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Adenine base editing can convert sickle hemoglobin (HbS, βΕ6V) to G-Makassar hemoglobin (HbG, βE6A), a naturally occurring variant that is clinically asymptomatic. However, the quality and functionality of purified HbG and of mature HbGG and HbGS red blood cells (RBC) has not been assessed. Here, we develop a mouse model to characterize HbG. Purified HbG appears normal and does not polymerize under hypoxia. The topology of the hemoglobin fold with the βΕ6Α mutation is similar to HbA in the oxy and deoxy states. However, RBC containing HbGS are dehydrated, showing altered function and increased sickling under hypoxia. Blood counts and mitochondrial retention measures place HbGS RBCs as intermediate in severity between HbAS and HbSS, while organ function is comparable to HbAS. HbGG resembles HbAA for most metrics. Our results highlight the importance of functionally assessing the mature red cell environment when evaluating novel gene editing strategies for hematologic disorders., Competing Interests: Competing interests: All authors from Beam Therapeutics disclose a conflict of interest and are shareholders of Beam Therapeutics. The remaining authors declare no competing interest., (© 2025. The Author(s).)

Background: Hb Chapel Hill [Alpha2 74(EF3) Asp > Gly] results from an GAC > GGC substitution at codon 74 of the HBA1 or HBA2 genes. Hb Chapel Hill has not been reported since 1986., Methods: A heterozygous mutation, HBA2: c.224A > G, was identified in the proband, her father and sister. We compared the haematological and clinical data of this family with the data reported in the limited number of individuals., Results: Having excluded iron deficiency, the Hb Chapel Hill was asymptomatic in heterozygous state. The cases presented here characterize cases in new techniques including capillary electrophoresis (CE). Two aberrant peaks were identified by CE, a major peak migrating in the zone 7 that correspond to Hb Chapel Hill (α Chapel Hill 2 β 2 ) and a minor peak migrating in the zone 1 that correspond to Hb Chapel Hill2 (α Chapel Hill 2 δ 2 ). Focusing on the variant expression, the Hb Chapel Hill plus Hb A2 variant were around 18.9-20.6% of total Hb in three members., Conclusion: This data will be useful for providing up-to-date and high quality information on the Hb Chapel Hill.

A 37-year-old Chinese man complained of recurrent dyspnea, with low pulse oximetry (SpO 2 ) and normal partial pressure of oxygen (PaO 2 ) by blood gas analysis. The patient's P 50 was elevated at 47.3 mmHg, while hemoglobin fraction by capillary electrophoresis revealed an abnormal HbD quantified at 43.2%. Further DNA analysis by whole exome sequencing (WES) identified heterozygosity for a mutation in exon 2 at codon 38 ( HBB : c.112T>A, p.Trp38Arg), which decodes to a substitution of the amino acid tryptophan (Try) by arginine (Arg), known as Hb Rothschild. The patient's mother and daughter were also diagnosed to have low SpO 2 readings and his daughter was confirmed to carry Hb Rothschild. This is the first known familial case of Hb Rothschild in China. As Hb Rothschild may be underdiagnosed in Asian descent, better understanding in this area would help avoid unnecessary cardiorespiratory interventions.

We describe a first Dutch case of Hb M Saskatoon (HBB:c.190C > T p.His64Tyr) in a 47-year-old female Dutch patient who presented with cyanosis, hemolysis, and abnormal co-oximetry. A mean corpuscular volume (MCV) of 105 fL caused by reticulocytosis (160 × 10 9 /L) and low red blood cell count (3.6 × 10 12 /L) suggested an increased erythrocyte turnover. An HPLC glyco-globin analysis revealed a decreased HbA1c fraction of 12.3 mmol/mmol, HbA0 of 93.3% and an additional unidentified fraction at 1.2 min. DNA sequencing revealed a missense mutation in the HBB gene, (HBB:c.190C > T p.His64Tyr), known as Hb M Saskatoon, a variant which has been previously identified as an unstable hemoglobin variant leading to methemoglobinemia and anemia. In this report, we describe the clinical and remarkable laboratory aspects of our patient with Hb M Saskatoon, and the consequences for treatment and drug use.

Objective: To evaluate the effectiveness of placental volume measured by virtual organ computer-aided analysis (VOCAL) at 12 to 14 weeks of gestation in predicting fetal hemoglobin (Hb) Bart's disease among pregnancies at risk., Methods: This study involves 3-dimensional ultrasound (3D-US) volume datasets derived from pregnancies at risk of fetal Hb Bart's disease at 12 to 14 weeks of pregnancy. VOCAL technique was used to measure and calculate placental volume by the authors, who did not know the fetal diagnosis. Placental thickness was also measured. The diagnostic values of placental volume and placental thickness in prediction of fetal Hb Bart's disease were calculated., Results: Sixty-five volume datasets, including 22 datasets of the affected fetuses and 43 unaffected fetuses, were included. The mean placental volume (±SD) of the affected cases was significantly higher than that of the unaffected ones, 85.35 ± 20.84 cm 3 vs 52.24 ± 19.01 cm 3  (Student's t test, P < .001). In predicting Hb Bart's disease, placental volume and placental thickness had sensitivities of 77.3% and 72.7% respectively as well as specificities of 88.37% and 76.7% respectively., Conclusion: Of fetuses at risk of Hb Bart's disease, 3D-US VOCAL placental volume may be useful in early detection of affected fetuses. Its effectiveness is superior to that of conventional placental thickness measurement., (© 2021 Wiley Periodicals LLC.)

