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Objective: The use of mice as animal models in biomedical research allows the standardization of genetic background and environmental conditions, which both affect phenotypic variability. As the use of both sexes in experiments is strongly recommended, sex-specific phenotypic variability is discussed with regard to putative consequences on the group size which is necessary for achieving valid and reproducible results. In this study, the sex-specific variability of 25 clinical chemical and hematological parameters which represent a comprehensive blood screen of laboratory mice, was analyzed in data sets which have been submitted to the Mouse Phenome Database., Results: The overall analysis comprising all 25 clinical chemical and hematological parameters showed no evidence for substantial and robust general sex-specific variability. A large range of the ratio of the female and male coefficient of variation (CV) was found for every parameter among the respective strain data sets. This clearly demonstrated the appearance of unpredictable major interactions between genotype and environment regarding the sex-specific variability of the blood parameters analyzed., (© 2021. The Author(s).)

Background: Increased levels of blood plasma urea were used as phenotypic parameter for establishing novel mouse models for kidney diseases on the genetic background of C3H inbred mice in the phenotype-driven Munich ENU mouse mutagenesis project. The phenotypically dominant mutant line HST014 was established and further analyzed., Methods: Analysis of the causative mutation as well as the standardized, systemic phenotypic analysis of the mutant line was carried out., Results: The causative mutation was detected in the potassium channel tetramerization domain containing 1 (Kctd1) gene which leads to the amino acid exchange Kctd1 I27N thereby affecting the functional BTB domain of the protein. This line is the first mouse model harboring a Kctd1 mutation. Kctd1 I27N homozygous mutant mice die perinatally. Standardized, systemic phenotypic analysis of Kctd1 I27N heterozygous mutants was carried out in the German Mouse Clinic (GMC). Systematic morphological investigation of the external physical appearance did not detect the specific alterations that are described in KCTD1 mutant human patients affected by the scalp-ear-nipple (SEN) syndrome. The main pathological phenotype of the Kctd1 I27N heterozygous mutant mice consists of kidney dysfunction and secondary effects thereof, without gross additional primary alterations in the other phenotypic parameters analyzed. Genome-wide transcriptome profiling analysis at the age of 4 months revealed about 100 differentially expressed genes (DEGs) in kidneys of Kctd1 I27N heterozygous mutants as compared to wild-type controls., Conclusions: In summary, the main alteration of the Kctd1 I27N heterozygous mutants consists in kidney dysfunction. Additional analyses in 9-21 week-old heterozygous mutants revealed only few minor effects.

Background: Type I Bartter syndrome is a recessive human nephropathy caused by loss-of-function mutations in the SLC12A1 gene coding for the Na+-K+-2Cl- cotransporter NKCC2. We recently established the mutant mouse line Slc12a1I299F exhibiting kidney defects highly similar to the late-onset manifestation of this hereditary human disease. Besides the kidney defects, low blood pressure and osteopenia were revealed in the homozygous mutant mice which were also described in humans. Beside its strong expression in the kidney, NKCC2 has been also shown to be expressed in other tissues in rodents i.e. the gastrointestinal tract, pancreatic beta cells, and specific compartments of the ear, nasal tissue and eye., Results: To examine if, besides kidney defects, further organ systems and/or metabolic pathways are affected by the Slc12a1I299F mutation as primary or secondary effects, we describe a standardized, systemic phenotypic analysis of the mutant mouse line Slc12a1I299F in the German Mouse Clinic. Slc12a1I299F homozygous mutant mice and Slc12a1I299F heterozygous mutant littermates as controls were tested at the age of 4-6 months. Beside the already published changes in blood pressure and bone metabolism, a significantly lower body weight and fat content were found as new phenotypes for Slc12a1I299F homozygous mutant mice. Small additional effects included a mild erythropenic anemia in homozygous mutant males as well as a slight hyperalgesia in homozygous mutant females. For other functions, such as immunology, lung function and neurology, no distinct alterations were observed., Conclusions: In this systemic analysis no clear primary effects of the Slc12a1I299F mutation appeared for the organs other than the kidneys where Slc12a1 expression has been described. On the other hand, long-term effects additional and/or secondary to the kidney lesions might also appear in humans harboring SLC12A1 mutations.

