Author response: Biallelic mutations in calcium release activated channel regulator 2A (CRACR2A) cause a primary immunodeficiency disorder
Year: 2021
Type: peer-review
Abstract: Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract CRAC channel regulator 2 A (CRACR2A) is a large Rab GTPase that is expressed abundantly in T cells and acts as a signal transmitter between T cell receptor stimulation and activation of the Ca2+-NFAT and JNK-AP1 pathways. CRACR2A has been linked to human diseases in numerous genome-wide association studies, however, to date no patient with damaging variants in CRACR2A has been identified. In this study, we describe a patient harboring biallelic variants in CRACR2A [paternal allele c.834 gaG> gaT (p.E278D) and maternal alelle c.430 Aga > Gga (p.R144G) c.898 Gag> Tag (p.E300*)], the gene encoding CRACR2A. The 33-year-old patient of East-Asian origin exhibited late onset combined immunodeficiency characterised by recurrent chest infections, panhypogammaglobulinemia and CD4+ T cell lymphopenia. In vitro exposure of patient B cells to a T-dependent stimulus resulted in normal generation of antibody-secreting cells, however the patient’s T cells showed pronounced reduction in CRACR2A protein levels and reduced proximal TCR signaling, including dampened SOCE and reduced JNK phosphorylation, that contributed to a defect in proliferation and cytokine production. Expression of individual allelic mutants in CRACR2A-deleted T cells showed that the CRACR2AE278D mutant did not affect JNK phosphorylation, but impaired SOCE which resulted in reduced cytokine production. The truncated double mutant CRACR2AR144G/E300* showed a pronounced defect in JNK phosphorylation as well as SOCE and strong impairment in cytokine production. Thus, we have identified variants in CRACR2A that led to late-stage combined immunodeficiency characterized by loss of function in T cells. Editor's evaluation The study is the first report and characterization of impaired immune function in an individual with biallelic mutations in the CRACR2A gene. The mechanistic insights from this study, are an important advance in the understanding of CRAC channels and the regulation of calcium dynamics in the T-cell lineage. The work will be of interest to cell biologists, immunologists and those with interests in intracellular signaling. https://doi.org/10.7554/eLife.72559.sa0 Decision letter eLife's review process Introduction T cell activation following antigen recognition involves several signaling events. Amongst others, these include Ca2+ influx and activation of the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) pathways. The Ca2+ influx is initiated shorty after T cell receptor (TCR) engagement, and involves activation of phospholipase Cγ and production of inositol-1,4,5-trisphosphate (IP3). The latter binds to IP3 receptors in the endoplasmic reticulum (ER) membrane, leading to receptor-mediated release of Ca2+ from the ER. The Ca2+ release is detected by stromal interaction molecules (STIM) - STIM1 and STIM2, which are EF-hand-containing proteins, located in the ER membrane (Roos et al., 2005; Liou et al., 2005). Depletion of the ER Ca2+ stores leads to Ca2+-loss from the STIM1 EF-hand causing it to multimerize and translocate to the ER in close proximity to the plasma membrane (PM). Subsequently, STIM1 binds to PM-resident ORAI1, which is the pore subunit of the Ca2+ release–activated Ca2+ (CRAC) channel, resulting in the opening of the channel and Ca2+ influx (Feske et al., 2006; Zhang et al., 2006; Vig et al., 2006). This process is otherwise known as store-operated Ca2+ entry (SOCE) since it depends on the filling state of the ER Ca2+ stores. The importance of Ca2+ influx for lymphocyte proliferation, differentiation and function, and the role that STIM1 and ORAI1 play in these processes, is illustrated by rare primary immunodeficiency syndromes caused by autosomal recessive (AR) loss of function (LOF) or null mutations in ORAI1 and STIM1 genes. The affected patients tend to present from early age with varying degrees of immune defects, ranging from severe combined immunodeficiency (SCID), to milder forms, where the immune impairment is functionally evident, but not associated