Wiskott-Aldrich Syndrome: A study on 577 patients defining the genotype as a predictive biomarker for disease severity.

WAS is a multifaceted monogenic disorder with a broad disease spectrum and variable disease severity and a variety of treatment options including allogeneic hematopoietic stem cell transplantation (HSCT) and gene therapy (GT). No reliable biomarker exists to predict disease course and outcome for individual patients. A total of 577 patients with a WAS variant from 26 countries and a median follow-up of 8.9 years (0.3-71.1), totaling 6118 patient-years, were included in this international retrospective study. Overall survival (OS) of the cohort (censored at HSCT or GT) was 82% (95% CI 78-87) at 15 years and 70% (61-80) at 30 years of age. The type of variant was predictive of outcome: patients with a missense variant in exons 1 or 2 or with the intronic hotspot variant c.559+5G>A (class I variants) had a 15-year OS of 93% (89-98) and a 30-year OS of 91% (86-97), compared to 71% (62-81) and 48% (34-68) in patients with any other variant (class II; p<0.0001). The cumulative incidence rates of disease-related complications such as severe bleeding (p=0.007), life-threatening infection (p<0.0001), and autoimmunity (p=0.004) occurred significantly later in patients with a class I variant. The cumulative incidence of malignancy (p=0.6) was not different between classes I and II. This study represents the largest cohort of WAS patients studied so far. It confirms the spectrum of disease severity and quantifies the risk for specific disease-related complications. The class of variant is a biomarker to predict the outcome for WAS patients.

Chronic granulamatous disease: Two decades of experience from a paediatric immunology unit in a country with high rate of consangineous marriages.

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by susceptibility to bacterial and fungal infections resulting from the inadequacy of phagocytic leucocytes to produce reactive oxygen radicals. CGD is a genetically heterogeneous disease with an X-linked recessive (XR-CGD) form caused by mutations in the CYBB (OMIM #300481) gene encoding the gp91(phox) protein, and an autosomal recessive (AR-CGD) form caused by mutations in the CYBA (OMIM #608508), NCF1 (OMIM #608512), NCF2 (OMIM #608515) and NCF4 (OMIM #601488) genes encoding p22(phox), p47(phox), p67(phox) and p40(phox), respectively. The genetic mutation of one of the cytosolic p47phox/p67phox proteins and membrane-bound gp91phox/p22phox proteins, which constitutes the NADPH oxidase enzyme complex, causes the disease. In this study, we evaluated the clinical, laboratory and genetic findings and the prognostic effects of molecular inheritance of our 24 CGD cases (14 XR, 10 autosomal recessive-AR). Consanguinity (three XR and all AR cases) showed statistically significant relationship with the type of hereditary inheritance (P < 0.001). 83% patients had an infection since early infancy. The mean age of initiation of symptoms was earlier in XR cases, and 78% patients had respiratory tract infections. Bone marrow transplantation was performed in five XR cases (two ex) and four AR (one ex) cases. Three of nine XR and two of six AR cases deceased on medical follow-up. In countries especially with high consanguinity rates, the early diagnosis for appropriate prophylactic treatment of CGD is quietly important to avoid from recurrent severe infections, early death and fatal complications of late transplantation.

Targeted Gene Panel Sequencing for Early-onset Inflammatory Bowel Disease and Chronic Diarrhea.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Dedicator of cytokinesis 8 regulates signal transducer and activator of transcription 3 activation and promotes TH17 cell differentiation.

