Down Syndrome Trisomy 21:
Understanding the Problem
Richard C. Deth, PhDDepartment of Pharmaceutical SciencesNortheastern University
Key Points:
• DS (trisomy of Chr21) is a result of Chr21 nondisjunction during meiosis (cell division of egg/sperm)
• Impaired methylation contributes to DS risk
• Epigenetic regulation of gene expression is the driver of development
•Genes on Chr21 can affect development via their effect on cellular oxidative state and methylation status
• Metabolic support of methylation may help optimize the abilities of DS individuals
Down Syndrome Trisomy Chr 21:
Trisomy 21 Causes Down SyndromeBy: Clare O'Connor, Ph.D. (Biology Department, Boston College) © 2008 Nature Education
Nondisjunction of linked sister chromatids can occur at two different stages of egg (or sperm) development:Meiosis I and Meiosis II
Trisomy begins with maternal gametogenesis, whichoccurs within the maternal grandmother’s body,during her pregnancy.
FaultyGamete
Production(mainly ova)
Ovulationand
Conception
PrenatalDevelopment
PostnatalDevelopment
Mother and
Placenta
Breastfeedingand
Nutrition
Methylation plays an important role in all phases of conception and development
Meiosis I Meiosis IIOocytes
formduring
maternal fetal
development
Motheror
Father
MaternalGrandmother
Years
Prophase
DNA Methyltransferase
Cytosine 5-Me Cytosine
DNA Methylation
Sites for Histone Methylation
Epigenetic Changes: Methylation of DNA or Histone
Regulation of gene expression during development
X
Gene sequence
Start site for mRNA synthesis
TF
Growth Factors
Neurotransmitters Hormones
RNA polymerase mRNA
Protein(e.g. enzyme)
DNA
TranscriptionTranslation
DNADNA + Histone = HeterochromatinGenes are silenced and transcription is blocked
Me Me
MBDP(e.g. MeCP2) Histone
proteins
HMT
Me Me
Me MeDNMT
SAM
SAMCpG CpG
Transcription Factor Regulation:
Epigenetic Regulation:
TF binding region
TF binding region
CpG CpG
No mRNAX
METHIONINE CYCLE
HCY
MET
SAH
SAM
~1,000MethylationReaction
svia 209
MTases
( - )
ATP PP + Pi
DIETARY PROTEIN
THF
MethylTHF
MethionineSynthase
Adenosine
Vitamin B12(Cobalamin)
A reversible reaction!
( + )
Global methylation =
SAMSAH( )
Glutathione (GSH) Redox
Statusγ-
Glutamylcysteine
Cysteine
Cystathionine
HCY
MethionineCycle
SAH
Adenosine
> 1,000Methylatio
nReactions
SAM
( - )
ATP PP + Pi
THF
Methyl-THF
MethionineSynthase
MET
Adenosine
D4-SAH
ATPPP + Pi
Dopamine (Attention)
PhospholipidMethylation
D4-HCY
D4-METD4-SAM
Methyl-THF
THF THF
D4 DopamineReceptor PLM Cycle CBS
GSHGSSG
=
Transsulfuration
24% decrease in HCY in DS47% decrease in MET in DS 350% increase in cystathionine
15% decrease in cysteine in DS
Consistent with a decrease in methioninesynthase activity and an increase in CBS activity
25% decrease in combined SAM and SAH in DS33% increase in adenosine in DS
Consistent with a decrease in methioninesynthase activity and an increase in CBS activity
Glutathione (GSH) Redox
Statusγ-
Glutamylcysteine
Cysteine
Cystathionine
HCY
↓Methionine
CycleSAH
Adenosine
> 1,000Methylatio
nReactions
SAM
( - )
ATP PP + Pi
THF
Methyl-THF
MethionineSynthase
MET
Adenosine
D4-SAH
ATPPP + Pi
Dopamine (Attention)
PhospholipidMethylation
D4-HCY
D4-METD4-SAM
Methyl-THF
THF THF
D4 DopamineReceptor PLM Cycle CBS
GSHGSSG
=
↑Transsulfurat
ion
In DS: Increased transsulfurationDecreased methylation
Methionine synthase has five domains + cobalamin (Vitamin B12)Domains alternate interacting with cobalamin during turnover
SAM Domain
CobalaminDomain
CapDomain
Cobalamin(vitamin B12)
SAM Domain
CobalaminDomain
CapDomain
5-Methyl THF Domain
HCY Domain
Cobalamin(vitamin B12)
1
23
B12 serves as a sensor of
redox status
REDOXSTATUS:
