Browsing by Subject "Assoziation"

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Bioinformatische Analyse und funktionelle Charakterisierung von strukturellen Genvarianten in ADME-Genen in humaner Leber
(2016) Tremmel, Roman; Zanger, Ulrich M.
Pharmacogenetics is the study about inter-individual genetic variation that influences the response to drugs and other xenobiotics. A major part of this variation is due to hepatic drug metabolism with enzymes, transporters and receptors involved in the ab-sorption, distribution, metabolism and the excretion of drugs, xenobiotics and endoge-nous substances and collectively defined as ADME-genes. Genetic factors along with environmental and endogenous factors, including gender, age, inflammation processes and others are known to influence the expression and activity of ADME-genes. These influences can affect drug response, side effects or toxicity. According to newly published data, the human genome of any subject differs from a reference genome at 4.1 to 5.0 million positions. More than 99.9% of these differences are single nucleotide polymorphisms (SNP) or short insertions or deletions. Further-more, a person carries up to 2,500 structural variants, including copy number variations (CNV) affecting ~20 million bases (1000 Genomes Project Consortium et al., 2015). Thus structural variants affect more bases than SNPs. Per definition the CNVs are du-plicated or deleted DNA segments greater than 1kb and it was shown that they cover at least 12-30% of the human genome. Genome-wide studies investigating the function-ality of CNVs in the fruit fly, the mouse and in humans showed that there are genes whose expression is clearly affected by CNVs (dosage-sensitve), but also genes show-ing lower expression with increased copy number (dosage reversed) or genes without any expression alterations despite different copy number (dosage-insensitive). A prominent example of CNVs influencing drug metabolism is the phase I gene CYP2D6. Carriers of reduced or amplified gene copies show significantly altered ex-pression and enzyme activity levels and also a different drug metabolism of substrates like codeine (opioid) or tamoxifen (selective estrogen receptor antagonist) in compari-son to carriers with normal copy status of two. Genotyping of CYP2D6 gene copy num-ber may thus help to adjust drug dosage in a genotype dependent manner. In this work I investigated if further ADME-genes are affected by CNVs and if these variants have a functional impact on the expression phenotype and drug metabolism. The distribution of CNVs in the most important ADME-genes (n=340) was investigated in three independent cohorts using CNV data in a public accessible database of ge-nomic variants (DGV; dgv.tcag.ca), processed SNP microarray data of paired samples of healthy (n=269) and tumor (n=351) liver tissue of the TCGA project (http://cancergenome.nih.gov/) and ADME-panel based exon next generation sequenc-ing (NGS) applied on 150 well documented human liver samples of an in-house cohort (IKP148). For the NGS data analysis a method was developed and optimized to esti-mate the relative copy number of the ADME genes or every single exon via the read depth. The results were validated using qPCR with specific TaqMan assays. RNA-sequencing data of 50 healthy TCGA liver samples, and normalised expression data from microarray experiments applied to lymphoblastoid cell lines (LCL) from the HapMap samples and the 150 human liver samples (IKP148) were used to analyse the association between CNVs and the mRNA expression. Furthermore, in the IKP148 liver samples protein and enzymatic activity levels were available or measured using West-ernBlot and mass spectrometry for selected ADME-genes. All pharmacologically important CNVs of phase I and phase II genes, including CYP2A6, CYP2D6, GSTM1, GSTT1, SULT1A1 and UGT2B17 could be confirmed in all datasets. CNVs which were known, but so far not functionally assessed were found in the phase I and II genes CES1, CYP2E1, CYP21A2, UGT2B15 and UGT2B28. In this work rare CNVs (<1%) were mainly found for transporters like ABCA2, SLC2A4 and SLC47A1. The analysis of the read depth in the IKP148 samples data revealed hybrid genes for CYP2A6 and CYP2D6 with their pseudogenes and allowed a fine mapping of the different alleles. The functional analysis further confirmed the positive association between CNVs and the mRNA expression of CYP2A6, CYP2D6, GSTM1, GSTT1, SULT1A1 and UGT2B17 in all three cohorts. The combination of all data from the NGS project in the IKP148 liver subjects, including SNP and CNV genotypes showed that 11% and 53% of the variability of CYP2A6 and CYP2D6 enzyme activity were explained by the genetic factors. In contrast the mRNA expression of the genes CES1 and CYP2E1 was not dependent of the CNV pattern in healthy liver tissue (IKP148 and TCGA) and lymphoblastoid cell lines. A detailed analysis of the protein and enzyme activity levels (chlorzoxazone-6-hydroxylation) of CYP2E1 confirmed the dosage-insensitivity in the IKP148 liver