Whole-genome sequencing of bladder cancers reveals somatic CDKN1A mutations and clinicopathological associations with mutation burden.

Bladder cancers are a leading cause of death from malignancy. Molecular markers might predict disease progression and behaviour more accurately than the available prognostic factors. Here we use whole-genome sequencing to identify somatic mutations and chromosomal changes in 14 bladder cancers of different grades and stages. As well as detecting the known bladder cancer driver mutations, we report the identification of recurrent protein-inactivating mutations in CDKN1A and FAT1. The former are not mutually exclusive with TP53 mutations or MDM2 amplification, showing that CDKN1A dysfunction is not simply an alternative mechanism for p53 pathway inactivation. We find strong positive associations between higher tumour stage/grade and greater clonal diversity, the number of somatic mutations and the burden of copy number changes. In principle, the identification of sub-clones with greater diversity and/or mutation burden within early-stage or low-grade tumours could identify lesions with a high risk of invasive progression.

Global microRNA expression profiles in insulin target tissues in a spontaneous rat model of type 2 diabetes.

AIMS/HYPOTHESIS: MicroRNAs regulate a broad range of biological mechanisms. To investigate the relationship between microRNA expression and type 2 diabetes, we compared global microRNA expression in insulin target tissues from three inbred rat strains that differ in diabetes susceptibility. METHODS: Using microarrays, we measured the expression of 283 microRNAs in adipose, liver and muscle tissue from hyperglycaemic (Goto-Kakizaki), intermediate glycaemic (Wistar Kyoto) and normoglycaemic (Brown Norway) rats (n = 5 for each strain). Expression was compared across strains and validated using quantitative RT-PCR. Furthermore, microRNA expression variation in adipose tissue was investigated in 3T3-L1 adipocytes exposed to hyperglycaemic conditions. RESULTS: We found 29 significantly differentiated microRNAs (p(adjusted) < 0.05): nine in adipose tissue, 18 in liver and two in muscle. Of these, five microRNAs had expression patterns that correlated with the strain-specific glycaemic phenotype. MiR-222 (p(adjusted) = 0.0005) and miR-27a (p(adjusted) = 0.006) were upregulated in adipose tissue; miR-195 (p(adjusted) = 0.006) and miR-103 (p(adjusted) = 0.04) were upregulated in liver; and miR-10b (p(adjusted) = 0.004) was downregulated in muscle. Exposure of 3T3-L1 adipocytes to increased glucose concentration upregulated the expression of miR-222 (p = 0.008), miR-27a (p = 0.02) and the previously reported miR-29a (p = 0.02). Predicted target genes of these differentially expressed microRNAs are involved in pathways relevant to type 2 diabetes. CONCLUSION: The expression patterns of miR-222, miR-27a, miR-195, miR-103 and miR-10b varied with hyperglycaemia, suggesting a role for these microRNAs in the pathophysiology of type 2 diabetes, as modelled by the Gyoto-Kakizaki rat. We observed similar patterns of expression of miR-222, miR-27a and miR-29a in adipocytes as a response to increased glucose levels, which supports our hypothesis that altered expression of microRNAs accompanies primary events related to the pathogenesis of type 2 diabetes.

Enhanced insulin secretion and cholesterol metabolism in congenic strains of the spontaneously diabetic (Type 2) Goto Kakizaki rat are controlled by independent genetic loci in rat chromosome 8.

AIMS/HYPOTHESIS: Genetic investigations in the spontaneously diabetic (Type 2) Goto Kakizaki (GK) rat have identified quantitative trait loci (QTL) for diabetes-related phenotypes. The aims of this study were to refine the chromosomal mapping of a QTL ( Nidd/gk5) identified in chromosome 8 of the GK rat and to define a pathophysiological profile of GK gene variants underlying the QTL effects in congenics. METHODS: Genetic linkage analysis was carried out with chromosome 8 markers genotyped in a GKxBN F2 intercross previously used to map diabetes QTL. Two congenic strains were designed to contain GK haplotypes in the region of Nidd/gk5 transferred onto a Brown Norway (BN) genetic background, and a broad spectrum of diabetes phenotypes were characterised in the animals. RESULTS: Results from QTL mapping suggest that variations in glucose-stimulated insulin secretion in vivo, and in body weight are controlled by different chromosome 8 loci (LOD3.53; p=0.0004 and LOD4.19; p=0.00007, respectively). Extensive physiological screening in male and female congenics at 12 and 24 weeks revealed the existence of GK variants at the locus Nidd/gk5, independently responsible for significantly enhanced insulin secretion and increased levels of plasma triglycerides, phospholipids and HDL, LDL and total cholesterol. Sequence polymorphisms detected between the BN and GK strains in genes encoding ApoAI, AIV, CIII and Lipc do not account for these effects. CONCLUSIONS/INTERPRETATION: We refined the localisation of the QTL Nidd/gk5 and its pathophysiological characteristics in congenic strains derived for the locus. These congenic strains provide novel models for testing the contribution of a subset of GK alleles on diabetes phenotypes and for identifying diabetes susceptibility genes.

