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We describe three unrelated girls who had an immunodeficiency disease with granulomas in the skin, mucous membranes, and internal organs. All three girls had severe complications after viral infections, including B-cell lymphoma associated with Epstein–Barr virus (EBV). Other findings were hypogammaglobulinemia, a diminished number of T and B cells, and sparse thymic tissue on ultrasonography. Molecular analysis revealed that the patients were compound heterozygotes for mutations in recombination activating gene 1 or 2 (RAG1 or RAG2). In each case, both parents were heterozygous carriers of a RAG mutation. The mutations were associated with reduced function of RAG in vitro (3 to 30% of normal activity). The parents and one sibling in the three families were healthy.
We describe a type of primary immunodeficiency disease with RAG1 or RAG2 mutations that diminish RAG activity and allow the maturation of a limited number of T and B cells. Three girls under the age of 10 years from three different families presented with extensive granulomatous disease involving the skin, mucous membranes, and internal organs.
Case Reports
Patient 1
At the age of 2.5 years, Patient 1, the daughter of nonconsanguineous parents, was referred to the University Children's Hospital Düsseldorf because of papulonodular skin lesions on the face and limbs. During the previous year, the lesions had failed to respond to treatment with topical corticosteroids (Table 1 and Figure 1A). The medical history was otherwise unremarkable, and the patient had received standard immunizations without complications.
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In immunohistochemical studies, the epithelioid cells were positive for CD68, and the lymphocytes were positive for CD3 and CD8 and negative for CD79a and CD4. Paraffin-fixed skin-biopsy samples, examined by seminested, multiplex polymerase-chain-reaction (PCR) assay, showed clonal rearrangements of the gene encoding T-cell receptor 
. These findings supported a diagnosis of small-cell pleomorphic T-cell lymphoma. Biopsies of bone marrow and liver did not reveal clear evidence of systemic involvement. Immunologic abnormalities were considered to be secondary to the presumed lymphoma.
The child was treated with chemotherapy for T-cell lymphoma, but numerous new papulonodular efflorescences developed, and the existing lesions progressed. After the lesions failed to improve after 4 months of chemotherapy, the treatment was stopped and the diagnosis of T-cell lymphoma was reconsidered. A primary immunodeficiency was suspected, since there was profound hypogammaglobulinemia (Table 2), and tissue infiltrates of clonal CD8+ T cells had been described in a patient with common variable idiopathic immunodeficiency.7 Periodic acid–Schiff (PAS), Giemsa, and Ziehl–Neelsen staining of skin-biopsy specimens showed no evidence of fungi, leishmania, or mycobacteria, and PCR assays for mycobacterium tuberculosis and Whipple's bacilli were negative (Table 2 of the Supplementary Appendix, available with the full text of this article at www.nejm.org).
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At the age of 7.5 years, Patient 2, the daughter of nonconsanguineous parents, was referred to the University Children's Hospital Ulm for evaluation of an immunodeficiency. Her 6-year-old brother was healthy. At the age of 9 months, she had severe varicella with hepatitis and bacterial superinfection. At 27 months of age, multiple, treatment-resistant ulcerative skin lesions developed on her limbs and face (Fig. 1 of the Supplementary Appendix). Skin-biopsy specimens showed changes similar to those in Patient 1: nodular collections of epithelioid cells surrounded by moderately dense lymphocytic infiltrates with some neutrophils. However, no necrosis was observed. Extensive testing failed to conclusively reveal microbiologic agents (Table 2 of the Supplementary Appendix). In an attempt to treat the lesions, regular subcutaneous immunoglobulin injections were started when the patient was 4 years old. At 6.5 years of age, nodular lesions developed on her tongue; these lesions had a histologic similarity to the cutaneous lesions (Fig. 1 of the Supplementary Appendix). PAS, Giemsa, and Ziehl–Neelsen staining showed no pathogens in either skin or tongue. PCR assays for mycobacterium tuberculosis and Whipple's bacilli that were performed on a tongue-biopsy specimen were negative. Similar granulomatous changes were also noted in biopsy specimens of the adenoids and the lung, which were obtained because of respiratory complications (pneumonias, bronchiectasis, and atelectasis).
Results of immunohistochemical analysis of the tongue lesion were similar to findings in Patient 1. Studies of the tongue-biopsy specimens with seminested, multiplex PCR showed a polyclonal population of T cells (Table 1 of the Supplementary Appendix). Therapy with corticosteroids, dapsone, and clofazimine was ineffective. Cyclosporine treatment led to deterioration of skin lesions. Repeated skin grafting was necessary after partial destruction of the Achilles tendon by progressive ulceration. Immunophenotyping of blood cells revealed low numbers of T and B cells but a normal number of natural killer cells (Table 2). Thymic tissue was undetectable on ultrasonography. Molecular studies revealed compound heterozygous RAG1 mutations.
