and Immunity (2014) 15, 190–194 & 2014 Macmillan Publishers Limited All rights reserved 1466-4879/14 www.nature.com/gene

SHORT COMMUNICATION SPAG7 is a candidate for the periodic fever, aphthous stomatitis, pharyngitis and adenopathy (PFAPA) syndrome

S Bens1, T Zichner2, AM Stu¨tz2, A Caliebe1, R Wagener1, K Hoff1,3, JO Korbel2, P von Bismarck4 and R Siebert1

Periodic fever, aphthous stomatitis, pharyngitis and adenopathy (PFAPA) syndrome is an auto-inflammatory disease for which a genetic basis has been postulated. Nevertheless, in contrast to the other periodic fever syndromes, no candidate genes have yet been identified. By cloning, following long insert size paired-end sequencing, of a de novo chromosomal translocation t(10;17)(q11.2;p13) in a patient with typical PFAPA syndrome lacking mutations in genes associated with other periodic fever syndromes we identified SPAG7 as a candidate gene for PFAPA. SPAG7 is expressed in tissues affected by PFAPA and has been functionally linked to antiviral and inflammatory responses. Haploinsufficiency of SPAG7 due to a microdeletion at the translocation breakpoint leading to loss of exons 2–7 from one allele was associated with PFAPA in the index. Sequence analyses of SPAG7 in additional patients with PFAPA point to genetic heterogeneity or alternative mechanisms of SPAG7 deregulation, such as somatic or epigenetic changes.

Genes and Immunity (2014) 15, 190–194; doi:10.1038/gene.2013.73; published online 23 January 2014 Keywords: SPAG7; PFAPA; periodic fever; chromosomal translocation

INTRODUCTION of 84 (45%) patients there was a family history of recurrent fever.9 Periodic fever, aphthous stomatitis, pharyngitis and adenopathy The high frequency of a recurrent fever other than PFAPA in the (PFAPA) syndrome is an auto-inflammatory disorder first described families of the patients is in line with the observation that patients by Marshall et al. in 1987.1 It is characterised by fever episodes with PFAPA syndrome show an increased probability to be carriers lasting 3–6 days with a recurrence every 3–8 weeks, exudative of heterozygous mutations in one of the genes associated with tonsillitis, cervical adenopathy and malaise in all and aphthae in the other familial periodic fever syndromes.10 In particular, the majority of patients.2–5 Headache, abdominal pain and heterozygous mutations in the MEFV gene associated with FMF arthralgia are also recurrently observed. are frequent in PFAPA patients. Such MEFV mutations might Owing to the recurrent fever episodes PFAPA has been assigned modify the clinical course of the disease.11 to the clinical spectrum of (periodic) fever syndromes. These also Overall, molecular data, along with the absence of a clear include familial Mediterranean fever (FMF MIM #134610/#249100, monogenetic trait despite significant familial clustering, might associated gene MEFV*608107), TNF receptor-associated periodic point to a polygenetic or multifactorial origin of PFAPA rather than fever syndrome (TRAPS #142680, gene TNFRSF1A*191190), hyper- a monogenetic predisposition. Part of this predisposition might be immunoglobulinaemia D (HIDS #260920, gene MVK*251170), conferred by sequence variants in inflammasome-associated cryopyrin-associated periodic syndrome (CAPS) including familial genes like NLRP3. These have been described in B20% of cold auto-inflammatory syndrome (FCAS1 #120100, gene patients with PFAPA.10 Such variants might increase the NLRP3*606416, and FCAS2 #611762, NLRP12*609648), Muckle– probability of dysregulated interleukin 1b production by Wells syndrome (MWS #191900, gene NLRP3*606416) and monocytes during febrile episodes typical for PFAPA neonatal onset multisystem inflammatory disease (NOMID/CINCA syndrome.10 Overall, the hitherto published functional studies #607115, gene NLRP3*606416).6,7 Remarkably, all of these fever point to a pathophysiologic model of PFAPA syndrome in which syndromes are monogenetically inherited disorders for which the microbial triggers activate a cascade that begins with the innate responsible genes have been identified. Interestingly, this does immune system and ultimately recruits activated T cells to the not hold true yet for PFAPA syndrome. periphery.12 Thus, PFAPA syndrome could be due to an abnormal Despite the current lack of a candidate gene for PFAPA adaptive immune response to viral or other infectious agents, syndrome, various studies point to the fact that this auto- likely at the level of lymphoid organs, able to induce a rapid inflammatory disease is not a sporadic disorder but based on a activation of cells of innate immunity.8,13 This infectious-origin genetic predisposition.8 Major epidemiologic evidence for this hypothesis at the level of lymphoid organs would be well in line assumption comes from the observations by Cochard et al.9 These with the histology of tonsils of patients with PFAPA syndrome, authors showed that a significant percentage of PFAPA patients which shows non-specific chronic inflammation, characterised by presents a positive family history of recurrent fever in general and lymphoid and follicular immunoblastic hyperplasia, focal of PFAPA in particular.9 Indeed, in this study, 10 of 84 (12%) histiocytic clusters, hyalinising fibrosis, crypt abscesses and patients had a relative affected with PFAPA syndrome. Even in 38 keratinising debris.14

