Inborn Errors of RNA Lariat Metabolism in Humans with Brainstem Viral Infection.

Viruses that are typically benign sometimes invade the brainstem in otherwise healthy children. We report bi-allelic DBR1 mutations in unrelated patients from different ethnicities, each of whom had brainstem infection due to herpes simplex virus 1 (HSV1), influenza virus, or norovirus. DBR1 encodes the only known RNA lariat debranching enzyme. We show that DBR1 expression is ubiquitous, but strongest in the spinal cord and brainstem. We also show that all DBR1 mutant alleles are severely hypomorphic, in terms of expression and function. The fibroblasts of DBR1-mutated patients contain higher RNA lariat levels than control cells, this difference becoming even more marked during HSV1 infection. Finally, we show that the patients' fibroblasts are highly susceptible to HSV1. RNA lariat accumulation and viral susceptibility are rescued by wild-type DBR1. Autosomal recessive, partial DBR1 deficiency underlies viral infection of the brainstem in humans through the disruption of tissue-specific and cell-intrinsic immunity to viruses.

IL-35 promotes IL-35+IL-10+ Bregs and Conventional LAG3+ Tregs in the lung tissue of OVA-Induced Asthmatic Mice.

AIMS: This study aimed to investigate the effect of interleukin-35 (IL-35) on inflamed lung tissue in a murine model of asthma. IL-35 was examined for its potential to induce regulatory lymphocytes during ovalbumin (OVA)-induced acute lung injury. METHODS: Female BALB/c mice sensitized with OVA and were treated with recombinant IL-35 (rIL-35) via intranasal or intraperitoneal routes and were administered 4 h before OVA challenge. The effects of rIL-35 treatment on the lung and blood levels of regulatory B cells (Bregs) and regulatory T cells (Tregs), as well as their production of immunosuppressive cytokines, were determined using flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: Treatment of OVA-sensitized asthmatic mice with rIL-35, whether administered intranasally or intraperitoneally, resulted in reduced lung inflammation and injury. This reduction was accompanied by an increase in the frequency of IL-35 producing Bregs, IL-35 and IL-10 producing Bregs, and conventional LAG3+ Tregs in the lung tissues and blood. This increase was more pronounced with intranasal rIL-35. Furthermore, there was a positive correlation between the levels of these regulatory cells and lung gene expression of IL-35 and IL-10, and an inverse correlation with both lung gene expression and plasma level of IL-17. CONCLUSIONS: The results of this study suggest that IL-35, through its ability to increase Bregs and Tregs, is effective in reversing lung inflammation in the context of asthma. Since the increase was more pronounced with intranasal administration, this highlights the therapeutic potential of its local intrapulmonary application in managing asthma-related inflammation.

Inherited deficiency of stress granule ZNFX1 in patients with monocytosis and mycobacterial disease.

Human inborn errors of IFN-γ underlie mycobacterial disease, due to insufficient IFN-γ production by lymphoid cells, impaired myeloid cell responses to this cytokine, or both. We report four patients from two unrelated kindreds with intermittent monocytosis and mycobacterial disease, including bacillus Calmette-Guérin-osis and disseminated tuberculosis, and without any known inborn error of IFN-γ. The patients are homozygous for ZNFX1 variants (p.S959* and p.E1606Rfs*10) predicted to be loss of function (pLOF). There are no subjects homozygous for pLOF variants in public databases. ZNFX1 is a conserved and broadly expressed helicase, but its biology remains largely unknown. It is thought to act as a viral double-stranded RNA sensor in mice, but these patients do not suffer from severe viral illnesses. We analyze its subcellular localization upon overexpression in A549 and HeLa cell lines and upon stimulation of THP1 and fibroblastic cell lines. We find that this cytoplasmic protein can be recruited to or even induce stress granules. The endogenous ZNFX1 protein in cell lines of the patient homozygous for the p.E1606Rfs*10 variant is truncated, whereas ZNFX1 expression is abolished in cell lines from the patients with the p.S959* variant. Lymphocyte subsets are present at normal frequencies in these patients and produce IFN-γ normally. The hematopoietic and nonhematopoietic cells of the patients tested respond normally to IFN-γ. Our results indicate that human ZNFX1 is associated with stress granules and essential for both monocyte homeostasis and protective immunity to mycobacteria.

Fulminant Viral Hepatitis in Two Siblings with Inherited IL-10RB Deficiency.