with any apparent immunodeficiency (Feske et al., 2006; Chou et al., 2015; Fuchs et al., 2012; McCarl et al., 2009; Picard et al., 2009; Parry et al., 2016; Rice et al., 2019; Byun et al., 2010; Schaballie et al., 2015). Patients with SCID-like phenotype present with severe viral, bacterial, and fungal infections. In many cases there is associated immune dysregulation manifesting as lymphoproliferation or autoimmunity (Picard et al., 2009). In most cases it is the function rather than numbers of lymphocytes that is affected. Lastly, non-immunological manifestations are common, such as congenital muscular hypotonia, defects in dental enamel development, and anhidrosis, due to critical role of SOCE for these physiological functions (Parry et al., 2016; Rice et al., 2019; Wang et al., 2014; Lian et al., 2018). CRAC Channel Regulator 2 A (CRACR2A) was identified as a protein that binds ORAI1/STIM1 and stabilizes their interaction to mediate SOCE in T cells (Srikanth et al., 2010a). Two isoforms of the protein are expressed in T cells, a short cytoplasmic isoform CRACR2A-c, that was originally shown to bind ORAI1/STIM1 and stabilize their interaction, and a longer isoform CRACR2A-a (hereafter referred to as CRACR2A), that is a Rab GTPase. CRACR2A (also called EFCAB4B and Rab46) is abundantly expressed in T cells and belongs to a family of large Rab GTPases that also includes Rab44 and Rab45. It is a protein with multiple functional domains including an N-terminal Ca2+-binding EF-hand (also present in the shorter CRACR2A-c isoform), a protein-interacting coiled coil domain and proline-rich domain, and a C-terminal Rab GTPase domain. This long isoform is prenylated at its C terminus and predominantly localizes to vesicles and Golgi in T cells. Both the isoforms interact with ORAI1 and STIM1 and are involved in regulation of SOCE (Srikanth et al., 2010a). In addition to regulation of SOCE, CRACR2A has also been shown to interact with Vav1 and regulate JNK phosphorylation. The GTPase domain of CRACR2A and prenylation have been shown to be necessary for its role in JNK activation (Srikanth et al., 2016). The human CRACR2A (formerly EFCAB4B) gene is located on chromosome 12. Associations between polymorphisms in CRACR2A and human diseases have been identified from numerous genome-wide association studies (GWAS) of Parkinson disease, nonalcoholic fatty liver disease, atrial fibrillation, and chronic infection of HIV type (Fung et al., 2006; Chalasani and Björnsson, 2010; Tan et al., 2013; Iglesias-Ussel et al., 2013; Edelman et al., 2015). However, until now, CRACR2A has not directly been linked with a primary immunodeficiency (PID) in humans. Here, we describe a single case of late onset combined immunodeficiency due to biallelic variants in CRACR2A which encodes CRACR2A. One of the alleles encodes a truncated protein containing another point mutation in the EF-hand domain (CRACR2A p.[R144G/E300*], hereafter referred to as double mutant DM), whereas the other allele encodes a point mutant (CRACR2A p.E278D), with the mutation residing in the protein-interacting coiled coil region of the protein. Exogenous expression of individual allelic mutations in T cells shows that the DM impairs both SOCE as well as JNK phosphorylation, whereas p.E278D selectively impairs SOCE. Results Case presentation and immunological work up We evaluated a 33-year-old male of East-Asian origin. He originally presented to gastroenterology services at the age of 19 years with chronic diarrhea. Following initial assessment, which included normal colonoscopy and negative coeliac screen, he was found to have severe panhypogammaglobulinaemia and was referred to clinical immunology for further investigations. On further questioning the patient reported a 5-year long history of recurrent lower respiratory tract infections requiring treatment with antibiotics. He also reported having a left lower lobe pneumonia at 15 years of age. He had no other significant infection history and reported being generally well during his childhood. His only other past medical history of note was appendicectomy at the age of 10 years. He had no history of developmental difficulties, dental, skin or any musculoskeletal abnormalities. He