Ligase IV Immunodeficiency Due to Mutations in DNA a Severe Form Of

A Severe Form of Human Combined Immunodeficiency Due to Mutations in DNA Ligase IV

This information is current as Anselm Enders, Paul Fisch, Klaus Schwarz, Ulrich Duffner, of October 2, 2021. Ulrich Pannicke, Elisabeth Nikolopoulos, Anke Peters, Marzenna Orlowska-Volk, Detlev Schindler, Wilhelm Friedrich, Barbara Selle, Charlotte Niemeyer and Stephan Ehl J Immunol 2006; 176:5060-5068; ; doi: 10.4049/jimmunol.176.8.5060 Downloaded from http://www.jimmunol.org/content/176/8/5060

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A Severe Form of Human Combined Immunodeﬁciency Due to Mutations in DNA Ligase IV1

Anselm Enders,* Paul Fisch,† Klaus Schwarz,‡ Ulrich Duffner,* Ulrich Pannicke,‡ Elisabeth Nikolopoulos,† Anke Peters,* Marzenna Orlowska-Volk,† Detlev Schindler,§ Wilhelm Friedrich,¶ Barbara Selle,ʈ Charlotte Niemeyer,* and Stephan Ehl2*

DNA ligase IV (LigIV) deficiency was identified as the molecular basis for a severe form of combined immunodeficiency in two microcephalic siblings with cellular radiosensitivity. In one patient the diagnosis was made directly after birth, allowing analysis of the role of LigIV in the development of specific immune cells. Absolute numbers of B cells were reduced 100-fold and ␣␤ T cells 10-fold, whereas ␥␦ T cells were normal. Spectratyping of all three cell populations showed a diverse repertoire, but sequencing of IgH V(D)J junctions revealed shorter CDR3 regions due to more extensive nucleotide deletions among D and J elements and

fewer N nucleotide insertions. Clonal restriction of IgG-expressing, but not IgM-expressing, B cells and the lack of primary and Downloaded from secondary lymph node follicles indicated impaired class switch recombination. Observations in the older sibling showed that this rudimentary immune system was able to mount specific responses to infection. However, partial Ab responses and extensive amplification of ␥␦ T cells could not prevent a life-threatening course of viral and bacterial infections, the development of an EBV-induced lymphoma, and immune dysregulation reflected by severe autoimmune cytopenia. Impaired generation of immune diversity under conditions of limited LigIV activity can cause a human SCID variant with a characteristic immunological

phenotype. The Journal of Immunology, 2006, 176: 5060–5068. http://www.jimmunol.org/

he V(D)J recombination process induces double-strand complementing protein 4 (XRCC4)/ligase IV (LigIV) complex, DNA breakage and rejoining. It forms the basis for the which finally rejoins the DNA ends (reviewed in Ref. 3). T generation of diversity in the specific immune system (1). The key role for some of the enzymes involved in V(D)J re- Early lymphoid progenitors express the RAG-1 and RAG-2 pro- combination for human lymphocyte development is well illus- teins, which bind at the recombination signal sequences flanking trated in patients with primary immunodeficiencies (4). Patients the genetic V, D, or J elements, induce double-strand DNA breaks with null mutations in RAG-1, RAG-2, or Artemis completely lack to excise intervening DNA sequences, and generate molecular B and T cells (5, 6) and present with SCID, a uniformly lethal hairpin structures at the remaining (coding) ends. DNA rejoining is disorder unless treated by bone marrow transplantation or gene by guest on October 2, 2021 then conducted by nonhomologous DNA end joining (NHEJ),3 a therapy (7). Sensitivity to ionizing irradiation differentiates Arte- double-strand break repair pathway active in all nucleated cells (2). mis-deficient patients from RAG-deficient patients (5), illustrating As a first step in NHEJ, the Ku proteins bind to the DNA ends and a more general role of the NHEJ pathway in the repair of double- associate with the DNA-dependent protein kinase (PK) catalytic strand DNA breaks. Patients with null mutations in the other genes subunit. The resulting DNA-PK complex associates with Artemis involved in V(D)J recombination have not been reported. This may and activates its endonuclease activity. The Artemis/DNA-PK in part be explained by their essential role in development. Mice complex opens the hairpinned V, D, or J ends. End processing by with a targeted deletion of DNA LigIV die at an early embryonic the endonuclease activity of Artemis in combination with tem- stage, suggesting other essential roles of that enzyme in develop- plate-dependent as well as template-independent polymerase ac- ment (8). tivity generates junctional diversity. The DNA-dependent PK cat- Although the expected presentation of patients with null muta- alytic subunit then also acts as a scaffold for the x-ray repair cross- tions in genes involved in V(D)J recombination is a TϪBϪ SCID, patients with hypomorphic mutations may retain some recombi- *Department of Pediatrics and Adolescent Medicine, University of Freiburg, nation activity and develop a rudimentary specific immune system † Freiburg, Germany; Institute for Pathology, University of Freiburg, Freiburg, Ger- (9, 10). Secondary modification by an infectious or self Ag can many; ‡Transfusion Medicine, University Hospital and Institute for Clinical Trans- fusion Medicine and Immunogenetics, Ulm, Germany; §Institute of Human Genetics, then induce characteristic phenotypes such as Omenn syndrome Wu¨rzburg, Germany; ¶Children’s Hospital, University of Ulm, Ulm, Germany; and ʈ (10–12), a disorder characterized by generalized erythroderma, St. Annastiftskrankenhaus, Ludwigshafen, Germany hepatosplenomegaly, eosinophilia, elevated IgE, and oligoclonal T Received for publication September 22, 2005. Accepted for publication January cell expansion. Both hypomorphic Artemis and RAG mutations 25, 2006. can lead to Omenn syndrome (10, 13), indicating that the partially The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance developed specific immunity and not the particular genetic defect with 18 U.S.C. Section 1734 solely to indicate this fact. may be responsible for the phenotype. Thus, patients with hypo- 1 This work was supported by German Research Foundation Grant SFB620 for morphic mutations can provide important insights on how the hu- projects A4 (to S.E.) and Z2 (to P.F.). man immune system develops and acts under limiting conditions. 2 Address correspondence and reprint requests to Dr. Stephan Ehl, Department of Reports on six patients with hypomorphic mutations in LigIV Pediatrics and Adolescent Medicine, University of Freiburg, Mathildenstrasse 1, 79106 Freiburg. E-mail address: [email protected] (14, 15) have been published to date. The first patient developed 3 Abbreviations used in this paper: NHEJ, nonhomologous end joining; LigIV, DNA leukemia at the age of 14 and was diagnosed on the basis of his ligase IV; PK, protein kinase; XRCC4, x-ray repair cross-complementing protein 4. increased sensitivity to radiation therapy (16). Another patient in a

