Comparison of novel and existing methods for detecting differentially methylated regions

Background

Single-probe analyses in epigenome-wide association studies (EWAS) have identified associations between DNA methylation and many phenotypes, but do not take into account information from neighboring probes. Methods to detect differentially methylated regions (DMRs) (clusters of neighboring probes associated with a phenotype) may provide more power to detect associations between DNA methylation and diseases or phenotypes of interest.

Results

We proposed a novel approach, GlobalP, and perform comparisons with 3 methods—DMRcate, Bumphunter, and comb-p—to identify DMRs associated with log triglycerides (TGs) in real GAW20 data before and after fenofibrate treatment. We applied these methods to the summary statistics from an EWAS performed on the methylation data. Comb-p, DMRcate, and GlobalP detected very similar DMRs near the gene CPT1A on chromosome 11 in both the pre- and posttreatment data. In addition, GlobalP detected 2 DMRs before fenofibrate treatment in the genes ETV6 and ABCG1. Bumphunter identified several DMRs on chromosomes 1 and 20, which did not overlap with DMRs detected by other methods.

Conclusions

Our novel method detected the same DMR identified by two existing methods and detected two additional DMRs not identified by any of the existing methods we compared.

     

Incorporating methylation genome information improves prediction accuracy for drug treatment responses

Background

Association studies using a single type of omics data have been successful in identifying disease-associated genetic markers, but the underlying mechanisms are unaddressed. To provide a possible explanation of how these genetic factors affect the disease phenotype, integration of multiple omics data is needed.

Results

We propose a novel method, LIPID (likelihood inference proposal for indirect estimation), that uses both single nucleotide polymorphism (SNP) and DNA methylation data jointly to analyze the association between a trait and SNPs. The total effect of SNPs is decomposed into direct and indirect effects, where the indirect effects are the focus of our investigation. Simulation studies show that LIPID performs better in various scenarios than existing methods. Application to the GAW20 data also leads to encouraging results, as the genes identified appear to be biologically relevant to the phenotype studied.

Conclusions

The proposed LIPID method is shown to be meritorious in extensive simulations and in real-data analyses.

     

New molecular evidence of wine yeast-bacteria interaction unraveled by non-targeted exometabolomic profiling

Introduction

Bacterial malolactic fermentation (MLF) has a considerable impact on wine quality. The yeast strain used for primary fermentation can systematically stimulate (MLF+ phenotype) or inhibit (MLF?) bacteria and the MLF process as a function of numerous winemaking practices, but the underlying molecular evidence still remains a mystery.

Objectives

The goal of the study was to elucidate such evidence by the direct comparison of extracellular metabolic profiles of MLF+ and MLF? phenotypes.

Methods

We have applied a non-targeted metabolomic approach combining ultrahigh-resolution FT-ICR-MS analysis, powerful statistical tools and a comprehensive wine metabolite database.

Results

We discovered around 2500 unknown masses and 800 putative biomarkers involved in phenotypic distinction. For the putative biomarkers, we also developed a biomarker identification workflow and elucidated the exact structure (by UPLC-Q-ToF–MS²) and/or exact physiological impact (by in vitro tests) of several novel biomarkers, such as D-gluconic acid, citric acid, trehalose and tripeptide Pro-Phe-Val. In addition to valid biomarkers, molecular evidence was reflected by unprecedented chemical diversity (around 3000 discriminant masses) that characterized both the yeast phenotypes. While distinct chemical families such as phenolic compounds, carbohydrates, amino acids and peptides characterize the extracellular metabolic profiles of the MLF+ phenotype, the MLF? phenotype is associated with sulphur-containing peptides.

Conclusion

The non-targeted approach used in this study played an important role in finding new and unexpected molecular evidence.

     

Plant growth-promoting bacterium <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> FR1 secrets a novel type of extracellular polyhydroxybutyrate polymerase involved in abiotic stress response in plants

Objectives

Identification of novel microbial factors contributing to plant protection against abiotic stress.

Results

The genome of plant growth-promoting bacterium Pseudomonas fluorescens FR1 contains a short mobile element encoding a novel type of extracellular polyhydroxybutyrate (PHB) polymerase (PhbC) associated with a type I secretion system. Genetic analysis using a phbC mutant strain and plants showed that this novel extracellular enzyme is related to the PHB production in planta and suggests that PHB could be a beneficial microbial compound synthesized during plant adaptation to cold stress.

Conclusion

Extracellular PhbC can be used as a new tool for improve crop production under abiotic stress.

