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Microarray Technology for Genotyping in Pharmacogenetics
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The studies in this thesis describe the development of a microarray based minisequencing system and its application to highly parallel genotyping of single nucleotide polymorphisms. The technical developments included identification of a three-dimensional microarray surface coating with high binding capacity for oligonucleotides modified with amino groups as the most optimal one for the system. The system was also established for multiplexed, reproducible quantitative analysis of SNP alleles both on the level of DNA and RNA. The sensitivity of the system to distinguish SNP alleles present as a minority in a mixed sample was found to be 1-6%.

The microarray based minisequencing system was applied in a pharmacogenetic study on antihypertensive drug response. A panel of 74 SNPs located in candidate genes related to blood pressure regulation were genotyped in DNA samples from hypertensive patients that had been treated with the antihypertensive drugs irbesartan or atenolol. Multiple regression analysis of the genotype data against the reduction in blood pressure identified genotype combinations of four to five SNPs that explain 44-56% of the reduction in blood pressure in the two treatment groups. The genotypes of two individual SNPs in the angiotensinogen (AGT) gene and a SNP in the low density lipoprotein receptor (LDLR) gene appeared to be associated to reduced blood pressure after treatment with atenolol, while a SNP in the apolipoprotein B (APOB) gene was associated to blood pressure reduction after irbesartan treatment. The genotype of one SNP in the adrenergic alpha-2A-receptor gene (ADRA2A) was related to the reduction in left ventricular mass following atenolol treatment while the genotypes of two SNPs, one in the APOB gene and one in the AGT gene were related to the reduction in left ventricular mass in the patients treated with irbesartan.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2004. , p. 69
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1342
Keywords [en]
Molecular medicine, microarray, genotyping, pharmacogenetics, molecular medicine, single nucleotide polymorphism, hypertension
Keywords [sv]
Molekylärmedicin
National Category
Medical Genetics
Identifiers
URN: urn:nbn:se:uu:diva-4222ISBN: 91-554-5937-4 (print)OAI: oai:DiVA.org:uu-4222DiVA, id: diva2:164425
Public defence
2004-05-13, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15
Opponent
Supervisors
Available from: 2004-04-20 Created: 2004-04-20 Last updated: 2018-01-13Bibliographically approved
List of papers
1. Minisequencing on oligonucleotide microarrays: comparison of immobilisation chemistries
Open this publication in new window or tab >>Minisequencing on oligonucleotide microarrays: comparison of immobilisation chemistries
2001 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 29, no 13, p. e69-e69Article in journal (Refereed) Published
Abstract [en]

In the microarray format of the minisequencing method multiple oligonucleotide primers immobilised on a glass surface are extended with fluorescent ddNTPs using a DNA polymerase. The method is a promising tool for large-scale single nucleotide polymorphism (SNP) detection. We have compared eight chemical methods for covalent immobilisation of the oligonucleotide primers on glass surfaces. We included both commercially available, activated slides and slides that were modified by ourselves. In the comparison the differently derivatised glass slides were evaluated with respect to background fluorescence, efficiency of attaching oligonucleotides and performance of the primer arrays in minisequencing reactions. We found that there are significant differences in background fluorescence levels among the different coatings, and that the attachment efficiency, which was measured indirectly using extension by terminal transferase, varied largely depending on which immobilisation strategy was used. We also found that the attachment chemistry affects the genotyping accuracy, when minisequencing on microarrays is used as the genotyping method. The best genotyping results were observed using mercaptosilane-coated slides attaching disulfide-modified oligonucleotides.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-91621 (URN)11433045 (PubMedID)
Available from: 2004-04-20 Created: 2004-04-20 Last updated: 2017-12-14Bibliographically approved
2. A microarray minisequencing system for pharmacogenetic profiling of antihypertensive drug response
Open this publication in new window or tab >>A microarray minisequencing system for pharmacogenetic profiling of antihypertensive drug response
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2003 (English)In: Pharmacogenetics, ISSN 0960-314X, E-ISSN 1473-561X, Vol. 13, no 1, p. 7-17Article in journal (Refereed) Published
Abstract [en]

