Integrative morphometric study exploring the effects of Epigallocatechin 3-gallate on craniofacial development in Down syndrome

Abstract

Introduction: Down syndrome (DS) is a genetic condition resulting from trisomy of chromosome 21 that is characterized by cognitive impairment, immunodeficiency and congenital heart diseases, along with skeletal and facial dysmorphologies. DS phenotypes are associated with genetic imbalance, and overexpression of specific genes located in the DS critical region, such as DYRK1A, seem to have a more important contribution. DYRK1A encodes a protein kinase whose downstream targets are involved in neurogenesis but also skeletal development. Epigallocatechin-3-gallate (EGCG), a green tea flavonoid, is a specific DYRK1A inhibitor that can normalize the kinase activity of DYRK1A and improve cognitive deficits in both humans with DS and trisomic Ts65Dn mice. Since DYRK1A is expressed within the facial prominences during embryonic development, we hypothesized that DYRK1A overexpression in craniofacial primordia may contribute to craniofacial dysmorphology in DS, and that normalizing DYRK1A kinase activity using an EGCG treatment may influence DS craniofacial morphology. Materials & Methods: We have analyzed the impact of DYRK1A kinase activity normalization using EGCG treatment in craniofacial development in DS, both in humans and mice. In humans, we relied on patients of different DS associations to follow the typical development in DS. A number of those patients had taken green tea extracts not under medical supervision that we also included, even though the dosage and time of treatment were not controlled. To characterize the human facial shape, we recorded the 3D coordinates of facial landmarks using a non-invasive photogrammetric technique and performed a General Procrustes analysis (GPA). In mice we performed a shape analysis comparing the craniofacial morphology of euploid and trisomic mice along the early postnatal period. In addition, in a controlled preclinical longitudinal study, we administered EGCG to the mother and the pups from embryonic day 9 (E9) to postnatal day 29. To characterize the cranial shape, we used a μCT scanning, from which we collected a set of 49 skull landmarks. Results: In humans, Principal Component Analysis (PCA) based on the Procrustes coordinates adjusted for age, revealed that the DS population is different from the euploid population, and that DS individuals treated with EGCG present an attenuated DS facial phenotype closer to the euploid average shape configuration. In mice, the PCA based on the Procrustes coordinates suggests that an overinhibition of Dyrk1A may have deleterious effects on Ts65Dn craniofacial development suggesting a dosage-dependent DYRK1A phenotype. Conclusion: Our results indicate the capacity of the EGCG to modulate the craniofacial morphology. We highlight the importance to find an optimal therapeutic window in order to avoid the adverse effects.

Introduction: Down syndrome (DS) is a genetic condition resulting from trisomy of chromosome 21 that is characterized by cognitive impairment, immunodeficiency and congenital heart diseases, along with skeletal and facial dysmorphologies. DS phenotypes are associated with genetic imbalance, and overexpression of specific genes located in the DS critical region, such as DYRK1A, seem to have a more important contribution. DYRK1A encodes a protein kinase whose downstream targets are involved in neurogenesis but also skeletal development. Epigallocatechin-3-gallate (EGCG), a green tea flavonoid, is a specific DYRK1A inhibitor that can normalize the kinase activity of DYRK1A and improve cognitive deficits in both humans with DS and trisomic Ts65Dn mice. Since DYRK1A is expressed within the facial prominences during embryonic development, we hypothesized that DYRK1A overexpression in craniofacial primordia may contribute to craniofacial dysmorphology in DS, and that normalizing DYRK1A kinase activity using an EGCG treatment may influence DS craniofacial morphology. Materials & Methods: We have analyzed the impact of DYRK1A kinase activity normalization using EGCG treatment in craniofacial development in DS, both in humans and mice. In humans, we relied on patients of different DS associations to follow the typical development in DS. A number of those patients had taken green tea extracts not under medical supervision that we also included, even though the dosage and time of treatment were not controlled. To characterize the human facial shape, we recorded the 3D coordinates of facial landmarks using a non-invasive photogrammetric technique and performed a General Procrustes analysis (GPA). In mice we performed a shape analysis comparing the craniofacial morphology of euploid and trisomic mice along the early postnatal period. In addition, in a controlled preclinical longitudinal study, we administered EGCG to the mother and the pups from embryonic day 9 (E9) to postnatal day 29. To characterize the cranial shape, we used a μCT scanning, from which we collected a set of 49 skull landmarks. Results: In humans, Principal Component Analysis (PCA) based on the Procrustes coordinates adjusted for age, revealed that the DS population is different from the euploid population, and that DS individuals treated with EGCG present an attenuated DS facial phenotype closer to the euploid average shape configuration. In mice, the PCA based on the Procrustes coordinates suggests that an overinhibition of Dyrk1A may have deleterious effects on Ts65Dn craniofacial development suggesting a dosage-dependent DYRK1A phenotype. Conclusion: Our results indicate the capacity of the EGCG to modulate the craniofacial morphology. We highlight the importance to find an optimal therapeutic window in order to avoid the adverse effects.