Impact of ITPA gene polymorphism for predicting anemia and treatment outcome in HCV infected patients taking Sofosbuvir Ribavirin therapy.

BACKGROUND: Globally, 80 million people are suffering from chronic Hepatitis C virus (HCV) infection. Sofosbuvir ribavirin-based anti-HCV therapy is associated with anemia and other adverse effects. Polymorphisms of Inosine triphosphatase (ITPA) gene may cause functional impairment in the Inosine triphosphate pyrophosphatase enzyme, resulting in enhanced sustained viral response (SVR) and protection from ribavirin-associated anemia in patients on therapy. The study objective was to investigate the effect of Inosine triphosphatase gene polymorphism on SVR achievement, hemoglobin decline and ribavirin dose reduction in patients on therapy. METHODS: This prospective cohort study was of 170 hepatitis C infected patients received 6-month sofosbuvir ribavirin therapy. Patient viral load, reduction in ribavirin amount, liver function test, and complete blood count were noted monthly. Inosine triphosphatase variants rs1127354 and rs7270101 were assessed through the restriction fragment length polymorphism and confirmed using Sanger sequencing. The impact of polymorphism on cumulative reduction of ribavirin, and anti-HCV therapy outcome were studied. RESULTS: A total of 74.3% of patients had ITPA rs1127354 CC genotype, 25.7% were CA and AA 0%. The frequency of ITPA genotype rs7270101-AA was 95%, AC 5%, and CC was 0%. ITPA rs1127354-CA had a notably positive impact on SVR achievement with a zero-relapse rate. ITPA rs1127354-CA genotype was significantly (P ˂0.05) protective against ≥ 2 g/dl Hb reduction from baseline to 1st, 2nd and 6th months of therapy. During treatment, Hb reduction ≥ 10 g/dl was frequently observed in rs1127354-CC genotype and rs7270101-AA genotype patients. Ribavirin dose reduction was significantly (P ˂0.05) high in rs1127354-CC genotype as compared to genotype CA whereas no significant difference was observed in ribavirin dose reduction in rs7270101 AA and non-AA genotype. Patient baseline characteristics such as age, body mass index, rs1127354-CC genotype, and baseline Hb were significantly associated with significant Hb reduction. CONCLUSION: Pretreatment evaluation of ITPA polymorphism can be a diagnostic tool to find out patients at risk of anemia and improve treatment adherence. ITPA genotype rs1127354-CA contributes to improved compliance with ribavirin dose and protects against hemoglobin decline in HCV patients while taking ribavirin-based therapy. However, ITPA rs1127354, rs7270101 polymorphism have no significant impact on SVR achievement.

Evaluating sheep hemoglobins with MD simulations as an animal model for sickle cell disease.

Sickle cell disease (SCD) affects millions worldwide, yet there are few therapeutic options. To develop effective treatments, preclinical models that recapitulate human physiology and SCD pathophysiology are needed. SCD arises from a single Glu-to-Val substitution at position 6 in the ß subunit of hemoglobin (Hb), promoting Hb polymerization and subsequent disease. Sheep share important physiological and developmental characteristics with humans, including the same developmental pattern of fetal to adult Hb switching. Herein, we investigated whether introducing the SCD mutation into the sheep ß-globin locus would recapitulate SCD's complex pathophysiology by generating high quality SWISS-MODEL sheep Hb structures and performing MD simulations of normal/sickle human (huHbA/huHbS) and sheep (shHbB/shHbS) Hb, establishing how accurately shHbS mimics huHbS behavior. shHbS, like huHbS, remained stable with low RMSD, while huHbA and shHbB had higher and fluctuating RMSD. shHbB and shHbS also behaved identically to huHbA and huHbS with respect to ß2-Glu6 and ß1-Asp73 (ß1-Asn72 in sheep) solvent interactions. These data demonstrate that introducing the single SCD-causing Glu-to-Val substitution into sheep ß-globin causes alterations consistent with the Hb polymerization that drives RBC sickling, supporting the development of a SCD sheep model to pave the way for alternative cures for this debilitating, globally impactful disease.

Misdiagnosis of ß-Thalassemia Major Due to Chinese Gγ+(Aγδß)0-Thalassemia Combined with ß0-Thalassemia.