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 5061 recently published study also developed T cell leukemia at the age of a primer extension reaction with a fluorescent (5Ј FAM-labeled) nested of 4.5 years and showed a phenotype resembling Nijmegen break- antisense primer specific for the constant region was performed (runoff age syndrome with microcephaly and growth retardation (17). The reaction). The products were then analyzed on an ABI 3100 capillary sequencer (Applied Biosystems) to determine the length distribution of the other four patients were diagnosed between 9 and 48 years of age fluorescent fragments. Clonal populations generally appear as sharp peaks, with various abnormalities, including developmental delay, micro- whereas polyclonal populations typically show Gaussian distributions but cephaly, and pancytopenia (14). Although in two patients the pan- may show one or several prominent peaks within the polyclonal peaks if cytopenia was severe enough to warrant bone marrow transplan- the repertoire is dominated by a few clones. The PCR products were cloned tation, none of the patients has been described as being severely into the pCR2.1-TOPO vector (Invitrogen Life Technologies), amplified, and sequenced. Ig gene segments were identified using the international immunodeficient (18). Although the crucial role of LigIV for hu- ImMunoGeneTics (IMGT) database (imgt.cines.fr) (30). Gene junctions man V(D)J recombination has been established in a gene-targeted were analyzed using the IMGT/JunctionAnalysis tool (31). All N nucleo- human pre-B cell line (19) and in cell lines obtained from LigIV- tide sequences of seven or more nucleotides were individually screened for deficient patients (20–23), the phenotypic consequences of LigIV the presence of a second D element in both orientations. A homology of at deficiency for human lymphocyte development and immunity to least 7 bp and appropriate chromosomal localization relative to the first D element were required for identification of a second D element. infection remain poorly defined. Here we describe the immunological profile of two siblings who were found to be compound heterozygous for a null allele and a Histology hypomorphic mutation in DNA LigIV and who presented with During placement of a central i.v. catheter, a cervical lymph node from severe immunodeficiency. One patient was identified after multiple patient 2 was removed for histological analysis. The sample was fixed in severe infections and the other was identified directly after birth, 4% buffered formalin, embedded in paraffin, cut at 4 ␮m, and stained with Downloaded from providing the opportunity to study the impact of LigIV deficiency H&E. For immunohistochemistry, the paraffin sections were put on albu- both on early lymphocyte development and on immune defense. minized glass slides following heat-mediated Ag retrieval. The primary Abs were obtained from DakoCytomation and included CD20 (clone L26), CD3 (clone F7.2.38), CD4 (clone MT310), CD8 (clone C8/144B), CD21 Materials and Methods (clone 1F8), CD30 (clone Ber-H2), and CD68. Visualization of the Abs was Informed consent performed using the ChemMate detection kit, AP/RED (DakoCytomation). Informed consent for the performed studies was obtained from the patient’s http://www.jimmunol.org/ family in accordance with the guidelines of the local ethics committee. Irradiation experiments Results Clinical course of two siblings with microcephaly, Primary diploid fibroblast-like cells were grown from skin biopsy speci- radiosensitivity, and severe immunodeficiency mens using standard cell culture techniques. Confluent, quiescent (G1 phase) cultures were trypsinized, and aliquots were irradiated at the indi- A 2-year-old girl (P1) born to nonconsanguineous parents of Ger- cated dosage levels using a 6-MV linear accelerator (Siemens) at a dose man origin was admitted to our hospital with a history of chronic Ϫ1 rate of 2 Gy min . For colony survival studies, aliquots of trypsinized diarrhea, failure to thrive, autoimmune cytopenia, and recurrent fibroblasts were irradiated at 0, 0.5, 1, 1.5, 2, 3, 4, 6, and 8 Gy and seeded in triplicate into petri dishes as described (24). Eagle’s MEM with 15% infections. At birth the girl