Matthias Klaften

Dominant-Negative Effects of a Novel Mutated Ins2 Allele Causes Early-Onset Diabetes and Severe β-Cell Loss in Munich Ins2C95S Mutant Mice

The novel diabetic mouse model Munich Ins2C95S was discovered within the Munich N-ethyl-N-nitrosourea mouse mutagenesis screen. These mice exhibit a T→A transversion in the insulin 2 (Ins2) gene at nucleotide position 1903 in exon 3, which leads to the amino acid exchange C95S and loss of the A6-A11 intrachain disulfide bond. From 1 month of age onwards, blood glucose levels of heterozygous Munich Ins2C95S 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 indices of heterozygous mutants were significantly lower than those of wild-type littermates. The initial blood glucose decrease during an insulin tolerance test was lower and HOMA insulin resistance indices were significantly higher in mutant mice, indicating the development of insulin resistance in mutant mice. 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. 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 Ins2C95S mutant mice are considered a valuable model to study the mechanisms of β-cell dysfunction and death during the development of diabetes.

Novel missense mutation of uromodulin in mice causes renal dysfunction with alterations in urea handling, energy, and bone metabolism.

Uromodulin-associated kidney disease is a heritable renal disease in humans caused by mutations in the uromodulin (UMOD) gene. The pathogenesis of the disease is mostly unknown. In this study, we describe a novel chemically induced mutant mouse line termed Umod(A227T) exhibiting impaired renal function. The A227T amino acid exchange may impair uromodulin trafficking, leading to dysfunction of thick ascending limb cells of Henles loop of the kidney. As a consequence, homozygous mutant mice display azotemia, impaired urine concentration ability, reduced fractional excretion of uric acid, and a selective defect in concentrating urea. Osteopenia in mutant mice is presumably a result of chronic hypercalciuria. In addition, body composition, lipid, and energy metabolism are indirectly affected in heterozygous and homozygous mutant Umod(A227T) mice, manifesting in reduced body weight, fat mass, and metabolic rate as well as reduced blood cholesterol, triglycerides, and nonesterified fatty acids. In conclusion, Umod(A227T) might act as a gain-of-toxic-function mutation. Therefore, the Umod(A227T) mouse line provides novel insights into consequences of disturbed uromodulin excretion regarding renal dysfunction as well as bone, energy, and lipid metabolism.

Morphologic and molecular characterization of two novel Krt71 (Krt2-6g) mutations: Krt71rco12 and Krt71rco13

We have analyzed two novel mouse mutant strains, Rco12 and Rco13, displaying a wavy pelage and curly vibrissae that have been identified in an ENU screen for dominant mutations affecting the pelage. The mutations were mapped to mouse Chromosome 15 and identified as missense point mutations in the first exon of the Krt71 (formerly called Krt2-6g) gene causing alterations of amino acid residue 143 from alanine to glycine (Rco12) and residue 146 from isoleucine to phenylalanine. The morphologic analyses demonstrated that both mutations cause identical phenotypes leading to the formation of filamentous aggregates in Henle’s and Huxley’s layers of the inner root sheath (IRS) of the hair follicle that leads to the bending of the hair shaft. Both novel mutations are located in the immediate vicinity of previously identified mutations in murine Krt71 that cause similar phenotypes and alter the helix initiation motif of the keratin. The characterization of these mutants demonstrates the importance of this Krt71 domain for the formation of linear IRS intermediate filaments.

Mutation of the Na+-K+-2Cl− cotransporter NKCC2 in mice is associated with severe polyuria and a urea-selective concentrating defect without hyperreninemia

The bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter NKCC2, located in the thick ascending limb of Henles loop, plays a critical role in the kidneys ability to concentrate urine. In humans, loss-of-function mutations of the solute carrier family 12 member 1 gene (SLC12A1), coding for NKCC2, cause type I Bartter syndrome, which is characterized by prenatal onset of a severe polyuria, salt-wasting tubulopathy, and hyperreninemia. In this study, we describe a novel chemically induced, recessive mutant mouse line termed Slc12a1(I299F) exhibiting late-onset manifestation of type I Bartter syndrome. Homozygous mutant mice are viable and exhibit severe polyuria, metabolic alkalosis, marked increase in plasma urea but close to normal creatininemia, hypermagnesemia, hyperprostaglandinuria, hypotension,, and osteopenia. Fractional excretion of urea is markedly decreased. In addition, calcium and magnesium excretions are more than doubled compared with wild-type mice, while uric acid excretion is twofold lower. In contrast to hyperreninemia present in human disease, plasma renin concentration in homozygotes is not increased. The polyuria observed in homozygotes may be due to the combination of two additive factors, a decrease in activity of mutant NKCC2 and an increase in medullary blood flow, due to prostaglandin-induced vasodilation, that impairs countercurrent exchange of urea in the medulla. In conclusion, this novel viable mouse line with a missense Slc12a1 mutation exhibits most of the features of type I Bartter syndrome and may represent a new model for the study of this human disease.

A Genetic Screen for Modifiers of the Delta1-Dependent Notch Signaling Function in the Mouse

The Notch signaling pathway is an evolutionarily conserved transduction pathway involved in embryonic patterning and regulation of cell fates during development. Recent studies have demonstrated that this pathway is integral to a complex system of interactions, which are also involved in distinct human diseases. Delta1 is one of the known ligands of the Notch receptors. Mice homozygous for a loss-of-function allele of the Delta1 gene Dll1lacZ/lacZ die during embryonic development. Here, we present the results of two phenotype-driven modifier screens. Heterozygous Dll1lacZ knockout animals were crossed with ENU-mutagenized mice and screened for dysmorphological, clinical chemical, and immunological variants that are dependent on the Delta1 loss-of-function allele. First, we show that mutagenized heterozygous Dll1lacZ offspring have reduced body weight and altered specific clinical chemical parameters, including changes in metabolites and electrolytes relevant for kidney function. In our mutagenesis screen we have successfully generated 35 new mutant lines. Of major interest are 7 mutant lines that exhibit a Dll1lacZ/+-dependent phenotype. These mutant mouse lines provide excellent in vivo tools for studying the role of Notch signaling in kidney and liver function, cholesterol and iron metabolism, cell-fate decisions, and during maturation of T cells in the immune system.

Reduced Corneal Thickness and Enlarged Anterior Chamber in a Novel ColVIIIa2G257D Mutant Mouse

PURPOSE The purpose of this study was the morphologic and genetic characterization of the novel eye size mutant Aca23 in the mouse. METHODS The eyes of the mutants were characterized in vivo by optical low-coherence interferometry, Scheimpflug imaging, and funduscopy. Visual acuity was examined using a virtual optomotor system. Morphology was studied by histology, in situ hybridization, and immunohistochemistry. Linkage analysis was performed using genomewide scans with single nucleotide polymorphisms and microsatellite markers. RESULTS Aca23 is a new semidominant eye size mutant that was discovered in an ENU mutagenesis screen. The phenotype includes increased anterior chamber depths, extended axial lengths, and reduced thickness of corneal layers. Aca23 was mapped to chromosome 4. A G-->A point mutation was identified at cDNA position 770 of Col8a2 encoding collagen VIII alpha2. The transition results in a G257D amino acid exchange affecting a highly conserved glycine residue in the collagenous domain. Proliferation of corneal endothelium, eye fundus, and visual acuity are not affected. CONCLUSIONS The mouse mutant Aca23 described here offers the first point mutation of the Col8a2 gene in the mouse. The results of this study suggest that a functional collagen VIII alpha2 is essential for the correct assembly of the Descemets membrane and for corneal stability. Aca23 might be used as a novel model for keratoglobus.

A Mutation in the Enamelin Gene in a Mouse Model

Amelogenesis imperfecta is an inherited disorder affecting tooth enamel formation. We previously isolated a mouse strain with an amelogenesis imperfecta phenotype (ATE1 mice) from a dominant ethylnitrosourea screen and mapped the disease-causing defect to a 9-cM region of mouse chromosome 5. In the current study, we tested the hypothesis that there is a mutation in enamelin (ENAM) or ameloblastin (AMBN), both of which are located wihin the linkage region, by sequencing these two candidate genes. Analysis of our data shows that the amelogenesis imperfecta phenotype is linked to a C > T transition in exon 8 of the enamelin gene. The mutation predicts a C826T transition, which is present in the enamelin transcript and changes the glutamine (Gln) codon at position 176 into a premature stop codon (Gln176X). Conversely, no mutation could be detected in the ameloblastin gene. These results define the ATE1 mice as a model for local hypoplastic autosomal-dominant amelogenesis imperfecta (AIH2), which is caused by enamelin truncation mutations in humans.

