1. 1 Diagnosis and treatment of autism considering features of the genetic background and metabolic status Ukrainian Institute of Clinical Genetics of KNMU Member-correspondent of NAMS of Ukraine, M.D., professor E.Y. Grechanina

2. 2 Genetic bases of many human diseases are successfully studied for last 20 years. Confession of World Health Organization that the basis of somatic, psychic and reproductive health is genomic health contributed this success. In opinion of H.Y. Zoghbi et al., Beaudet (2010) studying relationship between genotype and phenotype gives challenge for clinicists and researchers because some observations can’t be explained so easily.

3. 3 GENOMIC HEALTH GENE INTERACTION EPIGENETIC FACTORS ENVIRONMENTAL FACTORS Mitochondrial DNA Nuclear DNA Gene 1 ACGTAGCTAG Gene 2 ACGTAGCTAG Substitution of gene fragment Gene 2 ACGAGСCTAG

4. 4 Genomic health = somatic, psychic, reproductive health

5. 5 The role of the epigenome (changes of genetic information without changes of DNA nucleotides sequence) in normal as well as in pathological physiology of the genome.

6. 6 Many researchers proved that the causes of many inherent diseases are epigenetic mutations, which can change DNA methylation.

7. 7 By Ellis’ data, relationship between human genome and epigenome extended type range of molecular events, which cause human diseases. They can be de novo mutations or inherited from previous generations, genetic or epigenetic and can be a result of the influence of environmental factors.

8. 8 The appearance of convincing information about that environmental factors (at the first place – nutritional pattern) change the epigenome (DNA methylation) gave us better understanding the pathogenesis of human multifactorial diseases and at the first place – neurological disorders and psychic diseases.

9. 9 At the beginning of genetics creation, the well known psychiatrist, professor Bocherikov asked me, beginner genetician, to prove that psychic disorders are material. Very long professional way led to this understanding.

10. 10 We have to solve problems of thousands of children «…we have no alternative –we have to look at these problems, try to find out all details, to consider the point of view of everybody, whom this regards and out best for agreement achievement.The necessity to achieve the success is the another cause, where we need to use the antagonism between different points of view of the problem and the sooner the better. Let (all) voices be heart in discussion, let they try to come to understanding, and don’t try to shout down each other».

11. 11 Autism becomes one of the global human problem. It influences on many sides of physical life and spiritual life. It requires from us emergent development and introduction of a new paradigm of medicine 4 х «Р» -predictive, prognostic, preventive, partner.

12. 12 Parents of children with autism and doctors become partners. The sooner this partnership is achieved, the sooner this problem will be solved. Parents – all day persons on duty for their children,that’s why their information is invaluable, although it sometimes require medical correction. As soon as the resonance between partners is established, the next autistic child will begin to speak.

13. 13 A doctor, who received information from analysis and phenotype assessment of a patient, has to be at the head of the triangle «child-parents-doctor» with all responsibility in the process of search of the truth. Considering this I allow myself to analyze our way to understanding autism and desire to help. Everybody who will hear us, will be heard by us.

14. 14 Autism – heterogeneous syndrome, which is characterized by disorders in в 3 central domains (): Autism – heterogeneous syndrome, which is characterized by disorders in в 3 central domains (fr. Domaine- —area): 1.Social interaction 2.Speech 3.Interests and expressed genetic and phenotypic heterogeneity.

15. 15 Autism – the most severe result of disorders of nervous system development which is referred to autistic spectrum disorders (ASD). The frequency of incidence of ASD 37 in 10 000 Boys are prevalent, especially in clinically severe cases. The frequency of autism 13 in 10 000. Women/men ratio 4:1 (in severe forms 1:1) The frequency of Asperger syndrome 2,6:10 000 Women/men ratio 8:1

16. 16 The main feature of modern knowledge about ASD – its uncertainty. Many parallel approaches are necessary to understand genetic factors which underlie ASD: 1.Studies of the whole genome; 2.Associative studies; 3.Revealing mutation; 4.Expansion of clinical genetic examination of probands and their relatives

17. 17 Established genetic basis of autism: 1.Increase of the number of publications confirming that mutation and structural changes in any of several genes can significantly increase the risk of this disease. 2.If the diagnosis of autism is established in a child, the risk for the family will be 25 tomes higher. 3.Cognitive behavior features, which are similar to those observed in probands, are more likely observed in sibs and parents of an ill child than in controls. 4.Independent studies of twins show concordance for monozygotic twins 70-90 %, for dizygotic twins from 0 to 10%.

18. 18 Molecular studies of genes identified by nowadays show that no one molecular explanation will be enough.

19. 19 Many studie indicate system course of disorders in ASD development. Many studie indicate system course of disorders in ASD development. Different influence of maternal and paternal 15q11 in ASD is an important confirmation of cytogenetic disorders. Different influence of maternal and paternal 15q11 in ASD is an important confirmation of cytogenetic disorders. It is supposed that different molecular events are at the level of systems. It is supposed that different molecular events are at the level of systems.

20. 20 In recent years greater number of associated with autism syndromes are found (Tab. 1).

