Journal of Clinical Immunology (2020) 40:987–1000 https://doi.org/10.1007/s10875-020-00794-7 ORIGINAL ARTICLE Severe Autoinflammatory Manifestations and Antibody Deficiency Due to Novel Hypermorphic PLCG2 Mutations Andrea Martín-Nalda 1,2 & Claudia Fortuny 3,4,1 & Lourdes Rey 5 & Tom D. Bunney 6 & Laia Alsina 4,7,8 & Ana Esteve-Solé 4,7,8 & Daniel Bull 9 & Maria Carmen Anton 10 & María Basagaña 11 & Ferran Casals 12 & Angela Deyá 4,7,8 & Marina García-Prat 1,2 & Ramon Gimeno 13 & Manel Juan 10,14,15 & Helios Martinez-Banaclocha 16 & Juan J Martinez-Garcia 16 & Anna Mensa-Vilaró 10 & Raquel Rabionet 4,17 & Nieves Martin-Begue 18 & Francesc Rudilla 19,20 & Jordi Yagüe 10,14,15 & Xavier Estivill 21 & Vicente García-Patos 22 & Ramon M. Pujol 23 & Pere Soler-Palacín 1,2,24 & Matilda Katan 6 & Pablo Pelegrín 16 & Roger Colobran 2,25,26 & Asun Vicente 27 & Juan I. Arostegui 10,14,15 Received: 10 January 2020 / Accepted: 20 May 2020 / Published online: 15 July 2020 # The Author(s) 2020 Abstract Autoinflammatory diseases (AIDs) were first described as clinical disorders characterized by recurrent episodes of seemingly unprovoked sterile inflammation. In the past few years, the identification of novel AIDs expanded their phenotypes toward more complex clinical pictures associating vasculopathy, autoimmunity, or immunodeficiency. Herein, we describe two unrelated patients suffering since the neonatal period from a complex disease mainly characterized by severe sterile inflammation, recurrent bacterial infections, and marked humoral immunodeficiency. Whole-exome sequencing detected a novel, de novo heterozygous PLCG2 variant in each patient (p.Ala708Pro and p.Leu845_Leu848del). A clear enhanced PLCγ2 activity for both variants was demon- strated by both ex vivo calcium responses of the patient’s B cells to IgM stimulation and in vitro assessment of PLC activity. These data supported the autoinflammation and PLCγ2-associated antibody deficiency and immune dysregulation (APLAID) diagnosis in both patients. Immunological evaluation revealed a severe decrease of immunoglobulins and B cells, especially class-switched memory B cells, with normal T and NK cell counts. Analysis of bone marrow of one patient revealed a reduced immature B cell fraction compared with controls. Additional investigations showed that both PLCG2 variants activate the NLRP3-inflammasome through the alternative pathway instead of the canonical pathway. Collectively, the evidences here shown expand APLAID diversity toward more severe phenotypes than previously reported including dominantly inherited agammaglobulinemia, add novel data about its genetic basis, and implicate the alternative NLRP3-inflammasome activation pathway in the basis of sterile inflammation. Keywords Autoinflammatory diseases . APLAID . PLCγ2 . inflammasome . caspase-1 . interleukin-1 . agammaglobulinemia Introduction Autoinflammatory diseases (AIDs) constitute a specific group of primary immunodeficiency diseases (PID) characterized by re- Andrea Martin-Nalda and Claudia Fortuny are first co-authors who current episodes of systemic sterile inflammation mainly medi- contributed equally to this article. ated by cells of innate immunity [1]. At present, around 30 dif- Pablo Pelegrín, Roger Colobran, Asun Vicente, and Juan I. Arostegui ferent monogenic AIDs have been molecularly elucidated, with contributed equally to this article as senior authors. some of the novel ones also displaying features of dysregulated B Electronic supplementary material The online version of this article cell response either as circulating autoantibodies or as reduced (https://doi.org/10.1007/s10875-020-00794-7) contains supplementary antibody production [2–5]. The extremely rare, dominantly material, which is available to authorized users. inherited PLCγ2-associated antibody deficiency and immune dysregulation (PLAID) and autoinflammation and PLCγ2- * Juan I. Arostegui associated antibody deficiency and immune dysregulation jiaroste@clinic.cat (APLAID) may be included among those rare monogenic Extended author information available on the last page of the article AIDs combining sterile inflammation and humoral 988 J Clin Immunol (2020) 40:987–1000 immunodeficiency. Both diseases are linked to hypermorphic, was performed as previously described to evaluate parental pathogenic variants in the PLCG2 gene encoding the key signal gene mosaicism [10]. For Sanger sequencing, specific exons transduction enzyme PLCγ2. They are clinically characterized of the PLCG2 gene were amplified by in house–designed PCR by early-onset skin inflammation and recurrent infections. In (primers listed in Supplementary Table S2), purified with addition, patients with APLAID often develop ocular and lung Illustra ExoStar 1-Step kit (GE Healthcare, USA), bidirectional inflammation, enterocolitis, and interstitial pneumonitis but un- sequenced using ABI BigDye® Terminator v3.1 Cycle like PLAID, they do not present with cold urticaria or autoim- Sequencing Kit (Applied Biosystems, USA) and run on an munity [6–9]. However, because only a few APLAID cases have automated ABI 3730XL analyzer (Applied Biosystems, USA). been characterized so far, it is not clear to what extent their clinical manifestations could differ or overlap with other immune Analyses of Ca2+ Flux in B Cells and Measurements of disorders. Similarly, the range of genetic changes (in PLCG2 or Phosphoslipase-C Activity in genes regulating associated signal transduction pathways) and their impact on immune cell functions have not been defined. Intracellular Ca2+ flux was measured by flow cytometry after In the present study we describe two unrelated patients with labeling with FLUO-3 AM (Invitrogen, USA) as previously early-onset severe skin and eye inflammation, and recurrent described with slight modifications [6]. bacterial infections secondary to antibody deficiency. Further For the measurements of phosphoslipase-C (PLC) activity, genetic and functional studies are consistent with APLAID COS-7 cells were cultured in DMEM (Sigma-Aldrich, USA) con- and consolidate