Starbuck JM, Llambrich S, Gonzalez R, et al. Epigallocetechin-3-gallate improves facial dysmorphology associated with Down syndrome. BioRxiv 276493 [Preprint]. https//dx.doi.org.10.1101/276493. Posted March 5, 2018. Accessed June 20, 2018.
To determine if epigallocatechin-3-gallate (EGCG) can positively treat the facial dysmorphologies associated with Trisomy 21 (T21), commonly known as Down syndrome.
This was a mouse treatment study and a human observation study.
Mice consisting of 4 wild type (WT) males and 6 Ts65Dn (TS or Down syndrome) females served as the breeding colony. Fifty-five mice from 8 litters were included in the experiments. Mice had to be within the variation from WT to Ts65Dn with disparate facial phenotypes.
Human participants were 288 children from Europe and North America aged 0 to 18 years (divided into 3 groups: 0-3 years, 4-12 years, and 13-18 years) recruited between 2009 and 2017. Children had to have either Down syndrome, or Down syndrome mosaic with a quantified percentage of trisomic cells (50%-80%), or be euploid (normal number of chromosomes).
Mice were randomly treated with low-dose EGCG at 9 mg/kg/d or high dose EGCG at 30 mg/kg/d from embryonic day 9 to postnatal day 29. The EGCG was from Mega Green Tea Extract, Life Extension, USA with 326.25 mg EGCG per capsule, which was dissolved in water. A 20 g mouse drinks about 2 mL of water per day.
In children with T21 the effect of EGCG on facial dysmorphology depended on the age when treatment began.
The parents of the children who participated in the study were aware that EGCG had procognitive effects in Down syndrome. The use of EGCG in the children was not homogeneous or standardized. The children received a dose that was approximately equivalent to 8 cups of green tea per day or the 9 mg/kg/d that had been shown to improve adaptive functionality and cognitive deficits associated with Down syndrome.1,2
Study Parameters Assessed
Mouse and human facial shapes were assessed using geometric morphometric methods, including assessment of 3-dimensional (3D) facial shape. Facial images in 3D were acquired from euploid children, mosaic children, and Down syndrome children. Euclidean distance matrix analysis (EDMA) was used to calculate linear distances between facial landmarks for each mouse and human.
Primary Outcome Measures
Euclidean distance matrix analysis (EDMA) was used to assess local differences between pairs and across the sample in both mice and humans. Relative facial improvement scores (FIS) based on the EDMA were used to assess improvement in percentages. Multivariate regression of shape on age was done within each of the 3 age groups. Principal components analysis (PCA) was used for facial morphology variation and for grouping by genotype and treatment group.
In mice, the procrustes landmark (generalized procrustes analysis [GPA]) only slightly overlapped in the facial shapes of wild type and TS that were untreated. EGCG GPA varied depending on the dose with high variability, in some cases potentially harmful, in the high-dose group. The low-dose EGCG group had a moderate variation in facial shapes, more like the WT face shape. The WT demonstrated no effect on facial shape and 80% of the TS mice fell within the range of variation found in untreated WT mice. Facial improvement scores, although not significant, confirmed the negative effect of high-dose EGCG and the positive effect of low-dose EGCG.
In children with T21 the effect of EGCG on facial dysmorphology depended on the age when treatment began. Children aged 13 to 18 years completely overlapped with T21 that had never received treatment. Of the 2 children aged 4 to 12 years who were treated with EGCG, 1 fell within the euploid range of variation and the other in the overlap between T21 and euploid. In children aged 0 to 3 years, 6 of 7 were in the intermediate space between T21 and euploid, with 3 of the 6 in the range of variation for euploids and 3 of the 6 having milder T21 facial features. One of the 7 children had facial features in the range of untreated T21. In the children aged 0 to 3 years, the mosaic case with the lowest percentage of trisomic cells (50%) was in the middle of the range for euploid children, the 2 mosaic children with 70% and 80% trisomic cells overlapped with euploid and T21 facial phenotypes but had much less T21 facial features.
In children aged 13 to 18 years, comparing euploid and T21, 61% of linear facial measurements differed significantly, while EGCG treatment reduced this to 53% compared to euploid. In children 4 to 12 years, comparing euploid and T21, 59% of linear facial distances were significantly different while EGCG treatment reduced this to 46%. In children 0 to 3 years, comparing euploid and T21, 57% of facial linear distances were significantly different, while EGCG treatment reduced this to 25% compared to euploid, or an improvement in over half of the facial measurements compared to baseline. Facial improvement scores were positive in age groups 13-18 and 4-12, but they were not statistically significant. In the 0-3 group, the FIS was 58%, significant (P=0.008), and beneficial.
In mice, high-dose EGCG (30 mg/kg/d) had negative effects on facial changes while the low dose (9 mg/kg/d) had positive effects. In humans, the low-dose equivalent had the greatest positive effect on children aged 0 to 3 years compared to children in the other age groups (4-12 years and 13-18 years). Mice received the EGCG from day 9 post-conception, while the children received it after birth from 0 to 18 years old.
