Comparative Functional Genomics Research Articles

Compensatory evolution in NusG improves fitness of drug-resistant M. tuberculosis

Drug-resistant bacteria are emerging as a global threat, despite frequently being less fit than their drug-susceptible ancestors1–8. Here we sought to define the mechanisms that drive or buffer the fitness cost of rifampicin resistance (RifR) in the bacterial pathogen Mycobacterium tuberculosis (Mtb). Rifampicin inhibits RNA polymerase (RNAP) and is a cornerstone of modern short-course tuberculosis therapy9,10. However, RifR Mtb accounts for one-quarter of all deaths due to drug-resistant bacteria11,12. We took a comparative functional genomics approach to define processes that are differentially vulnerable to CRISPR interference (CRISPRi) inhibition in RifR Mtb. Among other hits, we found that the universally conserved transcription factor NusG is crucial for the fitness of RifR Mtb. In contrast to its role in Escherichia coli, Mtb NusG has an essential RNAP pro-pausing function mediated by distinct contacts with RNAP and the DNA13. We find this pro-pausing NusG–RNAP interface to be under positive selection in clinical RifR Mtb isolates. Mutations in the NusG–RNAP interface reduce pro-pausing activity and increase fitness of RifR Mtb. Collectively, these results define excessive RNAP pausing as a molecular mechanism that drives the fitness cost of RifR in Mtb, identify a new mechanism of compensation to overcome this cost, suggest rational approaches to exacerbate the fitness cost, and, more broadly, could inform new therapeutic approaches to develop drug combinations to slow the evolution of RifR in Mtb.

PlantFUNCO: Integrative Functional Genomics Database Reveals Clues into Duplicates Divergence Evolution.

Evolutionary epigenomics and, more generally, evolutionary functional genomics, are emerging fields that study how non-DNA-encoded alterations in gene expression regulation are an important form of plasticity and adaptation. Previous evidence analyzing plants' comparative functional genomics has mostly focused on comparing same assay-matched experiments, missing the power of heterogeneous datasets for conservation inference. To fill this gap, we developed PlantFUN(ctional)CO(nservation) database, which is constituted by several tools and two main resources: interspecies chromatin states and functional genomics conservation scores, presented and analyzed in this work for three well-established plant models (Arabidopsis thaliana, Oryza sativa, and Zea mays). Overall, PlantFUNCO elucidated evolutionary information in terms of cross-species functional agreement. Therefore, providing a new complementary comparative-genomics source for assessing evolutionary studies. To illustrate the potential applications of this database, we replicated two previously published models predicting genetic redundancy in A. thaliana and found that chromatin states are a determinant of paralogs degree of functional divergence. These predictions were validated based on the phenotypes of mitochondrial alternative oxidase knockout mutants under two different stressors. Taking all the above into account, PlantFUNCO aim to leverage data diversity and extrapolate molecular mechanisms findings from different model organisms to determine the extent of functional conservation, thus, deepening our understanding of how plants epigenome and functional noncoding genome have evolved. PlantFUNCO is available at https://rocesv.github.io/PlantFUNCO.

