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- Olsson Lindvall, Martina, et al.
(författare)
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A Comprehensive Sequencing-Based Analysis of Allelic Methylation Patterns in Hemostatic Genes in Human Liver
- 2020
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Ingår i: Thrombosis and Haemostasis. - : Georg Thieme Verlag KG. - 0340-6245 .- 2567-689X. ; 120:2, s. 229-242
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Tidskriftsartikel (refereegranskat)abstract
- Characterizing the relationship between genetic, epigenetic (e.g., deoxyribonucleic acid [DNA] methylation), and transcript variation could provide insights into mechanisms regulating hemostasis and potentially identify new drug targets. Several hemostatic factors are synthesized in the liver, yet high-resolution DNA methylation data from human liver tissue is currently lacking for these genes. Single-nucleotide polymorphisms (SNPs) can influence DNA methylation in cis which can affect gene expression. This can be analyzed through allele-specific methylation (ASM) experiments. We performed targeted genomic DNA- and bisulfite-sequencing of 35 hemostatic genes in human liver samples for SNP and DNA methylation analysis, respectively, and integrated the data for ASM determination. ASM-associated SNPs (ASM-SNPs) were tested for association to gene expression in liver using in-house generated ribonucleic acid-sequencing data. We then assessed whether ASM-SNPs associated with gene expression, plasma proteins, or other traits relevant for hemostasis using publicly available data. We identified 112 candidate ASM-SNPs. Of these, 68% were associated with expression of their respective genes in human liver or in other human tissues and 54% were associated with the respective plasma protein levels, activity, or other relevant hemostatic genome-wide association study traits such as venous thromboembolism, coronary artery disease, stroke, and warfarin dose maintenance. Our study provides the first detailed map of the DNA methylation landscape and ASM analysis of hemostatic genes in human liver tissue, and suggests that methylation regulated by genetic variants in cis may provide a mechanistic link between noncoding SNPs and variation observed in circulating hemostatic proteins, prothrombotic diseases, and drug response.
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| 2. |
- Olsson Lindvall, Martina, et al.
(författare)
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Comparison of DNA Methylation Profiles of Hemostatic Genes between Liver Tissue and Peripheral Blood within Individuals
- 2021
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Ingår i: Thrombosis and Haemostasis. - : Georg Thieme Verlag KG. - 0340-6245 .- 2567-689X. ; 121:5, s. 573-583
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Tidskriftsartikel (refereegranskat)abstract
- DNA methylation has become increasingly recognized in the etiology of complex diseases, including thrombotic disorders. Blood is often collected in epidemiological studies for genotyping and has recently also been used to examine DNA methylation in epigenome-wide association studies. DNA methylation patterns are often tissue-specific, thus, peripheral blood may not accurately reflect the methylation pattern in the tissue of relevance. Here, we collected paired liver and blood samples concurrently from 27 individuals undergoing liver surgery. We performed targeted bisulfite sequencing for a set of 35 hemostatic genes primarily expressed in liver to analyze DNA methylation levels of >10,000 cytosine-phosphate-guanine (CpG) dinucleotides. We evaluated whether DNA methylation in blood could serve as a proxy for DNA methylation in liver at individual CpGs. Approximately 30% of CpGs were nonvariable and were predominantly hypo- (<25%) or hypermethylated (>70%) in both tissues. While blood can serve as a proxy for liver at these CpGs, the low variability renders these unlikely to explain phenotypic differences. We therefore focused on CpG sites with variable methylation levels in liver. The level of blood-liver tissue correlation varied widely across these variable CpGs; moderate correlations (0.5 <= r <0.75) were detected for 6% and strong correlations ( r 0.75) for a further 4%. Our findings indicate that it is essential to study the concordance of DNA methylation between blood and liver at individual CpGs. This paired blood-liver dataset is intended as a resource to aid interpretation of blood-based DNA methylation results.
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| 3. |
- Olsson Lindvall, Martina, et al.
