Loss of B Cells in Patients with Heterozygous Mutations in IKAROS

The new england journal of medicine

Original Article

H.S. Kuehn, B. Boisson, C. Cunningham‑Rundles, J. Reichenbach, A. Stray‑Pedersen, E.W. Gelfand, P. Maffucci, K.R. Pierce, J.K. Abbott, K.V. Voelkerding, S.T. South, N.H. Augustine, J.S. Bush, W.K. Dolen, B.B. Wray, Y. Itan, A. Cobat, H.S. Sorte, S. Ganesan, S. Prader, T.B. Martins, M.G. Lawrence, J.S. Orange, K.R. Calvo, J.E. Niemela, J.-L. Casanova, T.A. Fleisher, H.R. Hill, A. Kumánovics, M.E. Conley, and S.D. Rosenzweig

ABSTRACT

BACKGROUND The authors’ full names, academic de- Common variable immunodeficiency (CVID) is characterized by late-onset hypo- grees, and affiliations are listed in the gammaglobulinemia in the absence of predisposing factors. The genetic cause is Appendix. Address reprint requests to Dr. Conley at St. Giles Laboratory of Human unknown in the majority of cases, and less than 10% of patients have a family Genetics of Infectious Diseases, Rocke- history of the disease. Most patients have normal numbers of B cells but lack feller University, 1230 York Ave., Box 163, plasma cells. New York, NY 10065-6399, or at mconley@ rockefeller.edu. METHODS Drs. Kuehn and Boisson and Drs. Conley We used whole-exome sequencing and array-based comparative genomic hybrid- and Rosenzweig contributed equally to this article. ization to evaluate a subset of patients with CVID and low B-cell numbers. Mutant proteins were analyzed for DNA binding with the use of an electrophoretic mobility- N Engl J Med 2016;374:1032-43. DOI: 10.1056/NEJMoa1512234 shift assay (EMSA) and confocal microscopy. Flow cytometry was used to analyze Copyright © 2016 Massachusetts Medical Society. peripheral-blood lymphocytes and bone marrow aspirates.

RESULTS Six different heterozygous mutations in IKZF1, the gene encoding the transcription factor IKAROS, were identified in 29 persons from six families. In two families, the mutation was a de novo event in the proband. All the mutations, four amino acid substitutions, an intragenic deletion, and a 4.7-Mb multigene deletion involved the DNA-binding domain of IKAROS. The proteins bearing missense mutations failed to bind target DNA sequences on EMSA and confocal microscopy; however, they did not inhibit the binding of wild-type IKAROS. Studies in family members showed progressive loss of B cells and serum immunoglobulins. Bone marrow aspirates in two patients had markedly decreased early B-cell precursors, but plasma cells were present. Acute lymphoblastic leukemia developed in 2 of the 29 patients.

CONCLUSIONS Heterozygous mutations in the transcription factor IKAROS caused an autosomal dominant form of CVID that is associated with a striking decrease in B-cell numbers. (Funded by the National Institutes of Health and others.)

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he genetic cause of many primary tions. Patients B4, F1, and F6 had recurrent sinus- immunodeficiencies remains unknown. itis, bronchitis, or both. Patient A2 had had two TCommon variable immunodeficiency (CVID) episodes of idiopathic thrombocytopenic pur- comprises a heterogeneous group of disorders pura. B-cell acute lymphoblastic leukemia (ALL) characterized by the late onset of recurrent infec- developed in Patients B7 and F12 at 3 and 5 years tions, hypogammaglobulinemia, and poor anti- of age, respectively. Patient B1 died from pneu- body response to vaccine antigens that cannot monia at 74 years of age, and Patient B7 died be explained by previous exposures, treatment, from a relapse of B-cell ALL at 5 years of age. No or infections. Some patients also have autoim- patients showed evidence of increased suscepti- munity, granulomatous disease, or cancer.1,2 bility to viral or fungal infections. Most of the Genomic approaches, including whole-exome patients with hypogammaglobulinemia have done sequencing and high-resolution array-based com- relatively well, despite inadequate treatment with parative genomic hybridization (CGH), have ac- gamma globulin in many of them. Additional celerated the identification of genetic causes in clinical data and the methods used for genetic patients with primary immunodeficiencies, includ- analysis, functional studies, and flow cytometry ing CVID.3-5 Mutations in several genes, including are described in the Supplementary Appendix. ICOS, CD19, CD81, CD20, CD21, TWEAK, CTLA4, LRBA, GATA2, CXCL12, NFKB1, and NFKB2, have Results been associated with a CVID-like phenotype in a small proportion of patients.6,7 A number of Mutation Detection these new disorders are autosomal dominant Whole-exome sequencing was performed on with incomplete penetrance. Often, there is DNA samples from Patients A1 and A2 and the marked variation in the clinical severity, even unaffected mother of Patient A1; Patients B1 and among family members with the same genetic B6; Patients C1, C2, and C3 and the healthy defect.3-7 We used whole-exome sequencing and daughter of Patient C1; and Patients D2 and E1. high-resolution array-based CGH to evaluate a Mutations in 269 genes known to be associated subset of patients with CVID and low B-cell with immunodeficiency (Table S2 in the Supple- numbers. mentary Appendix), including BTK (encoding Bruton’s tyrosine kinase), GATA2, CTLA4, and Methods TNFRS13B (encoding TACI [transmembrane acti- vator and calcium-modulator and cyclophilin- Study Participants ligand interactor]), were not identified in any of All study participants or their parents provided these persons. A G→T substitution at c.485 in written informed consent. The study was ap- Family A resulted in a substitution of leucine for proved by the local review boards. The probands arginine at codon 162 (R162L) in IKZF1, the gene in this study, Patients A1, B5, C1, D2, E1, and F2, encoding the transcription factor IKAROS. A G→A were first evaluated for immunodeficiency at substitution at c.485 in Family B resulted in a 3 to 32 years of age, after recurrent or severe substitution of glutamine for arginine (R162Q) bacterial infections that were often due to Strep- at the same mutated codon found in Family A. tococcus pneumoniae. All six patients had hypo- In Family C, an A→G substitution in IKZF1 at gammaglobulinemia and decreased numbers of c.500 was found, resulting in a substitution of ar- B cells, findings consistent with the diagnosis of ginine for histidine at codon 167 (H167R). A G→A CVID (Table 1, and Table S1 in the Supplemen- substitution at c.551 in Family D resulted in a tary Appendix, available with the full text of this substitution of glutamine for arginine at codon article at NEJM.org). CVID was subsequently 184 (R184Q). Sanger sequencing confirmed the diagnosed in Patients A2, B1, B6, C2, C3, F1, F3, mutations in the patients and detected muta- and F4. tions in additional affected family members. The After IKZF1 mutations had been identified in mutations were de novo in Patients A1 and C1; the probands, they were detected in additional however, Patient D1, the asymptomatic mother affected family members. Patients B1, B2, B3, of Patient D2, carried the same mutation as her F5, and F6 had a history of severe bacterial infec- daughter (Fig. 1).

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Table 1. Clinical and Laboratory Characteristics of 29 Patients with Heterozygous IKAROS Mutations.*

Patient Clinical Age at No. Manifestations Onset Immunoglobulin B Cells T Cells

IgG IgA IgM CD19+ CD27+† CD3+ CD3+CD4+ CD3+CD8+

yr mg/dl % (absolute lymphocyte count/mm3) A1 Infections 9 <33 <7 21 0.5 (17) NA 95 (3180) 34 (1128) 59 (1982) ITP 3 A2 727‡ 7 5 0.3 (12) NA 86 (3129) 47 (1831) 34 (1319) Infections 6 B1 Infections 12 386 30 60 NA NA NA NA NA B2 Infections 6 461 36 45 3.0 (80) 15 (12) 85 (2350) 34 (950) 51 (1410) B3 Infections 10 283 15 71 7.0 (260) 72 (187) 91 (3330) 31 (1140) 61 (2230) B4 Infections 43 433 19 56 2.0 (50) 45 (22) 86 (2030) 23 (540) 65 (1530) B5 Infections 3 1430‡ 21 22 2.0 (50) 3 (1) 76 (1900) 36 (900) 38 (950) B6 Infections 1 1260‡ 12 21 1.0 (20) 17 (3) 79 (1560) 38 (750) 36 (710) B7 B-cell ALL 3 NA NA NA NA NA NA NA NA C1 Infections 30 887‡ 73 11 1.0 (26) NA 83 (2177) 31 (806) 50 (1300) C2 Infections 13 1052‡ 157 12 0.2 (5) NA 83 (2049) 26 (627) 50 (1225) C3 Infections 4 1030‡ 12 <6 0.4 (18) NA 82 (3712) 26 (1168) 48 (2147) D1 Asymptomatic — 637 5 45 7.0 (160) NA 89 (2022) 29 (632) 63 (1397) D2 Infections 9 42 <1 7 0.3 (9) NA 96 (3388) 40 (1416) 54 (1888) E1 Infections 3 860‡ <1 10 0.2 (5) NA 93 (2817) 25 (787) 67 (1966) E2 Asymptomatic — NA NA NA NA NA NA NA NA F1 Infections 57 113 <6 14 0.8 (11) 22 (2) 66 (866) 26 (343) 38 (498) F2 Infections 29 <7 <4 <2 1.0 (18) NA 94 (1709) 59 (1068) 35 (640) F3 Infections 21 1010‡ <6 5 0.6 (8) 24 (2) 79 (1020) 40 (522) 38 (486) F4 Infections 22 1010‡ <6 7 0.1 (2) 58 (1) 95 (2489) 31 (787) 65 (1686) F5 Infections 31 382 29 21 0.5 (12) 19 (2) 87 (2078) 35 (822) 52 (1238) F6 Infections 19 148 32 39 5.5 (138) 4 (5) 82 (2115) 48 (1231) 34 (886) F7 Asymptomatic — 528 <6 18 0.5 (9) 18 (2) 84 (1464) 58 (996) 27 (469) F8 Asymptomatic — 789 64 46 2.7 (89) NA 86 (2851) 54 (1525) 32 (907) F9 Asymptomatic — 844 28 36 15.0 (354) 6 (22) 77 (1940) 34 (866) 40 (955) F10 Asymptomatic — 606 118 32 7.5 (187) 22 (42) 76 (1864) 44 (1068) 31 (758) F11 Asymptomatic — 541 120 53 17.1 (544) 15 (79) 68 (2026) 43 (1286) 22 (656) B-cell ALL 5 F12 590 36 13 13.7 (452) 9 (42) 75 (2292) 34 (1032) 42 (1269) Infections 6 F13 Asymptomatic — 629 28 40 15.4 (291) 12 (34) 78 (1356) 55 (953) 22 (378)

* The most recent, comprehensive, and representative laboratory data are shown for each patient; complete laboratory data are provided in Table S1 in the Supplementary Appendix. Numbers in italics indicate values above the normal range, and numbers in boldface indicate values below the normal range for age-matched controls in the laboratory in which the study was performed: the National Institutes of Health Clinical Center (Families A and C), University Children’s Hospital Zurich (Family B), Quest Diagnostics (Family D), Oslo University Hospital and University of Oslo (Family E), and ARUP Laboratories (Family F). ALL denotes acute lymphoblastic leukemia, CVID common variable immunodeficiency, ITP idiopathic thrombocytopenic purpura, and NA not available. † CD27+ B cells are expressed as the percentage among CD19+ B cells. Values are included only if at least 50 CD19+ B cells were analyzed. ‡ The patient was receiving IgG replacement therapy.

