Hepatocyte Nuclear Factor-1α Gene Research Articles

HNF-1a promotes pancreatic cancer growth and apoptosis resistance via its target gene PKLR.

Pancreatic ductal adenocarcinoma is one of the deadliest malignant tumors, and many genes play important roles in its development. The hepatocyte nuclear factor-1a (HNF-1a) gene encodes HNF-1a, which is a transcriptional activator. HNF-1a regulates the tissue-specific expression of multiple genes, especially in pancreatic islet cells and in the liver. However, the role of the HNF-1a gene in the development of pancreatic cancer is still unclear. Here, we used immunohistochemical staining and real-time PCR to analyze HNF-1a expression in pancreatic cancer tissue. Stable cell lines with HNF-1a knockdown or overexpression were established to analyze the role of HNF-1a in pancreatic cancer cell proliferation and apoptosis by colony formation assay and flow cytometry. We also analyzed the L-type pyruvate kinase (PKLR) promoter sequence to identify the regulatory effect of HNF-1a on PKLR transcription and confirmed the HNF-1a binding site in the PKLR promoter via a chromatin immunoprecipitation assay. HNF-1a was found to be overexpressed in pancreatic cancer and promoted proliferation while inhibiting apoptosis in pancreatic cancer cells. PKLR was identified as the downstream target gene of HNF-1a and binding of HNF-1a at two sites in PKLR (-1931/-1926 and -966/-961) regulated PKLR transcription. In conclusion, HNF-1a is overexpressed in pancreatic cancer, and the transcription factor HNF-1a can promote pancreatic cancer growth and apoptosis resistance via its target gene PKLR.

Expression of mutant mRNA and protein in pancreatic cells derived from MODY3- iPS cells.

Maturity-onset diabetes of the young (MODY) is a heterozygous monogenic diabetes; more than 14 disease genes have been identified. However, the pathogenesis of MODY is not fully understood because the patients’ pancreatic beta cells are inaccessible. To elucidate the pathology of MODY, we established MODY3 patient-derived iPS (MODY3-iPS) cells using non-integrating Sendai virus (SeV) vector and examined the mutant mRNA and protein of HNF1A (Hepatocyte Nuclear factor 1A) after pancreatic lineage differentiation. Our patient had a cytosine insertion in the HNF1A gene (P291fsinsC) causing frameshift and making a premature termination codon (PTC). We confirmed these MODY3-iPS cells possessed the characteristics of pluripotent stem cells. After we differentiated them into pancreatic beta cells, transcripts of HNF1A gene were cloned and sequenced. We found that P291fsinsC mutant transcripts were much less frequent than wild ones, but they increased after adding cycloheximide (CHX) to the medium. These results suggested that mutant mRNA was destroyed by nonsense-mediated mRNA decay (NMD). Moreover, we were not able to detect any band of mutant proteins in pancreatic lineage cells which were differentiated from MODY3-iPSCs by western blot (WB) analysis. A scarcity of the truncated form of mutant protein may indicate that MODY3 might be caused by a haplo-insufficiency effect rather than a dominant negative manner.

The HNF1A mutant Ala180Val: Clinical challenges in determining causality of a rare HNF1A variant in familial diabetes.

Heterozygous mutations in hepatocyte nuclear factor-1A (HNF1A) cause maturity-onset diabetes of the young type 3 (MODY3). Our aim was to compare two families with suspected dominantly inherited diabetes and a new HNF1A variant of unknown clinical significance. The HNF1A gene was sequenced in two independently recruited families from the Norwegian MODY Registry. Both familes were phenotyped clinically and biochemically. Microsatellite markers around and within the HNF1A locus were used for haplotyping. Chromosomal linkage analysis was performed in one family, and whole-exome sequencing was undertaken in two affected family members from each family. Transactivation activity, DNA binding and nuclear localization of wild type and mutant HNF-1A were assessed. The novel HNF1A variant c.539C>T (p.Ala180Val) was found in both families. The variant fully co-segregated with diabetes in one family. In the other family, two subjects with diabetes mellitus and one with normal glucose levels were homozygous variant carriers. Chromosomal linkage of diabetes to the HNF1A locus or to other genomic regions could not be established. The protein functional studies did not reveal significant differences between wild type and variant HNF-1A. In each family, whole-exome sequencing failed to identify any other variant that could explain the disease. The HNF1A variant p.Ala180Val does not seem to cause MODY3, although it may confer risk for type 2 diabetes mellitus. Our data demonstrate challenges in causality evaluation of rare variants detected in known diabetes genes.

