Abnormal splicing of hepatocyte nuclear factor 1 alpha in maturity-onset diabetes of the young.

AIMS/HYPOTHESIS: Mutations in the HNF-1 alpha gene result in maturity-onset diabetes of the young (MODY); an early-onset, dominantly inherited form of diabetes caused by pancreatic beta-cell dysfunction. Splice site mutations represent approximately 10% of reported HNF-1 alpha mutations. No studies to date have investigated the effect of splice site mutations on mRNA processing because the tissues with abundant HNF-1alpha expression (liver, pancreas, kidney and gut) are not easily accessible for analysis. This study aimed to define the pathogenic mechanism in three novel splice site mutations by analysing illegitimate transcripts. METHODS: To assess the consequence of potential HNF-1 alpha splice site mutations we developed a nested reverse transcriptase PCR (RT-PCR) assay for the amplification of illegitimate HNF-1 alpha transcripts in Epstein Barr virus transformed lymphoblastoid cell lines. RESULTS: Sequencing the illegitimate HNF-1 alpha transcripts showed that the splice donor site mutation IVS8nt+1G>A leads to complete skipping of exon 8, the splice acceptor site mutation IVS4nt-2A>G causes skipping of exon 5 with the recruitment of a cryptic splice acceptor site within intron 5 and the cryptic splice acceptor site mutation (IVS7nt-6G>A) resulted in the skipping of exon 7. All three changes are predicted to result in premature termination of the HNF-1alpha protein, providing further evidence for their role as pathogenic mutations. CONCLUSION/INTERPRETATION: We conclude that the sequencing of illegitimate transcripts from lymphoblastoid cell lines is helpful in the assessment of intronic variation in HNF-1 alpha that could alter splicing. This analysis of the mRNA is required to define mutational mechanisms and confirm pathogenic status.

beta-cell genes and diabetes: quantitative and qualitative differences in the pathophysiology of hepatic nuclear factor-1alpha and glucokinase mutations.

Mutations in the beta-cell genes encoding the glycolytic enzyme glucokinase (GCK) and the transcription factor hepatocyte nuclear factor (HNF)-1alpha are the most common causes of maturity-onset diabetes of the young (MODY). Studying patients with mutations in these genes gives insights into the functions of these two critical beta-cell genes in humans. We studied 178 U.K. and French MODY family members, including 45 GCK mutation carriers and 40 HNF-1alpha mutation carriers. Homeostasis model assessment of fasting insulin and glucose showed reduced beta-cell function in both GCK (48% controls, P<0.0001) and HNF-1alpha (42% controls, P<0.0001). Insulin sensitivity was similar to that of control subjects in the GCK subjects (93% controls, P = 0.78) but increased in the HNF-1alpha subjects (134.5% controls, P = 0.005). The GCK patients showed a similar phenotype between and within families with mild lifelong fasting hyperglycemia (fasting plasma glucose [FPG] 5.5-9.2 mmol/l, interquartile [IQ] range 6.6-7.4), which declined slightly with age (0.017 mmol/l per year) and rarely required pharmacological treatment (17% oral hypoglycemic agents, 4% insulin). HNF-1alpha patients showed far greater variation in fasting glucose both between and within families (FPG 4.1-18.5 mmol/l, IQ range 5.45-10.4), with a marked deterioration with age (0.06 mmol/l per year), and 59% of patients required treatment with tablets or insulin. Proinsulin-to-insulin ratios are increased in HNF-1alpha subjects (29.5%) but not in GCK (18.5%) subjects. In an oral glucose tolerance test, the 0- to 120-min glucose increment was small in GCK patients (2.4+/-1.8 mmol/l) but large in HNF-1alpha patients (8.5+/-3.0 mmol/l, P< 0.0001). This comparison shows that the clear clinical differences in these two genetic subgroups of diabetes reflect the quantitative and qualitative differences in beta-cell dysfunction. The defect in GCK is a stable defect of glucose sensing, whereas the HNF-1alpha mutation causes a progressive defect that alters beta-cell insulin secretion directly rather than the sensing of glucose.

beta-cell genes and diabetes: molecular and clinical characterization of mutations in transcription factors.

