BACKGROUND: V(D)J recombination takes place during lymphocyte development to generate a large repertoire of T- and B-cell receptors. Mutations in recombination-activating gene 1 (RAG1) and RAG2 result in loss or reduction of V(D)J recombination. It is known that different mutations in RAG genes vary in residual recombinase activity and give rise to a broad spectrum of clinical phenotypes. OBJECTIVE: We sought to study the immunologic mechanisms causing the clinical spectrum of RAG deficiency. METHODS: We included 22 patients with similar RAG1 mutations (c.519delT or c.368_369delAA) resulting in N-terminal truncated RAG1 protein with residual recombination activity but presenting with different clinical phenotypes. We studied precursor B-cell development, immunoglobulin and T-cell receptor repertoire formation, receptor editing, and B- and T-cell numbers. RESULTS: Clinically, patients were divided into 3 main categories: T(-)B(-) severe combined immunodeficiency, Omenn syndrome, and combined immunodeficiency. All patients showed a block in the precursor B-cell development, low B- and T-cell numbers, normal immunoglobulin gene use, limited B- and T-cell repertoires, and slightly impaired receptor editing. CONCLUSION: This study demonstrates that similar RAG mutations can result in similar immunobiological effects but different clinical phenotypes, indicating that the level of residual recombinase activity is not the only determinant for clinical outcome. We postulate a model in which the type and moment of antigenic pressure affect the clinical phenotypes of these patients.

Department of Bioinformatics Erasmus University Medical Center Rotterdam Rotterdam The Netherlands

Department of Blood Cell Research Stichting Sanquin Bloedvoorziening Amsterdam The Netherlands

Department of Cell Biology and Genetics Erasmus MC University Medical Center Rotterdam Rotterdam The Netherlands

Department of Clinical Immunology Polish American Institute of Pediatrics Jagiellonian University Medical College Krakow Poland

Department of Clinical Immunology Russian State Children's Hospital Moscow Russia

Department of Immunology Children's Memorial Health Institute Warsaw Poland

Department of Immunology Erasmus MC University Medical Center Rotterdam Rotterdam The Netherlands

Department of Immunology Erasmus MC University Medical Center Rotterdam Rotterdam The Netherlands; Department of Pediatrics Erasmus MC University Medical Center Rotterdam Rotterdam The Netherlands

Department of Pediatric Hematology and Oncology Teaching Hospital Motol and 2nd Medical School Charles University Prague Czech Republic

Department of Pediatric Hematology Oncology and Bone Marrow Transplantation Wroclaw Medical University Wroclaw Poland

Department of Pediatric Immunology and Infectious Diseases University Medical Center Utrecht and Wilhelmina Children's Hospital Utrecht The Netherlands

Department of Pediatrics Erasmus MC University Medical Center Rotterdam Rotterdam The Netherlands

Department of Pediatrics Leiden University Medical Center Leiden The Netherlands

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Schatz D.G. V(D)J recombination. Immunol Rev. 2004;200:5–11. PubMed

Schwarz K., Gauss G.H., Ludwig L., Pannicke U., Li Z., Lindner D. RAG mutations in human B cell-negative SCID. Science. 1996;274:97–99. PubMed

Moshous D., Callebaut I., de Chasseval R., Corneo B., Cavazzana-Calvo M., Le Deist F. Artemis, a novel DNA double-strand break repair/V(D)J recombination protein, is mutated in human severe combined immune deficiency. Cell. 2001;105:177–186. PubMed

Ahnesorg P., Smith P., Jackson S.P. XLF interacts with the XRCC4-DNA ligase IV complex to promote DNA nonhomologous end-joining. Cell. 2006;124:301–313. PubMed

Buck D., Malivert L., de Chasseval R., Barraud A., Fondaneche M.C., Sanal O. Cernunnos, a novel nonhomologous end-joining factor, is mutated in human immunodeficiency with microcephaly. Cell. 2006;124:287–299. PubMed