     

Longitudinal analytical approaches to genetic data

Background

Longitudinal phenotypic data provides a rich potential resource for genetic studies which may allow for greater understanding of variants and their covariates over time. Herein, we review 3 longitudinal analytical approaches from the Genetic Analysis Workshop 19 (GAW19). These contributions investigated both genome-wide association (GWA) and whole genome sequence (WGS) data from odd numbered chromosomes on up to 4 time points for blood pressure–related phenotypes. The statistical models used included generalized estimating equations (GEEs), latent class growth modeling (LCGM), linear mixed-effect (LME), and variance components (VC). The goal of these analyses was to test statistical approaches that use repeat measurements to increase genetic signal for variant identification.

Results

Two analytical methods were applied to the GAW19: GWA using real phenotypic data, and one approach to WGS using 200 simulated replicates. The first GWA approach applied a GEE-based model to identify gene-based associations with 4 derived hypertension phenotypes. This GEE model identified 1 significant locus, GRM7, which passed multiple test corrections for 2 hypertension-derived traits. The second GWA approach employed the LME to estimate genetic associations with systolic blood pressure (SBP) change trajectories identified using LCGM. This LCGM method identified 5 SBP trajectories and association analyses identified a genome-wide significant locus, near ATOX1 (p?=?1.0E^?8). Finally, a third VC-based model using WGS and simulated SBP phenotypes that constrained the β coefficient for a genetic variant across each time point was calculated and compared to an unconstrained approach. This constrained VC approach demonstrated increased power for WGS variants of moderate effect, but when larger genetic effects were present, averaging across time points was as effective.

Conclusion

In this paper, we summarize 3 GAW19 contributions applying novel statistical methods and testing previously proposed techniques under alternative conditions for longitudinal genetic association. We conclude that these approaches when appropriately applied have the potential to: (a) increase statistical power; (b) decrease trait heterogeneity and standard error; (c) decrease computational burden in WGS; and (d) have the potential to identify genetic variants influencing subphenotypes important for understanding disease progression.

     

Measuring phenotype-phenotype similarity through the interactome

Background

Recently, measuring phenotype similarity began to play an important role in disease diagnosis. Researchers have begun to pay attention to develop phenotype similarity measurement. However, existing methods ignore the interactions between phenotype-associated proteins, which may lead to inaccurate phenotype similarity.

Results

We proposed a network-based method PhenoNet to calculate the similarity between phenotypes. We localized phenotypes in the network and calculated the similarity between phenotype-associated modules by modeling both the inter- and intra-similarity.

Conclusions

PhenoNet was evaluated on two independent evaluation datasets: gene ontology and gene expression data. The result shows that PhenoNet performs better than the state-of-art methods on all evaluation tests.

     

A metabolomic approach to characterize the acid-tolerance response in <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis>

Introduction

Sinorhizobium meliloti establishes a symbiosis with Medicago species where the bacterium fixes atmospheric nitrogen for plant nutrition. To achieve a successful symbiosis, however, both partners need to withstand biotic and abiotic stresses within the soil, especially that of excess acid, to which the Medicago-Sinorhizobium symbiotic system is widely recognized as being highly sensitive.

Objective

To cope with low pH, S. meliloti can undergo an acid-tolerance response (ATR(+)) that not only enables a better survival but also constitutes a more competitive phenotype for Medicago sativa nodulation under acid and neutral conditions. To characterize this phenotype, we employed metabolomics to investigate the biochemical changes operating in ATR(+) cells.

Methods

A gas chromatography/mass spectrometry approach was used on S. meliloti 2011 cultures showing ATR(+) and ATR(?) phenotypes. After an univariate and multivariate statistical analysis, enzymatic activities and/or reserve carbohydrates characterizing ATR(+) phenotypes were determined.

Results

Two distinctive populations were clearly defined in cultures grown in acid and neutral pH based on the metabolites present. A shift occurred in the carbon-catabolic pathways, potentially supplying NAD(P)H equivalents for use in other metabolic reactions and/or for maintaining intracellular-pH homeostasis. Furthermore, among the mechanisms related to acid resistance, the ATR(+) phenotype was also characterized by lactate production, envelope modification, and carbon-overflow metabolism.

Conclusions

Acid-challenged S. meliloti exhibited several changes in different metabolic pathways that, in specific instances, could be identified and related to responses observed in other bacteria under various abiotic stresses. Some of the observed changes included modifications in the pentose-phosphate pathway (PPP), the exopolysaccharide biosynthesis, and in the myo-inositol degradation intermediates. Such modifications are part of a metabolic adaptation in the rhizobia that, as previously reported, is associated to improved phenotypes of acid tolerance and nodulation competitiveness.