MAT gene deregulation and liver cancer

Rodent models with low heterogeneity premalignant and malignant lesions provide a valuable contribution to hepatocellular carcinoma (HCC) pathogenesis in the field of research. Studies have been performed in transgenic mice, and rodent strains on signaling pathways deregulation in hepatocarcinogenesis with varying susceptibilities, and human HCC subtypes. Because ethionine, an antagonist of methionine, causes cancer and methyl-deficient diets cause steatohepatitis, followed by the development of HCC, researchers began to look into mechanisms regulating availability of S-adenosylmethionine (SAM) and its role in liver injury, including HCC development. Cirrhotic livers have decreased methionine adenosyltransferase (MATI/III) levels due to oxidation of cysteine residues in the ATP-binding site. MATII upregulation is inhibited by its reaction product, leading to MATI/III downregulation. Decreased MATI/III: MATII activity ratio, along with increased SAM decarboxylation for polyamine synthesis, results in decreased SAM. Some of the molecular pathways associated with specific cancer phenotypes are evolutionarily conserved, according to previous comparative functional genomics research. Cell cycle regulators (WNT/FZD, PI3K/AKT, and MAPK) and key genes (MAPK, IKK/NF-kB) are upregulated in human and rodent HCC progression. Pseudoprognostic markers for HCC include MAT1A/MAT2A switch. Changes in MAT1A expression and SAM levels occur during hepatocarcinogenesis. No evidence of SAM’s therapeutic effect on HCC has been found. The effects of stable MAT1A overexpression or MAT2A/MAT2B inhibition in vivo should be studied first. MAT2A or MAT2B-silenced HepG2 cells proliferate less in leptin. However, intracellular viral vector transduction in vivo has many limitations. This review interprets recent advances in SAM metabolism deregulation in liver injury predisposing to HCC and determining HCC prognosis.

Candidate Regulatory Genes for Hindlimb Development in the Embryos of the Chinese Alligator (Alligator sinensis).

Crocodilians, which are a kind of animal secondary adaptation to an aquatic environment, their hindlimb can provide the power needed to engage in various life activities, even in low-oxygen water environments. The development of limbs is an important aspect of animal growth and development, as it is closely linked to body movement, support, heat production, and other critical functions. For the Chinese alligator, the hindlimb is one of the main sources of power, and its development and differentiation will directly influence the survival ability in the wild. Furthermore, a better understanding of the hindlimb developmental process will provide data support for the comparative evolutionary and functional genomics of crocodilians. In this study, the expression levels of genes related to hindlimb development in the Chinese alligator embryos during fetal development (on days 29, 35, 41, and 46) were investigated through transcriptome analysis. A total of 1675 differentially expressed genes (DEGs) at different stages were identified by using limma software. These DEGs were then analyzed using weighted correlation network analysis (WGCNA), and 4 gene expression modules and 20 hub genes were identified that were associated with the development of hindlimbs in the Chinese alligator at different periods. The results of GO enrichment and hub gene expression showed that the hindlimb development of the Chinese alligator embryos involves the development of the embryonic structure, nervous system, and hindlimb muscle in the early stage (H29) and the development of metabolic capacity occurs in the later stage (H46). Additionally, the enrichment results showed that the AMPK signaling pathway, calcium signaling pathway, HIF-1 signaling pathway, and neuroactive ligand-receptor interaction are involved in the development of the hindlimb of the Chinese alligator. Among these, the HIF-1 signaling pathway and neuroactive ligand-receptor interaction may be related to the adaptation of Chinese alligators to low-oxygen environments. Additionally, five DEGs (CAV1, IRS2, LDHA, LDB3, and MYL3) were randomly selected for qRT-PCR to verify the transcriptome results. It is expected that further research on these genes will help us to better understand the process of embryonic hindlimb development in the Chinese alligator.

In silico Mining of Protein-coding and Non-coding RNA (ncRNA) Specific Genes in Exotic versus Indigenous Gaddi Dogs

