Understanding the genetics of neuropsychiatric disorders: the potential role of genomic regulatory blocks.

Recent genome-wide association studies have identified numerous loci associated with neuropsychiatric disorders. The majority of these are in non-coding regions, and are commonly assigned to the nearest gene along the genome. However, this approach neglects the three-dimensional organisation of the genome, and the fact that the genome contains arrays of extremely conserved non-coding elements termed genomic regulatory blocks (GRBs), which can be utilized to detect genes under long-range developmental regulation. Here we review a GRB-based approach to assign loci in non-coding regions to potential target genes, and apply it to reanalyse the results of one of the largest schizophrenia GWAS (SWG PGC, 2014). We further apply this approach to GWAS data from two related neuropsychiatric disorders-autism spectrum disorder and bipolar disorder-to show that it is applicable to developmental disorders in general. We find that disease-associated SNPs are overrepresented in GRBs and that the GRB model is a powerful tool for linking these SNPs to their correct target genes under long-range regulation. Our analysis identifies novel genes not previously implicated in schizophrenia and corroborates a number of predicted targets from the original study. The results are available as an online resource in which the genomic context and the strength of enhancer-promoter associations can be browsed for each schizophrenia-associated SNP.

Gut Microbiota beyond Bacteria-Mycobiome, Virome, Archaeome, and Eukaryotic Parasites in IBD.

The human microbiota is a diverse microbial ecosystem associated with many beneficial physiological functions as well as numerous disease etiologies. Dominated by bacteria, the microbiota also includes commensal populations of fungi, viruses, archaea, and protists. Unlike bacterial microbiota, which was extensively studied in the past two decades, these non-bacterial microorganisms, their functional roles, and their interaction with one another or with host immune system have not been as widely explored. This review covers the recent findings on the non-bacterial communities of the human gastrointestinal microbiota and their involvement in health and disease, with particular focus on the pathophysiology of inflammatory bowel disease.

IntPred: a structure-based predictor of protein-protein interaction sites.

MOTIVATION: Protein-protein interactions are vital for protein function with the average protein having between three and ten interacting partners. Knowledge of precise protein-protein interfaces comes from crystal structures deposited in the Protein Data Bank (PDB), but only 50% of structures in the PDB are complexes. There is therefore a need to predict protein-protein interfaces in silico and various methods for this purpose. Here we explore the use of a predictor based on structural features and which exploits random forest machine learning, comparing its performance with a number of popular established methods. RESULTS: On an independent test set of obligate and transient complexes, our IntPred predictor performs well (MCC = 0.370, ACC = 0.811, SPEC = 0.916, SENS = 0.411) and compares favourably with other methods. Overall, IntPred ranks second of six methods tested with SPPIDER having slightly better overall performance (MCC = 0.410, ACC = 0.759, SPEC = 0.783, SENS = 0.676), but considerably worse specificity than IntPred. As with SPPIDER, using an independent test set of obligate complexes enhanced performance (MCC = 0.381) while performance is somewhat reduced on a dataset of transient complexes (MCC = 0.303). The trade-off between sensitivity and specificity compared with SPPIDER suggests that the choice of the appropriate tool is application-dependent. AVAILABILITY AND IMPLEMENTATION: IntPred is implemented in Perl and may be downloaded for local use or run via a web server at www.bioinf.org.uk/intpred/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

BCL11B mutations in patients affected by a neurodevelopmental disorder with reduced type 2 innate lymphoid cells.

The transcription factor BCL11B is essential for development of the nervous and the immune system, and Bcl11b deficiency results in structural brain defects, reduced learning capacity, and impaired immune cell development in mice. However, the precise role of BCL11B in humans is largely unexplored, except for a single patient with a BCL11B missense mutation, affected by multisystem anomalies and profound immune deficiency. Using massively parallel sequencing we identified 13 patients bearing heterozygous germline alterations in BCL11B. Notably, all of them are affected by global developmental delay with speech impairment and intellectual disability; however, none displayed overt clinical signs of immune deficiency. Six frameshift mutations, two nonsense mutations, one missense mutation, and two chromosomal rearrangements resulting in diminished BCL11B expression, arose de novo. A further frameshift mutation was transmitted from a similarly affected mother. Interestingly, the most severely affected patient harbours a missense mutation within a zinc-finger domain of BCL11B, probably affecting the DNA-binding structural interface, similar to the recently published patient. Furthermore, the most C-terminally located premature termination codon mutation fails to rescue the progenitor cell proliferation defect in hippocampal slice cultures from Bcl11b-deficient mice. Concerning the role of BCL11B in the immune system, extensive immune phenotyping of our patients revealed alterations in the T cell compartment and lack of peripheral type 2 innate lymphoid cells (ILC2s), consistent with the findings described in Bcl11b-deficient mice. Unsupervised analysis of 102 T lymphocyte subpopulations showed that the patients clearly cluster apart from healthy children, further supporting the common aetiology of the disorder. Taken together, we show here that mutations leading either to BCL11B haploinsufficiency or to a truncated BCL11B protein clinically cause a non-syndromic neurodevelopmental delay. In addition, we suggest that missense mutations affecting specific sites within zinc-finger domains might result in distinct and more severe clinical outcomes.

