Structural and biophysical characterization of transcription factor HNF-1A as a tool to study MODY3 diabetes variants.

Hepatocyte nuclear factor 1A (HNF-1A) is a transcription factor expressed in several embryonic and adult tissues, modulating the expression of numerous target genes. Pathogenic variants in the HNF1A gene are known to cause maturity-onset diabetes of the young 3 (MODY3 or HNF1A MODY), a disease characterized by dominant inheritance, age of onset before 25 to 35 years of age, and pancreatic ß-cell dysfunction. A precise diagnosis can alter management of this disease, as insulin can be exchanged with sulfonylurea tablets and genetic counseling differs from polygenic forms of diabetes. Therefore, more knowledge on the mechanisms of HNF-1A function and the level of pathogenicity of the numerous HNF1A variants is required for precise diagnostics. Here, we structurally and biophysically characterized an HNF-1A protein containing both the DNA-binding domain and the dimerization domain, and determined the folding and DNA-binding capacity of two established MODY3 HNF-1A variant proteins (P112L, R263C) and one variant of unknown significance (N266S). All three variants showed reduced functionality compared to the WT protein. Furthermore, while the R263C and N266S variants displayed reduced binding to an HNF-1A target promoter, we found the P112L variant was unstable in vitro and in cells. Our results support and mechanistically explain disease causality for these investigated variants and present a novel approach for the dissection of structurally unstable and DNA-binding defective variants. This study indicates that structural and biochemical investigation of HNF-1A is a valuable tool in reliable variant classification needed for precision diabetes diagnostics and management.

Hepatocyte nuclear factor HNF1A is a potential regulator in shaping the super-enhancer landscape in colorectal cancer liver metastasis.

Super-enhancers (SEs) play essential roles in colorectal cancer (CRC) progression. However, how the SE landscape is orchestrated by transcriptional regulators and evolves is not clear. Using de novo motif analysis, we show that the hepatocyte nuclear factor 1 (HNF1)-binding motif is enriched in SEs in cell lines derived from liver metastases, but not in those from primary tumors. This finding was further validated by extending the method to pancreatic cancer and a pair of isogenic CRC lines. Next, we revealed HNF1-alpha (HNF1A) was majorly expressed and upregulated in CRC liver metastatic cell lines. Clinically, HNF1A was remarkably upregulated in synchronous liver metastases as compared to localized tumors. Collectively, our study implicates HNF1A as a key regulator in shaping the SE landscape in CRC liver metastasis.

Decreased GLUT2 and glucose uptake contribute to insulin secretion defects in MODY3/HNF1A hiPSC-derived mutant ß cells.

Heterozygous HNF1A gene mutations can cause maturity onset diabetes of the young 3 (MODY3), characterized by insulin secretion defects. However, specific mechanisms of MODY3 in humans remain unclear due to lack of access to diseased human pancreatic cells. Here, we utilize MODY3 patient-derived human induced pluripotent stem cells (hiPSCs) to study the effect(s) of a causal HNF1A+/H126D mutation on pancreatic function. Molecular dynamics simulations predict that the H126D mutation could compromise DNA binding and gene target transcription. Genome-wide RNA-Seq and ChIP-Seq analyses on MODY3 hiPSC-derived endocrine progenitors reveal numerous HNF1A gene targets affected by the mutation. We find decreased glucose transporter GLUT2 expression, which is associated with reduced glucose uptake and ATP production in the MODY3 hiPSC-derived ß-like cells. Overall, our findings reveal the importance of HNF1A in regulating GLUT2 and several genes involved in insulin secretion that can account for the insulin secretory defect clinically observed in MODY3 patients.

Central memory CD8+ T cells derive from stem-like Tcf7hi effector cells in the absence of cytotoxic differentiation.

