
by bizarre
threshold
abnormalities
tryptic
found
The
family
congenital
its classic
form
gy, a lowered
identical
all
of
as demonstrated
Co-Inheritance
A. Lane,
spectrin
resulted
sphering
features
both
Peter
was
37CC
singly
a poikilocytic
the
alpha’#{176} defect
sibling.
in
compared
classic
for
was
of inheritance
with
cells,
spectrin
has
Mohandas,
distinct
or homozygous
of spectrin
alone
two
the
Abnormalities
Narla
inherited
of
alpha’#{176} defects
another
consequences
many
(HPP)
defect
and
in which
were
who
shares
analysis
alphaUb
family
spectrin
heterozygote
spectrin
electrophoretic
Gail M. Wagner,
propositus,
that
Spectrin
With
AND
METHODS
reagents were purchased
from Bio-Rad Laboratodiisopropylfluonophosphate
(DFP) and phenylmethylsulfonyl
fluoride (PMSF)
from Sigma Chemical
Company,
St Louis; Seakem aganose from FMC Corporation,
Rockland,
ME;
Trypsin-TPCK
from Worthington/Cooper
Biomedical,
Inc, Malvern, PA. Horseradish
peroxidase-conjugated
goat anti-rabbit
IgG
antibodies
were obtained
from Pd-Freeze
Biologicals,
Rogers, AR.
The study
protocol
was approved
by the Committee
on Human
Research
of the University
of California,
San Francisco.
Phlebotomy
and routine
hematology.
Routine blood counts and
reticulocyte
determination
were performed
on EDTA
anticoagulated blood.
For all other studies, samples
were anticoagulated
in
acid citrate dextrose and stored at 4#{176}C
until studied. All studies were
performed
within 12 to 24 hours of phlebotomy.
Red cell membrane
protein
analysis.
Red cell membrane ghosts
were prepared
at 0 to 4#{176}C
using techniques
that we used previously.7
PMSF
0.2 mmol/L,
was added
to all preparative
solutions
to
minimize
proteolysis.
Red cell membrane
proteins were analyzed
by
one dimensional
SDS-PAGE8
using either a 4.5% acrylamide
stacking gel and a 4% to 15% nonlinear
acrylamide
separating
gel or a
nonlinear
3.5% to 17% polyacrylamide
gradient
SDS slab gel.9 The
spectnin to band 3 ratio was determined
by excising the Coomassie
blue stained
bands (spectrin and band 3), eluting the dye in 25%
pynidine overnight,
and reading the absonbance
at 605 nm.’#{176}
Spectrin
analysis.
A crude spectrin extract was obtained
from
fresh ghosts by low ionic strength
extraction
for 16 hours at 0 to
1 The
percentage
of spectnin
dimers
(expressed
as dimers/
dimers plus tetramers)
present in this extract was determined
by low
temperature
(0 to 4#{176}C),
nondenatuning
2.5% acrylamide,
0.3%
agarose composite gel electrophonesis.’2
Spectnin dimens and tetnamens were quantitated
by densitometry
at 545 nm.
An aliquot of the crude spectnin extract was subjected
to limited
tryptic
digestion.
PMSF
was removed
by overnight
dialysis
in 200
vol of 20 mmol/L
Tnis buffer, pH 8.0. The protein concentration
of
the extracts was determined
with the Bio-Rad protein assay (BioRad Laboratories)
using bovine serum albumin as the standard.
The
spectnin
extracts
were digested for 16 hours at 4#{176}C
using an enzyme
to substrate
ratio of 1:200 (wt:wt).’3
To more completely
charactenize the kinetics of digestion spectnin was also digested
for periods
from 30 minutes to 4 hours at 4#{176}C
using an enzyme to substrate
ratio
of 1:20 (wt:wt).
Digestion
was terminated
by adding diisopropylfluonophosphate
to a 1 mmol/L
final concentration.
The spectnin
tryptic fragments were analyzed by two dimensional
electrophoresis.
