Case Report: α-Spectrin Mutation Associated with αLELY Polymorphism Responsible for Hereditary Pyropoikilocytosis

Hereditary pyropoikilocytosis (HPP) is characterised by severe hemolytic anemia due to membrane instability. We report the case of a 13-day-old boy with neonatal jaundice and severe hemolytic anemia. A peripheral smear examination showed severe anisopoikylocytosis. DNA sequencing revealed compound double heterozygous for mutant α-spectrin SPTA1 (Arg28His) and homozygous αLELY polymorphism (low expression α-spectrin allele), compatible with diagnosis of HPP.The patient required a blood transfusion initially, but spontaneously improved after two years. Our case illustrates that, despite the presence of the allele αLELY in homozygous, the clinical phenotype is similar to cases with a mutation in SPTA1 associated with αLELY in trans.


Introduction
Hereditary pyropoikilocytosis (HPP) is a subset of hereditary elliptocytosis (HE) due to homozygous or compound heterozygous mutations in spectrin, leading to severe disruption of spectrin self-association due to homozygous or compound heterozygous mutations in α-spectrin, leading to severe disruption of α-β spectrin dimer self-association. This leads to a disruption of the horizontal junctions of the cytoskeleton of the red blood cell membrane, with loss of elasticity and fragmentation. HPP is characterised by severe hemolytic anemia with microspherocytes, poikilocytes, and an unusual thermal sensitivity of red cells because of membrane instability [1,2].
α-spectrin is the predominant component of the membrane skeleton of the red cell, and it is essential in determining the properties of the membrane. Different α-spectrin abnormalities are involved in spherocytosis type 3 (phenotype MIM, number 270970), HE type 2 (phenotype MIM number 130600) and HPP (phenotype MIM number 266140) [3].
Normally, α -spectrin synthesis is threefold greater than beta-spectrin. Heterozygote defects (HE) produce sufficient α-spectrin to balance normal β-spectrin production, and, therefore, biallelic involvement is necessary for the severe phenotypic expression in HPP. Biallelic spectrin involvement is rare, but more common is the presence of widespread polymorphisms that can modulate the phenotypic expression of the disease. The most frequent is the αLELY allele (20-30% of the population), a low-expression allele of spectrin α-spectrin gene.
This allele has carrier mutations that impair the recruitment of α-chains by β-chains, and would eventually account for the low-expression character. When αLELY polymorphism is present, and it is also present in trans, the propensity of the normal allele to associate with the corresponding β-chain diminishes, favouring the attachment of the elliptocytogenic α-spectrin allele. In HPP, clinical severity correlates with the amount of mutant spectrin and excess of spectrin dimer in the red cell membrane. In contrast, the αLELY component in cis to the alpha spectrin mutation counteracts the similar component in trans, and, therefore, the clinical presentation is very different. However, the clinical phenotype in the presence of the homozygous αLELY component has not been reported [2,[4][5][6][7].

Case Report
A 13-year-old Senegalese boy was referred to our hospital for neonatal jaundice and severe anemia. The blood test revealed microcytic regenerative anemia with hemoglobin levels of 60 gr/L, an increase of hemolysis markers and a negative direct antiglobulin test (Table 1). A peripheral smear examination showed severe anisopoikylocytosis with fragmented erythrocytes, spherocytes and ovalocytes consistent with hereditary membrane disorder ( Figure 1). Capillary electrophoresis did not detect HBS or any other variant. The enzymatic assays of glucose-6-phosphate dehydrogenase and pyruvate kinase were normal. We performed next-generation sequencing (NGS) in a panel of 40 genes for a patient with suspected congenital hemolytic anemia. DNA sequencing revealed compound double heterozygous for mutant α-spectrin SPTA1 (Arg28His) and homozygous αLELY polymorphism (low expression α-spectrin allele), with a final diagnosis of HPP. A more complete investigation was made of his parents ( Figure 2). The patient required blood transfusion initially but spontaneously improved after two years (hemoglobin levels 110 gr/L) (Figure 3).  The patient required blood transfusion initially but spontaneously improved after two years (hemoglobin levels 110 gr/L) (Figure 3).   The patient required blood transfusion initially but spontaneously improved after two years (hemoglobin levels 110 gr/L) (Figure 3).

