J Clin Immunol DOI 10.1007/s10875-015-0166-0

ASTUTE CLINICIAN REPORT

Symptomatic Males and Female Carriers in a Large Caucasian Kindred with XIAP Deficiency Magdalena Dziadzio 1 & Sandra Ammann 2,3 & Claire Canning 1 & Fiona Boyle 1 & Amel Hassan 4 & Cathy Cale 4 & Mamoun Elawad 5 & Berthe Katrine Fiil 6 & Mads Gyrd-Hansen 6 & Ulrich Salzer 2 & Carsten Speckmann 2,7 & Bodo Grimbacher 1,2

Received: 30 September 2014 / Accepted: 31 March 2015 # Springer Science+Business Media New York 2015

Abstract Purpose X-linked inhibitor of apoptosis (XIAP) deficiency caused by mutations in BIRC4 was originally described in male patients with X-linked lymphoproliferative syndrome type 2 (XLP2). Recent observations have highlighted a critical role of XIAP for the regulation of NOD2 signaling and are probably the molecular basis for increasingly recognized further immune dysregulatory symptoms of XIAP deficient patients, such as inflammatory bowel disease (IBD). We describe a large Caucasian family in which IBD and erythema nodosum (EN) also manifested in female carriers of XIAP mutations. Methods Clinical data and laboratory findings including flow cytometric analysis of XIAP protein expression and sequencing of the BIRC4 gene. NOD2 signaling was investigated by determination of TNFα production in monocytes upon L18MDP stimulation in vitro.

Results The BIRC4 nonsense mutation p.P225SfsX226 was identified as the genetic cause of XIAP deficiency in our family. Surprisingly, clinical symptoms were not restricted to male patients, but also occurred in several female carriers. The most severely affected carrier demonstrated random X-inactivation, leading to a significant expression of mutated XIAP protein in monocytes, and consequently to impaired NOD2 responses in vitro. Conclusion Our report provides further evidence that clinical symptoms of XIAP deficiency are not restricted to male patients. Random X-inactivation may be associated with EN and mild IBD also in female carriers of BIRC4 mutations. Analysis of the X-inactivation pattern reflected by XIAP protein expression can identify such carriers and the analysis of NOD2 signaling by flow cytometry can confirm the functional significance. XIAP expression patterns should be investigated in female patients with a family history of EN and/or IBD.

Magdalena Dziadzio, Sandra Ammann, Carsten Speckmann and Bodo Grimbacher contributed equally and are considered aequo loco. Electronic supplementary material The online version of this article (doi:10.1007/s10875-015-0166-0) contains supplementary material, which is available to authorized users. * Carsten Speckmann [email protected]

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Faculty of Biology, University of Freiburg, Freiburg, Germany

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* Bodo Grimbacher [email protected]

Department of Pediatric Immunology, Great Ormond Street Hospital, London, UK

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Department of Pediatric Gastroenterology, Great Ormond Street Hospital, London, UK

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Department of Immunology, Institute of Immunology and Transplantation, Royal Free Hospital, London, UK

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Ludwig Institute for Cancer Research, University of Oxford, Oxford, UK

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Center for Chronic Immunodeficiency (CCI), University Medical Center, Freiburg, Germany

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Department of Pediatrics and Adolescent Medicine, University Medical Center, Freiburg, Germany

J Clin Immunol

Keywords XIAP . BIRC4 . NOD2 . inflammatory bowel disease . Crohn’s disease . immunodeficiency

Introduction Primary immunodeficiencies (PID) may present with a wide range of clinical phenotypes. This includes susceptibility to infections, but increasingly recognized also variable immune dysregulatory symptoms. Among those inflammatory bowel disease (IBD) has been identified as a hallmark of several recently discovered PID. As summarized by Uhlig et al. [1], IBD may be a clinical feature of a multi-system disease (e.g. chronic granulomatous disease), or the leading clinical manifestation (e.g. IL-10 receptor deficiency). X-linked inhibitor of apoptosis (XIAP) deficiency, caused by mutations in BIRC4, was initially reported in patients with X-linked lymphoproliferative syndrome type 2 (XLP2). However, observations from larger clinical cohorts have highlighted that XIAP deficiency may also present with multiple other inflammatory symptoms, e.g. uveitis, arthritis and IBD [2–8]. In fact, in a recently published European XIAP cohort of 27 patients, IBD was the most common manifestation [4, 9]. Moreover, observations in two independent cohorts identified private variants in BIRC4 in about four percent of male patients with pediatric onset IBD [7, 8]. The phenotype of IBD in XIAP deficiency may resemble Crohn’s disease (CD) and is curable by hematopoietic stem cell transplantation (HSCT) [5, 8–10]. XIAP-deficient patients demonstrate a critical impairment of NOD2 signaling, which may explain the frequent incidence of IBD [11, 12]. Here we describe a Caucasian family with XIAP deficiency due to a BIRC4 nonsense mutation (p.P225SfsX226). The mutation affects the BIR2 domain of XIAP and leads to disruption of NOD2 signaling in monocytes in vitro. Four out of five affected males suffered from severe IBD and five out of six carrier mothers suffered from chronic erythema nodosum (EN) and variable bowel symptoms. One female carrier is so far asymptomatic. The most severely affected carrier with IBD and EN demonstrated significant expression of mutated XIAP protein in her monocytes, leading to impaired NOD-2 responses in vitro.