Central memory CD8+ T cells (Tcm) control systemic secondary infections and can protect from chronic infection and cancer as a result of their stem-cell-like capacity to expand, differentiate, and self-renew. Central memory is generally thought to emerge following pathogen clearance and to form based on the de-differentiation of cytolytic effector cells. Here, we uncovered rare effector-phase CD8+ T cells expressing high amounts of the transcription factor Tcf7 (Tcf1) that showed no evidence of prior cytolytic differentiation and that displayed key hallmarks of Tcm cells. These effector-phase Tcf7hi cells quantitatively yielded Tcm cells based on lineage tracing. Mechanistically, Tcf1 counteracted the differentiation of Tcf7hi cells and sustained the expression of conserved adult stem-cell genes that were critical for CD8+ T cell stemness. The discovery of stem-cell-like CD8+ T cells during the effector response to acute infection provides an opportunity to optimize Tcm cell formation by vaccination.

Defective functions of HNF1A variants on BCL2L1 transactivation and beta-cell growth.

Maturity-onset diabetes of the young type 3 (MODY3) is caused by mutations in a gene encoding transcription factor hepatocyte nuclear factor 1-alpha (HNF1A). Although the roles of HNF1A in regulation of hepatic and pancreatic genes to maintain glucose homeostasis were investigated, the functions of HNF1A are not completely elucidated. To better understand the functions of HNF1A, we characterized mutations of HNF1A in Thai MODY3 patients and studied the functions of wild-type HNF1A and variant proteins. We demonstrate for the first time that HNF1A upregulates transactivation of an anti-apoptotic gene BCL2 Like 1 (BCL2L1) and that all the identified HNF1A variants including p.D80V, p.R203C, p.P475L, and p.G554fsX556, reduce this ability. The four HNF1A variants impair HNF1A function in promoting INS-1 cell transition from G1 to S phase of cell cycle, which thereby retard cell growth. This finding indicates the role of HNF1A in beta-cell viability by upregulation of anti-apoptotic gene expression and also reaffirms its role in beta-cell growth through cell cycle control.

Defining the genetic control of human blood plasma N-glycome using genome-wide association study.

Glycosylation is a common post-translational modification of proteins. Glycosylation is associated with a number of human diseases. Defining genetic factors altering glycosylation may provide a basis for novel approaches to diagnostic and pharmaceutical applications. Here we report a genome-wide association study of the human blood plasma N-glycome composition in up to 3811 people measured by Ultra Performance Liquid Chromatography (UPLC) technology. Starting with the 36 original traits measured by UPLC, we computed an additional 77 derived traits leading to a total of 113 glycan traits. We studied associations between these traits and genetic polymorphisms located on human autosomes. We discovered and replicated 12 loci. This allowed us to demonstrate an overlap in genetic control between total plasma protein and IgG glycosylation. The majority of revealed loci contained genes that encode enzymes directly involved in glycosylation (FUT3/FUT6, FUT8, B3GAT1, ST6GAL1, B4GALT1, ST3GAL4, MGAT3 and MGAT5) and a known regulator of plasma protein fucosylation (HNF1A). However, we also found loci that could possibly reflect other more complex aspects of glycosylation process. Functional genomic annotation suggested the role of several genes including DERL3, CHCHD10, TMEM121, IGH and IKZF1. The hypotheses we generated may serve as a starting point for further functional studies in this research area.

The E3 SUMO ligase PIASγ is a novel interaction partner regulating the activity of diabetes associated hepatocyte nuclear factor-1α.

The transcription factor hepatocyte nuclear factor-1α (HNF-1A) is involved in normal pancreas development and function. Rare variants in the HNF1A gene can cause monogenic diabetes, while common variants confer type 2 diabetes risk. The precise mechanisms for regulation of HNF-1A, including the role and function of post-translational modifications, are still largely unknown. Here, we present the first evidence for HNF-1A being a substrate of SUMOylation in cellulo and identify two lysine (K) residues (K205 and K273) as SUMOylation sites. Overexpression of protein inhibitor of activated STAT (PIASγ) represses the transcriptional activity of HNF-1A and is dependent on simultaneous HNF-1A SUMOylation at K205 and K273. Moreover, PIASγ is a novel HNF-1A interaction partner whose expression leads to translocation of HNF-1A to the nuclear periphery. Thus, our findings support that the E3 SUMO ligase PIASγ regulates HNF-1A SUMOylation with functional implications, representing new targets for drug development and precision medicine in diabetes.

