Analyses of phylogeny, evolution, conserved sequences and genome-wide expression of the ICK/KRP family of plant CDK inhibitors

Background and Aims

The cell cycle is controlled by cyclin-dependent kinases (CDKs), and CDK inhibitors are major regulators of their activities. The ICK/KRP family of CDK inhibitors has been reported in several plants, with seven members in arabidopsis; however, the phylogenetic relationship among members in different species is unknown. Also, there is a need to understand how these genes and proteins are regulated. Furthermore, little information is available on the functional differences among ICK/KRP family members.

Methods

We searched publicly available databases and identified over 120 unique ICK/KRP protein sequences from more than 60 plant species. Phylogenetic analysis was performed using 101 full-length sequences from 40 species and intron–exon organization of ICK/KRP genes in model species. Conserved sequences and motifs were analysed using ICK/KRP protein sequences from arabidopsis (Arabidopsis thaliana), rice (Orysa sativa) and poplar (Populus trichocarpa). In addition, gene expression was examined using microarray data from arabidopsis, rice and poplar, and further analysed by RT-PCR for arabidopsis.

Key Results and Conclusions

Phylogenetic analysis showed that plant ICK/KRP proteins can be grouped into three major classes. Whereas the C-class contains sequences from dicotyledons, monocotyledons and gymnosperms, the A- and B-classes contain only sequences from dicotyledons or monocotyledons, respectively, suggesting that the A- and B-classes might have evolved from the C-class. This classification is also supported by exon–intron organization. Genes in the A- and B- classes have four exons, whereas genes in the C-class have only three exons. Analysis of sequences from arabidopsis, rice and poplar identified conserved sequence motifs, some of which had not been described previously, and putative functional sites. The presence of conserved motifs in different family members is consistent with the classification. In addition, gene expression analysis showed preferential expression of ICK/KRP genes in certain tissues. A model has been proposed for the evolution of this gene family in plants.     

High Prevalence and Clinical Relevance of Genes Affected by Chromosomal Breaks in Colorectal Cancer

Background

Cancer is caused by somatic DNA alterations such as gene point mutations, DNA copy number aberrations (CNA) and structural variants (SVs). Genome-wide analyses of SVs in large sample series with well-documented clinical information are still scarce. Consequently, the impact of SVs on carcinogenesis and patient outcome remains poorly understood. This study aimed to perform a systematic analysis of genes that are affected by CNA-associated chromosomal breaks in colorectal cancer (CRC) and to determine the clinical relevance of recurrent breakpoint genes.

Methods

Primary CRC samples of patients with metastatic disease from CAIRO and CAIRO2 clinical trials were previously characterized by array-comparative genomic hybridization. These data were now used to determine the prevalence of CNA-associated chromosomal breaks within genes across 352 CRC samples. In addition, mutation status of the commonly affected APC, TP53, KRAS, PIK3CA, FBXW7, SMAD4, BRAF and NRAS genes was determined for 204 CRC samples by targeted massive parallel sequencing. Clinical relevance was assessed upon stratification of patients based on gene mutations and gene breakpoints that were observed in >3% of CRC cases.

Results

In total, 748 genes were identified that were recurrently affected by chromosomal breaks (FDR <0.1). MACROD2 was affected in 41% of CRC samples and another 169 genes showed breakpoints in >3% of cases, indicating that prevalence of gene breakpoints is comparable to the prevalence of well-known gene point mutations. Patient stratification based on gene breakpoints and point mutations revealed one CRC subtype with very poor prognosis.

Conclusions

We conclude that CNA-associated chromosomal breaks within genes represent a highly prevalent and clinically relevant subset of SVs in CRC.     

Pathway Implications of Aberrant Global Methylation in Adrenocortical Cancer

Context

Adrenocortical carcinomas (ACC) are a rare tumor type with a poor five-year survival rate and limited treatment options.

Objective

Understanding of the molecular pathogenesis of this disease has been aided by genomic analyses highlighting alterations in TP53, WNT, and IGF signaling pathways. Further elucidation is needed to reveal therapeutically actionable targets in ACC.

