CIDs are caused by defects in T cell development and activation as well as compromised B cell activation with or without impaired B cell development. Phenotypes of CIDs are diverse, including infection, malignancy, allergy, autoimmunity and auto-inflammation. In order to identify the genetic basis of PID patients, we screened a panel of PID related genes by targeted next-generation sequencing.
By targeted NGS we identified a novel CARMIL2 mutation. Expression of CARMIL2 at protein level was characterized by flow cytometry and Western blot analysis. Functional analyzes were performed using flow cytometry.
We report a novel homozygous splice site mutation in CARMIL2 gene in 3 affected individuals from a consanguineous family that presented with selective antibody deficiency, psoriasis, molluscum contagiosum as well as warts.
CARMIL2 expression was absent in naïve CD4+ T cells of all patients. Absolute numbers of CD4+ T cells and the proportion of CD4+ naïve T cells were increased, while percentages of CD4+ T memory and CD4+ T follicular-like T cells were reduced. The phosphorylation of NFκB subunit p65 in CD4+ T cells was abolished indicating the contribution of CARMIL2 in activation of NF-κB signaling.
These cases underline the role of CARMIL2 in immunity and suggest that CARMIL2 mutations should be considered in patients with disseminated and/or persistent warts. Differences in clinical and immunological phenotypes among our patients highlight the variable clinical presentations of CARMIL2-deficiency and suggest the pathogenic relevance of additional genetic and/or epigenetic modifying factors.
ARPC1B is a key factor for ARP2/3 complex that is involved in actin branching from an existing filament. Bi-allelic mutations in the ARPC1B gene cause ARPC1B deficiency characterized by combined immunodeficiency (CID). ARPC1B deficiency is characterized by recurrent invasive infections, eczema, skin vasculitis, colitis, bleeding tendency, leukocytosis, T cell lymphopenia, eosinophilia, thrombocytopenia and hyper-IgE. Here we report a case of ARPC1B deficiency.
We used flow cytometry to characterize defects in T cells. Genetic analysis was performed by next-generation sequencing and Sanger sequencing.
A two-month-old boy was referred to our hospital with persistent oral moniliasis, pneumonia, cleft palate and congenital heart defects. His parents are non-consanguineous. The patient had a sister with cleft palate who died from sepsis. Laboratory evaluation showed anemia, marked leukocytosis, eosinophilia and normal platelet count. IgG, IgA, IgM and IgE levels were high. Direct-Coombs were positive. Percentages of CD3+, CD4+, CD4+CD45RA+ and CD4+CD45+CD31+T cells were low. T cell activation with PHA was inadequate. Echocardiography showed PFO and pulmonary stenosis. The patient was diagnosed with CID and autoimmune hemolytic anemia. IVIG and antimicrobial prophylaxis were initiated. HSCT could not be performed, because he did not have matched family donor. The patient died due to sepsis. Genetic analysis revealed a homozygous frameshift mutation in ARPC1B gene (p.H206YfsTer222), which was confirmed by Sanger sequencing. Both parents were heterozygous for the variant.
Unlike other patients, our patient had cleft palate and cardiac anomalies, and no thrombocytopenia. Early diagnosis and HSCT is crucial for survival of these patients.
Hypomorphic defects in RAG1 underlie leaky SCID, ranging from Omenn syndrome to atypical presentations of combined immunodeficiency. Hypomorphic RAG1 mutations also cause an atypical SCID characterized by chronic CMV infection, autoimmunity and expansion of TCRγδ T cells.
We report the clinical course and outcome of a child with hypomorphic RAG1 defects.
A 16-month-old girl with failure to thrive presented with pneumonia and ARDS due to H1N1 Influenza. A month later, she developed CMV and Pneumocystis jirovecii pneumonia, hepatosplenomegaly, autoimmune hemolytic anemia (AHIA) and pancreatitis. Previously, she had suffered from bronchiolitis requiring O2-therapy and had developed vaccine-related disease after Rotavirus and VZV vaccine. Her work-up showed moderate T and B lymphopenia with normal NK cells, excess of memory T and of TCRγδ T cells (53% of T cells), hypergammaglobulinemia, absent thymus on CT scan, and restricted TCR-V-beta repertoire. Whole exome sequencing revealed compound heterozygous missense mutations in RAG1 (p.R410Q, p.H612R). She received haploidentical HSCT after myeloablative conditioning, resulting in full engraftment. Three months post-HSCT she experienced graft loss with CMV reactivation and recurrence of AHIA, not responding to unconditioned stem cell boost and a course of bortezomib. She then developed fulminant hemophagocytic lymphohistiocytosis which was sadly fatal despite high dose steroid therapy, a second haploidentical HSCT and extensive antiviral therapy.
