Jonathan M. Locke

Recessive mutations in the INS gene result in neonatal diabetes through reduced insulin biosynthesis

Heterozygous coding mutations in the INS gene that encodes preproinsulin were recently shown to be an important cause of permanent neonatal diabetes. These dominantly acting mutations prevent normal folding of proinsulin, which leads to beta-cell death through endoplasmic reticulum stress and apoptosis. We now report 10 different recessive INS mutations in 15 probands with neonatal diabetes. Functional studies showed that recessive mutations resulted in diabetes because of decreased insulin biosynthesis through distinct mechanisms, including gene deletion, lack of the translation initiation signal, and altered mRNA stability because of the disruption of a polyadenylation signal. A subset of recessive mutations caused abnormal INS transcription, including the deletion of the C1 and E1 cis regulatory elements, or three different single base-pair substitutions in a CC dinucleotide sequence located between E1 and A1 elements. In keeping with an earlier and more severe beta-cell defect, patients with recessive INS mutations had a lower birth weight (−3.2 SD score vs. −2.0 SD score) and were diagnosed earlier (median 1 week vs. 10 weeks) compared to those with dominant INS mutations. Mutations in the insulin gene can therefore result in neonatal diabetes as a result of two contrasting pathogenic mechanisms. Moreover, the recessively inherited mutations provide a genetic demonstration of the essential role of multiple sequence elements that regulate the biosynthesis of insulin in man.

Persistent Hyperinsulinemic Hypoglycemia and Maturity-Onset Diabetes of the Young Due to Heterozygous HNF4A Mutations

OBJECTIVE—Mutations in the human HNF4A gene encoding the hepatocyte nuclear factor (HNF)-4α are known to cause maturity-onset diabetes of the young (MODY), which is characterized by autosomal-dominant inheritance and impaired glucose-stimulated insulin secretion from pancreatic β-cells. HNF-4α has a key role in regulating the multiple transcriptional factor networks in the islet. Recently, heterozygous mutations in the HNF4A gene were reported to cause transient hyperinsulinemic hypoglycemia associated with macrosomia. RESEARCH DESIGN AND METHODS—Three infants presented with macrosomia and severe hypoglycemia with a positive family history of MODY. The hypoglycemia was confirmed to be due to hyperinsulinism, and all three patients required diazoxide therapy to maintain normoglycemia. Two of the three infants are still requiring diazoxide therapy at 8 and 18 months, whereas one of them had resolution of hyperinsulinemic hypoglycemia at 32 months of age. RESULTS—Sequencing of the HNF4A gene identified heterozygous mutations in all three families. In family 1, a frameshift mutation L330fsdel17ins9 (c.987 1003del17ins9; p.Leu330fs) was present in the proband; a mutation affecting the conserved A nucleotide of the intron 2 branch site (c.264–21A>G) was identified in the proband of family 2; and finally a nonsense mutation, Y16X (c.48C>G, p.Tyr16X), was found in the proband of family 3. CONCLUSIONS—Heterozygous HNF4A mutations can therefore cause both transient and persistent hyperinsulinemic hypoglycemia associated with macrosomia. We recommend that macrosomic infants with transient or persistent hyperinsulinemic hypoglycemia should be screened for HNF4A mutations if there is a family history of youth-onset diabetes.

Increased expression of miR-187 in human islets from individuals with type 2 diabetes is associated with reduced glucose-stimulated insulin secretion

Aims/hypothesisType 2 diabetes is characterised by progressive beta cell dysfunction, with changes in gene expression playing a crucial role in its development. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression and therefore alterations in miRNA levels may be involved in the deterioration of beta cell function.MethodsGlobal TaqMan arrays and individual TaqMan assays were used to measure islet miRNA expression in discovery (n = 20) and replication (n = 20) cohorts from individuals with and without type 2 diabetes. The role of specific dysregulated miRNAs in regulating insulin secretion, content and apoptosis was subsequently investigated in primary rat islets and INS-1 cells. Identification of miRNA targets was assessed using luciferase assays and by measuring mRNA levels.ResultsIn the discovery and replication cohorts miR-187 expression was found to be significantly increased in islets from individuals with type 2 diabetes compared with matched controls. An inverse correlation between miR-187 levels and glucose-stimulated insulin secretion (GSIS) was observed in islets from normoglycaemic donors. This correlation paralleled findings in primary rat islets and INS-1 cells where overexpression of miR-187 markedly decreased GSIS without affecting insulin content or apoptotic index. Finally, the gene encoding homeodomain-interacting protein kinase-3 (HIPK3), a known regulator of insulin secretion, was identified as a direct target of miR-187 and displayed reduced expression in islets from individuals with type 2 diabetes.Conclusions/interpretationOur findings suggest a role for miR-187 in the blunting of insulin secretion, potentially involving regulation of HIPK3, which occurs during the pathogenesis of type 2 diabetes.

