Further Characterization of Hb Bronovo [α103(G10)His→Leu; HBA2: c.311A>T] and First Report of the Homozygous State

Hb Bronovo [α103(G10)His→Leu, HBA2: c.311A>T] is an α-globin variant that interferes with and decreases binding efficiency to α hemoglobin (Hb) stabilizing protein (AHSP), a chaperone molecule. The histidine residue at position 103 is integral to the AHSP hydrogen bond formation where disruption results in an increased quantity of cytotoxic free α-globin chains, thereby creating a similar pathophysiology as β-thalassemia (β-thal). We report a family with Hb Bronovo, including a homozygous proband, which resulted from maternal uniparental disomy (UPD). Although not detected by routine studies in previous reports, the variant protein is visible by intact mass spectrometry (MS).

Keywords: α-Globin; α hemoglobin (Hb) stabilizing protein (AHSP); Hb Bronovo; microcytic anemia; thalassemia

α-Globin, encoded by HBA1 and HBA2 located on chromosome 16, is a protein chain that interacts with other globins and the heme moiety to form the hemoglobin (Hb) tetramer. When in excess, insoluble free α-globin chains precipitate and damage the erythrocyte membrane. To ensure solubility and mitigate cell damage, α Hb stabilizing protein (AHSP) dimerizes with free α-globin chains prior to Hb tetramer assembly, at which point it is replaced by γ-, δ- or β-globin chains to form Hb F, Hb A2 and Hb A, respectively. When β-globin chains are relatively decreased in quantity such as in β-thalassemia (β-thal), excess α chains overwhelm AHSP capacity leading to red blood cell (RBC) destruction [[1]]. Conversely, α-thalassemia (α-thal), a clinically variable condition, results from a decreased quantity of α-globin chains creating an excess of β-like globin chains. Free β- and γ-globin chains form partially soluble and less damaging tetramers of Hb H (β4) and Hb Bart's (γ4), respectively. Unlike unpaired α chains, Hbs H and Bart's are detectable by protein methods, which can aid diagnoses of some α-thal conditions. α-Globin mutations that interfere with AHSP binding sites (G and H helix regions) increase free α chains and paradoxically result in α-globin inclusions and pathophysiology similar to β-thal [[2]].

Hb Bronovo is a Hb variant that results from a CAC>CTC transversion in HBA2, coding for an α-globin chain in which the native histidine residue is substituted for leucine at position 103 of the G helix [[3]]. The only report of the variant is in two heterozygous siblings of Turkish origin who were noted to have mild microcytic anemia (Table 1). The authors discussed the lack of protein detection by multiple methods [alkaline gel electrophoresis, cation exchange high performance liquid chromatography (HPLC) and capillary electrophoresis (CE)] and suggested the mutant chain may be unable to form stable multimers. Subsequent laboratory studies demonstrated in vitro Hb Bronovo protein expression in Escherichia coli, comparable in quantity to wild-type α-globin levels and predicted the leucine substitution to inhibit AHSP binding to half of the wild-type protein [[4]]; however, detection of the Hb Bronovo protein has not been confirmed in a clinical case. We report a family with Hb Bronovo to further clarify the nature of this unusual variant.

Table 1. Current and previous reports of Hb Bronovo (HBA2: c.311A>T).

1 Hb: hemoglobin; RBC: red blood cell count; MCV: mean corpuscular volume; MCH: mean corpuscular Hb; NA: not available; N/Abn: normal/abnormal; N/N: normal/normal.

• 2 Bold values: abnormal indices based on the original publication or the Mayo Clinic age-specific reference ranges.
• 3 aAssumed genotype for sister, MLPA/dosage not performed.
• 4 bHb Bronovo percentage estimated from MS (peak height of Hb Bronovo/peak heights of total α-globin chains). Harteveld et al. [[3]]: protein assessment included alkaline gel electrophoresis, HPLC (VARIANT II™; Bio-Rad Laboratories) and CE (CapillaryS2; Sebia).