was microcephalic with no further dys- FBS was replaced every 3–4 days. After a growth period of 2 wk, colonies plastic stigmata. She developed mild intermittent diarrhea and by guest on October 2, 2021 (Ͼ50 cells) were stained with 1% crystal violet in 20% ethanol. Means and growth delay. At 1 year of age she suffered from pneumococcal SDs of the ratio of colony number relative to the number of seeded cells sepsis two weeks after measles, mumps, and rubella vaccination. were calculated individually and plotted as colony survival vs radiation ␮ dosage for the two patient and the two normal control strains. Dose-re- Three months later she presented with severe neutropenia (80/ l) sponse curves were fitted to the linear quadratic model SF ϭ exp(Ϫ␣X Ϫ and thrombocytopenia (18,000/␮l). PCR was positive for human ␤X2), where SF is the survival fraction, X the radiation dose, and ␣ and ␤ herpes virus 6 from bone marrow and peripheral blood. She had are fitted parameters. Curves and statistics were generated using the Origin varying lymphopenia with reduced T cells, almost absent B cells, software (MicroCal). For 72-h cell cycle analysis, fibroblasts irradiated with 0 and 1.5 Gy were seeded at a density of 4000 cells/cm2 into parallel and normal NK cells. The thymus was small for her age but cultures. Growth medium was Eagle’s MEM substituted with 15% FBS, present. Motor development and cognitive development were nor- 1 ϫ 10Ϫ4 M BrdU, and 1 ϫ 10Ϫ4 M 2-deoxycytidine. After the growth mal, and all developmental milestones had been achieved in time. period, the cells were trypsinized and stained with Hoechst 33258 and At 20 mo of age she was diagnosed with persistent Norwalk virus- propidium iodide. Bivariate histograms were recorded on an LSR flow cytometer (BD Biosciences) and analyzed quantitatively using MULTI2D positive diarrhea with pronounced failure to thrive and the need for and MCYCLE software (25). parenteral nutrition. Four months later, she was referred to our hospital with high fever, cervical lymphadenopathy, hepatomeg- Genetic analysis aly, and a pronounced necrotizing mucositis. PCR for EBV was The LigIV gene was amplified with primer pairs yielding overlapping am- positive in peripheral blood (2,900 genome equivalents/ml) and plimers. These amplimers were sequenced in both directions. Primer se- bone marrow (640,000 genome equivalents/ml). Imaging studies quences for amplification and sequencing are available on request ([email protected]). Nucleotide and amino acid counts revealed multiple ovaloid mass lesions in the brain and the lung. A are according to RefSeq database sequences NM_002312 and NP_002303, biopsy showed an EBV-encoded latent membrane protein 1-positive, respectively. monoclonal, diffuse, large cell non-Hodgkin B cell lymphoma. Inten- Immunophenotyping and IgH and T cell CDR3 spectratyping sive chemotherapy reduced the tumor mass but was followed by severe mucositis with oral and gastrointestinal bleeding. The patient Lymphocyte phenotyping was performed with four-color flow cytometry developed rapidly progressive aspergillosis and died during prepara- using Abs from BD Pharmingen and Immunotech as described previously (26). TCR CDR3 spectratyping was performed as described by Pannetier et tive therapy for allogeneic stem cell transplantation. al. (27, 28) for analysis of the TCR ␤-chain. The primers and details for While the first patient was being treated for the lymphoma, a spectratyping of the TCR ␦-chains have been published (26). IgH spectra- sister was born (P2) who was also microcephalic (Fig. 1). At 2 wk typing was established according to previously published methods (29) and of age she had an absolute lymphocyte count of 936/␮l with a new work (E. Nikodopoulos, and P. Fisch, manuscript in preparation). In similar subset distribution as her older sister. At 4 mo of age she brief, the CDR3 regions were amplified using 35 cycles of PCR with a forward (sense) primer specific for the V region family and a backward received a bone marrow transplant from a matched unrelated donor (antisense) primer for the constant region. In a second round, three cycles following nonmyeloablative conditioning with fludarabine and 5062 SEVERE IMMUNODEFICIENCY IN LIGASE IV SYNDROME

