Rare SH2B3 coding variants in lupus patients impair B cell tolerance and predispose to autoimmunity.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease with a clear genetic component. While most SLE patients carry rare gene variants in lupus risk genes, little is known about their contribution to disease pathogenesis. Amongst them, SH2B3-a negative regulator of cytokine and growth factor receptor signaling-harbors rare coding variants in over 5% of SLE patients. Here, we show that unlike the variant found exclusively in healthy controls, SH2B3 rare variants found in lupus patients are predominantly hypomorphic alleles, failing to suppress IFNGR signaling via JAK2-STAT1. The generation of two mouse lines carrying patients' variants revealed that SH2B3 is important in limiting the number of immature and transitional B cells. Furthermore, hypomorphic SH2B3 was shown to impair the negative selection of immature/transitional self-reactive B cells and accelerate autoimmunity in sensitized mice, at least in part due to increased IL-4R signaling and BAFF-R expression. This work identifies a previously unappreciated role for SH2B3 in human B cell tolerance and lupus risk.

A Novel Targeted Amplicon Next-Generation Sequencing Gene Panel for the Diagnosis of Common Variable Immunodeficiency Has a High Diagnostic Yield: Results from the Perth CVID Cohort Study.

With the advent of next-generation sequencing (NGS), monogenic forms of common variable immunodeficiency (CVID) have been increasingly described. Our study aimed to identify disease-causing variants in a Western Australian CVID cohort using a novel targeted NGS panel. Targeted amplicon NGS was performed on 22 unrelated subjects who met the formal European Society for Immunodeficiencies-Pan-American Group for Immunodeficiency diagnostic criteria for CVID and had at least one of the following additional criteria: disease onset at age <18 years, autoimmunity, low memory B lymphocytes, family history, and/or history of lymphoproliferation. Candidate variants were assessed by in silico predictions of deleteriousness, comparison to the literature, and classified according to the American College of Medical Genetics and Genomics-Association for Molecular Pathology criteria. All detected genetic variants were verified independently by an external laboratory, and additional functional studies were performed if required. Pathogenic or likely pathogenic variants were detected in 6 of 22 (27%) patients. Monoallelic variants of uncertain significance were also identified in a further 4 of 22 patients (18%). Pathogenic variants, likely pathogenic variants, or variants of uncertain significance were found in TNFRSF13B, TNFRSF13C, ICOS, AICDA, IL21R, NFKB2, and CD40LG, including novel variants and variants with unexpected inheritance pattern. Targeted amplicon NGS is an effective tool to identify monogenic disease-causing variants in CVID, and is comparable or superior to other NGS methods. Moreover, targeted amplicon NGS identified patients who may benefit from targeted therapeutic strategies and had important implications for family members.

Rewiring of gene networks underlying mite allergen-induced CD4 + Th-cell responses during immunotherapy.

BACKGROUND: Multiple regulatory mechanisms have been identified employing conventional hypothesis-driven approaches as contributing to allergen-specific immunotherapy outcomes, but understanding of how these integrate to maintain immunological homeostasis is incomplete. OBJECTIVE: To explore the potential for unbiased systems-level gene co-expression network analysis to advance understanding of immunotherapy mechanisms. METHODS: We profiled genome-wide allergen-induced Th-cell responses prospectively during 24 months subcutaneous immunotherapy (SCIT) in 25 rhinitis, documenting changes in immunoinflammatory pathways and associated co-expression networks and their relationships to symptom scores out to 36 months. RESULTS: Prior to immunotherapy, mite-induced Th-cell response networks involved multiple discrete co-expression modules including those related to Th2-, type1 IFN-, inflammation- and FOXP3/IL2-associated signalling. A signature comprising 109 genes correlated with symptom scores, and these mapped to cytokine signalling/T-cell activation-associated pathways, with upstream drivers including hallmark Th1/Th2- and inflammation-associated genes. Reanalysis after 3.5 months SCIT updosing detected minimal changes to pathway/upstream regulator profiles despite 32.5% symptom reduction; however, network analysis revealed underlying merging of FOXP3/IL2-with inflammation-and Th2-associated modules. By 12 months SCIT, symptoms had reduced by 41% without further significant changes to pathway/upstream regulator or network profiles. Continuing SCIT to 24 months stabilized symptoms at 47% of baseline, accompanied by upregulation of the type1 IFN-associated network module and its merging into the Th2/FOXP3/IL2/inflammation module. CONCLUSIONS: Subcutaneous immunotherapy stimulates progressive integration of mite-induced Th cell-associated Th2-, FOXP3/IL2-, inflammation- and finally type1 IFN-signalling subnetworks, forming a single highly integrated co-expression network module, maximizing potential for stable homeostatic control of allergen-induced Th2 responses via cross-regulation. Th2-antagonistic type1 IFN signalling may play a key role in stabilizing clinical effects of SCIT.

