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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Br J Haematol. Author manuscript; available in PMC 2012 March 1.
Published in final edited form as:
PMCID: PMC3105174

Compound heterozygosity for two novel mutations in the erythrocyte protein 4.2 gene causing spherocytosis in a Caucasian patient

The term hereditary spherocytosis (HS) covers a range of genetically and phenotypically variable red blood cell (RBC) cytoskeleton disorders, caused by defects in proteins that link the membrane skeleton to the lipid bilayer. Nonsense and frameshift mutations of ankyrin, band 3, and β-spectrin are often responsible for dominant HS, while homozygosity or compound heterozygosity of defects in ankyrin, α-spectrin, or protein 4.2 cause recessive HS (Eber and Lux 2004, Iolascon and Avvisati 2008). Protein 4.2 is an important component of the RBC cytoskeleton, representing approximately 5% of the membrane protein content (Satchwell, et al 2009). It binds the N-terminal cytoplasmic domain of band 3 and regulates the avidity of the interaction between band 3 and ankyrin within the band 3 macrocomplex (Bruce, et al 2003). Protein 4.2 is encoded by the EPB42 gene, which encompasses approximately 20 kb, containing 13 exons and 12 introns, and maps to 15q15-q21 (Korsgren and Cohen 1991).

Recessive HS caused by protein 4.2 deficiency accounts for less than 5% of all HS cases and is common in Japan but rare in other populations. Eleven mutations have been reported so far in the literature, summarized by Satchwell, et al (2009). Here we report a child of Northern European descent diagnosed with HS due to protein 4.2 deficiency. Molecular analysis demonstrated two novel mutations of EPB42 gene in a compound heterozygous state.

A 16-month-old Caucasian girl was referred to our clinic for severe anaemia. Past medical history revealed a 3-day postnatal hospitalization for hyperbilirubinaemia requiring phototherapy. Family history was negative for anaemia, gallstones, or splenectomy. Nutritional history revealed that her diet contained excessive cow-milk intake (24 oz daily). Her complete blood cont revealed haemoglobin of 41 g/l, mean cell volume 62.6 fl and an absolute reticulocyte count (ARC) of 251 × 109/l (normal range 44-111 × 109/l). Her smear showed microcytic, hypochromic anaemia with polychromasia and significant anisocytosis. There were occasional ovalocytes and teardrops, and occasional macrocytes. No significant spherocytosis was noted. Further evaluation at that time revealed evidence of iron deficiency anaemia (decreased ferritin at 3 μg/l with elevated total iron-binding capacity [TIBC] 100.4 μmol/l) but also indicated an underlying haemolytic process with significantly elevated aspartate transaminase (200 u/l) and lactate dehydrogenase (1861 u/l) levels, although bilirubin levels were normal (12 μmol/l, all unconjugated). Direct Coombs test was negative; haemoglobin electrophoresis, glucose-6-phosphate dehydrogenase and pyruvate kinase screen were within normal limits. The patient was transfused to a haemoglobin concentration of 80 g/l and started on iron replacement therapy at 6 mg/kg elemental iron daily. After approximately 10 weeks of iron therapy her ferritin and TIBC normalized. The patient achieved and maintained an almost normal haemoglobin level (110-118 g/l) but ARC remained elevated at 160-240 × 109/L. A peripheral blood smear at 6 months after presentation revealed rare spherocytes (Figure 1A). Her mean cell haemoglobin concentration was increased (355-367 g/l) indicating the possibility of spherocytosis. Osmotic fragility was mildly increased (Figure 1B). Ektacytometry revealed a slightly decreased maximal deformability index (DImax) and increased Omin (osmolality at which 50% of the cells haemolyse), indicative of a spherocytic membrane disorder (Figure 1C). RBC membrane protein gel electrophoresis demonstrated absence of protein 4.2, confirmed by immunoblotting, thereby establishing the diagnosis of HS (Figure 1D and E).

Figure 1
A. Blood smear (obtained when patient at baseline health status) showing a slight poikilocytosis and anisocytosis and a few spherocytes (arrowheads). B. Osmotic fragility was borderline increased. C. Ektacytometry indicated the possibility of mild spherocytosis ...

CD47 has been shown to be concomitantly decreased with 4.2 deficiency (Bruce, et al 2002), while an increase in the cell adhesion molecule CD44 has been noted (van den Akker, et al 2010). Indeed, immunoblotting demonstrated decreased CD47 and increased CD44 in the patient's RBC membrane (Figure 1E). Flow cytometry revealed a 2.5- to 3-fold decrease in CD47 surface expression on the patient's RBCs as compared to normal control RBCs (Figure 1F).

Informed consent was obtained from the family under an Institutional Review Board approved protocol for EPB42 gene analysis. DNA sequencing revealed that the child was compound heterozygous for two novel mutations in the EBP42 gene. The paternal copy of EBP42 carried a C→T nucleotide change in exon 6, resulting in a non-conservative substitution of the hydrophilic threonine-307 by the hydrophobic isoleucine (T307I), which we propose to name 4.2Cincinnati (Figure 2A). The region 306-CTVLRCLG-313 has been highly conserved evolutionarily (Toye, et al 2005). SIFT software ( analysis (Ng and Henikoff 2003) revealed that 10 of the 15 amino acid residues from 306-320 can tolerate no substitutions, including residues 307, 310, and 317. In fact, four of the known mutations in protein 4.2 occur in this area: missense mutations 4.2Tozeur (R310Q) and 4.2Shiga (R317C), as well as nonsense mutations 4.2Nancy (frameshift 317/term 319) and 4.2Notame (del exon 6/frameshift 308). It is likely that protein 4.2Cincinnati behaves similarly to protein 4.2Tozeur, which is more susceptible to proteolysis, because it is not incorporated normally within the band 3 macrocomplex due to alteration of the domain containing the band 3-binding hairpin (Hayette, et al 1995, Satchwell, et al 2009). The fact that CD47 is decreased in our patient's RBCs confirms prior observations that protein 4.2 acts as a linker between band 3 and CD47 (Bruce, et al 2002). The maternal copy of EBP42 carried a deletion of 32 nucleotides within exon 10, resulting in a frame shift starting at codon L505T. The resulting protein, which we named 4.2Ohio, terminates at amino acid 517 instead of 691, with 13 amino acids modified at the C-terminus (Figure 2B).

Figure 2
The 13 exons of EPB42 in genomic DNA extracted from whole blood were amplified individually by polymerase chain reaction and sequenced bidirectionally using ByDye v1.1 chemistry on an ABI 3737xl DNA Analyzer. A. DNA sequencing of exon 6 revealed a heterozygote ...

While mutations in protein 4.2 are much more common in Japan, we present here a Caucasian patient who is compound heterozygous for two previously undescribed mutations in EBP42, resulting in protein 4.2 absence accompanied by decreased CD47 and increased CD44 in the membrane. Protein 4.2 deficiency results in HS inherited through an autosomal recessive pattern, with a mildly abnormal smear and modest decreases in osmotic resistance and deformability. In addition to the functional assays (osmotic fragility and ektacytometry) traditionally used to diagnose erythrocyte cytoskeleton disorders, membrane protein analysis, flow cytometry, and gene sequencing can be employed to offer a definitive diagnosis of such atypical cases.


We thank the family studied here for their kind cooperation. We thank Dr. Carolyn Hoppe in Children's Hospital Oakland Research Institute for the ektacytometry testing and interpretation.

This work was supported by the USA National Institutes of Health grant NHLBI K08 HL088126.


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