Methylation Alterations Of LINE-1 & Imprinting Genes .

Methylation alterations of LINE-1 &imprinting genes related to folatedeficiency and risk of neural tube defectsWang Li PhDMedical Genetic Department,Capital Institute of Pediatrics, China

Neural Tube DefectsNeural tube defects (NTDs) are a group of disorders in which an opening in thespinal cord or brain remains from early in human development.Failure of the tube to close properly can result in a number of NTDs .

Neural Tube Defects－Complex diseasesenviroment5-10%Genetics25-30%Interaction of geneticand enviroment60-70%NTDs have several suspected causes and have been linked togenetic, ethnic, nutritional, drug, and environmental factors. It isprobably the combination of several of these that produces anabnormality in any particular fetus.Lower concentrations of folate and vitamin B-12 duringpregnancy are higher risk factors of NTDs .

Preventive effect of folic acid on NTDsThe preventive effect of folic acid on NTD risk has been subjectedto extensive epidemiologic research.The preventive effect of folic acid on NTD risk in China‰85% decrease inNorth China,40% decrease inSouth ChinaWithoutFAWith FANorth ChinaSouth China中华医学杂志， 80：493，2000NEJM,2001

Folic Acid, Methylation and NTDsIntracellular folic acid and Bvitamins are key players in providing anadequate source of methyl groups formethylation of DNA and proteins, inaddition to their involvement in purineand pyrimidine metabolism.Life Extension Magazine August 2009

Prevalence of NTDs in Shanxi provinceShanxi province:BD (189.9/10,000),NTDs (102.3/10,000)Lvliang mountain region:BD (814.8/10,000),NTDs (186.0/10,000)------the highest area in ShanxiOthers 66.64NTDs 11.96CL/CP 10.00CHD 11.40

Food consumption of residents and child bearing agewomen in two counties compared with national average(g/reference/day)China(2002)Rural areasAverageResidentAverageRice and Its Products248.4239.921.625.1Wheat and Its Products141.0138.5317.2318.2Other Cereals25.923.363.263.3Beans4.84.24.24.2Bean Products11.411.81.11.1Dark Green Vegetables92.891.526.829.6Green 36.64.8Other Livestock Meat6.89.20.10.1Poultry Meat11.114.30.10.1Egg and Its Products19.923.627.722.2Fish and Shrimp24.430.10.10.0Vegetable Oil29.932.718.319.2Animal Fat10.58.70.40.4FoodCBA WomenAverage

Dietary pattern and the risks of NTD incidence

functional shortage of one-carbon nutrientsExpNeurol 2008;212:515–21.

functional shortage of one-carbon nutritionFolateB12?One carbon unit metabolism in association with Genomic regulative events

Methylation modification and NTDsTransposons and imprinted genes are 2 classes ofelements within the human genome that have methylation patternsthat are sensitive to malnutrition in early embryonic development.－－Nutrition 2004;20:63–8.Nature Review

long interspersed nucleotide element-1LINE-1 retrotransposon isregulated by epigenetic mechanisms.LINE-1 elements are normallysilenced by hypermethylation.Activation of LINE-1retrotransposition can affect ownstream gene expression.Hypomethylation of LINE-1 in somecancers is thought to be connected togenomic instability and endogenousDNA doublestrand breaks.

The structure of LINE-1Schematic structure of the LINE-1 element Am J Clin Nutr 2010;91:1359–67.

DNA hypomethylation in NTDsMethylation level of LINE-1 and genomic DNA between cephalicmalformations, spina bifida and control group (Mean SD)NTDs(n 48)Cephalic malformations(n 27)spina bifida(n 21)Controls(n 49)n: sample numbers †n 45Line-1 methylationlevel(%)Global methylationlevel(%)54.57 7.63**5.01 2.12*†52.43 8.84**4.79 2.27§57.32 4.605.26 1.9859.03 4.435.95 2.38§n 24**p 0.001 * p 0.05 Am J Clin Nutr 2010;91:1359–67.

