Tarmo Annilo

Meta-analysis of microRNA expression in lung cancer†

The prognostic and diagnostic value of microRNA (miRNA) expression aberrations in lung cancer has been studied intensely in recent years. However, due to the application of different technological platforms and small sample size, the miRNA expression profiling efforts have led to inconsistent results between the studies. We performed a comprehensive meta‐analysis of 20 published miRNA expression studies in lung cancer, including a total of 598 tumor and 528 non‐cancerous control samples. Using a recently published robust rank aggregation method, we identified a statistically significant miRNA meta‐signature of seven upregulated (miR‐21, miR‐210, miR‐182, miR‐31, miR‐200b, miR‐205 and miR‐183) and eight downregulated (miR‐126‐3p, miR‐30a, miR‐30d, miR‐486‐5p, miR‐451a, miR‐126‐5p, miR‐143 and miR‐145) miRNAs. We conducted a gene set enrichment analysis to identify pathways that are most strongly affected by altered expression of these miRNAs. We found that meta‐signature miRNAs cooperatively target functionally related and biologically relevant genes in signaling and developmental pathways. We have shown that such meta‐analysis approach is suitable and effective solution for identification of statistically significant miRNA meta‐signature by combining several miRNA expression studies. This method allows the analysis of data produced by different technological platforms that cannot be otherwise directly compared or in the case when raw data are unavailable.

Identification of miR-374a as a prognostic marker for survival in patients with early-stage nonsmall cell lung cancer†

Lung cancer is one of the deadliest types of cancer proven by the poor survival and high relapse rates after surgery. Recently discovered microRNAs (miRNAs), small noncoding RNA molecules, play a crucial role in modulating gene expression networks and are directly involved in the progression of a number of human cancers. In this study, we analyzed the expression profile of 858 miRNAs in 38 Estonian nonsmall cell lung cancer (NSCLC) samples (Stage I and II) and 27 adjacent nontumorous tissue samples using Illumina miRNA arrays. We found that 39 miRNAs were up‐regulated and 33 down‐regulated significantly in tumors compared with normal lung tissue. We observed aberrant expression of several well‐characterized tumorigenesis‐related miRNAs, as well as a number of miRNAs whose function is currently unknown. We show that low expression of miR‐374a in early‐stage NSCLC is associated with poor patient survival. The combinatorial effect of the up‐ and down‐regulated miRNAs is predicted to most significantly affect pathways associated with cell migration, differentiation and growth, and several signaling pathways that contribute to tumorigenesis. In conclusion, our results demonstrate that expression of miR‐374a at early stages of NSCLC progression can serve as a prognostic marker for patient risk stratification and may be a promising therapeutic target for the treatment of lung cancer.

Natural antisense transcript of natriuretic peptide precursor A (NPPA): structural organization and modulation of NPPA expression.

BackgroundMammalian transcriptome contains a large proportion of diverse and structurally complex noncoding RNAs. One class of such RNAs, natural antisense transcripts (NATs), are derived from the opposite strand of many protein-coding genes. Although the exact structure and functional relevance of most of the NATs is unknown, their emerging role as gene expression regulators raises the hypothesis that NATs might contribute to development of complex human disorders. The goal of our study was to investigate the involvement of NATs in regulation of candidate genes for blood pressure.ResultsFirst we analysed blood pressure candidate genes for the presence of natural antisense transcripts. In silico analysis revealed that seven genes (ADD3, NPPA, ATP1A1, NPR2, CYP17A1, ACSM3, SLC14A2) have an antisense partner transcribed from the opposite strand. We characterized NPPA and its antisense transcript (NPPA-AS) in more detail. We found that NPPA-AS is expressed in a number of human tissues as a collection of alternatively spliced isoforms and that NPPA-AS and NPPA can form RNA duplexes in vivo. We also demonstrated that a specific NPPA-AS isoform is capable of down-regulating the intron-retained NPPA mRNA variant. We studied the evolutionary conservation of NPPA-AS and were able to detect the presence of Nppa-as transcript in mouse.ConclusionOur results demonstrate functional interaction of NPPA-AS with NPPA at the RNA level and suggest that antisense transcription might be an important post-transcriptional mechanism modulating NPPA expression.

