Blood, Vol. 94 No. 5 (September 1), 1999:
pp. 1825-1826
Identification of the Molecular Genetic Defect of Patients With
Methemoglobin M Kankakee (M-Iwate), 
87 (F8) His 
Tyr:
Evidence for an Electrostatic Model of M Hemoglobin Assembly
By
A. Ameri,
V.F. Fairbanks,
G.A. Yanik,
F. Mahdi,
S.N. Thibodeau,
D.J. McCormick,
L.A. Boxer, and
K.T. McDonagh
From the Division of Pediatric Hematology-Oncology and the Department
of Internal Medicine, University of Michigan, Ann Arbor, MI; and the
Department of Laboratory Medicine and Pathology, Mayo Clinic,
Rochester, MN.
 |
ABSTRACT |
We determined that the molecular defect of 2 patients with
hemoglobin (Hb) M-Kankakee [Hb M-Iwate, 
87 (F8) His 
Tyr] resides in the 1-globin gene. The proportion of Hb M observed
is higher than that predicted for an 1-globin variant. Our evidence
suggests that the greater-than-expected proportion of Hb M-Kankakee
results from preferential association of the electronegative 
-globin chains with the M-globin chains that are more
electropositive than normal -globin chains.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
HEMOGLOBIN (Hb) M-KANKAKEE (Iwate) is a
variant Hb that presents clinically as congenital cyanosis due to
methemoglobinemia.1 Hb M-Kankakee is identical to Hb
M-Iwate, described in a Japanese kindred in which the proximal
histidine in the patients' 
-globin chain was replaced by tyrosine
(87 His Tyr), and with Hb M-Oldenburg and Hb
M-Sendai.2
Hb M-Iwate has been well characterized with respect to its abnormal
functional properties in oxygen transport,3-5 whereas no
conclusive molecular genetic data have been reported. The location of
the molecular defect to either the 2-or 1-globin gene should be
reflected in the observed proportion of Hb M in red blood cell lysates.
The 2-globin gene is transcribed at a higher rate than the
1-globin gene (2.6-3.1:1)6-8 and, therefore, normally
contributes 75% of -globin chains. Each 1-globin gene directs
approximately 12.5% of -globin chain synthesis. In this study, we
report on 2 patients with Hb M-Kankakee (M-Iwate) with Hb M levels
exceeding 20%. We establish that the molecular genetic defect of Hb
M-Kankakee resides in a single 1-globin gene. The
higher-than-predicted level of Hb M can be best explained by
preferential assembly of electropositive M-globin chains
with electronegative -globin chains, consistent with an
electrostatic model of Hb assembly.9
| |
MATERIALS AND METHODS |
Blood was obtained by venipuncture after informed consent.
Hb M protein studies.
Hb M quantification by cation exchange high-performance liquid
chromatography (HPLC) and isoelectric focusing were performed as
previously described.10 The isopropanol and heat methods were used to test for Hb stability.11
DNA studies.
Genomic DNA was extracted from 10 mL of whole blood using
a commercial kit (Boehringer Mannheim, Indianapolis, IN).
Polymerase chain reaction (PCR) primers were designed to selectively
amplify the human 2-and 1-globin genes.12 The
5' primer (5'-agtatggtgcggaggccctgg-3') is
complementary to a conserved region in exon 1 of the 2- and 1-globin genes. The 3' primers were complementary to a
nonhomologous region in the 3' untranslated region (UTR) of the
2- and 1-globin genes (5'-agcgggcaggaggaacggct-3' for
2-globin gene and 5'-aaggggcaagaagcatggcc-3' for
1-globin gene). PCR was performed on 50 ng of genomic DNA using a
high-fidelity PCR-kit (Boehringer Mannheim). The reaction was carried
out at 95°C × 1 minute, 65°C × 2 minutes, and
72°C × 2 minutes for 35 cycles in the presence of 0.5%
dimethyl sulfoxide. Southern analysis was performed as
described.11
| |
RESULTS AND DISCUSSION |
The patients are 2 sisters of a previously reported kindred with Hb
M-Kankakee and of Northern European descent.1 Their hematologic parameters are summarized in
Table 1. On isoelectric focusing, Hb M was
9.1 mm cathodic to Hb A, consistent with Hb M-Iwate.13 The
isoelectric point was calculated to be 7 (pI = 7). Hb quantification by
HPLC showed the relative proportion of Hb M to be 27.2% and 22.4% in
V.W. and K.W., respectively (Table 1). Stability tests of hemolysates
were normal.
