A rapid screening method for hepatocyte nuclear factor 1 alpha frameshift mutations; prevalence in maturity-onset diabetes of the young and late-onset non-insulin dependent diabetes

Non-insulin dependent diabetes (NIDDM) is a polygenic heterogeneous disorder of glucose homeostasis. Maturity-onset diabetes of the young (MODY) is a monogenic subtype of NIDDM characterised by early-onset (< 25 years) and autosomal dominant inheritance. Mutations in the hepatocyte nuclear factor 1 alpha (HNF-1α) gene have recently been shown to cause MODY. The incidence of mutations in this gene in MODY and late-onset NIDDM is not known. We have developed a rapid specific polymerase chain reaction test for HNF-1α mutations; this test involves the use of fluorescently labelled forward primers and modified reverse primers to detect length polymorphisms resulting from frameshift mutations. With this method, we have screened 102 MODY probands, viz. 60 defined according to strict diagnostic criteria (autosomal dominant inheritance and at least one member diagnosed age < 25 years) and 95 late-onset NIDDM probands (diagnosed 35–70 years with ≥ 1 affected relative), for the presence of 9 known HNF-1α frameshift mutations, including 6 that occur at two sites for recurring mutation (residues 291/292 and 379). Mutations were detected in 11 of the strictly defined MODY probands and one mutation was also found in a single subject with early-onset NIDDM but no family history of the disease. The HNF-1α frameshift mutations were not detected in any late-onset NIDDM subjects, suggesting these mutations do not have a major role in the pathogenesis of NIDDM. Our results indicate that the prevalence of the nine frameshift mutations in strictly defined UK MODY is 18%, with the P291fsinsC mutation alone having a frequency of 13%. Received: 13 May 1997 / Accepted: 13 August 1997     

Functional characterization of two novel splicing mutations of glucokinase gene associated with maturity-onset diabetes of the young type 2 (MODY2)

Two novel mutations in the glucokinase gene (GCK) have been identified in patients with maturity-onset diabetes of the young type-2 (MODY2), i.e., a C-for-G substitution at position ?1 of the acceptor splice site of intron 7 (c. 864-1G>C) and a synonymous c.666C>G substitution (GTC>GTG, p.V222V) at exon 6. An analysis of the splicing products obtained upon the transfection of human embryonic HEK293 cells with GCK minigene constructs carrying these mutations showed that both substitutions impaired normal splicing. As a result of c.864-1G>C, the usage of the normal acceptor site was blocked, which activated cryptic acceptor splice sites within intron 7 and generated several aberrant RNAs containing fragments of intron 7. The synonymous substitution c.666C>G created a novel donor splice site in exon 6, which results in the formation of an abnormal GCK mRNA with a 16-nucleotide deletion in exon 6. In vitro experiments on minigene splicing confirmed the inactivating effect of these mutations on glucokinase gene expression.     

Characterization of glucokinase polymorphisms associated with Maturity-Onset Diabetes of the Young (MODY2) in Jordanian population

Maturity-Onset Diabetes of the Young (MODY) is a monogenic form of Diabetes Mellitus (DM) characterized by an autosomal dominant inheritance, onset usually before 25 years of age and a primary defect in glucose-stimulated insulin secretion, Glucokinase (GCK) acts as a glucose sensor in the pancreatic beta cell and regulates insulin secretion. The mutation in the gene encoding GCK results in enzyme inactivation cause MODY2. Functional studies of naturally occurring GCK mutations associated with hyperglycaemia provide further insight into the biochemical basis of glucose sensor regulation. In this study 100 diabetic Jordanian patients with MODY2 phenotype and 150 Normal control subjects were screened for the presence of GCK gene mutations including the missense mutations at position Thr228Ala in exon 7, Gly299Arg in exon 8 and nonsense mutation Ser383Ter in exon 9, utilizing polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) analysis. The results shows no Thr228Ala, Gly299Arg and Ser383Ter mutations were detected in both groups, which was differ from the results obtained for Italian and Caucasian from the Oxford region in UK MODY2 patients. Our data indicated that the previously studied mutations in Italian and Caucasian patients in the GCK gene are not common in MODY Jordanian population, suggesting a racial difference can be found in the frequency of the GCK polymorphism.     