Localization, cDNA sequence and genomic organization of the rat seipin gene (Bscl2) and sequence analysis in inbred rat models of Type 2 diabetes mellitus.

Mutations in the gene encoding seipin cause Berardinelli-Seip congenital lipodystrophy 2, with symptoms including near-absence of adipose tissue and altered glucose tolerance. Radiation hybrid analysis localized the seipin gene (Bscl2) in rat to a major quantitative trait locus in rat chromosome 1 linked to glucose intolerance in the Goto-Kakizaki (GK) rat model of Type 2 diabetes. We determined the genomic organization of Bscl2 and screened coding exons and flanking intron sequences for mutations in GK, Wistar and Brown Norway rats, as well as in the Otsuka Long-Evans Tokushima Fatty (OLETF) diabetic rat. Two silent single nucleotide polymorphisms that were identified also were found in non-diabetic rat strains. We conclude that mutations in the gene for seipin are unlikely to contribute to diabetes in GK and OLETF rats.

A high-resolution consensus linkage map of the rat, integrating radiation hybrid and genetic maps.

We have constructed a high-resolution consensus genetic map of the rat in a single large intercross, which integrates 747 framework markers and 687 positions of our whole-genome radiation hybrid (RH) map of the rat. We selected 136 new gene markers from the GenBank database and assigned them either genetically or physically to rat chromosomes to evaluate the accuracy of the integrated linkage-RH maps in the localization of new markers and to enrich existing comparative mapping data. These markers and 631 D-Got- markers, which are physically mapped but still uncharacterized for evidence of polymorphism, were tested for allele variations in a panel of 16 rat strains commonly used in genetic studies. The consensus linkage map constructed in the GK x BN cross now comprises 1620 markers of various origins, defining 840 resolved genetic positions with an average spacing of 2.2 cM between adjacent loci, and includes 407 gene markers. This whole-genome genetic map will contribute to the advancement of genetic studies in the rat by incorporating gene/EST maps, physical mapping information, and sequence data generated in rat and other mammalian species into genetic intervals harboring disease susceptibility loci identified in rat models of human genetic disorders.

Detailed comparative gene map of rat chromosome 1 with mouse and human genomes and physical mapping of an evolutionary chromosomal breakpoint.

We report the localization of 92 new gene-based markers assigned to rat chromosome 1 by linkage or radiation hybrid mapping. The markers were chosen to enrich gene mapping data in a region of the rat chromosome known to contain several of the principal quantitative trait loci in rodent models of human multifactorial disease. The composite map reported here provides map information on a total of 139 known genes, including 80 that have been localized in mouse and 109 that have been localized in human, and integrates the gene-based markers with anonymous microsatellites. The evolutionary breakpoints identifying 16 segments that are homologous regions in the human genome are defined. These data will facilitate genetic and comparative mapping studies and identification of novel candidate genes for the quantitative trait loci that have been localized to the region.

A gene map of the rat derived from linkage analysis and related regions in the mouse and human genomes.

We report the localization by linkage analysis in the rat genome of 148 new markers derived from 128 distinct known gene sequences, ESTs, and anonymous sequences selected in GenBank database on the basis of the presence of a repeated element. The composite linkage map of the rat contributed by our group integrates mapping information on a total of 370 different known genes, ESTs, and anonymous mouse or human sequences, and provides a valuable tool for comparative genome analysis. 206 and 254 homologous loci were identified in the mouse and human genomes respectively. Our linkage map, which combines both anonymous markers and gene markers, should facilitate the advancement of genetic studies for a wide variety of rat models characterized for complete phenotypes. The comparative genome mapping should define genetic regions in human likely to be homologous to susceptibility loci identified in rat and provide useful information for the identification of new potential candidates for genetic disorders.

