|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
IMMUNOBIOLOGY
From the Department of Pediatric Oncology, Institute of
Development, Aging and Cancer, Tohoku University, Sendai, Japan;
Department of Human Genome and Multifactorial Disease, Instituto di
Tecnologie Biomediche Avanzate, Consiglio Nazionale delle Ricerche,
Milano, Italy; Miyagi Red Cross Blood Center, Sendai, Japan; and
Division of Transfusion, Tohoku University Hospital, Sendai, Japan.
Mutations of the RAG1 or RAG2 protein that eliminate their
recombination activity result in T-B-severe combined immunodeficiency (SCID), whereas mutations retaining partial recombination activity lead
to Omenn syndrome, a peculiar SCID characterized by increased host T
cells and absence of circulating B cells. The prognosis of this disease
is fatal, unless hematopoietic stem cell transplantation is performed.
This study reports a case of atypical SCID, carrying RAG1 mutations.
The patient survived for 6 years without hematopoietic stem cell
transplantation. The missense mutation, tested by in vivo recombination
assay, revealed residual recombination activity. By the age of 5 years,
the patient developed host B cells, but not T cells, possibly
due to engrafted maternal T cells. In addition, the host B cells were
able to produce antibodies, including anti-herpes simplex
virus-antibodies. The fact that host B cells could produce antibodies
in this patient could explain not only the mild phenotype observed but
also, at least in part, how patients with Omenn syndrome produce
immunoglobulin E and sometimes immunoglobulin M, as the same missense
mutation of RAG1 gene has been reported in a patient with
Omenn syndrome.
(Blood. 2001;98:1464-1468) Two lymphoid specific genes, RAG1
and RAG2, are essential for V(D)J recombination of the
antigen receptors.1,2 Mutations of the RAG1 or RAG2
protein that eliminate the recombination activity result in T-B-severe
combined immunodeficiency (SCID).3 Missense mutations of
these proteins with partial recombination activity have been
demonstrated to lead to Omenn syndrome, a SCID characterized by failure
to thrive, diarrhea, diffuse erythrodermia, hepatosplenomegaly, lymphadenopathy, hypereosinophilia, elevated serum immunoglobulin E
(IgE), increased host T cells that show oligoclonal expansion with an
activated phenotype, and absence of circulating B
cells.4,5 These diseases are fatal; the patients die early
in life, unless treated by bone marrow transplantation, because of
susceptibility to opportunistic organisms.6-8 Most
recently, 41 patients carrying RAG mutations were analyzed and a
subgroup, termed atypical SCID/OS, was identified. The patients in this
subgroup were characterized by the presence of some T and occasionally
B cells but did not fulfill the criteria of Omenn
syndrome.9 However, the immunopathologic mechanism of this
subgroup has not been well characterized. A similar phenotype to Omenn
syndrome has also been reported due to engraftment of maternal T cells,
leading to graft-versus-host disease (GVHD).10,11
In the present study, we report the case of a Japanese girl, bearing
RAG1 mutations, who presented with peripheral host B cells in
combination with maternal T cells, and who survived for 6 years without
stem cell transplantation. Furthermore, at least a subset of her B
cells was demonstrated to be functional, as the patient produced
anti-herpes simplex virus (HSV)-antibodies.
