Hb AHVAZ [α83(F4)Leu→Arg, CTG>CGG (α2);HBA2: c.251T>G],A New Hemoglobin Variant of theα2-Globin Gene

Abstract Hemoglobin Hana [α2β2 63 (E7) His-Asn)] was described in a Moravian family from the Czech Republic as an unstable hemoglobin variant with mild Heinz body hemolytic anemia, elevated levels of methemoglobin (12–13%), and variable reticulocytosis (1.8–11%) in the proband and her sister.1 Surprisingly, their mother is asymptomatic, although she expresses the same aberrant variant of the β-globin gene. We did not detect any differences in the globin gene expression (based on wild-type and mutant globin mRNA from reticulocytes and βA and βHana chains measured by HPLC).1 Iron metabolism indices were within normal range in all family members. G6PD status in both parents was normal. To test whether the erythrocyte antioxidant capacity affects severity of this unstable hemoglobinopathy, key antioxidant parameters, including content of reduced (GSH) and oxidized glutathione (GSSG), activity of superoxide dismutase (SOD), catalase (CAT), and NADH-methemoglobin reductase (MetHb RED) were examined. Our results revealed that erythrocytes of both children exhibited lower GSH content, the ratio of GSSG/GSH was increased 6 to 7 times, and their catalase activity was decreased to 71–77% of the average control activity (Table 1). The increased ratio of GSSG/GSH could be related to the accumulation of peroxides in erythrocytes of the children and reflects lower ability of the enzyme antioxidant system to eliminate free oxygen radicals. In contrast, erythrocytes of their mother exhibited normal values of all these measured parameters except the activity of SOD, which was increased by 32% above normal level (Table 1). Activities of some antioxidant enzymes are altered in cigarette smokers2 and the mother is a heavy smoker (with elevated CO-Hb to 5.5%). We hypothesize she might benefit from higher SOD activity caused by activation of “oxidative stress genes” and that could, in turn, enhance the ability of her erythrocytes to deal with superoxide and hydrogen peroxide accumulation which results in more efficient prevention of hemoglobin oxidation. In the children, the decrease in hydrogen peroxide catabolizing capacity due to lower erythrocyte catalase activity strongly associates with a more severe clinical manifestation of this unstable hemoglobinopathy. Our findings suggest that alterations (either inherent or caused by extrinsic factors) in the levels of red blood cell antioxidant defense enzymes represent modifying factors affecting the clinical manifestation of Heinz body hemolytic anemia associated with an unstable hemoglobin variant. Table 1: Levels of erythrocyte antioxidant systems in the family members Unaffected Mother Daughter 1 with HA Daughter 2 with HA Normal controls (n = 10) Range a - spectrophotometry, b - HPLC-MS GSHa(μmol/g Hb) 3.7 2.9 2.7 3.9±0.6 3.3–4.5 GSSG/GSHb 0.35 1.9 1.5 0.27±0.2 0.12–0.42 MetHb RED (U/g Hb) 15 15 14 16±2.8 13–19 SOD (U/g Hb) 2707 2325 1933 2052±311 1741–2363 CAT (U/g Hb) 282 227 245 320±57 263–377

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A Novel Case of Co-Inheritance of Rare Hemoglobin Variant; Hb Rancho Mirage [βCd 143 (CAC→GAC) His→Asp] with Hb D-Punjab

Blood ◽

10.1182/blood.v120.21.4759.4759 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4759-4759

Author(s):

Eiman A. Al Zaabi ◽

Amira Mahmoud ◽

Erol Baysal ◽

David G. Spence

Keyword(s):