and expand the key features and underpinning taining 10% (v/v) FBS and 2.5 mM glutamine (growth media). molecular mechanisms for this diagnosis. Cells were grown as a monolayer at 37 °C in 5% CO2. COS-7 cells were seeded into 96-well plates at a density of 7500 cells/well in 0.1 mL of growth media and incubated overnight. Fresh media Patients and Methods was applied and the cells transfected with plasmid DNA at 100 ng/ well that had been diluted in 5 μL jetPRIME® buffer and 0.2 μL The ethics committees of Hospital Sant Joan de Déu, Hospital jetPRIME® (Polyplus Transfection, France) that was prepared as Universitari Vall d’Hebron and Hospital Clínic, all in instructed by the manufacturer. The DNA concentration was kept Barcelona (Spain), approved the study. Written informed con- constant by adding empty plasmid. For the PLCγ2 expression sent for participation in the study was obtained from patients’ plasmids, the full-length ORF of human PLCG2 was cloned into parents. Blood samples from patients and unaffected relatives the vector pTriEx4 using Gateway technology (Thermo Fisher). were collected for molecular studies, which were performed in Mutations and deletions were prepared using the site-directed mu- accordance with the Declaration of Helsinki. tagenesis kit (Agilent) following manufacturer’s instructions. The ORFs of all constructs were fully sequenced prior to a Maxiprep Flow Cytometry Studies being performed to generate the plasmid DNA for transfection. Each PLCγ2 construct was transfected at 4 concentrations in trip- Peripheral blood mononuclear cells (PBMCs) were isolated by licate as outlined in the figures. Twenty-four hours post-transfec- Ficoll gradient (Fresenius-Kabi Norge, Norway) and stained for tion, the media was removed and replaced with growth media cell surface markers using fluorochrome-conjugated antibodies without FBS but containing 0.25% (w/v) fatty acid free BSA. (Supplementary Table S1). Samples were acquired using a The COS-7 cells were then incubated for further 24 h. FACSCanto II cytometer (BD Biosciences, USA), and data were Subsequently, the media was replaced with growth media without analyzed with CellQuest software (BD Biosciences, USA). FBS but containing 50 mM LiCl with and without 100 ng/mL EGF and incubated for further 1 h. The media was aspirated and Genetic Analyses replaced by 25 μL of stimulation buffer (20 mM HEPES.OH, 2 mM CaCl2, 1 mM MgCl2, 8.4 mM KCl, 292 mM NaCl, DNA samples were extracted from peripheral blood using a 11 mM glucose and 100 mM LiCl, pH 7.4) followed by 25 μL QIAmp DNA Blood Mini Kit (Qiagen, Germany). Libraries of lysis buffer (50 mM HEPES.OH, 0.8 M KF, 0.2% (w/v) BSA were prepared using TruSight One kit (Illumina, USA) in and 1% (v/v) Triton-X-100, pH 7.0). The cells were lysed for Family 1 and SureSelect Human All exon V2 kit (Agilent 30 min at room temperature on an orbital shaker. Seven microli- Technologies, USA) in Family 2 following manufacturer’s in- ters of the cell lysate was pipetted in duplicate into a white 384- structions. Paired-end sequencing was performed on an well plate (Greiner Bio-One GmbH, Austria) followed by 1.6 μL Illumina Genome Analyzer II platform (Illumina, USA). of IP1-d2 (Cisbio, France). After 5 min, 1.6 μL of anti-IP1- Reads were mapped against the human reference genome Cryptate (Cisbio, France) was added and the plate sealed and hg19 using the BWA software and analyzed with the GATK incubated at room temperature for 1 h. The plate was read on a Unified Genotyper v2.8. Amplicon-based deep sequencing of PHERAstar (BMG Labtech, Germany) plate reader in HTRF specific exons of the PLCG2 gene (RefSeq NM_002661.3) mode, and the data converted to IP1 concentration using a standard J Clin Immunol (2020) 40:987–1000 989 curve generated following manufacturer’s instructions. Data for of gestation by cesarean section due to loss of fetal wellbeing the measured PLC activity represent the standard error of the mean (pedigree in Fig. 1a). The main features of her disease includ- of transfections performed in triplicate. ed early-onset severe cutaneous and eye inflammation and Quantities of expressed proteins were measured using a WES recurrent respiratory infections (Table 1). Western Blotting system (Protein Simple, USA). For this, a Skin lesions appeared as early as the first day of life further 96-well plate was transfected identically to the plate used as numerous papulo-vesicular lesions, which became in the IP1 assay described above. Forty-eight hours post-trans- generalized during the following days requiring admis- fection, the cells were washed once in ice-cold PBS and subse- sion into pediatric intensive care unit. These lesions have quently lysed in 20 μL RIPA buffer (Thermofisher, USA) con- been nearly continuously present, with exacerbations, taining a protease and phosphatase inhibitor cocktail occasionally hemorrhagic and complicated with infec- (Thermofisher, USA). The cells were freeze-thawed at − 20 °C tions, ulcerated lesions, and ulcerative granulomata and subsequently 4 μL of each well loaded on a WES Western (Fig. 2a). In recent years, large areas of cutis laxa and Blotting. Proteins were detected with a 1:150 dilution of the anti- hyperpigmentation were detected (Fig. 2b). At 2.5 years PLCγ2 antibody sc5283 (Santa Cruz) and a 1:150 dilution of of age, bilateral conjunctivitis, corneal erosions, and the anti-β-actin antibody 13E5 (Cell Signaling Technology). nodules appeared (Fig. 2c, d). For the comparison of PLC activity of different PLCγ2 variants, The absence of circulating immunoglobulins was de- the expression levels were quantitated and points with the same tected during the first year of life (Table 2). Intravenous protein expression used, as previously described [11]. The dif- immunoglobulin (IVIG) replacement therapy (IVIGs; ferences were confirmed using the 2 tailed t test. 400 mg/kg q3w) was then started and has been main- tained until present. Despite this treatment, multiple infec- Detection of Intracellular ASC Specking and Active tions were detected (cutaneous infections, acute