Exploring the stage-specific roles of Tcf-1 in T cell development and malignancy at single-cell resolution.

Tcf-1 (encoded by Tcf7) not only plays critical roles in promoting T cell development and differentiation but also has been identified as a tumor suppressor involved in preventing T cell malignancy. However, the comprehensive mechanisms of Tcf-1 involved in T cell transformation remain poorly understood. In this study, Tcf7fl/fl mice were crossed with Vav-cre, Lck-cre, or Cd4-cre mice to delete Tcf-1 conditionally at the beginning of the HSC, DN2-DN3, or DP stage, respectively. The defective T cell development phenotypes became gradually less severe as the deletion stage became more advanced in distinct mouse models. Interestingly, consistent with Tcf7-/- mice, Tcf7fl/flVav-cre mice developed aggressive T cell lymphoma within 45 weeks, but no tumors were generated in Tcf7fl/flLck-cre or Tcf7fl/flCd4-cre mice. Single-cell RNA-seq (ScRNA-seq) indicated that ablation of Tcf-1 at distinct phases can subdivide DN1 cells into three clusters (C1, C2, and C3) and DN2-DN3 cells into three clusters (C4, C5, and C6). Moreover, Tcf-1 deficiency redirects bifurcation among divergent cell fates, and clusters C1 and C4 exhibit high potential for leukemic transformation. Mechanistically, we found that Tcf-1 directly binds and mediates chromatin accessibility for both typical T cell regulators and proto-oncogenes, including Myb, Mycn, Runx1, and Lyl1 in the DN1 phase and Lef1, Id2, Dtx1, Fyn, Bcl11b, and Zfp36l2 in the DN2-DN3 phase. The aberrant expression of these genes due to Tcf-1 deficiency in very early T cells contributes to subsequent tumorigenesis. Thus, we demonstrated that Tcf-1 plays stage-specific roles in regulating early thymocyte development and transformation, providing new insights and evidence for clinical trials on T-ALL leukemia.

Direct reprogramming of human umbilical vein- and peripheral blood-derived endothelial cells into hepatic progenitor cells.

Recent advances have enabled the direct induction of human tissue-specific stem and progenitor cells from differentiated somatic cells. However, it is not known whether human hepatic progenitor cells (hHepPCs) can be generated from other cell types by direct lineage reprogramming with defined transcription factors. Here, we show that a set of three transcription factors, FOXA3, HNF1A, and HNF6, can induce human umbilical vein endothelial cells to directly acquire the properties of hHepPCs. These induced hHepPCs (hiHepPCs) propagate in long-term monolayer culture and differentiate into functional hepatocytes and cholangiocytes by forming cell aggregates and cystic epithelial spheroids, respectively, under three-dimensional culture conditions. After transplantation, hiHepPC-derived hepatocytes and cholangiocytes reconstitute damaged liver tissues and support hepatic function. The defined transcription factors also induce hiHepPCs from endothelial cells circulating in adult human peripheral blood. These expandable and bipotential hiHepPCs may be useful in the study and treatment of human liver diseases.

Redundant and additive functions of the four Lef/Tcf transcription factors in lung epithelial progenitors.

In multicellular organisms, paralogs from gene duplication survive purifying selection by evolving tissue-specific expression and function. Whether this genetic redundancy is also selected for within a single cell type is unclear for multimember paralogs, as exemplified by the four obligatory Lef/Tcf transcription factors of canonical Wnt signaling, mainly due to the complex genetics involved. Using the developing mouse lung as a model system, we generate two quadruple conditional knockouts, four triple mutants, and various combinations of double mutants, showing that the four Lef/Tcf genes function redundantly in the presence of at least two Lef/Tcf paralogs, but additively upon losing additional paralogs to specify and maintain lung epithelial progenitors. Prelung-specification, pan-epithelial double knockouts have no lung phenotype; triple knockouts have varying phenotypes, including defective branching and tracheoesophageal fistulas; and the quadruple knockout barely forms a lung, resembling the Ctnnb1 mutant. Postlung-specification deletion of all four Lef/Tcf genes leads to branching defects, down-regulation of progenitor genes, premature alveolar differentiation, and derepression of gastrointestinal genes, again phenocopying the corresponding Ctnnb1 mutant. Our study supports a monotonic, positive signaling relationship between CTNNB1 and Lef/Tcf in lung epithelial progenitors as opposed to reported repressor functions of Lef/Tcf, and represents a thorough in vivo analysis of cell-type-specific genetic redundancy among the four Lef/Tcf paralogs.