O'Driscoll M., Gennery A.R., Seidel J., Concannon P., Jeggo P.A. An overview of three new disorders associated with genetic instability: LIG4 syndrome, RS-SCID and ATR-Seckel syndrome. DNA Repair (Amst) 2004;3:1227–1235. PubMed

van der Burg M., Ijspeert H., Verkaik N.S., Turul T., Wiegant W.W., Morotomi-Yano K. A DNA-PKcs mutation in a radiosensitive T-B- SCID patient inhibits Artemis activation and nonhomologous end-joining. J Clin Invest. 2009;119:91–98. PubMed PMC

van der Burg M., van Veelen L.R., Verkaik N.S., Wiegant W.W., Hartwig N.G., Barendregt B.H. A new type of radiosensitive T-B-NK+ severe combined immunodeficiency caused by a LIG4 mutation. J Clin Invest. 2006;116:137–145. PubMed PMC

Niehues T., Perez-Becker R., Schuetz C. More than just SCID—the phenotypic range of combined immunodeficiencies associated with mutations in the recombinase activating genes (RAG) 1 and 2. Clin Immunol. 2010;135:183–192. PubMed

Kuijpers T.W., Ijspeert H., van Leeuwen E.M., Jansen M.H., Hazenberg M.D., Weijer K.C. Idiopathic CD4+ T lymphopenia without autoimmunity or granulomatous disease in the slipstream of RAG mutations. Blood. 2011;117:5892–5896. PubMed

Noordzij J.G., de Bruin-Versteeg S., Verkaik N.S., Vossen J.M., de Groot R., Bernatowska E. The immunophenotypic and immunogenotypic B-cell differentiation arrest in bone marrow of RAG-deficient SCID patients corresponds to residual recombination activities of mutated RAG proteins. Blood. 2002;100:2145–2152. PubMed

Noordzij J.G., Verkaik N.S., van der Burg M., van Veelen L.R., de Bruin-Versteeg S., Wiegant W. Radiosensitive SCID patients with Artemis gene mutations show a complete B-cell differentiation arrest at the pre-B-cell receptor checkpoint in bone marrow. Blood. 2003;101:1446–1452. PubMed

Noordzij J.G., Verkaik N.S., Hartwig N.G., de Groot R., van Gent D.C., van Dongen J.J. N-terminal truncated human RAG1 proteins can direct T-cell receptor but not immunoglobulin gene rearrangements. Blood. 2000;96:203–209. PubMed

Boeckx N., Willemse M.J., Szczepanski T., van der Velden V.H., Langerak A.W., Vandekerckhove P. Fusion gene transcripts and Ig/TCR gene rearrangements are complementary but infrequent targets for PCR-based detection of minimal residual disease in acute myeloid leukemia. Leukemia. 2002;16:368–375. PubMed

van Dongen J.J., Langerak A.W., Bruggemann M., Evans P.A., Hummel M., Lavender F.L. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia. 2003;17:2257–2317. PubMed

Lefranc M.P. IMGT databases, web resources and tools for immunoglobulin and T cell receptor sequence analysis. Leukemia. 2003;17:260–266. http://imgt.cines.fr. PubMed

Lefranc M.P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. 2003;31:307–310. PubMed PMC

Alamyar E., Duroux P., Lefranc M.P., Giudicelli V. IMGT® tools for the nucleotide analysis of immunoglobulin (IG) and T cell receptor (TR) V-(D)-J repertoires, polymorphisms, and IG mutations: IMGT/V-QUEST and IMGT/HighV-QUEST for NGS. Methods Mol Biol. 2012;882:569–604. PubMed

Goecks J., Nekrutenko A., Taylor J., Galaxy T. Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol. 2010;11:R86. PubMed PMC

Blankenberg D., Von Kuster G., Coraor N., Ananda G., Lazarus R., Mangan M. Galaxy: a web-based genome analysis tool for experimentalists. Curr Protoc Mol Biol. 2010 Chapter 19:Unit 19.10.1-21. PubMed PMC

Giardine B., Riemer C., Hardison R.C., Burhans R., Elnitski L., Shah P. Galaxy: a platform for interactive large-scale genome analysis. Genome Res. 2005;15:1451–1455. PubMed PMC

R: a language and environment for statistical computing. R Foundation for Statistical Computing; Vienna: 2013.

