Long-term remission after allogeneic hematopoietic stem cell transplantation in LPS-responsive beige-like anchor (LRBA) deficiency
2014; Elsevier BV; Volume: 135; Issue: 5 Linguagem: Inglês
10.1016/j.jaci.2014.10.048
ISSN1097-6825
AutoresMarkus G. Seidel, Tatjana Hirschmugl, Laura Gámez‐Díaz, Wolfgang Schwinger, Nina K. Serwas, Andrea Deutschmann, Gregor Gorkiewicz, Werner Zenz, Christian Windpassinger, Bodo Grimbacher, Christian Urban, Kaan Boztuğ,
Tópico(s)Cystic Fibrosis Research Advances
ResumoLPS-responsive vesicle trafficking, beach and anchor containing protein (LRBA) deficiency has been identified as a primary immunodeficiency (PID) characterized by recurrent infections associated with autoimmunity, such as inflammatory bowel disease and autoimmune cytopenias (see Fig E1 in this article's Online Repository at www.jacionline.org).1Alangari A. Alsultan A. Adly N. Massaad M.J. Kiani I.S. Aljebreen A. et al.LPS-responsive beige-like anchor (LRBA) gene mutation in a family with inflammatory bowel disease and combined immunodeficiency.J Allergy Clin Immunol. 2012; 130: 481-488.e2Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 2Burns S.O. Zenner H.L. Plagnol V. Curtis J. Mok K. Eisenhut M. et al.LRBA gene deletion in a patient presenting with autoimmunity without hypogammaglobulinemia.J Allergy Clin Immunol. 2012; 130: 1428-1432Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 3Lopez-Herrera G. Tampella G. Pan-Hammarstrom Q. Herholz P. Trujillo-Vargas C.M. Phadwal K. et al.Deleterious mutations in LRBA are associated with a syndrome of immune deficiency and autoimmunity.Am J Hum Genet. 2012; 90: 986-1001Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar A wide range of immunosuppressive treatment measures have only induced temporary relief in affected subjects. Although allogeneic hematopoietic stem cell transplantation (HSCT) is the current treatment for many forms of PIDs, HSCT is less established in patients with autoimmune disease4Gratwohl A. Baldomero H. Sureda A. Indications for and current practice of allogeneic and autologous HSCT.in: Apperley J. Carreras E. Gluckman E. Masszi T. The EBMT handbook—haematopoietic stem cell transplantation: European Group for Blood and Marrow Transplantation & European School of Hematology. European School of Hematology, Paris, France. 2012: 302-315Google Scholar, 5Snowden J.A. Saccardi R. Farge D. Indications for HSCT in adults—Autoimmune diseases.in: Apperley J. Carreras E. Gluckman E. Masszi T. The EBMT handbook—haematopoietic stem cell transplantation: European Group for Blood and Marrow Transplantation & European School of Hematology. European School of Hematology, Paris, France. 2012: 458-469Google Scholar and has not yet been reported in LRBA-deficient patients.We studied a consanguineous family of Kurdish origin with a systemic autoimmune disorder. Patient 1's symptoms started at 2 years of age with immune thrombocytopenia (ITP; Fig 1, A). Serum immunoglobulin concentrations were slightly increased, and the cellular immunophenotype was normal (Table I and see Table E1 in this article's Online Repository at www.jacionline.org). A lymph node biopsy performed because of generalized lymphoproliferative disease (LPD) revealed a follicular lymphatic hyperplasia with abundant (about 20% to 30%) CD3+ and CD4− and CD8− double-negative T lymphocytes (DNT cells; Fig 1, C), suggesting an immune dysregulation, lymphocyte maturation, or apoptosis defect compatible with autoimmune lymphoproliferative syndrome (ALPS).6Canale V.C. Smith C.H. Chronic lymphadenopathy simulating malignant lymphoma.J Pediatr. 1967; 70: 891-899Abstract Full Text PDF PubMed Scopus (174) Google Scholar, 7Price S. Shaw P.A. Seitz A. Joshi G. Davis J. Niemela J.E. et al.Natural history of autoimmune lymphoproliferative syndrome associated with FAS gene mutations.Blood. 