Genotype and Tissue-Specific Effects on Alternative Splicing of the Transcription Factor 7-Like 2 Gene in Humans

Abstract

Context: Noncoding single-nucleotide polymorphisms (SNPs) within the TCF7L2 gene are confirmed risk factors for type 2 diabetes, but the mechanism by which they increase risk is unknown. Objective: We hypothesized that associated SNPs alter TCF7L2 splicing and that splice forms have altered biological roles. Design: Splice forms and 5′ and 3′ untranslated regions were characterized in sc adipose, muscle, liver, HepG2 cells, pancreas, and islet. Isoform-specific transcript levels were quantified in sc adipose. Alternative splice forms were characterized in HepG2 liver cells under glucose and insulin conditions and in SGBS cells with differentiation. Major isoforms were characterized by transfection. Setting: The study was conducted at an ambulatory general clinical research center. Patients: Patients included 78 healthy, nondiabetic study subjects characterized for insulin sensitivity and secretion. Results: We identified 32 alternatively spliced transcripts and multiple-length 3′ untranslated region transcripts in adipose, muscle, islet, and pancreas. Alternative exons 3a, 12, 13, and 13a were observed in all tissues, whereas exon 13b was islet specific. Transcripts retaining exons 13 and 13a but not total TCF7L2 transcripts were significantly correlated with both obesity measures (P < 0.01) and rs7903146 genotype (P < 0.026) in sc adipose. Insulin (5–10 nM) suppressed all TCF7L2 isoforms in SGBS cells but suppressed exon 13a-containing isoforms most significantly (P < 0.001). The isoform distribution differed throughout SGBS cell differentiation. Isoforms with predicted early stop codons yielded stable proteins of the predicted size, bound β-catenin, and targeted correctly to the nucleus. Conclusions: Intronic TCF7L2 variants may regulate alternative transcript isoforms, which in turn may have distinct physiologic roles. a a a Page 1 of 16 Genotype and Tissue-Specific Effects on Alternative Splicing of the Transcription Factor ... 6/19/2014 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2841530/?report=printable Type 2 diabetes (T2D) is among the common, complex diseases for which genetic predisposition is well accepted (1). Grant et al. (2) reported the association of single-nucleotide polymorphisms (SNPs) in the transcription factor 7-like 2 gene (TCF7L2), and this association has been convincingly replicated as the strongest genetic risk factor for T2D (3). Nonetheless, the SNPs implicated are intronic (4) and are not associated with total TCF7L2 transcripts in adipose and muscle (5). Physiological studies of TCF7L2 risk variants have most consistently demonstrated reduced insulin secretion in large epidemiology studies (6,7,8), but other findings suggest a broader role consistent with widespread gene expression (5,9,10,11). TCF7L2 plays a key role in the Wnt signaling pathway (12) and may be important in the expansion and maintenance of β-cell mass (9). However, both increased and decreased TCF7L2 expression in β-cells has been associated with altered insulin secretion and apoptosis in human islets (11,13). Other studies suggested reduced glucagon-like peptide (GLP)-1 induced insulin secretion among carriers (14) or altered proinsulin processing (15). GLP-1 and GLP-1 agonists may act through TCF7L2 and the Wnt pathway to expand and maintain β-cell mass (12). Whereas recent data strongly support a role for TCF7L2 in the β-cell, the means by which intronic variants alter β-cell function or mass remain unclear. Wnt signaling is known to be important in other tissues, including adipose (16), and the proinflammatory cytokine TNFα may inhibit adipogenesis by way of β-catenin/TCF7L2 signaling (17). Studies from our laboratory and others supported a role for TCF7L2 SNPs in altered insulin sensitivity (10), but we and others (5) failed to find a genotype effect on total adipose TCF7L2 transcript levels (5,18,19). Reports of differences in TCF7L2 expression between visceral and sc adipose are inconsistent (18,20,21). The T2D-associated TCF7L2 variants are located in a linkage disequilibrium block that encompasses introns 3 and 4. The T allele of SNP rs7903146 showed reduced binding to protein factors in several cell lines (22), but the relevance of this finding to T2D risk is uncertain. Previous studies suggested multiple TCF7L2 splice forms in tumor tissue (23) and while this work was in preparation, tissue-specific splicing was confirmed in pancreatic islets, lymphocytes, skeletal muscle, and adipose tissue in nondiabetic human subjects (24,25). We hypothesized that TCF7L2 susceptibility variants act by altering the levels of specific transcript isoforms and that splice forms differ in biological function. We characterized the transcript isoforms and untranslated regions in human tissues relevant to diabetes and quantified isoform-specific transcript levels by genotype in nondiabetic subjects. To examine the physiological role of transcript isoforms, we examined the isoform distribution in response to glucose and insulin and during adipocyte cell differentiation. Finally, we determined whether transcript isoforms yielded stable, nuclear targeted proteins that properly bound β-catenin. Materials and Methods