δß-thalassemia is a rare type of thalassemia characterized by increased Hb F levels, including mainly Chinese Gγ(Aγδß)0-thalassemia, Yunnanese Gγ(Aγδß)0-thalassemia, Cantonese Gγ(Aγδß)0-thalassemia in China. Due to the low rate of δß-thalassemia carriers, there are few reports of δß-thalassemia combined with ß-thalassemia causing ß-thalassemia major. Herein, we described the combination of Chinese Gγ(Aγδß)0-thalassemia and ß-thalassemia leading to ß-thalassemia major in a Chinese patient. Hemoglobin analysis was performed by capillary electrophoresis (CE). Routine genetic analysis was carried out by gap-polymerase chain reaction (Gap-PCR) and PCR and reverse dot blot (PCR-RDB). Multiple ligation-dependent probe amplification (MLPA) was used to detect the large deletion, and Gap-PCR confirmed the deletion. A CE result showed an elevated Hb F level of 98.7% and 11.7% in the proband and her mother, but the proband was diagnosed with ßCD17M/ßCD17M using routine genetic analysis. However, her father was heterozygous for CD17 in ß-globin, and her mother was detected as SEA heterozygous. The further analysis presented that the proband had actually missed the diagnosis of Chinese Gγ(Aγδß)0-thalassemia by MLPA and PCR-RDB. Finally, the genotype of the proband was corrected from ßCD17M/ßCD17M to ßCD17M/ßGγ(Aγδß)0. This is the first report of Chinese Gγ(Aγδß)0-thalassemia combined with ß-thalassemia resulting in ß-thalassemia major in China. Screening for δß-thalassemia by Hb analysis could be an effective method.

EPAS1-mutated paragangliomas associated with haemoglobin disorders.

We report a large series of 40 patients presenting EPAS1-mutated paraganglioma (PGL) in whom we investigated a cause underlying chronic hypoxia. Four patients suffered from hypoxaemic heart disease. In patients with available haemoglobin electrophoresis results, 59% presented with a haemoglobin disorder, including six with sickle cell disease, five with sickle cell trait and two with heterozygous haemoglobin C disease. Histological and transcriptomic characterization of EPAS1 tumours revealed increased angiogenesis and high similarities with pseudohypoxic PGLs caused by VHL gene mutations. Sickle haemoglobinopathy carriers could thus be at increased risk for developing EPAS1-PGLs, which should be taken into account in their management and surveillance.

Unresolved laboratory issues of the heterozygous state of ß-thalassemia: a literature review.

Although considered a mild clinical condition, many laboratory issues of the carrier state of ß-thalassemia remain unresolved. Accurate laboratory screening of ß-thalassemia traits is crucial for preventing the birth of a ß-thalassemia major child. Identification of carriers in the laboratory is affected by factors that influence red cell indices and HbA2 quantification. Silent mutations and co-inheriting genetic and non-genetic factors affect red cell indices which decreases the effectiveness of the conventional approach. Similarly, the type of ß mutation, co-inheriting genetic and non-genetic factors, and technical aspects, including the analytical method used and variations in the HbA2 cut-off values, affect the HbA2 results, leading to further confusion. However, the combination of mean corpuscular volume, mean corpuscular hemoglobin, and hemoglobin analysis increases the diagnostic accuracy. Diagnostic problems arising from non-genetic factors can be eliminated by carefully screening the patient's clinical history. However, issues due to certain genetic factors, such as Krüppel-like factor 1 gene mutations and α triplication still remain unresolved. Each laboratory should determine the population-specific reference ranges and be wary of machine-related variations of HbA2 levels, the prevalence of silent mutations in the community.

Dynamics of hemoglobins during nodule development, nitrate response, and dark stress in Lotus japonicus.

Legume nodules express multiple leghemoglobins (Lbs) and non-symbiotic hemoglobins (Glbs), but how they are regulated is unclear. Here, we study the regulation of all Lbs and Glbs of Lotus japonicus in different physiologically relevant conditions and mutant backgrounds. We quantified hemoglobin expression, localized reactive oxygen species (ROS) and nitric oxide (NO) in nodules, and deployed mutants deficient in Lbs and in the transcription factors NLP4 (associated with nitrate sensitivity) and NAC094 (associated with senescence). Expression of Lbs and class 2 Glbs was suppressed by nitrate, whereas expression of class 1 and 3 Glbs was positively correlated with external nitrate concentrations. Nitrate-responsive elements were found in the promoters of several hemoglobin genes. Mutant nodules without Lbs showed accumulation of ROS and NO and alterations of antioxidants and senescence markers. NO accumulation occurred by a nitrate-independent pathway, probably due to the virtual disappearance of Glb1-1 and the deficiency of Lbs. We conclude that hemoglobins are regulated in a gene-specific manner during nodule development and in response to nitrate and dark stress. Mutant analyses reveal that nodules lacking Lbs experience nitro-oxidative stress and that there is compensation of expression between Lb1 and Lb2. They also show modulation of hemoglobin expression by NLP4 and NAC094.

