Proc. Natl. Acad. Sci. USA Vol. 91, pp. 12496-12500, December 1994 Cell Biology Hereditary eosinophil peroxidase deficiency: Immunochemical and spectroscopic studies and evidence for a compound heterozygosity of the defect MAURIZIO ROMANO*, PIERLUIGI PATRIARCA*, CARLOS MELOt, FRANCIsco E. BARALLEt, AND PIETRO DRI** *Istituto di Patologia Generale, Universita di Trieste, Via A. Fleming, 22, 34127 Trieste, Italy; and tInternational Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34012, Trieste, Italy Communicated by Paul B. Beeson, July 22, 1994 ABSTRACT Hereditary eosinophil peroxidase (EPO; EC cursors derived from blood progenitor cells reveals a com- 1.11.1.7) deficiency is a rare abnormality of eosinophil gran- pound heterozygosity for the defect, with mutations consist- ulocytes characterized by decreased or absent peroxidase ac- ing of a base transition leading to an amino acid substitution tivity and decreased volume of the granule matrix. The mo- (Arg286 -- His) and an insertion in an intron-exon junction lecular basis of the defect is not known. We report here its that, by shifting the reading frame, generates a premature molecular characterization in an EPO-deficient subject and his stop codon. The family studies are compatible with an family members. The EPO-deficient eosinophils contained autosomal recessive mode of inheritance of the defect.§ EPO-related material as determined immunochemically using either monoclonal or polyclonal anti-EPO antibodies but had MATERIALS AND METHODS no spectroscopic evidence of EPO. Eosinophil precursors from Cell Isolation. Granulocytes were isolated from acid citrate the EPO-deficient subject contained normally sized EPO dextrose-anticoagulated peripheral blood (7). Cell popula- mRNA, which was reverse transcribed into the corresponding tions enriched in eosinophils were prepared by an adaptation cDNA clones encompassing the whole gene. Sequencing ofthese ofthe method ofRoberts and Gallin (8). Briefly, after dextran clones disclosed two mutations, a G -* A transition causing a sedimentation oferythrocytes, the white cell-rich plasma was nonconservative replacement of an arginine residue with a incubated with 5 ,.M fMet-Leu-Phe for 15 min at 37°C under histidine and an insertion causing a shift in the reading frame gentle agitation. The cell suspensions were then centrifuged with the appearance of a premature stop codon. The two on Lymphoprep (Nycomed, Oslo); most eosinophils local- mutations were located on different chromosomes indicating a ized with the erythrocytes at the bottom ofthe tube, whereas compound heterozygosity for the defect. Both the son and the mononuclear cells and most neutrophils remained at the cell daughter of the proband inherited the G -* A transition, and suspension/Lymphoprep interface. Eosinophil-enriched cell their eosinophils contained a peroxidase activity intermediate preparations were freed from erythrocytes by a brief hypo- between that of control subjects and the proband, suggesting tonic lysis. that the transition is a deficiency-causing mutation. Eosinophil Spectroscopy. The reduced-minus-oxidized difference precursors from the EPO-deficient subject were found to spectra of cell homogenates were obtained with a double- actively synthesize an EPO that was apparently normal in beam spectrophotometer (9). Cells [3-6 x 106 per ml in terms of cytochemical reaction for peroxidase and immunore- phosphate-buffered saline (PBS)] were sonicated and added activity with monoclonal and polyclonal anti-EPO antibodies, to both the sample and the reference cuvette. The absorption but spectroscopically abnormal. The cytochemical reaction for spectrum was then recorded after addition of a few grains of peroxidase tended to decrease or disappear in the eosinophil dithionite to the sample cuvette. precursors of the EPO-deficient subject but not of a normal Cytochemistry. Cytochemical staining for peroxidase and subject as differentiation went on, suggesting that the Arg -+ Sudan black staining were performed as described (10, 11). His substitution causes the production of an unstable EPO that Determination ofEPO Activity. EPO activity was measured undergoes progressive degradation as the cells mature. by a method based on oxidation of homovanillic acid (12). Antibodies. Anti-EPO antiserum was obtained by immu- nizing rabbits with purified EPO. The anti-EPO monoclonal Eosinophil peroxidase (EPO; donor:hydrogen-peroxide oxi- antibodies (mAbs) H2G3 and B2G8 were prepared and char- doreductase, EC 1.11.1.7) is a highly basic heme protein acterized as described (13). Antiserum against major basic contained in the specific granules ofeosinophil granulocytes. protein (MBP) and the anti-myeloperoxidase (MPO) mAb It is a 70-kDa dimer composed of a 15-kDa light chain and a MPO3 (14) were kindly donated by G. Gleich (Mayo Clinic, 55-kDa heavy chain held together by a disulfide bond (1). Rochester, MN) and Dr. C. E. van der Schoot (Central EPO deficiency was described for the first time by Pre- Laboratory of the Netherlands Red Cross Blood Transfusion sentey in 1968 (2) and about 100 subjects with the abnormality Service, Amsterdam, The Netherlands), respectively. have been reported (3-6). Cytochemical and biochemical Immunocytochemistry. Cytospin preparations were fixed family studies have suggested an autosomal recessive pattern with 0.125% glutaraldehyde in PBS and washed with 1% oftransmission ofthe defect in some subjects, while in others human serum in PBS to neutralize excess fixative. After a definite pattern of transmission has not been established. permeabilization with methanol/acetone (1:1, vol/vol), the The molecular mechanisms underlying the defect of EPO endogenous peroxidase was inactivated by incubating the are unknown. We report here that the eosinophils from an cytospins for 60 min at 37°C with a solution containing 10 mM EPO-deficient subject lack the characteristic EPO absorption glucose, 1 unit of glucose oxidase per ml, 2 mM NaN3 (15), spectrum but contain peptides immunochemically related to EPO. Analysis of the cDNA obtained from eosinophil pre- Abbreviations: EPO, eosinophil peroxidase; mAb, monoclonal an- tibody; MBP, major basic protein; MPO, myeloperoxidase. The publication costs of this article were defrayed in part by page charge 1To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" §The sequences reported in this paper have been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession nos. Z37727 and Z37728). 12496 Downloaded by guest on October 2, 2021 Cell Biology: Romano et al. Proc. Natl. Acad. Sci. USA 91 (1994) 12497 and 5 mM resorcinol (16). The cytospins were then incubated with a rabbit anti-EPO antiserum or the corresponding pre- immune serum diluted 1:1000 in PBS or with anti-EPO mAbs as ascites diluted 1:500 in PBS. Binding of antibodies was revealed by the avidin/biotin complex immunoperoxidase or alkaline phosphatase method (Vectastain ABC system; Vec- tor Laboratories) and counterstained with Mayer's hemalum. Eosinophil Precursors. These were obtained from liquid cultures of peripheral blood mononuclear cells (17). Molecular Genetic Techniques. RNA was extracted (18) from eosinophil precursors. Poly(dT)-primed cDNA was syn- thesized with a first-strand cDNA synthesis kit (Pharmacia). EPO cDNA was specifically amplified by nested PCR. In 400 450 500 550 400 450 500 550 the first round of amplification, two primers spanning the Wavelength, nm whole coding mRNA (nt -34 to 2247) were used. The second FIG. 1. Reduced-minus-oxidized absorption spectra of eosino- round used primer couples designed to amplify overlapping phils from the EPO-deficient subject F.V. (Left), from a control portions ofthe 2.3-kb EPO cDNA (nt -34 to 625, 339 to 1127, subject (Center), and from an erythrocyte lysate (Right). The soni- 1050 to 1721, and 1659 to 2247). A fragment comprising nt 625 cates from 4 x 106 granulocytes per ml containing 90o eosinophils to 1608 was also amplified, to establish whether the two (Left and Center) and the lysate from about 106 erythrocytes per ml mutations detected (see Results) were located on the same (Right) were used. allele or on different alleles. Standard PCR was carried out for 35 cycles (1 min at 93°C, 1 min at 56°C, 3 min at 72°C) in minus-oxidized absorption spectra ofthe eosinophil homoge- 100-,ul reaction volumes containing 1.5 mM MgCl2, 3% nates from a control subject and from F.V. and of an (vol/vol) dimethyl sulfoxide, and 2 units of Taq DNA poly- erythrocyte lysate. The spectrum of the control subject (Fig. merase (Perkin-Elmer/Cetus). The PCR products were 1 Center) shows a broad asymmetric peak with a maximum blunt-end cloned in pUC18 Sma I/BAP (Pharmacia) and at 436 nm and a shoulder at 444 nm. The 436-nm peak is sequenced with a T7 sequencing kit (Pharmacia). contributed mainly by hemoglobin that contaminates the A 260-bp genomic DNA fragment of EPO containing exon eosinophil sonicate (Fig. 1 Right). A minor contribution to 10 and its flanking regions and a 370-bp genomic DNA this peak may be given by the low-potential cytochrome b, fragment containing exon 7 and its flanking regions were which is known to be present in eosinophils and to have a amplified from DNA extracted from circulating leukocytes; characteristic absorption peak at 428 nm (21). The shoulder for this, PCR was carried out for 35 cycles (1 min at 93°C, 1 at 444 nm is contributed by EPO, which has an absorption min at 56°C, 1 min at 72°C), 100 ,ul with 1.5 mM MgC92. The peak at 448 nm (22). The slight shift ofthe EPO peak from 448 amplified fragments were directly sequenced. nm to 444 nm is caused by hemoglobin and cytochrome b in For Southern blot analysis, the amplified 370-bp genomic the sample. In the EPO-deficient subject (Fig. 1 Left) the DNA fragment was electrophores d in a 1% agarose gel and EPO peak at 444 nm is absent. blotted to a nitrocellulose membrane (19).