The spectrum of HNF1A gene mutations in patients with MODY 3 phenotype and identification of three novel germline mutations in Turkish Population

BackgroundMaturity-onset diabetes of the young (MODY) is a monogenic form of diabetes mellitus characterized by autosomal dominant inheritance, early age of onset, and pancreatic beta cell dysfunction. Heterozygous mutations in several genes may cause MODY. MethodsIn the present study, we investigated the molecular spectrum of HNF1A (hepatocyte nuclear factor 1a) mutations, in the individuals referred to a reference center for molecular genetic analysis. Mutations screening was performed in a group of 136 unrelated patients (average age 17.22 years) selected by clinical characterization of MODY. Mutation screening involved direct sequencing of the HNF1A gene. ResultsAmong 136 individuals analyzed, 10 were carrying heterozygous HNF1A mutations, 3 of them being novel. Clinical features, such as age of diabetes at diagnosis or severity of hyperglycemia, were not related to the mutation type or location. No clear phenotype – genotype correlations were identified. ConclusionsAs a conclusion MODY resulted from HNF1A mutations shows heterogeneity at both phenotypic and molecular levels in Turkish population.

Common variants of HNF1A gene are associated with diabetic retinopathy and poor glycemic control in normal-weight Japanese subjects with type 2 diabetes mellitus

AimThis study investigated the associations between the common hepatocyte nuclear factor-1A (HNF1A) variants and the risk of diabetic retinopathy (DR) in relation to the glycemic control and weight status. MethodsA retrospective longitudinal analysis was conducted among 354 Japanese patients with type 2 diabetes mellitus (T2DM) (mean follow-up duration: 5.8±2.5 years). The multivariable-adjusted hazard ratio (HR) for the cumulative incidence of DR was calculated using a Cox proportional hazard model. During the observation period, the longitudinal associations of the HNF1A diplotypes with the risk of DR and the clinical parameters were also analyzed using the generalized estimating equations approach. ResultsThe combination of risk variants, i.e., rs1169288-C, rs1183910-A and rs2464196-A, was defined as the H1 haplotype. The incidence of DR was higher in the H1/H1 diplotype cases than in the others (HR 2.75 vs. non-H1/non-H1; p=0.02). Only in normal-weight subjects, the risks of DR and poor glycemic control were higher in the H1/H1 diplotype cases than in the others [odds ratio 4.08 vs. non-H1/non-H1, p=0.02; odds ratio 3.03, p=0.01; respectively]. ConclusionsThis study demonstrated that the common HNF1A diplotype of three risk variants may be an independent risk factor for the development of DR resulting from poor glycemic control in normal-weight patients with T2DM. These results need to be replicated in larger and more varied study populations.

Studies of genetic variability of the hepatocyte nuclear factor-1α gene in an Indian maturity-onset diabetes of the young family.

Maturity-onset diabetes of the young (MODY), one of the specific types of diabetes mellitus, is a monogenetic disorder characterized by an autosomal dominant (AD) inheritance and β-cell dysfunction. To study an Indian family with clinical diagnosis of MODY and detect the genetic mutations in the aspect of molecular mechanism, seven blood samples were obtained from the diabetic patients of this pedigree and genomic DNA was extracted from peripheral leukocytes. The exon1, exon2 and exon4 of hepatocyte nuclear factor-1α (HNF-1α) gene were amplified by polymerase chain reaction. Then the products were sequenced and compared with standard sequences on gene bank. As a result, two mutations were detected in exon1. That was CTC → CTG (Leu → Leu) in codon17 and ATC → CTC (Ile → Leu) in codon27. I27L was speculated to have a close relationship with the glycometabolism and the pathogenesis of diabetes mellitus together with the putative novel mutation existed in this Indian pedigree. Meanwhile, one mutation of GGG → GGC (Gly → Gly) in codon288 of exon4 was detected in the proband. No mutations were found in exon2 but a G → T base substitution in the intron4 region among all seven samples was detected. It may have some potential effects on the onset of diabetes in this family, but we do not have any evidence right now. Although it requires further investigation on the function of mutations found in the intron region, our research may provide some clue for this issue and it deserves more attention.

Hepatocyte nuclear factor 1A (HNF1A) as a possible tumor suppressor in pancreatic cancer.

