Objective
We aimed to test the relevance of deficiency of adenosine deaminase 2 (DADA2) in patients with antibody deficiency and describe the clinical picture of the disease in adulthood.
Methods
We ...screened for DADA2 in a cohort of 181 patients with antibody deficiency with or without vascular lesions using next‐generation sequencing and targeted Sanger sequencing. All mutations were confirmed by determining the ADA2 enzymatic activity levels in dried plasma spots. Clinical data and laboratory values were collected in a standardized format.
Results
Following the diagnosis of 2 siblings in the index family, we identified 9 additional affected patients with compound heterozygous or homozygous CECR1 mutations, containing 6 novel and 4 previously published mutations. The patients' age at evaluation ranged from 13 to 51 years, with a median age of 22 years. Clinically, we saw a broad phenotype, ranging from isolated antibody deficiency to recurrent strokes. All but 1 patient had low numbers of memory B cells. Moreover, B cell function seemed to correlate with inflammation.
Conclusion
Taken together, our findings indicate that DADA2 presents not only with vasculopathy but also with an immunodeficiency of the B cell compartment. Therefore, patients with antibody deficiency should be screened for DADA2. Anti–tumor necrosis factor treatment might improve immunologic features over time and might be considered in patients without vascular manifestations but with elevated inflammation markers. Conservative management has so far proven to be the choice for our less severely affected adolescent and adult DADA2 patients; however, in patients with severe cytopenias and bone marrow failure, hematopoietic stem cell transplantation should be considered.
Hyper-IgE syndromes and chronic mucocutaneous candidiasis constitute rare primary immunodeficiency syndromes with an overlapping clinical phenotype. In recent years, a growing number of underlying ...genetic defects have been identified. To characterize the underlying genetic defects in a large international cohort of 275 patients, of whom 211 had been clinically diagnosed with hyper-IgE syndrome and 64 with chronic mucocutaneous candidiasis, targeted panel sequencing was performed, relying on Agilent HaloPlex and Illumina MiSeq technologies. The targeted panel sequencing approach allowed us to identify 87 (32 novel and 55 previously described) mutations in 78 patients, which generated a diagnostic success rate of 28.4%. Specifically, mutations in
DOCK8
(26 patients),
STAT3
(21),
STAT1
(15),
CARD9
(6),
AIRE
(3),
IL17RA
(2),
SPINK5
(3),
ZNF341
(2),
CARMIL2
/
RLTPR
(1),
IL12RB1
(1), and
WAS
(1) have been detected. The most common clinical findings in this cohort were elevated IgE (81.5%), eczema (71.7%), and eosinophilia (62.9%). Regarding infections, 54.7% of patients had a history of radiologically proven pneumonia, and 28.3% have had other serious infections. History of fungal infection was noted in 53% of cases and skin abscesses in 52.9%. Skeletal or dental abnormalities were observed in 46.2% of patients with a characteristic face being the most commonly reported feature (23.1%), followed by retained primary teeth in 18.9% of patients. Targeted panel sequencing provides a cost-effective first-line genetic screening method which allows for the identification of mutations also in patients with atypical clinical presentations and should be routinely implemented in referral centers.
Objective
Common variable immunodeficiency (CVID) is the most common clinically relevant entity of inborn errors of immunity. In these patients, an altered gut microbiome composition with reduced ...diversity has been described. We sought to investigate the fecal immunoglobulin levels and their impact on the gut microflora in patients with CVID.
Methods
We analyzed the gut microbiome of 28 CVID patients and 42 healthy donors (HDs), including 21 healthy household controls, by sequencing the V3 and V4 regions of the bacterial 16S rRNA gene extracted from stool samples. The fecal levels of immunoglobulin A, M, and G of 27 CVID patients and 41 HDs were measured in the supernatant by ELISA and normalized for protein concentration.
Results
We measured decreased IgA and increased IgG in stool samples from CVID patients compared to HDs. Decreased levels of fecal IgA and IgM were associated with reduced microbial diversity and increased dysbiosis. We identified a large number of significantly differentially abundant taxa, especially in patients with decreased IgA levels, but also in patients with decreased IgM levels compared to their counterparts.