Fulminant viral hepatitis (FVH) caused by hepatitis A virus (HAV) is a life-threatening disease that typically strikes otherwise healthy individuals. The only known genetic etiology of FVH is inherited IL-18BP deficiency, which unleashes IL-18-dependent lymphocyte cytotoxicity and IFN-γ production. We studied two siblings who died from a combination of early-onset inflammatory bowel disease (EOIBD) and FVH due to HAV. The sibling tested was homozygous for the W100G variant of IL10RB previously described in an unrelated patient with EOIBD. We show here that the out-of-frame IL10RB variants seen in other EOIBD patients disrupt cellular responses to IL-10, IL-22, IL-26, and IFN-λs in overexpression conditions and in homozygous cells. By contrast, the impact of in-frame disease-causing variants varies between cases. When overexpressed, the W100G variant impairs cellular responses to IL-10, but not to IL-22, IL-26, or IFN-λ1, whereas cells homozygous for W100G do not respond to IL-10, IL-22, IL-26, or IFN-λ1. As IL-10 is a potent antagonist of IFN-γ in phagocytes, these findings suggest that the molecular basis of FVH in patients with IL-18BP or IL-10RB deficiency may involve excessive IFN-γ activity during HAV infections of the liver. Inherited IL-10RB deficiency, and possibly inherited IL-10 and IL-10RA deficiencies, confer a predisposition to FVH, and patients with these deficiencies should be vaccinated against HAV and other liver-tropic viruses.

JAK Inhibitor Therapy in a Child with Inherited USP18 Deficiency.

Deficiency of ubiquitin-specific peptidase 18 (USP18) is a severe type I interferonopathy. USP18 down-regulates type I interferon signaling by blocking the access of Janus-associated kinase 1 (JAK1) to the type I interferon receptor. The absence of USP18 results in unmitigated interferon-mediated inflammation and is lethal during the perinatal period. We describe a neonate who presented with hydrocephalus, necrotizing cellulitis, systemic inflammation, and respiratory failure. Exome sequencing identified a homozygous mutation at an essential splice site on USP18. The encoded protein was expressed but devoid of negative regulatory ability. Treatment with ruxolitinib was followed by a prompt and sustained recovery. (Funded by King Saud University and others.).

Germline CYBB mutations that selectively affect macrophages in kindreds with X-linked predisposition to tuberculous mycobacterial disease.

Germline mutations in CYBB, the human gene encoding the gp91(phox) subunit of the phagocyte NADPH oxidase, impair the respiratory burst of all types of phagocytes and result in X-linked chronic granulomatous disease (CGD). We report here two kindreds in which otherwise healthy male adults developed X-linked recessive Mendelian susceptibility to mycobacterial disease (MSMD) syndromes. These patients had previously unknown mutations in CYBB that resulted in an impaired respiratory burst in monocyte-derived macrophages but not in monocytes or granulocytes. The macrophage-specific functional consequences of the germline mutation resulted from cell-specific impairment in the assembly of the NADPH oxidase. This 'experiment of nature' indicates that CYBB is associated with MSMD and demonstrates that the respiratory burst in human macrophages is a crucial mechanism for protective immunity to tuberculous mycobacteria.

Genetic, Immunological, and Clinical Features of 32 Patients with Autosomal Recessive STAT1 Deficiency.

Autosomal recessive (AR) STAT1 deficiency is a severe inborn error of immunity disrupting cellular responses to type I, II, and III IFNs, and IL-27, and conferring a predisposition to both viral and mycobacterial infections. We report the genetic, immunological, and clinical features of an international cohort of 32 patients from 20 kindreds: 24 patients with complete deficiency, and 8 patients with partial deficiency. Twenty-four patients suffered from mycobacterial disease (bacillus Calmette-Guérin = 13, environmental mycobacteria = 10, or both in 1 patient). Fifty-four severe viral episodes occurred in sixteen patients, mainly caused by Herpesviridae viruses. Attenuated live measles, mumps, and rubella and/or varicella zoster virus vaccines triggered severe reactions in the five patients with complete deficiency who were vaccinated. Seven patients developed features of hemophagocytic syndrome. Twenty-one patients died, and death was almost twice as likely in patients with complete STAT1 deficiency than in those with partial STAT1 deficiency. All but one of the eight survivors with AR complete deficiency underwent hematopoietic stem cell transplantation. Overall survival after hematopoietic stem cell transplantation was 64%. A diagnosis of AR STAT1 deficiency should be considered in children with mycobacterial and/or viral infectious diseases. It is important to distinguish between complete and partial forms of AR STAT1 deficiency, as their clinical outcome and management differ significantly.