Cell lines
Lymphocyte activation
Sequencing analysis of genomic DNA Genomic DNAs were isolated from fractionated PBMCs or the EBV-transformed B-LCLs by using the QIAamp Blood Kit (QIAGEN, Chatsworth, CA). The RAG1 gene was amplified in 4 segments (Table 1). Sequencing was performed directly on the polymerase chain reaction (PCR) products purified by QIAquick PCR Purification Kit (QIAGEN). Mutations were confirmed by analysis of several clones from PCR amplification products cloned into the TA vector (Invitrogen, Carlsbad, CA) and sequenced by ABI Taq DyeDeoxy Terminator Cycle Sequencing kit on an automated Applied Biosystems DNA sequencer, model 373A (Perkin-Elmer, Foster City, CA) as previously described.12
Genomic DNAs from the patient and healthy individuals were obtained after informed consent according to the Declaration of Helsinki. Expression plasmid construction and transfection The mutant complementary DNAs (cDNAs) were generated by site-directed mutagenesis, and they were then cloned into an expression vector, pCDM8. The wild-type human RAG1 vector was a generous gift from Dr K. Schwarz (University Ulm, Germany). Sequences of the mutant cDNAs were confirmed by sequencing. The expression vectors were transiently transfected into 293T cells by using FuGene 6 (Boehringer Mannheim, Mannheim, Germany). RAG1 protein from the transfected 293T cells was detected by immunoblotting with rabbit polyclonal anti-human RAG1 antibodies (Santa Cruz, Santa Cruz, CA) as previously described.13Magnetic cell sorting PBMCs were resuspended in phosphate-buffered saline (PBS) supplemented with 0.5% bovine serum albumin and 2 mM EDTA and incubated with anti-CD4, anti-CD8, anti-CD19, and anti-CD56 microbeads at 4°C for 30 minutes. The cells were purified by positive magnetic immunoselection by using automated magnetic cell sorting (autoMACS; Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. The selected fractions contained 98.7% CD4+ cells, 95.1% CD8+ cells, 91.8% CD19+ cells, and 96.1% CD56+ cells (data not shown).In vivo recombination assay PCR products bearing the RAG1 mutations found in the patients were cloned in pEBG vector containing aa 330-1043 of human RAG1 (RAG1 active core14,15).The mutant proteins were tested for recombination activity in 293T cells. Human RAG1 active core (aa 330-1043) carrying the mutations were cotransfected with mouse wild-type RAG2 active core (aa 1-388) and with the recombination substrate pJH200.16 Cells were transfected by calcium phosphate precipitation and harvested 48 hours later. Recombined products were isolated and analyzed for recombination frequency by PCR analysis by using primers OOP2 and CR3.17 The reactions incorporated 32P dCTP. Reaction products were analyzed on a 5% polyacrylamide gel and visualized by autoradiography. Detection of anti-HSV antibody-producing cells Anti-HSV antibody-producing cells were screened by an enzyme-linked immunosorbent assay (ELISA) kit (Herpes Simplex IgG detection kit; Denka Seiken, Tokyo, Japan) according to the manufacturer's protocol. Briefly, the assay was performed by incubating 1:1 dilutions of culture supernatants from the patient's EBV-transformed B-LCLs for 1 hour at room temperature in 96-well microtiter plates coated with HSV antigen. After rinsing with 0.05% Tween 20 in PBS, bound human antibody was detected with goat anti-human IgG antibody labeled with horseradish peroxidase and 3,3',5,5'-tetramethylbentidine in H2O2. The reaction was measured at 450 nm with an ELISA reader (Kokusai Shiyaku, Kobe, Japan). The antibody-producing cells were isolated by 2 rounds of limiting dilution. Limiting dilution was performed by co-culturing, in 96-well plates, 0.3 cells per well of an EBV-transformed B-LCL established from the patient with 1 × 104 EBV-transformed B-LCL, irradiated at 30 Gy, established from an X-SCID patient who lacks antibody production.