The diabetic phenotype in HNF4A mutation carriers is moderated by the expression of HNF4A isoforms from the P1 promoter during fetal development

OBJECTIVE—Mutations in the alternatively spliced HNF4A gene cause maturity-onset diabetes of the young (MODY). We characterized the spatial and developmental expression patterns of HNF4A transcripts in human tissues and investigated their role as potential moderators of the MODY phenotype. RESEARCH DESIGN AND METHODS—We measured the expression of HNF4A isoforms in human adult tissues and gestationally staged fetal pancreas by isoform-specific real-time PCR. The correlation between mutation position and age of diagnosis or age-related penetrance was assessed in a cohort of 190 patients with HNF4A mutations. RESULTS—HNF4A was expressed exclusively from the P2 promoter in adult pancreas, but from 9 weeks until at least 26 weeks after conception, up to 23% of expression in fetal pancreas was of P1 origin. HNF4A4–6 transcripts were not detected in any tissue. In whole pancreas, HNF4A9 expression was greater than in islets isolated from the endocrine pancreas (relative level 22 vs. 7%). Patients with mutations in exons 9 and 10 (absent from HNF4A3, HNF4A6, and HNF4A9 isoforms) developed diabetes later than those with mutations in exons 2–8, where all isoforms were affected (40 vs. 24 years; P = 0.029). Exon 9/10 mutations were also associated with a reduced age-related penetrance (53 vs. 10% without diabetes at age 55 years; P < 0.00001). CONCLUSIONS—We conclude that isoforms derived from the HNF4A P1 promoter are expressed in human fetal, but not adult, pancreas, and that their presence during pancreatic development may moderate the diabetic phenotype in individuals with mutations in the HNF4A gene.

Genome-Wide Homozygosity Analysis Reveals HADH Mutations as a Common Cause of Diazoxide-Responsive Hyperinsulinemic-Hypoglycemia in Consanguineous Pedigrees

HADH mutations are common in consanguineous pedigrees with diazoxide-responsive hyperinsulinaemichypoglycemia; therefore, genetic testing is recommended, even in the absence of abnormal fatty acid oxidation.

An alternative polyadenylation signal in TCF7L2 generates isoforms that inhibit T cell factor/lymphoid-enhancer factor (TCF/LEF)-dependent target genes

Aims/hypothesisIntronic single nucleotide polymorphisms within the transcription factor 7-like 2 (TCF7L2) gene are associated with risk of type 2 diabetes. It is widely hypothesised that the predisposing variation is involved in cis-regulation of TCF7L2 activity. The aim of this study was to seek evidence for the existence of novel TCF7L2 isoforms encoded within the type 2 diabetes-associated genomic region.MethodsWe searched expressed sequence tag (EST) databases for novel TCF7L2 transcripts and sought to validate the function and integrity of any isoforms found using a combination of RT-PCR, western blotting and reporter gene techniques.ResultsAnalysis of EST databases suggested the presence of an alternative polyadenylation site located in intron 4 of TCF7L2. We used 3′ rapid amplification of cDNA ends and real-time PCR to validate the integrity of this polyadenylation signal and show its wide use across human tissues. Western blotting results are consistent with the use of this polyadenylation signal to generate novel protein isoforms. The alternative polyadenylation signal results in the production of isoforms that retain the β-catenin binding domain but do not possess the high-mobility group box DNA-binding domain. Promoter–reporter gene assays suggest that these isoforms inhibit TCF7L2-dependent target genes by sequestering β-catenin.Conclusions/interpretationWe have identified a novel polyadenylation signal within TCF7L2 that can result in the production of isoforms that act to repress TCF/LEF-dependent target genes. These findings may provide new insights into the association of TCF7L2 with susceptibility to type 2 diabetes.