sub-jects. The dosage compensation can be principally explained by different mechanisms and could be tissue or tumor specific. Furthermore, CNV-linked genetic variants, altered miRNA regulation, incomplete inclusion of regulatory elements or coding sequences, hybridgenes, monoallelic expression, feedback loops or epigenetics could be factors which mask the CNV effect. In this work a haplotype analysis of the CYP2E1 region identified SNPs which were linked to the duplication and a reduced expression phenotype in persons with European ancestry. Using in silico prediction tools we found a relation of one of the linked SNPs in the 3’UTR with additional predicted miRNA bind-ing sites potentially regulating additional CYP2E1 gene copies. The CNV influence on the mRNA expression of the genes CYP21A2, UGT2B25 and UGT2B28 was inconsistent. Although CYP21A2 deletions were associated with a de-creased expression, gene duplications showed normal expression levels compared to samples with two copies. A significant influence of UGT2B28 CNVs was found in LCLs but not in human liver samples (IKP148 and TCGA). In total 7 of 17, 2 of 12 and 3 of 14 ADME genes showed a significant association between expression and CNV type in the IKP148, TCGA and LCLs of the HapMap samples, respectively. In the TCGA cancer tissue nearly all ADME-genes carry CNVs and in 30% of the genes a significant correlation was observed. With cooperation partners further polymorphisms and phenotypes of SULT1A1 and CYP2E1 were analyzed. CYP2E1: In this part of the thesis factors influencing the risk of developing differentiat-ed thyroid carcinoma (DTC) were investigated. Known risk factors for the progression of DTC are genetic and environmental factors, including ionizing radiations, previous thyroid diseases, and hormone factors. It has been speculated that dietary acrylamide intake correlates with the DTC formation. The acrylamide molecule is metabolized by CYP2E1 to the reactive carcinogenic glycidamide. The enzymatic reaction is probably dependent on the CYP2E1 genotype. Together with a cooperation partner (Prof. Dr. Landi, University of Pisa, Pisa, Italy) we investigated, whether CYP2E1 variants influ-ence the DTC risk. Prof. Landi and colleagues used a case-control-cohort and a haplo-type approach and observed a significant association between a tag-SNP rs2480258 (A allele), which covers variants in intron eight and the 3’UTR, and an increased DTC risk. In the human liver samples (IKP148) the rs2480258 genotypes were assessed using an imputation analysis and it was shown that particularly the A allele of the SNP reduce significantly the mRNA and protein expression and the enzyme activity. An in silico prediction for the molecular mechanism suggested that miR570 specifically down regulates the transcripts in carriers of the A allele. These results indicated that the inter-individual CYP2E1 activity as well as acrylamide (similar to glycidamide) influences the risk for DTC. SULT1A1: Methyleugenol, a secondary metabolite present in herbs such as basil or laurel is metabolized in humans by sulfation to a reactive product which can covalently bind to DNA. The resulting DNA adducts are mutagenic and can promote carcinogene-sis. Our cooperation partner Prof. Dr. Hans-Rudolf Glatt (German Institute for Human Nutrition Potsdam-Rehbruecke (DIfE), Nuthetal, Germany) had shown, that the meth-yleugenol metabolism takes place in the liver of mice and is mainly catalyzed by the phase II enzyme SULT1A1. To investigate these facts in humans, the methyleugenol DNA-adduct levels were measured by the cooperation partner in liver tissues (n=121; IKP148) using mass spectrometry. In this work the SULT1A1 protein levels were de-termined using western blot analysis and the relation between the DNA adducts as well as the SULT1A1 expression and the SULT1A1 CNVs was assessed. The SULT1A1 mRNA and protein expression were significantly correlated to the DNA adducts, e.g. higher SULT1A1 expression resulted in higher adduct levels. This emphasized the role of SULT1A1 in the in vivo metabolism in human liver samples. As mentioned above, there were individuals (IKP148) carrying one, two, three, four and five copies of SULT1A1. Deletions were found less frequent (4%) than duplications (36%). The CNVs were significantly associated with the SULT1A1 mRNA and protein expression. This result was consistent to previous studies investigating the association between SULT1A1 CNVs and enzyme activity. The methyleugenol DNA adduct levels were also significantly associated to the SULT1A1 copy number. Carriers of at least three gene copies exhibited a 2.8-fold higher DNA adduct level compared to donors carrying only one SULT1A1 gene copy. As a consequence this could mean that individuals with mul-tiple SULT1A1 copies reach faster, more often and more easily critical and ultimate adduct levels which increase the risk for developing cancer. Future studies should clari-fy whether methyleugenol intake as well as the individual SULT1A1 CNV make-up in-fluences the risk of cancer.
