H e m o g l o b i n
a
Qmdia*
/3-Thalassemia
w
in
ROBERT M. SCHMIDT, M.D.,
( E l 3 )
a
A s p ^
C a n a d i a n
His,
a n d
Family
M.P.H., M. A. M. A L I , M.D.,
PH.D.,
KATHERINE C. BECHTEL, B.S., AND
WINSTON F. MOO-PENN, P H . D .
Hematology Division, Center for Disease Control, Public Health Service, U.S. Department of Hea
Education and Welfare, Atlanta, Georgia 30333, and Hematology Dix'ision,
St. Joseph's Hospital, Hamilton, Ontario, Canada
ABSTRACT
Schmidt, Robert M., Ali, M. A. M., Bechtel, Katherine C , and Moo-Penn,
Winston F.: Hemoglobin QIn(iia, a 64 (E13) Asp —» His, and /3-thalassemia in a
Canadian family. Am J Clin Pathol 66: 4 4 6 - 4 4 8 , 1976. Cellulose acetate
electrophoresis at/;H 8.4 showed a hemoglobin variant with the mobility of
hemoglobin S in a Canadian family. Sequence analysis revealed that
histidine was substituted for aspartic acid at position 64 in the a-chain.
This variant was found in association with a ^-thalassemia trait condition. (Key words: Hemoglobin; Hemoglobin Q Im i ia ; Thalassemia; Hemoglobinopathies.)
A 4-YEAR-OLD BOY, undergoing a presurgical evaluation, was found by cellulose acetate electrophoresis at /;H 8.4 to have a
variant hemoglobin migrating like hemoglobin (Hb) S. T h e concentration of the
variant was less than 10% of the total
hemoglobin. In addition, the boy was
found to have /^-thalassemia trait. The
variant hemoglobin and /3-thalassemia trait
were also found in his father. This paper
describes this uncommon variant, hemoglobin Q,miia, am (E13) Asp-> His, in a
Canadian family of mixed French and
Iranian ancestry.
Received March 17, 1976; accepted for publication
March 17, 1976.
Use of trade names is for identification only and
does not constitute endorsement by the PHS or by
the U.S. HEW.
Address reprint requests to Dr. Schmidt: Hematology Division, Cenier for Disease Control, Atlanta,
Georgia 30333.
Dr. Ali's present address is Hematology Division,
St. Joseph's Hospital, Hamilton, Ontario, Canada.
Materials and Methods
Electrophoretic and quantitative methods for detecting hemoglobins have
been described. 6 T h e abnormal hemoglobin was separated on DEAE-Sephadex
by the method of Huisman and Dozy3; a
gradient of T r i s - H C l (0.05 M), /;H 8.4 to
6.4, was used. The variant a-chain was
isolated by the Clegg procedure, 2 and
after aminoethylation and tryptic digestion
the peptides were separated by column
chromatography. 5 Amino acid compositions were determined with a Beckman
Model 121 Amino Acid Analyzer by the
method of Spackman, Stein and Moore. 7
A Beckman Model 890C Sequencer was
used to determine the peptide sequence
after the peptide was modified with 4sulfophenylisothiocyanate (SPITC). 1 ' 4
Hematologic procedures were performed
by standard methods. All enzymes were obtained from Worthington Biochemical
446
August 1976
447
HEMOGLOBIN Qm„hl AND /3-THALASSEMIA
Table 1. Hematologic Data
Hemoglobin (Hb)
Hematocrit (Hct)
Erythrocyte count (RBC)
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC)
Leukocyte count. (WBC)
Platelet count
Differential
Corporation, and reagents were of the
highest purity available.
Results and Discussion
The propositus is a 4-year-old boy admitted to the hospital for a tonsillectomy.
Results of routine blood analyses are shown
in Table 1. An initial screening for abnormal hemoglobins by cellulose acetate
electrophoresis at/;H 8.4 showed bands at
the positions of Hb A and Hb S. Starch
gel electrophoresis confirmed this finding
and, with heme-specific stain, an additional
band was observed migrating in a more
cathodal position than Hb A2. These results
indicated the presence of a mutant enchain contributing to the abnormal hemoglobin at the Hb S position and also to
the abnormal Hb A2, since the a-chains
are common to both hemoglobins. Hb F
was 4.5% as determined by the Singer
method. Hb A 2 was 6.2%, whereas the
normal range is 2 - 3 . 5 % . The solubility
test was negative. Heterozygosity for /3thalassemia and an a-chain abnormality
were provisionally diagnosed, and family
studies were performed.
T h e boy's father was born in Iran, and
the mother is of French ancestry. Clinical
examination showed no physical abnormality in either parent, in particular no
hepatosplenomegaly or jaundice. Results
of hematologic investigation of the father
are shown in Table 1. Hemoglobin electrophoresis on cellulose acetate at pH 8.4
showed a slow-moving band in the Hb S
Propositus
Father
lOg/cll (1.55 mmol/l)
33%
5.23 x 10«//u.l
62.0 cu fim (II)
19 pg (0.29 fmol)
31%
7,800/jnl
268,000/ftl
Normal
10.7 g/dl (1.65 mmol/l)
33.8%
5.37 x 10'V/xl
6<1.0 cu /Mm (II)
19.6 pg (0.30 fmol)
31.7%
8,100/MI
235,000//xl
Normal
position similar to the band detected in the
propositus. Starch gel electrophoresis gave
similar results. T h e mother's hematologic
data were normal, with no evidence of
hypochromia or an abnormal hemoglobin.