Objectives: With improved access to intrauterine transfusion (IUT), more fetuses with haemoglobin Bart's hydrops fetalis (HBHF; homozygous α 0 -thalassaemia) will survive., Design: To evaluate the long-term outcome of affected fetuses with and without IUT in Ontario, Canada, we retrospectively collected data on IUTs and pregnancy outcomes in all cases of HBHF, from 1989 to 2014. Clinical outcome and neurocognitive profiles of long-term survivors were also collected and compared with data from 24 patients with transfusion-dependent β-thalassaemia (TDT-β)., Results: Of the 99 affected pregnancies (93 prenatally diagnosed), 68 resulted in miscarriage or elective termination of pregnancy. Twelve mothers (12%) continued their pregnancies without IUT, and none of those newborns survived the first week of life. All 13 fetuses that received IUT(s) were live-born, but 3 died due to severe hydrops at birth and 1 died due to infection. The remaining nine survivors, in comparison with TDT-β patients, had earlier iron overload requiring iron chelation therapy. Endocrinopathies and short stature were more frequent in these patients. Neurocognitive outcome was not significantly affected in five patients who were assessed, and none were diagnosed with intellectual impairment. In three patients, MRI studies demonstrated brain white matter changes in keeping with 'silent' ischaemic infarcts., Conclusions: In patients with HBHF, IUT is associated with improved survival. While acceptable neurocognitive outcome can be expected, these patients have more clinical complications compared with their TDT-β counterparts. The clinical and neurocognitive outcomes of HBHF should be discussed in detail when counselling and offering IUT for patients., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Severe fetal anemias can cause high output cardiac failure. Mitochondria are key regulators of cardiac function. However, the effects of an early phase of fetal anemia on the fetal heart and cardiac mitochondrial function are not known., Objective: The aim of this study is to compare mitochondrial function and cardiac biochemical alterations in the fetal cardiac tissue between anemic and non-anemic fetuses., Materials and Methods: A cross-sectional study was conducted in Fetuses affected by Hb Bart's disease (n=18) and non-anemic fetuses (n=10) at 17-20 weeks. Echocardiograms had been carried out in all cases to assess prenatal cardiac function. Cardiac tissues were collected after pregnancy termination for the determination of cardiac iron accumulation, mitochondrial function, including mitochondrial ROS production, mitochondrial depolarization and mitochondrial swelling, mitochondrial dynamics, inflammation, and apoptosis., Results: Prenatal cardiac function evaluated by ultrasound was comparable between the Hb Bart's and non-anemic groups. In Bart's group, the levels of cardiac mitochondrial depolarization and swelling, and the TNF-α level were significantly higher, compared to the non-anemic group. On the contrary, anti-inflammatory (IL-10) levels were significantly lower in the Hb Bart's group. Additionally, active caspase-3 and Bcl-2 expression were also significantly higher (P= 0.001, P=0.035) in Bart's group. The mitochondrial fission protein expression, including p-DRP1/total DRP1, was significantly higher in Bart's group. However, there was no difference in cardiac iron accumulation levels between these two groups., Conclusion: Despite equivalent prenatal cardiac function and comparable cardiac iron accumulation in the Bart's and non-anemic groups, fetal anemia is significantly associated with cardiac mitochondrial dysfunction, increased mitochondrial fission, and increased inflammation and apoptosis. These findings indicate that an early phase of fetal anemia without cardiac iron overload can lead to cardiac mitochondrial dysfunction in fetuses with Hb Bart's., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

We identified two unstable variants in the third exon of α-globin genes: Hb Bernalda/Groene Hart (HBA1:c.358C>T), and Hb Caserta (HBA2:c.79G>A) in cis to Hb Sun Prairie (HBA2:c.391G>C), also named Hb Southern Italy. These mutations occurred in the H helix of the α-globin that is involved in heme contacting, specific recognition of α-hemoglobin-stabilizing protein (AHSP), and α 1 β 1 interactions. The carriers showed α-thalassemia phenotype, but one also jaundice and cholelithiasis. Molecular identification of clusters of families in Southern Italy encouraged molecular characterization of mRNA, globin chain analyses, molecular modeling studies, and comparison with globin variants to understand the mechanisms causing the α-thalassemia phenotype. A normal amount of Hb Bernalda/Groene Hart mRNA were found, and molecular modeling highlighted additional H bonds with AHSP. For Hb Southern Italy, showing an unexpected α/β biosynthetic ratio typical of the β-thalassemia type, two different molecular mechanisms were shown: Reduction of the variant mRNA, likely due to the No-Go Decay for the presence of unused triplet ACG at cod 26, and protein instability due to the impairment of AHSP interaction. The UDP glucuronosyltransferase 1A (UGT1A1) genotyping was conclusive in the case of jaundice and cholelithiasis. Multiple approaches are needed to properly identify the mechanisms leading to unstable variants and the effect of a mutation.

