YCLIM-07457; No. of pages: 9; 4C: Clinical Immunology (2015) xx, xxx–xxx
available at www.sciencedirect.com
Clinical Immunology
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Shoshana Revel-Vilk a,⁎,1 , Ute Fischer b,1 , Barbel Keller c , Schafiq Nabhani b , Laura Gámez-Díaz c , Anne Rensing-Ehl c , Michael Gombert b , Andrea Hönscheid b , Hani Saleh d , Avraham Shaag e , Arndt Borkhardt b , Bodo Grimbacher c , Klaus Warnatz c,2 , Orly Elpeleg e,2 , Polina Stepensky a,2
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Pediatric Hematology/Oncology and Bone Marrow Transplantation, Hadassah-Hebrew University Medical Center, Jerusalem, Israel Department of Pediatric Oncology, Hematology and Clinical Immunology, University Children’s Hospital, Medical Faculty, Heinrich-Heine-University, Düsseldorf, Germany c Center for Chronic Immunodeficiency, University Medical Center and University of Freiburg, Freiburg, Germany d Pediatric Hemato-Oncology Unit, Augusta Victoria Hospital, Jerusalem, Israel e Monique and Jacques Roboh Department of Genetic Research, Hadassah, Hebrew University Medical Center, Jerusalem, Israel
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Received 10 November 2014; accepted with revision 19 April 2015
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Autoimmune lymphoproliferative disorder; Immunodeficiency
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Abstract Mutations in LPS-responsive and beige-like anchor (LRBA) gene were recently described in patients with combined immunodeficiency, enteropathy and autoimmune cytopenia. Here, we extend the clinical and immunological phenotypic spectrum of LRBA associated disorders by reporting on three patients from two unrelated families who presented with splenomegaly and lymphadenopathy, cytopenia, elevated double negative T cells and raised serum Fas ligand levels resembling autoimmune lymphoproliferative syndrome (ALPS) and one asymptomatic patient. Homozygous loss of function mutations in LRBA were identified by whole exome analysis. Similar to ALPS patients, Fas mediated apoptosis was impaired in LRBA deficient patients, while apoptosis in response to stimuli of the intrinsic mitochondria mediated apoptotic pathway was even enhanced. This manuscript illustrates the phenotypic overlap of other primary immunodeficiencies with ALPS-like disorders and strongly underlines the necessity of genetic diagnosis in order to provide early correct diagnosis and subsequent care. © 2015 Published by Elsevier Inc.
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Autoimmune lymphoproliferative syndrome-like disease in patients with LRBA mutation
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⁎ Corresponding author at: Pediatric Hematology/Oncology Department, Hadassah – Hebrew University Hospital, POB 12000, Jerusalem, 91200, Israel. Fax: +972 2 6777833. E-mail address: [email protected] (S. Revel-Vilk). 1 The first two authors had equal contribution to the manuscript. 2 The last three authors had equal contribution to the manuscript.
http://dx.doi.org/10.1016/j.clim.2015.04.007 1521-6616/© 2015 Published by Elsevier Inc. Please cite this article as: S. Revel-Vilk, et al., Autoimmune lymphoproliferative syndrome-like disease in patients with LRBA mutation, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.04.007
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S. Revel-Vilk et al.
1. Introduction
2.2. Immunophenotyping
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Immunophenotyping was performed using the following antibodies: CD19 APC-Cy7, CD38 PerCp-Cy5.5, CD28 PerCPCy5.5, CD25 PerCP-Cy5.5, CD31 PE and CD4 Brilliant Violet 421 were purchased from Biolegend (San Diego, CA); CD21 PE-Cy7, IgG Alexa Fluor 700, CD8 APC, CD127 Alexa Fluor 647, CD27 Horizon V450 and CD10 Brilliant Violet 605 were obtained from BD Biosciences (San Jose, CA); IgD FITC and IgA PE were purchased from Southern Biotech (Birmingham, AL); CD45RA FITC and CD3 PE-Cy7 were purchased from Beckman Coulter (Nyon, Switzerland); CCR7 PE was obtained from R&D Systems (Minneapolis, MN) and IgM Alexa Fluor 647 from Jackson ImmunoResearch Laboratories (Suffolk, UK). The DNT cell number in peripheral blood was measured according to standard protocols employing the following antibodies: CD3-APC (clone UCHT1), TCR α/β-FITC (clone T10B9.1A-31) (both from BD Biosciences), CD4-PerCP (clone VIT4), and CD8-PerCP (BW135/80, Miltenyi, Bergisch-Gladbach, Germany). Measurements were performed on a FACSCalibur equipped with CellQuestPro software (BD Biosciences).
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Primary immunodeficiencies (PIDs) comprise a large group of over 200 distinct disorders. The definition of the exact diagnosis is not only complicated by the overlapping presentation of different disorders, but also by the increasingly recognized variation in the clinical presentation of patients with the same genetic defect. The molecular basis has been partially identified in most of the over 200 PIDs, but among all defined PID syndromes a variable percentage of patients remain without definite molecular diagnosis. Classical methods for identifying causative mutations in genetic disorders include positional cloning based on linkage mapping or candidate gene sequencing. Recently, this methodological approach was revolutionized by whole exome sequencing (WES) which is a powerful tool for discovery of genes causing monogenic Mendelian disorders. The results obtained by this approach not only underscore the importance of this technology for the identification of new disease-causing genes but also for the delineation and redefinition of the clinical phenotype of known syndromes [1]. The autoimmune lymphoproliferative syndrome (ALPS; synonym: Canale–Smith syndrome) is an inherited disorder that is caused by defects in the Fas induced apoptotic pathway [2]. The definitive diagnosis of ALPS requires the presence of chronic splenomegaly or lymphadenopathy, elevated number of TCRαβ+CD4−CD8− double negative T cells (DNT) with defective lymphocyte apoptosis and/or somatic or germline mutation in FAS, FASLG or CASP10 [3]. Autoimmune cytopenia with polyclonal hypergammaglobulinemia, elevated sFasL, vitamin B12, plasma IL10 or IL18 levels, typical histological findings or family history provide accessory criteria for the diagnosis of ALPS. Somatic or germline mutations in FAS, FASLG or CASP10 were found in patients with ALPS and their identification provides additional support for the diagnosis. Recently, several unrelated patients who presented with combined immunodeficiency, enteropathy and autoimmune cytopenia were found to have mutations in the LPS-responsive and beige-like anchor (LRBA) gene [4–6] (Table 1). So far none of the patients have been reported to fulfill the criteria established for the diagnosis of ALPS. Here we describe three patients from two unrelated families with the clinical presentation resembling ALPS and one asymptomatic patient associated with LRBA mutation thus expanding the phenotypic spectrum of the LRBA associated phenotype and the growing list of genes underlying ALPS-like diseases.
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2. Patients and methods
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2.1. Patients
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The medical records of three patients from two Palestinian kindred were reviewed who presented with symptoms typical of ALPS and who were subsequently found to harbor LRBA gene mutations. Data concerning clinical presentation, immunological features, genetic findings, treatment and final outcome were collected. All experiments were performed after obtaining parental written informed consent and were approved by the Hadassah, the Israeli Ministry of Health and the Düsseldorf University Clinic's Ethical Review Boards.
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2.3. Western blotting
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Epstein Barr virus (EBV)-transformed B cell lines were generated as described previously [7]. Protein lysates were obtained from resting EBV-transformed B cells of the patients and controls using RIPA buffer. Forty micrograms of solubilized proteins were size-fractionated by SDS-PAGE (12%, 8% and 4% gradient gel), electro-transferred to a PVDF membrane for 2 h at 40 V. LRBA protein was detected employing a rabbit polyclonal anti-LRBA antibody (Sigma-Aldrich, St. Louis, MO) and secondary HRP-coupled anti-rabbit IgG (Cell Signaling Technology, Danvers, MA). LRBA protein was observed as a band of ~ 319 kDa. GAPDH and tubulin were used as loading controls and detected employing a mouse monoclonal anti-GAPDH antibody or a rabbit polyclonal anti-tubulin antibody (both from Abcam, Cambridge, UK), respectively, visualized subsequently by secondary HRP-coupled anti-mouse or anti-rabbit IgG (Cell Signaling Technology) and chemiluminescence (GE Healthcare, Freiburg, Germany).
