First Observation of HbM-Saskatoon at the Origin of Neonatal Cyanosis in a Tunisian Baby.

Several causes are known to be at the origin of neonatal cyanosis among them methemoglobinemia is by inheritance of an hemoglobin (Hb) M variant. This is a rare condition never been reported in Tunisia so far. Here, we report a Tunisian newborn with refractory cyanosis since birth. As cardiac and respiratory diseases were ruled out, methemoglobinemia was suspected. Hematological parameters, concentration of methemoglobin, capillary electrophoresis, and amplification sequencing of the HBB gene were performed. Computational analysis was achieved by different in silico tools to investigate the mutation effect. The diagnosis was established by a raised MetHb, confirmed by the presence HbM-Saskatoon [Beta63 (E7) His>Tyr] by capillary electrophoresis and molecular analysis. The identified mutation occurred as a de novo mutation. In silico analysis confirmed the pathogenicity of the mutation. To our knowledge, this is the first time that this mutation has been reported in the Tunisian population. In view of its low incidence rate, clinicians might misdiagnose cyanosis caused by HbM, which can lead to inappropriate treatment and clinical complications. An up-to-date literature review of HbM disease is presented in this study.

Failing blood gas measurement due to methemoglobin forming hemoglobin variants: a case report and review of the literature.

INTRODUCTION: We present a case of an arterial blood gas sample analysis from a 33-year old woman where no oximetry results could be obtained using the Radiometer ABL800 FLEX device. Clinical history of this patient learned that she was carrier of a methemoglobin forming hemoglobin variant type Hyde Park (HbM Hyde Park) and raised the question whether or not this variant could be the cause of the errors obtained during analysis. MATERIALS AND METHODS: A literature search was performed, focusing on methemoglobin forming hemoglobin variants and their influence on oxygenation measurements. An overview of the currently described methemoglobin forming hemoglobin variants is also included. RESULTS AND DISCUSSION: In the presence of dyshemoglobins such as methemoglobin, techniques used to obtain parameters that reflect the patient oxygenation status, such as pulse oximetry and CO-oximetry can be influenced. In these cases, CO-oximetry is the preferred technique because it can compensate for this, in contrast to pulse oximetry. In case of the presence of methemoglobin originating from a hemoglobin variant, it is possible that CO-oximetry data cannot be calculated because the absorbance spectrum of this methemoglobin can differ from regular methemoglobin. Moreover, pulse oximetry devices are actually prone to erroneous results since pulse oximetry data will be calculated in these cases, but unreliable and should be avoided. CONCLUSION: Methemoglobin forming hemoglobin variants are rare genetic mutations. However, they can possibly interfere with the calculation of CO-oximetry values. In these cases, pulse oximetry data should be avoided because they could lead to incorrect medical decisions.

Hemoglobin M Disease as a Cause of Cyanosis in a Newborn.

Methemoglobinemia, including the inherited or congenital form, is a known but infrequent cause of neonatal cyanosis. We present the case of a newborn patient with neonatal cyanosis, who was diagnosed with F-M-Osaka methemoglobinemia, and an up-to-date literature review of the disease.

De novo alpha 2 hemoglobin gene (HBA2) mutation in a child with hemoglobin M Iwate and symptomatic methemoglobinemia since birth

Cyanosis in an apparently healthy newborn baby may be caused by hemoglobin variants associated with the formation of methemoglobin, collectively known as M hemoglobins. They should not be confused with genetic alterations in methemoglobin reductase enzyme systems of red cells since treatment and prognosis are completely different. A newborn male child was noted to be significantly cyanotic at birth and is the basis for this report. Hemoglobin isoelectric focusing, acid and alkaline gel electrophoresis, and HBA/HBB gene sequencing were performed for the child, both parents and a sister. The newborn child was treated with methylene blue in an intensive care unit fearing that he had a defective reductase system and exposure to oxidant drugs or toxins. Newborn hemoglobin screening with high performance liquid chromatography was abnormal on the 10th and 45th days but no conclusive diagnosis was reached. Cyanosis persisted up to four years of age with no other symptoms. Hemoglobin M Iwate [alpha2 87(F8) His>Tyr, HBA2:c.262C>T] was detected. It was not present in the child's presumed mother, father, sister, and brother. The analysis of 15 short tandem repeats in the trio demonstrated a de novo mutation occurrence (p-value < 1 × 10 -8). The family was reassured that no further action was necessary and genetic counseling was provided. Methemoglobins should be considered for differential diagnosis of cyanosis in newborns even if no familial cases are detected. Except for cosmetic consequences, the clinical course of patients with hemoglobin M Iwate is unremarkable.