Functional rare and low frequency variants in BLK and BANK1 contribute to human lupus.

Systemic lupus erythematosus (SLE) is the prototypic systemic autoimmune disease. It is thought that many common variant gene loci of weak effect act additively to predispose to common autoimmune diseases, while the contribution of rare variants remains unclear. Here we describe that rare coding variants in lupus-risk genes are present in most SLE patients and healthy controls. We demonstrate the functional consequences of rare and low frequency missense variants in the interacting proteins BLK and BANK1, which are present alone, or in combination, in a substantial proportion of lupus patients. The rare variants found in patients, but not those found exclusively in controls, impair suppression of IRF5 and type-I IFN in human B cell lines and increase pathogenic lymphocytes in lupus-prone mice. Thus, rare gene variants are common in SLE and likely contribute to genetic risk.

Clinical and molecular characterization of non-syndromic retinal dystrophy due to c.175G>A mutation in ceroid lipofuscinosis neuronal 3 (CLN3).

PURPOSE: Mutation of the CLN3 gene, associated with juvenile neuronal ceroid lipofuscinosis, has recently been associated with late-onset, non-syndromic retinal dystrophy. Herein we describe the multimodal imaging, immunological and systemic features of an adult with compound heterozygous CLN3 mutations. METHODS: A 50-year-old female with non-syndromic retinal dystrophy from the age of 36 years underwent multimodal retinal imaging, electroretinography, neuroimaging, immunological studies and genetic testing. CLN3 transcripts were amplified from patient leukocytes by reverse transcriptase polymerase chain reaction and characterized by Sanger sequencing. RESULTS: Visual acuity declined to 6/12 and 6/76 due to asymmetrical central scotoma. ERG responses became electronegative and patient's serum contained anti-retinal antibodies. Final visual acuity stabilized at 6/60 bilaterally 3 years after peri-ocular steroid and rituximab infusion. Genetic testing revealed compound heterozygous CLN3 mutations: the 1.02 kb deletion and a novel missense mutation (c.175G>A). In silico, analyses predicted the c.175G>A mutation disrupted an exonic splice enhancer site in exon 3. In patient leukocytes, CLN3 expression was reduced and novel CLN3 transcripts lacking exon 3 were detected. CONCLUSIONS: Our case study shows that (1) non-syndromic CLN3 disease leads to rod and delayed primary cone degeneration resulting in constricting peripheral field and enlarging central scotoma and, (2) the c.175G>A CLN3 mutation, altered splicing of the CLN3 gene. Overall, we provide comprehensive clinical characterization of a patient with non-syndromic CLN3 disease.

Dysregulation of innate immunity in ulcerative colitis patients who fail anti-tumor necrosis factor therapy.

AIM: To study the innate immune function in ulcerative colitis (UC) patients who fail to respond to anti-tumor necrosis factor (TNF) therapy. METHODS: Effects of anti-TNF therapy, inflammation and medications on innate immune function were assessed by measuring peripheral blood mononuclear cell (PBMC) cytokine expression from 18 inflammatory bowel disease patients pre- and 3 mo post-anti-TNF therapy. Toll-like receptor (TLR) expression and cytokine production post TLR stimulation was assessed in UC "responders" (n = 12) and "non-responders" (n = 12) and compared to healthy controls (n = 12). Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels were measured in blood to assess disease severity/activity and inflammation. Pro-inflammatory (TNF, IL-1ß, IL-6), immuno-regulatory (IL-10), Th1 (IL-12, IFNγ) and Th2 (IL-9, IL-13, IL-17A) cytokine expression was measured with enzyme-linked immunosorbent assay while TLR cellular composition and intracellular signalling was assessed with FACS. RESULTS: Prior to anti-TNF therapy, responders and non-responders had similar level of disease severity and activity. PBMC's ability to respond to TLR stimulation was not affected by TNF therapy, patient's severity of the disease and inflammation or their medication use. At baseline, non-responders had elevated innate but not adaptive immune responses compared to responders (P < 0.05). Following TLR stimulation, non-responders had consistently reduced innate cytokine responses to all TLRs compared to healthy controls (P < 0.01) and diminished TNF (P < 0.001) and IL-1ß (P < 0.01) production compared to responders. This innate immune dysfunction was associated with reduced number of circulating plasmacytoid dendritic cells (pDCs) (P < 0.01) but increased number of CD4+ regulatory T cells (Tregs) (P = 0.03) as well as intracellular accumulation of IRAK4 in non-responders following TLR-2, -4 and -7 activation (P < 0.001). CONCLUSION: Reduced innate immunity in non-responders may explain reduced efficacy to anti-TNF therapy. These serological markers may prove useful in predicting the outcome of costly anti-TNF therapy.

