Aplastic anaemia in a case of hereditary neutrophil Fcgamma receptor IIIb deficiency

British Journal of Haematology, 1997, 99, 422–425

SHORT REPORT

Aplastic anaemia in a case of hereditary neutrophil Fcg receptor IIIb deficiency O L I VI E R T OURNIL HAC , J E A N -JACQ U E S K I L A D J I A N , J E A N -M I C HE L C AYUE L A , M A R I A -E L E´ NA N O G U E R A , J E A N -M A RC Z INI ,† M A R I E -T H E´ R E` S E DA N I E L , O D I L E M A A RE K , E´ L IAN E G L U C K M A N ,* G E´ RA R D S OC IE´ * A ND F R A N C¸ OIS S I G AU X Laboratoire Central d’He´matologie et Unite´ INSERM 462, Hopital Saint-Louis, *Unite´ Fonctionnelle de Greffe de Moelle, Hoˆpital Saint-Louis, and †Service d’He´matologie, Hopital Lariboisie`re, Paris, France Received 22 April 1997; accepted for publication 28 July 1997

Summary. CD16 antibodies recognize Fcg receptors III of a and b types. In a patient with severe idiopathic aplastic anaemia (AA), polymorphonuclear cells, which in normal subjects express FcgRIIIb, were found to be CD16 negative. The FcgRIIIb gene configuration was analysed by PCR on peripheral blood mononuclear cells. Bi-allelic deletion encompassing at least part of the coding exon 5 was found in the patient and his brother, suggesting a hereditary defect. The patient underwent successful bone marrow transplantation from his HLA-matched brother despite a similar

Membrane receptors for the Fc region of immunoglobulins (FcRs) form a family of homologous receptors including the low-affinity receptors (FcgRII and FcgRIII) and the highaffinity (FcgRI) receptor for IgG (reviewed by Hulett & Hogarth, 1994; Ravecht, 1994). The FcgRs, expressed as hetero-oligomeric or monomeric glycoproteinic chains, have an a subunit with highly conserved extracellular ligandbinding immunoglobulin-like domain, and are involved in phagocytosis of IgG-opsonized particles. CD16 monoclonal antibodies detect the two described isoforms of FcgRIII (FcgRIIIa and FcgRIIIb) which are encoded by two very homologous genes (FcgRIIIA and FcgRIIIB, respectively). FcgRIIIa is a transmembrane isoform expressed mostly on natural killer cells and macrophages. FcgRIIIb is a monomeric truncated form, anchored to the plasma membrane of polymorphonuclear neutrophils by a glycosylphosphatidylinositol (GPI) moiety, and displays a genetically determined polymorphism with two different alleles encoding isoforms NA-1 and NA-2. As for other GPI-anchored molecules, lack of FcgRIIIb isoform can be due either to an anchorage defect – as in Correspondence: Dr J. M. Cayuela, Laboratoire Central d’He´matologie, Hoˆpital Saint-Louis, 1 av. Claude Vellefaux, 75475 Paris Cedex 10, France.

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phenotype and genotype. This observation suggests that FcgRIIIb hereditary deficiency in donor and/or recipient does not impair engraftment and justifies the use of other monoclonal antibodies in addition to CD16 in the study of GPI-anchored antigen expression. Keywords: FcgRIII, CD16, aplastic anaemia, paroxysmal nocturnal haemoglobinuria, allogeneic bone marrow transplantation.

