Abstract Patients with biallelic truncating mutations in PALB2 have a severe form of Fanconi anaemia (FA-N), with a predisposition for developing embryonal-type tumours in infancy. Here we describe two unusual patients from a single family, carrying biallelic PALB2 mutations, one truncating, c.1676_1677delAAinsG;(p.Gln559ArgfsTer2), and the second, c.2586+1G>A; p.Thr839_Lys862del resulting in an in frame skip of exon 6 (24 amino acids). Strikingly, the affected individuals did not exhibit the severe developmental defects typical of FA-N patients and initially presented with B cell non-Hodgkin lymphoma. The expressed p.Thr839_Lys862del mutant PALB2 protein retained the ability to interact with BRCA2, previously unreported in FA-N patients. There was also a large increased chromosomal radiosensitivity following irradiation in G2 and increased sensitivity to mitomycin C. Although patient cells were unable to form Rad51 foci following exposure to either DNA damaging agent, U2OS cells, in which the mutant PALB2 with in frame skip of exon 6 was induced, did show recruitment of Rad51 to foci following damage. We conclude that a very mild form of FA-N exists arising from a hypomorphic PALB2 allele.

Author Summary PALB2 is a protein that creates a molecular bridge that promotes the recruitment of Homologous Recombination Repair (HRR) proteins BRCA1, BRCA2 and Rad51 to sites of DNA damage. Cells with functional loss of PALB2 show a defect in HRR and are characterized by an increased number of spontaneous chromosome breaks and hypersensitivity to cross-linking agents. Typically, inherited mutations in PALB2 are associated with a severe Fanconi Anaemia phenotype. Here we describe for the first time the effect of biallelic mutations of PALB2 in which one hypomorphic allele resulted in a low level of expression of the mutant PALB2 protein with some retained function, as shown by its interaction with BRCA2, as well as its ability to facilitate Rad51 focus formation following damage. This resulted in a considerably milder clinical phenotype with increased longevity compared with biallelic PALB2 null patients and an altered tumour spectrum towards development of lymphoid malignancies.

Citation: Byrd PJ, Stewart GS, Smith A, Eaton C, Taylor AJ, Guy C, et al. (2016) A Hypomorphic PALB2 Allele Gives Rise to an Unusual Form of FA-N Associated with Lymphoid Tumour Development. PLoS Genet 12(3): e1005945. https://doi.org/10.1371/journal.pgen.1005945 Editor: Peter McKinnon, St Jude Children's Research Hospital, UNITED STATES Received: December 4, 2015; Accepted: February 26, 2016; Published: March 18, 2016 Copyright: © 2016 Byrd et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: We thank Cancer Research UK for their continued support (www.cancerresearchuk.org Grant Number C1016/A14346 Grant holder: AMRT). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction BRCA2, PALB2, BRCA1, Rad51 and the Rad51 paralogs form the principal constituents of the homologous recombination (HR) machinery utilised by the cell not only to repair deleterious DNA lesions, such as DNA double strand breaks (DSBs) in an error-free manner, but also to stabilise, protect and restart damaged replication forks. PALB2 [1] functions as a molecular bridge by simultaneously binding both BRCA1 and BRCA2 via its N-terminal coiled-coil domain and C-terminal WD repeats respectively [2–4]. It has been proposed that BRCA1 plays a role in targeting the BRCA2-Rad51 recombination complex to sites of DNA damage directly through its ability to bind PALB2 [3]. However, since PALB2 and Rad51 foci are not completely ablated in the absence of BRCA1 and PALB2 has been shown to bind chromatin directly through its ChAM domain [5], it is likely that PALB2 acts as an essential conduit linking the pro-resection, anti-recombinogenic and the cell cycle checkpoint activities of the various known BRCA1 containing complexes (typified by the presence of RAP80, MERIT40, Abraxas, BRCC36/45, CtIP or FANCJ) with the recombination machinery [6]. It has been known for some time that heterozygosity for a germline mutation in either BRCA1 or BRCA2 strongly predispose women to the development