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Whole-exome sequencing and customized C were in zinc finger 2, and the mutation in array-based CGH were performed on a DNA sam- Family D was in zinc finger 3; these two highly ple from Patient E1. In silico copy-number varia- conserved domains (Fig. S1 in the Supplemen- tion analysis of data from whole-exome sequenc- tary Appendix) are essential for DNA binding.15 ing8 showed an IKZF1 intragenic heterozygous The deletion in Patient E1 resulted in the loss of deletion. Sanger sequencing identified the exact zinc fingers 1, 2, and 3. Previously published mutation breakpoints (Fig. S1 in the Supplemen- structural studies indicate that missense muta- tary Appendix). This 16.8-kb deletion (Chr7 [hu- tions R162L, R162Q, and R184Q occur at DNA man assembly GRCh37]:g.50435843_50452713del; contact residues, whereas the H167R mutation NM_006060.5(IKZF1):c.1618388_589+2308del) re- involves one of the C2H2 zinc-finger canonical sults in the in-frame deletion of IKZF1 exons 4 histidines (see Fig. S2 in the Supplementary Ap- and 5. The same heterozygous deletion was de- pendix for the structure of IKAROS).15 Further- tected in Patient E2 (the son of Patient E1) by more, these four mutations are predicted to be means of CGH. highly damaging to protein function (Fig. S3 in CGH analysis of DNA samples from Patient the Supplementary Appendix). These point mu- F1, her husband, and her four children revealed tations were not seen in more than 64,000 ge- a 4.7-Mb heterozygous deletion on chromosome nomes included in the 1000 Genomes Project 7 in Patient F1 and three of her children (Patients and the Exome Aggregation Consortium (ExAC) F3, F4, and F5). The 7p12.3-p12.1 deletion involves database. No premature stop codons in IKZF1 11 genes (ABCA13, CDC14C, VWC2, ZPBP, C7orf7, were found in these databases, and the few mis- IKZF1, FIGNL1, DDC, GRB10, COBL, and POM121L12). sense mutations reported were either very rare Only IKZF1 is known to play a role in B-cell devel- or predicted to be benign. In addition, IKZF1 is opment and function. Multiplex ligation-depen- under strong purifying selection (Fig. S4 in the dent probe amplification revealed the IKZF1 dele- Supplementary Appendix), meaning that persons tion in nine additional family members (Fig. 1). carrying dysfunctional variants tend not to sur- No germline constitutional deletion of this re- vive to reproductive age. Finally, DNA from 132 gion was reported in more than 8000 healthy additional patients with CVID, including 21 pa- controls.9 However, patients with complex genetic tients with low B-cell numbers, was analyzed for syndromes associated with total or subtotal mutations in IKZF1 by means of whole-exome chromosome 7p deletions, including one child sequencing or Sanger sequencing of exons, but with Greig cephalopolysyndactyly and ALL, have no additional mutations were identified. been described.10 IKAROS is a member of a family of hemato- Functional Analysis of IKZF1 Mutations poietic zinc-finger transcription factors.11 It was Flow cytometry showed that the amount of first identified on the basis of its ability to bind IKAROS in T cells and B cells from the study regulatory regions of genes encoding terminal participants with amino acid substitutions was deoxynucleotidyl transferase (TdT) and CD3δ.12,13 equal to that in T cells and B cells from controls, It also binds pericentromeric DNA as part of the whereas T cells and B cells from the participants NuRD (nucleosome remodeling and histone with the large deletion had approximately half deacetylase) complex and can both enhance and the normal amount of IKAROS (Fig. S5 in the repress gene transcription.14-16 The four N-termi- Supplementary Appendix). To examine the sta- nal zinc fingers of IKAROS form the DNA- bility of the proteins resulting from the four binding domain, and the two C-terminal zinc missense mutations in IKZF1 (R162L, R162Q, fingers act as a dimerization domain.11,17 Multi- H167R, and R184Q) and the ability of these ple splice variants of IKAROS are produced; those proteins to dimerize with wild-type IKAROS and that have lost the DNA-binding zinc fingers but enter the nucleus, we transfected HEK293T cells have retained the dimerization zinc fingers act with wild-type and mutant expression vectors. as dominant negative regulators of IKAROS Western blot analysis of extracts from the cyto- family members.18 plasm and nucleus of the transfected cells The missense mutations in Families A, B, and showed that these mutant proteins were stable,

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A Family A Family B Family C Family D c.485G→T c.485G→A c.500A→G c.551G→A (p.R162L) (p.R162Q) (p.H167R) (p.R184Q)

B1 D1 WT/WT WT/WT WT/Mut WT/WT WT/WT WT/WT WT/WT WT/Mut

A1 B2 B3 B4 C1 D2 WT/WT WT/Mut WT/WT WT/Mut WT/WT WT/WT WT/WT WT/Mut WT/Mut WT/WT WT/Mut WT/Mut

A2 B5 B6 B7 C2 C3 WT/Mut WT/WT WT/Mut WT/Mut WT/Mut WT/Mut WT/Mut WT/WT

Family E Family F 16.8-kb deletion 4.7-Mb deletion on chromosome 7 on chromosome 7

WT/WT

E1 F1 6 F2 WT/Mut WT/WT WT/WT WT/WT WT/Mut WT/Mut WT/WT

E2 F3 F4 F5 F6 WT/Mut WT/Mut WT/WT WT/Mut WT/WT WT/Mut WT/WT WT/WT WT/Mut WT/WT

F7 F8 F9 3 F10 F11 F12 F13 WT/Mut WT/Mut WT/WT WT/WT WT/Mut WT/WT WT/Mut WT/Mut WT/Mut WT/Mut

B A BC D

WT ZF1 ZF3 ZF5 ZF2 ZF4 ZF6

IKZF1 DNA-Binding Domain Dimerization Domain

E ZF5 ZF4 ZF6

Chromosome 7 WT

7p 7q

were dimerized with wild-type IKAROS, and were from transfected cells were used in an electro- able to migrate to the nucleus (Fig. S6 in the phoretic mobility-shift assay (EMSA) to evaluate Supplementary Appendix). The nuclear extracts the ability of the mutant proteins to bind an

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Figure 1 (facing page). Mutation in IKAROS (IKZF1) mutant IKAROS, the staining pattern was simi- in Families with Common Variable Immunodeficiency. lar to that seen in cells transfected with only the Panel A shows the pedigrees of the six families. Circles vector expressing wild-type IKAROS (Fig. S7D in and squares denote female and male family members, the Supplementary Appendix). Similar results respectively. Black symbols represent family members were obtained when HEK293T cells were trans- with a heterozygous mutation in IKZF1, and slashes fected (Fig. S8 in the Supplementary Appendix). indicate deceased family members. In Family F, the diamond with the number in it indicates the number The combined results of the EMSAs and confo- of family members (male or female) who were not cal microscopy are consistent with the hypothe- screened. Arrows indicate the probands. The specific sis that the four proteins resulting from mis- complementary DNA (IKZF1 transcript variant sense mutations do not have a dominant negative NM_006060) and protein mutation are indicated effect. above each pedigree. Mut denotes mutation, and WT wild type. Panel B, top, shows the exon structure of To compare the mutations found in these full-length IKAROS isoform 1 in light-gray boxes (IKZF1 patients with previously described mutations in transcript variant NM_006060), with the zinc fingers IKZF1, we performed an EMSA and confocal (ZFs) indicated in dark-gray boxes. The sites of the microscopy with the use of vectors expressing mutations in Families A, B, C, and D and the deletion N159A, an experimentally generated in vitro of c.161-8388_589+2308del in IKZF1 in Family E are 15 shown. The lower part of the panel shows a schematic mutation ; H191R, an ethylnitrosourea-induced 20 representation of the 4.7-Mb deletion on the short arm murine mutation ; and Y210C, a mutation in of chromosome 7 (7p12.3-p12.1) in Family F, with the zinc finger 4 that was identified in a critically ill deletion indicated in red. premature infant with pancytopenia.21 The abnormal EMSA results and perinuclear localization for the N159A and H191R mutations were IKAROS consensus-binding sequence (IK-bs4)15 similar to the findings with the R162L, R162Q, and a probe from the pericentromeric region of H167R, and R184Q mutations. The Y210C muta- human chromosome 8 (γSat8).19 The four pro- tion showed decreased but not absent DNA bind- teins resulting from missense mutations failed ing on an EMSA. Primary T cells from the in- to bind these probes (Fig. S7A and S7B in the fant, who was heterozygous for the Y210C allele, Supplementary Appendix). To determine whether were reported to show abnormal subcellular lo- the amino acid substitutions resulted in a domi- calization of IKAROS; however, we saw normal nant negative effect and to mimic the heterozy- pericentromeric localization of the protein with gous state, we performed EMSAs with nuclear the Y210C mutation when expressed alone or in extracts from human embryonic kidney 293T the presence of the wild-type allele in transfect- (HEK293T) cells transfected with vector expressed NIH-3T3 cells (Fig. S9 in the Supplementary ing 100% wild-type IKAROS or vectors express- Appendix). ing 50% wild-type and 50% mutant IKAROS. DNA binding was reduced by 38 to 74% in the Laboratory Findings cells transfected with the 50% wild-type and There was some variation in laboratory findings 50% mutant vectors, as compared with the cells among the study participants. However, 26 of transfected with 100% wild-type vector. This the 27 patients for whom pretreatment data were finding was consistent with the reduction in the available had a marked decrease in at least two amount of wild-type vector used in transfection of the three major immunoglobulin isotypes (Fig. S7A and S7B). (IgG, IgM, and IgA); 23 had a decrease in all Confocal microscopy showed that transfected three isotypes, and 6 with panhypogamma- NIH-3T3 cells expressing wild-type IKAROS had globulinemia had a serum IgG level of less than the punctate staining pattern that is characteris- 150 mg per deciliter before treatment with tic of pericentromeric heterochromatin binding gamma globulin was started (Table 1). Only 1 pa- and localization (Fig. S7C in the Supplementary tient (at 5 years of age) had normal findings for Appendix). In contrast, the four proteins result- the three major immunoglobulin isotypes. Assess- ing from missense mutations had diffuse nuclear ment of antibodies to vaccine antigens, which staining. When the NIH-3T3 cells were trans- was performed in 8 patients, showed an absence fected with equal amounts of the vector express- of antibodies in 6 patients, progressive loss of ing wild-type IKAROS and the vector expressing antibodies in 1, and normal titers in 1. At their

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A Families A–E B Family F A1 B1 C1 D1 F1 F4 F7 F10 A2 B2 C2 D2 F2 F5 F8 F11 B3 C3 E1 F3 F6 F9 F12 B4 F13 B5 B6

1200 1200

900 900

600 600 IgG (mg/dl) IgG (mg/dl) 300 300

0 0 0 10 20 30 40 50 60 70 0 5 10 15 20 25 30 35 40 45 50 55 60 65 Age (yr) Age (yr)

600 600 ) ) 3 3

400 400

200 200 CD19+ B Cells (no./mm CD19+ B Cells (no./mm 0 0 0 5 10 15 20 25 30 35 40 45 50 55 0 5 10 15 20 25 30 35 40 45 50 55 60 65 Age (yr) Age (yr)

5000 5000 ) ) 3 3 4000 4000

3000 3000

2000 2000

1000 1000 CD4+ T Cells (no./mm CD4+ T Cells (no./mm 0 0 0 5 10 15 20 25 30 35 40 45 50 55 0 5 10 15 20 25 30 35 40 45 50 55 60 65 Age (yr) Age (yr)

4000 4000 ) ) 3 3 3000 3000

2000 2000

1000 1000 CD8+ T Cells (no./mm CD8+ T Cells (no./mm

0 0 0 5 10 15 20 25 30 35 40 45 50 55 0 5 10 15 20 25 30 35 40 45 50 55 60 65 Age (yr) Age (yr)

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Figure 2 (facing page). Low Serum IgG Levels and bers of CD34+ and CD138+ plasma cells were Progressive Loss of Peripheral-Blood B-Cell Counts found in the bone marrow (Fig. 3A), but there in Patients with IKZF1 Mutations. was a marked decrease in pro-B cells (defined by The symbols represent individual family members, coexpression of CD34 and CD19) and earlier with black symbols indicating those who were asymp- precursors (expressing surface CD34 and cyto- tomatic; gray shading represents mean values ±1 SD plasmic TdT in the absence of CD19) (Fig. 3B).22 in healthy controls. Only patients who were not being treated with gamma globulin are included in the data Both the number of TdT+ cells and the intensity shown for IgG levels. of TdT expression were decreased. Because TdT is a direct target of IKZF1,12 we compared the number of TdT-mediated additions in immuno- most recent evaluation, 14 patients had a CD19+ globulin heavy-chain transcripts from comple- cell count of less than 1% in peripheral blood; mentary DNA obtained from Patients B5 and C1 however, CD19+CD27+ memory B cells were with the number in four healthy controls and readily detectable in the 14 patients in whom two patients with mutations in BTK who had a they were analyzed. When plotted against age, similar number of peripheral-blood B cells. In the serum IgG level showed a progressive de- the patients and the controls, the number of cline (Fig. 2). A similar finding was noted when TdT-mediated additions was within the normal the number of CD19+ B cells was evaluated range23 (Table S3 in the Supplementary Appen- (Table 1, and Table S1 in the Supplementary Ap- dix). The small number of CD34−CD19+ cells in pendix). bone marrow aspirates from Patients C1 and C2 T-cell studies showed a consistent increase in were equally divided between pre-B cells that CD8+ T cells with reversed CD4:CD8 ratios in 10 were negative for surface immunoglobulin and of 13 participants with DNA-binding defects in immature B cells that were positive for surface IKAROS (e.g., amino acid substitutions or exon immunoglobulin (Fig. 3B), indicating that the 4 and 5 deletion) and in 3 of 13 patients with block in B-cell differentiation was not complete complete deletion of IKZF1 (P = 0.01 on the basis and some precursors were able to differentiate of a two-tailed Fisher’s exact test) (Table 1 and into B cells. Fig. 2, and Table S1 in the Supplementary Ap- pendix). The CD8+ T cells were predominantly Discussion naive cells (CD62L+CD45RA+) and central memory cells (CD62L+CD45RA−) in the 6 patients CVID is characterized by late-onset hypogam- who were assessed for these markers. Both maglobulinemia and a poor antibody response CD4+ and CD8+ cells appeared to be polyclonal to infectious and vaccine antigens.1,2 The genetic on cytometric analysis of Vβ flow (Fig. S10 in cause is unknown in the majority of cases, and the Supplementary Appendix) and studies of T- less than 10% of patients have a family history cell receptor gamma-chain rearrangement. Pro- of the disease. Most patients have normal num- liferation of T-cells and Fas-mediated apoptosis bers of B cells but lack plasma cells. This study were similar in patients and controls (Fig. S11 in documents the progressive loss of serum immu- the Supplementary Appendix). Polymerase- noglobulins and B cells in a subset of patients chain-reaction assays for cytomegalovirus and with CVID associated with heterozygous muta- Epstein–Barr virus were negative in the 6 pa- tions in IKZF1. Clinical and laboratory findings tients with reversed CD4:CD8 ratios who were varied among these persons, particularly during evaluated. The number of natural killer cells childhood. However, 13 of the 14 study partici- ranged from the high end to the low end of the pants who were older than 25 years of age were normal range (Table S1 in the Supplementary clinically symptomatic, and all these adults had Appendix). laboratory evidence of immunodeficiency. Some Bone marrow aspirates were available from of the children and adults had a surprisingly mild two members of Family C (Patient C1, at the age clinical course despite low concentrations of se- of 45 years, and Patient C2, at the age of 16 rum immunoglobulins, decreased numbers of years), both of whom had a CD19+ B-cell count B cells, and inadequate treatment with gamma of 1% or less in peripheral blood. Normal num- globulin. One adult patient (D1) and several chil-