BACKGROUND: The autosomal recessive hyper-IgE syndrome (HIES) caused by dedicator of cytokinesis 8 (DOCK8) deficiency shares clinical features with autosomal dominant HIES because of signal transducer and activator of transcription 3 (STAT3) mutations, including recurrent infections and mucocutaneous candidiasis, which are suggestive of TH17 cell dysfunction. The mechanisms underlying this phenotypic overlap are unclear. OBJECTIVE: We sought to elucidate common mechanisms operating in the different forms of HIES. METHODS: We analyzed the differentiation of CD4+ TH cell subsets in control and DOCK8-deficient subjects. We also examined the role of DOCK8 in regulating STAT3 activation in T cells. TH cell differentiation was analyzed by ELISA, flow cytometry, and real-time PCR measurements of cytokines and TH cell transcription factors. The interaction of DOCK8 and STAT3 signaling pathways was examined by using flow cytometry, immunofluorescence, coimmunoprecipitation, and gene expression analysis. RESULTS: There was a profound block in the differentiation of DOCK8-deficient naive CD4+ T cells into TH17 cells. A missense mutation that disrupts DOCK8 guanine nucleotide exchange factor (GEF) activity while sparing protein expression also impaired TH17 cell differentiation. DOCK8 constitutively associated with STAT3 independent of GEF activity, whereas it regulated STAT3 phosphorylation in a GEF activity-dependent manner. DOCK8 also promoted STAT3 translocation to the nucleus and induction of STAT3-dependent gene expression. CONCLUSION: DOCK8 interacts with STAT3 and regulates its activation and the outcome of STAT3-dependent TH17 differentiation. These findings might explain the phenotypic overlap between DOCK8 deficiency and autosomal dominant HIES.

A novel disease-causing CD40L mutation reduces expression of CD40 ligand, but preserves CD40 binding capacity.

Mutations in CD40 ligand (CD40L) that permit residual CD40L expression typically impair binding of CD40. We report a male patient who presented with recurrent bacterial respiratory tract infections, normal IgM, decreased IgG, absent IgA levels, and CD40L expression at ~50% of the level observed in the normal control. He subsequently developed autoimmunity, inflammatory bowel disease, severe opportunistic infections suggestive of a combined immunodeficiency, and a cervical spine schwannoma. Whole exome sequencing of the patient's genomic DNA revealed a novel missense mutation (p.H47Y) in CD40L. Although this mutation was predicted to be benign in silico, flow cytometry at 13 years of age demonstrated markedly decreased CD40L expression (~32% of normal control) that retained the capacity to bind soluble CD40-Ig, suggesting that the mutation impairs CD40L surface expression without affecting its affinity for CD40. This case highlights the variability in the clinical evolution and phenotype of CD40L deficiency.

Three different classifications, B lymphocyte subpopulations, TNFRSF13B (TACI), TNFRSF13C (BAFF-R), TNFSF13 (APRIL) gene mutations, CTLA-4 and ICOS gene polymorphisms in Turkish patients with common variable immunodeficiency.

B lymphocyte subpopulations, previously defined classification schemes (Freiburg, Paris, EuroClass), TNFRSF13B (TACI), TNFRSF13C (BAFF-R), TNFSF13 (APRIL) gene mutations, CTLA-4 and ICOS gene polymorphisms were analyzed in 25 common variable immunodeficiency (CVID) patients and 25 healthy controls. Patients were also divided into two subgroups due to some disease severity criteria. SG (severe disease group) (n:11) included patients who have splenomegaly and/or granulomatous diseases and/or bronchiectasis and/or lower baseline IgG values (<270 mg/dl). MG (moderate disease group) (n:14) patients diagnosed as having ESID/PAGID criteria but does not fulfill SG inclusion criteria. The onset of infectious symptoms and age at diagnosis were 50.0 ± 45.7 and 78.5 ± 54.5 months, respectively. Parental consanguinity rate was 54.5% in SG and 7.1% in MG. Switched-memory B cells (CD19 + 27 + IgD-IgM-) showed significant decrease in CVID patients and these cells were also significantly lower in SG compared to MG. CVID patients had significantly higher percentages of CD19 + κ + B cells and CD19 + λ + B cells than healthy controls. Freiburg classification: 87.5% of patients (n:21) were in group I and 12.5% were in Group II. Eighteen (75%) CVID patients with a low percentage of CD21(low) B cells were in Group Ib while three patients classified as Group Ia. The significantly lower levels of IgG and IgA in Group Ia is a novel finding. The percentages of patients for Paris Classification groups MB0, MB1, MB2 were 88%, 4% and 8%, respectively. There was a significant increase of splenomegaly, lymphadenopathy and autoimmune cytopenia in Group MB0. EuroClass: 45.8% of patients were smB+ and 54.2% were smB-. Splenomegaly and lymphadenopathy were significantly higher in smB- group. TACI: One patient carried heterozygous C104R mutation which was known as disease causing. APRIL: G67R and N96S SNPs were detected in most of the patients and healthy controls. BAFF-R: P21R/H159Y compound heterozygous mutation (n:1) and P21R heterozygous mutations (n:3) were detected. +49 A > G changes in exon 1 of CTLA-4 gene: GG and AG genotypes increase the risk of CVID development 1.32 and 2.18 fold, respectively. 1564 T > C polymorphisms on 3'UTR region in exon 2 of ICOS gene was not found to be significantly different in CVID patients. CVID classifications were not helpful in determining the genetic etiology of CVID.

