Early Onset of Severe Anemia Caused by Hb Calgary (HBB: C.194G > T): Another Case Report.

Unstable hemoglobin (Hb) variants are a rare cause of congenital hemolytic anemia. We describe a Chinese girl who presented with transfusion-dependent anemia in early infancy. Her diagnosis of Hb Calgary [ß64(E8)Gly > Val; HBB:c.194G > T] was not made until molecular testing was performed at the age of 5 years. Our case highlights the importance of early genetic testing in order to make the diagnosis, which may not only be useful for patient management and family counseling, but also for avoiding further unnecessary investigative attempts.

A 6-Year Follow-up of a Chinese Child with Homozygous ß0-Thalaasemia and a Heterozygous KLF1 Mutation.

Patients with the genotype of ß0/ß0 for ß-thalassemia (ß-thal) usually behave as ß-thal major (ß-TM) phenotype which is transfusion-dependent. The pathophysiology of ß-thal is the imbalance between α/ß-globin chains. The degree of α/ß-globin imbalance can be reduced by the more effective synthesis of γ-globin chains, and increased Hb F levels, modifying clinical severity of ß-TM. We report a Chinese child who had homozygous ß0-thal and a heterozygous KLF1 mutation. The patient had a moderate anemia since 6 months old, keeping a baseline Hb value of 8.0-9.0 g/dL. She had normal development except for a short stature (3rd percentile) until 6 years old, when splenomegaly and facial bone deformities occurred. Although genetic alteration of KLF1 expression in ß0/ß0 patients can result in some degree of disease alleviation, our case shows that it is insufficient to ameliorate satisfactorily the presentation. This point should be borne in mind for physicians who provide the genetic counseling and prenatal diagnosis to at-risk families.

A reliable and high throughput HPLC-HRMS method for the rapid screening of ß-thalassemia and hemoglobinopathy in dried blood spots.

OBJECTIVES: Traditional methods for ß-thalassemia screening usually rely on the structural integrity of hemoglobin (Hb), which can be affected by the hemolysis of red blood cells and Hb degradation. Here, we aim to develop a reliable and high throughput method for rapid detection of ß-thalassemia using dried blood spots (DBS). METHODS: Hb components were extracted from a disc (3.2 mm diameter) punched from the DBS samples and digested by trypsin to produce a series of Hb-specific peptides. An analytical system combining high-resolution mass spectrometry and high-performance liquid chromatography was used for biomarker selection. The selected marker peptides were used to calculate delta/beta (δ/ß) and beta-mutated/beta (ßM/ß) globin ratios for disease evaluation. RESULTS: Totally, 699 patients and 629 normal individuals, aged 3 days to 89 years, were recruited for method construction. Method assessment showed both the inter-assay and intra-assay relative standard deviation values were less than 10.8%, and the limits of quantitation for the proteo-specific peptides were quite low (1.0-5.0 µg/L). No appreciable matrix effects or carryover rates were observed. The extraction recoveries ranged from 93.8 to 128.7%, and the method was shown to be stable even when the samples were stored for 24 days. Prospective applications of this method in 909 participants also indicated good performance with a sensitivity of 100% and a specificity of 99.6%. CONCLUSIONS: We have developed a fast, high throughput and reliable method for screening of ß-thalassemia and hemoglobinopathy in children and adults, which is expected to be used as a first-line screening assay.

Fist Detection of Hb Suqian[ß42(CD1) Phe→Leu, HBB:c.129T > A] in Han Chinese.

We have identified a variant on the ß-globin gene in a Chinese female. Sequencing of the HBB gene revealed a Phe→Leu substitution at codon 42[ß42(CD1) Phe→Leu, HBB:c.129T > A] which has been named Hb Suqian for where the proband was born.

α- and ß-Globin Gene Mutations in Individuals with Hemoglobinopathies in the Chattogram and Sylhet Regions of Bangladesh.