of breast and/or ovarian cancer. More recently, heterozygosity for a germline mutation in PALB2 [7], RAD51C [8] and RAD51D [9] have also been associated with an increased risk of developing breast and ovarian cancer. These observations highlight a critical role for the HR pathway in protecting against cellular transformation, particularly of hormone-responsive, epithelial cells that have a high proliferative capacity. Interestingly, however, it has been shown that biallelic, null germline mutations in BRCA2 [10–14] and PALB2 [15–17], hypomorphic mutations in BRCA1 [18,19] and Rad51C [20], or in the case of RAD51 [21] a dominant mutation, all give rise to a spectrum of clinical symptoms that have features of Fanconi Anaemia (FA). Unlike Fanconi anaemia patients with mutations of one of the core complex Fanconi proteins, those with inherited mutations in the HR machinery, including PALB2 mutant patients, typically display severe developmental abnormalities, such as microcephaly, growth retardation, intellectual impairment as well as skeletal abnormalities. These patients also commonly exhibit kidney malformations, microphthalmia, skin hypo/hyper-pigmentation, hypoplastic thumbs and gonadal dysgenesis, although unlike typical FA patients, bone marrow failure is almost never observed. Cells derived from these patients are almost invariably hyper-sensitive to DNA cross-linking agents, e.g. mitomycin C (MMC) and diepoxybutane, as well as ionising radiation (IR), consistent with a defect in homologous recombination. Given the clinical similarity between FA and the consequences of mutations in HR genes, patients with biallelic mutations in BRCA2, PALB2, BRCA1, RAD51C and the RAD51 dominant mutation have been designated FA-D1, FA-N, FA-S, FA-O and FA-R respectively. The severity of the defects exhibited by HR-defective patients serves to emphasise the importance of the recombination pathway in dealing with DSBs and damaged replication forks that occur naturally with fairly high frequency during development. In contrast to the consequences of mono-allelic mutation of these genes in carriers, biallelic null mutations predispose patients to embryonal tumours, e.g. Wilms’ tumour, neuroblastoma, medulloblastoma and AML in infancy. The different spectrum of tumours that develops in the FA-like patients, compared with carriers heterozygous for a germline mutation, probably arises as a consequence of inactivating HR early in embryonal cells versus inactivation in an already differentiated cell type. Given the rarity of patients with biallelic mutations in genes involved in the HR machinery, the range of the clinical symptoms displayed by the affected individuals is limited. Indeed, all hitherto described FA-N patients have two truncating PALB2 mutations and a very similar, severe clinical presentation. Here we describe a family with two PALB2 mutations where the affected individuals presented with few of the clinical features typically exhibited by FA-N patients. Unlike FA-N individuals who present in infancy, patients in this study presented at an older age and did not exhibit most of the developmental abnormalities associated with this FA complementation group. Interestingly, although the affected patients developed tumours at a relatively early age, these were B cell lymphomas rather than embryonal tumours. We demonstrate that the PALB2 protein produced from one of the mutant alleles carried by these patients retains both its N- and C-terminal domain required to interact with BRCA1 and BRCA2 respectively. We also show that this mutant PALB2 preserves some function compared with previously identified patient-associated mutant PALB2 proteins and allows low level recruitment of Rad51 foci at sites of DNA damage. We propose that the presence of this mutant protein accounts for the milder MMC hyper-sensitivity exhibited by cells from these patients and the lack of defining clinical characteristics. Lastly, the identification of this family implicates PALB2 as a potential suppressor of lymphoid tumourigenesis and raises the possibility that somatic mutations in this gene may contribute to the development of sporadic lymphomas.

