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FRONTIERS

Diabetes models by screen for hyperglycemia in phenotype-driven ENU mouse mutagenesis projects

¹Institute of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilians-University, Munich; ²Institute of Veterinary Pathology, Ludwig-Maximilians-University, Munich; ³Institute of Experimental Genetics, GSF Research Center for Environment and Health, Neuherberg; and ⁴Laboratory for Functional Genome Analysis, Gene Center, Ludwig-Maximilians-University, Munich, Germany

Submitted 11 September 2007 ; accepted in final form 28 November 2007

ABSTRACT

More than 150 million people suffer from diabetes mellitus worldwide, and this number is expected to rise substantially within the next decades. Despite its high prevalence, the pathogenesis of diabetes mellitus is not completely understood. Therefore, appropriate experimental models are essential tools to gain more insight into the genetics and pathogenesis of the disease. Here, we describe the current efforts to establish novel diabetes models derived from unbiased, phenotype-driven, large-scale N-ethyl-N-nitrosourea (ENU) mouse mutagenesis projects started a decade ago using hyperglycemia as a high-throughput screen parameter. Mouse lines were established according to their hyperglycemia phenotype over several generations, thereby revealing a mutation as cause for the aberrant phenotype. Chromosomal assignment of the causative mutation and subsequent candidate gene analysis led to the detection of the mutations that resulted in novel alleles of genes already known to be involved in glucose homeostasis, like glucokinase, insulin 2, and insulin receptor. Additional ENU-induced hyperglycemia lines are under genetic analysis. Improvements in screen for diabetic animals are implemented to detect more subtle phenotypes. Moreover, diet challenge assays are being employed to uncover interactions between genetic and environmental factors in the pathogenesis of diabetes mellitus. The new mouse mutants recovered in phenotype-driven ENU mouse mutagenesis projects complement the available models generated by targeted mutagenesis of candidate genes, all together providing the large resource of models required for a systematic dissection of the pathogenesis of diabetes mellitus.

clinical chemistry; N-ethyl-N-nitrosourea; glucose; insulin

Existing Rodent Diabetes Models

Suitable animal models mirror some of the multiform symptoms of human diabetes. Diabetes models were generated in different genetic backgrounds by dietary induction, toxic β-cell destruction, surgical manipulations, and/or combinations thereof. Specific models appeared by spontaneous mutations that were subsequently analyzed using forward genetics methods. On the other hand, reverse genetics techniques were used to produce genetically modified mice with defined alterations of genes that were suggested to play a role in glucose homeostasis (8, 44, 46). Furthermore, the genome-wide search for causative loci was carried out by examining the genetic polymorphisms that are linked to different plasma glucose phenotypes of the already-existing mouse strains. Mapping of quantitative trait loci (QTL) controlling plasma glucose levels was done in independent and combined crosses of inbred mouse strains (8–10). In addition, genetic polymorphisms of heterogeneous stock mice influencing the plasma glucose homeostasis were examined by high-resolution whole-genome association studies of quantitative traits (51).

Phenotype-driven ENU Mouse Mutagenesis Projects

Random chemical mutagenesis and subsequent screening for clinically relevant phenotypes without a priori assumptions is a powerful approach to derive novel mouse models for biomedical research. The alkylating agent N-ethyl-N-nitrosourea (ENU) is currently the most powerful mutagen for the production of mutant mice. ENU shows mutagenic action on premeiotic spermatogonial stem cells. This allows the production of a large number of randomly mutant offspring from treated males (20). Compared with other mutagenesis methods, ENU predominantly induces point mutations, thereby leading to allelic series for the functional analysis of genes (47). Mutations induced by ENU are not tagged molecularly as is the case in other random mutagenesis projects e.g., using gene traps. This is initially a disadvantage as the causative mutations have to be analyzed by linkage analysis. However, the availability of point mutations is important for the detailed functional analysis of genes. The function of a given gene in glucose homeostasis cannot be described by analyzing only one mutant allele; multiple alleles of the same gene are necessary for this goal. The ENU mouse mutagenesis projects established during the last 10 years serve as platforms for the systematic, genome-wide, large-scale production and analysis of mouse mutants as a model system for inherited human diseases, thereby facilitating the identification and functional characterization of genes that are relevant for the prevention, diagnosis, and therapy of diseases (20).