MicroRNA-34a Attenuates Metastasis and Chemoresistance of Bladder Cancer Cells by Targeting the TCF1/LEF1 Axis.

BACKGROUND/AIMS: Chemoresistance is largely responsible for relapses of bladder cancer during clinical therapy. However, the molecular mechanisms involved in the chemoresistance of bladder cancer are unclear. Growing evidence supports the theory that microRNAs (miRNAs) play an important role in chemotherapeutic drug resistance because they are downregulated in many malignancies that have been implicated in the regulation of diverse processes in cancer cells. More specifically, the extent and precise mechanism of the involvement of miR-34as in chemoresistance to epirubicin (EPI) in the treatment of bladder cancer remains unclear. METHODS: In this study, real-time quantitative polymerase chain reaction (PCR) was used to analyze the expression of miR-34a in bladder cancer cell line BIU87 and its EPI chemoresistant cell line BIU87/ADR. The miR-34a profiles in bladder cancer tissues were obtained from The Cancer Genome Atlas database. The effect of miR-34a on chemosensitivity was evaluated by cell viability assays, colony formation assays, and in vivo experimentation. Apoptosis and the cell cycle were examined by flow cytometry. A luciferase reporter assay was used to assess the target genes of miR-34a. Western blot and qPCR were used to analyze the expression of target proteins and downstream molecules. RESULTS: The downregulation of miR-34a in bladder cancer serves as an independent predictor of reduced patient survival. The CCK-8 assay showed that miR-34a overexpression resulted in increased sensitivity to EPI, while miR-34a downregulation resulted in chemoresistance to EPI in vitro. Moreover, it was found that miR-34a increased the sensitivity of BIU87/ADR cells to chemotherapy in vivo. The luciferase reporter assay ascertained that TCF1 and LEF1 are direct target genes of miR-34a. It was found that miR-34a increased chemosensitivity in BIU87/ADR cells by inhibiting the TCF1/LEF1 axis. CONCLUSIONS: The results of this study indicate that miR-34a contributes to the chemosensitivity of BIU87/ADR by inhibiting the TCF1/LEF1 axis. Consequently, miR-34a is a determinant of BIU87 chemosensitivity and may therefore serve as a potential therapeutic target in bladder cancer treatment.

The Common HNF1A Variant I27L Is a Modifier of Age at Diabetes Diagnosis in Individuals With HNF1A-MODY.

There is wide variation in the age at diagnosis of diabetes in individuals with maturity-onset diabetes of the young (MODY) due to a mutation in the HNF1A gene. We hypothesized that common variants at the HNF1A locus (rs1169288 [I27L], rs1800574 [A98V]), which are associated with type 2 diabetes susceptibility, may modify age at diabetes diagnosis in individuals with HNF1A-MODY. Meta-analysis of two independent cohorts, comprising 781 individuals with HNF1A-MODY, found no significant associations between genotype and age at diagnosis. However after stratifying according to type of mutation (protein-truncating variant [PTV] or missense), we found each 27L allele to be associated with a 1.6-year decrease (95% CI -2.6, -0.7) in age at diagnosis, specifically in the subset (n = 444) of individuals with a PTV. The effect size was similar and significant across the two independent cohorts of individuals with HNF1A-MODY. We report a robust genetic modifier of HNF1A-MODY age at diagnosis that further illustrates the strong effect of genetic variation within HNF1A upon diabetes phenotype.

Comprehensive screening for monogenic diabetes in 89 Japanese children with insulin-requiring antibody-negative type 1 diabetes.