2014; 123: 1989-1999Crossref PubMed Scopus (154) Google Scholar HSCT was performed with the clinically healthy HLA-identical mother as the donor (see the additional text in this article's Online Repository at www.jacionline.org), leading to complete remission with persisting full donor chimerism and without signs of acute or chronic graft-versus-host disease (GvHD). Four years after HSCT, ITP relapsed but responded well to high-dose intravenous immunoglobulin (IVIG) treatment. When romiplostim was started, platelet counts normalized, and administration of romiplostim (5 μg/kg, every 4 to 6 weeks) without further need for immunosuppression or IVIG has led to sustained but treatment-dependent remission.8Seidel M.G. Urban C. Sipurzynski J. Beham-Schmid C. Lackner H. Benesch M. High response rate but short-term effect of romiplostim in paediatric refractory chronic immune thrombocytopenia.Br J Haematol. 2014; 165: 419-421Crossref PubMed Scopus (14) Google ScholarFig 1Clinical course of a familial autoimmunity syndrome caused by LRBA deficiency, immunohistochemical analysis of lymph node specimens (patient 1), and histologic assessment of gastrointestinal biopsy specimens (patient 2). A, Clinical course of a now 19-year-old girl, patient 1, including treatment and HSCT at the age of 10 years. B, Symptoms and treatment outline of patient 2. C, C.1, Triple immunohistochemical staining of T-cell markers showing increased double-negative T-cell numbers marked only with an antibody against CD3 (light blue/gray, dashed arrow), which is reminiscent of CD95 deficiency; CD4+ (brown, solid arrow) and CD8+ (purple, open arrow) T cells are also shown. C.2, Duodenal biopsy specimens showing focal villous flattening and intraepithelial lymphocytosis. C.3, Colon mucosa with moderate crypt distortion and sparse apoptotic bodies. C.4, Signs of vasculitis indicated by abundant neutrophilic granulocytes within and migrating through the lamina propria capillaries of the colon mucosa. Plasma cells were absent in all sections. AdV, Adenovirus; AIHA, autoimmune hemolytic anemia; ATG-F, anti-thymocyte globulin-Fresenius (Fresenius Medical Care, Vienna, Austria); AZT, azidothymidine; cITP, chronic immune thrombocytopenia; CsA, cyclosporin A; ENT, ear, nose, and throat; IVIG, intravenous immunoglobulin subsitution; LPD, lymphoproliferative disease; MFD-BMT, matched family donor bone marrow transplantation; MMF, mycophenolate mofetil; PEG tube, percutaneous enterogastral tube; TPN, total parenteral nutrition; SCIG, subcutaneous immunoglobulin subsitution; UTI, urinary tract infection.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table ILaboratory parameters of 2 patients with LRBA deficiencyPatient 1Patient 2Before HSCTAfter HSCTBefore rituximabAfter rituximabHumoral immune system99.98% donor chimerism‖At 14 years (4 years after stem cell transplantation).At 19 years of age (9 years after HSCT).∗At 19.5 years of age (10 years after stem cell transplantation).(IVIG substituted) IgG (g/L)16.7 (6.5-14.1)∗At 6 years of age.11.3 (7-16)∗At 19.5 years of age (10 years after stem cell transplantation).4.61 (5.5-12)At 5.5 years before immunosuppression/rituximab.NA IgG1 (g/L)9.1 (3.5-9.1)∗At 6 years of age.7.85 (4.05-10.11)∗At 19.5 years of age (10 years after stem cell transplantation).NANA IgG2 (g/L)3.06 (0.85-3.30)∗At 6 years of age.3.69 (1.69-7.86)∗At 19.5 years of age (10 years after stem cell transplantation).NANA IgG3 (g/L)1.83 (0.2-1.04)∗At 6 years of age.0.879 (0.11-0.85)∗At 19.5 years of age (10 years after stem cell transplantation).NANA IgG4 (g/L)0.01 (0.03-1.58)∗At 6 years of age.0.481 (0.03-2.01)∗At 19.5 years of age (10 years after stem cell transplantation).NANA IgA (g/L)1.24 (0.83-2.17)∗At 6 years of age.2.61 (0.7-4.0)∗At 19.5 years of age (10 years after stem cell transplantation).0.28 (0.21-2.92)At 5.5 years before immunosuppression/rituximab.