Protein-based characterization performed at the Mayo Clinic (Rochester, MN, USA) included cation exchange high performance liquid chromatography (HPLC) [VARIANT II™; Bio-Rad Laboratories, Hercules, CA, USA], β-Thalassemia Short Program (Bio-Rad Laboratories), capillary electrophoresis (CE) (Hemoglobin E program, CapillaryS; Sebia, Lisses, France), isoelectric focusing (IEF) (RESOLVE Systems Hemoglobin Kit; Perkin Elmer, Waltham, MA, USA), and Hb stability studies (heat and isopropanol) using standard methods. In addition, intact globin chain mass spectrometry (MS) was performed using electrospray ionization (ESI) quadrupole time-of-flight MS (Agilent G6520 Quadrupole Time-of-Flight Detector; Agilent Technologies, Santa Clara, CA, USA) and results were analyzed using MassHunter Suite software (https://www.agilent.com/en/products/software-informatics/mass-spectrometry-software) (Agilent Technologies).

Molecular characterization was performed after DNA was extracted from whole blood leukocytes using the Qiagen EZ1 (Qiagen Inc., Valencia, CA, USA) system, and included sequencing of HBA1 and HBA2 genes in all family members and the HBB gene in the proband as previously described [[5]]. A multiplex ligation-dependent probe amplification (MLPA) assay to identify α-globin gene deletions/duplications covering the HBA1 and HBA2 gene cluster including the hypersensitive site-40 (HS-40) region was performed on the proband and both of his parents. The MLPA assay targeting the β-globin gene cluster including the locus control region (LCR), HBG1, HBG2, HBD and HBB genes was also performed in the proband. The previously described MLPA assay with Luminex-based detection and deletion polymerase chain reaction (PCR) technology was used to identify α- and β-globin gene cluster deletions/duplications using controls probes and 15 MLPA probes that spanned the 5′ regulatory region to the 3′ hypervariable region of the α-globin cluster or 40 MLPA probes that spanned the 5′ LCR upstream of HBE to the 3′ hypersensitivity site downstream from HBB for the β-globin cluster (Integrated DNA Technologies, Coralville, IA, USA), respectively [[6]].

Uniparental disomy (UPD) analysis of chromosomes 11 and 16 were also performed for the proband and his parents. Uniparental disomy studies were performed by a previously described technique [[7]]. In brief, samples were genotyped using PCR of chromosome-specific microsatellite markers (dinucleotide repeats). The markers used for chromosome 16 were D16S521, D16S418, D16S500, D16S3041, D16S3100, D16S3034, D16S3057, D16S503, D16S515, D16S516, D16S505 and D16S520; the markers used for chromosome 11 were D11S1363, D11S4046, D11S4146, D11S1760, D11S1338, D11S4116, D11S935, D11S987, D11S1314, D11S937, D11S901, D11S898, D11S4151, D11S1320 and D11S968. Diagnosis of UPD required that the proband carries at least two informative markers representing uniparental inheritance of chromosome 16, in addition to all informative markers for chromosome 11 showing biparental inheritance [[9]].

A 3-year-old male patient presented with persistent microcytosis and anemia. The proband and his 9-year-old sister are offspring of a Turkish mother and English father. The past medical history and physical examination were otherwise unremarkable, with no evidence of splenomegaly. The boy had two previously normal Hb electrophoreses performed at another institution. The complete blood count (CBC) data showed: RBC count of 4.9 × 1012/L, Hb of 9.7 g/dL, packed cell volume (PCV) of 0.31 L/L, mean corpuscular volume (MCV) of 63.4 fL, and RBC distribution width of 15.0%. Iron studies showed a mildly decreased ferritin of 10.2 μg/L (normal range 24.0–336.0 μg/L), iron of 82.0 μg/dL, and total iron binding capacity of 395.0 μg/dL. The degree of microcytosis and anemia was not felt to be fully explained by the mixed iron study results and further evaluation was performed.

Table 1 summarizes the data for each family member with comparison to previously published data. The proband's mother and sister showed hematological parameters consistent with mild thalassemia; however, the three affected family members showed low or low normal ferritin levels obscuring the clinical picture. The proband's father showed normal hematological parameters and normal ferritin. High performance liquid chromatography, CE and IEF were normal in the entire family. However, MS detected an abnormal peak at 15,103.0 amu (Figure 1) in the proband, his mother, and his sister but showed a normal result in the father. The estimated MS variant peak percentage was 23.0% of total α-globins in the proband, 2.7% in his mother and 3.4% in his sister. Similar results were obtained on repeated MS testing on subsequent blood samples.