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2.4. Measurement of apoptotic cell death
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Apoptosis was measured in primary T cells and EBV-transformed B cell lines from patients, family members and healthy controls. Mutated and wild type B cells were cultured in RPMI1640 supplemented with 20% FCS, 2 mM L-glutamine, 1% penicillin/streptomycin (Life Technologies, Darmstadt, Germany). Primary T cells were cultured in RPMI1640 (Life Technologies) and Panserin 401 (PAN-Biotech, Aidenbach, Germany) mixed 1:1, supplemented with 10% FCS, 100 μg gentamycin (Life Technologies) and 30 U/ml IL2 (Miltenyi). Prior to apoptosis induction T cells were activated with 7 μg/ml phytohemagglutinin (PHA, Life Technologies) for 4 days and cultivated for at least 1 week. Apoptosis was triggered by addition of recombinant SuperFasL (50 ng/ml, Enzo Life Sciences, Loerrach, Germany), 0.25 or 0.5 μM staurosporine (LC Laboratories, Woburn, MA) or by serum deprivation for 24 h, respectively. Untreated cells were used
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Please cite this article as: S. Revel-Vilk, et al., Autoimmune lymphoproliferative syndrome-like disease in patients with LRBA mutation, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.04.007
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Autoimmune lymphoproliferative syndrome-like disease in patients with LRBA mutation t1:1 t1:2 t1:3
Table 1
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10
No. of patients AIHA Leukopenia ITP Lymphadenopathy Splenomegaly Infections
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Clinical and laboratory characteristics of patients with LRBA mutation. Lopez-Herrera et al. [4]
Alangari et al. [5]
Burns et al. [6]
Current report
5 (1 family) 2/5 1/5 2/5 1/5 0/5 1: 5y EBV-associated lymphoproliferative disease 2: no infection 3: 4y pneumonia, recurrent otitis media
1 (1 family) 1 1 1 1 1 Psoas abscess secondary to chronic neutropenia
4 (2 families) 3/4 2/4 3/4 3/4 3/4 1: 14y pneumonia
t1:19 t1:20
AIHA, autoimmune hemolytic anemia; ITP, immune thrombocytopenia; EBV, Epstein–Barr virus; GI, gastroenteritis; DNT, double negative t-cell; ND, not done; FTT, failure to thrive; GH, growth hormone; IVIG, intravenous immunoglobulin; MMF, mycophenolate mofetil.
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2.6. Whole exome analysis
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Genomic DNA was extracted from blood samples of four affected children, parents and siblings. Next generation sequencing of DNA samples from the patients was carried out after targeted enrichment of whole exonic regions using the
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Normal ND 0/1 1/1 1/1 0/1 Erythema nodosum, arthritis
Elevated Increased 1/4 1/4 1/4 0/4 None
Steroids, rituximab, MMF
IVIG (1/4), steroids, MMF, sirolimus
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2.5. Sanger sequencing of germline mutations in known ALPS causing genes Exons including exon/intron borders of FAS, FASLG and CASP10 were amplified by PCR employing the Phusion High Fidelity PCR Master Mix (New England Biolabs), specific forward and reverse primers (listed in Supplemental Table 1, 0.5 μM each) and 20 ng of template DNA. Cycling conditions were 30 s at 98 °C followed by 30 cycles of 7 s at 98 °C, 23 s at 55–65 °C, 30 s at 72 °C and a final extension of 10 min at 72 °C. Capillary sequencing was carried out by a core facility at the BMFZ (Biological and Medical Research Center, University of Duesseldorf).
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ND ND 3/5 1/5 1/5 5/5 Nephrotic syndrome, GH deficiency, Vitamin B12 deficiency, arteritis IVIG (3/5), steroids, azathioprine, rituximab
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as controls. Apoptosis was subsequently assessed by flow cytometric measurement of Annexin V-FITC (BD Biossciences) and propidium iodide (PI, Sigma-Aldrich) staining. Specific apoptosis was calculated as AnnexinV+/PI− cells (%) measured in treated samples − AnnexinV+/PI− cells (%) measured in corresponding untreated controls.
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5 (4 families) 2/5 0/5 4/5 1/5 2/5 1: 5y Pneumonia, brain granuloma 2: 9y serious otitis media, massive pneumonia, empyema 3: 12y warts, perineal molluscum contagiosum, respiratory infection, brain granuloma 4: 2 y respiratory infection, pneumonia, GI infections 5: 2y respiratory infection, pneumonia DNT ND Apoptosis Increased Hypogammaglobulinemia 5/5 Chronic lung disease 5/5 FTT 5/5 Enteropathy 5/5 Other non-hematological Monoarthritis , atrophic gastritis, autoimmune urticarial, allergic dermatitis, phenomena hypothyroidism Therapy IVIG (5/5), steroids, infliximab
SeqCap EZ Exome Library 2.0 kit (Roche/Nimblegen, Madison, WI) according to the manufacturer's protocol as described previously [8]. Sequences were determined on a HiSeq2000 (Illumina, San Diego, CA) and 100-bp were read paired-end. 45.94 and 47.3 million reads were generated for P2 and P1, respectively. Reads alignment and variant calling were performed with DNAnexus software (Palo Alto, California, USA) using the default parameters with the human genome assembly hg19 (GRCh37) as a reference. We removed variants which were called less than X6, were off-target, heterozygous, synonymous, MAFN0.1% at dbSNP138 and MAFN1% in the Hadassah in-house dbSNP. Following this filtering, 26 and 20 homozygous variants remained in P2 and P1, respectively (online supplementary table). Nonetheless pathogenicity prediction (Mutation Taster software) narrowed the candidate list to 12 and 6 in P2 and P1, respectively. At this stage we focused on the LRBA mutations, because it was already reported in the case of ALPS5 and was immune relevant and markedly deleterious in ALPS15. Each mutation was validated by Sanger sequencing and segregation in the respective pedigree.
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2.7. Bioinformatic data analysis
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Sequencing data analysis including read alignment and variant calling was performed by DNAnexus software (Palo
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Please cite this article as: S. Revel-Vilk, et al., Autoimmune lymphoproliferative syndrome-like disease in patients with LRBA mutation, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.04.007
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3.1. LRBA mutations can present with chronic non-infectious lymphadenopathy, splenomegaly, pancytopenia and hypogammaglobulinaemia
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3.2. LRBA deficient patients present with elevated DNT cells and serum Fas ligand (sFasL)
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We present four male patients from two different consanguineous families of Arab Palestinian origin. Family 1: P1 presented at the age of 3 years with autoimmune hemolytic anemia, thrombocytopenia and neutropenia, lymphadenopathy and splenomegaly. Clinical diagnosis of ALPS was made. He responded well to prednisone therapy, but remained steroid dependent. Therapy with mycophenolate mofetil (MMF) was added for steroid sparing with partial response. He is currently 14 years of age (11 years of follow-up), treated with Sirolimus and low dose prednisone with stable hematological parameters and mild splenomegaly. He has no history of recurrent infections, enteropathy or other (non-hematological) autoimmune phenomena. The immunoglobulin levels that were normal at presentation gradually decreased to low IgG levels (total IgG b 250, IgA 118, IgM 90, IgG1 134, IgG2 b 67, IgG3106, IgG4 0 mg/dl) and at the age of 13 years he started IVIG replacement therapy. Recently, he started with recurrent episodes of cough. Chest CT scan showed the onset of lung bronchiectasis. Bronchoscopy alveolar lavage was positive for infection with Hemophilus influenza. Mutations in FAS, FASLG and CASP10 were excluded. Whole exome sequencing revealed a single homozygous base exchange in the region of the LRBA gene (c.7937TNG, referring to transcript ENST00000510413) coding for the WD40 domain of the protein and leading to an exchange of an isoleucine for a serine (p.Ile2646Ser, referring to the corresponding protein sequence of ENSP00000421552.1) (Fig. 1A). The homozygous mutation segregated with the disease state in the family and was previously reported as a deleterious mutation [4]. Family 2: P2 presented at the age of 6 years with autoimmune hemolytic anemia and a year later, he developed immune thrombocytopenia, lymphadenopathy and splenomegaly. Clinical diagnosis of ALPS was made. He also responded well to prednisone therapy, but required therapy with MMF for steroid sparing with partial response. He is currently 9 years of age (3 years of follow-up) and treated with Sirolimus and low dose prednisone with stable hematological parameters and mild splenomegaly. He has no history of recurrent infections, enteropathy, lung disease or other (non-hematological) autoimmune phenomena. The first immunoglobulin levels tested at the age of 7 years were IgG 651, IgA b 42, IgM 43 mg/dl (age adjusted normal values IgG 598–1379, IgA 33–258, IgM 41–200 mg/dl). Mutations in FAS, FASLG and CASP10 were excluded. Whole exome sequencing detected a homozygous insertion c.8139_8142dupCATG in the LRBA gene leading to a frameshift (p.Asn2715HisfsX13) in the AKAP domain (Fig. 1B). The mutation segregated with the disease state in the family (see P4 and P3, below).
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P4 is the younger brother of P2. He presented at the age of 1.5 years with autoimmune hemolytic anemia, immune thrombocytopenia, lymphadenopathy and splenomegaly. Clinical diagnosis of ALPS was made. He was treated with steroids with good clinical response. MMF was added for steroid sparing. He is currently 3.5 years of age (2 years of follow-up) and treated with MMF and low dose prednisone with stable hematology parameters and mild lymphadenopathy and splenomegaly. He had no history of recurrent infections, enteropathy, lung disease or additional signs of autoimmunity. The immunoglobulin levels first tested at the age of 1.5 years were IgG 508, IgA b 42, IgM 48 mg/dl (age adjusted normal values IgG 400–1250, IgA 24–192, IgM 35–200 mg/dl). Sanger sequencing confirmed the homozygous insertion c.8139_8142dupCATG detected in the older brother. P3, brother of P2 and P4, is currently 7 years of age (2 years of follow-up). He is asymptomatic without lymphadenopathy or splenomegaly with normal blood counts and immunoglobulin levels. He was found to be homozygous for the same insertion in the LRBA gene in the segregation analysis. The summary of clinical and laboratory characteristics of our patients are presented in Tables 1 and 2. All patients failed to express LRBA protein in EBV cell lysates (Fig. 1C).
