Publications by authors named "Neeme Tõnisson"

28 Publications

  • Page 1 of 1

Genotype-first approach to the detection of hereditary breast and ovarian cancer risk, and effects of risk disclosure to biobank participants.

Eur J Hum Genet 2020 Nov 23. Epub 2020 Nov 23.

Estonian Genome Center, Institute of Genomics, University of Tartu, Tartu, Estonia.

Genotype-first approach allows to systematically identify carriers of pathogenic variants in BRCA1/2 genes conferring a high risk of familial breast and ovarian cancer. Participants of the Estonian biobank have expressed support for the disclosure of clinically significant findings. With an Estonian biobank cohort, we applied a genotype-first approach, contacted carriers, and offered return of results with genetic counseling. We evaluated participants' responses to and the clinical utility of the reporting of actionable genetic findings. Twenty-two of 40 contacted carriers of 17 pathogenic BRCA1/2 variants responded and chose to receive results. Eight of these 22 participants qualified for high-risk assessment based on National Comprehensive Cancer Network criteria. Twenty of 21 counseled participants appreciated being contacted. Relatives of 10 participants underwent cascade screening. Five of 16 eligible female BRCA1/2 variant carriers chose to undergo risk-reducing surgery, and 10 adhered to surveillance recommendations over the 30-month follow-up period. We recommend the return of results to population-based biobank participants; this approach could be viewed as a model for population-wide genetic testing. The genotype-first approach permits the identification of individuals at high risk who would not be identified by application of an approach based on personal and family histories only.
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http://dx.doi.org/10.1038/s41431-020-00760-2DOI Listing
November 2020

Creating basis for introducing non-invasive prenatal testing in the Estonian public health setting.

Prenat Diagn 2019 12 6;39(13):1262-1268. Epub 2019 Nov 6.

Competence Centre on Health Technologies, Tartu, Estonia.

Objective: The study aimed to validate a whole-genome sequencing-based NIPT laboratory method and our recently developed NIPTmer aneuploidy detection software with the potential to integrate the pipeline into prenatal clinical care in Estonia.

Method: In total, 424 maternal blood samples were included. Analysis pipeline involved cell-free DNA extraction, library preparation and massively parallel sequencing on Illumina platform. Aneuploidies were determined with NIPTmer software, which is based on counting pre-defined per-chromosome sets of unique k-mers from sequencing raw data. SeqFF was implemented to estimate cell-free fetal DNA (cffDNA) fraction.

Results: NIPTmer identified correctly all samples of non-mosaic trisomy 21 (T21, 15/15), T18 (9/9), T13 (4/4) and monosomy X (4/4) cases, with the 100% sensitivity. However, one mosaic T18 remained undetected. Six false-positive (FP) results were observed (FP rate of 1.5%, 6/398), including three for T18 (specificity 99.3%) and three for T13 (specificity 99.3%). The level of cffDNA of <4% was estimated in eight samples, including one sample with T13 and T18. Despite low cffDNA level, these two samples were determined as aneuploid.

Conclusion: We believe that the developed NIPT method can successfully be used as a universal primary screening test in combination with ultrasound scan for the first trimester fetal examination.
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http://dx.doi.org/10.1002/pd.5578DOI Listing
December 2019

Detection of a balanced translocation carrier through trophectoderm biopsy analysis: a case report.

Mol Cytogenet 2019 18;12:28. Epub 2019 Jun 18.

1Competence Centre on Health Technologies, Tiigi 61b, 50410 Tartu, Estonia.

Background: Balanced translocation carriers are burdened with fertility issues due to improper chromosome segregation in gametes, resulting in either implantation failure, miscarriage or birth of a child with chromosomal disorders. At the same time, these individuals are typically healthy with no signs of developmental problems, hence they often are unaware of their condition. Yet, because of difficulties in conceiving, balanced translocation carriers often turn to assisted reproduction, some of whom may also undergo preimplantation genetic testing for aneuploidy (PGT-A) to improve the likelihood of achieving a successful pregnancy.

Case Report: We describe a female patient, who pursued in vitro fertilization (IVF) treatment coupled with PGT-A following two consecutive miscarriages, unaware of her genetic condition. PGT-A was performed on blastocyst-stage embryos and the results of comprehensive chromosome screening from a first IVF cycle demonstrated reciprocal segmental aberrations on chromosome 7 and chromosome 10 in two out of four embryos. Due to distinct embryo profiles, the couple was then referred for genetic counselling and subsequent parental karyotyping revealed the presence of a previously undetected balanced translocation in the mother.

