Human genetic defects in SRP19 and SRPRA cause severe congenital neutropenia with distinctive proteome changes.

The mechanisms of coordinated changes in proteome composition and their relevance for the differentiation of neutrophil granulocytes are not well studied. Here, we discover 2 novel human genetic defects in signal recognition particle receptor alpha (SRPRA) and SRP19, constituents of the mammalian cotranslational targeting machinery, and characterize their roles in neutrophil granulocyte differentiation. We systematically study the proteome of neutrophil granulocytes from patients with variants in the SRP genes, HAX1, and ELANE, and identify global as well as specific proteome aberrations. Using in vitro differentiation of human induced pluripotent stem cells and in vivo zebrafish models, we study the effects of SRP deficiency on neutrophil granulocyte development. In a heterologous cell-based inducible protein expression system, we validate the effects conferred by SRP dysfunction for selected proteins that we identified in our proteome screen. Thus, SRP-dependent protein processing, intracellular trafficking, and homeostasis are critically important for the differentiation of neutrophil granulocytes.

The Human Phenotype Ontology in 2021.

The Human Phenotype Ontology (HPO, https://hpo.jax.org) was launched in 2008 to provide a comprehensive logical standard to describe and computationally analyze phenotypic abnormalities found in human disease. The HPO is now a worldwide standard for phenotype exchange. The HPO has grown steadily since its inception due to considerable contributions from clinical experts and researchers from a diverse range of disciplines. Here, we present recent major extensions of the HPO for neurology, nephrology, immunology, pulmonology, newborn screening, and other areas. For example, the seizure subontology now reflects the International League Against Epilepsy (ILAE) guidelines and these enhancements have already shown clinical validity. We present new efforts to harmonize computational definitions of phenotypic abnormalities across the HPO and multiple phenotype ontologies used for animal models of disease. These efforts will benefit software such as Exomiser by improving the accuracy and scope of cross-species phenotype matching. The computational modeling strategy used by the HPO to define disease entities and phenotypic features and distinguish between them is explained in detail.We also report on recent efforts to translate the HPO into indigenous languages. Finally, we summarize recent advances in the use of HPO in electronic health record systems.

FARS1-related disorders caused by bi-allelic mutations in cytosolic phenylalanyl-tRNA synthetase genes: Look beyond the lungs!

Aminoacyl-tRNA synthetases (ARSs) catalyze the first step of protein biosynthesis (canonical function) and have additional (non-canonical) functions outside of translation. Bi-allelic pathogenic variants in genes encoding ARSs are associated with various recessive mitochondrial and multisystem disorders. We describe here a multisystem clinical phenotype based on bi-allelic mutations in the two genes (FARSA, FARSB) encoding distinct subunits for tetrameric cytosolic phenylalanyl-tRNA synthetase (FARS1). Interstitial lung disease with cholesterol pneumonitis on histology emerged as an early characteristic feature and significantly determined disease burden. Additional clinical characteristics of the patients included neurological findings, liver dysfunction, and connective tissue, muscular and vascular abnormalities. Structural modeling of newly identified missense mutations in the alpha subunit of FARS1, FARSA, showed exclusive mapping to the enzyme's conserved catalytic domain. Patient-derived mutant cells displayed compromised aminoacylation activity in two cases, while remaining unaffected in another. Collectively, these findings expand current knowledge about the human ARS disease spectrum and support a loss of canonical and non-canonical function in FARS1-associated recessive disease.

SmartPhase: Accurate and fast phasing of heterozygous variant pairs for genetic diagnosis of rare diseases.

There is an increasing need to use genome and transcriptome sequencing to genetically diagnose patients suffering from suspected monogenic rare diseases. The proper detection of compound heterozygous variant combinations as disease-causing candidates is a challenge in diagnostic workflows as haplotype information is lost by currently used next-generation sequencing technologies. Consequently, computational tools are required to phase, or resolve the haplotype of, the high number of heterozygous variants in the exome or genome of each patient. Here we present SmartPhase, a phasing tool designed to efficiently reduce the set of potential compound heterozygous variant pairs in genetic diagnoses pipelines. The phasing algorithm of SmartPhase creates haplotypes using both parental genotype information and reads generated by DNA or RNA sequencing and is thus well suited to resolve the phase of rare variants. To inform the user about the reliability of a phasing prediction, it computes a confidence score which is essential to select error-free predictions. It incorporates existing haplotype information and applies logical rules to determine variants that can be excluded as causing a recessive, monogenic disease. SmartPhase can phase either all possible variant pairs in predefined genetic loci or preselected variant pairs of interest, thus keeping the focus on clinically relevant results. We compared SmartPhase to WhatsHap, one of the leading comparable phasing tools, using simulated data and a real clinical cohort of 921 patients. On both data sets, SmartPhase generated error-free predictions using our derived confidence score threshold. It outperformed WhatsHap with regard to the percentage of resolved pairs when parental genotype information is available. On the cohort data, SmartPhase enabled on average the exclusion of approximately 22% of the input variant pairs in each singleton patient and 44% in each trio patient. SmartPhase is implemented as an open-source Java tool and freely available at http://ibis.helmholtz-muenchen.de/smartphase/.

