HbVar Database Research Articles

Characterization of Hemoglobin Variants Due to in-Frame Deletions in the β-Globin Gene from CRISPR/Cas9 Gene Correction for Sickle Cell Disease

Introduction: Sickle cell disease (SCD) is caused by a single nucleotide mutation in the β-globin gene (HBB). CRISPR/Cas9 based gene correction approaches rely on inducing a double-strand break (DSB) at or near the sickle mutation, which subsequently results in gene correction via the homology-directed repair (HDR) or insertions/deletions (Indels) via non-homologous end joining (NHEJ). Frameshift Indels induce a premature stop codon and disrupt HBB production. Indels that preserve the reading frame may yield structurally abnormal β-globin chain with altered amino acid sequences and protein structures that can affect the globin chain stability, leading to thalassemia of uncertain pathogenic significance. The high rate and diversity of in-frame Indels as an unintended by-product of gene correction therapy confers a challenge for predicting therapeutic outcomes when it is not possible to frame the clinical behavior of hemoglobin (Hb) variants. Here we provided the molecular and structural characterization of the most commonly identified Hb variants due to CRISPR-induced in-frame deletions and proposed a putative pathogenic mechanism for the observed phenotype using human umbilical cord-derived erythroid progenitor cells (HUDEP2). Methods: HUDEP2 clonal cell lines carrying mutated Hb variants from in-frame deletions on HBB exon1 introduced by R-66S and R-02 CRISPR sgRNAs currently in use in preclinical and clinical studies for correcting sickle mutation, respectively, were established, and clonal genotypes validated by next-generation sequencing and droplet digital PCR based copy number assay. The Hb variants derived from R-66S include p.Val7_Ser10del (-12bp), p.Val7del (-3bp), p.Glut8del (-3bp), p.Glut8_Lys9del (-6bp), pVal7Thr13del (-21bp), p.His3_Lys9delinsGln (-18bp), and p.Thr5_Lys18del (-42bp), in the order of allelic abundance observed in the bulk population of edited cells. The Hb variants derived from R-02 include p.Val12_Ala14del (-9bp), p.Val12_Thr13del (-6bp), p.Ala14del (-3bp), p.Thr13_Gly17del (-15bp), and p.Thr13_Val19delinsMet (-18bp). Selected ten clonal cell lines carrying a unique combination of genotypes and the parent HUDEP2 were cultured for expansion in serum-free media for one week. All clones were subsequently subjected to erythroid differentiation media for ten days. Samples were collected on days 4, 7, and 10 of differentiation for analysis via high-performance liquid chromatography (HPLC) using an ionic column to access the Hb profile and spectrophotometry via a Hb assay kit to assess the total Hb quantity. Results: HPLC analysis demonstrated a significant qualitative variation in Hb profile in HUDEP2 clones carrying different Hb variants. p.Val7del is Hb Leiden, and the rest of Hb variants are not listed in the HbVar database. In clones carrying Hb Leiden in homozygous or heterozygous states, Hb Leiden eluted in the position of sickle Hb (HbS). A low percentage of multiple abnormal Hb X fractions was observed in all clones carrying Hb variants compared to parent HUDEP2, which hinted at the presence of unstable Hb variants or alpha precipitates. Levels of fetal Hb (HbF) increased in mutant clones, suggesting globin chain imbalance and hematopoietic stress. Clones missing multiple amino acids showed ineffective erythropoiesis and cell death by day 4 of differentiation, most likely exacerbated by the relative excess of the α-chains. Discussion: We found that unintended gene modifications can significantly impact Hb quality and quantity, reinforcing concerns about the safety of gene-editing therapy for SCD. In patients, cells carrying in-frame deletions in the β-globin chain could produce an unstable Hb and may contribute to a β-thalassemia-like phenotype. More work is required to further assess the consequences of the diverse Hb variants in combination with other unintended modifications that disrupt HBB in a bulk population of gene-edited cells. Detecting, profiling, and reducing these unintended modifications remains a priority to increase the efficacy and safety of gene-editing therapy for treating SCD or β-thalassemia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A New Mutation, Hb A2-Canakkale [δ10(A7)Ala→Val; HBD: c.32C>T], and Other Well-Known δ Variants Identified in a Selected Cohort with Low Hb A2 Levels

