A novel homozygous mutation in recombination activating gene 2 in 2 relatives with different clinical phenotypes: Omenn syndrome and hyper-IgM syndrome.

Recombination activating genes (RAG) 1 and 2 encode proteins necessary for T- and B-cell antigen receptor rearrangement. Complete deficiency of either RAG1 or RAG2 results in classical severe combined immunodeficiency lacking T and B cells, since RAG1 mediates DNA binding and cleavage, while RAG2 is an essential cofactor for RAG1 function.1Schatz D.G. Ji Y. Recombination centres and the orchestration of V(D)J recombination.Nat Rev Immunol. 2011; 11: 251-263Crossref PubMed Scopus (369) Google Scholar Hypomorphic missense mutations that preserve residual RAG activity and allow the development of oligoclonal T cells, but virtually no B cells, result in recurrent infections, erythroderma, hepatomegaly, colitis, and αβ T-cell expansion (Omenn syndrome).2Niehues 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: 183-192Crossref PubMed Scopus (81) Google Scholar RAG1/2 mutations can also cause γδ T-cell expansion and immunodeficiency with granulomas.3Schuetz C. Huck K. Gudowius S. Megahed M. Feyen O. Hubner B. et al.An immunodeficiency disease with RAG mutations and granulomas.N Engl J Med. 2008; 358: 2030-2038Crossref PubMed Scopus (167) Google ScholarHyper-IgM syndrome is characterized by normal or increased IgM levels with decreased IgG and IgA levels. It results from defects in class switch recombination caused by mutations in CD40 ligand, CD40, activation-induced deaminase, uracil-DNA glycosylase, and nuclear factor kappa B essential modifier.4Vale A.M. Schroeder Jr., H.W. Clinical consequences of defects in B-cell development.J Allergy Clin Immunol. 2010; 125: 778-787Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar In addition to defective humoral immunity, T-cell function is affected in CD40L, CD40, and nuclear factor kappa B essential modifier deficiency and is associated with opportunistic infections.4Vale A.M. Schroeder Jr., H.W. Clinical consequences of defects in B-cell development.J Allergy Clin Immunol. 2010; 125: 778-787Abstract Full Text Full Text PDF PubMed Scopus (47) Google ScholarWe report a novel homozygous mutation in RAG2 resulting in 2 different phenotypes: Omenn syndrome and hyper-IgM syndrome.Patient 1 was born to first-degree consanguineous parents and presented at 4 months of age with lymphopenia (absolute lymphocyte count of 869 cells/μL), erythroderma, Pseudomonas aeruginosa pneumonia, and Coombs' positive hemolytic anemia. She had recurrent pulmonary infections and developed onchomycosis by 1 year of age. Lymphocyte subset analysis at 16 and 19 months of age revealed lymphopenia with severely decreased T and B cells and normal natural killer cell numbers (Table I). The majority of her T cells were CR45RO+ memory cells (not shown). HLA typing revealed no evidence of maternal cell engraftment. Lymphocyte proliferation to PHA and anti-CD3 mAb was severely decreased (Table I). She had low serum IgG levels and undetectable serum IgA level, while her serum IgM level was significantly decreased by 19 months of age (Table I). Because her phenotype was consistent with Omenn syndrome, RAG1 and RAG2 were sequenced. A previously unreported homozygous missense mutation in RAG2 (c.1375A>C) was identified, causing a methionine to leucine change at position 459 (M459L) in the protein's plant homeodomain. Patient 1 subsequently underwent a successful matched sibling bone marrow transplant.Table IImmune profilesPatient 1Patient 216 mo19 mo16 mo5 y 5 moLymphocyte counts (cells/μL)5Comans-Bitter W.M. de Groot R. van der Beemd R. Neijens H.J. Hop W.C. Groeneveld K. et al.Immunophenotyping of blood lymphocytes in childhood: reference values for lymphocyte subpopulations.J Pediatr. 