Trisomy 21, commonly known as Down syndrome, occurs in approximately 1 per 800 live births and is the most common chromosomal malformation in newborns.3 It was named after Jack Langdon Down, MD, who fully described it in 1866, although some aspects of it were noted in 1838 and 1844.4 The oldest known skeleton is a child who died about 1,500 years ago and was buried in a 5th and 6th century necropolis near a church in Chalon-sur-Saone in eastern France.5 Children with Down syndrome are at risk for congenital heart defects, respiratory infections, gastrointestinal tract problems, celiac disease, cataracts and other ocular abnormalities, thyroid disorders, orthopedic disorders, leukemia and testicular cancer, and neurobehavioral and psychiatric problems. Median life expectancy has increased to 49 years in the last 2 decades.4
Epigallocatechin-3-gallate is a kinase inhibitor, specifically tyrosine-Y-phosphorylation regulated 1A DYRK1A; DYRK1A is overexpressed in T21 and affects memory recognition, working memory, and quality of life.1 It may also have a role in cranial morphology and long-bone skeletal architecture. Work by the de la Torre group demonstrated in both mice and humans that EGCG had a positive effect on transgenic trisomy-16 mice, transgenic mice overexpressing DRYK1A, and ECGC was associated with fewer cognitive deficits in T21 humans.2 They also found that it reduced elevated homocysteine levels that were correlated with DRYK1A over expression. Lastly, EGCG in mice has been shown to improve cognitive function associated with triplication of DYRK1A.2
Epigallocatechin-3-gallate restores hippocampal development. The fetus with T21 has a smaller-than-normal cerebellum, frontal cortex, and hippocampus,2 which reduces the total number of neurons. There are also fewer dendritic branches, shorter dendrites, and reduced myelination of the nerve axon.2 The result is slower conduction of nerve action potentials and increased cross-talk among neurons. Treatment of DYRK1A transgenic mice at 21 days old with EGCG restored hippocampal neurogenesis and improved the prefrontal cortex.6-8 Treatment of adult mice (3-4 months old) with EGCG for 1 month restored gamma-aminobutyric acid (GABA)–ergic and glutamatergic pathways in the cortex and hippocampus and improved behavior deficits.9 The restoration of GABA-ergic and glutamatergic pathways with EGCG demonstrated that it was decaffeinated EGCG, and not caffeine itself, that produced these effects.
A separate double-blind, placebo-controlled phase 2 study of EGCG and cognitive training was done in adults with T21, aged 16 to 34 years old. There were 43 participants in the treatment group and 41 in the placebo (rice flour) group.1 Both received cognitive training and either EGCG at 9 mg/kg/d or placebo for 12 months. At 12 months the EGCG group had higher scores in visual recognition memory (P=0.039), inhibitory control (P=0.041), cat and dog response time tests (P=0.024), adaptive behavior (P=0.002), cholesterol (P=0.019), and homocysteine (P=0.015). There were no significant changes in social skills (P=0.071), social functioning and relation with peers (P=0.078), or liver enzyme levels (aspartate transaminase [AST]; P=0.831 and alanine transaminase [ALT]; P=0.623). Adverse events were rated mild and not thought to be related to the treatment. Positive effects of EGCG and cognitive training on memory and executive functions continued at 6 months after treatment but cholesterol and homocysteine improvements did not. Functional MRIs done at baseline, 6 months, and 12 months were significant in the frontal cortex of the EGCG group.
Why was EGCG chosen as the experimental agent? Cell and kinetic studies demonstrated that EGCG is a potent inhibitor of DYRK1A,10 a proline/arginine-directed serine/threonine kinase strongly implicated in the learning deficits of T21.11 Extra copies of DYRK1A in mice produce learning impairments encountered in T21. Histological and MRI studies have shown that DYRK1A is involved in the development and control of brain volume and cell density in some regions of the brain.11 Because EGCG is a known and safe inhibitor of DYRK1A, it was logical to assess the effects of EGCG on mouse T21 models and humans with trisomy-21. The facial changes in the 0-3 age group versus the 13-18 age group correlates with closure of bone growth as the face matures at about 16 to 18 years.1 What is fascinating is both the speed and outcomes that the above studies have demonstrated to date.
The treatment dose for the children was not specifically stated in this paper but was determined by reading the several other papers by this research group.1,2 The degree of compliance was also unfortunately not specifically stated but again gathered from reviewing this group’s other papers.1,2
In this complex mouse and human study, Euclidean facial morphology measurements were taken of WT mice, Ts65Dn mice, and their offspring after treatment with either EGCG at 30 mg/kg/d or 9 mg/kg/d from day 9 until after conception. The Ts65Dn mice in both treatment groups had facial changes with greater normalization of facial features with the low dose and significant but not always beneficial changes with the high dose The children took EGCG at the 9 mg/kg/d dose and were divided into age groups, 0-3, 4-12, and 13-18 years. The greatest changes were seen in the youngest age group and the least changes were seen in the oldest group. This is most likely related to facial bone plasticity that decreases with maturation. No adverse facial changes were report in the human portion of the trial.