gastroen- Caspase-1 teritis, periodontitis, herpetic stomatitis, bronchitis, and pneumonia). At 4 years of age, multiple central bronchi- PBMCs were treated with either nothing or E. coli LPS serotype ectases were detected on a CT scan (Fig. 2e), which sub- 055:B5 (Sigma-Aldrich, USA; 1 μg/mL, 2 h at 37 °C). LPS- sequently progressed and provoked recurrent episodes of primed PBMCs were then stimulated with either nothing, ATP acute hemoptysis that required urgent embolization. As (3 mM), or nigericin (10 μM) for 30 min at 37 °C. Stimulated consequence of these lesions, the medium right lung lobe PBMCs were fixed with 2% paraformaldehyde (Sigma-Aldrich, was surgically excised at the age of 13 years. USA) and stained for the detection of intracellular ASC specks by The patient received multiple treatments including antibiotics, TOFIE as previously described [12], using the rabbit polyclonal retinoids, corticosteroids, etanercept (25 mg q1w for 5 years), and antibody anti-ASC (N-15)-R (Santa Cruz Biotechnology, USA). anakinra (100 mg q1d for 1.5 years). With the use of etanercept For active caspase-1 detection, PBMCs were incubated for and anakinra, a partial control of skin inflammation was detected, 1 h with FLICA660 reagent (ImmunoChemistry Technologies, with no improvement of ocular manifestations or immune de- USA) and fixed following manufacturer recommendations. fects. By contrast, a marked decrease of plasma C-reactive pro- Monocytes were detected with the APC-vio770-conjugated tein (CRP) was detected with etanercept (mean 6.86 mg/L; range anti-human CD33 antibody (Miltenyi Biotec, Germany) and 0.08–21-8) and anakinra (mean 0.10 mg/L; range 0.05–0.16) with the APC-Cy7-conjugated anti-human CD14 antibody compared with periods in which these treatments were not ad- (TONBO Biosciences, USA). Stained cells were acquired on a ministered (mean 13.32 mg/L; range 1.3–31.63). FACSCanto cytometer (BD Biosciences, USA). Cytokines were measured in cell supernatants using a custom bead-based Family 2 multiplex Luminex immunoassay (eBioscience, USA). Heat maps representing cytokine expression profiles were created Patient 2 is a 9-year-old boy born from healthy parents using Morpheus software (Broad Institute, Cambridge, USA). (pedigree in Fig. 1a). Skin manifestations started during the first week of life as multiple erythematous macules, papules, and large plaques, mainly located at arms, abdo- Results men, and thighs (Table 1). These lesions recurred with no periodicity, sometimes presenting as urticaria-like, vesicu- Clinical Description lar or pustular lesions (Fig. 2f) or ulcerated or exudative plaques. Infections, minor traumas, vaccinations, and heat Family 1 were identified as triggering or worsening factors. They were successfully treated with oral or topical corticoste- Patient 1 has been first described in 2007 [13] and is now a 16- roids (1 mg/kg q1d), and partially with dapsone (1 mg/kg year-old girl. She was born from healthy parents at 40 weeks q1d), and they healed leaving focal, wrinkled-appearing 990 J Clin Immunol (2020) 40:987–1000 Fig. 1 Familial pedigrees and features of PLCG2 variants. Panel a the patients. Panel e Scheme of structural domains of phospholipase Pedigrees of enrolled families. Black filled symbols represent affected Cγ2 protein. The already known APLAID-associated PLCG2 individuals, open symbols unaffected individuals, squares male mutations are showed in red fonts, while the PLCG2 variants described individuals and circles female individuals. Panels b, c Schemes of in the present work are displayed in blue fonts. Panel f Multiple sequence filtering of the next-generation sequencing strategies used in the alignment of human phospholipase Cγ2 and sixteen orthologues. The enrolled families. Panel d Sanger sense chromatograms of the PLCG2 single asterisk represents the amino acid residue 708 of human gene from patients (upper boxes) and from wild-type healthy subjects phospholipase Cγ2, while two asterisks indicate the amino acid (bottom boxes). Gray arrows indicate the PLCG2 variants detected in residues 845–848 patches of cutis laxa and residual hyperpigmentation (Fig. Hematological and Immunological Parameters 2g). Ocular inflammatory manifestations have recently ap- peared as bilateral red eye, corneal limbitis, and episcleritis Laboratory monitoring revealed increased inflammatory (Fig. 2h). markers (CRP, platelet count) and reduced hemoglobin in Infections started at the age of 2 months as recurrent, both patients (Supplementary Fig. S1). Immunological tests mild viral bronchitis. Since the age of 4 years, recurrent repeatedly revealed low-to-absent immunoglobulins and bacterial infections were detected, mainly at ear (> 10 ep- marked decrease of B cells in both patients, with T and NK isodes) and lung (4 pneumonias), leading to mild bronchi- cell counts repeatedly normal (Table 2). A comprehensive ectasis (Fig. 2e). All infections were successfully treated analysis of circulating B cells revealed their near complete with oral antibiotics, without hospitalization. IVIG re- absence in patient 1 and an overall decrease of all B cell placement therapy (500 mg/kg q3w) was started in subpopulations, a decreased response to polysaccharide vac- January 2016, which resulted in a decrease of the frequen- cination, and the absence of autoantibodies in patient 2 cy of respiratory infections and in a marked improvement (Table 2 and Supplementary Table S3). Bone marrow aspira- of patient’s health status. tion was once performed in patient 1, which revealed that B Table 1 Summary of clinical and immunological features of enrolled patients and comparison with reported APLAID patients (Zhou et al. [7]; Neves et al. [8]; Morán-Villaseñor et al. [9] Present study Zhou et al. Neves et al. Moran-Villaseñor et al. Patient 1 Patient 2 Patient II-1 Patient III-2 Patient 1 Patient 1 Clinical features Age at disease onset Birth 5th day of life Infancy Infancy 1st week of life 3rd day of life J Clin Immunol (2020) 40:987–1000 Cutaneous lesions Erythematous plaques, Maculo-papular