A low renal threshold for glucose in diabetic patients with a mutation in the hepatocyte nuclear factor-1alpha (HNF-1alpha) gene.

One form of maturity-onset diabetes of the young, Type 3 (MODY3), results from mutations in the gene coding for hepatocyte nuclear factor-1alpha (HNF-1alpha), a transcription factor first described in the liver. MODY3 is characterized by a defective glucose-stimulated insulin secretion. Earlier observations of glycosuria with normal blood glucose levels in some MODY families suggest an additional renal manifestation of the respective genetic defect. We measured the renal threshold for glucose in five diabetic carriers of a missense mutation (Arg 272 His) in HNF-1alpha and, for comparison, in eight Type 1 diabetic patients, applying a non-invasive protocol of frequent parallel blood and urine sampling during a slow shift in blood glucose levels. We found that the mean renal threshold for glucose was lowered in the HNF-1alpha diabetic patients compared to those with Type 1 diabetes (6.5 +/- 0.9 mmol l(-1) vs 10.7 +/- 0.5 mmol l(-1); p < 0.01). This lowered glucose threshold might be an indication of an extra-pancreatic effect of HNF-1alpha gene mutations in humans. Defects in HNF-1alpha may lead to an altered tubular glucose reabsorption, possibly due to decreased expression of the renal glucose transporter proteins involved in reabsorption of glucose from the urine.

Construction and characterization of a 10-fold genome equivalent rat P1-derived artificial chromosome library.

A rat PAC library was constructed in the vector pPAC4 from genomic DNA isolated from female Brown Norway rats. This library consists of 215,409 clones arrayed in 614,384-well microtiter plates. An average insert size of 143 kb was estimated from 217 randomly isolated clones, thus representing approximately 10-fold genome coverage. This coverage provides a very high probability that the library contains a unique sequence in genome screening. Tests on randomly selected clones demonstrated that they are very stable, with only 4 of 130 clones showing restriction digest fragment alterations after 80 generations of serial growth. FISH analysis using 70 randomly chosen PACs revealed no significant chimeric clones. About 7% of the clones analyzed contained repetitive sequences related to centromeric regions that hybridized to some but not all centromeres. DNA plate pools and superpools were made, and high-density filters each containing an array of 8 plates in duplicate were prepared. Library screening on these superpools and appropriate filters with 10 single-locus rat markers revealed an average of 8 positive clones, in agreement with the estimated high genomic coverage of this library and representation of the rat genome. This library provides a new resource for rat genome analysis, in particular the identification of genes involved in models of multifactorial disease. The library and high-density filters are currently available to the scientific community.

Maturity-onset diabetes of the young due to a mutation in the hepatocyte nuclear factor-4 alpha binding site in the promoter of the hepatocyte nuclear factor-1 alpha gene.

Recent studies have shown that mutations in the transcription factor hepatocyte nuclear factor (HNF)-1 alpha are the cause of one form of maturity-onset diabetes of the young (MODY3). These studies have identified mutations in the mRNA and protein coding regions of this gene that result in the synthesis of an abnormal mRNA or protein. Here, we report an Italian family in which an A-->C substitution at nucleotide-58 of the promoter region of the HNF-1 alpha gene cosegregates with MODY. This mutation is located in a highly conserved region of the promoter and disrupts the binding site for the transcription factor HNF-4 alpha, mutations in the gene encoding HNF-4 alpha being another cause of MODY (MODY1). This result demonstrates that decreased levels of HNF-1 alpha per se can cause MODY. Moreover, it indicates that both the promoter and coding regions of the HNF-1 alpha gene should be screened for mutations in subjects thought to have MODY because of mutations in this gene.

Identification of nine novel mutations in the hepatocyte nuclear factor 1 alpha gene associated with maturity-onset diabetes of the young (MODY3).