Sphaerotilus natans hemoglobins have an NADH oxidation activity and promote the yield of limonene in an engineered E. coli strain.

Bacterial hemoglobins play important roles inside the cell. Phylogenetically, they belong to three different families: the single domain hemoglobin, flavohemoglobin and truncated hemoglobin. Vitreoscilla hemoglobin (VHb) is the first characterized bacterial hemoglobin, and belongs to the single domain hemoglobin family. Heterologous expression of VHb promotes the growth of host cells under microaerobic conditions, and enhances the yield of products during fermentation. Although VHb has been widely applied in the biotechnology field, other bacterial hemoglobins have not demonstrated similar applications. In this study, we identified four bacterial hemoglobins from the microaerobic growing bacterium Sphaerotilus natans, including one flavohemoglobins (FHB) and three truncated hemoglobins (THB1, THB2 and THB3). Absorption spectrum studies validate the existent of the Soret peak and Q-band characteristic to heme and suggest heme groups in FHB and THB1 are hexa- or penta-coordinated, respectively. Our studies demonstrate that FHB and all three truncated hemoglobins have NADH oxidation and radical production activities, which is surprising since truncated hemoglobins do not have a reductase domain that could bind NADH. However, the M. tuberculosis HbN does not show these activities, indicating they are not universal among truncated hemoglobins. Docking studies suggest the nicotinamide ring of NADH may bind to the distal heme pocket of THB1, suggesting the direct electron transfer from NADH to heme might be possible. Our truncated hemoglobins also show peroxidase activities that in THB2 and THB3 could be inhibited by FdR, indicating possible interactions between FdR and truncate hemoglobins. Expression of FHB and THB1 in E. coli could promote cell growth. THB1 also enhances the production of limonene in an engineered E. coli strain, while VHb does not have this effect, which suggests that studies on truncated hemoglobins may lead to the discovery of new and more powerful tools that could have profound impact on biotechnology.

A mass spectrometry assay for detection of endogenous and lentiviral engineered hemoglobin in cultured cells and sickle cell disease mice.

Sickle cell disease (SCD) results from a sequence defect in the ß-globin chain of adult hemoglobin (HbA) leading to expression of sickle hemoglobin (HbS). It is traditionally diagnosed by cellulose-acetate hemoglobin electrophoresis or high-performance liquid chromatography. While clinically useful, these methods have both sensitivity and specificity limitations. We developed a novel mass spectrometry (MS) method for the rapid, sensitive and highly quantitative detection of endogenous human ß-globin and sickle hß-globin, as well as lentiviral-encoded therapeutic hßAS3-globin in cultured cells and small quantities of mouse peripheral blood. The MS methods were used to phenotype homozygous HbA (AA), heterozygous HbA-HbS (AS) and homozygous HbS (SS) Townes SCD mice and detect lentiviral vector-encoded hßAS3-globin in transduced mouse erythroid cell cultures and transduced human CD34+ cells after erythroid differentiation. hßAS3-globin was also detected in peripheral blood 6 weeks post-transplant of transduced Townes SS bone marrow cells into syngeneic Townes SS mice and persisted for over 20 weeks post-transplant. As several genome-editing and gene therapy approaches for severe hemoglobin disorders are currently in clinical trials, this MS method will be useful for patient assessment before treatment and during follow-up.

Inherited disorders of hemoglobin: A review of old and new diagnostic methods.