reconstitution. At 1 year of age she has a signiﬁcant neurodevel- opmental delay.

Compound heterozygosity for LigIV mutations is the genetic basis of the severe immunodeficiency The lymphocyte subset distribution in the two patients (BlowT- lowNKϩ) suggested a genetic defect affecting both T and B cell differentiation. After adenosine deaminase, RAG1/2, and Artemis deficiency had been excluded by enzymatic testing or genomic sequencing, we considered LigIV syndrome. Two consistent features of LigIV syndrome are microcephaly and increased radiosensitivity. Clinically, the B cell lymphoma and the toxicity of chemotherapy observed in P1 were consistent with a defect in DNA repair. Fibroblasts strains from both siblings with LigIV deficiency proved significantly hypersensitive to ionizing irradiation regarding their markedly reduced long-term (14 d) clonogenic survival (Fig. 1A). In short-term (72 h) growth studies, the patients’

ﬁbroblasts showed arrest in the G2 phase of the cell cycle follow-

ing ionizing radiation (Fig. 1B). This finding explains the poor Downloaded from clonogenic survival. The degree of radiosensitivity was compara- ble to that of a paradigm genetic radiosensitivity disorder, ataxia telangiectasia (32) (Fig. 1B). Both patients showed normal length development along the 25th to 50th percentile (Fig. 1C, upper panels). By contrast, the head circumference was disproportionally small and was below the 3rd percentile in both patients (Fig. 1C, http://www.jimmunol.org/ lower panel). Although no severe immunodeficiency had been reported in the six previously published patients (14, 15, 17, 18), we decided to sequence the LIG4 gene. We identified a point mutation (1118A3T, resulting in H282L) on the maternal allele and a deletion of five bases (1544AAAGA1548) on the paternal allele, leading to a frame shift and an altered amino acid sequence starting after D423 and premature termination of translation after amino acid 442 (Fig. 2A) in both patients. The parents were heterozygous

for these mutations. by guest on October 2, 2021

Impaired B cell development, but remaining specific Ab production in LigIV deficient patients The absolute numbers of peripheral B cells were reduced ϳ100- FIGURE 1. Sensitivity to ionizing radiation in two patients with micro- fold in both siblings (Table I). Nevertheless, serum Igs and specific cephaly and immunodeficiency. A, Colony survival. Dose vs radiation re- Abs could be detected. When analyzed at 4 wk of age in the ab- sponse curves of fibroblasts from the two patients, P1 (filled triangle) and sence of obvious infections, P2 had low amounts of IgM, whereas P2 (inverted filled triangle), are compared with those of two random nor- IgA was absent. IgG levels could not be interpreted due to remain- mal controls, C1 (open circle) and C2 (closed circles), and studied in par- ing maternal Abs. Nevertheless, IgM hemagglutinins were detect- allel. Error bars denote one SD. B,G phase accumulation. 72 h after 2 able. Moreover, 7% of the B cells expressed the memory marker irradiation with 1.5 Gy, fibroblasts were stained with Hoechst 33258 and CD27, similar to age-matched controls. P1 was analyzed after se- propidium iodide, and cell cycle status was determined by flow cytometry. vere infections and showed surprisingly high levels of serum Igs. The percentage of cells in G1/G2 phase is shown as a function of the ratio Total IgG and IgM were elevated, but IgA was in the normal of cells in G2 phase among all cycling cells. The ratios from fibroblasts of patient P1 (filled triangle) and patient P2 (inverted filled triangle) are range. Despite three vaccinations, the patient did not have specific within the range of radiosensitive ataxia telangiectasia fibroblast strains Abs to tetanus or diphtheria and showed only low titers to pneu- (gray diamonds; n ϭ 18), whereas normal control strains (gray circles; n ϭ mococcus despite having undergone culture-proven sepsis. By 15) show a much lower ratio. The high noncycling cell fractions of the contrast, significant IgG responses could be detected against the patient cultures relative to positive (ataxia telangiectasia) and negative measles and mumps vaccines and to the human herpes virus 6 (normal) controls indicate their poor growth. C, Growth charts showing infection documented by PCR. Response to the rubella vaccine development of length and head circumference. The bold line represents was limited to IgM, and no Ab response could be detected against the 50th percentile for German children, and the lines beneath it show the EBV. In addition to these partial antimicrobial Ab responses, the 25th, 10th, and 3rd percentiles. patient developed high titers of autoantibodies against platelets, resulting in severe thrombopenia. thiotepa. Three months after transplantation she developed a se- Reduced BCR diversity in LigIV deficiency vere hemolytic-uremic syndrome, presumably triggered by cyclo- To analyze the B cell repertoire generated under conditions of sporine that responded to plasmapheresis but that left her with limited LigIV activity, we performed CDR3 spectratyping of the compensated renal insufficiency. She is presently clinically stable VH genes using IgM- and IgG-specific primers in P2 and a healthy 8 mo after transplantation with full chimerism and ongoing thymic age-matched control. The spectratyping analysis was performed The Journal of Immunology 5063