Background: Comparative functional genomics will aid in the molecular identification of diverse dog breeds. Methods: The current proposal aimed at conducting a differential study between the genomes of exotic canines (Labrador, Basenji, Tasha-Boxer breed, Mischka breed German Shepherd, Zoey breed Great Dane) and indigenous (Gaddi) breeds through whole genome annotation. Results: The prediction analysis by GeneMark tool yielded an average of 46484 transcripts, in Gaddi dogs and exotic breeds ranging from 29669 to 30956. A total of 57 miRNAs were discovered in exotic breeds and 22 miRNAs in Gaddi dogs, 18 are common in both, while 4 were unique to Gaddi dogs. lncRNA was predicted using the PLEK, CPAT, and LGC tools, resulting in 3201, 396, and 4188 noncoding sequences in exotic breeds, respectively. Approximately, 31 thousand lncRNA transcripts were identified in the Gaddi dog genome. Microsatellites were found to be distributed through approximately 0.3% of both genomes. The average island length of CpG ranged between 24246.48 to 28080.66 in exotic breeds at chromosome level assembly and 697.15 in indigenous Gaddi dogs at contig level assembly. The predicted protein-coding genes were subjected to pathway analysis by DAVID and PANTHER. Five genes that are expressed in the blood (INSL3, CLDN3, MYH1, CLN5, and GALC) were selected for validation through qPCR. The results indicated that the genes were expressed in both groups. Conclusion: The study is the maiden report on the comparative genome analysis between indigenous Gaddi dogs and exotic dog breeds. The findings set the stage for further research into the known and novel genes, which might be employed as biomarkers for disease diagnosis and to investigate their regulatory role.

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor.

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome neurofibromatosis type 1 (NF1), MPNSTs are the most common malignancy and the leading cause of death. These rare and aggressive soft-tissue sarcomas offer a stark future, with 5-year disease-free survival rates of 34-60%. Treatment options for individuals with MPNSTs are disappointingly limited, with disfiguring surgery being the foremost treatment option. Many once-promising therapies such as tipifarnib, an inhibitor of Ras signaling, have failed clinically. Likewise, phase II clinical trials with erlotinib, which targets the epidermal growth factor (EFGR), and sorafenib, which targets the vascular endothelial growth factor receptor (VEGF), platelet-derived growth factor receptor (PDGF), and Raf, in combination with standard chemotherapy, have also failed to produce a response in patients. In recent years, functional genomic screening methods combined with genetic profiling of cancer cell lines have proven useful for identifying essential cytoplasmic signaling pathways and the development of target-specific therapies. In the case of rare tumor types, a variation of this approach known as cross-species comparative oncogenomics is increasingly being used to identify novel therapeutic targets. In cross-species comparative oncogenomics, genetic profiling and functional genomics are performed in genetically engineered mouse (GEM) models and the results are then validated in the rare human specimens and cell lines that are available. This paper describes how to identify candidate driver gene mutations in human and mouse MPNST cells using whole exome sequencing (WES). We then describe how to perform genome-scale shRNA screens to identify and compare critical signaling pathways in mouse and human MPNST cells and identify druggable targets in these pathways. These methodologies provide an effective approach to identifying new therapeutic targets in a variety of human cancer types.

Domain Architecture Based Methods for Comparative Functional Genomics Toward Therapeutic Drug Target Discovery.

Genes duplicate, mutate, recombine, fuse or fission to produce new genes, or when genes are formed from de novo, novel functions arise during evolution. Researchers have tried to quantify the causes of these molecular diversification processes to know how these genes increase molecular complexity over a period of time, for instance protein domain organization. In contrast to global sequence similarity, protein domain architectures can capture key structural and functional characteristics, making them better proxies for describing functional equivalence. In Prokaryotes and eukaryotes it has proven that, domain designs are retained over significant evolutionary distances. Protein domain architectures are now being utilized to categorize and distinguish evolutionarily related proteins and find homologs among species that are evolutionarily distant from one another. Additionally, structural information stored in domain structures has accelerated homology identification and sequence search methods. Tools for functional protein annotation have been developed to discover, protein domain content, domain order, domain recurrence, and domain position as all these contribute to the prediction of protein functional accuracy. In this review, an attempt is made to summarise facts and speculations regarding the use of protein domain architecture and modularity to identify possible therapeutic targets among cellular activities based on the understanding their linked biological processes.

Phylogenetic modeling of enhancer shifts in African mole-rats reveals regulatory changes associated with tissue-specific traits.

