Folate And Methylation - (Mar/31/2005 )

Hi,

Your comments are very helpful to me. I am also fascinated by the one carbon metabolism. Although I know nothing about NTD, but to me, if you don't have enough folate, which might cause hypomethylation, and low folate may also cause oxidative stress since they all in the same pathway and I bet hyperhomocysteine might be there too. And this could be another reason.

absolutely Kestutis,

your questions are very valid, I suspect that methylation, albeit DNA or histones are involved in the developmental programming of the embryo, there are precise waves of methylation that reprogram the epigenetic marks on the genome.

as for NTD, the issue is that during develpoment if there is low folate the neural tube does not close properly, now there are lots of mechanisms involved, and I think it has to do with the way the cell is determined to undergo a certain fate or developmental pathway.....what makes a skin cell different to a lymphocyte? Simplisitically, they both have the same genome but certain genes are switched off or on depending on the cell type, through I think, epigenetics.

Now folate is involved in DNA sysnthesis and this may cause NTD, however the exact mechanism is not known, but I think it is the abberant epigenetic programming of the cells that were destined to migrate and close the neural tube that has caused NTD. How is this so.....I figure that the control of the genes that determine the cell fate is lost through low folate....however it must be a severe folate loss for this to happed as Kestutis rightly said there are other complex biochemical mechanisms that could compensate for the OCU deficency.......it is all a black box and needs to be determined.

hn37041, you are abosultely correct to suggest oxidative stress and hyperhomcystemia, these are things that have been looked at and are casued by low folate.

I would like to invite others to put their two cents into this topic....don't be scared!!!

Hi,
First, I would like to correct my statement: " NTD is very rare". Sorry, statistics I've found is: 1 NTD for 1000 and cancer rate is aproximately 5 for 1000, so both these problems are really comparable and extremly important not only in scientific point of view.
Second, yeasterday in Pubmed I've found more than thirty abstracts considering NTD and methylation (but hundred about NTD and Met!!!) and found some interesting facts - I would like to share, because I was lucky find out some answers for my questions.
There were observed some cases of NTD with normal folate but problematic B12 too. When studying the exelent website www.genome.ad.jp (what are the others of such a kind???) ( study I've used in this website was- pathway, Met metabolism, hommo sapiens, go, 2.1.1.37, EC 2.1.1.37 (in the light blue), disease, and information presented:

The absence of endogenous methylation in Drosophila facilitates
detection of experimentally induced methylation changes. Lyko et al.
(1999) expressed Dnmt1 and Dnmt3a in transgenic Drosophila melanogaster.
In this system, Dnmt3a functioned as a de novo methyltransferase,
whereas Dnmt1 had no detectable de novo methylation activity. When
coexpressed, Dnmt1 and Dnmt3a cooperated to establish and maintain
methylation patterns. Genomic DNA methylation impaired the viability of
transgenic flies, suggesting that cytosine methylation has functional
consequences for Drosophila development. The expression of Dnmt3a but
not Dnmt1 caused developmental defects in Drosophila, with the majority
dying in the pupal stage. Tissue-specific expression of Dnmt3a in the
Drosophila eye resulted in small or absent eyes.

Okano et al. (1999) generated mice with targeted disruption of the
Dnmt3a and Dnmt3b (602900) genes. Inactivation of both genes blocked de
novo methylation in embryonic stem cells and early embryos but had no
effect on maintenance of imprinted methylation patterns. Dnmt3a -/- mice
developed to term and appeared to be normal at birth. However, most
homozygous mutant mice became runted and died at about 4 weeks of age.
In contrast, no viable Dnmt3b -/- mice were recovered at birth.
Dissection of embryos at different stages of development revealed that
Dnmt3b -/- embryos had multiple developmental defects, including growth
impairment and rostral neural tube defects with variable severity at
later stages of development, though most of them appeared to develop
normally before E9.5. Dnmt3a and Dnmt3b also exhibited nonoverlapping
functions in development, with Dnmt3b specifically required for
methylation of centromeric minor satellite repeats. These results
indicated that both Dnmt3a and Dnmt3b are required for genomewide de
novo methylation and are essential for mammalian development.

