Role of the single nucleotide polymorphism rs7903146 of TCF7L2 in inducing nonsense-mediated decay.

Background The single nucleotide polymorphism (SNP) rs7903146 (C/T), located in intron 4 of the transcription factor 7-like 2 gene (TCF7L2), has been associated with an increased risk of developing Type 2 Diabetes, although the molecular mechanism remain elusive. The TCF7L2 gene is alternatively spliced but an association between genotype and splice variants has not been shown convincingly. We hypothesized that a yet unknown extra exon, containing either the C or T genotype of the SNP rs7903146, could introduce a premature stop codon and consequently result in nonsense-mediated decay (NMD). Findings Running the sequences C and T of the SNP region in different servers we found that the two alleles could display differential recognition by splicing factors. The C variant showed the possible inclusion of an unknown exon. This unknown exon contained a stop codon and thus could induce NMD. We then determined that the splicing pattern in isolated mouse islets and MIN6 cells was similar to that in human pancreatic islets. Therefore, we used MIN6 cells to study the splicing of human intron 4: two mini-genes of intron 4 containing either the C/C genotype or the T/T genotype were transfected into MIN6 cells. Our constructs were spliced normally, excluding intron 4, but we did not observe the presence of an extra exon with either construct. Conclusions We found that an extra exon could theoretically exist, although we were not able to capture it in our model. A better model is needed to determine whether a theoretical extra exon can induce NMD

Differential transcriptional and post-translational transcription factor 7-like regulation among nondiabetic individuals and type 2 diabetic patients.

Human genetic studies have revealed that the T minor allele of single nucleotide polymorphism rs7903146 in the transcription factor 7-like 2 (TCF7L2) gene is strongly associated with an increased risk of diabetes by 30%-40%. Molecular and clinical studies are of great importance for understanding how this unique variation in TCF7L2 influences type 2 diabetes (T2D) onset and progression. At the molecular level, some studies have been performed in diabetic mice and pancreatic islets from healthy human donors. Whereas TCF7L2 mRNA levels are up-regulated in islets, protein levels are down-regulated. We performed studies on TCF7L2 splicing, mRNA expression, and protein levels in immortalized human lymphocytes from nondiabetic individuals and T2D patients carrying the C/C or the at-risk T/T genotype. Our results show differential expression of TCF7L2 splice variants between nondiabetic and T2D patients carrying the at-risk genotype, as well as differences in protein levels. Therefore, we investigated the regulation of splice variants, and our results propose that splicing of exon 4 is under control of the serine-arginine-rich factor transformer 2 β (TRA2B). Finally, we studied the endoplasmic reticulum stress pathways, looking for a posttranslational explanation. We saw a shift in the activation of these pathways between nondiabetic individuals and T2D patients carrying the at-risk genotype. These results suggest that, in human immortalized lymphocytes carrying the at-risk T/T genotype, first the differential expression of TCF7L2 splice variants implies a regulation, at least for exon 4, by TRA2B and second, the differential protein levels between both T/T carriers point to a different activation of endoplasmic reticulum stress pathways

New Splice Site Acceptor Mutation in AIRE Gene in Autoimmune Polyendocrine Syndrome Type 1

Autoimmune polyglandular syndrome type 1 (APS-1, OMIM 240300) is a rare autosomal recessive disorder, characterized by the presence of at least two of three major diseases: hypoparathyroidism, Addison's disease, and chronic mucocutaneous candidiasis. We aim to identify the molecular defects and investigate the clinical and mutational characteristics in an index case and other members of a consanguineous family. We identified a novel homozygous mutation in the splice site acceptor (SSA) of intron 5 (c.653-1G>A) in two siblings with different clinical outcomes of APS-1. Coding DNA sequencing revealed that this AIRE mutation potentially compromised the recognition of the constitutive SSA of intron 5, splicing upstream onto a nearby cryptic SSA in intron 5. Surprisingly, the use of an alternative SSA entails the uncovering of a cryptic donor splice site in exon 5. This new transcript generates a truncated protein (p.A214fs67X) containing the first 213 amino acids and followed by 68 aberrant amino acids. The mutation affects the proper splicing, not only at the acceptor but also at the donor splice site, highlighting the complexity of recognizing suitable splicing sites and the importance of sequencing the intron-exon junctions for a more precise molecular diagnosis and correct genetic counseling. As both siblings were carrying the same mutation but exhibited a different APS-1 onset, and one of the brothers was not clinically diagnosed, our finding highlights the possibility to suspect mutations in the AIRE gene in cases of childhood chronic candidiasis and/or hypoparathyroidism otherwise unexplained, especially when the phenotype is associated with other autoimmune diseases

Role of the single nucleotide polymorphism rs7903146 of TCF7L2 in inducing nonsense-mediated decay.

Background The single nucleotide polymorphism (SNP) rs7903146 (C/T), located in intron 4 of the transcription factor 7-like 2 gene (TCF7L2), has been associated with an increased risk of developing Type 2 Diabetes, although the molecular mechanism remain elusive. The TCF7L2 gene is alternatively spliced but an association between genotype and splice variants has not been shown convincingly. We hypothesized that a yet unknown extra exon, containing either the C or T genotype of the SNP rs7903146, could introduce a premature stop codon and consequently result in nonsense-mediated decay (NMD). Findings Running the sequences C and T of the SNP region in different servers we found that the two alleles could display differential recognition by splicing factors. The C variant showed the possible inclusion of an unknown exon. This unknown exon contained a stop codon and thus could induce NMD. We then determined that the splicing pattern in isolated mouse islets and MIN6 cells was similar to that in human pancreatic islets. Therefore, we used MIN6 cells to study the splicing of human intron 4: two mini-genes of intron 4 containing either the C/C genotype or the T/T genotype were transfected into MIN6 cells. Our constructs were spliced normally, excluding intron 4, but we did not observe the presence of an extra exon with either construct. Conclusions We found that an extra exon could theoretically exist, although we were not able to capture it in our model. A better model is needed to determine whether a theoretical extra exon can induce NMD

Differential transcriptional and post-translational transcription factor 7-like regulation among nondiabetic individuals and type 2 diabetic patients.