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A B CD34 CD138 Control C2

68.7% 9.0% 7.7% 2.2%

Control

0.0% 22.3% 0.0% 90.1% CD19 CD34

C1 2.4% 9.2% 25.2% 1.0%

20.3% 22.3% 40.0% 12.1% TdT C2 CD19

20.3% 25.2%

CVID

38.1% 41.6% 40.0% 34.8% Lambda

Kappa

Figure 3. Immunohistochemical Analysis of Bone Marrow–Biopsy Specimens and Flow-Cytometric Analysis of Bone Marrow Aspirates. Panel A shows immunohistochemical staining for CD34+ cells (hematopoietic stem cells) and CD138+ cells (plasma cells) in bone marrow–biopsy specimens from a healthy control, Patients C1 and C2, and a patient with typical common variable immunodeficiency (CVID). CD34+ and CD138+ cells are present in the control and in Patients C1 and C2 (red staining); CD34+ cells are present but CD138+ cells are absent in the patient with typical CVID. Panel B shows the results of flow-cytometric analysis of bone marrow aspirates from a healthy control and Patient C2. A total of 500,000 events were analyzed for both persons. A gate was used that collected all cells that were positive for CD34, CD19, or both (top and middle graphs) and cells that were negative for CD34 but positive for CD19 (bottom graph). The graph at the top shows a marked decrease in CD34+CD19+ pro-B cells in Patient C2 as compared with the control. The middle graph shows that precursors of pro-B cells that express CD34+ and cTdT+ (cytoplasmic terminal deoxynucleotidyl transferase) but lack CD19 are also decreased. The bottom graph shows that the relative numbers of pre-B cells (CD34−CD19+sIg− [surface immunoglobulin]) to immature B cells (CD34−CD19+sIg+), as documented by the expression of surface kappa or surface lambda, in the patient is similar to that in the control, although the total number of cells in both populations is decreased in the patient.

dren with IKAROS deficiency were asymptom- selection against these mutations in evolution. atic, which suggests that penetrance is incom- In addition, IKZF1 ranks second to RPSA (and plete (Fig. S12 in the Supplementary Appendix); higher than CTLA4 and GATA2) in terms of nega- however, clinical manifestations may appear as tive selection among the 15 known autosomal late as the sixth decade of life. Nevertheless, dominant primary immunodeficiencies caused several observations indicate that the clinical by haploinsufficiency.24-27 penetrance of IKZF1 mutations is very high. The The mechanism of dominance in Family F is occurrence of de novo mutations in two of the most likely haploinsufficiency on the basis of six kindreds and the strong purifying (or nega- the complete deletion of one of the IKZF1 alleles; tive) selection for IKZF1 indicate that there is however, the other genes included in the dele-

1040 n engl j med 374;11 nejm.org March 17, 2016 The New England Journal of Medicine Downloaded from nejm.org at NIH Libary on March 17, 2016. For personal use only. No other uses without permission. Copyright © 2016 Massachusetts Medical Society. All rights reserved. B-Cell Loss with Heterozygous Mutations in IKAROS tion may contribute to the phenotype. The situ- instances, modifying genetic factors, environ- ation is also complex in Families A, B, C, and D, mental or infectious exposures, or even the age in which the mutant allele results in a stable at which environmental or infectious exposures protein that is able to dimerize with wild-type occur may influence the clinical features.7 IKAROS but is unable to bind target DNA (Fig. S. pneumoniae is a common infectious organ- S6 and S7, respectively, in the Supplementary ism in patients with hypogammaglobulinemia, Appendix). On the basis of previously reported poor antibody function, and low B-cell num- data,15 IKAROS protein lacking zinc fingers 1, 2, bers30; it also was common in our patients who and 3, as in Family E, behaves similarly to the had mutations in IKZF1. There was no evidence missense proteins in Families A through D. Be- of increased susceptibility to viral or fungal in- cause IKAROS functions as a dimer, one might fections in any of these patients. Thus, like expect these proteins to act in a dominant nega- other types of CVID, IKAROS deficiency should tive fashion, similar to that recently reported for be viewed as a “predominantly antibody defi- patients with heterozygous mutations in the ciency” on the basis of the International Union DNA-binding domain of E47,28 another tran- of Immunological Societies classification of pri- scription factor required for B-cell development. mary immunodeficiency diseases.4 However, both EMSAs and confocal microscopy There are several murine models of Ikaros showed that the mutant proteins do not inhibit deficiency.20,31-33 The most severe phenotype is the DNA binding of wild-type IKAROS (Fig. S7). associated with an amino acid substitution Furthermore, the clinical and laboratory find- (H191R) in zinc finger 3 of the DNA-binding ings in the patients with defects restricted to the domain.20 Homozygosity for this mutation is DNA-binding domain of IKAROS are similar to lethal to the embryo because of anemia. Hetero- those seen in the patients with complete dele- zygous mice have normal numbers of peripheral- tion of the gene. blood B cells but reduced numbers of B-cell In contrast, the patients with mutations in precursors in the bone marrow. Heterozygous the DNA-binding domain of IKAROS had a more Ikzf1H191R mice or Ikzf1del(ex3-ex4) mice (mice with a striking increase in the number of CD8+ cells mutation that deletes three of the N-terminal than did the patients with complete gene dele- zinc fingers) have a high incidence of T-cell leu- tion. A recent study suggests that murine CD8+ kemia.20,34 In contrast, somatic loss-of-function cells that are haploinsufficient for Ikaros pro- and dominant negative mutations in IKZF1 are duce increased amounts of autocrine interleu- predominantly associated with B-cell leukemias kin-2 when stimulated.29 This may explain the in humans.35-38 Furthermore, polymorphic vari- increased numbers of CD8+ cells, suggesting a ants in IKZF1 that decrease the number of tran- relatively T-cell–specific dominant negative effect scripts constitute a risk factor for both pediatric in patients with mutations resulting in stable and adult B-cell ALL.39 In our study, typical child- proteins that fail to bind DNA. hood B-cell ALL developed in 2 of the 29 pa- Y210C, a different heterozygous de novo mu- tients with germline heterozygous mutations in tation in IKZF1, was reported in a premature IKZF1. Thus, the penetrance for acquired hypo- infant with pancytopenia and complete loss of gammaglobulinemia was higher than that for B cells and natural killer cells.21 The pathophys- leukemia. This finding is compatible with data iological features and severity of the phenotype suggesting that mutations in IKZF1 are not the in this infant cannot be readily explained but initiating event in leukemias.38 Leukemias associ- may have been due to defects in functions of ated with somatic mutations in IKZF1 lead to over- IKAROS that are unrelated to DNA binding or to expression of hematopoietic stem-cell genes.37,40 additional genetic or nongenetic modifying fac- Overexpression of stem-cell genes in patients tors. One of the more striking features of re- with germline heterozygous mutations in IKZF1 cently identified autosomal dominant genetic may result in stem-cell exhaustion (i.e., excessive defects is the marked heterogeneity in phenotype proliferation of stem cells, resulting in prema- caused by mutations in the same gene. In some ture senescence or anergy). This in turn could instances, this heterogeneity results from the fact cause an acceleration of the normal decrease in that different mutations in the same gene have B-cell production that occurs with age.41 very different effects on protein function. In other We speculate that the relatively mild clinical

n engl j med 374;11 nejm.org March 17, 2016 1041 The New England Journal of Medicine Downloaded from nejm.org at NIH Libary on March 17, 2016. For personal use only. No other uses without permission. Copyright © 2016 Massachusetts Medical Society. All rights reserved. The new england journal of medicine

phenotype in the patients with heterozygous und Julia Bangerter-Rhyner-Stiftung, and Fondazione Ettore e mutations in IKZF1 may be due to the production Valeria Rossi (all to Dr. Reichenbach); National Jewish Health; NIH (AI-101093, AI-086037, AI-48693, and T32-GM007280, to of functional antibodies early in life, with the Dr. Cunningham-Rundles; AI-094004, to Drs. Voelkerding, Hill, generation of some CD27+ memory B cells and and Kumánovics; AI-104857, to Dr. Conley; and AI-061093 and the persistence of long-lived plasma cells in the TR-000043, to Drs. Boisson and Casanova); the National Human Genome Research Institute (U54HG006542, to the Baylor–Hop- bone marrow. The presence of these CD27+ kins Center for Mendelian Genomics); and Rockefeller University, memory B cells and plasma cells, in the context INSERM, and Paris Descartes University (all to Dr. Casanova). of very low numbers of peripheral-blood B cells, Disclosure forms provided by the authors are available with the full text of this article at NEJM.org. reversed CD4:CD8 ratios, and a family history We thank the patients and their families for their contribu- compatible with autosomal dominant inheri- tions to the study; Kimberley Lemberg, Joseph Monsale, Shakun- tance, distinguishes patients with IKAROS mu- tala Rampertaap, Jennifer Stoddard, Anahita Agharahimi, and Ashleigh Hussey for their technical and nursing assistance at the tations from other patients with CVID. National Institutes of Health; Jeannette Rejali and Ashley Bun- The content of this article does not necessarily reflect the ker for organizing and collecting the clinical samples at the views or policies of the Department of Health and Human Ser- University of Utah and ARUP Laboratories; Dr. Marko Radic for vices, nor does mention of trade names, commercial products, help with confocal microscopy; Drs. Rodney Miles and Joshua or organizations imply endorsement by the U.S. government. Schiffman for help with the analysis of the leukemia samples Supported in part by the Intramural Research Program of the from Patient F12; Drs. David Bahler, Sergey Preobrazhensky, and National Institutes of Health (NIH) Clinical Center and the Na- Tiffany J. Whitney for helping with flow cytometry; Dr. Jeremy P. tional Institute of Allergy and Infectious Diseases and by grants Crim, Leslie Rowe, and Jack Stephens for help with array-based from the Gebert Rüf Stiftung program Rare Diseases — New CGH, multiplex ligation-dependent probe amplification, and Approaches (GRS-046/10), the European Union’s Seventh Frame- Sanger sequencing; Elliot Kramer for help with the TdT studies; work Program for Research and Technological Development Pubudu Saneth Samarakoon and Robert Lyle for their assistance (EU-FP7 CELL-PID HEALTH-2010-261387 and EU-FP7 NET4CGD), with the genetic testing in the Norwegian family; and Laurent the Zurich Center for Integrative Human Physiology, Gottfried Abel for his contribution to the penetrance studies.