Targeted Allelic Expression Profiling in Human Islets Identifies cis-Regulatory Effects for Multiple Variants Identified by Type 2 Diabetes Genome-Wide Association Studies

Genome-wide association studies (GWAS) have identified variation at >65 genomic loci associated with susceptibility to type 2 diabetes, but little progress has been made in elucidating the molecular mechanisms behind most of these associations. Using samples heterozygous for transcribed single nucleotide polymorphisms (SNPs), allelic expression profiling is a powerful technique for identifying cis-regulatory variants controlling gene expression. In this study, exonic SNPs, suitable for measuring mature mRNA levels and in high linkage disequilibrium with 65 lead type 2 diabetes GWAS SNPs, were identified and allelic expression determined by real-time PCR using RNA and DNA isolated from islets of 36 white nondiabetic donors. A significant allelic expression imbalance (AEI) was identified for 7/14 (50%) genes tested (ANPEP, CAMK2B, HMG20A, KCNJ11, NOTCH2, SLC30A8, and WFS1), with significant AEI confirmed for five of these genes using other linked exonic SNPs. Lastly, results of a targeted islet expression quantitative trait loci experiment support the AEI findings for ANPEP, further implicating ANPEP as the causative gene at its locus. The results of this study support the hypothesis that changes to cis-regulation of gene expression are involved in a large proportion of SNP associations with type 2 diabetes susceptibility.

Role of microRNAs in the age-associated decline of pancreatic beta cell function in rat islets

Aims/hypothesisAgeing can lead to reduced insulin sensitivity and loss of pancreatic beta cell function, predisposing individuals to the development of diabetes. The aim of this study was to assess the contribution of microRNAs (miRNAs) to age-associated beta cell dysfunction.MethodsThe global mRNA and miRNA profiles of 3- and 12-month-old rat islets were collected by microarray. The functional impact of age-associated differences in miRNA expression was investigated by mimicking the observed changes in primary beta cells from young animals.ResultsBeta cells from 12-month-old rats retained normal insulin content and secretion, but failed to proliferate in response to mitotic stimuli. The islets of these animals displayed modifications at the level of several miRNAs, including upregulation of miR-34a, miR-124a and miR-383, and downregulation of miR-130b and miR-181a. Computational analysis of the transcriptomic modifications observed in the islets of 12-month-old rats revealed that the differentially expressed genes were enriched for miR-34a and miR-181a targets. Indeed, the induction of miR-34a and reduction of miR-181a in the islets of young animals mimicked the impaired beta cell proliferation observed in old animals. mRNA coding for alpha-type platelet-derived growth factor receptor, which is critical for compensatory beta cell mass expansion, is directly inhibited by miR34a and is likely to be at least partly responsible for the effects of this miRNA.Conclusions/interpretationChanges in the level of specific miRNAs that occur during ageing affect the proliferative capacity of beta cells. This might reduce their ability to expand under conditions of increased insulin demand, favouring the development of type 2 diabetes.

MicroRNA expression profiling of human islets from individuals with and without Type 2 diabetes: promises and pitfalls

Recent studies in mouse, involving the β-cell-specific deletion of Dicer1, have highlighted the crucial role of miRNAs (microRNAs) in regulating insulin secretion and consequently Type 2 diabetes. Identifying the individual miRNAs involved in human islet dysfunction may be of diagnostic and therapeutic interest. miRNA expression profiling of human islets isolated from donors with and without Type 2 diabetes may represent one of the first steps in the discovery of these specific miRNAs. The present review discusses some of the potential pitfalls and promises of such an approach.

A cautionary tale: the non-causal association between type 2 diabetes risk SNP, rs7756992, and levels of non-coding RNA, CDKAL1-v1

Aims/hypothesisIntronic single nucleotide polymorphisms (SNPs) in the CDKAL1 gene are associated with risk of developing type 2 diabetes. A strong correlation between risk alleles and lower levels of the non-coding RNA, CDKAL1-v1, has recently been reported in whole blood extracted from Japanese individuals. We sought to replicate this association in two independent cohorts: one using whole blood from white UK-resident individuals, and one using a collection of human pancreatic islets, a more relevant tissue type to study with respect to the aetiology of diabetes.MethodsLevels of CDKAL1-v1 were measured by real-time PCR using RNA extracted from human whole blood (n = 70) and human pancreatic islets (n = 48). Expression with respect to genotype was then determined.ResultsIn a simple linear regression model, expression of CDKAL1-v1 was associated with the lead type 2 diabetes-associated SNP, rs7756992, in whole blood and islets. However, these associations were abolished or substantially reduced in multiple regression models taking into account rs9366357 genotype: a moderately linked SNP explaining a much larger amount of the variation in CDKAL1-v1 levels, but not strongly associated with risk of type 2 diabetes.Conclusions/interpretationContrary to previous findings, we provide evidence against a role for dysregulated expression of CDKAL1-v1 in mediating the association between intronic SNPs in CDKAL1 and susceptibility to type 2 diabetes. The results of this study illustrate how caution should be exercised when inferring causality from an association between disease-risk genotype and non-coding RNA expression.