1997; 130: 388-393Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar CD3+149 (1900-6200)205 (1900-6200)691 (1900-6200)777 (1400-6200)CD3+CD4+85 (1300-3400)149 (1300-3400)311 (1300-3400)443 (700-2200)CD3+CD8+61 (620-2000)42 (620-2000)173 (620-2000)293 (490-1300)CD4+/CD8+ ratio1.4 (1.3-3.9)3.5 (1.3-3.9)1.7 (1.3-3.9)1.5 (0.9-3.7) CD19+4 (610-2600)1 (610-2600)173 (610-2600)15 (390-1400) CD16+/CD56+279 (160-1100)208 (160-1100)657 (160-1100)1111 (130-720)Immunoglobulins (mg/dL)6Jolliff C.R. Cost K.M. Stivrins P.C. Grossman P.P. Nolte C.R. Franco S.M. et al.Reference intervals for serum IgG, IgA, IgM, C3, and C4 as determined by rate nephelometry.Clin Chem. 1982; 28: 126-128PubMed Google Scholar IgG193 (400-1300)147 (400-1300)<152 (400-1300)213 (600-1500) IgAUndetectable (20-230)Undetectable (20-230)Undetectable (20-230)1 (50-150) IgM54.9 (30-120)<17.3 (30-120)171 (30-120)1048 (22-100)Proliferation (cpm) PHAND16700 (116187)ND1980 (57182) Anti-CD3 mAbND14310 (107265)ND12558 (82957)The values in parentheses represent the normal range for age of lymphocyte counts, immunoglobulin concentrations, and 3H-thymidine incorporation into DNA measured as radioactive counts per minute (cpm). For lymphocyte proliferation studies, a normal healthy control was studied the same day as the patient.ND, Not determined. Open table in a new tab Patient 2 was born to first-degree consanguineous parents. He presented at 4 months of age with recurrent skin abscesses. He subsequently developed recurrent pneumonias, P aeruginosa sepsis with disseminated intravascular coagulopathy, colitis, cytomegalovirus viremia, oral candidiasis, hepatosplenomegaly, autoimmune hemolytic anemia, and antiphospholipid antibodies. Lymphocyte subset analysis at 16 months of age revealed lymphopenia with low T and B cell numbers and normal natural killer cell numbers (Table I). Serum IgG level was very low, serum IgA level was undetectable, and serum IgM level was normal. By 5 years 5 months of age, his IgM level increased to 1048 mg/dL (Table I), prompting a diagnosis of hyper-IgM syndrome. The patient was referred to us for further investigation. His T cells upregulated CD40L normally following activation with phorbol 12-myristate 13-acetate and ionomycin (not shown). However, he had low numbers of T cells and virtually no B cells (Table I). The majority of his T cells were CR45RO+ memory cells (not shown). T-cell proliferation to PHA and anti-CD3 mAb was severely decreased (Table I). These results suggested that patient 2 had a combined immunodeficiency rather than hyper-IgM syndrome.A detailed family history revealed that the great-grandparents of patients 1 and 2 were cousins. This raised the possibility that they might share the same RAG2 mutation. Patient 2 was indeed homozygous for the RAG2 mutation found in patient 1. The healthy sister of patient 1 has a normal RAG2 sequence, while the parents of both patients and 2 healthy brothers of patient 2 were heterozygous for the RAG2 mutation.The M459 residue is highly conserved and located in the zinc-binding loop region of the RAG2 plant homeodomain. Mutations in this domain impair RAG2 protein stability, translocation to the nucleus, interaction with histones, and recombination capacity, resulting in severe combined immunodeficiency or Omenn syndrome.7Couedel C. Roman C. Jones A. Vezzoni P. Villa A. Cortes P. Analysis of mutations from SCID and Omenn syndrome patients reveals the central role of the Rag2 PHD domain in regulating V(D)J recombination.J Clin Invest. 2010; 120: 1337-1344Crossref PubMed Scopus (26) Google Scholar The recombination activity of the M459L RAG2 mutant was analyzed by using Abelson-transformed Rag2−/− tg.bcl2 pro-B cells containing an intrachromosomal inverted green fluorescent protein (GFP) cassette flanked by recombination signal sequences. Cells were transduced with retroviral vectors encoding either wild-type or mutant RAG2 and human CD2 as a reporter and then treated with the Abl inhibitor STI-571 to promote cell differentiation and induce RAG activity. The expressed RAG2, in combination with endogenous RAG1, causes inversion of the GFP cassette, and the resultant GFP expression correlates with the level of recombination activity.8De Ravin S.S. Cowen E.W. Zarember K.A. Whiting-Theobald N.L. Kuhns D.B. Sandler N.G. et al.Hypomorphic Rag mutations can cause destructive midline granulomatous disease.Blood. 