1Institute of Human Genetics, Christian-Albrechts-University Kiel & University Hospital Schleswig-Holstein, Kiel, Germany; 2European Molecular Biology Laboratory (EMBL), Genome Biology Research Unit, Heidelberg, Germany; 3Department of Congenital Heart Disease and Pediatric Cardiology, Christian-Albrechts-University Kiel & University Hospital Schleswig-Holstein, Kiel, Germany and 4Department of Pediatrics, Christian-Albrechts-University Kiel & University Hospital Schleswig-Holstein, Kiel, Germany. Correspondence: Dr S Bens, Institute of Human Genetics, Christian-Albrechts-University Kiel & University Hospital Schleswig-Holstein, Campus Kiel, Arnold-Heller Str. 3 (Haus 10), D-24105 Kiel, Germany. E-mail: [email protected] Received 9 November 2013; revised 15 December 2013; accepted 16 December 2013; published online 23 January 2014 SPAG7 in PFAPA syndrome S Bens et al 191 It has recently been shown that cloning of breakpoints of de analyses on the peripheral blood of the parents novo balanced chromosomal translocations in patients with revealed normal karyotypes in both mother and father. Thus, the phenotypic abnormalities of unknown origin can lead to the t(10;17) was a de novo event in the patient. Given that both identification of disease-causing genes.15,16 We have adapted this parents are generally healthy and particularly have neither signs of strategy here to PFAPA and characterised the breakpoints of a de PFAPA syndrome nor any other reported autoimmune or auto- novo chromosomal translocation in a patient with PFAPA inflammatory disease, the de novo appearance of both PFAPA syndrome without family history of recurrent fevers and lacking syndrome and the translocation might point to a causal relation- mutations in known genes associated with recurrent fever ship. Indeed, it has been shown that carriers of a de novo balanced syndromes, with the use of massively parallel DNA sequencing. chromosomal rearrangement have a two- to threefold increased By this approach we have identified herein SPAG7 as a novel risk for congenital anomalies.15,17 Consequently, cloning the candidate gene for PFAPA syndrome. The finding of de novo breakpoints of such de novo balanced chromosomal haploinsufficiency of the SPAG7 gene in the patient, the rearrangements in patients with phenotypic abnormalities of expression of the SPAG7 protein in tonsils and lymph nodes, as unknown origin has been successfully applied to identify disease- well as the evidence for its involvement in antiviral and causing genes.15,16 inflammatory responses are in full agreement with the above- In order to exclude a submicroscopic chromosomal imbalance outlined models of PFAPA pathogenesis. related or unrelated to the chromosomal translocation we performed array-comparative genomic hybridisation with a functional resolution of 0.1 Mb. Besides well-known copy number RESULTS AND DISCUSSION variations, no imbalances were detected at the resolution applied. Aiming at identifying potential genetic causes of PFAPA syndrome In particular, we did not detect any imbalances around the we investigated whether a chromosomal aberration had been cytogenetically assigned breakpoints. Moreover, pathogenic diagnosed in any of the patients presented at our institution with sequence variants in genes associated with periodic fever this auto-inflammatory disorder. Using this approach, we identi- syndromes or auto-inflammatory diseases, that is, MEFV, NLRP3, fied a girl with typical clinical features of PFAPA syndrome in NLRP12, ELA2, TNFRSF1A, MVK and CARD15, as a cause or modifier whom prior to this diagnosis a chromosome analysis on peripheral of the phenotype were excluded. blood lymphocytes had been performed due to growth retarda- Given the cytogenetically and molecular cytogenetically tion, facial dysmorphisms and dysplastic brachymesophalangy V balanced-appearing change and the lack of mutations in genes (for a detailed clinical description see the clinical synopsis in associated with periodic fever syndromes, we wondered whether Materials and methods). G-banded karyotyping at 400 bphs the translocation might lead to disruption or otherwise deregula- revealed a cytogenetically balanced translocation between the tion of a gene potentially associated with PFAPA. Thus, we aimed long arm of one chromosome 10 and the short arm of one at cloning the fusions between 10 and 17 on both chromosome 17 in all 20 analysed metaphases (Figure 1). The derivative chromosomes, der(10) and der(17). To this end, we karyotype was described as: 46,XX,t(10;17)(q11.2;p13). Remarkably, generated a long insert size paired-end18 (mate-pair) library with a