The Role of Vitamin D in Inflammatory Bowel Disease - Assessing Therapeutic and Preventive Potential of Supplementation and Food Fortification.

Inflammatory bowel diseases are a group of chronic inflammatory conditions that affect gastrointestinal tract due to inapt and continuous immune activation in response to a myriad of predisposing factors (most notably genetics, environmental impact and gut microbiota composition). It has been shown that vitamin D status can also play a role in the disease pathogenesis, as its deficiency is commonly observed in two major forms of inflammatory bowel diseases - Crohn's disease and ulcerative colitis. Mounting evidence supports the concept of intricate relationship between gut dysbiosis and vitamin D metabolism, while suboptimal levels of this vitamin have been linked to increased clinical disease relapse rates, inadequate response to drugs, as well as decreased quality of life in patients with Crohn's disease and ulcerative colitis. Consequently, the pertinent question is whether increased vitamin D supplementation and (on a population level) food fortification may bring significant benefit to the affected individuals. In this short review we discuss the synthesis, functions, status and food sources of vitamin D, appraise biotechnological facets of vitamin D status analysis and food fortification, and concentrate on novel developments in the field that describe its influence on intestinal microbiota and inflammatory bowel disease.

GenomicInteractions: An R/Bioconductor package for manipulating and investigating chromatin interaction data.

BACKGROUND: Precise quantitative and spatiotemporal control of gene expression is necessary to ensure proper cellular differentiation and the maintenance of homeostasis. The relationship between gene expression and the spatial organisation of chromatin is highly complex, interdependent and not completely understood. The development of experimental techniques to interrogate both the higher-order structure of chromatin and the interactions between regulatory elements has recently lead to important insights on how gene expression is controlled. The ability to gain these and future insights is critically dependent on computational tools for the analysis and visualisation of data produced by these techniques. RESULTS AND CONCLUSION: We have developed GenomicInteractions, a freely available R/Bioconductor package designed for processing, analysis and visualisation of data generated from various types of chromosome conformation capture experiments. The package allows the easy annotation and summarisation of large genome-wide datasets at both the level of individual interactions and sets of genomic features, and provides several different methods for interrogating and visualising this type of data. We demonstrate this package's utility by showing example analyses performed on interaction datasets generated using Hi-C and ChIA-PET.

Integrative analysis in head and neck cancer reveals distinct role of miRNome and methylome as tumour epigenetic drivers.

Head and neck cancer is the sixth most common malignancy worldwide, with the relatively low 5-year survival rate, mainly because it is diagnosed at a late stage. Infection with HPV is a well known aetiology, which affects the nature of these cancers and patients' survival. Besides, it is considered that the main driving force for this type of cancer could be epigenetics. In this study we aimed to find potential epigenetic biomarkers, by integrating miRNome, methylome, and transcriptome analyses. From the fresh head and neck cancer tissue samples, we chose a group for miRNome, methylome and transcriptome profiling, in comparison to adequate control samples. Bioinformatics analyses are performed in R v4.2.2. Count normalisation and group differential expression for mRNA and the previously obtained miRNA count data was performed with DESeq2 v1.36. Gene set enrichment analysis was performed and visualised using gProfiler2 v0.2.1 Identification of miRNA targets was performed by querying in miRTarBase using multiMiR v1.18.0. Annotation of CpG sites merging into islands was obtained from RnBeads.hg19 v1.28.0. package. For the integrative analysis we performed kmeans clustering using stats v4.2.2 package, using 8-12 clusters and nstart 100. We found that transcriptome analysis divides samples into cancers and controls clusters, with no relation to HPV status or cancer anatomical location. Differentially expressed genes (n = 2781) were predominantly associated with signalling pathways of tumour progression. We identified a cluster of genes under the control of the transcription factor E2F that are significantly underexpressed in cancer tissue, as well as T cell immunity genes and genes related to regulation of transcription. Among overexpressed genes in tumours we found those that belong to cell cycle regulation and vasculature. A small number of genes were found significantly differentially expressed in HPV-positive versus HPV-negative tumours (for example NEFH, ZFR2, TAF7L, ZNF541, and TYMS). In this comprehensive study on an overlapping set of samples where the integration of miRNome, methylome and transcriptome analysis were performed for head and neck cancer, we demonstrated that the majority of genes were associated exclusively with miRNome or methylome and, to a lesser extent, under the control of both epigenetic mechanisms.