So, in some cases there exists:
B12 dependent NTD PHENOTYPE
and Folate deficiance dependent NTD PHENOTYPE
and it was found, that
Dnmt3a and Dnmt3b disruption dependent NTD PHENOTYPE exists too...
Well, I suppose that those possible reasons are not only the side effects, so the last case makes very probable epigenetic DNA involvement, and it's time to conclude that treatment using not only folate but B12 and even I hope SAM could help. It depends!!
The simmilar clasiification of cancers I proposed when speaking to oncologists in ninethies.
When there will be obtained the answer about some histon methylation involvment, more precise PHENOTYPE clasification could be defined! Wide Folate treatment helped according to statistics, I'm trying to remember, aproximately for one half. So, perhaps in 50% we have Folate deficient (because of nutrition) with later consequencies phenotypes, and the others are slyghtly different I hope. It was the decision of nature...
I would like to rise one more question: what development program break causes embryo carcinoma and what NTD?
Some similarities - like DNA hypomethylation must be considered, but some differencies - I think the TIME MOMENT - makes this different. Were there observed embryo carcinomas on the 1-7 days of embryo existance? Perhaps no. Is it possible theoretically? Perhaps yes. What about 8-28 days of embryo?
I wish, dear Nick - You are the best hacker of cell control program starting from centromeras and publish this one day. Unfortunately not tomorrow.

Kestutis Urba
P.S. results about NTD and DNA methylases the role of oxydative stress not allow to (hyper) evaluate imediatelly, but its role must be revealed. Why there is stress only on tube (if) but not more wide?

Kestitus,

very valid questions you have pointed out. indeed DNMT defectiveness can cause NTD, my point of view is that if you deplete the cell of the OCU...SAM which is what DNMT uses to methylated DNA you are also effectively reducing the effectiveness of DNMT, there are histone methyltranferases (HMT) or Su(Var) homologs that also use SAM to methylate histones, as SAM can be derived from folate (and other pathways as you have found....very nice website by the way thanks for sharing that ) We thought this would be our point of focus.

Indeed there are embryonic carcinoma cell lines available and I am sure this could happen in nature although rare, and it is all a matter of timing and how sensitive the organism as a whole is to a change in the epigenetic marks. There may well be other events (molecular) occuring in folate deficent embryos apart from NTD that we are unable to detect phentypically, and this is what we would like to investigate.

I am really glad you are here to share your thoughts Kestutis, and I hope this will continue.

Nick

Hi Nick,
I've got aquaintance with NTD, folate, methylation problem -thanks, and this experience is very useful when trying to study cell malignisation processes having "hot point" - folate, methylation, OCU metabolism... secrets.
I would like to present very little summary (the main ideas) of PUBMED materials, obtained when searching "neural tube defect methylation" - 34 articles.
I've made the conclusion from this summary (bottom of message), that experiments on NTD problem shows such logical chain about involvment of:
A) folate deficience (food problem), folate metabolism sytem disturbances (something abnormal enzyms...), choline- betaine path, B12 problems, DNMT. This leads to HYPOTHESYS that some OCU controling and providing and continuing events are really involved in NTD, and some genes were investigated, but I've found some little contradictive statement -
Hmc affects cell movement neccesary for NTD - but how to explain DNMT influence?
I'll try to consider this question more deep, because to describe NTD development we need to use some other sciences too: fluid dynamics, electrophysiology....
SUMMARY:
PUBMED neural tube defect methylation -34

Folate nutrition

An overwhelming body of evidence for a protective effect of periconceptional
folic acid supplementation against neural tube defects (NTDs) led to mandatory
folic acid fortification in the United States Preliminary reports also suggest
a significant reduction ( approximately 15-50%) in NTDs in the United States.
folic acid supplementation may enhance the development and progression of already
existing, undiagnosed premalignant and malignant lesions.

Folate metabolism system

The purpose of this study is to investigate whether gene polymorphisms of methionine
synthase (MTR) and methionine synthase reductase (MTRR) are involved in the
risk for NTDs, specifically spina bifida. Our results indicate that MTRR and
MTR genes may interact to increase the infants' NTD risks.
Analysis of data on variants of two genes involved in homocysteine
remethylation/methionine
biosynthesis--methionine synthase (MTR) A2756G and methionine synthase reductase
(MTRR) A66G--provided evidence that both variants influence the risk of spina
bifida via the maternal rather than the embryonic genotype
Altered folate status and homozygous TT mutation in the MTHFR gene in both mother
and child would be expected to increase the risk of neural tube defects.
One of the most consistent, and possibly specific, differences between participant
groups is a statistically significant elevation of 5,10-methenyl-H(4)folate
in NTD mothers (affects three genotypes).

