Neeme Tõnisson

DNA methylome profiling of human tissues identifies global and tissue-specific methylation patterns

BackgroundDNA epigenetic modifications, such as methylation, are important regulators of tissue differentiation, contributing to processes of both development and cancer. Profiling the tissue-specific DNA methylome patterns will provide novel insights into normal and pathogenic mechanisms, as well as help in future epigenetic therapies. In this study, 17 somatic tissues from four autopsied humans were subjected to functional genome analysis using the Illumina Infinium HumanMethylation450 BeadChip, covering 486 428 CpG sites.ResultsOnly 2% of the CpGs analyzed are hypermethylated in all 17 tissue specimens; these permanently methylated CpG sites are located predominantly in gene-body regions. In contrast, 15% of the CpGs are hypomethylated in all specimens and are primarily located in regions proximal to transcription start sites. A vast number of tissue-specific differentially methylated regions are identified and considered likely mediators of tissue-specific gene regulatory mechanisms since the hypomethylated regions are closely related to known functions of the corresponding tissue. Finally, a clear inverse correlation is observed between promoter methylation within CpG islands and gene expression data obtained from publicly available databases.ConclusionsThis genome-wide methylation profiling study identified tissue-specific differentially methylated regions in 17 human somatic tissues. Many of the genes corresponding to these differentially methylated regions contribute to tissue-specific functions. Future studies may use these data as a reference to identify markers of perturbed differentiation and disease-related pathogenic mechanisms.

Evaluating the arrayed primer extension resequencing assay of TP53 tumor suppressor gene

Identification of mutations in the tumor suppressor gene TP53 has implications for the molecular epidemiology and for the molecular pathology of human cancer. We have developed and evaluated an arrayed primer extension assay for covering both strands of a region of the coding sequence containing more than 95% of the mutations described so far in TP53. On average, 97.5% of the arrayed TP53 gene sequence can be analyzed from either sense or antisense strands, and 81% from both strands. A patient DNA sample is amplified and annealed to arrayed primers, which then promote DNA polymerase extension reactions with four fluorescently labeled dideoxynucleotides. The TP53 gene chip spans exons 2–9 plus two introns from both strands. The performance of the assay was evaluated by using freshly extracted genomic DNA, as well as DNA extracted from archival (paraffin-embedded) DNA samples. The arrayed primer extension-based TP53 gene test provides an accurate and efficient tool for DNA sequence analysis of this frequently mutated gene for both research and clinical applications.

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.

Unravelling genetic data by arrayed primer extension.

Abstract We have developed a method for arrayed primer extension (APEX) on an oligonucleotide microchip together with the 4-color fluoresence imaging equipment and supporting software, that allows analysis of the DNA sequence and changes in it. Mutation analysis of BRCA1 gene and single nucleotide polymorphism (SNP) chip for genotyping were used as a model system. Chip surface chemistry, template preparation and APEX reaction conditions were optimised and the assay is ready to be implemented in variety of DNA analysis from SNP testing to DNA resequencing.

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.