Isoelectnic
focusing tube gels were prepared
with a pH gradient
of
Electrophoresis
ries, Richmond,
CA;
Blood,
Vol 71. No 5 (May). 1988:
pp 1390-1396
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
HEREDITARY
POIKILOCYTIC
ANEMIA
1391
3.5 to 10 by the method
ofO’Fanrell’4
with modifications.’5
Each gel
was loaded with I 50 ig of protein. For electrophonesis
of the focused
proteins
in the second dimension
we used a linear
10% to 15%
polyacnylamide
gradient
SDS slab gel.8
For immunologic
analysis
of the crude spectnin digests, spectnin
tryptic fragments
were transferred
from SDS slab gels onto nitrocellulose paper
(0.45
nm pores)
and subsequently
reacted
with a
polyclonal
rabbit anti-alpha
spectnin antibody
(specific for the alpha
I tryptic domain)
as previously
described.7
Red cell thermal
fragility.
Morphologic
changes (membrane
fragmentation
and budding)
were assessed after heating washed red
cells to various temperatures
between 37 and 50#{176}C
for ten minutes as
previously
CASE
The
propositus
the
tribe,
exhibited
severe
hyperbilirubinemia
totherapy.
hemoglobin
rose
to 9.9 g/dL
was
transfusion
and
costal
State,
06/1
8/83)
Nigeria.
required.
black
At
by 6 months
administered.
tip
margin.
Red
remains
cell
ofage.
fell precipitously
of fever (Tmax
Splenomegaly
the spleen
had
poikilocytosis
elliptocytic,
but was not
count
3%).
resolved
like
that
is shown
An
and
of the
in Fig
unrelated,
anemic
(Hb
By 7 months
red
cell
mother.
17.4 g/dL,
of age the
morphology
The
was
pedigree
of the
I.
23-year-old,
splenectomized
(patient
purposes
K.B.)
who
has HPP
in many of the studies
features
of HPP,
namely
black
was used
for
of this family.
an alpha
spectrin
woman
comparative
The classic
mutation,
spec-
deficiency,
a greatly
increased
fraction
of spectrin
dimers,
poikilocytic
and spherocytic
red cell morphology,
a
lowered
threshold
for thermal
fragmentation
of red cells,
No
was
palpable
morphology
red cell
extreme
=
noted
Representative
member
and
presented
in Table
which
was
morphology
severe
Her
hemolytic
clinical
course
(elliptocytes,
1 . Only
obtained
who has
KB.
and
was
developed
elliptocytosis
Red
exhibited
cell
cells
microfragments),
in the blood
poikilocytosis
itus.
amounts
previously
seen
at birth
the
(Fig
with
of
in the younger
morphology
protein
protein
reported
projections,
and
were
2).
sister,
seen
Mild
113, who
of common
heredi-
2).
membrane
to band
spectnin
112, were
microcytosis,
spherocytes
of K.B.
(Fig
composition,
assessed
was normal
in all family
members
His pattern
and that of patient
KB.
PAGE,
on each
family
classic
HPP,
are
the propositus,
Both
microelliptocytes,
shaped
frequently
subsequently
tary
counts
K.B.,
particularly
striking
in KB.
Although
red cell
was distinctly
abnormal
in both
individuals
irregularly
far more
4.5, protein
8,
in other
patients
3 ratio,
assessed
except
revealed
by SDSthe proposincreased
and
globin,
findings
with
HPP.’62’
The
by
Fairbanks
non-linear
acrylamide
gel electrophoresis,
was normal
in the propositus
and his family,
indicating
that spectrin
was present
in normal
amounts
(Table
2). In contrast,
the spectrin
to band 3 ratio
was
decrased
present
he
in patient
in cold
KB.
low
ionic
The
percentage
strength
of spectnin
spectnin
extracts
I
The
(7.4
followa blood
transfusions
in early
have
II
infancy
the
markedly
1
left
poiki-
Fig 1 .