We aimed to develop a microarray genotyping system for multiplex analysis of a panel of single nucleotide polymorphisms (SNPs) in genes encoding proteins involved in blood pressure regulation, and to apply this system in a pilot study demonstrating its feasibility in the pharmacogenetics of hypertension. A panel of 74 SNPs in 25 genes involved in blood pressure regulation was selected from the SNP databases, and genotyped in DNA samples of 97 hypertensive patients. The patients had been randomized to double-blind treatment with either the angiotensin II type 1 receptor blocker irbesartan or the beta 1-adrenergic receptor blocker atenolol. Genotyping was performed using a microarray based DNA polymerase assisted 'minisequencing' single nucleotide primer extension assay with fluorescence detection. The observed genotypes were related to the blood pressure reduction using stepwise multiple regression analysis. The allele frequencies of the selected SNPs were determined in the Swedish population. The established microarray-based genotyping system was validated and allowed unequivocal multiplex genotyping of the panel of 74 SNPs in every patient. Almost 7200 SNP genotypes were generated in the study. Profiles of four or five SNP-genotypes that may be useful as predictors of blood pressure reduction after antihypertensive treatment were identified. Our results highlight the potential of microarray-based technology for SNP genotyping in pharmacogenetics.

Keywords
Adrenergic beta-Antagonists/therapeutic use, Antihypertensive Agents/*therapeutic use, DNA Primers/genetics, Databases; Genetic, Double-Blind Method, Female, Gene Expression/*drug effects, Gene Expression Profiling, Gene Frequency/*drug effects, Genotype, Humans, Hypertension/*drug therapy/genetics, Male, Middle Aged, Oligonucleotide Array Sequence Analysis/*methods, Pharmacogenetics/*methods, Polymorphism; Single Nucleotide/*genetics, Random Allocation, Receptor; Angiotensin; Type 1, Receptors; Angiotensin/antagonists & inhibitors/metabolism, Research Support; Non-U.S. Gov't
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-73226 (URN)12544508 (PubMedID)
Available from: 2007-03-13 Created: 2007-03-13 Last updated: 2017-12-14Bibliographically approved
3. Angiotensinogen gene polymorphisms: relationship to blood pressure response to antihypertensive treatment. Results from the Swedish Irbesartan Left Ventricular Hypertrophy Investigation vs Atenolol (SILVHIA) trial
Open this publication in new window or tab >>Angiotensinogen gene polymorphisms: relationship to blood pressure response to antihypertensive treatment. Results from the Swedish Irbesartan Left Ventricular Hypertrophy Investigation vs Atenolol (SILVHIA) trial
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2004 (English)In: American Journal of Hypertension, ISSN 0895-7061, E-ISSN 1941-7225, Vol. 17, no 1, p. 8-13Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: The renin-angiotensin-aldosterone system (RAAS) is important for the development of hypertension, and several antihypertensive drugs target this system. Our aim was to determine whether specific single nucleotide polymorphisms (SNPs) in RAAS genes were related to the blood pressure (BP) lowering effect of antihypertensive treatment. METHODS: Patients with mild to moderate primary hypertension and left ventricular hypertrophy were randomized in a double-blind fashion to treatment with either the angiotensin II type 1 receptor antagonist irbesartan (n = 48) or the beta(1)-adrenergic receptor blocker atenolol (n = 49) as monotherapy. A microarray-based minisequencing system was used to genotype 30 SNPs in seven genes in the RAAS. These polymorphisms were related to the antihypertensive response after 12 weeks treatment. RESULTS: The BP reductions were similar in the atenolol and the irbesartan groups. Presence of the angiotensinogen (AGT) -6A allele or the AGT 235T allele were both associated with the most pronounced systolic BP response to atenolol treatment (P =.001 when -6 AA+AG was compared with GG and P =.008 for presence of the 235T variant compared with 235 MM). CONCLUSIONS: We found that SNPs in the angiotensinogen gene were associated with the BP lowering response to atenolol. This study is limited by a relatively small sample size, and the results should therefore be viewed as preliminary. Despite this limitation, these results illustrate the potential of using SNP genotyping as a pharmacogenetic tool in antihypertensive treatment.