21. 21 Table 1: Syndromes associated with ASD № SyndromesGenes which are associated with syndromes Proportions of patients with syndromes followed by ASD Proportions of patients with ASD, who have these syndromes 1 15q dup Angelman syndrome UBI3A (and other) >401,2% 216p11 del Gene is unknown high-1% 3 22q del SHANK3high-1% 4 Syndrome of cortical dysplasia of focal epilepsy CNTNAP2~ 70%rare 5Fragile X- chromosome FMR125% of men; 6% of women 1-2%

22. 22 Table 1 (continuation): Syndromes associated with ASD № SyndromesGenes associated with syndromes Proportions of patients with syndromes followed by ASD Proportions of patients with ASD, who have these syndromes 6Hobart syndrome GOUBIRT, Many loci 25%rare 7Potocki-Lupski syndrome Chromosomes 17 р 11 ~ 90%unknown 8 Smith-Lemli-Opitz syndrome DHSA750%rare 9Rett syndromeMISP2 All individuals who have Rett syndrome ~ 0,5% 10Timothy syndromeSASNAIS60-80%unknown 11Tuberous sclerosisTSC1, TSC220%~1%

23. 23 Gene polymorphism – a genetic event in which building of genes changes and this influence on protein function. If only one letter changes in genetic code, this is called single nucleotide polymorphism.

24. 24 Name of enzyme GENE COENZY ME MUTATIONSDISEASES Methyltetra-hydrofolate reductase MTHFR Vit.B9 Vit.B2 C677T (Ala to Val) A1298C (Asp to Gly) Thromboembolia Neural tube defects Diabetes mellitus Methionine synthase MTR Vit.B12A2756G Thromboembolia Colorectal cancer Malignant lymphoma Methionine synthase reductase MTRR A66G Cardiovascular Сystathionine-β- synthase CBS Vit.B6 Ile to Thr Gly to Ser Homocystinuria Cystathionine-γ-lyaseCSE / CBL Congenital cystathionineuria Methionine adenosyltransferase MAT I / IIIHypermethioninemia Glycine N- methyltransferase deficiency GNMTLiver pathology S-Adenosyl-homocysteine hydrolase SAHHPsychomotor development delay, neurological abnormalities, hepatitis, myopathy Genetic enzymatic polymorphism of homocysteine metabolism (G.R. Akopyan)

25. 25 Polymorphisms of genes of folate and methionine cycle  Hyperhomocysteinemia was found in every third from examined patients with IHD and preeclampsia  Genotypes with high predisposition to homocysteine-associated thrombophilia in the case of their assessment by four polymorphic loci MTHFR С677T_A1298C / MTR 2756 AG / MTRR 66 AG: CT_AA/AA/GG, CT_AC/AA/GG, CС_AA/AA/GG, CT_AC/AA/AA, CС_AC/GG/GG, CC_AC/AA/AG, CC_AA/AA/AG were found.  The risk of hyperhomocysteinemia is likely associated with AA carrier of MTR genotype and GG of MTRR genotype  Carriers of four and more mutant alleles (MTHFR, MTR, MTRR) need screening for homocysteine content in blood plasma.  There is necessity of hyperhomocysteinemia verification using methionine loading test.

26. 26 All biochemical processes in a cell are performed with the help of cycles, among them there is folate cycle, which achieved key positions: folate metabolism is the basis of cellular metabolism (G.R. Akopyan)

27. 27 The following events are performed in this cycle: - Synthesis of nucleic acids; - Synthesis of biologically active substances: adrenaline, melatonin, creatinine, phospholipids, polyamines (spermicides and spermines), glutamic acid, dihydro- tetrahydrobiopterin, nitric oxide ; - Epigenetic changes of DNA, DNA (methylation), RNA, chromatin, amino acids, proteins, lipids.

28. 28 We supposed and confirmed that if there is enzymatic activity of folate cycle in human body – methylentetrahydrofolatereductase is low, this leads to methylation disorder (switching on and off gene activity) and then it causes a lot of inherent and multifactorial syndromes.

29. 29 The following was underlain the basis of this study: creation of single information database including all levels of prevention of inherent pathology at all levels of ontogenesis. The following was underlain the basis of this study: creation of single information database including all levels of prevention of inherent pathology at all levels of ontogenesis.

30. 30 Service of urgent biochemical diagnosis KHARKIV SPECIALIZED MEDICAL GENETIC CENTRE (practical basis) UKRAINIAN INSTITUTE OF CLINICAL GENETICS, DEPARTMENT OF MEDICAL GENETICS OF KhNMU (scientific basis) REGIONAL METABOLIC CENTRE REGIONAL PULMONARY CENTRE Centre of connective tissue pathology ONCOGENETIC CENTRE PRENATAL CENTRE Association of families with phenylketonuria Association of families with mitochondrial diseases Association of families with cystic fibrosis Association of families with spinal muscle atrophy Association of families with organic acidurias Association of families with cystic fibrosis Centre of prenatal education Association of families with chromosomal pathology Centre of studying epigenetic diseases

