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Human genetic studies of critical COVID-19 pneumonia have revealed the essential role of type I interferon-dependent innate immunity to SARS-CoV-2 infection. Conversely, an association between the HLA-B∗15:01 allele and asymptomatic SARS-CoV-2 infection in unvaccinated individuals was recently reported, suggesting a contribution of pre-existing T cell-dependent adaptive immunity. We report a lack of association of classical HLA alleles, including HLA-B∗15:01, with pre-omicron asymptomatic SARS-CoV-2 infection in unvaccinated participants in a prospective population-based study in the United States (191 asymptomatic vs. 945 symptomatic COVID-19 cases). Moreover, we found no such association in the international COVID Human Genetic Effort cohort (206 asymptomatic vs. 574 mild or moderate COVID-19 cases and 1,625 severe or critical COVID-19 cases). Finally, in the Human Challenge Characterisation study, the three HLA-B∗15:01 individuals infected with SARS-CoV-2 developed symptoms. As with other acute primary infections studied, no classical HLA alleles favoring an asymptomatic course of SARS-CoV-2 infection were identified., Competing Interests: Declaration of interests E.T.C., K.M.S.B., and A.B. are employees of Helix., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

We describe a novel, severe autoinflammatory syndrome characterized by neuroinflammation, systemic autoinflammation, splenomegaly, and anemia (NASA) caused by bi-allelic mutations in IRAK4 . IRAK-4 is a serine/threonine kinase with a pivotal role in innate immune signaling from toll-like receptors and production of pro-inflammatory cytokines. In humans, bi-allelic mutations in IRAK4 result in IRAK-4 deficiency and increased susceptibility to pyogenic bacterial infections, but autoinflammation has never been described. We describe 5 affected patients from 2 unrelated families with compound heterozygous mutations in IRAK4 (c.C877T (p.Q293*)/c.G958T (p.D320Y); and c.A86C (p.Q29P)/c.161 + 1G>A) resulting in severe systemic autoinflammation, massive splenomegaly and severe transfusion dependent anemia and, in 3/5 cases, severe neuroinflammation and seizures. IRAK-4 protein expression was reduced in peripheral blood mononuclear cells (PBMC) in affected patients. Immunological analysis demonstrated elevated serum tumor necrosis factor (TNF), interleukin (IL) 1 beta (IL-1β), IL-6, IL-8, interferon α2a (IFN-α2a), and interferon β (IFN-β); and elevated cerebrospinal fluid (CSF) IL-6 without elevation of CSF IFN-α despite perturbed interferon gene signature. Mutations were located within the death domain (DD; p.Q29P and splice site mutation c.161 + 1G>A) and kinase domain (p.Q293*/p.D320Y) of IRAK-4. Structure-based modeling of the DD mutation p.Q29P showed alteration in the alignment of a loop within the DD with loss of contact distance and hydrogen bond interactions with IRAK-1/2 within the myddosome complex. The kinase domain mutation p.D320Y was predicted to stabilize interactions within the kinase active site. While precise mechanisms of autoinflammation in NASA remain uncertain, we speculate that loss of negative regulation of IRAK-4 and IRAK-1; dysregulation of myddosome assembly and disassembly; or kinase active site instability may drive dysregulated IL-6 and TNF production. Blockade of IL-6 resulted in immediate and complete amelioration of systemic autoinflammation and anemia in all 5 patients treated; however, neuroinflammation has, so far proven recalcitrant to IL-6 blockade and the janus kinase (JAK) inhibitor baricitinib, likely due to lack of central nervous system penetration of both drugs. We therefore highlight that bi-allelic mutation in IRAK4 may be associated with a severe and complex autoinflammatory and neuroinflammatory phenotype that we have called NASA (neuroinflammation, autoinflammation, splenomegaly and anemia), in addition to immunodeficiency in humans., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Cooray, Price-Kuehne, Hong, Omoyinmi, Burleigh, Gilmour, Ahmad, Choi, Bahar, Torpiano, Gagunashvili, Jensen, Bellos, Sancho-Shimizu, Herberg, Mankad, Kumar, Kaliakatsos, Worth, Eleftheriou, Whittaker and Brogan.)