A 659-bp DNA fragment encompassing exon 1 and portions of the
3'UTR of the patients 2- and 1-globin genes was separately amplified by PCR. Sequencing of the amplified DNA showed replacement of
CAC (His) by TAC (Tyr) in codon 87 of one 1-globin gene, while the
second 1-globin gene encoded the normal histidine residue. Southern
blot analysis of genomic DNA was negative for deletional -thalassemia.
It has been previously established that the ratio of -globin chains
derived from the 2-globin allele compared with the 1-globin allele is approximately 3:1 (range, 2.6 to 3.1).6-8
Increased synthesis of 2-globin is caused by preferential
transcription of the 2-globin gene. Translation of the 2-globin
and 1-globin mRNAs is equivalent.6,7 Therefore, the
inheritance of a single variant 1-globin gene should be associated
with a variant Hb level of 12% to 14% [1/(21 + 22) = 1/2 + 6.2 = 1/8.2, 12%]. Our patients have Hb M levels of 22% to 28%, a
value substantially higher than predicted. We excluded the possibility
that coinheritance of a deletion-type -thalassemia allele is
responsible for the increased proportion of M-globin
chains contributing to the -globin pool.
Why is the percentage of Hb M in this kindred greater than predicted
for an 1-globin variant? A possible explanation for the increased
proportion of Hb M may be preferential association of
M-globin with the -globin chain caused by
electrostatic protein surface interactions. An electrostatic model for
Hb assembly was proposed to explain the proportion of -globin
variant observed in individuals with Hb S, Hb C, Hb D, Hb J-Baltimore,
and Hb J-Iran.9,14-16 Reduced levels of the variant are
observed in cases wherein the amino acid substitution renders the
-globin chain more electropositive (Hb S, Hb C, Hb D). In -globin
variants in which the amino acid substitution renders the -globin
chain more electronegative, increased association with the
electropositively charged -chain occurs, resulting in elevated
proportions of the variant Hb (Hb J-Baltimore, Hb J-Iran
).14,16
This model is not restricted to -globin variants. A potential role
for an electrostatic model of Hb assembly in -globin variants was
predicted.9 In -globin variants, the presence of 4 -globin genes makes predictions of variant Hb levels more complex,
mandating a precise understanding of variant -globin gene locus
assignment (2 v 1), the number of affected genes, and
knowledge of the presence of -thalassemia or duplicated -globin genes. In Hb M-Kankakee, replacement of histidine (pK 6.5) by tyrosine
(pK 10) results in net increased positive charge of the M-globin chain at physiologic pH. This is confirmed on
isoelectric focusing, where Hb M is electropositive to Hb A. Analogous
to the observation that electronegative -globin variants exhibit preferential assembly with -globin, electropositive
M-globin variants may exhibit preferential assembly with
-globin. Our review of the literature on M-globin
variants and the similarity in observed proportions of the other Hb M
variants support this hypothesis.2 In patients with Hb
M-Boston, the proportion of Hb M is 20% to 30%,2 similar to patients with Hb M-Iwate, and we predict the mutation to reside in
the 1-globin gene.
Localization of the genetic defect of the M-globin
variants to the 1-globin gene may not be coincidental.
M-globin variants resulting from mutations in the
2-globin gene have not been reported to date. If an
M-globin mutation were located in a single 2-globin
gene, the predicted proportion of M-globin transcripts
would be approximately 3.1:8.2 (38%). Postulating an approximate
2-fold increased preference in assembly, the proportion of Hb M might
exceed 75%, a level likely incompatible with fetal viability. Neonates
with acquired methemoglobinemia and levels of methemoglobin above 60%
may have severe vital compromise.17
In conclusion, we have revisited the molecular defect in patients with
methemoglobin M-Kankakee 37 years from the initial description.1 Correlation of in vivo levels of Hb M, locus assignment of the genetic defect to the 1-globin gene, exclusion of
concomitant deletion -thalassemia, and knowledge of relative protein
charge provide in vivo evidence of preferential assembly of
electropositive -globin variants, as predicted by the electrostatic model of Hb assembly.
| |
FOOTNOTES |
Submitted February 10, 1999; accepted May 10, 1999.
