PMC3105174_01
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 DImax (0.44, normal range 0.54+-0.06), and increased Omin (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), CD44 (SantaCruz Biotechnology), and beta-actin (GenScript, Piscataway, NJ), 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) 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.
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F1
ARC remained elevated at 160-240 x 109/L. A peripheral blood smear at 6 months after presentation revealed rare spherocytes (Figure 1A). 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). 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).
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 missense mutation T>C 2bp 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.
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F2
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. 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).