Quantification of allele-specific G-protein β3 subunit mRNA transcripts in different human cells and tissues by Pyrosequencing

Abstract

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(À350)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 0 -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 allelespecific 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(À350)A genotypes (P40.05). 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.

Sex-specific association of X-linked Toll-like receptor 7 (TLR7) with male systemic lupus erythematosus

“…The significant elevation of TLR7 transcripts in PBMCs from individuals carrying G risk allele, along with the notable higher level of G allele-containing TLR7 transcripts in heterozygous participants, supported a functional role rs3853839 played in the regulation of TLR7. The allelic specific expression analysis by pyrosequencing as we presented here is an accurate and valid approach that represents the biologically functional consequence of a certain SNP in vivo; without subjecting to various confounding factors frequently associated with in vitro assays, such as inappropriate reporter gene construct or cell line chosen or lack of suitable condition or agonists applied (14). Further bioinformatic analysis based on current databases did not provide a clue on how the SNP regulates mRNA expression: rs3853839 is not located in the binding site of any transcriptional factors or miRNAs; neither is it in the AU-rich sequence that is usually involved in mRNA decay.…”

“…RPLP13A was used as internal control. We performed allelic-specific transcription quantification assay to measure the allelic expression of rs3853839 by using pyrosequencing as previously described (14). The allelic ratio for each cDNA and gDNA from each individual was calculated by using PSQMA software (version 2.1; Biotage) and compared by paired Student t test.…”

Mouse model implicates GNB3 duplication in a childhood obesity syndrome

“…However, the T haplotype is linked to a shorter in-frame splice variant, GNB3-s, which lacks part of exon 9 (34). Although total mRNA expression and protein levels are not significantly altered by the C825T polymorphism (34,35), cell culture assays have shown that cells derived from CT heterozygotes or TT homozygotes have increased activation of G proteins compared with CC homozygotes (34). We reasoned that an extra copy of GNB3 because of the der (8)t(8;12) could, like the C825T polymorphism, alter G protein signaling and confer increased risk of obesity.…”

A haplotype of the catalase gene confers an increased risk of essential hypertension in Chinese Han

“…To answer this question, in vivo AEI was pursued, and the T-20C polymorphism located in 5 0 -UTR could serve as a marker for AEI resulting from the CATH2 and CATH1. A pyrosequencing-based method was employed to quantify allele-specific transcript expression [Sun et al, 2005] and the allelic expression difference was examined to test the in vivo functionality of the T-20C polymorphism located in the 5 0 untranslated region of CAT. We genotyped the T-20C polymorphism of 25 persons with age r60 years, and six cell lines, quantified mRNA transcripts from different alleles using 13 heterozygous samples, 11 of them were normal persons peripheral blood, and 2 of them were cell lines .…”