Hepatic Nuclear Factor 1 Alpha (HNF-1α) In Human Physiology and Molecular Medicine.

The transcription factors (TFs) play a crucial role in the modulation of specific gene transcription networks. One of the hepatocyte nuclear factors (HNFs) family's member, hepatocyte nuclear factor-1α (HNF-1α) has continuously become a principal TF to control the expression of genes. It is involved in the regulation of a variety of functions in various human organs including liver, pancreas, intestine, and kidney. It regulates the expression of enzymes involved in endocrine and xenobiotic activity through various metabolite transporters located in the above organs. Its expression is also required for organ-specific cell fate determination. Despite two decades of its first identification in hepatocytes, a review of its significance was not comprehended. Here, the role of HNF-1α in the above organs at the molecular level to intimate molecular mechanisms for regulating certain gene expression whose malfunctions are attributed to the disease conditions has been specifically encouraged. Moreover, the epigenetic effects of HNF-1α have been discussed here, which could help in advanced technologies for molecular pharmacological intervention and potential clinical implications for targeted therapies. HNF-1α plays an indispensable role in several physiological mechanisms in the liver, pancreas, intestine, and kidney. Loss of its operations leads to the non-functional or abnormal functional state of each organ. Specific molecular agents or epigenetic modifying drugs that reactivate HNF-1α are the current requirements for the medications of the diseases.

Defining 'T cell exhaustion'.

'T cell exhaustion' is a broad term that has been used to describe the response of T cells to chronic antigen stimulation, first in the setting of chronic viral infection but more recently in response to tumours. Understanding the features of and pathways to exhaustion has crucial implications for the success of checkpoint blockade and adoptive T cell transfer therapies. In this Viewpoint article, 18 experts in the field tell us what exhaustion means to them, ranging from complete lack of effector function to altered functionality to prevent immunopathology, with potential differences between cancer and chronic infection. Their responses highlight the dichotomy between terminally differentiated exhausted T cells that are TCF1- and the self-renewing TCF1+ population from which they derive. These TCF1+ cells are considered by some to have stem cell-like properties akin to memory T cell populations, but the developmental relationships are unclear at present. Recent studies have also highlighted an important role for the transcriptional regulator TOX in driving the epigenetic enforcement of exhaustion, but key questions remain about the potential to reverse the epigenetic programme of exhaustion and how this might affect the persistence of T cell populations.

TCF1 and LEF1 Control Treg Competitive Survival and Tfr Development to Prevent Autoimmune Diseases.

CD4+ Foxp3+ T regulatory (Treg) cells are key players in preventing lethal autoimmunity. Tregs undertake differentiation processes and acquire diverse functional properties. However, how Treg's differentiation and functional specification are regulated remains incompletely understood. Here, we report that gradient expression of TCF1 and LEF1 distinguishes Tregs into three distinct subpopulations, particularly highlighting a subset of activated Treg (aTreg) cells. Treg-specific ablation of TCF1 and LEF1 renders the mice susceptible to systemic autoimmunity. TCF1 and LEF1 are dispensable for Treg's suppressive capacity but essential for maintaining a normal aTreg pool and promoting Treg's competitive survival. As a consequence, the development of T follicular regulatory (Tfr) cells, which are a subset of aTreg, is abolished in TCF1/LEF1-conditional knockout mice, leading to unrestrained T follicular helper (Tfh) and germinal center B cell responses. Thus, TCF1 and LEF1 act redundantly to control the maintenance and functional specification of Treg subsets to prevent autoimmunity.