cfDNA methylation in liquid biopsies as potential testicular seminoma biomarker.

Background: Seminoma is a testicular tumor type, routinely diagnosed after orchidectomy. As cfDNA represents a source of minimally invasive seminoma patient management, this study aimed to investigate whether cfDNA methylation of six genes from liquid biopsies, have potential as novel seminoma biomarkers. Materials & methods: cfDNA methylation from liquid biopsies was assessed by pyrosequencing and compared with healthy volunteers' samples. Results: Detailed analysis revealed specific CpGs as possible seminoma biomarkers, but receiver operating characteristic curve analysis showed modest diagnostic performance. In an analysis of panels of statistically significant CpGs, two DNA methylation panels emerged as potential seminoma screening panels, one in blood CpG8/CpG9/CpG10 (KITLG) and the other in seminal plasma CpG1(MAGEC2)/CpG1(OCT3/4). Conclusion: The presented data promote the development of liquid biopsy epigenetic biomarkers in the screening of seminoma patients.

Seminoma belongs to testicular cancer, which represents a common malignancy among men of reproductive age. Diagnosis of seminoma is a multistep process that also includes checking tumor biomarkers from blood. However, these biomarkers are not specific for seminoma and to conclude a definite diagnosis of seminoma immunohistochemical analysis is needed, which requires the removal of a whole or partial testicle. Therefore, there is a need for novel, noninvasive biomarkers. cfDNA is the most extensively investigated source of minimally invasive tumor markers. Therefore, this study investigated cfDNA methylation of six genes as potential noninvasive biomarkers for the management of seminoma patients. By examining CpG sites of selected genes by pyrosequencing, the authors detected significant differences. However, receiver operating characteristic curve analysis showed modest results. Therefore, the authors tested possible panels of significantly different CpGs and detected two possible DNA methylation panels for seminoma screening. These findings suggest the further investigation of possible epigenetic biomarkers for seminoma patient management from liquid biopsies.

Gut microbiota in mucosa and feces of newly diagnosed, treatment-naïve adult inflammatory bowel disease and irritable bowel syndrome patients.

The knowledge on how gut microbes contribute to the inflammatory bowel disease (IBD) at the onset of disease is still scarce. We compared gut microbiota in newly diagnosed, treatment-naïve adult IBD (Crohn's disease (CD) and ulcerative colitis (UC)) to irritable bowel syndrome (IBS) patients and healthy group. Mucosal and fecal microbiota of 49 patients (13 UC, 10 CD, and 26 IBS) before treatment initiation, and fecal microbiota of 12 healthy subjects was characterized by 16S rRNA gene sequencing. Mucosa was sampled at six positions, from terminal ileum to rectum. We demonstrate that mucosal microbiota is spatially homogeneous, cannot be differentiated based on the local inflammation status and yet provides bacterial footprints superior to fecal in discriminating disease phenotypes. IBD groups showed decreased bacterial diversity in mucosa at all taxonomic levels compared to IBS. In CD and UC, Dialister was significantly increased, and expansion of Haemophilus and Propionibacterium characterized UC. Compared to healthy individuals, fecal microbiota of IBD and IBS patients had increased abundance of Proteobacteria, Enterobacteriaceae, in particular. Shift toward reduction of Adlercreutzia and butyrate-producing taxa was found in feces of IBD patients. Microbiota alterations detected in newly diagnosed treatment-naïve adult patients indicate that the microbiota changes are set and detectable at the disease onset and likely have a discerning role in IBD pathophysiology. Our results justify further investigation of the taxa discriminating between disease groups, such as H. parainfluenzae, R. gnavus, Turicibacteriaceae, Dialister, and Adlercreutzia as potential biomarkers of the disease.

Characterization of pathogenic germline mutations in human protein kinases.

