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

Article (PDF Available)inBritish Journal of Haematology 152(6):780-3 · March 2011with24 Reads
DOI: 10.1111/j.1365-2141.2010.08516.x · Source: PubMed
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 b-spectrin are
often responsible for dominant HS, while homozygosity or
compound heterozygosity of defects in ankyrin, a-spectrin,
or protein 4.2 cause recessive HS (Eber & Lux, 2004;
Iolascon & Avvisati, 2008). Protein 4.2 is an important
component of the RBC cytoskeleton, representing approxi-
mately 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 & Cohen,
Recessive HS caused by protein 4.2 deficiency accounts for
<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 demon-
strated 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 photothera-
py. Family history was negative for anaemia, gallstones, or
splenectomy. Nutritional history revealed that her diet con-
tained excessive cow-milk intake (24 oz daily). Her complete
blood count revealed haemoglobin of 41 g/l, mean cell volume
62Æ6 and an absolute reticulocyte count (ARC) of 251 · 10
(normal range 44–111 · 10
/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 lg/l with elevated
total iron-binding capacity (TIBC) 100Æ4 lmol/l] but also
indicated an underlying haemolytic process with significantly
elevated aspartate transaminase (200 u/l) and lactate dehydro-
genase (1861 u/l) levels, although bilirubin levels were normal
(12 lmol/l, all unconjugated). Direct Coombs test was nega-
tive; haemoglobin electrophoresis, glucose-6-phosphate dehy-
drogenase and pyruvate kinase screen were within normal
limits. The patient was transfused to a haemoglobin concen-
tration 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 · 10
A peripheral blood smear at 6 months after presentation
revealed rare spherocytes (Fig 1A). Her mean cell haemoglobin
concentration was increased (355–367 g/l) indicating the
possibility of spherocytosis. Osmotic fragility was mildly
increased (Fig 1B). Ektacytometry revealed a slightly decreased
maximal deformability index (DI
) and increased O
(osmolality at which 50% of the cells haemolyse), indicative of
a spherocytic membrane disorder (Fig 1C). RBC membrane
protein gel electrophoresis demonstrated absence of protein
4.2, confirmed by immunoblotting, thereby establishing the
diagnosis of HS (Fig 1D, E).
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 (Fig 1E).
Flow cytometry revealed a 2Æ5- to threefold decrease in CD47
surface expression on the patient’s RBCs as compared to
normal control RBCs (Fig 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 hydro-
phobic isoleucine (T307I), which we propose to name
(Fig 2A). The region 306-CTVLRCLG-313 has
been highly conserved evolutionarily (Toye et al, 2005). SIFT
software ( analysis (Ng & Henikoff, 2003) revealed
that 10 of the 15 amino acid residues from 306 to 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 4.2
(R310Q) and 4.2
(R317C), as well as nonsense mutations 4.2
ª 2011 Blackwell Publishing Ltd, British Journal of Haematology doi:10.1111/j.1365-2141.2010.08516.x
317/term 319) and 4.2
(del exon 6/frameshift 308). It is
likely that protein 4.2
behaves similarly to protein
, 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 4.2
, terminates at amino acid 517 instead
of 691, with 13 amino acids modified at the C-terminus
(Fig 2B).
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
(A) (B)
(C) (D)
(E) (F)
Fig 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 with decreased DI
(0.44, normal range 0.54 ± 0.06), and increased O
(164.7, normal range 139.8 ± 16.0). (D) Red blood cell membrane electrophoresis (4–16%
gradient gel) was the diagnostic test demonstrating protein 4.2 deficiency in the patient’s sample (lane P) in comparison with samples from the
patient’s mother (M), father (F), and an adult normal healthy control (C). Protein 4.2 on the Coomassie stained gel is indicated with an arrow on the
left and the migration of the 75 kD molecular weight standard is indicated on the right. (E) Immunoblots, using primary antibodies for protein 4.2
(Abnova, Walnut, CA), CD47 (SantaCruz Biotechnology, Santa Cruz, CA, USA), CD44 (SantaCruz Biotechnology) and b-actin (GenScript,
Piscataway, NJ, USA), confirmed complete protein 4.2 deficiency in the patient’s sample (P) along with decreased CD47 and increased CD44. Actin is
shown as loading control. F. Flow cytometry using PE-conjugated anti-CD47 antibody (BD Biosciences, Franklin Lakes, NJ, USA) confirmed decrease
of CD47 in the membrane of the patient’s RBCs (red line) compared with control RBCs (blue line). Background fluorescence indicated by the dark
grey filled histogram.