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Alto, California, USA) using default parameters and the human genome assembly hg19 (GRCh37) as a reference, as previously described [9,10].
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All but one patient with homozygous LRBA mutations had elevated numbers of double negative (CD4− CD8−) TCRαβ+ CD3+ T cells (1.1–5.5%, 11–46 cells/μl) and elevated serum levels of sFasL (Table 2). Plasma vitamin B12 levels, the percentage of B220+ DNT cells and the ratio of CD25+/ HLA-DR+ lymphocytes were within the normal range for all patients (Table 2). Based on the serum levels of sFasL and plasma vitamin B12 levels the probability for FAS mutation was calculated (Table 2) [11] rendering FAS mutations very unlikely in patient 1 while patients 2 and 4 were in the gray zone suggesting the evaluation of additional markers.
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3.3. Altered B cell homeostasis in LRBA deficiency partially correlates with the presence of clinical manifestations
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Flow cytometric analysis of the four LRBA deficient patients revealed reduced B cell numbers in all patients (Table 2). Additionally, reduction of IgG+ and IgA+ CD27+ switched memory B cells was found in all symptomatic patients (P1, P2 and P4) while the levels were normal in the asymptomatic child (P3). Other B cell subpopulations were variably affected. All patients of the second family displayed an accumulation of CD21low B cells. IgM only memory B cells were generally decreased compared to age matched reference values. As an expression of combined immunodeficiency in LRBA deficiency all patients displayed low CD4+CD45RA+ naive T cells while the percentage of naive CD8 T cells was normal. Numbers of terminally differentiated CD8 T cells were low compared to age matched reference values. The heterozygous parents and siblings had no clinical symptoms and immune phenotyping revealed normal parameters (data not shown).
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Please cite this article as: S. Revel-Vilk, et al., Autoimmune lymphoproliferative syndrome-like disease in patients with LRBA mutation, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.04.007
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Figure 1 Molecular diagnosis of LRBA deficiency. (A) c.7937TNG mutation in the DNA sequence of exon 54 of the human LRBA gene in a patient 1 (upper row) and obligate heterozygote (lower row) is shown by the arrow. (B) 8139_8142dupCATG insertion in the DNA sequence of exon 56 of the human LRBA gene in patients 2, 3 and 4 (upper row), obligate heterozygote (middle row), and a healthy control (lower row). The mutation is indicated by the arrow. (C) Expression of LRBA protein in patients with homozygous mutations in LRBA. Western blot analysis revealed the absence of the LRBA protein in EBV cell lysates from P1, P2, P4 (left) and P3 (right), compared to the ~ 319 kDa LRBA band observed in EBV cell lysates from healthy donors. GAPDH (left) and tubulin (right) served as loading controls. Please cite this article as: S. Revel-Vilk, et al., Autoimmune lymphoproliferative syndrome-like disease in patients with LRBA mutation, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.04.007
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t2:1 t2:2 t2:3Q1
Table 2
Age at time of analysis
P1 13y
P2 8y
P3 6.5y
P4 2.5y
t2:4
Vitamin B12, pg/ml Normal values (200–900 pg/ml) Serum FAS ligand, pg/ml Normal values b 200 pg/ml Calculated probability for Fas mutation [11] Lymphocytes, cells/μl Normal values ⁎
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821
NA
760
520 b 1%
789 26%
312 –
599 6.9%
t2:44 t2:45 t2:46 t2:47 t2:48 t2:49 t2:50 t2:51 t2:52 t2:53
15 25–64 351 300–1300 106 78–640 57 2–86 54 16–810 16 35–420 11
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4 18–86 303 200–1700 83 42–1300 25 6–43 46 45–410 19
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57–340 23
307 641–1453 51 375–1096
1200–4700 absolute (cells/μl) 103 296–784 9 13–63 62 154–413 13 24–135 1 7–65 5 5–35 4 13–74
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15–41 2.8–2.9 b 2.5
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490 608–1217 170 311–781
1300 relative (%) 7.9 8.5–20.2 8.9 3.4–9.0 60.4 48–70 12.9 6–22 1.2 2–12 4.6 1.1–6.1 3.9 2.7–14.0 11.1 0.8–7.7 16.9 26.5–41.4 32.3 55.6–75.8 67.7 75.5 61.0–84.2 5.7 2.3–7.7 20.1 13–47 48.9 16–100 10.7 1–6 3.6 5–100 0.7
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1200–4700 absolute (cells/μl) 48 296–784 1 13–63 40 154–413 6 24–135 0 7–65 0 5–35 0 13–74
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1100 relative (%) 4.4 8.5–20.2 1.1 3.4–9.0 82.4 48–70 12.8 6–22 0.4 2–12 0.3 1.1–6.1 0.6 2.7–14.0 16.8 0.8–7.7 27.9 26.5–41.4 16.5 55.6–75.8 83.5 46.0 61.0–84.2 1.4 2.3–7.7 27.5 13–47 27.3 16–100 8.1 1–6 15.2 5–100 6.2
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1400–4200 absolute (cells/μl) 42 119–578 2 2–41 36 83–398 3 10–74 0 3–39 0 4–13 0 7–32
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1300 relative (%) B cells 3.2 Normal values ⁎ 7.8–23.7 Transitional B cells 3.6 Normal values ⁎ 1.5–7.3 Naive B cells 86.2 Normal values ⁎ 64.6–80.1 IgM Memory B cells 6.3 Normal values ⁎ 4.7–15.5 IgM only B cells 0.3 Normal values ⁎ 1.6–11.3 IgA switched mem (27pos) B cells 0.2 Normal values ⁎ 1.2–3.8 IgG switched mem (CD27pos) B cells 0.9 Normal values ⁎ 2.1–9.4 CD21low B cells 6.8 Normal values ⁎⁎ 0.8–7.7 CD4 37.7 Normal values ⁎ 30.4–52.9 Naive CD45RA cells 34.7 Normal values ⁎ 49.3–72.0 Memory CD45R0 cells 65.3 RTE % of CD45RApos CD4 T cells 84.0 Normal values ⁎ 50.7–78.9 Treg cells 3.1 Normal values ⁎ 2.8–7.2 CD8 27 Normal values ⁎⁎⁎ 14–40 Naive CD8 T cells 30.1 Normal values ⁎⁎⁎ 20–95 Central memory CD8 T cells 16.2 Normal values ⁎⁎⁎ 0.42–18 Effector memory CD8 T cells 15.5 Normal values ⁎⁎⁎ 4–100 Terminally differentiated 4.6 effector cells Normal values ⁎⁎⁎ 9–65 1.1–5.5 DN T cells of αγ+ CD3+ T cells Normal values b 2.5
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Serological and cellular parameters in peripheral blood of patients.
15–41 1.7–1.9 b 2.5
220 641–1453 71 375–1096
13 18–86 261 200–1700 128 42–1300 28 6–43 9 45–410 2 57–340 11
1900 relative (%) 5.7 17.3–30 23.2 3.6–21.4 63.0 50–79 9.6 5–12 0.3 1.6–6.6 0.4 0.7–2.9 0.6 0.8–6.3 10.3 0.8–7.7 24.7 28.1–43. 33.5 71.5–84.2 66.5 52.6 65.2–79.5 3.0 2.9–7.4 29 9–49 58.8 19–100 8.8 1–9 2.9 10–55 0.4
1400–5500 absolute (cells/μl) 108 686–1732 25 41–248 68 346–1356 10 50–148 0 19–53 0 6–57 1 8–26
6–83 2.2–3.3 b 2.5
25–530 46
469 925–2477 157 685–2055
14 42–69 551 200–1800 324 53–1100 48 4–64 16 24–590 2
If not indicated differently percentages refer to the respective parental population. Naive CD8 T cells are characterized by CCR7+ CD45RA+ CD27+, central memory by CCR7+CD45RA−CD27+, effector memory by CCR7−CD45RA−CD27− and terminally differentiated by CCR7−CD45RA+ CD27−. RTE = recent thymic emigrants. DN T cells = double negative (CD4−CD8−) cells of TCRαβ+CD3+ T cells. DN T cells were measured at two different time points. Pathological values are in bold print. ⁎ Normal values refer to age matched controls published in van Gent, R., et al. Refined characterization and reference values of the pediatric T- and B-cell compartments. Clinical Immunology 133, 95–107. ⁎⁎ Internal reference values for adults. ⁎⁎⁎ Schatorje, E.J., et al. Paediatric reference values for the peripheral T cell compartment. Scand J Immunol 75, 436–444.