GSHGSSH
MethylationStatus:
SAMSAH
~ 1,000 MethylationReactions
Nitric OxideSynthesis
PhospholipidMethylation
DNA/HistoneMethylation
GeneExpression
ArginineMethylation
MembraneProperties
CreatineSynthesis
CognitiveStatus
EnergyStatus
CatecholamineMethylation
SerotoninMethylation
Melatonin
Sleep
HCY FOL CAP COB SAM
187 bp 197 bp 419 bp
3' 5'
Exon 19 252420
188 bp 122 bp
21 22 23
HCY FOL CAP COB SAM
187 bp 197 bp 419 bp
3' 5'
Exon 19 252420
188 bp 122 bp
21 22 23
Dr. Christina Muratore - 2010 Research Fellow in Neurology Harvard Medical School
Domain-specific PCR analysis of MS mRNA in postmortem frontal cortex
Age-dependent decrease of Cob and Cap domain mRNA in postmortem frontal cortex in 49 subjects
Cobalamin-binding Domain Cap Domain
Methionine synthase mRNA is lower but protein levels are not different in cortex of autistic subjects
100
150
MW(kD)
250C1 C2 C3 C4 A3 A4 A5A1 A2C5
AutisticControl
MS DNA
MS pre-RNA
MS RNA
MSHCY
MET SAM
SAH
[SAM]
[SAH]
CystathionineCysteineGSH
CellularRedox Status
DNATranscription
MethylationRNA
Splicing
TranslationMET t-RNA
Protein
Ubiquitination
TNF-α( - )
Methylation( - )
( + )Amino Acids
Global Metabolic
Coordination
METHIONINE SYNTHASE REGULATION
GLOBAL REGULATION
AlternativeSplicing
DNA Methylation( - )
Methionine synthase provides redox-sensitiveglobal coordination of metabolism: HOMEOSTASIS
RISK OF DS
In a number of studies (but not all) impaired methylation status and/or elevated homocysteine in the mother was shown to influence the risk of Down Syndrome
(i.e. poor methylation may increase the risk of nondisjunction)
But, what about grandma?
After 10 years (1999–2009) of active research in the field thequestion of whether or not polymorphisms in folate/Hcymetabolizing genes are associated with increased DS risk is stilllargely debated in literature, and none of the studied polymorphisms can be firmly considered as an independent DS risk factor[15–40].
Even if MTHFR 677C > T, MTHFR 1298A > C and MTRR66A > G gene polymorphisms gave positive results in severalindependent studies, results are still conflicting and inconclusive
Increased HCY in mothers of DS individuals was found in a number of studies,but was not found in other studies (e.g. in France). MTHFR 677C > T correlateswith increased HCY.
CBS(833 ins 68)
Genetic variants in methylation pathway associated with DS
Combinations of methylation-related SNPs increase the maternal risk of having a DS baby in some studies (e.g. up to a 7-fold increase).
Higher HCY levels are associated with lower IQ.
Zampieri et al. (2012) found significant risk associationsfor maternal age, MTHFR 677 C>T,and Transcobalamin 766 C>G, butdecreased risk for BHMT 742 G>A.
DS children have a lower SAM/SAH, indicative of impaired methylation capacity
Role of oxidative stress and methylation during development
Eggs are richIn cysteine
Sperm are rich in selenium
GSH
GSH
GSH
GSH
GSH
GSH
∆ DNA and Histone Methylation
∆ GeneExpression
Does Redox ControlDevelopment ViaEpigenetic Effects?
juanv.wordpress.com/
Which chromosome 21 genes contribute to DS?
Answer: All of them contribute something.
Which genes are more important for causing Down Syndrome?
Answer: Methylation-related genes
Which genes are more important for cognitive development?
Answer: Methylation-related genes
Chromosome 21 Genes and Down Syndrome
• Chromosome 21 has about 400 genes. Increased gene dosage from each of them probably contributes to Down Syndrome characteristics.
• Certain genes deserve special attention for their relationship to oxidative stress and methylation, which are the foundation of development, especially brain development.