Design

In this study, global DNA methylation levels were assessed by the Infinium HumanMethylation450 BeadChip Array on 18 ACC tumors and 6 normal adrenal tissues. A new, non-linear correlation approach, the discretization method, assessed the relationship between DNA methylation/gene expression across ACC tumors.

Results

This correlation analysis revealed epigenetic regulation of genes known to modulate TP53, WNT, and IGF signaling, as well as silencing of the tumor suppressor MARCKS, previously unreported in ACC.

Conclusions

DNA methylation may regulate genes known to play a role in ACC pathogenesis as well as known tumor suppressors.     

Association of Common Variants in LOX with Keratoconus: A Meta-Analysis

Background

Several case-control studies have been performed to examine the association of genetic variants in lysyl oxidase (LOX) with keratoconus. However, the results remained inconclusive and great heterogeneity might exist across populations.

Method

A comprehensive literature search for studies that published up to June 25, 2015 was performed. Summary odds ratios (OR) and 95% confidence intervals (CI) of each single nucleotide polymorphism (SNP) were estimated with fixed effects model when I ²<50% in the test for heterogeneity or random effects model when I ²>50%. Publication bias was evaluated using funnel plots and Egger’s test.

Results

A total of four studies including 1,467 keratoconus cases and 4,490 controls were involved in this meta-analysis. SNPs rs2956540 and rs10519694 showed significant association with keratoconus, with ORs of 0.71 (95% CI: 0.63–0.80, P = 1.43E-08) and 0.77 (95% CI: 0.61–0.97, P = 0.026), respectively. In contrast, our study lacked sufficient evidences to support the association of rs1800449/rs2288393 with keratoconus across populations.

Conclusion

This meta-analysis suggested that two LOX variants, rs2956540 and rs10519694, may affect individual susceptibility to keratoconus, while distinct heterogeneity existed within this locus. Larger-scale and multi-ethnic genetic studies on keratoconus are required to further validate the results.     

Pathway Analysis Based on a Genome-Wide Association Study of Polycystic Ovary Syndrome

Background

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women of reproductive age, and it is affected by both environmental and genetic factors. Although the genetic component of PCOS is evident, studies aiming to identify susceptibility genes have shown controversial results. This study conducted a pathway-based analysis using a dataset obtained through a genome-wide association study (GWAS) to elucidate the biological pathways that contribute to PCOS susceptibility and the associated genes.

Methods

We used GWAS data on 636,797 autosomal single nucleotide polymorphisms (SNPs) from 1,221 individuals (432 PCOS patients and 789 controls) for analysis. A pathway analysis was conducted using meta-analysis gene-set enrichment of variant associations (MAGENTA). Top-ranking pathways or gene sets associated with PCOS were identified, and significant genes within the pathways were analyzed.

Results

The pathway analysis of the GWAS dataset identified significant pathways related to oocyte meiosis and the regulation of insulin secretion by acetylcholine and free fatty acids (all nominal gene-set enrichment analysis (GSEA) P-values < 0.05). In addition, INS, GNAQ, STXBP1, PLCB3, PLCB2, SMC3 and PLCZ1 were significant genes observed within the biological pathways (all gene P-values < 0.05).

Conclusions

By applying MAGENTA pathway analysis to PCOS GWAS data, we identified significant pathways and candidate genes involved in PCOS. Our findings may provide new leads for understanding the mechanisms underlying the development of PCOS.     

Changes in PTGS1 and ALOX12 Gene Expression in Peripheral Blood Mononuclear Cells Are Associated with Changes in Arachidonic Acid,Oxylipins, and Oxylipin/Fatty Acid Ratios in Response to Omega-3 Fatty Acid Supplementation

Introduction

There is a high degree of inter-individual variability among people in response to intervention with omega-3 fatty acids (FA), which may partly explain conflicting results on the effectiveness of omega-3 FA for the treatment and prevention of chronic inflammatory diseases. In this study we sought to evaluate whether part of this inter-individual variability in response is related to the regulation of key oxylipin metabolic genes in circulating peripheral blood mononuclear cells (PBMCs).