Immunoglobulin light chain levels can be used to determine disease stage in children with juvenile idiopathic arthritis.

OBJECTIVE: Patients with some inflammatory diseases have been shown to have increased levels of immunoglobulin light chains. In this study, we measured the concentrations of immunoglobulin kappa and lambda light chains in sera of patients with juvenile idiopathic arthritis (JIA) (study group), familial mediterranean fever (FMF) (disease control group) and in healthy children. Our aim was to compare immunoglobulin light chain levels with other well-known markers of inflammation, such as the erythrocyte sedimentation rate (ESR) and the acute phase reactants (APRs), serum amyloid A (SAA) and C-reactive protein (CRP), to find out if immunoglobulin light chain determinations have any discriminating value in the follow-up of these patients. RESULTS: ESR, CRP, SAA, kappa and lambda chain levels and lambda/IgG ratio showed a statistically significant difference between active and remission stages in JIA patients. Kappa correlated very well with SAA and ESR in both stages. On the other hand, lambda correlated with SAA and ESR only in the remission period. There was no significant difference in kappa and lambda chain levels between active and remission stages in FMF patients. In addition, kappa and lambda chain concentrations showed no correlation with other markers of inflammation and immunoglobulin levels neither in entire FMF group nor in different subgroups with respect to clinical status. Immunoglobulin light chains kappa and lambda as well as levels of three markers of inflammation were found to be significantly higher in JIA patients who were in the active stage of disease when compared to data of healthy children CONCLUSION: Ig light chains especially kappa chain concentrations are helpful to determine disease stage in JIA patients but with our current data, they do not exhibit superiority to any of the classical tests for inflammation.

Human soluble tumor necrosis factor receptor I (sTNF-RI) and interleukin-I receptor antagonist (IL-I Ra) in different stages of acute rheumatic fever.

OBJECTIVE: Acute rheumatic fever (ARF) results from an autoimmune response to infection with group A streptococci. Serum concentrations of two anti-inflammatory cytokines, interleukin-I receptor antagonist (IL-IRa) and human soluble tumor necrosis factor receptor I (sTNF-RI) were determined in patients with ARF at the time of admission and 3 months after treatment in order to evaluate changes in cytokine concentrations occurring during different stages of the disease. METHODS: Serum concentrations of two anti-inflammatory cytokines, IL-I Ra and sTNF-RI , were investigated in children with ARF at the time of admission (n=21) and after 3 months following the cessation of treatment (n=15). The sTNF-RI and sIL-IRa were measured quantitatively in serum using enzyme-linked immunosorbent assay (ELISA). RESULTS: Levels of IL-1Ra and sTNF-RI were found to be significantly higher during acute phase and remission period of ARF when compared to age-matched healthy controls (p=0.001 and p=0.0001, respectively). CONCLUSION: Our study demonstrated that two anti-inflammatory cytokines, serum sTNFRI and IL-1Ra, are increased in acute and remission stages of ARF reflecting activation of the cellular immune response. We suggest this increase might probably be generated in an effort to counteract the already increased concentrations of proinflammatory cytokines.