Changes in gene regulation are thought to underlie most phenotypic differences between species. For subterranean rodents such as the naked mole-rat, proposed phenotypic adaptations include hypoxia tolerance, metabolic changes, and cancer resistance. However, it is largely unknown what regulatory changes may associate with these phenotypic traits, and whether these are unique to the naked mole-rat, the mole-rat clade, or are also present in other mammals. Here, we investigate regulatory evolution in the heart and liver from two African mole-rat species and two rodent outgroups using genome-wide epigenomic profiling. First, we adapted and applied a phylogenetic modeling approach to quantitatively compare epigenomic signals at orthologous regulatory elements and identified thousands of promoter and enhancer regions with differential epigenomic activity in mole-rats. These elements associate with known mole-rat adaptations in metabolic and functional pathways and suggest candidate genetic loci that may underlie mole-rat innovations. Second, we evaluated ancestral and species-specific regulatory changes in the study phylogeny and report several candidate pathways experiencing stepwise remodeling during the evolution of mole-rats, such as the insulin and hypoxia response pathways. Third, we report nonorthologous regulatory elements overlap with lineage-specific repetitive elements and appear to modify metabolic pathways by rewiring of HNF4 and RAR/RXR transcription factor binding sites in mole-rats. These comparative analyses reveal how mole-rat regulatory evolution informs previously reported phenotypic adaptations. Moreover, the phylogenetic modeling framework we propose here improves upon the state of the art by addressing known limitations of inter-species comparisons of epigenomic profiles and has broad implications in the field of comparative functional genomics.

Comparative Functional Genomics Studies for Understanding the Hypothetical Proteins in Mycobacterium Tuberculosis Variant Microti 12

Comparative Functional Genomics Studies for Understanding the Hypothetical Proteins in Mycobacterium Tuberculosis Variant Microti 12

Analysis of multipartite bacterial genomes using alignment free and alignment-based pipelines

Since the discovery of second chromosome in Rhodobacter sphaeroides 2.4.1 in 1989, multipartite genomes have been reported in over three hundred bacterial species under nine different phyla. This has shattered the unipartite (single chromosome) genome dogma in bacteria. Since then, many questions on various aspects of multipartite genomes in bacteria have been addressed. However, our understanding of how multipartite genomes emerge and evolve is still lacking. Importantly, the knowledge of genetic factors underlying the differences in multipartite and single-chromosome genomes is lacking. In this work, we have performed comparative evolutionary and functional genomics analyses to identify molecular factors that discriminate multipartite from unipartite bacteria, with the goal to decipher taxon-specific factors, and those that are prevalent across the taxa, underlying these traits. We assessed the roles of evolutionary mechanisms, specifically gene gain, in driving the divergence of bacteria with single and multiple chromosomes. In addition, we performed functional genomic analysis to garner support for our findings from comparative evolutionary analysis. We found genes such as those encoding conserved hypothetical proteins in Deinococcus radiodurans R1, and putative phage phi-C31 gp36 major capsid like and hypothetical proteins in Rhodobacter sphaeroides 2.4.1, which are located on accessory chromosomes in these bacteria but were not found in the inferred ancestral sequences, and on the primary chromosomes, as well as were not found in their closest relatives with single chromosome within the same clade. Our study shines a new light on the potential roles of the secondary chromosomes in helping bacteria with multipartite genomes to adapt to specialized environments or growth conditions.

Comparative functional genomics identifies unique molecular features of EPSCs.

Extended pluripotent or expanded potential stem cells (EPSCs) possess superior developmental potential to embryonic stem cells (ESCs). However, the molecular underpinning of EPSC maintenance in vitro is not well defined. We comparatively studied transcriptome, chromatin accessibility, active histone modification marks, and relative proteomes of ESCs and the two well-established EPSC lines to probe the molecular foundation underlying EPSC developmental potential. Despite some overlapping transcriptomic and chromatin accessibility features, we defined sets of molecular signatures that distinguish EPSCs from ESCs in transcriptional and translational regulation as well as metabolic control. Interestingly, EPSCs show similar reliance on pluripotency factors Oct4, Sox2, and Nanog for self-renewal as ESCs. Our study provides a rich resource for dissecting the regulatory network that governs the developmental potency of EPSCs and exploring alternative strategies to capture totipotent stem cells in culture.