Please cite this article as: S. Revel-Vilk, et al., Autoimmune lymphoproliferative syndrome-like disease in patients with LRBA mutation, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.04.007
Autoimmune lymphoproliferative syndrome-like disease in patients with LRBA mutation
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In this study we expand the observed variability in the clinical and immunological presentation of children with mutations in LRBA. Three children of our study presented with clinical and laboratory features of ALPS-like disease and one child is still asymptomatic up to the age of 7 years. At presentation none of the children had clinical symptoms of recurrent infections, but all three symptomatic children presented with autoimmune cytopenia, splenomegaly and lymphadenopathy resembling the clinical phenotype of ALPS. Thus, according to the revised criteria for ALPS our patients were initially diagnosed with probable diagnosis of ALPS and therapy with steroids and MMF was initiated with stable response [13]. The diagnosis of ALPS remains challenging especially in centers that lack expertise in immunology testing. The specificity of elevated DNT cell number, a test that is widely available and commonly used for the diagnosis of ALPS, has recently been challenged [14]. An elevation of DNT as observed in our patients is found in 30% of children with autoimmune diseases [14] as well as in combined variable immunodeficiency (CVID) patients [15,16]. In healthy controls the 95th percentile for TCR αβ+ DNT was used to define a normal range up to 2.3% of total lymphocytes or 3.4% of T cells [15]. In the study by Oliveira et al. cut-offs for DNT were elevated DNT ≥ 1.5% of total lymphocytes or 2.5% of CD3+ lymphocytes [3]. However, in 163 patients with clinically suspected ALPS, the predictive value of DNT was found to be low (positive and negative predicative values of 61% and 77%, respectively) [11]. In the same cohort, the best predictor of FAS mutations was the combination of elevated vitamin B12 (N 1255 pg/ml) and sFasL (N 559 pg/ml) [11]. Unfortunately, sFasL and cytokine measurement like IL10 or IL18 are not attainable tests in most centers and therefore currently don't serve as a routine markers for the diagnostic evaluation. sFasL serum levels were strongly elevated in three of our four patients with LRBA mutations. Vitamin B12 levels, however, were normal in our patients. Based on the FAS mutation probability online calculator the probability for FAS mutations in our patients ranged between 0.1% and 26% rendering the probability of causative FAS mutations low in all of the analyzed patients [11]. Consistently, no germline mutations in FAS and other genes known to be associated with ALPS were found. Genetic analysis of sorted DNT in search for somatic FAS mutations was not done because of familial involvement and low FAS mutation probability and whole exome analysis
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staurosporine, another inducer of the intrinsic mitochondria mediated apoptotic pathway. Similar as in response to serum deprivation, LRBA deficient T cells of symptomatic patients were more sensitive to staurosporine treatment than controls (Fig. 2C). Notably, the asymptomatic patient (P3) showed an intermediate susceptibility to staurosporine and serum starvation induced apoptosis compared to healthy controls and affected patients. Similar results were obtained employing EBV transformed B cell lines derived from the patients and controls. The levels of apoptosis in response to staurosporine treatment and serum deprivation were significantly higher in LRBA-deficient EBV-transformed B cells compared to healthy controls (Fig. 2D+E).
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LRBA plays a role in the regulation of apoptosis [4,10]. Mechanistically there are two main pathways of apoptotic cell death: 1) the extrinsic pathway regulated by death receptors such as Fas and 2) the mitochondria mediated pathway regulated by proteins of the Bcl-2 family. In patients with ALPS accumulation of DNT cells is due to defective extrinsic Fas mediated apoptosis. To test whether mutation of LRBA impairs this apoptotic pathway activated primary T cells from the patients and controls were treated with recombinant Fas ligand (FasL) in vitro. Cells from patients with complete LRBA deficiency were clearly less responsive to apoptosis induced by recombinant FasL compared to their siblings with a wildtype genotype (healthy controls) as shown in Fig. 2A. Heterozygous carriers had an intermediate response (data not shown). In contrast, similar to a previous report [4] T cells derived from the patients showed increased intrinsic apoptosis after 24 h of serum starvation compared to healthy controls (Fig. 2B). In addition, we tested the response of these cells to
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Figure 2 LRBA mutations differentially affect extrinsic and intrinsic apoptosis pathways. Activated primary T cells from the patients and healthy controls were treated with 50 ng/ml recombinant FasL (A), serum deprivation (B) or 0.25 μM staurosporine (C) for 24 h. Immortalized B cells from the patients and a healthy control were deprived of serum (D) or treated with staurosporine (E) as described in (B, C). Subsequently apoptosis was measured by flow cytometric detection of Annexin V-FITC/ propidium iodide staining (A–E).
Please cite this article as: S. Revel-Vilk, et al., Autoimmune lymphoproliferative syndrome-like disease in patients with LRBA mutation, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.04.007
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Conflict of interest statement
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The authors declare that there are no conflicts of interest.
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Acknowledgments
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This study was supported by the German Federal Ministry of Education and Research (BMBF 01EO1303). PS was supported (in part) by a grant from the Joint Research Fund of the Hebrew University and the Hadassah Hebrew University Hospitals.
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Appendix A. Supplementary data
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Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.clim.2015.04.007.
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contribute to the autoimmune-lymphoproliferative phenotype of the affected patients. The follow up of our patients for 3 to 11 years allowed us to observe a progressive, but variable course of their disease with progression of both the clinical and laboratory features over the years. The immunosuppressive therapy probably modulated the clinical course of the disease and may have significantly influenced the rate of complications and quality of life. Based on the identified mutation in LRBA, the previously reported prognosis of patients with LRBA deficiency and with the availability of a fully matched family donor, we decided to go ahead with stem cell transplantation in patient P1 because of the smoldering progression of bronchiectasis; a step, which we would have not performed based purely on the clinical diagnosis of probable ALPS. In summary, this study emphasizes the complexity of diagnosing specific PID in patients who present with the clinical phenotype of ALPS. Several other clinical entities may also present with autoimmune cytopenia and lymphoproliferation. The recently suggested score to identify patients with ALPS and mutation in FAS was helpful to distinguish LRBA deficient patients from ALPS-FAS cases, but unfortunately in many centers laboratory biomarkers sFasL and IL-10 are not testable. The effect of disturbed autophagy on the extrinsic apoptosis pathway may contribute to the ALPS like presentation in some LRBA deficient patients. Thus, LRBA deficiency needs to be considered in the differential diagnosis of ALPS like patients. We believe that comprehensive genetic evaluation is important for arriving at the true diagnosis enabling proper care and follow-up of patients presenting with ALPS-like symptoms.