ª 2011 Blackwell Publishing Ltd, British Journal of Haematology
to the functional assays (osmotic fragility and ektacytometry)
traditionally used to diagnose erythrocyte cytoskeleton disor-
ders, 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 interpre-
tation. This work was supported by the USA National
Institutes of Health grant NHLBI K08 HL088126.
Adrienne M. Hammill
Mary A. Risinger
Clinton H. Joiner
Mehdi Keddache
Theodosia A. Kalfa
Cancer and Blood Diseases Institute, Cincinnati Children’s Hospital
Medical Center and University of Cincinnati College of Medicine, and
Department of Genetics, Cincinnati Children’s Hospital Medical Center
and University of Cincinnati College of Medicine, Cincinnati, OH, USA
Keywords: spherocytosis, protein 4.2, EPB42, erythrocyte
cytoskeleton, band 3 macrocomplex.
Fig 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 3737·l DNA Analyzer. A. DNA sequencing of exon 6 revealed a heterozygote missense mutation
T>C 2 bp from the end of the exon (red arrow) leading to the amino acid change T307I. B. DNA sequencing of exon 10 revealed two different
sequences in the patient’s sample (P), which were deconvoluted, using SoftGenetics’ MutationSurveyor v3.3 software, into the father’s wild type allele
(F) and the mother’s mutant allele (M). A deletion of 32 bases in the mother’s allele causes a frameshift resulting in the early termination of the
protein 13 amino acids post L505, after position 517.
ª 2011 Blackwell Publishing Ltd, British Journal of Haematology 3
van den Akker, E., Satchwell, T.J., Pellegrin, S., Flatt,
J.F., Maigre, M., Daniels, G., Delaunay, J., Bruce,
L.J. & Toye, A.M. (2010) Investigating the key
membrane protein changes during in vitro
erythropoiesis of protein 4.2 (-) cells (mutations
Chartres 1 and 2). Haematologica, 95, 1278–1286.
Bruce, L.J., Ghosh, S., King, M.J., Layton, D.M.,
Mawby, W.J., Stewart, G.W., Oldenborg, P.A.,
Delaunay, J. & Tanner, M.J. (2002) Absence of
CD47 in protein 4.2-deficient hereditary sphe-
rocytosis in man: an interaction between the Rh
complex and the band 3 complex. Blood, 100,
Bruce, L.J., Beckmann, R., Ribeiro, M.L., Peters,
L.L., Chasis, J.A., Delaunay, J., Mohandas, N.,
Anstee, D.J. & Tanner, M.J. (2003) A band 3-
based macrocomplex of integral and peripheral
proteins in the RBC membrane. Blood, 101,
Eber, S. & Lux, S.E. (2004) Hereditary spherocytosis
defects in proteins that connect the membrane
skeleton to the lipid bilayer. Seminars in Hema-
tology, 41, 118–141.
Hayette, S., Morle, L., Bozon, M., Ghanem, A., Ri-
singer, M., Korsgren, C., Tanner, M.J., Fattoum,
S., Cohen, C.M. & Delaunay, J. (1995) A point
mutation in the protein 4.2 gene (allele 4.2
Tozeur) associated with hereditary haemolytic
anaemia. British Journal of Haematology, 89, 762–
Iolascon, A. & Avvisati, R.A. (2008) Genotype/
phenotype correlation in hereditary spherocyto-
sis. Haematologica, 93, 1283–1288.
Korsgren, C. & Cohen, C.M. (1991) Organization of
the gene for human erythrocyte membrane pro-
tein 4.2: structural similarities with the gene for
the a subunit of factor XIII. Proceedings of the
National Academy of Sciences of the United States
of America, 88, 4840–4844.
Ng, P.C. & Henikoff, S. (2003) SIFT: predicting
amino acid changes that affect protein function.
Nucleic Acids Research, 31, 3812–3814.
Satchwell, T.J., Shoemark, D.K., Sessions, R.B. &
Toye, A.M. (2009) Protein 4.2: a complex linker.
Blood Cells, Molecules and Diseases, 42, 201–210.
Toye, A.M., Ghosh, S., Young, M.T., Jones, G.K.,
Sessions, R.B., Ramauge, M., Leclerc, P., Basu, J.,
Delaunay, J. & Tanner, M.J. (2005) Protein-4.2
association with band 3 (AE1, SLCA4) in Xeno-
pus oocytes: effects of three natural protein-4.2
mutations associated with hemolytic anemia.