Appendix The authors’ full names and academic degrees are as follows: Hye Sun Kuehn, Ph.D., Bertrand Boisson, Ph.D., Charlotte Cunningham‑ Rundles, M.D., Ph.D., Janine Reichenbach, M.D., Asbjørg Stray‑Pedersen, M.D., Ph.D., Erwin W. Gelfand, M.D., Patrick Maffucci, B.A., Keith R. Pierce, B.S., Jordan K. Abbott, M.D., Karl V. Voelkerding, M.D., Sarah T. South, Ph.D., Nancy H. Augustine, B.A., Jeana S. Bush, M.D., William K. Dolen, M.D., Betty B. Wray, M.D., Yuval Itan, Ph.D., Aurelie Cobat, M.D., Ph.D., Hanne Sørmo Sorte, M.S., Sundar Ganesan, Ph.D., Seraina Prader, M.D., Thomas B. Martins, M.S., Monica G. Lawrence, M.D., Jordan S. Orange, M.D., Ph.D., Katherine R. Calvo, M.D., Ph.D., Julie E. Niemela, M.S., Jean‑Laurent Casanova, M.D., Ph.D., Thomas A. Fleisher, M.D., Harry R. Hill, M.D., Attila Kumánovics, M.D., Mary Ellen Conley, M.D., and Sergio D. Rosenzweig, M.D., Ph.D. The authors’ affiliations are as follows: the Department of Laboratory Medicine, National Institutes of Health Clinical Center (H.S.K., K.R.C., J.E.N., T.A.F., S.D.R.), and the Primary Immunodeficiency Clinic (S.D.R.) and Biological Imaging Section, Research Technolo- gies Branch (S.G.), National Institute of Allergy and Infectious Diseases, Bethesda, MD; St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller University (B.B., Y.I., A.C., J.-L.C., M.E.C.), Howard Hughes Medical Institute (J.-L.C.), and the Depart- ment of Medicine and the Immunology Institute, Icahn School of Medicine at Mount Sinai (C.C.-R., P.M.) — all in New York; the Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM Unité 1163 and Paris Descartes University, Imagine Institute, Paris (A.C., J.-L.C.); the Division of Immunology, University Children’s Hospital Zurich (J.R., S.P.), Children’s Research Cen- ter (J.R., S.P.), and University of Zurich (J.R.) — all in Zurich, Switzerland; the Center for Human Immunobiology, Texas Children’s Hospital (A.S.-P., J.S.O.), and the Departments of Pediatrics (A.S.-P., J.S.O.) and Molecular and Human Genetics (A.S.-P.), Baylor–Hop- kins Center for Mendelian Genomics, Baylor College of Medicine, Houston; the Norwegian Unit for National Newborn Screening (A.S.-P.) and the Department of Medical Genetics (H.S.S.), Oslo University Hospital, Oslo; University of Tennessee College of Medicine, Memphis (K.R.P.); the Division of Allergy and Immunology, Department of Pediatrics, National Jewish Health, Denver (E.W.G., J.K.A.); the Departments of Pathology (K.V.V., S.T.S., N.H.A., T.B.M., H.R.H., A.K.) and Pediatrics and Medicine (H.R.H.), University of Utah School of Medicine and ARUP (Associated Regional and University Pathologists) Institute for Clinical and Experimental Pathology, ARUP Laboratories (T.B.M.) — both in Salt Lake City; the Division of Allergy–Immunology and Pediatric Rheumatology, Department of Pediatrics, Medical College of Georgia, Augusta University, Augusta (J.S.B., W.K.D., B.B.W.); and the Division of Asthma, Allergy, and Immunology, Department of Medicine, University of Virginia, Charlottesville (M.G.L.).

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Georgopoulos K, Moore DD, Derfler Mol Immunol 2007;44: 2173-83. Deletion of IKZF1 and prognosis in acute B. Ikaros, an early lymphoid-specific 24. Spinner MA, Sanchez LA, Hsu AP, et lymphoblastic leukemia. N Engl J Med transcription factor and a putative media- al. GATA2 deficiency: a protean disorder 2009;360: 470-80. tor for T cell commitment. Science 1992; of hematopoiesis, lymphatics, and immu- 37. Iacobucci I, Iraci N, Messina M, et al. 258:808-12. nity. Blood 2014;123: 809-21. IKAROS deletions dictate a unique gene 14. Avitahl N, Winandy S, Friedrich C, 25. Rieux-Laucat F, Casanova JL. Immu- expression signature in patients with adult Jones B, Ge Y, Georgopoulos K. Ikaros nology: autoimmunity by haploinsuffi- B-cell acute lymphoblastic leukemia. PLoS sets thresholds for T cell activation and ciency. Science 2014;345: 1560-1. One 2012;7(7):e40934. regulates chromosome propagation. Im- 26. Kuehn HS, Ouyang W, Lo B, et al. Im- 38. Kastner P, Dupuis A, Gaub MP, Her- munity 1999;10:333-43. mune dysregulation in human subjects brecht R, Lutz P, Chan S. Function of 15. Cobb BS, Morales-Alcelay S, Kleiger G, with heterozygous germline mutations in Ikaros as a tumor suppressor in B cell Brown KE, Fisher AG, Smale ST. Targeting CTLA4. Science 2014;345: 1623-7. acute lymphoblastic leukemia. Am J Blood of Ikaros to pericentromeric heterochro- 27. Bolze A, Mahlaoui N, Byun M, et al. Res 2013;3: 1-13. matin by direct DNA binding. Genes Dev Ribosomal protein SA haploinsufficiency 39. Papaemmanuil E, Hosking FJ, Vijaya 2000;14:2146-60. in humans with isolated congenital asple- krishnan J, et al. Loci on 7p12.2, 10q21.2 16. Brown KE, Guest SS, Smale ST, Hahm nia. Science 2013;340:976-8. and 14q11.2 are associated with risk of K, Merkenschlager M, Fisher AG. Associa- 28. Boisson B, Wang YD, Bosompem A, childhood acute lymphoblastic leukemia. tion of transcriptionally silent genes with et al. A recurrent dominant negative E47 Nat Genet 2009;41: 1006-10. Ikaros complexes at centromeric hetero- mutation causes agammaglobulinemia 40. Tonnelle C, Imbert MC, Sainty D, chromatin. Cell 1997;91: 845-54. and BCR(-) B cells. J Clin Invest 2013;123: Granjeaud S, N’Guyen C, Chabannon C. 17. John LB, Ward AC. The Ikaros gene 4781-5. Overexpression of dominant-negative family: transcriptional regulators of hema- 29. O’Brien S, Thomas RM, Wertheim Ikaros 6 protein is restricted to a subset topoiesis and immunity. Mol Immunol GB, Zhang F, Shen H, Wells AD. Ikaros of B common adult acute lymphoblastic 2011;48: 1272-8. imposes a barrier to CD8+ T cell differen- leukemias that express high levels of the 18. Sun L, Liu A, Georgopoulos K. Zinc tiation by restricting autocrine IL-2 pro- CD34 antigen. Hematol J 2003;4:104-9. finger-mediated protein interactions mod- duction. J Immunol 2014;192:5118-29. 41. Nuñez C, Nishimoto N, Gartland GL, ulate Ikaros activity, a molecular control 30. Winkelstein JA, Marino MC, Leder- et al. B cells are generated throughout life of lymphocyte development. EMBO J 1996; man HM, et al. X-linked agammaglobu- in humans. J Immunol 1996;156:866-72. 15:5358-69. linemia: report on a United States registry Copyright © 2016 Massachusetts Medical Society.

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n engl j med 374;11 nejm.org March 17, 2016 1043 The New England Journal of Medicine Downloaded from nejm.org at NIH Libary on March 17, 2016. For personal use only. No other uses without permission. Copyright © 2016 Massachusetts Medical Society. All rights reserved. Supplementary Appendix

This appendix has been provided by the authors to give readers additional information about their work.

Supplement to: Kuehn HS, Boisson B, Cunningham-Rundles C, et al. Loss of B cells in patients with heterozygous mutations in IKAROS. N Engl J Med 2016;374:1032-43. DOI: 10.1056/NEJMoa1512234 Supplementary Appendix

Supplement to: Kuehn HS, Boisson B, et al. Loss of B Cells in Patients with

Heterozygous Mutations in IKAROS

Contents

Authors and affiliations page 2

Supplementary Clinical Information page 3

Supplementary Materials and Methods page 11

Supplementary Figures S1-S12 page 20

Supplementary Tables S1-S3 page 33

References page 37

1 Loss of B Cells in Patients with Heterozygous Mutations in IKAROS

Hye Sun Kuehn*, Ph.D., Bertrand Boisson*, Ph.D., Charlotte Cunningham-Rundles, M.D., Ph.D., Janine Reichenbach, M.D., Asbjørg Stray-Pedersen, M.D., Ph.D., Erwin W. Gelfand, M.D., Patrick Maffucci, B.A., Keith R. Pierce, B.S., Jordan K. Abbott, M.D., Karl V. Voelkerding, M.D., Sarah T. South, Ph.D., Nancy H. Augustine, B.A., Jeana S. Bush, M.D., William K. Dolen, M.D., Betty B. Wray, M.D., Yuval Itan, Ph.D., Aurelie Cobat, M.D., Ph.D., Hanne Sørmo Sorte, M.S., Sundar Ganesan, Ph.D., Seraina Prader, M.D., Thomas B. Martins, M.S., Monica G. Lawrence, M.D., Jordan S. Orange, M.D., Ph.D., Katherine R. Calvo, M.D., Ph.D., Julie E. Niemela, M.S., Jean-Laurent Casanova, M.D., Ph.D., Thomas A. Fleisher, M.D., Harry R. Hill, M.D., Attila Kumánovics, M.D., Mary Ellen Conley, M.D.,#§, Sergio D. Rosenzweig, M.D., Ph.D.#

* Drs. Kuehn and Boisson contributed equally, # Drs. Conley and Rosenzweig contributed equally, § Corresponding author

Department of Laboratory Medicine, NIHCC (H.S.K., J.E.N., K.R.C., T.A.F., S.D.R.) and Primary Immunodeficiency Clinic, NIAID (S.D.R), National Institutes of Health, Bethesda, MD; St. Giles Laboratory of Human Genetics of Infectious Diseases, The Rockefeller University, New York City, NY (B.B., Y.I., A.C., J.-L.C., M.E.C.); Laboratory of Human Genetics of Infectious Diseases, Necker Branch, Inserm U1163 and Paris Descartes University, Imagine Institute, Paris, France (A.C., J.-L. C.), and Howard Hughes Medical Institute, New York, NY, and Laboratory of Human Genetics of Infectious Diseases, Necker Branch, Inserm U1163 and Pediatric Hematology- Immunology Unit, Necker Hospital for Sick Children, Paris, France, and Paris Descartes University, Imagine Institute, Paris, France (J.-L.C); Department of Medicine and the Immunology Institute, Icahn School of Medicine at Mount Sinai, NY (C.C.-R., P.M.); Division of Immunology, University Children’s Hospital Zurich and Children’s Research Center (J.R., S.P.) and University Zurich, Switzerland (J.R); Center for Human Immunobiology of Texas Children’s Hospital/Department of Pediatrics, Baylor College of Medicine, Houston, TX (A.S.-P., J.S.O), Baylor-Hopkins Center for Mendelian Genomics of the Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX and Norwegian Unit for National Newborn Screening, Oslo University Hospital, Oslo, Norway (A.S.-P.); Division of Allergy and Immunology, Department of Pediatrics, National Jewish Health, Denver, CO (J.K.A., E.W.G.); University of Tennessee College of Medicine, Memphis, TN (K.R.P.); Department of Pathology (K.V.V., S.T.S., N.H.A., T.B.M, H.R.H., A.K.) and Department of Pediatrics and Medicine (H.R.H), University of Utah School of Medicine and ARUP Institute for Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, UT; Division of Allergy-Immunology and Pediatric Rheumatology, Department of Pediatrics, Medical College of Georgia at Georgia Regents University, Augusta, GA (J.S.B.,W.K.D., B.B.W.); Department of Medical Genetics, Oslo University Hospital and University of Oslo, Norway (H.S.S.); Biological Imaging Section, Research Technologies Branch, NIAID, NIH (S.G.), Division of Asthma, Allergy and Immunology, Department of Medicine, University of Virginia, Charlottesville, VA (M.G.L.)