DNA hypomethylation in NTDsMethylation level of CpG sites in LINE-1100Methyaltion ＊＊20CpG sitesite1site2Controlsite3site4Spinal bifidasite5site6,7site8,9site11,12Cephalic malformations Am J Clin Nutr 2010;91:1359–67.

Assessment of risk of NTDsNTDs risks assessments with methylation level of genomic DNA and LINE-1OR(95% CI)Adjusted OR§(95% %)1.056(0.299-3.728)0.727(0.183-2.889) 568-22.704)Global methyaltion4549 75%8(17.8%)13(26.5%)1(reference)1(reference)25% -3.078) -9.872)NTDsn(%)Controln(%)LINE-1 methyaltion level4849 75%5(10.4%)25% 75%NTDs – neural tube defects; OR: odds ratio; CI: confidence interval*p 0.01；Defined 25and75 percentile of control group methylation level as cutoff value§Adjusted with sex and gestation week Am J Clin Nutr 2010;91:1359–67.

Methylation changes in tissues*

ORF1p expression and methylation* Increased expression of ORF1p was detected in samples from female NTDsbrain tissues .ORF1p expression increased with decreasing level of L1Hs methylation(r 1.61, p 0.006, in HCT-15 cells with 5-Aza treatment: 0µm (Lines 1-3), 15µm(Lines 4-6), 50µm (Lines 7-9) )

DNA damage and chr. accessibility

methylation level and maternal nutritionComparison of maternal vitamin B12, folate and tHcy between NTDsand control group (Mean SD)NTDs(n 32)Controls(n 36)p-valuevitamin B12 (pmol L-1)70.16 19.0388.62 38.920.015Folate (nmol L-1)8.38 3.2310.57 8.830.172tHcy (μmol L-1)15.21 7.30†12.09 7.330.068Correlations between methylation level and maternal nutrition†Pearson correlationLINE-1 methylationGlobal methylationFolate (case/control)0.026 (36/32)0.147 (28/30)vitamin B12(case/control)0.047 (36/32)0.073 (28/30)tHcy(case/control)0.015 (36/29)0.147 (36/29)† Correlationwas performed including all NTDs and controls, similar results were obtained whencorrelation was performed in cases and controls respectively Am J Clin Nutr 2010;91:1359–67.

Summary 1 Hypomethylation of LINE-1 and genomic DNA was associated with anincreased risk of NTDs. LINE-1 hypomethylation in NTDs was associated with a significantincrease in expression level, DNA instability and chromatinsaccessibility. Functional insufficiency of maternal plasma vitamin B-12 and folatewere associated with NTDs, although no significant correlationestablished between maternal nutrition and LINE-1 methylation.----the target genes? that folate regulated through methylationmodifications and increase risk of NTD

Mice and dietsDiet formulation based on the AIN-93G （g/kg）ControlFDCornstarch380.500380.500Amino acids mix﹡200.000200.000Dextrinized an oil70.00070.000Fiber50.00050.000Mineral mix﹡35.00035.000Vitamin Mix﹡10.000\\10.0002.5002.500Ingredientw/o Folate Vitamin Mix﹡Choline bitartrate

Reproductive outcomesGroup(n)MaternalWeightNo. ofFetusRatio ofresorptedfetus(%)Crownrump Length(cm)Weight ofFetus (mg)Weight ofPlacenta (mg)Group 1(F/F)(17)36.9 6.08.1 2.88.2 13.22.4 0.11173 70106 217Group 2(C/F)(9)35.8 4.66.0 3.122.0 34.52.3 0.11300 101120 20Group3(F/C)(16)35.3 4.97.1 2.34.4 8.62.0 0.11214 112113 30Group 4(C/C)(13)34.1 4.97.2 2.43.2 6.02.4 0.21241 119100 24