Bridging genomics and genetic diversity : association between sequence polymorphism and trait variation in a spring barley collection
(2009) Haseneyer, Grit; Geiger, Hartwig H.
Association analysis has become common praxis in plant genetics for high-resolution mapping of quantitative trait loci (QTL), validating candidate genes, and identifying important alleles for crop improvement. In the present study the feasibility of association mapping in barley is investigated by associating DNA polymorphisms in selected candidate genes with variation in grain quality traits, plant height, and flowering time to gain further understanding of gene functions involved in the control of these traits. (1) As a starting point a worldwide collection of spring barley (Hordeum vulgare L.) accessions has been established to serve as an association platform for the present and possible further studies. This collection of 224 accessions, sampled from the IPK genebank, consists of 109 European, 45 West Asian and North African, 40 East Asian and 30 American entries. Forty-five EST derived polymorphic SSRs were used to determine the genetic structure. The markers were equally distributed over all seven chromosomes. Phenotypic data were assessed in field experiments performed at three locations in 2004 and 2005 in Germany. (2) Seven candidate genes were considered. Fragments of these genes were amplified and sequenced in the established collection. Single nucleotide polymorphisms (SNPs), haplotype variants, and linkage disequilibrium (LD) were investigated. (3) One gene was additionally analysed in 42 bread wheat (Triticum aestivum L.) accessions in order to compare barley and wheat for nucleotide diversity and LD. (4) Association analysis between SNPs and haplotype variants of the selected candidate genes and the phenotypic variation in thousand-grain weight, crude protein content, starch content, plant height, and flowering time was used to identify candidate genes influencing the variation of these traits in spring barley. A mixed model association-mapping method was employed for this purpose. In the established collection, significant genotypic variation was observed for all traits under study. Genotype×environment interaction variances were much smaller than the genotypic variances and heritability coefficients exceeded 0.9. Statistical analyses of population stratification revealed two major subgroups, mainly comprising two-rowed and six-rowed accessions, respectively. Within the sequenced fragments (13kb) of the seven candidate genes, 216 polymorphic sites and 93 haplotypes were detected demonstrating a moderate to high level of nucleotide and haplotype diversity in the germplasm collection. Most haplotypes (74.2%) occurred at a low frequency (<0.05) and therefore were rejected in the candidate gene-based association analysis. Pair-wise LD estimates between the detected SNPs revealed different intra-gene linkage patterns. The 45 SSR markers used for analysing the population structure revealed low intra- and interchromosomal LD (r²<0.2). Significant marker-trait associations between the candidate genes and the respective target traits were identified. The barley and wheat genes showed a high level of nucleotide identity (>95%) in the coding sequences, the distribution of polymorphisms was also similar in the two species, and both map to a syntenic position on chromosome 3. However, the genes were different in both collections with respect to LD and Tajima?s D statistic. In the barley collection only a moderate level of LD was observed whereas in wheat, LD was absolute between polymorphic sites located in the first intron while it decayed by distance between the former sites and those located downstream the first intron. Differences in Tajima?s D values indicate a lower selection pressure on the gene in barley than in wheat. In conclusion, the established association platform represents an excellent resource for marker-trait association studies. The germplasm collection displays a wide range of genotypic and phenotypic diversity providing phenotypic data for economically important traits and comprehensive information about the nucleotide and haplotype polymorphism of seven candidate genes. Association results demonstrate that the candidate gene-based approach of association mapping is an appropriate tool for characterising gene loci that have a significant impact on plant development and grain quality in spring barley.