BackgroundHNF1A (Hepatocyte nuclear factor 1 alpha) is a transcription factor that is known to regulate pancreatic differentiation and maintain homeostasis of endocrine pancreas. Recently, genome-wide association studies have implicated HNF1A as a susceptibility gene for pancreatic cancer. However, the functional significance and molecular mechanism of HNF1A in pancreatic carcinogenesis remains unclear.MethodsUsing RT-PCR, Western blot and immunohistochemistry methods, we examined HNF1A gene expression in eight pancreatic carcinoma cell lines and in paired tumor and normal tissue samples from patients with resected pancreatic ductal adenocarcinoma. We knocked down the HNF1A gene expression in two cancer cell lines using three siRNA sequences. The impacts on cell proliferation, apoptosis, and cell cycle as well as the phosphorylation of Akt signaling transduction proteins were examined using ATP assay, flow cytometry and Western blot.Results HNF1A was expressed in three out of eight pancreatic adenocarcinoma cell lines and the level of HNF1A mRNA and protein expression was significantly lower in tumors than in normal adjacent tissues by both RT-PCR and Western Blot analyses. Immunohistochemistry revealed that the level of HNF1A expression was significantly lower in tumor tissues than in non-tumor tissues. Selective blocking of HNF1A by specific siRNA conferred a 2-fold higher rate of cell proliferation, 20% increased S phase and G2 phase cells, and 30-40% reduced apoptosis in pancreatic cancer cell lines. We further demonstrated that HNF1A knockdown activated Akt and its downstream target, the mammalian target of rapamycin (mTOR) in pancreatic cancer cells.ConclusionThese observations provide experimental evidence supporting a possible tumor suppressor role of HNF1A in pancreatic cancer.

Intima-media thickness and endothelial dysfunction in GCK and HNF1A-MODY patients.

Mutations in the glucokinase (GCK) gene, along with hepatocyte nuclear factor 1A (HNF1A) gene mutations, are the most frequent cause of maturity-onset diabetes of the young (MODY). GCK-MODY patients are typically characterized by a moderate fasting hyperglycemia; however, little is known about atherosclerosis and intermediate-related phenotypes in these subjects. To examine carotid artery intima-media thickness (IMT) and endothelial function assessed by brachial artery flow-mediated dilatation (FMD) in GCK gene mutations carriers and HNF1A-MODY. A total of 64 subjects with GCK gene mutations, and 52 HNF1A gene mutation carriers as well as 53 nondiabetic controls were examined. IMT and FMD were assessed by ultrasonography. Appropriate statistical tests were performed to assess differences between the groups, and multivariate linear regression was done for the association with IMT and FMD. The clinical characteristics of all groups were similar with the mean age at examination of 35.1, 41.1, and 39.5 years for GCK, HNF1A and the control group respectively. The highest mean IMT value was in the HNF1A-MODY group: 7.0±1.4 mm, whereas it reached 6.3±1.4 mm in GCK mutation carriers and 6.3±1.3 mm in controls (P=0.008). After adjustment for possible clinical and biochemical cofounders, IMT remained higher in HNF1A-MODY patients as compared with GCK-MODY patients (P=0.02) and controls (P=0.0003). FMD was significantly lower in HNF1A (9.9±4.6%) and GCK-MODY (11.1±4.6%) patients in comparison with controls (13.9±4.7%; P=0.0001). After adjustment, FMD remained lower in HNF1A-MODY (P=0.0005) and GCK-MODY patients (P=0.01) as compared with controls. Both examined MODY groups demonstrated evidence of endothelial dysfunction. In addition, HNF1-MODY patients seem to be more prone to an early atherosclerotic phenotype.

Japanese boy with maturity-onset diabetes of the young type 3 who developed diabetes at 19 months old

Maturity-onset diabetes of the young type 3 (MODY3) is caused by hepatocyte nuclear factor 1α gene mutation and is clinically characterized by young onset and insufficient insulin secretion. We report a 19-month-old Japanese boy with a family history of young-onset diabetes who was initially diagnosed with type 1 diabetes. Mutational analysis of the hepatocyte nuclear factor 1α gene revealed a novel heterozygous frameshift mutation (c.593delA p.Lys198fs) resulting in a truncated protein in the patient and his father. The patient was diagnosed as having MODY3 and was successfully treated with insulin glargine. We could not determine the genetic or environmental factors to explain the difference in the age of disease onset within the same family. This is the youngest case of a MODY3 child presenting with overt diabetes. Our experience suggests that clinicians should always consider the possible diagnosis of MODY3 in a diabetic child with a family history of young-onset diabetes and should perform molecular investigations.

