Modification of Hemoglobin H Disease by Sickle
Trait
Katherine K. Matthay, … , Yuet Wai Kan, Dorothy F. Bainton
J Clin Invest.
1979;64(4):1024-1032. https://doi.org/10.1172/JCI109539.
The rarity of hemoglobin (Hb) H disease in combination with sickle trait may be due in part
to the absence of actual Hb H in individuals who, nonetheless, have inherited the deletion
of three
a
-globin genes. We describe here a boy with persistent microcytic, hypochromic
anemia despite adequate iron stores, who exhibited splenomegaly with a normal
reticulocyte count and only rare inclusions in circulating erythrocytes. Starch gel
electrophoresis and isoelectric focusing at age 5 yr showed 21% Hb S, persistent Hb Bart's,
but no Hb H. Recticulocyte
a
/non-
a
globin chain synthesis ratio was 0.58 at age 5. The
mother (Asian) had laboratory evidence of
a
-thalassemia trait and the father (Black) had
sickle trait. The nature of
a
-thalassemia in this patient was investigated both by liquid
hybridization and by the Southern method of gene mapping, in which DNA is digested with
restriction endonucleases and the DNA fragments that contained the
a
-globin structural
gene identified by hybridization with complementary DNA. The patient had only one
a
-globin structural gene, located in a DNA fragment shorter than that found in normal or
a
-thalassemia trait individuals, but similar to that present in other patients with Hb H disease.
Morphologic studies of bone marrow by light and electron microscopy revealed erythroid
hyperplasia with inclusions in polychromatic and orthochromatic erythroblasts, suggesting
early precipitation of an unstable hemoglobin. […]
Research Article
Find the latest version:
Modification of Hemoglobin
H
Disease
by
Sickle Trait
KATHERINE K. MATTHAY, WILLIAM C. MENTZER, Jr.,ANDREE M. Dozy,
YUET WAIKAN,
and
DOROTHY F.BAINTON, Departments
of Pediatrics,
Medicine,
and
Pathology, University of California, San Francisco, San Francisco,
California 94143
A B S T R A C T
The rarity
of hemoglobin (Hb)
Hdis-ease in
combination with sickle
traitmay be
due
inpart
to
the absence
of actual Hb
Hinindividuals
who,
none-theless, have inherited the deletion of three a-globin
genes. We
describe here
a
boy
with persistent
micro-cytic, hypochromic
anemia
despite adequate
ironstores,
who exhibited
splenomegaly
with a normal
re-ticulocyte
count
and
only
rare
inclusions
incirculating
erythrocytes.
Starch
gel electrophoresis and isoelectric
focusing
at
age
5yr
showed
21%
Hb S, persistent Hb
Bart's, but no Hb H.
Recticulocyte a/non-a
globin
chain
synthesis
ratio was
0.58 at
age 5.
The mother
(Asian)
had laboratory evidence of a-thalassemia
trait
and
the
father (Black) had sickle
trait.
The nature
of
a-thalassemia
inthis
patient
wasinvestigated both by
liquid hybridization
and by the Southern
method
of
gene
mapping,
inwhich
DNA isdigested
with
restric-tion
endonucleases and the
DNAfragments that
con-tained the a-globin structural
gene
identified
by
hy-bridization with complementary
DNA.The
patient
had
only
one
a-globin structural
gene,
located
in a DNAfragment
shorter than
that
found
in
normal
or
a-thalas-semia
trait
individuals, but similar
to
that
present
inother
patients
with
Hb
Hdisease.
Morphologic studies
of
bone
marrow by light and electron
microscopy
re-vealed erythroid
hyperplasia
with inclusions in
poly-chromatic and
orthochromatic erythroblasts,
suggest-ing
early precipitation
of
an
unstable
hemoglobin. The
lack
of demonstrable
Hb H may be the result
of
both
diminished
amounts of (A
available for Hb
H
formation
(since one
8-globin
gene
is(3s)
and the
greater
affinity
of a-chains for (3A than
8s-globin
chains leading to the
formation of relatively
more Hb A than Hb S. The
pres-This workwaspresentedinpart totheAmericanSocietyof Hematology, NewOrleans, La.,December 1978.