We report a patient presenting with life-threatening flu, which led to the diagnosis of leaky SCID associated with AIHA, pancreatitis and HLH. This highlights the importance of timely diagnosis of SCID and the wide spectrum of presentation of hypomorphic RAG mutations.
Patients with STAT1/STAT3 imbalanced primary immunodeficiencies frequently suffer from chronic microbial skin and respiratory infections caused by a compromised Th17 immunity. The environmental sensor aryl hydrocarbon receptor (AHR) has immune-modulating functions by interfering with Th17 immunity, with potential beneficial effect in STAT‑imbalanced patients. Here, we assess the effect of Th17 immunity and AHR pathway on microbiobal challenges in STAT-imbalanced patients.
Activated human peripheral blood mononuclear cells of healthy subjects and STAT-imbalanced patients were assessed for the expression of AHR pathway related targets and Th17 cytokines upon treatment with AHR agonists. Responses of human epithelial cells to environmental signals in combination with STAT1/STAT3 activating cytokines were analyzed regarding the production of antimicrobial peptides and pro-inflammatory mediators.
AHR activation increased the Th17 cytokine IL-22 and AHR pathway related genes in PBMCs of healthy subjects and STAT-imbalanced patients. Despite the increase, IL-22 concentration in patients do not reach the level found in healthy subjects. Challenge of epithelial cells by environmental triggers increased the production of antimicrobial peptides whereas pro-inflammatory chemokines were reduced.
The observed induction of IL-22 in STAT-imbalanced patients by environmentally-triggered AHR signaling changed Th17 immunity with possible beneficial effects of an increased epithelial defense.
IKZF2 gene codes for a zinc-finger protein Helios that is an activator and repressor of transcription. Helios knockout mice develop autoimmunity by the age of 6 month. Helios is upregulated in T cells in response to T cell receptor stimulation and it is identified as an important transcription factor in exhausted T cells. Highest expression of Helios is found from regulatory T cells (Treg), where it stabilizes their suppressive phenotype. Though studies in mice suggest that Helios has an important role in controlling immune responses, the full range of its effects in humans remains to be shown. We have identified, to our knowledge, first germline loss of function mutation described in humans with immunodeficiency phenotype.
Characterization of immunophenotype using array of techniques including interactome analysis, flow cytometry, RNA sequencing, and cell cultures.
A Finnish adult was exome sequenced due to hypogammaglobulinemia and heterozygous loss-of function mutation in IKZF2 (Helios) was found. Same variant was also detected from her first degree relative. Both patients had hypothyreosis, but otherwise signs of autoimmunity were relatively mild. The T cell phenotype of patients was proinflammatory and markedly more mature, even senescent. We found the patients’ Treg phenotype to be more inflammatory and Treg numbers were reduced.
In line with data from Helios deficient murine models heterozygous loss of Helios expression in patients leads to more proinflammatory T cell phenotype. This most likely is the result of Helios’ effect on both effector and regulatory T cells.
The BCG ( Bacillus Calmette-Guérin) vaccine is effective in preventing early-life infections with Mycobacterium Tuberculosis. The WHO recommends BCG vaccination as soon as posible after birth for all infants in high-burden countries. All Iranian newborns received BCG vaccine immediately after birth. Although BCG vaccine is safe, but some complications may occur especially in immune-compromised infants. Disseminated BCG disease, also called BCGosis, is a rare but life-threatening complication.Here we present a young infant with recurrent vomiting due to gastric outlet obstruction.
A 12 month-old infant boy was admitted in our tertiary children hospital due to severe failure to thrive and recurrent vomiting. After through investigations, we performed upper gastrointestinal series and an external pressure effect was detected on C-loop of duodenum. After resuscitation with fluids, patient was candidate for surgery and underwent emergent laparotomy. There was severe inflammation of small intestine mesentery and too many prominent lymphadenopathy and fibrosis of mesentery and retro-peritoneum. Some fibrotic bands attached to jejunum. These pathologies result in complete obstruction of small intestine at proximal of jejunum.
The pathology report shows severe histiocytic infiltration of lymph nodes and was positive for numerous acid fast bacilli.
Although very rare, but we should consider anatomical bowel obstruction due to external pressure effect in patients with BCGosis
The human thymus shows a transient involution along the neonatal period and starts a continuous process of decline between the 1st and 2nd years. The immune alterations associated to thymic aging in humans have been extensively investigated, but the genomic mechanisms underlying this process remain largely unknown. Here we conducted a temporal gene co-expression network analysis in the newborn and infant thymic tissue aiming to identify transcriptional modules associated to age.
