A Graves’ Disease-Associated Kozak Sequence Single-Nucleotide Polymorphism Enhances the Efficiency of CD40 Gene Translation: A Case for Translational Pathophysiology

Abstract

We analyzed the mechanism by which a Graves' disease-associated C/T polymorphism in the Kozak sequence of CD40 affects CD40 expression. CD40 expression levels on B cells in individuals with CT and TT genotypes were decreased by 13.3 and 39.4%, respectively, compared with the levels in CC genotypes (P = 0.012). Similarly, Rat-2 fibroblasts transfected with T-allele cDNA expressed 32.2% less CD40 compared with their C-allele-transfected counterparts (P = 0.004). Additionally, an in vitro transcription/translation system showed that the T-allele makes 15.5% less CD40 than the C-allele (P < 0.001), demonstrating that the effect of the single-nucleotide polymorphism (SNP) on CD40 expression is at the level of translation. However, the SNP did not affect transcription, because the mRNA levels of CD40, as measured by quantitative RT-PCR, were independent of genotype. Therefore, our results may suggest that the C allele of the CD40 Kozak SNP, which is associated with Graves' disease, could predispose to disease by increasing the efficiency of translation of CD40 mRNA.

A CD40 Kozak sequence polymorphism and susceptibility to antibody-mediated autoimmune conditions: the role of CD40 tissue-specific expression

“…Recently, our group demonstrated that the C allele of this polymorphism caused a 15-32% increase in CD40 protein expression 21 relative to the T allele. Our functional and genetic data, coupled with the strong link that the CD40 molecule and its pathway possess to the potentiation of B-cell responses, allowed us to hypothesize that increased expression levels of CD40 could predispose individuals to antibody-mediated autoimmunity, in general.…”

“…Human peripheral B cells were isolated from volunteers, as described previously. 21 Human skeletal muscle was obtained from the National Disease Research Interchange (Philadelphia, PA, USA). Human thyroidal tissue was obtained from the healthy lobe of cancerous thyroids that were excised, during surgery (kindly provided by Dr Yuri Nikiforov, University of Cincinnati).…”

“…Using in vitro studies, we have demonstrated that the C allele of the CD40 Kozak SNP increases the translational efficiency of nascent CD40 mRNA transcripts, resulting in 15-32% more CD40 protein than that seen in the context of the T allele. 21 Hence, we proposed that there exists a translational pathophysiological aspect to GD, owing to subtle changes in the levels of CD40 protein, with increased CD40 expression contributing to disease etiology.…”

The Relationship between CD40 Gene Polymorphism and Unstable Coronary Atherosclerotic Plaques

“…6 A C/T in the 5 untranslated region of CD40 located at the −1 position within the Kozak sequence (rs1883832) has been associated with CD40 protein expression. 10 Recently, CD40 (−1C/T) polymorphism was found to be associated with ACS in Chinese people. 11 In this study, we focus on identifying a genetic marker that may help refine the disruption risk profile of vulnerable atheromatous plaque.…”

Length of the ORF, position of the first AUG and the Kozak motif are important factors in potential dual-coding transcripts

“…Additionally, although it was thought that if an AUG codon is flanked by R in the position of −3 and G in +4, the rest of the consensus sequence contributes only marginally [9], some exceptions were reported. CD40 is an example that contains a G in both −3 and +4 positions, neverthe-less, the C/T SNP in the −1 site of CD40 was reported to be associated with Grave's disease, probably because the protein expression levels with CT and TT genotypes were lower when compared with the levels in CC genotypes [28,29]. Based on our criteria, the transcript of GNAS belongs to the "ss" subtype, and the short ORF encoding ALEX should not be translated.…”