31. 31 Comparative characteristics of IDD by screening data on Kharkiv region for 2000-2008 Diseases 20002001 2002200320042005200620072008 Ab. amo unt In. val ue A b. a m o u nt In. val ue A b. a m o u nt In. val ue A b. a m o u nt In. val ue A b. a m o u nt In. val ue Ab. am oun t In. val ue A b. a m o u nt In. val ue Ab. am oun t In. value A b. a m o u nt In. value 1.Anencephaly 20,9 9 3 1,5 3 3 1,4 5 3 1,3 8 1 0,4 4 2 0,9 0 1 0,4 1 10,3910,36 2.Myelocele 73,4 9 5 2,5 5 6 2,9 0 9 4,1 6 1010 4,4 0 11 4,7 5 5 2,0 7 124,7282,9 3.Meningocele -- 1 0,5 3 1 0,4 8 1 0,4 6 ------31,1820,72 4.Hydrocephaly 31,4 9 9 4,5 9 1414 6,7 8 1010 4,6 3 1313 5,7 2 7 3,5 6 1313 5,3 8 124,7282,9 5.Microcephaly 52,4 9 3 1,5 3 --4 1,8 5 5 2,2 0 5 2.9 7 9 3,7 2 51,9651,8 6.Anotia -- ----2 0,9 2 1 0,4 4 2 0,9 0 4 1,6 4 51,9631,09 7.Anophthalmia 10,4 9 --1 0,4 8 --------10,39-- 8.Microphthalmia 20,9 9 1 0,5 3 1 0,4 8 --1 0,4 4 4 1,7 9 4 1,6 4 10,3920,72

32. 32 The frequency of genotypes and alleles of polymorphic gene variants C677T MTHFR И A66G MTRR (n=4586)

33. 33 Since 2008 we have been conducting the stage of the scientific search according to the following hypothesis. Hypothesis:The influence of mtDNA polymorphisms on MTChD is a result of pathological transformation of mtDNA polymorphisms against the background of the changed status of methylation as the main genome modificator and the presence of triggers.

34. 34 DNA methylation Cytosine Guanine

35. 35 Methylation of biologically active substances

36. 36 Betaine is a donor of methyl groups in the reaction of remethylation of homocysteine in participation of betaine-homocysteine-methyltransferase. (G.R. Akopyan ) CBL (B6) MAT I/III SAHH Folate and methionine cycle ! BHMT GNMT (B6) (B12) (B2) All reactions of methionine cycle are connected with tanssulfuration of homocysteine

37. 37 Folate and methionine cycle CBL (B 6 ) The level of development of hyperhomocycteinemia depends on the content of folic acid (B12), pyridoxine (B6), riboflavin (B2), serine, glycine, choline, betaine, cysteine. (G.R. Akopyan) B6 B2 B12 B6

38. 38 Hyperhomocysteinemia and methylation disorder (G.R. Akopyan)

39. 39 О2О2 Decreases the level and activity of thioredoxin, superoxide dismutase, syntase NO Increases NAD(P)H-oxidase activity Homocysteine thiolactone Acts in the normal level of homocysteine and in less concentrations (10 nmol/l) !!! Protein modification Homocysteine thiolactonase or paraoxonase (PON 1) can hydrolyze homocysteine thiolactone!!! LDL oxidation Endothelium damage + thrombogenesis Atheromatous plague formation Homocysteine – a strong oxidant and protein modificator

40. 40 Homocystination of proteins by homocysteine thiolactone: (albumine, hemoglobine, fibrinogene, apoliprotein B, fibriline !!!) Consequences: protein breakdown by multimerization and precipitation and change of their antigen possibilities that contributes to disease chronization independently from homocysteine level Binding carbamyl group of homocysteine thiolactone with ε-amino groups of lysine residues and SH-groups of cysteine residues in protein molecule S-homocystination N-homocystination Binding with SH-group of cysteine Binding with ε-amino group of lysine

41. 41 Methylation has been admitted the main genome modificator, central pathway of all metabolic events in organism life Optimization of methylation function in A. Yasko’s opinion (2010) becomes a model for management of genetic polymorphism, which influences on many important biological events in the body.

42. 42 METHYLATION FUNCTION: 1.DNA methylation is necessary for support of differential expression of paternal and maternal gene copy susceptible to the genome imprinting. 2. For stable gene silencing on inactive X- chromosome.

43. 43 3. Stable transcriptional repression of provirus genomes and endogen retrotransposons depends on DNA methylation DNA methylation takes part in management and support tissue-specific patterns of gene expression in development DNA methylation takes part in management and support tissue-specific patterns of gene expression in development Absence of DNA methylation decreases reliability of support of chromosomes number that leads to chromosomal aberrations Absence of DNA methylation decreases reliability of support of chromosomes number that leads to chromosomal aberrations DNA hypomethylation in consequence of influence of DNA-methyltransferase inhibitors leads to elimination of tumors DNA hypomethylation in consequence of influence of DNA-methyltransferase inhibitors leads to elimination of tumors Formation of other types of tumors increases in DNA hypomethylation Formation of other types of tumors increases in DNA hypomethylation