Establishment of gene copy number-specific normal ranges for serum C4 and its utility for interpretation in patients with chronically low serum C4 concentrations.

OBJECTIVE: To establish gene copy number (GCN)-specific normal ranges for serum C4 genes and to determine their utility with respect to the interpretation of chronically low serum C4 concentrations in patients with clinically quiescent systemic lupus erythematosus (SLE). METHODS: C4 serum concentrations were estimated by automated turbidimetry, and C4 GCNs were determined using the TaqMan real-time polymerase chain reaction (PCR) analysis in 184 unselected individuals and in 10 patients with type 1 diabetes mellitus (DM) who were selected for the presence of only 2 copies of the C4 gene. C4 GCNs were also determined in 11 patients with clinically quiescent SLE who had chronically low serum C4 concentrations. RESULTS: A total of 33% of the variation in serum C4 concentrations could be accounted for by both C4A and C4B GCNs (R(2) = 0.30, P ≤ 0.0001). There was a median of 2 gene copies at the C4A locus (53.8%) and 2 at the C4B locus (58.7%). The median total number of C4 genes was 4 (55.4%). C4 GCN-specific normal ranges were established. A chronically low serum C4 concentration was explained by a low C4 GCN in 3 of 11 patients tested. CONCLUSION: This study establishes the feasibility of establishing C4 GCN-specific normal ranges using the TaqMan real-time PCR assay. Chronically low serum C4 concentrations in SLE patients are sometimes explained by low C4 GCNs.

Pentoxifylline improves haemoglobin and interleukin-6 levels in chronic kidney disease.

AIM: To assess whether pentoxifylline improves anaemia of chronic kidney disease (CKD) via suppression of interleukin-6 (IL-6) and improved iron mobilization. BACKGROUND: CKD patients may have elevated IL-6 and tumour necrosis factor alpha levels. These cytokines can increase hepcidin production, which in turn reduces iron release from macrophages resulting in reduced availability of iron for erythropoiesis. In experimental models, pentoxifylline was shown to reduce IL-6 expression. METHODS: We studied 14 patients with stages 4-5 CKD (glomerular filtration rate <30mL/min per 1.73 m(2)) due to non-inflammatory renal diseases. None of the patients had received immunosuppressive or erythropoietin-stimulating agents or parenteral iron. Patients had weekly blood tests for iron studies and cytokines during a control run-in period of 3 weeks and during 4 weeks of pentoxifylline treatment. RESULTS: Ten patients (eGFR 23 + or - 6 mL/min) completed the study. At the end of the run-in period average haemoglobin was 111 + or - 5 g/L, ferritin 92 + or - 26 microg/L, transferrin saturation 15 + or - 3% and circulating IL-6 10.6 + or - 3.8 pg/mL. Tumour necrosis factor alpha values were below threshold for detection. Treatment with pentoxifylline reduced circulating IL-6 (6.6 + or - 1.6 pg/mL, P < 0.01), increased transferrin saturation (20 + or - 5%, P < 0.003) and decreased serum ferritin (81 + or - 25 microg/L, P = NS). Haemoglobin increased after the second week of pentoxifylline, reaching 123 + or - 6 g/L by week 4 (P < 0.001). CONCLUSIONS: Pentoxifylline reduces circulating IL-6 and improves haemoglobin in non-inflammatory moderate to severe CKD. These changes are associated with changes in circulating transferrin saturation and ferritin, suggesting improved iron release. It is hypothesized that pentoxifylline improves iron disposition possibly through modulation of hepcidin.

Screening for coeliac disease using anti-tissue transglutaminase antibody assays, and prevalence of the disease in an Australian community.