Production of Diabetic Offspring Using Cryopreserved Epididymal Sperm by <i>In Vitro</i> Fertilization and Intrafallopian Insemination Techniques in Transgenic Pigs

Somatic cell nuclear transfer (SCNT) is a useful technique for creating pig strains that model human diseases. However, production of numerous cloned disease model pigs by SCNT for large-scale experiments is impractical due to its complexity and inefficiency. In the present study, we aimed to establish an efficient procedure for proliferating the diabetes model pig carrying the mutant human hepatocyte nuclear factor-1α gene. A founder diabetes transgenic cloned pig was generated by SCNT and treated with insulin to allow for normal growth to maturity, at which point epididymal sperm could be collected for cryopreservation. In vitro fertilization and intrafallopian insemination using the cryopreserved epididymal sperm resulted in diabetes model transgenic offspring. These results suggest that artificial reproductive technology using cryopreserved epididymal sperm could be a practical option for proliferation of genetically modified disease model pigs.

Variants of the HNF1α gene: A molecular approach concerning diabetic patients from southern Brazil.

Maturity Onset Diabetes of the Young (MODY) presents monogenic inheritance and mutation factors which have already been identified in six different genes. Given the wide molecular variation present in the hepatocyte nuclear factor-1α gene (HNF1α) MODY3, the aim of this study was to amplify and sequence the coding regions of this gene in seven patients from the Campos Gerais region, Paraná State, Brazil, presenting clinical MODY3 features. Besides the synonymous variations, A15A, L17L, Q141Q, G288G and T515T, two missense mutations, I27L and A98V, were also detected. Clinical and laboratory data obtained from patients were compared with the molecular findings, including the I27L polymorphism that was revealed in some overweight/obese diabetic patients of this study, this corroborating with the literature. We found certain DNA variations that could explain the hyperglycemic phenotype of the patients.

Assessing the association of the HNF1A G319S variant with C-reactive protein in Aboriginal Canadians: a population-based epidemiological study

BackgroundC-reactive protein (CRP), a biomarker of inflammation, has been associated with increased risk of developing cardiovascular disease. Common variants of the hepatocyte nuclear factor 1A (HNF1A) gene encoding HNF-1α have been associated with plasma CRP in predominantly European Caucasian samples. HNF1A might therefore have an impact on vascular disease and diabetes risk that is mediated by CRP. In an Aboriginal Canadian population, a private polymorphism, HNF1A G319S, was associated with increased prevalence of type 2 diabetes. However, it has not been investigated whether this association is mediated by CRP. We aimed to investigate whether CRP was mediating the association between HNF1A G319S and type 2 diabetes in an Aboriginal Canadian population with a high prevalence of diabetes.MethodsA total of 718 individuals who participated in a diabetes prevalence and risk factor survey were included in the current analysis. Participants were genotyped for HNF1A G319S. Fasting plasma samples were analyzed for CRP. Fasting plasma glucose and a 75-g oral glucose tolerance test were obtained to determine type 2 diabetes.ResultsThe prevalence rate of type 2 diabetes was 17.4% (125/718) using the 1999 World Health Organization definition and was higher among S319 allele carriers compared to G/G homozygotes (p < 0.0001). Among participants without type 2 diabetes, CRP levels were higher among G/G homozygotes (1.64 [95% confidence interval 1.35-2.00] mg/l) than in S319 carriers (1.26 [1.04-1.54] mg/l) (p = 0.009) after adjustment for age, sex, 2-h post-load glucose, waist circumference, and serum amyloid A. CRP levels were elevated among those with diabetes after similar adjustment (4.39 [95% confidence interval 3.09-6.23] and 4.44 [3.13-6.30] mg/L, respectively), and no significant difference in CRP was observed between S319 carriers and non-carriers (p = 0.95).ConclusionsCRP levels were lower in S319 allele carriers of the HNF1A gene compared to non-carriers among individuals without diabetes, but this difference was not present among those with diabetes, who uniformly had elevated CRP levels. Therefore, while HNF1A appears to influence CRP concentrations in the non-diabetic state, chronic elevation of CRP is unlikely mediating the association between the HNF1A polymorphism and the high prevalence of type 2 diabetes in this Aboriginal population.