44. 44  Entirety of methylation systems determines genome and it means psychic, physic, reproductive health.  Studies, which explain how environmental factors can induce epigenetic changes and biologic effects, have appeared. En Li, Adrian Bira (2010)

45. 45 DNA methylation and chromosomal instability Ehrlich, 2003; Dobge et al, 2005 established that DNMT3B mutations in patients with ICF syndrome or Dnmt 3binactivation in mice lead to various chromosomal aberrations (structural and quantitative) Ehrlich, 2003; Dobge et al, 2005 established that DNMT3B mutations in patients with ICF syndrome or Dnmt 3b inactivation in mice lead to various chromosomal aberrations (structural and quantitative) There is the hypothesis that DNA methylation contributes exact chromosomal disjunction and in its absence more often there is leading to chromosomal disordersnondisjunction (hypomethylation, demethylation) There is the hypothesis that DNA methylation contributes exact chromosomal disjunction and in its absence more often there is leading to chromosomal disorders nondisjunction (hypomethylation, demethylation)

46. 46 Alternative possibility means that DNA methylation can inhibit expression and recombination of retrotransposons in animals’ genome, thus defending chromosomes from harmful recombinations. Alternative possibility means that DNA methylation can inhibit expression and recombination of retrotransposons in animals’ genome, thus defending chromosomes from harmful recombinations.

47. 47 Identification of folate cycle disorders include: 1.Inherent malabsorption of folic acid caused by mutations in the gene which encodes folic acid transporter. 2.Deficiency of formiminotransferase caused by the mutation in FTCD gene. 3.Deficiency of methylentetrahydrofolate reductase caused by the mutation in MTHFR gene.

48. 48 4-5. Deficiency of functional methionine synthase as a result of mutations in MTR gene affecting methionine synthase (cblG) or mutations affecting methionine synthase reductase (cblE due to the mutation in MTRР gene). 6. Cerebral deficiency of folic acid caused by mutations in FOLR1 gene. 7. Deficiency of thrifunctional enzyme containing methylenetetragydrofolate cyclohydrogenase and formyltetrahydrofolate synthase caused by mutations in MTHFD1 gene (Mac Gill, Rosenblatt et al.).

49. 49 It is necessary to note that homozygotous pattern of the polymorphism means more expressed level of enzymatic activity decrease. If a human is a carrier of a specific mutation, it not always means that function activity certainly will decease, SNP are indicators of potential problem areas which can manifest independently or under the influence of triggers or gene interaction

50. 50 Defects in 5-methyltetrahydrofolat homocysteine methyltransferase can disturb detoxification process, meanwhile toxic substances, for example, mercurous can worse the effect because of decreased activity methionione synthase (MTR) and decreased detoxification effectiveness

51. 51 There is summary of the genes that are included in a complex panel of methylation analysis (Amy Yasko, 2010) Mutations or single nucleotide polymorphisms : Gene mutations - changes affecting the sequence of a single gene. Mutations vary in size from one affecting base pair to large segments of chromosomes. Single nucleotide polymorphisms are small genetic changes or variations that may occur in the DNA sequence. The genetic code is denoted by 4 "letters": A, C, G and T. SNP variation is due to the replacement of one nucleotide for another. The presence of mutations in genes encoding enzymes affects their productivity. Homozygous mutations are those mutations that affect both copies of the gene, heterozygous mutations are those mutations that affect only one of the copies of the gene. Each of us has two copies of each gene obtained from each parent. Some mutations enhance the activity of enzymes (such as CBS) while others may decrease the activity (such as MTHFR 677 1298 COMT)

52. 52 COMT V158M, H62H, 61 The main function of this gene is involved in the breakdown of dopamine. Dopamine - a neurotransmitter that is involved in the formation of behavioral reactions and attention. Dopamine contributes to the appearance of good feeling, because it causes a feeling of pleasure influencing the processes of motivation and learning. Dopamine is produced during positive thinking. COMT exposed cleavage leads to the formation of another neurotransmitter - norepinephrine. The correspondence between the level of epinephrine and dopamine levels is involved in ADD / ADHD; dopamine levels is important in the development of diseases such as Parkinson's disease. COMT is also involved in transformation of the corresponding estrogen in the body. COMT activity is often associated with sensitivity to pain. COMT homozygotes may be more sensitive to pain.

53. 53 VDR/Taq and VDR/Fok (vitamin D receptor) The panel contains some receptors of vitamin D, Taq and Fok sites. While Fok change was due to the regulation of blood sugar, modified Taq may affect the level of dopamine. For this reason it is important to watch the status of COMT VDR / Taq and draw conclusions based on the totality of the results of these two sites. Focus on changes part of the VDR in the Fok against supplements that support the pancreas and assist in the maintenance of blood sugar in the normal healthy range.

54. 54 MAO A R297R (monamine oxidase A): Mao is involved in the cleavage of serotonin in the body. Like dopamine, serotonin - neurotransmitter. It is associated with mood, an imbalance of serotonin levels is associated with depression, aggression, anxiety and OCD behavior. MAO A is localized on chromosome X and is considered X-linked trait that does not appear in men. Because the X chromosome in a man can come only from the mother, it means that Mao mutations of father (or their absence) plays no role in the son. For women, as one chromosome is inherited from each parent, geneticians, tend to reflect the status of Mao in both parents.