Dissection of the genetic architecture of stalk mechanical strength and in vivo haploid induction in maize
(2016) Hu, Haixiao; Melchinger, Albrecht E.
Stalk lodging causes yield losses in maize cultivation ranging from 5 to 20% annually worldwide and stalk mechanical strength is widely accepted as an indirect indicator for its measurement. QTL mapping can reveal the genetic basis of stalk strength and provide information about markers suitable for marker-assisted selection (MAS). Constantly increasing market demands urge maize geneticists and breeders not only to enhance the field performance of new hybrids, but also to improve the breeding process. During the last decade, advances in the double haploid (DH) technology based on in vivo haploid induction (HI) shifted the breeding paradigm and greatly accelerated the breeding process in maize. Further spread of DH technology urgently demands a simple but efficient way for developing new inducers, which could be achieved by introducing the mandatory QTL/gene(s) of HI to advanced breeding lines. Therefore, the main goal of my thesis was to dissect the genetic architecture of stalk strength and detect the mandatory genomic region(s) of HI using genome-wide molecular markers. Several methods have been developed and applied in the literature to evaluate stalk mechanical strength, among which the rind penetrometer resistance (RPR) is a simple, rapid and non-destructive measurement during data collection, whereas stalk bending strength (SBS) is more closely associated with stalk lodging in the field. According to common knowledge in the mechanics of materials, SBS is reflected by the maximum load exerted to breaking (Fmax), the breaking moment (Mmax) and the critical stress (σmax). Thus, to have a complete understanding of the genetic architecture of stalk strength in maize, RPR and SBS (measured by Fmax, Mmax and σmax) were used to characterize stalk strength in our study. Utilizing a segregating population with 216 recombinant inbred lines, our analysis showed that stalk strength traits, RPR and SBS, have high heritability, ranging from 0.75 to 0.91. Nine QTL and one epistatic interaction between QTL were detected for RPR. Two, three and two QTL were detected for Fmax, Mmax and σmax, respectively. All QTL showed minor effects and only one QTL on chromosome 10 had overlapping support intervals between RPR and SBS. Co-locations of QTL and high positive correlations between stalk strength traits and other stalk traits suggested presence of pleiotropism and a complex genetic architecture of stalk strength. Owing to lack of major QTL, MAS solely based on molecular markers was found to be less effective than classical phenotypic selection for stalk strength. However, for SBS we observed considerably higher proportions of genetic variance explained by a genomic selection approach than obtained in QTL mapping with cross validation. Therefore, genomic selection might be a promising tool to improve the efficiency of breeding for stalk strength. All QTL mapping studies conducted hitherto for unraveling the genetic architecture of HI rate detected a major QTL, termed qhir1, in bin 1.04. Dong et al. (2013) further narrowed down this QTL to a 243 kb region. Considering the complex genetic architecture of HI and genetic background noise possibly affecting fine mapping of qhir1, we attempted to validate these results with an alternative approach before embarking on map-based gene isolation. Utilizing 51 maize haploid inducers and 1,482 non-inducers collected worldwide, we were able to investigate the genetic diversity between inducers and non-inducers and detect genomic regions mandatory for HI. The genetic diversity analyses indicated that the inducer group was clearly separated from other germplasm groups and had high familial relatedness. Analyzing our data by a case-control association approach failed because the segregation of HI was heavily confounded with population structure. Moreover, selective sweep approaches commonly used in the literature that are designed for capturing selective sweeps in a long-term evolutionary context failed due to high familial relatedness among inducers. To solve this problem, we developed a novel genome scan approach to detect fixed segments among inducers. With this approach, we detected a segment, termed qhir12, 4.0 Mb in length, within the support interval of the qhir1. This segment was the longest genomic segment detected by our novel approach and was entirely absent in all non-inducers analyzed. However, qhir12 has no overlap with the fine mapping region of Dong et al. (2013), termed qhir11. This indicates that the genomic region harboring the mandatory gene of HI should be confirmed by further experiments to corroborate its existence and identify its location in the maize genome.