2 Supplementary Clinical Information

Family A

A1 is a Hispanic female who was evaluated for immunodeficiency at 10 years of age because of a history of recurrent respiratory tract infections and a recent episode of pneumococcal meningitis. At 2 weeks of age she was hospitalized with fever and a seizure. Starting at 1 year of age, she had 10-15 episodes of bronchitis or otitis per year. In the year preceding evaluation, she had 2 episodes of pneumonia, one requiring hospitalization. One month preceding evaluation she was hospitalized with meningoencephalitis. Spinal fluid was purulent and cultures grew S. pneumoniae. Sequelae included temporary right-sided hearing loss and mild intellectual impairment. Laboratory evaluation at 10 years of age revealed mild thrombocytopenia, hypogammaglobulinemia, absent antibodies to vaccine antigens and markedly reduced numbers of B cells (Table 1). She was started on gammaglobulin replacement but compliance was intermittent. She was hospitalized at 20 years of age for pneumonia and treated as an outpatient for pneumonia several times. At 24 years of age, high resolution CT demonstrated mild airway disease but no bronchiectasis or nodules. A2, the son of A1, was evaluated for immunodeficiency at 6 years of age because of recurrent upper respiratory infections, acute otitis media and sinusitis. At age 3 years he developed immune thrombocytopenia (ITP; platelets

46,000/µl). He did not respond to steroid therapy but Anti-D Ig therapy controlled his ITP for 2 years. At 5 years of age, he presented with a second episode of ITP and was successfully treated with 4 doses of rituximab, (platelets 388,000/µL). Before rituximab treatment, his serum IgG concentration was low at 431mg/dL (IgA, IgM and B cells were not determined) and he was placed on gammaglobulin replacement. At age 6 years his IgG was normal, IgA was low as was

IgM. B cells in peripheral blood were also very low (CD19 0.3%, 12/µL); a residual effect of

3 rituximab treatment could not be completely ruled out. A2 has done well, with no hospitalizations and no recurrences of ITP for the last 4 years.

Family B

B5 is a Swiss female who was evaluated for immunodeficiency at 3 years of age because of recurrent otitis, cough, dental caries and apthous ulcers (Table 1). Serum immunoglobulins were reported to be decreased and antibody titers to pneumococcus were low despite repeated vaccinations. She continued to have frequent episodes of otitis and bronchitis and was started on gammaglobulin therapy at 15 years of age, which resulted in a decrease in the incidence of otitis and bronchitis. At 16 years of age she had an episode of diarrhea due to Giardia lamblia, which responded to treatment. She has had recurrent herpes labialis since age 20 and she had an episode of diarrhea associated with

Blastocystis hominis at 23 years of age. B6, the brother of B5 was recognized to have hypogammaglobulinemia at 1 year of age after multiple episodes of otitis, bronchitis and apthous ulcers (Table 1). He was hospitalized for pneumonia at 2 and 6 years of age, and again at 9 years of age, when he had pneumococcal sepsis and pneumonia. He was started on gammaglobulin therapy at 9 years of age. He has done well since then but continues to have recurrent apthous ulcers. B1, the paternal grandfather of B5 and B6 had a long history of recurrent respiratory tract infections and was sent to a TB sanitarium at 12 years of age because of a chronic cough. He had a cerebral hemorrhage at 30 years of age and a cerebral infarction at 52 years of age. He was evaluated for immunodeficiency at

71 years of age and given the diagnosis of CVID (Table 1). He was treated with gammaglobulin for less than a year and died of pneumonia at 74 years of age. The DNA

4 sample from this individual was obtained at the time of his diagnosis with CVID. B2, B3,

B4 and B7 were recognized to have immunodeficiency during the family studies.

B2, the son of B1 and the father of B5 and B6, had recurrent otitis and bronchitis as a small child. He had a severe pneumonia requiring partial pneumonectomy at 6 years of age (Table 1). He had the onset of recurrent purulent skin infections at 20 years of age and he continues to have recurrent bronchitis. B3, the daughter of B1, had a basal skull fracture as the result of a scooter accident at 6 years of age and developed meningitis with hearing loss. She had a second episode of meningitis at 10 years of age but has done well otherwise. B4, the daughter of B1, has had recurrent otitis and bronchitis since early childhood. B7, the daughter of B3, developed B cell acute lymphoblastic leukemia (ALL at 3 years of age and died of relapsed ALL at 5 years of age. The leukemia was described as having strong expression of CD19 and CD10, weak expression of CD20 and CD22 with some cells positive for CD34 and HLA-DR. Five of 16 metaphases were positive for an interstitial deletion of the region around Chromosome 13q.14. The DNA sample from

B7 was obtained from bone marrow taken when she was diagnosed as having ALL.

Family C

C1 is a European-American who was evaluated for immunodeficiency at 32 years of age. She was reportedly a healthy child, although she had 2 episodes of “walking pneumonia” at less than

13 years of age. Her spleen was removed after an automobile accident when she was 27 years old. Three years later, she was hospitalized with pneumococcal meningitis, with full recovery after adequate antibiotic treatment. At 32 years of age, she had two separate episodes of pneumococcal sepsis requiring admissions to the ICU. After the second septic episode, immune evaluation demonstrated hypogammaglobulinemia affecting IgG, IgA and IgM (Table 1) and

5 “very few B cells in peripheral blood”. She was started on gammaglobulin replacement.

Prophylactic antibiotics were added when she was 40 years old and she has not had major infections since that time.

Patient C2 is the eldest daughter of C1. She was a healthy child but given her mother’s history was immunologically evaluated at age 9 months (Table 1). Because her serum immunoglobulins were below normal for age with low B cell numbers, she was placed on gammaglobulin replacement. By 4 years of age her B cells dropped below 2%. Because she remained asymptomatic, a trial off gammaglobulin replacement was attempted at age 5 years. The patient maintained adequate IgG and IgA, but not IgM levels for 8 years, but was unable to mount protective responses to tetanus toxoid, diphtheria toxoid, varicella and rubella after proper and repetitive immunizations. During the same period her B cell numbers continued to decline. At age 13 years of age, after one episode of pneumonia, the patient was restarted on gammaglobulin replacement with good response. Patient C3 is the second daughter of C1. During her neonatal period she had a short NICU admission because of mild bronchiolitis; she also developed 4 episodes of acute otitis media during her first 18 months of life. At that age, per her mother’s request, she had her first immune evaluation showing slightly low IgG and IgM, while IgA was normal (Table 1). B cells were present although low for age and protective tetanus toxoid and diphtheria toxoid titers were also detected. For the following 3 years she had repeated, although not severe, upper respiratory infections. At 4 years of age she had pneumonia and was placed on gammaglobulin replacement. Previous to initiating replacement, her laboratory tests showed hypogammaglobulinemia, no response to recall immunizations with tetanus toxoid and diphtheria toxoid, and B cells still present in peripheral blood although at low number per age.

During follow up, B cells continued to decline reaching <2% by the age of 11y. On gammaglobulin replacement the patient has had no recurrence of infections for a follow up

6 period of almost 10 years.

Family D

D2 is a European-American who was reportedly well until she developed pneumonia at 9 years of age. At that time she had severe hypogammaglobulinemia of the three major isotypes and her medical records indicate that she had “normal B cells” (Table 1). No further studies or specific prophylactic therapy were initiated. She was started on gammaglobulin replacement at 12 years of age after a second pneumonia; but discontinued therapy at 16 years of age. At 23 years of age, chronic cough prompted a new evaluation that revealed severe hypogammaglobulinemia and markedly decreased B cells. For the last 2 years she has been on IgG replacement therapy and has not had significant infections. D1 is the mother of D2. She was identified during family studies analyzing the segregation of mutation c.551G>A, p.R184Q in family D. She is a self- reported healthy 50-year-old woman. Her immune evaluation showed mild hypogammaglobulinemia with normal B cells.

Family E

E1 is a Norwegian male who was treated with penicillin in the first month of life for facial skin infections. He had the onset of recurrent respiratory infections in early childhood with multiple episodes of pneumonia, two of which were associated with sepsis (blood cultures positive for S. pneumoniae and H. influenzae, respectively). At

3 years of age he developed a S. pneumonia right hip osteoarthritis. Immune evaluation at 4 years of age revealed severe hypogammaglobulinemia and low B cells (Table 1) and he was started on gammaglobulin replacement therapy. This significantly reduced the number and severity of his infections He continues to have

7 sporadic upper respiratory infections and diarrhea but he is otherwise well. His son,

E2, is 6 years old and reportedly healthy. He was identified during family studies analyzing the segregation of the deletion in family E.

Family F

F2 is a European-American who was evaluated for immunodeficiency at 29 years of age because of recurrent pneumonias and severe sinusitis. Her serum immunoglobulins at that time were undetectable (Table 1). She was started on suboptimal doses of intramuscular gammaglobulin

(2 mL/month) but continued to have severe sinus infections and multiple episodes of pneumonia.

At 52 years of age she had surgery for maxillary sinusitis and was treated with multiple antibiotics. Intravenous gammaglobulin was recommended but she was unable to secure funding for this. Her serum immunoglobulins at that time were undetectable (Table 1). At 56 years of age, she suffered pneumonias and had increasing diarrhea and weight loss. C. difficile toxin was discovered in her stool and she was treated with vancomycin. At 57 years of age, she had not had gammaglobulin therapy for the previous 4 years, and her IgG, IgA and IgM were still undetectable. After vaccination, she had no detectable antibody to 14 pneumococcal serotypes.

X-rays obtained at that time revealed clear lung fields and normal cardiac size. She was placed on 400 mg/kg of IV gammaglobulin replacement every month and dramatically improved.

During the following years, and while on intermittent gammaglobulin replacement, she had 2-4 sinus infections per year, but suffered from no invasive pneumonias, chronic diarrhea or other significant problems. Currently at 70 years of age, she is on 30 grams of IV gammaglobulin replacement every four weeks and has had occasional sinusitis and one episode of vaginitis but otherwise is doing well.

F1, the sister of F2, was first investigated as part of the evaluation of F2's extended family. At

8 that time, she was 57 years. Her IgG and IgM were low with normal IgA (Table 1). She had very low B cells, normal CD3 cells, and normal CD4/8 ratio. She has refused gammaglobulin replacement and was doing well except for 2-3 sinus infections per year with no other infections.

Patient F3, the 37-year-old son of F1, suffered frequent upper respiratory infections, as a child but had no ear infections or chronic diarrhea. At the 21 years of age, his serum immunoglobulins were extremely low, B cells were <2% while T and NK cells were normal (Table 1). He was started on IV gammaglobulin replacement at 400 mg/kg. At his present age of 40 years he is on regular gammaglobulin replacement, he has 1-2 episodes of sinusitis per year but has no other infections.

Patient F4, the son of F1 suffered recurrent upper respiratory infections since he was a teenager with no documented pneumonias, hospitalizations, sinusitis, or diarrhea. At 24 years of age his serum immunoglobulins were extremely low (Table 1). He was reevaluated at 25 years of age and his immunoglobulins were still extremely low; B cells were also very low, while T and NK cells were normal. Chest X-rays and pulmonary function tests were normal. He was placed on

400 mg/kg of IV gammaglobulin replacement every 4 weeks and has done well except for periodic sinusitis and cough treated with oral antibiotics. His children all remain entirely asymptomatic.

Patient F5 was first studied at 28 years of age as part of the evaluation of the extended family

(Table 1). At that time she was essentially healthy, as she reported no pneumonias, sinusitis, draining ear infection, or chronic diarrhea. Her three major serum immunoglobulins were low.

She refused gammaglobulin replacement at that time. When contacted in 2015 at 31 years of age, she reported a history of a walking pneumonia, but no diarrhea, sinusitis, or other infections were reported. She still refuses gammaglobulin replacement therapy.

F6 is the son of F2. He had one pneumonia as a child and was noted to have low

9 immunoglobulins at 19 years of age. He has continued to have 3-5 episodes of sinusitis per year but he has not had significant gastrointestinal complications or additional pneumonias. His three major serum immunoglobulin levels were low when enrolled in the family study at 42 years of age (Table 1). He has refused gammaglobulin replacement.

F12, the son of F6, developed B-cell acute lymphocytic leukemia at 5 years of age after presenting with bruising and joint pain (Table 1). His white blood cell count at that time was

22,000/µl and the leukemia was characterized as being positive for CD19, CD10, CD22, CD24,

HLA-DR, CD34 and TdT with normal cytogenetics. The leukemia was successfully treated with chemotherapy. At 6 years of age, he developed fever and a left cheek nodule as well as left upper thigh nodules and had cultures positive for Mycobacterium chelonae. He was treated for several months with antibacterial therapy but continued to have additional skin nodules. Because of persistent neutropenia and bone marrow suppression, he was treated with bone marrow transplant from his HLA-matched brother, F13. At 13 years of age, his IgG, IgA, and IgM were low. He has not suffered serious infections since that time and is currently not on immunoglobulin therapy at 17 years of age.

F7, F8, F9, F10, F11, F13 have remained asymptomatic up until July of 2015 with none having pneumonia, sinusitis, or chronic diarrhea in spite of low levels of IgG, IgA, and/or IgM.

10 Supplementary Materials and Methods

Patients and samples

All patients or their guardians provided informed consent in accordance with the

Declaration of Helsinki under institutional review boards. Blood from healthy donors and patients was obtained under approved protocols, which also allow for the collection and use of patients’ family history and pedigrees for publication. All procedures were based on standard of care and established clinical guidelines were followed.