BACKGROUND: Protein Kinases are a superfamily of proteins involved in crucial cellular processes such as cell cycle regulation and signal transduction. Accordingly, they play an important role in cancer biology. To contribute to the study of the relation between kinases and disease we compared pathogenic mutations to neutral mutations as an extension to our previous analysis of cancer somatic mutations. First, we analyzed native and mutant proteins in terms of amino acid composition. Secondly, mutations were characterized according to their potential structural effects and finally, we assessed the location of the different classes of polymorphisms with respect to kinase-relevant positions in terms of subfamily specificity, conservation, accessibility and functional sites. RESULTS: Pathogenic Protein Kinase mutations perturb essential aspects of protein function, including disruption of substrate binding and/or effector recognition at family-specific positions. Interestingly these mutations in Protein Kinases display a tendency to avoid structurally relevant positions, what represents a significant difference with respect to the average distribution of pathogenic mutations in other protein families. CONCLUSIONS: Disease-associated mutations display sound differences with respect to neutral mutations: several amino acids are specific of each mutation type, different structural properties characterize each class and the distribution of pathogenic mutations within the consensus structure of the Protein Kinase domain is substantially different to that for non-pathogenic mutations. This preferential distribution confirms previous observations about the functional and structural distribution of the controversial cancer driver and passenger somatic mutations and their use as a proxy for the study of the involvement of somatic mutations in cancer development.

The Role of Gut, Vaginal, and Urinary Microbiome in Urinary Tract Infections: From Bench to Bedside.

The current paradigm of urinary tract infection (UTI) pathogenesis takes into account the contamination of the periurethral space by specific uropathogens residing in the gut, which is followed by urethral colonization and pathogen ascension to the urinary bladder. Consequently, studying the relationship between gut microbiota and the subsequent development of bacteriuria and UTI represents an important field of research. However, the well-established diagnostic and therapeutic paradigm for urinary tract infections (UTIs) has come into question with the discovery of a multifaceted, symbiotic microbiome in the healthy urogenital tract. More specifically, emerging data suggest that vaginal dysbiosis may result in Escherichia coli colonization and prompt recurrent UTIs, while urinary microbiome perturbations may precede the development of UTIs and other pathologic conditions of the urinary system. The question is whether these findings can be exploited for risk reduction and treatment purposes. This review aimed to appraise the three aforementioned specific microbiomes regarding their potential influence on UTI development by focusing on the recent studies in the field and assessing the potential linkages between these different niches, as well as evaluating the state of translational research for novel therapeutic and preventative approaches.

An integrated approach to the interpretation of single amino acid polymorphisms within the framework of CATH and Gene3D.

BACKGROUND: The phenotypic effects of sequence variations in protein-coding regions come about primarily via their effects on the resulting structures, for example by disrupting active sites or affecting structural stability. In order better to understand the mechanisms behind known mutant phenotypes, and predict the effects of novel variations, biologists need tools to gauge the impacts of DNA mutations in terms of their structural manifestation. Although many mutations occur within domains whose structure has been solved, many more occur within genes whose protein products have not been structurally characterized. RESULTS: Here we present 3DSim (3D Structural Implication of Mutations), a database and web application facilitating the localization and visualization of single amino acid polymorphisms (SAAPs) mapped to protein structures even where the structure of the protein of interest is unknown. The server displays information on 6514 point mutations, 4865 of them known to be associated with disease. These polymorphisms are drawn from SAAPdb, which aggregates data from various sources including dbSNP and several pathogenic mutation databases. While the SAAPdb interface displays mutations on known structures, 3DSim projects mutations onto known sequence domains in Gene3D. This resource contains sequences annotated with domains predicted to belong to structural families in the CATH database. Mappings between domain sequences in Gene3D and known structures in CATH are obtained using a MUSCLE alignment. 1210 three-dimensional structures corresponding to CATH structural domains are currently included in 3DSim; these domains are distributed across 396 CATH superfamilies, and provide a comprehensive overview of the distribution of mutations in structural space. CONCLUSION: The server is publicly available at http://3DSim.bioinfo.cnio.es/. In addition, the database containing the mapping between SAAPdb, Gene3D and CATH is available on request and most of the functionality is available through programmatic web service access.

Methodology challenges in studying human gut microbiota - effects of collection, storage, DNA extraction and next generation sequencing technologies.