A continuous supply of oxygen is essential for the survival of multicellular organisms. The understanding of how this supply is regulated in the microvasculature has evolved from viewing erythrocytes (red blood cells [RBCs]) as passive carriers of oxygen to recognizing the complex interplay between Hb (hemoglobin) and oxygen, carbon dioxide, and nitric oxide-the three-gas respiratory cycle-that insures adequate oxygen and nutrient delivery to meet local metabolic demand. In this context, it is blood flow and not blood oxygen content that is the main driver of tissue oxygenation by RBCs. Herein, we review the lines of experimentation that led to this understanding of RBC function; from the foundational understanding of allosteric regulation of oxygen binding in Hb in the stereochemical model of Perutz, to blood flow autoregulation (hypoxic vasodilation governing oxygen delivery) observed by Guyton, to current understanding that centers on S-nitrosylation of Hb (ie, S-nitrosohemoglobin; SNO-Hb) as a purveyor of oxygen-dependent vasodilatory activity. Notably, hypoxic vasodilation is recapitulated by native S-nitrosothiol (SNO)-replete RBCs and by SNO-Hb itself, whereby SNO is released from Hb and RBCs during deoxygenation, in proportion to the degree of Hb deoxygenation, to regulate vessels directly. In addition, we discuss how dysregulation of this system through genetic mutation in Hb or through disease is a common factor in oxygenation pathologies resulting from microcirculatory impairment, including sickle cell disease, ischemic heart disease, and heart failure. We then conclude by identifying potential therapeutic interventions to correct deficits in RBC-mediated vasodilation to improve oxygen delivery-steps toward effective microvasculature-targeted therapies. To the extent that diseases of the heart, lungs, and blood are associated with impaired tissue oxygenation, the development of new therapies based on the three-gas respiratory system have the potential to improve the well-being of millions of patients.

We report the case of a 61-year-old Canadian male of Maltese descent investigated for unexplained polycythemia. Decreased p50 suggested the presence of a high oxygen affinity hemoglobin (Hb) variant. Molecular genetic testing demonstrated that he carries a novel missense mutation ( HBB : c.258T>G), resulting in a Phe→Leu substitution at position 85 of the β chain. The novel Hb variant has been designated Hb Kennisis in recognition of where the proband resides. Two other missense mutations have been reported at this position [Hb Bryn Mawr or Hb Buenos Aires, β85(F1)Phe→Ser ( HBB : c.257T>C); Hb Grantham, β85(F1)Phe→Cys; ( HBB : c.257T>G)], both of which have increased oxygen affinity.

Hb Dompierre [β29(B11)Gly→Arg, HBB : c.88G>C] is a rare β-globin gene variant that was previously described in the heterozygous state in a 24-year-old female patient. It is defined in the HbVar database as being clinically and biologically asymptomatic. A few years after the first description, we had an opportunity of reassessing the index case because she presented with splenomegaly and clinical and biological manifestations of hemolysis. After ruling out the most common causes of hemolysis, further analyses on the variant hemoglobin (Hb) using brilliant cresyl blue staining, indicated that it showed mild instability, which may explain the clinical and biological manifestations. A structural bioinformatic analysis on the Hb variant suggested that the amino acid replacement may be deleterious to the integrity of the Hb. This report confirms the importance of completely characterizing all new Hb variants in order to guide the patients' clinical management and follow-up, as well as to provide the probands and their family members with appropriate genetic counseling.

Introduction: In sickle cell disease (SCD) patients, among the predictors of survival, HbF levels play a significant role in lowering the morbidity and mortality. Coinheritance of δβ thalassemia and hereditary persistence of fetal hemoglobin (HPFH) may contribute to variable HbF levels in SCD patients, thus influencing their clinicopathological profile. Such cases are sparsely documented in the literature and thus, we screened the presence of δβ thalassemia and HPFH in 126 cases of SCD with high HbF., Material and Methods: A total 126 SCD individuals with raised HbF levels were the study subject. Capillary zone electrophoresis (CZE) was done for the quantitative assessment of hemoglobin variants. HbSC, HbSD, HbAS and HbSE cases were excluded. Asian Indian Gγ(Aγδβ)0-thal, δβ0-thal (Sicilian, 13.4 kb), (Chinese, 100 kb), HPFH-1 (Black, 106 kb), HPFH-2 (Ghanaian, 105 kb), HPFH-3 (Indian, 48.5 kb) were done by GAP-PCR., Results: Out of 126, 78 cases (62%) were homozygous for SCD. The remaining 48 cases suspected to be heterozygous were furthered screened and 6/48 cases (12.5%) were found to be compound heterozygous. Out of these 6 cases,4(66.66%) had HbS/ δβ- Gγ(Aγδβ)0 and 2(33%) had HbS/HPFH compound heterozygous condition. None of the patients had δβ0-thal (Sicilian, 13.4 kb), (Chinese, 100 kb), HPFH-1 (Black, 106 kb), HPFH-2 (Ghanaian, 105 kb)., Conclusion: This study highlights the importance of understanding the complex patho-physiology of compound heterozygous cases of HbS/HPFH and HbS/δβ thalassemia, as these infrequent conditions lead to change in phenotype and clinical severity of the disease. Insight into more such cases will open the window to better analyze the disease pathogenesis in these rare compound heterozygous conditions, as this will be beneficial to formulate proper management protocol in these patients.

Haemoglobin (Hb) H-constant spring (CS) alpha thalassaemia (- -/-α CS ) is the most common type of nondeletional Hb H disease in southern China. The CRISPR/Cas9-based gene correction of patient-specific induced pluripotent stem cells (iPSCs) and cell transplantation now represent a therapeutic solution for this genetic disease. We designed primers for the target sites using CRISPR/Cas9 to specifically edit the HBA2 gene with an Hb-CS mutation. After applying a correction-specific PCR assay to purify the corrected clones followed by sequencing to confirm the mutation correction, we verified that the purified clones retained full pluripotency and exhibited a normal karyotype. This strategy may be promising in the future, although it is far from representing a solution for the treatment of HbH-CS thalassemia now.

