Disrupted Ca2+ homeostasis and immunodeficiency in patients with functional IP3 receptor subtype 3 defects

Calcium signaling is essential for lymphocyte activation, with genetic disruptions of store-operated calcium (Ca2+) entry resulting in severe immunodeficiency. The inositol 1,4,5-trisphosphate receptor (IP3R), a homo- or heterotetramer of the IP3R1-3 isoforms, amplifies lymphocyte signaling by releasing Ca2+ from endoplasmic reticulum stores following antigen stimulation. Although knockout of all IP3R isoforms in mice causes immunodeficiency, the seeming redundancy of the isoforms is thought to explain the absence of variants in human immunodeficiency. In this study, we identified compound heterozygous variants of ITPR3 (a gene encoding IP3R subtype 3) in two unrelated Caucasian patients presenting with immunodeficiency. To determine whether ITPR3 variants act in a nonredundant manner and disrupt human immune responses, we characterized the Ca2+ signaling capacity, the lymphocyte response, and the clinical phenotype of these patients. We observed disrupted Ca2+ signaling in patient-derived fibroblasts and immune cells, with abnormal proliferation and activation responses following T-cell receptor stimulation. Reconstitution of IP3R3 in IP3R knockout cell lines led to the identification of variants as functional hypomorphs that showed reduced ability to discriminate between homeostatic and induced states, validating a genotype–phenotype link. These results demonstrate a functional link between defective endoplasmic reticulum Ca2+ channels and immunodeficiency and identify IP3Rs as diagnostic targets for patients with specific inborn errors of immunity. These results also extend the known cause of Ca2+-associated immunodeficiency from store-operated entry to impaired Ca2+ mobilization from the endoplasmic reticulum, revealing a broad sensitivity of lymphocytes to genetic defects in Ca2+ signaling.

stool. Additional investigations were normal including a sweat test, transfontanellar ultrasound, OPG, gastroscopy with biopsies, x-ray of the upper gastro-intestinal tract and impedance-pH monitoring. While his first bone marrow analysis was unremarkable, at age 2 years bone marrow examination demonstrated a normochrome normocytic anemia and leukopenia with absolute lymphopenia. Common gamma chain expression (CD132), VDJ recombination, DHR oxidation, and CD40 ligand expression were within normal limits. Pneumococcal polysaccharide antibody response was abnormal and repeat TLR testing was suboptimal. Combined immune deficiency was diagnosed and he was started on subcutaneous immunoglobulin replacement therapy at age 2.5 years. Serial bronchoscopy revealed frail mucosae and secretions. BAL bacterial, mycobacterial and fungal cultures were unrevealing on multiple occasions. The patient was treated with empiric antibiotics, respiratory physiotherapy and started on azithromycin at an antiinflammatory dosage. At age 4 years a mycobacterial infection was suspected due to worsening CT thorax with consolidation, unilateral hilar lymph nodes and compression on the left main bronchus, with a caseous lesion viewed with bronchoscopy. Despite negative quantiferon test and negative gastric lavage cultures, a 6-month course of antimycobacterial therapy was administered with good effect. IGF-1 was on the lower limit of normal with a delay in bone age of 4 years at age 7 years. However, glucagon response testing and IGF-1 generation tests were normal, with insufficient response to exogenous growth hormone. Hypercaloric nutrition was started. Sequential sanger sequencing had ruled out RMRP, NEMO, RAG1/2, ADA, SBDS and JAK3 mutations. Karyotyping and FISH for 22q11 were normal.
The patient received an allogenic hematopoietic stem cell transplant from a matched unrelated donor at the age of 6 years (Supplementary Table 2) after conditioning with treosulfan 42g/m², fludarabine 150mg/m², Campath 5x0.2mg/kg. Full donor chimerism was observed on day 30 with no signs of GvHD. Pre-transplant screening showed a positive EBV PCR (2.92 log EBV IU/ml). Aspergillus antigen proved positive on blood with negative fungal cultures and negative aspergillus antigen on BAL 3 months post-transplant. Chest CT didn't show any typical characteristics of angio-invasive aspergillosis. Amphotericine B was initiated. 5 months posttransplant P1 developed diarrhea and red blood loss, treated with anti-TNF after ruling out GvHD (EBV PCR positive on biopsy). Immune hemolysis with negative antinuclear antibodies 6 months post-transplant was treated with high dose CS and responded to rituximab treatment. P1 developed multiple endobronchial EBV-induced leiomyomas approximately one year after transplantation necessitating multiple debulking surgeries. Both mollusca and leiomyomas resolved with immune reconstitution. Recognition of the patient's areflexia and progressive distal muscular weakness were delayed by the onset of bilateral avascular necrosis of the hip at the age of 10 years requiring therapeutic use of a wheelchair. Upon presentation with peripheral neuropathy at the age of 11 years, EMG revealed reduced ulnar and medial sensory and motor nerve conduction velocities as well as typical EMG patterns for an axonal form of Charcot-Marie-Tooth. He is now 6 years post-HSCT with full donor chimerism and good immune reconstitution but with severely impaired lung function. Whole exome sequencing on genomic DNA extracted from pre-transplantation blood cells of the patient and both parents was performed and led to the identification of two candidate variants in ITPR3, inherited in a compound heterozygous way (c.5549G>A:p.Arg1850Gln inherited from father and c.7570C>T:p.Arg2524Cys de novo).