The target landscape of clinical kinase drugs

An atlas for drug interactions Kinase inhibitors are an important class of drugs that block certain enzymes involved in diseases such as cancer and inflammatory disorders. There are hundreds of kinases within the human body, so knowing the kinase “target” of each drug is essential for developing successful treatment strategies. Sometimes clinical trials can fail because drugs bind more than one target. Yet sometimes off-target effects can be beneficial, and drugs can be repurposed for treatment of additional diseases. Klaeger et al. performed a comprehensive analysis of 243 kinase inhibitors that are either approved for use or in clinical trials. They provide an open-access resource of target summaries that could help researchers develop better drugs, understand how existing drugs work, and design more effective clinical trials. Science, this issue p. eaan4368 The druggable kinome is unraveled. INTRODUCTION Molecularly targeted drugs such as imatinib and crizotinib have revolutionized the treatment of certain blood and lung cancers because of their remarkable clinical success. Over the past 20 years, protein kinases have become a major class of drug targets because these signaling biomolecules are often deregulated in disease, particularly in cancer. Today, 37 small kinase inhibitors (KIs) are approved medicines worldwide and more than 250 drug candidates are undergoing clinical evaluation. RATIONALE Although it is commonly accepted that most KIs target more than one protein, the extent to which this information is available to the public varies greatly between drugs. It would seem important to thoroughly characterize the target spectrum of any drug because additional off-targets may offer opportunities, not only for repurposing but also to explain undesired side effects. To this end, we used a chemical proteomic approach (kinobeads) and quantitative mass spectrometry to characterize the target space of 243 clinical KIs that are approved drugs or have been tested in humans. RESULTS The number of targets for a given drug differed substantially. Whereas some compounds showed exquisite selectivity, others targeted more than 100 kinases simultaneously, making it difficult to attribute their biological effects to any particular mode of action. Also of note is that recently developed irreversible KIs can address more kinases than their intended targets epidermal growth factor receptor (EGFR) and Bruton’s tyrosine kinase (BTK). Collectively, the evaluated KIs targeted 220 kinases with submicromolar affinity, offering a view of the druggable kinome and enabling the development of a universal new selectivity metric termed CATDS (concentration- and target-dependent selectivity). All drug profiles can be interactively explored in ProteomicsDB and a purpose-built shinyApp. Many uses of this unique data and analysis resource by the scientific community can be envisaged, of which we can only highlight a few. The profiles identified many new targets for established drugs, thus improving our understanding of how these drugs might exert their phenotypic effects. For example, we evaluated novel salt-inducible kinase 2 (SIK2) inhibitors for their ability to modulate tumor necrosis factor–α (TNFα) and interleukin-10 (IL-10) production, which may allow repurposing these drugs for inflammatory conditions. Integrating target space information with phosphoproteomic analysis of several EGFR inhibitors enabled the identification of drug response markers inside and outside the canonical EGFR signaling pathway. Off-target identification may also inform drug discovery projects using high-value clinical molecules as lead compounds. We illustrate such a case by a novel structure-affinity relationship analysis of MELK inhibitors based on target profiles and cocrystal structures. To assess the repurposing potential of approved or clinically advanced compounds, we used cell-based assays and mouse xenografts to show that golvatinib and cabozantinib may be used for the treatment of acute myeloid leukemia (AML) based on their FLT3 inhibitory activity. CONCLUSION This work provides a rich data resource describing the target landscape of 243 clinically tested KIs. It is the most comprehensive study to date and illustrates how the information may be used in basic research, drug discovery, or clinical decision-making. Schematic representation of identifying the druggable kinome. A chemical proteomic approach revealed quantitative interaction profiles of 243 clinically evaluated small-molecule KIs covering half of the human kinome. Results can be interactively explored in ProteomicsDB and inform basic biology, drug discovery, and clinical decision-making. Kinase inhibitors are important cancer therapeutics. Polypharmacology is commonly observed, requiring thorough target deconvolution to understand drug mechanism of action. Using chemical proteomics, we analyzed the target spectrum of 243 clinically evaluated kinase drugs. The data revealed previously unknown targets for established drugs, offered a perspective on the “druggable” kinome, highlighted (non)kinase off-targets, and suggested potential therapeutic applications. Integration of phosphoproteomic data refined drug-affected pathways, identified response markers, and strengthened rationale for combination treatments. We exemplify translational value by discovering SIK2 (salt-inducible kinase 2) inhibitors that modulate cytokine production in primary cells, by identifying drugs against the lung cancer survival marker MELK (maternal embryonic leucine zipper kinase), and by repurposing cabozantinib to treat FLT3-ITD–positive acute myeloid leukemia. This resource, available via the ProteomicsDB database, should facilitate basic, clinical, and drug discovery research and aid clinical decision-making.

Evaluation of arrayed primer extension for TP53 mutation detection in breast and ovarian carcinomas

Mutations in the tumor suppressor gene TP53 are associated with a wide range of different cancers and may have prognostic and therapeutic implications. Methods for rapid and sensitive detection of mutations in this gene are therefore required. In order to make screening more effective, a commercially available TP53 genotyping microarray from Asper Biotech has been constructed by arrayed primer extension (APEX). The present study is the first report that blindly evaluates the efficiency of the second generation APEX TP53 genotype chip outside the Asper laboratory and compares it to temporal temperature gradient electrophoresis (TTGE) and sequencing of TP53 for mutation detection in ovarian and breast cancer samples. All nucleotides in the TP53 gene from exon 2-9 are included on the chip by synthesis and application of sequence-specific oligonucleotides. The chip was validated by screening 48 breast and 11 ovarian cancer cases, all of which had previously been analyzed by TTGE and sequencing. APEX scored 17 of 20 sequence variants, missing one deletion, one insertion, and a missense mutation. Resequencing efficiency using APEX was 92% for both DNA strands and 99.5% for sense and/or antisense strand. We conclude that the APEX TP53 microarray is a robust, rapid, and comprehensive screening tool for sequence alterations in tumors.