FIGURE 2. Compound heterozygosity for LigIV mutations. A, Struc- ture of the LigIV gene with protein domains and localization of the LigIV mutations identiﬁed previously and in this report. B, Key clinical features of LigIV deﬁcient patients reported to date. The abbrevi- ation “homoz.” indicates homozygos- ity for the given mutation. The three patients published during revision of this manuscript are denoted by italics. Downloaded from

three times from the same RNA samples and found to be repro- than six nucleotides revealed the presence of two D elements in http://www.jimmunol.org/ ducible, especially for the more commonly used VH1, VH3, and 1/70 sequences from the patient and 1/51 sequences from the con- VH4 families (33). Several differences were noted in comparison to trol. Fig. 4 shows an alignment of the V(D)J regions obtained with the control (Fig. 3A). First, diversity was more restricted among primers for VH3 and the constant region of IgM (upper panel)or some VH families such as VH5 and VH6. Second, there were more the constant region of IgG (lower panel) for P2 and the age- seemingly clonal expansions, e.g., in the VH2, VH3, VH5, and VH6 matched control. The differences were subtle when single VH fam- families. Third, the sizes of the CDR3 fragments were smaller in ilies were compared but became clearer after 70 sequences from the patient than in the healthy control, most obvious in the VH3 and the patient and 51 sequences from the control had been analyzed. VH4 families. CDR3 length diversity was less pronounced in both

infants when IgG-specific primers were used (Fig. 3B). Because by guest on October 2, 2021 the analysis was performed 8 wk after birth, this finding may reflect the largely naive state of the B cell system.

Reduced V(D)J junctional diversity in LigIV deficiency To test whether the limited LigIV activity resulted in a particular pattern of V(D)J recombination, we cloned and sequenced 63 PCR products from IgM-specific CDR3 amplifications and 58 products from IgG-specific CDR3 amplifications. V, D, and J elements as well as N and P nucleotides were identified using the IMGT database. Additional analysis of N/P nucleotide sequences longer

Table I. B cell compartmenta

P1 P2

CD19ϩ/␮l 14–21 (600–1900) 1–10 (600–3100) CD27ϩ ND 7 (percentage of CD19) IgG (g/L) 13.2 (3.8–12.4) 4.3 (1.4–8.6) IgM (g/L) 1.7 (0.5–1.9) 0.06 (0.2–1.0) IgA (g/L) 0.6 (0.2–1.3) Ͻ0.06 (0.1–0.7) IgE (U/ml) Ͻ1.5 ND Anti-tetanus Absent ND FIGURE 3. IgH repertoire analysis. CDR3 length profile of IgH popu- Anti-diphteria Absent ND lations. RNA was isolated from PBMCs obtained from P2 and an age- Isohemagglutinins ND Anti-A, 1:4; anti-B, 1:64 matched healthy control, reverse transcribed, and amplified using IgM b Anti-viral IgM Rubella ND VH1–6 (upper panels) and IgG VH1–6 specific primers (lower panels). Anti-viral IgG HHV-6, measlesb, ND The size distribution of PCR products was determined by an automated mumpsb sequencer and GeneScan software. Dark peaks represent size markers, and Auto-antibodies Platelets ND light peaks represent PCR products. The fluorescence intensity reflects the a Age-matched normal values are given in parentheses. frequency of PCR fragments of a given size and is plotted as a function of b Vaccine responses. the size of the PCR fragments. 5064 SEVERE IMMUNODEFICIENCY IN LIGASE IV SYNDROME Downloaded from http://www.jimmunol.org/

ϩ ϩ FIGURE 4. VH3 junctional sequences among IgM and IgG B cells. PCR products from the IgH CDR3 spectratyping (see Fig. 3) were isolated, cloned into the pCR2.1-TOPO vector, amplified, and sequenced. Sequences amplified using VH3- and IgM-specific (upper panel)orVH3- and IgG-specific primers (lower panel) are shown for P2 and a healthy age-matched control. The sequences are shown as displayed by the IMGT software. The dots in the aligned V and J regions as well as in the D regions reflect deletions of the germline end nucleotides. The far right column shows the number of amino acids in the CDR3 region. by guest on October 2, 2021

First, there was signiﬁcant clonal identity among IgG sequences patient (Fig. 5A). This could be seen among both IgM and IgG from the patient. Although among IgM sequences 23/24 control sequences (Fig. 5 and data not shown). By contrast, the number of and 37/39 patient sequences were unique, only 15/31 IgG se- sequences that harbored P nucleotides was similar in both infants quences from the patient compared with 26/27 from the control (15/52 in P2 vs 16/49 in the control). Third, more deletions of the showed a unique sequence. Thus, in contrast to IgM, the clonal end nucleotides could be observed among the J and D elements of diversity of IgG-expressing B cells appeared to be signiﬁcantly the patient. Fig. 5 shows the number of nucleotides deleted from more restricted in P2 than in the control. Second, N nucleotide the J elements (Fig. 5B) and the D elements (Fig. 5C) among the sequences were used less often and, if used, were shorter in the unique IgM and IgG sequences from P2 and the control.