Hemoglobinopathies are the most common single-gene disorders, and β-thalassemia (β-thal) imposes a tremendous health burden on Turkey. Thus, premarital carrier screening is obligatory in Turkey, as it is in some other countries. As a result of this mandatory procedure, at routine clinical checkups, many individuals who had undergone premarital screening but did not have any clinical symptoms and/or hematological findings, have compulsorily been required to undergo further evaluation due to abnormal levels of hemoglobin (Hb) fractions (Hb A, Hb A2 and Hb F). Many consequences, such as mutations in unrelated gene(s) or someone’s nutritional status, have been reported to affect the Hb fractions levels. In the present study, we aimed to determine whether HBD has a molecular causative role in patients with low Hb A2 levels (below 1.8%). The study was conducted with 20 individuals with low Hb A2 levels who had applied to our outpatient clinic. All DNA samples were analyzed for the HBD gene. Nineteen of the 20 subjects were diagnosed to carry a mutation with one of four different δ-globin variants. Three of them had been described previously [Hb A2-Yialousa (HBD: c.82G>T), Hb A2-Bornova (HBD: c.350G>C) and Hb A2-Yokoshima (HBD: c.77G>A)]. The novel [δ10(A7)Ala→Val, HBD: c.32C>T] mutation was defined as a new δ variant and reported to the HbVar database as Hb A2-Canakkale. In conclusion, the molecular characterization of Hb A2 low levels has been suggested to be significant for a definite diagnosis and counseling.

Assessment of molecular and clinical characteristics in blood transfusion dependent HbE/β-thalassemia patients

Hb E/β-thalassemia is one of the most common forms of hemoglobinopathies worldwide. This life-threatening genetic disorder is not completely curable. This study aims to investigate the correlation of mutations with clinical manifestations among the transfusion dependent Hb E/β- thalassemia patients using molecular, hematological and biochemical methods. A total of 60 blood samples were collected from regular blood transfusion-dependent HbE/β-thalassemia patients. Mutations within HBB gene were analysed by Sanger sequencing, BLAST 2.0, HbVar database and Alibaba 2.1. Clinical data were analysed based on CBC, Hemoglobin Electrophoresis and Serum Ferritin assay. Results demonstrate that a total of three mutationsdeletion of A (+23bp in HBB promoter region), c.9 T>C and c.79 G>A were detected among the study population, where deletion of A (+23bp in HBB promoter region) is a novel mutation. A total of three (USF, AP-1 and GATA-1) important putative binding sites were identified within the mutated region of HBB gene. Hematological analysis also showed remarkable correlation between the mutations and clinical manifestations in the patients. Reduced levels of RBC, Hb, MCV and MCH were found among the patients than the normal. Highly increased level of serum ferritin was also found among the patients due to the frequent destruction of RBCs. In conclusion, the findings of this study will be helpful to understand the mutational and hematological status of HbE/β-thalassemia patients. And this study will also be beneficial for effective molecular drug designing, drug response and other therapeutic approach for HbE/β-thalassemia patients.
 Jahangirnagar University J. Biol. Sci. 9(1 & 2): 109-121, 2020 (June & December)

Clinically relevant updates of the HbVar database of human hemoglobin variants and thalassemia mutations.

HbVar (http://globin.bx.psu.edu/hbvar) is a widely-used locus-specific database (LSDB) launched 20 years ago by a multi-center academic effort to provide timely information on the numerous genomic variants leading to hemoglobin variants and all types of thalassemia and hemoglobinopathies. Here, we report several advances for the database. We made clinically relevant updates of HbVar, implemented as additional querying options in the HbVar query page, allowing the user to explore the clinical phenotype of compound heterozygous patients. We also made significant improvements to the HbVar front page, making comparative data querying, analysis and output more user-friendly. We continued to expand and enrich the regular data content, involving 1820 variants, 230 of which are new entries. We also increased the querying potential and expanded the usefulness of HbVar database in the clinical setting. These several additions, expansions and updates should improve the utility of HbVar both for the globin research community and in a clinical setting.