Blood was collected from the father and
mailed to the Center for Disease Control
(CDC) for further investigation. The presence of bands migrating in the positions
of Hb S, Hb A, Hb A2 and Hb G2
(variant Hb A2) on cellulose acetate electrophoresis at/;H 8.4 was confirmed. Citrate
agar electrophoresis at />H 6.2 indicated
only the presence of Hb A. T h e hemoglobin fractions were purified and quantitated by column chromatography on
DEAE-Sephadex, and the values of the
various fractions were: Hb G2, 0.5%; Hb
A2, 3.8%; Hb Q India , 8.6%; Hb A, 87.1%.
The Hb F value was 2.8%, as determined
by the Singer method.
After the chains were separated, the
mutant a-chain was aminoethylated and
trypsinized, and the peptides were purified. Chromatography of the tryptic digest
on the cationic resin Aminex A-5 showed
that peptide a T p IX eluted between peptides a T p I — II and IV instead of in its
normal position as the first eluting and
most acidic peptide. T h e change in the net
charge on the peptide is reflected in the
altered elution time of abnormal a T p IX;
a T p V I I I - I X was also found between
a T p IV and VI.
T h e amino acid composition of abnormal
a T p IX is shown in Table 2. The composi-
448
SCHMIDT ETAL.
A J.C.P.—Vol.
66
Table 2. Amino Acid Composition of
Abnormal a T p IX*
boy was available. The child and the father
were given weekly doses of folic acid.
Eight weeks later the boy's hemoglobin
aTp IX
Obtained
Amino Acid
value was 11.5 g/dl (1.78 /imol/1) and his
MCV was 64 fl. For the father, these
1
0.9
Lysine
values
were 13.8 g/dl (2.139 /umol/1) and 67
Histidine
3
3.4
Aspartic acid
6
4.7
fl, respectively. These data are compatible
Threonine
1
0.9
with /3-thalassemia trait.
Serine
2
1.9
Both father and son have hemoglobin
Proline
1
1.0
Alanine
7
6.8
Qmdia. as well as ^-thalassemia trait. T h e
Valine
3
2.4
first reported instance of Hb Q I n d i a was in a
Methionine
1
1.0
Hindu family in which /3-thalassemia
Leucine
4
3.9
major was also present. 8 In that study,
•The peptide was hydrolyzed in 6 N HC1 for 24 hours at HOC
. Asparagine is converted toaspartic acid during acid hydrolysis.the proportions of Hb QIndia ranged from 8
ini'firuo
to 20%; the proportions in those with fithalassemia minor tended to be lower than
Table 3. Automated Edman Degradation
proportions in those without ^-thalasof Abnormal a T p IX*
semia.
Cycle No.
Sequence No.
I.D.
nmol
1
2
3
4
62
63
64
65
Valt
Ala
Hist
Ala
77.3
53.0
66.2
* Initial sample: 200 nmol.
t Modified with SPITC and not determined.
$ Identified on Water's high-pressure liquid chromatograph.
tion shows a loss of one residue of aspartic
acid with a corresponding gain of half a
residue of histidine. In a T p IX there are
four residues of aspartic acid and two
residues of asparagine. Sequence analysis
was performed to determine which of these
six possibles sites was the site of mutation.
The sequencer data are given in Table 3.
T h e data show that histidine is substituted
for aspartic acid at position 64 in the achain. This substitution has previously
been reported as Hb Qindta-8
Because the father was slightly anemic,
further tests were done. Serum ion was
95 /xg/dl (17.01 (imo\/\), total iron-binding
capacity was 384 /x,g/dl (68.74 /xmol/1),
serum B 12 was 491 pg/ml (363.34 pmol/1),
and serum folate was 4.2 ng/ml (9.53
nmol/1). T h e erythrocytic folate was low,
89 ng/ml (202.03 nmol/1) (normal, 1 4 0 670 ng/ml 317.8-1520.9 nmol/1). No
information about the folate status of the
Acknowledgments. Effie Brosious, Barbara Morrison,
Solomon Holland, and Jane Wright performed the
reference screen and preliminary diagnostic tests.
Danny Jue and Mary Johnson assisted in the biochemical characterization of the hemoglobin variant.
References
1. Beckman Instruments, Inc.: Instruction Manual
and Sequencer Programs (#071872). Beckman Instruments, Palo Alto, Cal., 1972
2. Clegg JB, Naughton MA, Weatherall DJ: Abnormal hemoglobins. J Mol Biol 19:91-108,
1966
3. Huisman THJ, Dozy AM: Studies on the heterogeneity of hemoglobin. IX. The use of tris
(hydroxymethyl) aminomethane-HCl buffers
in the anion-exchange chromatography of
hemoglobins. J Chromatogr 19:160-169, 1965
4. Inman JK, Hannon JE, Appella E: Demonstration of a simple method for reducing losses of
tryptic peptides during automatic sequencing:
Biochem Biophys Res Commun 46:20752081, 1972
5. Jones RT: Structural studies of aminoethylated
hemoglobins by automatic peptide chromatography. Cold Spring Harbor Symp Quant Biol
19:297-308, 1964
6. Schmidt RM, Brosious EM: Basic Laboratory
Methods of Hemoglobinopathy Detection.
Atlanta, Ga., DHEW Publication No. (CDC)
76-8266, 1975
7. Spackman DH, Stein WH, Moore S: Automatic
recording apparatus for use in the chromatography of amino acids. Anal Chem 30:11901206, 1958
8. Sukumaran PK, Merchant SM, Desai MP, et al:
Haemoglobin QIndla (a64(E13) aspartic acid
—> histidine) associated with /3-thalassaemia
observed in three Sindi families. Med Gen
9:436-442, 1972