Keywords
Angiotensin II Type 1 Receptor Blockers, Angiotensinogen/*genetics, Antihypertensive Agents/*therapeutic use, Atenolol/*therapeutic use, Biphenyl Compounds/*therapeutic use, Blood Pressure, Double-Blind Method, Female, Genotype, Humans, Hypertension/complications/drug therapy/*genetics, Hypertrophy; Left Ventricular/etiology, Male, Middle Aged, Polymorphism; Single Nucleotide, Renin-Angiotensin System/*genetics, Research Support; Non-U.S. Gov't, Tetrazoles/*therapeutic use, Treatment Outcome
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-73304 (URN)10.1016/j.amjhyper.2003.09.009 (DOI)14700505 (PubMedID)
Available from: 2005-09-07 Created: 2005-09-07 Last updated: 2017-12-14Bibliographically approved
4. Single nucleotide polymorphisms in the apolipoprotein B and low density lipoprotein receptor genes affect response to antihypertensive treatment
Open this publication in new window or tab >>Single nucleotide polymorphisms in the apolipoprotein B and low density lipoprotein receptor genes affect response to antihypertensive treatment
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2004 (English)In: BMC Cardiovascular Disorders, ISSN 1471-2261, E-ISSN 1471-2261, Vol. 4, no 1, p. 16-Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Dyslipidemia has been associated with hypertension. The present study explored if polymorphisms in genes encoding proteins in lipid metabolism could be used as predictors for the individual response to antihypertensive treatment. METHODS: Ten single nucleotide polymorphisms (SNP) in genes related to lipid metabolism were analysed by a microarray based minisequencing system in DNA samples from ninety-seven hypertensive subjects randomised to treatment with either 150 mg of the angiotensin II type 1 receptor blocker irbesartan or 50 mg of the beta1-adrenergic receptor blocker atenolol for twelve weeks. RESULTS: The reduction in blood pressure was similar in both treatment groups. The SNP C711T in the apolipoprotein B gene was associated with the blood pressure response to irbesartan with an average reduction of 19 mmHg in the individuals carrying the C-allele, but not to atenolol. The C16730T polymorphism in the low density lipoprotein receptor gene predicted the change in systolic blood pressure in the atenolol group with an average reduction of 14 mmHg in the individuals carrying the C-allele. CONCLUSIONS: Polymorphisms in genes encoding proteins in the lipid metabolism are associated with the response to antihypertensive treatment in a drug specific pattern. These results highlight the potential use of pharmacogenetics as a guide for individualised antihypertensive treatment, and also the role of lipids in blood pressure control.

Keywords
Antihypertensive treatment, pharmacogenetics, lipids, minisequencing, genotyping
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-73315 (URN)10.1186/1471-2261-4-16 (DOI)15453913 (PubMedID)
Available from: 2005-06-02 Created: 2005-06-02 Last updated: 2017-12-14Bibliographically approved
5. Single nucleotide polymorphisms predict the change in left ventricular mass in response to antihypertensive treatment
Open this publication in new window or tab >>Single nucleotide polymorphisms predict the change in left ventricular mass in response to antihypertensive treatment
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2004 (English)In: Journal of Hypertension, ISSN 0263-6352, E-ISSN 1473-5598, Vol. 22, no 12, p. 2321-8Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Our aim was to determine whether the change in left ventricular (LV) mass in response to antihypertensive treatment could be predicted by multivariate analysis of single nucleotide polymorphisms (SNPs) in candidate genes reflecting pathways likely to be involved in blood pressure control. METHODS: Patients with mild to moderate primary hypertension and LV hypertrophy were randomized in a double-blind fashion to treatment with either the angiotensin II type 1 receptor antagonist irbesartan (n = 48) or the beta1 adrenoreceptor blocker atenolol (n = 49). A microarray-based minisequencing system was used for genotyping 74 SNPs in 25 genes. These genotypes were related to the change in LV mass index by echocardiography, after 12 weeks treatment as monotherapy, using stepwise multiple regression analysis. RESULTS: The blood pressure reductions were similar and significant in both treatment groups. Two SNPs in two separate genes (the angiotensinogen T1198C polymorphism, corresponding to the M235T variant and the apolipoprotein B G10108A polymorphism) for those treated with irbesartan, and the adrenoreceptor alpha2A A1817G for those treated with atenolol, significantly predicted the change in LV mass. The predictive power of these SNPs was independent of the degree of blood pressure reduction. CONCLUSION: SNPs in the angiotensinogen, apolipoprotein B, and the alpha2 adrenoreceptor gene predicted the change in LV mass during antihypertensive therapy. These results illustrate the potential of using microarray-based technology for SNP genotyping in predicting individual drug responses.