B12

Reduced vitamin B12 binding by transcobalamin II increases the risk of neural
tube defects
From studies with the rat embryo in vitro, it can be concluded that the de-
and remethylation cycle of methionine, being folate and vitamin B12 dependent,
is crucial for embryonic and fetal growth probably via generation of DNA, proteins
and polyamines.
Morphological changes induced by valproate and its administration concomitant
with folinic acid or S-adenosylmethionine in pregnant rats

the aetiology of NTDs. This genetic metabolic defect can be overcome by treatment
with folic acid and/or vitamin B12.
Mothers of children with neural-tube defects had significantly higher homocysteine
values (8.62 [SD 2.8] mumol/L) than did B12-matched

Chol-Bet

NTD risk estimates were lowest for women whose diets were rich in choline,
betaine, and methionine.
Inhibition of choline uptake and metabolism during neurulation results in growth
retardation and developmental defects that affect the neural tube and face.

HYPOTHESYS
HMC

Disturbance of maternal and fetal homocysteine metabolism has been associated
with fetal neural tube defects,
Thus, HoCys-induced changes in brain membrane composition correlated with
HoCys-induced
apoptosis and reduced embryo viability.
In order to unravel morphogenetic mechanisms involved in neural tube closure,
critical cell movements that are fundamental to remodelling of the cranial neural
tube in the chick embryo were studied in vitro by quantitative time-lapse video
microscopy. Two main directions of movements were observed
Homocysteine is adversely involved in human neural tube closure defects. After
application of a single dose of homocysteine to chick embryos, a closure delay
at the initial closure site and at the neuropores, flattening of the head fold
and neural tube, and a halt of cell movements was seen. A possible interference
of Hcy with actin microfilaments is discussed.
In persons inheriting the variant alleles of MTHFR the increase in the level
of homocysteine is noted resulting in the increased susceptibility to vascular
diseases and the neural tube defects in the progeny. The procedure recommended
for the prevention of effects of deficiency of MTHFR activity consists of the
supplementation of the diet with 0.4 mg of folic acid daily.

................................................................................
.....
Genes

NAP1L2/Nap1l2 expression may therefore depend on the genetic-environmental factors
that are frequently associated with NTDs.

Mouse models for neural tube closure defects.
Department of Medical Genetics, University of British Columbia, 6174 University
Boulevard, Vancouver, British Columbia, Canada. juriloff@interchange.ubc.ca


Neural tube closure defects (NTDs), in particular anencephaly and spina bifida,
are common human birth defects (1 in 1000), their genetics is complex and their
risk is reduced by periconceptional maternal folic acid supplementation. There
are > 60 mouse mutants and strains with NTDs, many reported within the past
2 years. Not only are NTD mutations at loci widely heterogeneous in function,
but also most of the mutants demonstrate variable low penetrance and some show
complex inheritance patterns (e.g. SELH/Bc, Abl / Arg, Mena / Profilin1 ). In
most of these mouse models, the NTDs are exencephaly (equivalent to anencephaly)
or spina bifida or both, reflecting failure of neural fold elevation in well
defined, mechanistically distinct elevation zones. NTD risk is reduced in various
models by different maternal nutrient supplements, including folic acid ( Pax3,
Cart1, Cd mutants), inositol ( ct ) and methionine ( Axd ). Lack of de novo
methylation in embryos ( Dnmt3b -null) leads to NTD risk, and we suggest a potential
link between methylation and the observed female excess among cranial NTDs in
several models. Some surprising NTD mutants ( Gadd45a, Terc, Trp53 ) suggest
that genes with a basic mitotic function also have a function specific to neural
fold elevation. The genes mutated in several mouse NTD models involve actin
regulation ( Abl/Arg, Macs, Mena/Profilin1, Mlp, Shrm, Vcl ), support the postulated
key role of actin in neural fold elevation, and may be a good candidate pathway
to search for human NTD genes.
THANKS FOR ALL THE EXPERIMENTATORS!!!

Kestutis Urba

P.S. This morning I've found changed lock in the door and was waiting till midday...
When I was a kid I had a dream - to study some problems having the hole in the roof looking at the rain like French scientists Cury, when investigating radioactivity phenomena. My dream was fulfilled...I'm only laughing at this situation now!

