Novel mechanism of hereditary pyropoikilocytosis phenotype due to co‐inheritance of β globin and α spectrin mutations
Authors: Soo Jin Kim, Jihyun Song, N. Scott Reading, Julio Lautersztain, Abdullah Kutlar, Archana M. Agarwal, Theresa L. Coetzer, Josef T. Prchal
Journal: American Journal of Hematology (2021)

To the Editor: Co-existence of red blood cell (RBC) membrane disorders and hemoglobinopathies are rare and may present a diagnostic challenge due to overlap of clinical findings. We describe a novel mechanism for the phenotype of hereditary pyropoikilocytosis (HPP), associated with dominant β-thalassemia due to a novel β-globin gene (HBB) mutation, exacerbated by co-inheritance of two α-spectrin gene (SPTA1) mutations in trans. Beta-thalassemia is generally an autosomal recessive disorder caused by HBB mutations, which affect the production of the β-globin subunit of hemoglobin. Frameshift or nonsense mutations in HBB exons 1 and 2 are common, resulting in abnormal termination codons, which trigger nonsense-mediated mRNA decay (NMD) that eliminates the defective mRNA.1 Individuals who are heterozygous for mutations in HBB generally have β-thalassemia minor or a trait with microcytosis and mild or no anemia. However, there are rare HBB mutations that lead to a dominantly inherited form of β-thalassemia causing moderate to severe symptomatic anemia. Splenomegaly is often present and hemolysis may be prominent. More than 40 of these mutations have been described, ranging from heterogeneous missense mutations to frameshift mutations, caused by small insertions and deletions, resulting in truncated or elongated β-globin variants.1 Most of these mutations are located in the distal part of HBB exon 2 or 3 and tend to escape NMD. This leads to an accumulation of defective peptides, which are hyper-unstable, non-functional and toxic when combined with normal α/β globin dimers. These variants may exhibit heterogeneous clinical phenotypes within families.2 Hereditary pyropoikilocytosis is a rare severe hemolytic disorder characterized by unique poikilocytic and microspherocytic erythrocytes.3 The HPP patients are typically homozygous or compound heterozygotes for mutations in SPTA1, which markedly reduce the “horizontal interactions” of spectrin αβ heterodimers to form tetramers, the main structural unit of the RBC membrane skeleton, that regulates the shape and deformability of the cell.4 These abnormalities severely weaken the skeleton resulting in poikilocyte formation during circulatory shear stress. Hereditary pyropoikilocytosis patients also exhibit a decreased amount of spectrin, which interferes with the “vertical interactions” between the skeleton and the lipid bilayer and leads to loss of membrane and microspherocytes. This combination of qualitative and quantitative defects of spectrin is a hallmark of HPP.4 Here we describe a 59-year-old Dominican male who presented with life-long history of severe microcytic hypochromic anemia and jaundice, with his hemoglobin ranging from 5–7 g/dL requiring occasional RBC transfusions, without iron deficiency. The clinical and laboratory findings are shown in Figure 1(A). His peripheral blood smear (Figure 1(B)) revealed typical HPP RBC morphology. After initial evaluation, the proband became progressively severely anemic. Ten months later, he underwent bone marrow biopsy that was compatible with myelodysplastic syndrome, confirmed by cytogenetic abnormalities. He was then lost to follow up. We performed several investigations as outlined in supplementary data. Sequencing of the HBB gene revealed a novel pathogenic frameshift β-thalassemia mutation in exon 2, HBB282delT, coding for β-globin Cys93fs. No large deletions or duplications were detected by multiplex ligation-dependent probe amplification (MLPA) analysis. Computational algorithms predicted that intron 2 splicing is not affected, and the mutant HBB transcript was confirmed by Sanger sequencing using cDNA obtained from the patient's peripheral blood granulocyte RNA. As we could not obtain sufficient reticulocyte RNA, we performed in vitro expansion of erythroid progenitors in 3-week liquid erythroid cultures. Initially, most of the peripheral blood mononuclear cells were lymphoid cells. At day 7, most lymphoid cells were dead, and the remaining cells were erythroid progenitors. The cells expressing CD71 at day 7 were counted showing that the difference in cell number between samples was minimal. Progenitor cells were harvested at day 14 and we detected a mutant HBB transcript missing the T in codon 93 of exon 2. Equal amounts of the mutant and wild type HBB transcripts were present, indicating that NMD was not induced. The mutation is predicted to result in an elongated protein of 156 amino acids, referred to as Hb Santo Domingo, containing ten additional residues at the C-terminal end, and altering all the amino acids downstream of the frameshift until a new stop codon terminates translation (Figure S1). The mutant sequence is enriched in hydrophobic amino acids, including nine tryptophan residues, which have bulky side chains that influence the structural aspects and stability of the peptide. Secondary structure predictions revealed that the α helices normally present in the C-terminal end of β-globin are disrupted and replaced by random coils and extended strands. Four acidic amino acids (two aspartic and two glutamic acid residues) that are important for electrostatic interactions with α-globin are lacking, and ten other amino acids that are critical for α/β dimer formation have been lost, implying that the elongated mutant β-globin is unable to participate in the assembly of a stable globin tetramer. The new hydrophobic C-terminal may render the mutant peptide less susceptible to effective proteolysis, resulting in an accumulation of defective peptides. The excess single β-globin chains form insoluble aggregates that precipitate, together with redundant α-globin, as intracellular inclusions,5 evidenced by the presence of numerous Heinz bodies in the patient. The isopropanol test was positive for unstable hemoglobin. However, no mutant hemoglobin could be detected by cation exchange HPLC, reverse phase HPLC or capillary electrophoresis, most likely due to the hyper-instability of the aberrant β-globin molecule and Heinz body formation, which prevents in vitro solubility and laboratory detection. These inclusions likely