Conclusions: These results confirm previous reports that genome-wide PGT-A can facilitate the identification of balanced translocation carriers in IVF patients, providing explanation for poor reproductive outcome and allowing adjustments in treatment strategies.
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http://dx.doi.org/10.1186/s13039-019-0444-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582470PMC
June 2019

Polygenic prediction of breast cancer: comparison of genetic predictors and implications for risk stratification.

BMC Cancer 2019 Jun 10;19(1):557. Epub 2019 Jun 10.

Estonian Genome Center, Institute of Genomics, University of Tartu, Riia 23b, 51010, Tartu, Estonia.

Background: Published genetic risk scores for breast cancer (BC) so far have been based on a relatively small number of markers and are not necessarily using the full potential of large-scale Genome-Wide Association Studies. This study aimed to identify an efficient polygenic predictor for BC based on best available evidence and to assess its potential for personalized risk prediction and screening strategies.

Methods: Four different genetic risk scores (two already published and two newly developed) and their combinations (metaGRS) were compared in the subsets of two population-based biobank cohorts: the UK Biobank (UKBB, 3157 BC cases, 43,827 controls) and Estonian Biobank (EstBB, 317 prevalent and 308 incident BC cases in 32,557 women). In addition, correlations between different genetic risk scores and their associations with BC risk factors were studied in both cohorts.

Results: The metaGRS that combines two genetic risk scores (metaGRS - based on 75 and 898 Single Nucleotide Polymorphisms, respectively) had the strongest association with prevalent BC status in both cohorts. One standard deviation difference in the metaGRS corresponded to an Odds Ratio = 1.6 (95% CI 1.54 to 1.66, p = 9.7*10) in the UK Biobank and accounting for family history marginally attenuated the effect (Odds Ratio = 1.58, 95% CI 1.53 to 1.64, p = 7.8*10). In the EstBB cohort, the hazard ratio of incident BC for the women in the top 5% of the metaGRS compared to women in the lowest 50% was 4.2 (95% CI 2.8 to 6.2, p = 8.1*10). The different GRSs were only moderately correlated with each other and were associated with different known predictors of BC. The classification of genetic risk for the same individual varied considerably depending on the chosen GRS.

Conclusions: We have shown that metaGRS that combined on the effects of more than 900 SNPs, provided best predictive ability for breast cancer in two different population-based cohorts. The strength of the effect of metaGRS indicates that the GRS could potentially be used to develop more efficient strategies for breast cancer screening for genotyped women.
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http://dx.doi.org/10.1186/s12885-019-5783-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6558751PMC
June 2019

Search for Early Pancreatic Cancer Blood Biomarkers in Five European Prospective Population Biobanks Using Metabolomics.

Endocrinology 2019 07;160(7):1731-1742

Department of Human Genetics, Leiden University Medical Center, RC Leiden, Netherlands.

Most patients with pancreatic cancer present with advanced disease and die within the first year after diagnosis. Predictive biomarkers that signal the presence of pancreatic cancer in an early stage are desperately needed. We aimed to identify new and validate previously found plasma metabolomic biomarkers associated with early stages of pancreatic cancer. Prediagnostic blood samples from individuals who were to receive a diagnosis of pancreatic cancer between 1 month and 17 years after sampling (N = 356) and age- and sex-matched controls (N = 887) were collected from five large population cohorts (HUNT2, HUNT3, FINRISK, Estonian Biobank, Rotterdam Study). We applied proton nuclear magnetic resonance-based metabolomics on the Nightingale platform. Logistic regression identified two interesting hits: glutamine (P = 0.011) and histidine (P = 0.012), with Westfall-Young family-wise error rate adjusted P values of 0.43 for both. Stratification in quintiles showed a 1.5-fold elevated risk for the lowest 20% of glutamine and a 2.2-fold increased risk for the lowest 20% of histidine. Stratification by time to diagnosis suggested glutamine to be involved in an earlier process (2 to 5 years before diagnosis), and histidine in a process closer to the actual onset (<2 years). Our data did not support the branched-chain amino acids identified earlier in several US cohorts as potential biomarkers for pancreatic cancer. Thus, although we identified glutamine and histidine as potential biomarkers of biological interest, our results imply that a study at this scale does not yield metabolomic biomarkers with sufficient predictive value to be clinically useful per se as prognostic biomarkers.
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http://dx.doi.org/10.1210/en.2019-00165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594461PMC
July 2019

Recall by genotype and cascade screening for familial hypercholesterolemia in a population-based biobank from Estonia.