Case Presentations The family index patient is V.24 (Fig. 1a). He presented at the age of 3 years with severe diarrhea and failure to thrive. He had a strong family history of male relatives with IBD and females with EN (Fig. 1a). At the age of 8 years a colonoscopy showed colitis and a few months later he developed EN over his legs. Intestinal biopsies were compatible with a diagnosis o f C r o h n ’s d i s e a s e ( C D ) ( F i g u r e S 1 ) . Va r i a b l e

immunosuppressive treatment regimens resulted only in transient control of his symptoms. At 9 years of age he developed arthritis of knees and ankles. His condition improved only temporarily under adalimumab and prolonged courses of steroids. He was eventually considered for hematopoietic stem cell transplantation (HSCT) because of an assumed familial X-linked immunodeficiency. He received peripheral blood stem cells from a matched unrelated donor, following reduced intensity conditioning. Cyclosporine was introduced for GvHD prophylaxis. He initally engrafted well, but was readmitted at day +119 with cough, and again arthritis of ankles and knees. Bronchoalveolar lavage could not identify an infectious trigger. He remained neutropenic and had a poor immune reconstitution, despite having 100 % donor chimerism. After stopping cyclosporine, he developed acute skin GvHD and was readmitted at day +220. He developed fatigue, muscle weakness and memory loss. He was diagnosed with acute viral encephalitis with evidence of VZV, HHV6, BK and JC viruses in CSF and died at the age of 12 years as a result of acute renal failure and progressive multifocal leucoencephalopathy. Over the years he was repeatedly tested negative for Epstein barr virus (EBV) by PCR. Positive PCR screens during his last 2 months of life (Table S1) were interpreted as asymptomatic reactivation under immunosuppressive treatment. He never experienced any (partial) HLH epsiodes. Genetic testing carried out from his archived DNA revealed a hemizygous duplication mutation in exon 2 of the BIRC4 gene, causing a frameshift and preterminal termination of the XIAP protein, p.P225SfsX226 (Fig. 1b and c). V.24 is part of the cohort published by Speckmann et al. (patient #9) [9]. V.24 had two brothers: V.25 and V.26. V.25 was well until the age of 2.5 years, when he developed cough and spiking fever. His respiratory status deteriorated despite antibiotic treatment on day three with onset of hepatosplenomegaly. Investigations showed thrombocytopenia, abnormal coagulation and elevated CRP. On day 14 he developed pancytopenia, disseminated intravascular coagulation, multi-organ failure and irreversible cardiac arrest. On that day, adenoviral infection was identified. Retrospectively, the clinical picture was most likely consistent with a virally triggered hemophagocytic lymphohistiocytosis (HLH). He was later confirmed to have the same BIRC4 mutation as his brother V.24. The brother V.26 is 9 years old and well. He tested negative for the BIRC4 mutation. His mother (IV.10), 47 years old, has a history of intermittent gastrointestinal (GI) symptoms since childhood, diagnosed as irritable bowel syndrome. She has no history of EN. Her three sisters (IV.6, IV.8 and IV.9) all have had episodes of EN and GI symptoms, as did their mother (III.4) and their maternal grandmother (II.2). IV.6 (67 years old), first developed EN at the age of 11 years; she had multiple flare-ups until her thirties.