was an only child from non-consanguineous parents with no family history of PID. Shortly after presenting to immunology, he developed a left hip and iliac fossa pain and was found to have multiloculated left psoas abscess which required a computerized tomography (CT) guided drainage and prolonged antibiotic treatment. This infective episode was complicated by pulmonary embolism (incidental finding on a repeat CT scan) for which he was anticoagulated for a period of 6 months. He had no subsequent history of thromboembolic events. A detailed CT scan of his chest showed cylindrical bronchiectasis of the left lower lobe, lingua and R middle lobe. This appearance remains unchanged as shown on a repeat scan 11 years later in 2016 (Figure 1A). Figure 1 with 1 supplement see all Download asset Open asset Clinical and immunological features of index patient. (A) A computerized tomography scan of the chest showing area of cylindrical bronchiectasis at the basal segment of the left lower lobe. (B) TCR repertoire as assessed by T receptor spectraphenotyping. Data are representative of one independent assay. (C) Phytohaemagglutinin (PHA) and (D) anti-CD3 T cell proliferation. CPM (counts per minute). Data are representative of three independent assays. The initial immunological investigations showed profound panhypogammaglobulinaemia with drastically reduced IgG and undetectable IgA and IgM levels (Table 1). The basic lymphocyte profiling showed mild CD4+ T cell lymphopenia with adequate numbers of B and NK cells. Subsequent detailed phenotyping revealed reduced proportion of naïve T cells and seemingly adequate percentages of central and effector memory T cells. The proportion of TH1/TH2 and TH17 T cells was comparable to healthy controls (Figure 1—figure supplement 1) and TCR phenotyping showed a normal TCR repertoire (Figure 1B). The patient also had reduced proportion of class switched memory B cells (Table 1). Functional testing showed reduced T cell proliferation to both PHA and anti-CD3 stimulation (Figure 1C and D). Over the next 15 years, there was evidence of progressive CD4+ T cell and B cell lymphopenia. Interestingly, biopsy of his large bowel, which was performed in 2011 for investigation of intermittent diarrhoea, showed mild non-specific inflammatory changes, but also absence of the plasma cells. Table 1 Immunological characteristics of patient with variants in EFCAB4B. Date/year20052011201720192021 (Feb)2021 (Nov)RefTotal lymphocyte count2,8809901,2011,5721,4721,2681000–2800 cells/µlCD3+ T cells 2,505733891990952846700–1200 cells/µlCD4+ T cellTotal216235148165122112300–1400 cells/µl CD27+ CD45RA+ (naïve)%20.9212.6525.514–65% CD27+ CD45RA- (memory)%42.3447.0442.915–52% CD27-CD45RA- (memory effector) %36.0137.6720.58–35% CD27+ CD45RA-2.6411.10–22% CD25+ CD127- (Treg)%0.731.62.93.53–10%CD8+ T cellsTotal2080475738840772689200–900 cells/µl CD27+ CD45RA+ (naïve)17.6931.31.5–65.5% CD27+ CD45RA- (memory)25.1619.13.5–28.6% CD27- CD45RA- (memory effector)16.0911.10.7–72.6% CD27- CD45RA+ (effector)41.0738.61.6–53%CD19+ (B cells)Total24510273778771100–500 cells/µl CD27- IgM+ IgD + (naïve) %63.189.1867.77844–84% CD24hi CD38hi (Transitional) %6.710.38.2112–14% CD27+ IgM + IgD+ (non-switched) %15.43.6922.5115–32% CD27+ IgM- IgD- (switched memory) %14.77.155.755–33% Plasmablasts %0.20.41.110.2–5%CD56+ CD16+ (NK cells)14413929048642934090–600 cells/µlImmunoglobulin profileIgG 1.48.410.38.39.96–16 g/lIgA < 0.06< 0.06< 0.06< 0.06< 0.060.8–4 g/lIgM < 0.05< 0.05< 0.05< 0.05< 0.050.5–2 g/lIgE < 0.21–120 ku/l Following the initial assessment, the patent was given a provisional diagnosis of common variable immunodeficiency (CVID) and treated with regular intravenous immunoglobulin (IVIG) replacement therapy. His clinical progress since the diagnosis has been uncomplicated. His chronic diarrhoea resolved following commencement of IVIG and the ongoing treatment with IVIG has resulted in excellent control of respiratory infections. A rare biallelic variants in CRACR2A identified by exome sequencing The patient was consented for genetic testing and whole-exome sequencing was performed. Full details regarding candidate variant selection are provided in methods. Three potentially damaging rare