eruption, Epidermolysis Epidermolysis bullosa– Vesiculopustular rash, Erythematous pustules, vesiculopustular and erythematous plaques, bullosa–like eruption, like eruption, cutaneous granulomas, yellow-pink papules, ulcerative lesions, urticarial-like lesions, erythematous plaques, erythematous plaques, blistering skin rash pseudovesicles, acral ulcerative vesiculo-pustular lesions, vesiculopustular vesiculopustular lesions cutis laxa hemorrhagic blisters, granulomata, hyperpigmentation lesions cutaneous granulomas hyperpigmentation cutis laxa cutis laxa Eye inflammation Bilateral corneal erosions, Bilateral episcleritis, - Corneal small blisters, Recurrent eye Non-purulent corneal limbitis, corneal limbitis, bilateral corneal erosions, inflammation, conjunctival erythema corneal nodules, haze episcleritis corneal ulcerations, posterior uveitis, bilateral conjunctivitis, intraocular ocular hypertension bilateral episcleritis hypertension cataracts Lung involvement Bronchiectasis, recurrent - Interstitial pneumonitis Interstitial pneumonitis Interstitial pneumonitis - episodes of hemoptysis Joint involvement - - Arthralgias Arthralgias - Arthralgias Gastrointestinal - - Enterocolitis Recurrent abdominal pain, Bloody diarrhea, Recurrent episodes involvement bloody diarrhea, early-onset of diarrhea ulcerative colitis inflammatory bowel disease Infections Recurrent sinopulmonary Recurrent bronchitis, Recurrent sinopulmonary Cellulitis Recurrent infections Recurrent upper infections, herpetic recurrent suppurative infections, cellulitis (pneumonia, ear, sinus) respiratory infections stomatitis, recurrent otitis, pneumonias, bacterial and fungal perineal dermatitis, acute skin infections gastroenteritis (C. jejuni) Immunodeficiency Yes Yes Yes Yes Yes Yes Immunological features T cells Normal Normal Normal Normal Normal Normal B cells Very low/absent Very low Normal Low Low Low NK cells Normal Normal Normal Normal Normal Low IgG Very low Normal Normal Normal Low Low IgA Very low Normal Low Low Low Low IgM Very low Low-Very low Low Low Low Low IgE Very low Low n.a. n.a. n.a. n.a. Autoantibodies Negative Negative Negative Negative Negative Negative Genetic features PLCG2 genotype p.L845_L848del/wt p.A708P/wt p.S707Y/wt p.S707Y/wt p.L848P/wt p.L848P/wt 991 992 J Clin Immunol (2020) 40:987–1000 Fig. 2 Description of cutaneous, pulmonary, and ocular inflammatory manifestations in patients. Panel a Multiple papules and serosal and hemorrhagic vesicles on the hands and palms detected in patient 1 at the age of 4 years. Panel b Large areas of cutis laxa in the abdominal region detected in patient 1 at the age of 7 years. Panels c, d Ocular inflammatory lesions including intense bilateral conjunctivitis, keratitis, episcleritis, and nodules in the sclera detected over the course of the disease in patient 1. Panel e Bronchiectasis detected in patient 1. Panel f Blistering inflammatory cutaneous lesions in the leg detected in patient 2 at the age of 6 months. Panel g Areas of cutis laxa detected in patient 2 at the age of 6 years. Panel h Ocular inflammatory lesions including conjunctivitis and corneal limbitis detected in patient 2 at the age of 7 years cell lineage represented 6.2% of total leukocytes and 28.4% of PLCG2 variants (Supplementary Table S5). According to total lymphocytes, with the presence of all B cell stages. the consensus joint recommendations of the American However, a reduction of the immature B cell stage compared College of Medical Genetics and Genomics and the with healthy controls was detected (Supplementary Fig. S2). Association of Molecular Pathology [14], these two PLCG2 variants were classified as pathogenic on the basis of different criteria including their de novo nature, their absence in healthy Molecular Genetics controls, their location in regulatory domains and in conserved residues of the protein (Fig. 1e–f), and the results of different Genetic analyses identified rare candidate variants in both bioinformatics and functional analyses (Table 3). patients (Fig. 1b, c shows candidate filtering strategies; Supplementary Table S4 lists the gene variants detected in patient 1). Assuming a dominant inheritance pattern for the Functional Characterization of PLCγ2 Variants disease, patients only shared heterozygous variants at PLCG2 gene (p.Leu845_Leu848del in Family 1 and p.Ala708Pro in Analyses of Ca2+ flux in CD19+ cells after IgM crosslinking Family 2) (Fig. 1d and Table 3). Additional investigations and ionomycin stimulation were performed in both patients confirmed that they were novel, de novo, and germline using Fluo-3 flow cytometric assay. In patient 1, no firm Table 2 Summary of immunological investigations performed in enrolled patients. Figures in brackets indicate the normal range of each parameter adjusted per age. Italic fonts indicate values lower than the normal range Patient 1 Patient 2 Age at measurement 9m 3y8m 4y 3 m 5 y 11 m 11 y 6 m 5m 1y1m 3y7m 4y7m 5y6m Lymphocyte subpopulations Total lymphocytes n.a. 3.46 (2.0–8.0) 2.9 (2.0–8.0) 2.9 (2.0–8.0) 2.1 (1.2–5.2) 6.6 (4.0–13.5) 4.1 (4.0–10.5) 3.1 (2.0–8.0) 2.2 (2.0–8.0) 3.1 (2.0–8.0) J Clin Immunol (2020) 40:987–1000 (103/μL) CD3+ (cels/μL) n.a. 3044 2639 2726 1806 5742 3649 2666 1936 2759 (1400–3700) (1400–3700) (1400–3700) (1200–2600) (2500–5600) (2100–6200) (1400–3700) (1400–3700) (1400–3700) CD3+ CD4+ (cels/μL) n.a. 2145 1827 1783 1197 4290 2706 1364 1056 1364 (700–2200) (700–2200) (700–2200) (650–1500) (1800–4000) (1300–4300) (700–2200) (700–2200) (700–2200) CD3+ CD8+ (cels/μL) n.a. 1038 754 (490–1300) 899 (490–1300) 546 (370–1100) 1452 902 (620–2000) 1209 792 (490–1300) 1209 (490–1300) (590–1600) (490–1300) (490–1300) CD19+ (cels/μL) n.a. 35 (390–1400) 17 (390–1400) 6 (390–1400) 13 (270–860) 330 (430–3000) 246 (720–2600) 62 (390–1400) 44 (390–1400) 31 (390–1400) CD16/56+ (cels/μL) n.d. 138 (130–720) 208 (130–720) 130 (130–720) 182 (100–480) 462 (170–830) 164 (180–920) 310 (130–720) 176 (130–720) 217 (130–720) Immunoglobulin plasma levels IgG (mg/dL) 125 439* n.d. 871* 638* 173 (172–814) 537 (345–1213) 732 (441–1135) 696 (463–1236) 819 (463–1236) (217–904) (441–1135) (463–1236) (639–1349) IgA (mg/dL) 11 (11–90) 4.9* (22–159) n.d. < 10* (25–154) < 10* (70–312) 21 (8–68) 58.8 (14–106) 139 (22–159) 83 (25–154) 102 (25–154) IgM (mg/dL) 4 (34–126) 8* (47–200) n.d. < 10* (43–196) < 10* (56–352) 8.2 (33–109) 82.9 (43–173) 26 (47–200) 21 (43–196) 31.5 (43–196) IgE (kU/L) n.d. < 10.0 (0.0–7.3) < 10.0 (0.0–7.3) < 10.0 (0.0–7.3) n.d. < 10.0 (0–140) n.d. < 10.0 (0–140) n.d. < 10.0 (0–140) IgG subclass plasma levels IgG1 (mg/dL) n.d. n.v.