Dr. Kan is an investigatorof the Howard Hughes Medical
Institute. Dr. Mentzeristherecipientofa U.S.Public Health
Service ResearchCareerDevelopment Award.
Received forpublication9April 1979andinrevisedform
8 June 1979.
ence
of
a(3s gene may thus
modify
the usual
clinical
expression
of Hb
Hdisease.
I
NTRODUCTION
Simultaneous
inheritance of
a-thalassemia
and
struc-tural abnormalities of the
(3-chain
frequently
modifies
the usual
clinical
expression
of the
f8-globin
mutation
(
1-13).
For
example, observation of the
natureof sickle
cell
anemia
inseveral individuals
who
have also
in-herited a-thalassemia trait
has suggested
in
some cases
(4-6),
but
not
all
(7, 8), a
favorable effect
of the
a-thalassemia
gene on
the clinical
severity
of sickle cell
anemia. a-Thalassemia with sickle cell
traitconsis-tently
results
ina
lower than
usual
percentage
of
hemo-globin (Hb)S
(9-13). We
have
investigated a
child
with
sickle
trait
and
microcytic
anemia
whose
Asian
mother
had a-thalassemia
trait
and whose
Black
father had
sickle trait.
The
very
low percentage of Hb S suggested
that
both a-thalassemia
trait
and sickle
cell
trait
werepresent, but
the
presence
of splenomegaly,
extreme
microcytosis, and bizarre erythrocyte morphology,
fea-tures
not seen
ina-thalassemia
trait,
stimulated further
investigation.
Analysis
of
DNA
from
this
child by molecular
hy-bridization and
gene-mapping
techniques revealed
that three
of the four a-globin
genes
normally
found
were
deleted, thus unequivocally demonstrating the
presence
of the
Hb H genotype. Paradoxically, Hb H
was
not
identified
inhemolysate from either peripheral
blood or bone marrow,
although inclusion bodies could
be
found
inoccasional
erythrocytes in
both places.
The
biochemical
natureof
these
inclusions
was
not
deter-mined, but the appearance
inearly
erythroid
pro-genitors
suggested
agreater
degree of instability
than
that
associated
with Hb
H(14-16). This patient,
who
represents
the unusual coincidence of Hb H disease
with sickle trait, demonstrates how the inheritance of
apicture. In
such
patients, accurate genetic analysis is
impossible unless the new
techniques of molecular
hybridization
and gene mapping are used.
METHODS
Hematologic values. Hemoglobin levels and erythrocyte indices were obtained with a model S Coultercounter(Coulter Electronics Inc., Hialeah, Fla.). Bone marrow differential counts wereperformed by countingat least500cells.
Cellu-lose acetate electrophoresis was performed in
Tris-EDTA-borate buffer at pH 8.4 (17). Hb A2 wasdetermined by
micro-chromatography (18). Fetal hemoglobin was determined by
alkali denaturation (19). Hb H preparations were made by mixing one partof fresh whole blood with two parts of1% brilliantcresyl blue (BCB)l in 0.9% NaCl andincubating at 37°Cfor 1-3h.
Hemoglobinand globinanalyses. Globin chainsvnthesis
wvas
measuredin vitro with fresh heparinizedperipheralblood orbone marrow,asdescribed byKan etal.(20). L-[3H]leucine-labeledglobin chains precipitated from cell-free hemolvsate were separated on carboxymethyl cellulose columns in 8 MIurea at pH 7.2 using a linear Na2HPO4 gradient. The rela-tive synthesis of a-chainto
,-
andy-chainwasexpressedastheratioof the totalradioactivityof thea-tonon-a-globinpeaks.
Starch gel electrophoresis was carriedout at 4°C for3h at
25 mA and 200Vaccording to the method of Smithies (21) with
0.006 \I Na2HPO4 buffer for the gel and 0.04NI Na2HPO4 buffer forthe electrodes, both at pH 7.0. The gel was then slicedhorizontally and stained withbenzidine.
Thin-layer isoelectric focusing in polyacrylamide gel was
carried outwith freshhemolysate according to Basset et al.