Methods

Plasma FA and oxylipin profiles from 12 healthy individuals were compared to PBMC gene expression profiles following six weeks of supplementation with fish oil, which delivered 1.9 g/d eicosapentaenoic acid (EPA) and 1.5 g/d docosahexaenoic acid (DHA). Fold changes in gene expression were measured by a quantitative polymerase chain reaction (qPCR).

Results

Healthy individuals supplemented with omega-3 FA had differential responses in prostaglandin-endoperoxide synthase 1 (PTGS1), prostaglandin-endoperoxide synthase 2 (PTGS2), arachidonate 12-lipoxygenase (ALOX12), and interleukin 8 (IL-8) gene expression in isolated PBMCs. In those individuals for whom plasma arachidonic acid (ARA) in the phosphatidylethanolamine (PE) lipid class decreased in response to omega-3 intervention, there was a corresponding decrease in gene expression for PTGS1 and ALOX12. Several oxylipin product/FA precursor ratios (e.g. prostaglandin E₂ (PGE₂)/ARA for PTGS1 and 12-hydroxyeicosatetraenoic acid (12-HETE)/ARA for ALOX12) were also associated with fold change in gene expression, suggesting an association between enzyme activity and gene expression. The fold-change in PTGS1 gene expression was highly positively correlated with ALOX12 gene expression but not with PTGS2, whereas IL-8 and PTGS2 were positively correlated.

Conclusions

The regulation of important oxylipin metabolic genes in PBMCs varied with the extent of change in ARA concentrations in the case of PTGS1 and ALOX12 regulation. PBMC gene expression changes in response to omega-3 supplementation varied among healthy individuals, and were associated with changes in plasma FA and oxylipin composition to different degrees in different individuals.

Trial Registration

clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01838239","term_id":"NCT01838239"}}NCT01838239     

In-Silico Computing of the Most Deleterious nsSNPs in HBA1 Gene

Background

α-Thalassemia (α-thal) is a genetic disorder caused by the substitution of single amino acid or large deletions in the HBA1 and/or HBA2 genes.

Method

Using modern bioinformatics tools as a systematic in-silico approach to predict the deleterious SNPs in the HBA1 gene and its significant pathogenic impact on the functions and structure of HBA1 protein was predicted.

Results and Discussion

A total of 389 SNPs in HBA1 were retrieved from dbSNP database, which includes: 201 non-coding synonymous (nsSNPs), 43 human active SNPs, 16 intronic SNPs, 11 mRNA 3′ UTR SNPs, 9 coding synonymous SNPs, 9 5′ UTR SNPs and other types. Structural homology-based method (PolyPhen) and sequence homology-based tool (SIFT), SNPs&Go, PROVEAN and PANTHER revealed that 2.4% of the nsSNPs are pathogenic.

Conclusions

A total of 5 nsSNPs (G60V, K17M, K17T, L92F and W15R) were predicted to be responsible for the structural and functional modifications of HBA1 protein. It is evident from the deep comprehensive in-silico analysis that, two nsSNPs such as G60Vand W15R in HBA1 are highly deleterious. These “2 pathogenic nsSNPs” can be considered for wet-lab confirmatory analysis.     

Novel Immortal Cell Lines Support Cellular Heterogeneity in the Human Annulus Fibrosus

Introduction

Loss of annulus fibrosus (AF) integrity predisposes to disc herniation and is associated with IVD degeneration. Successful implementation of biomedical intervention therapy requires in-depth knowledge of IVD cell biology. We recently generated unique clonal human nucleus pulposus (NP) cell lines. Recurring functional cellular phenotypes from independent donors provided pivotal evidence for cell heterogeneity in the mature human NP. In this study we aimed to generate and characterize immortal cell lines for the human AF from matched donors.

Methods

Non-degenerate healthy disc material was obtained as surplus surgical material. AF cells were immortalized by simian virus Large T antigen (SV40LTAg) and human telomerase (hTERT) expression. Early passage cells and immortalized cell clones were characterized based on marker gene expression under standardized culturing and in the presence of Transforming Growth factor β (TGFβ).