<0.08 (0.31-3.06)At 11 years of age. IgM (g/L)1.43 (0.55-2.10)∗At 6 years of age.1.64 (0.4-2.3)∗At 19.5 years of age (10 years after stem cell transplantation).0.18 (0.37-1.41)At 5.5 years before immunosuppression/rituximab.0.02 (0.47-1.0)At 11 years of age. IgE (kU/L)24.7∗At 6 years of age. (<110)44 (0-100)∗At 19.5 years of age (10 years after stem cell transplantation).<19‡On repeated occasions. 15%)8.5At 19 years of age (9 years after HSCT). | 6∗At 19.5 years of age (10 years after stem cell transplantation). (>10%)57#At 2 years. (>15)26At 10 years of age. (>10%) αβTCR+CD3+ cells/μLND638‖At 17 months.2,578#At 2 years.1,097At 10 years of age. γδTCR+CD3+ cells/μLND10‖At 17 months.137#At 2 years.39At 10 years of age. αβTCRCD3+CD4−CD8− (DNT cells [% of CD3+ cells])0.95% to 3%∗At 6 years of age.†At 8 years. (<2)0.03%∗At 19.5 years of age (10 years after stem cell transplantation). (<2)0.4%#At 2 years. (<2)3.24%At 10 years of age. ( 0.01)ND0.02%At 11 years of age. (>0.01) CD19+ B cells/μL335-118∗At 6 years of age.†At 8 years. (200-1,600)213At 19 years of age (9 years after HSCT). (100-500)413#At 2 years. (200-2,100)32At 7.5 years of age. (200-1,600) CD19+IgD+CD27+ cells (% of CD19+ cells)ND∗At 6 years of age.†At 8 years.0.38‖At 17 months. | 30At 19 years of age (9 years after HSCT). (>2)ND#At 2 years.0.02At 7.5 years of age. (>2) CD19+IgD−CD27+ cells (% of CD19+ cells)ND∗At 6 years of age.†At 8 years.0.08‖At 17 months. | 31At 19 years of age (9 years after HSCT). (>2)ND#At 2 years.0.00At 7.5 years of age. (>2) Lymphocyte stimulation in vitro detected based on tritiated thymidine incorporation (trigger and antigens in parentheses)†See the Methods section in this article's Online Repository.Normal†At 8 years. (PHA, SEB, CD3, PMA/ionomycin)NDNDNormalAt 9 years of age. unstimulated: 1,716 cpm (1,650-7,162 cpm)PHA: 16,513 cpm (14,218-39,235 cpm)Concanavalin A: 11,384 cpm (4,928-29,519 cpm)CD3/CD28: 17,746 cpm (12,181-31,490 cpm)Footnotes indicate time point of analysis. Pathologic results are shown in boldface (normal ranges are shown in parentheses).AMA, Anti-mitochondrial antibodies; ANA, antinuclear antibody; dsDNA, double-stranded DNA; iNKT, invariant natural killer T; NA, not applicable under IVIG substitution and not done before IVIG; ND, not done; NK, natural killer; PHA, phytohemagglutinine; PMA, phorbol 12-myristate 13-acetate; SCT, stem cell transplantation; SEB, staphylococcal enterotoxin B; SMA, smooth muscle autoantibodies; TCR, T-cell receptor.∗ At 6 years of age.† At 8 years.§ At 10 years.‖ At 14 years (4 years after stem cell transplantation).¶ At 17 months.# At 2 years.∗∗ At 5.5 years before immunosuppression/rituximab.†† At 7.5 years of age.‡‡ At 9 years of age.§§ At 10 years of age.‖‖ At 11 years of age.¶¶ At 19 years of age (9 years after HSCT).## Subfraction of antimitochondrial antibodies directed against the M2 fraction of liver cell mitochondrial antigens located on inner mitochondrial membranes (comprising proteins of the 2-oxo-acid dehydrogenase complex).∗∗∗ At 19.5 years of age (10 years after stem cell transplantation).††† See the Methods section in this article's Online Repository.‡‡‡ On repeated occasions. Open table in a new tab Patient 2, the now 11-year-old younger sister of patient 1, became symptomatic at 5 years of age (fulminant autoimmune hemolytic anemia; Fig 1, B). Immunosuppression was started immediately (corticosteroids, mycophenolate mofetil, and vincristine), leading to a sustained remission (Fig 1, B). Rituximab was administered (4 × 375 mg/m2; Fig 1, B) to secure the treatment response, especially given the severe course of her sister. Before treatment, immunoglobulin concentrations were mildly reduced (4.61 g/L IgG, normal IgA level, and 0.18 g/L