Genet Med 2019 05 1;21(5):1173-1180. Epub 2018 Oct 1.

Estonian Genome Center, Institute of Genomics, University of Tartu, Tartu, Estonia.

Purpose: Large-scale, population-based biobanks integrating health records and genomic profiles may provide a platform to identify individuals with disease-predisposing genetic variants. Here, we recall probands carrying familial hypercholesterolemia (FH)-associated variants, perform cascade screening of family members, and describe health outcomes affected by such a strategy.

Methods: The Estonian Biobank of Estonian Genome Center, University of Tartu, comprises 52,274 individuals. Among 4776 participants with exome or genome sequences, we identified 27 individuals who carried FH-associated variants in the LDLR, APOB, or PCSK9 genes. Cascade screening of 64 family members identified an additional 20 carriers of FH-associated variants.

Results: Via genetic counseling and clinical management of carriers, we were able to reclassify 51% of the study participants from having previously established nonspecific hypercholesterolemia to having FH and identify 32% who were completely unaware of harboring a high-risk disease-associated genetic variant. Imaging-based risk stratification targeted 86% of the variant carriers for statin treatment recommendations.

Conclusion: Genotype-guided recall of probands and subsequent cascade screening for familial hypercholesterolemia is feasible within a population-based biobank and may facilitate more appropriate clinical management.
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http://dx.doi.org/10.1038/s41436-018-0311-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6443485PMC
May 2019

The target landscape of clinical kinase drugs.

Science 2017 12;358(6367)

Chair of Proteomics and Bioanalytics, Technical University of Munich (TUM), Freising, Germany.

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.
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http://dx.doi.org/10.1126/science.aan4368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6542668PMC
December 2017

Genome-wide association study and meta-analysis in Northern European populations replicate multiple colorectal cancer risk loci.

Int J Cancer 2018 02 12;142(3):540-546. Epub 2017 Oct 12.

Department of Medical and Clinical Genetics, Medicum, University of Helsinki, Helsinki, Finland.

Genome-wide association studies have been successful in elucidating the genetic basis of colorectal cancer (CRC), but there remains unexplained variability in genetic risk. To identify new risk variants and to confirm reported associations, we conducted a genome-wide association study in 1,701 CRC cases and 14,082 cancer-free controls from the Finnish population. A total of 9,068,015 genetic variants were imputed and tested, and 30 promising variants were studied in additional 11,647 cases and 12,356 controls of European ancestry. The previously reported association between the single-nucleotide polymorphism (SNP) rs992157 (2q35) and CRC was independently replicated (p = 2.08 × 10 ; OR, 1.14; 95% CI, 1.06-1.23), and it was genome-wide significant in combined analysis (p = 1.50 × 10 ; OR, 1.12; 95% CI, 1.08-1.16). Variants at 2q35, 6p21.2, 8q23.3, 8q24.21, 10q22.3, 10q24.2, 11q13.4, 11q23.1, 14q22.2, 15q13.3, 18q21.1, 20p12.3 and 20q13.33 were associated with CRC in the Finnish population (false discovery rate < 0.1), but new risk loci were not found. These results replicate the effects of multiple loci on the risk of CRC and identify shared risk alleles between the Finnish population isolate and outbred populations.
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http://dx.doi.org/10.1002/ijc.31076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6383773PMC
February 2018

Somatic mosaicism for copy-neutral loss of heterozygosity and DNA copy number variations in the human genome.

BMC Genomics 2015 Sep 16;16:703. Epub 2015 Sep 16.

Competence Centre on Health Technologies, Tiigi 61b, 50410, Tartu, Estonia.

Background: Somatic mosaicism denotes the presence of genetically distinct populations of somatic cells in one individual who has developed from a single fertilised oocyte. Mosaicism may result from a mutation that occurs during postzygotic development and is propagated to only a subset of the adult cells. Our aim was to investigate both somatic mosaicism for copy-neutral loss of heterozygosity (cn-LOH) events and DNA copy number variations (CNVs) in fully differentiated tissues.

Results: We studied panels of tissue samples (11-12 tissues per individual) from four autopsy subjects using high-resolution Illumina HumanOmniExpress-12 BeadChips to reveal the presence of possible intra-individual tissue-specific cn-LOH and CNV patterns. We detected five mosaic cn-LOH regions >5 Mb in some tissue samples in three out of four individuals. We also detected three CNVs that affected only a portion of the tissues studied in one out of four individuals. These three somatic CNVs range from 123 to 796 kb and are also found in the general population. An attempt was made to explain the succession of genomic events that led to the observed somatic genetic mosaicism under the assumption that the specific mosaic patterns of CNV and cn-LOH changes reflect their formation during the postzygotic embryonic development of germinal layers and organ systems.