beta-Cell transcription factor genes are important in the pathophysiology of the beta-cell, with mutations in hepatocyte nuclear factor (HNF)-1alpha, HNF-4alpha, insulin promoter factor (IPF)-1, HNF-1beta, and NeuroD1/BETA2, all resulting in early-onset type 2 diabetes. We assessed the relative contribution of these genes to early-onset type 2 diabetes using linkage and sequencing analysis in a cohort of 101 families (95% U.K. Caucasian). The relative distribution of the 90 families fitting maturity-onset diabetes of the young (MODY) criteria was 63% HNF-1alpha, 2% HNF-4alpha, 0% IPF-1, 1% HNF-1beta, 0% NeuroD1/ BETA2, and 20% glucokinase. We report the molecular genetic and clinical characteristics of these patients including 29 new families and 8 novel HNF-1alpha gene mutations. Mutations in the transactivation domain are more likely to be protein truncating rather than result in amino acid substitutions, suggesting that a relatively severe disruption of this domain is necessary to result in diabetes. Mutations in the different transcription factors result in clinical heterogeneity. IPF-1 mutations are associated with a higher age at diagnosis (42.7 years) than HNF-1alpha (20.4 years), HNF-1beta (24.2 years), or HNF-4alpha (26.3 years) gene mutations. Subjects with HNF-1beta mutations, in contrast to the other transcription factors, frequently present with renal disease. A comparison of age at diagnosis between subjects with different types and locations of HNF-1alpha mutations did not reveal genotype-phenotype correlations. In conclusion, mutations in transcription factors expressed in the beta-cell are the major cause of MODY, and the phenotype clearly varies with the gene that is mutated. There is little evidence to indicate that different mutations within the same gene have different phenotypes.

Mutations in the hepatocyte nuclear factor-1beta gene are associated with familial hypoplastic glomerulocystic kidney disease.

Familial glomerulocystic kidney disease (GCKD) is a dominantly inherited condition characterized by glomerular cysts and variable renal size and function; the molecular genetic etiology is unknown. Mutations in the gene encoding hepatocyte nuclear factor (HNF)-1beta have been associated with early-onset diabetes and nondiabetic renal disease-particularly renal cystic disease. We investigated a possible role for the HNF-1beta gene in four unrelated GCKD families and identified mutations in two families: a nonsense mutation in exon 1 (E101X) and a frameshift mutation in exon 2 (P159fsdelT). The family members with HNF-1beta gene mutations had hypoplastic GCKD and early-onset diabetes or impaired glucose tolerance. We conclude that there is genetic heterogeneity in familial GCKD and that the hypoplastic subtype is a part of the clinical spectrum of the renal cysts and diabetes syndrome that is associated with HNF-1beta mutations.

Proposed mechanism for a novel insertion/deletion frameshift mutation (I414G415ATCG-->CCA) in the hepatocyte nuclear factor 1 alpha (HNF-1 alpha) gene which causes maturity-onset diabetes of the young (MODY).

Maturity-onset diabetes of the young (MODY) is a monogenic subgroup of non-insulin dependent diabetes (NIDDM) characterized by an early age of diagnosis (usually < 25 years) and an autosomal dominant mode of inheritance. Mutations in the hepatocyte nuclear factor 1 alpha (HNF-1alpha) [MODY3] gene represent the most common cause of MODY in the UK and a common cause of MODY in many other populations. Sixty-three different mutations have been described in a total of 112 families worldwide. This report describes two families, not known to be related, who carry a novel insertion/deletion mutation (I414G415ATCG-->CCA) and a 6bp intronic deletion of the HNF-1alpha gene in cis. We propose that the insertion/deletion mutation has arisen by formation of a hairpin loop due to the presence of a quasi-palindromic sequence, followed by insertion of CC and deletion of TCG resulting in the increased stability of the hairpin loop.

Mutations in the human delta homologue, DLL3, cause axial skeletal defects in spondylocostal dysostosis.

Spondylocostal dysostosis (SD, MIM 277300) is a group of vertebral malsegmentation syndromes with reduced stature resulting from axial skeletal defects. SD is characterized by multiple hemivertebrae, rib fusions and deletions with a non-progressive kyphoscoliosis. Cases may be sporadic or familial, with both autosomal dominant and autosomal recessive modes of inheritance reported. Autosomal recessive SD maps to a 7.8-cM interval on chromosome 19q13.1-q13.3 that is homologous with a mouse region containing a gene encoding the Notch ligand delta-like 3 (Dll3). Dll3 is mutated in the X-ray-induced mouse mutant pudgy (pu), causing a variety of vertebrocostal defects similar to SD phenotypes. Here we have cloned and sequenced human DLL3 to evaluate it as a candidate gene for SD and identified mutations in three autosomal recessive SD families. Two of the mutations predict truncations within conserved extracellular domains. The third is a missense mutation in a highly conserved glycine residue of the fifth epidermal growth factor (EGF) repeat, which has revealed an important functional role for this domain. These represent the first mutations in a human Delta homologue, thus highlighting the critical role of the Notch signalling pathway and its components in patterning the mammalian axial

Missense mutations in the insulin promoter factor-1 gene predispose to type 2 diabetes.