Human genetic studies have revealed that the T minor allele of single nucleotide polymorphism rs7903146 in the transcription factor 7-like 2 (TCF7L2) gene is strongly associated with an increased risk of diabetes by 30%-40%. Molecular and clinical studies are of great importance for understanding how this unique variation in TCF7L2 influences type 2 diabetes (T2D) onset and progression. At the molecular level, some studies have been performed in diabetic mice and pancreatic islets from healthy human donors. Whereas TCF7L2 mRNA levels are up-regulated in islets, protein levels are down-regulated. We performed studies on TCF7L2 splicing, mRNA expression, and protein levels in immortalized human lymphocytes from nondiabetic individuals and T2D patients carrying the C/C or the at-risk T/T genotype. Our results show differential expression of TCF7L2 splice variants between nondiabetic and T2D patients carrying the at-risk genotype, as well as differences in protein levels. Therefore, we investigated the regulation of splice variants, and our results propose that splicing of exon 4 is under control of the serine-arginine-rich factor transformer 2 β (TRA2B). Finally, we studied the endoplasmic reticulum stress pathways, looking for a posttranslational explanation. We saw a shift in the activation of these pathways between nondiabetic individuals and T2D patients carrying the at-risk genotype. These results suggest that, in human immortalized lymphocytes carrying the at-risk T/T genotype, first the differential expression of TCF7L2 splice variants implies a regulation, at least for exon 4, by TRA2B and second, the differential protein levels between both T/T carriers point to a different activation of endoplasmic reticulum stress pathways

New Splice Site Acceptor Mutation in <i>AIRE</i> Gene in Autoimmune Polyendocrine Syndrome Type 1

<div><p>Autoimmune polyglandular syndrome type 1 (APS-1, OMIM 240300) is a rare autosomal recessive disorder, characterized by the presence of at least two of three major diseases: hypoparathyroidism, Addison’s disease, and chronic mucocutaneous candidiasis. We aim to identify the molecular defects and investigate the clinical and mutational characteristics in an index case and other members of a consanguineous family. We identified a novel homozygous mutation in the splice site acceptor (SSA) of intron 5 (c.653-1G>A) in two siblings with different clinical outcomes of APS-1. Coding DNA sequencing revealed that this <i>AIRE</i> mutation potentially compromised the recognition of the constitutive SSA of intron 5, splicing upstream onto a nearby cryptic SSA in intron 5. Surprisingly, the use of an alternative SSA entails the uncovering of a cryptic donor splice site in exon 5. This new transcript generates a truncated protein (p.A214fs67X) containing the first 213 amino acids and followed by 68 aberrant amino acids. The mutation affects the proper splicing, not only at the acceptor but also at the donor splice site, highlighting the complexity of recognizing suitable splicing sites and the importance of sequencing the intron-exon junctions for a more precise molecular diagnosis and correct genetic counseling. As both siblings were carrying the same mutation but exhibited a different APS-1 onset, and one of the brothers was not clinically diagnosed, our finding highlights the possibility to suspect mutations in the <i>AIRE</i> gene in cases of childhood chronic candidiasis and/or hypoparathyroidism otherwise unexplained, especially when the phenotype is associated with other autoimmune diseases.</p></div

Clinical features and analytical data.

<p>Abbreviations: IDDM (Insulin-dependent Diabetes Mellitus), T2D (Type 2 Diabetes), PTH (Parathyroid hormone), ACTH (Adrenocorticotropic hormone), GAD (Glutamic acid decarboxylase), IA-2 (Islet Antigen 2) and Ab (antibodies).</p

AIRE overview family pedigree and detection of the c.653-1G>A AIRE mutation.

<p>(A) Schematic AIRE protein representation showing the different protein domains: HSR domain (HSR), conserved bipartite nuclear localization signal (NLS), PHD zinc finger motif (PHD), proline-rich region (PRR), LXXLL motif (L), and SAND domain (SAND). (B) Schematic <i>AIRE</i> gene representation, where rectangles indicate exons and the dashed line the introns. Finally, (C) schematic representation of the consensus sequences for the 5′ splice site donor (SSD), branch site and 3′ splice site acceptor (SSA). The star indicates the mutation. (D) Pedigree of the Spanish consanguineous family. Genotypes were shown as wild-type (G/G), heterozygous (G/A) and homozygous (A/A) of the c.653-1G>A <i>AIRE</i> mutation. The arrow indicates the index case. (E) Above, the schematic representation of the junction between intron 5 and exon 6, and below, the direct sequence analysis of the <i>AIRE</i> gene identified homozygous carriers of the c.653-1G>A mutation in APS-1 patients, while heterozygous carriers were found in unaffected relatives (I-1, I-2, III-2) of the family.</p

Nonsense pre-mRNA mediated decay analysis.

<p>(A) Schematic representation of the non-mutated and mutated allele analysis, with the specifically designed primers positions, including the non-mutated forward (white), the mutated forward (black) and the common reverse (grey). (B) Agarose gel electrophoresis of the qRT-PCR products revealed that the cDNA fragment of 76-bp corresponds to the predicted wild-type transcript, whereas the cDNA large fragment of 129-bp corresponds to the aberrant transcript. (C) Expression results of each allele normalized to that of the control G/G carriers for the individuals studied. The table below includes the mean relative expression and the standard error mean (SEM) for the non-mutated (NM) and mutated (M) alleles.</p