Conclusions: Our results give further support to the idea that somatic mosaicism for CNVs, and also cn-LOHs, is a common phenomenon in phenotypically normal humans. Thus, the examination of only a single tissue might not provide enough information to diagnose potentially deleterious CNVs within an individual. During routine CNV and cn-LOH analysis, DNA derived from a buccal swab can be used in addition to blood DNA to get information about the CNV/cn-LOH content in tissues of both mesodermal and ectodermal origin. Currently, the real frequency and possible phenotypic consequences of both CNVs and cn-LOHs that display somatic mosaicism remain largely unknown. To answer these questions, future studies should involve larger cohorts of individuals and a range of tissues.
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http://dx.doi.org/10.1186/s12864-015-1916-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4573927PMC
September 2015

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

Genome Biol 2014 Apr 1;15(4):r54. Epub 2014 Apr 1.

Background: DNA 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.

Results: Only 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.

Conclusions: This 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.
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http://dx.doi.org/10.1186/gb-2014-15-4-r54DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4053947PMC
April 2014

Tissue-specific mitochondrial heteroplasmy at position 16,093 within the same individual.

Curr Genet 2014 Feb 11;60(1):11-6. Epub 2013 Jul 11.

Competence Centre on Reproductive Medicine and Biology, Tartu, Estonia,

Human mitochondrial DNA (mtDNA) research has entered a massively parallel sequencing (MPS) era, providing deep insight into mtDNA genomics and molecular diagnostics. Analysis can simultaneously include coding and control regions, many samples can be studied in parallel, and even minor heteroplasmic changes can be detected. We investigated heteroplasmy using 16 different tissues from three unrelated males aged 40-54 years at the time of death. mtDNA was enriched using two independent overlapping long-range PCR amplicons and analysed by employing illumina paired-end sequencing. Point mutation heteroplasmy at position 16,093 (m.16093T > C) in the non-coding regulatory region showed great variability among one of the studied individuals; heteroplasmy extended from 5.1 % in red bone marrow to 62.0 % in the bladder. Red (5.1 %) and yellow bone marrow (8.9 %) clustered into one group and two arteries and two aortas from different locations into another (31.2-50.9 %), giving an ontogenetic explanation for the formation of somatic mitochondrial heteroplasmy. Our results demonstrate that multi-tissue screening using MPS provides surprising data even when there is a limited number (3) of study subjects and they give reason to speculate that mtDNA heteroplasmic frequency, distribution, and even its possible role in complex diseases or phenotypes seem to be underestimated.
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http://dx.doi.org/10.1007/s00294-013-0398-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3895442PMC
February 2014

Methylation markers of early-stage non-small cell lung cancer.

PLoS One 2012 29;7(6):e39813. Epub 2012 Jun 29.

Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0039813PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3387223PMC
November 2012

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

Genes Chromosomes Cancer 2011 Oct 11;50(10):812-22. Epub 2011 Jul 11.

Department of Biotechnology, Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia.

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.
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http://dx.doi.org/10.1002/gcc.20902DOI Listing
October 2011

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

Hum Mutat 2011 Jul 10;32(7):806-14. Epub 2011 May 10.

Human Molecular Genetics Research Group, Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.

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.
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http://dx.doi.org/10.1002/humu.21508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3298642PMC
July 2011

Molecular diagnosis of Down syndrome using quantitative APEX-2 microarrays.

Prenat Diagn 2010 Dec;30(12-13):1170-7

Estonian Biocentre, Tartu, Estonia.

Objective: To develop a new rapid and high-throughput microarray-based prenatal diagnostic test for the detection of trisomy 21 (T21).

Methods: The T21 arrayed primer extension-2 (APEX-2) assay discriminates between trisomy and euploid DNA samples by comparing the signal intensities of allelic fractions of heterozygous single nucleotide polymorphisms (SNPs) after APEX reaction. After preliminary validation using DNA samples from Down syndrome patients, we analyzed DNA samples from cultured and uncultured amniocytes and chorionic villus for 90 SNPs with high heterozygosity from the 21(q21.1q22.2) region. Differences in allelic ratios of heterozygous SNPs in normal and T21 individuals were verified by t-test.

