A disorder clinically resembling cystic fibrosis caused by biallelic variants in the AGR2 gene.

PURPOSE: We sought to describe a disorder clinically mimicking cystic fibrosis (CF) and to elucidate its genetic cause. METHODS: Exome/genome sequencing and human phenotype ontology data of nearly 40 000 patients from our Bio/Databank were analysed. RNA sequencing of samples from the nasal mucosa from patients, carriers and controls followed by transcriptome analysis was performed. RESULTS: We identified 13 patients from 9 families with a CF-like phenotype consisting of recurrent lower respiratory infections (13/13), failure to thrive (13/13) and chronic diarrhoea (8/13), with high morbidity and mortality. All patients had biallelic variants in AGR2, (1) two splice-site variants, (2) gene deletion and (3) three missense variants. We confirmed aberrant AGR2 transcripts caused by an intronic variant and complete absence of AGR2 transcripts caused by the large gene deletion, resulting in loss of function (LoF). Furthermore, transcriptome analysis identified significant downregulation of components of the mucociliary machinery (intraciliary transport, cilium organisation), as well as upregulation of immune processes. CONCLUSION: We describe a previously unrecognised autosomal recessive disorder caused by AGR2 variants. AGR2-related disease should be considered as a differential diagnosis in patients presenting a CF-like phenotype. This has implications for the molecular diagnosis and management of these patients. AGR2 LoF is likely the disease mechanism, with consequent impairment of the mucociliary defence machinery. Future studies should aim to establish a better understanding of the disease pathophysiology and to identify potential drug targets.

Th-17 regulatory cytokines IL-21, IL-23, and IL-6 enhance neutrophil production of IL-17 cytokines during asthma.

BACKGROUND: In a subset of severe asthma patients, chronic airway inflammation is associated with infiltration of neutrophils, Th-17 cells and elevated expression of Th-17-derived cytokines (e.g., interleukin [IL]-17, IL-21, IL-22). Peripheral neutrophils from allergic asthmatics are known to express higher IL-17 cytokine levels than those from healthy subjects, but the regulatory mechanisms involved are not well understood. We hypothesize that Th-17 regulatory cytokines could modulate IL-17 expression in neutrophils. METHODS: Peripheral blood neutrophils isolated from asthmatics were stimulated with IL-21, IL-23, and IL-6 cytokines and their ability to produce IL-17A and IL-17F was determined relative to healthy controls. Signal transducer and activator of transcription 3 (STAT3) phosphorylation levels were measured in stimulated neutrophil using flow cytometry. The requirement for STAT3 phosphorylation was determined by blocking its activation using a specific chemical inhibitor. RESULTS: Stimulating asthmatic neutrophils with IL-21, 23, and 6 enhanced the production of IL-17A and IL-17F at significantly higher levels comparatively to healthy controls. Stimulating neutrophils with IL-21, IL-23, and IL-6 cytokines enhanced STAT3 phosphorylation, in all cases. Interestingly, inhibiting STAT3 phosphorylation using a specific chemical inhibitor dramatically blocked the ability of neutrophils to produce IL-17, demonstrating that STAT3 activation is the major factor mediating IL-17 gene expression. CONCLUSIONS: These findings suggest that neutrophil infiltration in lungs of severe asthmatics may represent an important source of pro-inflammatory IL-17A and -F cytokines, a production enhanced by Th-17 regulatory cytokines, and thus providing a feedback mechanism that sustains inflammation. Our results suggest that STAT3 pathway could be a potential target for regulating neutrophilic inflammation during severe asthma.

Th-17 regulatory cytokines inhibit corticosteroid induced airway structural cells apoptosis.