2010; 116: 1263-1271Crossref PubMed Scopus (90) Google Scholar, 9Gapud E.J. Lee B.S. Mahowald G.K. Bassing C.H. Sleckman B.P. Repair of chromosomal RAG-mediated DNA breaks by mutant RAG proteins lacking phosphatidylinositol 3-like kinase consensus phosphorylation sites.J Immunol. 2011; 187: 1826-1834Crossref PubMed Scopus (14) Google Scholar The recombination activity of the RAG2(M459L) mutant was 29.7% ± 3.3% of wild-type RAG2 (Fig 1).We describe 2 related patients with a novel mutation in RAG2 causing different clinical phenotypes. Patient 1 had Omenn syndrome with erythroderma, low T and B cell numbers, and opportunistic infections. Patient 2 lacked erythroderma and had persistently high IgM levels, leading to a presumptive diagnosis of hyper-IgM syndrome. Of note, the phenotype of patient 2 also evolved over time; the progressive loss of B cells and lack of T-cell proliferation to PHA and anti-CD3 seen at age 5 years are consistent with a mutation in RAG2. Phenotypic heterogeneity due to the same RAG2 mutation has been attributed to differences in genetic background, epigenetic factors, and environmental exposures.10Marrella V. Poliani P.L. Casati A. Rucci F. Frascoli L. Gougeon M.L. et al.A hypomorphic R229Q Rag2 mouse mutant recapitulates human Omenn syndrome.J Clin Invest. 2007; 117: 1260-1269Crossref PubMed Scopus (87) Google Scholar, 11Dalal I. Tabori U. Bielorai B. Golan H. Rosenthal E. Amariglio N. et al.Evolution of a T-B-SCID into an Omenn syndrome phenotype following parainfluenza 3 virus infection.Clin Immunol. 2005; 115: 70-73Crossref PubMed Scopus (36) Google Scholar The low-level recombinase activity in vitro, and the presence of residual autologous T and B lymphocytes in the patients, indicate that the M459L RAG2 variant may retain some V(D)J recombination activity in vivo. Combined with different antigen exposure, this may contribute to our patients' different phenotypes. Thirty percent recombination activity was also found in a patient with RAG2 mutations resulting in hypogammaglobulinemia, lymphopenia, and recurrent infections, indicating that this level of reduced recombination activity results in clinically significant disease.3Schuetz C. Huck K. Gudowius S. Megahed M. Feyen O. Hubner B. et al.An immunodeficiency disease with RAG mutations and granulomas.N Engl J Med. 2008; 358: 2030-2038Crossref PubMed Scopus (167) Google ScholarTo our knowledge, hyper-IgM has not been previously reported with mutations in RAG2. It is therefore important to consider RAG mutations in patients with elevated levels of IgM and progressive lymphopenia. Recombination activating genes (RAG) 1 and 2 encode proteins necessary for T- and B-cell antigen receptor rearrangement. Complete deficiency of either RAG1 or RAG2 results in classical severe combined immunodeficiency lacking T and B cells, since RAG1 mediates DNA binding and cleavage, while RAG2 is an essential cofactor for RAG1 function.1Schatz D.G. Ji Y. Recombination centres and the orchestration of V(D)J recombination.Nat Rev Immunol. 2011; 11: 251-263Crossref PubMed Scopus (369) Google Scholar Hypomorphic missense mutations that preserve residual RAG activity and allow the development of oligoclonal T cells, but virtually no B cells, result in recurrent infections, erythroderma, hepatomegaly, colitis, and αβ T-cell expansion (Omenn syndrome).2Niehues 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: 183-192Crossref PubMed Scopus (81) Google Scholar RAG1/2 mutations can also cause γδ T-cell expansion and immunodeficiency with granulomas.3Schuetz C. Huck K. Gudowius S. Megahed M. Feyen O. Hubner B. et al.An immunodeficiency disease with RAG mutations and granulomas.N Engl J Med. 2008; 358: 2030-2038Crossref PubMed Scopus (167) Google Scholar Hyper-IgM syndrome is characterized by normal or increased IgM levels with decreased IgG and IgA levels. It results from defects in class switch recombination caused by mutations in CD40 ligand, CD40, activation-induced deaminase, uracil-DNA glycosylase, and nuclear factor kappa B essential modifier.4Vale A.M. Schroeder Jr., H.W. Clinical consequences of defects in B-cell development.J Allergy Clin Immunol. 