We report two cases of hemoglobin Sendagi in a Romanian family residing in Spain: a four-year-old boy and his mother, who had been previously diagnosed with another type of congenital hemolytic anemia and had undergone splenectomy in her country during childhood. The unstable hemoglobin variant, hemoglobin Sendagi, is characterized by decreased oxygen affinity caused by replacement of one of the critical amino acid residues, phenylalanine beta 42 (CD1) of the beta-chain, with valine in the heme pocket, resulting in methemoglobin formation. As a result of migratory movements in Europe, new disease-causing hemoglobin variants are emerging in our country. Here, capillary electrophoresis enabled the identification of the variant and a molecular study was used to establish an accurate diagnosis., (Copyright © 2019 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Healthy human blood contains only a trace amount of methemoglobin (Hb M), less than 1%. In Hb M iron is present in the oxidized ferric state (Fe 3+ ) not in the reduced ferrous form (Fe 2+ ) and this reduces the ability of hemoglobin to bind oxygen. The described rare hemoglobin variant Hb M‑Hyde Park (also known as Hb M-Akita) results from the substitution of amino acid tyrosine by histidine at position 93 of the beta-globin chain of hemoglobin. The rare Hb variant Hb M‑Hyde Park (Hb M‑Akita) is mainly inherited autosomal dominant and causes methemoglobinemia. Due to the low frequency of inherited Hb M variants, the diagnosis is challenging. Here, we here report on a family with Hb M‑Hyde Park (Hb M‑Akita) whose members demonstrated Hb M > 10%, but were, asymptomatic except for chronic cyanosis. Due to human mobility and migration other hemogobin variants, such as beta-thalassemia minor have spread to Austria . A genetic combination of two different hemoglobin variants may result in severe anemia. Genetic counseling for patients with hemoglobin variants, including Hb M‑Hyde Park (Hb M‑Akita) and beta-thalassemia minor, is essential.

Covalent wrapping of recombinant human hemoglobin (Cys-β93→Ala) variant rHb(βC93A) by human serum albumin (HSA) yielded the rHb(βC93A)-HSA 3 cluster as an artificial O 2 carrier as a red blood cell substitute. Complexation of inositol hexaphosphate to the central rHb(βC93A) core reduced the O 2 affinity moderately, in much the same way as that of naked hemoglobin. This reduction might be attributable to the inert, small Ala-β93 residue, which cannot be reacted with the bulky maleimide crosslinker., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Hemoglobin (Hb) and iron are prooxidants in nature and sources of free radicals in the biological system of all Hb phenotypes. Recent evidence linked abnormal hemoglobin S and C (HbSC) in sickle cell disease (SCD) to various complications in multiple oxidative processes. However, similar studies in relation to abnormal Hb traits are sparse. Besides, reports on activities of antioxidant enzymes and iron status in SCDs are still contradictory. This study assessed the interplay between lipid peroxidation and antioxidant defense capacity in various Hb variants. We enrolled 193 participants with different Hb phenotypes. They were consecutive patients with sickle cell anemia (HbSS, n = 32) and hemoglobin SC (HbSC) disease (n = 28) regularly followed up in a steady state. Other participants were subjects with abnormal Hb traits (HbAS, n = 50; HbAC, n = 33) and normal controls (HbAA, n = 50). The hematocrit (Hct) level, hemoglobin (Hb) concentration, iron status, and biochemical parameters including malondialdehyde (MDA), total antioxidant status (TAS), superoxide dismutase (SOD), and glutathione peroxidase (GPx) enzymes were investigated simultaneously. The MDA and SOD levels were significantly higher (P < 0.05) in Hb variants in order of HbSS>HbSC>HbAC>HbAS when compared with controls. Conversely, GPx and TAS levels showed significant reductions (P < 0.05). Similarly, Hct, Hb, and iron concentrations showed significant reductions (P < 0.05) sequentially following HbAC > HbAS > HbSC > HbSS compared with controls. The results suggest that both SCDs and the carriers were relatively more vulnerable to systemic oxidative stress against normal phenotype, and may be owing to ineffective antioxidant mechanisms needed for keeping spontaneous generations of free radicals in control without necessarily iron-mediated., (© 2019 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia on behalf of Kaohsiung Medical University.)

Altered oxygen affinity variant hemoglobins (Hbs) are caused by mutations of the globin genes. Changes in Hb oxygen affinity shift the oxygen dissociation curve, and can be identified by abnormal p50 measurements of patient red blood cells. Variants are categorized as either low oxygen affinity (high p50) or high oxygen affinity (low p50). Accurate diagnosis requires recognition of typical clinical and laboratory findings. In this case-based review, we present two patients with altered oxygen affinity variants, illustrating barriers to prompt and accurate diagnosis, and issues in management. We then review pathophysiology, diagnostic tests, clinical features, and management strategies., (© 2019 Wiley Periodicals, Inc.)