Results

The AF-specific expression signature included COL1A1, COL5A1, COL12A1, SFRP2 and was largely maintained in immortal AF cell lines. Remarkably, TGFβ induced rapid 3D sheet formation in a subgroup of AF clones. This phenotype was associated with inherent differences in Procollagen type I processing and maturation, and correlated with differential mRNA expression of Prolyl 4-hydroxylase alpha polypeptide 1 and 3 (P4HA1,3) and Lysyl oxidase (LOX) between clones and differential P4HA3 protein expression between AF cells in histological sections.

Conclusion

We report for the first time the generation of representative human AF cell lines. Gene expression profile analysis and functional comparison of AF clones revealed variation between immortalized cells and suggests phenotypic heterogeneity in the human AF. Future characterization of AF cellular (sub-)populations aims to combine identification of additional specific AF marker genes and their biological relevance. Ultimately this knowledge will contribute to clinical application of cell-based technology in IVD repair.     

Complement System in the Pathogenesis of Benign Lymphoepithelial Lesions of the Lacrimal Gland

Objective

We aimed to examine the potential involvement of local complement system gene expression in the pathogenesis of benign lymphoepithelial lesions (BLEL) of the lacrimal gland.

Methods

We collected data from 9 consecutive pathologically confirmed patients with BLEL of the lacrimal gland and 9 cases with orbital cavernous hemangioma as a control group, and adopted whole genome microarray to screen complement system-related differential genes, followed by RT-PCR verification and in-depth enrichment analysis (Gene Ontology analysis) of the gene sets.

Results

The expression of 14 complement system-related genes in the pathologic tissue, including C2, C3, ITGB2, CR2, C1QB, CR1, ITGAX, CFP, C1QA, C4B|C4A, FANCA, C1QC, C3AR1 and CFHR4, were significantly upregulated while 7 other complement system-related genes, C5, CFI, CFHR1|CFH, CFH, CD55, CR1L and CFD were significantly downregulated in the lacrimal glands of BLEL patients. The microarray results were consistent with RT-PCR analysis results. Immunohistochemistry analysis of C3c and C1q complement component proteins in the resected tissue were positive in BLEL patients, while the control group had negative expression of these proteins. Gene ontology (GO) analysis revealed that activation of the genes of complement system-mediated signaling pathways were the most enriched differential gene group in BLEL patients.

Conclusions

Local expression of complement components is prominently abnormal in BLEL, and may well play a role in its pathogenesis.     

The phytocyanin gene family in rice (Oryza sativa L.): genome-wide identification, classification and transcriptional analysis

Background

Phytocyanins (PCs) are plant-specific blue copper proteins involved in electron transport, and a large number of known PCs are considered to be chimeric arabinogalactan proteins (AGPs). To date there has not been a genome-wide overview of the OsPC gene family. Therefore, as the first step and a useful strategy to elucidate the functions of OsPCs, there is an urgent need for a thorough genome-wide analysis of this gene family.

Methodology/Principal Findings

In this study, a total of 62 OsPC genes were identified through a comprehensive bioinformatics analysis of the rice (Oryza sativa L.) genome. Based on phylogeny and motif constitution, the family of OsPCs was classified into three subclasses: uclacyanin-like proteins (OsUCLs), stellacyanin-like proteins (OsSCLs) and early nodulin-like proteins (OsENODLs). Structure and glycosylation prediction indicated that 46 OsPCs were glycosylphosphatigylinositol-anchored proteins and 38 OsPCs were chimeric AGPs. Gene duplication analysis revealed that chromosomal segment and tandem duplications contributed almost equally to the expansion of this gene family, and duplication events were mostly happened in the OsUCL subfamily. The expression profiles of OsPC genes were analyzed at different stages of vegetative and reproductive development and under abiotic stresses. It revealed that a large number of OsPC genes were abundantly expressed in the various stages of development. Moreover, 17 genes were regulated under the treatments of abiotic stresses.

Conclusions/Significance

The genome-wide identification and expression analysis of OsPC genes should facilitate research in this gene family and give new insights toward elucidating their functions in higher plants.