I27L Polymorphism in hepatocyte nuclear factor-1α gene and type 2 diabetes mellitus: A meta-analysis of studies about orient population (Chinese and Japanese)

AimsThe aim of the study was to investigate the relationship between I27L variant of HNF-1α gene and type 2 diabetes mellitus (T2DM) in an/the oriental population. MethodsWe recruited 149 T2DM patients and 96 non-diabetes controls from China. The I27L polymorphism in HNF-1α gene was detected by PCR–RFLP analysis. A mete-analysis of previously published studies on I27L and T2DM of orient population and our new study was performed. Databases of MEDLINE, CBM, and the Cochrane Library (CD-ROM) were electronically searched from January 1980 to April 2008. Analysis was performed by RevMan 4.2 software which was downloaded from website of Cochrane collaboration. Results(1). The genotype distribution of I27L/exon1 polymorphism in the HNF-1α gene was in Hardy–Weinberg equilibrium (χ2=2.34, 0.05<P<0.1). The IL, LL genotype frequencies and L allelic frequency were slightly higher in T2DM group than in controls (0.52, 0.14 and 0.40 in T2DM vs. 0.49, 0.08 and 0.33 in controls), but the difference were not statistically significant, which indicated that 27L variant did not increase the risk of T2DM in our small sample Chinese population. (2). Three published studies concerning the Chinese population, two studies involving the Japanese population and our present study, providing information on a total of 1225 unique subjects, were included in the meta-analysis. The results showed that the 27L variant increased the prevalence of T2DM (OR 1.22, 95% CI 1.03–1.44, p=0.02). ConclusionI27L polymorphism in the HNF-1α gene increases the risk of T2DM in the orient population (Chinese and Japanese).

Identification of Novel Variants in the Hepatocyte Nuclear Factor-1α Gene in South Indian Patients with Maturity Onset Diabetes of Young

Mutations in the HNF 1A gene are the most common cause of maturity-onset diabetes of the young (MODY) in most populations. India currently has the largest number of people with diabetes in the world, and onset of type 2 diabetes occurs at a younger age with possible overlap with MODY. There are very few data on MODY mutations from India. The objective was to screen coding and promoter regions of HNF1A gene for mutations in unrelated South Indian subjects in whom a clinical diagnosis of MODY was made. This was an observational cross-sectional study. The study was conducted at a diabetes specialties centre in Chennai in southern India. Ninety-six unrelated south Indian subjects in whom clinical diagnosis of MODY was made were included in the study. The control population comprised of 57 unrelated nondiabetic subjects selected from the Chennai Urban Rural Epidemiology Study, a study conducted on a representative population (aged > or =20 yr) of Chennai. We identified nine novel variants comprising seven mutations (one novel mutation -538G>C at promoter region and six novel coding region mutations) and two polymorphisms in the HNF1A gene. Functional studies revealed reduced transcriptional activity of the HNF1A promoter for two promoter variants. We also observed cosegregation with diabetes of the Arg263His coding region mutation in eight members of one MODY family, whereas it was absent in nondiabetic subjects of this family. This study suggests that mutations in the HNF1A gene comprise about 9% of clinically diagnosed MODY subjects in southern India and a novel Arg263His mutation cosegregates with MODY in one family.

Differentiation therapy of hepatocellular carcinoma in mice with recombinant adenovirus carrying hepatocyte nuclear factor-4α gene