Hematopoietic stem-cell transplantation from an HLA-matched unrelated donor was performed when the patient was 8.5 years old. More than a year after transplantation, no new skin lesions have developed, and the patient is receiving immunosuppressive therapy for chronic graft-versus-host disease.
Patient 3
At the age of 9.9 years, Patient 3, the only child of nonconsanguineous parents, was referred to the University Children's Hospital Düsseldorf with a history of recurrent pneumonia beginning at the age of 2 years. Otherwise, she had an uneventful early childhood. At 8.5 years of age, she had a severe varicella infection complicated by encephalitis and the acute respiratory distress syndrome. An immunologic evaluation that was performed when she was 9.9 years old showed profound hypogammaglobulinemia with low numbers of T and B cells (Table 2). At the age of 10.5 years, she was found to have massive splenomegaly with hypodense infiltrates on ultrasonography. Splenic biopsy revealed granulomas with epithelioid cells. Staining with PAS, Giemsa, and Ziehl–Neelsen was negative. PCR assays for mycobacterium tuberculosis and Whipple's bacilli that were performed on the splenic specimen were negative (Fig. 2 of the Supplementary Appendix).
Immunophenotyping of the lesion showed a polyclonal population of T cells (Table 2). Small nodular infiltrates consistent with granulomatous disease were seen on computed tomography of the lungs. The results of bronchoalveolar lavage were inconclusive. Molecular analysis, which was performed when the patient was 10.7 years old, revealed compound heterozygous mutations in RAG2. The patient's condition is stable on regular IgG infusions, and she is being evaluated for stem-cell transplantation.
Molecular Findings
The parents of each of the three children gave informed consent to carry out the investigations described below.
Compound heterozygous mutations in RAG1 or RAG2 were identified in all three patients (Figure 2 and Table 3). None of the mutations were detected in 105 healthy control subjects who were matched for white race (data not shown). The functional significance of the mutations was evaluated in primary human dermal fibroblasts that were cotransfected with substrate vectors that allow scoring for V(D)J recombination efficiency and expression plasmids coding for wild-type or mutant RAG1 or RAG2 proteins (Table 4). The percentages of recombination-positive cells in the subpopulation of transfected fibroblasts varied from less than 5% to approximately 30% of the positive control. Lacking suitable monoclonal antibodies against human RAG proteins, we measured RNA stability of the in vitro transfectants as a surrogate. All RNAs were equally well expressed, as assessed by semiquantitative reverse-transcriptase PCR.
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Discussion
We describe three girls with a primary immunodeficiency disease associated with hypomorphic mutations in one of the two recombinase activating genes (RAG1 and RAG2). Two of the three patients (Patients 1 and 3) had either no or insignificant infections in infancy or early childhood.
The histologic findings in various organs were notable for granulomas containing CD68+ epithelioid cells and CD8+ T cells, which were polyclonal in two of the three patients. In peripheral blood, there were diminished numbers of T and B cells but a normal number of natural killer cells. The proportion of activated T cells (HLA-DR+) was increased, and most T cells were of the memory type (CD45RO+).
The clinical findings in the three children were clearly distinct from those of classical SCID (null mutations of both RAG alleles and no RAG activity), the Omenn syndrome (a missense mutation of at least one RAG allele with retention of some RAG activity), and atypical SCID (a missense mutation of at least one RAG allele but with features that are not typical of the Omenn syndrome).8,9 In contrast to our patients, those with atypical SCID are children of consanguineous parents, present with threatening infections during infancy, and do not have granulomatous disease. Of five such patients who underwent hematopoietic stem-cell transplantation in infancy or early childhood, two survived and three died of severe cytomegalovirus infection.
The mutations we found in RAG genes have been detected in other patients with a different phenotype.10 The R507W mutation in Patient 1 has also been detected in an infant who had a skin rash, eosinophilia, slight lymphopenia, and normal levels of IgG but decreased IgA and IgM. Another child with a heterozygous R737H mutation (found in Patient 1) presented at the age of 1 month with typical Omenn syndrome. One of the mutations found in Patient 2 (R975W, RAG1) has been reported in a patient with the Omenn syndrome but with a different amino acid exchange (R975Q).3 The other mutation in Patient 2 and both mutations in Patient 3 (RAG2) have not been reported previously.