Standardized genome-wide function prediction enables comparative functional genomics: a new application area for Gene Ontologies in plants.

BackgroundGenome-wide gene function annotations are useful for hypothesis generation and for prioritizing candidate genes potentially responsible for phenotypes of interest. We functionally annotated the genes of 18 crop plant genomes across 14 species using the GOMAP pipeline.ResultsBy comparison to existing GO annotation datasets, GOMAP-generated datasets cover more genes, contain more GO terms, and are similar in quality (based on precision and recall metrics using existing gold standards as the basis for comparison). From there, we sought to determine whether the datasets across multiple species could be used together to carry out comparative functional genomics analyses in plants. To test the idea and as a proof of concept, we created dendrograms of functional relatedness based on terms assigned for all 18 genomes. These dendrograms were compared to well-established species-level evolutionary phylogenies to determine whether trees derived were in agreement with known evolutionary relationships, which they largely are. Where discrepancies were observed, we determined branch support based on jackknifing then removed individual annotation sets by genome to identify the annotation sets causing unexpected relationships.ConclusionsGOMAP-derived functional annotations used together across multiple species generally retain sufficient biological signal to recover known phylogenetic relationships based on genome-wide functional similarities, indicating that comparative functional genomics across species based on GO data holds promise for generating novel hypotheses about comparative gene function and traits.

Comparative functional genomics identifies an iron-limited bottleneck in a Saccharomyces cerevisiae strain with a cytosolic-localized isobutanol pathway

Metabolic engineering strategies have been successfully implemented to improve the production of isobutanol, a next-generation biofuel, in Saccharomyces cerevisiae. Here, we explore how two of these strategies, pathway re-localization and redox cofactor-balancing, affect the performance and physiology of isobutanol producing strains. We equipped yeast with isobutanol cassettes which had either a mitochondrial or cytosolic localized isobutanol pathway and used either a redox-imbalanced (NADPH-dependent) or redox-balanced (NADH-dependent) ketol-acid reductoisomerase enzyme. We then conducted transcriptomic, proteomic and metabolomic analyses to elucidate molecular differences between the engineered strains. Pathway localization had a large effect on isobutanol production with the strain expressing the mitochondrial-localized enzymes producing 3.8-fold more isobutanol than strains expressing the cytosolic enzymes. Cofactor-balancing did not improve isobutanol titers and instead the strain with the redox-imbalanced pathway produced 1.5-fold more isobutanol than the balanced version, albeit at low overall pathway flux. Functional genomic analyses suggested that the poor performances of the cytosolic pathway strains were in part due to a shortage in cytosolic Fe–S clusters, which are required cofactors for the dihydroxyacid dehydratase enzyme. We then demonstrated that this cofactor limitation may be partially recovered by disrupting iron homeostasis with a fra2 mutation, thereby increasing cellular iron levels. The resulting isobutanol titer of the fra2 null strain harboring a cytosolic-localized isobutanol pathway outperformed the strain with the mitochondrial-localized pathway by 1.3-fold, demonstrating that both localizations can support flux to isobutanol.

Comprehensive analysis of 1R- and 2R-MYBs reveals novel genic and protein features, complex organisation, selective expansion and insights into evolutionary tendencies.