Clinical findings A 4-month-old girl from non-consanguinous parents was referred to our hospital with diffuse papular scaling rash with lymphadenopathy. The patient had experienced recurrent infections, including otitis media and impetigo since the age of 2 months. Laboratory evaluation at onset revealed elevated serum levels of IgM (3.47 g/L [347 mg/dL]) and IgE (611 U/mL), hypereosinophilia (9.308 × 109/L [9308/µL]), increased numbers of T cells (6.1 × 109/L [6100/µL]) with activated phenotype, and markedly reduced B cells because of a developmental block in the bone marrow (Table 2). With corticosteroid therapy, the skin lesions and lymphadenopathy were improved, although the skin was secondarily infected with Staphylococcus aureus resistant to methicillin. At 18 months, she experienced periodic thrombocytopenia followed by spontaneous recovery and a transient neutropenia with antineutrophil antibodies at the age of 4 years. Since then, the patient had recurrent episodes of sino-pulmonary infections caused by Streptococcus pneumoniae resistant to penicillin, and antibiotic therapy was required to control the infection. HLA analysis of the patient and her family revealed maternal T-cell engraftment in the patient's peripheral blood (Table 3). The paternal HLA was not detected in her CD4+ and CD8+ T-cell population. In addition, all the CD4+ T cells were CD4+CD45RO+ memory phenotype, and CD4+CD45RA+ naive T cells were not detected in her periphery (data not shown). Responses of PBMCs to various mitogens such as phytohemagglutinin and poke weed mitogen were reduced, whereas response to IL-2 was increased (Figure 1). The result indicated that T cells of this patient were in an anergic state. As shown in Table 2, the patient had few circulating B cells at age 4 months, but the number increased with age. The blood group of the patient was A, and isoagglutinin was positive in her serum. In addition, specific antibodies against diphtheria toxin and HSV were detected after diphtheria toxin vaccination and HSV infection, respectively, although monoclonal gammopathy was present.
Identification of RAG1 mutations in the patient Because the patient did not have circulating B cells and her own T cells at her first appearance, we sequenced her RAG1 genes. For this purpose, we used genomic DNA isolated from a B-LCL of the patient, having demonstrated that the B-LCL was of host origin by HLA analysis.The patient was a compound heterozygote bearing a G1789T transition
causing a missense mutation, R559S, located to the RAG2 binding domain,
and a C2801T mutation leading to a nonsense mutation, R897X, mapping to
the core domain responsible for the catalytic activity14,15
(Figure 2A). The former mutation comes
from her mother and the latter from her father. To rule out a possible polymorphism of the G1789T transition, we sequenced 100 chromosomes from Japanese controls and verified that the mutation is not a representative polymorphism (data not shown).
To determine whether the RAG mutations of this patient affect steady-state RAG protein levels, an immunoblot for RAG1 protein was performed. The mutant RAG1 cDNAs were generated by site-directed mutagenesis, and expression vectors containing the cDNAs were transiently transfected into 293T cells. Protein from the transfected 293T cells was detected by immunoblotting with polyclonal anti-RAG1 antibodies. The size and expression level of the RAG1 mutant containing the R559S substitution is similar to that of the wild type, whereas the size of the RAG1 mutant containing R897X is shorter as predicted (Figure 2B). From the results of HLA analysis, T cells of the patient were of maternal origin, whereas B cells were of host origin. To confirm the result of HLA analysis in each cell population, RAG1 genes were sequenced by using fractionated CD4, CD8, CD19, and CD56 cells. In accordance with the HLA analysis, the paternal mutation (C2801T) was detected in CD19+ cells but not in CD4+ or CD8+ cells (Figure 2C). In addition, CD56+ cells were demonstrated to be of host origin. These results demonstrated that maternal T cells were present with host B and natural killer cells in her periphery. Recombination activity of the RAG1 mutants on an extrachromosomal substrate To test the ability of the RAG1 mutants to mediate recombination of extrachromosomal substrates in vivo, RAG1, RAG2, and the recombination substrate, pJH200 (deletional), were cotransfected into 293T cells. Recombination products were determined by PCR analysis. The nonsense mutant, R897X, abolished the ability of the mutant protein to mediate recombination of the substrate, whereas the missense mutant, R559S, shows a weaker but clearly detectable signal compared with the wild-type RAG1 (Figure 3).