Novel polymorphic AluYb8 insertion in the WNK1 gene is associated with blood pressure variation in Europeans

Mutations in WNK1 and WNK4 cause familial hypertension, the Gordon syndrome. WNK1 and WNK4 conserved noncoding regions were targeted to polymorphism screening using DHPLC and DGGE. The scan identified an undescribed polymorphic AluYb8 insertion in WNK1 intron 10. Screening in primates revealed that this Alu‐insertion has probably occurred in human lineage. Genotyping in 18 populations from Europe, Asia, and Africa (n = 854) indicated an expansion of the WNK1 AluYb8 bearing chromosomes out of Africa. The allele frequency in Sub‐Saharan Africa was ∼3.3 times lower than in other populations (4.8 vs. 15.8%; P = 9.7 × 10−9). Meta‐analysis across three European sample sets (n = 3,494; HYPEST, Estonians; BRIGHT, the British; CADCZ, Czech) detected significant association of the WNK1 AluYb8 insertion with blood pressure (BP; systolic BP, P = 4.03 × 10−3, effect 1.12; diastolic BP, P = 1.21 × 10−2, effect 0.67). Gender‐stratified analysis revealed that this effect might be female‐specific (n = 2,088; SBP, P = 1.99 × 10−3, effect 1.59; DBP P = 3.64 × 10−4, effect 1.23; resistant to Bonferroni correction), whereas no statistical support was identified for the association with male BP (n = 1,406). In leucocytes, the expressional proportions of the full‐length WNK1 transcript and the splice‐form skipping exon 11 were significantly shifted in AluYb8 carriers compared to noncarriers. The WNK1 AluYb8 insertion might affect human BP via altering the profile of alternatively spliced transcripts. Hum Mutat 32:1–9, 2011.

Prevalence of c.35delG and p.M34T mutations in the GJB2 gene in Estonia.

OBJECTIVE The purpose of this study was to determine the prevalence of c.35delG and p.M34T mutations in the GJB2 gene among children with early onset hearing loss and within a general population of Estonia. METHODS Using an arrayed primer extension assay, we screened 233 probands with early childhood onset hearing loss for 107 different mutations in the GJB2 gene. We then looked for the two most common mutations, c.35delG and p.M34T, in a population of 998 consecutively born Estonian neonates to determine the frequency of these mutations in the general population. RESULTS In 115 (49%) of the patients with early onset hearing loss, we found a mutation in at least one allele of the GJB2 gene. Seventy-three (31%) were homozygous for the c.35delG mutation, seven (3%) were homozygous for the p.M34T mutation, and five (2%) had c35delG/p.M34T compound heterozygosity. Other six identified mutations in GJB2 gene occurred rarely. Among the 998 anonymous newborn samples, we detected 45 who were heterozygous for c.35delG, 2 individuals homozygous for c.35delG, and 58 who were heterozygous for p.M34T. Additionally, we detected two c.35delG/p.M34T compound heterozygotes. CONCLUSION The most common GJB2 gene mutations in Estonian children with early onset hearing loss were c.35delG and p.M34T, with c.35delG accounting for 75% of GJB2 alleles. The carrier frequency for c.35delG and p.M34T in a general population of Estonia was 1 in 22 and 1 in 17, respectively, and was higher than in most other countries.

Detection of small genomic imbalances using microarray-based multiplex amplifiable probe hybridization

Array-based genome-wide screening methods were recently introduced to clinical practice in order to detect small genomic imbalances that may cause severe genetic disorders. The continuous advancement of such methods plays an extremely important role in diagnostic genetics and medical genomics. We have modified and adapted the original multiplex amplifiable probe hybridization (MAPH) to a novel microarray format providing an important new diagnostic tool for detection of small size copy-number changes in any locus of human genome. Here, we describe the new array-MAPH diagnostic method and show proof of concept through fabrication, interrogation and validation of a human chromosome X-specific array. We have developed new bioinformatic tools and methodology for designing and producing amplifiable hybridization probes (200–600 bp) for array-MAPH. We designed 558 chromosome X-specific probes with median spacing 238 kb and 107 autosomal probes, which were spotted onto microarrays. DNA samples from normal individuals and patients with known and unknown chromosome X aberrations were analyzed for validation. Array-MAPH detected exactly the same deletions and duplications in blind studies, as well as other unknown small size deletions showing its accuracy and sensitivity. All results were confirmed by fluorescence in situ hybridization and probe-specific PCR. Array-MAPH is a new microarray-based diagnostic tool for the detection of small-scale copy-number changes in complex genomes, which may be useful for genotype–phenotype correlations, identification of new genes, studying genetic variation and provision of genetic services.