TCF-1 limits the formation of Tc17 cells via repression of the MAF-RORγt axis.

Interleukin (IL)-17-producing CD8+ T (Tc17) cells have emerged as key players in host-microbiota interactions, infection, and cancer. The factors that drive their development, in contrast to interferon (IFN)-γ-producing effector CD8+ T cells, are not clear. Here we demonstrate that the transcription factor TCF-1 (Tcf7) regulates CD8+ T cell fate decisions in double-positive (DP) thymocytes through the sequential suppression of MAF and RORγt, in parallel with TCF-1-driven modulation of chromatin state. Ablation of TCF-1 resulted in enhanced Tc17 cell development and exposed a gene set signature to drive tissue repair and lipid metabolism, which was distinct from other CD8+ T cell subsets. IL-17-producing CD8+ T cells isolated from healthy humans were also distinct from CD8+IL-17- T cells and enriched in pathways driven by MAF and RORγt Overall, our study reveals how TCF-1 exerts central control of T cell differentiation in the thymus by normally repressing Tc17 differentiation and promoting an effector fate outcome.

Lineage-Determining Transcription Factor TCF-1 Initiates the Epigenetic Identity of T Cells.

T cell development is orchestrated by transcription factors that regulate the expression of genes initially buried within inaccessible chromatin, but the transcription factors that establish the regulatory landscape of the T cell lineage remain unknown. Profiling chromatin accessibility at eight stages of T cell development revealed the selective enrichment of TCF-1 at genomic regions that became accessible at the earliest stages of development. TCF-1 was further required for the accessibility of these regulatory elements and at the single-cell level, it dictated a coordinate opening of chromatin in T cells. TCF-1 expression in fibroblasts generated de novo chromatin accessibility even at chromatin regions with repressive marks, inducing the expression of T cell-restricted genes. These results indicate that a mechanism by which TCF-1 controls T cell fate is through its widespread ability to target silent chromatin and establish the epigenetic identity of T cells.

Transcription Factor 7 Limits Regulatory T Cell Generation in the Thymus.

Regulatory T cells (Tregs) differentiate in the thymus, but the mechanisms that control this process are not fully understood. We generated a comprehensive quantitative and differential proteome of murine Tregs and conventional T cells. We identified 5225 proteins, 164 of which were differentially expressed in Tregs. Together with the comparative analysis of proteome and gene expression data, we identified TCF7 as a promising candidate. Genetic elimination of transcription factor 7 (TCF7) led to increased fractions of Tregs in the thymus. Reduced levels of TCF7, found in the heterozygote, resulted in a greater potential for Treg precursors to differentiate into the Treg lineage. In contrast, activation of TCF7 through ß-catenin had the opposite effect. TCF7 levels influenced the required TCR signaling strength of Treg precursors, and TCF7 deficiency broadened the repertoire and allowed lower TCR affinities to be recruited into the Treg lineage. FOXP3 was able to repress TCF7 protein expression. In summary, we propose a regulatory role for TCF7 in limiting access to the Treg lineage.

Role of Specificity Protein 1, Hepatocyte Nuclear Factor 1α, and Pregnane X Receptor in the Basal and Rifampicin-Induced Transcriptional Regulation of Porcine Cytochrome P450 3A46.