55. 55 ACAT 102 (acetyl coenzyme A acetyltransferase): ACAT plays a role in lipid metabolism, helps to prevent the accumulation of excess cholesterol in certain parts of a cell in the body. ACAT is also involved in the production of energy in the body. Contributes to the breakdown of proteins, fats and carbohydrates from food, energy and then will be used in life. Furthermore, the absence of ACAT may also lead to depletion of B12, which is required in methylation cycle. ACE (angiotensin converting enzyme): Considered for all - No longer testing Various factors, including diet can affect the activity of ACE gene, changes which can lead to high blood pressure. The connection between gene activity disorders with increased anxiety, memory loss and learning decrease has been revealed in animal studies. Increased activity of ACE may also lead to the removal of minerals in the body by decreasing excretion of sodium in urine and increased elimination of potassium. This reaction is also associated with stress in a situation of chronic stress can lead to additional sodium accumulation and an increase excretion of potassium. This excess of potassium is in body if the kidneys function properly. In the case of impaired renal function, it may result in retention of potassium in the body.

56. 56 MTHFR A1298C, C677T, 3 (methylenetetrahydrofolate reductase): MTHFR gene product is at a critical point in the methylation cycle. Participates in the normalization of the level of homocysteine. Some mutations in MTHFR were well characterized, and are associated with the risk of cardiovascular diseases and cancer and may play a role in the level of neurotransmitters of serotonin and dopamine MTR A2756G/MTRR A66G, H595Y, K350A, R415T, S257T, 11 (methionine synthase/ methionine synthase reductase): These two gene product work together and are involved in the conversion of homocysteine to methionine. Elevated homocysteine levels are risk factors in a number of pathologies including heart disease, Alzheimer's disease, etc. As in the case of COMT and VDR / Taq, MTR and MTRR should be studied together. Mutations in the MTR can increase the activity of the gene product in a way that leads to greater consumability of B12 as the enzyme. On the other hand, recent publications show that MTRR A66G mutation reduces the activity of the enzyme. Regardless of which theory is correct breaking B12 cycle or methylation function activity disorder at this point, B12 is used as an additive in all the cases.

57. 57 BHMT 1,2,4,8 (betaine homocysteine methyltransferase): The product of this gene is central in the short pathway of methylation, performs remethylation of homocysteine to methionine. This gene product activity may influence on stress, cortisol level and may play the role in the ADD / ADHD, affecting the levels of norepinephrine. AHCY 1,2,19 (S adenosylhomocysteine hydrolase): Different mutations in the AHCY can affect the levels of homocysteine and ammonia in the body. CBS C699T, A360A, N212N (cystathionine-beta-synthase): CBS enzyme basically acts as a gateway between homocysteine and lower part of the path which generates ammonia in the body. There are some positive end-products that are generated by the lower part of methylation pathway, such as glutathione and taurine, there are also negative side-products such as ammonia and excess sulfite. Because of increased activity of CBS, sulfites that are toxic for the body present an additional upload for SUOX gene product.

58. 58 SHMT C1420T (serine hydroxymethyltransferase): The product of this gene is involved in the setting blocks needed for synthesis of new DNA and transformation of homocysteine to methionine. While DNA blocks are important, mutations that affect the ability to regulate the gene product and thereby affecting the methylation process can cause the accumulation of homocysteine and imbalance in the other intermediate compounds in the body. NOS D298E (nitric oxide synthase): NOS enzyme plays an important role in the detoxification of ammonia in the urea cycle. Individuals who are homozygotous for NOS have the enzyme with decreased activity. NOS mutations can affect the regulation of CBS until increase of ammonia, which is generated by CBS.

59. 59 SUOX S370S (sulfite oxidase): The product of this gene promotes detoxification of sulfites in the body. Sulfites are generated as a natural by-product of the methylation cycle, and enter the body with food. Sulfites, sulfur-based preservatives that are used to prevent or reduce discoloration of light colored fruits and vegetables to prevent the appearance of black spots on the shrimps and lobsters, inhibit the growth of microorganisms in fermented foods (e.g., wine), and are able to maintain the activity of certain medications. Sulfites may also be used for bleaching edible starch, rust and scale prevention in boilers used for steam cooking food, and even in the production of cellophane, for packing food products. FDA considers that one from hundred sulfites is sensitive, approximately 5% of individuals suffer from asthma. A person can face the problem of sulfite sensitivity at any time of life.

60. 14.05.2015 Many cases of sulfite sensitivity have been registered, and therefore the FDA requires that the labels have information about product content of these substances. Scientists don’t note the smallest concentration of sulfites needed to cause a reaction. Shortness of breath is the most common symptom. Sulfites release sulfur dioxide gas, which can cause irritation in the lungs and cause severe asthma attack for those who suffer from asthma. Sulfites can cause chest tightness, nausea, urticaria, and in rare cases, more severe allergic reactions. Mutations in SUOX may be a risk factor for developing certain types of cancer, including leukemia.