Screening of mutations and polymorphisms in the glucokinase gene in Czech diabetic and healthy control populations

Glucokinase (GCK) plays a key role in glucose metabolism. GCK mutations are known as a pathogenic cause of maturity-onset diabetes of the young type 2 (MODY2). These mutations are also found in gestational diabetics. The aim of our study was to assess the variability of the GCK gene in the Czech diabetic and control populations. We screened all 10 exons specific for the pancreatic isoform of glucokinase (1a and 2-10) including the intron flanking regions in 722 subjects (in 12 patients with an unrecognised type of MODY and their 10 family members, 313 patients with diabetes mellitus type 2 (DM2), 141 gestational diabetics (GDM), 130 healthy offspring of diabetic parents, and 116 healthy controls without family history of DM2). In two MODY families we identified two mutations in exon 2 of the GCK gene: a novel mutation Val33Ala and the previously described mutation Glu40Lys. In other subgroups (excluding MODY families) we detected only intronic variants and previously described polymorphisms in exons 6 (Tyr215Tyr) and 7 (Ser263Ser), we did not find any known GCK pathogenic mutation. We observed no difference in the frequencies of GCK polymorphisms between Czech diabetic (DM2, GDM) and non-diabetic populations.     

Mutant HNF-1alpha and mutant HNF-1beta identified in MODY3 and MODY5 downregulate DPP-IV gene expression in Caco-2 cells

Dipeptidylpeptidase IV (DPP-IV) is a well-documented drug target for the treatment of type 2 diabetes. Hepatocyte nuclear factors (HNF)-1alpha and HNF-1beta, known as the causal genes of MODY3 and MODY5, respectively, have been reported to be involved in regulation of DPP-IV gene expression. But, it is not completely clear (i) that they play roles in regulation of DPP-IV gene expression, and (ii) whether DPP-IV gene activity is changed by mutant HNF-1alpha and mutant HNF-1beta in MODY3 and MODY5. To explore these questions, we investigated transactivation effects of wild HNF-1alpha and 13 mutant HNF-1alpha, as well as wild HNF-1beta and 2 mutant HNF-1beta, on DPP-IV promoter luciferase gene in Caco-2 cells by means of a transient experiment. Both wild HNF-1alpha and wild HNF-1beta significantly transactivated DPP-IV promoter, but mutant HNF-1alpha and mutant HNF-1beta exhibited low transactivation activity. Moreover, to study whether mutant HNF-1alpha and mutant HNF-1beta change endogenous DPP-IV enzyme activity, we produced four stable cell lines from Caco-2 cells, in which wild HNF-1alpha or wild HNF-1beta, or else respective dominant-negative mutant HNF-1alphaT539fsdelC or dominant-negative mutant HNF-1betaR177X, was stably expressed. We found that DPP-IV gene expression and enzyme activity were significantly increased in wild HNF-1alpha cells and wild HNF-1beta cells, whereas they decreased in HNF-1alphaT539fsdelC cells and HNF-1betaR177X cells, compared with DPP-IV gene expression and enzyme activity in Caco-2 cells. These results suggest that both wild HNF-1alpha and wild HNF-1beta have a stimulatory effect on DPP-IV gene expression, but that mutant HNF-1alpha and mutant HNF-1beta attenuate the stimulatory effect.     

Incipient mitochondrial evolution in yeasts. II. The complete sequence of the gene coding for cytochrome b in Saccharomyces douglasii reveals the presence of both new and conserved introns and discloses major differences in the fixation of mutations in evolution.