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.

Enhanced Infiltration of Central Memory T Cells to the Lung Tissue during Allergic Lung Inflammation.

Memory T cells play a central role in regulating inflammatory responses during asthma. However, tissue distribution of effector memory (TEM) and central memory (TCM) T-cell subtypes, their differentiation, and their contribution to the persistence of lung tissue inflammation during asthma are not well understood. Interestingly, an increase in survival and persistence of memory T cells was reported in asthmatic lungs, which may suggest a shift toward the more persistent TCM phenotype. In this report, we investigated the differential distribution of memory T-cell subtypes during allergic lung inflammation and the mechanism regulating that. Using an OVA-sensitized asthma mouse model, we observed a significant increase in the frequency of TCM cells in inflamed lungs compared to healthy controls. Interestingly, adoptive transfer techniques confirmed substantial infiltration of TCM cells to lung tissues during allergic airway inflammation. Expression levels of TCM homing receptors, CD34 and GlyCAM-1, were also significantly upregulated in the lung tissues of OVA-sensitized mice, which may facilitate the increased TCM infiltration into inflamed lungs. Moreover, a substantial increase in the relative expression of TCM profile-associated genes (EOMES, BCL-6, ID3, TCF-7, BCL-2, BIM, and BMI-1) was noted for TEM cells during lung inflammation, suggesting a shift for TEM into the TCM state. To our knowledge, this is the first study to report an increased infiltration of TCM cells into inflamed lung tissues and to suggest differentiation of TEM to TCM cells in these tissues. Therapeutic interference at TCM infiltration or differentiations could constitute an alternative treatment approach for lung inflammation.

Clinical and Immunological Characterization of Combined Immunodeficiency Due to TFRC Mutation in Eight Patients.

PURPOSE: Combined immunodeficiency (CID), due to mutations in TFRC gene that encodes the transferrin receptors (TfR1), is a rare monogenic disorder. In this study, we further characterize the clinical and immunological phenotypes in a cohort of eight patients. METHODS: A retrospective review of clinical and immunological features of patients diagnosed with a TFRC gene mutation between 2015 and 2019 in three tertiary centers. RESULTS: Eight patients from six unrelated families were enrolled. The patients had a median age of 7 years (4-32 years). All patients presented with recurrent sinopulmonary infections, chronic diarrhea, and failure to thrive in early life. Less common features were skin abscesses, conjunctivitis, global developmental delay, optic nerve atrophy, vitiligo, multinodular goiter, and hemophagocytic lymphohistiocytosis-like symptoms. All patients had intermittent neutropenia and 87% of the patients had recurrent thrombocytopenia. Anemia was found in 62%. All patients had hypogammaglobinemia and one had a persistent high IgM level. All patients had impaired function of T cells. The same homozygous missense mutation c.58T>C:p.Y20H, in the TFRC gene, was detected in all patients. Stem cell transplantation from matched donors was successful in two patients. Five patients did not receive stem cell transplantation, and they are on prophylactic treatment. One patient died due to severe sepsis and neurological complications. CONCLUSION: This report provides a large cohort with a long follow up of patients with this disease. Our cohort showed variable disease severity.

Graft Versus Host Disease Following HLA-Matched Sibling Donor Compared with Matched Related Donor for Hematopoietic Stem Cell Transplantation for the Treatment of Severe Combined Immunodeficiency Disease.

BACKGROUND: One of the limiting factors for successful hematopoietic stem cell transplantation (HSCT) is graft versus host disease (GVHD). The EBMT/ESID guidelines for HSCT in severe combined immunodeficiency (SCID) recommend no GVHD prophylaxis for a matched sibling donor (MSD). OBJECTIVE: To determine the risk of GVHD in MSD HSCT for SCID patients compared to matched related donor (MRD). METHODS: This retrospective cohort study compares MSD with MRD and the outcome of GVHD in all SCID patients who underwent HSCT between 1993 and 2013. All statistical analyses were done using IBM SPSS statistics software. RESULTS: One hundred forty-five SCID patients underwent 152 HSCTs while 82 (54%) received GVHD prophylaxis. GVHD occurred in 48 patients (31.5%); 20/48 (42%) had GVHD prophylaxis compared to 28/48 (58%) that did not, P = 0.022. Acute GVHD occurred at a higher trend in MSD, 37/120 (30.8%), compared to MRD, 6/32 (18.8%), P = 0.17. We also analyzed the outcome according to the period of HSCT. The first period was 1993 to 2003, 48 HSCTs, 43 MSD, 5 MRD; all patients had GVHD prophylaxis, and there was no difference in GVHD. The second period was 2004 to 2013: of 104 HSCTs, 77 had MSD and 27 had MRD; GVHD prophylaxis was used in 22.1% of MSD and 63% of MRD, P = 0.000. GVHD was significantly higher in the MSD (40.2%) compared to MRD (18.5%) patients, P = 0.041. CONCLUSION: GVHD prophylaxis in MSD transplant should be considered in SCID patients.