After the ENU-mutagenized males were mated to wild-type females, specific pathological states were identified in the offspring by appropriate routine procedures that allow the screen of large numbers of mice for a broad spectrum of parameters. High numbers of screen parameters are usually used to optimize the cost/benefit calculation of ENU mouse mutagenesis projects. Phenotypic screens in major ENU mouse mutagenesis projects often include dysmorphology, clinical chemistry, immunology, allergy, and behavior as well as other screens with a high number of test parameters. However, smaller laboratories have also efficiently set up specialized projects. Screen profiles of clinical chemical blood parameters were established to detect phenotypic variants with defects of various organ systems or changes in metabolic pathways. Breeding of the phenotypically affected mice and screen of the offspring confirmed the transmission of the altered phenotype to subsequent generations, thereby revealing a mutation as cause for the aberrant phenotype. The underlying mutations are then identified by forward genetics strategies and lead to the identification of genes/alleles that may have counterparts relevant for human diseases (Fig. 1). Mutant lines with the causative mutation already identified are successfully used in different areas of biomedical research (19, 20, 37).

View larger version (23K): [in this window] [in a new window]   Fig. 1. Generation of novel disease models in phenotype-driven N-ethyl-N-nitrosourea (ENU) mouse mutagenesis projects. After intraperitoneal treatment of male mice with ENU, defined mating schemes produced the G1 and G3 offspring (A), which were phenotypically examined for alterations, e.g., hyperglycemia (B). Phenotypical G1 and G3 variants were mated to test the inheritance of the altered phenotype caused by dominant or recessive mutations, respectively (C). The final step of establishing new disease models includes identification of the causative mutation by linkage analysis (D) and subsequent candidate gene analysis (E), as well as in-depth phenotypic analysis.

ENU mouse mutagenesis projects are done on different inbred genetic backgrounds (BALB/c, C3H, C57BL/6). The chemically mutagenized mice are subsequently mated with mice of the same or of different inbred strains. In both cases, the G1 animals harbor identical genetic backgrounds (inbred vs. hybrid), which is not the case in the animals of the subsequent generations after mating the chemically mutagenized mice with mice of another inbred strain. A defined phenotype may occur in a reproducible manner through subsequent generations in an identical genetic background. A heterogenous genetic background may reveal various interactions of the ENU-induced mutations in different genotypes. Different alleles may cause diabetes in various genetic backgrounds. By using mice with increased diabetes susceptibility like C57BL/6J, which lack the Nnt protein and therefore display glucose intolerance and reduced insulin secretion, sensitized/modifier screens may be carried out to detect additive diabetes susceptibility alleles (12).

Mutation rates in early ENU mouse mutagenesis experiments have been determined by the specific locus test as well as other functional tests, including a limited number of specific genes and loci, which led to results with restricted common validity (Ref. 20 and references therein). Using similar mutagenesis protocols and various inbred strains, studies of different chromosomal regions of ENU-mutagenized mice with different molecular genetic methods [denaturing HPLC, temperature gradient capillary electrophoresis (TGCE), direct sequencing] observed one mutation in 100,000 bp (5) and one mutation in 1.0–2.5 Mbp (3, 11, 33, 39, 43). Thus, molecular genetic analysis may be the most effective approach for the determination of mutation frequencies in ENU mouse mutagenesis projects. After confirmation of the inheritance of the mutant phenotype to the subsequent generations, back-cross of the phenotypic mutant animal to wild-type mice leads to the loss of noncausative mutations. After ten back-cross generations, 20 cM of the originally mutagenized genomic DNA remains, which harbors about 25 mutations, including the causative mutation (45). Considering only a small number of the more than 1,000 mutations per animal as potentially functional by causing phenotypic consequences, at least the largest part of them, except for the causative mutation, is segregated (5, 24, 42).