(normal
tologically
mother
alpha
sister.
blood
patient
on
and had reticulocytosis.
anemic
history
has
and hema-
as is his 5-year-old
and
At the age of
1 to 2 cm below
remains
fragility,
RESULTS
and
pho-
birth.
of age
to 5.9 g/dL
104#{176})
and
other
first
mechanical
anemia
were all found
in this individual.
has been summarized
elsewhere.3
locytic
at 4 years of age. A portion
of his clinical
previously
been reported.2#{176} His father
is clinically
normal
at birth
reticulocyte
boy
birth,
poikilocytosis,
hemolytic
anemia,
requiring
exchange
transfusion
and
2 years the hemoglobin
ing a two-day
history
been
(BD
Kwara
Hemolytic
anemia
persisted
after
level reached
a nadir
at 19 weeks
then
g/dL)
poikilocytosis
MCV
100,
family
elliptocytosis
without
evidence
of hemolsister,
born in 1986, exhibited
mild red cell
hereditary
younger
His
dimers
HISTORY
is a 4’/4-year-old
Yoruba
ysis.
tnin
6
Incubation
oferythrocytes
at 37’ C.
The rapidity and extent to
which membrane
budding and fragmentation
occurred
during longterm incubation
of RBC at 37#{176}C
was evaluated in vitro.’ Washed
erythrocytes
(hematocnit
20%) were incubated
at 37#{176}C
in 10 mmol/
L Tnis buffered saline, pH 7.4 containing
penicillin and streptomycin
to inhibit
bacterial
growth.’7
During
incubation,
samples
were
removed
and fixed in 3% glutaraldehyde
in normal saline. Cellular
morphology
was examined
by interference
phase microscopy
(Carl
Zeiss Inc. Oberkochen,
West Germany).
Deformability
measurements.
The defonmability
of intact red
cells was measured
in the ektacytometer,
a couette
viscometer,
which applies
a well defined laminar
shear stress to the cell while
simultaneously
monitoring
the extent of cell deformation
by laser
defractometry.
The deformability
of the red cells was continuously
recorded
as the suspending
medium
osmolanity
was linearly
increased
from 50 to 500 mosm/kg
in order to obtain information
about initial cell surface area, surface area to volume ratio, and cell
water content.’#{176}
Erythrocyte
ghost
mechanical
stability.
Red cell ghost fragmentation
was assayed in a Technicon
digital processing
ektacytometer.’92#{176} Resealed
ghosts were suspended in a dextran solution of 97
cp viscosity
(dextran
40,000, 35 g/dL
wt/vol,
290 mosm/kg,
pH
7.4). Laser diffraction
patterns
produced
by the ghosts in suspension
give a signal proportional
to the average
cellular
ellipticity.
This
signal, designated
as the deformability
index (DI), decreases
with
time as the samples are subjected
to a continuous
shear stress of 750
dynes/cm2.
In order to compare
different samples of ghosts, the time
required
for ghost defonmability
to decrease
to 60% of its maximal
value (T)
was determined.
The ratio of the observed To,, to that of a
simultaneously
nun control
sample
was then used to calculate
relative
ghost mechanical
stability,
expressed as a percentage
of
normal.
from
common
His
has
(HE);
.
2
Family pedigree.
red cell morphology);
compound
defects.
I,
t.
heterozygote
3
Carrier
of spectrin
carrier
of spectrin
for both
alpha”
alpha”
spectrin
defect
defect
alphal1e
and
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1392
IAROCCI
Table
Subject
Hb (g/dL)
Father(l)
Sister(ll,)
1 . Hematologic
MCV
MCHC
35
34
Mother(I2)
12.4
83
Sister(II3)
Propositus(1I2)
HPP(patientK.B.)
12.0
10.7
9.9
73
61
52
approximately
and
threefold
tenfold
other
in
in KB., sevenfold
family
members
in the
(Table
2).
Electrophoretic
trin
from
analysis
family
that
of the younger
ized by a modest
decrease
an increase
in the
father
3B)
(Fig
a decrease
appearance
and
of limited
members
revealed
in Fig 3. The maternal
shown
and
spectnin
sister
in the
of new
(not
tryptic
shown)
digest
were
in the 80 kd alpha
Spectnin
sister
the older
80 kd fragment
fragments
with
propositus
tryptic
digests
of specdistinct
patterns,
as
three
65 kd fragment.
and
21 kd. The
showed
(not
(Fig
3C)
character-
I fragment
digests
shown)
both
accompanied
molecular
weights
a marked
and
from
decrease
the
showed
by
analysis
were
kd fragments
domain
I tryptic
sented
spectnin,
enzyme
the
of
I spectnin
(Fig
4). To evaluate
fragment
residual
noted
undigested
whether
in the
mutant
digestion
was carried
to substrate
ratio)
the residual
propositus
alpha
out using
for periods
(Fig
spectrin
3D)
repre-
or normal
more trypsin
(1:20
from
30 to 240
minutes.
