Unique splicing pattern of the TCF7L2 gene in human pancreatic islets
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Intronic variation in the TCF7L2 gene exhibits the strongest association to type 2 diabetes observed to date, but the mechanism whereby this genetic variation translates into altered biological function is largely unknown. A possible explanation is a genotype-dependent difference in the complex splicing pattern; however, this has not previously been characterised in pancreatic or insulin target tissues. Here, the detailed TCF7L2 splicing pattern in five human tissues is described and dependence on risk genotype explored.
RT-PCR and quantitative real-time PCR were employed to characterise TCF7L2 splicing in pancreatic islets, blood lymphocytes, skeletal muscle and subcutaneous and visceral adipose tissue from non-diabetic individuals.
The mapping of TCF7L2 splice variants shows a specific pattern in pancreatic islets, with four predominant transcripts and high usage of the variable exons 4 and 15. The overall concentration of TCF7L2 mRNA is highest in islets and fat and lower in blood and muscle. No significant difference in overall amount or splicing pattern was observed between carriers and non-carriers of the rs7903146 risk (T) allele. However, incorporation of exon 4 in islets correlates positively with plasma HbA1c levels (r = 0.758; p = 0.018).
There were pronounced tissue-specific differences in the splicing of TCF7L2 with forms containing exon 4 and 15 being most abundant in islets. The incorporation of exon 4 in islets correlated with HbA1c levels. Further experiments will be needed to determine the direction of this correlation, and larger cohorts needed to unequivocally resolve whether there is a relationship between genotype and splicing in islets.
KeywordsGenetics of type 2 diabetes Human Islets Splicing TCF7L2 Transcription factors
C-terminal binding protein 1
Subcutaneous adipose tissue
Transcription factor 7-like 2
Visceral adipose tissue
Of all common risk variants identified for type 2 diabetes, variants in the TCF7L2 gene, which encodes a transcription factor in the morphogenic wingless-type MMTV integration site family (WNT) signalling pathway, confer the highest risk of developing the disease (reviewed in ). The exact role of TCF7L2 in the development of diabetes has not been determined, but several links between WNT signalling and insulin secretion and proliferation of human beta cells have been established (reviewed in ).
All tissue samples were from non-diabetic individuals. Islets were obtained from the Human Tissue Laboratory at Lund University Diabetes Centre from deceased donors (six female, 11 male), BMI 17.6–29.0 kg/m2, aged 26–73 years. Purity varied from 13% to 90%. The islets were culture in CMRL 1066 (ICN Biomedicals, Costa Mesa, CA, USA) supplemented with 10 mmol/l HEPES, 2 mmol/l l-glutamine, 50 μg/ml gentamicin, 0.25 μg/ml Fungizone (GIBCO, BRL, Gaithersburg, MD, USA), 20 μg/ml ciprofloxacin (Bayer Healthcare, Leverkusen, Germany), and 10 mmol/l nicotinamide at 37°C (5% CO2) for 1–9 days prior to RNA preparation. HbA1c levels were available for nine islet donors. SAT and VAT were obtained from bariatric surgery of obese individuals (19 female, two male), BMI 32.6–55.5 kg/m2, aged 20–61 years. Muscle biopsies and blood samples were collected from 18 individuals (all male), BMI 22.6–34.0 kg/m2, aged 30–46 years. Informed consent was obtained from all study participants. All islet donors had given consent to donate organs for medical research. All procedures were approved by the ethical committees at Uppsala and Lund Universities.
RNA was isolated from islets using the AllPrep DNA/RNA Mini Kit (Qiagen, Valencia, CA, USA), from muscle using the RNeasy Fibrous Tissue Kit (Qiagen), from fat using the RNeasy Mini Kit (Qiagen), and from blood using the Tempus 12-Port RNA Isolation Kit and an ABI Prism 6100 Nucleic Acid PrepStation (Applied Biosystems, Foster City, CA, USA). Concentration and purity was measured using a NanoDrop ND-1000 spectrophotometer (A260/A280 > 1.8 and A260/A230 > 1.0) (NanoDrop Technologies, Wilmington, DE, USA). No sign of degradation was observed using agarose gel electrophoresis and Experion DNA 1K gel chips (Bio-Rad, Hercules, CA, USA).
Analysis of expression and splicing of the TCF7L2 gene
A detailed description of the procedure can be found in the Electronic supplementary material (ESM). Briefly, reverse transcription was performed using 1 µmol/l dT18 oligomer and 3 µmol/l random hexamer primers. Quantitative real-time PCR was performed using TaqMan chemistry according to the manufacturer’s recommendation (Applied Biosystems) on an ABI 7900HT sequence detection system (see ESM Table 1). All samples were analysed in triplicates (maximum accepted variation in Ct value: 0.1 cycles). The absolute quantity was calculated using a dilution standard curve of an oligonucleotide template of known concentration  (see ESM Table 2).
Data are expressed as means ± SD. Differences between genotypes and tissues were analysed using non-parametric Kruskal–Wallis and non-parametric Mann–Whitney U tests. Correlations were analysed using non-parametric Spearman’s tests. In all tests p < 0.05 was considered statistically significant. Statistical tests were performed with SPSS 16.0 software (SPSS, Chicago, IL, USA).
Large differences in splicing patterns between different tissues
cDNA was prepared from human pancreatic islets, blood lymphocytes, skeletal muscle, SAT and VAT. Sequencing, RT-PCR and restriction cleavage analysis of TCF7L2 cDNA species reveals clear tissue-dependent differences in the splicing pattern (see ESM Figs 1 and 2, ESM Table 3).