* n.v.* n.v.* n.v.* 158 (170–950) n.d. n.d. n.d. n.d. IgG2 (mg/dL) n.d. n.v.* n.v.* n.v.* n.v.* 19 (21–440) n.d. n.d. n.d. n.d. IgG3 (mg/dL) n.d. n.v.* n.v.* n.v.* n.v.* 25.6 (12.7–55.5) n.d. n.d. n.d. n.d. IgG4 (mg/dL) n.d. n.v.* n.v.* n.v.* n.v.* 4 (5–16) n.d. n.d. n.d. n.d. Post-vaccine antibodies Pneumovax-23 n.d. n.v.* n.v.* n.v.* n.v.* n.d. n.d. n.d. n.d. Negative Diphtheria n.d. n.v.* n.v.* n.v.* n.v.* n.d. n.d. n.d. n.d. Negative Tetanus n.d. n.v.* n.v.* n.v.* n.v.* n.d. n.d. n.d. n.d. Negative Autoantibodies n.d. Negative [1] Negative [2] n.d. Negative [3] n.d. Negative [4] Negative [5] Negative [6] n.d. *Values obtained during intravenous immunoglobulin therapy [1]N.egative results for anti-transglutaminase (IgG and IgA) and antiendomisio (IgA) antibodies [2]N.egative results for antinuclear antibodies (ANA), anti-Ro, anti-La, anti-RNP, anti-Sm, anti-smooth muscle, anti-LKM, and anti-mitochondrial antibodies [3]N.egative results for antinuclear antibodies (ANA), anti-DNA, anti-Ro, anti-La, anti-RNP, anti-Sm, anti-Scl70, and anti-JO-1 antibodies [4]N.egative results for anti-transglutaminase antibodies [5]N.egative results for antinuclear antibodies (ANA), anti-DNA autoantibodies, and anti-neutrophil cytoplasmatic autoantibodies [6]N.egative results for anti-neutrophil cytoplasmatic autoantibodies. m, months; y, years; n.a., not available; n.d., not done; n.v., not valuable 993 994 J Clin Immunol (2020) 40:987–1000 Molecular Pathology [4].Pt, patient; Chr, chromosome; 1000 GP, 1000 Genomes Project Phase 3; ExAc, Exome Aggregation Consortium; gnomAD, Genome Aggregation Database; GERP, Genomic Evidence2 RefSeq: NM_002661.3. 2 On the basis of standards and guidelines proposed in the consensus recommendations of the American College of Medical Genetics and Genomics and the Association of Pathogenic Pathogenic conclusions were drawn from these analyses due to the nearly complete absence of circulating B cells (data not shown). By contrast, analyses performed in patient 2 revealed a signifi- cantly higher release of Ca2+ into the cytosol of B cells after IgM crosslinking stimulation than in cells from control sub- GERP Score 5.43 4.99 jects, whereas no significant differences were observed after ionomycin stimulation (Supplementary Fig. 3A-C). Further analysis of novel APLAID variants was per- Mutation Dis Caus formed in a standard PLC assay, similar to that previously Taster used to measure the activity of PLAID and APLAID var- Pol iants [6–8]. In this type of analysis, using model cell sys- Bioinformatics tems, the substrate is presented in native membranes and ExAC gnomAD Polyphen-2 (Hum Div) Prob Dam (0.997) the production of inositol phosphates measured under bas- al or stimulated conditions. As shown in Fig. 3a, both variants have higher PLC activity compared with the wild - type with the p.Ala708Pro substitution showing a greater increase in basal and stimulated activities. When analyzed Population Genetics 0 0 in the context of CLL resistance to ibrutinib, p.Ala708Pro mutation had a pronounced effect [15], consistent with 0 0 our observations. 1000 GP 0 0 NLRP3-Inflammasome Activation and Cytokine Predicted amino Secretion acid alteration Leu848del) p.(Ala708Pro) Evolutionary Rate Profiling; Prob Dam, probably damaging; Pol, polymorphism; Dis Caus, disease causing p.(Leu845_ PBMCs from APLAID patients showed an increased pro- duction of different cytokines after LPS treatment compared with healthy controls, which was similar to that detected in TTAGGGTCTCTT Exon cDNA alteration1 PBMCs from cryopyrin-associated periodic syndrome c.2533_2544del (CAPS) patients. The upregulated cytokines included proin- c.2122G>C flammatory cytokines such as TNF-α and different members of IL-1 family (IL-1α, IL-1β, IL-1Ra, IL-18) (Fig. 4a). The increased release of IL-1 cytokines occurred simultaneously to an increase of ASC speck formation and activation of Characteristics of PLCG2 variants detected in enrolled patients PLCG2 24 PLCG2 20 caspase-1 on LPS-treated monocytes from APLAID and Gene CAPS patients when compared with monocytes from healthy individuals (Fig. 4b). IL-1β and IL-18 release from APLAID patients’ PBMCs after LPS treatment was compa- Variant rable to that of CAPS patients (Fig. 4c). The release of IL- allele 1β and the activation of caspase-1 induced by LPS in C TTAGGGTCTCTT - APLAID PBMCs were reduced when intracellular calcium Reference allele was chelated with BAPTA-AM or when the widely used PLC inhibitor U73122 was used (Fig. 4d). Canonical acti- vation of the NLRP3-inflammasome by adding ATP or nigericin after LPS priming resulted in an equal formation G of intracellular ASC specks in monocytes and similar release Chr 16: 81962181– of IL-1β in samples from healthy donors, APLAID patients, Chr 16: 81953156 Patient Chromosome and CAPS patients (Fig. 4e). Collectively, these results sug- 81962192 gest an over-activation of the alternative NLRP3 position inflammasome pathway in monocytes from APLAID pa- tients, which could be enhanced by the elevated levels of Table 3 intracellular calcium associated with the increased activity of Pt 1 Pt 2 mutated PLCγ2. 