The genetic regulation of hemoglobin is complex and there are a number of genetic abnormalities that result in clinically important hemoglobin disorders. Here, we review the molecular pathophysiology of hemoglobin disorders and review both old and new methods of diagnosing these disorders. Timely diagnosis of hemoglobinopathies in infants is essential to coordinate optimal life-saving interventions, and accurate identification of carriers of deleterious mutations allows for genetic counseling and informed family planning. The initial laboratory workup of inherited disorders of hemoglobin should include a complete blood count (CBC) and peripheral blood smear, followed by carefully selected tests based on clinical suspicion and available methodology. We discuss the utility and limitations of the various methodologies to fractionate hemoglobin, including cellulose acetate and citrate agar hemoglobin electrophoresis, isoelectric focusing, high-resolution high-performance liquid chromatography, and capillary zone electrophoresis. Recognizing that most of the global burden of hemoglobin disorders exists in low- and middle-income countries, we review the increasingly available array of point-of-care-tests (POCT), which have an increasingly important role in expanding early diagnosis programs to address the global burden of sickle cell disease, including Sickle SCAN, HemoTypeSC, Gazelle Hb Variant, and Smart LifeLC. A comprehensive understanding of the molecular pathophysiology of hemoglobin and the globin genes, as well as a clear understanding of the utility and limitations of currently available diagnostic tests, is essential in reducing global disease burden.

The Spectrum of α-Thalassemia Mutations in Syrian Patients.

α-Thalassemia (α-thal) is a globally prevalent genetic disorder of hemoglobin (Hb) structure where the rate of α-globin chain synthesis is reduced or absent due to the presence of α-globin mutation(s). The aim of this study is to define the spectrum of α-globin gene mutations and evaluate their allele frequency in a group of α-thal carriers. A total of 55 individuals with possible α-thal patients were referred from the thalassemia centers in Syria. They have unexplained hypochromia and microcytosis. All patients were genetically tested for 21 common α-globin gene mutations using reverse hybridization kit. Seven different α-globin gene mutations and 13 different genotypes were detected in 55 patients. The two most frequently encountered mutations were -α3.7 deletion (47.1%) and --MED mutation (21.4%). The most commonly observed genotype was -α3.7/αα (40%), followed by --MED/αα genotype (21.8%). We determined the most common α thalassemia mutations in the Syrian patients. α-Thalassemia mutations with deletions were mostly observed in our study.

Cold-Driven Hemoglobin Evolution in Antarctic Notothenioid Fishes Prior to Hemoglobin Gene Loss in White-Blooded Icefishes.

Expression of multiple hemoglobin isoforms with differing physiochemical properties likely helps species adapt to different environmental and physiological conditions. Antarctic notothenioid fishes inhabit the icy Southern Ocean and display fewer hemoglobin isoforms, each with less affinity for oxygen than temperate relatives. Reduced hemoglobin multiplicity was proposed to result from relaxed selective pressure in the cold, thermally stable, and highly oxygenated Antarctic waters. These conditions also permitted the survival and diversification of white-blooded icefishes, the only vertebrates living without hemoglobin. To understand hemoglobin evolution during adaptation to freezing water, we analyzed hemoglobin genes from 36 notothenioid genome assemblies. Results showed that adaptation to frigid conditions shaped hemoglobin gene evolution by episodic diversifying selection concomitant with cold adaptation and by pervasive evolution in Antarctic notothenioids compared to temperate relatives, likely a continuing adaptation to Antarctic conditions. Analysis of hemoglobin gene expression in adult hematopoietic organs in various temperate and Antarctic species further revealed a switch in hemoglobin gene expression underlying hemoglobin multiplicity reduction in Antarctic fish, leading to a single hemoglobin isoform in adult plunderfishes and dragonfishes, the sister groups to icefishes. The predicted high hemoglobin multiplicity in Antarctic fish embryos based on transcriptomic data, however, raises questions about the molecular bases and physiological implications of diverse hemoglobin isoforms in embryos compared to adults. This analysis supports the hypothesis that the last common icefish ancestor was vulnerable to detrimental mutations affecting the single ancestral expressed alpha- and beta-globin gene pair, potentially predisposing their subsequent loss.

Characterization and Expression Analysis of Genes from Megalobrama amblycephala Encoding Hemoglobins with Extracellular Microbicidal Activity.

