Targeted next generation sequencing identifies a novel β‐spectrin gene mutation A2059P in two Omani children with hereditary pyropoikilocytosis
Authors: Arwa Z. Al‐Riyami, Achille Iolascon, Shoaib Al‐Zadjali, Immacolata Andolfo, Sahima Al‐Mammari, Francesco Manna, AbdulHakim Al Rawas, May‐Jean King, Roberta Russo
Journal: American Journal of Hematology (2017)

Hereditary pyropoikilocytosis (HPP) is a red blood cell (RBC) membrane disorder characterized by RBC fragmentation, poikilocytosis and mean corpuscular volume of 50–60 fL. During the neonatal period and early childhood, patients often present with transfusion-dependent hemolytic anemia.1 HPP is a bi-allelic disorder due to homozygous mutations in spectrin gene (SpHE) or inheritance of one SpHE allele in trans to a low-expression α-spectrin SPTA1 allele (SpαLELY).2 The mutated genes encode spectrin variants that possess trypsin resistant sites. Common α-spectrin variants account for a weakening of the integrity of the spectrin-based network when their defective domain(s) forms a part of or in the vicinity of the spectrin heterodimer self-association site. Both codon 28 of α-spectrin gene SPTA1 (the hotspot for mutations) and exon 30 of β spectrin gene SPTB can give rise to Spectrin (Sp) αI/74, which is detected as a tryptic peptide with a molecular weight (Mr) of 74,000 in gel electrophoresis after limited trypsin digestion.3 Diagnosis of HPP can be challenging with coexistence of underlying hemoglobinopathy especially in infants. Herein we describe the clinicopathological profile of two related Omani families with co-presence of HPP and hemoglobinopathies. Two probands (1-a and 2-a) presented with history of hemolytic anemia at 3 months of age requiring transfusion support. Both infants had history of neonatal jaundice requiring phototherapy, and so were the older siblings of child 1-a and two siblings of child 2-a (2-b and 2-e). The parents of proband 2-a are first degree cousins, and they are also first degree cousins of the father of child 1-a (Supporting Information Figure 1). Blood films of children 1-a, 2-a, 2-b, and 2-e showed polychromasia and poikilocytosis including spherocytes, elliptocytes, ovalocytosis, and tear drop cells. They also had evident signs of hemolysis (Table 1). Blood films of the parents showed numerous elliptocytes, in keeping with hereditary elliptocytosis (HE). All subjects, except father 2-d, had variable microcytosis most striking in child 2-b (Table 1). Age at testing Hb (g dL−1) Retic (absolute count ×109/L) male: 11.5–15.5 female: 11–14.5 The RBCs of both probands gave a broad fluorescence peak in the Eosin-5′-maleimide (EMA) Binding test (Supporting Information Figure 2A). Proband 1-a had more affected RBCs than proband 2-a because her EMA-labeled RBCs produced a lower fluorescence reading and the histogram showed a slight left shoulder to the known hereditary spherocytosis (HS) sample, indicating a subpopulation of more fragile RBCs.4 By contrast, the parents of both probands gave a single uniform peak with a florescence reading within the normal range, as expected for HE. In spectrin analysis, a significant increase of dimer content was found in both probands: child 1-a had a greater amount than child 2-a (Supporting Information Figure 2B). Although the dimer contents for both sets of parents were within the HE range, the RBCs of parents of proband 1-a had a greater dimer content than those for the parents of proband 2-a. In limited trypsin digestion of spectrin, a single intensely stained peptide with Mr of 74 000 was obtained for both probands (Supporting Information Figure 2C). Because there was no residual αI/80 peptide in their tryptic digests, both probands were considered homozygous for the SpαI/74 variant. The detection of SpαI/74 variant and the marked increase of dimer content suggested HPP. None of the probands and parents had reductions in α-spectrin and protein 4.1. In overall membrane protein quantitation after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of erythrocyte membrane proteins, elevated ankyrin:band-3 ratio was obtained for both probands: 0.277 for 1-a and 0.261 for 2-a (range for normal controls = 0.214–0.238). Although the detection of SpαI/74 variant and the marked increase of dimer content were positive results for HPP, the normal α-spectrin content in both probands was not consistent with this diagnosis. Thus, it was necessary to determine the molecular basis for the increase of the SpαI/74 variant. DNA samples from both probands, and parents 1-b and 1-c were tested by direct sequencing analysis for exon 30 of SPTB, exons 1, 2, 3, 4, 5, 6, and 40 of SPTA1, and intron 45 of SPTA1 for the SpLELY allele. No mutations were found. A second round of analysis was performed using a targeted next generation sequencing including 34 loci causative of different hereditary hemolytic anemias.5 Both probands showed a homozygous c.6175G > C, p.Ala2059Pro mutation in SPTB gene, confirming HPP. This novel mutation is predicted to be "damaging" by prediction analysis using two web-server tools, PolyPhen2 and SIFT. The parents were heterozygous for this mutation. An additional heterozygous mutation c.5029G > A, p.Gly1677Arg in SPTA1 gene was found in proband 1-a and his mother 1-b, but not in proband 2-a. All family members with exception of both fathers were found to have αα/-α3.7 genotype, while no β-globin mutations in both probands and their fathers were found. The mother 2-c confirmed to be a β-thalassemia carrier with IVS1,5(G > C) mutation. The co-inheritance of SPTB mutation (A2059P) and thalassemia in proband 1-a produced a more severe microcytosis that markedly exceeded that observed in her mother and approximates what is observed in her father (Table 1). Proband 2-a had a similar degree of microcytosis, while child 2-b presented the most striking microcytosis of all family members, probably resulting from the combined effect of the ß-thalassemia trait and HPP. Coinheritance of β-thalassemia and HS is known to occur in the Mediterranean countries.1 However, little is known about the prevalence of coinheritance of other membranopathies with hemoglobinopathies in the Arabian Peninsula. Conducting a thorough investigation when family members present with suspicious clinicopathological features can provide more insight on the prevalence of membranopathy in populations having high incidence of hemoglobinopathies. The novel SPTB mutation A2059P identified is located within the subdomain repeat 17 of β-spectrin, involved in spectrin self-association. Amino-acid substitution by proline in this subdomain can disrupt the functional integrity of dimer cross-linkage due to conformational changes.6 Three other amino-acid substitutions have already been described in the same domain, namely, spectrin Kayes-2 (A2053P), Cagliari (A2018G), and Providence (S2019P). Similarly to our probands, homozygous individuals for these variants exhibited severe hemolysis and anemia, while heterozygous subjects were found clinically asymptomatic.6 To the best of our knowledge, this study is the first reporting HPP in Oman. The authors acknowledge all who were involved in making the diagnosis in this family. This work was supported by grants from the Italian Ministry of University and Research, by PRIN to AI (20128PNX83), by SIR to RR (RBSI144KXC), by grants from Regione Campania (DGRC2362/07). The Local Ethics Board at the College of Medicine and Health Sciences (Sultan Qaboos University, Oman) has given approval for undertaking molecular genetics investigations on the probands and their parents. The authors declare no conflict of interest. Additional Supporting Information may be found in the online version of this article. 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.