The purpose of this case study is to describe the physiological characteristics of a patient with the low-oxygen affinity Titusville hemoglobin variant. A 46-yr-old man with exertional dyspnea was diagnosed with a mediastinal lymphadenopathy of unknown origin and, to obtain definitive diagnosis by biopsy, underwent endobronchial ultrasound-guided transbronchial needle aspirate under sedation and video-assisted thoracoscopy under general anesthesia. High inspired fraction of oxygen ( F I O 2 ) was used to guarantee adequate oxygenation even during the one-lung ventilation needed for thoracoscopy. Following radial and pulmonary arterial catheterization, continuous mixed-venous oxygen saturation ( S V O 2 ), cardiac output, oxygen delivery (DO 2 ), oxygen consumption (V̇o 2 ), and oxygen extraction ratio (ERO 2 ) were measured. Serial blood gas analyses were obtained at different FI O 2 . Anesthesia and surgery were carried out safely. Data obtained during the clinical case were utilized to 1) construct an in vivo Titusville hemoglobin dissociation curve and 2) describe oxygen delivery and consumption of a human with Titusville mutation. Titusville hemoglobin showed relatively high P50 (i.e., 30 vs. normal of 27) and very low cooperativity (Hill coefficient of 1.45 vs. normal 2.27), which was compensated in our patient by increases in cardiac output, rather than by augmenting oxygen extraction.

We report two members of a French family who are carriers of a rare hemoglobin (Hb) variant leading to erythrocytosis: Hb Saint Nazaire [β103(G5)Phe→Ile; HBB : c.310T>A]. The proband is a 38-year-old woman referred to our institution for a moderate but persistent polycythemia without any clinical consequence. As her mother had a similar blood count, a diagnosis of a Hb variant with high oxygen affinity was proposed. The variant was difficult to detect by capillary electrophoresis (CE) and not distinguishable by high performance liquid chromatography (HPLC) and isoelectric focusing. Finally, a heterozygous mutation on the HBB gene corresponding to Hb Saint Nazaire was identified. This case report illustrates that this rare cause of erythrocytosis can be easily under or misdiagnosed unless several Hb separation techniques are used.

The α-thalassemia trait, associated with deletions removing both α-globin genes from 1 chromosome (genotype ζ αα/ζ--), is common throughout Southeast Asia. Consequently, many pregnancies in couples of Southeast Asian origin carry a 1 in 4 risk of producing a fetus inheriting no functional α-globin genes (ζ--/ζ--), leading to hemoglobin (Hb) Bart's hydrops fetalis syndrome (BHFS). Expression of the embryonic α-globin genes (ζ-globin) is normally limited to the early stages of primitive erythropoiesis, and so when the ζ-globin genes are silenced, at ∼6 weeks of gestation, there should be no α-like globin chains to pair with the fetal γ-globin chains of Hb, which consequently form nonfunctional tetramers (γ 4 ) known as Hb Bart's. When deletions leave the ζ-globin gene intact, a low level of ζ-globin gene expression continues in definitive erythroid cells, producing small amounts of Hb Portland (ζ 2 γ 2 ), a functional form of Hb that allows the fetus to survive up to the second or third trimester. Untreated, all affected individuals die at these stages of development. Prevention is therefore of paramount importance. With improvements in early diagnosis, intrauterine transfusion, and advanced perinatal care, there are now a small number of individuals with BHFS who have survived, with variable outcomes. A deeper understanding of the mechanism underlying the switch from ζ- to α-globin expression could enable persistence or reactivation of embryonic globin synthesis in definitive cells, thereby providing new therapeutic options for such patients., Competing Interests: Conflict-of-interest disclosure: The authors declare no competing financial interests., (© 2018 by The American Society of Hematology. All rights reserved.)

A new detection system for the endogenous gaseous transmitter and environmental pollutant hydrogen sulfide is presented. It is based on the modulation of the fluorescence spectrum of a coumarin dye by the absorption spectrum of the recombinant hemoglobin I from clam Lucina pectinata upon coordination of the analyte. While we establish that the reported affinity of rHbI for H 2 S has been overestimated, the association of the protein with an appropriate fluorophore allows fast, easy, and reversible detection and quantification of hydrogen sulfide in buffer as well as biological fluids such as human plasma, with a quantification limit around 200 nM at pH 7.4.

Human hemoglobin (Hb) Coimbra (βAsp99Glu) is one of the seven βAsp99 Hb variants described to date. All βAsp99 substitutions result in increased affinity for O 2 and decreased heme-heme cooperativity and their carriers are clinically characterized by erythrocytocis, caused by tissue hypoxia. Since βAsp99 plays an important role in the allosteric α1β2 interface and the mutation in Hb Coimbra only represents the insertion of a CH 2 group in this interface, the present study of Hb Coimbra is important for a better understanding of the global impact of small modifications in this allosteric interface. We carried out functional, kinetic and dynamic characterization of this hemoglobin, focusing on the interpretation of these results in the context of a growth of the position 99 side chain length in the α1β2 interface. Oxygen affinity was evaluated by measuring p50 values in distinct pHs (Bohr effect), and the heme-heme cooperativity was analyzed by determining the Hill coefficient (n), in addition to the effect of the allosteric effectors inositol hexaphosphate (IHP) and 2,3-bisphosphoglyceric acid (2,3-BPG). Computer simulations revealed a stabilization of the R state in the Coimbra variant with respect to the wild type, and consistently, the T-to-R quaternary transition was observed on the nanosecond time scale of classical molecular dynamics simulations., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Following a previous detailed investigation of the β subunit of α 2 β 2 human adult hemoglobin (Hb A), this study focuses on the α subunit by using three natural valency hybrid α(Fe 2+ -deoxy/O 2 )β(Fe 3+ ) hemoglobin M (Hb M) in which O 2 cannot bind to the β subunit: Hb M Hyde Park (β92His → Tyr), Hb M Saskatoon (β63His → Tyr), and Hb M Milwaukee (β67Val → Glu). In contrast with the β subunit that exhibited a clear correlation between O 2 affinity and Fe 2+ -His stretching frequencies, the Fe 2+ -His stretching mode of the α subunit gave two Raman bands only in the T quaternary structure. This means the presence of two tertiary structures in α subunits of the α 2 β 2 tetramer with T structure, and the two structures seemed to be nondynamical as judged from terahertz absorption spectra in the 5-30 cm -1 region of Hb M Milwaukee, α(Fe 2+ -deoxy)β(Fe 3+ ). This kind of heterogeneity of α subunits was noticed in the reported spectra of a metal hybrid Hb A like α(Fe 2+ -deoxy)β(Co 2+ ) and, therefore, seems to be universal among α subunits of Hb A. Unexpectedly, the two Fe-His frequencies were hardly changed with a large alteration of O 2 affinity by pH change, suggesting no correlation of frequency with O 2 affinity for the α subunit. Instead, a new Fe 2+ -His band corresponding to the R quaternary structure appeared at a higher frequency and was intensified as the O 2 affinity increased. The high-frequency counterpart was also observed for a partially O 2 -bound form, α(Fe 2+ -deoxy)α(Fe 2+ -O 2 )β(Fe 3+ )β(Fe 3+ ), of the present Hb M, consistent with our previous finding that binding of O 2 to one α subunit of T structure α 2 β 2 tetramer changes the other α subunit to the R structure.