Crooks G.E., Hon G., Chandonia J.M., Brenner S.E. WebLogo: a sequence logo generator. Genome Res. 2004;14:1188–1190. PubMed PMC

Schneider T.D., Stephens R.M. Sequence logos: a new way to display consensus sequences. Nucleic Acids Res. 1990;18:6097–6100. PubMed PMC

Corneo B., Moshous D., Gungor T., Wulffraat N., Philippet P., Le Deist F.L. Identical mutations in RAG1 or RAG2 genes leading to defective V(D)J recombinase activity can cause either T-B-severe combined immune deficiency or Omenn syndrome. Blood. 2001;97:2772–2776. PubMed

de Villartay J.P., Lim A., Al-Mousa H., Dupont S., Dechanet-Merville J., Coumau-Gatbois E. A novel immunodeficiency associated with hypomorphic RAG1 mutations and CMV infection. J Clin Invest. 2005;115:3291–3299. PubMed PMC

Santagata S., Gomez C.A., Sobacchi C., Bozzi F., Abinun M., Pasic S. N-terminal RAG1 frameshift mutations in Omenn's syndrome: internal methionine usage leads to partial V(D)J recombination activity and reveals a fundamental role in vivo for the N-terminal domains. Proc Natl Acad Sci U S A. 2000;97:14572–14577. PubMed PMC

Villa A., Sobacchi C., Notarangelo L.D., Bozzi F., Abinun M., Abrahamsen T.G. V(D)J recombination defects in lymphocytes due to RAG mutations: severe immunodeficiency with a spectrum of clinical presentations. Blood. 2001;97:81–88. PubMed

Martinez-Martinez L., Vazquez-Ortiz M., Gonzalez-Santesteban C., Martin-Nalda A., Vicente A., Plaza A.M. From severe combined immunodeficiency to Omenn syndrome after hematopoietic stem cell transplantation in a RAG1 deficient family. Pediatr Allergy Immunol. 2012;23:660–666. PubMed

van Zelm M.C., van der Burg M., de Ridder D., Barendregt B.H., de Haas E.F., Reinders M.J. Ig gene rearrangement steps are initiated in early human precursor B cell subsets and correlate with specific transcription factor expression. J Immunol. 2005;175:5912–5922. PubMed

Ohm-Laursen L., Nielsen C., Fisker N., Lillevang S.T., Barington T. Lack of nonfunctional B-cell receptor rearrangements in a patient with normal B cell numbers despite partial RAG1 deficiency and atypical SCID/Omenn syndrome. J Clin Immunol. 2008;28:588–592. PubMed

Ochs H.D., Davis S.D., Mickelson E., Lerner K.G., Wedgwood R.J. Combined immunodeficiency and reticuloendotheliosis with eosinophilia. J Pediatr. 1974;85:463–465. PubMed

Omenn G.S. Familial Reticuloendotheliosis with Eosinophilia. N Engl J Med. 1965;273:427–432. PubMed

Cavadini P., Vermi W., Facchetti F., Fontana S., Nagafuchi S., Mazzolari E. AIRE deficiency in thymus of 2 patients with Omenn syndrome. J Clin Invest. 2005;115:728–732. PubMed PMC

Poliani P.L., Facchetti F., Ravanini M., Gennery A.R., Villa A., Roifman C.M. Early defects in human T-cell development severely affect distribution and maturation of thymic stromal cells: possible implications for the pathophysiology of Omenn syndrome. Blood. 2009;114:105–108. PubMed PMC