     

A study of candidate genes for day blindness in the standard wire haired dachshund

Background

A genetic study was performed to identify candidate genes associated with day blindness in the standard wire haired dachshund. Based on a literature review of diseases in dogs and human with phenotypes similar to day blindness, ten genes were selected and evaluated as potential candidate genes associated with day blindness in the breed.

Results

Three of the genes, CNGB3, CNGA3 and GNAT2, involved in cone degeneration and seven genes and loci, ABCA4, RDH5, CORD8, CORD9, RPGRIP1, GUCY2D and CRX, reported to be involved in cone-rod dystrophies were studied. Polymorphic markers at each of the candidate loci were studied in a family with 36 informative offspring. The study revealed a high frequency of recombinations between the candidate marker alleles and the disease.

Conclusion

Since all of the markers were at the exact position of the candidate loci, and several recombinations were detected for each of the loci, all ten genes were excluded as causal for this canine, early onset cone-rod dystrophy. The described markers may, however, be useful to screen other canine resource families segregating eye diseases for association to the ten genes.

     

<Emphasis Type="Italic">massPix</Emphasis>: an R package for annotation and interpretation of mass spectrometry imaging data for lipidomics

Introduction

Mass spectrometry imaging (MSI) experiments result in complex multi-dimensional datasets, which require specialist data analysis tools.

Objectives

We have developed massPix—an R package for analysing and interpreting data from MSI of lipids in tissue.

Methods

massPix produces single ion images, performs multivariate statistics and provides putative lipid annotations based on accurate mass matching against generated lipid libraries.

Results

Classification of tissue regions with high spectral similarly can be carried out by principal components analysis (PCA) or k-means clustering.

Conclusion

massPix is an open-source tool for the analysis and statistical interpretation of MSI data, and is particularly useful for lipidomics applications.

     

Fungal Paronychia. Clinical,Diagnosis and Treatment Aspects

Purpose of Review

The purpose of this study is to recognize and expand the knowledge of mycotic paronychia as a variable clinical condition due to various predisposing factors and multiple fungal organisms.

Recent Findings

Candida-associated mycotic paronychia is common but other non-dermatophyte molds, such as Fusarium, are identified as potential agents of paronychia and onychomycosis.

Summary

Mycotic paronychia is characterized by inflammation of the proximal or lateral nail folds caused by certain fungi. Mycological analysis is necessary to identify the causal agent and prescribe an appropriate treatment. Further studies are needed to know the involved microorganisms in the disease and the pathogenicity factors involved in this localized area of the nail apparatus.

     

Iridoid and phenylethanoid/phenylpropanoid metabolite profiles of <Emphasis Type="Italic">Scrophularia</Emphasis> and <Emphasis Type="Italic">Verbascum</Emphasis> species used medicinally in North America

Introduction

Botanicals containing iridoid and phenylethanoid/phenylpropanoid glycosides are used worldwide for the treatment of inflammatory musculoskeletal conditions that are primary causes of human years lived with disability, such as arthritis and lower back pain.

Objectives

We report the analysis of candidate anti-inflammatory metabolites of several endemic Scrophularia species and Verbascum thapsus used medicinally by peoples of North America.

Methods

Leaves, stems, and roots were analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and partial least squares-discriminant analysis (PLS-DA) was performed in MetaboAnalyst 3.0 after processing the datasets in Progenesis QI.

Results

Comparison of the datasets revealed significant and differential accumulation of iridoid and phenylethanoid/phenylpropanoid glycosides in the tissues of the endemic Scrophularia species and Verbascum thapsus.

Conclusions

Our investigation identified several species of pharmacological interest as good sources for harpagoside and other important anti-inflammatory metabolites.

     

Metabolic response of porcine colon explants to in vitro infection by <Emphasis Type="Italic">Brachyspira hyodysenteriae</Emphasis>: a leap into disease pathophysiology

Introduction

Swine dysentery caused by Brachyspira hyodysenteriae is a production limiting disease in pig farming. Currently antimicrobial therapy is the only treatment and control method available.

Objective

The aim of this study was to characterize the metabolic response of porcine colon explants to infection by B. hyodysenteriae.

Methods

Porcine colon explants exposed to B. hyodysenteriae were analyzed for histopathological, metabolic and pro-inflammatory gene expression changes.

Results

Significant epithelial necrosis, increased levels of l-citrulline and IL-1α were observed on explants infected with B. hyodysenteriae.

