RIDDLE Immunodeficiency Syndrome Is Linked to Defects in 53BP1-Mediated DNA Damage Signaling

RIDDLE immunodeficiency syndrome is linked to defects in 53BP1-mediated DNA damage signaling Grant S. Stewart*†, Tatjana Stankovic*, Philip J. Byrd*, Thomas Wechsler‡, Edward S. Miller*, Aarn Huissoon§, Mark T. Drayson¶, Stephen C. West‡, Stephen J. Elledge†ʈ, and A. Malcolm R. Taylor* *Cancer Research UK, Institute for Cancer Studies, Birmingham University, Vincent Drive, Edgbaston, Birmingham B15 2TT, United Kingdom; ‡Cancer Research UK, Clare Hall Laboratories, London Research Institute, South Mimms, Hertfordshire EN6 3LD, United Kingdom; §Department of Immunology, Birmingham Heartlands Hospital, Birmingham, B9 5SS, United Kingdom; ¶Division of Immunity and Infection, Birmingham University Medical School, Vincent Drive, Edgbaston, Birmingham B15 2TT, United Kingdom; and ʈHoward Hughes Medical Institute, Department of Genetics, Harvard Partners Center for Genetics and Genomics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115 Contributed by Stephen J. Elledge, September 6, 2007 (sent for review July 6, 2007) Cellular DNA double-strand break-repair pathways have evolved with an alternative constant region (e.g., ␣, ␥, ␧) to generate to protect the integrity of the genome from a continual barrage of different Ig isotypes e.g., IgA, IgG, and IgE (2). potentially detrimental insults. Inherited mutations in genes that During the process of CSR, it has been hypothesized that two control this process result in an inability to properly repair DNA closely positioned single-stranded DNA nicks result in the damage, ultimately leading to developmental defects and also production of a DNA DSB. Although the mechanism of DNA cancer predisposition. Here, we describe a patient with a previ- DSB generation during this process remains unclear, there is a ously undescribed syndrome, which we have termed RIDDLE syn- requirement for the presence of a DNA DSB to be produced for drome (radiosensitivity, immunodeficiency, dysmorphic features the proper resolution of the switch recombination process, as and learning difficulties), whose cells lack an ability to recruit loss of components of the DSBR repair pathway, such as H2AX, 53BP1 to sites of DNA double-strand breaks. As a consequence, DNA-PKcs, ATM, and Nbs1, may affect the ability to efficiently cells derived from this patient exhibit a hypersensitivity to ionizing switch from IgM to other Ig isotypes (2). radiation, cell cycle checkpoint abnormalities, and impaired end- More recently it has been demonstrated that loss of the DSB joining in the recombined switch regions. Sequencing of TP53BP1 response protein, 53BP1, in mice also results in a CSR defect (3, and other genes known to regulate ionizing radiation-induced 4). In support of this role, it has been shown that 53BP1 rapidly 53BP1 foci formation in this patient failed to detect any mutations. relocalizes to sites of DNA damage marked by ␥-H2AX after Therefore, these data indicate the existence of a DNA double- exposure of cells to IR, as well as to the Ig heavy-chain (IgH) loci strand break-repair protein that functions upstream of 53BP1 and that have been induced to undergo switch recombination (5). contributes to the normal development of the human immune Here, we describe the analysis of cells derived from a newly system. described human syndrome, termed RIDDLE syndrome whose clinical features are an increased radiosensitivity, immunodefi- DNA repair ͉ cell cycle checkpoint ͉ radiosensitivity ͉ BRCA1 ciency, dysmorphic features, and learning difficulties. Cells from this patient exhibit a different pattern of S␮–S␣ junctions in CSR NA damage recognition and repair is a fundamental process compared with normal and 53BP1-mediated cellular responses Drequired to maintain the fidelity of the genome. Disruption to DNA damage, without any genetic alterations in the TP53BP1 of this process is implicated in the development of human disease gene. The increased sensitivity to IR is likely a result of a failure and tumorigenesis. The impact of defective DNA double-strand to properly relocalize 53BP1, Rif1, and BRCA1 to sites of DNA break repair (DSBR) on human development can be observed in DSBs. In addition, these cells exhibit protracted ATM- patients with inherited disorders such as ataxia-telangiectasia dependent DNA damage signaling, characterized by elevated (A-T), Nijmegen breakage syndrome (NBS), DNA ligase IV numbers of ␥-H2AX, MDC1, and Nbs1 foci, hyperphosphory- deficiency syndrome (LiDS), and radiosensitive severe com- lation of Nbs1, RPA2, and H2AX and a prolonged G2/M bined immunodeficiency (RS-SCID) who have mutations in the checkpoint. These data indicate the existence of a previously DSBR genes ATM, NBS1, DNA Ligase IV, and Artemis and uncharacterized component of the DSB repair pathway that Cernunnos, respectively. These patients manifest a multitude of facilitates 53BP1 recruitment to sites of DNA damage as well as clinical features, which include a marked hypersensitivity to promoting efficient repair of DNA DSBs that occurs after agents that induce DNA double-strand breaks (DSBs), such as