Supported in part by National Institutes of Health Grant
No. T32 HL07622.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to A. Ameri, MD, Division of Pediatric
Hematology-Oncology, University of Michigan, Ann Arbor, MI 48109;
e-mail: aameri{at}umich.edu.
| |
REFERENCES |
1.
Heller P, Weinstein HG, Yakulis VJ, Rosenthal IM:
Hemoglobin- M Kankakee: A new variant of hemoglobin M.
Blood
20:287, 1962[Abstract/Free Full Text]
2.
Huisman TJ, Carver MFH, Efremov GD:
A Syllabus of Human Hemoglobin Variants. Augusta, GA, The Sickle Cell Anemia Foundation, 1996.
3.
Hayashi N, Motokawa Y, Kikuchi G:
Studies on relationships between structure and function of hemoglobin M-Iwate.
J Biol Chem
241:79, 1966[Abstract/Free Full Text]
4.
Kikuchi G, Hayashi N, Tamura A:
Oxygen equilibrium of hemoglobin M-Iwate.
Biochim Biophys Acta
90:199, 1964
5.
Perutz MF, Lehman H:
Molecular pathology of human hemoglobins.
Nature
219:902, 1968[Medline]
[Order article via Infotrieve]
6.
Liebhaber SA, Cash FE, Ballas SK:
Human alpha-globin gene expression. The dominant role of the alpha 2-locus in mRNA and protein synthesis.
J Biol Chem
261:15327, 1986[Abstract/Free Full Text]
7.
Shakin SH, Liebhaber SA:
Translational profiles of alpha 1-, alpha 2-, and beta-globin messenger ribonucleic acids in human reticulocytes.
J Clin Invest
78:1125, 1986
8.
Higgs DR, Vickers MA, Wilkie AO, Pretorius IM, Jarman AP, Weatherall DJ:
A review of the molecular genetics of the human alpha-globin gene cluster.
Blood
73:1081, 1989[Free Full Text]
9.
Bunn HF, McDonald MJ:
Electrostatic interactions in the assembly of haemoglobin.
Nature
306:498, 1983[Medline]
[Order article via Infotrieve]
10.
Hutt PJ, Pisciotta AV, Fairbanks VF, Thibodeau SN, Green MM:
DNA sequence analysis proves Hb M-Milwaukee-2 is due to beta-globin gene codon 92 (CAC TAC), the presumed mutation of Hb M-Hyde Park and Hb M-Akita.
Hemoglobin
22:1, 1998[Medline]
[Order article via Infotrieve]
11.
Merritt D, Jones RT, Head C, Thibodeau SN, Fairbanks VF, Steinberg MH, Coleman MB, Rodgers GP:
Hb Seal Rock [(alpha 2)142 term Glu, codon 142 TAA GAA]: An extended alpha chain variant associated with anemia, microcytosis, and alpha-thalassemia-2 (-3.7 Kb).
Hemoglobin
21:331, 1997[Medline]
[Order article via Infotrieve]
12.
Michelson AM, Orkin SH:
The 3' untranslated regions of the duplicated human alpha-globin genes are unexpectedly divergent.
Cell
22:371, 1980[Medline]
[Order article via Infotrieve]
13.
Hocking DR,
Huisman THJ
(eds):
The Separation and Identification of Hemoglobin Variants by Isoelectric Focusing Electrophoresis: An Interpretative Guide. Akron, OH, Isolab Inc, 1997, p 1.
14.
Bunn HF:
Subunit assembly of hemoglobin: An important determinant of hematologic phenotype.
Blood
69:1, 1987[Abstract/Free Full Text]
15.
Mrabet NT, McDonald MJ, Turci S, Sarkar R, Szabo A, Bunn HF:
Electrostatic attraction governs the dimer assembly of human hemoglobin.
J Biol Chem
261:5222, 1986[Abstract/Free Full Text]
16.
Rahbar S, Bunn HF:
Association of hemoglobin H disease with Hb J-Iran (beta 77 His Asp): Impact on subunit assembly.
Blood
70:1790, 1987[Abstract/Free Full Text]
17.
Beutler E:
Methemoglobinemia and other causes of cyanosis, in
Beutler E,
Lichtman MA,
Coller BS,
Kipps TJ
(eds):
Williams Hematology (ed 5). New York, NY, McGraw-Hill, 1995, p 654.
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ABSTRACT
INTRODUCTION