Dna Analysis ◽

Conflicts Of Interest ◽

Globin Gene ◽

Oxygen Binding ◽

Beta Chain ◽

Hemoglobin Variant ◽

Current Case ◽

Binding Characteristics ◽

Arterial Blood ◽

First Case

Abstract Abstract 4759 Introduction Co-inheritance of Hb Rancho Mirage and Hb D-Punjab produces a clinically significant condition through increasing oxygen affinity that causes a compensatory erythrocytosis. We report a first case of co-inheritance of Rancho Mirage with Hb D-Punjab which differs in its clinical presentation from that described in the literature. Case presentation A 59-year-old Pakistani male was admitted to hospital because of chest tightness. The imaging studies showed no marked organomegaly or masses. Laboratory investigations revealed erythrocytosis with high hematocrit and borderline MCV and MCH. Arterial blood gas testing (ABG) showed low pO2 and JAK2 V617F mutation was not detected. Erythropoietin level was normal. Hemoglobin analysis through high performance liquid chromatography (HPLC) revealed two hemoglobin variants; Hb D-Punjab (32.5%) and the remaining Hb comprised of a fast moving variant. The variant was faster than Hb F expressing at a considerably high level. There was no detectable Hb A. (figure 1, HPLC shows both variants) At the molecular level, the β globin gene was amplified and sequenced using specific primers. DNA analysis demonstrated the presence of two mutations in Exon 3. The first one was a rare mutation in βCd 143 (CAC→GAC) causing a substitution of His→Asp that was identified as Hb-Rancho Mirage. It is speculated that the replacement of the ‘His‘ residue with that of ‘Asp’ would unequivocally alter the oxygen binding characteristics of the heme pocket. The other mutation was the common variant defined as Hb D-Punjab βCd 121 (GAA→CAA). (figure 2, DNA sequencing shows both variants). The patient had undergone regular venesection to alleviate erythrocytosis. Discussion The co-inheritance of Hb Rancho Mirage with Hb D-Punjab has never been reported previously. Both mutations exist in the β globin gene giving rise to concomitant beta chain variants. Carriers of Hb Rancho Mirage are anticipated with mild anemia. However, coexistence of both abnormal hemoglobins showed functional defects manifested as polycythemia, likely due to the disruption of the 2,3 DPG binding site which is actively involved in the allosteric equilibrium during oxygen transport. The current case depicts the first occurrence of a double heterozygosity for two beta chain variants affecting exon 3 of the β globin gene with a relatively mild to moderate phenotype. Family studies are recommended to understand further the structure and function relationship of this rare hemoglobin variant in association with a more common Hb D- Punjab. Disclosures: No relevant conflicts of interest to declare.

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Hemoglobin Ville-Marie: A New β-Globin Variant (del480C) Causing Oxydative Hemolysis.

Blood ◽

10.1182/blood.v106.11.3783.3783 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3783-3783

Author(s):

Celine Devaux ◽

Pierre Chagnon ◽

Claude Belisle ◽

Denis Soulieres ◽

Lambert Busque

Keyword(s):

Hemolytic Anemia ◽

Genomic Sequence ◽

Stop Codon ◽

Globin Gene ◽

Hemoglobin Variant ◽

History Of ◽

Chronic Hemolysis ◽

Haptoglobin Level ◽

Splice Junctions ◽

Unstable Hemoglobin

Abstract Introduction: A 26 year old male patient was investigated at Maisonneuve-Rosemont Hospital (Montreal, Canada) for chronic hemolysis. The patient was known for many years to have chronic hemolysis of unknow etiology and splenomegaly. He had previously undergone cholecystectomy. There was no family history of hemolytic anemia. The investigation revealed a normal automated CBC but the blood smear showed polychromatophilia, basophilic stippling and a few degmacytes (bite cells). The serum chemistry analysis revealed an increased LDH and bilirubin and a decreased haptoglobin level. The search for Heinz body was positive after exposure to acetyl phenylhydrazine. Unstable hemoglobin was demonstrated using the isopropanol test, but the heat instability test was negative. The dosages of erythrocyte enzymes (G-6PD, pyruvate kinase, gluthation reductase and acethylcholinesterase) were normal. Hb HPLC analysis was done using a VARIANT II, Bio-Rad. Results demonstrated the presence of normal HbA and an abnormal Hb migrating as Hb Hope at a level of 24% of total hemoglobin. Methods and Results: Blood DNA sample was obtained to perform automated sequencing of the β-globin gene (HBB). The HBB gene was sequenced using primers designed to amplify coding sequences of the three exons, splice junctions, introns and the promoter region. Five samples (the patient and four controls) were analysed. Sequence were compared to the cDNA sequence of the HBB gene (NM_000518) and from the genomic sequence of the region (AC104389). In the case of the propositus, a 1 nucleotide deletion was detected at codon 142 (nucleotide 480 of GenBank entry NM_000518) in exon 3, causing a reading frameshift. This deletion (TGCCCTGGCC [C/del] ACAAGTATCA) was never detected before and eliminates the regular stop codon (TAA) at position 147. A new stop codon is therefore produced after the addition of 14 residues, identical to those observed in Hbs Saveme, Trento and TAK, all of which result from a similar reading frameshift secondary to a deletion. However, a threonine residu is present at position 142 in the case of the propositus. Conclusion: Hemoglobin Ville-Marie is an elongated C-terminal hemoglobin variant causing mild compensated hemolytic anemia with some degree of hemoglobin instability. Others analysis are being performed to better characterise this new hemoglobin variant, the results of which will be presented at the meeting.