paroxysmal nocturnal haeoglobinuria – or to the absence of the molecule itself, as in hereditary deficiencies. Some haematological diseases have been reported in association with hereditary FcgRIIIb deficiency; however, no association between aplastic anaemia (AA) and this congenital disorder has previously been reported. We report the first case of such an association. CASE REPORT A 21-year-old man was referred for diagnosis and treatment of pancytopenia. Full blood count at diagnosis was as follows: haemoglobin 5.7 g/dl, white blood cells 2.2 × 109/l, neutrophils 10%, eosinophils 1%, lymphocytes 86%, monocytes 3%, platelets 20.0 × 109/l. There was no sign of myelodysplasia, and bone marrow trephine biopsy was hypocellular. Cytogenetic analysis of bone marrow was normal. The search for a known cause of the disease was negative and he was therefore considered to have an idiopathic AA. As he met the criteria for the severity of the disease, and since he had an HLA-identical brother, he underwent bone marrow transplantation (BMT) early in September 1996, after cytoxan plus antithymocyte globulin conditioning. Cyclosporine and methotrexate were used as graft-versus-host prophylaxis. The early period post-transplant was uneventful. Engraftment took place as expected. q 1997 Blackwell Science Ltd

Short Report METHODS AND RESULTS Before BMT, in order to eliminate an association between paroxysmal nocturnal haemoglobinuria (PNH) and aplastic anaemia (AA), flow cytometry was performed on leucocytes from peripheral blood (Griscelli-Bennaceur, 1995), using an XL Coulter cytometer, and Immunotech monoclonal antibodies (MoABs) specific for three GPI-linked antigens CD16 (3G8), CD66b (80H3), CD13 (SJ1D1) and specific for CD56 (T199). Surprisingly, we found an isolated and complete lack of CD16 expression on neutrophils, whereas expression of CD13 (data not shown) and CD66b (Fig 1B) was normal, showing that absence of CD16 was not due to a GPI defect. Furthermore, we were able to detect a small population of both CD56 and CD16 positive cells, consistent with a normal

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expression of FcgRIIIa isoform on NK cells (Fig 1A). Complete lack of CD16 expression was also found on neutrophils derived from the patient’s brother, whereas his sister and a healthy volunteer displayed a normal phenotype. Confirmation of these data was obtained by an immunoenzymatic assay on smears of leucoconcentration (data not shown). Eight genes encoding the FcgRs have been mapped on chromosome 1. FcgRIIIA and FcgRIIIB genes are very closely related genes located 250 kb apart on chromosome band 1q22. They comprise five exons and display a high degree of identity (Peltz et al, 1989; Ravecht & Perussia, 1989). However, a 12 nucleotide difference in their coding sequence enables easy recognition of both FcgRIII genes by restriction fragment length polymorphism (RFLP) analysis using restriction endonucleases Dra I, Taq I or BamHI (Clark

Fig 1. Double-staining flow-cytometry analysis in patient with aplastic anaemia and control samples. (A) Lymphocytes analysis, using monoclonal antibodies specific for CD16 and CD56. In the control sample (healthy volunteer), 39% of lymphocytes were positive for both CD16 and CD56, which is consistent with the presence of NK cells. In the patient sample, only 2.5% and 0.5% of lymphocytes were positive for CD16 alone or both CD16 and CD56, respectively. (B) Polymorphonuclear cells analysis using CD16 and CD66b. In the control sample, 99% of polymorphonuclear cells were positive for both CD16 and CD66b (neutrophils). No CD16-positive polymorphonuclear cells were detected in the patient sample. These data are consistent with a lack of FcgRIIIb expression in the patient’s polymorphonuclear cells, associated with a low but persistent expression of FcgRIIIa in lymphocytes. q 1997 Blackwell Science Ltd, British Journal of Haematology 99: 422–425

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Short Report

Fig 2. RFLP analysis. (A) Position of PCR primers (horizontal arrows) in exon 5, and restriction endonuclease DraI sites, shared by FcgRIIIA and B genes (DraIA-B) and specific of FcgRIIIA (DraIA). (B) Agarose gel electrophoresis of PCR fragments specific for FcgRIIIA and B genes, amplified from genomic DNA of the propositus (lane 4), his brother (lane 3), his sister (lane 2) and a healthy volunteer (lane 1), after digestion by Dra I. Negative control (no DNA) (lane 5), molecular weight marker V (Boehringer Mannheim, France) (lane 6). Fragments sizes are indicated by arrows. (C) Agarose gel electrophoresis of the same PCR fragments before digestion by Dra I.