Functional analysis of RAG alleles showed V(D)J recombination activity varying between less than 5% in RAG mutants of Patients 1 and 2 and approximately 30% in RAG mutants of Patients 1 and 3, making it highly likely that the RAG mutations caused the impaired V(D)J recombination. Residual functional RAG1 or RAG2 allows a low level of T-cell production.3,10,11 The presence of CD4+/CD45RA+ cells in all three of our patients may reflect production of naive T cells by the thymus. Spectratyping of T-cell receptors in Patients 1 and 3 revealed a diverse repertoire. This finding was unexpected, since patients with SCID or the Omenn syndrome have no T cells or a population of T cells with a very restricted receptor repertoire.12 The number of T cells in our patients is decreased, but the late onset and low incidence of repeated or life-threatening infections may indicate that the T-cell repertoire is sufficiently broad and competent for protection against microbes, at least for a limited period.
The late onset and the presence of functional T cells could be related to somatic mosaicism in T cells or engraftment of maternal T cells. Somatic mosaicism has been observed repeatedly in patients with SCID that is caused by adenosine deaminase deficiency or with X-linked SCID (a deficiency in the interleukin-2–receptor common chain). The mosaicism results from a mutation that reverts the mutation in adenosine deaminase or X-linked SCID to a wild-type mutant.13,14,15 In our three patients, revertants were excluded by genomic sequencing of sorted CD3, CD19, and CD14 cells in which we found only the disease-causing mutations. Engraftment of maternal T cells, which can also lead to partial immunologic reconstitution resulting in a milder phenotype, was excluded by HLA typing of CD3 T cells in all three patients.
It is unclear whether the granulomas in our patients are the reactive sarcoid-like granulomas found in other primary immunodeficiency disorders, especially in granulomatous common variable immunodeficiency disease.7,16,17,18,19 In a cohort of 19 adults with this disease, we found no functionally relevant RAG1 or RAG2 mutations (data not shown). Furthermore, we did not see resolution of granulomas after treatment with intravenous immunoglobulin, as has been noted in some patients with common variable immunodeficiency.18,19 Extensive and repeated tests revealed no pathogen in the granulomas, but it is possible that the lesions were a reaction to an undetected low-virulence pathogen, comparable to "sterile" granuloma formation seen in patients with chronic granulomatous disease. It is also possible that, apart from the RAG mutations, other mutations in modifier genes may be responsible for the granulomas.
The late onset and atypical clinical picture of the forms of RAG1 or RAG2 deficiency we describe here may prompt the inappropriate use of chemotherapy or immunosuppression. RAG deficiency should be a consideration in older patients with evidence of combined humoral and cellular immunodeficiency and otherwise unexplained granulomatous lesions.
Supported by grants from the Jeffrey Modell Foundation (to Dr. Niehues), the Elterninitiative Kinderkrebsklinik (to Dr. Göbel and Dr. Niehues), the Forschungskommission der Medizinischen Fakultät, University of Düsseldorf (to Dr. Feyen), and the Deutsche Forschungsgemeinschaft and the Else Kröner Fresenius Foundation (to Dr. Borkhardt).
No potential conflict of interest relevant to this article was reported.
We thank E.M. Rump, S. Braun, T. Kersten, and I. Janz at the Institute for Clinical Transfusion Medicine and Immunogenetics and S. Bellert, W. Kus, G. Göbel, and E. Oellers in Düsseldorf for excellent technical assistance; D. Diallo, H. Kreipe, and P. Racz in Düsseldorf, Hannover, and Hamburg for their evaluations of histologic analyses; U. Salzer of the Department of Clinical Immunology and Rheumatology, University of Freiburg, for contributing samples from patients with common variable immunodeficiency; and J. Schaper, Düsseldorf, for evaluation of spleen sonography.
Source Information
From the Departments of Pediatrics and Adolescent Medicine (C.S., A.S., W.F.) and Internal Medicine (B.M.) and the Institute for Transfusion Medicine (U.P.), University Hospital Ulm, and the Institute for Clinical Transfusion Medicine and Immunogenetics (K.S.) — all in Ulm; the Department of Pediatric Oncology, Hematology, and Clinical Immunology, Children's Hospital, Universitäts Klinikum Düsseldorf, Heinrich Heine University, Düsseldorf (K.H., S.G., O.F., B.H., D.T.S., U.G., A.B., T.N.); and the Departments of Pathology (R.K.) and Dermatology (M.M.), University Hospital Aachen, Aachen — all in Germany; and the Department of Dermatology, Leiden University Medical Center, Leiden, the Netherlands (R.W.).
Address reprint requests to Dr. Niehues at the Center for Child and Adolescent Health, HELIOS Klinikum Krefeld, Academic Hospital, Heinrich Heine University of Düsseldorf, Lutherpl. 40, 47805 Krefeld, Germany, or at tim.niehues{at}helios-kliniken.de.
References
T cells. J Clin Invest 2005;115:3140-3148. [CrossRef][ISI][Medline]
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