J Clin Immunol Fig. 1 Mutation detection. a Pedigree of family, symbols: circles- female; squares- male; filled squares- affected individual; dotted circles- carrier; slashdeceased, # not tested. b sequence chromatograms of genomic DNA sequences of BIRC4 (encoding XIAP) exon 2, arrow indicates site of duplication. c Protein domain structure of XIAP and localization of the mutation

IV.8 (62 years old) developed EN in her twenties and has experienced several episodes since. She has recurrent GI symptoms, diagnosed as diverticulitis 5 years ago. Flow cytometry analysis of lymphocyte subsets and monocytes from carriers IV.6, IV.8 and IV.10 revealed preferential expression of XIAP wildtype protein and normal NOD2 function in vitro (Fig. 2). In contrast, carrier IV.9 clinically presented with more pronounced inflammatory symptoms, demonstrated a random Xinactivation pattern with expression of mutated XIAP protein in up to 42 % of monocytes, which was associated with markedly reduced NOD2 signalling in vitro (Fig. 2). She is 57 years

old and continues to experience flares of Sweet's syndrome, EN, vasculitis, and IBD (confirmed on a rectal biopsy). III.4 (91 years old) had EN at the age of 20 years and was diagnosed with diverticular disease in her eighties. II.2, who died at the age of 78 from stroke, had an episode of EN at the age of 50 years. Carrier IV.8 has an asymptomatic daughter (V.21, tested negative for the BIR4 mutation) and two sons (V.19 and V.20) with severe IBD since childhood. V.19 died at the age of 16 from fulminant Crohn’s colitis. V.20 is part of the cohort published by Speckmann et al. (patient #8) [9]. Flow cytometry of PBMC from V.20 demonstrated absent XIAP protein

J Clin Immunol B cells

NK cells

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Fig. 2 XIAP expression and function a Overlay of staining with antiXIAP (white) and an isotype control (gray) antibody, gated on CD19+ B cells, CD56+CD3- NK cells, CD3+ T lymphocytes and CD14+ Monocytes. XIAP expression is shown against percentage of maximal analyzed cells in XIAP and isotype sample. V.21: female, non-carrier; V.20: male, symptomatic; IV.8: female, carrier, symptomatic (Erythema nodosum—EN, gastrointestinal—GI); IV.10: female, carrier, symptomatic (GI); IV.6: female, carrier, symptomatic (EN, GI); IV.9: female, carrier, symptomatic (EN, GI). b Example evaluation of the L18-MDP test as published [12]. The percentage indicates the increase of TNFα expression upon monocyte stimulation, calculated by subtracting the percentage of monocytes producing TNFα after incubation in medium alone. Monocytes are gated for CD14 and HLA-

DR. Monocytes from V.20 and IV.9 demonstrated an impaired TNFα production after stimulation with L18-MDP (via NOD2), but normal production after stimulation with LPS (via TLR4). c Summary of results from the L18-MDP stimulation in our family, shown together with previously reported healthy controls, female XIAP carriers and disease controls (XIAP deficient patients)—all indicated by black circles [12]. Disease controls further include the XIAP deficient family patient V.20 (white circle) and a NOD2 deficient patient (grey circle). Carrier IV.9 (white square) demonstrated reduced TNFα production in 2 independent experiments. The other carriers of the family (white symbols) showed a response in range of previously reported carriers [12]. XIAP staining and L18MDP analyses were performed as previously reported [9, 12]

(Fig. 2a) and genetic testing showed the presence of the p.P225SfsX226 mutation. TNFα production by monocytes in response to NOD2 stimulation by muramyl dipeptides (L18-MDP) was severely abrogated in vitro (Fig. 2b and c). V.20 showed asymptomatic EBV seroconversion (including anti-EBNA-1), a recent EBV PCR showed a low-level EBV replication of 4,222 copies/ml in absence of any obvious organ involvement or inflammatory symptoms.

Carrier IV.6 has two daughters (V.14 and V.15), both healthy. V.15 tested negative for XIAP, V.14 was not tested. IV.6 has one son (V.16) aged 31, who does not wish to be tested. He has had severe IBD since infancy, poorly responding to steroids and underwent ileostomy at age 17 followed by further surgical procedures at the age of 24. He was treated with 6-mercaptopurin and infliximab with benefit. As V.20 he subsequently developed generalized boils.

J Clin Immunol

Infliximab was changed to Adalimumab and his boils have improved. Carrier IV.9 has one daughter (V.23) who has just been identified as a carrier and who is currently asymptomatic, and one son (V.22). V.22 (30 years old), tested positive for the XIAP mutation. He had one episode of an unusual febrile illness at the age of 15 and suffers from acne and folliculitis. Supplementary table S1 provides a comparative overview on the clinical phenotypes of male and female patients from the reported family. A more detailed description of the patients’ phenotype is deposited as supplement S2 in the online repository of the Journal.