A 25-year-old Caucasian man with a history of spherocytosis, splenectomy, recurrent blood transfusion, and no cardiopulmonary disease presented for an emergent laparoscopic cholecystectomy with a baseline pulse oximetric saturation (SpO2) of 88% while breathing room air. The SpO2 increased to only 89% during preoxygenation with an FIO2 1.0. Multiple arterial blood samples revealed SaO2 as high as 100% with PaO2 averaging 390 mm Hg. He was subsequently diagnosed with a dyshemoglobin, hemoglobin Köln. The simultaneous presentation of a stable patient from a cardiopulmonary perspective with normal arterial oxygen tension and saturation in the blood gas analyses despite a low SpO2 measurement outlines the importance of integrating the history of present illness and both the importance and the limitation of the pulse oximetry.

Many globins convert • NO to innocuous NO 3 - through their nitric oxide dioxygenase (NOD) activity. Mycobacterium tuberculosis fights the oxidative and nitrosative stress imposed by its host (the toxic effects of O 2 •- and • NO species and their OONO - and • NO 2 derivatives) through the action of truncated hemoglobin N (trHbN), which catalyzes the NOD reaction with one of the highest rates among globins. The general NOD mechanism comprises the following steps: binding of O 2 to the heme, diffusion of • NO into the heme pocket and formation of peroxynitrite (OONO - ), isomerization of OONO - , and release of NO 3 - . Using quantum mechanics/molecular mechanics free-energy calculations, we show that the NOD reaction in trHbN follows a mechanism in which heme-bound OONO - undergoes homolytic cleavage to give Fe IV ═O 2 - and the • NO 2 radical but that these potentially harmful intermediates are short-lived and caged by the heme pocket residues. In particular, the simulations show that Tyr33(B10) side chain is shielded from Fe IV ═O 2 - and • NO 2 (and protected from irreversible oxidation and nitration) by forming stable hydrogen bonds with Gln58(E11) side chain and Leu54(E7) backbone. Aromatic residues Phe46(CD1), Phe32(B9), and Tyr33(B10) promote NO 3 - dissociation via C-H···O bonding and provide stabilizing interactions for the anion along its egress route.

We report a case of an extremely rare hemoglobin (Hb) variant-Hb Broomhill, which has been only reported once in the literature. Hemoglobin fractions were determined by capillary electrophoresis (Sebia Capillarys 2 Flex piercing) and high performance liquid chromatography (HPLC) (Bio-Rad Variant™ II Hemoglobin Testing System), respectively. Complete blood count and DNA sequencing were also performed. The capillary electrophoregram revealed a tiny shoulder peak before the HbA peak and a subtle abnormal HbA 2 peak (slightly wider and lower), even though the percentage of each hemoglobin fraction was within the reference range (HbA, 97.4%; HbA 2 , 2.6%). On HPLC, not only the percentage but also the peak shape of each hemoglobin fraction was normal (HbA 88.2%, HbA 2 2.5%, HbF 0.6%). Eventually, sequencing analysis of α genes confirmed a missense mutation (CCC>GCC at codon 114 in alpha1 gene) which caused Hb Broomhill variant. Our report suggest that capillary electrophoresis may be an accurate tool for screening and diagnosis of Hb disorders., (© 2017 by the Association of Clinical Scientists, Inc.)

Objective: To date in Italy, there is paucity on data about the prevalence, clinical and haematological features of patients carrying the haemoglobin (Hb) Lepore variant in homozygous or in association with other haemoglobinopathies., Methods: Here we report the results of a retrospective analysis on 33 patients from Campania, a region of Southern Italy, historically followed at 'UOSD Malattie Rare del Globulo Rosso' of Cardarelli hospital, Naples, Italy., Results: We described 33 patients carrying the Hb Lepore variant: 21 compound heterozygotes with a common thalassaemia allele, six patients with homozygous state for Hb Lepore, five patients with Hb Lepore/Hb S and one patient with Hb Lepore/Hb Neapolis were identified. All individuals carried haplotype I or V., Discussion: These thalassaemic patients showed different phenotypes ranging from severe disease with early blood transfusion dependency to moderate form of thalassaemia intermedia. In most cases, thalassaemia mutation type determined the severity of the disease., Conclusion: A great variability of clinical phenotype among the same genotypes was also observed suggesting the presence of unknown genetic modifiers acting in combination with Hb Lepore.

