GERMAN MOUSE CLINIC, Martin Hrab ě de Angelis , Director

Mouse Line DUF1220KO(Domain Deletion from PDE4DIP: phosphodiesterase 4D interacting protein (myomegalin))

GERMAN MOUSE CLINIC,Martin Hrabě de Angelis, Director

Collaboration withMajesta O‘Bleness, Jonathon Keeney and James SikelaBiochemistry and Molecular GeneticsUniversity of Colorado Anschutz Medical Campus/USA

Body Composition (qNMR)

female male Linear model Linear model Linear model

control mutant control mutant sex genotype body mass

n=15 n=15 n=15 n=13

mean ± sd mean ± sd mean ± sd mean ± sd p-value p-value p-value

Body mass 20.7 ± 1.5 20.8 ± 1.9 25.8 ± 2.4 24.9 ± 2.7 < 0.001 0.49 NA

Fat mass 4.6 ± 0.6 4.7 ± 0.6 5.1 ± 0.6 5.2 ± 0.9 < 0.001 0.035 < 0.001

Lean mass 12.9 ± 0.8 13 ± 1.2 17 ± 1.6 16.1 ± 1.7 < 0.001 0.121 < 0.001

female male Linear model Linear model Linear model

control mutant control mutant sex genotype body mass

n=15 n=15 n=12 n=13


Body mass 23.8 ± 2.4 23.1 ± 2.1 29.1 ± 2.6 28.5 ± 4 < 0.001 0.386 NA

Fat mass 5.9 ± 1.2 5.3 ± 0.8 6.5 ± 1.1 6.7 ± 1.8 < 0.001 0.629 < 0.001

Lean mass 14.5 ± 1.1 14.5 ± 1.2 18.7 ± 1.6 18.2 ± 2.3 < 0.001 0.519 < 0.001

Body Composition (qNMR)

Linear mixed effects model

sex genotype measurement body mass genotype:measurement

p-value p-value p-value p-value p-value

Body mass

< 0.001 0.467 < 0.001 NA 0.388

Fat mass < 0.001 0.205 0.555 < 0.001 0.055

Lean mass

< 0.001 0.683 0.161 < 0.001 0.005

Body Composition (qNMR) – Only Young

Energy Metabolism: Results

Indirect calorimetry

Female male Linear model Linear model Linear model Linear model

control mutant control mutant sex genotype body mass sex:genotype

n=15 n=15 n=15 n=13

mean ± sd mean ± sd mean ± sd mean ± sd p-value p-value p-value p-value

avg. mass [g]

21 ± 1.5 21.1 ± 1.8 26.1 ± 2.4 25.2 ± 2.7 < 0.001 0.52 NA 0.41

Food intake [g]

2.4 ± 0.4 2.6 ± 0.4 2.7 ± 0.7 2.5 ± 0.6 0.427 0.994 0.677 0.106

avg. VO2 [ml/(h animal)]

81.77 ± 7.44 84.61 ± 8.78 87.01 ± 7.51 84.95 ± 6.45 0.004 0.465 < 0.001 0.378

min. VO2 [ml/(h animal)]

56.27 ± 5.60 57.13 ± 5.01 58.8 ± 7.61 55 ± 6.81 < 0.001 0.544 < 0.001 0.27

max. VO2 [ml/(h animal)]

110.4 ± 10.55 113.6 ± 11.95 121.53 ± 9.91 119.23 ± 11.45 0.075 0.485 < 0.001 0.586

avg. RER [VCO2/VO2]

0.89 ± 0.03 0.89 ± 0.03 0.90 ± 0.04 0.90 ± 0.04 0.42 0.884 NA 0.576

avg. distance [cm] 4909 ± 1576 6236 ± 1459 4585 ± 1769 5589 ± 1802 0.269 0.01 NA 0.711

avg. rearing [counts] 127 ± 92 194 ± 130 98 ± 51 157 ± 122 0.237 0.024 NA 0.883

Energy Metabolism: Results

Energy Metabolism: Conclusions

Duf1220 ko mice were hyperactive both regarding locomotor activity as well as rearing behavior. The hyperactivity had no major effects on energy metabolism related parameters.

Results IpGTT

Fasting induced body mass loss vs. Initial body weight

DUF1220ko mutant mice

Body mass loss due to overnight food withdrawal mainly dependedon initial body mass.No clear genotype effect was detected for body mass loss.

DUF1220ko Estimate CI p-value

(Intercept) -0.3 [ -1.52 , 0.91 ] 0.62

genotypemutant 0.18 [ -0.19 , 0.55 ] 0.33

sexm -0.36 [ -0.82 , 0.09 ] 0.11

bw_before 0.12 [ 0.06 , 0.17 ] <0.001

genotypemutant:sexm 0.12 [ -0.41 , 0.65 ] 0.65

Results IpGTTPlasma glucose concentration [mmol/l] during IpGTT

AUC 0-30 AUC 30-120

We see a trend towards higher AUC values in male mutants, but the differences are quite small with largely overlapping ranges. Therefore this result might be accidental.

DUF1220ko female male Linear model Linear model Linear model

control mutant control mutant genotype sex genotype:sex

n=15 n=14 n=15 n=13


Glucose (T=0) 4.82 ± 0.71 4.53 ± 0.55 5.15 ± 0.71 5.35 ± 0.77 0.804 0.003 0.19

AUC 0-30 274.04 ± 37.57 290.16 ± 51.13 280.51 ± 66.73 325.07 ± 55.39 0.038 0.152 0.323

AUC 30-120 435.58 ± 88.13 463.38 ± 99.17 568.93 ± 109.89 693.61 ± 177.25 0.022 < 0.001 0.139

Clinical Chemistry Results, Fasting

Means, standard deviation and p-values calculated by a linear model(parameter ~ genotype * sex)

DUF1220ko female maleLinear model

Linear model

Linear model

control mutant control mutant genotype sexgenotype:

sex

n=15 n=15 n=15 n=14


fasting Glucose [mmol/l] 10.95 ± 1.59a 13.05 ± 2.92a 14.9 ± 3.62 12.48 ± 2.28 0.832 0.026 0.003

fasting Cholesterol [mmol/l] 2.447 ± 0.224a 2.324 ± 0.289a 2.689 ± 0.311 2.738 ± 0.205 0.604 < 0.001 0.231

fasting HDL-cholesterol [mmol/l] 1.824 ± 0.173a 1.735 ± 0.212a 2.031 ± 0.277 2.143 ± 0.15 0.845 < 0.001 0.084

fasting non-HDL Cholesterol [mmol/l]

0.623 ± 0.071a 0.589 ± 0.089a 0.657 ± 0.157 0.595 ± 0.1 0.116 0.505 0.629

fasting Triglycerides [mmol/l] 0.716 ± 0.134a 0.704 ± 0.166a 0.784 ± 0.107 0.753 ± 0.154 0.57 0.131 0.794

fasting NEFA [mmol/l] 0.58 ± 0.14a 0.6 ± 0.23a 0.58 ± 0.15 0.67 ± 0.16 0.236 0.495 0.423

fasting Glycerol [mmol/l] 0.192 ± 0.048a 0.162 ± 0.049a 0.153 ± 0.03 0.165 ± 0.055 0.471 0.157 0.088

Four female animals – 1 control, 3 mutant – not tested because of too low body weight.Glucose

Clinical Chemistry Results

Means, standard deviation and p-values calculated by a linear model(parameter ~ genotype * sex)

Sodium

One female Mut excluded ID 30264548 > severe kidney disease

Chloride


Linear model

Linear model


sex

n=15 n=14 n=12 n=13


Sodium [mmol/l] 143 ± 1 143 ± 1 147 ± 1 145 ± 2 0.031 < 0.001 0.032

Potassium [mmol/l] 3.8 ± 0.4 3.8 ± 0.3 4.3 ± 0.2 4.1 ± 0.2 0.059 < 0.001 0.273

Chloride [mmol/l] 106.8 ± 1.3 106.9 ± 2.1 108.5 ± 1.2 106.9 ± 1.7 0.104 0.067 0.066

Total protein [g/l] 52.7 ± 2.8 52.8 ± 2 53.9 ± 1.1 53.8 ± 1.9 0.989 0.057 0.857

Albumin [g/l] 28.3 ± 1.5 28.3 ± 1.3 27.5 ± 0.9 27 ± 1.3 0.399 0.003 0.519

Creatinine enz. [mumol/l] 14.73 ± 0.98 15.2 ± 1.61 15.12 ± 2.21 15.25 ± 1.42 0.496 0.618 0.699

Urea [mmol/l] 10.58 ± 1.72 10.14 ± 0.96 11.81 ± 1.34 11.76 ± 1.27 0.508 < 0.001 0.606

Potassium

Clinical Chemistry: Results

Means, standard deviation and p-values calculated by a linear model (parameter ~ genotype * sex)

ASATALAT LDH


Linear model

Linear model


sex

n=15 n=14 n=12 n=13


Cholesterol [mmol/l] 2.024 ± 0.265 2.024 ± 0.284 2.343 ± 0.237 2.288 ± 0.188 0.684 < 0.001 0.688

Triglyceride [mmol/l] 0.975 ± 0.185 0.888 ± 0.205 1.255 ± 0.305 1.255 ± 0.434 0.588 < 0.001 0.587

ALAT/GPT [U/l] 25 ± 4 27 ± 5 36 ± 15 66 ± 57 0.04 0.003 0.084

ASAT/GOT [U/l] 53 ± 14 57 ± 15 56 ± 14 87 ± 71 0.081 0.097 0.177

alpha-Amylase [U/l] 572.42 ± 59.29 577.08 ± 61.36 776.37 ± 153.99 790.11 ± 171.33 0.779 < 0.001 0.89

Glucose [mmol/l] 13.3 ± 1.4 13.32 ± 1.53 13.18 ± 2.08 13.66 ± 1.43 0.576 0.808 0.605

LDH [U/l] 125.7 ± 38.3 137.9 ± 33.9 172.5 ± 56.7 255 ± 168.1 0.061 0.002 0.161One female Mut excluded ID 30264548 > severe kidney disease

Clinical Chemistry: Results


ALPOne female Mut excluded ID 30264548 > severe kidney disease


Linear model

Linear model


sex

n=15 n=14 n=12 n=13


Calcium [mmol/l] 2.45 ± 0.08 2.43 ± 0.08 2.34 ± 0.1 2.34 ± 0.08 0.75 < 0.001 0.728

Inorganic phosphate [mmol/l] 1.6 ± 0.2 1.6 ± 0.3 1.6 ± 0.3 1.6 ± 0.3 0.55 0.857 0.99

Iron [mumol/l] 24.9 ± 4.3 23.8 ± 4.1 19.6 ± 2.9 20.7 ± 2.4 0.986 < 0.001 0.26

ALP [U/l] 164 ± 26 176 ± 22 106 ± 15 95 ± 15 0.91 < 0.001 0.046


Hematology: Results

DUF1220ko female male Linear model Linear model Linear model

control mutant control mutant genotype sex genotype:sex

n=15 n=15 n=12 n=12


RBC [Mio/mm³] 11.16 ± 0.58 11.29 ± 0.58 11.19 ± 0.65 11.08 ± 0.61 0.972 0.592 0.459

HGB [g/dl] 17.27 ± 0.76 17.53 ± 0.87 16.57 ± 1.12 16.48 ± 1.17 0.752 0.002 0.531

HCT [percent] 53.79 ± 2.63 54.49 ± 2.84 54.73 ± 2.53 54.95 ± 2.18 0.516 0.331 0.738

MCV [fl] 48.33 ± 0.9 48.4 ± 0.74 49 ± 2.09 49.75 ± 1.29 0.259 0.007 0.344

MCH [pg] 15.49 ± 0.55 15.53 ± 0.37 14.79 ± 0.54 14.87 ± 0.49 0.688 < 0.001 0.877

MCHC [g/dl] 32.13 ± 0.83 32.16 ± 0.66 30.3 ± 1.64 29.98 ± 1.48 0.662 < 0.001 0.59

RDW [percent] 14.89 ± 0.41 14.81 ± 0.46 15.24 ± 0.37 15.25 ± 0.72 0.794 0.005 0.748

WBC [10³/mm³] 12.23 ± 3.04 11.49 ± 3.27 11.32 ± 2.69 11.08 ± 3.48 0.573 0.449 0.776

PLT [10³/mm³] 967.2 ± 95.54 948.8 ± 123.16 1246.75 ± 191.89 1262.67 ± 253.32 0.979 < 0.001 0.715

MPV [fl] 6.12 ± 0.15 6.13 ± 0.1 6 ± 0.17 6 ± 0.18 0.936 0.004 0.936

PDW [fL] 5.37 ± 0.24 5.37 ± 0.16 5.25 ± 0.18 5.19 ± 0.24 0.609 0.013 0.609

PLCR [percent] 1.78 ± 0.39 2 ± 0.33 1.59 ± 0.61 1.66 ± 0.53 0.266 0.043 0.55

PCT [percent] 0.59 ± 0.06 0.58 ± 0.07 0.75 ± 0.11 0.75 ± 0.15 0.929 < 0.001 0.857

Data of one animal excluded due to technical problems (30264526, male mutant)

No evidence of remarkable genotype-related differences.

Behaviour: OF Results

Forward locomotion

No significant genotype effects on locomotor activity


Vertical locomotion

No genotype effect on rearing activity


Anxiety-related behaviour

No genotype effects on centre time

Behaviour: Acoustic Startle Reactivity

Increased ASR by the female mutant mice

Behaviour: Prepulse Inhibition

No genotype effect on PPI

Neurology: SHIRPA-Results

No difference in SHIRPA parameters

Locomotor activity measured as the numbers of squares crossed in the first 30 seconds after transfer into the vieving arena.

No difference in grip strength

Neurology: Results

Since we are especially interested in mitochondria function we measure as a marker lactate levels in collaboration with the Clinical Chemistry Screen

Neurology: Results

White 2001, Sex Transm Infect 2001;77:158-173 doi:10.1136/sti.77.3.158

No difference

No difference in mean latencies, usual improvement over trials,But slightly more often falling

Neurology: Results

Hotplate Test

Nociception: Results

Type of reaction on hotplate

mean ± sd mean ± sd mean ± sd mean ± sd pairwise(Tukey)

pairwise(Tukey)

ANOVA ANOVA ANOVA

female female male male females malescontrol mutant control mutant mutant-

controlmutant-control

genotype sex sex:genotype

N=15 N=15 N=15 N=13 adj. p-value adj. p-value p-value p-value p-valueFirst response time

8.2 ± 2.65 9.26 ± 2.94 10.98 ± 3.48 8.78 ± 2.85 0.768 0.224 0.52 0.125 0.043

Second response time

20.84 ± 33.6 13.07 ± 2.77 15.64 ± 4.79 13.52 ± 3.89 0.616 0.988 0.275 0.618 0.541