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was performed instead. This revealed unexpectedly mutations in the LRBA gene, typically associated with a disorder of combined immunodeficiency, enteropathy and autoimmune disease [4]. Only prolonged follow-up revealed signs of immunodeficiency, i.e. progressive decrease of immunoglobulin levels and development of bronchiectasis in P1. Thus the correct diagnosis in children with ALPS like disorders, which is important both for management decisions and for genetic consultation, requires further unbiased genetic analysis after exclusion of known candidate genes underlying ALPS [17]. Interestingly, the mutation of LRBA gene that we found in P1 was previously described in two children of Arab origin who were treated in another hospital in Israel [4]. Like in many other PID, there is no clear genotype-phenotype correlation in patients with LRBA mutation. The previously published family with the same single base exchange (designated c.7970TNG referring to the gene transcript ENST00000357115) mutation presented with signs and symptoms of immunodeficiency like progressive lung disease, cerebral granuloma, lymphoid interstitial pneumonia and arthritis that were absent in our patient [4]. In the second family with an unrelated mutation in LRBA, two patients (P2, P4) had an ALPS-like clinical presentation while the 7 year old brother (P3) was asymptomatic. The immunological laboratory studies of our patients show significant variability. As described in previous cases, the three symptomatic patients demonstrated variable but marked decrease in class-switched B cells, while the asymptomatic patient presented with normal numbers. These differences between the symptomatic and asymptomatic patients may be partially related to the therapy with MMF and steroid given to the symptomatic patients. The progressive hypogammaglobulinemia and bronchiectasis on chest CT found in our oldest patient (P1) is of clinical importance and may point to the evolving nature of this disease. Normal immunoglobulins levels at presentation were reported also by other groups (Table 1). ALPS is usually caused by loss-of-function or dominantnegative mutations in components of the Fas signaling pathway that compromise Fas mediated apoptosis. Interestingly, also LRBA deficiency was associated with a deficient response to Fas triggered apoptosis while the sensitivity to apoptosis stimulated by intrinsic signals was even increased as previously reported. LRBA belongs to a family of BEACH domain containing proteins that are involved in cellular mechanisms including vesicular transport, autophagy, apoptosis, membrane dynamics and receptor signaling [18]. LRBA-deficient B cells were previously reported to have a significantly reduced ability to induce autophagy in response to starvation [4]. Autophagy as a major process involved in the degradation of cellular material is cytoprotective for hematopoietic cells [19] and essential for the homeostasis and activation of T cells [20]. Thus, deficiency in autophagy was suggested to underlie the increased cell death induced by intrinsic stressors in LRBA deficient patients. On the other hand, it was recently recognized that autophagy specifically facilitates Fas mediated apoptosis by degradation of the Fap-1 protein phosphatase, a critical negative regulator of apoptotic cell death signaled by Fas [21]. Thus, autophagy affects cell death in a cell-type and stimulus-depending manner [21] and defects in LRBA mediated autophagy may account for the observed divergent effects on extrinsic vs intrinsic cell death. The detected effect on the extrinsic pathway may
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Clinical Immunology
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Shoshana Revel-Vilk a,⁎,1 , Ute Fischer b,1 , Barbel Keller c , Schafiq Nabhani b , Laura Gámez-Díaz c , Anne Rensing-Ehl c , Michael Gombert b , Andrea Hönscheid b , Hani Saleh d , Avraham Shaag e , Arndt Borkhardt b , Bodo Grimbacher c , Klaus Warnatz c,2 , Orly Elpeleg e,2 , Polina Stepensky a,2
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Pediatric Hematology/Oncology and Bone Marrow Transplantation, Hadassah-Hebrew University Medical Center, Jerusalem, Israel Department of Pediatric Oncology, Hematology and Clinical Immunology, University Children’s Hospital, Medical Faculty, Heinrich-Heine-University, Düsseldorf, Germany c Center for Chronic Immunodeficiency, University Medical Center and University of Freiburg, Freiburg, Germany d Pediatric Hemato-Oncology Unit, Augusta Victoria Hospital, Jerusalem, Israel e Monique and Jacques Roboh Department of Genetic Research, Hadassah, Hebrew University Medical Center, Jerusalem, Israel
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Received 10 November 2014; accepted with revision 19 April 2015
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Abstract Mutations in LPS-responsive and beige-like anchor (LRBA) gene were recently described in patients with combined immunodeficiency, enteropathy and autoimmune cytopenia. Here, we extend the clinical and immunological phenotypic spectrum of LRBA associated disorders by reporting on three patients from two unrelated families who presented with splenomegaly and lymphadenopathy, cytopenia, elevated double negative T cells and raised serum Fas ligand levels resembling autoimmune lymphoproliferative syndrome (ALPS) and one asymptomatic patient. Homozygous loss of function mutations in LRBA were identified by whole exome analysis. Similar to ALPS patients, Fas mediated apoptosis was impaired in LRBA deficient patients, while apoptosis in response to stimuli of the intrinsic mitochondria mediated apoptotic pathway was even enhanced. This manuscript illustrates the phenotypic overlap of other primary immunodeficiencies with ALPS-like disorders and strongly underlines the necessity of genetic diagnosis in order to provide early correct diagnosis and subsequent care. © 2015 Published by Elsevier Inc.
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⁎ Corresponding author at: Pediatric Hematology/Oncology Department, Hadassah – Hebrew University Hospital, POB 12000, Jerusalem, 91200, Israel. Fax: +972 2 6777833. E-mail address: [email protected] (S. Revel-Vilk). 1 The first two authors had equal contribution to the manuscript. 2 The last three authors had equal contribution to the manuscript.
http://dx.doi.org/10.1016/j.clim.2015.04.007 1521-6616/© 2015 Published by Elsevier Inc. Please cite this article as: S. Revel-Vilk, et al., Autoimmune lymphoproliferative syndrome-like disease in patients with LRBA mutation, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.04.007
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1. Introduction
2.2. Immunophenotyping
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Immunophenotyping was performed using the following antibodies: CD19 APC-Cy7, CD38 PerCp-Cy5.5, CD28 PerCPCy5.5, CD25 PerCP-Cy5.5, CD31 PE and CD4 Brilliant Violet 421 were purchased from Biolegend (San Diego, CA); CD21 PE-Cy7, IgG Alexa Fluor 700, CD8 APC, CD127 Alexa Fluor 647, CD27 Horizon V450 and CD10 Brilliant Violet 605 were obtained from BD Biosciences (San Jose, CA); IgD FITC and IgA PE were purchased from Southern Biotech (Birmingham, AL); CD45RA FITC and CD3 PE-Cy7 were purchased from Beckman Coulter (Nyon, Switzerland); CCR7 PE was obtained from R&D Systems (Minneapolis, MN) and IgM Alexa Fluor 647 from Jackson ImmunoResearch Laboratories (Suffolk, UK). The DNT cell number in peripheral blood was measured according to standard protocols employing the following antibodies: CD3-APC (clone UCHT1), TCR α/β-FITC (clone T10B9.1A-31) (both from BD Biosciences), CD4-PerCP (clone VIT4), and CD8-PerCP (BW135/80, Miltenyi, Bergisch-Gladbach, Germany). Measurements were performed on a FACSCalibur equipped with CellQuestPro software (BD Biosciences).
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Primary immunodeficiencies (PIDs) comprise a large group of over 200 distinct disorders. The definition of the exact diagnosis is not only complicated by the overlapping presentation of different disorders, but also by the increasingly recognized variation in the clinical presentation of patients with the same genetic defect. The molecular basis has been partially identified in most of the over 200 PIDs, but among all defined PID syndromes a variable percentage of patients remain without definite molecular diagnosis. Classical methods for identifying causative mutations in genetic disorders include positional cloning based on linkage mapping or candidate gene sequencing. Recently, this methodological approach was revolutionized by whole exome sequencing (WES) which is a powerful tool for discovery of genes causing monogenic Mendelian disorders. The results obtained by this approach not only underscore the importance of this technology for the identification of new disease-causing genes but also for the delineation and redefinition of the clinical phenotype of known syndromes [1]. The autoimmune lymphoproliferative syndrome (ALPS; synonym: Canale–Smith syndrome) is an inherited disorder that is caused by defects in the Fas induced apoptotic pathway [2]. The definitive diagnosis of ALPS requires the presence of chronic splenomegaly or lymphadenopathy, elevated number of TCRαβ+CD4−CD8− double negative T cells (DNT) with defective lymphocyte apoptosis and/or somatic or germline mutation in FAS, FASLG or CASP10 [3]. Autoimmune cytopenia with polyclonal hypergammaglobulinemia, elevated sFasL, vitamin B12, plasma IL10 or IL18 levels, typical histological findings or family history provide accessory criteria for the diagnosis of ALPS. Somatic or germline mutations in FAS, FASLG or CASP10 were found in patients with ALPS and their identification provides additional support for the diagnosis. Recently, several unrelated patients who presented with combined immunodeficiency, enteropathy and autoimmune cytopenia were found to have mutations in the LPS-responsive and beige-like anchor (LRBA) gene [4–6] (Table 1). So far none of the patients have been reported to fulfill the criteria established for the diagnosis of ALPS. Here we describe three patients from two unrelated families with the clinical presentation resembling ALPS and one asymptomatic patient associated with LRBA mutation thus expanding the phenotypic spectrum of the LRBA associated phenotype and the growing list of genes underlying ALPS-like diseases.
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The medical records of three patients from two Palestinian kindred were reviewed who presented with symptoms typical of ALPS and who were subsequently found to harbor LRBA gene mutations. Data concerning clinical presentation, immunological features, genetic findings, treatment and final outcome were collected. All experiments were performed after obtaining parental written informed consent and were approved by the Hadassah, the Israeli Ministry of Health and the Düsseldorf University Clinic's Ethical Review Boards.
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Epstein Barr virus (EBV)-transformed B cell lines were generated as described previously [7]. Protein lysates were obtained from resting EBV-transformed B cells of the patients and controls using RIPA buffer. Forty micrograms of solubilized proteins were size-fractionated by SDS-PAGE (12%, 8% and 4% gradient gel), electro-transferred to a PVDF membrane for 2 h at 40 V. LRBA protein was detected employing a rabbit polyclonal anti-LRBA antibody (Sigma-Aldrich, St. Louis, MO) and secondary HRP-coupled anti-rabbit IgG (Cell Signaling Technology, Danvers, MA). LRBA protein was observed as a band of ~ 319 kDa. GAPDH and tubulin were used as loading controls and detected employing a mouse monoclonal anti-GAPDH antibody or a rabbit polyclonal anti-tubulin antibody (both from Abcam, Cambridge, UK), respectively, visualized subsequently by secondary HRP-coupled anti-mouse or anti-rabbit IgG (Cell Signaling Technology) and chemiluminescence (GE Healthcare, Freiburg, Germany).