Keywords
Adrenergic beta-Antagonists/therapeutic use, Angiotensin II Type 1 Receptor Blockers/therapeutic use, Angiotensinogen/genetics, Antihypertensive Agents/*therapeutic use, Apolipoproteins B/genetics, Atenolol/therapeutic use, Biphenyl Compounds/therapeutic use, Blood Pressure/*genetics, Double-Blind Method, Echocardiography, Female, Genotype, Humans, Hypertension/complications/*drug therapy/*genetics, Hypertrophy; Left Ventricular/etiology/*ultrasonography, Male, Middle Aged, Multivariate Analysis, Oligonucleotide Array Sequence Analysis, Pharmacogenetics, Polymorphism; Single Nucleotide, Predictive Value of Tests, Receptors; Adrenergic; beta-2/genetics, Research Support; Non-U.S. Gov't, Tetrazoles/therapeutic use
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-70033 (URN)15614026 (PubMedID)
Available from: 2005-09-08 Created: 2005-09-08 Last updated: 2017-11-21Bibliographically approved
6. Assessing hematopoietic chimerism after allogeneic stem cell transplantation by multiplexed SNP genotyping using microarrays and quantitative analysis of SNP alleles
Open this publication in new window or tab >>Assessing hematopoietic chimerism after allogeneic stem cell transplantation by multiplexed SNP genotyping using microarrays and quantitative analysis of SNP alleles
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2004 (English)In: Leukemia, ISSN 0887-6924, E-ISSN 1476-5551, Vol. 18, no 2, p. 255-266Article in journal (Refereed) Published
Abstract [en]

Single-nucleotide polymorphisms (SNPs) have the potential to be particularly useful as markers for monitoring of chimerism after stem cell transplantation (SCT) because they can be analyzed by accurate and robust methods. We used a two-phased minisequencing strategy for monitoring chimerism after SCT. First, informative SNPs with alleles differing between donor and recipient were identified using a multiplex microarray-based minisequencing system screening 51 SNPs to ensure that multiple informative SNPs were detected in each donor–recipient pair. Secondly, the development of chimerism was followed up after SCT by sensitive, quantitative analysis of individual informative SNPs by applying the minisequencing method in a microtiter plate format. Using this panel of SNPs, we identified multiple informative SNPs in nine unrelated and in 16 related donor–recipient pairs. Samples from nine of the donor–recipient pairs taken at time points ranging from 1 month to 8 years after transplantation were available for analysis. In these samples, we monitored the allelic ratios of two or three informative SNPs in individual minisequencing reactions. The results agreed well with the data obtained by microsatellite analysis. Thus, we conclude that the two-phased minisequencing strategy is a useful approach in the following up of patients after SCT.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-91626 (URN)10.1038/sj.leu.2403213 (DOI)
Available from: 2004-04-20 Created: 2004-04-20 Last updated: 2017-12-14Bibliographically approved
7. Detecting imbalanced expression of SNP alleles by minisequencing on microarrays
Open this publication in new window or tab >>Detecting imbalanced expression of SNP alleles by minisequencing on microarrays
2004 (English)In: BMC Biotechnology, ISSN 1472-6750, E-ISSN 1472-6750, Vol. 4, no 24, p. 1-10Article in journal (Refereed) Published
Abstract [en]

BACKGROUND:

Each of the human genes or transcriptional units is likely to contain single nucleotide polymorphisms that may give rise to sequence variation between individuals and tissues on the level of RNA. Based on recent studies, differential expression of the two alleles of heterozygous coding single nucleotide polymorphisms (SNPs) may be frequent for human genes. Methods with high accuracy to be used in a high throughput setting are needed for systematic surveys of expressed sequence variation. In this study we evaluated two formats of multiplexed, microarray based minisequencing for quantitative detection of imbalanced expression of SNP alleles. We used a panel of ten SNPs located in five genes known to be expressed in two endothelial cell lines as our model system.

RESULTS:

The accuracy and sensitivity of quantitative detection of allelic imbalance was assessed for each SNP by constructing regression lines using a dilution series of mixed samples from individuals of different genotype. Accurate quantification of SNP alleles by both assay formats was evidenced for by R2 values > 0.95 for the majority of the regression lines. According to a two sample t-test, we were able to distinguish 1-9% of a minority SNP allele from a homozygous genotype, with larger variation between SNPs than between assay formats. Six of the SNPs, heterozygous in either of the two cell lines, were genotyped in RNA extracted from the endothelial cells. The coefficient of variation between the fluorescent signals from five parallel reactions was similar for cDNA and genomic DNA. The fluorescence signal intensity ratios measured in the cDNA samples were compared to those in genomic DNA to determine the relative expression levels of the two alleles of each SNP. Four of the six SNPs tested displayed a higher than 1.4-fold difference in allelic ratios between cDNA and genomic DNA. The results were verified by allele-specific oligonucleotide hybridisation and minisequencing in a microtiter plate format.

CONCLUSIONS:

We conclude that microarray based minisequencing is an accurate and accessible tool for multiplexed screening for imbalanced allelic expression in multiple samples and tissues in parallel.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-92628 (URN)10.1186/1472-6750-4-24 (DOI)000225233200001 ()15500681 (PubMedID)
Available from: 2005-02-18 Created: 2005-02-18 Last updated: 2017-12-14Bibliographically approved

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