contribute to accelerated RBC destruction. The in vitro analysis of erythroid progenitors revealed a marked decrease in both proliferation (Figure 1(C)) and differentiation (Figure 1(D)) as evaluated by temporal changes of glycophorin and transferrin receptor positivity and intensity in the proband. The hemoglobinization of maturing erythroid progenitors was not discernible at day 14 while the control had readily discernible “red” erythroid progenitors. These data suggest that Hb Santo Domingo compromises erythropoiesis and that the mutant protein is toxic to erythroid progenitors. The HPP morphology of the proband prompted further investigations. Of note, EMA analysis showed a mild decrease in fluorescence of 13% indicative of an RBC membrane protein defect. By exome and targeted Sanger sequencing, two mutations were detected in SPTA1. High-throughput sequencing revealed no pathogenic mutations in genes coding for the other major RBC membrane proteins and enzymes, as well as other genes relevant to hemolytic anemia. Theproband was heterozygous for spectrin Jendouba (SPTA1D791E[c.2373C > A]),6 in the SPTA1 gene in exon 17. This mutation resides in the α8 repeat within the αII domain of α-spectrin, which is far away from the dimer self-association site at the N-terminal in the αI domain, and thus has minimal effect on spectrin tetramer formation. Morphologically, it is associated with infrequent elliptocytes.6 The SPTA1 hypomorphic αLELY polymorphism7 was present in trans to spectrin Jendouba. This αLELY is a low expression allele composed of three variants (c.5572C > G, c.6531-12C > T, c.6549-12G > A) in SPTA1 and has a frequency of 20%–31% in different populations.7 On its own αLELY does not cause disease, neither in heterozygotes nor homozygotes. The αLELY allele causes exon skipping in 50% of the SPTA1 transcripts, which results in only 50% of the normal amount of α-spectrin being produced, but since α-spectrin is synthesized in excess of β-spectrin, there is sufficient α-spectrin to interact with β-spectrin to form heterodimers and tetramers. Inheritance of αLELY in trans with other pathogenic SPTA1 mutations associated with defects in the spectrin self-association site, can lead to moderate to severe hemolysis. However, co-inheritance in trans with spectrin Jendouba only resulted in a marginal increase in spectrin dimers6 and would thus not give rise to the HPP morphology in our patient. A more plausible explanation for the RBC membrane budding and microspherocytosis is that the hyper-unstable β-globin thalassemia mutation promotes the formation of Heinz bodies, which bind to the RBC membrane8 and destabilize the underlying abnormal spectrin skeleton. It has been shown that there is a reduction in the quantity of spectrin in patients with unstable hemoglobin and Heinz bodies, due to oxidative damage to the membrane.9 A decreased amount of spectrin would thus explain the microspherocytes on the peripheral smear, as well as the relatively mild reduction in the binding of EMA to exposed residues of the macromolecular band 3 complex of the membrane. We propose that the combination of defects in β-globin and α-spectrin enhances the clinical severity and exacerbates the thalassemic RBC morphological abnormalities leading to an HPP phenotype. The inheritance of Hb Santo Domingo and the SPTA1 alleles was traced in family studies (Figure 1(A) and Figure S2), which were limited by the paucity of material obtained and inability to perform follow up studies. The mother had sickle cell trait and passed the HbS gene onto her three children, but only two children inherited Hb Santo Domingo. Their peripheral blood morphology was complicated by numerous sickle cells, but their RBCs did not show the typical HPP red cell morphology evident in the father. Child one showed delayed erythroid maturation compared to child two (Figure 1(D)), although the reason for this intriguing finding is not clear. Both children inherited Hb Santo Domingo and HbS, but they have different SPTA1 alleles, which, however, are either asymptomatic or have a minimal effect on the mature RBC membrane skeleton. The consequences during erythropoiesis are not known. This study illustrates the diagnostic challenge in a case of co-inheritance of defects in the RBC membrane and hemoglobin. A few such rare cases have been described10, 11 where the presence of hereditary spherocytosis or elliptocytosis enhances the severity of β-thalassemia, and also adversely influences the RBC morphology. In this we describe not only a novel HBB mutation leading to autosomal dominant β-thalassemia, but also provide a novel etiology of the HPP phenotype.10, 11 [Correction added on March 17, 2021, after first online publication: references #10 and #11 are added after original publication.] We propose that this is due to a complex interplay between mutant β-globin peptides and an abnormal RBC membrane skeleton, which reduces the spectrin content of the membrane and exacerbates the effect of the α-spectrin elliptocytosis mutation and low expression of the SPTA1 allele. S.K. and J.S. designed and performed most experiments, analyzed data and wrote the manuscript; S.R. designed and performed some experiments, and analyzed data. J.L. cared for the patient, obtained the sample and provided clinical data; A.K. performed some hemoglobinopathy experiments and analyzed data and edited and approved manuscript; A.A. assessed and evaluated clinical data, analyzed pathology, found novel beta globin mutation, and wrote the manuscript; T.L.C. performed computer modeling, analyzed data, conceptually analyzed RBC cytoskeleton data, and wrote the manuscript; J.T.P. conceived and designed the study, and interpreted all experimental and clinical data and wrote the manuscript. All authors read and approved the final version of the manuscript. This research was supported by VA Merit Review Award (J.T.P) for funding and the National Institutes of Health under Ruth L. Kirschstein National Research Service Award 2T32HL007576-31 from the National Heart, Lung, and Blood Institute (JS). The authors declare no conflict of interest. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants involved in the study. The data that support the findings of this study are available from the corresponding author upon reasonable request. Appendix S1 Supporting Information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.