Hi Nick again,
I've spent less time by the computer, so I've got the time to consider all Hypothesys about NTD. Amazing puzzle! I would like to try to be more sceptic for some moment and contradict presented ideas about NTD - verify how strong they are!
First, when studying pictures of cells before tube closing (easy obtain using google-images search) I've rised the question - what are consequances of biochemical changes in embryo cells in physical meaning - in terms of forces, mass, electrical charge...
The simpliest explanation of NTD - closing defect is: if there is some process like growing mountains at both sides of canyon (maybe better use imagination of mountains like yeast before pie cooking too, because mountain growth is controlled by the hell), then closure defect appears because of the lack of dividing cell mass and amount. The reason is - folate deficience slows effective nucleotides production in cell and this somehow (HOW) slows or stops for some time new cell appearance. For 50% cases of NTD prevented by additional folate (folate defficient NTD phenotype) this idea fits. What about the other phenotypes? This nucleotide production slowlinnes -NTD appears when some repression happens in the long chain of nucleotide production- possible explanation for the other not folate deficient NTD phenotype. If this is truuuuuuuuue, then we can study methylations and all nucleosomas over all genome and we could find nothing.
How to contradict or verify this simpliest explanation? Experiments slowing some embryo nucleotides producing at some moment are needed. Possibly someone have studied this?
How to contradict this simpliest explanation by tools of logic?
Well, those results when observed DNmtylases defects and NTD are really impressive and I can't derive easily and simply from this directly the feedback - THE nucleotides production slowing: if SAM when DNMT repressed was not used efficiently, then....not enough - less of AdHmc and Hmc was produced in Met cycle, less amount of 5mtTHF used for Met resynthesys...some methyl trap appears??...less folate for nucleotides production??? DNA Methylase (and its friends) affects folate system? Complicated problem of feedback, but some arguments maybe possible could be found - from some leucemias -i.m trying to remember. I can't say this is true now - only very little probably 20% (usualy NTD is related to hyperHmc - important argument against simplicitie, too).
So, if we denye such mountain growth stopping scenary, we must return to the logical chain of folate defficiency, (or chol-bet system deficience), Met cycle disregulating, SAM leackage in the cell, and changing methylation patterns in some nucleosomas?
Then which nucleosomas and genes are under suspicion? First, maybe - responsible for cell growth - promoting repressing.... Second- for cell adhesive features, if NTD reason is "mountain tops can't lay close over canyon". Among them some proteins from cell membranes and some glycolipids -gangliosydes. I've found 6 articles in Pubmed studying NTD- the last one about ganglyosides was impressive enough. If folate system is defficient, then less Ser is produced into Gly and more phosphoSerine produced - the greater production of phospholipids is expected and this somehow changes cell membranes forming and electrical characteristics too.... Very complicated questions, but I hope some ideas of such kind will help to reveal reasons and mechanism of NTD.
How to control NTD processes? The 50% efficient tool is folate. If I have my own lab, I study SAM too, because it even normalised some malignant foci in liver (I think normalised Met cycle) and if the reason is not DNMT damage but for example Methioninesynthetase-reductase problem, then this works theoretically making compensation. Maybe possible usage of B12(adenosyl cbl) in some cases - more effective Met cycle regulator? I think it is necessary more adaptive prevention of NTD - first - identification of OCU metabolism situation and only then - adaptive treatment because folate treatment is only 50% efficient.

Kestutis Urba
P.S. What does it mean Kia Ora and Fair Dinkum In which language - the origin of words is my hobby...

hey kestutis

some very great questions you have there once again, certianly food for thought and I will ponder on this for a little while today.

Kia ora, is Maori, The Maori are the natives of New Zealand, where I am from. Literally Kia Ora means live on or live well, it's a way of saying hello, can be used to say goodbye and also thank you.