Conclusions
CVID patients have an altered gut microbiota composition, which is most prevalent in patients with decreased fecal IgA and IgM levels. In this study, we identify fecal immunoglobulins as a potential modifier of the gut microbiome in CVID patients.
Precise spatiotemporal control of axon guidance factor expression is a prerequisite for formation of functional neuronal connections. Although Netrin/Dcc- and Robo/Slit-mediated attractive and ...repulsive guidance of commissural axons have been extensively studied, little is known about mechanisms controlling mediolateral positioning of longitudinal axons in vertebrates. Here, we use a genetic approach in zebrafish embryos to study pathfinding mechanisms of dopaminergic and neuroendocrine longitudinal axons projecting from the hypothalamus into hindbrain and spinal cord. The transcription factors Sim1a and Arnt2 contribute to differentiation of a defined population of dopaminergic and neuroendocrine neurons. We show that both factors also control aspects of axon guidance: Sim1a or Arnt2 depletion results in displacement of hypothalamo-spinal longitudinal axons towards the midline. This phenotype is suppressed in robo3 guidance receptor mutant embryos. In the absence of Sim1a and Arnt2, expression of the robo3 splice isoform robo3a.1 is increased in the hypothalamus, indicating negative control of robo3a.1 transcription by these factors. We further provide evidence that increased Robo3a.1 levels interfere with Robo2-mediated repulsive axon guidance. Finally, we show that the N-terminal domain unique to Robo3a.1 mediates the block of Robo2 repulsive activity. Therefore, Sim1a and Arnt2 contribute to control of lateral positioning of longitudinal hypothalamic-spinal axons by negative regulation of robo3a.1 expression, which in turn attenuates the repulsive activity of Robo2.
In contrast to adult-onset inflammatory bowel disease (IBD), where many genetic loci have been shown to be involved in complex disease etiology, early-onset IBD (eoIBD) and associated syndromes can ...sometimes present as monogenic conditions. As a result, the clinical phenotype and ideal disease management in these patients often differ from those in adult-onset IBD. However, due to high costs and the complexity of data analysis, high-throughput screening for genetic causes has not yet become a standard part of the diagnostic work-up of eoIBD patients.
We selected 28 genes of interest associated with monogenic IBD and performed targeted panel sequencing in 71 patients diagnosed with eoIBD or early-onset chronic diarrhea to detect causative variants. We compared these results to whole-exome sequencing (WES) data available for 25 of these patients.
Target coverage was significantly higher in the targeted gene panel approach compared with WES, whereas the cost of the panel was considerably lower (approximately 25% of WES). Disease-causing variants affecting protein function were identified in 5 patients (7%), located in genes of the IL10 signaling pathway (3), WAS (1), and DKC1 (1). The functional effects of 8 candidate variants in 5 additional patients (7%) are under further investigation. WES did not identify additional causative mutations in 25 patients.
Targeted gene panel sequencing is a fast and effective screening method for monogenic causes of eoIBD that should be routinely established in national referral centers.
Induced pluripotent stem cells (iPSCs) provide a unique opportunity for the generation of patient-specific cells for use in disease modelling, drug screening, and regenerative medicine. The aim of ...this study was to compare human-induced pluripotent stem cells (hiPSCs) derived from different somatic cell sources regarding their generation efficiency and cardiac differentiation potential, and functionalities of cardiomyocytes.
We generated hiPSCs from hair keratinocytes, bone marrow mesenchymal stem cells (MSCs), and skin fibroblasts by using two different virus systems. We show that MSCs and fibroblasts are more easily reprogrammed than keratinocytes. This corresponds to higher methylation levels of minimal promoter regions of the OCT4 and NANOG genes in keratinocytes than in MSCs and fibroblasts. The success rate and reprogramming efficiency was significantly higher by using the STEMCCA system than the OSNL system. All analysed hiPSCs are pluripotent and show phenotypical characteristics similar to human embryonic stem cells. We studied the cardiac differentiation efficiency of generated hiPSC lines (n = 24) and found that MSC-derived hiPSCs exhibited a significantly higher efficiency to spontaneously differentiate into beating cardiomyocytes when compared with keratinocyte-, and fibroblast-derived hiPSCs. There was no significant difference in the functionalities of the cardiomyocytes derived from hiPSCs with different origins, showing the presence of pacemaker-, atrial-, ventricular- and Purkinje-like cardiomyocytes, and exhibiting rhythmic Ca2+ transients and Ca2+ sparks in hiPSC-derived cardiomyocytes. Furthermore, spontaneously and synchronously beating and force-developing engineered heart tissues were generated.