variants were identified in CRACR2A GRCh37 12:3715799–3873985 ENSG00000130038 ENST00000440314 (c.430A > G; p.R144G, c.898G > T; p.E300*, exons 6/20 and 10/20), which were maternal and c.834G > T; p.E278D in exon 9/20 on the other allele. Only c.834G > T is present in the gnomAD database, though it is considered rare (heterozygous only; 0.00095 in East Asian). All three amino acids are highly conserved in other species. The variants were confirmed in the genomic DNA by Sanger sequencing the proband and the mother and maternal grandmother and the father, with the pedigree shown in Figure 2A. Compound heterozygous segregation of these variants was confirmed by subcloning of the proband cDNA to isolate the individual alleles. Sanger sequencing confirmed the c.834 G > T variant on a separate allele to the other variants (Figure 2B). The protein domains affected by the mutations are schematically depicted in Figure 2C. Figure 2 Download asset Open asset A rare biallelic mutation in CRACR2A segregates with the disease. (A) Pedigree showing the index case and p.[R144G/E300*] and p.E278D CRACR2A variant carriers. The circles and squares represent female and male members, respectively. Black shading shows affected individuals, either heterozygous (partial black) or homozygous (completely black). (B) Sanger sequencing data of the proband showing the indicated mutations. (C) Schematic of human CRACR2A showing the location of compound heterozygous mutations from the patient’s alleles. The maternal allele comprised of an p.R144G variant that was located in the EF-hand motif and an p.E300* truncation variant within the leucine-rich region (LR). The paternal allele contained the p.E278D variant that was located in the coiled-coil domain 2 (CC1 and CC2). CRACR2A also contains a proline-rich domain (PRD) and a C-terminal Rab GTPase domain. (D) Detection of CRACR2A in human PBMCs harvested from healthy control and patient by immunoblotting (left) and real-time quantitative RT-PCR (right). For qRT-PCR, cells were left unstimulated or re-stimulated with PMA plus ionomycin for 5 hr. β-actin was used as a loading control for immunoblotting. Immunoblot is representative of two independent experiments and mRNA analysis shows representative triplicate from two independent experiments. * p < 0.05. To check whether the mutant proteins are expressed, we performed immunoblot analyses using T cells from patient and healthy control. Peripheral blood mononuclear cells (PBMCs) harvested from the patient and healthy control were stimulated with plate-coated anti-CD3 and anti-CD28 antibodies for 48 hr, following which they were further expanded for a further 4 days and then harvested for making cellular lysates. Western blot analysis using anti-CRACR2A antibodies detected reduced amounts of only the higher molecular weight band (corresponding to the long isoform) in patient samples (Figure 2D, left panel). The antibody was targeting the N-terminal 160 amino acids of CRACR2A, and hence it should detect the ~30 KDa truncated form of the p.E300* variant, if expressed. However, we could not detect presence of any band at ~30 KDa, suggesting possibly degradation of the truncated form. To check for CRACR2A transcript levels, we used primers that were common to both isoforms, however, we detected pronounced reduction in CRACR2A transcript levels from patient samples (Figure 2D, right panel). Further, in healthy control samples, expression of CRACR2A transcripts increased after stimulation with PMA and ionomycin by more than two-fold, but it was not upregulated in patient T cells. These data suggest possible nonsense-mediated mRNA decay for CRACR2A transcripts. Patient B cells exhibit intact plasma cell generation Since the patient exhibited profound panhypogammaglobulinaemia and a relative decline in memory B and plasma cell populations, we next checked whether patient B cells were functional. To test if the panhypogammaglobulinaemia is due to an intrinsic B cell defect, we set up an in vitro culture system with a T cell-dependent (TD) stimulus that enables the generation of mature plasma cells from primary B cells (Figure 3A; Cocco et al., 2012). The capacity of the patient’s B cells to differentiate into plasma cells