Under these conditions,
the 80 kd alpha I fragment
could be identified
in normal
digests until 180 minutes,
while
35
12.7
2.3
35.7
35.7
36.7
14.6
38
34
1.0
10.3
12
absent
at all
time
points
In contrast,
spectnin.
and 21 kd tryptic
fragments
digests
(data not shown).
Thus,
fragment
tryptic
(Fig
3D)
Limited
tryptic
not performed
others
defect.3
was
noted
tnin.
was
to
in
undigested
several
changes
well
(Fig
5). These
of hemolysis
were
first
Normal
cells
and
red
not
that
spectrin
alphah/50a
function
cell
in this
stability
required
in
exhibited
at 44 to
to produce
or in erythrocytes
Prolonged
incubation
as well
changes
as visible
evidence
noted within
six hours of incubation.
red cells from
the parents
developed
changes
during
incubation,
but even after
28
budding,
fragmentation
or hemolysis
was noted.
Propositus
red cells became
echinocytic
more quickly
than
did normal cells, but by 28 hours had developed
only minimal
fragmentation,
spherocytosis,
and
mechanical
stability
of erythrocyte
propositus
a level
(112),
only
(patient
in
two
the
II,)
of
normal
displayed
(Fig
even
mechanical
6).
ofage,
greater
who
had
propositus,
HPP.
arm
that
toward
number
The
the alphah/soa
silent
of minimal
of the curve)
mean
osmotic
fragility
carrier
to
ghosts
(data
not
and
36%
to the
defect.
to volume
relaektacytometry
in red cells from
extent
deformability
seen
(on
the
was normal
in the propositus,
fragility
was normal,’6
but
greaten
tonicity
of spherocytes
flattening
not
although
point
the mother with
in the maximum
usual
HPP
was 1 7% to 29%
stability
the
was reduced
Classic
Whole
cell deformability
and cell surface
tionships
were evaluated
by osmotic
gradient
(Fig 7). Deformability
was severely
reduced
the
hemolysis.
ghosts from
family
members
(12, 113) who had the alpha”65
defect
and 39% and 43% in the two individuals
elliptocytosis
(I,,
18%
Ghost
at 33/4 years
measured
K.B.)
shown).
Wright-stained
peripheral
blood smears
(original
magnification
x 500).
Occasional
micropoikilocytes
and elliptocytes
were noted in the younger
sister.
113. at birth (A). By 7 months
of
age (B). only elliptocytes
and discocytes
were seen. Bizarre red cell
shapes,
elliptocytes.
fragments.
and budding
forms were seen in
the propositus.
112. (C) and in patient
K.B. with
classic
HPP (D) who
also exhibited
profound
spherocytosis.
spec-
echinocytic
hours
no
budding,
The
Fig 2.
normal
from patient
KB.
was reported
by
of red
erythrocytes
(Table
2).
I
digests
the propositus
first
and fragmentation)
below
kd,
in propositus
80 kd alpha
propositus
the
studies
from
50
red cells (patient
K.B.) in glucose
free buffer
budding,
fragmentation,
and spherocyte
extensive
formation
of
from
similar
changes
in normal
from other family
members
produced
trypsin
spectrin,
(budding
46#{176}C,a temperature
at 37#{176}C
of HPP
were evident
the residual
I :200
erythrocytes
morphologic
65 kd,
red cell membrane
performed
Heated
prepared
presence
to characterize
we
in digests
mutant
the
(%)
the abnormal
digestion
of spectnin
by us but previously
reveal
In order
vitro.