Cytochrome P450 (CYP) 3A46, one of human CYP3A4 homologs, functions as a key enzyme in the metabolism of xenobiotics in pigs. However, the regulatory mechanism for the transcriptional activation of CYP3A46 in porcine liver remains unknown. In this study, we confirmed that CYP3A46 is constitutively expressed in porcine primary hepatocytes, and its expression was significantly induced by rifampicin (RIF) instead of dexamethasone. We further found that a proximal GC box and a distal hepatocyte nuclear factor 1 (HNF1) binding site within the 5'-flanking region of CYP3A46 are the important cis-regulatory elements involved in regulating the constitutive expression of CYP3A46, via recruiting specificity protein 1 (Sp1) and HNF1α, respectively. Furthermore, we revealed that HNF1α and pregnane X receptor (PXR) activate the RIF-mediated transcription of CYP3A46 by binding to the distal HNF1 binding site and the proximal direct repeats of AGGTCA separated by 4 bases motif, respectively. Meanwhile, HNF1α is also involved in regulating RIF-induced expression of CYP3A4 through a novel distal HNF1 binding site identified in the xenobiotic-responsive enhancer module. In summary, our data demonstrate that several transcription factors, including Sp1, HNF1α, and PXR, function in the basal and RIF-mediated transcriptional regulation of CYP3A46 by binding to their related cis-regulatory elements in the proximal promoter and distal enhancer.

Premature expression of Foxp3 in double-negative thymocytes.

Peripheral immune regulation depends on the generation of thymic-derived regulatory T (tTreg) cells to maintain self-tolerance and to counterbalance overshooting immune responses. The expression of the Treg lineage defining transcription factor Foxp3 in developing tTreg cells depends on TCR signaling during the thymic selection process of these T cells. In this study, we surprisingly identify Foxp3+ immature thymocytes at the double-negative (DN) stage in transcription factor 7 (Tcf7)-deficient mice. These Foxp3+ cells did not express a TCR (ß or γδ chains), CD3 or CD5 and therefore these cells were true DN cells. Further investigation of this phenomenon in a transgenic TCR model showed that Foxp3-expressing DN cells could not respond to TCR stimulation in vivo. These data suggest that Foxp3 expression in these DN cells occurred independently of TCR signaling. Interestingly, these Foxp3+ DN cells were located in a transition state between DN1 and DN2 (CD4-CD8-CD3-TCR-CD44highCD25low). Our results indicate that Tcf7 is involved in preventing the premature expression of Foxp3 in DN thymocytes.

TG-interacting factor 1 acts as a transcriptional repressor of sterol O-acyltransferase 2.

Acat2 [gene name: sterol O-acyltransferase 2 (SOAT2)] esterifies cholesterol in enterocytes and hepatocytes. This study aims to identify repressor elements in the human SOAT2 promoter and evaluate their in vivo relevance. We identified TG-interacting factor 1 (Tgif1) to function as an important repressor of SOAT2. Tgif1 could also block the induction of the SOAT2 promoter activity by hepatocyte nuclear factor 1α and 4α. Women have ∼ 30% higher hepatic TGIF1 mRNA compared with men. Depletion of Tgif1 in mice increased the hepatic Soat2 expression and resulted in higher hepatic lipid accumulation and plasma cholesterol levels. Tgif1 is a new player in human cholesterol metabolism.

Pleiotropic effects of HNF1A rs1183910 in a population-based study of 60,283 individuals.