IgM; Table I), direct and indirect Coombs test and platelet antibody results were positive, and DNT cell numbers were increased (3.4% of CD3+ cells), with an otherwise normal cellular immune phenotype (Table I and see Table E1), suggesting a familial ALPS-like disorder. Chronic enteropathy with increased calprotectin levels, borderline reduced elastase levels, and chronic norovirus positivity in stool were diagnosed. Gastroduodenoscopy specimens of patient 2 revealed inflammatory bowel disease, absence of plasma cells, and vasculitis (Fig 1, C; and see Fig E2, D-I, in this article's Online Repository at www.jacionline.org). She is being treated with budesonide and IVIG (1 g/kg body weight twice per month; trough level, 8-10 g/L) and requires parenteral nutrition (12-14 hours per night).The fact that 2 patients born to consanguineous parents presented with a similar clinical phenotype prompted us to screen for an underlying (mono-) genetic defect. Homozygous intervals were mapped by applying the GeneChip Human-Mapping-250K-Nsp-Assay (Affymetrix, Santa Clara, Calif). Homozygous stretches were identified and overlaid with HomozygosityMapper.9Seelow D. Schuelke M. Hildebrandt F. Nurnberg P. HomozygosityMapper—an interactive approach to homozygosity mapping.Nucleic Acids Res. 2009; 37: W593-W599Crossref PubMed Scopus (295) Google Scholar Both patients had identical homozygous intervals on chromosomes 2, 3, 4, 9, 11, and 15 (Fig 2, A). Exome sequencing and subsequent computational analysis of patient 1 revealed 23,582 exonic variants, of which 30 were rare missense, nonsense, or splice-site variants located inside the shared homozygous regions of the 2 siblings (see Table E2 in this article's Online Repository at www.jacionline.org). Among the final variant list, one frameshift deletion was identified, resulting in a premature stop codon. This mutation (NM_001199282:c.7162delA; p.T2388Pfs*7) is located inside the gene encoding LRBA. Sanger sequencing confirmed the presence and segregation of the variant, suggesting an autosomal recessive defect with full penetrance (Fig 2, B). Expression of the corresponding protein product was near absent (Fig 2, C).Fig 2Representative depiction of single nucleotide polymorphism array–based homozygosity mapping and Sanger validation, pedigree of the core family, and LRBA protein detection by using fluorescence-activated cell sorting analysis. A, Chromosomal positions are plotted against the homozygosity score in a bar chart, with red bars indicating homozygous regions present in both affected siblings (top). The disease-causing mutation is localized in a homozygous interval (q22.2-q31.3) on the long arm of chromosome 4, as emphasized by the red box (bottom). B, Perfect segregation of the single base deletion (c.7162delA; p.T2388fs) is shown in the 2 patients, the nonaffected sibling, and the parents. Solid symbols indicate homozygous affected subjects, and half-filled symbols refer to the heterozygous carrier. Male and female subjects are distinguished by squares and circles, respectively. C, PBMCs were stimulated with PHA, as described in the Methods section in this article's Online Repository at www.jacionline.org. The increased LRBA protein expression after stimulation (black) compared with that in unstimulated cells (gray) is shown in the in-house control and in a travel control (1 and 2 asterisks, respectively; upper panel); is reduced in the LRBA-heterozygous mother, who was the stem cell donor, in patient 1 after HSCT; and is absent in patient 2 (lower panel). The plot is representative of 2 independent analyses.