1 J Clin Immunol (2020) 40:987–1000 995 Fig. 3 PLC activity analyses. Panel a The effect of p.Ala708Pro and Position of p.Ala708 and p.Leu845-Leu848 segment (red arrows) is p.Leu845_Leu848del variants on PLC activity was measured in mapped on the structure of cSH2 and spPH domain, respectively. transfected COS-7 cells under basal conditions (basal) or after stimulation Positions of other mutations, reported for the main PLCγ1- and PLCγ2- by EGF (stimulated). Each data point represents the mean of triplicates and linked pathologies that map to the same domains, are labeled using single error bars indicate standard error of the mean. Expression levels of PLCγ2, letters and numbers corresponding to PLCγ2 sequence. Residues so far corresponding to increasing concentrations of plasmids used for found to be mutated only in PLCγ1 are shown in gray. Other residues transfection, were measured using WES (top, inset). Further evaluation of mutated in APLAID (p.Ser707 and p.Leu848) and Ali14 mice (p.Tyr495) the differences in PLC activities between the WT and variants was are indicated by orange arrows performed for the points with an equal protein expression. Panel b Discussion that carry gain-of-function PLCG2 mutations [17, 18]. Another two families carrying the same PLCG2 variant in the C2 do- The patients here described were initially suspected of suffering main of the protein have been recently described during manu- from a rare disease combining humoral immunodeficiency and script reviewing [19]. The two unrelated patients described in inflammatory manifestations. Previous genetic studies did not our study enable us to make more extensive comparisons and identify defects in different genes causing monogenic antibody highlight the key changes that characterize the human syn- deficiencies or AIDs [1, 16]. Due to strong similarities, we drome and parallels with the mouse models. These include hypothesized that the patients’ disease might be a consequence severe inflammatory lesions at skin, ocular, and joints mediated of defects in the same gene. Our further genetic studies revealed by non-lymphoid hematopoietic cells and variable degree of that each patient carried a novel, de novo heterozygous PLCG2 immunodeficiency. With regard to the immune defects, all pathogenic variant. Furthermore, our extensive characterization known APLAID patients exhibited variable degrees of B cell of clinical manifestations, properties of immune cells, and lymphopenia and antibody deficiency, with no apparent impair- changes in function of the encoded enzyme, PLCγ2, support ment of T and NK functions (Table 1). Patient 1 carrying the a definitive diagnosis of PLCγ2-associated antibody deficiency p.Leu845_Leu848 PLCG2 deletion showed the complete ab- and immune dysregulation syndrome, designated as APLAID. sence of B cells and circulating immunoglobulins, an immune Previously, only 4 patients from three families have been phenotype compatible with a rare form of non-X-linked agam- diagnosed with APLAID [7–9] and have been found to share maglobulinemia [16] and quite similar to the immune pheno- some of the manifestations with Ali5 and Ali14 mouse strains type of the APLAID patients carrying the missense 996 J Clin Immunol (2020) 40:987–1000 J Clin Immunol (2020) 40:987–1000 997 ƒFig. 4 Involvement of NLRP3-inflammasome activation in sterile contrast, monoallelic PLCG2 mutations are associated with var- inflammation. Panel a Heat map of cytokine analysis from peripheral iable humoral immune deficiency and immune dysregulation in blood mononuclear cell (PBMCs) supernatants after LPS stimulation as indicated (1 μg/mL, 2 h) isolated from healthy controls (n = 5), patients PLAID and APLAID syndromes [6–9]. The few available data with CAPS (heterozygous for p.Arg260Trp NLRP3 mutation; n = 2) or about the defects of the B cell lineage in APLAID patients do APLAID carrying the heterozygous p.A708P (n = 1) or p.Leu845_ not permit to drawn firm conclusions about the precise molec- Leu848del (n = 1) PLCG2 variants. Representative of relative values of ular mechanisms underlying the humoral deficiency in these minimum and maximum concentrations measured per cytokine. Panel b Apoptosis-associated Speck-like protein containing a Caspase patients. Considering the relevant role of PLCγ2 in multiple recruitment domain (ASC) speck forming monocytes by flow signal transduction pathways in B cells and the gain-of-function cytometry and active caspase-1 by YVAD-Fluorochrome Inhibitor of nature of PLCG2 variants detected in APLAID syndrome, a Caspases (FLICA) staining on monocytes after LPS stimulation as mechanism that may explain the immune phenotype observed indicated (1 μg/mL, 2 h) isolated from healthy controls, patients with CAPS, or patients with APLAID. Showed results are representative of and that should be further investigated is the deletion and/or duplicate experiments. Panel c PBMCs IL-1β and IL-18 cytokine functional anergy of specific B cell progenitors due to enhanced production at baseline and after LPS stimulation as indicated (1 μg/mL, cellular signaling. 2 h) in healthy controls, patients with CAPS, and patients with APLAID. We were particularly interested in sterile inflammatory le- Showed results are representative of duplicate experiments. Panel d PBMCs IL-1β and percentage of monocytes stained for active caspase- sions and their molecular basis. From a clinical perspective, the 1 by FLICA at baseline and after LPS stimulation (1 μg/mL, 2 h) in the cutaneous manifestations were the most prominent in both of presence or absence of BAPTA-AM (20 μM) or U73122 (2.5 μM) as our patients (patient 1 and 2) since the onset of their diseases as indicated in APLAID patient 1 (p.Leu845_Leu848del PLCG2 variant) have been also described for the other four published APLAID and her healthy mother. Showed results are representative of experiments performed only once due to limited availability of samples. Panel e patients [7–9]. These manifestations were initially vesicular and PBMCs IL-1β and ASC speck forming monocytes upon canonical blistering lesions that subsequently evolved to the destruction NLRP3 activation by LPS priming (1 μg/mL, 2 h) followed by 30-min of elastin fibers leading to large areas of cutis laxa in both treatment with ATP (3 mM) or nigericin (10 μM) in healthy controls, patients, a phenomenon also described in the APLAID patient patients with CAPS, and patients with APLAID. Showed results are representative of duplicate experiments. ND, not