The establishment of lines showing the mutant phenotype indicates the penetrance of the mutant phenotype and the number of segregating mutations causing the mutant phenotype. The examination of the chromosomal position of the causative mutation is carried out by linkage analysis. Briefly, phenotypic mutants harboring the mutation are bred to wild-type mice of a second inbred strain; the resulting hybrid offspring are analyzed for the abnormal phenotype of the parent, and hybrid mutants are back-crossed to wild-type mice of the second inbred strain (for dominant mutations) or intercrossed (for recessive mutations). The resulting offspring are again analyzed for the abnormal phenotype of the parent, thereby dividing them into phenotypically mutant and nonmutant animals. Genome-wide linkage analysis of the genomic DNA samples using polymorphic markers subsequently reveals the chromosomal position of the mutation. Further linkage analysis on the determined chromosomal site and candidate gene analysis including sequencing techniques are then performed to identify the causative mutation (45). The probability that a confounding mutation is linked to a sequenced mutation in a determined chromosomal region with a length of 5 Mb was calculated to be low (P < 0.05). In total, most mutant phenotypes are effectively monogenic (23, 24). Novel genetic backgrounds appear in the G1 and G2 animals of the linkage analysis which might result in variations of the ENU-induced mutant phenotype. This can lead to the loss of the mutant phenotype; in this case linkage analysis has to be tried with another second inbred strain.

The functional proof that a mutation that lies in the correct genomic interval is responsible for the phenotype may be carried out by demonstrating that the affected protein is absent or inactive, by rescuing the phenotype via introduction of a wild-type copy of the gene, using reverse genetics methods, or by performing complementation tests with another mutant allele (6). The mutants may carry loss-of-function, dominant negative, hypomorphic, or gain-of-function alleles of the affected genes.

Phenotypic Screen for Hyperglycemia

Several ENU mouse mutagenesis projects used plasma glucose as parameter for the high-throughput screen of the mutagenized mice (Table 1). Multiple factors influence the outcome of the projects. Standardization of the phenotype analyses has been greatly improved by establishing specialized mouse phenotyping centers that use defined standard operating procedures (http://www.interphenome.org). As an example, in the Munich ENU mouse mutagenesis project (http://www.gsf.de/ieg/groups/genome/enu.html) (13), clinical chemical analysis, including plasma substrates, proteins, electrolytes and enzymes, was carried out with blood samples from 3-mo-old G1 and G3 C3H mice fasted overnight that were obtained by puncture of the retroorbital sinus under short-term general anesthesia. Plasma from Li-heparin-treated blood was analyzed using an Olympus AU400 autoanalyzer (Olympus, Hamburg, Germany) and the adapted reagents (Olympus). Plasma glucose concentrations were examined using the enzymatic hexokinase assay with the reagent OSR6121 (Olympus) in the linear measurement range of 10–800 mg/dl (0.6–45 mmol/l) (40, 41).

Comparison of the efficiency of the production of hyperglycemia lines between the ENU mouse mutagenesis projects might lead to an optimized protocol for the generation of ENU-induced hyperglycemia lines but this is practically prevented by several pitfalls. Interaction of unrecognized laboratory-specific factors in mouse genotype, husbandry and experimental procedure may lead to significant deviations in the results of highly standardized experiments (26). Major in vivo and in vitro interfering factors include age, body weight, activity, health status, nutrition, social interference, interference with humans, personnel, time point and site of blood collection, as well as sample preparation and analysis. Furthermore, use of stringent cut-off levels according to a determined wild-type physiologic data range may improve the establishment of mutant lines from the recognized variants but prevent the identification of additional variant animals.

In the four lines harboring a dominant mutation (GLS001, GLS004, GLS006, GLS007), mutant offspring showing pathological plasma glucose levels were produced by mating heterozygous mutants to wild-type animals except for line GLS006, which was previously suggested to harbor a recessive mutation (Table 3). The phenotypic mutants of the four lines showed mean plasma glucose levels between 245 and 363 mg/dl. The phenotypic penetrance of the hyperglycemia in the lines was analyzed by defining 100% phenotypic penetrance in the case of the appearance of 50 and 75% offspring exhibiting the phenotypic deviation after mating phenotypic mutants to wild-type mice and breeding heterozygous mutant mice, respectively. A suitable phenotypic penetrance of the pathological glucose levels above 50% was observed in all four lines, which facilitates the effective subsequent phenotypic and molecular genetic analyses. Reanalysis of animals after a few weeks increased the phenotypic penetrance of pathological glucose levels, thereby indicating that the penetrance was generally underestimated in the analysis after 12 wk of age. Further in-depth analysis of the line GLS004 revealed complete penetrance of the diabetic phenotype (16) (see below), which indicated the inherent impossibility of carrying out complete standardization in long-term, large-scale phenotyping projects. However, incomplete phenotypic penetrance was also seen in established diabetic lines of other ENU mutagenesis projects (17). The lines GLS001 and GLS004 had already been bred for more than ten generations starting from the ENU-mutagenized G0 founder animal without losing the abnormal phenotype, whereas the lines GLS006 and GLS007 were recently established. In all four lines, the numbers of animals exhibiting plasma glucose levels above the cut-off level were significantly (²-test, P < 0.001) higher than the expected 5% of the C3H mouse population (Table 3).

