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 β-globin chains with the M-globin chains that are more electropositive than normal -globin chains.
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’ α-globin chain was replaced by tyrosine (α87 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 α2-or α1-globin gene should be reflected in the observed proportion of Hb M in red blood cell lysates. The α2-globin gene is transcribed at a higher rate than the α1-globin gene (2.6-3.1:1)6-8 and, therefore, normally contributes 75% of α-globin chains. Each α1-globin gene directs approximately 12.5% of α-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 α1-globin gene. The higher-than-predicted level of Hb M can be best explained by preferential assembly of electropositive αM-globin chains with electronegative β-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.
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 α2-and α1-globin genes.12 The 5′ primer (5′-agtatggtgcggaggccctgg-3′) is complementary to a conserved region in exon 1 of the α2- and α1-globin genes. The 3′ primers were complementary to a nonhomologous region in the 3′ untranslated region (UTR) of the α2- and α1-globin genes (5′-agcgggcaggaggaacggct-3′ for α2-globin gene and 5′-aaggggcaagaagcatggcc-3′ for α1-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.
|.||VW .||KW .|
|.||VW .||KW .|
|Hb Concentration .||VW .||KW .||Normal .|
|Hb Concentration .||VW .||KW .||Normal .|
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.
A 659-bp DNA fragment encompassing exon 1 and portions of the 3′UTR of the patients α2- and α1-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 α1-globin gene, while the second α1-globin gene encoded the normal histidine residue. Southern blot analysis of genomic DNA was negative for deletional α-thalassemia.
It has been previously established that the ratio of α-globin chains derived from the α2-globin allele compared with the α1-globin allele is approximately 3:1 (range, 2.6 to 3.1).6-8Increased synthesis of α2-globin is caused by preferential transcription of the α2-globin gene. Translation of the α2-globin and α1-globin mRNAs is equivalent.6,7 Therefore, the inheritance of a single variant α1-globin gene should be associated with a variant Hb level of 12% to 14% [α1/(2α1 + 2α2) = 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 α-thalassemia allele is responsible for the increased proportion of αM-globin chains contributing to the α-globin pool.
Why is the percentage of Hb M in this kindred greater than predicted for an α1-globin variant? A possible explanation for the increased proportion of Hb M may be preferential association of αM-globin with the β-globin chain caused by electrostatic protein surface interactions. An electrostatic model for Hb assembly was proposed to explain the proportion of β-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 β-globin chain more electropositive (Hb S, Hb C, Hb D). In β-globin variants in which the amino acid substitution renders the β-globin chain more electronegative, increased association with the electropositively charged α-chain occurs, resulting in elevated proportions of the variant Hb (Hb J-Baltimore, Hb J-Iran ).14,16
This model is not restricted to β-globin variants. A potential role for an electrostatic model of Hb assembly in α-globin variants was predicted.9 In α-globin variants, the presence of 4 α-globin genes makes predictions of variant Hb levels more complex, mandating a precise understanding of variant α-globin gene locus assignment (α2 v α1), the number of affected genes, and knowledge of the presence of α-thalassemia or duplicated α-globin genes. In Hb M-Kankakee, replacement of histidine (pK 6.5) by tyrosine (pK 10) results in net increased positive charge of the αM-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 β-globin variants exhibit preferential assembly with α-globin, electropositive αM-globin variants may exhibit preferential assembly with β-globin. Our review of the literature on αM-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 α1-globin gene.
Localization of the genetic defect of the αM-globin variants to the α1-globin gene may not be coincidental. αM-globin variants resulting from mutations in the α2-globin gene have not been reported to date. If an αM-globin mutation were located in a single α2-globin gene, the predicted proportion of αM-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 α1-globin gene, exclusion of concomitant deletion α-thalassemia, and knowledge of relative protein charge provide in vivo evidence of preferential assembly of electropositive α-globin variants, as predicted by the electrostatic model of Hb assembly.
Supported in part by National Institutes of Health Grant No. T32 HL07622.
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Address reprint requests to A. Ameri, MD, Division of Pediatric Hematology-Oncology, University of Michigan, Ann Arbor, MI 48109; e-mail: email@example.com.