Fair dinkum is australian....or as the locals say it....'stralian. It can be used as an expletive or in any manner you wish really. but I could be wrong on this one because i have only been in australia for four years!!!

take it easy!

nick

Kia ora hey Nick,
I'm still thinking - considering NTD, cancer problems and still fighting for surviving. This morning I remembered- 13th October, Vilnius - the huge crowd of students attacked even The Lithuanian parliament after protest at governmental palace, demanding money for Universities. I helped them to organise demands and to avoid some sharp conflicts with police. The year street adventures...
It's not clear - what does it means cell moves to close the tube - move on the surface, move because some layers of embryo surface grow or some neighbour cells grow and split and...when studyiing corresponding images and photos (google search impressive) it's difficult to make decision. I've described for better understanding those problems schematically.
The most important conclusions and facts what is possible to obtain from this NTD-cancer research, I suppose is understanding and revealing the differences between NTD embryo cells and embryonal carcinoma cells. They could give the key for growth (the malignant too) regulation. The embrional teratomas cells I think are little less useful than carcinomas, because we need compare the embryonal surface layers, but teratomas must be of great interest in these problems too.

Kestutis Urba
P.S. I've made some suggestions for forum administration - posted them - to discuss the most valuable reviews and later - after the month - to make conferences with the authors online -scientific research process will become more progressive. They answered positive. Whats your opinion? Where is the list of reviews (maybe in some superreview) and when we start to discuss them in our topic? We can try to place some free available text link in forum?
Xmas is coming... Y mas for genetics too.

Kia ora hey Nick,
I would like to continue discussion considering very interesting information from recent review:

Am J Med Genet C Semin Med Genet. 2005 May 15;135(1):9-23.

Candidate gene analysis in human neural tube defects.

Boyles AL, Hammock P, Speer MC.

Duke University Program in Genetics and Genomics, USA.

Biochemical and developmental pathways, mouse models, and positional evidence have provided numerous candidate genes for the study of human neural tube defects. In a survey of 80 studies on 38 candidate genes, few found significant results in human populations through case-control or family-based association studies. While the folate pathway has been explored extensively, only the MTHFR 677C > T polymorphism was significant, and only in an Irish population. Developmental pathways such as the Wnt signaling pathway and Hox genes have also been explored without positive results. More than 90 mouse candidates have been identified through spontaneous and knockout mutations, but only the T locus (mouse Brachyury gene) showed association in an initial study that was not confirmed on follow-up.... Recent studies of an energy metabolism gene (UCP2) and vitamin B metabolism (Transcoalbumin) have produced promising results. Utilizing other model organisms may also be beneficial, as in a recent study from a chick model of NTDs in NCAM1. New approaches combined with traditional methods and increased sample sizes will help prioritize human NTD candidate genes and clarify the complex etiology of this condition.

So, 38 genes studied (more than 300 000 websites mentioning NTD) and no solution - only problems, problems. I can add, not only folate pathway (and more wide -OCU metabolism) was explored, but some cell adhesive features study was started too and development pathways study is under way...
Unfortunately my experience considering NTD is of beginner, but I hope, I have in this situation the advantage too - no standart opinion. I've tried to look at this problem without any predisposition and I'll try to explain some hypothetical questions, I must find the answer. Possibly, I'm building some unreal models, sorry, but I would like to know how to denye this one based more on physics but not genetics.
When looking at the photos and drawings describing NTD appearance I've tried to understand - why do embryo ectoderm changes its shape - rises before the connection over the tube. I've noticed that corresponding paraxial mesoderm under ectoderm is growing. Could this growth of paraxial mesodermal cells form some pressure and to change the form of ectodermal layer? On the contrary - could the ectodermal layer form some bows and this space then was fulfilled by paraxial mesodermal cells? I suppose first hypothesis is more possible, but I only can conclude now - the physics of neural tube closure is interesting enough and important, of course, too. If we can't find anything about this -please, Nick contact some proffesional in mechanics, forces, elasticity... This is not only the problem of genetics and biochemistry. So, the first model of neural tube close I consider is based on paraxial mesodermal cell growth mass force pressure into ectodermal layer:


I've even tried to evaluate - what cells are harder - mesodermal or ectodermal. I suppose(intuition) - mesodermal, but are they coated with calcium salts for hardness I have no information.
I've found some articles about retinoid (vitamine A) involvment into NTD, but these mechanisms I must study - A controls calcium...
The second possibility is if some forces makes pressure from the both ends of ectodermal layer to the centre and ectodermal layer changes its shape, but I have no understanding what reasons could create these tensions and change the shape needed for the tube closure.

Kestutis Urba
P.S. I've got the other computer at the other place not far from the former physics faculty Vilnius university...after attack I suffered. Life is life.