HBB gene mutation spectrum in an Indian cohort of 1530 cases using an in-house targeted next-generation sequencing assay

Beta thalassemia major is a common genetic disorder characterized by the reduced production or absence of beta globin, a product of the haemoglobin subunit beta (HBB) gene. Every year, approximately 10,000–12,000 children with thalassemia major are born in India. Molecular methodologies like ARMS (amplification-refractory mutation system)-PCR (polymerase chain reaction) and capillary sequencing are used to detect HBB gene mutations. It is, however, challenging to achieve comprehensive coverage of the HBB gene by these methods. Next-generation sequencing (NGS) can be used to circumvent these problems. Commercial NGS panels are prohibitively expensive and hence are not routinely implemented in most laboratories. We have developed a cost-effective, highly sensitive and specific, indigenous targeted NGS assay for detecting mutations in the HBB gene. Using this custom NGS assay, we processed 1530 samples (3017 alleles), in which we detected a spectrum of 48 pathogenic/likely pathogenic variants (mutations); IVS-I-5 (c.92+5G>C) was the most common mutation detected (allele frequency (AF) 44.55%), followed by the 619-bp deletion (AF 10.74%), c.92+1G>T (AF 6.99%), c.27_28insG (AF 6.23%), c.47G>A (AF 5.77%) and c.126_129delCTTT (AF 4.71%). Additionally, we discovered a novel mutation (c.7delC) that was submitted to the HbVar database (HbVar ID 3193) as a variant of unknown significance (VUS), probably pathogenic. The targeted NGS assay developed during this study was validated using orthogonal methods and showed excellent correlation with currently available molecular methods. Additionally, this targeted NGS assay was used to analyse the mutation spectrum of the largest beta thalassemia cohort from India.

First observation of hemoglobin G-Norfolk in the Turkish population

Abstract Objectives Hemoglobinopathies are inherited transition blood diseases associated with globin chains of the hemoglobin. However many mutations have been defined, there may be many of them not defined yet. We here report the first case of those mutations, named Hb G-Norfolk in Turkey. Case presentation A 15 years-old male patient with erythrocytosis was referred to our laboratory for the evaluation of hemoglobinopathy. In chromatographic analysis, an unidentified peak was observed. A similar identification for variant Hb could not be obtained from High-Performance Liquid Chromatography (HPLC) analyzer’s data library. No definitive diagnosis could also be made by different analyzer. Family screening and molecular genetic DNA sequence analysis were carried out. Conclusions Although there were not found any beta gene mutation of neither the patient nor his family, analyses of alpha genes A1 and A2 were performed and abnormal hemoglobines were detected for all of them. This change in the HbA2 gene was at codon85 GAC>AAC (Asp>Asn) in the heterozygous state, known as Hb G-Norfolk [HbA2:c256G>A p.Asp85Asn] based on HbVar database. Abnormal Hb bands detected by HPLC with clinical findings such as erythrocytosis or cyanosis should be investigated by sequence analysis to corroborate alpha and/or beta-globin gene mutations for avoiding misdiagnosis and misinterpretation.

The Importance of Characterizing the Hemoglobin Instability of New Variants: The Case of Hb Dompierre [β29(B11)Gly→Arg, HBB: c.88G>C

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.

Double Heterozygosity for Hb Durham-N.C. (HBB: c.344T>C) [β114(G16)Leu→Pro] and the IVS-I-110 (HBB: c.93-21G>A) Causing a Severe β-Thalassemia Phenotype

The evaluation of a 10-month-old girl of Sicilian origin with a clinical phenotype of severe thalassemia led to the identification of two β-globin gene defects, a β-thalassemia (β-thal), mutation at IVS-I-110 (HBB: c.93-21G>A) and a variant hemoglobin (Hb) mutation at codon 114 (HBB: c.344T>C) on the other allele, reported as Hb Durham-N.C. (also known as Hb Brescia) [β114(G16)Leu→Pro] in the HbVar database. A very low Hb level (Hb 3.5 g/dL), microcytosis [mean corpuscular volume (MCV) 63.2 fL] and hypocromia [mean corpuscular Hb (MCH) 19.6 pg], increased red blood cell (RBC) distribution width (RDW) (36.0%), higher reticulocytes (6.2%), anisocytosis, poikilocytosis, hypocromia, basophilic stippling and inclusion body formation, were present in the affected subject. Analysis of other family components showed the presence of HBB: c.93-21G>A defect in the mother and in her brother, while Hb Durham-N.C. was absent in all other relatives, thus, this mutation has arisen as a de novo defect. This is the first case described as a severe thalassemic phenotype in a compound heterozygote carrier of this unstable Hb and a common β-thalassemic allele. The important information gained from this case is that a rare dominant or recessive mutation may arise in every individual, even if this is a very rare event.