• These genes is located in the region of chromosome 21 (21q21-22) that has been implicated as being most important for DS.
• Increased activity of the proteins produced by these genes is likely to contribute to DS.
Amyloid precursor protein (APP)
Cystathionine-beta-synthase (CBS)
DNA methyltransferase 3L (DNMT3L)
Formiminotransferase cyclodeaminase (FTCD)
Superoxide dismutase 1 (SOD1)
Formiminotransferase cyclodeaminase (FTCD):
Removes an imino group from forminoglutamate (FIGLU), attaches it to THF, and converts it to methenylTHF.
This provides a back-up source of methylTHF to support methionine synthase and methylation.
Formiminotransferase cyclodeaminase
FIGLU
In trisomy, a higher than normal activity of FTCD might provide extra methyl groups to compensate for decreased MTHFR activity.
MTHFR
AMYLOID PRECURSOR PROTEIN (APP):
A cleavage product of amyloid precursor protein (APP), known as Aβ, is though to be the primary cause of Alzheimer’s disease (AD).
Amyloid plaques rich in Aβ are found at autopsy in AD brain, but the neurodegeneration is thought to be caused by small Aβ oligomers, starting decades before the onset of AD symptoms.
AD is much more common in DS, presumably because the extra APP gene leads to increased Aβ.
• Early onset familial AD▫ APP▫ Presenilin 1 and 2
• Late onset AD▫ APOE4▫ LRP1▫ A2M▫ AD5-8▫ HLA-A▫ NOS3▫ PAXIP1▫ MS▫ MTHFR
Current Genetics of Alzheimer’s Disease
OMIM.org
Amyloid Processing Amyloid Processing
http://www.bath.ac.uk/bio-sci/research/profiles/brown-d.html
Neurons have impaired transsulfuration and low GSH levels that depend upon growth factor-stimulated cysteine uptake
MethionineSynthase
HCY
MET
SAH
SAM
>1,000Methylation Reactions
ATP PP+Pi
Adenosine
MethylTHF
THF
Cystathionine
Cysteine
GSH
γ-Glutamylcysteine
D4HCY
D4SAM
D4SAH
D4METATPPP+Pi
MethylTHF
THF
PhospholipidMethylation
Adenosine
Dopamine
Cysteine
( - )
PI3-kinase ( + )
PARTIALLY BLOCKED IN NEURONAL CELLS
EAAT3
AstrocytesCysteinylglycine GSH
GSSG
NeurotrophicGrowth Factors
GSCbl
MeCbl
SAMOHCbl
GSH
NEURON
Cystine
EAAT3
Soluble Aβ oligomers inhibit cysteine uptake, increase oxidative stress and
decrease methylation capacity in neuroblastoma cells
0
50
100
150
*
Intr
acel
lula
r C
yste
ine
nM
ol/m
g p
rote
in
0
10
20
30
40
50
*
GS
H/G
SS
G R
atio
0
2
4
6
8
*
SA
M/S
AH
Rat
io
0.0
0.5
1.0
1.5
*
*
7PA2CHO
L-[
35S
]-C
yste
ine
Up
take
(n
mo
l/m
g p
rote
in)
0.0
0.5
1.0
1.5
*
L-[
35S
]-C
yste
ine
Up
take
(nm
ol/m
g pr
otei
n)
A-β oligomers decrease DNA methylation and alter expression of redox/methylation
pathway genes in neuroblastoma cells
Control
CHO-CM
7PA2-
CMIG
F-1
CHO-CM
+ IG
F-1
7PA2-
CM +
IGF-1
7PA2-
CM +
IGF-1
+ W
MN
LBTBA
LBTBA + IG
F-1
CHO-CM
+ A
W7
7PA2-
CM +
AW
70.0
0.1
0.2
0.3
0.4
0.5
*
*
*
#
*
**
5-M
eth
ylcy
tosi
ne
(ng
)
Our Amyloid Hypothesis
Cysteine
EAAT3
SAH
SAM
HCY
MET
Cystathionine
Cysteine
GSH
γ-Glutamylcysteine
GSSG
Cystine
Homocystine
MethylTHF
THF
ATP PP+P
MethionineSynthase
Adenosine
DNA
Methyl-DNA
GSH GSCblSAM
OHCbl
MeCbl
Cysteinylglycine GSHHealthy
Glial Cells(Astrocytes)
( - )
PI3-kinase
( + )
Abeta Oligomers
Epigenetic Changes
Nate HodgsonPhD Aug 2012
• A-β inhibits cysteine uptake, decreasesDNA methylation and alters gene expression.