Hemoglobinopathies, including α- and ß-thalassemias and sickle cell disease, are among the most widely disseminated hereditary blood disorders worldwide. Bangladesh is considered a hotspot for hemoglobinopathies, and these diseases cause a significant health concern in the country. However, the country has a dearth of knowledge on the molecular etiology and carrier frequency of thalassemias, primarily due to a lack of diagnostic facilities, limited access to information, and the absence of efficient screening programs. This study sought to investigate the spectrum of mutations underlying hemoglobinopathies in Bangladesh. We developed a set of polymerase chain reaction (PCR)-based techniques to detect mutations in α- and ß-globin genes. We recruited 63 index subjects with previously diagnosed thalassemia. Along with age- and sex-matched control subjects, we assessed several hematological and serum indices and genotyped them using our PCR-based methods. We identified that parental consanguinity was associated with the occurrence of these hemoglobinopathies. Our PCR-based genotyping assays identified 23 HBB genotypes, with the codons 41/42 (-TTCT) (HBB: c.126_129delCTTT) mutation leading the spectrum. We also observed the presence of cooccurring HBA conditions, of which the participants were not aware. All index participants in this study were on iron chelation therapies, yet we found they had very high serum ferritin (SF) levels, indicating inefficient management of the individuals undergoing such treatments. Overall, this study provides essential information on the hemoglobinopathy mutation spectrum in Bangladesh and highlights the need for nationwide screening programs and an integrated policy for diagnosing and managing individuals with hemoglobinopathies.

Impact of Anemia on The Quality of Life of Chronic Kidney Disease Patients: A Single Institution Experience.

BACKGROUND: Anemia is a common complication of chronic kidney disease (CKD) and has been shown to worsen as CKD advances. CKD negatively impacts patients' health-related quality of life. It is therefore necessary to determine the impact of anemia on the quality of life in patients with CKD. OBJECTIVES: We assessed the relationship between the severity of anemia and its impact on the quality of life of anemic CKD patients attending nephrology clinics. METHODOLOGY: A cross-sectional study of one hundred and sixty-three subjects which included 102 CKD patients with anemia and sixty-one CKD subjects without anemia, was done between April 2016 and January 2017. Karnofsky's structured questionnaire was used for the quality of life, while the packed cell volume was used to determine the severity of anemia. RESULTS: The prevalence of anemia among CKD subjects was 102(62.6%), and it significantly worsens as CKD advances, which ranged from 42.3% in stage 3 to 93% in stage 5 (p < 0.001). The mean physical performance score was significantly lower among anemic CKD subjects than among controls, which was 73.17 ± 12.95 and 84.59 ± 11.04 respectively (P < 0.001). Furthermore, the mean physical performance score decreases significantly with the advancing CKD among both study groups. CONCLUSION: This study showed that CKD patients with anemia had significant impairment in their physical ability than CKD patients without anemia.

CONTEXTE: L'anémie est une complication fréquente de la maladie rénale chronique (MRC) et a tendance à s'aggraver à mesure que la MRC progresse.La MRC a un impact négatif sur la qualité de vie liée à la santé des patients. Il est donc nécessaire de déterminer l'impact de l'anémie sur la qualité de vie des patients atteints de MRC. OBJECTIFS: Nous avons évalué la relation entre la gravité de l'anémie et son impact sur la qualité de vie des patients atteints de MRC anémiques fréquentant les cliniques de néphrologie. MÉTHODOLOGIE: Une étude transversale portant sur cent soixante-trois sujets, dont 102 patients atteints de MRC avec anémie et soixante et un sujets atteints de MRC sans anémie, a été réalisée entre avril 2016 et janvier 2017. Le questionnaire structuré de Karnofsky a été utilisé pour évaluer la qualité de vie, tandis que le volume globulaire a été utilisé pour déterminer la gravité de l'anémie. RÉSULTATS: La prévalence de l'anémie chez les sujets atteints de MRC était de 102 (62,6 %), et elle s'aggrave significativement à mesure que la MRC progresse, passant de 42,3 % au stade 3 à 93 % au stade 5 (p < 0,001). Le score moyen de performance physique était significativement plus bas chez les sujets atteints de MRC anémiques que chez les témoins, soit 73,17 ± 12,95 et 84,59 ± 11,04 respectivement (p < 0,001). De plus, le score moyen de performance physique diminue significativement avec la progression de la MRC dans les deux groupes d'étude. CONCLUSION: Cette étude a montré que les patients atteints de MRC avec anémie présentaient une altération significative de leur capacité physique par rapport aux patients atteints de MRC sans anémie. Mots-clés: MRC,Anémie, Qualité de vie (QdV), Hémoglobine (Hb).