Previous molecular analyses of α-thalassemia (α-thal) in the Republic of Macedonia have identified the following genetic defects: -α 3.7 (rightward), -(α) 20.5 and - - MED I deletions and Hb Icaria [α142, Term→Lys (α2), HBA2: c.427T>A] and polyadenylation signal (polyA) [AATAAA>AATGAA (α2), HBA2: c.*92A>G] point mutations. Here, we report two unrelated patients from the Romani population in the Republic of Macedonia, homozygotes for the α2-globin gene variant Hb Agrinio [α29(B10)Leu→Pro; HBA2: c.89T>C]. To date, Hb Agrinio has been described only in individuals of Greek, Cypriot and Spanish origin. Both of our patients had early presentation of the disease (3.5 years and 2 months, respectively) with frequent blood transfusions from early infancy. They have a severe intermediate phenotype of thalassemia (Hb H disease) with hemoglobin (Hb) levels of 7.8 and 7.7 g/dL, respectively. Although the HBA2: c.89T>C mutation results in an α + allele, the severe phenotype of the homozygotes is due to the production of hyperunstable α chains that undergo post translational precipitation. This leads to a greater degree of red cell damage and hemolytic anemia. The detection of Hb Agrinio in two unrelated families of Romani ethnic origin, may suggest it is a founder mutation in this population living in the Republic of Macedonia. Considering the severity of the clinical presentation of the homozygotes or compound heterozygotes for this rare Hb variant, a targeted molecular screening for Hb Agrinio mutation carriers should be considered in all patients of Romani ethnic origin with manifested microcytosis.

We report a novel HBB: c.114G>C mutation in a Chinese family. This mutation resulted in a β37(C3)Trp→Cys amino acid substitution and was synonymous with Hb Kent, a hemoglobin (Hb) variant that was reported exclusively in patients of European descent. Though Hb Kent has a normal oxygen affinity and molecular stability, it has a characteristic dual variant appearance on cellulose acetate electrophoresis (CAE) and high performance liquid chromatography (HPLC) caused by the posttranslational modification of cysteine. We also report the phenotypic expression of this variant when coinherited with the Southeast Asian (- - SEA ) double α-globin gene deletion.

Background: In most routine laboratories in Spain, the commonly used method for evaluating HbA1c is ion-exchange high performance liquid chromatography (HPLC). The presence of a variant of Hb may interfere with the quantification of HbA1c., Aims: Here, we report a novel haemoglobin variant, named Hb Moncloa, which was found during a routine health check at the Hospital Clínico San Carlos in Moncloa (Madrid), Spain., Methods and Results: Molecular characterization of β gene identified a novel transversion mutation [β80(EF4)Asn>Ser; HBB:c.242A>G]., Conclusions: When there is no correlation between clinical, glycemic status and glycated haemoglobin of the patient, the chromatogram of HbA1c should be carefully checked to detect the possible presence of variants that cause interference in their measurement., (Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Congenital erythrocytosis (CE) is a rare and heterogeneous disease. The high oxygen affinity hemoglobin (Hb) variants are the most common cause of CE. Herein, we report a Korean patient with isolated erythrocytosis. A 25-year-old man was referred to our hospital for evaluation of high Hb level (Hb 20.4 g/dL, hematocrit 58%, reticulocyte count 2.90%, white blood cell count 6.83×10⁹/L, and platelet count 195×10⁹/L). Bone marrow biopsy revealed normocellular marrow without myeloproliferative features. JAK2 (V617F, exon 12), CALR (exon 9), and MPL W515K/L mutations were not detected. P₅₀ (partial pressure at which Hb is half saturated with oxygen), which is an indicator of left-shift of oxygen dissociation curve (high oxygen affinity state), was 14.3 mm Hg (reference value 22.6-29.4 mm Hg). He was suspected to have CE. Mutation analysis of the HBB gene revealed the known Hb variant, Hb Heathrow [β103(G5)Phe→Leu]. This is the first report of Hb Heathrow in Asian., Competing Interests: The authors have no financial conflicts of interest., (© Copyright: Yonsei University College of Medicine 2017.)

Introduction: Haemoglobin Bart's (Hb Bart's) level is associated with α-thalassaemia traits in neonates, enabling early diagnosis of α-thalassaemia. The study aimed to detect and quantify the Hb Bart's using Cord Blood (CB) and CE Neonat Fast Hb (NF) progammes on fresh and dried blood spot (DBS) specimen respectively by capillary electrophoresis (CE)., Methods: Capillarys Hemoglobin (E) Kit (for CB) and Capillarys Neonat Hb Kit (for NF) were used to detect and quantify Hb Bart's by CE in fresh cord blood and dried blood spot (DBS) specimens respectively. High performance liquid chromatography (HPLC) using the β-Thal Short Programme was also performed concurrently with CE analysis. Confirmation was obtained by multiplex ARMS Gap PCR., Results: This study was performed on 600 neonates. 32/600 (5.3%) samples showed presence of Hb Bart's peak using the NF programme while 33/600 (5.5%) were positive with CB programme and HPLC methods. The range of Hb Bart's using NF programme and CB programme were (0.5-4.1%) and (0.5-7.1%), respectively. Molecular analysis confirmed all positive samples possessed α-thalassaemia genetic mutations, with 23/33 cases being αα/-- SEA , four -α 3.7 /-α 3.7 , two αα/-α 3.7 and three αα/αα CS . Fifty Hb Bart's negative samples were randomly tested for α-genotypes, three were also found to be positive for α-globin gene mutations. Thus, resulting in sensitivity of 91.7% and 88.9% and specificity of 100% for the Capillarys Cord Blood programme and Capillarys Neonat Fast programme respectively., Conclusion: Both CE programmes using fresh or dried cord blood were useful as a screening tool for α-thalassaemia in newborns. All methods show the same specificity (100%) with variable, but acceptable sensitivities in the detection of Hb Bart.