Characterization of the IVS-II-821 (A>C) (HBB: c.316-30A>C) Mutation in a β-Thalassemia Phenotype in Iran

β-Thalassemia (β-thal) is the most frequently observed hereditary blood disorder that results from genetic defects causing deficient synthesis of hemoglobin (Hb) polypeptide chains. Detecting thalassemia mutations are necessary for prenatal diagnosis (PND) programs leading a better quality of life for the patients, as well as a reduction in the cost of their medical care. There are more than 900 different genomic mutations of the β-globin gene described in the human hemoglobin variant (HbVar) database. In this study, we identified a mid-intronic mutation at IVS-II-821 (A>C) (HBB: c.316-30A>C) position in the HBB gene of an Iranian proband and two of her siblings that was associated with β-thal clinical features. Direct DNA sequence analysis was performed by mutation scanning of the β-globin gene. Based on the observed β-thal phenotype and bioinformatics analysis results, we concluded that this β-globin gene mutation was associated with a mild phenotype of β-thal through activating potential splice sites by creating exonic splicing enhancers (ESEs), exon-identity element (EIE) and exonic splicing regulatory sequences (ESRs) sites.

The Believe Trial: Results of a Phase 3, Randomized, Double-Blind, Placebo-Controlled Study of Luspatercept in Adult Beta-Thalassemia Patients Who Require Regular Red Blood Cell (RBC) Transfusions

The Believe Trial: Results of a Phase 3, Randomized, Double-Blind, Placebo-Controlled Study of Luspatercept in Adult Beta-Thalassemia Patients Who Require Regular Red Blood Cell (RBC) Transfusions

Short in-Frame Insertions/Deletions in the Coding Sequence of the α-Globin Gene. Consequences of the 3D Structure and Resulting Phenotypes: Hb Choisy as an Example

A small group of hemoglobin (Hb) variants result from ‘in-frame’ deletion/insertion (del/ins). We describe a new variant of this group (Hb Choisy), found on the α1 gene, which is the exact counterpart of a previously published deletional variant, Hb J-Biskra [codons 51-58 (or codons 52-59) (–24 bp) (-TCTGCCCAGGTTAAGGGCCACGGC); HBA1: c.157_180del (or HBA2)]. In Hb J-Biskra, the sequence Ser-Ala-Gln-Val-Lys-Gly-His-Gly located from positions α52(E1) to α59(E8) is deleted, while in Hb Choisy the same sequence (Ser-Ala-Gln-Val-Lys-Gly-His-Gly) is inserted at position α52(E1). The variant carrying the insertion appears to be less damaging than the one with the deletion. A possible explanation could be that the additional sequence is located in the C to E interhelical region, and is less disturbing to the general structure of the globin chain. This insertion/deletion (ins/del) is likely favored by the repetition, at an interval of 16 nucleotides, of an eight nucleotide sequence. Comparison of variants of this group, found in the HbVar database, shows that structural modifications resulting from insertions are frequently less damaging than that caused by deletions.

A Novel β-Thalassemia Mutation in a Chinese family: IVS-II-203-205 (TCT>CC) (HBB: c.315+203TCT>CC)

β-Thalassemia (β-thal) is one of the most common inherited disorders in southern China. More than 300 β-globin gene mutations around the world have been reported in the HbVar database. In this study, a novel mutation in a 30-year-old Chinese woman [IVS-II-203-205 (TCT>CC), HBB: c.315+203TCT>CC] was first found by direct sequencing. Subsequently, investigation of her parents’ genetic codes was completed, and the results showed that her father also carried this mutation. Based on the features observed in clinical practice, this novel mutation was regarded as a mild phenotype of β-thal.

Hb Mozhaisk [β92(F8)His→Arg; HBB: c.278A>G] as a De Novo Mutation in a Child of Mixed Ethnic Origins

Approximately 150 variants described in the HbVar database have been found to be unstable and about 80.0% of these are on the β-globin gene. We describe the case of a 3-year-old child who presented at the emergency room with fever and asthenia. Hematological data suggested severe hemolytic anemia. Sequencing of the β-globin gene revealed the mutation HBB: c.278A>G at codon 92 in a heterozygous state, reported as Hb Mozhaisk in the HbVar database. Other family members did not have Hb Mozhaisk, thus, this variant is due to a de novo mutation. Because of the rarity of this globin variant, we believe it is important to report similar cases, to have a more complete phenotype description of the pathology and define an adequate reproductive risk for couples, considering the dominant inheritance pattern (hence an inheritance risk of 50.0%).