Myeloblastosis (MYB) family, the largest plant transcription factor family, has been subcategorised based on the number and type of repeats in the MYB domain. In spite of several reports, evolution of MYB genes and repeats remains enigmatic. Brassicaceae members are endowed with complex genomes, including dysploidy because of its unique history with multiple rounds of polyploidisation, genomic fractionations and rearrangements. The present study is an attempt to gain insights into the complexities of MYB family diversity, understand impacts of genome evolution on gene families and develop an evolutionary framework to understand the origin of various subcategories of MYB gene family. We identified and analysed 1129 MYBs that included 1R-, 2R-, 3R- and atypical-MYBs across sixteen species representing protists, fungi, animals and plants and exclude MYB identified from Brassicaceae except Arabidopsis thaliana; in addition, a total of 1137 2R-MYB genes from six Brassicaceae species were also analysed. Comparative analysis revealed predominance of 1R-MYBs in protists, fungi, animals and lower plants. Phylogenetic reconstruction and analysis of selection pressure suggested ancestral nature of R1-type repeat containing 1R-MYBs that might have undergone intragenic duplication to form multi-repeat MYBs. Distinct differences in gene structure between 1R-MYB and 2R-MYBs were observed regarding intron number, the ratio of gene length to coding DNA sequence (CDS) length and the length of exons encoding the MYB domain. Conserved as well as novel and lineage-specific intron phases were identified. Analyses of physicochemical properties revealed drastic differences indicating functional diversification in MYBs. Phylogenetic reconstruction of 1R- and 2R-MYB genes revealed a shared structure-function relationship in clades which was supported when transcriptome data was analysed in silico. Comparative genomics to study distribution pattern and mapping of 2R-MYBs revealed congruency and greater degree of synteny and collinearity among closely related species. Micro-synteny analysis of genomic segments revealed high conservation of genes that are immediately flanking the surrounding tandemly organised 2R-MYBs along with instances of local duplication, reorganisations and genome fractionation. In summary, polyploidy, dysploidy, reshuffling and genome fractionation were found to cause loss or gain of 2R-MYB genes. The findings need to be supported with functional validation to understand gene structure-function relationship along the evolutionary lineage and adaptive strategies based on comparative functional genomics in plants.

A study of the flexibility of the carbon catabolic pathways of extremophilic P. aeruginosa san ai exposed to benzoate versus glucose as sole carbon sources by multi omics analytical platform

Polyextremophilic, hydrocarbonoclastic Pseudomonas aeruginosa san ai can survive under extreme environmental challenges in the presence of a variety of pollutants such as organic solvents and hydrocarbons, particularly aromatics, heavy metals, and high pH. To date, the metabolic plasticity of the extremophilic P. aeruginosa, has not been sufficiently studied in regard to the effect of changing carbon sources. Therefore, the present study explores the carbon metabolic pathways of polyextremophilic P. aeruginosa san ai grown on sodium benzoate versus glucose and its potential for aromatic degradation. P. aeruginosa san ai removed/metabolised nearly 430 mg/L of benzoate for 48 h, demonstrating a high capacity for aromatic degradation. Comparative functional proteomics, targeted metabolomics and genomics analytical approaches were employed to study the carbon metabolism of the P. aeruginosa san ai. Functional proteomic study of selected enzymes participating in the β-ketoadipate and the Entner-Doudoroff pathways revealed a metabolic reconfiguration induced by benzoate compared to glucose. Metabolome analysis implied the existence of both catechol and protocatechuate branches of the β-ketoadipate pathway. Enzymatic study of benzoate grown cultures confirmed the activity of the ortho- catechol branch of the β-ketoadipate pathway. Even high concentrations of benzoate did not show increased stress protein synthesis, testifying to its extremophilic nature capable of surviving in harsh conditions. This ability of Pseudomonas aeruginosa san ai to efficiently degrade benzoate can provide a wide range of use of this strain in environmental and agricultural application.

Engineering, decoding and systems-level characterization of chimpanzee cytomegalovirus.