61. 61 EPIGENETICS Epigenetics (επί-over) – the section of medical biological science studying principles of changes of gene expressions or cell phenotype caused by mechanisms not associated with DNA sequence. Epigenetics characterizes the process of body and environment interaction in genotype formation. К. Uodington 1947 61

62. 62 Factors which lead to switching on epigenesis: - Nutrition; - Infection; - Smoking; - Stress; - Trauma; - Operation; - Alcohol.

63. 63 Triggers (provocateurs) of switching on epigenesis: nutrition, infection, smoking, stress, alcohol The presence of gene predispositions (mediators) Methylation is the main epigenetic mark and key reaction of epigenesis.

64. 64 Cryshtof Bokk generalized scientific facts about epigenetic regulation occurrence and how it influence on human diseases. T. Kouzarides thinks that such epigenetic mechanisms how DNA methylation and histone modifications (acetylation), regulate gene expression by DNA modulation in cellular nuclei.

65. 65 Such environmental factors as nutrition and stress influence are able to cause changes of epigenetic status (Yeijmans B.T. et al., 2007). These circumstances form opinion of many scientists about that human epigenome can be considered as the biochemical record of life events, accumulated changes throughout life.

66. 66 Effects of epigenetics Genome imprinting (and its disorders) Cellular differentiation Transgenerative epigenetic effects Mutation process Blastemas Organism ageing Conservatism of genetic information 66

67. 67 Mechanisms of epigenetics DNA methylation Chromatine remodeling RNA-mediated modifications Protein preionization X-chromosome inactivation 67

68. 68 It is established that many epigenetic changes may not followed by phenotypic changes, meanwhile some changes caused by environment factor action modulate gene activity (expression) (Herst M, Marra M.A.,2009; Feinberg A.P., 2007; Bijornson H.T., 2004) that’s why abnormal epigenetic status can be associated with a number of diseases (e.g. rheumatoid arthritis, SLE).

69. 69 It is shown that neural activity in the brain is regulated epigenetically, and potential relevance of epigenetic changes in schizophrenia, biopolar disorders and alcoholism allow us to see the problem in a different way (Esteller M., 2007; Jones P.H, Baylin S.B., 2007; Feinberg A.P. et al. 2006).

70. 70 EPIGENETIC DISEASES INCLUDE (HUDS Y.ZOGHBI, ARTHUR L. BEANDET): 1.Genome imprinting disorder. 1.1. Sister syndromes; Prader-Willi syndrome. 1.2. Beckwith-Wiedemann syndrome 1.3. Silver-Russell syndrome 1.4. Pseudohypoparathyroidism. 2. Disorders influencing on chromatin structure in transconfiguration: 2.1. Rubinshtein-Taybi syndrome 2.2. Rett syndrome 2.3. X-linked ά-thalassemia followed by mental delay. The syndrome of immunodeficiency, instability of the centromeric region and facial anomalies 2.4. Spondyloepiphyseal dysplasia of Schimke. 2.5. Methylenetetrahydrofolate reductase deficiency. 3. Disorders influencing on chromatin structure in cis-configuration. 3.1. άδβ-δβ-thalassemia 3.2. X-fragile syndrome 3.3. Facioscapulohumeral muscular dysrtrophy

71. 71 Classification of epigenetic human diseases (S.А. Nazarenko, 2004) Epigenetic status disorder of separate regions of the genome (locate effect) Disorder of epigenetic status of the whole genome (global effect) 1. Diseases caused by inherited mutations disturbing monoallele gene expression – diseases of genome imprinting (Beckwith-Wiedemann syndrome, Prader-Willi syndrome, Engelman syndrome) 1. Diseases caused by inherited mutations of genes, products of which are involved in the support of DNA methylation level or modification of methionine structure - ICF syndrome, Rett syndrome, ATR-X syndrome, Rubinshtein-Taybi syndrome, Coffin- Lowry syndrome 2. Diseases caused by methylation status disorder of separate genes in the result of de novo mutations in somatic cells - a)cancer areas connected with imprinting loss leading to inactive gene activation or inhibition of active gene expression; b)cancer diseases caused by hypermethylation of promoters of tumor suppressor gene 2. Diseases caused by global disorders of genome methylation in the result of de novo mutations in somatic cells – cancer disease connected with the global genome hypomethylation leading to activation of oncogenes, retrotransposons and chromosomal instability

72. 72 Methionine Methionine – an essential amino acid, is contained in proteins. Is methyl group donor (in composition of S-adenosyl- methionine) in synthesis of choline, adrenaline and other; Source of sulfur in cysteine synthesis. Has 52 biochemical synonyms. Chemical name of methionine – (2S)-2-amino-4-methylsulfanyl- butanoic acid. Chemical formula- C5H11NO2S.

73. 73 Unique functions of methionine Takes part in trasmethylation reactions; Is a donor of methyl groups; In synthesis of biologically active substances; Takes part in synthesis of nucleic acids; Is an acceptor of methyl for 5-methylenehydrofolate- homocysteine methyltransferase (methionine syntase).