Maturity-onset diabetes of the young (MODY) is a genetically heterogeneous subtype of non-insulin-dependent diabetes mellitus (NIDDM) characterised by early onset, autosomal dominant inheritance and a primary defect in insulin secretion. Recent studies have shown that mutations in the two functionally related transcription factors, hepatocyte nuclear factor 4 alpha (HNF-4alpha) and hepatocyte nuclear factor 1 alpha (HNF-1alpha) are associated with the MODY1 and MODY3 forms of diabetes respectively, whereas mutations in the enzyme glucokinase are the cause of the MODY2 form. We have examined 10 unrelated Caucasian families in which MODY/NIDDM co-segregated with markers for MODY3 for mutations in the HNF-1alpha gene (TCF1). Ten different mutations were observed in these families, all of which co-segregated with diabetes. There were no obvious relationships between the nature of the mutations observed (i.e. frameshift, nonsense, or missense) or their location in the gene with clinical features of diabetes (age at onset, severity) in these families. The mechanisms by which mutations in the HNF-1alpha gene cause diabetes mellitus are unclear but might include abnormal pancreatic islet development during foetal life thereby limiting their later function, as well as impaired transcriptional regulation of genes that play a key role in normal pancreatic beta cell function.

Genetic variation in the hepatocyte nuclear factor-1 alpha gene in Danish Caucasians with late-onset NIDDM.

Non-insulin-dependent diabetes mellitus (NIDDM) is a phenotypically and genetically heterogeneous disorder. A recent random genome mapping study has localized a locus termed NIDDM2 that maps to the region of chromosome 12 that includes MODY3, one of the three genes responsible for maturity-onset diabetes of the young, a monogenic form of NIDDM characterized by early age of onset and autosomal dominant inheritance. These findings suggest that NIDDM2 and MODY3 may represent different alleles of the same gene. MODY3 has recently been shown to be the gene encoding the transcription factor hepatocyte nuclear factor-1 alpha (HNF-1 alpha) thereby allowing us to determine whether mutations in the HNF-1 alpha gene are present in subjects with late-onset NIDDM. We screened 84 white NIDDM patients of Danish ancestry and found four nucleotide substitutions that changed the sequence of HNF-1 alpha, Ile27-->Leu, Ala98-->Val, Ser487-->Asn and Arg583-->Gln, five nucleotide substitutions that were silent and did not change the amino acid, Leu17, Gly288, Leu459 and Thr515, and five substitutions in the intron regions. The frequencies of the codon 27, 98 and 487 amino acid variants were similar in 245 unrelated NIDDM patients and 242 age-matched control subjects. The Arg583-->Gln mutation was found in 2 of 245 NIDDM patients and in none of the control subjects. Thus, genetic variation in the HNF-1 alpha gene is not a common factor contributing to NIDDM susceptibility in white subjects of Danish ancestry.

Mutations in the hepatocyte nuclear factor-1alpha gene are a common cause of maturity-onset diabetes of the young in the U.K.

Mutations in the hepatocyte nuclear factor-1alpha (HNF1alpha) gene have recently been shown to cause maturity-onset diabetes of the young (MODY). We have examined 15 U.K. MODY families for mutations in the coding region of the HNF-1alpha gene. Eight different mutations, three frameshift (P291fsinsC, P379fsdelCT, and A443fsdelCA) and five missense mutations (P129T, R131W, R159W, P519L, and T620I), were identified in eleven families (73%). The previously reported mutation P291fsinsC was found in four pedigrees. A screen of a further 32 probands with early onset (<40 years of age) NIDDM showed the mutation in two additional families. This common mutation was present on at least three different haplotypes, suggesting that its high frequency is due to recurrent mutation rather than a founder effect. We have demonstrated that mutations in the HNF-1alpha gene are a common cause of MODY in U.K. families and result in early onset NIDDM with a progressive clinical course. Mutation-based genetic counseling can now be considered for the majority of patients with MODY.

Mutations in the hepatocyte nuclear factor-1alpha gene in MODY and early-onset NIDDM: evidence for a mutational hotspot in exon 4.

We have recently shown that mutations in the gene encoding the transcription factor hepatocyte nuclear factor (HNF)-1alpha are the cause of one form of maturity-onset diabetes of the young (MODY3). Here, we report the exon-intron organization and partial sequence of the human HNF-1alpha gene. In addition, we have screened the ten exons and flanking introns of this gene for mutations in a group of 25 unrelated white subjects from Germany who presented with NIDDM before 35 years of age and had a first-degree relative with NIDDM. Mutations were identified in nine of these individuals, suggesting that mutations in the HNF-1alpha gene are a common cause of diabetes in German subjects with early-onset NIDDM and a family history of diabetes. Thus, screening for mutations in this gene may be indicated in subjects with early-onset NIDDM. Interestingly, three of the nine mutations occurred at the same site in exon 4 with insertion of a C in a polyC tract, centered around codon 290 (designated Pro291fsinsC), thereby resulting in a frameshift during translation and premature termination. Analyses of linked DNA polymorphisms in the HNF-1alpha gene indicated that the Pro291fsinsC mutation was present on a different haplotype in each subject, implying that the polyC tract represents a mutational hot spot. We have also identified the mutation in the HNF-1alpha gene in the Jutland pedigree, one of the original MODY pedigrees reported in the literature, as being a T-->G substitution in codon 241, resulting in the replacement of a conserved Cys by Gly (C241G). The information on the sequence of the HNF-1alpha gene and its promoter region will facilitate the search for mutations in other subjects and studies of the role of the gene in determining normal beta-cell functions.