Neisseria meningitidis protects itself from complement-mediated killing by binding complement factor H (FH). Previous studies associated susceptibility to meningococcal disease (MD) with variation in CFH, but the causal variants and underlying mechanism remained unknown. Here we attempted to define the association more accurately by sequencing the CFH-CFHR locus and imputing missing genotypes in previously obtained GWAS datasets of MD-affected individuals of European ancestry and matched controls. We identified a CFHR3 SNP that provides protection from MD (rs75703017, p value = 1.1 × 10 -16 ) by decreasing the concentration of FH in the blood (p value = 1.4 × 10 -11 ). We subsequently used dual-luciferase studies and CRISPR gene editing to establish that deletion of rs75703017 increased FH expression in hepatocyte by preventing promotor inhibition. Our data suggest that reduced concentrations of FH in the blood confer protection from MD; with reduced access to FH, N. meningitidis is less able to shield itself from complement-mediated killing., Competing Interests: Declaration of interests R.B.P., M.C.B., D.W., and T.W.K. are co-inventors of patents or patent applications describing FH potentiating antibodies and uses thereof. A.J.P. is chair of the UK Department of Health and Social Care’s Joint Committee on Vaccination and Immunisation. F.M.-T. has received honoraria from GSK group of companies, Pfizer Inc, Sanofi Pasteur, MSD, Seqirus, Biofabri, and Janssen for taking part in advisory boards and expert meetings and for acting as a speaker in congresses outside the scope of the submitted work. F.M.-T. has also acted as principal investigator in randomized controlled trials of the above-mentioned companies as well as Ablynx, Gilead, Regeneron, Roche, Abbott, Novavax, and MedImmune, with honoraria paid to his institution. All other authors declare no relevant competing interest related to the contents of this manuscript., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The role of primary immunodeficiencies (PID) in susceptibility to sepsis remains unknown. It is unclear whether children with sepsis benefit from genetic investigations. We hypothesized that sepsis may represent the first manifestation of underlying PID. We applied whole-exome sequencing (WES) to a national cohort of children with sepsis to identify rare, predicted pathogenic variants in PID genes., Methods: We conducted a multicenter, population-based, prospective study including previously healthy children aged ≥28 days and <17 years admitted with blood culture-proven sepsis. Using a stringent variant filtering procedure, analysis of WES data was restricted to rare, predicted pathogenic variants in 240 PID genes for which increased susceptibility to bacterial infection has been reported., Results: There were 176 children presenting with 185 sepsis episodes who underwent WES (median age, 52 months; interquartile range, 15.4-126.4). There were 41 unique predicted pathogenic PID variants (1 homozygous, 5 hemizygous, and 35 heterozygous) found in 35/176 (20%) patients, including 3/176 (2%) patients carrying variants that were previously reported to lead to PID. The variants occurred in PID genes across all 8 PID categories, as defined by the International Union of Immunological Societies. We did not observe a significant correlation between clinical or laboratory characteristics of patients and the presence or absence of PID variants., Conclusions: Applying WES to a population-based cohort of previously healthy children with bacterial sepsis detected variants of uncertain significance in PID genes in 1 out of 5 children. Future studies need to investigate the functional relevance of these variants to determine whether variants in PID genes contribute to pediatric sepsis susceptibility., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America.)

Background: Neisseria meningitidis (Nm) is a nasopharyngeal commensal carried by healthy individuals. However, invasive infections occurs in a minority of individuals, with devastating consequences. There is evidence that common polymorphisms are associated with invasive meningococcal disease (IMD), but the contributions of rare variants other than those in the complement system have not been determined., Methods: We identified familial cases of IMD in the UK meningococcal disease study and the European Union Life-Threatening Infectious Disease Study. Candidate genetic variants were identified by whole-exome sequencing of 2 patients with familial IMD. Candidate variants were further validated by in vitro assays., Results: Exomes of 2 siblings with IMD identified a novel heterozygous missense mutation in BPIFA1/SPLUNC1. Sequencing of 186 other nonfamilial cases identified another unrelated IMD patient with the same mutation. SPLUNC1 is an innate immune defense protein expressed in the nasopharyngeal epithelia; however, its role in invasive infections is unknown. In vitro assays demonstrated that recombinant SPLUNC1 protein inhibits biofilm formation by Nm, and impedes Nm adhesion and invasion of human airway cells. The dominant negative mutant recombinant SPLUNC1 (p.G22E) showed reduced antibiofilm activity, increased meningococcal adhesion, and increased invasion of cells, compared with wild-type SPLUNC1., Conclusions: A mutation in SPLUNC1 affecting mucosal attachment, biofilm formation, and invasion of mucosal epithelial cells is a new genetic cause of meningococcal disease., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.)

Mycobacterium tuberculosis (M. tuberculosis) survives and multiplies inside human macrophages by subversion of immune mechanisms. Although these immune evasion strategies are well characterised functionally, the underlying molecular mechanisms are poorly understood. Here we show that during infection of human whole blood with M. tuberculosis, host gene transcriptional suppression, rather than activation, is the predominant response. Spatial, temporal and functional characterisation of repressed genes revealed their involvement in pathogen sensing and phagocytosis, degradation within the phagolysosome and antigen processing and presentation. To identify mechanisms underlying suppression of multiple immune genes we undertook epigenetic analyses. We identified significantly differentially expressed microRNAs with known targets in suppressed genes. In addition, after searching regions upstream of the start of transcription of suppressed genes for common sequence motifs, we discovered novel enriched composite sequence patterns, which corresponded to Alu repeat elements, transposable elements known to have wide ranging influences on gene expression. Our findings suggest that to survive within infected cells, mycobacteria exploit a complex immune "molecular off switch" controlled by both microRNAs and Alu regulatory elements.

Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) is an autosomal-dominant cancer-predisposition syndrome with a significant risk of gastric, but not colorectal, adenocarcinoma. We mapped the gene to 5q22 and found loss of the wild-type allele on 5q in fundic gland polyps from affected individuals. Whole-exome and -genome sequencing failed to find causal mutations but, through Sanger sequencing, we identified point mutations in APC promoter 1B that co-segregated with disease in all six families. The mutations reduced binding of the YY1 transcription factor and impaired activity of the APC promoter 1B in luciferase assays. Analysis of blood and saliva from carriers showed allelic imbalance of APC, suggesting that these mutations lead to decreased allele-specific expression in vivo. Similar mutations in APC promoter 1B occur in rare families with familial adenomatous polyposis (FAP). Promoter 1A is methylated in GAPPS and sporadic FGPs and in normal stomach, which suggests that 1B transcripts are more important than 1A in gastric mucosa. This might explain why all known GAPPS-affected families carry promoter 1B point mutations but only rare FAP-affected families carry similar mutations, the colonic cells usually being protected by the expression of the 1A isoform. Gastric polyposis and cancer have been previously described in some FAP-affected individuals with large deletions around promoter 1B. Our finding that GAPPS is caused by point mutations in the same promoter suggests that families with mutations affecting the promoter 1B are at risk of gastric adenocarcinoma, regardless of whether or not colorectal polyps are present., (Copyright © 2016 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Targeted resequencing technologies have allowed for efficient and cost-effective detection of genomic variants in specific regions of interest. Although capture sequencing has been primarily used for investigating single nucleotide variants and indels, it has the potential to elucidate a broader spectrum of genetic variation, including copy number variants (CNVs). Various methods exist for detecting CNV in whole-genome and exome sequencing datasets. However, no algorithms have been specifically designed for contiguous target sequencing, despite its increasing importance in clinical and research applications. We have developed cnvCapSeq, a novel method for accurate and sensitive CNV discovery and genotyping in long-range targeted resequencing. cnvCapSeq was benchmarked using a simulated contiguous capture sequencing dataset comprising 21 genomic loci of various lengths. cnvCapSeq was shown to outperform the best existing exome CNV method by a wide margin both in terms of sensitivity (92.0 versus 48.3%) and specificity (99.8 versus 70.5%). We also applied cnvCapSeq to a real capture sequencing cohort comprising a contiguous 358 kb region that contains the Complement Factor H gene cluster. In this dataset, cnvCapSeq identified 41 samples with CNV, including two with duplications, with a genotyping accuracy of 99%, as ascertained by quantitative real-time PCR., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Motivation: Exome sequencing technologies have transformed the field of Mendelian genetics and allowed for efficient detection of genomic variants in protein-coding regions. The target enrichment process that is intrinsic to exome sequencing is inherently imperfect, generating large amounts of unintended off-target sequence. Off-target data are characterized by very low and highly heterogeneous coverage and are usually discarded by exome analysis pipelines. We posit that off-target read depth is a rich, but overlooked, source of information that could be mined to detect intergenic copy number variation (CNV). We propose cnvOffseq, a novel normalization framework for off-target read depth that is based on local adaptive singular value decomposition (SVD). This method is designed to address the heterogeneity of the underlying data and allows for accurate and precise CNV detection and genotyping in off-target regions., Results: cnvOffSeq was benchmarked on whole-exome sequencing samples from the 1000 Genomes Project. In a set of 104 gold standard intergenic deletions, our method achieved a sensitivity of 57.5% and a specificity of 99.2%, while maintaining a low FDR of 5%. For gold standard deletions longer than 5 kb, cnvOffSeq achieves a sensitivity of 90.4% without increasing the FDR. cnvOffSeq outperforms both whole-genome and whole-exome CNV detection methods considerably and is shown to offer a substantial improvement over naïve local SVD., Availability and Implementation: cnvOffSeq is available at http://sourceforge.net/p/cnvoffseq/., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2014. Published by Oxford University Press.)

Insertion and deletion polymorphisms (indels) are an important source of genomic variation in plant and animal genomes, but accurate genotyping from low-coverage and exome next-generation sequence data remains challenging. We introduce an efficient population clustering algorithm for diploids and polyploids which was tested on a dataset of 2000 exomes. Compared with existing methods, we report a 4-fold reduction in overall indel genotype error rates with a 9-fold reduction in low coverage regions.

Recent advances in sequencing technologies provide the means for identifying copy number variation (CNV) at an unprecedented resolution. A single next-generation sequencing experiment offers several features that can be used to detect CNV, yet current methods do not incorporate all available signatures into a unified model. cnvHiTSeq is an integrative probabilistic method for CNV discovery and genotyping that jointly analyzes multiple features at the population level. By combining evidence from complementary sources, cnvHiTSeq achieves high genotyping accuracy and a substantial improvement in CNV detection sensitivity over existing methods, while maintaining a low false discovery rate. cnvHiTSeq is available at http://sourceforge.net/projects/cnvhitseq.