FIGURE 5. Fewer N nucleotides and more gene end deletions among J and D elements in LigIV deficiency. The unique VH3 junctional sequences amplified using IgM- and IgG-specific primers from the patient (52 unique/70 total sequences) and the control (49 unique/51 total sequences) were aligned using the IMGT software. A, The number of N nucleotides was determined in the individual sequences. B and C, J and D elements were compared with the germline sequences, and the number of nucleotides deleted from 5Ј and 3Ј ends during V(D)J recombination was determined for the individual sequences. B, Number of nucleotides deleted from the J elements. C, Number of nucleotides deleted from the D elements. Data are displayed as box plots with the median, 25/75th, and 10/90th percentiles indicated as horizontal bars. The Journal of Immunology 5065

Absence of primary and secondary lymph node follicles A cervical lymph node from P2 was removed. H&E staining revealed a poorly structured ﬁbrotic node with pronounced lymphopenia, lack of follicular areas, and interfollicular zones (Fig. 6A). Immunohistochemical analysis using B cell Abs (CD20; Fig. 6, B and C) showed a proper number of follicles (normal anlage) but of small size and with different severity of B cell depletion. The follicles lacked CD3ϩ T cells but contained abundant CD68ϩ macrophages (Fig. 6D). These cells can frequently be observed in the granuloma-like structures forming in lymph nodes of SCID patients. There were no secondary follicles, no centroblasts, and no signs of germinal center activity as shown by negative staining for the proliferation marker Ki-67 (data not shown).

Impaired T cell development but normal T cell repertoire diversity The number of circulating CD4ϩ and CD8ϩ T cells was reduced in both patients with a reduced proportion of CD45RAϩ naive T ϩ cells among total CD4 T cells. T cell proliferation assessed by Downloaded from [3H]thymidine incorporation was significantly reduced in response to PHA and anti-CD3 stimulation, and there was no response to tetanus in P1. Using a CFSE dilution, we could demonstrate sig- FIGURE 7. Normal ␣␤ T cell repertoire. A and B,V␤ chain expression ϩ nificant proliferation of CD4 T cells obtained from P2 in re- among CD4ϩ (A) and CD8ϩ T cells (B) as determined by flow cytometry. sponse to PHA and anti-CD3/anti-CD28, whereas there was little C, CDR3 length profile of the indicated V␤ populations. cDNA was pre- ϩ ␤ pared from PBMC RNA, amplified using V␤-specific primers, and ana- response from CD8 T cells (data not shown). All of the seven V http://www.jimmunol.org/ chains analyzed were used by T cells from P2, and there were lyzed by spectratyping (see legend to Fig. 3). The frequency of PCR frag- only limited shifts compared with published controls (Fig. 7, A ments of a given size is plotted as a function of the size of the PCR and B). CDR3 spectratyping of the V␤ chain revealed a poly- fragment. Dark peaks represent size markers, and light peaks represent PCR products. clonal repertoire that resembled the diversity of an age-matched control (Fig. 7C).

Predominance of ␥␦ T cells 8), arguing against Ag-driven oligoclonal expansion as the basis Both patients showed a predominance of ␥␦ T cells (Table II). In ␥␦ ϩ for the T cell predominance in P2. the absence of any obvious infection in P2, 29% of CD3 lym- by guest on October 2, 2021 ␥␦ ϩ phocytes expressed the TCR. Up to 89% of CD3 lymphocytes Discussion expressed the ␥␦ TCR in P1 after a history of severe bacterial and In this report LigIV deficiency has been identified as a new genetic viral infections. The diversity of ␦-chains was analyzed in T cells cause for a variant of severe combined immunodeficiency in hu- from P2 by TCR CDR3 spectratyping analysis (Fig. 8). Significant mans. It adds to the wide phenotypic spectrum of LigIV syndrome genetic diversity could be documented among the more common (Online Mendelian Inheritance in Man no.601837), a previously V␦1, V␦2, and V␦3 chains, whereas the more rare V␦4, V␦5, and described heterogenous disorder with radiosensitivity, growth fail- V␦6 chains showed an oligoclonal spectrum. The overall pattern of ure, microcephaly, developmental delay, predisposition to lym- CDR3 sizes was as diverse as that of an age-matched control (Fig. phoma, and milder forms of immunodeficiency. In all patients described it is caused by hypomorphic LigIV mutations (14, 15, 17, 34). This event is not unexpected, because mice with a targeted deletion of LigIV die during embryonal development (8). Neither of the LigIV mutations identified in this study have been reported previously (Fig. 2A). The first mutation leads to a frameshift after D423 and a premature stop after amino acid 442, presumably representing a null allele. The most similar mutation that has been characterized in detail is the R580X mutation, likely representing a null allele, because the protein is not stably expressed, does not interact with XRCC4, and does not enter the nucleus (14). The second most likely hypomorphic mutation (H282L) is close to the conserved ligase motif that includes the active site K273. It is in the vicinity of the R278H mutation (15) that leads to 5–10% re- sidual adenylation and double-strand ligation activity (20, 22, 23). It has been reported that this activity is further reduced below 1% by the additional polymorphisms A3V and T9I (34). These polymorphisms were not present in our patients. Because the functional consequences of the H282L mutation have not yet been studied by FIGURE 6. Absence of primary and secondary lymph node follicles. in vitro assays, we cannot exclude the possibility that additional Lymph node sections were either stained with H&E or immunostained with uncharacterized defects may also contribute to the impaired class the indicated mAbs. switch recombination. 5066 SEVERE IMMUNODEFICIENCY IN LIGASE IV SYNDROME