Figure 1. Cytogenetic and molecular characterisation of the translocation t(10;17)(q11.2;p13)dn. (a) Partial karyotype showing chromosomes 10 and 17 of the index patient with PFAPA and t(10;17). The normal chromosomes 10 and 17 are shown on the left side of each pair. The derivative chromosomes 10 and 17, respectively, of the t(10;17)(q11.2;p13) are shown on the right side of each pair. (b) Schematic representation of the results of sequence analysis of the translocation. Top and bottom ideograms of normal chromosomes 10 (grey) and 17 (violet), respectively. The middle panel shows the molecular junctions with the der(10) junction on the left and the der(17) junction on the right side. Deletions on both chromosomes at the breakpoint regions (middle part) lead to loss of most of the SPAG7 gene from chromosome 17. Numbers give breakpoint mapping coordinates in base pairs. Genes (blue) are indicated in the respective exon–intron structure, with arrows indicating transcriptional direction. (c) Confirmation of the breakpoint junctions by Sanger sequencing. The middle part shows the derivative chromosomes 10 and 17 with the annotated breakpoints. Top and bottom panels show electropherograms from Sanger sequencing on both derivative chromosomes with the derivation of the sequences and the breakpoints indicated.

& 2014 Macmillan Publishers Limited Genes and Immunity (2014) 190 – 194 SPAG7 in PFAPA syndrome S Bens et al 192 median insert size of 4600 bp, followed by sequencing the library variants were detected. Thus, the haploinsufficiency of SPAG7 using 36 bp reads. After mapping and quality filtering, 24.1 million might be associated with PFAPA in the proposita. non-redundant read pairs remained, resulting in a physical SPAG7 encodes the sperm-associated antigen 7 (aliases ACRP, genome coverage of 38.7x. High-confidence structural variation FSA-1, MGC20134; MIM*610056). The gene has been identified by calls for inter-chromosomal variations were identified and filtered sequencing cDNAs from CD34 þ hematopoietic stem cells as a against germline variants identified in samples of the 1000 homologue of fox sperm acrosomal protein-1.21,22 It is highly genomes project19 and an additional whole-genome sequencing conserved and encodes a protein with 227 amino acids study.20 By this approach, high-confidence balanced fusions of (NP_004881), which is widely expressed. Given the typical chromosomes 10 and 17 near the cytogenetically assigned presentation of PFAPA syndrome, nuclear expression in cells of breakpoints were inferred for both derivative chromosomes. The lymph nodes and tonsils is notable (http://www.proteinatlas.org). junctions were amplified by PCR using different primer The SPAG7 protein contains two nuclear localisation signals (AA35– combinations and validated by Sanger sequencing (Figure 1). This 51 and 122–139) and a R3H domain (AA46–109) named from the resulted in the exact definition of the fusion breakpoints: on the characteristic spacing of the most conserved arginine and histidine der(10) chromosome, chr10:43,844,710 was fused to residues.23 This domain is predicted to bind ssDNA or ssRNA in a chr17:4,861,900. On the der(17), chr17:4,867,779 was fused to sequence-specific manner.23 This might indicate a potential role of chr10:43,850,287 (all hg19, see Figure 1). At both junctions there the protein in response to viruses or free (cellular) DNAs or RNAs. In was one base identical from both chromosomes. Remarkably, on this context it is remarkable that SPAG7 was shown by array- and the molecular level the translocation is not balanced but rather qPCR-based analyses to be significantly overexpressed in RNA from leads to loss of genetic material on both affected chromosomes, peripheral blood mononuclear cells from 57 parvovirus B19- that is, a deletion of 5577 bp from chromosome 10 and of 5879 bp seropositive as compared with 13 parvovirus B19-seronegative from chromosome 17. These losses could also be identified in donors, pointing to a role in reaction to viral infections.24 read-depth analyses of the short read data (Figure 2), which Given the identification of haploinsufficiency of the SPAG7 gene indicates that the