BACKGROUND: Mutations in causative genes for neonatal diabetes or maturity-onset diabetes of the young have been identified in multiple patients with autoantibody-negative type 1 diabetes (T1D). OBJECTIVES: We aimed to clarify the prevalence and phenotypic characteristics of monogenic abnormalities among 89 children with autoantibody-negative insulin-requiring T1D. METHODS: Mutations in 30 genes were screened using next-generation sequencing, and copy-number alterations of 4 major causative genes were examined using multiplex-ligation-dependent probe amplification. We compared the clinical characteristics between mutation carriers and non-carriers. RESULTS: We identified 11 probable pathogenic substitutions (6 in INS , 2 in HNF1A , 2 in HNF4A , and 1 in HNF1B ) in 11 cases, but no copy-number abnormalities. Only 2 mutation carriers had affected parents. De novo occurrence was confirmed for 3 mutations. The non-carrier group, but not the carrier group, was enriched with susceptible HLA alleles. Mutation carriers exhibited comparable phenotypes to those of non-carriers, except for a relatively normal body mass index (BMI) at diagnosis. CONCLUSIONS: This study demonstrated significant genetic overlap between autoantibody-negative T1D and monogenic diabetes. Mutations in INS and HNF genes, but not those in GCK and other monogenic diabetes genes, likely play critical roles in children with insulin-requiring T1D. This study also suggests the relatively high de novo rates of INS and HNF mutations, and the etiological link between autoimmune abnormalities and T1D in the non-carrier group. Carriers of monogenic mutations show non-specific phenotypes among all T1D cases, although they are more likely to have a normal BMI at diagnosis than non-carriers.

Determining the role of missense mutations in the POU domain of HNF1A that reduce the DNA-binding affinity: A computational approach.

Maturity-onset diabetes of the young type 3 (MODY3) is a non-ketotic form of diabetes associated with poor insulin secretion. Over the past years, several studies have reported the association of missense mutations in the Hepatocyte Nuclear Factor 1 Alpha (HNF1A) with MODY3. Missense mutations in the POU homeodomain (POUH) of HNF1A hinder binding to the DNA, thereby leading to a dysfunctional protein. Missense mutations of the HNF1A were retrieved from public databases and subjected to a three-step computational mutational analysis to identify the underlying mechanism. First, the pathogenicity and stability of the mutations were analyzed to determine whether they alter protein structure and function. Second, the sequence conservation and DNA-binding sites of the mutant positions were assessed; as HNF1A protein is a transcription factor. Finally, the biochemical properties of the biological system were validated using molecular dynamic simulations in Gromacs 4.6.3 package. Two arginine residues (131 and 203) in the HNF1A protein are highly conserved residues and contribute to the function of the protein. Furthermore, the R131W, R131Q, and R203C mutations were predicted to be highly deleterious by in silico tools and showed lower binding affinity with DNA when compared to the native protein using the molecular docking analysis. Triplicate runs of molecular dynamic (MD) simulations (50ns) revealed smaller changes in patterns of deviation, fluctuation, and compactness, in complexes containing the R131Q and R131W mutations, compared to complexes containing the R203C mutant complex. We observed reduction in the number of intermolecular hydrogen bonds, compactness, and electrostatic potential, as well as the loss of salt bridges, in the R203C mutant complex. Substitution of arginine with cysteine at position 203 decreases the affinity of the protein for DNA, thereby destabilizing the protein. Based on our current findings, the MD approach is an important tool for elucidating the impact and affinity of mutations in DNA-protein interactions and understanding their function.

Identification of HNF4A Mutation p.T130I and HNF1A Mutations p.I27L and p.S487N in a Han Chinese Family with Early-Onset Maternally Inherited Type 2 Diabetes.

Maturity-onset diabetes of the young (MODY) is characterized by the onset of diabetes before the age of 25 years, positive family history, high genetic predisposition, monogenic mutations, and an autosomal dominant mode of inheritance. Here, we aimed to investigate the mutations and to characterize the phenotypes of a Han Chinese family with early-onset maternally inherited type 2 diabetes. Detailed clinical assessments and genetic screening for mutations in the HNF4α, GCK, HNF-1α, IPF-1, HNF1ß, and NEUROD1 genes were carried out in this family. One HNF4A mutation (p.T130I) and two HNF1A polymorphisms (p.I27L and p.S487N) were identified. Mutation p.T130I was associated with both early-onset and late-onset diabetes and caused downregulated HNF4A expression, whereas HNF1A polymorphisms p.I27L and p.S487N were associated with the age of diagnosis of diabetes. We demonstrated that mutation p.T130I in HNF4A was pathogenic as were the predicted polymorphisms p.I27L and p.S487N in HNF1A by genetic and functional analysis. Our results show that mutations in HNF4A and HNF1A genes might account for this early-onset inherited type 2 diabetes.