AIMS/HYPOTHESIS: According to genome-wide association studies (GWAS) a locus in the HNF1A gene has pleiotropic effects on several metabolic traits. In a large single-centre study we used the intronic variant rs1183910 located in a region with no or low recombination rate as an instrument for the HNF1A locus to evaluate pleiotropic effects of this locus on the risk of developing type 2 diabetes, as well as on body composition and levels of non-fasting glucose, lipids, acute-phase reactants, and biomarkers of liver and pancreas function. METHODS: We investigated 60,283 individuals from the Danish general population who were all examined in the same laboratory, comprising the Copenhagen General Population Study. RESULTS: We confirm previous GWAS findings, namely that the minor rs1183910 A allele is associated with an increased risk of developing type 2 diabetes (p(trend) = 0.003), decreased levels of C-reactive protein (CRP; p(trend) = 6 × 10(-76)) and γ-glutamyltransferase (p(trend) = 4 × 10(-48)), and increased levels of total cholesterol (p(trend) = 3 × 10(-10)) and LDL-cholesterol (p(trend) = 3 × 10(-11)). For the first time, we report that the minor rs1183910 A allele is associated with increased levels of non-fasting plasma glucose (p(trend) = 3 × 10(-5)), apolipoprotein B (ApoB; p(trend) = 1 × 10(-4)) and alkaline phosphatase (p(trend) = 5 × 10(-14)), and decreased levels of bilirubin (p trend = 3 × 10(-5)). Our results suggest that the association with increased risk of type 2 diabetes is driven by high non-fasting glucose levels. CONCLUSIONS/INTERPRETATION: The minor rs1183910 A allele prompts a potential adverse metabolic profile with increased levels of non-fasting glucose, total cholesterol, LDL-cholesterol, ApoB, and alkaline phosphatase, but simultaneously has potential beneficial effects through decreased levels of CRP, γ-glutamyltransferase and bilirubin.

The transcription factor HNF1α induces expression of angiotensin-converting enzyme 2 (ACE2) in pancreatic islets from evolutionarily conserved promoter motifs.

Pancreatic angiotensin-converting enzyme 2 (ACE2) has previously been shown to be critical for maintaining glycemia and ß-cell function. Efforts to maintain or increase ACE2 expression in pancreatic ß-cells might therefore have therapeutic potential for treating diabetes. In our study, we investigated the transcriptional role of hepatocyte nuclear factor 1α (HNF1α) and hepatocyte nuclear factor 1ß (HNF1ß) in induction of ACE2 expression in insulin-secreting cells. A deficient allele of HNF1α or HNF1ß causes maturity-onset diabetes of the young (MODY) types 3 and 5, respectively, in humans. We found that ACE2 is primarily transcribed from the proximal part of the ACE2 promoter in the pancreas. In the proximal part of the human ACE2 promoter, we further identified three functional HNF1 binding sites, as they have binding affinity for HNF1α and HNF1ß and are required for induction of promoter activity by HNF1ß in insulinoma cells. These three sites are well-conserved among mammalian species. Both HNF1α and HNF1ß induce expression of ACE2 mRNA and lead to elevated levels of ACE2 protein and ACE2 enzymatic activity in insulinoma cells. Furthermore, HNF1α dose-dependently increases ACE2 expression in primary pancreatic islet cells. We conclude that HNF1α can induce the expression of ACE2 in pancreatic islet cells via evolutionarily conserved HNF1 binding sites in the ACE2 promoter. Potential therapeutics aimed at counteracting functional HNF1α depletion in diabetes and MODY3 will thus have ACE2 induction in pancreatic islets as a likely beneficial effect.

Cigarette smoke mediates epigenetic repression of miR-487b during pulmonary carcinogenesis.

MicroRNAs are critical mediators of stem cell pluripotency, differentiation, and malignancy. Limited information exists regarding microRNA alterations that facilitate initiation and progression of human lung cancers. In this study, array techniques were used to evaluate microRNA expression in normal human respiratory epithelia and lung cancer cells cultured in the presence or absence of cigarette smoke condensate (CSC). Under relevant exposure conditions, CSC significantly repressed miR-487b. Subsequent experiments demonstrated that miR-487b directly targeted SUZ12, BMI1, WNT5A, MYC, and KRAS. Repression of miR-487b correlated with overexpression of these targets in primary lung cancers and coincided with DNA methylation, de novo nucleosome occupancy, and decreased H2AZ and TCF1 levels within the miR-487b genomic locus. Deoxy-azacytidine derepressed miR-487b and attenuated CSC-mediated silencing of miR-487b. Constitutive expression of miR-487b abrogated Wnt signaling, inhibited in vitro proliferation and invasion of lung cancer cells mediated by CSC or overexpression of miR-487b targets, and decreased growth and metastatic potential of lung cancer cells in vivo. Collectively, these findings indicate that miR-487b is a tumor suppressor microRNA silenced by epigenetic mechanisms during tobacco-induced pulmonary carcinogenesis and suggest that DNA demethylating agents may be useful for activating miR-487b for lung cancer therapy.