(22). Hemoglobin
columiin
chromatographywasperformedoncarboxymethyl cellulose (CM-52,
Whatman,
Inc., Clifton,N. J.) accordingto Schroeder etal. (23). 1-ml of stroma-free
hemoly
satewasprepared from fresh peripheral bloodorbone marrow and then dialyzed for 18 h against 0.01 MI Bis-tris[N,N-Bis-(2-hydroxymethyl)-iminotris-(hydroxymethyl)-methane], pH 6.1, plus 0.01% KCN. 50-60 mg of dialvzed hemoglobinwasappliedto a0.9x 20-cmcolumn equilibrated with thedialysis buffer. The
hemoglobin
waseluted with 0.03 M Bis-trisand 0.01%KCN, pH 6.1, using a linear salt gradient (0-0.08MI
NaCl,total volume, 1,200 ml) over 24 h. The posi-tion of each elutedhemoglobin
typewas determined byre-cording the optical densitv at 415 nm of each fraction as a
function ofsalt concentration, measured from conductance.
To identifv each hemoglobin eluted, two or three fractions from eachpeak werethenpooled and centrifuged for18h at 50,000
rpm
in aBeckmnan
ultracentrifuge (Beckman Instru-ments, Inc., Spinco Div.,PaloAlto,Calif.)toconcentrate thehemoglobininto a 0.5-ml vol. Globin was then prepared from
each concentrated portion by acid-acetone precipitation,
lyophilized,thenresuspendedand
electrophoresed
oncellu-lose acetate in Bis-tris-EDTA-borate-urea buffer at pH 6.4
accordinig
tothemethod ofSalmon et al.(24).The stripswerestained withPonceauSanddecolorizedwith 3%aceticacid.
Electron microscopy. Specimens of bone marrow and blood
w%ere
immediately fixed in 1.5% buffered glutaralde-hydeat220C,
postfixedin OS04,andprocessed forexaminia-tion by transmission electron microscopy, as described
(25).
Other
samiiples
were prepared for scanning electron micros-copy bycritical-pointdryingandcoated withgold-platinum.'Abbreviations
used in this paper: BCB, brilliant cresyl blue;cDNA,
complementary DNA;kb,
kilobase pairs;HI),
hemoglobin.
Liquid
hybridization. DNA from the patient, his mother, and controlswithhydrops fetalis, Hb H disease,a-thalassemiatrait, and normals was prepared from leukocytes, liver, or spleen by sodium dodecyl sulfate-pronase digestion and phenol extraction. The RNA was digested with ribonuclease and the DNA reduced to 2 x 106 daltons by limited depurina-tion (26, 27). a-globin complementary DNA (cDNA) was pre-pared as described (26, 28). The a-cDNA (1,000 cpm) was incubated for 76 h at78°Cin duplicate20-1.lreaction mixtures containing 100 ,ug cellular DNA, 8 pga-cDNA, 500 cpm of
[3H]dCTP-labeled
unique sequence HeLa DNA as internal control, 0.5MI NaCl, 0.002 NI EDTA, and 0.04 I Tris-HCl (pH 7.4). The percentage ofa-cDNA annealed was assayedbybatchwise elutioi wvithhydroxylapatite (29).
Restriction endonuclease analysis
of
DNA. DNA was pre-pared from the leukocytes of the patient, his mother, a-thalas-semia trait, silent carrier, Hb H disease, and normal controls, as described (30). 10ygofhuman DNAand 1 ug ofX-DNA(as internal size marker) were digested for 4h at 370C with
1.25 U of Eco RI or Hpa I per microgram of DNA. The buffer for Eco RI digestion contained 100mX1 Tris-HCI (pH 7.5), 50 mM NaCl, 6 mM MIgCl2, and 6 mnM 2-mercaptoethanol,
and forHpaI digestion,6mnM Tris-HCI(pH 7.5), 26mMNaCl, 6 mM \1gCI2, and 6 mnM
2-mercaptoethaniol.