PHOTO (COLOR): Figure 1. Mass spectrometry of Hb Bronovo in the proband (A), his mother (B) and his father (C). Normal α- and β-globin peaks are present (151,126.7 and 15,867.5 amu, respectively). The y-axis (height of peak) represents the abundance of each respective molecule. A peak present at approximately 15,103.0 amu (arrow) correlates with a His>Leu amino acid substitution. (A) The proband has a peak at 15,102.69 amu that approximates 23.0% of total α-globin chains. (B) The proband's mother has a peak at 15,103.53 amu that approximates 2.7% of total α-globin chains. (C) The proband's father does not have a peak detected at this mass and is a normal tracing. Images are adapted from MassHunter Suite software.

α-Globin gene sequence analysis confirmed the presence of Hb Bronovo [α103(G10)His→Leu; HBA2: c.311A>T] in three family members including an apparently homozygous result in the proband [Figure 2(A)], a heterozygous result in his mother and sister [Figure 2(B)], and a wild-type result in his father. Multiplex ligation-dependent probe amplification α-globin gene cluster deletion/duplication analyses performed on the proband and both parents were negative. β-Globin gene sequence analysis and β-globin gene cluster deletion/duplication analyses performed on the proband were negative.

PHOTO (COLOR): Figure 2. Sequence of Hb Bronovo in the proband (A) and his mother (B). Sanger sequence reads are presented in the forward and reverse direction. Arrows on the right designate the T nucleotide (variant), and arrows on the left designate the A nucleotide (wild-type). The proband's mother has both bases present at this position (W), which represents the heterozygous state. A wild-type allele is not detected in the proband. Images are adapted from Mutation Surveyor Version 5.0.1.

Due to the inheritance pattern discrepancy, further testing was performed to confirm paternity and evaluate for the possibility of UPD (inheritance of a chromosome pair from one parent). Subsequent studies confirmed biparental inheritance in 10 informative markers on chromosome 11 and revealed full maternal UPD across informative markers on chromosome 16 [Figure 3(A)] most consistent with segmented isodisomy with heterodisomy [Figure 3(B)].

MAP: Figure 3. Uniparental disomy studies. (A) Map of chromosome 16 with the D16 markers used in the analysis. The HBA2 gene is located near the tip of the p arm. (B) Results of the UPD study in the proband (denoted by the arrow) and his parents. Six markers were informative for chromosome 16 and confirm maternal UPD most consistent with segmented isodisomy with heterodisomy. Analysis shows only maternal markers in the genomic coordinates flanking the HBA2 gene (markers D16S521 and D16S418).

Hb Bronovo is an incompletely characterized α Hb variant that was first described in two siblings of Turkish descent with a mild thalassemia phenotype. Because the variant was not detected by routine methods, the authors postulated the variant could possibly be hyperunstable [[3]]. We report the identification of another family of Turkish descent with Hb Bronovo, one in a homozygous state. Similar to the previous report, the variant was not detectable by routine methods including CE, cation exchange HPLC and IEF; however, intact MS detected an abnormal variant peak. For unknown reasons, the homozygous state was associated with a much higher estimated variant percentage than the heterozygous state. Stability studies showed variable results in the affected family members but did not appear to be the cause of the comparatively larger percentage of variant protein in the homozygous proband. Other causes such as ferritin levels were considered but low-to-borderline low normal levels were seen in all three affected family members.

Histidine at position 103 impacts α-globin binding to AHSP, a chaperone protein that stabilizes and solubilizes free α-globin chains. In normal Hb molecules, His103 is involved in a hydrogen bond with Asp43 of AHSP [[10]]. A disruptive amino acid substitution would decrease protein binding affinity and results in increased free α-globin chain quantities. Excess insoluble free α-globin chains precipitate and damage the erythrocyte membrane and result in a mechanism of thalassemia similar to β-thal, albeit with normal Hb A2 levels.

The proband, his mother, and his sister all displayed a mild thalassemia phenotype with more pronounced microcytosis in the homozygote. The low or borderline low ferritin levels may support an element of concurrent iron deficiency. Other AHSP-related Hb variants near position 103 that present with a thalassemia phenotype include Hb Sallanches [α104(G11)Cys→Tyr; HBA2: c.314G>A] and Hb Oegstgeest [α104(G11)Cys→Ser; HBA1: c.313T>A] [[11]].

Although the proband is homozygous for Hb Bronovo, his father had neither the variant (Figure 1) nor any α-thal deletions and further study was performed. Uniparental disomy testing confirmed paternity and revealed maternal UPD involving the HBA2 gene [Figure 3(A) and (B)].

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.