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Familial Polycythemia Likely Due to Novel Hemoglobin Variant Hemoglobin Hyden

Blood ◽

10.1182/blood-2018-99-119590 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4925-4925

Author(s):

Sandhya Kolagatla ◽

Nagabhishek Moka ◽

Samuel D. Bailey

Keyword(s):

Social History ◽

Conflicts Of Interest ◽

Globin Gene ◽

Laboratory Data ◽

Peripheral Blood Smear ◽

Beta Globin ◽

Hemoglobin Variant ◽

Molecular Alterations ◽

Globin Chains ◽

Normal Limits

Abstract Adult hemoglobin (HbA) is made up of two pairs of globin chains. Some rare mutations of the globin chains can result in high affinity towards hemoglobin molecule thus changing the equilibrium of normal oxygen loading in lungs and the delivery of same to the tissues. Because of change in the affinity to the oxygen these mutations can result in erythrocytosis (polycythemia). Here we discuss a case of Familial Polycythemia likely due to novel hemoglobin variant. 42-year Caucasian male presents to the clinic with high hemoglobin for several years but otherwise denies any symptoms of headache, vision changes, chest pain. He had history of multiple phlebotomies. His past medical history is significant for Polycythemia, PICC line associated clot, Type2 Diabetes, Hypertension, Epidural abscess. Social history is significant for smokeless tobacco but otherwise non-smoker, non-alcoholic, not an IVDA and doesn't use testosterone. Family history is significant for his sister, her two 14year old daughters and multiple other family members with elevated hemoglobin and undergoes phlebotomies, maternal grandfather died of cancer in his 30s. Physical examination is only significant for BMI of 32.46 otherwise no skin discoloration or cyanosis. Laboratory data WBC 9.4 with normal differential, hemoglobin 18.2, hematocrit 55.4, platelet count 180,000, peripheral blood smear was within normal limits, negative for JAK-2, normal erythropoietin, negative for hemochromatosis, Oxygen dissociation p50 of 19 which is low indicating left shifted dissociation curve, hemoglobin electrophoresis HbA: 61.2%, HbA2: 3%, HbF: 0%, Beta variant: 35.8%. In order to further characterize beta variant Bidirectional sequence analysis for Molecular alterations was performed and the following alterations were detected Gene: HBB, DNA change: Codon 39, heterozygous CAG>CCG Protein change: P.G1n39Pro. [glutamine (Q) to proline (P)]. HGVS: c.119A>C, p.Q40P, Classification: Likely deleterious variant. (GenBank accession number NM_000518.4). This is a previously unreported beta chain hemoglobin variant present. This hemoglobin variant is named as Hemoglobin Hyden based on the place where this is found in Hyden, Kentucky. There have been four variants reported at codon 40 of the beta globin gene, which is an external contact site between beta globin and alpha-2 globin. One variant, Hb vassa, is associated with mild hemolytic anemia. The three other variants (Hb Alabama, Hb Tianshui and Hb San Bruno) are not associated with clinical or hematological abnormalities. In our opinion p.Q40P is likely a cause of erythrocytosis as the same mutation is found in atleast three other patients with polycythemia who are unrelated but all of them presenting from same county. In order to further establish the causality it may be beneficial to test first degree relatives in this family in order to determine whether the p.Q40P alteration tracks with disease and is not present in unaffected individuals. Hemoglobin threshold for phlebotomy to lower the risk of thrombosis and cardiovascular events is yet to be defined. Disclosures No relevant conflicts of interest to declare.

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DNA Sequence of Hemoglobin Chico.

Blood ◽

10.1182/blood.v106.11.3784.3784 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3784-3784

Author(s):

Aaron M. Fond ◽

Stephen Neville ◽

John Gotchall ◽

Enli Liu ◽

Ching-Nan Ou ◽

...