Several hyperglycemia lines derived from phenotype-driven ENU mouse mutagenesis projects with the causative mutation already identified have been published (Table 4). All lines harbor a dominant mutation. Most of them are glucokinase (Gck) mutants. Gck phosphorylates glucose in the first step of glycolysis and plays a critical role in the glucose-responsive insulin secretion of pancreatic β-cells by functioning as a "glucose sensor". In humans, almost 200 heterozygous mutations in the GCK gene have been reported to cause maturity-onset diabetes of the young type 2 (MODY2), characterized by mild hyperglycemia. Homozygous inactivating GCK mutations result in permanent neonatal diabetes mellitus (PNDM). In addition, heterozygous activating GCK mutations causing hypoglycemia have been observed (14, 31). In mice, targeted global Gck knockout mutations resulted in mild hyperglycemia in the heterozygous mutants and extreme hyperglycemia and embryonic to postnatal lethality during the first week after birth in the homozygous mutants (4, 15).

In another ENU mutagenesis project using C57BL/6J x DBA/2J mice, 20 dominant hereditary diabetic mutants were obtained from 43 hyperglycemic G1 variants that had been subjected to the inheritance tests. Eleven independent Gck mutant lines were observed, including six missense mutations, two nonsense mutations, and three splice site mutations. One additional Gck mutant harboring a missense mutation (M100960: Gck^S127P) has been published online (http://www.gsc.riken.go.jp/Mouse). The cut-off glucose level determining hyperglycemia was 200 mg/dl. Ad libitum-fed serum glucose levels of the 11-wk-old G1 mutant founder mice were between 199 and 369 mg/dl. Four (Gck^V182M, Gck^T206M, Gck^T228A, Gck^IVS1A+1GT) of the 11 ENU mutations have also been found in MODY2 patients and another mutation (Gck^IVS8+2TC) in PNDM patients. Gck mRNA and protein expression were further analyzed in six mutant lines (Gck^V182M, Gck^C220Y, Gck^M224R, Gck^Y273Stop, Gck^R345Stop, Gck^IVS1A+1GT). The heterozygous mutants of all lines examined showed mild hyperglycemia. Homozygous mutants were analyzed in the three lines Gck^V182M, Gck^R345Stop, and Gck^IVS1A+1GT. They suffered from severe hyperglycemia soon after birth. Depending on the line, the homozygous mutants died within the first week after birth or survived at least 5 wk without insulin treatment. In total, the six mutant lines, like the human patients, suffered from impaired glucose-responsive insulin secretion, which causes hyperglycemia. The high frequency of 12 Gck mutations in 20 dominant hereditary diabetic mutants may be due to the phenotype monogenically associated with Gck mutations, the high penetrance of Gck gene defects, or a greater sensitivity of glucokinase activity to amino acid changes, or it may be due to the possibility that haploinsufficiency leads to the phenotypes caused by Gck mutations (21).

We recently identified a novel insulin 2 (Ins2) allele in the hyperglycemic line GLS004 derived from the Munich ENU mouse mutagenesis screen. In humans, insulin gene mutations were found as rare disorders (48, 49). In mice, homozygous knockout animals lacking either the Ins1 or the Ins2 gene had normal plasma insulin levels and normal values in the glucose tolerance test (27). The Akita mouse serves as an Ins2 mutant model on the C57BL/6N genetic background, which dominantly develops early onset diabetes mellitus without insulitis or obesity. The animals exhibit a spontaneous GA transition at nt 1907 in exon 3 of Ins2, leading to the amino acid exchange C96Y, the disruption of the A7-B7 interchain disulfide bond, and the appearance of a severe defect in insulin secretion and hypoinsulinemia in heterozygous mutants. Males show a more severe phenotype. The accumulation of mutant insulin in β-cells is thought to be responsible for the onset of diabetes mellitus, rather than the initial lack of active insulin (52). Homozygous Akita mice showed increased postnatal lethality (22).

