A Novel ß-Globin Variant, Hb Raklev [ß 75(E19) HBB:c.227T > A (Leu→Gln)].

We present a new hemoglobin variant, Hb Raklev, characterized by the substitution of leucine with glutamine at position 75 in the ß-globin chain. This variant was discovered inadvertently during an HbA1c evaluation using high performance liquid chromatography in a symptomless 54-year-old Caucasian woman, with the same variant also identified in her 16-year-old daughter. Purification of the hemoglobin revealed possibly diminished 2,3-bisphosphoglycerate (2,3-BPG) sensitivity, which may result in heightened oxygen affinity. Notably, two variants have been previously documented at this location: the unstable Hb Atlanta and the high-affinity Hb Pasadena.

Hb Ryazan: An Elongated C-Terminal ß-Chain Due to a New Frameshift Mutation, HBB: c.396delG p.Val133Trpfs*25.

We identified a novel abnormal hemoglobin variant caused by a frameshift mutation at nucleotide position 396 in exon 3 of the ß-globin gene (HBB): NM_000518:c.396delG. This variant causes an emergence of alternative amino acid sequence starting at codon 133 and a new stop codon formed in the 3' untranslated region (3'UTR) of the HBB gene at amino acid position 158. This ß-globin gene variant was identified in a woman with a long history of hemolytic anemia. We named this variant Hb Ryazan after the proband's city of origin.

In silico analysis of substitution mutations in the ß-globin gene in Turkish population of ß-thalassemia.

Beta-thalassemia is a genetic blood disorder represented by anomalies in hemoglobin's beta chain production. Most hemoglobin defects are a result of mutations of the structural ß-globin gene. Many diseases, including ß-thalassemia, benefit from computational studies that aid researchers in investigating the association of genotype and phenotype. In this study, the alanine substitution mutations of the ß-globin protein sub-units in the Turkish population (Hb Ankara, Hb Siirt and Hb Izmir) and the effects of those mutations on the ß-globin protein structure and performance are examined using molecular dynamics simulation. While Hb Ankara variant showed a non-conservative mutation, Hb Siirt and Hb Izmir showed a semi-conservative mutation. RMSF values of Hb Siirt, between residues 95 and 99, were higher than wild-type and the other mutant proteins. The residues of Hb Ankara showed lower fluctuation compared to the other structures. The mean ROG values were 1.47 nm, 1.46 nm, 1.49 nm and 1.48 and the average number of the hydrogen bonds were 92, 100, 99, and 89 for Hb Ankara, Hb Siirt and Hb Izmir, respectively. Moreover, a significant increase in overall motion in Hb Siirt was observed based on PCA analysis. Hb Siirt substitution mutation might cause an effect in ß-globin proteins which could impact the protein function. This indicates a major role on beta globin subunit's stability for alanine on 27th position. However, Hb Ankara and Hb Izmir variants may act as a silent mutation, since these two mutations did not show a large change in the dynamics of the protein.Communicated by Ramaswamy H. Sarma.

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 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.

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.

Transcription factor competition at the γ-globin promoters controls hemoglobin switching.

BCL11A, the major regulator of fetal hemoglobin (HbF, α2γ2) level, represses γ-globin expression through direct promoter binding in adult erythroid cells in a switch to adult hemoglobin (HbA, α2ß2). To uncover how BCL11A initiates repression, we used CRISPR-Cas9, dCas9, dCas9-KRAB and dCas9-VP64 screens to dissect the γ-globin promoters and identified an activator element near the BCL11A-binding site. Using CUT&RUN and base editing, we demonstrate that a proximal CCAAT box is occupied by the activator NF-Y. BCL11A competes with NF-Y binding through steric hindrance to initiate repression. Occupancy of NF-Y is rapidly established following BCL11A depletion, and precedes γ-globin derepression and locus control region (LCR)-globin loop formation. Our findings reveal that the switch from fetal to adult globin gene expression within the >50-kb ß-globin gene cluster is initiated by competition between a stage-selective repressor and a ubiquitous activating factor within a remarkably discrete region of the γ-globin promoters.

DNA methylation patterns of ß-globin cluster in ß-thalassemia patients.