Whole exome sequencing and bioinformatics

For family A, B and D, massively parallel sequencing was performed. Genomic DNA extracted from the patient’s peripheral blood cells was sheared with a Covaris S2

Ultrasonicator. An adapter-ligated library was prepared with the Paired-End Sample Prep kit V1 (Illumina). Exome capture was performed with the SureSelect Human All Exon kit

(Agilent Technologies). Paired-end sequencing was performed on a HiSeq 2500, generating 100-base reads. We used BWA aligner 1 to align sequences with the human genome reference sequence (hg19 build). Downstream processing was carried out with the Genome analysis toolkit (GATK) 2, SAMtools 3 and Picard Tools

(http://picard.sourceforge.net). Substitution and indel calls were identified with a GATK

Unified Genotyper and a GATK Indel GenotyperV2, respectively. All calls with a read coverage ≤2x and a Phred-scaled SNP quality of ≤20 were filtered out. All the variants were annotated with the GATK Genomic Annotator. We used several genomic databases:

Exome Aggregation Consortium (ExAC), Cambridge, MA (exac.broadinstitute.org),

1000 genome project (www.1000genomes.org) and dbSNP

11 (www.ncbi.nlm.nih.gov/SNP/). CADD scores were calculated from

(http://cadd.gs.washington.edu/score).

For family C, genomic DNA was submitted to Otogenetics for whole-exome capture

[Agilent V4 (51 Mbp)] and next-generation sequencing on an Illumina HiSeq2000. The

DNAnexus interface was used for alignment of the Illumina reads to the hg19 human reference genome and for discovery and genotyping of single-nucleotide polymorphisms and insertions or deletions. To prioritize variant calls, we implemented the ANNOVAR functional annotation package, filtering the output by gene–amino acid annotation, functional prediction scores, nucleotide conservation scores and allele frequencies according to the NCBI dbSNP database (build 137), The 1000 Genomes Project (2012

April release) and the NHLBI GO Exome Sequencing Project (ESP6500). Only nonsynonymous novel variants or variants with population frequencies less than 1% were considered further. Exomic variants were finally prioritized by clinical correlation and analysis of protein structure and relevant protein motifs.

Sanger sequencing

Selected next-generation sequencing results were confirmed by Sanger sequencing.

Specifically, IKZF1 coding exons were amplified by PCR with exon-specific oligonucleotide primers and GoTaq Hot Start Polymerase (Promega) (primer sequences and cycling conditions available upon request). Purified PCR products were directly sequenced using BigDye Terminators (version 1.1) and were analyzed on a 3130xL

Genetic Analyzer (Applied Biosystems).

Detection of intragenic deletion on IKZF1

12 Genomic DNA from the proband in family E was analyzed by whole exome sequencing as previously described.4 Copy number variants were predicted from the whole-exome file using ExCopyDepth and the predicted deletion affecting IKZF1 was confirmed using a custom high-resolution exon-tiling array, exaCGH.4 Probes for aCGH were prepared from genomic DNA according to Agilent protocol v.6.3. Agilent Genomic Workbench

(v7.0) was used for analysis of exaCGH. PCR to identify deletion breakpoints was performed using AmpliTaq Gold® 360 (Applied Biosystems) and primers spanning the region of the deletion. The purified, amplified product was further Sanger sequenced using BigDye terminators (version 3.1) and analyzed on a 3730 sequencer (Applied

Biosystems) to yield the exact breakpoint of the deletion (primers available upon request). 5-8

Detection of deletion on Chromosome 7

4.7 Mb deletion on the short arm of chromosome 7 (7p12.3-p12.1; shown in red in Figure

1B) testing was done by array comparative genomic hybridization (aCGH) and/or by

Multiplex Ligation-Dependent Probe Amplification (MLPA) from MRC Holland, and

Molecular Inversion Probe (MIP) panel. Among the 11 genes included in the 7p12.3- p12.1; ABCA13, CDC14C, VWC2, ZPBP, C7orf7, IKZF1, FIGNL1, DDC, GRB10,

COBL, POM121L12, only IKZF1 is selectively expressed in hematopoietic cells; moreover, IKAROS is expressed at all stage of lymphoid development, from HSC through to mature B and T cells, as well as in natural killer (NK) and thymic dendritic antigen presenting cells9-11.

Data analysis

13 The data from Family A were analyzed based on the assumption that the mutation in A1 was a de novo event. Sequence data from Families B and D were screened for mutations in IKZF1 based on the findings in Family A. The data from Family C and F were independently analyzed based on the hypothesis that the affected family members would share a rare or unreported genetic variant. The data from Family E were analyzed with the knowledge that mutations in IKZF1 had been identified in the other families.

Plasmid preparation

Human IKAROS family zinc finger 1 (IKAROS) (IKZF1; NM_006060) DNA were synthesized (GeneCopoeia and GeneScript) and subcloned into the mammalian expression vector pcDNA3-HA or CMV driven N-terminal DDK-Myc-flag tagged expression vectors (pCMV6-AC, Origene). Indicated mutants for the IKZF1 were generated based on the site directed mutagenesis protocol using Quick Change II kit

(Agilent) or AccuPrime Pfx DNA Polymerase, followed by DpnI treatment (Life technologies).

Confocal microscopy

NIH3T3 cells (0.8-1×105) were seeded onto cover slips in 6 well plates. The next day, cells were transfected with indicated plasmid using Turbofect (Thermo Scientific) or

Effectene (Qiagen) according to the manufacturer’s instruction. The cells were washed twice in PBS, fixed for 10 min in 4% paraformaldehyde and permeablized for 15 min in

0.1% Triton X-100 in PBS at RT. The cells were then incubated for 30 min in blocking buffer (PBS with 10% FBS and 0.1% Triton X-100) then incubated for 2 hours with mouse anti-HA monoclonal antibody (Covance), and/or rabbit anti-Flag polyclonal

14 affinity antibody (Sigma)). The cells were then washed with PBS and incubated for 1 hour with either Alexa Fluore 488 (green color) or Alexa Fluore 568 (red color)- conjugated secondary antibodies in blocking buffer. Cells were washed, mounted on slides using VECTASHIELD Mounting Medium (Vector Laboratories). Images were collected on a Leica SP5 inverted confocal microscope with a 63x oil immersion objective NA 1.4 (Leica Microsystems, Buffalo Grove, IL). Post processing and image analysis were performed using, Huygens (SVI imaging, Nederland) Imaris software

(Bitplane Inc., South Windsor, CT).

Western blots

Expression vectors (pCMV6-AC-Myc-DDK) were transfected into HEK-293 cells by

Lipofectamine LTX (Invitrogen). Nuclear extracts were obtained with NE-PER nuclear and cytoplasmic extraction reagent (Thermo Scientific) 19 hours after transfection.

Proteins were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with antibodies to anti-DDK (Flag. Origene), Lamin A/C

(Santa Cruz), or GAPDH (Santa Cruz) and visualized by chemiluminescence using species-specific HRP-linked antibodies (GE Healthcare).

EMSA assay

Nuclear extracts were incubated 10 min at room temperature in 20mM HEPES, 100mM

KCl, 10% glycerol with 100nM final 32P-labeled probes in presence of poly-dIdC

(100ng/µl). DNA-protein complexes were separated on 5% acrylamide gel with 0.5x

TBE. The probes were derived from the regulatory regions of IK-bs4

15 (Forward: 5’-gtcaTGACAGGGAATACACATTCCCAAAAGC, Reverse: 5’- gtcaGCTTTTGGGAATGTGTATTCCCTGTCA) or Hs-2mer-bs (Forward: 5’- gtcaATCCATAGGGAAAATGTTTTCCCTGACATC, Reverse: 5’- gtcaGATGTCAGGGAAAACATTTTCCCTATGGAT)12. Images have been obtained using the Amersham Biosciences Typhoon 9400 Variable Imager and quantify with

ImageQuant V.5.0.

Bone marrow staining

Bone marrow flow cytometric analysis was performed on a FACSCanto II flow cytometer and immunohistochemistry was performed on bone marrow core biopsy sections using the Ventana automated staining system as previously described.13 For the flow cytometric analysis, 500,000 total events were captured per bone marrow sample. Cells depicted are gated on all CD34+ cells and all CD19+ cells in bone marrow aspirate from a healthy control marrow (37,000 gated events) and patient

B2 (14,000 gated events). The following antibodies (clone) were used: CD34 (8G12),

CD19 (SJ25C1), TdT (E17-1519).

TdT mediated additions in VDJ recombination of Ig heavy chains

Total RNA was extracted from frozen peripheral blood lymphocytes from four healthy adult controls, 2 patients with mutations in BTK (R255X and L452P) and less than 0.2%

CD19+ B cells in the peripheral circulation, and B5 and C1 using an RNeasy Mini kit

(QIAGEN Inc). cDNA was prepared with random hexamers and SuperScript III First-

Strand (Life Technologies Corporation). The VDJ segment of immunoglobulin heavy chain transcripts belonging to the VH3 family was amplified using the following primers:

16 GGGGTACCATGGAGTTTGGGCTGAG (forward) and

GGAATTCTCACAGGAGACGAG (reverse). PCR conditions were 5 minutes at 95 oC, followed by 35 cycles of 95 oC melting for 45 seconds, 60 oC annealing for 30 seconds, and 72 oC extension for 30 seconds with a final extension of 5 minutes at 72 C. PCR products were cloned into a TA vector (TOPO TA Cloning Kit for Sequencing, Life

Technologies Corporation, Norwalk, CT, USA) and sequenced using M13 vector primers. The VDJ sequenced was analyzed using IMGT website tools which indicate the

N additions at both the V to D and D to J junctions. Between 8 and 32 unique sequences were analyzed for each control and for each patient.

Evaluation of cell proliferation

Human PBMCs were isolated by density-gradient centrifugation with Ficoll-Paque PLUS

(GE Healthcare), washed twice in phosphate buffered saline (PBS) and cultured in complete RPMI-1640 medium containing 10% FBS (FBS), Penicillin-Streptomycin-

Glutamine (Life technology) at 37°C in a humidified 5% CO2 incubator. PBMCs, either freshly isolated or thawed from liquid nitrogen−stored samples, were incubated with

CellTrace violet Cell Proliferation Kit (1µM; Invitrogen). After 20 minutes, 10 volumes of RPMI/10% fetal bovine serum (FBS) were added, and the cells were centrifuged and washed twice more with RPMI/10% FBS. A total of 1 × 105 cells were seeded into 96- well plates, stimulated with anti-CD3 antibody and anti-CD28 antibody (1 µg/mL each, eBioscience) as indicated in the figures, and cultured for 3 days. Cells were stained with fluorochrome-conjugated CD4 and CD8 antibodies (BD Biosciences) for 30 minutes at

4°C (dark). Cells were washed with PBS twice, and cells were acquired and analyzed by flow cytometry (Becton Dickinson FACSCanto II) and FlowJo software (TreeStar).

Apoptosis measurement

Total PBMCs were stimulated with anti-CD3 and CD28 (1µg/mL) for 3 days, and then cultured cells with IL-2 (10ng/ml). At day 11, cells were aliquoted in duplicate into 96 well plates and stimulated with Apo1.3 (1 µg/ml; ENZO Life Sciences) in the presence of protein A (1 µg/ml; Sigma), or 5µM staurosporine (Sigma).

After 20 h, cells were incubated with propidium iodide (Life Technology), and live cells

(PI negative) were counted by flow cytometry (BD FACSCanto II; BD Biosciences) by constant time acquisition. The percentage of cell loss was calculated according to the following formula: (number of live cells without APO-1-3 treatment − number of live cells with APO-1-3 treatment/number of live cells without APO-1-3 treatment) × 100.

IKAROS intracellular staining

For IKAROS intracellular staining (family A, B, and C), PBMCs were fixed and permeabilized after surface staining (CD3+ and CD19+) using FoxP3 staining kit

(eBioscience) following the manufacturer’s instructions, and then incubated with anti-

IKAROS antibodies (BD, clone R32-1149) on ice for 30 min. All samples were acquired and analyzed by flow cytometry (Becton Dickinson FACSCanto II) and FlowJo software

(TreeStar). For family D (bottom panel), goat polyclonal anti-IKAROS antibodies or normal goat serum (R&D Systems) were used in permeabilized peripheral blood mononuclear cells to measure IKAROS expression.