Novel dominant ß-thalassemia: Hb Boston-Kuwait [codon 139/140(+T)].

Dominant ß-thalassemias exhibit a hybrid phenotype of unstable hemoglobin and ineffective erythropoiesis. Most arise from heterozygous ß-globin gene mutations in exons 3 or 2 and present in adulthood as thalassemia intermedia. We report a novel, de novo ß-globin mutation presenting in a toddler with features of thalassemia major and chromaturia. Hemoglobin Boston-Kuwait is an elongated ß-chain variant (163 amino acids) that results from a frameshift mutation caused by a thymidine insertion in codons 139/140. Hematopoietic stem cell transplant provided a successful alternative therapy for this severe form of dominant ß-thalassemia.

Transient neonatal cyanosis associated with a new Hb F variant: Hb F viseu.

Neonatal cyanosis in healthy newborns can be associated either with methemoglobin due to cytochrome b5 reductase deficiency or to M-hemoglobin, a group of hemoglobin variants resulting from mutations in the globin chain genes. We report the clinical case of a neonate with cyanosis and normal cardiac and respiratory function. At birth the hematological parameters were normal; however, the methemoglobinemia was 16%. Spontaneously, the cyanosis gradually decreased and by the fifth month of age the methemoglobin level was normal. A heterozygous Gγ-globin gene (HBG2) missense mutation 87 C-A (Leu28Met) was identified. His father, with a history of transfusion in the neonatal period, is heterozygous for the same mutation. This hemoglobin variant, not previously described, was called Hb F Viseu and is the sixth Gγ-chain variant reported in association with neonatal cyanosis.

Familial congenital cyanosis caused by Hb-M Yantai (alfa-76 GAC - TAC, Asp - Tyr)

Methemoglobin (Hb-M) is a rare hemoglobinopathy in China. We hereby report on a family living in Yantai, East China, with congenital cyanosis due to Hb-M mutation. The proband, a 65-year-old female, presented 63 percent oxygen saturation. Both Hb-M concentration and arterial oxygen saturation remained unchanged, even following intravenous treatment with methylene blue. There was also no change in blood-color (chocolate-brown) after adding 0.1 percent KCN. A fast-moving band (Hb-X) in hemolysates was found by cellulose acetate electrophoresis, the Hb-X/Hb-A ratio exceeding 10 percent. GT transition at 131nt of exon 2, although present in one of the alfa2-globin alleles, was not found in alfa1-globin alleles as a whole. This mutation leads to the aspartic acid to tyrosine substitution (Asp76Tyr). In this family, the novel mutation in the alfa2-globin gene resulted in a rare form of congenital cyanosis due to Hb-M. This hemoglobin was named Hb-M Yantai.

Serum levels of TGF-beta and fibronectin in autosomal dominant osteopetrosis in relation to underlying mutations and well-described murine counterparts.