Transcriptomic analysis reveals the regulatory mechanism underlying the indirubin-mediated amelioration of dextran sulfate sodium-induced colitis in mice.

CONTEXT: Aryl hydrocarbon receptor (AhR) agonists are potential therapeutic agents for ulcerative colitis (UC). Indirubin (IDR), which is a natural AhR ligand approved for leukemia treatment, ameliorates dextran sulfate sodium (DSS)-induced colitis in mice. However, the therapeutic mechanisms of IDR are unknown, limiting its application. OBJECTIVE: This study explores the therapeutic mechanisms of IDR in DSS-induced colitis using transcriptomic analysis. MATERIALS AND METHODS: Male BALB/c mice were categorized to six groups: normal, DSS model (2% DSS), IDR treatment (10, 20 and 40 mg/kg), and sulfasalazine (520 mg/kg) groups. The drugs were intragastrically administered for 7 consecutive days. The disease activity index (DAI) was recorded. After euthanasia, the colon length was measured, and histopathological examination, immunohistochemistry staining using F4/80, and colonic transcriptomic analysis were conducted. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting (WB) were conducted to verify our findings. RESULTS: Compared with DSS, IDR treatment decreased the DAI score by 64.9% and increased colon length by 26.2%. Moreover, it alleviated mucosal injury and reduced macrophage infiltration. Transcriptomic analysis identified several downregulated genes (Igkvs and Nlrp3), as well as Nlrp3/Il1ß and hemoglobin gene networks, after IDR treatment. The abundances of NF-κB p65, NLRP3, IL-1ß, and HBA decreased by 69.1, 59.4, 81.1, and 83.0% respectively, after IDR treatment. DISCUSSION AND CONCLUSION: Apart from the well-documented NF-κB signalling pathway, IL-17A, and NLRP3-IL-1ß, the suppression of haemoglobin-induced lipid peroxidation could be a previously unknown mechanism of IDR. Our study can help improve its application for UC treatment.

Influence of hemoglobin on blood pressure among people with GP.Mur blood type☆.

BACKGROUND/PURPOSE: GP.Mur is a clinically important red blood cell (RBC) type. GP.Mur and band 3 interact on the RBCs. We previously observed that healthy adults with GP.Mur type present slightly higher blood pressure (BP). Because band 3 and Hb comodulate nitric oxide (NO)-dependent vasodilation and hemoglobin (Hb) is positively associated with BP, we aimed to test whether these could contribute to higher BP in GP.Mur+ people. METHODS: We recruited 989 non-elderly adults (21% GP.Mur) free of catastrophic illness and not on cardiovascular or anti-hypertensive medication. Their body indices, blood lab data and lifestyle data were collected for analyses of potential BP-related factors (BMI, age, smoking, Hb, and GP.Mur). RESULTS: BMI and age remained the most significant contributors to BP. GP.Mur slightly increased systolic BP (SBP). The direct correlation between Hb and BP was only found in Taiwanese non-anemic men, not women. After age and BMI adjusted, we estimated an increase of 1.8 mmHg and 2.6 mmHg of SBP by 1 g/dL Hb among men without and with GP.Mur type, respectively. Hb was generally lower among people expressing GP.Mur, which likely limited their larger impact on BP. CONCLUSION: GP.Mur contributed to BP in both Hb-dependent and Hb-independent fashion. A pronounced impact of hemoglobin on BP likely requires sufficient Hb, as GP.Mur increased the sensitivity of SBP to Hb only in non-anemic Taiwanese men, and not in Taiwanese women or anemic men. The mechanism through which GP.Mur affected BP independent of Hb is unknown.

Noninvasive Hemoglobin Measurement Reduce Invasive Procedures in Thalassemia Patients.

