CDG: an online server proposing biologically closest disease-causing genes and pathologies and its application to primary immunodeficiency

Summary: In analyses of exome data, candidate gene selection can be challenging in the absence of variants in known disease-causing genes. We calculated the putative biologically closest known disease-causing genes for 13,005 human genes not currently reported to be disease-causing. We used these data to construct the Closest Disease-Causing Genes (CDG) server, which can be used to infer the closest associated disease-causing genes and phenotypes for lists of candidate genes. This resource will be a considerable asset for ascertaining the poten-tial relevance of lists of genes to specific diseases of interest

SeqTailor: a user-friendly webserver for the extraction of DNA or protein sequences from next-generation sequencing data

Human whole-genome-sequencing reveals about 4 000 000 genomic variants per individual. These data are mostly stored as VCF-format files. Although many variant analysis methods accept VCF as input, many other tools require DNA or protein sequences, particularly for splicing prediction, sequence alignment, phylogenetic analysis, and structure prediction. However, there is no existing webserver capable of extracting DNA/protein sequences for genomic variants from VCF files in a user-friendly and efficient manner. We developed the SeqTailor webserver to bridge this gap, by enabling rapid extraction of (i) DNA sequences around genomic variants, with customizable window sizes and options to annotate the splice sites closest to the variants and to consider the neighboring variants within the window; and (ii) protein sequences encoded by the DNA sequences around genomic variants, with built-in SnpEff annotator and customizable window sizes. SeqTailor supports 11 species, including: human (GRCh37/GRCh38), chimpanzee, mouse, rat, cow, chicken, lizard, zebrafish, fruitfly, Arabidopsis and rice. Standalone programs are provided for command-line-based needs. SeqTailor streamlines the sequence extraction process, and accelerates the analysis of genomic variants with software requiring DNA/protein sequences. It will facilitate the study of genomic variation, by increasing the feasibility of sequence-based analysis and prediction. The SeqTailor webserver is freely available at http://shiva.rockefeller.edu/SeqTailor/

Genome-wide detection of human variants that disrupt intronic branchpoints

The search for candidate variants underlying human disease in massive parallel sequencing data typically focuses on coding regions and essential splice sites, mostly ignoring noncoding variants. The RNA spliceosome recognizes intronic branchpoint (BP) motifs at the beginning of splicing and operates mostly within introns to define the exon-intron boundaries; however, BP variants have been paid little attention. We established a comprehensive genome-wide database and knowledgebase of BP and developed BPHunter for systematic and informative genome-wide detection of intronic variants that may disrupt BP and splicing, together with an effective strategy for prioritizing BP variant candidates. BPHunter not only constitutes an important resource for understanding BP, but should also drive discovery of BP variants in human genetic diseases and traits. Pre-messenger RNA splicing is initiated with the recognition of a single-nucleotide intronic branchpoint (BP) within a BP motif by spliceosome elements. Forty-eight rare variants in 43 human genes have been reported to alter splicing and cause disease by disrupting BP. However, until now, no computational approach was available to efficiently detect such variants in massively parallel sequencing data. We established a comprehensive human genome-wide BP database by integrating existing BP data and generating new BP data from RNA sequencing of lariat debranching enzyme DBR1-mutated patients and from machine-learning predictions. We characterized multiple features of BP in major and minor introns and found that BP and BP-2 (two nucleotides upstream of BP) positions exhibit a lower rate of variation in human populations and higher evolutionary conservation than the intronic background, while being comparable to the exonic background. We developed BPHunter as a genome-wide computational approach to systematically and efficiently detect intronic variants that may disrupt BP recognition. BPHunter retrospectively identified 40 of the 48 known pathogenic BP variants, in which we summarized a strategy for prioritizing BP variant candidates. The remaining eight variants all create AG-dinucleotides between the BP and acceptor site, which is the likely reason for missplicing. We demonstrated the practical utility of BPHunter prospectively by using it to identify a novel germline heterozygous BP variant of STAT2 in a patient with critical COVID-19 pneumonia and a novel somatic intronic 59-nucleotide deletion of ITPKB in a lymphoma patient, both of which were validated experimentally. BPHunter is publicly available from an

Genome-wide detection of human intronic AG-gain variants located between splicing branchpoints and canonical splice acceptor sites

Human genetic variants that introduce an AG into the intronic region between the branchpoint (BP) and the canonical splice acceptor site (ACC) of protein-coding genes can disrupt pre-mRNA splicing. Using our genome-wide BP database, we delineated the BP-ACC segments of all human introns and found extreme depletion of AG/YAG in the [BP+8, ACC-4] high-risk region. We developed AGAIN as a genome-wide computational approach to systematically and precisely pinpoint intronic AG-gain variants within the BP-ACC regions. AGAIN identified 350 AG-gain variants from the Human Gene Mutation Database, all of which alter splicing and cause disease. Among them, 74% created new acceptor sites, whereas 31% resulted in complete exon skipping. AGAIN also predicts the protein-level products resulting from these two consequences. We performed AGAIN on our exome/genomes database of patients with severe infectious diseases but without known genetic etiology and identified a private homozygous intronic AG-gain variant in the antimycobacterial gene SPPL2A in a patient with mycobacterial disease. AGAIN also predicts a retention of six intronic nucleotides that encode an in-frame stop codon, turning AG-gain into stop-gain. This allele was then confirmed experimentally to lead to loss of function by disrupting splicing. We further showed that AG-gain variants inside the high-risk region led to misspliced products, while those outside the region did not, by two case studies in genes STAT1 and IRF7. We finally evaluated AGAIN on our 14 paired exome-RNAseq samples and found that 82% of AG-gain variants in high-risk regions showed evidence of missplicing