Discussion Here we describe an unusual family in which the affected individuals have biallelic mutations in PALB2, yet do not display the clinical features typically associated with the disease. We propose that the mild clinical phenotype exhibited by the affected patients in this family results from the presence of an expressed mutant PALB2 protein that retains a sufficient level of WT activity. PALB2 functions as a molecular bridge linking BRCA1-containing DNA repair complexes with the core HR machinery consisting of BRCA2, Rad51 and the Rad51 paralogs. The C-terminal WD40 domain of PALB2 is the major binding site for BRCA2 and as a consequence is essential for the formation of Rad51 foci in response to DNA damage. The N-terminal coiled-coil domain of PALB2 is required for its ability to interact with BRCA1 [3,4]. However, the functional relevance of this interaction is unclear. It has been reported that BRCA1 targets the PALB2-BRCA2 complex to sites of DNA damage. Whether this occurs directly through its ability to bind PALB2, or indirectly via its role in promoting DNA end-resection, is not known. Unlike loss of BRCA2, compromising BRCA1 expression does not completely abrogate the ability of PALB2 to relocalise to sites of DNA damage or promote Rad51 nucleofilament formation, suggesting that this interaction may be required to increase the efficiency of HR. Despite this, there is conflicting evidence as to whether the BRCA1-PALB2 interaction is actually required for DNA repair or not. Simhadri et al, [24] reported that an engineered triple point mutation in the murine PALB2 gene that reduced its ability to bind BRCA1 gave rise to a mild DNA repair defect. In contrast Xia et al, [15] showed that a C-terminally truncated PALB2 that still retains its BRCA1 binding site could not complement the DNA repair defects exhibited by a FA-N patient-derived cell line. Based on these observations and the fact that we could co-precipitate the T839_K862del but not the Q559RfsTer2 patient-associated mutant PALB2 protein with BRCA2, we suggest that the T839_K862del mutant PALB2 allele retained sufficient function to alleviate the clinical symptoms associated with a PALB2 deficiency. Structural modelling of this hypomorphic patient mutation indicated that, whilst the loss of residues Thr839-Lys862 located proximal to a small hydrophobic pocket within blade 7 of the WD40 repeat domain is likely to destabilise the protein due to loss of several critical hydrogen bonds, repositioning of upstream hydrophobic residues Cys828, His832 and Val836 may in part compensate for the loss of the missing amino acids and provide some increased stability of the mutant protein (Fig 6). As a consequence, expression of this mutant protein, albeit at reduced levels, may be sufficient to alleviate the clinical symptoms and cellular defects exhibited by the affected patients. Interestingly, cells derived from the affected patients from this family do not form Rad51 foci in response to either DNA DSBs or cross-links. In contrast, following either IR or MMC exposure Rad51 foci were observed in U2OS cells that expressed the exon 6 deleted T839_K862del mutant PALB2. Therefore, the T839_K862del mutant PALB2 does retain residual function, but it is not expressed at levels sufficient to result in detectable Rad51 foci in patient cells. Rather, it appears that Rad51 is recruited to sites of DNA damage in these patient cells at a level that is below detection by standard immunofluorescence. The presence of the p.Thr839_Lys862del mutant PALB2 impacts on the ability of the cell to repair DNA damage by HR as shown by the large increase in G2 chromosome damage following IR exposure. The disparity between the IR-induced chromosomal breakage and the colony survival analysis of the affected patient cells is readily explained. The chromosomal analysis assay, described here, predominantly measures G2 phase DSB repair, which will detect defects in both NHEJ and HR. In contrast, the colony survival assay will primarily detect abnormalities in the NHEJ pathway since the DNA damage is induced in an asynchronous culture in which the vast majority of the fibroblasts will be in G1 phase of the cell cycle. Absence of increased radiosensitivity by colony forming assay has