Genetic approaches to dissect iron efficiency in maize
(2015) Benke, Andreas; Stich, Benjamin
Maize is susceptible to severe Fe-deficiency symptoms when growing on soils with high pH. Therefore, development of Fe-efficient maize genotypes would aid to overcome Fe limitation on these soils. However, Fe-efficiency is a quantitative trait depending on complex mechanism interactions. The determination of these mechanisms would provide a better understanding of the complex trait Fe-efficiency. In the actual study, the determination of Fe-efficiency involved mechanisms were tackled by population and quantitative genetics. In fact, population genetics facilitate the discovery of genes being important to crop improvement based on a comparison of gene evolution and its ancestral genetic material. Linkage mapping and association analyses require both phenotypic variation and polymorphic markers to determine important quantitative trait loci (QTL). The objective of this research was to dissect the genetic architecture of Fe-efficiency in maize by applying different genetic approaches. Artificial selection during domestication and (or) crop improvement can result in limitation of sequence variation at candidate genes that could limit their detection by quantitative genetic approaches. The objectives of our study were to (i) describe patterns of sequence variation of 14 candidate genes for mobilization, uptake, and transport of Fe in maize, as well as regulatory function and (ii) determine if these genes were targets of selection during domestication. This study was based on 14 candidate genes sequences of 27 diverse maize inbreds, 18 teosinte inbreds, and one Zea luxurians strain as an outgroup. The experimental results suggested that the majority of candidate genes for Fe-efficiency examined in this study were not target of artificial selection. Nevertheless, the genes NAAT1, NAS1, and MTK coding for enzymes involved in phytosiderophore production, NRAMP3 responsible for Fe remobilization during germination, and YS1 transporting PS-Fe-complexes into the root showed signatures of selection. These genes might be important for the adaptation of maize to diverse environments with different Fe availabilities. This in turn suggests, that Fe-efficiency was an adaptive trait during maize domestication from teosinte. Identification of QTL provides information on the chromosomal locations contributing to the quantitative variation of complex traits. The benefit of QTL mapping compared to mutant screenings is the possibility to detect multiple genes which may be associated with the phenotypic trait. The objectives of our studies were to (i) identify QTLs for morphological and physiological traits related to Fe homeostasis, (ii) analyze Fe-dependent expression levels of genes known to be involved in Fe homeostasis as well as positional candidate genes from QTL analysis, and (iii) identify QTLs which control the mineral nutrient concentration difference. Our studies were based on experimental data of 85 genotypes from the IBM population cultivated in a hydroponic system. The QTL mapping of morphological and physiological traits provided new putative candidate genes like Ferredoxin 1, putative ferredoxin PETF, MTP4, and MTP8 which complement the genes already known as being responsible for efficient Fe homeostasis at both, deficient and sufficient Fe regime. Furthermore, the candidate gene expression indicated a trans-acting regulation for DMAS1, NAS3, NAS1, FDH1, IDI2, IDI4, and MTK. The mineral element trait QTL confidence intervals comprised candidate genes that sequestrate Cd in vacuoles (HMA3), transport Fe2+into the root cells (ZIP10), protect the cell against oxidative stress (glutaredoxin), ensure micro nutrient homeostasis during sufficient iron regime (NRAMP2), regulate protein activities (PP2C), and prevent deleterious accumulation and interaction of specific elements within cells (PHT1;5, ZIP4). Association mapping is promising to overcome the limitations of low allele diversity and absent recombinations events causing poor resolution in detecting QTL by linkage mapping. In order to unravel the genetic architecture of Fe-efficiency a vast association mapping panel comprising 267 maize inbred lines was used to (i) detect polymorphisms affecting the morphological/physiological trait formation and (ii) fine map QTL confidence intervals determined according to linkage mapping. Some of the SNPs located beyond coding regions of genes that might be important cis-binding-sites for transcription factors. Furthermore, genes detected at the Fe-deficient regime indicate to be involved in universal stress response. However, genes linked to SNPs detected at Fe-sufficient regime might comprise alleles of Fe inefficient genotypes causing inferior trait expression. The combination of several approaches provided a valuable resource of candidate genes which might aid to increase our understanding of the mechanisms of Fe-efficiency in maize and foster the efforts in breeding superior cultivars by applying molecular marker techniques.