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2.4. Measurement of apoptotic cell death
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Apoptosis was measured in primary T cells and EBV-transformed B cell lines from patients, family members and healthy controls. Mutated and wild type B cells were cultured in RPMI1640 supplemented with 20% FCS, 2 mM L-glutamine, 1% penicillin/streptomycin (Life Technologies, Darmstadt, Germany). Primary T cells were cultured in RPMI1640 (Life Technologies) and Panserin 401 (PAN-Biotech, Aidenbach, Germany) mixed 1:1, supplemented with 10% FCS, 100 μg gentamycin (Life Technologies) and 30 U/ml IL2 (Miltenyi). Prior to apoptosis induction T cells were activated with 7 μg/ml phytohemagglutinin (PHA, Life Technologies) for 4 days and cultivated for at least 1 week. Apoptosis was triggered by addition of recombinant SuperFasL (50 ng/ml, Enzo Life Sciences, Loerrach, Germany), 0.25 or 0.5 μM staurosporine (LC Laboratories, Woburn, MA) or by serum deprivation for 24 h, respectively. Untreated cells were used
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Autoimmune lymphoproliferative syndrome-like disease in patients with LRBA mutation t1:1 t1:2 t1:3
Table 1
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10
No. of patients AIHA Leukopenia ITP Lymphadenopathy Splenomegaly Infections
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Clinical and laboratory characteristics of patients with LRBA mutation. Lopez-Herrera et al. [4]
Alangari et al. [5]
Burns et al. [6]
Current report
5 (1 family) 2/5 1/5 2/5 1/5 0/5 1: 5y EBV-associated lymphoproliferative disease 2: no infection 3: 4y pneumonia, recurrent otitis media
1 (1 family) 1 1 1 1 1 Psoas abscess secondary to chronic neutropenia
4 (2 families) 3/4 2/4 3/4 3/4 3/4 1: 14y pneumonia
t1:19 t1:20
AIHA, autoimmune hemolytic anemia; ITP, immune thrombocytopenia; EBV, Epstein–Barr virus; GI, gastroenteritis; DNT, double negative t-cell; ND, not done; FTT, failure to thrive; GH, growth hormone; IVIG, intravenous immunoglobulin; MMF, mycophenolate mofetil.
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Genomic DNA was extracted from blood samples of four affected children, parents and siblings. Next generation sequencing of DNA samples from the patients was carried out after targeted enrichment of whole exonic regions using the
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Steroids, rituximab, MMF
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2.5. Sanger sequencing of germline mutations in known ALPS causing genes Exons including exon/intron borders of FAS, FASLG and CASP10 were amplified by PCR employing the Phusion High Fidelity PCR Master Mix (New England Biolabs), specific forward and reverse primers (listed in Supplemental Table 1, 0.5 μM each) and 20 ng of template DNA. Cycling conditions were 30 s at 98 °C followed by 30 cycles of 7 s at 98 °C, 23 s at 55–65 °C, 30 s at 72 °C and a final extension of 10 min at 72 °C. Capillary sequencing was carried out by a core facility at the BMFZ (Biological and Medical Research Center, University of Duesseldorf).
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as controls. Apoptosis was subsequently assessed by flow cytometric measurement of Annexin V-FITC (BD Biossciences) and propidium iodide (PI, Sigma-Aldrich) staining. Specific apoptosis was calculated as AnnexinV+/PI− cells (%) measured in treated samples − AnnexinV+/PI− cells (%) measured in corresponding untreated controls.
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5 (4 families) 2/5 0/5 4/5 1/5 2/5 1: 5y Pneumonia, brain granuloma 2: 9y serious otitis media, massive pneumonia, empyema 3: 12y warts, perineal molluscum contagiosum, respiratory infection, brain granuloma 4: 2 y respiratory infection, pneumonia, GI infections 5: 2y respiratory infection, pneumonia DNT ND Apoptosis Increased Hypogammaglobulinemia 5/5 Chronic lung disease 5/5 FTT 5/5 Enteropathy 5/5 Other non-hematological Monoarthritis , atrophic gastritis, autoimmune urticarial, allergic dermatitis, phenomena hypothyroidism Therapy IVIG (5/5), steroids, infliximab
SeqCap EZ Exome Library 2.0 kit (Roche/Nimblegen, Madison, WI) according to the manufacturer's protocol as described previously [8]. Sequences were determined on a HiSeq2000 (Illumina, San Diego, CA) and 100-bp were read paired-end. 45.94 and 47.3 million reads were generated for P2 and P1, respectively. Reads alignment and variant calling were performed with DNAnexus software (Palo Alto, California, USA) using the default parameters with the human genome assembly hg19 (GRCh37) as a reference. We removed variants which were called less than X6, were off-target, heterozygous, synonymous, MAFN0.1% at dbSNP138 and MAFN1% in the Hadassah in-house dbSNP. Following this filtering, 26 and 20 homozygous variants remained in P2 and P1, respectively (online supplementary table). Nonetheless pathogenicity prediction (Mutation Taster software) narrowed the candidate list to 12 and 6 in P2 and P1, respectively. At this stage we focused on the LRBA mutations, because it was already reported in the case of ALPS5 and was immune relevant and markedly deleterious in ALPS15. Each mutation was validated by Sanger sequencing and segregation in the respective pedigree.
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Sequencing data analysis including read alignment and variant calling was performed by DNAnexus software (Palo
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Please cite this article as: S. Revel-Vilk, et al., Autoimmune lymphoproliferative syndrome-like disease in patients with LRBA mutation, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.04.007
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3.2. LRBA deficient patients present with elevated DNT cells and serum Fas ligand (sFasL)
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We present four male patients from two different consanguineous families of Arab Palestinian origin. Family 1: P1 presented at the age of 3 years with autoimmune hemolytic anemia, thrombocytopenia and neutropenia, lymphadenopathy and splenomegaly. Clinical diagnosis of ALPS was made. He responded well to prednisone therapy, but remained steroid dependent. Therapy with mycophenolate mofetil (MMF) was added for steroid sparing with partial response. He is currently 14 years of age (11 years of follow-up), treated with Sirolimus and low dose prednisone with stable hematological parameters and mild splenomegaly. He has no history of recurrent infections, enteropathy or other (non-hematological) autoimmune phenomena. The immunoglobulin levels that were normal at presentation gradually decreased to low IgG levels (total IgG b 250, IgA 118, IgM 90, IgG1 134, IgG2 b 67, IgG3106, IgG4 0 mg/dl) and at the age of 13 years he started IVIG replacement therapy. Recently, he started with recurrent episodes of cough. Chest CT scan showed the onset of lung bronchiectasis. Bronchoscopy alveolar lavage was positive for infection with Hemophilus influenza. Mutations in FAS, FASLG and CASP10 were excluded. Whole exome sequencing revealed a single homozygous base exchange in the region of the LRBA gene (c.7937TNG, referring to transcript ENST00000510413) coding for the WD40 domain of the protein and leading to an exchange of an isoleucine for a serine (p.Ile2646Ser, referring to the corresponding protein sequence of ENSP00000421552.1) (Fig. 1A). The homozygous mutation segregated with the disease state in the family and was previously reported as a deleterious mutation [4]. Family 2: P2 presented at the age of 6 years with autoimmune hemolytic anemia and a year later, he developed immune thrombocytopenia, lymphadenopathy and splenomegaly. Clinical diagnosis of ALPS was made. He also responded well to prednisone therapy, but required therapy with MMF for steroid sparing with partial response. He is currently 9 years of age (3 years of follow-up) and treated with Sirolimus and low dose prednisone with stable hematological parameters and mild splenomegaly. He has no history of recurrent infections, enteropathy, lung disease or other (non-hematological) autoimmune phenomena. The first immunoglobulin levels tested at the age of 7 years were IgG 651, IgA b 42, IgM 43 mg/dl (age adjusted normal values IgG 598–1379, IgA 33–258, IgM 41–200 mg/dl). Mutations in FAS, FASLG and CASP10 were excluded. Whole exome sequencing detected a homozygous insertion c.8139_8142dupCATG in the LRBA gene leading to a frameshift (p.Asn2715HisfsX13) in the AKAP domain (Fig. 1B). The mutation segregated with the disease state in the family (see P4 and P3, below).
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P4 is the younger brother of P2. He presented at the age of 1.5 years with autoimmune hemolytic anemia, immune thrombocytopenia, lymphadenopathy and splenomegaly. Clinical diagnosis of ALPS was made. He was treated with steroids with good clinical response. MMF was added for steroid sparing. He is currently 3.5 years of age (2 years of follow-up) and treated with MMF and low dose prednisone with stable hematology parameters and mild lymphadenopathy and splenomegaly. He had no history of recurrent infections, enteropathy, lung disease or additional signs of autoimmunity. The immunoglobulin levels first tested at the age of 1.5 years were IgG 508, IgA b 42, IgM 48 mg/dl (age adjusted normal values IgG 400–1250, IgA 24–192, IgM 35–200 mg/dl). Sanger sequencing confirmed the homozygous insertion c.8139_8142dupCATG detected in the older brother. P3, brother of P2 and P4, is currently 7 years of age (2 years of follow-up). He is asymptomatic without lymphadenopathy or splenomegaly with normal blood counts and immunoglobulin levels. He was found to be homozygous for the same insertion in the LRBA gene in the segregation analysis. The summary of clinical and laboratory characteristics of our patients are presented in Tables 1 and 2. All patients failed to express LRBA protein in EBV cell lysates (Fig. 1C).
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Alto, California, USA) using default parameters and the human genome assembly hg19 (GRCh37) as a reference, as previously described [9,10].