Life-threatening influenza pneumonitis in a child with inherited IRF9 deficiency

Life-threatening pulmonary influenza can be caused by inborn errors of type I and III IFN immunity. We report a 5-yr-old child with severe pulmonary influenza at 2 yr. She is homozygous for a loss-of-function IRF9 allele. Her cells activate gamma-activated factor (GAF) STAT1 homodimers but not IFN-stimulated gene factor 3 (ISGF3) trimers (STAT1/STAT2/IRF9) in response to IFN-α2b. The transcriptome induced by IFN-α2b in the patient's cells is much narrower than that of control cells; however, induction of a subset of IFN-stimulated gene transcripts remains detectable. In vitro, the patient's cells do not control three respiratory viruses, influenza A virus (IAV), parainfluenza virus (PIV), and respiratory syncytial virus (RSV). These phenotypes are rescued by wild-type IRF9, whereas silencing IRF9 expression in control cells increases viral replication. However, the child has controlled various common viruses in vivo, including respiratory viruses other than IAV. Our findings show that human IRF9- and ISGF3-dependent type I and III IFN responsive pathways are essential for controlling IAV

Immunodeficiency, auto-inflammation and amylopectinosis in humans with inherited HOIL-1 and LUBAC deficiency

We report the clinical description and molecular dissection of a new fatal human inherited disorder characterized by chronic auto-inflammation, invasive bacterial infections and muscular amylopectinosis. Patients from two kindreds carried biallelic loss-of-expression and loss-of-function mutations in HOIL1, a component the linear ubiquitination chain assembly complex (LUBAC). These mutations resulted in impairment of LUBAC stability. NF-κB activation in response to interleukin-1β (IL-1β) was compromised in the patients’ fibroblasts. By contrast, the patients’ mononuclear leukocytes, particularly monocytes, were hyperresponsive to IL-1β. The consequences of human HOIL-1 and LUBAC deficiencies for IL-1β responses thus differed between cell types, consistent with the unique association of auto-inflammation and immunodeficiency in these patients. These data suggest that LUBAC regulates NF-κB-dependent IL-1β responses differently in different cell types

Systemic Type I IFN Inflammation in Human ISG15 Deficiency Leads to Necrotizing Skin Lesions