74. 74 74 Metabolism

75. 75 Methionine – cysteine precursor which gives it sulfur. Has 52 biochemical synonyms. Chemical name of methionine – (2S)- 2-amino-4-methylsulfanyl-butanoic acid. Chemical formula - C5H11NO2S.

76. 76 Biological function of methionine An essential acid A component of aminoacyl tRNA biosyntase A component of glycine metabolism, serine and trianine A component of histidine metabolism A component of methionine metabolism A component of selenium amino acid metabolism A component of tyrosine metabolism

77. 77 Enzymes of methionine metabolism are presented by Methionine syntase Thyrosine amino transferase S-adenosyl methionine synthetase isoform II type Arsenit methyltransferase Indomethylamine N-methyltransferase S-adenosyl methionine synthetase isoform I type Betaine homocysteine S-methyltransferase I. Methionine-tRNA synthetase, cytoplasmic Methionine adenosyltranferase II beta

78. 78 Disorder of processes remethylation (formation of methionine from homocysteine), in the result of deficiency of MTHFR и MTRR enzymes leads to a number of pathological conditions such as: atherosclerosis; atherothrombosis; Neural tube closure defect; infarcts; Chromosome disjunction defect in oogenesis and the risk of birth of children with Down syndrome.

79. 79 Folate and methionine cycles

80. 80 Classification (N. Blau et al., 1996) Disorder — Affected component Tissue distribution Chromoso me localisatio n № MIM 10.1 Methionine adenosyltransferase (МАТ) of the liver Liver250850 10.2 Cystathionine beta- synthase (CBS) Liver, brain, lymphoblasts, cultured fibroblasts, amniocytes and choroidal fibers 21q22.3236200 10.3 Gamma- cystatathionase(СТН) Liver, lymphoblasts16219500 10.4 Sulfitoxidase, isolated or molybdenum cofactor 10.4.1. Type А 10.4.2 Type В Liver, kidneys, lungs, heart, lymphoblasts, choroidal fibers, cultured fibroblasts and amniocytes 272300 252150 252160

81. 81 Classification (N. Blau et al., 1996) Disorder — Affected component Tissue distributionChromoso me localizatio n № MIM 10.5 5,10- Methylenetetrahydrofolate reductase (MTHFR) Liver, lymphocytes, lymphoblasts, choroidal fibers, cultured fibroblasts 1р36.3236250 10.6 Methionine synthase (methyl cobalamin) cblE, cblG 236270 250940 10.7 Methylmalonyl-СоА-mutase (adenosylcobalamine) and methionine synthase methylcobalamin) Liver, cultured fibroblasts, amniocytes 277400 277410 277380 Liver, cultured fibroblasts, amniocytes

82. 82 Depending on the frequency, separate genotypes can compose bases for development of common pathology, other can be factors of development of rare (orphan) diseases.

83. 83 Spectrum of nosologies in combination of polymorphisms С677Т MTHFR / А66G MTRR in patient selection ( n=1938) Nosology spectrum С677Т MTHFR / А66G MTRR Hmzg /Hmz g Hmzg /Htzg Htzg/ Htzg Htzg/ Hmz g N/ Hmz g N/ Htzg Hmzg /N Htzg /N Deficiency of folate cycle enzymes (16%)61136376999536 Deficiency of folate cycle enzymes3311133751115 Homocysteine remethylation disorder1133 Spina bifida12 HCU1371081928 HCU (in relatives)11 Thromboembolias/thrombosis1142376 (in relatives )13121 Varicose vein dilatation16611 15 Varicose vein dilatation (in relatives )12434412

84. 84 Spectrum of nosologies in combination of polymorphisms С677Т MTHFR / А66G MTRR in patient selection ( n=1938) Nosology spectrum С677Т MTHFR / А66G MTRR Hmzg/H mzg Hmzg/H tzg Htzg/H tzg Htzg/H mzg N/ Hmzg N/ HtzgHmzg/N Htzg/ N IMD (7.8%)9107164711241044 IMD210111323413 IMD (in relatives)2221 IMD of amino acids1121223 IMD of sulfur-containing amino acids48591713 IMD of sulfur-containing amino acids (in relatives)211 IMD of fatty acids3124 IMD of methionine278831 CTD1321191333211 CTD (in relatives) DMD1131 Disorder of tryptophan metabolism1 Disaccharidose deficiency1

85. 85 Spectrum of nosologies in combination of polymorphisms С677Т MTHFR / А66G MTRR in patient selection ( n=1938) Nosology spectrum С677Т MTHFR / А66G MTRR Hmzg /Hmz g Hmzg /Htzg Htzg/ Htzg Htzg/ Hmzg N/ Hmzg N/ Htzg Hmzg /N Htzg /N Sulfite oxidase deficiency 213 Metabolism disorder in urea cycle 1 Maple syrup disease 1 Hyperprolinemia 211 Aminoacidemia 1 Aciduria 2241 Hypothyrodism 1 Autism 2 Mitochondrial diseases 1189101917 Mitochondrial diseases (in relatives) 1 Kearns-Sayre syndrome 1 Epilepsy 113151012