Identification and characterization of a novel ABCA subfamily member, ABCA12, located in the lamellar ichthyosis region on 2q34

The ABCA subfamily of ABC transporters includes ten members to date. In this study, we describe an additional gene, ABCA12. Four full-length cDNA sequences have been obtained from human placenta that contain two different polyadenylation sites and two splicing forms, coding for ABCA12 isoforms of 2,595 and 2,516 amino acid residues. Both isoforms are predicted to have two ATP-binding domains (nucleotide binding domain, NBD) and two transmembrane (TM) domains, features shared by all other ABCA subfamily proteins. ABCA12 is most closely related to ABCA1, with an amino acid similarity of 47%. Northern blot analysis demonstrates that a 9.5-kb transcript is mainly expressed in the stom- ach. ABCA12 was mapped to human chromosome 2q34. Two other genes from ABCA subfamily are associated with human inherited diseases, ABCA1 with the cholesterol transport disorders Tangier disease and familial hypoalphalipoproteinemia, and ABCA4 with several retinal degeneration disorders. The ABCA12 gene is located in a region of chromosome 2q34 that harbors the genes for lamellar ichthyosis, polymorphic congenital cataract, and insulin-dependent diabetes mellitus (IDDM13), and therefore is a positional candidate for these pathologies.

Comparative genome analysis of potential regulatory elements in the ABCG5-ABCG8 gene cluster

The excretion of sterols from the liver and intestine is regulated by the ABCG5 and ABCG8 transporters. To identify potential regulatory elements, 152 kb of the human ABCG5-ABCG8 gene cluster was sequenced and comparative genome analysis was performed. The two genes are oriented in a head-to-head configuration and are separated by a 374-bp intergenic region, which is highly conserved among several species. Using a reporter construct, the intergenic region was found to act as a bidirectional promoter. A conserved GATA site in the intergenic region was shown by site-directed mutagenesis to act as a repressor for the ABCG5 promoter. The intergenic region was also shown to be partially responsive to treatment by LXR agonists. In summary, several potential regulatory elements were found for the ABCG5 and ABCG8 genes, and the intergenic region was found to act as a bidirectional promoter.

Human and mouse orthologs of a new ATP-binding cassette gene, ABCG4

We characterized a new ATP-binding cassette (ABC) transporter gene from human and mouse that is highly expressed in the brain. The gene, ABCG4, produces several transcripts that differ at the 5′ end and encode proteins of various lengths. The ABCG4 protein is closely related to the Drosophila white and human ABCG1 genes, and belongs to the ABCG subfamily several members of which are involved in cholesterol transport. All representatives of this “reverse transporter” subfamily, including ABCG4, have a single ATP-binding domain at the N-terminus and a single C-terminal set of transmembrane segments. ABCG4 maps to human chromosome 11q23, between the markers D11S939 and D11S924, and Abcg4 to a conserved syntenic region on mouse chromosome 9. The abundant expression of this gene in the brain and close evolutionary relationship to the other members of the subfamily suggests a potential role for ABCG4 in cholesterol transport processes in this tissue.

De novo SCN8A mutation identified by whole-exome sequencing in a boy with neonatal epileptic encephalopathy, multiple congenital anomalies, and movement disorders.

Epileptic encephalopathies represent a clinically and genetically heterogeneous group of disorders, majority of which are of unknown etiology. We used whole-exome sequencing of a parent-offspring trio to identify the cause of early infantile epileptic encephalopathy in a boy with neonatal seizures, movement disorders, and multiple congenital anomalies who died at the age of 17 months because of respiratory illness and identified a de novo heterozygous missense mutation (c.3979A>G; p.Ile1327Val) in SCN8A (voltage-gated sodium-channel type VIII alpha subunit) gene. The variant was confirmed in the proband with Sanger sequencing. Because the clinical phenotype associated with SCN8A mutations has previously been identified only in a few patients with or without epileptic seizures, these data together with our results suggest that mutations in SCN8A can lead to early infantile epileptic encephalopathy with a broad phenotypic spectrum. Additional investigations will be worthwhile to determine the prevalence and contribution of SCN8A mutations to epileptic encephalopathies.