Our ENU-induced diabetic line GLS004 on the C3H genetic background was named Munich Ins2^C95S and exhibits a TA transversion in the Ins2 gene at nt 1903 in exon 3, which leads to the amino acid exchange C95S and loss of the A6-A11 intrachain disulfide bond. From 1 mo of age onward, blood glucose levels of heterozygous Munich Ins2^C95S mutant mice were significantly increased compared with controls. The fasted and postprandial serum insulin levels of the heterozygous mutants were indistinguishable from those of wild-type littermates. However, serum insulin levels after glucose challenge, pancreatic insulin content, and homeostasis model assessment (HOMA) β-cell indexes of heterozygous mutants were significantly lower than those of wild-type littermates. Initial blood glucose decrease during insulin tolerance tests was lower and HOMA insulin resistance indexes were significantly higher in male mutants, indicating the development of insulin resistance. The total islet volume, the volume density of β-cells in the islets, and the total β-cell volume of heterozygous male mutants was significantly reduced compared with wild-type mice (Fig. 2). Electron microscopy of the β-cells of male mutants showed virtually no secretory insulin granules, the endoplasmic reticulum was severely enlarged, and mitochondria appeared swollen. Thus, Munich Ins2^C95S mutant mice may serve as a model to study the pathophysiological alterations of β-cells during the development of diabetes mellitus (16).

View larger version (39K): [in this window] [in a new window]   Fig. 2. Heterozygous mutant Munich Ins2C95S mice as an example for the establishment of novel diabetes models in ENU mouse mutagenesis projects. Plasma glucose (A) and insulin (B) levels 10 min after glucose challenge at 1, 3, and 6 mo of age in mutant (mt) mice vs. wild-type controls (wt). Quantitative stereological analysis of the pancreas at 6 mo of age is shown by total islet volume [V(Islet, Pan); C] and the calculated total β-cell volume [V(β-cells, Islet); D]; m, male; f, female; n, no. of animals examined. Data represent means and SE, *P < 0.05 (16).

In total, the dominant ENU-induced mutations identified to date caused novel alleles of genes already known to be involved in glucose homeostasis. All but one of the glucokinase mutations are derived from a single project. It is assumed that the high number of glucokinase mutations is caused by the specific interaction of factors of the genetic background, environment and experiment in this project and therefore will not appear in other projects. However, the predominant appearance of mutants of another single gene may occur within a given project with constant in vivo and in vitro factors. The predominant appearance of mutations in a single gene may be controlled by modifying various parameters of the project. After the establishment of mutant lines, the ad hoc prescreen of the lines for mutations in this gene may be done.

Additional ENU-Induced Diabetic Lines

In addition to the hyperglycemia lines with the causative mutation already identified, ENU-induced hyperglycemia lines from screens for dominant and recessive mutations with the causative mutation still unknown are published online (Table 5). Causative mutations in the coding region of suitable candidate genes that lie in the linked chromosomal region of mouse lines derived from phenotype-driven ENU mutagenesis projects are inherently identified earlier compared with mutations in genes or noncoding functional sequences previously unknown to influence glucose homeostasis. This is obviously more time consuming. The potential use of high numbers of SNPs as polymorphic markers (http://www.ncbi.nlm.nih.gov/SNP) improves the linkage analysis. Expression analysis of genes in the linked chromosomal region by microarray techniques may assist the selection of suitable candidate genes. The current progress of the sequencing techniques may be used in future analyses by sequencing extensive parts of ENU-mutagenized mouse genomes after breeding them for a low number of generations to wild-type mice.

