BRIEF REPORT

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
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.
r 1999 by The American Society of Hematology.

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 38UTR 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 2globin 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 associa-

EMOGLOBIN (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 58 primer
(58-agtatggtgcggaggccctgg-38) is complementary to a conserved region
in exon 1 of the 2- and 1-globin genes. The 38 primers were
complementary to a nonhomologous region in the 38 untranslated
region (UTR) of the 2- and 1-globin genes (58-agcgggcaggaggaacggct-38 for 2-globin gene and 58-aaggggcaagaagcatggcc-38 for 1globin gene). PCR was performed on 50 ng of genomic DNA using a
high-fidelity PCR-kit (Boehringer Mannheim). The reaction was carried
out at 95C 1 minute, 65C 2 minutes, and 72C 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
Blood, Vol 94, No 5 (September 1), 1999: pp 1825-1826

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.
Submitted February 10, 1999; accepted May 10, 1999.
Supported in part by National Institutes of Health Grant No. T32
HL07622.
Address reprint requests to A. Ameri, MD, Division of Pediatric
Hematology-Oncology, University of Michigan, Ann Arbor, MI 48109;
e-mail: aameri@umich.edu.
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.
r 1999 by The American Society of Hematology.
0006-4971/99/9405-0033$3.00/0
1825

1826

AMERI ET AL

Table 1. Hematological Data of Patients With Hb M-Kankakee

RBC 106/L
HB g/dL
MCV fL
MCH pg
MCHC
RDW %

VW

KW

4.23
12.6
88.1
29.9
33.9
12.3

4.85
13.5
81.3
27.9
34.2
11.9

Hb Concentration

VW

KW

Normal

HbA %
HbA2%
HbF %
HbM %

71.2
1.3
.3
27.2

75.9
1.4
.3
22.4

95-98
2-3.3
0-2
0

Abbreviations: RBC, red blood cell count; MCV, mean corpuscular

volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration g/dL RBC; RDW, red blood cell distribution width.

tion 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 JBaltimore, 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 incompat-

ible 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.
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