Cassani B., Poliani P.L., Marrella V., Schena F., Sauer A.V., Ravanini M. Homeostatic expansion of autoreactive immunoglobulin-secreting cells in the Rag2 mouse model of Omenn syndrome. J Exp Med. 2010;207:1525–1540. PubMed PMC

Walter J.E., Rucci F., Patrizi L., Recher M., Regenass S., Paganini T. Expansion of immunoglobulin-secreting cells and defects in B cell tolerance in Rag-dependent immunodeficiency. J Exp Med. 2010;207:1541–1554. PubMed PMC

Pascual V., Victor K., Lelsz D., Spellerberg M.B., Hamblin T.J., Thompson K.M. Nucleotide sequence analysis of the V regions of two IgM cold agglutinins. Evidence that the VH4-21 gene segment is responsible for the major cross-reactive idiotype. J Immunol. 1991;146:4385–4391. PubMed

Silberstein L.E., Jefferies L.C., Goldman J., Friedman D., Moore J.S., Nowell P.C. Variable region gene analysis of pathologic human autoantibodies to the related i and I red blood cell antigens. Blood. 1991;78:2372–2386. PubMed

de Saint-Basile G., Le Deist F., de Villartay J.P., Cerf-Bensussan N., Journet O., Brousse N. Restricted heterogeneity of T lymphocytes in combined immunodeficiency with hypereosinophilia (Omenn's syndrome) J Clin Invest. 1991;87:1352–1359. PubMed PMC

Breit T.M., Verschuren M.C., Wolvers-Tettero I.L., Van Gastel-Mol E.J., Hahlen K., van Dongen J.J. Human T cell leukemias with continuous V(D)J recombinase activity for TCR-delta gene deletion. J Immunol. 1997;159:4341–4349. PubMed

RAG1 mutation database. Available at: http://www.uta.fi/imt/bioinfo/RAG1base. Accessed December 27, 2013.

Villa A., Santagata S., Bozzi F., Giliani S., Frattini A., Imberti L. Partial V(D)J recombination activity leads to Omenn syndrome. Cell. 1998;93:885–896. PubMed

Chou J., Hanna-Wakim R., Tirosh I., Kane J., Fraulino D., Lee Y.N. A novel homozygous mutation in recombination activating gene 2 in 2 relatives with different clinical phenotypes: Omenn syndrome and hyper-IgM syndrome. J Allergy Clin Immunol. 2012;130:1414–1416. PubMed PMC

Pasic S., Djuricic S., Ristic G., Slavkovic B. Recombinase-activating gene 1 immunodeficiency: different immunological phenotypes in three siblings. Acta Paediatr. 2009;98:1062–1064. PubMed

Haq I.J., Steinberg L.J., Hoenig M., van der Burg M., Villa A., Cant A.J. GvHD-associated cytokine polymorphisms do not associate with Omenn syndrome rather than T-B- SCID in patients with defects in RAG genes. Clin Immunol. 2007;124:165–169. PubMed

Klonowski K.D., Primiano L.L., Monestier M. Atypical VH-D-JH rearrangements in newborn autoimmune MRL mice. J Immunol. 1999;162:1566–1572. PubMed

Wardemann H., Yurasov S., Schaefer A., Young J.W., Meffre E., Nussenzweig M.C. Predominant autoantibody production by early human B cell precursors. Science. 2003;301:1374–1377. PubMed

Partial RAG deficiency in humans induces dysregulated peripheral lymphocyte development and humoral tolerance defect with accumulation of T-bet+ B cells

The Clinical and Genetic Spectrum of 82 Patients With RAG Deficiency Including a c.256_257delAA Founder Variant in Slavic Countries

EuroFlow Standardized Approach to Diagnostic Immunopheneotyping of Severe PID in Newborns and Young Children

EuroFlow-Based Flowcytometric Diagnostic Screening and Classification of Primary Immunodeficiencies of the Lymphoid System

The EuroFlow PID Orientation Tube for Flow Cytometric Diagnostic Screening of Primary Immunodeficiencies of the Lymphoid System