This study was conducted to investigate the agreement between laboratory hemoglobin (LabHb) measured in venous blood and noninvasive, spectrophotometric hemoglobin (SpHb) measurement and the usability of SpHb measurement in the transfusion decision-making in patients with thalassemia whose hemoglobin (Hb) was monitored by taking blood samples at frequent intervals and who were transfused. Cardiac pulse, oxygen saturation, Pleth variability index (PVI), and SpHb values were measured in patients who came to the hematology outpatient clinic for a control visit and whose Hb levels were planned to be measured. Venous blood samples were taken for LabHb measurement, which we accept as the gold standard. Cohen's kappa value was calculated for the agreement between SpHb measurements and LabHb values. The relationship and predictability between both measurement methods were evaluated by Pearson correlation analysis, a modified Bland-Altman plot and the linear regression model. In the study conducted with a total of 110 children with thalassemia, a moderate level of agreement between the two measurement methods (kappa = 0.370, p < 0.0001) and a significantly high correlation between the two tests (r = 0.675) were found. The mean bias between the differences was found to be 0.3 g/dL (-1.27 to 1.86 g/dL). The sensitivity and the specificity of SpHb in identifying patients who needed transfusions (Hb <10.0 g/dL) were calculated as 92.2 and 57.1%, respectively. Our results suggest SpHb measurement may be used to screen anemia in hemodynamically stable hemoglobinopathy patients and even for transfusion decision-making with combination clinical findings.

Hb Wanjiang: A New ß-Globin Chain Variant with Two Amino Acid Substitutions (HBB: c.255_264delinsTTTTTCTCAG).

We report a new hemoglobin (Hb) variant that we have named Hb Wanjiang (HBB: c.255_264delinsTTTTTCTCAG). We identified this variant in a Chinese man by the next-generation sequencing (NGS) method. The father of the proband also carried the same variant. This variant results from a 10 bp deletion at codons 84-87 of the ß-globin chain, replaced with 10 nucleotides coming from the δ-globin gene at the same position, leading to the substitution of two amino acids in the peptide chain with no change in the ß-globin chain length. The heterozygotes had a normal hematological feature with no abnormal Hb variant detectable on capillary electrophoresis (CE) and high performance liquid chromatography (HPLC). The combination of Hb Wanjiang and ß-thalassemia (ß-thal) was not found to aggravate anemia.

Hb Nivaria: A New Hemoglobin Variant with a Shortened α-Globin Chain [α139(HC1)Lys→Stop; HBA1: c.418A>T].

We report a novel hemoglobin (Hb) variant found in a Spanish individual from Santa Cruz de Tenerife, the Canary Islands in Spain. The proband was a 39-year-old male. High performance liquid chromatography (HPLC) displayed an unknown peak (19.3%) at a retention time of 1.3 min. eluting before Hb A0. Capillary zone electrophoresis (CZE) showed an abnormal peak (20.0%) in zone 12. Direct DNA sequencing of the α-globin genes revealed heterozygosity for a nonsense mutation at codon 139 (AAA>TAA), causing a lysine to stop codon substitution [α139(HC1)Lys→Stop; HBA1: c.418A>T]. We decided to name the variant Hb Nivaria (Tenerife) for the place of birth and residence of the proband.

Use of Capillary Electrophoresis Migration Position for the Presumptive Identification of Hemoglobin Variants Prevalent in China.

Capillary electrophoresis (CE) is increasingly being used to screen for hemoglobinopathies. We here report the migration position of hemoglobin (Hb) variants prevalent in China determined by CE. The CE migration position showed excellent precision and minor inter-individual variation. We conclude that the CE migration position can be used as a reliable indicator for identifying Hb variants and can also help to establish a well-characterized library of Hb variants for their refined presumptive identification.

A New Case of Hb Headington (HBB: c.217A>C) Due to a New DNA Transversion, Found in a Patient with Type 2 Diabetes Mellitus.