detected with the p.Leu848Pro PLCG2 variant [8]. Later in life both patients also developed severe eye inflammation, mainly affect- ing conjunctiva and episclera. The beneficial effect of topic anakinra in the ocular lesions in PLAID [23] suggests the po- p.Leu848Pro PLCG2 variant [8, 9]. The humoral immunodefi- tential combination of topical ocular treatments to the systemic ciency of this patient (patient 1) was clinically characterized by therapies that both patients are already receiving. severe and recurrent bacterial infections, structural lung lesions, We have further investigated the molecular mechanisms recurrent episodes of acute hemoptysis, and the requirement of underlying sterile inflammation in APLAID. We found that surgical lung lobe resection. To our knowledge, this patient the novel PLCγ2 variants here identified were able to induce represents the severest APLAID patient described to date and activation of the NLRP3 inflammasome, with ASC aggrega- clearly expands the diversity of the immune deficiency of this tion, caspase-1 activation, and IL-1β and IL-18 release in disease toward phenotypes compatible with dominantly monocytes. Previous evidence suggested a potential connec- inherited agammaglobulinemia. Since allogeneic hematopoietic tion between hypermorphic PLCG2 mutations and NLRP3- stem cell transplantation has been proposed as a curative option inflammasome activation throughout the increase of intracel- in severe PID [20, 21], a major unsolved question is whether lular calcium [7, 16, 24, 25]. Consequently, it is possible that this particular patient would have benefited from this therapeu- in the patients described here, the sterile inflammation mani- tic approach at a younger age, even with the causative genetic festations could be also linked to NLRP3-inflammasome. Our defect not elucidated. With regard to patient 2 who carried the results showed that canonical activation pathway of NLRP3- missense p.Ala708Pro PLCG2 variant, his immunological phe- inflammasome in patient’s monocytes did not result in an notype (low IgM and B cell count and decreased circulating increase of IL-1β release or ASC-specking as observed for class-switched memory B cells) could be classified as mild-to- healthy monocytes. Therefore, the inflammasome pathway moderate and displayed marked similarities with the phenotype that is potentiated in APLAID might be related to the alterna- of the first described APLAID family carrying the missense tive NLRP3 activation pathway, similar to that described in p.Ser707Tyr PLCG2 variant [7]. The B cell disturbances ob- human monocytes upon LPS activation [26], rather than the served in patients with APLAID are variable ranging from pa- canonical pathway that requires ATP or nigericin as second tients displaying low circulating B cells to patients with absent stimuli after LPS priming [27]. B cells. PLCG2 was proposed as a good candidate gene to Finally, our identification of two new pathogenic variants in explain some B cell deficiencies in humans because the mouse PLCG2 linked to APLAID (p.Ala708Pro and model of PLCγ2 deficiency showed a decrease of mature B cell p.Leu845_Leu848) expands the range of genetic changes that and a block in the pro-B cell differentiation [22]. However, the cause this syndrome. Consistent with previous studies [7–9], human deficiency of PLCγ2 has not been yet described. By we show that the new PLCG2 variants have higher PLC 998 J Clin Immunol (2020) 40:987–1000 activity compared with the wild type. Interestingly, p.Ala708Pro PLCG2 variant has been also discovered in Funding Information This work has been partially funded by the follow- ing: CERCA Programme/Generalitat de Catalunya (JIA), SAF2015- ibrutinib-resistant chronic lymphocytic leukemia (CLL) in the 68472-C2-1-R grant from the Spanish Ministry of Economy and form of somatic variant [28–30]. Two other somatic PLCG2 Competitiveness co-financed by European Regional Development Fund variants detected in resistant CLL, p.Ser707Tyr and (ERDF) (JIA), RTI2018-096824-B-C21 grant from the Spanish Ministry p.Asp993Gly, are also shared with immune disorders in of Science, Innovation and Universities co-financed by ERDF (JIA), AC15/00027 grant from the Instituto de Salud Carlos III/Transnational APLAID [7] and Ali5 mice [17], respectively. More broadly, Research Projects on Rare Diseases (JIA), PI14/00405 grant from the the new variants map to distinct regions in the cSH2 domain, Instituto de Salud Carlos III co-financed by ERDF (RC), PI13/00174 and spPH domain and its vicinity, the regions that appear to grant from the Spanish Ministry of Economy and Competitiveness co- harbor mutations across different PLCγ-linked pathologies financed by ERDF (PP), SAF2017-88276-R grant from the Spanish Ministry of Economy, Industry and Competitiveness co-financed by with higher frequencies (Fig. 3b). Therefore, the molecular ERDF (PP), ERC-2013-CoG project 614578 from the European mechanisms that lead to an increase in PLC activity in diverse Research Council (PP), 20859/PI/18 grant from Fundación Séneca (PP), pathologies could be similar. Considerable supporting evidence SAF2015-68472-C2-2-R grant from the Spanish Ministry of Economy is consistent with a model where PLCγ2 is kept in an inactive and Competitiveness co-financed by ERDF (FC) and RTI2018-096824- B-C22 grant from the Spanish Ministry of Science, Innovation and state by extensive intramolecular interactions between the reg- Universities co-financed by ERDF (FC). MK acknowledges support from ulatory domains (including cSH2 and spPH domains) and the CRUK (A16567) and MRC (P028160). H M-B is a Rio Hortega fellow- PLC-core domains [11, 31]. Release of this autoinhibition by ship from Instituto de Salud Carlos III (CM14/00008) and DB acknowl- physiological stimulation (via phosphorylation) or by mutations edges support from the UCL Impact Studentship. represents an important step leading to an increase in PLC activity. Therefore, our data showing that the novel variants Compliance with Ethical Standards are hypermorphic and their