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Allogeneic bone marrow transplantation has the potential to cure genetic disorders affecting marrow-derived cells, such as 
-thalassemia,16 severe combined immunodeficiency,17 the Wiskott–Aldrich syndrome,18 the X-linked lymphoproliferative syndrome,19 and leukocyte-adhesion deficiency.20 We performed allogeneic bone marrow transplantation in a child with the X-linked hyper-IgM syndrome who was at risk for life-threatening opportunistic infections.
Case Report
A five-month-old boy (Subject III-3 in Figure 1) was given a diagnosis of X-linked hyper-IgM syndrome on the basis of low serum concentrations of IgG and IgA (Table 1) and the family history. Two maternal uncles (Subjects II-6 and II-7 in Figure 1) had died of protracted diarrhea at the respective ages of six months and two years; both had had hypogammaglobulinemia. A first cousin (Subject III-1) was also given a diagnosis of X-linked hyper-IgM syndrome on the basis of low serum IgG and IgA concentrations. He has persistent diarrhea caused by cryptosporidium and cholangitis associated with liver cirrhosis. He receives parenteral nutrition and is under consideration for liver transplantation.
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Because of the family history of two fatal cases of X-linked hyper-IgM syndrome and the occurrence of an opportunistic infection in the patient, the parents gave informed consent for the patient to undergo bone marrow transplantation, with his sister as the donor. The conditioning regimen consisted of busulfan (5 mg per kilogram of body weight per day for four days beginning nine days before transplantation) and cyclophosphamide (Endoxan) (50 mg per kilogram per day for four days beginning five days before transplantation). Prophylaxis against graft-versus-host disease consisted of cyclosporine (initial dose, 3 mg per kilogram per day as a continuous infusion, followed by a dose of 6 mg per kilogram per day orally beginning 1 day before transplantation and continuing for 180 days afterward) and methotrexate (10 mg per square meter of body-surface area on days 1, 3, 6, and 11 after transplantation). Because of the incompatibility between red-cell groups (the donor was A-positive and the recipient O-negative), the donor's marrow inoculum was depleted of erythrocytes on Plasmagel (gelatin in glucose). On December 8, 1993, the patient received 3.2x108 nucleated marrow cells per kilogram from his sister, who was negative for cytomegalovirus and Epstein–Barr virus. Prophylaxis against infection included hospitalization in a Trexler's isolation unit, oral administration of nonabsorbable antibiotics, and weekly treatment with intravenous immune globulin (200 mg per kilogram) for four months.
Methods
Polymorphonuclear cells and mononuclear cells were isolated from heparin-treated blood by exposure to dextran followed by Ficoll–Hypaque centrifugation. An E-rosette assay was performed by incubating mononuclear cells with neuraminidase-treated red cells from sheep.
Monocytes and B lymphocytes were isolated by sorting with a CD14-specific LeuM3 antibody (Becton Dickinson, San Diego, Calif.) and a CD19-specific antibody (Becton Dickinson), respectively, in a FACStar Plus cell sorter (Becton Dickinson).
The expression of the CD40 ligand was evaluated with a CD40–immunoglobulin fusion protein (CD40-Fc)22 as described previously,3 after a five-hour incubation of E-rosette–forming cells with phorbol myristate acetate and ionomycin. A polymorphism of the CD40 ligand microsatellite CA repeat21 was studied after amplification with the polymerase chain reaction with two primers flanking the CA repeat located in the 3' untranslated region of the CD40 ligand gene. Products were analyzed on 5 percent denaturing polyacrylamide gels.
Fluorescence in situ hybridization was performed with an X-chromosome 
-satellite probe (DX21) and a Y-chromosome–cocktail probe (DY23 and DY21) labeled with biotin and digoxigenin, respectively (Oncor, Gaithersburg, Md.). Hybridization was carried out according to the manufacturer's recommendations. After overnight hybridization and post-hybridization washes, the slides were incubated in blocking solution (0.1 percent phosphate-buffered saline, 20.5 percent Tween, and nonfat dry milk). The X-chromosome probe was detected with avidin–Texas red (Vector Laboratories, Burlingame, Calif.), and the Y-chromosome probe with mouse antidigoxigenin fluorescein-labeled antibody (Boehringer–Mannheim, Mannheim, Germany). The slides were mounted with an antifade solution containing 1 µg of 4',6-diamidino-2-phenylindole per milliliter, examined with a Leitz microscope (model DM, Leitz, Rockleigh, N.J.), and analyzed with a Cytovision computer (Imaging International, Sunderland, United Kingdom).
Results
The patient's clinical course after bone marrow transplantation was uneventful. The absolute granulocyte count exceeded 500 per cubic millimeter by day 26, and the last platelet transfusion was given on day 20. Manifestations of neither acute nor chronic graft-versus-host disease occurred. No infectious complications were seen. The patient was sent home on day 35. Seventeen months after bone marrow transplantation, he is doing well, with normal blood counts and no need for therapy.