Immunoprecipitation

18 HEK293T cells were co-transfected with pcDNA3-HA- IKAROS (WT or mutants) and pFlag-CMV2- IKAROS WT using Effectene transfection reagent (Qiagen). Cell lysates were prepared 20h after transfection in lysis buffer (50mM Tris [pH 7.4], 150mM NaCl,

2mM EDTA, 0.5% Triton X-100, and halt protease and phosphatase inhibitor cocktail

[Thermo fisher scientific]). Total protein (500 µg) was incubated with rabbit anti-FLAG polyclonal affinity antibody (Sigma) or rabbit IgG (Cell signaling). After 2h incubation at

4 °C on a rotating wheel, 50 µl Protein A/G-agarose beads (Pierce) were added to each reaction and incubation was continued for another hour. Beads were washed three times with lysis buffer, resuspended in NuPAGE LDS sample buffer along with NuPAGE®

Sample Reducing Agent (Life technology), boiled and separated on a NuPAGE®

Novex® 4-12% Bis-Tris Protein Gels. Subsequent western blot analysis was performed with the mouse anti-HA monoclonal antibody (Covance), and rabbit anti-FLAG polyclonal affinity antibody.

Statistical Analysis

When indicated, data were analyzed using GraphPad Prism software (GraphPad). The differences were considered significant when P < 0.05.

19 Supplementary Figures

S1. Mutations and sequence conservation in IKZF1

S2. IKAROS protein structure and homology modeling

S3. In silico prediction of the damaging effects of the amino acid substitutions in

IKZF1.

S4. Selective pressure on genes underlying autosomal dominant primary

immunodeficiency by haploinsufficiency.

S5. IKAROS protein expression levels in T and B cells

S6. The stability of the mutant IKAROS proteins and their ability to dimerize

with wild type IKAROS

S7. DNA binding and pericentromeric targeting of the mutant IKAROS proteins,

EMSA and confocal microscopy

S8. Pericentromeric targeting of the mutant IKAROS proteins in HEK293T

cells, confocal microscopy

S9. Pericentromeric targeting and DNA binding of the mutant IKAROS proteins

S10. Flow cytometric TCR-Vβ repertoire analysis

S11. T cell proliferation and Fas-mediated apoptosis

S12. Penetrance of clinical and laboratory findings in patients with heterozygous

mutations in IKZF1

Figure S1. Mutations and sequence conservation in IKZF1

A! Family A! Family B! Family C! Family D! c.485G>T ! c.485G>A! c.500A>G ! c.551G>A ! (p.R162L)! (p.R162Q)! (p.H167R)! (p.R184Q)!

WT! WT! WT! WT!

A1! B5! C1! D2!

Family E Chromosome(7(GRCh37):g.50435843_50452713del( ! WT: Intron 3!

E1!

WT: Intron 5!

B! ZF2$ ZF3$

Homo.sapiens !GERPFQCNQCGASFTQKGNLLRHIKLHSGEKPFKCHLCNYACRRRDALTGHLRTHS ! P.Troglodytes !GERPFQCNQCGASFTQKGNLLRHIKLHSGEKPFKCHLCNYACRRRDALTGRLRTHS ! M.Mulatta ! !GERPFQCNQCGASFTQKGNLLRHIKLHSGEKPFKCHLCNYACRRRDALTGRLRTHS ! C.Lupus! !!GERPFQCNQCGASFTQKGNLLRHIKLHSGEKPFKCHLCNYACRRRDALTGRLRTHS ! B.Taurus ! !-ERPFQCNQCGASFTQKGNLLRHIKLHSGEKPFKCHLCNYACRRRDALTGRLRTHS ! M.Musculus ! !GERPFQCNQCGASFTQKGNLLRHIKLHSGEKPFKCHLCNYACRRRDALTGRLRTHS ! G.Gallus ! !GERPFQCNQCGASFTQKGNLLRHIKLHSGEKPFKCHLCNYACRRRDALTGRLRTHS ! D.Rerio! !!GERPFQCNQCGASFTQKGNLLRHIKLHSGEKPFKCHLCNYACRRRDALTGRLRTHS ! X.Tropicalis !-ERPFQCNQCGASFTQKGNLLRHIKLHSGEKPFKCHMCNYACRRRDALTGRLRTHS!

Panel A. Sequence chromatograms from the probands in family A-E. Chromatograms show the sequence of a healthy donor (wild type, WT) and heterozygous carriers of the mutation. Sanger sequencing over the breakpoints for the deletion in patient E1. The first part of the E1's sequence (middle sequence) matches the top sequence showing normal sequence for the region of the breakpoint in intron 3. The last part of E1's sequence matches the lower sequence showing normal sequence for the region of the breakpoint in intron 5. Panel B. Sequence conservation of zinc-finger domains 2 and 3 in IKZF1. The mutated amino acids found in Families A, B, C and D (R162L, R162Q, H167R and R184Q respectively) are indicated in red.

21 Figure S2. IKAROS protein structure and homology modeling

GOL R162

R184

H167

The structure of human IKAROS has not yet been determined. Therefore, to determine the approximate locations of the IKAROS variants R162L, R162Q, H167R, R184Q relative to the zinc ion coordination site and DNA binding pocket, we used UCSF Chimera14 to superimpose a 3D protein structure homology model of wild-type human IKZF1 (UniProtKB - Q13422) from the SWISS-MODEL repository15 onto its structural homology template, Mus musculus Aart (template)(PDB ID 2I13) bound to DNA.16 The target-template sequence identity was 44%, indicating the potential for alignment errors in non-conserved segments of the target protein; however, the side chains of the highly conserved residues of interest do overlap with those of the corresponding template residues, suggesting accurate modeling in the regions of interest. This model suggests that the IKAROS variant H167R directly affects a zinc ion coordination site, while variants R162L, R162Q, and R184Q may affect direct or indirect (glycerol/water- mediated) binding of IKAROS to DNA due to non-conservative substitution of a positively charged arginine in the DNA binding pocket. Figure legend: Section of model of wild-type human IKAROS (tan) provided by SWISSMODEL superimposed on PDB template 2I13 (gray)[Mus musculus Aart, a six finger zinc finger designed to recognize ANN triplets] bound to DNA (cyan, blue, red). Element colors: red oxygen, blue nitrogen, yellow sulphur, purple zinc. GOL=glycerol. Water molecules are shown as red spheres.

22 Figure S3. In silico prediction of the damaging effects of the amino acid substitutions in IKZF1.

30 pa#ents( 25 1kg( 20 EXaC( EVS( CADD 15

0 private 10-5 10-4 10-3 10-2 10-1 1

Minor allele frequency (MAF) in an exponential scale

The mutations in IKAROS, R162L, R162Q, H167R and R184Q are predicted to be damaging by PolyPhen. R162L, R162L, and H167R are predicted to be deleterious and R184Q is predicted to be borderline deleterious by SIFT analysis. The Combined Annotation Dependent Depletion (CADD, v1.3) score, a method that integrates several different annotations was calculated for R162L, R162L, H167R and R184Q (red squares) and 94 variants found in the EXaC (The Exome Aggregation Consortium) database, (gray diamonds), the EVS database (green diamonds) or the 1000 genome project (blue diamonds). The ninety-four variants shown in the figure correspond to 92 missense variants, 1 in-frame deletion (minor allele frequency (MAF), 0.000008288), and 1 frameshift deletion six codons before the stop codon (Q513SfsX6; MAF, 0.000008601). The four missense mutations from unrelated patients were not found in these databases. Their MAF is therefore < 0.00001. The same applies to the deletions, not represented here.

23 Figure S4. Selective pressure on genes underlying autosomal dominant primary immunodeficiency by haploinsufficiency.

A" ! NeutralityIndex

Human genes (ranked)!

"B" ! S /D N D

Human genes (ranked)!

Panel A. The selective pressure acting on IKZF1 and other genes associated with autosomal dominant immunodeficiency was determined by estimating the neutrality 17,18,19 index (NI) at the population level: NI=(PNPS)/(DNDS), where PN and PS are the number of non-synonymous and synonymous alleles, respectively, at population level (1000 Genomes Project) and DN and DS are the number of non-synonymous and synonymous fixed sites, respectively, for the gene’s coding sequence. Human genes (18,423 entries) have been ranked based on their score. Panel B. The DN/DS, ratio for human genes is compared with mouse (blue) or chimpanzee (red) genes respectively.20,21

24 Figure S5. IKAROS protein expression levels in T and B cells

+ + CD3CD3+ Cells cells ! CD19 +cells Cells! 8000 8000

C1 C1 6000 6000 C2,C3 C3 C2, D2 A2 A1 A1 4000 D2 4000

A2 IKAROS (MFI) IKAROS IKAROS (MFI) IKAROS 2000 2000

0 0 Patients HD Patients HD (Family A,C,D) (Family A,C,D)

CD3+ cells CD19+ cells p=0.0002 p=0.0112 6000 6000

5000 5000

4000 4000

3000 3000

2000 2000 IKAROS (MFI) IKAROS IKAROS (MFI) IKAROS 1000 1000

0 0 Patients HD Patients HD Deletion Wild type (FamilyDeletion F) Wild type (Family F)

IKAROS expression levels were evaluated in affected family members and healthy donors (HD). For family D (bottom panel), ten affected members and six unaffected family members as well as four healthy donors were evaluated by flow cytometry. PE- conjugated IKAROS antibodies (for family A-C) or goat anti-IKAROS antibodies (family D) were used in permeabilized peripheral blood mononuclear cells to measure IKAROS expression (Mean fluorescence intensity [MFI]). Error bars represent the SEM; the Student’s t test p values are shown above the bars.

25 Figure S6. The stability of the mutant IKAROS proteins and their ability to dimerize wild type IKAROS

cytoplasm! nucleus! A! ! ! ! ! ! ! ! ! ! ! ! ! Vector R162Q H167R R184Q WT Vector R162Q R162L H167R R184Q WT R162L

Flag!

Lamin A/C!

GAPDH!

B! Total Cell lysate! IP: Flag Ab! IP: IgG!

Flag%WT% -! +! +! +! +! +! -! +! +! +! +! +! +! ! ! ! ! ! ! ! ! ! ! ! ! ! HA% WT WT WT R162Q R184Q V con V R162Q R184Q V con V R162L H167R R162L H167R

WB: HA!

IKAROS% WB: Flag! Ig%heavy%chain%%

Panel A. 293T cells were transfected with Myc-tagged vectors expressing the indicated mutations and the cell lysate was divided into cytoplasmic and nuclear fractions for western blot analysis. Antibodies to Myc, Lamin A/C and GAPDH were used to document the stability of the mutant proteins, the ability of those proteins to migrate to the nucleus, and to evaluate the purity of the nuclear and cytoplasmic fractions. Panel B. 293T cells were transfected with pFlag-CMV2-IKAROS wild type (WT) and pcDNA3-HA-IKAROS WT or mutants (R162L, R162Q, H167R, and R184Q). V indicates empty vector control. Immunoprecipitations were performed using anti-Flag antibodies or rabbit IgG. Western blot analysis of the IP samples with anti-HA (upper panels) and anti-Flag antibodies (lower panels) is shown. Total cell lysates was loaded with 5% of the protein extract used for each IP reaction. Data shown are representative of 3 experiments.

26 FigureFigure S72! . DNA binding and pericentromeric targeting of the mutant IKAROS proteins, EMSA and confocal microscopy

A! WT! R162Q! R162L! H167R! R184Q! B! WT! R162Q! R162L! H167R! R184Q! WT! 1! ½! 0! 1! ½! 0! 1! ½! 0! 1! ½! 0! 1! ½! 0! WT! 1! ½! 0! 1! ½! 0! 1! ½! 0! 1! ½! 0! 1! ½! 0! Mutant! 0! 0! 0! 0! ½! 1! 0! ½! 1! 0! ½! 1! 0! ½! 1! Mutant! 0! 0! 0! 0! ½! 1! 0! ½! 1! 0! ½! 1! 0! ½! 1! Empty vector! !0! !½! !1! 0! 0! 0! 0! 0! 0! 0! 0! 0! 0! 0! 0! Empty vector! !0! !½! !1! 0! 0! 0! 0! 0! 0! 0! 0! 0! 0! 0! 0!

Ik-bs4! ϒSat8!

Gamma-sat8 IKBS4 1.25 1.25$1.25 1.25$

1.00 1.00$1.00 1.00$

0.75 0.75$0.75 0.75$

0.50 0.50$0.50 0.50$

0.25 0.25$0.25 0.25$

0.00 0.00$0.00 0.00$ WT Hz WT Wt Wt WT WT Hz WT WT WT WT Empty Empty Hz-162 ho-162 Hz-167 Ho-167 Hz-184 Ho-184 Hz-162 ho-162 Hz-167 Ho-167 Hz-184 Ho-184 hz-162QHo-162Q hz-162QHo-162Q H167R! C! WT! R162Q! R162L! R184Q! ! HA

5 μm!

HA-WT! HA-R162Q! HA-R162L! HA-H167R! HA-R184Q! D! ! HA ! ! Flag (Flag-WT) ! Merge

5 μm!