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig 2Representative depiction of single nucleotide polymorphism array–based homozygosity mapping and Sanger validation, pedigree of the core family, and LRBA protein detection by using fluorescence-activated cell sorting analysis. A, Chromosomal positions are plotted against the homozygosity score in a bar chart, with red bars indicating homozygous regions present in both affected siblings (top). The disease-causing mutation is localized in a homozygous interval (q22.2-q31.3) on the long arm of chromosome 4, as emphasized by the red box (bottom). B, Perfect segregation of the single base deletion (c.7162delA; p.T2388fs) is shown in the 2 patients, the nonaffected sibling, and the parents. Solid symbols indicate homozygous affected subjects, and half-filled symbols refer to the heterozygous carrier. Male and female subjects are distinguished by squares and circles, respectively. C, PBMCs were stimulated with PHA, as described in the Methods section in this article's Online Repository at www.jacionline.org. The increased LRBA protein expression after stimulation (black) compared with that in unstimulated cells (gray) is shown in the in-house control and in a travel control (1 and 2 asterisks, respectively; upper panel); is reduced in the LRBA-heterozygous mother, who was the stem cell donor, in patient 1 after HSCT; and is absent in patient 2 (lower panel). The plot is representative of 2 independent analyses.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Taken together, we describe a clinical, immunologic, and genetic analysis of 2 patients presenting with multiorgan autoimmunity and severe infections caused by a novel mutation in LRBA, the clinical spectrum of which both recapitulates and extends the previously described phenotypes (see additional text in this article's Online Repository).1Alangari A. Alsultan A. Adly N. Massaad M.J. Kiani I.S. Aljebreen A. et al.LPS-responsive beige-like anchor (LRBA) gene mutation in a family with inflammatory bowel disease and combined immunodeficiency.J Allergy Clin Immunol. 2012; 130: 481-488.e2Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 2Burns S.O. Zenner H.L. Plagnol V. Curtis J. Mok K. Eisenhut M. et al.LRBA gene deletion in a patient presenting with autoimmunity without hypogammaglobulinemia.J Allergy Clin Immunol. 2012; 130: 1428-1432Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 3Lopez-Herrera G. Tampella G. Pan-Hammarstrom Q. Herholz P. Trujillo-Vargas C.M. Phadwal K. et al.Deleterious mutations in LRBA are associated with a syndrome of immune deficiency and autoimmunity.Am J Hum Genet. 2012; 90: 986-1001Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar The fact that patient 1 had a profound immunodeficiency with life-threatening infections and refractory autoimmunity justified the approach of allogeneic matched family donor HSCT according to international guidelines.4Gratwohl A. Baldomero H. Sureda A. Indications for and current practice of allogeneic and autologous HSCT.in: Apperley J. Carreras E. Gluckman E. Masszi T. The EBMT handbook—haematopoietic stem cell transplantation: European Group for Blood and Marrow Transplantation & European School of Hematology. European School of Hematology, Paris, France. 2012: 302-315Google Scholar, 5Snowden J.A. Saccardi R. Farge D. Indications for HSCT in adults—Autoimmune diseases.in: Apperley J. Carreras E. Gluckman E. Masszi T. The EBMT handbook—haematopoietic stem cell transplantation: European Group for Blood and Marrow Transplantation & European School of Hematology. European School of Hematology, Paris, France. 