Obesity-associated insulin resistance is a major risk factor for T2DM. Therefore, "secondary" hyperglycemia may be observed in ENU-induced mouse mutants that were selected and bred using obesity as a parameter in dysmorphology screens. Search for obese phenotypes in published chemically induced (ENU) mutants (as of august 24, 2007: 1,392 alleles, 1,164 genes/markers) of the "phenotypes and alleles" database in the Mouse Genome Informatics website (http://www.informatics.jax.org/searches/allele_form.shtml) revealed a number of lines showing obesity, e.g., in mice harboring novel alleles for the alstrom syndrome 1 homolog [Alms1^bbb (http://www.apf.edu.au), Alms1^L2131X (28)], leptin receptor [Lepr^db-1R (http://tnmouse.org), Lepr^m1Btlr (http://www.mmrrc.org)], melanocortin 4 receptor [Mc4r^Glu3 = Glu^m03Jus (http://www.mouse-genome.bcm.tmc.edu), Mc4r^m1Btlr (http://www.mmrrc.org) (32)], and proprotein convertase subtilisin/kexin type 1 (Pcsk1^N222D) (29). In addition, lines showing obesity with the mutation not yet linked and/or discovered are listed: Bgby, Mity, Pkcp (Center for Functional Genomics, Northwestern University); Blb2 (http://www.mouse-genome.bcm.tmc.edu); Hlb44, Hlb52, Hlb80, Hlb81, Hlb93, Hlb124, Hlb125, Hlb131, Hlb132, Hlb147, Hlb163, Hlb181, Hlb197, Hlb199, Hlb230, Hlb349A (http://pga.jax.org); Nmf15 (http://www.jax.org/nmf).

Improvement of the detection of diabetic variants in the screening procedure of the G1 and G3 offspring may be achieved by using more sophisticated screening parameters as indicators of insulin resistance, including elevated fasting insulin levels, increased fasting glucose, and impaired glucose tolerance. This may be done using the HOMA, meal tolerance test (MTT), intraperitoneal or oral glucose tolerance test (IPGTT, OGTT), and intraperitoneal insulin sensitivity test (IPIST) (7). For example, detection of insulin resistance was used as a high-throughput method to establish mutant lines in an ENU mutagenesis project (http://www.mgu.har.mrc.ac.uk/research/diabetes/sens.html).

The screening procedure may also be improved by analyzing the animals more frequently and/or at a higher age. Screening mice at an increased age might lead to a higher level of phenotypic penetrance of the mutation as well as to additional deviations in the plasma glucose due to alternative ENU-induced mutations. Diet challenge tests may reveal interactions between genetic and environmental factors in the development of hyperglycemia. Change of environmental factors is used or intended to be used in ongoing ENU mouse mutagenesis projects, and the outcome may be published in the future. Strain-specific susceptibility to diabetes and/or strain-specific variations in the plasma glucose concentration (38) (http://www.interphenome.org) have been found. Therefore, generation and subsequent analysis of hybrids and/or congenic strains may give rise to variations in the appearance of diabetes.

Summary

Using hyperglycemia as a high-throughput screening parameter, novel dominant alleles of genes already known to be involved in glucose homeostasis, like glucokinase (Gck), insulin 2 (Ins2), and insulin receptor (Insr), were identified in phenotype-driven, large-scale ENU mouse mutagenesis projects started a decade ago. This led to allelic series for the functional analysis of the respective genes. Additional ENU-induced hyperglycemia lines harboring dominant and recessive mutations are currently under genetic and phenotypic analysis. Due to the triggering of random mutations by ENU, new genes and/or alleles not yet known to be involved in glucose homeostasis may be discovered with these mouse mutants. Ongoing ENU mutagenesis projects also use more sophisticated screen methods and breeding strategies, which will reveal additional ENU-derived diabetic mouse models for biomedical research.

GRANTS

This work was supported by the German Human Genome Project (DHGP), the National Genome Research Network (NGFN) (M. Hrabé de Angelis, E. Wolf) and by the German Research Council (Research Training Unit 1029 "Functional Genome Research in Veterinary Medicine") (B. Aigner, N. Herbach, R. Wanke, E. Wolf).

FOOTNOTES

Address for reprint requests and other correspondence: B. Aigner, Inst. of Molecular Animal Breeding and Biotechnology, Hackerstr. 27, D-85764 Oberschleißheim, Germany (e-mail: b.aigner{at}gen.vetmed.uni-muenchen.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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