been observed in FA-D1 cells [25], although a chromosome assay of FA-D1 lymphocytes exposed at G2 showed them to be very radiosensitive. A similar insensitivity to IR by colony forming ability, but clear increased sensitivity to chromosome damage following G2 irradiation, has also been observed in both fibroblasts and lymphoblasts from patients with Cornelia de Lange Syndrome [26] with a defect in sister chromatid cohesion. Interestingly, our observations, which are consistent with deficient DNA DSB repair as a result of a defect in HR in these hypomorphic PALB2 patients, using the same endpoint (Table 1) show an effect greater than seen in classical ataxia telangiectasia patients. The G2 chromosomal radiosensitivity of our patients with hypomorphic PALB2 mutations is also greater than seen in Fanconi patients with likely mutations in the FA core complex proteins [27]. In contrast, following exposure to MMC the colony forming ability of fibroblasts from patients II-4 and II-5, while impaired compared with normal, appeared not to be as deficient as cells from core complex protein Fanconi patients, PALB2 patients [15,16] or FA-D1 patients [25,26]. Published data for chromosomal sensitivity to MMC does not appear to be available for FA-D1 or FA-N patient lymphocytes compared with core complex patients. We assume that the chromosomal sensitivity to MMC of our hypomorphic PALB2 patient was less than that of a null patient. While null PALB2 (and BRCA2) patients have a defect in both DNA double strand break repair and also the resolution of DNA crosslinks leading to their profound clinical presentation, the presence of the hypomorphic PALB2 mutation alleviates this considerably. The clinical features of the two siblings (II-4 and II-5) shown to have the biallelic PALB2 mutations included abnormally small size, dysmorphic facies and lymphoid tumour development. It is less clear whether the learning difficulty and speech impairment are part of the PALB2 syndrome in this family, because of their occurrence in other family members without biallelic mutation of PALB2. Most strikingly, these sisters did not present in infancy with the much more severe developmental features and embryonal type tumours associated with biallelic truncating mutations of PALB2 or, indeed, biallelic truncating mutation of BRCA2. However, the sisters shared the Fanconi anaemia patient characteristic of small size. The absence of radial aplasia or abnormalities of the thumbs would not preclude a diagnosis of FA, as not all FA patients show these features. Unlike typical Fanconi anaemia patients, however, 80% of whom have a mutation in a core Fanconi anaemia core protein/gene, they did not have aplastic anaemia, a common presentation of FA between ages 5–10 years. Therefore, although some milder clinical features of FA were present, the phenotype of the affected sisters was clearly distinct from either typical PALB2 (FA-N) patients or typical FA. A particularly important aspect of the clinical phenotype of the patients with a PALB2 defect described in this study is that both affected individuals developed B cell non-Hodgkin lymphoma (B-NHL). This tumour type clearly differs from the embryonal-type tumours that normally develop in FA-N or FA-D1 patients. The occurrence of B-NHL in this family could be specific to the particular inherited PALB2 mutation or could arise as a consequence of the slightly older age of the affected patients in this family as compared to most classical FA-N patients. Nevertheless, the presence of a lymphoid tumour in both affected individuals does raise the interesting possibility that PALB2 may represent a novel tumour suppressor of lymphoid malignancies in a manner similar to ATM and NBN.