Structure-function studies of HNF1A (MODY3) gene mutations in South Indian patients with monogenic diabetes.

Maturity-onset diabetes of the young (MODY) is a genetically heterogeneous monogenic form of diabetes characterized by onset of diabetes below 25 years of age, autosomal dominant mode of inheritance and primary defect in insulin secretion. Mutations in the gene (HNF1A) encoding transcription factor hepatocyte nuclear factor 1A (HNF-1A) results in one of the most common forms of MODY (MODY3). HNF-1A is mainly enriched in pancreatic ß-cells and hepatocytes and important for organ development and normal pancreatic function. We here report on the functional interrogation of eight missense HNF1A mutations associated with MODY3 in South Indian subjects, and the contributing effect of common variant (S487N) within HNF1A. Of the eight mutations, three mutations (p.R171G, p.G245R and p.R263H), in particular, affected HNF-1A function in transfected HeLa cells by reducing both transcriptional activity and nuclear localization, possibly due to disruption of the integrity of the three dimensional structure. The common variant p.S487N contributed further to the loss-of-function of p.R271Q (p.R271Q+p.S487N double mutant), in vitro, on both activity and localization. Our data on the first functional study of HNF1A mutations in South India subjects confers that the defect of the HNF-1A mutant proteins are responsible for MODY3 diabetes in these patients.

Repression of HNF1α-mediated transcription by amino-terminal enhancer of split (AES).

HNF1α (Hepatocyte Nuclear Factor 1α) is one of the master regulators in pancreatic beta-cell development and function, and the mutations in Hnf1α are the most common monogenic causes of diabetes mellitus. As a member of the POU transcription factor family, HNF1α exerts its gene regulatory function through various molecular interactions; however, there is a paucity of knowledge in their functional complex formation. In this study, we identified the Groucho protein AES (Amino-terminal Enhancer of Split) as a HNF1α-specific physical binding partner and functional repressor of HNF1α-mediated transcription, which has a direct link to glucose-stimulated insulin secretion in beta-cells that is impaired in the HNF1α mutation-driven diabetes.

Berry Phenolic Compounds Increase Expression of Hepatocyte Nuclear Factor-1α (HNF-1α) in Caco-2 and Normal Colon Cells Due to High Affinities with Transcription and Dimerization Domains of HNF-1α.

Hepatocyte nuclear factor-1α (HNF-1α) is found in the kidneys, spleen, thymus, testis, skin, and throughout the digestive organs. It has been found to promote the transcription of various proteins involved in the management of type II diabetes, including dipeptidyl peptidase-IV (DPP-IV). Phenolic compounds from berries and citrus fruits are known to inhibit DPP-IV, but have not been tested for their interactions with wild-type HNF-1α. By studying the interactions of compounds from berries and citrus fruits have with HNF-1α, pre-transcriptional mechanisms that inhibit the expression of proteins such as DPP-IV may be elucidated. In this study, the interactions of berry phenolic compounds and citrus flavonoids with the dimerization and transcriptional domains of HNF-1α were characterized using the molecular docking program AutoDock Vina. The anthocyanin delphinidin-3-O-arabinoside had the highest binding affinity for the dimerization domain as a homodimer (-7.2 kcal/mol) and transcription domain (-8.3 kcal/mol) of HNF-1α. Anthocyanins and anthocyanidins had relatively higher affinities than resveratrol and citrus flavonoids for both, the transcription domain and the dimerization domain as a homodimer. The flavonoid flavone had the highest affinity for a single unit of the dimerization domain (-6.5 kcal/mol). Nuclear expression of HNF-1α was measured in Caco-2 and human normal colon cells treated with blueberry and blackberry anthocyanin extracts. All extracts tested increased significantly (P < 0.05) the nuclear expression of HNF-1α in Caco-2 cells by 85.2 to 260% compared to a control. The extracts tested increased significantly (P < 0.02) the nuclear expression of HNF-1α in normal colon cells by 48.6 to 243%. It was confirmed that delphinidin-3-O-glucoside increased by 3-fold nuclear HNF-1α expression in Caco-2 cells (P < 0.05). Anthocyanins significantly increased nuclear HNF-1α expression, suggesting that these compounds might regulate the genes HNF-1α promotes.