Whole transcriptional profiles of newborn and infant thymus surgical explants (31 males and 23 females) grouped by age intervals – 0-30d (A), 31d-6mo (B), 7-12mo (C), 13-18mo (D), and 19-31mo (E) - were analyzed using the Weighted Gene Co-expression Networks Analysis (WGCNA). The genomic analyses were centered in: i) identification of transcriptional modules; ii) node categorization (hubs, HGS genes); iii) integrative mRNA-miRNA-transcription factors (TFs) co-expression networks.
Three transcriptional modules were correlated with A (neonate) and/or E groups. The two modules positively correlated with the E group (mean age 25 months) harbor comparatively more hubs related to T-cell and thymic stromal functions. Interestingly, most of the HGS genes hyper-expressed in group A (A vs E comparison) are involved in T-cell development and stromal functions. Integrative network analysis revealed that HGS genes have more links with miRNAs and TFs when compared with hubs.
Our results show that hubs are related to network/module robustness. Conversely, the differentially expressed HGS genes, acting as bridges between modules or as border genes, may well be markers of age-related thymic processes.
FAPESP 2014/50489-9; 2015/22308-2
Severe Combined Immune Deficiency (SCID) is a life-threatening disease of the immune system that is caused by genetic defects. SCID patients suffer from opportunistic infections as a result of abrogation of T lymphocyte development and/or function in which B and NK cell development might be affected as well. This results in a heterogeneous spectrum of phenotypes that often complicates diagnosis. Furthermore, the identification of the underlying genetic defect is often confounded by incomplete penetrance, genetic modifiers and ongoing infections. To improve the genotype-phenotype correlation, we evaluated the use of OP9-DLL1 and MS5 stromal cocultures to study the in vitro immune cell differentiation potential of CD34+Lin- hematopoietic precursor cells (HPCs) from SCID patients.
In this particular case, HPCs were used from a patient that was characterized by a novel splice acceptor mutation in the DCLRE1C gene encoding for Artemis.
The differentiation of T and B cells showed a clear stage-specific developmental defect, associated with the dependency on Artemis function. As anticipated, the differentiation of NK and myeloid cells was not disturbed.
These results thus clearly show the potential of using patient-derived HSCs in these in vitro differentiation cultures to model SCID patients. By defining these genotype-phenotype correlations, the treatment, diagnosis and counseling of these patients can be facilitated. From a fundamental perspective, the in vitro differentiation of patient-derived HPCs can be exploited to address the developmental impact of SCID genes to further elucidate the molecular mechanisms that drive human immune cell development.
Severe Combined Immunodeﬁciency is an uncommon but most serious form of primary immunodeﬁciencies. Mutations in genes that encode proteins necessary for the development of the immune recognition receptors on T and B-lymphocytes calles ,recombinase activating genes 1 and 2 (RAG1 and RAG2) lead to a very rare type of SCID known as Omenn syndrome.
A 11 month old girl who was admitted because of aphthous stomatitis and diarrhea. On examination, the patient's skin was dry and the patient was suffering from severe respiratory distress and stridor that improved by nebulized bronchodilator. Laboratory studies reported that the patient was B negative, T negative and because of severe dermatitis with leukocytosis was diagnosed as Omenn syndrome. Patient frequently refered to hospital due to diarrhea, and suffer from severe FTT. The patient eventually died due to the severity of symptoms.
Genetic investigations showed the mutation in gene JAK3.
This is a very interesting conflict between the genetic results and clinical symptoms of two different types of SCID.
Progressive multifocal leukoencephalopathy(PML) is a demyelinating disease caused by John Cunningham(JC) virus reactivation in the central nervous system. Especially in the presence of primary or secondary immunodeficiency, it may manifest itself with various neurological findings.
A 13-year-old male patient with a diagnosis of atopic dermatitis for nine years; admitted with recurrent wound on nose. There was first-degree consanguineous marriage between his parents. Valaciclovir treatment and excision was performed for 4 times. However, alpha interferon treatment was given because of persistent herpesvirus infections and the lesions scared. Genetic analysis performed at University Medical Center in Freiburg revealed heterozygous missense mutation in TRAF3IP2 and TYK2 regions and he was diagnosed with DOC 8 deficiency. In the first year of the diagnosis, soft tissue lesion started on his scrotum. The excision sample was found compatible with squamous cell carcinoma. The patient was admitted with a generalized tonic clonic seizure, who received intravenous immunoglobulin supplementation with three-week intervals. No pathology was detected in the serebrospinal fluid sample except JC virus copy(23600 copies/ml). Radiological evaluation was consistent with PML. Sidofovir and maravirok treatment was started with the literature review.
After 28 doses of sidofovir, the patient had radiological and neurological regression and sidofovir was discontinued. He transferred to the bone marrow transplantation unit.
JC virus, which is latent in the body, should be considered in any neurological finding, especially in patients with cellular immune suppression. Although there is no proven treatment, it is possible to prolong survival based on the examples in the literature.