The sampleswvere precipitated in alcohol, dried and resuspended in 30
ILI
of 5 mMI Tris (pH 7.5), 0.1 mM EDTA. The digested DNA's wereelectrophoresedin 0.8%agaroseand then transferred to nitrocellulose filters (31). The filters were hybridized with 32P-labeled a- and 8-globin cDNA (2 x 108
epm/pLg)
for 2 d and extensively washed and autoradiographed, as de-scribed (30).RESULTS
Case report.
S.H. is a 5 9/12-yr-old Black/Chinese
male with marked microcvtic anemia in the presence
of adequate iron
stores.Physical
examinationhas been
notable for persistent
splenomegaly.
Hematologic
dataobtained
over a 5-yrperiod of observation
areshown
in
Table
I.Erythrocyte morphology has consistently
resemiibled that of Hb
Hdisease, with hypochromia,
poikilocytosis,
fragments, spherocytes, and
targetcells
evident
onthe peripheral blood
smear(Fig. 1Aand
B). BCB stainsrevealed
noHb
Hinclusions
inthe
circu-lating ervthrocytes
at age 1 or 2 yr. Anoccasionial
in-clusion-containiing cell
wasevident
atage 5 yr (Fig. ICand
D).Hemoglobin electrophoresis has been
consis-tentwith sickle
trait,but with
anunusually low
per-centageof
HbS,
persistenceof Hb Bart's and elevated
Hb F (Table I). Bone
marrow
aspirate and biopsy
showed erythroid hyperplasia (myeloid:erythroid ratio,
1:2), a
findinig
in apparent contradiction to the
re-peatedly normal reticulocyte
count.The
percenitageof early erythroid
precursors(proerythroblasts, 5%;
basophilic
erythroblasts,
22%) wasmarkedly increased,
while
polychromatophilic anid orthochromatic
erythro-blasts were
present
innormal numbers. Methyl violet
andcl
BCB stainisof the bone
marrowshowed
the
samnefrequeney of hemoglobin inclusions as in the
periph-eral blood. Famiily studies confirmed
that the BlackTABLE I
HematologicData inHbHDisease with SickleTrait
Patient
Mother Father
10mo 31mo 60mo (Asian) (Black)
Hb,gldl 9.4 9.3 9.4 11.3 13.6
PCV 0.29 0.29 0.27 0.34 0.39 MCH, pg 16.9 16.0 17.0 22.0 29.8 MCHC,gldl 33.3 31.9 33.8 33.0 35.1
MCV,fl
50 50 51 70 86Erythrocyte, xlOt2lliter 5.67 5.85 5.50 4.90 4.52 Retic, % 0.6 0.7 0.9 1.0 1.4 Fe/TIBC, ,umIliter 18.5/80.8 15.7/61.7
Hemoglobinelectrophoresis*
Hb A, % 65.9 70.4 71.8 96.9 64.3
A2, % 3.1 1.9 3.2 2.5 2.5
S, % 17.3 20.1 21.0 0 32.6
F, % 10.3 4.6 4.0 0.6 0.6
HbBart's, % 3.4 3.0 Trace 0 0
Globin synthesisratio
a/(6
+ y) 0.924 0.654 0.584 0.884t 0.96§Abbreviations usedinthistable: PCV, packed cell volume or hematocrit; MCH, mean corpuscular
hemoglobin;MCHC, MCH concentration; MCV, mean corpuscularvolume;TIBC, total ironbinding capacity.
*Relativehemoglobin percentages determined by densitometric scan of cellulose acetate strip, as well as separate quantitation of Hb A2 and Hb F.
t
Peripheral
blood.§ Bone marrow.
father
had
sickle
trait,whereas
the
Asianmother
had
the
hematologic features of a-thalassemia
trait(Table
I).
Globin chain synthesis.
The reduced
a/(,8
+y)-globin
chain
synthetic
ratiosinperipheral blood
reticu-locytes and bone
marrowof the
patient,shown
inTable
I,
were consistentwith either
a-thalassemia
trait orHb
Hdisease (32).
3A:13s
activity ratios were 6:1, 4:1,and
3:1 at 10,31, and
60 mo,respectively.
The
bone marrow PA:,s
activity(2:1)
was morenearly
normal.
The mother's
peripheral blood
a/ft-globin
chain
syn-thetic
ratio was0.88,
consistentwith a-thalassemia
trait.
