Immunoepidemiology of Dracunculus medinensis infections II. Variation in antibody responses in relation to transmission season and patency

Recent studies have shown that mutations in the hepatocyte nuclear factor (HNF)-4alpha gene give rise to maturity-onset diabetes of the young, type 1 (MODY1). HNF-4, an orphan member of the nuclear receptor superfamily, contains a DNA-binding domain (DBD) and a putative ligand-binding domain (LBD) that can act independently of each other. The first MODY1 mutation identified creates a stop codon at amino acid 268 in the LBD of HNF-4 (Q268X) that leaves the DBD intact, suggesting that the mutant protein may retain some of the properties of the wild-type protein. To determine the functional properties of this mutant, we constructed HNF4.Q268X and tested it in vitro and in vivo for DNA binding, protein dimerization, and transactivation activity. Results of an electrophoretic mobility shift assay showed that HNF4.Q268X neither binds DNA alone nor binds it as a dimer with wild-type HNF-4 (HNF4.wt). In contrast, a co-immunoprecipitation assay showed that HNF4.Q268X is capable of dimerizing in solution with HNF4.wt. Transient transfection assays, however, indicated that HNF4.Q268X does not affect transactivation by HNF4.wt in vivo, supporting the argument against a dominant negative effect. Additional results suggest that the lack of a dominant negative effect could be due to a striking differential subcellular localization of the HNF4.Q268X protein: HNF4.Q268X could be extracted from transfected cells only when treated with SDS. Taken together, our results suggest that the MODY1 phenotype is due to a loss of functional HNF-4 protein that is aggravated in tissues that express relatively low amounts of HNF-4, such as pancreas.     

A novel Phe75fsdelT mutation in the hepatocyte nuclear factor-4alpha gene in a Danish pedigree with maturity-onset diabetes of the young

Mutations in 5 different genes [the hepatocyte nuclear factor (HNF)-4alpha), glucokinase, HNF-1alpha, insulin promoter factor-1, and HNF-1beta genes] have been shown to cause maturity onset diabetes of the young (MODY). About 50% of all known MODY in Danish Caucasian MODY probands can be explained by mutations in the HNF-1alpha gene (MODY3). To estimate the prevalence of MODY caused by mutations in the HNF-4alpha gene (MODY1), we screened 10 non-MODY3 probands for mutations in the minimal promoter and the 12 exons of the HNF-4alpha gene. One of the probands had a novel frameshift mutation (Phe75fsdelT) in exon 2 of the HNF-4alpha gene, resulting in a premature termination of translation after 117 amino acids of the messenger RNA encoded by that allele. The mutation cosegregated with diabetes in the pedigree and was not detected in 84 unrelated Danish Caucasian healthy glucose-tolerant control subjects or in 84 type 2 diabetic patients. At the time of examination, 4 of 6 mutation carriers were treated with insulin and 2 with oral hypoglycemic medication. Two mutation carriers had late-diabetic complications. Even though the HNF-4alpha protein is known to be important in the regulation of genes involved in lipid metabolism, carriers of the mutation did not differ from age and sex-matched control subjects, in regard to levels of fasting serum total cholesterol, serum high-density lipoprotein-cholesterol, and serum triglyceride. In conclusion, by screening 10 non-MODY3 probands for mutations in the HNF-4alpha gene, we identified 1 diabetes-associated frameshift mutation (Phe75fsdelT), suggesting that defects in HNF-4alpha are a rare cause of MODY in Denmark.     

Mutations in the hepatocyte nuclear factor-1alpha gene in Caucasian families originally classified as having Type I diabetes

Mutations in the hepatocyte nuclear factor-1alpha (HNF-1alpha) gene are the cause of maturity-onset diabetes of the young type 3 (MODY3), which is characterised by a severe impairment of insulin secretion and an early onset of the disease. Also at onset of diabetes some MODY patients show similar clinical symptoms and signs as patients with Type I (insulin-dependent) diabetes mellitus. The objective of this study was to estimate the prevalence of MODY3 patients misclassified as Type I diabetic patients. From a large population-based sample of unrelated Danish Caucasian Type I diabetic patients with an affected first degree relative, 39 patients (6.7%) who did not carry any high-risk HLA-haplotypes, i.e. DR3 or DR4 or both were examined by single-strand conformational polymorphism scanning and direct sequencing of the coding region and the minimal promoter of the HNF-1alpha gene. Four of the 39 Type I diabetic patients (10%) were identified as carrying mutations in the HNF-1alpha gene. One patient carried a missense mutation (Glu48Lys) in exon 1, two patients carried a missense mutation (Cys241Gly) in exon 4 and one patient carried a frameshift mutation (Pro291fsdelA) in exon 4. The mutations were all identified in heterozygous form, segregated with diabetes, and were not identified in 84 unrelated, healthy subjects. Furthermore, family history in three of the four families showed diabetes in four consecutive generations, suggestive of an autosomal dominant inheritance. In conclusion, about 10% of Danish diabetic patients without a high-risk HLA-haplotype, originally classified as having Type I diabetes could have diabetes caused by mutations in the HNF-1alpha gene. Clinical awareness of family history of diabetes and mode of inheritance might help to identify and reclassify these diabetic subjects as MODY3 patients.     