A, B. EMSAs were performed using nuclear extracts from HEK293T cells transfected with 500 ng of vector expressing WT IKAROS, 250 ng of WT plus 250 ng of mutant IKAROS expression vectors, or 500 ng of mutant IKAROS expression vector. The extracts were allowed to bind to 2 different IKAROS probes: IK-bs4, an IKAROS consensus binding sequence and γ Sat 8, a sequence from the pericentromeric region of human Chromosome 8. IKAROS containing complexes are indicated with an arrow. Data are normalized using the binding of the extracts from the WT transfected cells as 100%. Errors bars represent the SEM (2 or 3 independent experiments). C. NIH3T3 cells were transfected with HA-tagged WT or mutant expression vectors and were labeled with anti- HA antibody and an Alexa 488-conjugated (green) secondary antibody. Cells were visualized by using a confocal microscope. D. NIH3T3 cells were co-transfected with HA-tagged WT or mutant vectors (green), plus FLAG-tagged vectors expressing WT IKAROS and developed using an anti-FLAG antibody followed by Alexa 568-conjugated (red) antibody. Cells that received both the mutant and WT vectors demonstrated the typical punctate staining. Data shown are representative of 3 confocal experiments.

27 Figure S8. Pericentromeric targeting of the mutant IKAROS proteins in HEK293T cells

Human embryo kidney (HEK) 293T cells were transfected with pCMV6-AC-Myc-DDK- IKAROS WT or indicated mutant. After 20 hours, cells were stained with a Flag antibody, followed by Alexa Fluor 488 antibody. Pictures were acquired using identical confocal settings (results on similar experiments done on mouse NIH3T3 cells are included for comparison).

28 Figure S9. Pericentromeric targeting and DNA binding of the mutant IKAROS proteins

cytoplasm! nucleus! WT! Y210C! N159A! H191R! ! ! ! ! ! ! ! !

A! ! ! D! ! ! ! R162L R162L H191R H191R N159A N159A Y210C Y210C Vector Vector WT WT

Myc! -Flag α

Lamin A/C!

GAPDH!

B! WT! Y210C! N159A! H181R! WT! 1! ½! 0! 1! ½! 0! 1! ½! 0! 1! ½! 0! Mutant! 0! ½! 1! 0! ½! 1! 0! ½! 1! 0! ½! 1! Empty vector! 0! 0! 0! 0! 0! 0! 0! 0! 0! 0! 0! 0! E! WT-Flag! Y210C-Flag! N159A-Flag! H191R-Flag!

Ik-bs4! ! -Flag

IKBS4 α 1.25$1.25

1.00$1.00

0.75$0.75 ! 0.50$0.50

0.25$0.25

0.00$0.00 WT Hz WT WT WT Empty Hz-210 Ho-210 Hz-159 Ho-159 Hz-191 Ho-191 -HA (WT) -HA α C! WT! Y210C! N159A! H181R! WT! 1! ½! 0! 1! ½! 0! 1! ½! 0! 1! ½! 0! Mutant! 0! ½! 1! 0! ½! 1! 0! ½! 1! 0! ½! 1! Empty vector! 0! 0! 0! 0! 0! 0! 0! 0! 0! 0! 0! 0! ! Merge ϒSat8!

Gamma-sat8 1.25$1.25

1.00$1.00

0.75$0.75

0.50$0.50

0.25$0.25

0.00$0.00 WT Hz Wt Empty Hz-210 Ho-210 WT-159 Hz-159 Ho-159 Hz-191 Ho-191 WT-191R

Panel A. 293T cells were transfected with pCMV6-AC-Myc-DDK-wild type or mutants. Western blot data shows the relative amount of each protein. Panels B and C. EMSAs were performed with nuclear extracts using 2 different probe for IKAROS binding sites. Data are normalized using the wild type transfected cells as 1. Errors bars represent the SEM (2 or 3 independent experiments). Panel D, The confocal images of NIH3T3 cells transfected with indicated mutant vector. In all images, cells were stained with a Flag antibody, followed by Alexa Fluor 568 antibody. E. NIH3T3 cells were co-transfected with pCMV6-AC-Myc-DDK-wild type or mutants, plus HA-tagged vectors expressing wild type IKAROS. In all images, cells were stained with an anti-HA antibody and an anti-Flag antibody, followed by fluorochrome-conjugated secondary antibodies. Data shown are representative of 3 confocal experiments.

29 Figure S10. Flow cytometric TCR-Vβ repertoire analysis

HD1! HD2!

A1! A2! C1!

C2! C3! D2!

Whole blood samples from two healthy donors (HD) or 6 patients were stained with fluorochrome-conjugated CD3, CD4, CD8, and TCR-VB families antibodies according to manufacturer’s instruction (Beckman Coulter; IOTest® Beta Mark TCR V beta Repertoire Kit). Cells were analyzed by flow cytometry (Becton Dickinson FACSCanto II). Blue indicates out of range for CD4 T cells, green is out of range for CD8 T cells

30 Figure S11. T cell proliferation and Fas-mediated apoptosis

HD1! HD2! A1! A2! C1! C2! C3! D2!

65 69.5 44.9 44.6 76.1 48.5 57.7 A! ! 86.3 + Max of CD4 %

65.7 68.6 61 87.4 57.1 81.7 55.2 73.5 ! + Max of CD8 %

CellTrace Violet

B! C!

Panel A. Peripheral blood mononuclear cells were stained with CellTrace Violet and stimulated with (black line) or without (solid gray) anti-CD3 and anti-CD28 antibodies (1µg/mL) for 3 days. Numbers indicate percentage of cells having undergone at least one cellular division, assessed by dye dilution. Proliferation data shown are representative of 3 experiments. Panel B. Graph indicates percentages of cells divided from healthy donors and patients. Data are means ± SEM of replicates of 6 patients and 10 healthy donors. Panel C. Total PBMCs were stimulated with anti-CD3 and CD28 for 3 days, and then cultured cells with IL-2. At day 11, cells were stimulated with Apo1.3 in the presence of protein A, or staurosporine for 20 hours. Cells were stained with PI and (%) of cell loss are calculated as described in the method. Data are means ± SEM from 3 healthy donors and 6 patients.

31 Figure S12. Penetrance of clinical and laboratory findings in patients with heterozygous mutations in IKZF1

We estimated clinical penetrance by excluding index cases, focusing on the 15 symptomatic and 8 asymptomatic genetically affected family members. The overall penetrance of clinical manifestations was found to increase progressively over time. x indicates the age of asymptomatic family members at the time of the most recent evaluation. Penetrance curves as a function of age were estimated by the Kaplan-Meier method, using the survival package of the R software v3.0.2 (http://cran.r-project.org/). The small number of patients accounts for the large confidence intervals.

32 Supplementary tables

S1. Clinical and laboratory features in patients with heterozygous IKAROS

mutations

S2. List of variants identified among the list of 269 genes associated with PID in

the patients evaluated by WES.

S3. TdT mediated N nucleotide additions within VH3 heavy chain CDR3

sequences

33 Table S1. Clinical and laboratory features in patients with heterozygous IKAROS mutations

Numbers in Red/Blue indicate values above/below the normal range (respectively) compared to age-matched controls in the laboratory in which the study was performed: NIH Clinical Center (Family A, C), University Children’s Hospital Zurich (Family B), Quest Diagnostics (Family D), Oslo University Hospital and University of Oslo, Norway (Family E), and ARUP Laboratories (Family F). Serum IgG, IgA and IgM are expressed in mg/dL; *Patient was receiving Immunoglobulin (IgG) replacement therapy; ALC, absolute lymphocyte count; &Values for CD27+ B cells are included only if at least 50 CD19+ or CD20+ B cells were analyzed. Vaccine titers: [+] Protective, [-] Non-protective, for pneumococcal vaccine, [serotype-specific protective titers/serotypes tested are depicted in parenthesis]; PPV, pneumococcal polysaccharides vaccine; TT, Tetanus toxoid; DT, Diphtheria Toxoid; Hib, Haemophilus influenzae type b vaccine; VZV, Varicella-zoster vaccine; Rub, rubella vaccine; Infect, Infections; ITP, Idiopathic thrombocytopenic purpura; B-ALL, B-cell Acute Lymphoblastic Leukemia; # Deceased patient 34 Table S2. List of 269 genes associated with PID that were screened in the patients analyzed by WES.

ACP5, ADA, ADAR, AICDA, AIRE, AK2, AP3B1, AP3D1, APOL1, ATM, B2M, BCL10, BLM, BLNK, BLOC1S6, BTK, C1QA, C1QB, C1QC, C1R, C1S, C2, C3, C4A, C4B, C5, C6, C7, C8A, C8B, C8G, C9, CARD11, CARD14, CARD9, CASP10, CASP8, CCBE1, CCBEB1, CD19, CD247, CD27, CD3D, CD3E, CD3G, CD40, CD40LG, CD46, CD59, CD70, CD79A, CD79B, CD81, CD8A, CEBPE, CFB, CFD, CFH, CFHR1, CFI, CFP, CIB1, CIITA, CLPB, COLEC11, COPA, CORO1A, CR2, CSF2RA, CSF2RB, CTLA4, CTPS1, CXCR4, CYBA, CYBB, DCLRE1B, DCLRE1C, DKC1, DNA ligase 1, DNMT3B, DOCK2, DOCK8, ELANE, EP45, FADD, FAS, FASLG, FCN3, FERMT3, FOXN1, FOXP3, G6PC3, G6PD, GATA2, GFI1, GINS1, HAX1, IFIH1, IFNGR1, IFNGR2, IGHM, IGKC, IGLL1, IKBKB, IKBKG, IKZF1, IL10, IL10RA, IL10RB, IL12B, IL12RB1, IL17F, IL17RA, IL17RC, IL18, IL1RN, IL21, IL21R, IL2RA, IL2RG, IL36RN, IL7R, INO80, IRAK4, IRF3, IRF4, IRF7, IRF8, ISG15, ITCH, ITGAX, ITGB2, ITK, JAGN1, JAK3, KRAS, LAMTOR2, LCK, LIG4, LIPA, LPIN2, LRBA, LYST, MAGT1, MALT1, MAP3K14, MASP1, MASP2, MBL2, MCM4, MEFV, MKL1, MOGS, MPO, MRE11A, MSN, MVK, MYD88, NBN, NCF1, NCF2, NCF4, NFAT5, NFKB1, NFKB2, NHEJ1, NHP2, NLRC4, NLRP12, NLRP3, NOD2, NOP10, NRAS, ORAI1, PCNA, PGM3, PIK3CD, PIK3R1, PLCG2, PMS2, PNP, POLE, PRF1, PRKCD, PRKDC, PSMB8, PSTPIP1, PTPN6, PTPRC, RAB27A, RAC1, RAC2, RAG1, RAG2, RBCK1, RFX5, RFXANK, RFXAP, RHOH, RLTPR, RMRP, RNASEH2A, RNASEH2C, RNASEL, RNF168, RNF31, RORC, RPSA, RTEL1, SAMHD1, SBDS, SERPING1, SH2D1A, SLC35C1, SMARCAL1, SP110, SPINK5, SPPL2A, STAT1, STAT2, STAT3, STAT5B, STIM1, STK4, STX11, STXBP2, TADA2A, TAP1, TAP2, TAPBP, TBK1, TBX1, TCC37, TCF3, TECR, TERC, TERT, TGFBR1, TGFBR2, TICAM1, TINF2, TLR3, TMC6, TMC8, TMEM173, TNFRSF13B, TNFRSF13C, TNFRSF1A, TNFRSF4, TNFSF12, TPP2 , TRAC, TRAF3, TRAF3IP2, TREX1, TTC37, TTC7A, TYK2, UNC119, UNC13D, UNC93B1, UNG, USB1, VPS45, WAS, WIPF1, XIAP, ZAP70, ZBTB24).

35 Table S3. TdT mediated N nucleotide additions within VH3 heavy chain CDR3 sequences

TdT#mediated#N#nucleo/de#addi/ons## within#VH3#heavy#chain#CDR3#sequences# Unique! CDR3! !Subject! N1! N2! Total!N! Sequences! Length!

Control!1!! 20! 5.7! 4.8! 10.6! 15.6!

Control!2! 12! 5.8! 7.3! 13.0! 14.3!

Control!3! 16! 7.5! 6.5! 14.0! 15.2!

Control!4! 12! 5.5! 7.1! 12.6! 16.1!

BTK!R255X! 8! 6.3! 3.7! 10.0! 13.8!

BTK!L452P! 10! 4.4! 4.8! 9.3! 14.4!

IKZF1! 32! 5.9! 8.5! 14.4! 16.5! H167R! IKZF1( 21! 5.3! 4.1! 9.4! 15.9! R162Q!

The IMGT/V-QUEST website tool was used for sequence analysis. N1 and N2 indicate the average number of nucleotide additions between V and D regions and between the D and J regions respectively. The length of the CDR3 region in amino acids is shown.

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