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All but one patient with homozygous LRBA mutations had elevated numbers of double negative (CD4− CD8−) TCRαβ+ CD3+ T cells (1.1–5.5%, 11–46 cells/μl) and elevated serum levels of sFasL (Table 2). Plasma vitamin B12 levels, the percentage of B220+ DNT cells and the ratio of CD25+/ HLA-DR+ lymphocytes were within the normal range for all patients (Table 2). Based on the serum levels of sFasL and plasma vitamin B12 levels the probability for FAS mutation was calculated (Table 2) [11] rendering FAS mutations very unlikely in patient 1 while patients 2 and 4 were in the gray zone suggesting the evaluation of additional markers.
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3.3. Altered B cell homeostasis in LRBA deficiency partially correlates with the presence of clinical manifestations
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Flow cytometric analysis of the four LRBA deficient patients revealed reduced B cell numbers in all patients (Table 2). Additionally, reduction of IgG+ and IgA+ CD27+ switched memory B cells was found in all symptomatic patients (P1, P2 and P4) while the levels were normal in the asymptomatic child (P3). Other B cell subpopulations were variably affected. All patients of the second family displayed an accumulation of CD21low B cells. IgM only memory B cells were generally decreased compared to age matched reference values. As an expression of combined immunodeficiency in LRBA deficiency all patients displayed low CD4+CD45RA+ naive T cells while the percentage of naive CD8 T cells was normal. Numbers of terminally differentiated CD8 T cells were low compared to age matched reference values. The heterozygous parents and siblings had no clinical symptoms and immune phenotyping revealed normal parameters (data not shown).
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Please cite this article as: S. Revel-Vilk, et al., Autoimmune lymphoproliferative syndrome-like disease in patients with LRBA mutation, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.04.007
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Figure 1 Molecular diagnosis of LRBA deficiency. (A) c.7937TNG mutation in the DNA sequence of exon 54 of the human LRBA gene in a patient 1 (upper row) and obligate heterozygote (lower row) is shown by the arrow. (B) 8139_8142dupCATG insertion in the DNA sequence of exon 56 of the human LRBA gene in patients 2, 3 and 4 (upper row), obligate heterozygote (middle row), and a healthy control (lower row). The mutation is indicated by the arrow. (C) Expression of LRBA protein in patients with homozygous mutations in LRBA. Western blot analysis revealed the absence of the LRBA protein in EBV cell lysates from P1, P2, P4 (left) and P3 (right), compared to the ~ 319 kDa LRBA band observed in EBV cell lysates from healthy donors. GAPDH (left) and tubulin (right) served as loading controls. Please cite this article as: S. Revel-Vilk, et al., Autoimmune lymphoproliferative syndrome-like disease in patients with LRBA mutation, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.04.007
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Table 2
Age at time of analysis
P1 13y
P2 8y
P3 6.5y
P4 2.5y
t2:4
Vitamin B12, pg/ml Normal values (200–900 pg/ml) Serum FAS ligand, pg/ml Normal values b 200 pg/ml Calculated probability for Fas mutation [11] Lymphocytes, cells/μl Normal values ⁎
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821
NA
760
520 b 1%
789 26%
312 –
599 6.9%
t2:44 t2:45 t2:46 t2:47 t2:48 t2:49 t2:50 t2:51 t2:52 t2:53
15 25–64 351 300–1300 106 78–640 57 2–86 54 16–810 16 35–420 11
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4 18–86 303 200–1700 83 42–1300 25 6–43 46 45–410 19
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57–340 23
307 641–1453 51 375–1096
1200–4700 absolute (cells/μl) 103 296–784 9 13–63 62 154–413 13 24–135 1 7–65 5 5–35 4 13–74
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15–41 2.8–2.9 b 2.5
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490 608–1217 170 311–781
1300 relative (%) 7.9 8.5–20.2 8.9 3.4–9.0 60.4 48–70 12.9 6–22 1.2 2–12 4.6 1.1–6.1 3.9 2.7–14.0 11.1 0.8–7.7 16.9 26.5–41.4 32.3 55.6–75.8 67.7 75.5 61.0–84.2 5.7 2.3–7.7 20.1 13–47 48.9 16–100 10.7 1–6 3.6 5–100 0.7
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1200–4700 absolute (cells/μl) 48 296–784 1 13–63 40 154–413 6 24–135 0 7–65 0 5–35 0 13–74
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1100 relative (%) 4.4 8.5–20.2 1.1 3.4–9.0 82.4 48–70 12.8 6–22 0.4 2–12 0.3 1.1–6.1 0.6 2.7–14.0 16.8 0.8–7.7 27.9 26.5–41.4 16.5 55.6–75.8 83.5 46.0 61.0–84.2 1.4 2.3–7.7 27.5 13–47 27.3 16–100 8.1 1–6 15.2 5–100 6.2
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1400–4200 absolute (cells/μl) 42 119–578 2 2–41 36 83–398 3 10–74 0 3–39 0 4–13 0 7–32
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1300 relative (%) B cells 3.2 Normal values ⁎ 7.8–23.7 Transitional B cells 3.6 Normal values ⁎ 1.5–7.3 Naive B cells 86.2 Normal values ⁎ 64.6–80.1 IgM Memory B cells 6.3 Normal values ⁎ 4.7–15.5 IgM only B cells 0.3 Normal values ⁎ 1.6–11.3 IgA switched mem (27pos) B cells 0.2 Normal values ⁎ 1.2–3.8 IgG switched mem (CD27pos) B cells 0.9 Normal values ⁎ 2.1–9.4 CD21low B cells 6.8 Normal values ⁎⁎ 0.8–7.7 CD4 37.7 Normal values ⁎ 30.4–52.9 Naive CD45RA cells 34.7 Normal values ⁎ 49.3–72.0 Memory CD45R0 cells 65.3 RTE % of CD45RApos CD4 T cells 84.0 Normal values ⁎ 50.7–78.9 Treg cells 3.1 Normal values ⁎ 2.8–7.2 CD8 27 Normal values ⁎⁎⁎ 14–40 Naive CD8 T cells 30.1 Normal values ⁎⁎⁎ 20–95 Central memory CD8 T cells 16.2 Normal values ⁎⁎⁎ 0.42–18 Effector memory CD8 T cells 15.5 Normal values ⁎⁎⁎ 4–100 Terminally differentiated 4.6 effector cells Normal values ⁎⁎⁎ 9–65 1.1–5.5 DN T cells of αγ+ CD3+ T cells Normal values b 2.5
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Serological and cellular parameters in peripheral blood of patients.
15–41 1.7–1.9 b 2.5
220 641–1453 71 375–1096
13 18–86 261 200–1700 128 42–1300 28 6–43 9 45–410 2 57–340 11
1900 relative (%) 5.7 17.3–30 23.2 3.6–21.4 63.0 50–79 9.6 5–12 0.3 1.6–6.6 0.4 0.7–2.9 0.6 0.8–6.3 10.3 0.8–7.7 24.7 28.1–43. 33.5 71.5–84.2 66.5 52.6 65.2–79.5 3.0 2.9–7.4 29 9–49 58.8 19–100 8.8 1–9 2.9 10–55 0.4
1400–5500 absolute (cells/μl) 108 686–1732 25 41–248 68 346–1356 10 50–148 0 19–53 0 6–57 1 8–26
6–83 2.2–3.3 b 2.5
25–530 46
469 925–2477 157 685–2055
14 42–69 551 200–1800 324 53–1100 48 4–64 16 24–590 2
If not indicated differently percentages refer to the respective parental population. Naive CD8 T cells are characterized by CCR7+ CD45RA+ CD27+, central memory by CCR7+CD45RA−CD27+, effector memory by CCR7−CD45RA−CD27− and terminally differentiated by CCR7−CD45RA+ CD27−. RTE = recent thymic emigrants. DN T cells = double negative (CD4−CD8−) cells of TCRαβ+CD3+ T cells. DN T cells were measured at two different time points. Pathological values are in bold print. ⁎ Normal values refer to age matched controls published in van Gent, R., et al. Refined characterization and reference values of the pediatric T- and B-cell compartments. Clinical Immunology 133, 95–107. ⁎⁎ Internal reference values for adults. ⁎⁎⁎ Schatorje, E.J., et al. Paediatric reference values for the peripheral T cell compartment. Scand J Immunol 75, 436–444.