2012: 458-469Google Scholar In our case allogeneic HSCT resulted in long-lasting partial remission in the patient with LRBA deficiency. The observation that mild autoimmune symptoms (ITP and vitiligo) have recurred in patient 1 years after HSCT despite full donor chimerism might be due to reduced LRBA expression compared with a healthy donor (in the same range as the heterozygous stem cell donor, who has detectable autoantibodies without clinical symptoms; see Fig 2, C, and additional text in this article's Online Repository), thus representing residual disease activity or late, limited chronic GvHD. These data show that HSCT might be a treatment option for patients with LRBA deficiency. LPS-responsive vesicle trafficking, beach and anchor containing protein (LRBA) deficiency has been identified as a primary immunodeficiency (PID) characterized by recurrent infections associated with autoimmunity, such as inflammatory bowel disease and autoimmune cytopenias (see Fig E1 in this article's Online Repository at www.jacionline.org).1Alangari A. Alsultan A. Adly N. Massaad M.J. Kiani I.S. Aljebreen A. et al.LPS-responsive beige-like anchor (LRBA) gene mutation in a family with inflammatory bowel disease and combined immunodeficiency.J Allergy Clin Immunol. 2012; 130: 481-488.e2Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 2Burns S.O. Zenner H.L. Plagnol V. Curtis J. Mok K. Eisenhut M. et al.LRBA gene deletion in a patient presenting with autoimmunity without hypogammaglobulinemia.J Allergy Clin Immunol. 2012; 130: 1428-1432Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 3Lopez-Herrera G. Tampella G. Pan-Hammarstrom Q. Herholz P. Trujillo-Vargas C.M. Phadwal K. et al.Deleterious mutations in LRBA are associated with a syndrome of immune deficiency and autoimmunity.Am J Hum Genet. 2012; 90: 986-1001Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar A wide range of immunosuppressive treatment measures have only induced temporary relief in affected subjects. Although allogeneic hematopoietic stem cell transplantation (HSCT) is the current treatment for many forms of PIDs, HSCT is less established in patients with autoimmune disease4Gratwohl A. Baldomero H. Sureda A. Indications for and current practice of allogeneic and autologous HSCT.in: Apperley J. Carreras E. Gluckman E. Masszi T. The EBMT handbook—haematopoietic stem cell transplantation: European Group for Blood and Marrow Transplantation & European School of Hematology. European School of Hematology, Paris, France. 2012: 302-315Google Scholar, 5Snowden J.A. Saccardi R. Farge D. Indications for HSCT in adults—Autoimmune diseases.in: Apperley J. Carreras E. Gluckman E. Masszi T. The EBMT handbook—haematopoietic stem cell transplantation: European Group for Blood and Marrow Transplantation & European School of Hematology. European School of Hematology, Paris, France. 2012: 458-469Google Scholar and has not yet been reported in LRBA-deficient patients. We studied a consanguineous family of Kurdish origin with a systemic autoimmune disorder. Patient 1's symptoms started at 2 years of age with immune thrombocytopenia (ITP; Fig 1, A). Serum immunoglobulin concentrations were slightly increased, and the cellular immunophenotype was normal (Table I and see Table E1 in this article's Online Repository at www.jacionline.org). A lymph node biopsy performed because of generalized lymphoproliferative disease (LPD) revealed a follicular lymphatic hyperplasia with abundant (about 20% to 30%) CD3+ and CD4− and CD8− double-negative T lymphocytes (DNT cells; Fig 1, C), suggesting an immune dysregulation, lymphocyte maturation, or apoptosis defect compatible with autoimmune lymphoproliferative syndrome (ALPS).6Canale V.C. Smith C.H. Chronic lymphadenopathy simulating malignant lymphoma.J Pediatr. 1967; 70: 891-899Abstract Full Text PDF PubMed Scopus (174) Google Scholar, 7Price S. Shaw P.A. Seitz A. Joshi G. Davis J. Niemela J.E. et al.Natural history of autoimmune lymphoproliferative syndrome associated with FAS gene mutations.Blood. 