We here report a novel case of Hb Headington [ß72(E16)Ser→Arg, HBB: c.217A>C, p.Ser73Arg], in a 68-year-old woman with type 2 diabetes mellitus (T2DM). Glycosylated hemoglobin (Hb) was measured by capillary electrophoresis (CE). The spectrum showed abnormal peaks between the A0 and A2 peaks. DNA sequencing demonstrated a mutation on the HBB gene, which predicted a substitution of serine to arginine at position 73 in the ß-globin chain. Moreover, this amino acid substitution occurs at the same position as Hb Headington [ß72(E16)Ser→Arg, HBB: c.219T>A, p.Ser73Arg], which showed increased oxygen affinity.

Hb Alessandria [ß37(C3)Trp→Leu; HBB: c.113G>T]: a Novel Variant on the ß-Globin Chain with Slightly Increased Affinity for Oxygen Detected by Capillary Electrophoresis.

We report a novel mutation on the ß-globin gene in a 68-year-old woman of Sicilian origin living in Alessandria, Italy. This mutation produces a hemoglobin (Hb) variant of Hb A that was detected by the capillary electrophoresis (CE) method during measurement of Hb A1c. The variant Hb did not separate from Hb A using different high performance liquid chromatography (HPLC) instruments. Direct DNA sequencing revealed a G>T transversion at codon 37 and subsequent substitution of a tryptophan residue for a leucine residue. The new Hb variant was named Hb Alessandria [ß37(C3)Trp→Leu; HBB: c.113G>T]. The p50 value was slightly decreased while the stability test at 37 °C in isopropyl alcohol and the main erythrocyte parameters were normal. Overall, the patient appeared clinically normal.

Dominantly Inherited ß-Thalassemia Caused by a Single Nucleotide Deletion in Exon 3 of the ß-Globin Gene: Hb Xiangyang (HBB: c.393delT).

We report a de novo frameshift mutation in exon 3 of the ß-globin gene that leads to a ß-thalassemia (ß-thal) intermedia (ß-TI) phenotype in a 6-year-old Chinese boy. This novel mutation with deletion of the last nucleotide (-T) at codon 130 results in a ß-globin chain that is extended to 156 amino acid residues. This study highlights the importance of considering dominantly inherited ß-thal in the investigation of anemia, even in patients with ethnic backgrounds not usually associated with ß-thal and hematologically normal parents.

Hb Laibin [ß96(FG3)Leu→Arg; HBB: c.290T>G]: A Novel Hemoglobin Variant Described in a Chinese Family.

We report a novel ß chain hemoglobin (Hb) variant found in a Chinese family. A high level of Hb F was observed on capillary electrophoresis (CE). However, high performance liquid chromatography (HPLC) showed a high level of Hb A2. DNA sequencing revealed a single base substitution (T>G) at codon 96 of exon 2 of the ß-globin gene. This alters the normally encoded leucine to arginine [ß96(FG3)Leu→Arg; HBB: c.290T>G] that we propose to name Hb Laibin for the region of origin of the proband. The pedigree study showed that it was inherited from his mother.

Treatment with Hydroxyurea Leads to Fetal Hemoglobin Reactivation through CA1 and LIN28B Genes: An In Vitro Study.

Hydroxyurea (HU) is an effective drug to increase fetal γ-globin gene (Hb F) expression, replacing the missing adult ß-globin gene. The mechanism of Hb F induction by HU and improvement in clinical symptoms are still poorly understood. The current study aimed to improve the molecular understanding of drug-induced alterations and reveals genes related to HU treatment responsiveness in ß-thalassemia (ß-thal). We analyzed the GSE109186 dataset using system biology and weighted gene coexpression network analysis (WGCNA) to identify and quantify gene expression changes reflected in the HU-treated human erythroblastic leukemia cells. The K562 cell line was treated in 50, 100, and 150 µM concentrations of HU for 24, 48, and 72 hours with three replications. The alteration of CA1, LIN28B and Hb F gene expression in HU-treated cells was evaluated using the real-time polymerase chain (real-time PCR) technique. The results showed that LIN28B has an increase of 4.27-fold on the first day of HU-treatment in 50 µM (p < 0.01). The CA1 expression showed a decrease at all times and doses of treatment, and the most decrease happened in 48 hours and 50 µM (p < 0.04). Hb F also showed the highest increase in 100 µM after 24 hours of treatment (5.18-fold). In summary, the data suggest that alteration of LIN28B and CA1 gene expression is associated with γ-globin increasing in HU-treated cells.