Hemoglobin (Hb) usually comprises two α and two ß subunits, forming a tetramer responsible for oxygen transportation and storage. Few studies have elucidated fish hemoglobin immune functions. Megalobrama amblycephala is a freshwater-cultured fish prevalent in China. We identified two M. amblycephala hemoglobin subunits and analyzed their expression patterns and antibacterial activities. The respective full-length cDNA sequences of the M. amblycephala Hb α (MaHbα) and ß (MaHbß) subunits were 588 and 603 bp, encoding 143 and 148 amino acids. MaHbα and MaHbß were highly homologous to hemoglobins from other fish, displaying typical globin-like domains, most heme-binding sites, and tetramer interface regions highly conserved in teleosts. In phylogenetic analyses, the hemoglobin genes from M. amblycephala and other cypriniformes clustered into one branch, and those from other fishes and mammals clustered into other branches, revealing fish hemoglobin conservation. These M. amblycephala Hb subunits exhibit different expression patterns in various tissues and during development. MaHbα is mainly expressed in the blood and brain, while MaHbß gene expression is highest in the muscle. MaHbα expression was detectable and abundant post-fertilization, with levels fluctuating during the developmental stages. MaHbß expression began at 3 dph and gradually increased. Expression of both M. amblycephala Hb subunits was down-regulated in most examined tissues and time points post-Aeromonas hydrophila infection, which might be due to red blood cell (RBC) and hematopoietic organ damage. Synthetic MaHbα and MaHbß peptides showed excellent antimicrobial activities, which could inhibit survival and growth in five aquatic pathogens. Two M. amblycephala hemoglobin subunits were identified, and their expression patterns and antibacterial activities were analyzed, thereby providing a basis for the understanding of evolution and functions of fish hemoglobins.

[Clinical and genotypic analysis of hereditary spherocytosis combined with cholestasis among pediatric patients].

Objective: To understand the clinical and genetic characteristics of hereditary spherocytosis (HS) combined with cholestasis among pediatric patients. Methods: 12 cases of HS children accompanied by cholestasis at Hunan Children's Hospital were selected as the research subjects between January 2013 and December 2022. Clinical data were collected. Whole-exome sequencing was performed by second-generation sequencing. Suspected pathogenic mutation sites were verified by Sanger sequencing. Results: All pediatric patients were admitted to the hospital due to their yellow skin tone. Eight cases (66.67%) had a positive family history. The clinical manifestations were jaundice, splenomegaly (12/12), abdominal pain, anemia (4/12), and hepatomegaly (5/12). All pediatric patients had decreased hemoglobin, an increased reticulocyte ratio, total bilirubin and direct bilirubin, a positive erythrocyte fragility test, and remarkable spherical erythrocytes in their peripheral blood. Seven cases had elevated aminotransferase; four cases had severely elevated aminotransferase and bilirubin; eight cases had biliary calculi; and two cases had a dilated biliary tract. Liver pathological examination showed mild damage to the liver cells (G1S1) in three pediatric cases. Five children had a total of six unreported mutations: SPTB gene c.2431_2450del, c.4974-2A > G, c.2575G > A, and exon 22-35 deletion; ANK1 gene: c.2379-2380delC; and c .6dupC. Children still had abnormal bilirubin levels following treatment. Two pediatric cases underwent splenectomy. Bilirubin and hemoglobin levels returned to normal after surgery. Conclusion: Children with HS may experience cholestasis, and those with poor treatment results may consider undergoing a splenectomy. Six new types of variants have expanded the HS gene mutation spectrum.

An extra-erythrocyte role of haemoglobin body in chondrocyte hypoxia adaption.

Although haemoglobin is a known carrier of oxygen in erythrocytes that functions to transport oxygen over a long range, its physiological roles outside erythrocytes are largely elusive1,2. Here we found that chondrocytes produced massive amounts of haemoglobin to form eosin-positive bodies in their cytoplasm. The haemoglobin body (Hedy) is a membraneless condensate characterized by phase separation. Production of haemoglobin in chondrocytes is controlled by hypoxia and is dependent on KLF1 rather than the HIF1/2α pathway. Deletion of haemoglobin in chondrocytes leads to Hedy loss along with severe hypoxia, enhanced glycolysis and extensive cell death in the centre of cartilaginous tissue, which is attributed to the loss of the Hedy-controlled oxygen supply under hypoxic conditions. These results demonstrate an extra-erythrocyte role of haemoglobin in chondrocytes, and uncover a heretofore unrecognized mechanism in which chondrocytes survive a hypoxic environment through Hedy.

Mutational analysis of hemoglobin genes and functional characterization of detected variants, through in-silico analysis, in Pakistani beta-thalassemia major patients.

