Hereditary methemoglobinemia is a relatively rare disorder usually manifesting with cyanosis at birth. The more common form results from the deficiency of the enzyme, NADH-Cytochrome b5 reductase (methemoglobin reductase, diaphorase) and displays an autosomal recessive inheritance pattern. Less common are the so-called M-hemoglobins with an autosomal dominant pattern, which result from amino acid substitutions in the heme binding pocket of α, β, or less commonly γ-globin chains. The majority of the M-hemoglobin (Hb) variants occur from substitutions in the E or F-helices, which constitute the heme binding pocket, most commonly from amino acid substitutions involving the conserved proximal (F8) or distal (E11) histidine residues. Here we report a new Hb variant due to a three nucleotide deletion (-GTG between codons 25 and 26 of the β globin gene causing a single amino acid (-Gly) deletion in the B helix (B7/B8) of the β-globin chain that leads to methemoglobinemia with a novel mechanism. The propositus is a 9 month old Caucasian boy from Dothan AL who was found to have a low O2 saturation prior to an ENT procedure. He was referred to cardiology at Children’s Health System, Birmingham, AL to rule out a congenital heart disease. A low O2 saturation (85–86%) was confirmed. Cardiac catheterization excluded the structural abnormality of the heart. Cooximetry showed a normal PaO2 but confirmed a low O2 saturation. Methemoglobin level was 20%, while methemoglobin reductase activity was in the low–normal range but when repeated was found to be normal. His growth and development have been normal. On alkaline electrophoresis an abnormal hemoglobin band was observed. The patient’s blood was sent to the Hemoglobinopathy Laboratory of the Sickle Cell Center at MCG, Augusta, GA for definite identification of the variant. CBC revealed a RBC of 4.3 M/mm3, HGB 13.6 g/dL, HCT 40.8 %, MCV 95.2 fl, MCH 31.8 pg MCHC 33.4 g/dL, Retics 4.4 %. Isoelectrofocusing (IEF) on agarose showed the presence of an abnormal Hb with approximately the same isoelectric point (pI) as Hb F. Quantitation of Hb components by Cation Exchange HPLC revealed 62.7% Hb A, 27.9% Hb X, 3.0% Hb A2, and 6.4% Hb F. By globin chain analyses with reversed phase HPLC, βχ was detected as 37.6% of the total beta chains. Isopropanol stability test gave strongly positive results. P50 was found to be 24.8 mm Hg in the patient and 26.4 in the control (slightly increased oxygen affinity). Peptide analysis was done using mass spectrometry (Alphalyse, Palo Alto, CA) where tryptic digests of purified Hb X (95.0% enriched) and normal control (97.0% Hb A) were analyzed and compared. Peptide 19–30 of helix-B fragment revealed 1314 Da mass in control, whereas peptide 19–29 (with –Gly) of helix-B fragment of Hb X gave 1257 Da mass, confirming the deletion of a Gly residue. The corresponding deletion of three nucleotides (-GTG) in the genomic DNA (codons 25–26: GTGGAG→GAG ) was demonstrated by polymerase chain reaction (PCR) amplification and direct sequencing of β-globin gene and confirmed by cDNA sequencing of β-globin mRNA. No abnormality was detected in the sequences of δ, Gγ, Aγ, α1 and α2 globin genes. The three nucleotide deletion between codons 25 and 26 (-GTG) of the β-globin gene causes a one amino acid (-Gly) deletion in the B helix (B7/B8) of the β-globin chain, however does not alter the amino acid composition of β-globin chain after the deletion point but results in a shorter (145 AA, instead of the normal 146) mutant β-globin chain. As a result close spatial contact of amino acids in tertiary structure of hemoglobin is altered completely. Most importantly, distal histidine at residue 63 of E7 helix now becomes Gly leading to methemoglobin formation. A similar variant was previously reported in a Japanese baby, Hb Higashitochigi (

Fujisawa et al,
) where a three nucleotide deletion in codons 24/25 also resulted in the loss of a single Gly residue with a similar outcome. These two cases differ from the known M-hemoglobins all of which result from single amino acid substitution in the E or F-helices thus altering the heme pocket. Hb Dothan and Hb Higashitochigi represent a novel mechanism for M-hemoglobin generation where an in frame deletion alters the tertiary structure of the globin chain with alterations in the structure of E-helix and loss of the distal histidine residue.

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