2010; 125: 778-787Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar In addition to defective humoral immunity, T-cell function is affected in CD40L, CD40, and nuclear factor kappa B essential modifier deficiency and is associated with opportunistic infections.4Vale A.M. Schroeder Jr., H.W. Clinical consequences of defects in B-cell development.J Allergy Clin Immunol. 2010; 125: 778-787Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar We report a novel homozygous mutation in RAG2 resulting in 2 different phenotypes: Omenn syndrome and hyper-IgM syndrome. Patient 1 was born to first-degree consanguineous parents and presented at 4 months of age with lymphopenia (absolute lymphocyte count of 869 cells/μL), erythroderma, Pseudomonas aeruginosa pneumonia, and Coombs' positive hemolytic anemia. She had recurrent pulmonary infections and developed onchomycosis by 1 year of age. Lymphocyte subset analysis at 16 and 19 months of age revealed lymphopenia with severely decreased T and B cells and normal natural killer cell numbers (Table I). The majority of her T cells were CR45RO+ memory cells (not shown). HLA typing revealed no evidence of maternal cell engraftment. Lymphocyte proliferation to PHA and anti-CD3 mAb was severely decreased (Table I). She had low serum IgG levels and undetectable serum IgA level, while her serum IgM level was significantly decreased by 19 months of age (Table I). Because her phenotype was consistent with Omenn syndrome, RAG1 and RAG2 were sequenced. A previously unreported homozygous missense mutation in RAG2 (c.1375A>C) was identified, causing a methionine to leucine change at position 459 (M459L) in the protein's plant homeodomain. Patient 1 subsequently underwent a successful matched sibling bone marrow transplant. The values in parentheses represent the normal range for age of lymphocyte counts, immunoglobulin concentrations, and 3H-thymidine incorporation into DNA measured as radioactive counts per minute (cpm). For lymphocyte proliferation studies, a normal healthy control was studied the same day as the patient. ND, Not determined. Patient 2 was born to first-degree consanguineous parents. He presented at 4 months of age with recurrent skin abscesses. He subsequently developed recurrent pneumonias, P aeruginosa sepsis with disseminated intravascular coagulopathy, colitis, cytomegalovirus viremia, oral candidiasis, hepatosplenomegaly, autoimmune hemolytic anemia, and antiphospholipid antibodies. Lymphocyte subset analysis at 16 months of age revealed lymphopenia with low T and B cell numbers and normal natural killer cell numbers (Table I). Serum IgG level was very low, serum IgA level was undetectable, and serum IgM level was normal. By 5 years 5 months of age, his IgM level increased to 1048 mg/dL (Table I), prompting a diagnosis of hyper-IgM syndrome. The patient was referred to us for further investigation. His T cells upregulated CD40L normally following activation with phorbol 12-myristate 13-acetate and ionomycin (not shown). However, he had low numbers of T cells and virtually no B cells (Table I). The majority of his T cells were CR45RO+ memory cells (not shown). T-cell proliferation to PHA and anti-CD3 mAb was severely decreased (Table I). These results suggested that patient 2 had a combined immunodeficiency rather than hyper-IgM syndrome. A detailed family history revealed that the great-grandparents of patients 1 and 2 were cousins. This raised the possibility that they might share the same RAG2 mutation. Patient 2 was indeed homozygous for the RAG2 mutation found in patient 1. The healthy sister of patient 1 has a normal RAG2 sequence, while the parents of both patients and 2 healthy brothers of patient 2 were heterozygous for the RAG2 mutation. The M459 residue is highly conserved and located in the zinc-binding loop region of the RAG2 plant homeodomain. Mutations in this domain impair RAG2 protein stability, translocation to the nucleus, interaction with histones, and recombination capacity, resulting in severe combined immunodeficiency or Omenn syndrome.7Couedel C. Roman C. Jones A. Vezzoni P. Villa A. Cortes P. Analysis of mutations from SCID and Omenn syndrome patients reveals the central role of the Rag2 PHD domain in regulating V(D)J recombination.J Clin Invest. 