Human-induced pluripotent stem cells can be reprogrammed from all three somatic cell types, but with different efficiency. All analysed iPSCs can differentiate into cardiomyocytes, and the functionalities of cardiomyocytes derived from different cell origins are similar. However, MSC-derived hiPSCs revealed a higher cardiac differentiation efficiency than keratinocyte- and fibroblast-derived hiPSCs.
Barth syndrome (BTHS) patients carrying mutations in tafazzin (TAZ1), which is involved in the final maturation of cardiolipin, present with dilated cardiomyopathy, skeletal myopathy, growth ...retardation and neutropenia. To study how mitochondrial function is impaired in BTHS patients, we generated induced pluripotent stem cells (iPSCs) to develop a novel and relevant human model system for BTHS. BTHS–iPSCs generated from dermal fibroblasts of three patients with different mutations in TAZ1 expressed pluripotency markers, and were able to differentiate into cells derived from all three germ layers both in vitro and in vivo. We used these cells to study the impact of tafazzin deficiency on mitochondrial oxidative phosphorylation. We found an impaired remodeling of cardiolipin, a dramatic decrease in basal oxygen consumption rate and in the maximal respiratory capacity in BTHS–iPSCs. Simultaneous measurement of extra-cellular acidification rate allowed us a thorough assessment of the metabolic deficiency in BTHS patients. Blue native gel analyses revealed that decreased respiration coincided with dramatic structural changes in respiratory chain supercomplexes leading to a massive increase in generation of reactive oxygen species. Our data demonstrate that BTHS–iPSCs are capable of modeling BTHS by recapitulating the disease phenotype and thus are important tools for studying the disease mechanism.
•We generate iPSCs from three patients with Barth syndrome (BTHS).•BTHS–iPSCs recapitulate biochemical features of the disease.•Impaired mitochondrial respiration of BTHS–iPSCs is not compensated by glycolysis.•BN-PAGE reveals respiratory chain complex remodeling in BTHS–iPSCs.•Reduced energy coupling causes generation of reactive oxygen species.
Human induced pluripotent stem cells (hiPSCs) could be used to generate autologous cells for therapeutic purposes, which are expected to be tolerated by the recipient. However, iPSC-derived grafts ...are at risk of giving rise to teratomas in the host, if residuals of tumorigenic cells are not rejected by the recipient. We have analyzed the susceptibility of hiPSC lines to allogeneic and autologous natural killer (NK) cells. IL-2-activated, in contrast to resting NK cells killed hiPSC lines efficiently (P = 1.69 x 10(-39)). Notably, the specific lysis of the individual hiPSC lines by IL-2-activated NK cells was significantly different (P = 1.72 x 10(-6)) and ranged between 46 % and 64 % in 51Cr-release assays when compared to K562 cells. The hiPSC lines were killed by both allogeneic and autologous NK cells although autologous NK cells were less efficient (P=8.63 x 10(-6)). Killing was partly dependent on the activating NK receptor DNAM-1 (P = 8.22 x 10(-7)). The DNAM-1 ligands CD112 and CD155 as well as the NKG2D ligands MICA and MICB were expressed on the hiPSC lines. Low amounts of human leukocyte antigen (HLA) class I proteins, which serve as ligands for inhibitory and activating NK receptors were also detected. Thus, the susceptibility to NK cell killing appears to constitute a common feature of hiPSCs. Therefore, NK cells might reduce the risk of teratoma formation even after autologous transplantations of pluripotent stem cell-derived grafts that contain traces of pluripotent cells.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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