We have determined the complete sequence of the mitochondrial gene coding for cytochrome b in Saccharomyces douglasii. The gene is 6310 base-pairs long and is interrupted by four introns. The first one (1311 base-pairs) belongs to the group ID of secondary structure, contains a fragment open reading frame with a characteristic GIY ... YIG motif, is absent from Saccharomyces cerevisiae and is inserted in the same site in which introns 1 and 2 are inserted in Neurospora crassa and Podospora anserina, respectively. The next three S. douglasii introns are homologous to the first three introns of S. cerevisiae, are inserted at the same positions and display various degrees of similarity ranging from an almost complete identity (intron 2 and 4) to a moderate one (intron 3). We have compared secondary structures of intron RNAs, and nucleotide and amino acid sequences of cytochrome b exons and intron open reading frames in the two Saccharomyces species. The rules that govern fixation of mutations in exon and intron open reading frames are different: the relative proportion of mutations occurring in synonymous codons is low in some introns and high in exons. The overall frequency of mutations in cytochrome b exons is much smaller than in nuclear genes of yeasts, contrary to what has been found in vertebrates, where mitochondrial mutations are more frequent. The divergence of the cytochrome b gene is modular: various parts of the gene have changed with a different mode and tempo of evolution.     

Liver-specific reactivation of the inactivated Hnf-1alpha gene: elimination of liver dysfunction to establish a mouse MODY3 model

Mice deficient in hepatocyte nuclear factor 1 alpha (HNF-1alpha) develop dwarfism, liver dysfunction, and type 2 diabetes mellitus. Liver dysfunction in HNF-1alpha-null mice includes severe hepatic glycogen accumulation and dyslipidemia. The liver dysfunction may appear as soon as 2 weeks after birth. Since the HNF-1alpha-null mice become diabetic 2 weeks after birth, the early onset of the liver dysfunction is unlikely to be due to the diabetic status of the mice. More likely, it is due directly to the deficiency of HNF-1alpha in liver. Although the HNF-1alpha-null mice have an average life span of 1 year, the severe liver phenotype has thwarted attempts to study the pathogenesis of maturity-onset diabetes of the young type 3 (MODY3) and to examine therapeutic strategies for diabetes prevention and treatment in these mice. To circumvent this problem, we have generated a new Hnf-1alpha mutant mouse line, Hnf-1alpha(kin/kin), using gene targeting to inactivate the Hnf-1alpha gene and at the same time, to incorporate the Cre-loxP DNA recombination system into the locus for later revival of the Hnf-1alpha gene in tissues by tissue-specifically expressed Cre recombinase. The Hnf-1alpha(kin/kin) mice in which the expression of HNF-1alpha was inactivated in germ line cells were indistinguishable from the HNF-1alpha-null mice with regard to both the diabetes and liver phenotypes. Intriguingly, when the inactivated Hnf-1alpha gene was revived in liver (hepatic Hnf-1alpha revived) by the Cre recombinase driven by an albumin promoter, the Hnf-1alpha(kin/kin) mice, although severely diabetic, grew normally and did not develop any of the liver dysfunctions. In addition, we showed that the expression of numerous genes in pancreas, including a marker gene for pancreas injury, was affected by liver dysfunction but not by the deficiency of HNF-1alpha in pancreas. Thus, our hepatic-Hnf-1alpha-revived mice may serve as a useful mouse model to study the human MODY3 disorder.     

Maturity-onset diabetes of the young

Maturity-onset diabetes of the young (MODY) is a subtype of noninsulin dependent diabetes mellitus (NIDDM). It is characterized by an early age of onset and autosomal dominant mode of inheritance. These features and the availability of large multigenerational pedigrees make MODY useful for genetic studies of diabetes. In the large, 5-generational RW pedigree, MODY is tightly linked to genetic markers on chromosome 20q. Affected subjects in this family show abnormalities of carbohydrate metabolism varying from impaired glucose tolerance (IGT) to severe diabetes. Approximately 30% of diabetic subjects become insulin requiring and vascular complications occur. MODY is also linked to the glucokinase gene on chromosome 7p and many different mutations associated with MODY have been identified in this gene. MODY due to mutations in the glucokinase gene is a relatively mild form of diabetes with mild fasting hyperglycemia and IGT in the majority. It is rarely insulin requiring and rarely has vascular complications. Clinical studies indicate that the genetic or primary defect in MODY is characterized by deranged and deficient insulin secretion and not by insulin resistance and that there are quantitative and qualitative differences in insulin secretory defects which differentiate subjects with MODY due to glucokinase mutations from those with mutations in the gene on chromosome 20q. These differences correlate with the severity of diabetes between these two genetic forms of MODY.