A single-amino-acid mutation in hepatitis C virus NS5A disrupts physical and functional interaction with the transcription factor HNF-1α.

Hepatitis C virus (HCV) infection often causes extrahepatic manifestations, such as type 2 diabetes. We previously reported that HCV infection induces the lysosomal degradation of the transcription factor HNF-1α via an interaction with viral NS5A, thereby suppressing GLUT2 gene expression. However, the molecular mechanism of NS5A-induced degradation of HNF-1α is largely unknown. We aimed to identify the determinants necessary for the degradation of HNF-1α induced by NS5A. Coimmunoprecipitation analysis revealed that the POU specific (POUs) domain spanning from aa 91 to 181 of HNF-1α is responsible for the interaction of NS5A. We also found that the region from aa 121 to 126 of NS5A, which is known as the binding motif of the HCV replication factor FKBP8, is important for the degradation of HNF-1α. A NS5A V121A mutation disrupted the NS5A-HNF-1α interaction as well as the degradation of HNF-1α. Our findings suggest that NS5A Val121 is crucial for viral pathogenesis.

The TCF C-clamp DNA binding domain expands the Wnt transcriptome via alternative target recognition.

LEF/TCFs direct the final step in Wnt/ß-catenin signalling by recruiting ß-catenin to genes for activation of transcription. Ancient, non-vertebrate TCFs contain two DNA binding domains, a High Mobility Group box for recognition of the Wnt Response Element (WRE; 5'-CTTTGWWS-3') and the C-clamp domain for recognition of the GC-rich Helper motif (5'-RCCGCC-3'). Two vertebrate TCFs (TCF-1/TCF7 and TCF-4/TCF7L2) use the C-clamp as an alternatively spliced domain to regulate cell-cycle progression, but how the C-clamp influences TCF binding and activity genome-wide is not known. Here, we used a doxycycline inducible system with ChIP-seq to assess how the C-clamp influences human TCF1 binding genome-wide. Metabolic pulse-labeling of nascent RNA with 4'Thiouridine was used with RNA-seq to connect binding to the Wnt transcriptome. We find that the C-clamp enables targeting to a greater number of gene loci for stronger occupancy and transcription regulation. The C-clamp uses Helper sites concurrently with WREs for gene targeting, but it also targets TCF1 to sites that do not have readily identifiable canonical WREs. The coupled ChIP-seq/4'Thiouridine-seq analysis identified new Wnt target genes, including additional regulators of cell proliferation. Thus, C-clamp containing isoforms of TCFs are potent transcriptional regulators with an expanded transcriptome directed by C-clamp-Helper site interactions.

Transcriptional networks driving enhancer function in the CFTR gene.

A critical cis-regulatory element for the CFTR (cystic fibrosis transmembrane conductance regulator) gene is located in intron 11, 100 kb distal to the promoter, with which it interacts. This sequence contains an intestine-selective enhancer and associates with enhancer signature proteins, such as p300, in addition to tissue-specific TFs (transcription factors). In the present study we identify critical TFs that are recruited to this element and demonstrate their importance in regulating CFTR expression. In vitro DNase I footprinting and EMSAs (electrophoretic mobility-shift assays) identified four cell-type-selective regions that bound TFs in vitro. ChIP (chromatin immunoprecipitation) identified FOXA1/A2 (forkhead box A1/A2), HNF1 (hepatocyte nuclear factor 1) and CDX2 (caudal-type homeobox 2) as in vivo trans-interacting factors. Mutation of their binding sites in the intron 11 core compromised its enhancer activity when measured by reporter gene assay. Moreover, siRNA (small interfering RNA)-mediated knockdown of CDX2 caused a significant reduction in endogenous CFTR transcription in intestinal cells, suggesting that this factor is critical for the maintenance of high levels of CFTR expression in these cells. The ChIP data also demonstrate that these TFs interact with multiple cis-regulatory elements across the CFTR locus, implicating a more global role in intestinal expression of the gene.

