Biomedical subjects

Abbas Assadzadeh

Publications and source records attributed to Abbas Assadzadeh.

4 recordsLinked to original sources

PubMed2006

Intra- and interindividual epigenetic variation in human germ cells.

Epigenetics represents a secondary inheritance system that has been poorly investigated in human biology. The objective of this study was to perform a comprehensive analysis of DNA methylation variation between and within the germlines of normal males. First, methylated cytosines were mapped using bisulphite modification-based sequencing in the promoter regions of the following disease genes: presenilins (PSEN1 and PSEN2), breast cancer (BRCA1 and BRCA2), myotonic dystrophy (DM1), and Huntington disease (HD). Major epigenetic variation was detected within samples, since the majority of sperm cells of the same individual exhibited unique DNA methylation profiles. In the interindividual analysis, 41 of 61 pairwise comparisons revealed distinct DNA methylation profiles (P=.036 to 6.8 x 10(-14)). Second, a microarray-based epigenetic profiling of the same sperm samples was performed using a 12,198-feature CpG island microarray. The microarray analysis has identified numerous DNA methylation-variable positions in the germ cell genome. The largest degree of variation was detected within the promoter CpG islands and pericentromeric satellites among the single-copy DNA fragments and repetitive elements, respectively. A number of genes, such as EED, CTNNA2, CALM1, CDH13, and STMN2, exhibited age-related DNA methylation changes. Finally, allele-specific methylation patterns in CDH13 were detected. This study provides evidence for significant epigenetic variability in human germ cells, which warrants further research to determine whether such epigenetic patterns can be efficiently transmitted across generations and what impact inherited epigenetic individuality may have on phenotypic outcomes in health and disease.

Confounding Factors, Epidemiologic↗

PubMed2006

Microarray-based DNA methylation profiling: technology and applications.

This work is dedicated to the development of a technology for unbiased, high-throughput DNA methylation profiling of large genomic regions. In this method, unmethylated and methylated DNA fractions are enriched using a series of treatments with methylation sensitive restriction enzymes, and interrogated on microarrays. We have investigated various aspects of the technology including its replicability, informativeness, sensitivity and optimal PCR conditions using microarrays containing oligonucleotides representing 100 kb of genomic DNA derived from the chromosome 22 COMT region in addition to 12 192 element CpG island microarrays. Several new aspects of methylation profiling are provided, including the parallel identification of confounding effects of DNA sequence variation, the description of the principles of microarray design for epigenomic studies and the optimal choice of methylation sensitive restriction enzymes. We also demonstrate the advantages of using the unmethylated DNA fraction versus the methylated one, which substantially improve the chances of detecting DNA methylation differences. We applied this methodology for fine-mapping of methylation patterns of chromosomes 21 and 22 in eight individuals using tiling microarrays consisting of over 340 000 oligonucleotide probe pairs. The principles developed in this work will help to make epigenetic profiling of the entire human genome a routine procedure.

Chromosome Mapping↗

PubMed2005

Search for somatic DNA variation in the brain: investigation of the serotonin 2A receptor gene.

Somatic DNA variation represents one of the most interesting but also one of the least investigated genetic phenomena. In addition to the classical case of DNA hypermutability at the V(D)J region, there is an increasing body of experimental evidence suggesting that genes other than immunoglobulin in tissues other than lymphocytes also exhibit nonuniformity of DNA sequence, which opens new opportunities for explaining various features of multicellular organisms. Identification of somatic DNA mutability, however, is not a trivial task and numerous confounding factors have to be taken into account. In this work we investigated putative DNA variation in the serotonin 2A receptor gene (HTR2A). A series of real-time PCR-based experiments was performed on DNA samples (n = 8) from human brain and peripheral leukocytes. Amplification of the target DNA sequences was carefully matched to that of the control plasmid containing the insert of HTR2A. Sequencing of nearly 500 clones containing a total of 150,000 nucleotides did not show any evidence for somatic DNA variation in the brain and peripheral leukocytes. It is argued in this article that although intraindividual DNA mutability may be a more common phenomenon than is generally accepted, some of the earlier claims of genetic nonidentity on the brain cells may be premature.

Brain↗

PubMed2005

Single nucleotide extension technology for quantitative site-specific evaluation of metC/C in GC-rich regions.

The development and use of high throughput technologies for detailed mapping of methylated cytosines (metC) is becoming of increasing importance for the expanding field of epigenetics. The single nucleotide primer extension reaction used for genotyping of single nucleotide polymorphisms has been recently adapted to interrogate the bisulfite modification induced 'quantitative' C/T polymorphism that corresponds to metC/C in the native DNA. In this study, we explored the opportunity to investigate C/T (and G/A) ratios using the Applied Biosystems (ABI) SNaPshot technology. The main effort of this study was dedicated to addressing the complexities in the analysis of DNA methylation in GC-rich regions where interrogation of the target cytosine can be confounded by variable degrees of methylation in other cytosines (resulting in variable C/T or G/A ratios after treatment with bisulfite) in the annealing site of the interrogating primer. In our studies, the mismatches of the SNaPshot primer with the target DNA sequence resulted in a biasing effect of up to 70% while these effects decreased as the location of the polymorphic site moved upstream of the target cytosine. We demonstrated that the biasing effect can be corrected with the SNaPshot primers containing degenerative C/T and G/A nucleotides. A series of experiments using various permutations of quantitative C/T and G/A polymorphisms at various positions of the target DNA sequence demonstrated that SNaPshot is able to accurately report cytosine methylation levels with <5% average SD from the true values. Given the relative simplicity of the method and the possibility to multiplex C/T and G/A interrogations, the SNaPshot approach may become a useful tool for large-scale mapping of metC.

Base Composition↗