Clinical and Mutation Description of the First Iranian Cohort of Infantile Inflammatory Bowel Disease: The Iranian Primary Immunodeficiency Registry (IPIDR).

We describe a cohort of 25 Iranian patients with infantile inflammatory bowel disease (IBD), 14 (56%) of whom had monogenic defects. After proper screening, patients were referred for whole exome sequencing (WES). Four patients had missense mutations in the IL10 RA, and one had a large deletion in the IL10 RB. Four patients had mutations in genes implicated in host:microbiome homeostasis, including TTC7A deficiency, and two patients with novel mutations in the TTC37 and NOX1. We found a novel homozygous mutation in the SRP54 in a deceased patient and the heterozygous variant in his sibling with a milder phenotype. Three patients had combined immunodeficiency: one with ZAP-70 deficiency (T+B+NK-), and two with atypical SCID due to mutations in RAG1 and LIG4. One patient had a G6PC3 mutation without neutropenia. Eleven of the 14 patients with monogenic defects were results of consanguinity and only 4 of them were alive to this date.

New Findings of Immunodysregulation, Polyendocrinopathy, and Enteropathy X-linked Syndrome (IPEX); Granulomas in Lung and Duodenum.

Immune dysregulation, polyendocrinopathy and enteropathy, X-linked (IPEX) syndrome is a rare disorder caused by loss-of-function mutations in the gene forkhead box protein 3 (FOXP3). IPEX patients frequently show chronic diarrhea (enteropathy) associated with villous atrophies in the small intestine. Our case is different from this classical information in the literature, since he presented with neonatal onset inflammatory bowel disease within the first months of life accompanied by deep ulcers throughout colonic mucosa. Moreover, he developed chronic lung disease during follow-up and histopathological examinations showed granulomas in both gastrointestinal tract and lung parenchyma. Genetic analysis revealed the diagnosis of IPEX syndrome with a germline mutation in FOXP3. Thus, our study provides an unusual presentation of IPEX syndrome with colitis and granulomas presence in histopathological examinations.

An RTEL1 Mutation Links to Infantile-Onset Ulcerative Colitis and Severe Immunodeficiency.

PURPOSE: More than 50 different monogenic disorders causing inflammatory bowel disease (IBD) have been identified. Our goal was to characterize the clinical phenotype, genetic workup, and immunologic alterations in an Ashkenazi Jewish patient that presented during infancy with ulcerative colitis and unique clinical manifestations. METHODS: Immune workup and whole-exome sequencing were performed, along with Sanger sequencing for confirmation. Next-generation sequencing of the TCRB and IgH was conducted for immune repertoire analysis. Telomere length was evaluated by in-gel hybridization assay. Mass cytometry was performed on patient's peripheral blood mononuclear cells, and compared with control subjects and patients with UC. RESULTS: The patient presented in infancy with failure to thrive and dysmorphic features, consistent with a diagnosis of dyskeratosis congenita and Hoyeraal-Hreidarsson syndrome. Severe ulcerative colitis manifested in the first year of life and proceeded to the development of a primary immunodeficiency, presenting as Pneumocystis jiroveci pneumonia and hypogammaglobulinemia. Genetic studies identified a deleterious homozygous C.3791G>A missense mutation in the helicase regulator of telomere elongation 1 (RTEL1), leading to short telomeres in the index patient. Immune repertoire studies showed polyclonal T and B cell receptor distribution, while mass cytometry analysis demonstrated marked immunological alterations, including a predominance of naïve T cells, paucity of B cells, and a decrease in various innate immune subsets. CONCLUSIONS: RTEL1 mutations are associated with significant alterations in immune landscape and can manifest with infantile-onset IBD. A high index of suspicion is required in Ashkenazi Jewish families where the carriage rate of the C.3791G>A variant is high.

DEUS: an R package for accurate small RNA profiling based on differential expression of unique sequences.