BACKGROUND: Reactivation of fetal hemoglobin (HbF, α2γ2) holds a therapeutic target for ß-thalassemia and sickle cell disease. Although many HbF regulators have been identified, the methylation patterns in ß-globin cluster driving the fetal-to-adult hemoglobin switch remains to be determined. RESULTS: Here, we evaluated DNA methylation patterns of the ß-globin cluster from peripheral bloods of 105 ß0/ß0 thalassemia patients and 44 normal controls. We also recruited 15 bone marrows and 4 cord blood samples for further evaluation. We identified that the CpG sites in the locus control region (LCR) DNase I hypersensitive site 4 and 3 (HS4-3) regions, and γ- and ß-globin promoters displayed hypomethylation in ß0/ß0-thalassemia patients, especially for the patients with high HbF level, as compared with normal controls. Furthermore, hypomethylations in most of CpG sites of the HS4-3 core regions were also observed in bone marrows (BM) of ß0/ß0-patients compared with normal controls; and methylation level of γ-globin promoter -50 and + 17 CpG sites showed lower methylation level in patients with high HbF level compared with those with low HbF level and a negative correlation with HbF level among ß0-thalassemia patients. Finally, γ-globin promoter + 17 and + 50 CpG sites also displayed significant hypomethylation in cord blood (CB) tissues compared with BM tissues from normal controls. CONCLUSIONS: Our findings revealed methylation patterns in ß-globin cluster associated with ß0 thalassemia disease and γ-globin expression, contributed to understand the epigenetic modification in ß0 thalassemia patients and provided candidate targets for the therapies of ß-hemoglobinopathies.

Curating the gnomAD database: Report of novel variants in the globin-coding genes and bioinformatics analysis.

Massive parallel sequencing technologies are facilitating the faster identification of sequence variants with the consequent capability of untangling the molecular bases of many human genetic syndromes. However, it is not always easy to understand the impact of novel variants, especially for missense changes, which can lead to a spectrum of phenotypes. This study presents a custom-designed multistep methodology to evaluate the impact of novel variants aggregated in the genome aggregation database for the HBB, HBA2, and HBA1 genes, by testing and improving its performance with a dataset of previously described alterations affecting those same genes. This approach scored high sensitivity and specificity values and showed an overall better performance than sequence-derived predictors, highlighting the importance of protein conformation and interaction specific analyses in curating variant databases. This study also describes the strengths and limitations of these structural studies and allows identifying residues in the globin chains more prone to tolerate substitutions.

Analysis of cysteine glutathionylation in hemoglobin of gastric cancer patients using nanoflow liquid chromatography/triple-stage mass spectrometry.

Glutathione is an intracellular antioxidant capable of scavenging free radicals and detoxifying electrophiles from endogenous and exogenous sources via the free thiol group. Post-translational glutathionylation at cysteine residues of proteins can affect the structure and cause a functional change of proteins. Protein glutathionylation has been proven to reflect the cellular redox status. Our previous report indicates that the levels of glutathionylation in hemoglobin from peripheral blood of smokers are significantly higher than in nonsmokers. In this study, a nanoflow liquid chromatography/nanospray ionization triple-stage mass spectrometric (nanoLC/NSI-MS3 ) method with a linear ion trap mass spectrometer was employed to quantify glutathionylated peptides in the trypsin digests of hemoglobin from gastric cancer patients. We compare the extent of glutathionylation in hemoglobin from nonsmoking gastric cancer patients with that from nonsmoking healthy adults. Using a carboxymethylated peptide as the reference peptide, the relative quantification of each glutathionylated peptide was measured as the peak area ratio of the modified peptide versus the sum of the peak areas of the modified and the carboxymethylated parent peptide in the selected reaction monitoring chromatograms. Using this method, we found that the extents of glutathionylation at Cys-104 of the α-globin and Cys-93 of ß-globulin hemoglobin from 10 gastric cancer patients were significantly higher than those from 14 normal individuals with p values <0.0001. Our results suggest the possibility of using the extent of cysteine glutathionylation at ß-93 of hemoglobin as an oxidative stress biomarker candidate for gastric cancer.

Hereditary xerocytosis - spectrum and clinical manifestations of variants in the PIEZO1 gene, including co-occurrence with a novel ß-globin mutation.