New mouse models for metabolic bone diseases generated by genome-wide ENU mutagenesis.

Metabolic bone disorders arise as primary diseases or may be secondary due to a multitude of organ malfunctions. Animal models are required to understand the molecular mechanisms responsible for the imbalances of bone metabolism in disturbed bone mineralization diseases. Here we present the isolation of mutant mouse models for metabolic bone diseases by phenotyping blood parameters that target bone turnover within the large-scale genome-wide Munich ENU Mutagenesis Project. A screening panel of three clinical parameters, also commonly used as biochemical markers in patients with metabolic bone diseases, was chosen. Total alkaline phosphatase activity and total calcium and inorganic phosphate levels in plasma samples of F1 offspring produced from ENU-mutagenized C3HeB/FeJ male mice were measured. Screening of 9,540 mice led to the identification of 257 phenodeviants of which 190 were tested by genetic confirmation crosses. Seventy-one new dominant mutant lines showing alterations of at least one of the biochemical parameters of interest were confirmed. Fifteen mutations among three genes (Phex, Casr, and Alpl) have been identified by positional-candidate gene approaches and one mutation of the Asgr1 gene, which was identified by next-generation sequencing. All new mutant mouse lines are offered as a resource for the scientific community.

A novel N‐ethyl‐N‐nitrosourea–induced mutation in phospholipase Cγ2 causes inflammatory arthritis, metabolic defects, and male infertility in vitro in a murine model

OBJECTIVE It is difficult to identify a single causative factor for inflammatory arthritis because of the multifactorial nature of the disease. This study was undertaken to dissect the molecular complexity of systemic inflammatory disease, utilizing a combined approach of mutagenesis and systematic phenotype screening in a murine model. METHODS In a large-scale N-ethyl-N-nitrosourea mutagenesis project, the Ali14 mutant mouse strain was established because of dominant inheritance of spontaneous swelling and inflammation of the hind paws. Genetic mapping and subsequent candidate gene sequencing were conducted to find the causative gene, and systematic phenotyping of Ali14/+ mice was performed in the German Mouse Clinic. RESULTS A novel missense mutation in the phospholipase Cγ2 gene (Plcg2) was identified in Ali14/+ mice. Because of the hyperreactive external entry of calcium observed in cultured B cells and other in vitro experiments, the Ali14 mutation is thought to be a novel gain-of-function allele of Plcg2. Findings from systematic screening of Ali14/+ mice demonstrated various phenotypic changes: an abnormally high T cell:B cell ratio, up-regulation of Ig, alterations in body composition, and a reduction in cholesterol and triglyceride levels in peripheral blood. In addition, spermatozoa from Ali14/+ mice failed to fertilize eggs in vitro, despite the normal fertility of the Ali14/+ male mice in vivo. CONCLUSION These results suggest that the Plcg2-mediated pathways play a crucial role in various metabolic and sperm functions, in addition to initiating and maintaining the immune system. These findings may indicate the importance of the Ali14/+ mouse strain as a model for systemic inflammatory diseases and inflammation-related metabolic changes in humans.

Generation of N-ethyl-N-nitrosourea-induced mouse mutants with deviations in hematological parameters

Research on hematological disorders relies on suitable animal models. We retrospectively evaluated the use of the hematological parameters hematocrit (HCT), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), red blood cell count (RBC), white blood cell count (WBC), and platelet count (PLT) in the phenotype-driven Munich N-ethyl-N-nitrosourea (ENU) mouse mutagenesis project as parameters for the generation of novel animal models for human diseases. The analysis was carried out on more than 16,000 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in the levels of the chosen parameters. Identification of animals exhibiting altered values and transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of mutant lines for the parameters MCV, RBC, and PLT. Analysis of the causative mutation was started in selected lines, thereby revealing a novel mutation in the transferrin receptor gene (Tfrc) in one line. Thus, novel phenotype-driven mouse models were established to analyze the genetic components of hematological disorders.