Quantification of allele-specific G-protein beta3 subunit mRNA transcripts in different human cells and tissues by Pyrosequencing.
The G-protein 825T allele is associated with altered drug responses while the underlying mechanism is not fully understood. Differential expression of transcripts from the C and T alleles could contribute to this process. The C825T polymorphism located in exon 10 is in close linkage disequilibrium with the A(-350)G promoter single nucleotide polymorphism (SNP) and the C1429T SNP and could therefore serve as a marker for allele-specific expression resulting from the promoter SNP. However, alternative splicing of exon 10 in 825T allele carriers may result in under-represented mRNA transcripts. We, therefore, established a novel method based on the Pyrosequencing technology to quantify allele-specific transcript expression and quantified the allelic variance of the C1429T polymorphism located in the 3'-untranslated region of GNB3. Validation of the method was performed using linear regression analysis of measured versus expected ratios of DNA mixed at different known concentrations as well as determining allele-specific mRNA expression of the partially imprinted IGF-2 gene. We genotyped the C1429T polymorphism of 83 samples derived from six different human tissues and cell lines and quantified mRNA transcripts from different alleles using heterozygous samples. No significantly different transcript amounts from the two alleles were found. There were also no significantly different transcript amounts associated with different G(-350)A genotypes (P>0.05). As a result, we could show that Pyrosequencing provides a sensitive tool to quantify allele-specific transcript expression. Our data do not support the hypothesis that differential G-protein activity associated with the C825T SNP results from different transcript amounts associated with specific GNB3 genotypes.
Sun
Aijun
A
Institute of Cardiovascular Disease, Zhongshan Hospital, Fudan University, P.R. China.
Ge
Junbo
J
Siffert
Winfried
W
Frey
Ulrich H
UH
D020413:3' Untranslated Regions / Q000235:genetics; D000273:Adipose Tissue / Q000378:metabolism; D000483:Alleles; D002460:Cell Line; D005819:Genetic Markers; D005838:Genotype; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics*; D006801:Humans; D011110:Polymorphism, Genetic; D020641:Polymorphism, Single Nucleotide; D021122:Protein Subunits; D012333:RNA, Messenger / Q000032:analysis; D012175:Retinoblastoma / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D014158:Transcription, Genetic
D020413:3' Untranslated Regions; C454420:G-protein beta3 subunit; D005819:Genetic Markers; D021122:Protein Subunits; D012333:RNA, Messenger; D020962:Heterotrimeric GTP-Binding Proteins