Hemoglobin analyses.
Starch gel electrophoresis of
hemolysate
obtained
from peripheral
blood, shown
in Fig. 2,revealed a trace amountof
Hb Bart's. No Hb H orother abnormal fast
hemoglobins
werefound.
Thin-layer isoelectric focusing also showed only
hemo-globins Bart's,
A, F, S,and
A2. Column chromatography,
(Fig. 3)
also
failed
todetect measurable
amountsof
Hb
H orother fast hemoglobins,
exceptfor Hb
Bart's.Globin electrophoresis
wasused
toconfirmthe identity
of each
hemoglobin
eluted.
The
firstpeak eluted
con-tained
y-chains;
the
second,
a-and
y-chains; the small
shoulder and the third peak contained
aand
AA;the
fourth,
aand
8; andthe
lastpeak,
a andBs.
Bs-chains
were found
only
inthe
Hb Sfraction, ruling
out thepossibility
that 8s tetramers were present elsewhere, but hidden becausethey
eluted withanother
hemo-globin. Only the Hb Bart's fraction
was composedof
asingle globin chain, which
migrated tothe
y position, asexpected. Hemoglobin analysis of
abone
marrowhemolysate by starch gel
andcolumn chromatography
gave virtually identicalresults to
those found
in periph-eral blood. The bone marrow percentages were HbBart's,
1.4%;Hb
F, 6.1%;Hb
A, 72.5%;Hb
A2, 3.6%;and Hb
S, 16.4%.Electron microscopy of bone marrow.
Numerous
dense
amorphous inclusions of
varying sizes,compara-ble
tothose
seeninthe
bone
marrowof
patientswith
B-thalassemia
major(33,
34)
were seen inreticulocytes
and
orthochromatic
(Fig.4A)
andpolychromatic
erythroblasts
(Fig. 4B).Although these
were generally notmembrane-bound
(Fig. 4A), in afew
instancesthey
were
contained within autophagic vacuoles
(Fig. 4B),as
has also been described
in8-thalassemia
(33, 35). Thefrequency
ofinclusions corresponded
tothat
seenon
methyl violet
stain.Many of the
mature erythro-cytesappeared
verythin and distorted,
ascanbe
seen inthe
scanning electron micrographs (Fig. 4C and D),000
Aw
r
A
d0
to
I*
C
v
'i.
10'a
.r 4, 4k
lu
a
a,.rt
*1
i
D
FIGURE 1 Peripheral blood (Wright's stain) of Propositus (A) and of individual with Hb H disease (B). Peripheral blood(BCB stain)ofPropositus (C) showing occasional(1/125) erythro-cytescontain inclusions and ofindividual with Hb H disease (D) showing inclusions in almost
everyerythrocyte.
in
thalassemia (34). Occasionally small
pits (Fig. 4C)could
be
detected.
Similar
pittingof erythrocytes
has
previously
been noted with Nomarski
optics inindi-viduals with Hb
Hdisease,
eventhose with
intactspleens
(14).
a-Globin-cDNA
liquid
hybridization.
Because
hematological, family,
and
globin chain
synthesis
H BART'S pH 7.0
SICKLE
TRAITHB H
DISEASE
PATIENT SH
NORMAL
NEWBORN
WITH HB BARTS+ _
ORIGIN
FIGURE
2 Starchgel hemoglobin electrophoresis atpH 7.0.Thepatient hashemoglobinBart's,aswell as A, F, and S.
studies
wereinconclusive, additional studies
weredone
toanalyze the
a-globin
genesdirectly.
Fig. 5shows
the
percentannealing
of32P-labeled a-globin
cDNA to DNA
from the
propositus,his mother, and
controls.
DNAfrom
the patient showedthe
same per-centannealing
as six AsianHb Hdisease
controls,
wholack three of four
a-globin
genes. Hismother
showed
the
samepercentageof
a-sequences asother
a-thalas-semia trait
individuals with
adeletion
of
twoof
the
four
a-globin
genes.Restriction
endonuclease
analysis
of
DNA.Figs.