Hereditary xerocytosis (HX) is a rare, autosomal dominant congenital hemolytic anemia (CHA) characterized by erythrocyte dehydration with presentation of various degrees of hemolytic anemia. HX is often misdiagnosed as hereditary spherocytosis or other CHA. Here we report three cases of suspected HX and one case of HX associated with ß-thalassemia. Sanger method was used for sequencing cDNA of the PIEZO1 gene. Variants were evaluated for potential pathogenicity by MutationTaster, PROVEAN, PolyPhen-2 and M-CAP software, and by molecular modeling. Four different variants in the PIEZO1 gene were found, including three substitutions (p.D669H, p.D1566G, p.T1732 M) and one deletion (p.745delQ). In addition, in the patient with the p.T1732 M variant we detected a 12-nucleotide deletion in the ß-globin gene leading to a deletion of amino acids 62AHGK65. The joint presence of mutations in two different genes connected with erythrocytes markedly aggravated the presentation of the disease. Bioinformatic analysis and molecular modeling strongly indicated likely deleterious effects of all four PIEZO1 variants, but co-segregation analysis showed that the p.D1566G substitution is in fact non-pathogenic. Identification of causative mutations should improve the diagnosis and management of HX and provide a new insight into the molecular basis of this complex red blood cell abnormality.

Urea Stress: Myo-inositol's efficacy to counteract destabilization of TIM-ß-globin complex by urea is as good as that of the methylamine.

Do kidney osmolytes counteract the deleterious effects of urea on kidney proteins? To answer this question, we measured guanidinium chloride (GdmCl)-induced denaturation of triose phosphate isomerase-ß-globin subunit complex (TIM-ß-globin) from sheep kidney in the presence of various concentrations of urea and each of kidney osmolytes (glycine betaine, myo-inositol and sorbitol) alone and in combination at pH 7.5 and 25 °C. Analysis of GdmCl-induced denaturation curve at a given osmolyte (or urea) concentration gave ΔGD0 (Gibbs free energy change in the absence of GdmCl). For a co-solute (CO), a linear plot of ΔΔGD0 (difference between values of ΔGD0 in the presence and absence of CO) against [CO] (molar concentration of CO) yielded m-value (=δΔΔGD0/δ[CO]). m-value for urea (murea) and that for the osmolyte (mosm) were used to determine [urea]:[osmolyte], the molar ratio for the perfect counteraction of the denaturing effect of urea on the protein stability by an osmolyte. For each osmolyte, we tested this ratio for the perfect counteraction. It was observed that glycine betaine and myo-inositol provide perfect counteraction at the predicted molar ratio whereas sorbitol fails to do so. Furthermore, urea and GdmCl induced a two-state denaturation, and both TIM and ß-globin have identical thermodynamic stability.

Dominant ß-thalassaemia with unusually high Hb A2 and Hb F caused by ßCD121(-G) (HBB:c.364delG) in exon 3 of ß-globin gene.

We describe a dominant ß-thalassaemia caused by a deletion of G at nucleotide position 364 in exon 3 of the ß-globin gene. The heterozygosity of this mutation was found in a 36-year-old Thai patient who had moderate hypochromic microcytic anaemia with haemolytic blood picture. Haemoglobin (Hb) analysis revealed relatively higher Hbs A2 (6.8%) and F (4.7%) as compared with those of ß0-thalassaemia (n=278) and ß+-thalassaemia (n=55) carriers in our series. Secondary structure prediction of the elongated ß-globin chain showed that the α-helix at the C-terminal is disrupted dramatically by the random coil and ß-sheet, which should result in a highly unstable ß-globin variant, undetectable in peripheral blood and a dominant clinical phenotypic feature.

The Spectrum of ß-Thalassemia Mutations in Siirt Province, Southeastern Turkey.