Please cite this article as: S. Revel-Vilk, et al., Autoimmune lymphoproliferative syndrome-like disease in patients with LRBA mutation, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.04.007
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In this study we expand the observed variability in the clinical and immunological presentation of children with mutations in LRBA. Three children of our study presented with clinical and laboratory features of ALPS-like disease and one child is still asymptomatic up to the age of 7 years. At presentation none of the children had clinical symptoms of recurrent infections, but all three symptomatic children presented with autoimmune cytopenia, splenomegaly and lymphadenopathy resembling the clinical phenotype of ALPS. Thus, according to the revised criteria for ALPS our patients were initially diagnosed with probable diagnosis of ALPS and therapy with steroids and MMF was initiated with stable response [13]. The diagnosis of ALPS remains challenging especially in centers that lack expertise in immunology testing. The specificity of elevated DNT cell number, a test that is widely available and commonly used for the diagnosis of ALPS, has recently been challenged [14]. An elevation of DNT as observed in our patients is found in 30% of children with autoimmune diseases [14] as well as in combined variable immunodeficiency (CVID) patients [15,16]. In healthy controls the 95th percentile for TCR αβ+ DNT was used to define a normal range up to 2.3% of total lymphocytes or 3.4% of T cells [15]. In the study by Oliveira et al. cut-offs for DNT were elevated DNT ≥ 1.5% of total lymphocytes or 2.5% of CD3+ lymphocytes [3]. However, in 163 patients with clinically suspected ALPS, the predictive value of DNT was found to be low (positive and negative predicative values of 61% and 77%, respectively) [11]. In the same cohort, the best predictor of FAS mutations was the combination of elevated vitamin B12 (N 1255 pg/ml) and sFasL (N 559 pg/ml) [11]. Unfortunately, sFasL and cytokine measurement like IL10 or IL18 are not attainable tests in most centers and therefore currently don't serve as a routine markers for the diagnostic evaluation. sFasL serum levels were strongly elevated in three of our four patients with LRBA mutations. Vitamin B12 levels, however, were normal in our patients. Based on the FAS mutation probability online calculator the probability for FAS mutations in our patients ranged between 0.1% and 26% rendering the probability of causative FAS mutations low in all of the analyzed patients [11]. Consistently, no germline mutations in FAS and other genes known to be associated with ALPS were found. Genetic analysis of sorted DNT in search for somatic FAS mutations was not done because of familial involvement and low FAS mutation probability and whole exome analysis
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staurosporine, another inducer of the intrinsic mitochondria mediated apoptotic pathway. Similar as in response to serum deprivation, LRBA deficient T cells of symptomatic patients were more sensitive to staurosporine treatment than controls (Fig. 2C). Notably, the asymptomatic patient (P3) showed an intermediate susceptibility to staurosporine and serum starvation induced apoptosis compared to healthy controls and affected patients. Similar results were obtained employing EBV transformed B cell lines derived from the patients and controls. The levels of apoptosis in response to staurosporine treatment and serum deprivation were significantly higher in LRBA-deficient EBV-transformed B cells compared to healthy controls (Fig. 2D+E).
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LRBA plays a role in the regulation of apoptosis [4,10]. Mechanistically there are two main pathways of apoptotic cell death: 1) the extrinsic pathway regulated by death receptors such as Fas and 2) the mitochondria mediated pathway regulated by proteins of the Bcl-2 family. In patients with ALPS accumulation of DNT cells is due to defective extrinsic Fas mediated apoptosis. To test whether mutation of LRBA impairs this apoptotic pathway activated primary T cells from the patients and controls were treated with recombinant Fas ligand (FasL) in vitro. Cells from patients with complete LRBA deficiency were clearly less responsive to apoptosis induced by recombinant FasL compared to their siblings with a wildtype genotype (healthy controls) as shown in Fig. 2A. Heterozygous carriers had an intermediate response (data not shown). In contrast, similar to a previous report [4] T cells derived from the patients showed increased intrinsic apoptosis after 24 h of serum starvation compared to healthy controls (Fig. 2B). In addition, we tested the response of these cells to
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3.4. LRBA mutation decreases Fas mediated apoptosis
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Figure 2 LRBA mutations differentially affect extrinsic and intrinsic apoptosis pathways. Activated primary T cells from the patients and healthy controls were treated with 50 ng/ml recombinant FasL (A), serum deprivation (B) or 0.25 μM staurosporine (C) for 24 h. Immortalized B cells from the patients and a healthy control were deprived of serum (D) or treated with staurosporine (E) as described in (B, C). Subsequently apoptosis was measured by flow cytometric detection of Annexin V-FITC/ propidium iodide staining (A–E).
Please cite this article as: S. Revel-Vilk, et al., Autoimmune lymphoproliferative syndrome-like disease in patients with LRBA mutation, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.04.007
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5. Uncited reference
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Conflict of interest statement
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The authors declare that there are no conflicts of interest.
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Acknowledgments
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This study was supported by the German Federal Ministry of Education and Research (BMBF 01EO1303). PS was supported (in part) by a grant from the Joint Research Fund of the Hebrew University and the Hadassah Hebrew University Hospitals.
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Appendix A. Supplementary data
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Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.clim.2015.04.007.
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contribute to the autoimmune-lymphoproliferative phenotype of the affected patients. The follow up of our patients for 3 to 11 years allowed us to observe a progressive, but variable course of their disease with progression of both the clinical and laboratory features over the years. The immunosuppressive therapy probably modulated the clinical course of the disease and may have significantly influenced the rate of complications and quality of life. Based on the identified mutation in LRBA, the previously reported prognosis of patients with LRBA deficiency and with the availability of a fully matched family donor, we decided to go ahead with stem cell transplantation in patient P1 because of the smoldering progression of bronchiectasis; a step, which we would have not performed based purely on the clinical diagnosis of probable ALPS. In summary, this study emphasizes the complexity of diagnosing specific PID in patients who present with the clinical phenotype of ALPS. Several other clinical entities may also present with autoimmune cytopenia and lymphoproliferation. The recently suggested score to identify patients with ALPS and mutation in FAS was helpful to distinguish LRBA deficient patients from ALPS-FAS cases, but unfortunately in many centers laboratory biomarkers sFasL and IL-10 are not testable. The effect of disturbed autophagy on the extrinsic apoptosis pathway may contribute to the ALPS like presentation in some LRBA deficient patients. Thus, LRBA deficiency needs to be considered in the differential diagnosis of ALPS like patients. We believe that comprehensive genetic evaluation is important for arriving at the true diagnosis enabling proper care and follow-up of patients presenting with ALPS-like symptoms.
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was performed instead. This revealed unexpectedly mutations in the LRBA gene, typically associated with a disorder of combined immunodeficiency, enteropathy and autoimmune disease [4]. Only prolonged follow-up revealed signs of immunodeficiency, i.e. progressive decrease of immunoglobulin levels and development of bronchiectasis in P1. Thus the correct diagnosis in children with ALPS like disorders, which is important both for management decisions and for genetic consultation, requires further unbiased genetic analysis after exclusion of known candidate genes underlying ALPS [17]. Interestingly, the mutation of LRBA gene that we found in P1 was previously described in two children of Arab origin who were treated in another hospital in Israel [4]. Like in many other PID, there is no clear genotype-phenotype correlation in patients with LRBA mutation. The previously published family with the same single base exchange (designated c.7970TNG referring to the gene transcript ENST00000357115) mutation presented with signs and symptoms of immunodeficiency like progressive lung disease, cerebral granuloma, lymphoid interstitial pneumonia and arthritis that were absent in our patient [4]. In the second family with an unrelated mutation in LRBA, two patients (P2, P4) had an ALPS-like clinical presentation while the 7 year old brother (P3) was asymptomatic. The immunological laboratory studies of our patients show significant variability. As described in previous cases, the three symptomatic patients demonstrated variable but marked decrease in class-switched B cells, while the asymptomatic patient presented with normal numbers. These differences between the symptomatic and asymptomatic patients may be partially related to the therapy with MMF and steroid given to the symptomatic patients. The progressive hypogammaglobulinemia and bronchiectasis on chest CT found in our oldest patient (P1) is of clinical importance and may point to the evolving nature of this disease. Normal immunoglobulins levels at presentation were reported also by other groups (Table 1). ALPS is usually caused by loss-of-function or dominantnegative mutations in components of the Fas signaling pathway that compromise Fas mediated apoptosis. Interestingly, also LRBA deficiency was associated with a deficient response to Fas triggered apoptosis while the sensitivity to apoptosis stimulated by intrinsic signals was even increased as previously reported. LRBA belongs to a family of BEACH domain containing proteins that are involved in cellular mechanisms including vesicular transport, autophagy, apoptosis, membrane dynamics and receptor signaling [18]. LRBA-deficient B cells were previously reported to have a significantly reduced ability to induce autophagy in response to starvation [4]. Autophagy as a major process involved in the degradation of cellular material is cytoprotective for hematopoietic cells [19] and essential for the homeostasis and activation of T cells [20]. Thus, deficiency in autophagy was suggested to underlie the increased cell death induced by intrinsic stressors in LRBA deficient patients. On the other hand, it was recently recognized that autophagy specifically facilitates Fas mediated apoptosis by degradation of the Fap-1 protein phosphatase, a critical negative regulator of apoptotic cell death signaled by Fas [21]. Thus, autophagy affects cell death in a cell-type and stimulus-depending manner [21] and defects in LRBA mediated autophagy may account for the observed divergent effects on extrinsic vs intrinsic cell death. The detected effect on the extrinsic pathway may
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[1] C. Platt, R.S. Geha, J. Chou, Gene hunting in the genomic era: approaches to diagnostic dilemmas in patients with primary immunodeficiencies, J. Allergy Clin. Immunol. 134 (2014) 262–268.
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Please cite this article as: S. Revel-Vilk, et al., Autoimmune lymphoproliferative syndrome-like disease in patients with LRBA mutation, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.04.007
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