• This is likely to be a natural role for A-β, promoting oxidative stress in neurons.
• The extra APP and A-β production occurring in trisomy 21 may produce excessive oxidative stress, with adverse epigenetic consequences.
Superoxide dismutase 1 (SOD1)
• Superoxide anion is a reactive oxygen species (ROS) produced by mitochondria as a by-product during ATP synthesis
• SOD converts superoxide to hydrogen peroxide
Data from Waly et al. (In Prep)
Interestingly, methionine synthase has B12-dependent SOD activity, which is essential for GSH-dependent reactivation of enzyme activity after B12 oxidation
Cystathionine beta synthase (CBS):
• CBS converts HCY to cystathionine in a vitamin CBS converts HCY to cystathionine in a vitamin B6-dependent reactionB6-dependent reaction
• CBS activity is increased by SAM but increased CBS activity is increased by SAM but increased by oxidative stress and TNF-alphaby oxidative stress and TNF-alpha
• Vitamin D was recently shown to increase the Vitamin D was recently shown to increase the level of CBS level of CBS
• Testosterone decreases CBS activity Testosterone decreases CBS activity
• CBS also converts cysteine to hydrogen sulfideCBS also converts cysteine to hydrogen sulfide
Transsulfuration
Pathway Glutathione (GSH) Redox
Statusγ-Glutamylcystein
e
Cysteine
Cystathionine
HCY SAH
Adenosine
> 1,000Methylatio
nReactions
SAM
( - )
ATP PP + Pi
THF
Methyl-THF
MethionineSynthase
MET
THF
GSHGSSG=~
CBS
Cysteine
HydrogenSulfide
SAMVitamin DOxidative
stressTHF-alpha
(+)
Testosterone(-)
Table 2 | Intellectual disability by gender (n=121)
Males FemalesTotalcount % count % count %
mild 9 12.9 14 27.5 23 19.0moderate 23 32.9 13 25.5 36 29.8severe 21 30.0 19 37.3 40 33.1profound 17 24.3 5 9.8 22 18.2
Males are affected more severely than females
Excessive CBS activity in DS limitsmethylation by removing HCY fromthe methionine cycle.
The critical balance between methylation and transsulfuration is therefore altered.
DNA methyltransferase 3L (DNMT3L):
• Does not directly attach methyl groups to DNA,but forms a complex with other methyltransferasesand alters their activity.
• Important for gender-specific DNA methylation
In the absence of Dnmt3L, neither methylation of most oocyte-methylatedgDMRs nor intragenic methylation was observed. There was also genome-wide hypomethylation, and partial methylation at particular retrotransposons, while maintaining global gene expression, in oocytes. Along with the identification of the many Dnmt3L-dependent gDMRs at intragenic regions, the present results suggest that oocyte methylation can be divided into 2 types: Dnmt3L-dependent methylation, which is required for maternal methylation imprinting, and Dnmt3L-independentmethylation, which might be essential for endogenous retroviral DNA silencing. The present data provide entirely new perspectives on the evaluation of epigenetic markers in germline cells.
In other words, DNMT3L is critical for oocyte methylation.
SUMMARY
• > 400 genes on Chr 21 contribute to Down syndrome.
• The relatively high survival rates for Chr 21 trisomy indicates that the higher expression of genes creates an acceptable alternative pathway for development.
• Impaired methylation, importantly involving altered DNA methylation, is a primary factor in causing DS.
• Altered patterns of methylation continue to be important throughout the lifespan.
• Metabolic interventions which address oxidative stress and improve methylation capacity may be beneficial.
• Controlled clinical trials of these interventions are needed.
Brain Samples:Autism Tissue ProgramHarvard Brain Tissue Resource CenterTissue Resource Center (Australia)Stanley Medical Research Foundationand donor families.
Collaborators:Antonio PersicoSuzanne De la MonteHamid AbdolmalekyMostafa WalyYahya Al-Farsi
Grant Support:Autism Research InstituteSafeMindsNational Autism AssociationAutism Speaks
ACKNOWLEDGEMENTS