A missense mutation in the hepatocyte nuclear factor 4 alpha gene in a UK pedigree with maturity-onset diabetes of the young

Maturity-onset diabetes of the young (MODY) is a monogenic subgroup of non-insulin dependent diabetes mellitus (NIDDM) characterised bylan early age of onset (< 25 years) and an autosomal dominant mode of inheritance. MODY is genetically heterogeneous with three different genes identified to date; hepatocyte nuclear factor 4 alpha (HNF-4 alpha) [MODY1], glucokinase [MODY2] and hepatocyte nuclear factor 1 alpha (HNF-1 alpha) [MODY3]. A nonsense mutation in the HNF-4 alpha gene has recently been shown to cause MODY in a single large North American pedigree (RW). We screened a large UK Caucasian MODY family which showed weak evidence of linkage to the MODY1 locus on chromosome 20q (lod score for ADA 0.68 at theta = 0) for mutations in the coding region of the HNF-4 alpha gene by direct sequencing. A missense mutation resulting in the substitution of glutamine for glutamic acid was identified in exon 7 (E276Q). The mutation was present in all of the diabetic members of the pedigree plus two unaffected subjects and was not detected in 75 normal control subjects or 95 UK Caucasian subjects with late-onset NIDDM. This is the first missense mutation to be described in the HNF-4 alpha gene.     

Swiss journey through the clinical and genetic characteristics of diabetes in young patients

The aim of this study is to understand better the genetic causes of type II diabetes and the phenotypic consequences of the genetic changes. We first investigated the relative prevalence of the different forms of diabetes in young adults and their clinical features. 51 non-obese patients were identified in whom diabetes had been diagnosed before age 40; cases of typical insulin-dependent type I diabetes were excluded. A search for mutations of the glucokinase and HNF-1 alpha genes and for mitochondrial DNA was made, anti-islet and anti-GAD antibodies were determined and HLA class II genotyping was performed. Patients were subdivided on clinical grounds into a MODY (maturity onset diabetes of the young) group (n = 19) and a non-MODY group (n = 32). MODY is a form of diabetes which has an autosomal dominant inheritance for which 3 genes have already been implicated (MODY1, HNF-4 gene; MODY2, glucokinase gene, and MODY3, HNF-1 alpha gene). In the MODY group we identified 3 patients with MODY2, 1 with MODY3, 1 with the 3243 mitochondrial mutation and a further patient with autoimmune diabetes. In the non-MODY group we found 5 patients with autoimmune diabetes and 1 with MODY2. No clinical parameter was helpful in classifying patients in one of these subclasses of diabetes; however, glucagon stimulated C-peptide was useful in discriminating between MODY2 patients and the others. Young and lean non-insulin-dependent diabetic patients thus constitute a very heterogeneous group, though presenting similar clinical features. In the second study we analyzed hepatic glucose metabolism in patients with a mutation of the glucokinase gene expressed in both liver and islet beta cells. We found that endogenous glucose production is inadequately inhibited by hyperglycemia, a fact which contributes to the pathogenesis of hyperglycemia in these patients.     

Heterodimerization preferences of thyroid hormone receptor alpha isoforms

Thyroid hormone receptors (TRs) are members of the steroid hormone receptor superfamily and are encoded by two different genes, alpha and beta. Three isoforms (alpha 1, alpha 2, and alpha 3) are created by alternative splicing of the TR alpha gene. In TR alpha 2 and alpha 3, the distal half of the putative dimerization domain is disrupted and the carboxy terminus of the protein is substituted with different amino acids. To evaluate the properties of these alterations in the dimerization region, DNA binding and dimerization of TR alpha isoforms were studied by electrophoretic mobility shift assays. TR alpha 1 formed a monomer or a homodimer on certain thyroid hormone responsive elements (TREs), whereas TR alpha 2 and alpha 3 did not bind effectively to any of the TREs studied. TR alpha 1 formed a heterodimer with 9-cis retinoic acid receptor alpha (RXR alpha) on all TREs studied. Although TR alpha 2 did not bind as a homodimer, it did bind as a heterodimer with RXR alpha to DR4 and MHC-TRE. TR alpha 3 bound as a heterodimer to a broader repertoire of TREs, including DR4, MHC, ME, and F2-TRE. These results indicate that the alterations in the dimerization region in TR alpha 2 and alpha 3 abrogated homodimer binding, but differentially affected heterodimerization with RXR alpha on various TREs.     