The transcription factor insulin promoter factor-1 (IPF-1) plays a central role in both the development of the pancreas and the regulation of insulin gene expression in the mature pancreatic beta cell. A dominant-negative frameshift mutation in the IPF-l gene was identified in a single family and shown to cause pancreatic agenesis when homozygous and maturity-onset diabetes of the young (MODY) when heterozygous. We studied the role of IPF-1 in Caucasian diabetic and nondiabetic subjects from the United Kingdom. Three novel IPF-1 missense mutations (C18R, D76N, and R197H) were identified in patients with type 2 diabetes. Functional analyses of these mutations demonstrated decreased binding activity to the human insulin gene promoter and reduced activation of the insulin gene in response to hyperglycemia in the human beta-cell line Nes2y. These mutations are present in 1% of the population and predisposed the subject to type 2 diabetes with a relative risk of 3.0. They were not highly penetrant MODY mutations, as there were nondiabetic mutation carriers 25-53 years of age. We conclude that mutations in the IPF-1 gene may predispose to type 2 diabetes and are a rare cause of MODY and pancreatic agenesis, with the phenotype depending upon the severity of the mutation.

A gene for autosomal recessive spondylocostal dysostosis maps to 19q13.1-q13.3.

In spondylocostal dysostosis (SD), vertebral-segmentation defects are associated with rib anomalies. This results in short-trunk short stature, nonprogressive kyphoscoliosis, and radiological features of multiple hemivertebrae and rib fusions. SD can be familial, and both autosomal dominant and autosomal recessive (AR) inheritance have been reported, but no genes have been identified or localized for nonsyndromic SD in humans. We performed genomewide scanning by homozygosity mapping in a large consanguineous ARSD Arab Israeli family with six definitely affected members. Significant linkage was found to chromosome 19q13, with a LOD score of 6.9. This was confirmed in a second Pakistani family with three affected members, with a LOD score of 2.4. The combined-haplotype data identify a critical region between D19S570 and D19S908, an interval of 8.5 cM on 19q13.1-19q13.3. This is the first study to localize a gene for nonsyndromic SD. ARSD is clinically heterogeneous and is likely to result from mutations in developmental genes or from regulating transcription factors. Identification of these genes will improve the understanding of the molecular processes contributing to both normal and abnormal human vertebral development.

Loss of HNF1alpha function in human renal cell carcinoma: frequent mutations in the VHL gene but not the HNF1alpha gene.

Human renal cell carcinoma (RCC) is a common malignant disease of the kidney characterized by dedifferentiation of renal epithelial cells. Our previous experiments showed that most RCCs have a loss of function of the tissue-specific transcription factor hepatocyte nuclear factor (HNF) 1alpha. Detailed analyses of the 10 exons encoding HNF1alpha in 32 human RCCs by single-strand conformation polymorphism analysis and direct DNA sequencing revealed no tumor-associated mutation, whereas with the same probes we frequently found mutations in the von Hippel-Lindau tumor suppressor gene. No mutation leading to loss of HNF1alpha function was detected by analyzing the integrity of the HNF1alpha transcripts in the RNA derived from RCCs by the protein truncation test. Investigating human RCC cell lines by western blotting and gel retardation assays showed a dramatic loss in the expression of the tissue-specific transcription factor HNF1alpha in eight of 10 cell lines. As the HNF1alpha-related transcription factor HNF1beta was expressed in all these tumor cell lines, the loss of HNF1alpha expression was a specific event and was maintained in RCC cell lines. The loss of HNF1alpha expression in RCC cell lines on the RNA level was confirmed by reverse transcription polymerase chain reaction. We propose that tumor-associated mutations in the HNF1alpha gene do not occur in human RCC and that the loss of function is partially due to a transcriptional inactivation of the HNF1alpha gene.

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.

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-1 alpha) 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-1 alpha 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 > or = 1 affected relative), for the presence of 9 known HNF-1 alpha 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-1 alpha 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%.