The chimpanzee cytomegalovirus (CCMV) is the closest relative of human CMV (HCMV). Because of the high conservation between these two species and the ability of human cells to fully support CCMV replication, CCMV holds great potential as a model system for HCMV. To make the CCMV genome available for precise and rapid gene manipulation techniques, we captured the genomic DNA of CCMV strain Heberling as a bacterial artificial chromosome (BAC). Selected BAC clones were reconstituted to infectious viruses, growing to similar high titers as parental CCMV. DNA sequencing confirmed the integrity of our clones and led to the identification of two polymorphic loci and a deletion-prone region within the CCMV genome. To re-evaluate the CCMV coding potential, we analyzed the viral transcriptome and proteome and identified several novel ORFs, splice variants, and regulatory RNAs. We further characterized the dynamics of CCMV gene expression and found that viral proteins cluster into five distinct temporal classes. In addition, our datasets revealed that the host response to CCMV infection and the de-regulation of cellular pathways are in line with known hallmarks of HCMV infection. In a first functional experiment, we investigated a proposed frameshift mutation in UL128 that was suspected to restrict CCMV's cell tropism. In fact, repair of this frameshift re-established productive CCMV infection in endothelial and epithelial cells, expanding the options of CCMV as an infection model. Thus, BAC-cloned CCMV can serve as a powerful tool for systematic approaches in comparative functional genomics, exploiting the close phylogenetic relationship between CCMV and HCMV.

Matching mouse models to specific human liver disease states by comparative functional genomics of mouse and human datasets

Omics has broadened our view of transcriptional and gene regulatory networks of multifactorial diseases, such as metabolism associated liver disease and its advanced stages including hepatocellular carcinoma. Identifying liver disease biomarkers and potential treatment targets makes use of experimental models, e.g. genetically engineered mice, which show molecular features of human pathologies but are experimentally tractable. We compared gene expression profiling data from human to our studies on transgenic mice with hepatocyte deletion of Cyp51 from cholesterol synthesis with the aim of identifying the human liver disease state best matched by the Cyp51 knockout model. Gene Expression Omnibus was used to identify relevant human datasets. We identified enriched and deregulated genes, pathways and transcription factors of mouse and human disease samples. Analysis showed a closer match of the Cyp51 knockout to the female patient samples. Importantly, CYP51 was depleted in both mouse and female human data. Among the enriched genes were the oxysterol-binding protein-related protein 3 (OSBPL3), which was enriched in all datasets, and the collagen gene COL1A2, which was enriched in both the mouse and one human dataset. KEGG and Reactome analyses revealed the most enriched pathway to be ECM-receptor interaction. Numerous transcription factors were differentially expressed in mice of both sexes and in the human female dataset, while depleted HNF4α and RXRα:PPARα-isoform1 were a hallmark in all cases. Our analysis exposed novel potential biomarkers, which may provide new avenues towards more personalized approaches and different targets in females and males. The analysis was only possible because of availability of open data resources and tools and broadly consistent annotation.

OVATE FAMILY PROTEINS (OFP) gene family across Brassicaceae: Comparative genomic analysis uncovers evolutionary relationships, extensive sequence and structural variation with a potential for functional diversification

Plant-specific Ovate Family Proteins (OFP), containing OVATE domain were identified as regulators of fruit shape. OFP homologs have been detected across plant kingdom as negative regulators of growth and adaptation, albeit with fragmentary information about evolutionary history, diversification, and functional significance. Phylogenetic position, evolutionary history, and availability of genome sequences from several species of Brassicaceae make them suitable for analysis of OFP gene family. Nineteen members of OFP gene family were previously identified from Arabidopsis thaliana. In the present study, we identified 390 homologs of 19 AtOFPs across thirteen Brassicaceae species and analysed their genomic distribution, organization, gene and protein structure, evolution, and possible mode of expansion. Comparative analysis of gene structure and protein architecture of 409 genes revealed conservation and variability in OVATE domain. This feature was used to classify OFPs as: i. OVATE-OFPs containing a canonical OVATE domain, ii. OVATE-like OFPs containing a partial or non-canonical OVATE domain, and, iii: OFP-Related which completely lacked OVATE domain. Two motifs that occur close to, or overlap with the OVATE domain were identified, and variations in amino acid composition that alter folding of OVATE domain were observed. Phylogenetic analysis showed that the paralogous pairs of OFPs found in A. thaliana were conserved across all species. Similar topologies of phylograms constructed from either full length protein, or only with OVATE domain indicate that the evolution of OFP gene family is driven by OVATE domain. A consequence of several post-polyploidization processes including sequence and structural variation among homeologs, and altered sub-cellular localization was observed. Analysis of selection pressure revealed purifying selection acting on seventeen out of nineteen OFPs, and positive selection on the remaining two OFPs. This present work thus lays the foundation for functional genomics of OFPs not only in agronomically important species of Brassicaceae but also in other multigene families to create a framework for future comparative functional genomics studies.