80 kd alpha
Reticulocytes
0.6
0.5
was
in the
tryptic
fragments
using
a polyclonal
antibody
confirmed
that the 65 and 50
derived
from the 80 kd alpha I spectrin
(%)
RDW
13.1
13.2
propositus
family,
kd alpha I tryptic
fragment
with a concomitant
increase
in the 65 kd fragment
and the appearance
of new fragments
with molecular
weights
of 50 and 21 kd (Fig 3D). Immunoanti-alpha
it
the
of 50
80
blot
(g/dL)
83
77
propositus,
AL
Data
(it)
16.2
11.2
was increased
El
of
indicating
was shifted
in patient
K.B., reflecting
present
in her blood.
Red
elliptocytosis
deformability
the
curve
(12) showed a modest
index
accompanied
characteristic
in classic
hypotonic
of
the
cells
large
from
reduction
by the
HE.
The
younger
sister’s (113) red cells, studied
in the first 2 weeks of
life showed a shift to the left in the hypotonic
arm, a normal
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
HEREDITARY
POIKILOCYTIC
1393
ANEMIA
Table
2.
Clinical
and Biochemical
Data
ABC Thermal
Subject
Father
(I)
Sister
(II)
Clinical
Presentation
Sensitivity
N
N
Spectrin
(‘C)
Dimes
(%)
Spectrin/Band
3
Spectrin Molecular
Defect
49
16.1
1.06
Sp alpha”#{176}’
-
20.5
1.03
Sp alpha”#{176}’
Mother
(12)
HE
49
20.5
1.16
Sp alpha”85
Sister
(113)
HE
48
17.4
1.13
Sp alpha”65
44-46
35.0
1 .05
Sp alpha”65#{176}’
59
0.81
Sp alpha”#{176}
Propositus
(112)
Patient
KB.
Normal
controls
Mild HPP or HHE
HPP
46
N
49
N
5.72
40
N. nor mal; HHE. homozygous
Abbreviations:
the
newborn
in
pattern
Similar
had changed
to that of her mother
reductions
in red cell deformability
and
By
the older
7 months
of
age
her
with elliptocytosis.
were found in the
sister
(II,),
neither
of
distinct
structural
alpha”74,
designated
alpha’14342,
and
in individuals
mutations
alpha”65,
proteins
both
present,
singly
and
spectnin
have
by SDS-PAGE
who
had
spectrin
in combination.
carriers
whose
red cell morphology
of the spectnin
alpha”5#{176}variant
carriers
of this
The
been identified
evaluation
of
of tryptic
peptides
here
were
mother
The
father
and
was normal,
and resembled
and
younger
the
were
other
sister,
were
elliptocytosis,
alpha65
tryptic
found
to
be carriers
The
variant.2228’29
of
younger
the
sister
exhibited
mild neonatal
poikilocytosis
which
subsequently
(by 7 months of age) evolved
to elliptocytosis.
The propositus
both
and
exhibited
mal
recessive
and the alpha’#{176} spectrin
the alpha”65
many
characteristics
inheritance,
46#{176}C,
striking
red
cell
of HPP
poikilocytic
of red cells
susceptibility
spectrin,
The family
reported
two of these mutations
sister,
defect.21’26’27
alpha
alpha”5#{176},alphaISOb,
or by analysis
following
limited
digestion.2’5”2’25
is of particular
interest
because
older
of
alpha”6’,
truncated
alpha
with HE or HPP
red cell membrane
45
inherited
DISCUSSION
Seven
1.1 1 ± .08
hereditary elliptocytosis.
finding
red cells of the father
whom had elliptocytosis.
period.
2.05
±
45
red
to thermal
membrane
defects
including
cell
autoso-
morphology,
fragmentation
mechanical
at 45 to
fragility,
and
the presence
of a greatly
increased
proportion
of spectrin
dimers.24
The failure
of these abnormalities
to resolve by 4
years of age precluded
the possibility
that
the propositus
actually
suffered
from transient
neonatal
poikilocytosis.3#{176}
Most of the clinical
and biochemical
features
of HPP may
also be found
phology,
cell
altered
erythrocyte
thermal
and
an
Unlike
dimers.
For example,
Garbarz
et
with homozygous
HE who had inherited
mutation
from each parent.6
Like mdithis child had poikilocytic
red cell mor-
deformability,
spectrin
HE.5’6
in homozygous
al described
a child
an alpha”65
spectrin
viduals
with HPP,
sensitivity,
increase
in
individuals
with
the
reduced
red
proportion
of
HPP,
red cell
the
spectrin
content
was normal
and spherocytes
were not a
prominent
feature
of the blood
smear.