ß-Thalassemia (ß-thal) is the most common hereditary genetic blood disorder. The aims of this study were: (i) to determine the mutation types and the frequency of these mutations in ß-thal patients to obtain the ethnic origins of the population in Siirt Province; (ii) to evaluate the pathogenicity of these mutations by performing in silico analysis; (iii) to reveal the genotype-phenotype correlation by comparing the clinical manifestation of our patients to the specific mutations in this population. This study included 34 patients (18 males and 16 females) with a mean age of 9.1 ± 3.6 years (range 3-16 years). All mutations were determined using sequence analysis methods, and the mutations were analyzed using bioinformatics tools. Thirteen different mutations were detected in the patients: IVI-I-110 (G>A) (HBB: c.93-21G>A) (38.9%); IVS-II-1 (G>A) (HBB: c.315_1G>A) (11.1%); -30 (T>A) (HBB: c.-80T>A) (9.25%) and IVS-I-1 (G>A) (HBB: c.92 + 1G>A) (9.25%), were the most common, and these mutations constituted 68.5% of the cases. Missense codon 6 (A>T) (HBB: c.20A>T) was not pathogenic; however, all the intronic mutations (IVS-I-1, IVS-I-110, IVS-II-1) and frameshift mutations [codon 44 (-C) (HBB: c.135delC) and codons 36/37 (-T) (HBB: c.112delT)] resulted in disease. These mutations can be used to determine the ethnic origin of the Siirt population and, in affected pregnant women, to develop prenatal strategies. A fatal phenotype can be identified by in silico analysis; however, mutations that are unknown prior to marriage, pregnancy, and childbirth or new mutations can be less accurately identified.

Quadrupole-Time-of-Flight Mass Spectrometric Identification of Hemoglobin Subunits α, ß, γ and δ in Unknown Peaks of High Performance Liquid Chromatography of Hemoglobin in ß-Thalassemias.

This is the first report of quadrupole time-of-flight (Q-TOF) mass spectrometric identification of the hemoglobin (Hb) subunits, α, ß, δ and γ peptides, derived from enzymatic-digestion of proteins in the early unknown peaks of the cation exchange chromatography of Hb. The objectives were to identify the unknown high performance liquid chromatography (HPLC) peaks in healthy subjects and in patients with ß-thalassemia (ß-thal). The results demonstrate the existence of pools of free globin chains in red blood cells (RBCs). The α-, ß-, δ- and γ-globin peptides were identified in the unknown HPLC peaks. The quantification and role of the free globin pool in patients with ß-thal requires further investigation. Identification of all types of Hb subunits in the retention time (RT) before 1 min. suggests that altered Hbs is the nature of these fast-eluting peaks. Relevancy of thalassemias to the protein-aggregation disorders will require review of the role of free globin in the pathology of the disease.

Computational Analysis of Protein Structure Changes as a Result of Nondeletion Insertion Mutations in Human ß-Globin Gene Suggests Possible Cause of ß-Thalassemia.

Beta-thalassemia is described as a group of hereditary blood disorders characterized by abnormalities in the synthesis of beta chains of hemoglobin. These anomalies result in different phenotypes ranging from moderate to severe clinical symptoms to no symptoms at all. Most of the defects in hemoglobin arise directly from the mutations in the structural ß-globin gene (HBB). Recent advances in computational tools have allowed the study of the relationship between the genotype and phenotype in many diseases including ß-thalassemia. Due to high prevalence of ß-thalassemia, these analyses have helped to understand the molecular basis of the disease in a better way. In this direction, a relational database, named HbVar, was developed in 2001 by a collective academic effort to provide quality and up-to-date information on the genomic variations leading to hemoglobinopathies and thalassemia. The database recorded details about each variant including the altered sequence, hematological defects, its pathology, and its occurrence along with references. In the present study, an attempt was made to investigate nondeletion mutations in the HBB picked up from HbVar and their effects using the in silico approach. Our study investigated 12 nucleotides insertion mutations in six different altered sequences. These 12 extra nucleotides led to the formation of a loop in the protein structure and did not alter its function. It appears that these mutations act as 'silent' mutations. However, further in vitro studies are required to reach definitive conclusions.

A Statistical Similarity/Dissimilarity Analysis of Protein Sequences Based on a Novel Group Representative Vector.

Similarity/dissimilarity analysis is a key way of understanding the biology of an organism by knowing the origin of the new genes/sequences. Sequence data are grouped in terms of biological relationships. The number of sequences related to any group is susceptible to be increased every day. All the present alignment-free methods approve the utility of their approaches by producing a similarity/dissimilarity matrix. Although this matrix is clear, it measures the degree of similarity among sequences individually. In our work, a representative of each of three groups of protein sequences is introduced. A similarity/dissimilarity vector is evaluated instead of the ordinary similarity/dissimilarity matrix based on the group representative. The approach is applied on three selected groups of protein sequences: beta globin, NADH dehydrogenase subunit 5 (ND5), and spike protein sequences. A cross-grouping comparison is produced to ensure the singularity of each group. A qualitative comparison between our approach, previous articles, and the phylogenetic tree of these protein sequences proved the utility of our approach.