Results: Analysis of the T21 APEX-2 assay results revealed that 90 SNPs were sufficient for reliable discrimination between T21 and euploid DNA samples (P≤0.05 for one or both strands). Using 134 clinical samples from cultured or uncultured fetal cells, both the sensitivity and the specificity of the assay were 100%.

Conclusion: Our study provides a proof of principle demonstration of the use of the modified APEX-2 assay as a new, fast and reliable method for prenatal diagnosis of fetal T21.
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http://dx.doi.org/10.1002/pd.2639DOI Listing
December 2010

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

Int J Pediatr Otorhinolaryngol 2010 Sep 18;74(9):1007-12. Epub 2010 Jun 18.

Department of Genetics, United Laboratories, Tartu University Hospital, Tartu, 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.
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http://dx.doi.org/10.1016/j.ijporl.2010.05.026DOI Listing
September 2010

Classical galactosemia in Estonia: selective neonatal screening, incidence, and genotype/phenotype data of diagnosed patients.

J Inherit Metab Dis 2010 Apr 12;33(2):175-6. Epub 2010 Feb 12.

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http://dx.doi.org/10.1007/s10545-010-9045-2DOI Listing
April 2010

Hypervariable intronic region in NCX1 is enriched in short insertion-deletion polymorphisms and showed association with cardiovascular traits.

BMC Med Genet 2010 Jan 28;11:15. Epub 2010 Jan 28.

Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.

Background: Conserved non-coding regions (CNR) have been shown to harbor gene expression regulatory elements. Genetic variations in these regions may potentially contribute to complex disease susceptibility.

Methods: We targeted CNRs of cardiovascular disease (CVD) candidate gene, Na(+)-Ca(2+) exchanger (NCX1) with polymorphism screening among CVD patients (n = 46) using DHPLC technology. The flanking region (348 bp) of the 14 bp indel in intron 2 was further genotyped by DGGE assay in two Eastern-European CVD samples: essential hypertension (HYPEST; 470 cases, 652 controls) and coronary artery disease, CAD (CADCZ; 257 cases, controls 413). Genotype-phenotype associations were tested by regression analysis implemented in PLINK. Alignments of primate sequences were performed by ClustalW2.

Results: Nine of the identified NCX1 variants were either singletons or targeted by commercial platforms. The 14 bp intronic indel (rs11274804) was represented with substantial frequency in HYPEST (6.82%) and CADCZ (14.58%). Genotyping in Eastern-Europeans (n = 1792) revealed hypervariable nature of this locus, represented by seven alternative alleles. The alignments of human-chimpanzee-macaque sequences showed that the major human variant (allele frequency 90.45%) was actually a human-specific deletion compared to other primates. In humans, this deletion was surrounded by other short (5-43 bp) deletion variants and a duplication (40 bp) polymorphism possessing overlapping breakpoints. This indicates a potential indel hotspot, triggered by the initial deletion in human lineage. An association was detected between the carrier status of 14 bp indel ancestral allele and CAD (P = 0.0016, OR = 2.02; Bonferroni significance level alpha = 0.0045), but not with hypertension. The risk for the CAD development was even higher among the patients additionally diagnosed with metabolic syndrome (P = 0.0014, OR = 2.34). Consistent with the effect on metabolic processes, suggestive evidence for the association with heart rate, serum triglyceride and LDL levels was detected (P = 0.04).

Conclusions: Compared to SNPs targeted by large number of locus-specific and genome-wide assays, considerably less attention has been paid to short indel variants in the human genome. The data of genome dynamics, mutation rate and population genetics of short indels, as well as their impact on gene expressional profile and human disease susceptibility is limited. The characterization of NCX1 intronic hypervariable non-coding region enriched in human-specific indel variants contributes to this gap of knowledge.
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http://dx.doi.org/10.1186/1471-2350-11-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2832636PMC
January 2010

Splice variant IVS2-2A>G in the SLC26A5 (Prestin) gene in five Estonian families with hearing loss.

Int J Pediatr Otorhinolaryngol 2009 Jan 22;73(1):103-7. Epub 2008 Nov 22.

Department of Oto-Rhino-Laryngology, University of Tartu, Tartu, Estonia.

Objective: The aim of our study was to identify the IVS2-2A>G sequence change in the SLC26A5 (Prestin) gene in Estonian individuals with hearing loss and in their family members.

