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Assessing hematopoietic chimerism after stem cell transplantation by multiplexed SNP genotyping using microarrays and quantitative analysis of SNP alleles
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular Medicine.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
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2004 (English)In: Leukemia, ISSN 0887-6924, E-ISSN 1476-5551, Vol. 18, no 2, 255-266 p.Article 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.

Place, publisher, year, edition, pages
2004. Vol. 18, no 2, 255-266 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-92626DOI: 10.1038/sj.leu.2403213PubMedID: 14671647OAI: oai:DiVA.org:uu-92626DiVA: diva2:165775
Available from: 2005-02-18 Created: 2005-02-18 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Using Minisequencing Technology for Analysing Genetic Variation in DNA and RNA
Open this publication in new window or tab >>Using Minisequencing Technology for Analysing Genetic Variation in DNA and RNA
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In this thesis, the four-color fluorescence tag-microarray minisequencing system pioneered by our group was further developed and applied for analysing genetic variation in human DNA and RNA. A SNP marker panel representing different chromosomal regions was established and used for identification of informative SNP markers for monitoring chimerism after stem cell transplantation (SCT). The success of SCT was monitored by measuring the allelic ratios of informative SNPs in follow-up samples from nine patients with leukaemia. The results agreed with data obtained using microsatellite markers. Further the same SNP marker panel was used for evaluation of two whole genome amplification methods, primer extension preamplification (PEP) and multiple displacement amplification (MDA) in comparison with genomic DNA with respect to SNP genotyping success and accuracy in tag-array minisequencing. Identical results were obtained from MDA products and genomic DNA.

The tag-microarray minisequencing system was also established for multiplexed quantification of imbalanced expression of SNP alleles. Two endothelial cell lines and a panel of ten coding SNPs in five genes were used as model system. Six heterozygous SNPs were genotyped in RNA (cDNA) from the cell lines. Comparison of the relative amounts of the SNPs alleles in cDNA to heterozygote SNPs in genomic DNA displayed four SNPs with significant imbalanced expression between the SNP alleles. Finally, the tag-array minisequencing system was modified for detection of splice variants in mRNA from five leukaemia cell lines. A panel of 20 cancer-related genes with 74 alternatively splice variants was screened. Over half of the splice variants were detected in the cell lines, and similar alternative splicing patterns were observed in each cell line. The results were verified by size analysis of the PCR product subjected to the minisequencing primer extension reaction. The data from both methods agreed well, evidencing for a high sensitivity of our system.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2005. 55 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 10
Keyword
Molecular medicine, microarray, minisequencing, molecular medicine, single nucleotide polymorphism, stem cell transplantation, whole genome amplification, allelic imbalance, alternative splicing, Molekylärmedicin
National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-4789 (URN)91-554-6152-2 (ISBN)
Public defence
2005-03-15, Rudbecksalen, Rudbecklaboratoriet, Uppsala, 09:15
Opponent
Supervisors
Available from: 2005-02-18 Created: 2005-02-18Bibliographically approved

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Syvänen, Ann-Christine

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