Identification of B cells producing anti-HSV antibodies The patient had complications with a self-limited HSV infection. After the episode, anti-HSV antibodies were readily detectable in her serum. Therefore, we established EBV-transformed B-LCL clones from the patient and analyzed the anti-HSV titer of their supernatants by using the same ELISA kit that had been used for detection of anti-HSV antibodies in her serum. The anti-HSV antibody-producing cells were isolated by 2 rounds of limiting dilution. As shown in Figure 4A, supernatants from 5 independent clones of 150 clones of the first round of limiting dilution were positive for HSV-antibodies. The positive clones were then selected in a second round of limiting dilution. Although the growth of the clones was gradually impaired during the second round of cloning, we could recover 3 positive second round clones from one of the initial clones (clone 67; Figure 4B). To determine the origin of the B cells, we performed direct sequencing of the RAG1 gene and demonstrated that the clone was of host origin (Figure 4C, and data not shown). Furthermore, we examined RAG1 genes in 40 independent clones after the first round of cloning and demonstrated that all of the B-cell clones were of host origin regardless of their production of anti-HSV antibodies (data not shown).
We describe a girl with an attenuated form of SCID resulting from mutations in the RAG1 gene. Genetic analysis of the patient's B cells revealed a G1789T transition causing an R559S substitution on one allele and a C2801T mutation leading to an R897X change in the other one. The former mutant RAG1 protein, R559S, showed a reduced, but still present, ability to mediate recombination of the deletion substrate pJH200, whereas the latter R897X, which has already been reported in a patient with T-B-SCID, completely impaired the recombination activity.3 Therefore, these amino acids are critical for V(D)J recombination activity. A previous report demonstrated that mutations of the RAG1 or RAG2 genes that eliminate the recombination activity result in T-B-SCID.3 Recently, Omenn syndrome has been demonstrated to arise from mutations of the RAG1 or RAG2 that decrease but do not abolish the efficiency of V(D)J recombination process.5 Because one of the RAG1 mutations of our patient has partial recombination activity, and the same mutation was found in a patient with Omenn syndrome, the patient would be expected to have Omenn syndrome.9 However, our patient did not have any detectable levels of host T cells in her peripheral blood, as assayed by reverse transcriptase-PCR. This finding could be due to the presence of engrafted maternal T cells interfering with host T cells, or the host T cells might disappear with age. One of the most prominent initial clinical symptoms of our patient was diffuse papular scalding rash with lymphadenopathy. Corticosteroid therapy was effective for this condition. Previous reports demonstrated that engraftment of maternal T cells lead to GVHD in some infants with SCID.10,11 Because CD4+ and CD8+ T cells from this patient were of maternal origin as assessed by HLA typing and RAG1 mutations, the initial clinical symptoms would have been caused by GVHD due to engrafted maternal T cells. Patients with T-B-SCID carrying RAG1 or RAG2 mutations or with Omenn
syndrome usually die within the first year of life because of severe
infections unless hematopoietic stem cell transplantation is
performed.6-8 Our patient is classified as a SCID with
transplacentally transferred maternal T cells but not Omenn syndrome
because of the lack of oligoclonal T cells, the presence of maternal T
cells, her lengthy survival, and a skin rash responsive to steroids. In
this regard, it is noteworthy that the allele bearing the missense RAG1
mutation showed a reduced but clearly detectable V(D)J recombination activity. She has not experienced serious viral infections, although she has had many episodes of bacterial infections. One of the reasons
for this mild phenotype was that she had mature B cells in her
periphery, and some of them could produce specific antibodies, although
monoclonal
Submitted February 12, 2001; accepted April 3, 2001.
Supported in part by grants for Pediatric Research from the Ministry of Health, Labor, and Welfare, Japan, and E0917 from Telethon.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Satoru Kumaki, Department of Pediatric Oncology, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-Machi, Aoba-Ku, Sendai 980-8575, Japan; e-mail: kumakis{at}idac.tohoku.ac.jp.
1. Schatz DG, Oettinger MA, Baltimore D. The V(D)J recombination activating gene, RAG-1. Cell. 1989;59:1035-1048[CrossRef][Medline] [Order article via Infotrieve].
2.
Oettinger MA, Schatz DG, Gorka C, Baltimore D.
RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination.
Science.
1990;248:1517-1523
3.
Schwarz K, Gauss GH, Ludwig L, et al.
RAG mutations in human B cell-negative SCID.
Science.