DCGR: feature extractions from protein sequences based on CGR via remodeling multiple information.

BACKGROUND: Protein feature extraction plays an important role in the areas of similarity analysis of protein sequences and prediction of protein structures, functions and interactions. The feature extraction based on graphical representation is one of the most effective and efficient ways. However, most existing methods suffer limitations from their method design. RESULTS: We introduce DCGR, a novel method for extracting features from protein sequences based on the chaos game representation, which is developed by constructing CGR curves of protein sequences according to physicochemical properties of amino acids, followed by converting the CGR curves into multi-dimensional feature vectors by using the distributions of points in CGR images. Tested on five data sets, DCGR was significantly superior to the state-of-the-art feature extraction methods. CONCLUSION: The DCGR is practically powerful for extracting effective features from protein sequences, and therefore important in similarity analysis of protein sequences, study of protein-protein interactions and prediction of protein functions. It is freely available at https://sourceforge.net/projects/transcriptomeassembly/files/Feature%20Extraction .

Diagnosis of Hemoglobinopathy and ß-Thalassemia by 21 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and Tandem Mass Spectrometry of Hemoglobin from Blood.

BACKGROUND: Hemoglobinopathies and thalassemias are the most common genetically determined disorders. Current screening methods include cation-exchange HPLC and electrophoresis, the results of which can be ambiguous because of limited resolving power. Subsequently, laborious genetic testing is required for confirmation. METHODS: We performed a top-down tandem mass spectrometry (MS/MS) approach with a fast data acquisition (3 min), ultrahigh mass accuracy, and extensive residue cleavage by use of positive electrospray ionization 21 Tesla Fourier transform ion cyclotron resonance-tandem mass spectrometry (21 T FT-ICR MS/MS) for hemoglobin (Hb) variant de novo sequencing and ß-thalassemia diagnosis. RESULTS: We correctly identified all Hb variants in blind analysis of 18 samples, including the first characterization of homozygous Hb Himeji variant. In addition, an Hb heterozygous variant with isotopologue mass spacing as small as 0.0194 Da (Hb AD) was resolved in both precursor ion mass spectrum (MS1) and product ion mass spectrum (MS2). In blind analysis, we also observed that the abundance ratio between intact δ and ß subunits (δ/ß) or the abundance ratio between intact δ and α subunits (δ/α) could serve to diagnose ß-thalassemia trait caused by a mutation in 1 HBB gene. CONCLUSIONS: We found that 21 T FT-ICR MS/MS provides a benchmark for top-down MS/MS analysis of blood Hb. The present method has the potential to be translated to lower resolving power mass spectrometers (lower field FT-ICR mass spectrometry and Orbitrap) for Hb variant analysis (by MS1 and MS2) and ß-thalassemia diagnosis (MS1).

A Case Report of Compound Heterozygosity for ß0/ß+-Thalassemia Resulting from under Diagnosed ß-Thalassemia Found in a Hb A'2 Sample.

Hb A'2 (or Hb B2) (HBD: c.49G>C) is the most frequent δ chain variant that has been described in Africa but not in Thailand. We report here a 10-month-old Thai infant with compound heterozygosity for ß0 codon 17 (A>T; HBB: c.52A>T) and ß+ IVS II-654 (C>T; HBB: c.316-197C>T). Under diagnosed ß-thalassemia (ß-thal) in her father, who carries Hb A'2 and a heterozygous ß0 codon 17 mutation, and the mother, who carries a heterozygous ß+ IVS II-654 mutation, was noted. Although Hb A'2 does not cause any problems, heterozygosity for Hb A'2 can lead to under diagnosis of ß-thal in Hb A'2 samples. This case highlights the importance of Hb A'2 in prenatal diagnosis (PND). Thus, molecular analysis for ß-thal mutations should be carried out when a small peak presents at the retention time (RT) of 4.71 min. on high performance liquid chromatography (HPLC) and the summation level of this peak and Hb A2 was equal or higher than 4.0%.