Hemoglobin Kirklareli (α H58L), a New Variant Associated with Iron Deficiency and Increased CO Binding

Mutations in hemoglobin can cause a wide range of phenotypic outcomes, including anemia due to protein instability and red cell lysis. Uncovering the biochemical basis for these phenotypes can provide new insights into hemoglobin structure and function as well as identify new therapeutic opportunities. We report here a new hemoglobin α chain variant in a female patient with mild anemia, whose father also carries the trait and is from the Turkish city of Kirklareli. Both the patient and her father had a His-58(E7) → Leu mutation in α1. Surprisingly, the patient's father is not anemic, but he is a smoker with high levels of HbCO (∼16%). To understand these phenotypes, we examined recombinant human Hb (rHb) Kirklareli containing the α H58L replacement. Mutant α subunits containing Leu-58(E7) autoxidize ∼8 times and lose hemin ∼200 times more rapidly than native α subunits, causing the oxygenated form of rHb Kirklareli to denature very rapidly under physiological conditions. The crystal structure of rHb Kirklareli shows that the α H58L replacement creates a completely apolar active site, which prevents electrostatic stabilization of bound O2, promotes autoxidation, and enhances hemin dissociation by inhibiting water coordination to the Fe(III) atom. At the same time, the mutant α subunit has an ∼80,000-fold higher affinity for CO than O2, causing it to rapidly take up and retain carbon monoxide, which prevents denaturation both in vitro and in vivo and explains the phenotypic differences between the father, who is a smoker, and his daughter.

IVS-II-648/649 (–T) (HBB: c.316−202del) Triggers a Novel β-Thalassemia Phenotype

Thalassemia is the most common inherited disorder in Iran. There are approximately 800 different genomic alterations of the β-globin gene described in the HbVar database. In this study, we identified a novel mutation in a 21-year-old woman [IVS-II-648/649 (–T); HBB: c.316−202del)] and describe its clinical implications. Two other members of this family, all with hematological and clinical features associated with β-thalassemia (β-thal), also carried this mutation. The molecular diagnosis of the β-globin gene mutation was performed by direct sequencing. Based on the observed β-thal phenotype and in silico analysis results, we concluded that this novel β-globin gene mutation was associated with the mild phenotype of β-thal.

Hb Midnapore [β53(D4)Ala→Val; HBB: c.161C>T]: A Novel Hemoglobin Variant with a Structural Abnormality Associated with IVS-I-5 (G>C) (HBB: c.92+5G>C) Found in a Bengali Indian Family

We describe a novel C>T substitution at codon 53 of the HBB gene (HBB: c.161C>T). The proband was a transfusion-dependent β-thalassemia major (β-TM) patient. DNA was extracted and subsequently, DNA sequencing was done to detect the mutations on the HBB gene. Capillary zone electrophoresis (CZE) revealed the presence of an unknown peak. She inherited this mutation from her grandmother through her mother. This mutation exists in cis with the common β0 mutation IVS-I-5 (G>C) (HBB: c.92+5G>C). The proband is homozygous for HBB: c.92+5G>C and needs monthly transfusions. On the other hand, her grandmother, mother and sister all possess this novel mutation cis with the heterozygous HBB: c.92+5G>C. They are carriers not thalassemic. This mutation produces the substitution β53(D4)Ala→Val; HBB: c.161C>T, a new structural hemoglobin (Hb) variant. As this variant was identified in a Bengali family from Paschim Midnapore district of West Bengal, India, it has been designated as Hb Midnapore. This variant has now been reported to the HbVar database.

Coinheritance of a Rare Nucleotide Substitution on the β-Globin Gene and Other Known Mutations in the Globin Clusters: Management in Genetic Counseling

A large number of methods for DNA analysis are available to identify defects in globin genes associated with hemoglobin (Hb) disorders. In this study, we report a rare nucleotide (nt) substitution on the β-globin gene, nt 781 in the second intron [IVS-II-781 (C > G); HBB: c.316-70C > G], identified in four patients. This nt substitution was previously described only as a personal communication to the HbVar database and indicated as a β0 or β+ mutation. The purpose of this study was to evaluate the clinical implication of this nt change, particularly when coinherited with severe β-thalassemia (β-thal), in order to be able to conduct appropriate genetic counseling. Genetic studies were performed on two subjects, one carried Hb S [β6(A3)Glu→Val; HBB: c.20A > T], and the other carried IVS-I-110 (G > A) (HBB: c.93-21G > A). All these subjects showed this new β nt substitution in association with Hb A2' (or Hb B2) [δ16(A13)Gly→Arg; HBD: c.49G > C]. Another 16 samples, carrying the same δ variant as the probands, were processed by β-globin gene sequencing in order to better understand the correlation between this Hb variant and the rare nt substitution reported in this study. The present investigation emphasizes the importance of sharing the observed nt changes in the globin gene cluster, especially in the case of new or rare undefined mutations, in order to facilitate the determination of their phenotypic expression, the possible interactions with known molecular defects and to formulate appropriate genetic counseling for at-risk couples.

