section vim
Laboratory Evaluation of Hemoglobin Disorders
Beta Chain Mutants
Hemoglobin
Structure
Mobility in Citrate Agar pH 6.0
*
:
i
A
e
~
s
0 6 G lu - Val
I
|
c
0
6 G lu -* Lys
C H arlem
0 6 G lu -♦ Val
I
0
7 3 A sp -*• Asn
G-San Jose
0
7 G lu ~ *G ly
■
J-Baltim ore
0
16 G ly - » Asp
à
G-Coushatta
0
22 G lu -*• Ala
■
E
0 2 6 G lu “ * Lys
■
Alabam a
0 3 9 G in - Lys
■
G-Galveston
0 4 3 G lu -*• Ala
■
W illiam ette
0 51 Pro
Arg
>
Osg Christiansborg
0 5 2 A sp ^ Asn
•
N-Seattle
0 6 1 Lys -*• G lu
è
K o rle 8u
0 73 A sp -* Asn
d
M ob ile
0 7 3 A sp -*• Val
■
D -lbadan
0 8 7 Thr
- *
Lys
■
G un H ilt
0 9 1 -9 5 deleted
■
N -Baltim ore
0 9 5 L y s -► G lu
f
M ai m o
0 9 7 H is -» G in
P
K o ln
0 9 8 V a l-*-M e t
P
Kempsey
0 99 A sp -* Asn
■
R ichm ond
0 1 0 2 Asn -*• Lys
Burke
0 107 G ly
Arg
[
P
0 1 1 7 H is
Arg
P
0 L .A . (Punjab)
0 1 2 1 G lu “ *G ln
O -Arab
0 1 2 1 G lu
Lys
Camden
0 131 G in -»G lu
Deaconess
0 1 3 1 G in - * 0
K-W oolw ich
0 1 3 2 Lys “ *• G in
Hope
0 1 3 6 G ly
Asp
Bethesda
0 1 4 5 T yr -*■ His
C ochin P o rt R oyal
0 1 4 6 H is-*-A rg
1
FIG U RE VII-4
M obilities o f /3-chain m utants on citrate agar electrophoresis relative to
hem oglobins C, S, A, and F. (Courtesy of Dr. Rose Schnieder. Reproduced
w ith perm ission from H elena Laboratories, B eaum ont, Texas.)
then precipitated by addition of ammonium sulfate, and
the undenatured HbF in the clear supernatant is measured
by spectrophotometry.
HbA
2
can be separated from most other common
hemoglobins by
anion exchange chromatography
using
diethylaminoethylcellulose. Separation results from dif-
ferences in the interactions of the charged groups of the
various hemoglobins with the positively charged groups
on the anion exchange resin. Following separation, the
HbA
2
can be quantitated by spectrophotometry. Although
several other hemoglobins (including C, E, O, and D)
are eluted with HbA2, it is unlikely that one of these
hemoglobins would be present in a person being tested for
elevated HbA2. FIbA
2
measurement is used in the diagno-
sis of 0 -thalassemia trait, in which the HbA
2
is elevated
to about twice the normal level (the upper normal limit is
about 3-3.5%).
HbH and many unstable hemoglobins spontaneously
precipitate within the red cells, forming
Heinz bodies,
which can be detected in splenectomized patients by stain-
ing with methylene blue. Alternatively,
precipitation
of
these hemoglobins can be induced and the precipitates
visualized by incubation of the erythrocytes with a redox
dye such as brilliant cresyl blue (Chapter 28).
Many abnormal hemoglobins are also much more
readily
denatured by heat
than normal hemoglobins.
Heating an unstable hemoglobin for 30 minutes at 60°C
usually causes complete denaturation, whereas HbA is
hardly precipitated at all under the same conditions. The
sulfhydryl groups of the mutant globins are generally
more exposed than those in normal hemoglobin, making
them
more reactive toward parachloromercuriben-
zoate
(PCMB). Treatment with PCMB for several hours
precipitates many mutant hemoglobins, whereas it only
causes dissociation of HbA.
A sensitive method for identifying changes in the pri-
mary structure of hemoglobin is
peptide mapping
or
fin-
gerprinting
(Chapter 3). High-performance liquid chro-
matographic techniques have been used as a sensitive
method to detect various hemoglobins, including fetal
hemoglobins
y A
and
y°.
Molecular biology techniques, such as Southern blot
analysis and polymerase chain reaction (PCR), have been
used in the diagnosis of hemoglobinopathies (see the fol-
lowing discussion).
Thalassemia Syndromes
Thalassemia syndromes (see
also Chapter 28) are a group of heterogeneous inheri-
ted disorders. They are relatively common in persons
of Mediterranean, African, and Southeast Asian ances-
try and are characterized by an unbalanced and defec-
tive rate of (absent or reduced) synthesis of one or more
globin chains of hemoglobin. Defects in a-globin chain
synthesis are designated as a-thalassemia and defects in
/3-globin chain synthesis are designated as
0
-thalassemia.
The clinical manifestations in thalassemia syndromes
occur due to inadequate hemoglobin synthesis and the
accumulation of unused globin subunits due to unbal-
anced synthesis. The former leads to hypochromic mi-
crocytic anemia and the latter leads to inaffective ery-
thropoiesis and hemolytic anemia. The clinical severity of
these disorders depends on the nature of mutation. They
range from asymptomatic mild hypochromic microcytosis
(a-thalassemia silent trait) to early childhood mortality
(homozygous 0 -thalassemia) to
in utero
death (Hb-Bart’s
hydrops fetalis).
/3-Thalassemia:
The
f3
and /3-like genes are located
in chromosome
1 1
and their organization is discussed in
Chapter 28. In homozygous /1-thalassemia syndrome the
0
-globin chain synthesis is either absent (/
1
°) or severely
reduced (
f3
+). The molecular defects are numerous and
include gene deletion, as well as defects in transcrip-
tion, processing, transport, and translation of mRNA.
The clinical symptoms manifest after
6
months of age,
when the switch of synthesis from HbF to HbA occurs.
Numerous clinical problems follow and require transfu-
sion therapy with its consequent complications (e.g., iron
overload; see Chapter 29). Heterozygous
/3
-thalassemia
{/3
-thalassemia trait) is accompanied by mild hypochromic
microcytic anemia without any significant clinical abnor-
malities. /3-Thalassemia trait can be identified by elevated
959