et al, 1990; de Haas et al, 1995). Genomic DNA was extracted from peripheral blood mononuclear cells and FcgRIIIA and FcgRIIIB genes were amplified by PCR, using primers located in exon 5, as described previously (de Haas et al, 1995). Both genes produce a 507 bp amplified fragment. RFLP analysis of these products using restriction endonuclease DraI (Promega, France), enable the detection of a 456 bp fragment specific for FcgRIIIB gene. This 456 bp fragment was detected in the sister and a healthy volunteer, but was absent in both the patient and his brother (Fig 2). DISCUSSION A congenital defect of neutrophil CD16 expression has already been reported with variable phenotypic associations. Most reported cases are of healthy individuals. Systematic serological screening of a population of 3377 blood donors revealed the absence of CD16 neutrophil expression in four donors (Fromont et al, 1992). Cases of CD16 deficiency have also been reported in healthy mothers whose newborns

developed transient neonatal alloimmune neutropenia as the result of transplacental transfer of maternal anti-FcgRIII alloantibodies (Schnell et al, 1989; Stroncek et al, 1991; Cartron et al, 1992; Fromont et al, 1992; de Haas et al, 1995). However, lack of CD16 expression on neutrophils has been reported in association with a broad spectrum of clinical manifestations including anaemia, neutropenia, autoimmune thyroiditis, recurrent infections, acute myeloblastic leukaemia (de Haas et al, 1995) and a case of systemic lupus erythematosus (Clark et al, 1990). Genomic analysis has been performed in 22 reported cases (Clark et al, 1990; de Haas et al, 1995), showing a deletion of at least a part of both alleles of the FcgRIIIB gene. A family study by semiquantitative Southern blotting showed that in six families both parents were heterozygote for FcgRIIIB deficiency, whereas in one family only one parent was a heterozygote, suggesting the occurrence of a mutation during spermatogenesis. In the case described here, we have the same result from RFLP analysis and the FcgRIIIb deficiency of the patient also results probably from a deletion of at least a part of both FcgRIIIB alleles. We report the first case of FcgRIIIb deficiency associated with idiopathic AA, but, as in the other reported cases, the clinical relevance of this defect remains unclear. Autoimmune disorders have been reported in association with FcgRIIIb deficiency and an autoimmune mechanism has also been proposed in AA. Despite this hypothesis, we must consider the important variability of phenotypic association with FcgRIIIB defect. Variable-sized deletions of chromosome 1 could be one explanation for this phenotypic variability. In this regard, deletion of the telomeric flanking FcgRIIC gene has been found in all of the 10 studied cases, but without any correlation with phenotype (de Haas et al, 1995). Using conventional banding methods, constitutional karyotyping failed to demonstrate any deletion of chromosome 1 in our patient, his brother or his sister. Bone marrow transplantation from FcgRIIIb positive donor to FcgRIIIb negative recipient have already been performed with success in a man in complete remission of acute myeloid leukaemia (Minchinton et al, 1995). In our case, both the donor and recipient were FcgRIIIb negative. After 6 months follow-up, engraftment remains uneventful. Whatever the role, if any, of FcgRIIIb deficiency in the pathogenesis of AA, it does not preclude geno-identical BMT. FcgRIIIb is a GPI-anchored surface antigen whose expression is often tested when searching for PNH clones, especially in exploration of AA (Griscelli-Bennaceur et al, 1995). It should be emphasized that in AA, lack of CD16 does not always indicate PNH, since it can result from hereditary deficiency of the GPI-linked protein instead of a defect in GPI anchorage. We used a combination of three MoABs (CD16, CD14 and CD66b) which identified this sole case of FcgRIIIb deficiency in more than a hundred AA cases tested over a 3-year period. ACKNOWLEDGMENT We thank Ve´ronique Ce´zard, Kenneth Cummins, Sophie Genyk, Laurence Grollet, Daniel Juanjiche, Christine Orange, Pascal

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