Methylation Markers of Early-Stage Non-Small Cell Lung Cancer

Background Despite of intense research in early cancer detection, there is a lack of biomarkers for the reliable detection of malignant tumors, including non-small cell lung cancer (NSCLC). DNA methylation changes are common and relatively stable in various types of cancers, and may be used as diagnostic or prognostic biomarkers. Methods We performed DNA methylation profiling of samples from 48 patients with stage I NSCLC and 18 matching cancer-free lung samples using microarrays that cover the promoter regions of more than 14,500 genes. We correlated DNA methylation changes with gene expression levels and performed survival analysis. Results We observed hypermethylation of 496 CpGs in 379 genes and hypomethylation of 373 CpGs in 335 genes in NSCLC. Compared to adenocarcinoma samples, squamous cell carcinoma samples had 263 CpGs in 223 hypermethylated genes and 513 CpGs in 436 hypomethylated genes. 378 of 869 (43.5%) CpG sites discriminating the NSCLC and control samples showed an inverse correlation between CpG site methylation and gene expression levels. As a result of a survival analysis, we found 10 CpGs in 10 genes, in which the methylation level differs in different survival groups. Conclusions We have identified a set of genes with altered methylation in NSCLC and found that a minority of them showed an inverse correlation with gene expression levels. We also found a set of genes that associated with the survival of the patients. These newly-identified marker candidates for the molecular screening of NSCLC will need further analysis in order to determine their clinical utility.

Evolutionary analysis of a cluster of ATP-binding cassette (ABC) genes.

To study the evolutionary history of ATP-binding cassette (ABC) transporters in mammals, we have characterized a cluster of five ABCA-subfamily genes localized on mouse Chromosome (Chr) 11. The genes, named Abca5, Abca6, Abca8a, Abca8b, and Abca9, are arranged in a head-to-tail fashion in a cluster that spans about 400 kb of the genomic DNA, each gene occupying about 70 kb. The transcripts of these genes contain an open reading frame from 4863 (for Abca8a and Abca8b) to 4929 (for Abca5) nucleotides, and have distinct tissue-specific expression pattern. The predicted proteins contain two transmembrane domains and two nucleotide binding domains, arranged similar to the other members of ABCA subfamily. Similarity of both the genomic organization and primary structure among the genes in this cluster suggests that the duplications generating the cluster occurred relatively recently compared with most of the ABC genes in present-day mammalian genomes. For instance, the Fugu rubripes genome contains an ortholog for only one gene, Abca5, from this cluster. Phylogenetic and comparative sequence analysis reveals that after the divergence of rodent and primate lineages, at least one gene has been lost in each genome. In addition, we found that both mouse and human clusters show evidence of a number of gene conversions, in several cases involving intron sequences.

The dMRP/CG6214 gene of Drosophila is evolutionarily and functionally related to the human multidrug resistance-associated protein family

ATP‐binding cassette (ABC) transporters are involved in the transport of substrates across biological membranes and are essential for many cellular processes. Of the fifty‐six Drosophila ABC transporter genes only white, brown, scarlet, E23 and Atet have been studied in detail. Phylogenetic analyses identify the Drosophila gene dMRP/CG6214 as an orthologue to the human multidrug‐resistance associated proteins MRP1, MRP2, MRP3 and MRP6. To study evolutionarily conserved roles of MRPs we have initiated a characterization of dMRP. In situ hybridization and Northern analysis indicate that dMRP is expressed throughout development and appears to be head enriched in adults. Functional studies indicate that DMRP is capable of transporting a known MRP1 substrate and establishes DMRP as a high capacity ATP‐dependent, vanadate‐sensitive organic anion transporter.