BACKGROUND: Although corticosteroid is a powerful anti-inflammatory drug that is used widely to control asthma, still severe asthmatics can develop steroid resistance. Airway fibroblasts are quite resistant to steroids during Idiopathic pulmonary fibrosis (IPF) and fibrosis in asthmatic lungs is not always controlled. Th-17 regulatory cytokine which are elevated in lung tissues of asthmatics were shown to enhance the survival of various types of cells. STAT factors are central to this anti-apoptotic function. However, it is not yet clear whether these cytokines contribute to steroid hypo-responsiveness in asthma. Therefore, in this study, we investigated the ability of Th-17 regulatory cytokines, specifically IL-21, IL22 and IL23, to protect structural airway cells against dexamethasone-induced apoptosis. METHODS: Primary human fibroblasts, ASM cells, and lung endothelial cells line were treated with IL-21, IL-22, and IL-23 cytokines before incubation with dexamethasone and the level of apoptosis was determined by measuring cellular Annexin-V using Flow cytometry. RESULTS: Our data indicated that treatment with Th-17 regulatory cytokines was effective in inhibiting induced apoptosis for both fibroblasts and endothelial cells but not ASM cells. STAT3 phosphorylation levels were also upregulated in fibroblasts and endothelial upon treatment with these cytokines. Interestingly, inhibiting STAT3 phosphorylation abrogated IL-21, IL-22, and IL-23 anti-apoptotic effect on fibroblasts and endothelial cells. CONCLUSIONS: This data suggest that Th-17 regulatory cytokines may play a critical role in regulating the survival of fibroblasts during asthma, IPF as well as other chronic lung inflammatory diseases leading to enhanced fibrosis. Accordingly, findings of this paper may pave the way for more extensive research on the role of these regulatory cytokines in fibrosis development in various chronic inflammatory diseases.

Rs37972 and rs37973 single-nucleotide polymorphisms in the glucocorticoid-inducible 1 gene are not associated with asthma risk in a Saudi Arabian population.

OBJECTIVE: Rs37972 and rs37973 variants in the glucocorticoid-induced transcript 1 gene have been associated with inhaled glucocorticosteroid responsiveness in asthmatics; however, some discrepancies have been also reported. This study aims to determine whether rs37972 and rs37973 SNPs are associated with asthma risk in Saudi Arabian asthmatics. METHODS: Two-hundred seventy-one diagnosed asthmatics (3-65 years old) and 387 healthy control subjects of equivalent age were recruited. DNA from peripheral blood was purified, and genotyping of rs37972 and rs37973 SNPs was performed by PCR amplification of segments of interest, followed by Sanger sequencing. RESULTS: The global frequencies of the minor (risk) alleles were 28% ("T" allele, rs37972) and 30% ("G" allele, rs37973). Yates-corrected Chi-square (χ(2)) tests revealed significant differences between asthmatic and healthy groups, in allele frequencies for rs37973 SNP only (χ(2) = 3.98, Yates' p value = 0.046). Regarding genotype frequencies, a significant difference between asthmatic and healthy groups was observed for variant rs37972 only (χ(2) = 8.19, Yates' p value = 0.016). To determine a possible association of the minor "T" and "G" alleles with asthma, both the recessive and dominant genetic models were tested. For rs37973, none of the genotypes were significantly associated with asthma. Concerning rs37972, the dominant model (C/T + T/T versus C/C) indicated a significant "protective" association with asthma, in which C/T + T/T individuals had lower odds of being asthmatics than C/C individuals (OR = 0.67; 95% CI = 0.48-0.94; p = 0.019*). CONCLUSIONS: The minor alleles "T" and "G" of rs37972 and rs37973 SNPs, respectively, were not significantly associated with increased asthma risk in asthma patients from Saudi Arabia.

Association of the STAT-6 rs324011 (C2892T) variant but not rs324015 (G2964A), with atopic asthma in a Saudi Arabian population.

BACKGROUND: The signal transducer and activator of transcription 6 (STAT6) transduces signals in response to IL-4 and IL-13 cytokine stimulations, resulting in many cell-specific responses. Some common STAT6 SNPs were associated with asthma predisposition and/or IgE levels, although discrepancies have also been reported. OBJECTIVE: To determine whether STAT6 rs324011 and rs324015 polymorphisms are associated with atopic asthma in Saudi Arabian patients. METHODS: A total of 536 Saudi individuals aged 11-70years old (230 atopic asthmatics, 306 healthy subjects) were recruited. DNA was purified from peripheral blood and genotyping for rs324011 and rs324015 polymorphisms was performed by PCR amplification, followed by cycle sequencing of the purified PCR fragments using BigDye chain terminator and capillary electrophoresis. RESULTS: By the contrast of alleles tests, no significant differences between asthma and healthy groups were detected for both variants (rs324011: X(2)=0.25, Pearson's P-value=0.617; rs324015: X(2)=0.068, Pearson's P=0.814).When testing for genotypes, rs324011 homozygous T/T genotype was significantly associated with asthma, when the Recessive model is considered (T/T vs. C/C+C/T) (adjusted, OR=2.49, 95% CI=1.18-5.25, Pearson's P=0.014(∗), Yates' P=0.022(∗)). In contrast, rs324015 variant was not significantly associated with asthma. CONCLUSIONS: Rs324011 homozygous T/T genotype was significantly associated with asthma risk whereas rs324015 genotypes were not in the Saudi population.