The study gives a further biochemical description of two different forms of autosomal dominant osteopetrosis (ADO) in relation to murine counterparts, with special attention to osteoblast function and the recent discovery of LRP5 gene mutations in ADO I. Patients and controls were investigated for markers of bone formation and resorption at baseline and following stimulation with thyroid hormone. Moreover, four different well-described murine models of osteopetrosis were investigated. Concerning the human forms, serum TSH levels decreased in all subjects, indicating effects on the target tissue. Osteocalcin and cross-linked collagen (NTx) were without significant differences among the groups. Significant increases in both markers were seen following stimulation. Baseline active TGF-beta1 levels were increased in both types of ADO (60% in ADO I [P = 0.006]; 46% in ADO II [P = 0.001], respectively), whereas fibronectin levels were decreased in both (ADO I 58% and ADO II 63% of normal, respectively [P = 0.012 and P = 0.001]). Following treatment, levels increased temporarily in all groups. In the murine models, active TGF-beta1 was significantly decreased in the tl- and ia-rat, whereas fibronectin levels were decreased in the mi-mouse, however, increased in the ia-rat. In conclusion, both types of ADO showed the same qualitative biochemical differences compared to controls, except that OPG levels were higher in ADO I. The decreased fibronectin levels in both types and in murine models reflect decreased bone resorption; however, this may also indicate hitherto unrecognized alterations in bone formation. Biochemical differences among known syndromes related to mutations in the LRP5 gene indicate different underlying pathogenetic mechanisms.

Combined mass spectrometric methods for the characterization of human hemoglobin variants localized within alpha T9 peptide: identification of Hb Villeurbanne alpha 89 (FG1) His-->Tyr.

Mutation-induced amino acid exchanges occurring on the large T9 peptide of the alpha-chain of human hemoglobin (residues 62-90) are difficult to identify. Despite their high m/z value (around m/z 3000), collision-induced dissociation spectra of liquid secondary ion mass spectrometrically generated protonated alpha T9 peptides were performed successfully. In parallel electrospray mass spectrometry (MS) was used both to measure the molecular mass of the intact proteins and to determine the number of protonatable sites in the alpha T9 peptides. Peptide ladder sequencing using carboxypeptidase digestions and analysis of the truncated peptides by matrix-assisted laser desorption ionization time-of-flight MS confirmed the interpretation. This set of methods allowed the characterization of three hemoglobin variants, with amino acid exchanges located in the alpha T9 part of the sequence. Two of them, Hb Aztec [alpha 76(EF5) Met-->Thr] and Hb M-Iwate [alpha 87(F8) His-->Tyr] were already known. The third [alpha 89(FG1) His-->Tyr] was novel and named Hb Villeurbanne.

Effect of ionizing radiation on haemoglobin: the oxy-derivative of haemoglobin Iwate.

It is well established that exposure of oxyhaemoglobin to ionizing radiation results in remarkably selective electron addition to the (FeO2) unit, giving a novel species, (FeO2)-, in which the extra electron is largely localized on iron and dioxygen. This work has now been extended to haemoglobin (Hb.) Iwate. The haemoglobin M. Iwate used is a mutant haemoglobin having only Fe(III) alpha-chains by oxy beta-chains (alpha 2 Met beta 2 oxy). The haem iron atoms in the alpha-chains are coordinated in the fifth site by a proximal tyrosine in place of histidine. This unit is a high-spin Fe(III) with axial symmetry and prominent electron spin resonance (ESR) features in the g = 6 and g = 2 regions. On exposure to 60Co gamma-rays at 77 K, efficient electron addition occurred at both types of iron centre, giving Fe(II) and (FeO2)- units. The former was monitored by the decrease of the g = 6 feature for Fe(III) and the latter by the growth of g-features at 2.254 (gx), 2.149 (gy) and 1.967 (gz). These values are close to those for the FeO2- centre formed in the beta-chains of normal oxyhaemoglobin. On annealing above 77 K, two changes occurred: first there was a small but clear increase in gx and gy, followed by a marked reduction in gx and gy giving g-values close to those for the centre formed directly in the alpha-chains of the normal protein. Finally, this intermediate species gave a centre having gx = 2.310, gy = 2.180 and gz = 1.935. These values are typical of low-spin Fe(III) haemoglobin and are assigned to the protonated complex, Fe(III)O2H. Ultimately at ca. room temperature, this was converted into the high-spin, met-form, with a gain in the g = 6 feature. These results established that the beta-chain centre in Hb. Iwate behave in the same way as isolated beta-chains. They also confirm that electron addition to the oxy-units is facile, even in the presence of Fe(III) units in each tetramer. The results also confirm that electron capture to give (FeO2)- units is not followed by internal electron-transfer to give Fe(II) from the Fe(III) centres in the alpha-chains.