was comparable to healthy controls, as judged by the acquisition of CD38 and CD138 surface expression, which is associated with the transition to an antibody-secreting state (Figure 3B). Furthermore, assessment of immunoglobin levels in cell culture showed adequate production of IgM and IgG (Figure 3C), suggesting that the failure of antibody production in this case might be secondary to inadequate in vivo T cell help. Figure 3 Download asset Open asset Patient B cell responses to T cell-dependent (TD) stimulation. (A) Schematic representation of the culture conditions used to assess plasma cell differentiation in vitro. PBs – Plasmablasts, PCs – Plasma cells (B) Representative flow plots showing CD38 and CD138 expression profile following TD stimulation with F(ab’)two anti-IgG/M and CD40L. Percentages are indicated within individual quadrants. Data are representative of 2 independent assays. (C) levels of IgM (left) and IgG from the cells in (B) at indicated data from independent healthy cells. Patient T cells reduced cytokine expression due to Ca2+ influx and JNK activation Since the patient B cells antibodies in in response to TD we that panhypogammaglobulinaemia is due to defect in T cell we showed that had a defect in effector of T cell functions et al., 2018). we further of patient T cells, including signaling and cytokine production. Patient CD4+ cells than the from a representative healthy (Figure We using cells from multiple healthy and of the patient cells by expression of and using quantitative RT-PCR (Figure and (Figure Expression of all these was impaired in patient T cells to control T cells from multiple healthy Since CRACR2A is known to interact with ORAI1 and to regulate SOCE and JNK phosphorylation (Srikanth et al., et al., we SOCE after anti-CD3 as well as of the ER Ca2+ stores using a In both these patient cells showed significant reduction in SOCE, to those from representative healthy controls (Figure and Further, patient T cells also showed a profound defect in JNK phosphorylation TCR (Figure To that reduced SOCE in patient cells is not due to expression of CRAC channel we checked expression of ORAI1 and STIM1 in of T cells from healthy controls and patient. Expression of ORAI1 and STIM1 were between control and patient T cells as judged by immunoblotting as well as real-time quantitative RT-PCR (Figure supplement 1). these the conserved role of CRACR2A in effector T cell responses by the Ca2+ and JNK in human effector T cells, with the finding in T cells et al., 2018). Figure 4 with 1 supplement see all Download asset Open asset Patient T cell responses to T cell receptor stimulation. (A) Representative flow plots showing expression of in human PBMCs from a healthy and the patient. PBMCs were stimulated with anti-CD3 and anti-CD28 antibodies for 48 and for further 4 days in the presence of with PMA plus ionomycin for 5 for cytokine were for CD4+ T cells. (B) mRNA expression analysis of indicated from human PBMCs as with or stimulation with PMA plus ionomycin for 5 hr. (C) of and in human PBMCs from culture of cells stimulated as (B) were by (D) Representative showing SOCE responses from healthy control and patient PBMCs as indicated in after stimulation with anti-CD3 antibody or ionomycin in the presence of containing 2 Ca2+ (left) as for anti-CD3 antibody or ionomycin from independent experiments (right). Representative showing SOCE responses from healthy control and patient PBMCs as indicated in after with stimulation in SOCE was by addition of 2 as indicated shows at the of SOCE from independent experiments (right). JNK levels in CD4+ T cells from healthy control and patient PBMCs as indicated in stimulated with anti-CD3 antibody for indicated from three independent experiments. p < p < of and to reduced cytokine production in T cells To into the of mutants from individual CRACR2A alleles in the we first the mutant cDNA in a in cells, and mutants at the molecular of and (Figure supplement 1A). To check their expression from CRACR2A proteins, we CRACR2A T cells using the system and found that and had a (Figure supplement 1B). we expressed and mutants in CRACR2A cells these mutants at their molecular with cells. after the expression of mutant protein