2010; 120: 1337-1344Crossref PubMed Scopus (26) Google Scholar The recombination activity of the M459L RAG2 mutant was analyzed by using Abelson-transformed Rag2−/− tg.bcl2 pro-B cells containing an intrachromosomal inverted green fluorescent protein (GFP) cassette flanked by recombination signal sequences. Cells were transduced with retroviral vectors encoding either wild-type or mutant RAG2 and human CD2 as a reporter and then treated with the Abl inhibitor STI-571 to promote cell differentiation and induce RAG activity. The expressed RAG2, in combination with endogenous RAG1, causes inversion of the GFP cassette, and the resultant GFP expression correlates with the level of recombination activity.8De Ravin S.S. Cowen E.W. Zarember K.A. Whiting-Theobald N.L. Kuhns D.B. Sandler N.G. et al.Hypomorphic Rag mutations can cause destructive midline granulomatous disease.Blood. 2010; 116: 1263-1271Crossref PubMed Scopus (90) Google Scholar, 9Gapud E.J. Lee B.S. Mahowald G.K. Bassing C.H. Sleckman B.P. Repair of chromosomal RAG-mediated DNA breaks by mutant RAG proteins lacking phosphatidylinositol 3-like kinase consensus phosphorylation sites.J Immunol. 2011; 187: 1826-1834Crossref PubMed Scopus (14) Google Scholar The recombination activity of the RAG2(M459L) mutant was 29.7% ± 3.3% of wild-type RAG2 (Fig 1). We describe 2 related patients with a novel mutation in RAG2 causing different clinical phenotypes. Patient 1 had Omenn syndrome with erythroderma, low T and B cell numbers, and opportunistic infections. Patient 2 lacked erythroderma and had persistently high IgM levels, leading to a presumptive diagnosis of hyper-IgM syndrome. Of note, the phenotype of patient 2 also evolved over time; the progressive loss of B cells and lack of T-cell proliferation to PHA and anti-CD3 seen at age 5 years are consistent with a mutation in RAG2. Phenotypic heterogeneity due to the same RAG2 mutation has been attributed to differences in genetic background, epigenetic factors, and environmental exposures.10Marrella V. Poliani P.L. Casati A. Rucci F. Frascoli L. Gougeon M.L. et al.A hypomorphic R229Q Rag2 mouse mutant recapitulates human Omenn syndrome.J Clin Invest. 2007; 117: 1260-1269Crossref PubMed Scopus (87) Google Scholar, 11Dalal I. Tabori U. Bielorai B. Golan H. Rosenthal E. Amariglio N. et al.Evolution of a T-B-SCID into an Omenn syndrome phenotype following parainfluenza 3 virus infection.Clin Immunol. 2005; 115: 70-73Crossref PubMed Scopus (36) Google Scholar The low-level recombinase activity in vitro, and the presence of residual autologous T and B lymphocytes in the patients, indicate that the M459L RAG2 variant may retain some V(D)J recombination activity in vivo. Combined with different antigen exposure, this may contribute to our patients' different phenotypes. Thirty percent recombination activity was also found in a patient with RAG2 mutations resulting in hypogammaglobulinemia, lymphopenia, and recurrent infections, indicating that this level of reduced recombination activity results in clinically significant disease.3Schuetz C. Huck K. Gudowius S. Megahed M. Feyen O. Hubner B. et al.An immunodeficiency disease with RAG mutations and granulomas.N Engl J Med. 2008; 358: 2030-2038Crossref PubMed Scopus (167) Google Scholar To our knowledge, hyper-IgM has not been previously reported with mutations in RAG2. It is therefore important to consider RAG mutations in patients with elevated levels of IgM and progressive lymphopenia. We thank Dr Barry P. Sleckman for providing the Abelson-transformed Rag2−/− tg.bcl2 pro-B cells.