A large multi-centre European study validates high-sensitivity C-reactive protein (hsCRP) as a clinical biomarker for the diagnosis of diabetes subtypes.

AIMS/HYPOTHESIS: An accurate molecular diagnosis of diabetes subtype confers clinical benefits; however, many individuals with monogenic diabetes remain undiagnosed. Biomarkers could help to prioritise patients for genetic investigation. We recently demonstrated that high-sensitivity C-reactive protein (hsCRP) levels are lower in UK patients with hepatocyte nuclear factor 1 alpha (HNF1A)-MODY than in other diabetes subtypes. In this large multi-centre study we aimed to assess the clinical validity of hsCRP as a diagnostic biomarker, examine the genotype-phenotype relationship and compare different hsCRP assays. METHODS: High-sensitivity CRP levels were analysed in individuals with HNF1A-MODY (n = 457), glucokinase (GCK)-MODY (n = 404), hepatocyte nuclear factor 4 alpha (HNF4A)-MODY (n = 54) and type 2 diabetes (n = 582) from seven European centres. Three common assays for hsCRP analysis were evaluated. We excluded 121 participants (8.1%) with hsCRP values >10 mg/l. The discriminative power of hsCRP with respect to diabetes aetiology was assessed by receiver operating characteristic curve-derived C-statistic. RESULTS: In all centres and irrespective of the assay method, meta-analysis confirmed significantly lower hsCRP levels in those with HNF1A-MODY than in those with other aetiologies (z score -21.8, p < 5 × 10(-105)). HNF1A-MODY cases with missense mutations had lower hsCRP levels than those with truncating mutations (0.03 vs 0.08 mg/l, p < 5 × 10(-5)). High-sensitivity CRP values between assays were strongly correlated (r (2) ≥ 0.91, p ≤ 1 × 10(-5)). Across the seven centres, the C-statistic for distinguishing HNF1A-MODY from young adult-onset type 2 diabetes ranged from 0.79 to 0.97, indicating high discriminative accuracy. CONCLUSIONS/INTERPRETATION: In the largest study to date, we have established that hsCRP is a clinically valid biomarker for HNF1A-MODY in European populations. Given the modest costs and wide availability, hsCRP could translate rapidly into clinical practice, considerably improving diagnosis rates in monogenic diabetes.

Crystallization of hepatocyte nuclear factor 4 alpha (HNF4 alpha) in complex with the HNF1 alpha promoter element.

Hepatocyte nuclear factor 4alpha (HNF4alpha) is a member of the nuclear receptor superfamily that plays a central role in organ development and metabolic functions. Mutations on HNF4alpha cause maturity-onset diabetes of the young (MODY), a dominant monogenic cause of diabetes. In order to understand the molecular mechanism of promoter recognition and the molecular basis of disease-causing mutations, the recombinant HNF4alpha DNA-binding domain was prepared and used in a study of its binding properties and in crystallization with a 21-mer DNA fragment that contains the promoter element of another MODY gene, HNF1alpha. The HNF4alpha protein displays a cooperative and specific DNA-binding activity towards its target gene-recognition elements. Crystals of the complex diffract to 2.0 A using a synchrotron-radiation source under cryogenic (100 K) conditions and belong to space group C2, with unit-cell parameters a = 121.63, b = 35.43, c = 70.99 A, beta = 119.36 degrees . A molecular-replacement solution has been obtained and structure refinement is in progress. This structure and the binding studies will provide the groundwork for detailed functional and biochemical studies of the MODY mutants.