The propositus
we
studied
does not fully meet the criteria
for classification
as
homozygous
were found
or doubly
heterozygous
HE,
in only one parent.
However,
biochemical
features
consistent
spherocytes
of the
and
than
based
largely
spectnin
features
HPP.
of classification
system
cell
that
The
of the
on clinical
of red
are
thermal
completely
the
content
more
closely
of
red
cells
resemble
of the traditional
poikilocytic
sensitivity
paucity
in the
limitations
features,
elliptocytes
clinical
and
otherwise
In particular,
normal
are
HE
case
diagnosis.
the
propositus
homozygous
tests
of the
this
with
since
the
anemias,
inheritance
have
been
which
is
pattern
and
pointed
out
previously2’3’
and were encounterd,
as discussed
above, in the
propositus
reported
here.
The genetic
basis of HPP is not fully understood.
In most
instances,
one parent
has an identifiable
defect
in alpha
spectnin
while the other is normal.2
It is speculated
that the
apparently
undetectable
of alpha
Fig 3.
after
Two-dimensional
eloctrophoretic
limited tryptic
digestion.
(A) Normal
cytic
father
(I,). (C) elliptocytic
mother
Abnormal
tryptic
fragments
are indicated
analysis
of spectrin
control.
(B) non-ellipto(12). (D) propositus
(112).
by arrows.
present
duced
normal
parent
abnormality
spectrin.
in isolation
in excess32
the requirements
Such
may
actually
have
resulting
in diminished
an abnormality
would
a clinically
synthesis
be silent
when
because
and even
alpha
reduced
spectrin
synthesis
is normally
will fully
of the cell.
When
co-inherited
with
promeet
another
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1394
IAROCCI El AL
Fig
4.
Immunoblots
of
limited
tryptic
digests
of
normal (A) and propositus
(B) spectrin.
Tryptic
digests
were
electrophoresed
in two dimensions
as in Fig 3.
transferred
to nitrocellulose
filters.
and reacted
with
polyclonal
anti-alpha
I spectrin
antibody.
gene
coding
defective
for an alpha
synthesis
the proportion
spectrin
of normal
of the structural
clinical
syndrome
of
between
the amount
of alpha
severity
is also
compound
Here
structural
alpha
evident
spectnin
amount
mutant(s)
rare
for two
individual
separate
of abnormal
increase
and thus
A
result
in a
a spectnin
alphah/Soa
mutation
is an example
of this type
propositus
clinical
were
to be a
positus
defects.’6’23
is increased
by
the propositus.
interest
was
Although
mal,
and
anemic
particularly
after
changes
rheologic
mild
clinical
transfusions
hyperbilirubinemia
infection-associated
the
the
observed
were
at 2 years
crisis,
first
course
year
on osmotic
needed
following
anemia
noted
was
of life.
at birth
an acute,
usually
mini-
Similarly,
gradient
in
the
ektacytome-
HPP
in the
cases.4
Although
proportion
of spectnin
not of the magnitude
were
as exemplified
by patient
poikilocytes
K.B.
were
observed
by other
and
abundant,
microspherocytes.
Prolonged
incubation
red
cells
at 37#{176}Cdid not lead
to the
budding,
fragmentation,
in red cells
described
classic
by
and
from
spherocyte
patient
formation
KB.
Zarkowsky
et
there
of proextensive
that
and that
in another
al
we
was previindividual
HPP.’
The two factors
most
likely
to have
modified
clinical
severity
are the degree
to which mutant
spectnin
participated
in tetramer
formation
and the lack of spectnin
deficiency.
The
Of particular
of increase
abnormal,
observed
with
that
we found
in the
of compound
heterozy-
extent
fewer
ously
gote.
for
in classic
and
found
the
while
reported
the presence
of a second
spectrin
structural
variant,
rather
than
by decreased
synthesis
of normal
spectrin.