Resonance raman studies of hemoglobins M: evidence for iron-tyrosine charge-transfer interactions in the abnormal subunits of Hb M Boston and Hb M Iwate.

Resonance Raman spectra have been obtained for Hb M Boston [His-E7(58) alpha leads to Tyr], Hb M Iwate [His-F8-(87) alpha leads to Tyr], and Hb M Milwaukee [Val-E11(67) beta leads to Glu]. The abnormal alpha subunits of Hb M Boston and Hb M Iwate exhibited the porphyrin nu10 band at 1628 and 1627 cm-1, respectively, which indicates that the ferric alpha hemes are five-coordinated in both Hb M Boston and Hb M Iwate. In addition to the porphyrin bands, four extra polarized lines were observed at 1607, 1506, 1278, and 603 cm-1 for the alpha abnormal subunit of Hb M Boston and at 1605, 1506, 1310, and 589 cm-1 for that of Hb M Iwate. By comparison with the vibrational spectra of Fe-tyrosine proteins and Fe-phenolate complexes, the 1605-1607- and 1506-cm-1 lines are assigned to the phenolate ring vibrations of the heme-coordinated tyrosine, and the 1278-cm-1 line of Hb M Boston and the 1310-cm-1 line of Hb M Iwate are assigned to the phenolate CO stretching mode. We propose that the 603-cm-1 line of Hb M Boston and the 589-cm-1 line of Hb M Iwate arise from the Fe-O(tyrosine) stretching mode. These four Raman lines are intensity enhanced upon the excitation around 475-520 nm, probably due to the presence of a charge-transfer interaction between Fe and Tyr. The dissimilarity of the Fe-O and phenolate CO stretching frequencies between Hb M Boston and Hb M Iwate, despite the similarity of frequencies of their porphyrin and phenolate ring modes, suggests that the heme-phenolate bonding angles differ between Hb M Boston and Hb M Iwate although both adopt the five-coordinate form with Tyr as the only axial ligand. The resonance Raman spectra of oxy- and deoxy-Hb M Milwaukee showed no anomaly and can be accounted for by those of the equimolar mixtures of aquomet- and oxy- or deoxy-Hb A.

Minor early embryonic chick hemoglobin M. Amino acid sequences of the epsilon and alpha D chains.

Erythrocytes of the early chick embryo contain four hemoglobins, two major and two minor. In this paper, we present amino acid sequences for the beta-like and alpha-like chains of HbM, the least abundant of the four early chick hemoglobins. The complete amino acid sequence of the beta-like chain of HbM is identical with that of the epsilon chain of HbE, the other minor early embryonic hemoglobin in the domestic chicken. Analysis of the alpha-like chain of HbM (92 of 141 residues) reveals a globin sequence closely related to the minor adult alpha D chain. Comparison of our sequence data with the nucleotide sequence of the alpha D globin gene suggests that a single gene encodes both the embryonic and adult alpha D globin polypeptides. We discuss the structure, possible function, and evolution of the HbM globin chains.

Co-binding studies on Hb M Iwate. Allostery of a T state haemoglobin.