Discussion We describe our observations in a family with XIAP deficiency, illustrating the clinical heterogeneity of this immune dysregulatory disease. IBD was a key manifestation in male patients, which is in agreement with other recent reports [3, 5, 7–9]. Only one out of six patients (V.25) developed a virally triggered XLP-like phenotype. The identified nonsense mutation in our family (p.P225SfsX226) is located within the BIR2 domain of XIAP, a region which is known to be critical for the interaction of XIAP with RIPK2 and signaling via the NOD2complex [11]. Indeed, TNFα production in patient derived monocytes in response to NOD2 stimulation by muramyl dipeptides (L18-MDP) was markedly reduced and in range of a NOD2-deficient patient with CD and previously reported XIAP patients [12]. Normal TNFα production upon TLR4 stimulation with lipopolysaccharide (LPS) indicated, that this finding was truly NOD2-specific and not due to a general impairment of monocyte activation or cytokine production. These observations provide further evidence that impaired NOD2 signaling is a driving pathophysiological mechanism of XIAP deficiency. Aside from CD, this may also explain other auto-inflammatory disease manifestations such as arthritis, uveitis or EN [9]. As previously shown by us, the p.P225SfsX226 mutation also results in an increased activation induced cell death (AICD) of patient-derived T-cell blasts in vitro, suggesting that the mutation also affects the antiapoptotic properties of XIAP [9]. Onset of disease and clinical courses were highly variable amongst our patients. This illustrates the absence of genotype/ phenotype correlation in XIAP deficiency, which is in line with previous observations [3, 6, 9, 13]. While HSCT may be advocated for severely affected patients, the complicated course of V.24 and several other patients summarized by Marsh et al. warrants caution with this procedure [10]. The evolving knowledge on how XIAP regulates inflammatory responses may identify potential conservative treatment options. In this regard, recent murine data suggests that antiTNFα treatment might be a targeted therapeutic option for

inflammatory complications in XIAP deficiency [14], and has indeed resulted in transient improvement of IBD, arthritis and boils in some of our patients. However, in other patients of our previously published cohort, anti-TNFα treatment was ineffective [9]. As XIAP deficiency is an X-linked inherited disorder, a survival advantage of XIAP wild-type expressing leukocytes has been suggested [8]. Therefore, female carriers usually demonstrate non-random X-chromosome inactivation and do not present clinical symptoms [2]. Surprisingly, inflammatory symptoms were not restricted to males in our family, but also occurred in several female carriers. Five out of seven carriers suffered from relapsing EN and variable bowel symptoms. The most severely affected carrier (IV.9) revealed a random Xinactivation pattern in her monocytes, which resulted in significant expression of mutated XIAP protein and reduced NOD2 signalling in vitro. Four carriers evidenced preferential expression of the wild-type protein in their leukocytes, displayed a normal TNFα response of monocytes upon L18-MDP stimulation and presented only mild bowel symptoms. Samples from the remaining three carriers (II.2, III.4, V.23) were not available. Recently, Aguliar et al. also described two unrelated symptomatic XIAP carriers where random X-inactivation led to a severely reduced XIAP expression in leukocytes, impaired NOD2 signalling and a clinically significant IBD [8]. This supports our interpretation that random X-inactivation can lead to inflammatory symptoms in female carriers of BIRC4 mutations. Whether the severity of their clinical presentation depends on the degree of X-inactivation and the corresponding residual expression of XIAP protein and function in female carriers has to be confirmed in larger systematic studies. Finding the molecular cause in this family with an X-linked disease was a decade-long diagnostic challenge. During the work-up we learned that flow cytometry enables a rapid and inexpensive screening for XIAP deficiency [9, 15]. In addition, our recently described L18-MDP test is a valuable additional diagnostic screen, which is more robust and diseasespecific than AICD analysis and also identifies those patients with residual protein expression due to missense mutation [12]. We encourage the use of these diagnostic tools in a wide clinical range of familial autoinflammatory conditions, not to miss this probably still under-diagnosed disease. Acknowledgments Prof. Stephan Ehl from the CCI in Freiburg, who pointed us towards the possible involvement of XIAP in this family. We sincerely thank all our patients and their family members who made this study possible and the referring consultants (Dr Claire Dolling and Dr Yusuf Karim) for their help. We also thank the technicians of the Center of Chronic Immunodeficiency Advanced Diagnostic for excellent technical assistance. Funding This study was supported by the German Federal Ministry of Education and Research (BMBF 01 EO 0803 and and BMBF 01GM1111B). The authors are responsible for the contents of this publication.

J Clin Immunol Financial Disclosure There is no financial disclosure.

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Conflict of Interest The authors have no conflicts of interest. 9.

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