Conclusions

The spirochete induces necrosis in vitro likely through an inflammatory process mediated by IL-1α and NO.

     

A fungal endophyte defensive symbiosis affects plant-nematode interactions in cotton

Background and aims

Most investigations of fungi as nematode antagonists have focused on their interactions with nematodes in the soil. This study tested a foliar-isolated endophytic Phialemonium inflatum for its effects against the root-knot nematode as an endophyte in cotton using a seed treatment inoculation.

Methods

Cotton seeds were inoculated with P. inflatum spore suspensions prior to planting. Nematode infection and reproduction were quantified at Day 12 and 6 weeks after nematode egg inoculation, respectively. To establish whether the observed negative effects on nematodes were due to P. inflatum in the soil or as an endophyte in the plant, we also applied a soil fungicide treatment at the seedling stage to kill the fungi outside the plant.

Results

Persistent suppression of nematode penetration and galling, as well as subsequent reproduction, were observed in endophyte-treated plants independent of fungicide treatment, consistent with an endophytic mode of nematode suppression; and these negative effects did not depend on the concentration of fungal inoculum used to treat to the seed.

Conclusions

Our study highlights a novel role for P. inflatum as part of a plant-fungal defensive symbiosis in cotton, as well as the need for a broader understanding of endophyte-plant-nematode ecological interactions.

     

integRATE: a desirability-based data integration framework for the prioritization of candidate genes across heterogeneous omics and its application to preterm birth

Background

The integration of high-quality, genome-wide analyses offers a robust approach to elucidating genetic factors involved in complex human diseases. Even though several methods exist to integrate heterogeneous omics data, most biologists still manually select candidate genes by examining the intersection of lists of candidates stemming from analyses of different types of omics data that have been generated by imposing hard (strict) thresholds on quantitative variables, such as P-values and fold changes, increasing the chance of missing potentially important candidates.

Methods

To better facilitate the unbiased integration of heterogeneous omics data collected from diverse platforms and samples, we propose a desirability function framework for identifying candidate genes with strong evidence across data types as targets for follow-up functional analysis. Our approach is targeted towards disease systems with sparse, heterogeneous omics data, so we tested it on one such pathology: spontaneous preterm birth (sPTB).

Results

We developed the software integRATE, which uses desirability functions to rank genes both within and across studies, identifying well-supported candidate genes according to the cumulative weight of biological evidence rather than based on imposition of hard thresholds of key variables. Integrating 10 sPTB omics studies identified both genes in pathways previously suspected to be involved in sPTB as well as novel genes never before linked to this syndrome. integRATE is available as an R package on GitHub (https://github.com/haleyeidem/integRATE).

Conclusions

Desirability-based data integration is a solution most applicable in biological research areas where omics data is especially heterogeneous and sparse, allowing for the prioritization of candidate genes that can be used to inform more targeted downstream functional analyses.

     

Novel mutation G324C in <Emphasis Type="Italic">WNT1</Emphasis> mapped in a large Pakistani family with severe recessively inherited <Emphasis Type="Italic">Osteogenesis Imperfecta</Emphasis>

Introduction

Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous disease with skeletal fragility and variable extra-skeletal manifestations. To date several point mutations in 18 different genes causing different types of OI have been identified. Mutations in WNT1 compromise activity of the osteoblasts leading to disturbed bone mass accrual, fragility fractures and progressive skeletal abnormalities. The present study was conducted to determine the underlying genetic cause of an autosomal recessive skeletal dysplasia in a large consanguineous family from Chinute, Pakistan.

Materials and methods

Blood was collected from 24 individuals of affected family along with clinical data. Homozygosity mapping was performed to confirm consanguinity. SNPs were identified, followed by whole exome and Sanger sequencing. In silico characterization of WNT1 mutation was performed using multiple platforms.

Results

Nine affected family members exhibited severe bone deformities, recurrent fractures, short stature and low bone mineral density. SNP array data revealed homozygous segments >?1 Mb in length accounting for 2.1–12.7% of the genome in affected individuals and their siblings and a single 6,344,821 bp homozygous region in all affected individuals on chromosome 12q12-q13. This region includes two potential OI candidate genes WNT1 and VDR. We did whole-exome sequencing for both genes in two patients and identified a novel damaging missense mutation in exon 4 of WNT1: c.1168G?>?T (NM_005430) resulting in p.G324C. Sanger sequencing confirmed segregation of mutation with the disease in family.