position in the protein highlight The ethics committees of Hospital Sant Joan de Déu, Hospital particular structural features within the regulatory array that Universitari Vall d’Hebron, and Hospital Clínic, all in Barcelona are the key regions involved in autoinhibition. (Spain), approved the study. Written informed consent for participation In conclusion, from the perspective of clinical practice, in the study was obtained from patients’ parents. Blood samples from patients and unaffected relatives were collected for molecular studies, the detection of a novel, de novo hypermorphic PLCG2 which were performed in accordance with the Declaration of Helsinki. pathogenic variant in each of the two patients displaying early-onset severe skin and eye inflammation and humoral Conflict of Interest The authors declare that they have no conflict of immunodeficiency supports their definitive diagnosis of interest. the extremely rare APLAID syndrome. Moreover, the pa- Open Access This article is licensed under a Creative Commons tients’ immunological features expand the APLAID diver- Attribution 4.0 International License, which permits use, sharing, sity toward most severe phenotypes than previously de- adaptation, distribution and reproduction in any medium or format, as scribed including complete B cell depletion and agamma- long as you give appropriate credit to the original author(s) and the globulinemia inherited as a dominant trait, which should source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article be considered when evaluating patients with severe defi- are included in the article's Creative Commons licence, unless indicated ciencies of antibody production. otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not Acknowledgments We would like to particularly acknowledge the pa- permitted by statutory regulation or exceeds the permitted use, you will tients and their families for their collaboration in this study. We would need to obtain permission directly from the copyright holder. To view a also like to acknowledge the Biobank of Vall d’Hebron Research Institute copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. (PT13/0010/0021), integrated in the Spanish National Biobanks Network, and its director Isabel Novoa for her help in this study. The authors would like to acknowledge Mr. William Sinclair for his help in language editing. Authorship Contributions JIA, PP, and MK designed and wrote the References manuscript. AM-N, CF, LR, MG-P, NM-B, VG-P, RMP, PS-P, and AV collected clinical data and provided samples of patients and relatives 1. 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Bunney 6 & Laia Alsina 4,7,8 & Ana Esteve-Solé 4,7,8 & Daniel Bull 9 & Maria Carmen Anton 10 & María Basagaña 11 & Ferran Casals 12 & Angela Deyá 4,7,8 & Marina García-Prat 1,2 & Ramon Gimeno 13 & Manel Juan 10,14,15 & Helios Martinez-Banaclocha 16 & Juan J Martinez-Garcia 16 & Anna Mensa-Vilaró 10 & Raquel Rabionet 4,17 & Nieves Martin-Begue 18 & Francesc Rudilla 19,20 & Jordi Yagüe 10,14,15 & Xavier Estivill 21 & Vicente García-Patos 22 & Ramon M. Pujol 23 & Pere Soler-Palacín 1,2,24 & Matilda Katan 6 & Pablo Pelegrín 16 & Roger Colobran 2,25,26 & Asun Vicente 27 & Juan I. Arostegui 10,14,15 1 15 Pediatric Infectious Diseases and Immunodeficiencies Unit, Vall School of Medicine, Universitat de Barcelona, Barcelona, Spain d’Hebron Institut de Recerca, Hospital Universitari Vall d’Hebron, 16 Instituto Murciano de Investigación Biosanitaria IMIB-Arrixaca, Barcelona, Spain Hospital Clínico Universitario Virgen de la Arrixaca, Murcia, Spain 2 Jeffrey Modell Diagnostic and Research Center for Primary 17 Department of Genetics, Microbiology and Statistics, Faculty of Immunodeficiencies, Barcelona, Spain Biology, University of Barcelona, IBUB, IRJSD, CIBERER, 3 Department of Pediatrics, Hospital Sant Joan de Deu, Barcelona, Spain Esplugues, Spain 18 Department of Pediatric Ophthalmology, Hospital Universitari Vall 4 Institut de Recerca Hospital Sant Joan de Déu, Universitat de d’Hebron, Vall d’Hebron Institut de Recerca, Barcelona, Spain Barcelona, Esplugues, Spain 19 Histocompatibility and Immunogenetics Laboratory, Blood and 5 Department of Pediatrics, Hospital Alvaro Cunqueiro, Vigo, Spain Tissue Bank, Barcelona, Spain 6 20 Institute of Structural and Molecular Biology, University College Transfusional Medicine Group, Vall d’Hebron Research Institute, London, London, UK Autonomous University of Barcelona, Barcelona, Spain 7 21 Department of Allergy and Clinical Immunology Clinical Quantitative Genomic Medicine Laboratories (qGenomics), Immunology and Primary, Immunodeficiencies Unit, Hospital Sant Esplugues del Llobregat, Barcelona, Catalonia, Spain Joan de Déu, Esplugues, Spain 22 Department of Pediatric Dermatology, Hospital Universitari Vall 8 Clinical Immunology Unit, Hospital Sant Joan de Déu-Hospital d’Hebron, Vall d’Hebron Institut de Recerca, Barcelona, Spain Clínic, Barcelona, Spain 23 Department of Dermatology, Hospital del Mar, Institut Mar 9 ARUK Drug Discovery Institute, University College London, d’Investigacions Mèdiques, Universitat Autonoma de Barcelona, London, UK Barcelona, Spain 10 24 Department of Immunology-CDB (esc 4-pl 0), Hospital Clínic, Universitat Autonoma de Barcelona, Barcelona, Spain Villarroel, 170, 08036 Barcelona, Spain 25 Immunology Division, Department of Clinical and Molecular 11 Allergy Section, Hospital Universitari Germans Trias i Pujol, Genetics, Hospital Universitari Vall d’Hebron, Vall d’Hebron Autonomous University of Barcelona, Badalona, Spain Research Institute, Barcelona, Spain 12 26 Genomics Core Facility, Experimental and Health Sciences Department of Cell Biology, Physiology and Immunology, Department, Universitat Pompeu Fabra, Barcelona, Spain Autonomous University of Barcelona, Barcelona, Spain 13 27 Department of Immunology, Hospital del Mar, Institut Mar Department of Pediatric Dermatology, Hospital Sant Joan de Deu, d’Investigacions Mèdiques, Barcelona, Spain Esplugues, Spain 14 Institut d’Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain