Additional file 1: of Standardized, systemic phenotypic analysis reveals kidney dysfunction as main alteration of Kctd1 I27N mutant mice

Time points of the phenotypic analyses for line Kctd1 I27N in the German Mouse Clinic (GMC) (DOCX 32 kb

Influence of main factors (enrichment, sex, cohort) on mean of parameters in 2 mouse strains.

<p>(A) The heatmap shows the results for all metric parameters (rows) and influencing factors and their double interactions (columns). The B6 and D2 data are on the left and right of each column, respectively. The influence of main factors was evaluated using linear models. A variable selection using the BIC was performed. Reference categories are enrichment none, sex female, and the first cohort. The differences between means of parameters are expressed as color-coded estimators β of the respective model in percent of the intercept of the model. The estimator β describes the change induced by the respective factors. The intercept is the mean of the parameter of the reference group and is set as 100%. Grey shade: variable selection using the BIC yielded nonrelevant influencing factor, thus no effect can be assumed; violet shade: factor is relevant for the model, i.e., mean is increased compared to reference group; yellow shade: factor is relevant for the model, i.e., mean is decreased compared to reference group; strength of color reflects size of difference. “X” marks parameters that were not analyzed. (B) Color grading scheme: the color grading illustrates the change in percent of the intercept; the strength of color reflects the size of difference. ABR, auditory brain stem response; ALAT/GPT, Alanine aminotransferase/Glutamat pyruvat transaminase; ALP, Alkaline phosphatase; ASAT/GOT, Aspartate aminotransferase/Glutamat oxalacetat transaminase; ASR, acoustic startle response; AU400, name of clinical chemistry analyzer; AUC, area under the curve; BIC, Bayesian Information Criterion; BN, background noise; CNPG3, substrate of test reaction (2-Chloro-4-Nitrophenyl-α-D-Maltotriosid); DEXA, dual-energy x-ray absorptiometry; HR, heart rate; HRV, heart rate variability; IgE, immunoglobulin E; IpGTT, intraperitoneal glucose tolerance test; LDH, Lactat-dehydrogenase; LVIDd, left ventricular end-diastolic internal diameter; LVIDs, left ventricular end-systolic internal diameter; LVPW, left ventricular posterior wall; P1-P4, startle amplitude following pulse of 67 (P1), 69 (P2), 73 (P3), 81 (P4) dB; PP (67, 69, 73, 81), startle amplitude and PPI following startle pulse (110 dB) with preceding pre-pulse of 67, 69, 73, 81 dB; PPI, pre-pulse inhibition; qNMR, quantitative nuclear magnetic resonance; QTc, corrected QT; R1, region analyzed (whole body excluding the skull); SHIRPA, Smithkline Beecham, MRC Harwell, Imperial College, the Royal London Hospital Phenotype Assessment; ST 110, acoustic startle response at 110 dB.</p

Workflow for the screens of this study.

<p>DEXA, dual-energy x-ray absorptiometry; IpGTT, intraperitoneal glucose tolerance test; NMR, nuclear magnetic resonance; PPI, pre-pulse inhibition; SHIRPA, Smithkline Beecham, MRC Harwell, Imperial College, the Royal London Hospital Phenotype Assessment.</p

Animal numbers used for this study.

<p>Animal numbers used for this study.</p

Raw data of selected parameters on the background of the biological range for B6.

<p>Raw data of the 4 main parameters of the open field test: (A) “distance traveled total,” (B) “number of rears total,” (C) “percent center distance total,” and (D) “center permanence time” as box- and whisker-plots. The box represents 25th percentile, median, and 75th percentile; the length of whiskers is maximally the 1.5-fold interquartile range but is determined by the last value within this range. All individual values are shown for each experimental group (“con,” nest, double) and every cohort (1, 2, 3) for female and male mice in the upper and lower plot, respectively, for each selected parameter (A–D). The range in the background gives 1 SD (dark shading) and 2 SD (bright shading) of >200 reference B6 female (red) and male (blue) mice. The reference mice were same-aged wild-type control mice from other phenotyping projects of the GMC and were measured within the same timespan as the mice used in this project. “con”, control; GMC, German Mouse Clinic.</p

Influence of main factors (enrichment, sex, cohort) on bootstrapped CVs in 2 strains.

<p>The heatmap shows the results for all metric parameters (rows) and influencing factors (columns) for B6 (left column) and D2 (right column). Reference categories are enrichment none, sex female, and the first cohort. Results of the bootstrap method are summarized by computed confidence intervals for estimators (β), and bootstrapped CVs are then classified into the following 3 categories: confidence interval for β includes 0, thus no effect is assumed (grey); confidence interval for β is greater than 0, i.e., bootstrapped CVs are increased compared to reference group (violet); confidence interval for β is below 0, i.e., bootstrapped CVs are decreased compared to reference group (yellow). “X” mark parameters that were not analyzed. ABR, auditory brain stem response; ALAT/GPT, Alanine aminotransferase/Glutamat pyruvat transaminase; ALP, Alkaline phosphatase; ASAT/GOT, Aspartate aminotransferase/Glutamat oxalacetat transaminase; ASR, acoustic startle response; AU400, name of clinical chemistry analyzer; AUC, area under the curve; BIC, Bayesian Information Criterion; BN, background noise; CNPG3, substrate of test reaction (2-Chloro-4-Nitrophenyl-α-D-Maltotriosid); CV, coefficient of variation; DEXA, dual-energy x-ray absorptiometry; HR, heart rate; HRV, heart rate variability; IgE, immunoglobulin E; IpGTT, intraperitoneal glucose tolerance test; LDH, Lactat-dehydrogenase; LVIDd, left ventricular end-diastolic internal diameter; LVIDs, left ventricular end-systolic internal diameter; LVPW, left ventricular posterior wall; P1-P4, startle amplitude following pulse of 67 (P1), 69 (P2), 73 (P3), 81 (P4) dB; PP (67, 69, 73, 81), startle amplitude and PPI following startle pulse (110 dB) with preceding pre-pulse of 67, 69, 73, 81 dB; PPI, pre-pulse inhibition; qNMR, quantitative nuclear magnetic resonance; QTc, corrected QT; R1, region analyzed (whole body excluding the skull); SHIRPA, Smithkline Beecham, MRC Harwell, Imperial College, the Royal London Hospital Phenotype Assessment; ST 110, acoustic startle response at 110 dB.</p

Small eye phenotype in <i>Zscan10</i> homozygous mutant mice.

<p>Lateral (A) and frontal (B) view of adult mutant (left) and wild type (right) mice. Lateral view of right eye of a wild type (C) and mutant (D) adult female. (E) Eye size measurements by laser interference biometry revealed significantly reduced axial eye length in mutants of both sexes (p<0.001). Anterior eye investigation by Scheimpflug imaging indicated significantly increased mean lens density between controls and mutants for left eyes in males (p = 0.002) (F) and right eyes in females (p = 0.016) (G). Virtual drum vision testing showed a reduced response with borderline significance in female mutants (p = 0.034) (H). (E) to (H) are displayed as boxplot split by sex and genotype. Outliers are indicated by open circles.</p

Hematological and clinical chemistry analysis.

<p>(A) Plasma electrolyte, protein, creatinine and urea concentrations of ad libitum fed mice. (B) Lipid and glucose levels as well as selected enzyme activities in plasma of ad libitum fed mice. (C) Plasma concentrations of minerals, iron and ALP activity of ad libitum fed mice. (D) Values obtained from EDTA-blood samples. Means, standard deviations and p-values for genotype, sex and genotype x sex interaction effects calculated by a linear model (A-D). Platelet (E) and mean platelet volume (F) boxplot strip chart, split by sex and genotype. (G) Concentration of steroid hormones in plasma: Medians, first and third quartile and p-values calculated by a Wilcoxon rank-sum test (n = 57). Points are individual animals; circles represent females (f); diamonds represent males (m) of <i>Zscan10</i> homozygous mutants (mut) and wild type littermates (con). Line within the boxplot represents median. Box represents the 25% and 75% quantile. Asterix represents the mean. Circled points: Values outside the upper whisker (min(max(x), 75% quantile +1.5*IQR) and outside the lower whisker (max(min(x), 25% quantile+1.5*IQR)  =  outliers. IQR  =  interquartile range (75% quantile–25% quantile).</p

Organ weight determined in pathology screen.

<p>(A) Normalized liver weight is significantly increased in male mutant mice (P = 0.008). (B) Absolute spleen weight is significantly reduced in mutant mice of both genders. (C) Normalized spleen weight is significantly reduced in female mutant mice (P = 0.002). (D) Absolute heart weight is significantly reduced in female mutant mice (p = 0.003). (E) Normalized heart weight is significantly reduced in female mutant mice (p = 0.016). All results demonstrated as boxplot with strip chart, split by sex and genotype. For tibia length refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104568#pone-0104568-g001" target="_blank">Figure 1F</a>. Points are individual animals; circles represent females (f); diamonds represent males (m) of <i>Zscan10</i> homozygous mutants (mut) and wild type littermates (con). Line within the boxplot represents median. Box represents the 25% and 75% quantile. Asterix represents the mean. Circled points: Values outside the upper whisker (min(max(x), 75% quantile +1.5*IQR) and outside the lower whisker (max(min(x), 25% quantile+1.5*IQR)  =  outliers. IQR  =  interquartile range (75% quantile–25% quantile).</p

Open Field Testing.

<p>Distance traveled (A), percent distance in the center (B) and percent time spent in the center (C). Total boxplot with strip chart, split by sex and genotype. Points are individual animals; circles represent females (f); diamonds represent males (m) of <i>Zscan10</i> homozygous mutants (mut) and wild type littermates (con). Line within the boxplot represents median. Box represents the 25% and 75% quantile. Asterix represents the mean. Circled points: Values outside the upper whisker (min(max(x), 75% quantile +1.5*IQR) and outside the lower whisker (max(min(x), 25% quantile+1.5*IQR)  =  outliers. IQR  =  interquartile range (75% quantile–25% quantile).</p