Methods: In the years 2005-2007 we have screened 194 probands with early onset hearing loss and 68 family members with an arrayed primer extension (APEX) microarray, which covers 201 mutations in six nuclear genes (GJB2, GJB6, GJB3, GJA1, SLC26A4, SLC26A5) and two mitochondrial genes encoding 12S rRNA and tRNA-Ser (UCN).

Results: In four probands with early onset hearing loss and in five unaffected family members from five families we identified the IVS2-2A>G change in one allele of the SLC26A5 gene. We did not find any homozygosity for this splice variant. IVS2-2A>G was identified in 2.1% of probands. One of these probands, however, is also homozygous for the 35delG mutation in the GJB2 gene and a second patient has Down syndrome, which is also associated with hearing impairment. Therefore, in those two cases the etiology of the hearing loss is probably not associated with the IVS2-2A>G sequence change in the SLC26A5 gene.

Conclusion: Our data support the hypothesis that heterozygosity for the mutation IVS2-2A>G in SLC26A5 gene may not, by itself, be sufficient to cause hearing loss.
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http://dx.doi.org/10.1016/j.ijporl.2008.10.003DOI Listing
January 2009

Array-MAPH: a methodology for the detection of locus copy-number changes in complex genomes.

Nat Protoc 2008 ;3(5):849-65

Department of Cytogenetics, The Cyprus Institute of Neurology & Genetics, Nicosia, Cyprus.

High-throughput genome-wide screening methods to detect subtle genomic imbalances are extremely important for diagnostic genetics and genomics. Here, we provide a detailed protocol for a microarray-based technique, applying the principle of multiplex amplifiable probe hybridization (MAPH). Methodology and software have been developed for designing unique PCR-amplifiable sequences (400-600 bp) covering any genomic region of interest. These sequences are amplified, cloned and spotted onto arrays (targets). A mixture of the same sequences (probes) is hybridized to genomic DNA immobilized on a membrane. Bound probes are recovered and quantitatively amplified by PCR, labeled and hybridized to the array. The procedure can be completed in 4-5 working days, excluding microarray preparation. Unlike array-comparative genomic hybridization (array-CGH), test DNA of specifically reduced complexity is hybridized to an array of identical small amplifiable target sequences, resulting in increased hybridization specificity and higher potential for increasing resolution. Array-MAPH can be used for detection of small-scale copy-number changes in complex genomes, leading to genotype-phenotype correlations and the discovery of new genes.
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http://dx.doi.org/10.1038/nprot.2008.49DOI Listing
August 2008

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

Eur J Hum Genet 2007 Feb 22;15(2):162-72. Epub 2006 Nov 22.

Department of Cytogenetics, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus.

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.
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http://dx.doi.org/10.1038/sj.ejhg.5201738DOI Listing
February 2007

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

Biotechniques 2005 Nov;39(5):755-61

The Norwegian Radium Hospital, Oslo, Norway.

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.
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http://dx.doi.org/10.2144/000112000DOI Listing
November 2005

Reliable detection of beta-thalassemia and G6PD mutations by a DNA microarray.

Clin Chem 2002 Nov;48(11):2051-4

IARC, International Agency for Research on Cancer, 150, Cours Albert Thomas, Lyon 69372, France.

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November 2002

A first-generation linkage disequilibrium map of human chromosome 22.

Nature 2002 Aug 10;418(6897):544-8. Epub 2002 Jul 10.

The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

DNA sequence variants in specific genes or regions of the human genome are responsible for a variety of phenotypes such as disease risk or variable drug response. These variants can be investigated directly, or through their non-random associations with neighbouring markers (called linkage disequilibrium (LD)). Here we report measurement of LD along the complete sequence of human chromosome 22. Duplicate genotyping and analysis of 1,504 markers in Centre d'Etude du Polymorphisme Humain (CEPH) reference families at a median spacing of 15 kilobases (kb) reveals a highly variable pattern of LD along the chromosome, in which extensive regions of nearly complete LD up to 804 kb in length are interspersed with regions of little or no detectable LD. The LD patterns are replicated in a panel of unrelated UK Caucasians. There is a strong correlation between high LD and low recombination frequency in the extant genetic map, suggesting that historical and contemporary recombination rates are similar. This study demonstrates the feasibility of developing genome-wide maps of LD.
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http://dx.doi.org/10.1038/nature00864DOI Listing
August 2002

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

Proc Natl Acad Sci U S A 2002 Apr;99(8):5503-8

Asper, Ltd., 3 Oru Street, 51014 Tartu, Estonia.

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.
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http://dx.doi.org/10.1073/pnas.082100599DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC122799PMC
April 2002