exposure to IR or during class switch recombination. ionizing radiation (IR), neurological abnormalities, mental re- tardation, growth defects, gonad dysfunction, and an elevated Results predisposition to the development of tumors. Because normal A clinical description of patient 15–9BI is given in supporting development of the immune system requires the introduction of information (SI) Text. DNA DSBs during antigen receptor gene assembly, defects in the repair of these specialized breaks can lead to profound immunodeficiency (1). This is most notable in LiDS and RS- Author contributions: G.S.S. designed research; G.S.S., T.S., P.J.B., T.W., E.S.M., S.C.W., and S.J.E. performed research; A.H. and M.T.D. contributed new reagents/analytic tools; G.S.S., SCID patients, although immune system abnormalities are also A.H., M.T.D., S.C.W., and S.J.E. analyzed data; and G.S.S. and A.M.R.T. wrote the paper. associated with A-T and NBS. The authors declare no conflict of interest. It is known that Artemis and DNA ligase IV, along with Freely available online through the PNAS open access option. XRCC4, Cernunnos/XLF, and the DNA-PK holoenzyme, com- Abbreviations: DSBs, DNA double strand breaks; DSBR, DSB repair; CSR, class switch prise the components of the nonhomologous DNA end-joining recombination; IR, ionizing radiation; IRIF, IR-induced foci; MEF, mouse embryo fibroblast. (NHEJ) machinery. The error-prone nature of this DNA repair †To whom correspondence may be addressed. E-mail: [email protected] or pathway is invaluable for creating genetic diversity when assem- [email protected]. bling immune system gene receptors. For example, class switch This article contains supporting information online at www.pnas.org/cgi/content/full/ recombination (CSR) results in the editing of a functional ␮-Ig 0708408104/DC1. heavy-chain gene, so that the ␮-constant region (C␮) is replaced © 2007 by The National Academy of Sciences of the USA 16910–16915 ͉ PNAS ͉ October 23, 2007 ͉ vol. 104 ͉ no. 43 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0708408104 Downloaded by guest on September 26, 2021 Cells Derived from Patient 15–9BI Show Evidence of Normal SHM but Altered Patterns of S␮–S␣ Junctions in CSR. There was no evidence for a defect in CSR affecting all isotypes in patient 15–9BI. Nevertheless, the absence of any gross abnormalities in B and T cell number suggested that the low serum levels of IgG observed in patient 15–9BI could have been caused by a defect in a component of class switch recombination. To address this pos- sibility, genomic DNA was extracted from a peripheral blood sample of a healthy donor and patient 15–9BI and the S␮–S␣ and S␮–S␥ switch junctions amplified. In normal donors, CSR is characterized by generation of a range of IgA or IgG fragments, which exhibit little or no sequence homology across the switch junctions between S␮ and S␣ or S␥ sequences respectively. Mutations or insertions around the switch break points are observed. Although the number of fragments obtained from 10 PCRs run in parallel were not different between cells from the healthy donor and patient 15–9BI, sequencing across the switch junctions revealed that cells derived from the patient utilized significantly increased levels of microhomology across the break region compared with the control; 94% (15 of 16) of S␮–S␣ switch products amplified Fig. 1. Skin fibroblasts derived from patient 15–9BI are radiosensitive, as measured by colony formation assay. Normal, classical A-T and 15–9BI primary from patient 15–9BI exhibited microhomology of 4 bp or over fibroblasts were plated at low density and irradiated with the doses indicated, ␹2 Ͻ compared with only 17% (3 of 18) in the control; , P 0.001 and the number of surviving colonies were counted. (SI Table 1). A reduced frequency of insertions and mutations around the S␮–S␣ break points was observed in the patient 15–9BI (1 of 16) genetic defect in patient 15–9BI may lie within a previously compared with the control (11 of 18); ␹2, P ϭ 0.001. Analysis of uncharacterized DNA DSBR gene. a restricted number of S␮–S␥3 15–9BI switches revealed a defect similar in the patient to that observed for the S␮–S␣ switch Abnormal Relocalization of DSBR Proteins in RIDDLE Syndrome Cells sequences (data not shown). After IR Exposure. One important aspect of the cellular response Taken together, our analysis of the patient’s switch recombi- to DNA damage is the ability of the repair and checkpoint nation fragments suggested the presence of a defect similar, proteins to efficiently relocalize to sites of damage; this has been although less severe, to that observed in patients with hypomor- clearly demonstrated in mice that lack histone variant, H2AX, phic DNA ligase IV mutations (6). which fail to properly relocalize 53BP1, BRCA1, and Nbs1 to To determine whether the resolution of other developmen- repair foci, subsequently resulting in increased genomic insta- tally programmed DNA DSBs was also affected in the patient, bility and a hypersensitivity to agents that induce DNA DSBs (7, we addressed the status of somatic hypermutation.

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