Conclusion

We report a novel mutation responsible for OI and our investigation expands the spectrum of disease-causing WNT1 mutations and the resulting OI phenotypes.

     

Expression,characterization of a novel nitrilase PpL19 from <Emphasis Type="Italic">Pseudomonas psychrotolerans</Emphasis> with <Emphasis Type="Italic">S</Emphasis>-selectivity toward mandelonitrile present in active inclusion bodies

Objectives

To identify a novel nitrilase with S-selectivity toward mandelonitrile that can produce (S)-mandelic acid in one step.

Results

A novel nitrilase PpL19 from Pseudomonas psychrotolerans L19 was discovered by genome mining. It showed S-selectivity with an enantiomeric excess of 52.7 % when used to hydrolyse (R, S)-mandelonitrile. No byproduct was observed. PpL19 was overexpressed in Escherichia coli BL21 (DE3) and formed inclusion bodies that were active toward mandelonitrile and stable across a broad range of temperature and pH. In addition, PpL19 hydrolysed nitriles with diverse structures; arylacetonitriles were the optimal substrates. Homology modelling and docking studies of both enantiomers of mandelonitrile in the active site of nitrilase PpL19 shed light on the enantioselectivity.

Conclusions

A novel nitrilase PpL19 from P. psychrotolerans L19 was mined and distinguished from other nitrilases as it was expressed as an active inclusion body and showed S-selectivity toward mandelonitrile.

     

Study on the Clinical Features and Prognosis of <Emphasis Type="Italic">Penicilliosis marneffei</Emphasis> Without Human Immunodeficiency Virus Infection

Objective

To improve the diagnosis and treatment of Penicilliosis marneffei without human immunodeficiency virus infection.

Methods

Analyze and review the clinical features, diagnosis and treatment of six cases of P. marneffei without human immunodeficiency virus infection at The First Affiliated Hospital of Fujian Medical University.

Results

Two cases were diagnosed in the ENT Department, three cases in the respiratory department and one case in the dermatological department. Penicillium marneffei infection was confirmed by sputum culture, blood culture and tissue biopsy. After definite diagnosis, one refused further treatment, and others showed significant improvement.

Conclusion

Penicilliosis marneffei is insidious onset and easy to be escaped and misdiagnosed. To achieve early diagnosis and appropriate treatment, doubtful cases should be alerted for the diagnoses as P. marneffei.

     

Phenotypic variability and identification of novel <Emphasis Type="Italic">YARS2</Emphasis> mutations in YARS2 mitochondrial myopathy,lactic acidosis and sideroblastic anaemia

Background

Mutations in the mitochondrial tyrosyl-tRNA synthetase (YARS2) gene have previously been identified as a cause of the tissue specific mitochondrial respiratory chain (RC) disorder, Myopathy, Lactic Acidosis, Sideroblastic Anaemia (MLASA). In this study, a cohort of patients with a mitochondrial RC disorder for who anaemia was a feature, were screened for mutations in YARS2.

Methods

Twelve patients were screened for YARS2 mutations by Sanger sequencing. Clinical data were compared. Functional assays were performed to confirm the pathogenicity of the novel mutations and to investigate tissue specific effects.

Results

PathogenicYARS2 mutations were identified in three of twelve patients screened. Two patients were found to be homozygous for the previously reported p.Phe52Leu mutation, one severely and one mildly affected. These patients had different mtDNA haplogroups which may contribute to the observed phenotypic variability. A mildly affected patient was a compound heterozygote for two novel YARS2 mutations, p.Gly191Asp and p.Arg360X. The p.Gly191Asp mutation resulted in a 38-fold loss in YARS2 catalytic efficiency and the p.Arg360X mutation did not produce a stable protein. The p.Phe52Leu and p.Gly191Asp/p.Arg360X mutations resulted in more severe RC deficiency of complexes I, III and IV in muscle cells compared to fibroblasts, but had relatively normal YARS2 protein levels. The muscle-specific RC deficiency can be related to the increased requirement for RC complexes in muscle. There was also a failure of mtDNA proliferation upon myogenesis in patient cells which may compound the RC defect. Patient muscle had increased levels of PGC1-α and TFAM suggesting mitochondrial biogenesis was activated as a potential compensatory mechanism.

Conclusion

In this study we have identified novel YARS2 mutations and noted marked phenotypic variability among YARS2 MLASA patients, with phenotypes ranging from mild to lethal, and we suggest that the background mtDNA haplotype may be contributing to the phenotypic variability. These findings have implications for diagnosis and prognostication of the MLASA and related phenotypes.