Patient 2 is a 36 year old male, born to non-consanguineous parents of Caucasian ethnicity. A summary of demographic, clinical, laboratory, technical and treatment data is shown in Supplementary Table 1. He presented at the age of 18 years with diffuse petechiae and severely decreased thrombocytes in peripheral blood (9x10 9 /L, reference 150-450x10 9 /L, Supplementary  Table 3) showed profound macrocytic anemia (nadir hemoglobin 2.6 g/dl, reference 14-18 g/dl), with high reticulocytes (107x10 9 /L, reference 20-100), elevated total bilirubin (2.14mg/dl, ref < 1 mg/dl) and low haptoglobin (<0.20 g/L, reference 0.3-2 g/L). Direct coombs test was positive, confirming the suspicion of auto-immune hemolytic anemia (AIHA). As underlying etiology a precedent infection in the past month before onset of AIHA (either gastrointestinal caused by Blastocystis hominis or respiratory caused by Mycoplasma pneumonia) was suspected. Bone marrow examination did not suggest the presence of a myeloproliferative disorder or lymphoma and additional clinical and laboratory tests were negative for autoimmune diseases (ANF, ANCA negative), paroxysmal nocturnal hemoglobinuria (normal expression of CD55/CD59 on erythrocytes and granulocytes) and viral infections (Parvovirus B19, HIV, EBV and HCV were negative). He was treated with red blood cell transfusions, erythropoietin (Aranesp 150 µg/week), anti-CD20 (Rituximab 700 mg IV once weekly for a total of 4 doses), cyclophosphamide (Endoxan 1000 mg IV once), intravenous immunoglobulins (Multigam 65 g for a total of 4 doses), corticosteroids (125 mg, tapered over time) and cyclosporin (Neoral, initiated after induction at a dose of 150 mg twice daily).
Hemoglobin levels recovered to a normal level after 9 months and cyclosporin was tapered to a dose of 50 mg twice daily. During follow up he reported recurrent upper respiratory tract infections (sinusitis), treated with multiple courses of antibiotics. Immunoglobulin G levels, two years after the last rituximab administration, were still decreased to a level of 6.83 g/dl (Supplementary Table 3). On immunophenotyping he had normal B cell numbers (352/µL, reference 82-476x10 9 /L) but severely decreased switched memory B cells (1.1 % CD27 + IgM -IgD of total B cells). At that time, he was given intravenous immunoglobulins monthly to prevent infections. Despite treatment he continued to suffer from respiratory tract infection and pulmonary CT scan showed multiple nodular infiltrates. Subsequent bronchoscopy with bronchoalveolar lavage (BAL) revealed a positive galactomannan test with negative microbial cultures and viral PCR. Galactomannan testing on peripheral blood was also positive. A lung biopsy was performed and demonstrated a predominantly lymphoplasmocytary infiltrate. Based on the revised EORTC criteria, probable invasive pulmonary aspergillosis was diagnosed.
Despite multiple antifungal treatments given for a total period of two years (voriconazole, posaconazol), he continued to have migrating pulmonary infiltrates. A new bronchoscopy was done and BAL did not reveal signs of aspergillosis nor other relevant pathogens by PCR or culture. In this period he also reported progressive gastro-intestinal symptoms of secretory diarrhea, nausea and anorexia with a weight loss of 8 kg. Ileocolonoscopy with biopsies was normal and gastroscopy showed signs of chronic gastritis and mildly increased intraepithelial lymphocytosis in the duodenal biopsy. No enteropathogens were identified on faecal culture.
Tissue transglutaminase was normal (<1.9, reference ≤20 CU). Subsequently he was referred to the immunology clinic for a suspicion of common variable immunodeficiency (CVID) with infectious susceptibility and associated autoimmunity (AIHA, ITP), granulomatous and lymphocytic interstitial lung disease (GLILD), and enteropathy. Clinical immunophenotyping at that time (Supplementary Table 3