Previous studies have shown that hepatocyte nuclear factor-4alpha (HNF4alpha) is a central regulator of differentiated hepatocyte phenotype and forced expression of HNF4alpha could promote reversion of tumors toward a less invasive phenotype. However, the effect of HNF4alpha on cancer stem cells (CSCs) and the treatment of hepatocellular carcinoma (HCC) with HNF4alpha have not been reported. In this study, an adenovirus-mediated gene delivery system, which could efficiently transfer and express HNF4alpha, was generated to determine its effect on hepatoma cells (Hep3B and HepG2) in vitro and investigate the anti-tumor effect of HNF4alpha in mice. Our results demonstrated that forced re-expression of HNF4alpha induced the differentiation of hepatoma cells into hepatocytes, dramatically decreased "stemness" gene expression and the percentage of CD133(+) and CD90(+) cells, which are considered as cancer stem cells in HCC. Meanwhile, HNF4alpha reduced cell viability through inducing apparent apoptosis in Hep3B, while it induced cell cycle arrest and cellular senescence in HepG2. Moreover, infection of hepatoma cells by HNF4alpha abolished their tumorigenesis in mice. Most interestingly, systemic administration of adenovirus carrying the HNF4alpha gene protected mice from liver metastatic tumor formation, and intratumoral injection of HNF4alpha also displayed significant antitumor effects on transplanted tumor models. The striking suppression effect of HNF4alpha on tumorigenesis and tumor development is attained by inducing the differentiation of hepatoma cells--especially CSCs--into mature hepatocytes, suggesting that differentiation therapy with HNF4alpha may be an effective treatment for HCC patients. Our study also implies that differentiation therapy may present as one of the best strategies for cancer treatment through the induction of cell differentiation by key transcription factors.

Expression of HNF-4α (MODY1), HNF-1β (MODY5), and HNF-1α (MODY3) proteins in the developing mouse pancreas

The type 1, 3, and 5 forms of maturity-onset diabetes of the young (MODY) are caused by mutations of the genes encoding hepatocyte nuclear factor (HNF)-4α, HNF-1α, and HNF-1β, respectively [Yamagata, K., Oda, N., Kaisaki, P.J., Menzel, S., Furuta, H., Vaxillaire, M., et al., 1996a. Mutations in the hepatocyte nuclear factor-1α gene in maturity-onset diabetes of the young (MODY3). Nature 384, 455–458; Yamagata, K., Furuta, H., Oda, N., Kaisaki, P.J., Menzel, S., Cox, N.J., et al., 1996b. Mutations in the hepatocyte nuclear factor-4α gene in maturity-onset diabetes of the young (MODY1). Nature 384, 458–460; Horikawa, Y., Iwasaki, N., Hara, M., Furuta, H., Hinokio, Y., Cockburn, B.N. et al., 1997. Mutation in hepatocyte nuclear factor-1β gene (TCF2) associated with MODY. Nat. Genet. 17, 384–385]. Among these transcription factors, the pattern of HNF-4α expression during pancreatic differentiation remains largely unknown. We performed an immunohistochemical study to investigate its expression in comparison with the expression of HNF-1α and HNF-1β. We found considerable variation in the level of HNF-4α expression by the individual epithelial cells in the pancreatic buds on E9.5. HNF-4α and HNF-1β were initially expressed by Pdx1 + common progenitor cells and neurogenin3 + (Ngn3 +) endocrine precursor cells during the first transition, but expression of HNF-1β and either HNF-4α or HNF-1α became complementary around the end of the second transition (E15.5). In the mature pancreas, HNF-4α was expressed by glucagon-positive α-cells, insulin-positive β-cells, somatostatin-positive δ-cells, and pancreatic polypeptide-positive PP-cells, as well as by pancreatic exocrine cells and ductal cells. Most of the HNF-4α + cells were also positive for HNF-1α, but HNF-4α expression in some non-β-cells was remarkably high, and this was not paralleled by high HNF-1α expression. These results indicate that the expression of MODY proteins in each of the pancreatic cell types is strictly regulated in accordance with the status of differentiation during pancreatic organogenesis.

Association of genetic variations near P2 promoter of the hepatocyte nuclear factor-4α gene and insulin secretion index in Thais

This study was aimed to assess the association of the two single nucleotide polymorphisms (SNPs) near P2 promoter (rs1884614 and rs2144908) of hepatocyte nuclear factor-4alpha (HNF4A) with insulin secretion index and type 2 diabetes in Thais. Participants were categorized into three groups; unrelated type 2 diabetes (N = 219), prediabetes subjects (N = 228), and normal glucose tolerant controls (N = 203). Homeostasis model assessment was calculated for individual insulin secretion and insulin sensitivity index. Genotyping of both SNPs was done by allele-specific PCR technique. Difference of SNP allele frequencies between groups were computed using the chi (2)-statistic. Multivariate regression analysis was performed to determine the effect of SNPs on insulin secretion index. The clinical features of all groups were similar. We demonstrated genotype TT at rs1884614, BMI, and waist circumference were significantly associated with insulin secretion index (P = 0.023) but not with diabetes phenotype.