Female Male

Control Mutant Control Mutant

n=15 n=15 n=15 n=13

1st response type

hind paw licking 4 8 9 4

hind paw shaking 11 7 6 9

2nd response type

hind paw licking 11 7 6 9

hind paw shaking 4 8 9 4

0 = no reaction at all

1 = no Preyer reflex

2 = retarded reaction

3 = normal reaction

4 = strong reaction

5 = particularly strong reaction

Dysmorphology: Results

Clickbox Test

Score Males Females

Control Mutant Control Mutant

0 7 4 12 13

1 - - - -

2 4 4 2 -

3 4 5 1 2

4 - - - -

5 - - - -

Median 2.00 2.00 0.00 0.00

BL6 background→ become deaf with age

→ no significance (Fisher’s Exact test)

Cardiovascular: Results Echocardiography

Cardiovascular: Results Electrocardiography

Cardiovascular: Results Electrocardiography

** *

Eye: Results

Scheimpflug Imaging

control Duf1220

Eye: Results

Scheimpflug Imaging

Eye: Results

Virtual Drum (vision test)

Levels of Immunoglobulins in Blood Plasma

Levels of Immunoglobulins in Blood Plasma [µg/ml]

Frequencies of Leukocyte Subsets in Blood [% of respective parent gate]

Frequencies of Leukocyte Subsets in Blood [% of all leukocytes]

Lower frequencies of T cells and a slightly lower CD4: CD8 T cell ratio in mutants of both sexes

IgE

female male

female male overallcontrol mutant control mutant

13 14 9 7median median median median

p-value p-value p-value[25%, 75%] [25%, 75%] [25%, 75%] [25%, 75%]

88.7 [57.3,114.8]

90.2 [46.5,182.4]

36.4 [16.7,46.7]

75.4 [32.95,81.35]

0.896 0.365 0.643

IgE Results

•Total plasma IgE in Duf1220KO mice (1st sample)Median, first and third quartile and p-values calculated by a Wilcoxon rank-sum test.

IgE Results

Box plot of total plasma IgE in Duf1220KO mice ng/mlNo statistically significant differences in the IgE levels between control and mutants mice

TEWL Measurement

female male female male overall

control mutant control mutant

n = 15 n = 15 n = 15 n = 13

[25%, 75%] [25%, 75%] [25%, 75%] [25%, 75%] p-value p-value p-value

28.1 [26.65,33.4]

30.3 [29.05,33.15]

33.2 [29.75,39.4]

36.1 [27.1,46.2]

0.34 0.892 0.856

Box plot of transepidermal water loss (TEWL) [g/(m²h)] in Duf1220KO miceMedian, first and third quartile and p-values calculated by a Wilcoxon rank-sum test.

NO statistically significant differences in the TEWL levels between control and mutants male mice.

TEWL Measurement

Lung Function Parameters: Duf1220KO Data are presented as mean SD.

Parameter Female Controln=6

Female Mutant n=5

Wilcoxon-test

Body Mass (BM) [g] 24 ± 3.8 23.9 ± 3.9 1

Volumetric Parameters

Tidal Volume (TV) [ml] 0.205 ± 0.009 0.209 ± 0.004 0.424

Inspiratory Capacity (IC) [ml] 0.79 ± 0.15 0.916 ± 0.059 0.082

Expiratory Reserve Volume (ERV) [ml] 0.16 ± 0.05 0.16 ± 0.04 0.894

Vital Capacity (VC) [ml] 0.96 ± 0.19 1.06 ± 0.06 0.128

Functional Residual Capacity (FRC) [ml] ND ND

Residual Volume (RV) [ml] ND ND

Total Lung Capacity (TLC) [ml] ND ND

Forced Vital Capacity (FVC) [ml] 0.973 ± 0.176 1.079 ± 0.059 0.082

Flow Parameters

Forced Expiratory Volume (FEV100) [ml] 0.944 ± 0.156 1.037 ± 0.05 0.082

Peak Expiratory Flow (PEF) [ml/sec] 30.7 ± 2.1 31.6 ± 3.9 0.266

Mechanical Parameters

Static Lung Compliance (Cchord) [ml/cm H2O] 0.0528 ± 0.0121 0.0569 ± 0.013 0.429

Dynamic Lung Compliance (Cdyn.) [ml/cm H2O] 0.0239 ± 0.005 0.025 ± 0.0024 0.452

Lung Resistance (R) [cm H2O/ml/sec] 1.4 ± 0.11 1.34 ± 0.08 0.329

Lung Function: Results

Pathology: Results Organ Weight

female male female male overall

control mutant control mutant

n=9 n=9 n=8 n=8

median [25%, 75%]

median [25%, 75%]

median [25%, 75%]

median [25%, 75%]

p-value

p-value

p-value

Heart weight [mg] 121

[119 , 128]