Hb Kalundborg [ß79(EF3)Asp→Glu; HBB: c.240C>a], a Possible Low-affinity Hemoglobin Variant Detected during Hb A1c Measurement.

A previously unknown hemoglobin (Hb) variant was detected during measurement of glycosylated Hb (Hb A1c) after the introduction of a new high performance liquid chromatography (HPLC) apparatus. Subsequent DNA sequencing revealed a heterozygous single nucleotide substitution at codon 79 (C>A) on the ß-globin gene changing an amino acid [ß79(EF3)Asp→Glu; HBB: c.240C>A]. The new Hb variant was named Hb Kalundborg after the place of origin of the proband. Heterozygosity for this mutation appears to have no clinical significance in itself except for a possibly slightly lower oxygen affinity. However, it interferes with Hb A1c measurement by HPLC, causing a falsely high Hb A1c concentration when using the G11 apparatus with clinical implications possibly to follow.

Hb Kirikiriroa [α57(E6)Gly→Cys; HBA1: c.172G>T]: A Novel Unstable α-Globin Variant with Oxidized Derivatives Interfering with Hb A1c.

We report the identification of a novel hemoglobin (Hb) variant [α57(E6)Gly→Cys; HBA1: c.172G>T], to be referred to as Hb Kirikiriroa. The variant was detected in five subjects from two families, with familial relationship established between the families following diagnosis. A persistently elevated Hb A1c over a 1-year period prompted hemoglobinopathy screening in an adolescent male of New Zealand (NZ) European descent (case 1). Capillary electrophoresis (CE) revealed the variant was negatively charged and susceptible to oxidation, with multiple abnormal peaks detected (0.4-5.1% total Hb). Hb A1c analysis by cation exchange high performance liquid chromatography (HPLC) was the first indication of the variant in a pregnant female of NZ European descent (case 2). Cases 1 and 2 had normal complete blood counts. Isopropanol stability testing provided evidence the variant was unstable. We herein describe the characterization of Hb Kirikiriroa and clinical significance of the variant for interference with Hb A1c analysis by CE and cation exchange HPLC.

Impact of diabetes mellitus on outcome after transcatheter aortic valve replacement: Identifying high-risk diabetic population from the OCEAN-TAVI registry.

OBJECTIVES: To identify the vulnerable diabetic cohort in patients undergoing transcatheter aortic valve replacement (TAVR). BACKGROUNDS: Considerable controversy remains about whether specific cohort exists in which presence of diabetes mellitus (DM) carries adverse risk of mortality after TAVR. METHODS: Of the 2588 patients who were enrolled in the OCEAN-TAVI registry, 2526 patients with glycohemoglobin data were analyzed. The individuals were divided into DM and non-DM groups according to previous medical history of DM or using diabetic medicine, and increased HbA1c values (≥6.5%) at baseline. The primary endpoint of this study was 2-year all-cause mortality after TAVR. RESULTS: The follow up rate of clinical outcome at 1-year was 2514/2526 (99.5%) and median follow-up period was 22.5 months. DM group had 699 (27.7%) patients, in which 153 (21.9%) was diagnosed by increased HbA1c levels without previous medical history of DM. Kaplan-Meier curve of 2-year all-cause mortality presented significant difference between patients with and without DM (p = 0.029). In addition, patients with low-density lipoprotein cholesterol (LDL-C) levels > 100 mg/dl and left ventricular ejection fraction (LVEF) < 40% had great risk of mortality after TAVR (LDL-C: hazard ratio [HR] 1.82, p < 0.001; LVEF: HR 2.61, p = 0.002, respectively). CONCLUSIONS: Presence of DM was significantly associated with poor outcome after TAVR and adverse effect of DM was remarkable in patients with relatively higher LDL-C levels and reduced LVEF under 40%. These subtypes may need intensive control of cardiovascular risk factors, including DM, before and after TAVR.