Genetic aspects

Low-resolution SNP genotyping of GLS001 and GLS006 mice and subsequent high- resolution linkage analysis of GLS001 mice mapped the diabetic phenotype of both strains to polymorphic DNA markers on chromosome 11 at 5.9 and 7.1 Mb, respectively. The mouse genome database query for positional diabetes-associated genes revealed the glucokinase (Gck) gene as potential candidate gene for sequencing. Sequence analysis of Gck revealed a defined point mutation in exon 6 of Gck in both strains. The ENU strain GLS001 exhibited a T to G transversion at nucleotide position (nt) 629, leading to an amino acid exchange from methionine to arginine at position 210. In GLS006 mice an A to T transversion at nt 650 was identified, thereby resulting in an amino acid exchange from aspartic acid to valine at position 217. According to the respective missense mutations, the laboratory names GLS001 and GLS006 were replaced by the official terms Munich GckM210R and

GckD217V mutant mouse. Mutations in the human glucokinase (GCK) gene are

associated with 2 distinct phenotypic traits. Mutations can either cause hypoglycaemia (when activating glucokinase function) or hyperglycaemia (when impairing glucokinase function). While heterozygous inactivating mutations are

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reported to cause maturity-onset diabetes of the young, type 2 (MODY 2), homozygous mutations are associated with neonatal diabetes mellitus (NDM) (Gloyn 2003). Four M210 mutations and 1 D217 mutation have already been detected in human diabetic subjects, whereas base exchanges at these two positions have not yet been detected in other animal models so far. Referring to the missense mutation M210 in human subjects, the base exchanges from methionine to lysine (M210K) has been discovered to cause MODY 2 and NDM, whereas the mutations M210T, M210V, and M210I (amino acid exchange to threonine, to valine, or to isoleucine, respectively) have only been identified in families with MODY features. At position D217, only the amino acid exchange from aspartic acid to glutamic acid (D217E) has been reported to be associated with MODY 2 (Njolstad et al. 2001; Osbak et al. 2009; Sagen et al. 2006; Velho et al. 1997). Various knockout mouse models for MODY 2 have already been generated by targeted global or tissue-specific disruption of Gck (Bali et al. 1995; Grupe et al. 1995; Postic et al. 2001; Postic et al. 1999; Terauchi et al. 1995) (http://www.informatics.jax.org/). However, monogenic diseases like MODY 2 in human beings usually arise from single point mutations and include a broad spectrum of phenotypic outcomes depending on the position and the nature of the mutation. Knockout strategies which target complete ablation of the respective gene transcript, might fail to produce animal models that recapitulate human disease phenotypes. Therefore, animal models exhibiting naturally occurring or induced mutations represent additional useful tools to dissect specific gene functions (Clark et al. 1994; Oliver et al. 2007; Peltonen and McKusick 2001). A total number of 15 ENU-induced glucokinase mutant mouse strains have been generated in various ENU-driven mutagenesis projects (Fenner et al. 2009; Inoue et al. 2004; Toye et al. 2004) (http://www.informatics.jax.org/). In the screen for dominant mutations of the RIKEN mutagenesis project (RIKEN BioResouces Center), 12 of 17 mouse strains exhibiting hyperglycaemia were identified to bear single-base pair substitutions in Gck (http://www.brc.riken.jp/lab/gsc/mouse/). This predominance of ENU-induced Gck mutations might be due to the high penetrance of Gck defects, a great sensitivity of the glucokinase protein to amino acid exchanges or may arise from the fact that Gck haploinsufficiency leads to abnormal phenotypic traits (Inoue et al. 2004). Another probable explanation for the plenitude of Gck mutations, identified in screens for hyperglycaemia in other ENU mutagenesis projects, might be founded in the structural properties of Gck. Exhibiting a surpassing coding sequence length

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(1,398 bp vs. 1,137 bp median coding sequence for the mouse genome), a high exon number (11 exons vs. a median exon number of 6), and an above-average G+C- content (57.3% vs. a median G+C-content of about 45%), Gck may be more frequently targeted by ENU-induced base exchanges than other genes for statistical reasons (Barbaric et al. 2007).

The occurrence of the diabetic phenotype in both Munich GckM210R _{and Gck}D217V

mutants remained unaltered in ensuing generations, giving proof of the heritability and the complete penetrance of the abnormal phenotype. Together with the linkage analysis carried out and the revelation of the functional consequences of the respective mutations, these findings indicate that the identified missense mutations in Gck are causative for the diabetic phenotype of both mouse strains.

The induction of different mutations in the same gene represents an extremely powerful tool to address the question of gene and protein function (Justice et al. 1999). An impressive example of an allelic series is the quaking locus. Prior to ENU mutagenesis, the quaking locus was defined by a single spontaneous allele, characterised by quaking and seizures as a result of defective CNS myelination in homozygous mutants. The identification of novel ENU-induced alleles indicated an additional involvement of quaking in embryogenesis (Justice and Bode 1988; Sidman et al. 1964).

Since the causative mutations in Munich GckM210R and GckD217V mutants are located in a distance of only 7 amino acids to each other, the creation of an allelic series in these mouse strains represents an extremely valuable genetic resource for detailed dissection of the impact of alterations at different amino acid residues of the glucokinase protein on phenotypic and pathomorphologic traits.

5.2 Functional consequences of the mutations