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

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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