P2 Promoter Variants of the Hepatocyte Nuclear Factor 4α Gene Are Associated With Type 2 Diabetes in Mexican Americans

Common and rare variants of the hepatocyte nuclear factor 4alpha (HNF4A) gene have been associated with type 2 diabetes and related traits in several populations suggesting the involvement of this transcription factor in diabetes pathogenesis. Single nucleotide polymorphisms (SNPs) within a large haplotype block surrounding the alternate P2 promoter, located approximately 45 kb upstream from the coding region, have been investigated in several populations of varying ethnicity with inconsistent results. Additionally, SNPs located within the P1 promoter and coding region have also been inconsistently associated with type 2 diabetes. Characterization of variation across this gene region in Mexican-American populations has not been reported. We therefore examined polymorphisms across the HNF4A gene in a cohort of Mexican-American pedigrees and assessed their association with type 2 diabetes. We observed evidence for association of SNPs in the P2 promoter region with type 2 diabetes (P = 0.003) and its age at diagnosis (P = 0.003). The risk allele frequency (53%) was intermediate to that reported in Caucasian populations (20-27%) and Pima Indians (83%). No other SNPs were associated with either trait. These results support the possibility that a variant in the P2 promoter region of HNF4A, or variants in linkage disequilibrium within this region, contributes to susceptibility to type 2 diabetes in many ethnic populations including Mexican Americans.

The Production of a Diabetic Mouse Using Constructs Encoding Porcine Insulin Promoter-Driven Mutant Human Hepatocyte Nuclear Factor-1.ALPHA.

A diabetic mouse model was produced using a mutant human hepatocyte nuclear factor-1alpha gene (HNF1alphaP291fsinsC) regulated by the porcine insulin promoter. The functionality of two different constructs containing HNF1alphaP291fsinsC, termed PD1 and PD2 (cytomegalovirus enhancer minus and plus), were examined in transgenic mice. The blood glucose levels and body weights of the PD1 transgenic mice did not differ from their non-transgenic littermates over the period from 3 to 8 weeks of age. Conversely, the PD2 transgenic mice exhibited hyperglycemia and decreased body weight. Western blot analysis demonstrated that mutant HNF-1alpha protein (HNF1alphaP291), derived from the PD2 transgene, was expressed in the PD2 mice. Morphometric studies of the pancreas of a PD2 mouse revealed that the number of pancreatic islets present was less than that in the non-transgenic mice, indicating disturbed islet neogenesis. These results suggest that impaired insulin secretion in disrupted islets causes hyperglycemia. In addition, the phenotype of PD2 transgenic mice similar to that of the HNF-1alpha gene-deficient mouse, which displays growth retardation and impaired viability. These results indicate that HNF1alphaP291 expression driven by the porcine insulin promoter, together with the cytomegalovirus enhancer, induces a diabetic phenotype in transgenic mice.

Identification of four novel mutations in the HNF-1A gene in Chinese early-onset and/or multiplex diabetes pedigrees

Mutations in the hepatocyte nuclear factor-1A gene cause the type 3 form of maturity-onset diabetes of the young (MODY3). This study was undertaken to determine mutations and sequence variations of the HNF-1A gene in Chinese with familial early-onset and/or multiplex diabetes mellitus. We screened all ten exons of the HNF-1A gene, including exon/intron junctions, by direct sequencing in 272 unrelated Chinese, including 80 healthy controls and 192 probands of early-onset and/or multiplex diabetes pedigrees. In addition to one silent mutation of c.864 G > C [p.G288G] in exon4 at codon 288, which had been reported previously, a total of four novel mutations including two missense mutations (c.245C > T [p.T82M] and c. 390 G > T [p.Q130H]) and one frameshift mutation P353fsdelACGGGCCTGGAGC and one silent mutation c.759 G > T [p.G253G] were identified. Moreover, eleven substitutions were identified in 192 probands. Of these, three variants (-8 G > A, -128 T > G and IVS2 + 21 G > A) were not observed in 80 healthy controls and one of them (-8 G > A) was not reported previously and the two promoter variants co-segregated with diabetes. The genotype and allele frequencies of the other eight variants in the diabetic patients were not significantly different from those in the healthy controls. No significant relationships were observed between the eight variants of the HNF-1A gene and clinical variables (plasma glucose, insulin, C-peptide and fasting lipid profile). The prevalence of structural mutations in the HNF-1A gene responsible for familial early-onset and/or multiplex diabetes appears to be rare among Chinese patients.