Discussion
The Arg28His mutation was originally reported in a family of French descent a later in a Tunisian family. This mutation is due to a CAT-to-CGT change in codon 22 exon 2 of the SPTA1 gene (which encodes amino acids 3 to 82 of the alpha-I domain). T defect resulted in decreased ability of the spectrin dimers to self-associate (elliptocy genic α-spectrin chain) [8,9].
When αLELY polymorphism is present (affecting 20-30% of the population), an is also present in trans (both mutations in different alpha-chain alleles), the propensity the normal allele to associate with the corresponding beta-chain diminishes, favour the attachment of the elliptocytogenic alpha-spectrin allele. In HPP, clinical severity c relates with the amount of mutant spectrin and excess of spectrin dimer in the red membrane. The phenotype improves with age, probably due to γ/β globin gene swit ing and a decrease of red blood cell 2,3-diphosphoglycerate [10].
HPP was originally described in 1975 as a distinct hemolytic anemia characteriz by microspherocytosis, poikilocytosis, and an unusual thermal sensitivity of red ce HPP is a subset of hereditary elliptocytosis due to homozygous or compound hetero gous mutations in spectrin, leading to severe disruption of spectrin self-association [4] Hereditary membrane erythrocyte disorder is a rare cause of neonatal jaundice a anemia. This case highlights the importance of correlating erythrocyte morphology, clinical course, and molecular analysis in cases of non-immune hemolytic jaundice. T finding of elliptocytogenic spectrin variants can predict the clinical course in these k of patients. Typically, when mutation in SPTA1 is associated with αLELY in trans, patients present transfusion needs in infancy, but the phenotype improves later in life 7].
Our case also illustrates that, despite the presence of the allele αLELY in homo gous, the clinical phenotype is similar to cases with trans presentation previously ported, and this clinical behavior supports the diagnosis of HPP rather than HE alone

Discussion
The Arg28His mutation was originally reported in a family of French descent and later in a Tunisian family. This mutation is due to a CAT-to-CGT change in codon 22, in exon 2 of the SPTA1 gene (which encodes amino acids 3 to 82 of the alpha-I domain). The defect resulted in decreased ability of the spectrin dimers to self-associate (elliptocytogenic α-spectrin chain) [8,9].
When αLELY polymorphism is present (affecting 20-30% of the population), and it is also present in trans (both mutations in different alpha-chain alleles), the propensity of the normal allele to associate with the corresponding beta-chain diminishes, favouring the attachment of the elliptocytogenic alpha-spectrin allele. In HPP, clinical severity correlates with the amount of mutant spectrin and excess of spectrin dimer in the red cell membrane. The phenotype improves with age, probably due to γ/β globin gene switching and a decrease of red blood cell 2,3-diphosphoglycerate [10].
HPP was originally described in 1975 as a distinct hemolytic anemia characterized by microspherocytosis, poikilocytosis, and an unusual thermal sensitivity of red cells. HPP is a subset of hereditary elliptocytosis due to homozygous or compound heterozygous mutations in spectrin, leading to severe disruption of spectrin self-association [4].
Hereditary membrane erythrocyte disorder is a rare cause of neonatal jaundice and anemia. This case highlights the importance of correlating erythrocyte morphology, the clinical course, and molecular analysis in cases of non-immune hemolytic jaundice. The finding of elliptocytogenic spectrin variants can predict the clinical course in these kind of patients. Typically, when mutation in SPTA1 is associated with αLELY in trans, the patients present transfusion needs in infancy, but the phenotype improves later in life [5][6][7].
Our case also illustrates that, despite the presence of the allele αLELY in homozygous, the clinical phenotype is similar to cases with trans presentation previously reported, and this clinical behavior supports the diagnosis of HPP rather than HE alone. Informed Consent Statement: Informed consent has been obtained from the legal guardian.

Conflicts of Interest:
The authors declare no conflict of interest.