Human TRAF3 adaptor molecule deficiency leads to impaired Toll-like receptor 3 response and susceptibility to herpes simplex encephalitis.

Tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3) functions downstream of multiple TNF receptors and receptors that induce interferon-α (IFN-α), IFN-ß, and IFN-λ production, including Toll-like receptor 3 (TLR3), which is deficient in some patients with herpes simplex virus-1 encephalitis (HSE). Mice lacking TRAF3 die in the neonatal period, preventing direct investigation of the role of TRAF3 in immune responses and host defenses in vivo. Here, we report autosomal dominant, human TRAF3 deficiency in a young adult with a history of HSE in childhood. The TRAF3 mutant allele is loss-of-expression, loss-of-function, dominant-negative and associated with impaired, but not abolished, TRAF3-dependent responses upon stimulation of both TNF receptors and receptors that induce IFN production. TRAF3 deficiency is associated with a clinical phenotype limited to HSE resulting from the impairment of TLR3-dependent induction of IFN. Thus, TLR3-mediated immunity against primary infection by HSV-1 in the central nervous system is critically dependent on TRAF3.

Genetic, immunological, and clinical features of patients with bacterial and fungal infections due to inherited IL-17RA deficiency.

Chronic mucocutaneous candidiasis (CMC) is defined as recurrent or persistent infection of the skin, nails, and/or mucosae with commensal Candida species. The first genetic etiology of isolated CMC-autosomal recessive (AR) IL-17 receptor A (IL-17RA) deficiency-was reported in 2011, in a single patient. We report here 21 patients with complete AR IL-17RA deficiency, including this first patient. Each patient is homozygous for 1 of 12 different IL-17RA alleles, 8 of which create a premature stop codon upstream from the transmembrane domain and have been predicted and/or shown to prevent expression of the receptor on the surface of circulating leukocytes and dermal fibroblasts. Three other mutant alleles create a premature stop codon downstream from the transmembrane domain, one of which encodes a surface-expressed receptor. Finally, the only known missense allele (p.D387N) also encodes a surface-expressed receptor. All of the alleles tested abolish cellular responses to IL-17A and -17F homodimers and heterodimers in fibroblasts and to IL-17E/IL-25 in leukocytes. The patients are currently aged from 2 to 35 y and originate from 12 unrelated kindreds. All had their first CMC episode by 6 mo of age. Fourteen patients presented various forms of staphylococcal skin disease. Eight were also prone to various bacterial infections of the respiratory tract. Human IL-17RA is, thus, essential for mucocutaneous immunity to Candida and Staphylococcus, but otherwise largely redundant. A diagnosis of AR IL-17RA deficiency should be considered in children or adults with CMC, cutaneous staphylococcal disease, or both, even if IL-17RA is detected on the cell surface.

Mendelian Susceptibility to Mycobacterial Disease Caused by a Novel Founder IL12B Mutation in Saudi Arabia.

PURPOSE: Mendelian susceptibility to mycobacterial disease (MSMD) is a rare primary immunodeficiency predisposing congenitally affected individuals to diseases caused by weakly virulent mycobacteria, such as Bacillus Calmette-Guérin (BCG) vaccine strains and environmental mycobacteria. IL-12p40 deficiency is a genetic etiology of MSMD resulting in impaired IL-12- and IL-23-dependent IFN-γ immunity. Most of the reported patients with IL-12p40 deficiency originate from Saudi Arabia (30 of 52) and carry the recurrent IL12B mutation c.315insA (27 of 30). METHODS: Whole-exome sequencing was performed on three patients from two unrelated kindreds from Saudi Arabia with disseminated disease caused by a BCG vaccine substrain. RESULTS: Genetic analysis revealed a homozygous mutation, p.W60X, in exon 3 of the IL12B gene, resulting in complete IL12p40 deficiency. This mutation is recurrent due to a new founder effect. CONCLUSIONS: This report provides evidence for a second founder effect for recurrent mutations of IL12B in Saudi Arabia.