Dimerization specificity of P22 and 434 repressors is determined by multiple polypeptide segments

The repressor protein of bacteriophage P22 binds to DNA as a homodimer. This dimerization is absolutely required for DNA binding. Dimerization is mediated by interactions between amino acids in the carboxyl (C)-terminal domain. We have constructed a plasmid, p22CT-1, which directs the overproduction of just the C-terminal domain of the P22 repressor (P22CT-1). Addition of P22CT-1 to DNA-bound P22 repressor causes the dissociation of the complex. Cross-linking experiments show that P22CT-1 forms specific heterodimers with the intact P22 repressor protein, indicating that inhibition of P22 repressor DNA binding by P22CT-1 is mediated by the formation of DNA binding-inactive P22 repressor:P22CT-1 heterodimers. We have taken advantage of the highly conserved amino acid sequences within the C-terminal domains of the P22 and 434 repressors and have created chimeric proteins to help identify amino acid regions required for dimerization specificity. Our results indicate that the dimerization specificity region of these proteins is concentrated in three segments of amino acid sequence that are spread across the C-terminal domain of each of the two phage repressors. We also show that the set of amino acids that forms the cooperativity interface of the P22 repressor may be distinct from those that form its dimer interface. Furthermore, cooperativity studies of the wild-type and chimeric proteins suggest that the location of cooperativity interface in the 434 repressor may also be distinct from that of its dimerization interface. Interestingly, changes in the dimer interface decreases the ability of the 434 repressor to discriminate between its wild-type binding sites, O(R)1, O(R)2, and O(R)3. Since 434 repressor discrimination between these sites depends in large part on the ability of this protein to recognize sequence-specific differences in DNA structure and flexibility, this result indicates that the C-terminal domain is intimately involved in the recognition of sequence-dependent differences in DNA structure and flexibility.     

Mutations affecting the alpha subunit of Bordetella pertussis RNA polymerase suppress growth inhibition conferred by short C-terminal deletions of the response regulator BvgA

The effects of short deletions of the C terminus of the BvgA response regulator protein of the BvgAS two-component system were examined in Bordetella pertussis. When present as a single copy in the chromosome, deletions removing as few as two amino acids conferred a completely Bvg- phenotype. When provided in trans, on the broad-host-range plasmid pRK290, under the control of the native bvgAS promoter, deletions of two or three amino acids conferred a profound growth inhibition which was dependent on the integrity and activity of the wild-type chromosomal bvgAS locus. It is proposed that this phenotype was the result of an inappropriate interaction of the mutant BvgA protein with the RNA polymerase enzyme, specifically the alpha subunit. Mutant strains in which this growth inhibition was relieved were isolated and characterized. Although most of the suppressor mutations affected either the mutant plasmid copy or the wild-type chromosomal bvg locus, three mutations which affected the alpha subunit of B. pertussis RNA polymerase were also isolated. Two of these resulted in increased levels of the alpha subunit, and one caused a substitution of glycine for the aspartic acid residue at position 171, in the N-terminal domain. All three mutations also resulted in a differential phenotype in that expression of fha was essentially normal, but expression of ptx was greatly reduced.     

N- and O-glycosylation and phosphorylation of the bar secretion leader derived from the barrier protease of Saccharomyces cerevisiae

A secretion leader derived from a domain of the extracellular Barrier protease of the yeast Saccharomyces cerevisiae has been expressed in wild-type and in mnn1, mnn9, and mnn1 mnn9 glycosylation mutant strains of S. cerevisiae. Structural comparison of the extracellular leader by mass spectrometry, peptide mapping, and elementary analysis proved that all strains produced a heterogeneous, heavily glycosylated polypeptide of 161 amino acids with both N- and O-glycosylation and phosphorylation. All three potential Asn N-linked sites were glycosylated to some extent with the expected structures. Neither the different growth media used nor the glycosylation mutations had significant effect on O-glycosylation with respect to both site selectivity and size of the carbohydrate structures. All 33 Ser and 21 Thr residues in the polypeptide were glycosylated at least partially, with an average of more than 2 mannoses/site. Although the mnn1 mutation blocks addition of alpha 1,3-linked mannose, the bar secretion domain expressed in the mnn1 and mnn1 mnn9 transformants unexpectedly contained some O-linked structures with at least 4 mannoses/chain. These O-linked structures were as large as when the leader was expressed in the mnn9 and wild-type strains. The bar secretion domain also had a previously undocumented phosphorylated O-linked structure.     

Defective pancreatic beta-cell glycolytic signaling in hepatocyte nuclear factor-1alpha-deficient mice

Mutations in the hepatocyte nuclear factor-1alpha (HNF-1alpha) gene cause maturity onset diabetes of the young type 3, a form of type 2 diabetes mellitus. In mice lacking the HNF-1alpha gene, insulin secretion and intracellular calcium ([Ca2+]i) responses were impaired following stimulation with nutrient secretagogues such as glucose and glyceraldehyde but normal with non-nutrient stimuli such as potassium chloride. Patch clamp recordings revealed ATP-sensitive K+ currents (KATP) in beta-cells that were insensitive to suppression by glucose but normally sensitive to ATP. Exposure to mitochondrial substrates suppressed KATP, elevated [Ca2+]i, and corrected the insulin secretion defect. NAD(P)H responses to glucose were substantially reduced, and inhibitors of glycolytic NADH generation reproduced the mutant phenotype in normal islets. Flux of glucose through glycolysis in islets from mutant mice was reduced, as a result of which ATP generation in response to glucose was impaired. We conclude that hepatocyte nuclear factor-1alpha diabetes results from defective beta-cell glycolytic signaling, which is potentially correctable using substrates that bypass the defect.