Table II. T cell compartmenta

P1 P2

Age 1.5–2.5 years 2 wk–4 mo Lymphocytes/␮l 360–1300 (2600–10,400) 583–936 (3700–9600) CD3ϩ/␮l 280–1074 (1600–6700) 176–228 (2300–6500) CD3ϩCD4ϩ/␮l 36–529 (1000–4600) 71–112 (1500–5000) CD3ϩCD8ϩ/␮l 72–542 (400–2100) 24–77 (500–1600) CD3ϪCD16ϩ56ϩ/␮l 32–198 (200–1200) 241–449 (100–1300) CD45RA (percentage of CD4) 1–4 14–22 (64–95) PHA Reduced Reduced Tetanus Reduced ND TCR ␥␦ (percentage of CD3) 65–81 (2–10) 23–29 (1–10) TCR ␥␦/␮l 72–262 (85–215) 51–68 (85–215)

a Age-matched normal values are given in parentheses.

Characteristic phenotypic features of most patients described reduction in the frequency of V(D)J recombination (14, 20, 23), with LigIV syndrome include microcephaly and radiosensitivity whereas it was reduced 1000-fold in the gene-targeted LigIV-de- (Fig. 2B). Growth defects and radiosensitivity can also be observed ficient human pre-B cell line N114P2 (19). In our patients, the in mice deficient in the NHEJ enzymes Ku (35–37), as well as in LigIV mutations led to a significant impairment of lymphocyte Downloaded from mice deficient in XRCC4 or LigIV, when bred on a p53-deficient development. Obviously, the repertoire of peripheral B and T cells background to overcome their embryonic lethality (38, 39). Al- detected in the periphery represents the complex result of devel- though microcephaly has not been reported in these mice, exces- opmental impairment, homeostatic proliferation, and peripheral sive neuronal apoptosis has been observed (8, 38, 39), indicating modification by infectious and other types of Ags. However, the the need for repair of double-strand breaks during nervous system younger sibling was studied only a few weeks after birth without development. Neuronal cell loss in the brain is a known etiologic obvious infections, and the spectratyping analysis revealed a http://www.jimmunol.org/ factor for microcephaly in humans. The clinical outcome of this largely undisturbed repertoire. The severely reduced number of neurological defect is, however, highly variable. Although LigIV- circulating B and T lymphocytes may, therefore, allow a rough deficient mice die during embryonal development (8), the devel- estimation of the efficiency of V(D)J recombination. Interestingly, opmental delay in patients with hypomorphic mutations ranges the impact was different on various cell populations. Although B from no defects (patient 118BR) (15) to significant developmental cell numbers were reduced ϳ100-fold, there was only a 10-fold and mental delay (patient 99P0149) (14). This can only in part be reduction in ␣␤ T cells and no reduction in ␥␦ T cells. This could explained by the localization of the individual mutations, because be due to cell type-specific differences in LigIV requirements, an our two siblings differed markedly in their cognitive development