Methods Cell culture Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCLs) were established from the younger sister (II-5), both parents (I-1 and I-2) and siblings II-3, II-6 and II-7) (Fig 1) as well as control lymphocytes. LCLs were cultured in RPMI 1640 medium (Sigma-Aldrich, Irvine, UK) supplemented with 10% foetal calf serum (FCS). Fibroblast cultures were also established from skin biopsies from both affected patients II-4 & II-5 and cells grown in DMEM with 10%FCS. Assays for γ-irradiation and mitomycin C sensitivity For colony forming assays, serial dilutions of primary skin fibroblasts from a normal individual, the two affected individuals (II-4 & II-5) in the family under investigation and TERT immortalised fibroblasts from an ataxia telangiectasia patient were irradiated with 1 to 5Gy of γ rays. Patient, normal control and Fanconi Anaemia fibroblasts were exposed for 1h to mitomycin C at concentrations of 0.05–0.40μgml-1. Treated fibroblasts were seeded on to a lethally irradiated (35Gy) feeder layer of normal fibroblasts (6x104 cells per 9.0cm dish) in DMEM (Sigma Aldrich) with 10% FCS. Cells were plated in quadruplicate and incubated in 5% CO 2 at 37°C for 18–21 days with medium changing. The medium was removed, colonies fixed with formaldehyde and stained with 0.5% Methylene Blue (Fisher Scientific, Loughborough, UK). The number of colonies on each plate was counted and percentage survival calculated. To assess chromosomal radiosensitivity, whole blood cultures in RPMI 1640 + FCS were stimulated with PHA for 72h and exposed to 1Gy 137Cs γ-rays 4h before harvesting. Colcemid was added 1h prior to harvesting. Chromosome spreads were made using standard methods and chromosome damage analysed by light microscopy. For chromosomal sensitivity to MMC, whole blood cultures were exposed to either 0.01 or 0.05μgml-1 for 30 minutes, washed out and cells harvested 48h later, again with colcemid added 1h prior to harvesting. Immunofluorescence Cells seeded on to coverslips were permeabilized in CSK100 buffer (10 mM PIPES, 20 mM NaCl, 3 mM MgCl 2 , 300 mM sucrose, 0.5% Triton X-100) for 5 min at 4°C and then fixed in 3.6% PFA/PBS for 10 min at 4°C. Coverslips were blocked for 1h in 10% FCS in PBS, incubated for 1h at room temperature with the primary antibody diluted in 2% FCS, washed three times in PBS, and then incubated for an additional 1h with the secondary antibody. Coverslips were washed again three times in PBS and then mounted onto a microscope slide with Vectashield containing DAPI (Vector Laboratories, Burlingame, CA). Primary antibodies used were anti-γH2AX (05–636; Merck Millipore), anti-Rad51 (PC130; Calbiochem), anti-BRCA1 (sc-6954; Santa Cruz Biotechnology), anti-MDC1 (made by G. Stewart), anti-53BP1 (NB100-904; Novus Biologicals), and anti-FANCD2 (sc-20022; Santa Cruz) and anti-RPA2 (NA18; Calbiochem). All secondary antibodies were either Alexa Fluor 488 or Alexa Fluor 594 coupled and purchased from Invitrogen (Carlsbad, CA). Fluorescence images were taken using a Nikon E600 Eclipse microscope 333 equipped with a 60X oil lens, and images were acquired and analysed using Volocity Software 334 v4.1 (Improvision). DNA sequence analysis DNA sequencing was performed on PCR products using the Applied Biosystems BigDye Terminator v3.1 Cycle Sequencing Kit (Part No. 4336917). Sequence analysis of purified products of sequencing reactions was performed using an Applied Biosystems 3500xL Genetic Analyzer. PALB2 sequence variations were designated according to the reference genomic (NG_007406.1), mRNA (NM_024675.3) and protein (NP_078951.2) sequences. Immunoblotting for PALB2 expression and ATM kinase activity assays To analyse the level of PALB2 expression in LCLs, cell pellets were resuspended in UTB buffer (8M urea, 50mM Tris pH 7.5, 150mM β-mercaptoethanol) and lysed on ice by sonication. 50μg of lysate was separated by SDS-polyacrylamide gel electrophoresis and the proteins transferred to nitrocellulose membrane (Pierce). Nitrocellulose strips were subjected to immunoblotting and protein bands visualised using the enhanced chemiluminescence (ECL) system and exposure to Hyper-film (GE Healthcare). PALB2 antibodies used for immunoblotting were rabbit polyclonals A301-246A (Bethyl Laboratories) that recognises an epitope between residues 200–250 of PALB2 and A301-247A that recognises an epitope between residues 675–725 (see Fig 5). Other antibodies used were anti-BRCA2 (OP95; Calbiochem), anti-BRCA1 (OP92; Calbiochem), anti-Rad51 (SC-8349; Santa Cruz), anti-Aprataxin (made in house) and anti-FLAG (F1804; Sigma-Aldrich). To analyse ATM signalling cells were either mock-irradiated or exposed to 5Gy ionising radiation and harvested after 30 minutes. Antibodies used for immunoblotting were: ATM 11G12 (made in house), anti-phospho