Association of IL-13 rs20541 and rs1295686 variants with symptomatic asthma in a Saudi Arabian population.

OBJECTIVE: Interleukin 13 (IL-13) plays a critical pro-inflammatory role in asthma. Several single nucleotide polymorphisms (SNPs) are associated with asthma susceptibility in specific populations; however, further replicative studies in other ethnic groups are mandatory. METHODS: The association between IL-13 SNPs rs762534, rs20541, rs1295686, and rs1800925 (risk alleles A, A, T, and A, respectively) and asthma predisposition in a Saudi Arabian cohort was examined via a case-control cross-sectional study. RESULTS: The frequencies of alleles between asthmatics and control populations were significantly different for rs20541 and rs1295686 SNPs (p < 0.001), whereas the frequencies of genotypes between asthmatics and controls were significantly different only for rs20541. The association of the risk (minor) alleles with asthma was examined using the dominant genetic model. Individuals with at least one copy of the risk alleles A (for rs20541) and T (for rs1295686) had significantly greater odds of being asthmatic (OR = 2.13, 95% CI = 1.39-3.26, p < 0.0001; OR = 1.69, 95% CI = 1.12-2.54, p = 0.008) relative to their most common homozygous genotypes. On the other hand, the minor A alleles for rs762534 and rs1800925 were not significantly associated with asthma risk. Regarding haplotype association analysis, individuals with at least one copy of the minor "risk" allele for both rs20541 and rs1295686 (CATG and CATA, respectively) had greater odds of being asthmatic relative to CGCG haplotype; however, this trend was not statistically significant (p > 0.3). CONCLUSIONS: IL-13 minor T and A alleles for rs1295686 and rs20541, respectively, were associated with significantly higher risk of asthma in the Saudi Arabian population.

Impaired intrinsic immunity to HSV-1 in human iPSC-derived TLR3-deficient CNS cells.

In the course of primary infection with herpes simplex virus 1 (HSV-1), children with inborn errors of toll-like receptor 3 (TLR3) immunity are prone to HSV-1 encephalitis (HSE). We tested the hypothesis that the pathogenesis of HSE involves non-haematopoietic CNS-resident cells. We derived induced pluripotent stem cells (iPSCs) from the dermal fibroblasts of TLR3- and UNC-93B-deficient patients and from controls. These iPSCs were differentiated into highly purified populations of neural stem cells (NSCs), neurons, astrocytes and oligodendrocytes. The induction of interferon-ß (IFN-ß) and/or IFN-λ1 in response to stimulation by the dsRNA analogue polyinosinic:polycytidylic acid (poly(I:C)) was dependent on TLR3 and UNC-93B in all cells tested. However, the induction of IFN-ß and IFN-λ1 in response to HSV-1 infection was impaired selectively in UNC-93B-deficient neurons and oligodendrocytes. These cells were also much more susceptible to HSV-1 infection than control cells, whereas UNC-93B-deficient NSCs and astrocytes were not. TLR3-deficient neurons were also found to be susceptible to HSV-1 infection. The rescue of UNC-93B- and TLR3-deficient cells with the corresponding wild-type allele showed that the genetic defect was the cause of the poly(I:C) and HSV-1 phenotypes. The viral infection phenotype was rescued further by treatment with exogenous IFN-α or IFN-ß ( IFN-α/ß) but not IFN-λ1. Thus, impaired TLR3- and UNC-93B-dependent IFN-α/ß intrinsic immunity to HSV-1 in the CNS, in neurons and oligodendrocytes in particular, may underlie the pathogenesis of HSE in children with TLR3-pathway deficiencies.

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.

Risk factors hindering asthma symptom control in Saudi children and adolescents.