The mutant haemoglobin Hb M Iwate alpha 2Mmet87His leads to Tyr beta 2, is characterized by a stable T structure and a low ligand affinity. Sigmoidal CO-binding isotherms of symmetrical shape with Hill coefficients of n = 1.4 at pH 6 to n = 1.9 at pH 10 and the differences in the mean affinity (PCO(1/2)) and the affinity of the first ligand-binding beta subunit (1/L1 greater than Pco(1/2)) are the evidence for the cooperativity. The comparison of the Bohr effects of the two valency hybrid states (alpha 2Mmet beta met beta deoxy alpha 2Mmet beta 2deoxy) in the absence of and in the presence of polyphosphates leads to an indirect proof of pH-dependent subunit-subunit interaction. Inositol hexaphosphate-binding suppresses cooperativity in the pH range 5.5-8 (n = 1). Above pH 8 hte cooperativity increases to a final value of n = 1.9 at pH greater than 10, which is identical to that of stripped Hb M Iwate. The CO binding to the first binding site exhibits a Bohr effect. Polyphosphate anions have no influence on the CO binding of the first binding site. The heterotropic effects are discussed as intrachain effects (Bohr effect of the first binding site) and interchain effects (Bohr effect of Pco(1/2); influence of polyphosphates).

Hb M Milwaukee in a German family.

The second occurrence of Hb M Milwaukee is reported in two members of a German family who had cyanosis since early childhood. Contrary to earlier reports, Hb M Milwaukee exhibits a distinct heat instability. It is suggested, that in this family the variant resulted from a new mutation.

Functional and physicochemical studies of hemoglobin St. Louis beta 28 (B10) Leu replaced by Gln: a variant with ferric beta heme iron.

Studies have been performed on a 20-yr-old man exhibiting methemoglobinemia and a severe hemolytic anemia involving formation of Heinz bodies. This condition was due to an abnormal Hb present in the red cells of the proband: Hb St. Louis, beta 28 (B10) replaced by Gln, whose structural characteristics have been previously reported. This unstable Hb represented 30% of the total and was isolated by starch block electrophoresis at pH 8.6. Electrophoretic and spectral studies showed Hb St. Louis to be a valency hybird, alpha 2 beta 2+. The presence of hemichrome in this Hb was detected by electron paramagnetic resonance studies. During this study, an electrophoretic technique was developed that allows study of the mobility of hemichrome. Oxygen equilibria performed on purified Hb St. Louis revealed a high oxygen affinity and a markedly reduced cooperativity. The Bohr effect was normal, but the interaction of this hemoglobin with 2,3-diphosphoglycerate was decreased. The oxidation rate of Hb St. Louis was normal. Hb St. Louis was completely reduced by dithionite and ferrous citrate, and the functional properties of this reduced form were normal. In contrast, Hb St. Louis was only partially reduced by diaphorase. The mechanism of the oxidation of Hb St. Louis therefore appears to differ markedly from that postulated for other Hbs M.

Altered erythropoiesis and increased hemolysis in hemoglobin M Akita (M Hyde Park beta92 His replaced by Tyr) disease.

Hb M Akita disease is a cyanotic hemoglobinopathy found in Akita Prefecture, Japan. The abnormal hemoglobin was found to be the same as Hb M Hyde Park (beta92 His replaced by Tyr) by chemical analysis in 1967. In this disease signs of accelerated hemolysis (serum bilirubin, 2.4 mg/dl; splenomegaly, 2 finger breadths; Hb, 10.7 g/dl; reticulocyte index, 2.7) were noted, but the causes of its slight anemia were revealed to be fairly complex by ferrokinetic study, RBC life-span measurement, and 99mTc myeloscintigram. The anemia in this disease is caused not only by shortened erythrocyte survival (T 1/2 = 11.5 days by 51Cr-tagging method) and sequestration of red cells in the spleen (Spleen: liver ratio = 2.5 approximately 3.0 by 51Cr-surface counting), but also by slow supply of erythrocytes to the peripheral blood from the bone marrow, presumably, related to the existence of unstable Hb M Akita and its derivative (Hb Akita) in the erythroid cells. Both Carrell's isopropanol test and Heinz body formation test were positive. In spite of maximally increased total erythropoiesis (8 times as high as the normal level; M:E ratio = 0.22:1.0), supply of red cells from the bone marrow to the peripheral blood was significantly decreased. The distribution of hematopoietic sites throughout the body was reasonably uniform.