Thalassemia is one of the most prevalent genetic disorders worldwide. The present study aimed to explore the mutational spectrum of all hemoglobin (HB) encoding genes and to identify the potentially damaging and pathogenic variants in the beta (ß)-thalassemia major patients and thalassemia minor carriers of Southern Punjab, Pakistan. A total of 49 ß-thalassemia major patients and 49 carrier samples were screened for the identification of HBA1, HBA2, HBB, HBD, HBE1, HBG1 and HBG2 variants by NGS. PCR was performed for the amplification of HB encoding genes and the amplified product of 13 patients and 7 carrier samples were processed for the Sanger sequencing. Various bioinformatics tools and databases were employed to reveal the functional impact and pathogenicity potential of the observed variants. Results depicted a total of 20 variants of HB-related genes by NGS and 5 by Sanger sequencing in thalassemia patients. While 20 variants by NGS and 3 by Sanger were detected in carriers. Few known genetic variants of HB-encoding genes are being reported for the first time in Pakistani thalassemia patients and carriers. However, two novel HBB variants c.375A>C (p.P125P) and c.*61T>G and a novel variant of HBE1 (c.37A>T (p.T13S)) were also documented. Pathogenicity analysis predicted the pathogenic potential of HBB variants (c.47G>A (p.W16*), c.27-28insG (p. S10fs), and c.92+5G>C) for ß thalassemia. The study of functional impact indicated that these HBB variants result in the premature termination of translation leading to the loss of functional ß-globin protein. It is therefore suggested that the pathogenic HBB variants, identified during present study, can be employed for the diagnosis, carrier screening, and planning therapy of thalassemia.

Elevated cardiac hemoglobin expression is associated with a pro-oxidative and inflammatory environment in primary mitral regurgitation.

BACKGROUND: Primary mitral regurgitation (PMR) is associated with oxidative and inflammatory myocardial damage. We reported greater exosome hemoglobin (Hb) in pericardial fluid (PCF) versus plasma, suggesting a cardiac source of Hb. OBJECTIVE: Test the hypothesis that Hb is produced in the PMR heart and is associated with increased inflammation. METHODS AND RESULTS: Hb gene expression for subunits alpha (HBA) and beta (HBB) was assessed in right atria (RA), left atria (LA) and left ventricular (LV) tissue from donor hearts (n = 10) and PMR patient biopsies at surgery (n = 11). PMR patients (n = 22) had PCF and blood collected for macrophage markers, pro-inflammatory cytokines, and matrix metalloproteinases (MMPs). In-situ hybridization for HBA mRNA and immunohistochemistry for Hb-alpha (Hbα) and Hb-beta (Hbß) protein was performed on PMR tissue. RESULTS: HBA and HBB genes are significantly increased (>4-fold) in RA, LA, and LV in PMR vs. normal hearts. In PMR tissue, HBA mRNA is expressed in both LV cardiomyocytes and interstitial cells by in-situ hybridization; however, Hbα and Hbß protein is only expressed in interstitial cells by immunohistochemistry. PCF oxyHb is significantly increased over plasma along with low ratios (<1.0) of haptoglobin:oxyHb and hemopexin:heme supporting a highly oxidative environment. Macrophage chemotactic protein-1, tumor necrosis factor-α, interleukin-6, and MMPs are significantly higher in PCF vs. plasma. CONCLUSION: There is increased Hb production in the PMR heart coupled with the inflammatory state of the heart, suggests a myocardial vulnerability of further Hb delivery and/or production during cardiac surgery that could adversely affect LV functional recovery.

Detecting rare thalassemia in children with anemia using third-generation sequencing.

BACKGROUND: In China, conventional genetic testing methods can only detect common thalassemia variants. Accurate detection of rare thalassemia is crucial for clinical diagnosis, especially for children that need long-term blood transfusion. This study aims to explore the application value of third-generation sequencing (TGS) in the diagnosis of rare thalassemia in children with anemia. METHODS: We enrolled 20 children with anemia, excluding from iron deficiency anemia (IDA). TGS was employed to identify both known and novel thalassemia genotypes, while sanger sequencing was used to confirm the novel mutation detected. RESULTS: Among the 20 samples, we identified 5 cases of rare thalassemia. These included ß-4.9 (hg38,Chr11:5226187-5231089) at HBB gene, α-91(HBA2:c.*91delT), αCD30(HBA2:c.91-93delGAG), Chinese Gγ+(Aγδß)0(NG_000007.3: g .48795-127698 del 78904) and delta - 77(T > C)(HBD:c.-127T>C). Notably, the -SEA/α-91α genotype associated with severe non-deletional hemoglobin H disease (HbH disease) has not been previously reported. Patients with genotypes ß654/ß-4.9 and -SEA/α-91α necessitate long-term blood transfusions, and those with the -SEA/αCD30α, Chinese Gγ+(Aγδß)0 and delta thalassemia demonstrate mild anemia. CONCLUSIONS: TGS demonstrates promising potential as a diagnostic tool for suspected cases of rare thalassemia in children, especially those suspected to have transfusion-dependent thalassemia (TDT).