SUMMARY: Despite their fundamental role in various biological processes, the analysis of small RNA sequencing data remains a challenging task. Major obstacles arise when short RNA sequences map to multiple locations in the genome, align to regions that are not annotated or underwent post-transcriptional changes which hamper accurate mapping. In order to tackle these issues, we present a novel profiling strategy that circumvents the need for read mapping to a reference genome by utilizing the actual read sequences to determine expression intensities. After differential expression analysis of individual sequence counts, significant sequences are annotated against user defined feature databases and clustered by sequence similarity. This strategy enables a more comprehensive and concise representation of small RNA populations without any data loss or data distortion. AVAILABILITY AND IMPLEMENTATION: Code and documentation of our R package at http://ibis.helmholtz-muenchen.de/deus/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

KNIME4NGS: a comprehensive toolbox for next generation sequencing analysis.

Summary: Analysis of Next Generation Sequencing (NGS) data requires the processing of large datasets by chaining various tools with complex input and output formats. In order to automate data analysis, we propose to standardize NGS tasks into modular workflows. This simplifies reliable handling and processing of NGS data, and corresponding solutions become substantially more reproducible and easier to maintain. Here, we present a documented, linux-based, toolbox of 42 processing modules that are combined to construct workflows facilitating a variety of tasks such as DNAseq and RNAseq analysis. We also describe important technical extensions. The high throughput executor (HTE) helps to increase the reliability and to reduce manual interventions when processing complex datasets. We also provide a dedicated binary manager that assists users in obtaining the modules' executables and keeping them up to date. As basis for this actively developed toolbox we use the workflow management software KNIME. Availability and Implementation: See http://ibisngs.github.io/knime4ngs for nodes and user manual (GPLv3 license). Contact: robert.kueffner@helmholtz-muenchen.de. Supplementary information: Supplementary data are available at Bioinformatics online.

TGF-ß Induction of miR-143/145 Is Associated to Exercise Response by Influencing Differentiation and Insulin Signaling Molecules in Human Skeletal Muscle.

Physical training improves insulin sensitivity and can prevent type 2 diabetes (T2D). However, approximately 20% of individuals lack a beneficial outcome in glycemic control. TGF-ß, identified as a possible upstream regulator involved in this low response, is also a potent regulator of microRNAs (miRNAs). The aim of this study was to elucidate the potential impact of TGF-ß-driven miRNAs on individual exercise response. Non-targeted long and sncRNA sequencing analyses of TGF-ß1-treated human skeletal muscle cells corroborated the effects of TGF-ß1 on muscle cell differentiation, the induction of extracellular matrix components, and identified several TGF-ß1-regulated miRNAs. qPCR validated a potent upregulation of miR-143-3p/145-5p and miR-181a2-5p by TGF-ß1 in both human myoblasts and differentiated myotubes. Healthy subjects who were overweight or obese participated in a supervised 8-week endurance training intervention (n = 40) and were categorized as responder or low responder in glycemic control based on fold change ISIMats (≥+1.1 or <+1.1, respectively). In skeletal muscle biopsies of low responders, TGF-ß signaling and miR-143/145 cluster levels were induced by training at much higher rates than among responders. Target-mining revealed HDACs, MYHs, and insulin signaling components INSR and IRS1 as potential miR-143/145 cluster targets. All these targets were down-regulated in TGF-ß1-treated myotubes. Transfection of miR-143-3p/145-5p mimics in differentiated myotubes validated MYH1, MYH4, and IRS1 as miR-143/145 cluster targets. Elevated TGF-ß signaling and miR-143/145 cluster induction in skeletal muscle of low responders might obstruct improvements in insulin sensitivity by training in two ways: by a negative impact of miR-143-3p on muscle cell fusion and myofiber functionality and by directly impairing insulin signaling via a reduction in INSR by TGF-ß and finetuned IRS1 suppression by miR-143-3p.

CARMIL2 Deficiency Presenting as Very Early Onset Inflammatory Bowel Disease.

BACKGROUND: Children with very early onset inflammatory bowel diseases (VEO-IBD) often have a refractory and severe disease course. A significant number of described VEO-IBD-causing monogenic disorders can be attributed to defects in immune-related genes. The diagnosis of the underlying primary immunodeficiency (PID) often has critical implications for the treatment of patients with IBD-like phenotypes. METHODS: To identify the molecular etiology in 5 patients from 3 unrelated kindred with IBD-like symptoms, we conducted whole exome sequencing. Immune workup confirmed an underlying PID. RESULTS: Whole exome sequencing revealed 3 novel CARMIL2 loss-of-function mutations in our patients. Immunophenotyping of peripheral blood mononuclear cells showed reduction of regulatory and effector memory T cells and impaired B cell class switching. The T cell proliferation and activation assays confirmed defective responses to CD28 costimulation, consistent with CARMIL2 deficiency. CONCLUSION: Our study highlights that human CARMIL2 deficiency can manifest with IBD-like symptoms. This example illustrates that early diagnosis of underlying PID is crucial for the treatment and prognosis of children with VEO-IBD.