OBJECTIVES: To determine (i) the prevalence of positive results of anti-tissue transglutaminase (anti-tTG) antibody assays and coeliac disease (CD) in a rural Australian community; and (ii) whether confirmatory testing of a positive assay result with an alternative anti-tTG assay improved the positive predictive value of the test in population screening for CD. DESIGN: Retrospective analysis in December 2004 of stored serum samples taken in 1994-1995 from 3011 subjects in the Busselton Health Study follow-up. Assays for IgA and IgG anti-tTG antibodies were performed, and positive or equivocal samples were retested with a different commercial anti-tTG assay. Available subjects with one or more positive assay results were interviewed, had serum collected for repeat anti-tTG assays and for HLA-DQ2 and HLA-DQ8 haplotyping and, if appropriate, gastroscopy and duodenal biopsy were performed. In unavailable subjects, HLA-DQ2 and -DQ8 haplotyping was performed on stored sera. Total serum IgA levels were assessed in subjects with initially negative assay results. MAIN OUTCOME MEASURE: Prevalence of anti-tTG positivity and biopsy-proven CD. RESULTS: In 47 of 3011 serum samples (1.56%), at least one anti-tTG assay gave positive results: 31 of the subjects who provided these sera were available for clinical review, and 21 were able to have a gastroscopy. Seventeen subjects (0.56%) were diagnosed with definite CD (14 were confirmed at gastroscopy, and three unavailable subjects had three positive results of anti-tTG assays and an HLA haplotype consistent with CD); in a further 12 unavailable subjects, CD status was considered equivocal, with one or more positive anti-tTG assay results and an HLA haplotype consistent with CD. If these subjects were regarded as having CD, the prevalence of CD would be 0.96%. The positive predictive value when all three anti-tTG assays gave positive results was 94%, but fell to 45.2% with only one positive result. CONCLUSIONS: The prevalence of anti-tTG antibodies in this population is 1.56%; the prevalence of CD is at least 0.56%. The utility of a single, positive result of an anti-tTG assay in screening for CD in the community is poor, and repeat and/or collateral assessment with different assays may decrease the need for gastroscopy and distal duodenal biopsy.

Skin prick testing predicts peanut challenge outcome in previously allergic or sensitized children with low serum peanut-specific IgE antibody concentration.

Peanut allergy is transient in some children but it is not clear whether quantitating peanut-specific IgE by Skin Prick Test (SPT) adds additional information to fluorescent-enzyme immunoassay (FEIA) in discriminating between allergic and tolerant children. To investigate whether SPT with a commercial extract or fresh foods adds additional predictive information for peanut challenge in children with a low FEIA (<10 k UA/L) who were previously sensitized, or allergic to peanuts. Children from a hospital-based allergy service who were previously sensitized or allergic to peanuts were invited to undergo a peanut challenge unless they had a serum peanut-specific IgE>10 k UA/L, a previous severe reaction, or a recent reaction to peanuts (within two years). SPT with a commercial extract, raw and roasted saline soaked peanuts was performed immediately prior to open challenge in hospital with increasing quantity of peanuts until total of 26.7 g of peanut was consumed. A positive challenge consisted of an objective IgE mediated reaction occurring during the observation period. 54 children (median age of 6.3 years) were admitted for a challenge. Nineteen challenges were positive, 27 negative, five were indeterminate and three did not proceed after SPT. Commercial and fresh food extracts provided similar diagnostic information. A wheal diameter of >or=7 mm of the commercial extract predicted an allergic outcome with specificity 97%, positive predictive value 93% and sensitivity 83%. There was a tendency for an increase in SPT wheal since initial diagnosis in children who remained allergic to peanuts while it decreased in those with a negative challenge. The outcome of a peanut challenge in peanut sensitized or previously allergic children with a low FEIA can be predicted by SPT. In this cohort, not challenging children with a SPT wheal of >or=7 mm would have avoided 15 of 18 positive challenges and denied a challenge to one out of 27 tolerant children.

Cytomegalovirus infection of a cutaneous ulcer in a patient with ANCA-positive vasculitis.

This case describes a patient in whom cytomegalovirus (CMV) infected a preexisting ulcer. The patient was immune-suppressed because of treatment for Wegener's granulomatosis. Specific antiviral therapy was delayed because of uncertainty as to the role of CMV, but the infection cleared and the ulcer improved promptly on institution of valganciclovir.

Allergy, atopy, and cancer: a prospective study of the 1981 Busselton cohort.

The associations among certain allergic disorders, atopy upon skin-prick testing, and specific cancers were evaluated in a prospective study. Information regarding history of asthma and hay fever was collected by questionnaire from 3,308 cancer-free participants in the 1981 Busselton Health Survey. A subset of 1,005 participants also underwent skin-prick testing. The cohort was followed for a new diagnosis of cancer or death until the end of 1999. Cox proportional hazards regression analysis was used to estimate adjusted hazard ratios (relative risks) for breast, prostate, colorectal, lung, and hematologic cancers and melanoma. Having a skin reaction to house dust mites nearly tripled the risk of prostate cancer (relative risk = 2.90, 95% confidence interval: 1.26, 6.68). History of asthma and hay fever were associated with a trend toward a reduced risk of colorectal cancer and increased risk of leukemia, but these results were not statistically significant. Hay fever was associated with melanoma risk in men but not in women. No association was found between breast and lung cancers and allergic disorders or atopy.