Monogenic Childhood Diabetes: Dissecting Clinical Heterogeneity by Next-Generation Sequencing in Maturity-Onset Diabetes of the Young

Diabetes is a common disorder with a heterogeneous clinical presentation and an enormous burden on health care worldwide. About 1-6% of patients with diabetes suffer from maturity-onset diabetes of the young (MODY), the most common form of monogenic diabetes with autosomal dominant inheritance. MODY is genetically and clinically heterogeneous and caused by genetic variations in pancreatic β-cell development and insulin secretion. We report here new findings from targeted next-generation sequencing (NGS) of 13 MODY-related genes. A sample of 22 unrelated pediatric patients with MODY and 13 unrelated healthy controls were recruited from a Turkish population. Targeted NGS was performed with Miseq 4000 (Illumina) to identify genetic variations in 13 MODY-related genes: HNF4A, GCK, HNF1A, PDX1, HNF1B, NEUROD1, KLF11, CEL, PAX4, INS, BLK, ABCC8, and KCNJ11. The NGS data were analyzed adhering to the Genome Analysis ToolKit (GATK) best practices pipeline, and variant filtering and annotation were performed. In the patient sample, we identified 43 MODY-specific genetic variations that were not present in the control group, including 11 missense mutations and 4 synonymous mutations. Importantly, and to the best of our knowledge, the missense mutations NEUROD1 p.D202E, KFL11 p.R461Q, BLK p.G248R, and KCNJ11 p.S385F were first associated with MODY in the present study. These findings contribute to the worldwide knowledge base on MODY and molecular correlates of clinical heterogeneity in monogenic childhood diabetes. Further comparative population genetics and functional genomics studies are called for, with an eye to discovery of novel diagnostics and personalized medicine in MODY. Because MODY is often misdiagnosed as type 1 or type 2 diabetes mellitus, advances in MODY diagnostics with NGS stand to benefit diabetes overall clinical care as well.

Comprehensive analysis of SSRs and database construction using all complete gene-coding sequences in major horticultural and representative plants

Simple sequence repeats (SSRs) are one of the most important genetic markers and widely exist in most species. Here, we identified 249,822 SSRs from 3,951,919 genes in 112 plants. Then, we conducted a comprehensive analysis of these SSRs and constructed a plant SSR database (PSSRD). Interestingly, more SSRs were found in lower plants than in higher plants, showing that lower plants needed to adapt to early extreme environments. Four specific enriched functional terms in the lower plant Chlamydomonas reinhardtii were detected when it was compared with seven other higher plants. In addition, Guanylate_cyc existed in more genes of lower plants than of higher plants. In our PSSRD, we constructed an interactive plotting function in the chart interface, and users can easily view the detailed information of SSRs. All SSR information, including sequences, primers, and annotations, can be downloaded from our database. Moreover, we developed Web SSR Finder and Batch SSR Finder tools, which can be easily used for identifying SSRs. Our database was developed using PHP, HTML, JavaScript, and MySQL, which are freely available at http://www.pssrd.info/. We conducted an analysis of the Myb gene families and flowering genes as two applications of the PSSRD. Further analysis indicated that whole-genome duplication and whole-genome triplication played a major role in the expansion of the Myb gene families. These SSR markers in our database will greatly facilitate comparative genomics and functional genomics studies in the future.