respective genomic material is not integrated in the index patient along with expression of the encoded protein elsewhere in the genome. in lymphatic tissues affected in PFAPA syndrome and functional Whereas the breakpoints and the associated deletion on links of SPAG7 to antiviral and inflammatory responses, we chromosome 10 affect no gene, the deletion in chromosome 17 consider SPAG7 a reasonable candidate for PFAPA syndrome. results in the loss of exons 2–7 and, thus, most of the coding Thus, we investigated whether other patients with this syndrome region of the SPAG7 gene (Figure 1). In order to investigate might also show alterations of the protein. To this end, we whether the second allele of SPAG7 was also altered in the patient, sequenced all exons and exon–intron junctions of the SPAG7 gene we sequenced all exons and exon–intron boundaries of this gene in four additional patients fulfilling the typical clinical criteria for in the patient. Besides known polymorphisms no sequence PFAPA syndrome (Table 1); one was of Turkish (ID 2) and three of German (ID 3–5) descent. Moreover, three patients had a positive family history for febrile episodes during early childhood (Table 1). The Turkish patient lacked a family history but was found to be heterozygous for the mutation c.2080A4G (p.Met694Val) in the MEFV gene associated with FMF. In this small cohort of four patients we did not find any potentially pathogenic mutations in the SPAG7 gene. In summary, through cloning of a molecularly unbalanced de novo chromosomal translocation we identified SPAG7 as a candidate gene for PFAPA. Nevertheless, our analyses on additional patients failed to identify recurrent haploinsufficiency though the cohort was limited in size. We cannot exclude somatic alterations of SPAG7 in the affected tissues or epigenetic changes leading to SPAG7 deregulation in these patients. Alternatively, and in line with previous reports, the syndrome might be genetically heterogeneous and associated with a polygenetic predisposition. In this context, it is notable that the SPAG7 protein has been shown to physically interact with the ABHD16A (abhydrolase domain containing 16A) protein by Y2H screening.25 ABHD16A is encoded by the BAT5 gene located in the HLA gene cluster. Remarkably, the BAT5 locus has also been linked to auto- inflammatory diseases like Kawasaki syndrome.26 This might point to a group of interacting involved in the pathogenesis of auto-inflammatory disorders.

MATERIALS AND METHODS Patient cohort Figure 2. Read-depth plot showing the deletions at the breakpoint The index patient presented with a ‘typical PFAPA’ syndrome.27 The girl junctions in chromosomes 10 and 17. The dark-grey bars represent (Table 1 ID 1) was 5 years old when she experienced first febrile episodes the number of sequencing reads mapping between the breakpoints with intermittent body temperatures between 39 and 40 1C. During the on chromosome 10 (5577 bp region) and chromosome 17 (5879 bp first two years the febrile temperatures lasted between 3 and 5 days region). The light-grey bars represent the number of sequencing without any concomitant symptoms and recurred with an interval of 8–12 reads mapping into adjacent regions of identical size up- and weeks. The girl was asymptomatic in between the fever episodes. An downstream of the breakpoints. The lower number of reads infectious disease, cyclic neutropenia and malignancies or a monogenic between the breakpoints compared with the surrounding regions hereditary fever syndrome were excluded and a symptomatic therapy with supports a heterozygous deletion on both chromosomes. antipyretic drugs was established. In the third year of the disease the girl

Genes and Immunity (2014) 190 – 194 & 2014 Macmillan Publishers Limited SPAG7 in PFAPA syndrome S Bens et al 193 Table 1. Clinical description of patients with PFAPA analysed in this study

ID Sex Age at Length of Interval PFAPA Additional Resolution of Positive family Genetic Analysis onset febrile between symptoms symptoms during episodes to history for Hereditary periodic (years) episodes episodes episodes therapy with: recurrent fever syndrome (days) (weeks) febrile episodes