The information on microbiota composition in the human gastrointestinal tract predominantly originates from the analyses of human faeces by application of next generation sequencing (NGS). However, the detected composition of the faecal bacterial community can be affected by various factors including experimental design and procedures. This study evaluated the performance of different protocols for collection and storage of faecal samples (native and OMNIgene.GUT system) and bacterial DNA extraction (MP Biomedicals, QIAGEN and MO BIO kits), using two NGS platforms for 16S rRNA gene sequencing (Ilumina MiSeq and Ion Torrent PGM). OMNIgene.GUT proved as a reliable and convenient system for collection and storage of faecal samples although favouring Sutterella genus. MP provided superior DNA yield and quality, MO BIO depleted Gram positive organisms while using QIAGEN with OMNIgene.GUT resulted in greatest variability compared to other two kits. MiSeq and IT platforms in their supplier recommended setups provided comparable reproducibility of donor faecal microbiota. The differences included higher diversity observed with MiSeq and increased capacity of MiSeq to detect Akkermansia muciniphila, [Odoribacteraceae], Erysipelotrichaceae and Ruminococcaceae (primarily Faecalibacterium prausnitzii). The results of our study could assist the investigators using NGS technologies to make informed decisions on appropriate tools for their experimental pipelines.

The mystery of extreme non-coding conservation.

Regions of several dozen to several hundred base pairs of extreme conservation have been found in non-coding regions in all metazoan genomes. The distribution of these elements within and across genomes has suggested that many have roles as transcriptional regulatory elements in multi-cellular organization, differentiation and development. Currently, there is no known mechanism or function that would account for this level of conservation at the observed evolutionary distances. Previous studies have found that, while these regions are under strong purifying selection, and not mutational coldspots, deletion of entire regions in mice does not necessarily lead to identifiable changes in phenotype during development. These opposing findings lead to several questions regarding their functional importance and why they are under strong selection in the first place. In this perspective, we discuss the methods and techniques used in identifying and dissecting these regions, their observed patterns of conservation, and review the current hypotheses on their functional significance.

Compensated pathogenic deviations.

Deleterious or 'disease-associated' mutations are mutations that lead to disease with high phenotype penetrance: they are inherited in a simple Mendelian manner, or, in the case of cancer, accumulate in somatic cells leading directly to disease. However, in some cases, the amino acid that is substituted resulting in disease is the wild-type native residue in the functionally equivalent protein in another species. Such examples are known as 'compensated pathogenic deviations' (CPDs) because, somewhere in the second species, there must be compensatory mutations that allow the protein to function normally despite having a residue which would cause disease in the first species. Depending on the nature of the mutations, compensation can occur in the same protein, or in a different protein with which it interacts. In principle, compensation can be achieved by a single mutation (most probably structurally close to the CPD), or by the cumulative effect of several mutations. Although it is clear that these effects occur in proteins, compensatory mutations are also important in RNA potentially having an impact on disease. As a much simpler molecule, RNA provides an interesting model for understanding mechanisms of compensatory effects, both by looking at naturally occurring RNA molecules and as a means of computational simulation. This review surveys the rather limited literature that has explored these effects. Understanding the nature of CPDs is important in understanding traversal along fitness landscape valleys in evolution. It could also have applications in treating diseases that result from such mutations.

Compensated pathogenic deviations: analysis of structural effects.

Pathogenic deviations (PDs) in humans are disease-causing missense mutations. However, in some cases, these disease-associated residues occur as the wild-type residues in functionally equivalent proteins in other species and these cases are termed 'compensated pathogenic deviations' (CPDs). The lack of pathogenicity in a non-human protein is presumed to be explained in most cases by the presence of compensatory mutations, most commonly within the same protein. Identification of structural features of CPDs and detection of specific compensatory events will help us to understand traversal along fitness landscape valleys in protein evolution. We divided mutations listed in the OMIM (Online Mendelian Inheritance in Man) database into PD and CPD data sets and performed two independent analyses: (i) We searched for potential compensatory mutations spatially close to the CPDs and, (ii) using our SAAPdb database, we examined likely structural effects to try to explain why mutations are pathogenic, comparing PDs and CPDs. Our data sets were obtained from a set of 245 human proteins of known structure and contained a total of 2328 mutations of which 453 (from 85 structures) were seen to be compensated in at least one functionally equivalent protein in another (non-human) species. Structural analysis results confirm previous findings that CPDs are, on average, 'milder' in their likely structural effects than uncompensated PDs and tend to be on the protein surface. We also showed that the residues surrounding the CPD residue in the folded protein are more often mutated than the residues surrounding an uncompensated mutation, supporting the hypothesis that compensation is largely a result of structurally local mutations.