126 [117 , 126]

155.5 [148.5 , 162.8]

143.5 [136.8 ,

149] 0.983 0.13 0.448

Tibia length [mm] 17.9

[17.8 , 18.2]

17.9 [17.6 , 18]

18.1 [17.9 , 18.3]

18.3 [18.1 , 18.4]

0.474 0.442 0.899

Liver weight [g] 1.182

[1.109 , 1.209]

1.135 [1.112 , 1.255]

1.685 [1.59 , 1.714]

1.618 [1.56 , 1.772]

0.863 1 0.939

Spleen weight [g] 0.095

[0.088 , 0.116]

0.098 [0.085 , 0.121]

0.084 [0.079 , 0.088]

0.087 [0.084 , 0.098]

0.65 0.456 0.438

Body weight [g] 23.176 [22.11 , 23.714]

22.43 [20.958 , 23.882]

32.517 [29.934 , 32.773]

29.596 [28.241 , 33.197]

0.605 0.505 0.474

Heart Weight / Tibia Length [mg/mm]

6.8 [6.51 , 7.36]

7 [6.64 , 7.16]

8.64 [8.19 , 8.96]

7.82 [7.54 , 8.07]

0.931 0.052 0.362

Heart Weight / Body Weight [mg/g]

5.4 [5.07 , 5.78]

5.59 [5.37 , 5.62]

4.89 [4.62 , 5.17]

4.92 [4.42 , 5.03]

0.589 0.798 0.925

Liver Weight / Tibia Length [mg/mm]

65.85 [62.94 , 66.43]

64.34 [61.78 , 69.99]

91.12 [88.08 , 92.81]

88.22 [85.81 , 94.39]

1 0.645 0.708

Liver Weight / Body Weight [mg/g]

50.88 [47.58 , 52.17]

52.89 [50.86 , 53.72]

52.72 [51.56 , 53.72]

53.09 [52.52 , 54.17]

0.113 0.328 0.073

Spleen Weight / Tibia Length [mg/mm]

5.27 [4.95 , 6.44]

5.71 [4.72 , 6.66]

4.67 [4.34 , 4.91]

4.8 [4.59 , 5.12]

0.649 0.524 0.546

Spleen Weight / Body Weight [mg/g]

4.14 [3.9 , 4.46]

4.11 [3.84 , 4.94]

2.73 [2.51 , 2.93]

2.94 [2.86 , 3.01]

0.605 0.234 0.518

Pathology: Results Organ Weight

No statistically significant difference of total and normalized heart weight was detected comparing mutant and control mice.

Slide 43

Skin NAD Pancreas NAD

Musculoskeletal system NAD Cervical lymph node NAD

Eyes NAD Thymus NAD

Brain NAD Spleen NAD

Cerebellum NAD Thyroid NAD

Heart / Arteries NAD Parathyroid NAD

Trachea NAD Adrenal gland NAD

Lung NAD Kidneys NAD

Teeth NAD Urinary bladder NAD

Salivary gland NAD Testes NAD

Esophagus NAD Epididymis NAD

Stomach NAD Funiculus spermaticus NAD

Small intestine NAD Ovaries NAD

Large intestine NAD Uterus NAD

Liver NAD Vagina NAD

Mamma NAD Body weight NAD

Pathology: Results Overview

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GERMAN MOUSE CLINIC, Martin Hrab ě de Angelis , Director