The previously unreported
combined
inheritance
of a spectrin
alpha”65
and
and
dimers,
relationship
spectrin
spectrin
try
however,
would
severity.2
in the
heterozygote
the total
mutant
greater
defect,
spectnin
abnormal
propositus
spectrins
are
tetramer
known
present
to
be
in
to a limited
formation
the
capable
red
of
extent.23’29
cells
in another
individual
with
HPP
the
in
As a result,
percentage
of spectnin
dimers
was increased
to only
mately
35% instead
of the >50% observed
in classic
contrast,
of
participating
who
was
the
approxiI-IPP. In
doubly
heterozygous
for the Sp alpha”5#{176} and
the Sp alpha”74
defects,32
spectrin
dimers were increased
to >50%, reflecting
the severely
impaired
ability
of the alpha”74
mutation
to
I-
form
tetramers.2
There
is a direct
correlation
between
the
percentage
of spectnin
dimers
present
in the membrane
and
diminished
red cell membrane
stability.7
Thus,
it is not
0
U
surprising
-J
0
z
fragmentation
in vitro
stability
one
to find in the propositus
that
in vivo and diminution
hand
and
fell somewhere
HE
or
between
asymptomatic
the degree of red cell
of ghost
mechanical
classic
carriers
HPP
of
on the
spectrin
(I,
on the
abnormalities
brane
spectnin
content,
other.
The
second
is a finding
quite
factor,
unlike
normal
the
memspectnin
08
aa.
z
06
U
04
12
U
0
02
0
0
18
24
HOURS OF INCUBATION
MINUTES
Fig 5.
Appearance
of red cells
under
interference
microscopy
during
prolonged
incubation
at 37’C.
Top
control
red
cells;
middle
panels.
propositus
red cells;
panels. classic HPP red cells from patient
K.B.
phase
panels.
bottom
Fig 6.
Erythrocyte
ghost mechanical
stability.
Shaded
area is
the normal range. The membrane
stability
of 113red cell ghosts was
measured
at birth (1 ), 2 weeks
(2). and 7 months
(3) of age.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
HEREDITARY
POIKILOCYTIC
ANEMIA
1395
the same to be true
in poikilocytic
homozygous
HE.
The abundance
of microspherocytes
>(
u.J
0
patients
spectrin
z
>-
such as K.B.
deficiency.4’33
spherocytes
0
LU
0
300
200
OSMOLALITY
Fig 7.
Red cell osmotic
(mosmol
gradient
kg)
ektacytometry
on all family
usually
tary
encountered
spherocytosis,
another
in classic
hemolytic
HPP.33
anemia
clinical
course34
and by analogy
on cell
processes
leading
lation,
spectrin
endocytosis,
deficiency.
a more
important
spectrin
in classic
or
HPP
to spherocyte
and/or
fragmentation)
However,
spectnin
factor
deficient,
with
That spherocytes
spectrin
may
in spherocyte
formation
the
normal
are seen at
have effects
(eg, vesicu-
similar
to those of
deficiency
seems
to be
formation
since
and the propositus),
only K.B., whose
exhibited
of
of
extensive
in the
both of
red cells
spherocyto-
ACKNOWLEDGMENT
characterized
one might
HPP
sis.
In herediThe
by spectrin
deficiency,
the extent
of the deficiency
correlates
well with clinical
severity.
The least deficient
subjects
have
the mildest
of his red cells.
his dysfunctional
also
found
is consistent
propositus
spectrin
content
all implies
that
were
like
may also be related
to the extent
Similarly,
the relative
paucity
two individuals
we studied
(K.B.
whom
had dysfunctional
spectrin,
members.
113 was studied
at birth (curve
1 ) and 7 months
of age
(curve
3). The normal
range is indicated
by the shaded area.
deficiency
in the
anemias
expect
authors
gratefully
acknowledge
V. Bible for manuscript
and the family described
in this report for their interest
and cooperation.
The polyclonal
anti alpha-I spectnin antibody
was a
gift from David Liu and un Palek.
assistance
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1988 71: 1390-1396
Hereditary poikilocytic anemia associated with the co-inheritance of two
alpha spectrin abnormalities
TA Iarocci, GM Wagner, N Mohandas, PA Lane and WC Mentzer
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