1 F 5 4–5 4–8 PF, A, P, À Cimetidine À normal index LA 2 F 2,5 4–6 3–6 PF, A, P, Abdominal pain tonsillectomy À (a) LA 3 F 3 3–5 8–10 PF, A, LA À Colchicine þ (father) (b) 4 F 9 4–5 2–4 PF, P, LA Abdominal pain, Cimetidine þ (father) (b, c) occ. vomiting 5 M 1,5 4–6 3–4 PF, A, P, À Colchicine þ (father) not done LA Abbreviations: A, aphthous stomatitis; F, female; LA, cervical lymphadenitis; M, male; occ, occasionally; P, pharyngitis; PF, periodic fever. Clinical characteristics of all five patients diagnosed with PFAPA syndrome. Age at onset corresponds to the start of febrile episodes. Hereditary periodic fever syndrome (HPF) was excluded by absence of typical clinical signs for HPF (skin rash, arthritis, serositis, splenomegaly, fever episodes longer than 7 days, hearing loss and symptoms secondary to cold exposure). Patient 2 had severe abdominal pain and an ethnic origin from Turkey, which led to a molecular testing for FMF. (a) This patient had a heterozygous mutation in the MEFV gene c.2080A4G (p.Met694Val). (b) Molecular testing of TNFRSF1A exons 2, 3, 4 and 6 including the corresponding exon–intron junctions, with normal results. (c) Molecular testing of MVK exons 9 and 11 including the corresponding exon–intron junctions, with normal results.

developed 2–3 days prior to febrile episodes abdominal pain, nausea and, the manufacturer’s instructions. Pooled peripheral blood DNA from 10 with the febrile temperature, an aphthous stomatitis. In the later course healthy donors with a female karyotype served as hybridisation control. she suffered additionally from cervical lymphadenitis and pharyngitis Analysis was performed using the CGH Analytics Software (Agilent during the days with fever. Oral prednisolone caused immediate fever Technologies). Regions that were determined as significantly deviated by reduction within 2 h but shortened the asymptomatic interval (2–3 weeks). the software and that included more than 10 single oligonucleotides were The diagnosis of PFAPA syndrome was made 28 months after the regarded as aberrant, thus reaching a functional resolution of around commencement of primary symptoms. A therapy with Cimetidine 0.1 Mb. The differentiation between pathologic and benign copy number (20 mg per kg per day) was started, which led to a complete cessation of variations was performed by using a database integrated into the software. fever episodes. The medication was tapered after one and a half year, which led to recurrence of febrile episodes. Prior to this auto-inflammatory Mate-pair structural rearrangement analysis disease the girl was presented to our hospital with signs of mild growth retardation. Her parents were Caucasian, overall healthy and not related. Mate-pair (MP, or long insert size) DNA library preparation was carried out The girl had during her first two years of life a proportional growth rate using the v2 protocol from Illumina Gmbh (Munich, Germany). In brief, shortly below the third percentile. A hypothyreosis and a metabolic 10 mg of genomic DNA was fragmented using a Covaris S2 instrument and disorder were excluded. Further mild stigmata like an ocular hypertelorism red miniTUBE (LGC Genomics Ltd., Hoddesdon, UK) with the manufac- B and short bones as well as dysplastic brachymesophalangy V of the hand turer’s recommended settings to 5 kb, followed by size selection led to karyotyping. through agarose gel excision. Deep sequencing was carried out with an In addition, four further children who fulfilled the typical clinical criteria Illumina GAIIx (2 Â 36 bp paired-end mode) instrument to reach an average for PFAPA syndrome were selected for molecular genetic testing. All physical coverage of at least 30 Â . Mate-pair reads were aligned to the except one had recurrent febrile episodes from an early age (p5 years). hg19 assembly of the human reference genome using the Illumina- None of the patients had signs of an infection during the episodes but provided alignment software (ELAND, version 2). Rearrangements based on mate-pair data were identified using the DELLY program.28 Structural presented at least one or two of the following symptoms: aphthous 19 stomatitis, cervical lymphadenitis or pharyngitis. The children were rearrangement calls that were also inferred from 1000 genomes project genome data or germline data of additional whole-genome sequencing additionally completely asymptomatic between the febrile episodes. Cyclic 20 neutropenia or malignancies (leukemia or neuroblastoma) were excluded. samples were removed to exclude polymorphic germline structural Three patients had a positive family history (one of the parents) for febrile variants as well as rearrangement calls caused by mapping artefacts. episodes during early childhood. In one patient (Table 1 ID: 2) we found a Furthermore, rearrangement calls with less than four supporting read pairs heterozygous mutation in the MEFV gene (c.2080A4G[p.Met694Val]). This as well as supporting pairs with an average mapping quality o20 were patient presented, except for recurrent fever, no further signs of FMF (rash, excluded. arthritis, serositis). All children were initially treated with Cimetidine; To assess whether the genomic regions between the translocation second-line therapy was Colchicine. One child who did not respond to the breakpoints on chromosome 10 and 17 are lost as a consequence of the medical therapy received a tonsillectomy. The clinical characteristics of all rearrangement, the number of reads that map within these regions was patients with PFAPA syndrome are summarised in Table 1. counted and compared with neighbouring regions of identical size All patients agreed to the study and the parents gave written informed (5577 bp on chromosome 10; 5879 bp on chromosome 17). consent. The procedures of the study were approved by the Ethics Commission of the Medical Faculty of the Christian-Albrechts-University in PCR and Sanger sequencing Kiel (AZ D401/08 and amendment 08 December 2011). For PCR amplification of the translocation breakpoint a primer walking strategy was applied. Primers leading to amplification of the breakpoint and respective sequencing primers are listed in Supplementary Table 1. For Karyotying and molecular karyotyping sequencing of the SPAG7 gene, primers flanking all exon–intron junctions Karyotyping was performed on phytohemagglutinin-stimulated cultures of of the gene were designed as outlined in Supplementary Table 1. PCR was peripheral blood using G-banding analyses according to standard performed according to standard protocols. Direct sequencing of the PCR techniques. Metaphases were captured using the IKAROS software products using BigDye Terminator technology (BigDye Terminator v1.1 (MetaSystems, Altlussheim, Germany). Karyotypes were described Cycle Sequencing Kit, Applied Biosystems, Foster City, CA, USA) was according to ISCN2013. DNA was extracted from the peripheral blood performed using the same primers as for PCR if not otherwise indicated of all patients using standard methods. Array-comparative genomic (Supplementary Table 1) on an ABI 3100 Genetic Analyzer (Applied hybridisation using the CGH microarray 244A platform Biosystems). Alternatively, sequencing at GATC Biotech AG (Constance, (Agilent Technologies, Santa Clara, CA, USA) was performed according to Germany) was performed.