2014; 123: 1989-1999Crossref PubMed Scopus (154) Google Scholar HSCT was performed with the clinically healthy HLA-identical mother as the donor (see the additional text in this article's Online Repository at www.jacionline.org), leading to complete remission with persisting full donor chimerism and without signs of acute or chronic graft-versus-host disease (GvHD). Four years after HSCT, ITP relapsed but responded well to high-dose intravenous immunoglobulin (IVIG) treatment. When romiplostim was started, platelet counts normalized, and administration of romiplostim (5 μg/kg, every 4 to 6 weeks) without further need for immunosuppression or IVIG has led to sustained but treatment-dependent remission.8Seidel M.G. Urban C. Sipurzynski J. Beham-Schmid C. Lackner H. Benesch M. High response rate but short-term effect of romiplostim in paediatric refractory chronic immune thrombocytopenia.Br J Haematol. 2014; 165: 419-421Crossref PubMed Scopus (14) Google Scholar Footnotes indicate time point of analysis. Pathologic results are shown in boldface (normal ranges are shown in parentheses). AMA, Anti-mitochondrial antibodies; ANA, antinuclear antibody; dsDNA, double-stranded DNA; iNKT, invariant natural killer T; NA, not applicable under IVIG substitution and not done before IVIG; ND, not done; NK, natural killer; PHA, phytohemagglutinine; PMA, phorbol 12-myristate 13-acetate; SCT, stem cell transplantation; SEB, staphylococcal enterotoxin B; SMA, smooth muscle autoantibodies; TCR, T-cell receptor. Patient 2, the now 11-year-old younger sister of patient 1, became symptomatic at 5 years of age (fulminant autoimmune hemolytic anemia; Fig 1, B). Immunosuppression was started immediately (corticosteroids, mycophenolate mofetil, and vincristine), leading to a sustained remission (Fig 1, B). Rituximab was administered (4 × 375 mg/m2; Fig 1, B) to secure the treatment response, especially given the severe course of her sister. Before treatment, immunoglobulin concentrations were mildly reduced (4.61 g/L IgG, normal IgA level, and 0.18 g/L IgM; Table I), direct and indirect Coombs test and platelet antibody results were positive, and DNT cell numbers were increased (3.4% of CD3+ cells), with an otherwise normal cellular immune phenotype (Table I and see Table E1), suggesting a familial ALPS-like disorder. Chronic enteropathy with increased calprotectin levels, borderline reduced elastase levels, and chronic norovirus positivity in stool were diagnosed. Gastroduodenoscopy specimens of patient 2 revealed inflammatory bowel disease, absence of plasma cells, and vasculitis (Fig 1, C; and see Fig E2, D-I, in this article's Online Repository at www.jacionline.org). She is being treated with budesonide and IVIG (1 g/kg body weight twice per month; trough level, 8-10 g/L) and requires parenteral nutrition (12-14 hours per night). The fact that 2 patients born to consanguineous parents presented with a similar clinical phenotype prompted us to screen for an underlying (mono-) genetic defect. Homozygous intervals were mapped by applying the GeneChip Human-Mapping-250K-Nsp-Assay (Affymetrix, Santa Clara, Calif). Homozygous stretches were identified and overlaid with HomozygosityMapper.9Seelow D. Schuelke M. Hildebrandt F. Nurnberg P. HomozygosityMapper—an interactive approach to homozygosity mapping.Nucleic Acids Res. 2009; 37: W593-W599Crossref PubMed Scopus (295) Google Scholar Both patients had identical homozygous intervals on chromosomes 2, 3, 4, 9, 11, and 15 (Fig 2, A). Exome sequencing and subsequent computational analysis of patient 1 revealed 23,582 exonic variants, of which 30 were rare missense, nonsense, or splice-site variants located inside the shared homozygous regions of the 2 siblings (see Table E2 in this article's Online Repository at www.jacionline.org). Among the final variant list, one frameshift deletion was identified, resulting in a premature stop codon. This mutation (NM_001199282:c.7162delA; p.T2388Pfs*
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