BACKGROUND: Maintaining good control of asthma symptoms can help to prevent exacerbations and its associated complications. The Asthma Control Test (ACT) can rapidly assess the effectiveness of asthma management plan and therapy. The aim of this study was therefore to identify risk factors associated with uncontrolled asthma symptoms in young Saudi asthmatic children (3-17 years old). METHODS: In this cross-sectional hospital-based survey, the ACT was administered to 297 asthmatic children/adolescents, recruited at the emergency department (ED) of two major hospitals. RESULTS: Most recruited patients had intermittent (63.5%) and mild persistent (27.6%) asthma; few had moderate persistent (8.9%) and none had severe asthma. These patients visited the ED four times (3.9 ± 3.2), on average. Almost half of the patients stated that they had not received education about asthma (47%) or education about medication use (43%). Most patients (60.3%) had uncontrolled symptoms (ACT score ≤19), of whom the intermittent asthma patients had better scores than those with more severe symptoms. Children ≤6 years old, with symptoms diagnosed <5 years previously and who were not attending school, had significantly worse control than older patients. Poor medication compliance and inappropriate inhaler device use were ascribed to younger patients (<12 years old) and worse scores; particularly in relation to stopping inhaled corticosteroid therapy when their symptoms improve. Patients with poor control also stated that they had not received education about inhaler device use. CONCLUSIONS: Most Saudi asthmatic children/adolescents visiting the ED had poor control of symptoms; indeed, none achieved complete control, which is related to deficient medication compliance and improper medication inhaler device use; deficient knowledge about asthma was also another factor hindering control.

Unbiased targeted next-generation sequencing molecular approach for primary immunodeficiency diseases.

BACKGROUND: Molecular genetics techniques are an essential diagnostic tool for primary immunodeficiency diseases (PIDs). The use of next-generation sequencing (NGS) provides a comprehensive way of concurrently screening a large number of PID genes. However, its validity and cost-effectiveness require verification. OBJECTIVES: We sought to identify and overcome complications associated with the use of NGS in a comprehensive gene panel incorporating 162 PID genes. We aimed to ascertain the specificity, sensitivity, and clinical sensitivity of the gene panel and its utility as a diagnostic tool for PIDs. METHODS: A total of 162 PID genes were screened in 261 patients by using the Ion Torrent Proton NGS sequencing platform. Of the 261 patients, 122 had at least 1 known causal mutation at the onset of the study and were used to assess the specificity and sensitivity of the assay. The remaining samples were from unsolved cases that were biased toward more phenotypically and genotypically complicated cases. RESULTS: The assay was able to detect the mutation in 117 (96%) of 122 positive control subjects with known causal mutations. For the unsolved cases, our assay resulted in a molecular genetic diagnosis for 35 of 139 patients. Interestingly, most of these cases represented atypical clinical presentations of known PIDs. CONCLUSIONS: The targeted NGS PID gene panel is a sensitive and cost-effective diagnostic tool that can be used as a first-line molecular assay in patients with PIDs. The assay is an alternative choice to the complex and costly candidate gene approach, particularly for patients with atypical presentation of known PID genes.

Prevalence of UDP-glucuronosyltransferase polymorphisms (UGT1A6∗2, 1A7∗12, 1A8∗3, 1A9∗3, 2B7∗2, and 2B15∗2) in a Saudi population.

Glucuronidation is an important phase II pathway responsible for many endogenous substances and drug metabolism. The present work evaluated allele frequencies of certain UDP-glucuronosyl-transferases (UGT 1A6∗2, A7∗12, A8∗3, A9∗3, 2B7∗2, and 2B15∗2) in Saudi Arabians that could provide essential ethnic information. Blood samples from 192 healthy unrelated Saudi males of various geographic regions were collected. Genomic DNA was isolated and genotyping of various UGTs was carried out using polymerase chain reaction (PCR) followed by direct sequencing. For UGT1A6∗2 A/G genotype, the most common variant was the homozygous repeat (AA) and the most common allele was (A) with a frequency of 46.5% and 67.3%, respectively. Similarly, the most common variant for UGT1A7∗12 T/C genotype was the heterozygous repeat (TC) with a frequency of 78.7% while the mutant allele (C) was present in 60.6% of the study population. Both UGT1A8∗3 (G/A) and UGT1A9∗3 (T/C) showed only a wild homozygous pattern in all screened subjects. For UGT2B7∗2, the heterozygous repeat (TC) was found with a frequency of 57.3% and the alleles (A) showed a frequency of 50.8%. In contrast, for UGT2B15∗2 (G253T), the heterozygous repeat (TG) presented 62.3% of the subjects where the most common allele (G) was with a frequency of 66.2%. In conclusion, our data indicate that Saudis harbor some important UGT mutations known to affect enzyme activity. Additional studies are therefore, warranted to assess the clinical implications of these gene polymorphisms in this ethnic group.