6and
7show the
diagrammatic
and
autoradiographic
re-sults of
DNAdigested with
Eco RIand
Hpa Iand
hybridized with
cDNA.The a-globin specific
fragment
in DNA
from the
patient wassmaller than normal after
Eco RI
digestion
and
wasidentical
tothe 19-kilobase
pairs
(kb) a-specific
DNAof
known Hb
Hdisease
con-trols. The
patient waslacking
the
4.0-kbfragment
after
Hpa I
digestion,
oneof the
twoa-specific fragments
normally
present,againcorresponding
toknown Hb
Hdisease controls. The
autoradiogram
of
hybridized
a-globin
DNAfrom the
patient's
mother
wasidentical
to
the
patternof controls with a-thalassemia
trait.The
patient's father
was notavailable
for
testing.E LU
C 0
WI o.8 A0C
5,, ~~0
H F
oLA0-
7% 3 U0~~~~~~~~~
O PATEiNTSH 0O
~20
0.08~-- 0.06
11.6
0.0*2.0L,,_----l 14% 002
1.2 0.00
0.8 -F 6
BARrs A2
04A 2% A~2%
200 400 600 800 1000 1200 EFFLUENT IN ml
FIGURE 3 Carboxymethyl cellulose chromatography of he-moglobin. The identity of each peak was confirmed with globin electrophoresis, including the
Ax
fraction (the small peak eluted just before themainhemoglobin
Apeak),
which contained a-andpA_globin
chains.DISCUSSION
This
patient wasinvestigated because of
manyclinical
findings
that did
notconform
with
the usual
features
of a-thalassemia
traitwith
sickle
trait.The
presenceof splenomegaly, anemia, marked
microcytosisand
strikingly
abnormal erythrocyte
morphology all
sug-gested
amore severea-chain
deficiency,
such
asHb
Hdisease, rather
than
a-thalassemia
trait. However,the
normal reticulocyte
count,the
absence of Hb
H inhemolysates, and the
paucityof inclusions
incirculat-ing
erythrocytes
were notcharacteristic of Hb
Hdis-ease.
The
possibility
that Hb
H wasabsent
as aresult
of
severe irondeficiency
(36)
wasruled
outby
re-peatedly
normal
serumand
bone
marrow ironstudies.
Although
the
globin chain synthesis studies
demon-strated
deficient
a-chain production,
the results
were consistentwith
either
atwo gene orthree
genedeletion
(32).
Adecreasing
a/non-a ratiowith
increasing age, as seen inthe propositus, has been previously noted
inpatients
with
a-thalassemia
(37) and may be related tothe switch from fetal
toadult hemoglobin production.
No
ready
explanation
is apparentfor
thedecreasing
13A:f8s
rationoted with
age.The
morenormal
a/non-a-chain
ratio seen inbone
marrow, ascompared
tope-ripheral blood, has also been
reported in some patientswith a-thalassemia
(38).A more
precise definition
of the nature ofthe
thalas-semia defect
wasobtained by molecular hybridization
studies. Liquid hybridization
witha-globin
cDNAwasclearly compatible
with deletion of threea-globin
genes,
thetypical genotype
of Asian Hb H disease(27).
Restrictionendonuclease gene-mapping
tech-niques also
clearly delineated the presence
ofonly
asingle a-globin gene.
It isimportant
to note thata-thalassemia
trait,with
deletion of the
twoa-globin
genes
intrans,
also results in a restriction endonu-cleasedigest
of twoidentical, but smaller than normal,
19kb
a-globin
DNAfragments,
likethe Eco
RIpattern
seen in the
patient
and inother
individuals with Hb
Hdisease (39).
However,the
patientcould
nothave
in-herited a-thalassemia trait
intrans,
sincethe
DNAdi-gest from
the mother showedonly
anormal size 23 kba-globin DNA fragment,
consistentwith
cis-a-thalas-semia
trait,
where onechromosome
has two intacta-globin
genes andthe
other
isdevoid of a-globin genes.
The
patient,therefore, inherited his single
intacta-globin
genefrom
hisBlack
father, who presumably
was a silent carrier
of
a-thalassemia (genotype, -alaa).