In heme proteins, the efficient transport of ligands such as NO or O2 to the binding site is achieved via ligand migration networks. A quantitative assessment of ligand diffusion in these networks is thus essential for a better understanding of the function of these proteins. For this, Xe migration in truncated hemoglobin N (trHbN) of Mycobacterium Tuberculosis was studied using molecular dynamics simulations. Transitions between pockets of the migration network and intra-pocket relaxation occur on similar time scales (10 ps and 20 ps), consistent with low free energy barriers (1-2 kcal/mol). Depending on the pocket from where Xe enters a particular transition, the conformation of the side chains lining the transition region differs which highlights the coupling between ligand and protein degrees of freedom. Furthermore, comparison of transition probabilities shows that Xe migration in trHbN is a non-Markovian process. Memory effects arise due to protein rearrangements and coupled dynamics as Xe moves through it.

Sickle cell disease (SCD) is a highly complex genetic blood disorder in which red blood cells (RBC) exhibit heterogeneous morphology changes and decreased deformability. We employ a kinetic model for cell morphological sickling that invokes parameters derived from patient-specific data. This model is used to investigate the dynamics of individual sickle cells in a capillary-like microenvironment in order to address various mechanisms associated with SCD. We show that all RBCs, both hypoxia-unaffected and hypoxia-affected ones, regularly pass through microgates under oxygenated state. However, the hypoxia-affected cells undergo sickling which significantly alters cell dynamics. In particular, the dense and rigid sickle RBCs are obstructed thereby clogging blood flow while the less dense and deformable ones are capable of circumnavigating dead (trapped) cells ahead of them by choosing a serpentine path. Informed by recent experiments involving microfluidics that provide in vitro quantitative information on cell dynamics under transient hypoxia conditions, we have performed detailed computational simulations of alterations to cell behavior in response to morphological changes and membrane stiffening. Our model reveals that SCD exhibits substantial heterogeneity even within a particular density-fractionated subpopulation. These findings provide unique insights into how individual sickle cells move through capillaries under transient hypoxic conditions, and offer novel possibilities for designing effective therapeutic interventions for SCD.

Objectives: Hemoglobin Le Lamentin (α20(B1)His→Gln) is a ubiquitous variant that has been previously described in a small number of isolated patients. We report the incidental observation of Hb Le Lamentin in a large population from the province of Albacete, in southeastern Spain. Our study investigates possible reasons for the elevated number of carriers of this variant and its implications for the management of diabetes in our region., Design & Methods: The subjects are 32 diabetic patients whose hemoglobin displayed an unusual peak while they were being tested for glycated hemoglobin at the laboratory of the University General Hospital of Albacete over a 3-year period. Measurements were made by high performance liquid chromatography using a Variant™ II Turbo Kit-2.0, and subsequently the samples of the 32 patients with anomalous peaks were sent to the Hospital Clínico San Carlos (Madrid, Spain) for molecular characterization of any Hb variants., Results: Molecular studies revealed 31 out of 32 patients heterozygous for Le Lamentin, and in one of them, Hb City of Hope was associated with Hb Le Lamentin. The remaining patient was homozygous for the Le Lamentin mutation. Additionally, most patients were native to the northeastern half of the province of Albacete and were unrelated., Conclusions: Our study describes the largest finding to date of hemoglobin Le Lamentin in a sample of patients. The fact that our region has been perpetually depopulated, with a population that has remained stable in small localities over the centuries, may have favored the survival of the mutation. Since the presence of this variant underestimates the true value of glycated hemoglobin measured by HPLC, it is necessary to systematically review chromatograms., (Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

We describe a case with a low oxygen affinity hemoglobin (Hb) variant who presented with cyanosis in the absence of cardiopulmonary disease. The patient, a 27-year-old pregnant female (P1G2), complained of a productive cough and bluish discoloration of the lips that started 3 days prior to seeking attention. She had no previous episodes and has generally been in good health. A positive family history of cyanosis was obtained in one sibling. Systematic examination, notably the cardiorespiratory system, revealed no abnormalities. The arterial Hb oxygen saturation (SpO 2 ) on pulse oximetry was 81.0% and Hb separation studies revealed an Hb variant identified as Hb Rothschild [β37(C3)Trp→Arg] (HBB: c.[112 T>A or 112 T>C]) by gene sequencing. The amino acid substitution (Trp→Arg) is an important contact point at the α1β2 interface and favors a T-quaternary state of the Hb tetramer. This leads to a low oxygen affinity state, which results in premature release of oxygen and drop in oxygen saturation. In the absence of cardiopulmonary disease, a decreased oxygen saturation reading, with or without cyanosis, should arouse suspicion for a possible dysHb.