Regulation of hepatic LDL receptors by mTORC1 and PCSK9 in mice.

Individuals with type 2 diabetes have an increased risk of atherosclerosis. One factor underlying this is dyslipidemia, which in hyperinsulinemic subjects with early type 2 diabetes is typically characterized by increased VLDL secretion but normal LDL cholesterol levels, possibly reflecting enhanced catabolism of LDL via hepatic LDLRs. Recent studies have also suggested that hepatic insulin signaling sustains LDLR levels. We therefore sought to elucidate the mechanisms linking hepatic insulin signaling to regulation of LDLR levels. In WT mice, insulin receptor knockdown by shRNA resulted in decreased hepatic mTORC1 signaling and LDLR protein levels. It also led to increased expression of PCSK9, a known post-transcriptional regulator of LDLR expression. Administration of the mTORC1 inhibitor rapamycin caused increased expression of PCSK9, decreased levels of hepatic LDLR protein, and increased levels of VLDL/LDL cholesterol in WT but not Pcsk9-/- mice. Conversely, mice with increased hepatic mTORC1 activity exhibited decreased expression of PCSK9 and increased levels of hepatic LDLR protein levels. Pcsk9 is regulated by the transcription factor HNF1α, and our further detailed analyses suggest that increased mTORC1 activity leads to activation of PKCδ, reduced activity of HNF4α and HNF1α, decreased PCSK9 expression, and ultimately increased hepatic LDLR protein levels, which result in decreased circulating LDL levels. We therefore suggest that PCSK9 inhibition could be an effective way to reduce the adverse side effect of increased LDL levels that is observed in transplant patients taking rapamycin as immunosuppressive therapy.

Regulation of tissue-specific expression of renal organic anion transporters by hepatocyte nuclear factor 1 α/ß and DNA methylation.

We have reported previously that the kidney- and liver-specific expression of transporters in mice involves the coordinated regulation by hepatocyte nuclear factor 1 (HNF1) and DNA methylation. The present study was aimed at investigating the role of this cascade in the transcriptional regulation of renal organic anion transporters (OATs) yet to be characterized in human and mouse. Luciferase assays and electrophoretic mobility-shift assays demonstrated that HNF1α/ß enhances the promoter activity of OAT4/SLC22A11 via binding to the HNF1 motif located near the transcriptional start site (TSS). DNA methylation profiles of human OAT1, OAT3, OAT4, and urate transporter 1 (URAT1) were determined in human liver and kidney cortex by bisulfite sequencing. Most of the CpG dinucleotides around the TSSs of OAT1 and OAT3 were highly methylated in the liver compared with kidney cortex, being consistent with their tissue specificity, whereas the difference in the DNA methylation status was less remarkable between the two tissues for OAT4 and URAT1. Mouse Oat1 gene also contained CpG dinucleotides hypomethylated in the kidney and hypermethylated in the liver downstream its TSS, whereas two of the seven CpG dinucleotides around the TSS of mouse Oat3 were significantly methylated in the liver compared with the kidney. Taken together, these findings underscored the central role of HNF1α/ß in the transcriptional regulation of OATs and highlighted DNA methylation-dependent gene silencing as one of the mechanisms underlying the tissue-specific transactivation by this master regulator.

The stable repression of mesenchymal program is required for hepatocyte identity: a novel role for hepatocyte nuclear factor 4α.