The silent
carrier state isactually
quite common inBlacks, with
afrequency of
24%by restriction
endo-nuclease
gene mapping(40).
Hb
Hdisease has
neverbefore
been reported
in apatient
withsickle
trait.Although the incidence of
Hb Hdisease
inBlacks
isadmittedly low (40-42), the
apparent rarity
of the
combined defect
mayalso be
aresult of the lack of
areadily available
meansof
de-tection, sinceHb
H is notalways
present inmeasurable
amounts.
Although other
patientswith
a,-chain
abnor-mality and
asuspected Hb
Hgenotype, but without
4A
tetramershave been
described (1, 5, 14, 43-46),
direct demonstration of the a-globin
genedefect has
not
previously
been possible.
There
areseveral
possible
reasonsthat Hb
Hdisease,
when
inherited with the
,-gene
mutationof
sickle
trait, mayproduce
asyndrome
of hemolytic
anemiawithout
detectable
f3-globin
tetramers. One isthe decreased
quantity
of
Aglobin chains available
for
Hb
Hforma-tion, since
only
onef8-globin
gene isactually
synthe-sizing
such chains.
Furthermore, a-globin chains have
agreater
affinity for
A_than
for s-chains.
Thus,
Hb
A isformed
morereadily than Hb
S,leaving
excessfree
PS-chains
whenever there
is arelative deficit of
achains. Such
adifferential
affinity
of a-chains for
I3A_chains
issuggested
by
the lower than
expected
per-centageof Hb
S seen in patientswith a-thalassemia
trait
and with
sickle
trait(9-13).
In vitrostudies of
the
reassociationof
a-chains
with pA-and
p5-chains
show diminished formation of
Hb Swhen
decreasedamounts
of a-globin chains
are present(47,
48).
frag--. Iv ~ ~ M
P*~~~~~~~~~~~~i-
:
@
U4
*-::k
- i,,. 4~~~~~~~~A
4i;.J**
*
c.i
FIGURE4 Transmission electron micrographs ofdeveloping erythroblasts from the bone
mar-row. (A)is an orthochromatic erythroblast andits cytoplasm containsabnormal amorphous
in-clusions (arrow).The nucleus and otherorganelles suchastheGolgi complex(G),mitochondria
(m), andpolyribosomes (p)appearnormal. x15,000. (B)is apolychromatic erythroblast which
alsocontainssimilar inclusions but withinamembrane-bound organelle,probablyanautophagic
vacuole (AV). x18,000.(C and D) Scanning electron micrographs ofcirculating blood
erythro-cytes. The cells arethinner than normal and there are manyirregularities inshape. Note the
knob-likeprojections in D.Occasional smallpits(arrow)areevident.C, x8,000; D, x7,200.
Hemoglobin HDisease 1029
A
kt
I
0
0
.A
z itD
my
4 2 > ...
4.~ 5
HYDROPS Hb-H a-THAL.1 SILENT NORMAL
DISEASE CARRIER
FIGURE5 Liquid hybridizationwith [a-32P]globin cDNA.
mentation
and splenomegaly,
as well as ineffectiveerythropoiesis.
It ispossible
that the excess1Bs-chain
present in
the bone
marrow andperipheral blood of
the
propositus may combine to form unstableB3s
L.,r-
.R
GAATTC40V--=X
'tNORMAL 23.0kb
7-LIZ-
HB H 19 0kO..W-9
RORIGIN
9 23.0 kb
-19.0kb
FIGURE 6 (A) Diagram representing the Eco RI restriction
endonuclease cleavage sites ofa-globin DNA in thenormal
individual anda person with Hb H disease. (B) Autoradio-gramiiofEco RIdigestionpatternof DNA frompatient,mother, andappropriatecontrols,using[a-32P]globincDNAas aprobe.
The normal DNA shows a single23kb a-specific fragment.
The patient has a smaller 19kb fragment, identical to the
Hb Hcontrol. TheDNAof the mother shows the23-kb
frag-menit seen in the a-thalassemia trait control, who has two
a-globingenesincis on onechromosome,the other chromo-somebeing devoid ofa-genes.Thehydrops DNA has no
a-specific fragment, while the silent carrier ofa-thalassemia,
withthreeoutof fourintacta-globingenes,hasbothanormal
23kbandasmall 19 kbfragment.