was lower than or CRACR2A, suggesting that the transcripts of this double mutant be to with patient cells (Figure to patient T cells, CRACR2A T cells showed reduced production (Figure expression of CRACR2A in these cells the production defect, that the was caused due to loss of CRACR2A. However, expression of or mutants showed of expression in CRACR2A T cells the We also these with primary human CD4+ cells. We found that with encoding to CRACR2A resulted in reduced production in primary cells, which was by expression of CRACR2A (Figure However, both and mutants did not cytokine production in the primary T cells, to with T cells. Exogenous expression of CRACR2A in primary T cells from healthy the presence of production the mutants did not (Figure On the of the showed significant reduction in cytokine suggesting that this mutant function of CRACR2A. cytokine analysis that expression of mutants from of the CRACR2A alleles could not cytokine production in CRACR2A T cells. Figure 5 with 1 supplement see all Download asset Open asset expression in T cells individual allelic mutations in CRACR2A. (A) Representative flow plots showing expression of in control or CRACR2A T cells CRACR2A, CRACR2AE278D or CRACR2AR144G/E300* after stimulation with PMA plus ionomycin for shows of from two independent experiments (right). (B) Representative flow plots showing expression of in primary human CD4+ T cells with encoding and those encoding of or indicated mutants of CRACR2A after stimulation with anti-CD3 and anti-CD28 antibodies for 5 shows of from three independent experiments. (C) Representative flow plots showing expression of in primary human CD4+ T cells from healthy and with encoding for or indicated mutant of CRACR2A after stimulation with anti-CD3 and anti-CD28 antibodies for 5 shows of from three independent experiments. * p < p < of and to reduced Ca2+ influx and JNK activation We next checked whether individual mutants could in CRACR2A T cells. we showed that loss of CRACR2A impaired TCR SOCE and JNK phosphorylation in T cells (Srikanth et al., 2016). CRACR2A T cells showed reduced Ca2+ entry by TCR stimulation (Figure Expression of CRACR2A the of Ca2+ entry in cells that of and mutants did We SOCE was by of intracellular Ca2+ stores using (Figure Interestingly, these two mutants in activation of the JNK signaling and the mutant phosphorylation of mutant to (Figure These that mutant has a defect in both Ca2+ entry and JNK activation due to truncation of the coiled proline-rich and Rab GTPase mutant has a defect in Ca2+ signaling. Figure 6 Download asset Open asset SOCE and JNK phosphorylation in T cells individual allelic mutations in CRACR2A. (A) Representative showing SOCE from control or T cells with an or those encoding CRACR2A CRACR2AE278D or CRACR2AR144G/E300* mutants were stimulated with anti-CD3 by ionomycin treatment in the presence of containing 2 SOCE from anti-CD3 antibody and ionomycin from three independent experiments (right). (B) Representative showing SOCE by treatment from control or T cells with or those encoding CRACR2A CRACR2AE278D or CRACR2AR144G/E300* mutants were stimulated with in to the intracellular stores and to containing SOCE levels at the or later point – from three independent experiments. (C) JNK levels in control or CRACR2A T cells and indicated mutants of CRACR2A, stimulated with anti-CD3 antibody for indicated from three independent experiments. * p < p < p < CRACR2AR144G/E300* has defect in the interaction with and shows cytoplasmic The C-terminal Rab GTPase domain of contains the conserved required for and as well as a prenylation for membrane (Figure The N-terminal EF-hand motif and coiled-coil domains are involved in intracellular Ca2+ levels and interaction (Srikanth et al., 2010a). the longer CRACR2A, which localizes to intracellular to the immunological and with a signaling its proline-rich domain (PRD) (Srikanth et al., 2016). To into the the function of the we checked the interaction of these mutants with three known ORAI1, and Vav1 using These showed that the R
Source: eLife
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Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
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