Blood, 105, 4088–4095.
ª 2011 Blackwell Publishing Ltd, British Journal of Haematology
  • [Show abstract] [Hide abstract] ABSTRACT: We present data on a patient of South Asian origin with recessive hereditary spherocytosis (HS) due to absence of protein 4.2 [4.2 (-) HS]. Protein 4.2 cDNA sequence analysis showed the presence of a novel 41-bp frameshift deletion that predicts a truncated peptide designated protein 4.2 Hammersmith. Quantitative reverse transcription-polymerase chain reaction indicated that the mutant mRNA was unstable. Sequencing of protein 4.2 genomic DNA revealed that the deletion stems from aberrant splicing. The proband was homozygous for a G>T substitution at position 1747 (cDNA numbering) that activates a cryptic acceptor splice site within exon 11 of the protein 4.2 gene (EPB42). The proband's mother was found to be heterozygous for this substitution. Unlike protein 4.2 null mice, the proband's red cells showed no evidence for abnormal cation permeability. Quantitation of red cell membrane proteins was carried out by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and flow cytometric measurement. CD47, a protein associated with the Rh complex, was markedly reduced to about 1% (in the proband) and 65% (in the mother) that found in healthy controls. The Rh-associated glycoprotein migrated with a higher than normal apparent molecular weight on SDS-PAGE. There was no obvious reduction in Rh polypeptides. These observations indicate that protein 4.2 and CD47 interact in the human red cell membrane. They provide further evidence for an association between the band 3 complex (band 3, ankyrin, protein 4.2, glycophorin A) and the Rh complex (Rh-associated glycoprotein, Rh polypeptides, glycophorin B, CD47, LW) and define a point of attachment between the Rh complex and the red cell cytoskeleton.
    Article · Sep 2002
  • [Show abstract] [Hide abstract] ABSTRACT: The molecular causes of hereditary spherocytosis (HS) have been unraveled in the past decade. No frequent defect is found, and nearly every family has a unique mutation. In dominant HS, nonsense and frameshift mutations of ankyrin, band 3, and beta-spectrin predominate. Recessive HS is most often due to compound heterozygosity of defects in ankyrin, alpha-spectrin, or protein 4.2. Common combinations include a defect in the promoter or 5'-untranslated region of ankyrin paired with a missense mutation, a low expression allele of alpha-spectrin plus a missense mutation, and various mutations in the gene for protein 4.2. In most patients' red cells, no abnormal protein is present. Only rare missense mutations, like ankyrin Walsrode (V463I) or beta-spectrin Kissimmee (W202R), have given any insight into the functional domains of the respective proteins. Although the eminent role of the spleen in the premature hemolysis of red cells in HS is unquestioned, the molecular events that cause splenic conditioning of spherocytes are unclear. Electron micrographs show that small membrane vesicles are shed during the formation of spherocytes. Animal models give further insight into the pathogenetic consequences of membrane protein defects as well as the causes of the variability of disease severity.
    Article · May 2004
  • [Show abstract] [Hide abstract] ABSTRACT: We have studied the membrane proteins of band 3 anion exchanger (AE1)-deficient mouse and human red blood cells. It has been shown previously that proteins of the band 3 complex are reduced or absent in these cells. In this study we show that proteins of the Rh complex are also greatly reduced (Rh-associated glycoprotein, Rh polypeptides, CD47, glycophorin B) or absent (LW). These observations suggest that the Rh complex is associated with the band 3 complex in healthy RBCs. Mouse band 3(-/-) RBCs differed from the human band 3-deficient RBCs in that they retained CD47. Aquaporin 1 was reduced, and its glycosylation was altered in mouse and human band 3-deficient RBCs. Proteins of the glycophorin C complex, and other proteins with independent cytoskeletal interactions, were present in normal or increased amounts. To obtain direct evidence for the association of the band 3 and the Rh protein complexes in the RBC, we examined whether Rh complex proteins were coimmunoprecipitated with band 3 from membranes. RhAG and Rh were found to be efficiently coimmunoprecipitated with band 3 from deoxycholate-solubilized membranes. Results suggest that band 3 forms the core of a macrocomplex of integral and peripheral RBC membrane proteins. The presence of these proteins in a single structural macrocomplex makes it likely that they have linked functional or regulatory roles. We speculate that this macrocomplex may function as an integrated CO(2)/O(2) gas exchange unit (metabolon) in the erythrocyte.
    Full-text · Article · Jun 2003
Show more