interpretation that is supported by the finding that the repertoire by guest on October 2, 2021 and motor development. Our observations also imply that the neu- diversity was more restricted among B cells than among T cells. rodevelopmental outcome cannot be predicted by the extent to which a particular mutation impairs immunological development. An additional factor may be thymic selection. Under conditions of The crucial role of LigIV in DNA double-strand repair and limiting production of precursor cells, a relatively higher propor- NHEJ identified in mice (8) and human cell lines (19) predicted the tion of T cells may be able to pass selection in the thymus such that possibility of a SCID phenotype in human LigIV syndrome. Cy- the lack of mature T cells may be relatively less pronounced than ␥␦ topenia of several hemopoietic lineages and an increased suscep- the lack of T cell precursors. The predominance of T cells tibility to infection have been reported in some LigIV-deficient observed in both patients may have been influenced by the less patients (14). However, despite various abnormalities in the fre- stringent thymic selection of ␥␦ T cells that is probably not influ- quency and fidelity of V(D)J recombination in vitro (14, 20–23), enced by MHC restriction. Following the infections in P1, the ␥␦ none of the mutations described to date caused a severe immuno- T cells may have been triggered and further expanded by microbial deficiency in vivo (14, 15). In plasmid-based assays, cell lines Ags recognized by the ␥␦ TCR (40–43) or other triggering mech- obtained from patients 180BR and 411BR only showed a 2–3-fold anisms of the innate immune system. LigIV deficiency significantly affected the fidelity of the joining reaction in V(D)J recombination. Previous studies (20) with trans- fected recombination substrates revealed signal joint, but not coding joint, deletions during recombination in the 180BR cell line. The defects in cell lines from patients 2304 and 411BR were more pronounced and included deletions, lack of end protection, and reduced insertion of N nucleotides by TdT (14, 20, 21). In the sequences obtained from peripheral B cells of P2 we found significant deletions of D and J elements and reduced N nucleotide insertion. This may suggest a disturbed interaction between LigIV and TdT, but a direct interaction between these two enzymes has not been proven biochemically. The increased number of nucleo- ␥␦ FIGURE 8. Normal T cell repertoire. CDR3 length profile of the tide deletions could be due to lack of end protection by LigIV (21). indicated V␦ populations. cDNA was prepared from PBMC RNA, amplified using V␦ specific primers, and analyzed by spectratyping (see legend Another possible explanation is that, due to limited LigIV activity, to Fig. 3). The frequency of PCR fragments of a given size is plotted as a the DNA ends are accessible to nucleases such as Artemis for a function of the size of the PCR fragment. Dark peaks represent size mark- longer period of time, leading to a relative overactivity of these ers, and light peaks represent PCR products. enzymes. The Journal of Immunology 5067

The severe immunodeficiency observed in our patients included deficiency, and we speculate that this could also include Omenn susceptibility to life-threatening bacterial and viral infections, de- syndrome (9, 13). Radiosensitivity and microcephaly are important velopment of an EBV-positive B cell lymphoma, and severe au- clues guiding rational genetic analysis in these patients. toimmune cytopenia. These features developed in the presence of normal to elevated total levels of serum Igs, indicating that a sig- Acknowledgments nificant number of Ab-producing plasma cells could be generated We are grateful to Alain Fischer (Institut National de la Santeétdela from the few developing B cells. However, specific IgM and IgG Recherche Me´dicale, Paris, France) for pointing out microcephaly as an responses were only detected against some vaccines and infec- important diagnostic clue for the identification of LigIV-deficient patients. tions, presumably reflecting an additional defect in the periphery. We thank Ralf Ku¨ppers (Institut fu¨r Zellbiologie, Essen, Germany) for In line with this interpretation, the frequent finding of identical helpful discussions on the sequence data. We thank all nurses and doctors sequences showed that the repertoire diversity of IgG Ig receptors involved in the dedicated clinical care of the patients. The excellent tech- was more limited than that of IgM. This could either reflect limited nical help by Annette Schult, Tatjana Kersten, Daniela Bukatz, and helper T cell activity or, more likely, indicate a role for LigIV in Sylvia Koch is kindly appreciated. class switch recombination. An altered pattern of recombination at the S␮-S␣ switch junctions has recently been observed in DNA Disclosures obtained from patients 2303/4 and 411BR (44). The absence of The authors have no financial conflict of interest. primary and secondary lymph node follicles in P2 was also consistent with a role for LigIV in class switch recombination. The References cytopenia in our patients was not due to ineffective hematopoiesis 1. Dudley, D. D., J. Chaudhuri, C. H. Bassing, and F. W. Alt. 2005. Mechanism and (data not shown) but was caused by autoantibodies with peripheral control of V(D)J recombination versus class switch recombination: similarities Downloaded from and differences. Adv. Immunol. 86: 43–112. destruction. It remains unclear to what extent autoimmunity con- 2. Lieber, M. R., Y. Ma, U. Pannicke, and K. Schwarz. 2004. The mechanism of tributed to the cytopenia observed in the previously reported Li- vertebrate nonhomologous DNA end joining and its role in V(D)J recombination. gIV-deficient patients. DNA Repair (Amst.) 3: 817–826. ␥␦ 3. Lieber, M. R., Y. Ma, U. Pannicke, and K. Schwarz. 2003. Mechanism and A predominance of T cells and Ab-mediated autoimmunity regulation of human non-homologous DNA end joining. Nat Rev. Mol. Cell Biol. despite minimal B cell and severely reduced ␣␤ T cell numbers 4: 712–720. has recently been reported as a characteristic phenotype of patients 4. de Villartay, J. P., A. Fischer, and A. Durandy. 2003. The mechanisms of immune http://www.jimmunol.org/ diversification and their disorders. Nat. Rev. Immunol. 3: 962–972. with a SCID variant due to hypomorphic RAG mutations and 5. Moshous, D., I. Callebaut, R. de Chasseval, B. Corneo, M. Cavazzana-Calvo, CMV infection (26, 45). This report describes another molecular F. Le Deist, I. Tezcan, O. Sanal, Y. Bertrand, N. 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