8叶酸缺乏组F/C7F/F正常对照组6*543210Size of brain(mm2)13.5 �μm）13.5 dpc胎鼠脑部面积（mm2 ）Developmental retardation of **100500端脑Telencephalon中脑mesencephalon

Rare developmental malformationsNo.GroupNumber offetus/resorptionPhenotype1Group 1 (F/F)11/2anophthalmia2Group 1 (F/F)10/0spinal bending3Group 1 (F/F)10/0thinness ，suspected spinabifida4Group 1 (F/F)8/1spin tail5Group 2 (C/F)9/0anophthalmia6Group 2 (C/F)8/4microphthalmus7Group 2 (C/F)9/1coil tail8Group3 (F/C)7/1suspected spina bifida9Group3 (F/C)7/0suspected spina bifida10Group3 (F/C)9/0stillborn foetusPicture

Rare developmental malformationsF/F groupF/C groupC/F group

imprinting regulationPaternal folate deficiency showed limitation of growth, such as decrease offetus weight or length; while maternal folate deficiency decreased live birth, instead ofsize of fetus.------ consistent with imprinting regulation？ The father's genes that encode for imprinting tend to be growth limiting; The mother‘s is often to conserve resources for her own and current and subsequentlitters

Gnas imprinting and NTDsGnas imprinting cluster : Gnas encodes the alpha subunit of a major heterotrimeric Gs signalingprotein, which takes part in fetal growth and development. Gene expression studies in IUGR placentas have demonstrated thedownregulation of Gnas.Three differentially methylated regions (DMRs) have been found in theGnas imprinting cluster, two of which are gDMRs. The difference of DNAmethylation on gDMRs is established during gametogenesis and maintainedthroughout development.

Structure of Gnas imprinting clusterThree differentially methylated regions (DMRs) in the Gnas imprinting cluster: germline maternally methylated region at the Nespas promoter – Nespas gDMR; germline maternally methylated region at the Exon1A promoter – Exon1A/Gnas gDMR; paternally methylated region spanning the Nesp promoter – Nesp DMR.

Imprinting aberrant in F/F & F/C fetusMethylation alternations of gDMRs in F/F groupMethylation alternations of gDMRs in F/C 3520301025020Nespas DMRExon1A/Gnas DMRNesp DMRNespas DMRExon1A/Gnas DMRNesp DMRNespas DMR(Mean SD, %)Exon1A/Gnas DMR(Mean SD, %)Nesp DMR(Mean SD, %)Group 1 (F/F)41.16 4.9934.50 3.2343.82 16.15Control (C/C)46.46 3.7940.70 10.9343.28 13.51p0.0300.0010.81Group 2 (F/C)43.49 10.1233.67 6.9040.75 4.44Control (C/C)42.68 9.0938.28 6.6741.58 4.30p0.5440.0130.708

Imprinting within Nespas DMR in F/C fetusMethylation level of CpG Unit in chr2:174,120,698–174,120,842 of Nespas DMR region

Folate deficiency directly induced loss of imprintingfolate content in EB (ng/106 cell)4.843.22.41.60.8**0FFFF/0D FAFNgroups

cAMP levels in fetus brain140*120100pmo 200pmo *20015080pmo olresorptedF/c with absorbed

Gene expression in imprinting sA: in brain tissue of F/F group; B: in brain tissue of F/C group

GNAS Imprinting in NTDs with low folate level

hypomethylation of Nespas gDMR and folate

summary Mice embryos developmental dysplasia in response to folatedeficiency. Parental folate deficiency was associated with loss of imprinting inGNAS gene. Parental folate deficiency increased expression levels of Nespasand Exon1A companied with aberrant cAMP signaling. NTDs with lower folate concentration had a reduced methylationlevel of the Nespas gDMR.

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imprinting regulation Paternal folate deficiency showed limitation of growth, such as decrease of fetus weight or length; while maternal folate deficiency decreased live birth, instead of size of fetus.----- consistent with imprinting regulation？ The father's ge