Bone marrow engraftment was demonstrated by several means. The patient's red-cell group changed from O-negative to A-positive. In this boy, fluorescence in situ hybridization with probes specific for the X and Y chromosomes showed that on day 360, 100 polymorphonuclear neutrophils and 100 peripheral-blood mononuclear cells were all positive for the X-chromosome probe and negative for the Y-chromosome probe. The same result was found on 50 separated E-rosette–forming cells and 50 cells that did not form E rosettes (data not shown).
Twelve months after bone marrow transplantation, examination of the CA-repeat polymorphism associated with the CD40 ligand showed a shift from allele 2 (associated with a mutation of the CD40 ligand gene) before bone marrow transplantation to alleles 2 and 3, which were present in the carrier donor (Figure 3). The same change was detected in E-rosette–forming cells, cells that did not form E rosettes, and granulocytes. Expression of the CD40 ligand by activated T cells from the recipient one year after bone marrow transplantation was equivalent to the expression of the ligand by the donor's T cells (Figure 2A, Figure 2B, Figure 2C, and Figure 2D). In both children, expression of the ligand was lower than in control subjects, because of the donor's carrier status (Figure 2A, Figure 2B, Figure 2C, and Figure 2D).
The patient had a full recovery of immune function six months after bone marrow transplantation. By then he had normal T-cell and B-cell counts; antigen-induced T-cell proliferation in vitro; normal serum concentrations of IgG, IgA, and IgE after the cessation of intravenous immune globulin therapy; and normal antibody responses to immunization with poliovirus and tetanus toxoid (Table 1).
Discussion
We report successful allogeneic bone marrow transplantation in a child with the X-linked hyper-IgM syndrome. To reduce the risks of long-term sequelae, the conditioning regimen consisted only of chemotherapy, in accordance with a regimen used in other patients with various immunodeficiencies.23 Full engraftment was shown by several means, including changes in red-cell antigens, the results of fluorescence in situ hybridization for X and Y chromosomes, polymorphism of the CD40 ligand gene, and expression of the CD40 ligand by activated T cells. After transplantation, expression of the ligand by the recipient's T cells was equivalent to that by the donor's T cells, which had reduced expression because of her carrier status. In female carriers of the mutant CD40 ligand gene, a variable fraction of T cells expresses the CD40 ligand because of random inactivation of the X chromosome.24 As expected on the basis of the normal immunologic and clinical status of carriers of the X-linked hyper-IgM syndrome, the reduced number of T cells expressing the CD40 ligand in the donor was nevertheless sufficient to provide signals for switching to the production of IgG, IgA, and IgE25 and normalizing immune functions.
Not all patients with X-linked hyper-IgM syndrome have life-threatening opportunistic infections like the family of the propositus.1 It has not been possible to correlate a severe clinical outcome with given genotypes.3,6,25 The frequency of opportunistic infections in X-linked hyper-IgM syndrome is not known. In a recent summary, P. carinii pneumonitis developed in 8 of 67 patients.1 Infections caused by H. capsulatum, aspergillus, cryptococcus, and toxoplasma have also been reported in this syndrome.1,8,9,10,11,12,13 In a recently reported series, 5 of 16 patients died of either encephalitis or cholangitis with liver failure in a 14-year period.13 In our own experience, severe cholangitis associated with cryptosporidial infections developed in 3 of 12 patients (including the first cousin of the child described in this report). One died of liver failure. Liver transplantation in patients with the X-linked hyper-IgM syndrome has been reported.13,26
The relation between the clinical severity of the X-linked hyper-IgM syndrome and the genotype is not obvious, because a large number of different mutations have been described in the disease. Therefore, the formulation of a prognosis in an individual case requires great caution. Because of the poor outcome in the patients with X-linked hyper-IgM syndrome in the family of the propositus and the occurrence of P. carinii pneumonitis in the patient, we assumed that he was susceptible to further opportunistic infections. Some unknown genetic factors might modify the clinical consequences of the X-linked hyper-IgM syndrome from a benign course to a severe T-cell immunodeficiency, as we observed in this family.
Supported by the Institut National de la Santé et de la Recherche Médicale and the Association Française contre les Myopathies.
We are indebted to the nurses who took care of the patient.
Source Information
From the Unité d'Immunohématologie (C.T., M.B., E.H., S.B., A.F.), INSERM Unité 429 (G.S.B., F.L.D., A.F.), and the Laboratoire de Cytogénétique, Hôpital des Enfants-Malades (D.T.) — all in Paris.
Address reprint requests to Dr. Fischer at INSERM Unité 429, Hôpital Necker, 149 Rue de Sevres, 75015 Paris, France.
References
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