Characterization of the HBB: c.*233G > C Variant: No Evidence of a β-Thalassemic Phenotype

β-Thalassemia (β-thal) results from homozygous or compound heterozygous inheritance of β-globin alleles that yield decreased or absent synthesis of the β chain. Disease is frequently severe, requiring lifelong transfusion therapy. Heterozygosity for a β-thal allele results in an asymptomatic carrier state with mild but characteristic hematological findings. More than 200 β-globin alleles have been demonstrated to produce β-thal. For populations with a high prevalence of β-thal, screening for carrier status, genetic counseling and prenatal diagnosis are important components of efforts to both reduce disease incidence and provide early diagnosis and treatment. It is therefore important to define and characterize potential β-thal alleles. We sought to further characterize the previously reported β-thal allele, HBB: c.*233G > C. This variant is provisionally included in the HbVar database based on a study of Palestinians in the Gaza Strip with β-thal disease or carrier status (known or suspected) where 4.2% of subjects were found to have HBB: c.*233G > C. In our patient population, we detected the HBB: c.*233G > C variant in 17.3% of individuals (17 heterozygotes, one homozygote) undergoing β hemoglobin (Hb) gene sequencing at our laboratory over a 25-month period. Hematological parameters were analyzed to determine if these individuals demonstrated findings consistent with inheritance of a β-thal allele. Individuals with the HBB: c.*233G > C variant did not demonstrate any abnormalities in hematological parameters characteristic of β-thal carrier state (17 heterozygotes) or clinical evidence of disease (homozygote). Our data demonstrate no evidence for pathogenicity of the HBB: c.*233G > C variant but rather demonstrate that this variant is a common benign polymorphism.

Two New γ Chain Variants: Hb F-Augusta GA [Gγ59(E3)Lys → Arg; HBG2: c.179A > G] and Hb F-Port Royal-II [Aγ125(H3)Glu → Ala; HBG1: c.377A > C

The total number of hemoglobin (Hb) variants so far reported to the HbVar database is 1598 (April 9 2014) and 130 of them are fetal Hb variants. Fetal Hb are categorized as two different subunits, Gγ- and Aγ-globin chains, and γ chain variants can be observed in both subunits. There are 72 Gγ- and 58 Aγ-globin chain variants. Most of them are clinically silent and detected during newborn screening programs in the USA and outside the USA. In this report, we discuss the molecular characteristics and diagnostic difficulties of two new γ-globin chain variants found in an African American baby with no clinical symptoms. One is a new Gγ-globin chain variant, Hb F-Augusta GA [Gγ59(E3)Lys → Arg; HBG2: c.179A > G] and the other one is Hb F-Port Royal-II [Aγ125(H3)Glu → Ala; HBG1: c.377A > C].

Updates of the HbVar database of human hemoglobin variants and thalassemia mutations

HbVar (http://globin.bx.psu.edu/hbvar) is one of the oldest and most appreciated locus-specific databases launched in 2001 by a multi-center academic effort to provide timely information on the genomic alterations leading to hemoglobin variants and all types of thalassemia and hemoglobinopathies. Database records include extensive phenotypic descriptions, biochemical and hematological effects, associated pathology and ethnic occurrence, accompanied by mutation frequencies and references. Here, we report updates to >600 HbVar entries, inclusion of population-specific data for 28 populations and 27 ethnic groups for α-, and β-thalassemias and additional querying options in the HbVar query page. HbVar content was also inter-connected with two other established genetic databases, namely FINDbase (http://www.findbase.org) and Leiden Open-Access Variation database (http://www.lovd.nl), which allows comparative data querying and analysis. HbVar data content has contributed to the realization of two collaborative projects to identify genomic variants that lie on different globin paralogs. Most importantly, HbVar data content has contributed to demonstrate the microattribution concept in practice. These updates significantly enriched the database content and querying potential, enhanced the database profile and data quality and broadened the inter-relation of HbVar with other databases, which should increase the already high impact of this resource to the globin and genetic database community.