(författare)
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Hemostatic Genes Exhibit a High Degree of Allele-Specific Regulation in Liver
- 2019
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Ingår i: Thrombosis and Haemostasis. - : Georg Thieme Verlag KG. - 0340-6245 .- 2567-689X. ; 119:7, s. 1072-1083
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Tidskriftsartikel (refereegranskat)abstract
- Objective Elucidating the genetic basis underlying hepatic hemostatic gene expression variability may contribute to unraveling genetic factors contributing to thrombotic or bleeding disorders. We aimed to identify novel cis-regulatory variants involved in regulating hemostatic genes by analyzing allele-specific expression (ASE) in human liver samples. Study Design Biopsies of human liver tissue and blood were collected from adults undergoing liver surgery at the Sahlgrenska University Hospital (n =20). Genomic deoxyribonucleic acid (gDNA) and total ribonucleic acid (RNA) were isolated. A targeted approach was used to enrich and sequence 35 hemostatic genes for single nucleotide polymorphism (SNP) analysis (gDNAseq) and construct individualized genomes for transcript alignment. The allelic ratio of transcripts from targeted RNAseq was determined via ASE analysis. Public expression quantitative trait loci (eQTL) and genome-wide association study (GWAS) data were used to assess novelty and importance of the ASE SNPs (and proxies, r(2) >= 0.8) for relevant traits/diseases. Results Sixty percent of the genes studied showed allelic imbalance across 53 SNPs. Of these, 7 SNPs were previously validated in liver eQTL studies. For 32 with eQTLs in other cell/tissue types, this is the first time genotype-specific expression is demonstrated in liver, and for 14 ASE SNPs, this is the first ever reported genotype-expression association. A total of 29 ASE SNPs were previously associated with the respective plasma protein levels and 17 ASE SNPs to other relevant GWAS traits including venous thromboembolism, coronary artery disease, and stroke. Conclusion Our study provides a comprehensive ASE analysis of hemostatic genes and insights into the regulation of hemostatic genes in human liver.
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| 4. |
- Olsson Lindvall, Martina
(författare)
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On the role of genetic variation and epigenetics in hemostatic gene regulation
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Many genetic variants have been identified to associate with circulating levels of hemostatic proteins and with thrombotic or hemorrhagic disorders. However, the underlying molecular mechanisms remain largely unknown. The overall aim of this thesis was to study how genetic variation and epigenetic mechanisms influence the regulation of hemostatic gene expression. The specific aims were to investigate epigenetic mechanisms regulating tissue-type plasminogen activator (t-PA) gene expression in the human brain (Paper I); to identify cis-acting variants involved in hemostatic gene regulation in liver (Papers II and III); and to investigate whether DNA methylation patterns in hemostatic genes in blood can reliably predict those in liver (Paper IV). In Paper I, human astrocytes and neurons were treated with histone deacetylase (HDAC) inhibitors. Protein and mRNA levels of t-PA were measured using ELISA and real-time qPCR, respectively. Histone modifications were assayed with chromatin immunoprecipitation, and DNA methylation analysis of the t-PA promoter was performed by bisulfite sequencing. In Papers II-IV, liver tissue and blood samples were collected from patients undergoing liver surgery and targeted DNA-, RNA- and methylation sequencing was performed for 35 hemostatic genes with predominant expression in the liver. These data were used in Papers II and III to performed allele-specific analyses of mRNA expression (ASE) and DNA methylation (ASM) in liver. In Paper IV, the extent to which blood can be used as a surrogate for DNA methylation of hemostatic genes in the liver was investigated. In Paper I, cell treatments with HDAC inhibitors resulted in an increase in t-PA mRNA and protein expression, and in a significant increase in histone H3 acetylation. DNA methylation analysis revealed that the t-PA promoter was hypomethylated in neurons, astrocytes, and in post-mortem brain tissue, which indicates active transcription. In Paper II, ASE was identified in 60% of the hemostatic genes studied and 14 novel genotype-expression associations were discovered. In Paper III, a detailed DNA methylation map of the targeted hemostatic genes in liver was created, and novel associations between SNPs and DNA methylation were identified. The analyses performed in Paper IV showed that the correlation of hemostatic gene methylation between liver and blood was generally low. However, about 3% of the investigated CpGs had methylation levels that were significantly correlated between the two tissues, and for these, blood may potentially be used as a surrogate tissue to detect liver methylation. Taken together, these findings highlight the importance of integrating genetic, epigenetic, and expression analyses in the relevant tissue, and demonstrate that this approach can contribute to new insights into the biological processes affecting hemostasis and thrombosis.
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