1996;274:97-99 4. Omenn GS. Familial reticuloendotheliosis with eosinophilia. New Engl J Med. 1965;273:427-432. 5. Villa A, Santagata S, Bozzi F, et al. Partial V(D)J recombination activity leads to Omenn syndrome. Cell. 1998;93:885-896[CrossRef][Medline] [Order article via Infotrieve]. 6. Hong R. Disorders of the T-cell system. In: Stiehm ER, ed. Immunologic Disorders in Infants and Children. 4th ed. Philadelphia, PA: Saunders; 1996:339-408.
7.
Buckley RH, Schiff SE, Schiff RI, et al.
Hematopoietic stem-cell transplantation for the treatment of severe combined immunodeficiency.
New Engl J Med.
1999;340:508-516
8.
Fischer A.
Thirty years of bone marrow transplantation for severe combined immunodeficiency.
New Engl J Med.
1999;340:559-561
9.
Villa A, Sobacchi C, Notarangelo LD, et al.
V(D)J recombination defects in lymphocytes due to RAG mutations: severe immunodeficiency with a spectrum of clinical presentations.
Blood.
2001;97:81-88 10. Wirt DP, Brooks EG, Vaidya S, Klimpel GR, Waldmann TA, Goldblum RM. Novel T-lymphocyte population in combined immunodeficiency with features of graft-versus-host disease. N Engl J Med. 1989;321:370-374[Medline] [Order article via Infotrieve]. 11. Knobloch C, Goldmann SF, Friedrich W. Limited T cell receptor diversity of transplacentally acquired maternal T cells in severe combined immunodeficiency. J Immunol. 1991;146:4157-4164[Abstract].
12.
Kumaki S, Ochs HD, Timour M, et al.
Characterization of B-cell lines established from two X-linked severe combined immunodeficiency patients: interleukin-15 binds to the B cells but is not internalized efficiently.
Blood.
1995;86:1428-1436
13.
Kumaki S, Ishii N, Minegishi M, et al.
Functional role of IL-4 and IL-7 in the development of X-linked severe combined immunodeficiency.
Blood.
1999;93:607-612
14.
Sadofsky MJ, Hesse JE, McBlane JF, Gellert M.
Expression and V(D)J recombination activity of mutated RAG-1 proteins.
Nucleic Acids Res.
1993;21:5644-5650
15.
Silver DP, Spanopoulou E, Mulligan RC, Baltimore D.
Dispensable sequence motifs in the RAG-1 and RAG-2 genes for plasmid V(D)J recombination.
Proc Natl Acad Sci U S A.
1993;90:6100-6104 16. Lieber MR, Hesse JE, Lewis S, et al. The defect in murine severe combined immune deficiency: joining of signal sequences but not coding segments in V(D)J recombination. Cell. 1988;55:7-16[CrossRef][Medline] [Order article via Infotrieve].
17.
Roman CAJ, Baltimore D.
Genetic evidence that the RAG1 protein directly participates in V(D)J recombination through substrate recognition.
Proc Natl Acad Sci U S A.
1996;93:2333-2338
18.
Haddad E, Le Deist F, Aucouturier P, et al.
Long-term chimerism and B-cell function after bone marrow transplantation in patients with severe combined immunodeficiency with B cells: a single-center study of 22 patients.
Blood.
1999;94:2923-2930
© 2001 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  M. Ege, Y. Ma, B. Manfras, K. Kalwak, H. Lu, M. R. Lieber, K. Schwarz, and U. Pannicke Omenn syndrome due to ARTEMIS mutations Blood, June 1, 2005; 105(11): 4179 - 4186. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||||
 |
 |
 |
 |
 |
 |
 |
 |
||||
| |||||||||||
| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||||
Top
Abstract
Introduction
), containing 2000-fold diluted
phytohemagglutinin-P (
), 5 ng/mL phorbol myristyl acetate plus 500 ng/mL ionomycin (
), 50 ng/mL human IL-2 (
), or 100-fold diluted
poke weed mitogen (
). Results represent the mean ± SD of
triplicate wells.