Observational and Genetic Evidence for Bidirectional Effects Between Red Blood Cell Traits and Diastolic Blood Pressure.

BACKGROUND: Previous studies have found associations of red blood cell (RBC) traits (hemoglobin and RBC count) with blood pressure; whether these associations are causal is unknown. METHODS: We performed cross-sectional analyses in the Lifelines Cohort Study (n = 167,785). Additionally, we performed bidirectional 2 sample Mendelian randomization (MR) analyses to explore the causal effect of the 2 traits on systolic (SBP) and diastolic blood pressure (DBP), using genetic instrumental variables regarding hemoglobin and RBC identified in UK Biobank (n = 350,475) and International Consortium of Blood Pressure studies for SBP and DBP (n = 757,601). RESULTS: In cross-sectional analyses, we observed positive associations with hypertension and blood pressure for both hemoglobin (odds ratio [OR] = 1.18, 95% confidence interval [CI]: 1.16-1.20 for hypertension; B = 0.11, 95% CI: 0.11-0.12 for SBP; B = 0.11, 95% CI: 0.10-0.11 for DBP, all per SD) and RBC (OR = 1.14, 95% CI: 1.12-1.16 for hypertension; B = 0.11, 95% CI: 0.10-0.12 for SBP; B = 0.08, 95% CI: 0.08-0.09 for DBP, all per SD). MR analyses suggested that higher hemoglobin and RBC cause higher DBP (inverse-variance weighted B = 0.11, 95% CI: 0.07-0.16 for hemoglobin; B = 0.07, 95% CI: 0.04-0.10 for RBC, all per SD). Reverse MR analyses (all per SD) suggested causal effects of DBP on both hemoglobin (B = 0.06, 95% CI: 0.03-0.09) and RBC (B = 0.08, 95% CI: 0.04-0.11). No significant effects on SBP were found. CONCLUSIONS: Our results suggest bidirectional causal relationships of hemoglobin and RBC with DBP, but not with SBP.

Genetic Modulation of the Erythrocyte Phenotype Associated with Retinopathy of Prematurity-A Multicenter Portuguese Cohort Study.

The development of retinopathy of prematurity (ROP) may be influenced by anemia or a low fetal/adult hemoglobin ratio. We aimed to analyze the association between DNA methyltransferase 3 ß (DNMT3B) (rs2424913), methylenetetrahydrofolate reductase (MTHFR) (rs1801133), and lysine-specific histone demethylase 1A (KDM1A) (rs7548692) polymorphisms, erythrocyte parameters during the first week of life, and ROP. In total, 396 infants (gestational age < 32 weeks or birth weight < 1500 g) were evaluated clinically and hematologically. Genotyping was performed using a MicroChip DNA on a platform employing iPlex MassARRAY®. Multivariate regression was performed after determining risk factors for ROP using univariate regression. In the group of infants who developed ROP red blood cell distribution width (RDW), erythroblasts, and mean corpuscular volume (MCV) were higher, while mean hemoglobin and mean corpuscular hemoglobin concentration (MCHC) were lower; higher RDW was associated with KDM1A (AA), MTHFR (CC and CC + TT), KDM1A (AA) + MTHFR (CC), and KDM1A (AA) + DNMT3B (allele C); KDM1A (AA) + MTHFR (CC) were associated with higher RDW, erythroblasts, MCV, and mean corpuscular hemoglobin (MCH); higher MCV and MCH were also associated with KDM1A (AA) + MTHFR (CC) + DNMT3B (allele C). We concluded that the polymorphisms studied may influence susceptibility to ROP by modulating erythropoiesis and gene expression of the fetal/adult hemoglobin ratio.