UNLABELLED: The concept that cellular terminal differentiation is stably maintained once development is complete has been questioned by numerous observations showing that differentiated epithelium may undergo an epithelial-to-mesenchymal transition (EMT) program. EMT and the reverse process, mesenchymal-to-epithelial transition (MET), are typical events of development, tissue repair, and tumor progression. In this study, we aimed to clarify the molecular mechanisms underlying these phenotypic conversions in hepatocytes. Hepatocyte nuclear factor 4α (HNF4α) was overexpressed in different hepatocyte cell lines and the resulting gene expression profile was determined by real-time quantitative polymerase chain reaction. HNF4α recruitment on promoters of both mesenchymal and EMT regulator genes was determined by way of electrophoretic mobility shift assay and chromatin immunoprecipitation. The effect of HNF4α depletion was assessed in silenced cells and in the context of the whole liver of HNF4 knockout animals. Our results identified key EMT regulators and mesenchymal genes as new targets of HNF4α. HNF4α, in cooperation with its target HNF1α, directly inhibits transcription of the EMT master regulatory genes Snail, Slug, and HMGA2 and of several mesenchymal markers. HNF4α-mediated repression of EMT genes induces MET in hepatomas, and its silencing triggers the mesenchymal program in differentiated hepatocytes both in cell culture and in the whole liver. CONCLUSION: The pivotal role of HNF4α in the induction and maintenance of hepatocyte differentiation should also be ascribed to its capacity to continuously repress the mesenchymal program; thus, both HNF4α activator and repressor functions are necessary for the identity of hepatocytes.

[Interest of immunohistochemistry for the diagnosis of benign hepatocellular tumors]. / Apport de l'immunohistochimie dans le diagnostic des tumeurs hépatocellulaires bénignes.

In this review, we focus on the interest of immunohistochemistry first, to differentiate the two types of benign hepatocellular nodules: focal nodular hyperplasia (FNH) and hepatocellular adenoma (HCA) and second, to recognize the different subtypes of HCA: HNF1α inactivated HCA, ß-catenin activated HCA and inflammatory HCA. This pathomolecular classication followed the study of genotype/phenotype correlations which led to the identification of immunohistochemical data characteristic of each subgroup. Immunohistochemical characteristics described on resected specimen are suitable on biopies of these tumors, facilitating their diagnosis and therefore allowing a better management of the patients.

Familial young-onset forms of diabetes related to HNF4A and rare HNF1A molecular aetiologies.

OBJECTIVE: We have dissected the rare molecular anomalies that may affect hepatocyte nuclear factor-1a (HNF1A) and hepatocyte nuclear factor-4a (HNF4A) in patients with familial young-onset diabetes for whom HNF1A mutations have been excluded by sequence analysis. METHODS: Eighty-four unrelatedHNF1A-negative patients with diabetes diagnosed before the age of 40 years, a family history of diabetes and the absence of features suggestive of Type 2 diabetes were included. We analysed by sequencing the HNF4A promoter and coding regions, the HNF1A promoter region and specific regions of HNF1A(B) and HNF1A(C) isoforms and searched for large deletions of HNF1A and HNF4A by multiplex ligation-dependent probe amplification (MLPA). RESULTS: We identified five novel HNF4A mutations (5 / 84, 6%), including four missense and one in-frame deletion, and one mutation of the HNF1A promoter (1 / 84). Sequence analysis of isoform-specific coding regions of HNF1A did not reveal any mutation. We next identified two whole gene deletions of HNF1A and HNF4A, respectively (2 / 84, 2.4%). CONCLUSIONS: Altogether, the search for rare molecular events in HNF1A and HNF4A led us to elucidate 8 / 84 (9.5%) of our HNF1A-negative cases.This study shows that genetic aetiologies remain to be elucidated in familial young-onset diabetes. It also highlights the difficulty of the differential diagnosis with Type 2 diabetes because of the wide clinical expression of monogenic young-onset diabetes and the absence of specific biomarkers.