Scientific Meeting on the Rarer Types of 19 November 2019 EDS: From Genetics to Management
Skin and Vascular Abnormalities in Chst14 Gene-Deleted Mice, an Animal Model of Musculocontractual Ehlers-Danlos Syndrome Takahiro Yoshizawa, Shuji Mizumoto, Yuki Takahashi, Shin Shimada, Kazuyuki Sugahara, Jun Nakayama, Shin’ichi Takeda, Yoshihiro Nomura, Yuko Nitahara-Kasahara, Takashi Okada, Kiyoshi Matsumoto, Shuhei Yamada, Tomoki Kosho
Musculocontractural Ehlers–Danlos Syndrome (mcEDS) is a new form of EDS, delineated clinically, molecularly, and biochemically
Symptoms of mcEDS
Multiple congenital malformations Progressive fragility-related Talipes deformities manifestations
Skin hyperextensibility and fragility Large hematoma (skin and vascular symptom) is one of the most serious complications accompanied by decreased QOL Large subcutaneous hematomas Spinal deformities and potential lethality. Kosho, 2016
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D4ST1 enzyme deficiency caused by CHST14 gene mutations
Gene mutation and change of glycosylation in mcEDS-CHST14
T. Kosho et al. CHST14 gene mutation Loss of D4ST1 (Dermatan 4-sulfotransferase 1) enzyme activity Abnormality of glycosylation (loss of DS chain connected with decorin)
Defect of D4ST1 via CHST14 gene mutation induced abnormality of glycosylation and affected connective tissues function in mcEDS.
So far, mcEDS reported characteristic symptoms including connective tissue malfunction and identified a causative gene.
T. Kosho et al. In the future, it is the long-term aim to develop the therapeutic drug of this disease… Model animal of this disease is necessary for clarify detailed mechanisms to reach a goal.
We want to clarify cause of the serious symptoms including large hematomas.
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Reproduction of Chst14 -/- mice
Genotyping (Parents: Chst14 +/-) +/+ +/- -/- Total
Adult 135(28%) 336(70%) 6(1%) 477(100%) Embryo(E18.5) 40(27%) 87(58%) 22(15%) 149(100%) l Chst14 gene deleted mouse is regarded as an useful model animal of mcEDS. l However, most of Chst14 -/- were died in perinatal stage. l On the other hand, Chst14 -/- embryos, just before delivery, were survived stably .
The plan of analysis of Chst14 -/- mice
① Analysis of the mice before delivery
②Trial of the birth rate improvement ③Analysis of the adult mice
①Analysis of the mice before delivery
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Analysis of Chst14 KO fetus
Crown-rump length (mm) ** 25 **
20 E18.5 15
10
5
0 +/+ +/- -/- ave
() Weight 5mm 1.6
Chst14 +/+ Chst14 +/- Chst14 -/- 1.2
0.8
0.4
0 +/+ +/- -/- 1
Morphological abnormality of the placenta
Appearances of the placenta (chorionic plate side)
Chst14 +/+ Chst14 +/- Chst14 -/- hypoxia necrosis Placental Weight (E18.5) (mg) 90
80 Chst14 gene could play an
70 important role in the development
60 and/or function of the placenta
50 +/- +/+ 1 -/-
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Blood vessel of the placenta
Appearances of chorionic plate
Chst14 +/+ Chst14 +/- Chst14 -/-
Disproportionation of the blood vessel diameter Blood vessel knob
Placenta of Chst14 -/- showed structural abnormalities of blood vessel.
Blood capillary of chorion frondosum
vascular lumen basement membrane endothelial cell
trophoblast
+/+ +/- -/-
0.20
m)
μ 0.16
0.12
0.08 Thickness of of Thickness 0.04
basement membrane ( membrane basement 0.00 +/+ +/1 - -/- Placenta of Chst14 -/- showed abnormal structures of capillary basement membrane, one of extracellular matrix.
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rupture of basement membrane Chst14/D4ST1 is important for development of extracellular matrix of blood vessel in the placenta. It suggested that abnormality of capillary basement membrane related with hematoma in mcEDS.
Fragility-related complications of mcEDS were observed in childhood and adulthood patients. However, phenotype of blood vessel has not been reported in the postnatal mice……
Analysis of skin phenotypes in adult Chst14-/- is necessary.
②Trial of the birth rate improvement
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BALB/c congenic Chst14 KO
Before backcrossing +/+ +/- -/- Total F0 x F0 135(28%) 336(70%) 6(1%) 477(100%)
129;B6 Chst14 KO (usual) Backcross into BALB/c strain
After backcrossing for 12 times +/+ +/- -/- Total F12 x F12 41(31%) 84(64%) 6(5%) 131(100%) BALB. Chst14 KO
BALB/c : inbreed Chst14 strain that shows Weaning rate of -/- was improved. high weaning rate.
③Analysis of the adult mice
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Pathological analysis of skin in BALB.Chst14 KO
H&E • Unclearly collagen fiber bundle • Disorder of collagen fibril assembly • Structural abnormality of TEM capillary basement membrane (derma)
Chst14 gene-deletion induced structural changes of TEM VEC VEC extracellular matrix. And it (skin capillary) may influence skin function RBC RBC
+/+ -/-
Wound healing model in BALB.Chst14 KO
+/+
-/-
0 3 6 9 12 15 (days after wounding) 0.25 * 10 0 )
2 * 0.20 75 ** ** 0.15 50 Chst14 gene-deletion induced ●: +/+ (n=10) significant delay of wound 0.10 * 25 ** ●: -/- (n=8) healing in vivo.
Wound area (cm 0.05 0 0 3 6 9 12 15 (day) % of wound% of closure (%) 0.00 -25 0 3 6 9 12 15 (day)
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Inflammation after the wound in BALB.Chst14 KO
Day 7 after wounding
H&E (×40)
Additional Explanation H&E (×100)
+/+ -/- Generally, neutrophil shows plural nucleuses. Increased inflammation may related with delayed wound healing
Summary & Conclusion
Animal models Patients
Placental Vascular abnormality abnormality ???
Mechanisms of ? mcEDS Blood vessel? Delay of Proliferation? wound healing Inflammation?
CHST14 may regulate function of extracellular matrix and support skin function (e.g. wound healing).
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