86. 86 Spectrum of nosologies in combination of polymorphisms С677Т MTHFR / А66G MTRR in patient selection ( n=1938) Nosology spectrum С677Т MTHFR / А66G MTRR Hmzg/H mzg Hmzg/H tzg Htzg/H tzg Htzg/H mzg N/ Hmzg N/ HtzgHmzg/N Htzg/ N Vascular pathology(8%)2513 4643222 Inborn heart defect2151 (in relatives)13 Cardiopathy112 Myocardial infarction111 Myocardial infarction (in relatives)11223 Ischemic heart disease11 Ischemic heart disease (in relatives)1111 Vascular pathology11188 Vascular pathology (in relatives)1962 Insults213 Insults (in relatives)2341374

87. 87 Spectrum of nosologies in combination of polymorphisms С677Т MTHFR / А66G MTRR in patient selection ( n=1938) Nosology spectrum С677Т MTHFR / А66G MTRR Hmzg/ Hmzg Hmzg /Htzg Htzg/ Htzg Htzg/ Hmzg N/ Hmzg N/ Htzg Hmzg /N Htzg /N Monogene pathology (5%)2411 202148 Ehlers-Danlos syndrome1214562 Prader-Willy syndrome1111 Louis-Bar syndrome11 Klippel-Trenaunay syndrome11 Tuberous sclerosis221 Rendu-Osler syndrome11 Silver-Russell syndrome1 Rubinstein-Taybi syndrome1 Hyperirritability syndrome121 Arnold-Kiari syndrome1 IDD syndrome1 AGS1 Anonichia-ectodactyly syndrome1 McCune-Albright syndrome1 Lesch-Nyhan syndrome1

88. 88 Spectrum of nosologies in combination of polymorphisms С677Т MTHFR / А66G MTRR in patient selection ( n=1938) Nosology spectrum С677Т MTHFR / А66G MTRR Hmzg /Hmz g Hmzg /Htzg Htzg/ Htzg Htzg/ Hmzg N/ Hmzg N/ Htzg Hmzg /N Htzg /N Hemorrhagic syndrome1 Erb-Rott myopathy1 Myopathy syndrome11 Duchenne muscular dystrophy2 PKU111 Cystic fibrosis3212 Reiter syndrome1 Marfan syndrome112 Dandy-Walker syndrome1 Fridreich’s ataxia1 Polyneuropathy11 Pertas disease1 Gilbert’s syndrome12 DHD syndrome1 Hypophyseal nanism1

89. 89 Spectrum of nosologies in combination of polymorphisms С677Т MTHFR / А66G MTRR in patient selection ( n=1938) Nosology spectrum С677Т MTHFR / А66G MTRR Hmzg /Hmz g Hmzg /Htzg Htzg/ Htzg Htzg/ Hmzg N/ Hmzg N/ Htzg Hmzg /N Htzg /N Chromosomal pathology(6%) 216102927214 Down syndrome 15151412 Down syndrome (in relatives) 6314616 Shereshevsky-Turner syndrome 2544 Various chromosomal pathologies/polymorphisms 136532

90. 90 Epigenetic disease (hypomethylation, chromosomal polymorphism (46,ХУ, 9 phqh ) and polymorphic gene variants of folate cycle (677 С-Т, А222 V mutation in heterozygotous state). Mild homocystinuria. Syndromal epilepsy.

91. 91 Rendu-Osler disease. Polymorphic variant of 677 C/T MTHFR gene in homozygotous state

92. 92 Epigenetic disease? Mosaic form of Shereshevsky-Turner syndrome. Disorders of active enzymes of folate cycle. Polymorphic variant of 677 С/Т MTHFR gene was found in heterozygotous state, gene of endothelial NO-synthase 4a/4b вin homozygotous state). Energy metabolism disorder (MNGIE?).

93. 93 Familial case of epigenetic disease ? DNA hypomethylation, folate cycle deficiency, methionine metabolism disorder (mosaic form of trisomy 21, chromosomal polymorphism of chromosome 1 ). Polymorphic variants of MTHFR 677 C/T gene in heterozygotous state, MTRR 66 G gene in homozygotous state

94. 94 Epigenetic disease?: glycoprotein metabolism disorder (defect of posttranslation of lysosomal enzymes). Disorder of folate cycle metabolism (66A→G (122М) polymorphism in MTRR gene in heterozygotous state). Chromosomal polymorphism: 46, ХY, 14 рs+.

95. 95 Saethre-Chotzen syndrome, secondary mitochondriopathy, folate cycle deficiency

96. 96 McCune-Albright syndrome in mother. Polymorphic variants of 677ТТ MTHFR/66A/G MTRR genes Polymorphic variants of 677ТТ MTHFR/66A/G MTRR genes. Healthy children

97. 97 Robinow syndrome. Multiple harmatose growth in the liver Polymorphic variants of677ТТ MTHFR/66GG MTRR genes Multiple harmatose growth in the liver Polymorphic variants of 677ТТ MTHFR/66GG MTRR genes

98. 98 Ukrainian Institute of Clinical Genetics KhNMU Kharkiv-22, Pravdu avenue, 13 Е-mail: mgc@ukr.net