Background: Genome- and population-wide re-sequencing would allow for most efficient detection of causal trait variants. However, despite a strong decrease of costs for next-generation sequencing in the last few years, re-sequencing of large numbers of individuals is not yet affordable. We therefore resorted to re-sequencing of a limited number of bovine animals selected to explain a major proportion of the population's genomic variation, so called key animals, in order to provide a catalogue of functional variants and a substrate for population- and genome-wide imputation of variable sites., Results: Forty-three animals accounting for about 69 percent of the genetic diversity of the Fleckvieh population, a cattle breed of Southern Germany and Austria, were sequenced with coverages ranging from 4.17 to 24.98 and averaging 7.46. After alignment to the reference genome (UMD3.1) and multi-sample variant calling, more than 17 million variant positions were identified, about 90 percent biallelic single nucleotide variants (SNVs) and 10 percent short insertions and deletions (InDels). The comparison with high-density chip data revealed a sensitivity of at least 92 percent and a specificity of 81 percent for sequencing based genotyping, and 97 percent and 93 percent when a imputation step was included. There are 91,733 variants in coding regions of 18,444 genes, 46 percent being non-synonymous exchanges, of which 575 variants are predicted to cause premature stop codons. Three variants are listed in the OMIA database as causal for specific phenotypes., Conclusions: Low- to medium-coverage re-sequencing of individuals explaining a major fraction of a population's genomic variation allows for the efficient and reliable detection of most variants. Imputation strongly improves genotype quality of lowly covered samples and thus enables maximum density genotyping by sequencing. The functional annotation of variants provides the basis for exhaustive genotype imputation in the population, e.g., for highest-resolution genome-wide association studies.

Background: Currently, genome-wide evaluation of cattle populations is based on SNP-genotyping using ~ 54,000 SNP. Increasing the number of markers might improve genomic predictions and power of genome-wide association studies. Imputation of genotypes makes it possible to extrapolate genotypes from lower to higher density arrays based on a representative reference sample for which genotypes are obtained at higher density., Methods: Genotypes using 639 214 SNP were available for 797 bulls of the Fleckvieh cattle breed. The data set was divided into a reference and a validation population. Genotypes for all SNP except those included in the BovineSNP50 Bead chip were masked and subsequently imputed for animals of the validation population. Imputation of genotypes was performed with Beagle, findhap.f90, MaCH and Minimac. The accuracy of the imputed genotypes was assessed for four different scenarios including 50, 100, 200 and 400 animals as reference population. The reference animals were selected to account for 78.03%, 89.21%, 97.47% and > 99% of the gene pool of the genotyped population, respectively., Results: Imputation accuracy increased as the number of animals and relatives in the reference population increased. Population-based algorithms provided highly reliable imputation of genotypes, even for scenarios with 50 and 100 reference animals only. Using MaCH and Minimac, the correlation between true and imputed genotypes was > 0.975 with 100 reference animals only. Pre-phasing the genotypes of both the reference and validation populations not only provided highly accurate imputed genotypes but was also computationally efficient. Genome-wide analysis of imputation accuracy led to the identification of many misplaced SNP., Conclusions: Genotyping key animals at high density and subsequent population-based genotype imputation yield high imputation accuracy. Pre-phasing the genotypes of the reference and validation populations is computationally efficient and results in high imputation accuracy, even when the reference population is small.

Background: Clinical chemical blood analysis including plasma electrolytes is routinely carried out for the diagnosis of various organ diseases. Phenotype-driven N-ethyl-N-nitrosourea (ENU) mouse mutagenesis projects used plasma electrolytes as parameters for the generation of novel animal models for human diseases., Methods: Here, we retrospectively evaluated the use of the plasma electrolytes calcium, chloride, inorganic phosphorus, potassium and sodium in the Munich ENU mouse mutagenesis project where clinical chemical blood analysis was carried out on more than 20,000 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in various plasma parameter levels., Results: We identified a small number of animals consistently exhibiting altered plasma electrolyte values. Transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of mutant lines for the parameters calcium and potassium. Published data from other phenotype-driven ENU projects also included only a small number of mutant lines which were generated according to altered plasma electrolyte levels., Conclusion: Thus, use of plasma electrolytes detected few mouse mutants in ENU projects compared to other clinical chemical blood parameters.