Human pancreatic islets show primarily splice isoforms containing exons 4 and 15
Pancreatic islets display a high incorporation of exon 4 (Fig. 2b and see ESM Table 4), which is located immediately upstream of the diabetes-associated polymorphism rs7903146 in the TCF7L2 gene (Fig. 1a, b). In islets, exon 4 is present in 62.3 ± 4.8% of the transcripts compared with 26.9 ± 2.7 to 33.2 ± 2.0% in the other tissues (p = 2.6 × 10−9). In the 3′-end, exons 14 and 15 tend to be mutually exclusive and tissue-specific, so that exon 15 predominates in islets and blood, whereas exon 14 is more common in fat and muscle tissues (Fig. 2c and see ESM Table 5). An overview of the major splice variants in each tissue is given in Fig. 1c.
Exon 4 incorporation in islets correlates with HbA1c levels
In islets, neither BMI (r = −0.109; p = 0.676) nor age (r = −0.173; p = 0.506) was significantly correlated to incorporation of exon 4. However, exon 4 incorporation in human pancreatic islets was significantly and positively correlated with HbA1c levels in blood (r = 0.758; p = 0.018; Fig. 2d) and with total amount of TCF7L2 (r = 0.583; p = 0.014). Neither BMI (r = 0.359; p = 0.157) nor age (r = 0.097; p = 0.711) was correlated with the total amount of TCF7L2 mRNA.
The incomplete knowledge of the complex and tissue-specific TCF7L2 splicing pattern has hampered endeavours to detail the biological role of the transcription factor in glucose homeostasis and type 2 diabetes. We show that the tissues examined exhibit distinct variations in splicing patterns and that only a few of the theoretically possible variants are represented in each tissue. In the 5′-end there is a tissue-dependent variation in the presence or absence of exon 4. In the 3′-end, exon 14 and 15 largely appear to be mutually exclusive in the transcripts. It is noteworthy that these two exons are of the same length and share 63% identity at the mRNA level and 67% identity at the protein level, and the presence of both introduces a stop codon in exon 15. This leaves the impression that the two exons can replace one another. Also, they tend to be tissue-specific, exon 15 being preferred in blood and islets and exon 14 in fat and muscle, which also display an enhanced incorporation of exon 13. A characteristic of the islets is the high incorporation of exon 15 and, most notably, of exon 4, which is retained in ~62% of the cDNA transcripts, compared with ~33% or less in the other tissues studied.
TCF7L2 interacts with numerous binding partners and several binding sites have been mapped to the sequence (reviewed in ; Fig. 1b). None of the known sites overlap with parts corresponding to alternative exons, but variants without either exon 14 or 15 are predicted to produce protein isoforms without the C-terminal binding protein 1 (CtBP1) site encoded by exon 17 (Fig. 1c). As CtBP1 is a co-repressor, these shorter forms will be expected to possess reduced repression capacities. Since exon 4 comprises 69 nucleotides its presence does not affect the reading frame or, consequently, the primary structure of the rest of the protein. However, a difference of 23 amino acids can influence the three-dimensional structure and, thereby, the interaction of TCF7L2 with its binding partners.
The key issue addressed was whether the intronic single-nucleotide polymorphism (SNP) rs7903146 influences the splicing pattern. Although the splicing pattern differed between islets and other tissues with exon 4 being more predominant in islets, the splicing pattern was not significantly influenced by the rs7903146 genotype (ESM Fig. 3). The variation observed in islet tissue may reflect a number of factors, i.e. purity of the preparations, material from unmatched donors and differences in cultivation times. Significant correlations between TCF7L2 expression levels and age and BMI were not found, and differences in purity of the islet preparations did not seem to have a major influence on TCF7L2 splicing or expression levels (ESM). It is premature, though, to exclude an effect of genotype on splicing pattern as the power was limited to detect such a presumably subtle effect, and because no individuals were homozygous for the T allele. Also, the positive correlation between HbA1c levels and retention of exon 4 in islets makes it likely that the genotype effect is revealed only after plasma glucose levels are taken into account. Although this correlation does not prove causality it suggests a link between TCF7L2 splicing and plasma glucose levels.
We conclude that islets of Langerhans display a unique splicing pattern predominantly consisting of isoforms containing exons 4 and 15, the former being located immediately upstream of the rs7903146 polymorphism. However, we did not observe a significant effect of genotype on splicing pattern. Interestingly, the amount of exon 4 in islets correlated with HbA1c levels, which suggests a possible link between glucose and TCF7L2 expression.
This work was supported by the Swedish Research Council, the Wallenberg Foundation, the Novo Nordisk Foundation, the UMAS Fonder, the Lund University Diabetes Centre (LUDC) and the Magn. Bergvalls Stiftelse and Syskonen Svenssons Fond. Human pancreatic islets were provided by the Nordic Network for Clinical Islet Transplantation by the courtesy of O. Korsgren, Uppsala, Sweden. The authors acknowledge the Human Tissue Laboratory at Lund University Diabetes Centre and J. Taneera for providing RNA from pancreatic islets, J. Hedenbro, M. Ridderstråle, L. Johansson and L. Johansson for providing RNA from fat tissues, K.-F. Eriksson and T. Elgzyri for muscle biopsies and blood samples and C. Ling and M. Dekker Nitert for assisting with RNA preparation. C. Ladenvall is thanked for assisting with statistical analysis. M. Svärdh, A. Berglund and M. Svensson are acknowledged for skilled technical assistance.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.