Most monogenic disorders have a primary clinical presentation. Inherited ISG15 deficiency, however, has manifested with two distinct presentations to date: susceptibility to mycobacterial disease and intracranial calcifications from hypomorphic interferon-II (IFN-II) production and excessive IFN-I response, respectively. Accordingly, these patients were managed for their infectious and neurologic complications. Herein, we describe five new patients with six novel ISG15 mutations presenting with skin lesions who were managed for dermatologic disease. Cellularly, we denote striking specificity to the IFN-I response, which was previously assumed to be universal. In peripheral blood, myeloid cells display the most robust IFN-I signatures. In the affected skin, IFN-I signaling is observed in the keratinocytes of the epidermis, endothelia, and the monocytes and macrophages of the dermis. These findings define the specific cells causing circulating and dermatologic inflammation and expand the clinical spectrum of ISG15 deficiency to dermatologic presentations as a third phenotype co-dominant to the infectious and neurologic manifestations.Fil: Martin Fernandez, Marta. Icahn School Of Medicine At Mount Sinai; Estados Unidos. King Saud University; Arabia SauditaFil: Bravo García Morato, María. Instituto de Investigacion del Hospital de la Paz.; EspañaFil: Gruber, Conor. Icahn School Of Medicine At Mount Sinai; Estados Unidos. King Saud University; Arabia SauditaFil: Murias Loza, Sara. Instituto de Investigacion del Hospital de la Paz.; EspañaFil: Malik, Muhammad Nasir Hayat. Twincore; Alemania. University Of Lahore; Países Bajos. Leibniz Universitat Hannover; Alemania. Helmholtz Gemeinschaft; AlemaniaFil: Alsohime, Fahad. King Saud University; Arabia SauditaFil: Alakeel, Abdullah. King Saud University; Arabia SauditaFil: Valdez, Rita. Gobierno de la Ciudad Autónoma de Buenos Aires. Hospital General de Agudos Doctor Cosme Argerich; ArgentinaFil: Buta, Sofija. Icahn School Of Medicine At Mount Sinai; Estados UnidosFil: Buda, Guadalupe. Bitgenia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Biología Celular e Histología; ArgentinaFil: Marti, Marcelo Adrian. Bitgenia; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Biología Celular e Histología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Larralde, Margarita. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Boisson, Bertrand. L'institut Des Maladies Génétiques Imagine; Francia. The Rockefeller University; Estados Unidos. Universite de Paris; FranciaFil: Feito Rodriguez, Marta. Instituto de Investigacion del Hospital de la Paz.; EspañaFil: Qiu, Xueer. Icahn School Of Medicine At Mount Sinai; Estados UnidosFil: Chrabieh, Maya. L'institut Des Maladies Génétiques Imagine; FranciaFil: Al Ayed, Mohammed. Najran University; Arabia SauditaFil: Al Muhsen, Saleh. King Saud University; Arabia SauditaFil: Desai, Jigar V.. National Institutes of Health; Estados UnidosFil: Ferre, Elise M.N.. National Institutes of Health; Estados UnidosFil: Rosenzweig, Sergio D.. National Institutes of Health; Estados UnidosFil: Amador-Borrero, Blanca. Icahn School Of Medicine At Mount Sinai; Estados UnidosFil: Bravo-Gallego, Luz Yadira. Instituto de Investigacion del Hospital de la Paz.; EspañaFil: Olmer, Ruth. Hannover Medical School; Alemania. German Center for Lung Research; AlemaniaFil: Merkert, Sylvia. Hannover Medical School; Alemania. German Center for Lung Research; AlemaniaFil: Bret, Montserrat. Instituto de Investigacion del Hospital de la Paz.; EspañaFil: Sood, Amika K.. University of North Carolina; Estados UnidosFil: Al-rabiaah, Abdulkarim. King Saud University; Arabia SauditaFil: Temsah, Mohamad Hani. King Saud University; Arabia SauditaFil: Halwani, Rabih. University of Sharjah; Emiratos Arabes UnidosFil: Hernandez, Michelle Marilyn. University of North Carolina; Estados UnidosFil: Pessler, Frank. Twincore; Alemania. Helmholtz Centre for Infection Research; AlemaniaFil: Casanova, Jean Laurent. The Rockefeller University; Estados Unidos. Necker Hospital for Sick Children; Francia. Howard Hughes Medical Institute; Estados Unidos. Universite de Paris; FranciaFil: Bustamante, Jacinta. The Rockefeller University; Estados Unidos. Necker Hospital for Sick Children; Francia. Universite de Paris; FranciaFil: Lionakis, Michail S.. National Institutes of Health; Estados UnidosFil: Bogunovic, Dusan. Icahn School Of Medicine At Mount Sinai; Estados Unido

NOF1 Encodes an Arabidopsis Protein Involved in the Control of rRNA Expression

The control of ribosomal RNA biogenesis is essential for the regulation of protein synthesis in eukaryotic cells. Here, we report the characterization of NOF1 that encodes a putative nucleolar protein involved in the control of rRNA expression in Arabidopsis. The gene has been isolated by T-DNA tagging and its function verified by the characterization of a second allele and genetic complementation of the mutants. The nof1 mutants are affected in female gametogenesis and embryo development. This result is consistent with the detection of NOF1 mRNA in all tissues throughout plant life's cycle, and preferentially in differentiating cells. Interestingly, the closely related proteins from zebra fish and yeast are also necessary for cell division and differentiation. We showed that the nof1-1 mutant displays higher rRNA expression and hypomethylation of rRNA promoter. Taken together, the results presented here demonstrated that NOF1 is an Arabidopsis gene involved in the control of rRNA expression, and suggested that it encodes a putative nucleolar protein, the function of which may be conserved in eukaryotes

Blacklisting variants common in private cohorts but not in public databases optimizes human exome analysis

Computational analyses of human patient exomes aim to filter out as many nonpathogenic genetic variants (NPVs) as possible, without removing the true disease-causing mutations. This involves comparing the patient's exome with public databases to remove reported variants inconsistent with disease prevalence, mode of inheritance, or clinical penetrance. However, variants frequent in a given exome cohort, but absent or rare in public databases, have also been reported and treated as NPVs, without rigorous exploration. We report the generation of a blacklist of variants frequent within an in-house cohort of 3,104 exomes. This blacklist did not remove known pathogenic mutations from the exomes of 129 patients and decreased the number of NPVs remaining in the 3,104 individual exomes by a median of 62%. We validated this approach by testing three other independent cohorts of 400, 902, and 3,869 exomes. The blacklist generated from any given cohort removed a substantial proportion of NPVs (11-65%). We analyzed the blacklisted variants computationally and experimentally. Most of the blacklisted variants corresponded to false signals generated by incomplete reference genome assembly, location in low-complexity regions, bioinformatic misprocessing, or limitations inherent to cohort-specific private alleles (e.g., due to sequencing kits, and genetic ancestries). Finally, we provide our precalculated blacklists, together with ReFiNE, a program for generating customized blacklists from any medium-sized or large in-house cohort of exome (or other next-generation sequencing) data via a user-friendly public web server. This work demonstrates the power of extracting variant blacklists from private databases as a specific in-house but broadly applicable tool for optimizing exome analysis