Ukrainian Institute of Clinical Genetics of KNMU Member-correspondent of NAMS of Ukraine ,

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 NAMSof Ukraine, M.D., professor E.Y. Grechanina 1

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. Zoghbiet al., Beaudet (2010) studying relationship between genotype and phenotype gives challenge for clinicists and researchers because some observations can’t be explained so easily. 2

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

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

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. 5

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

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. 7

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. 8

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. 9

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». 10

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. 11

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. 12

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. 13

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

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 ASD37 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 15

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

17. Established genetic basis of autism: • 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. • If the diagnosis of autism is established in a child, the risk for the family will be 25 tomes higher. • 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 . • Independent studies of twins show concordance for monozygotic twins 70-90 %, for dizygotictwins from 0 to 10%. 17

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

19. Many studie indicate system course of disorders in ASD development. • Different influence of maternal and paternal 15q11in ASDis an important confirmation of cytogenetic disorders. • It is supposed that different molecular events are at the level of systems. 19

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

21. Table 1: Syndromes associated withASD 21

22. Table 1 (continuation): Syndromes associated with ASD 22

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. 23

24. Genetic enzymatic polymorphism of homocysteine metabolism (G.R. Akopyan) 24

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/AGwere found. • The risk of hyperhomocysteinemia is likely associated with AA carrier of MTR genotypeand 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. 25

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) 26

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. 27

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. 28

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. 29

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

31. Comparative characteristics of IDDby screening data on Kharkiv region for 2000-2008 31

32. Polymorphisms Genotypes and alleles Population selection n=200, % Patient selection n=4586,% Expected genotype frequency, % С677Т MTHFR СТ 40.7 1994/43,48 1926.12/ 42 ТТ 7.04 416/9,07 412.74/ 9 СС 52.26 2175/47,42 2247.14/ 49 Т 27.39 30.81 А66G MTRR AG 43.0 2015/43,93 2248,05/ 49 GG 35.5 1615/35,21 1490,0/ 32,5 AA 21.5 955/20,82 847,95/ 18,5 G 57.0 57.18 AG 334/34,61 341,8/ 35,4 GG 55/5,69 51,0/ 5,3 AA 552/57,20 572,2/ 59,3 G 23.00 The frequency of genotypes and alleles of polymorphic gene variants C677T MTHFR И A66G MTRR (n=4586) 32

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. 33

34. DNA methylation Cytosine Guanine 34

35. Methylation of biologically active substances 35

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

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

38. Homocysteine – a strong oxidant and protein modificator Homocysteine thiolactone Acts in the normal level of homocysteine and in less concentrations (10 nmol/l) !!! Decreases the level and activity of thioredoxin, superoxide dismutase, syntase NO Increases NAD(P)H-oxidase activity LDL oxidation Endothelium damage + thrombogenesis Atheromatous plague formation О2 Protein modification Homocysteine thiolactonase or paraoxonase (PON 1) can hydrolyze homocysteine thiolactone!!! 39

39. 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. 41

40. METHYLATION FUNCTION: • 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. 42

41. 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 • 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 • Formation of other types of tumors increases in DNA hypomethylation 43

42. 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) 44

43. DNA methylation and chromosomal instability • Ehrlich, 2003; Dobge et al, 2005established that DNMT3B mutations in patients with ICF syndromeor Dnmt 3binactivation 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) 45

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

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

46. 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.). 48

47. 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 49

48. 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 50

49. 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) 51

50. 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. 52