ATM (S1981) (AF1655; R&D Systems), anti-SMC1 (A300-055A; Bethyl Laboratories), anti-phospho SMC1 (S966) (A300-050A; Bethyl), anti-KAP-1 (A300-274A; Bethyl), anti-phospho KAP-1 (S824) (A300-767A; Bethyl), anti-NBN (ab23996; Abcam), anti-phospho NBN (S343) (ab47272; Abcam), anti-phospho CHK2 (T68) (2661; Cell Signaling Technology, New England Biolabs), anti-CREB (48H2; Cell Signaling), anti-phospho CREB (S121) (NB100-410; Novus Biologicals), anti-γH2AX (05–636; Merck Millipore), anti-H2A (07–146; Merck Millipore), anti-phospho BRCA1 (S1423) (Bethyl), anti-BRCA1 (OP92; Calbiochem), anti-Chk2 (gift from Dr S. Elledge), anti-53BP1 (NB100-904; Novus Biologicals) and anti-phospho 53BP1 (S1778) (2675; Cell Signaling Technology). Creation of stable Flp-In/T-Rex U2OS cell lines Isogenic U2OS cell lines inducibly expressing either WT or mutant FLAG-tagged PALB2 were created using the Flp-In/T-Rex system. WT PALB2, or a series of PALB2 mutants were cloned into the pcDNA5/FRT/TO plasmid containing an N-terminal FLAG tag and then transfected into U2OS cells containing a single FRT recombination site. Transfected cells were selected with hygromycin and blasticidin and then cloned. Expression of the inducible FLAG-tagged PALB2 protein in each of the cell clones was determined by Western blotting. The PALB2 mutants generated in this study were a revertant PALB2 exon 4 deletion (Exon4delRev) and a FA-N patient-derived mutation Y551Ter, both previously described by Xia et al (15) and both exon 6 deletion (p.T839_K862del) (c. 2586+1G>A; p.Thr839_Lys862del) and Q559RfsTer2 (c.1676_1677delAAinsG; p.Gln559ArgfsTer2) identified in our patients. Knockdown of endogenous PALB2 using siRNA A PALB2 siRNA (GGAGAAUAUCUGAAUGACAdTdT) directed against the 3’ UTR region of PALB2 mRNA [4] was used to knockdown endogenous PALB2. Cells were transfected with siRNA using Lipofectamine RNAiMAX Reagent (Invitrogen) according to the manufacturers protocol. Four hours post-transfection 1μg/ml doxycycline was added to the cells to induce exogenous PALB2 protein expression. The PALB2 cDNA sequences cloned in the pcDNA5-FLAG/FRT/TO vector did not contain 3’-UTR sequences and transcripts from these constructs were not targeted by the siRNA. Cells were harvested 48h post-transfection. The efficiency of PALB2 knockdown was determined by Western blotting. Immunoprecipitation Immunoprecipitations were performed using a protocol published previously [28].

Supporting Information S1 Fig. Measurement of ATM signaling in cells from affected siblings. ATM signaling assay on fibroblasts from affected siblings measured by western blotting for both II-4 & II-5 reveals normal ATM signaling as indicated by phosphorylation of ATM targets. https://doi.org/10.1371/journal.pgen.1005945.s001 (PDF) S2 Fig. Intranuclear redistribution of DNA repair proteins in affected siblings. Following exposure to IR or mitomycin C fibroblasts from patients II-4 and II-5 were able to relocalise MDC1, 53BP1, BRCA1, FANCD2 and RPA2 to sites of DNA DSBs marked by γH2AX foci in a manner similar to the normal fibroblast cell line. Fluorescence images were taken using a Nikon E600 Eclipse microscope 333 equipped with a 60X oil lens, and images were acquired and analysed using Volocity Software 334 v4.1 (Improvision). https://doi.org/10.1371/journal.pgen.1005945.s002 (PDF) S3 Fig. Reduced level of BRCA2 in affected sibling. Western blot showing a reduced level of BRCA2 and the absence of full length PALB2 in cells from affected patient II-5 (performed in duplicate). Loading control is aprataxin. https://doi.org/10.1371/journal.pgen.1005945.s003 (PDF) S4 Fig. Doxycycline induced expression of the different PALB2 mutant proteins in U2OS cells. Western blot showing doxycycline induced expression of FLAG-tagged WT PALB2 protein and each of the mutant PALB2 proteins, Y551Ter, Q559RfsTer2, T839_K862del and Exon4delRev following siRNA knockdown of endogenous PALB2 in the same cell cultures as used for the Rad51 immunofluorescence (Fig 7). https://doi.org/10.1371/journal.pgen.1005945.s004 (PDF)

Author Contributions Conceived and designed the experiments: PJB GSS AMRT. Performed the experiments: PJB GSS MRT AS CE AJT CG IE PF JIL RH AWO DJ LD. Analyzed the data: PJB GSS AMRT. Wrote the paper: GSS TS AMRT.