Keyword(s):

Dna Sequence ◽

Globin Gene ◽

Oxygen Affinity ◽

Blood Gases ◽

Hemoglobin Concentration ◽

Hemoglobin Variant ◽

Arterial Blood ◽

Oxygen Dissociation ◽

Hplc Peak ◽

Abnormal Hemoglobin

Abstract We were asked to investigate a 54 year old male who was undergoing evaluation for sleep apnea. The patient had a low resting O2 saturation at 91%, but no signs of cardiac or pulmonary disease as indicated by a normal echocardiogram, normal spirometry, and normal high resolution CT of the chest. Arterial blood gases revealed a pO2 of 79 mm Hg on room air, but pCO2 was normal. The patient’s hemoglobin concentration was 13.7 g/dL. The possibility of a mutant hemoglobin was raised and HPLC revealed HbA2 = 2.6%, HbA = 51.4% and an unidentified mutant hemoglobin of 46%. Oxygen dissociation testing demonstrated a P50 of 39 mm Hg (normal 24–32) consistent with a low affinity hemoglobin. The isopropanol test showed that the hemoglobin variant was unstable. Trypsin digestion of the abnormal hemoglobin resulted in a smaller than normal HPLC peak for the β9 segment of β-globin, which is composed of amino acids 67 through 82, and the appearance of a new peak at 193 min. This peak did not match any of the known mutant hemoglobins contained in our reference bank. We PCR amplified and sequenced the β-globin gene. The patient was heterozygous for an A to C mutation in the second position of codon 66. This mutation changed the lysine to threonine and revealed that the hemoglobin variant was Hb Chico. This result was consistent with the loss of ~50% of the β9 peak in the trypsin digest due to loss of a cleavage site after amino acid 66, and the appearance of a new peak which was a hybrid segment of β8 and β9. This represents the first DNA sequence of hemoglobin Chico a mildly unstable hemoglobin variant with decreased oxygen affinity.

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A Novel Hemoglobin, Bonn, Causes Falsely Decreased Oxygen Saturation Measurements in Pulse Oximetry

Clinical Chemistry ◽

10.1373/clinchem.2007.095158 ◽

2008 ◽

Vol 54 (3) ◽

pp. 594-596 ◽

Cited By ~ 25

Author(s):

Berndt Zur ◽

Andreas Hornung ◽

Johannes Breuer ◽

Ulrike Doll ◽

Christine Bernhardt ◽

...

Keyword(s):

Oxygen Saturation ◽

Pulse Oximetry ◽

Globin Gene ◽

Blood Gas Analysis ◽

Gas Analysis ◽

Low Oxygen ◽

Hemoglobin Variant ◽

Arterial Blood Gas ◽

Arterial Blood ◽

Arterial Blood Gas Analysis

Abstract Background: A 4-year-old boy and his father exhibited low oxygen saturation measured transcutaneously by pulse oximetry, a finding that could not be confirmed by arterial blood gas analysis. Both patients exhibited slight hemolysis in their blood, and the boy had a microcytic anemia. There was no evidence of hypoxemia or methemoglobinemia. Despite the normal results from the arterial blood gas analysis, a right-to-left-shunt was assumed in the boy until a cardiology examination excluded this diagnosis. Sleep apnea syndrome was suspected in the father and treated with nocturnal positive pressure respiration based on the low oxygen saturation values obtained with pulse oximetry. Only after consultation with our laboratory was a hemoglobin variant suspected and investigated. Methods: We performed hemoglobin protein analysis by HPLC, electrophoretic separation, and spectrophotometry and DNA sequence analysis of the α-globin gene. Results: Both HPLC chromatographic separation and alkaline electrophoresis revealed a unique hemoglobin peak. In both patients, α-globin gene sequencing revealed a mutation resulting in a histidine-to–aspartatic acid substitution at position α87. The low oxygen saturation measurement by pulse oximetry was due to hemoglobin Bonn oxyhemoglobin having an absorption peak at 668 nm, near the 660 nm measured by pulse oximeters. Conclusion: Hemoglobin Bonn is a novel hemoglobin variant of the proximal α-globin that results in falsely low oxygen saturation measurements with pulse oximetry.

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