& 2014 Macmillan Publishers Limited Genes and Immunity (2014) 190 – 194 SPAG7 in PFAPA syndrome S Bens et al 194 CONFLICT OF INTEREST 13 Caorsi R, Pelagatti MA, Federici S, Finetti M, Martini A, Gattorno M. Periodic fever, The authors declare no conflict of interest. aphthous stomatitis, pharyngitis and adenitis syndrome. Curr Opin Rheumatol 2010; 22: 579–584. 14 Peridis S, Pilgrim G, Koudoumnakis E, Athanasopoulos I, Houlakis M, Parpounas K. ACKNOWLEDGEMENTS PFAPA syndrome in children: a meta-analysis on surgical versus medical treat- ment. Int J Pediatr Otorhinolaryngol 2010; 74: 1203–1208. We thank the technical staff of the cytogenetic and molecular genetic laboratories of 15 Higgins AW, Alkuraya FS, Bosco AF, Brown KK, Bruns GA, Donovan DJ et al. the Institute of Human Genetics for expert assistance, as well as all clinicians involved Characterization of apparently balanced chromosomal rearrangements from in the care of the patients for support and all individuals for their participation. JOK the developmental genome anatomy project. Am J Hum Genet 2008; 82: 712–722. was supported by an Emmy Noether Fellowship (KO 4037/1-1) from the German 16 Cacciagli P, Haddad MR, Mignon-Ravix C, El-Waly B, Moncla A, Missirian C et al. Research Foundation and RW is recipient of a stipend from the KinderKrebsInitiative Disruption of the ATP8A2 gene in a patient with a t(10;13) de novo balanced Buchholz/Holm-Seppensen. RS is a member of the Excellence Cluster ‘Inflammation translocation and a severe neurological phenotype. 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Supplementary Information accompanies this paper on Genes and Immunity website (http://www.nature.com/gene)

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