FIGuRE 7 (A)DiagramrepresentingHpa I restriction endo-nuclease cleavage sites ofa-globin DNAin the normal and the individual with Hb Hdisease. (B) Autoradiogram of Hpa
Idigestionin DNAfrom thepatient,his mother, and controls
usingbotha-and,1-cDNAasprobes. The normalDNAhastwo
a-specific fragments of 4.0 and 14.5 kb. The other fragments have been showntobey-,8-,and
,8-specific
(30).The hydropsDNAlacks both a-specific fragments. The patient'sDNAlacks the smaller4.0kba-specific fragmentasdoes the Hb H
con-trol. (The single a-specific fragmentinthe patient's DNA is
larger than the usual 14.5 kb fragment, but this corresponds
toavarianta-globinDNAfragmentseeninsome normal
in-dividuals without Hb H disease, probably because of poly-morphismn of thea-gene[Kan, Y. W.,unpublished data].The
very faint bandappearing in the 14.5-kb position is not
a-specific and was not present in a subsequent Hpa I digest
of thepatient's DNA.) The patient'sDNAalsocontainsa
13.0-kb p-specific fragment, which occurs in 80%of individuals
with sickle hemoglobin (30). The mother's DNAhas both a
specific fragments,asseen ina-thalassemiatrait.
tetramersthatthenprecipitaterapidly, resultinginloss of
erythroid
precursorsinthebone
marrowanddestruc-tionofprecipitate-containing
peripheral
bloodcells by
the
spleen. Alternatively,
the excess813-chain might
bind to the
erythrocyte membrane,
causingdamage
andsubsequent
hemolysis, sincepreferential binding
of
j3s
to stromahasbeen demonstrated (49). The pres-ence ofsome type ofunstablehemoglobin
wasindi-cated
by
the occasionalinclusion-containing
cellsseenin both peripheral
blood
and bone marrow by specialstains and electron microscopy.
The electrophoretic
and
chromatographic techniques employed
arenotsuf-ficiently
sensitive tounequivocally
rule outthepossi-bility
that theseoccasional inclusions
arecomposed
of Hb H.However, the distribution of these inclusions
was nottypical of Hb H disease. 8Atetramers
precipi-tateinmature
erythroblasts
andin peripheralerythro-cytes
(15)
causing membrane pittingandrigidity
with gradual extramedullary hemolysis, rather thanineffec-40
30
0
z
z
z 0
z
U1
a-20_
0 0 10 _
o
*CONTtOLS
O PATIENTSH
A MOTHER
IQ,
tive
erythropoiesis.
Onthe other hand,
f8-thalassemia
is
characterized
by
extensiveineffective
erythropoiesis
(14).
Inclusions, presumably
aresult
of unstable
a-chains,
canbe
seen inthe
bone
marrow in immatureerythroblasts with methyl violet
stains (50)orelectron
microscopy (14),
but do
not appearinperipheral blood exceptafter splenectomy. The distribution of
inclu-sions in our patient to some degree resembles both
,3-thalassemia
and
Hb
Hdisease.
As inf8-thalassemia,
some
inclusions
are seen inthe
youngerpolychroma-tophilic erythroblasts, and
maybe responsible for
rapidintramedullary hemolysis,
as indicated by the largeproportion of immature erythroid forms
in theface of a
low reticulocyte
count. However, a few cells containingunstable hemoglobin do reach the peripheral
blood (Fig.1), where they
arepresumably destroyed
in thespleen.
ACKNOWLE DG ME NTS
WethankDr.J. Rosa,HopitalHenriMondor, Creteil,France,
for kindly performing the thin-layer isoelectric focusing and
Mr.RickTrecartin forperforming the globin chain synthesis studies and assistingwith the column chromatography. We
alsogratefully acknowledge the secretarialassistance ofMs. TerryTschopp.
This investigation was supported by grants from the
Na-tionalInstitutesof Health(HL20985), the U. S.PublicHealth
Service (5 K04 HL00086), and the National Foundation-March of Dimes (6-49).
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