Mutations in the hepatocyte nuclear factor-1alpha gene in maturity-onset diabetes of the young (MODY3)

The disease non-insulin-dependent (type 2) diabetes mellitus (NIDDM) is characterized by abnormally high blood glucose resulting from a relative deficiency of insulin. It affects about 2% of the world's population and treatment of diabetes and its complications are an increasing health-care burden. Genetic factors are important in the aetiology of NIDDM, and linkage studies are starting to localize some of the genes that influence the development of this disorder. Maturity-onset diabetes of the young (MODY), a single-gene disorder responsible for 2-5% of NIDDM, is characterized by autosomal dominant inheritance and an age of onset of 25 years or younger. MODY genes have been localized to chromosomes 7, 12 and 20 (refs 5, 7, 8) and clinical studies indicate that mutations in these genes are associated with abnormal patterns of glucose-stimulated insulin secretion. The gene on chromosome 7 (MODY2) encodes the glycolytic enzyme glucokinases which plays a key role in generating the metabolic signal for insulin secretion and in integrating hepatic glucose uptake. Here we show that subjects with the MODY3-form of NIDDM have mutations in the gene encoding hepatocyte nuclear factor-1alpha (HNF-1alpha, which is encoded by the gene TCF1). HNF-1alpha is a transcription factor that helps in the tissue-specific regulation of the expression of several liver genes and also functions as a weak transactivator of the rat insulin-I gene.

Mutations in the hepatocyte nuclear factor-4alpha gene in maturity-onset diabetes of the young (MODY1)

The disease maturity-onset diabetes of the young (MODY) is a genetically heterogeneous monogenic form of non-insulin-dependent (type 2) diabetes mellitus (NIDDM), characterized by early onset, usually before 25 years of age and often in adolescence or childhood, and by autosomal dominant inheritance. It has been estimated that 2-5% of patients with NIDDM may have this form of diabetes mellitus. Clinical studies have shown that prediabetic MODY subjects have normal insulin sensitivity but suffer from a defect in glucose-stimulated insulin secretion, suggesting that pancreatic beta-cell dysfunction rather than insulin resistance is the primary defect in this disorder. Linkage studies have localized the genes that are mutated in MODY on human chromosomes 20 (MODY1), 7 (MODY2) and 12 (MODY3), with MODY2 and MODY3 being allelic with the genes encoding glucokinase, a key regulator of insulin secretion, and hepatocyte nuclear factor-1alpha (HNF-1alpha), a transcription factor involved in tissue-specific regulation of liver genes but also expressed in pancreatic islets, insulinoma cells and other tissues. Here we show that MODY1 is the gene encoding HNF-4alpha (gene symbol, TCF14), a member of the steroid/thyroid hormone receptor superfamily and an upstream regulator of HNF-1alpha expression.

Human gastric inhibitory polypeptide receptor: cloning of the gene (GIPR) and cDNA.

Gastric inhibitory polypeptide (GIP), which is released from the gastrointestinal tract, stimulates insulin secretion from pancreatic beta cells and plays a crucial role in the regulation of insulin secretion during the postprandial phase. We have isolated the human gene (GIPR) and cDNA encoding the GIP receptor by a combination of the conventional screening and polymerase chain reaction procedures. Human GIP receptor cDNA encodes a protein of 466 amino acids that is 81.5 and 81.2% identical to the previously cloned hamster and rat GIP receptor, respectively. Hydropathic analysis shows the presence of a signal peptide and seven potential transmembrane domains, a feature characteristic of the VIP/glucagon/secretin receptor family of G protein-coupled receptors. The human GIPR gene is about 13.8 kb long, consists of 14 exons, and carries 17 Alu repeats.