1 diagnosis and treatment of autism considering features of the genetic background and metabolic...

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Diagnosis and treatment Diagnosis and treatment of autism considering of autism considering

features of the genetic features of the genetic background and metabolic background and metabolic

status status

Ukrainian Institute of Clinical Genetics of KNMUUkrainian Institute of Clinical Genetics of KNMUMember-correspondent of NAMSMember-correspondent of NAMS of Ukraineof Ukraine, ,

M.D., professorM.D., professor

E.Y. GrechaninaE.Y. Grechanina

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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.

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GENOMIC HEALTH

GENE INTERACTION

EPIGENETIC FACTORS

ENVIRONMENTAL FACTORS

Mitochondrial DNANuclear DNA

Gene 1

ACGTAGCTAG

Gene 2

ACGTAGCTAG

Substitution of gene fragment

Gene 2

ACGAGСCTAG

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Genomic health = somatic, psychic, reproductive health

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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.

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

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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.

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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.

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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.

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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».

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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.

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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.

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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.

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AutismAutism – – heterogeneous syndrome heterogeneous syndrome, , which is characterized by disorders which is characterized by disorders in in в 3 в 3 central domains central domains ((fr. Domaine- —area):):

1.1.Social interactionSocial interaction

2.2.SpeechSpeech

3.3.InterestsInterests

and expressed genetic and and expressed genetic and phenotypic heterogeneity.phenotypic heterogeneity.

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AutismAutism – – the most severe result of the most severe result of disorders of nervous system development disorders of nervous system development which is referred to which is referred to autistic spectrum autistic spectrum disorders disorders ((ASDASD))..The frequency of incidence of ASDThe frequency of incidence of ASD 37 37 inin 10 10 000000

Boys are prevalentBoys are prevalent, , especially in clinically severe especially in clinically severe cases.cases.

The frequency of autism The frequency of autism 13 13 inin 10 000. 10 000.

Women/men ratio Women/men ratio 4:1 4:1

((in severe forms in severe forms 1:11:1))

The frequency of Asperger syndrome The frequency of Asperger syndrome 2,6:10 0002,6:10 000

Women/men ratio Women/men ratio 8:1 8:1

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The main feature of modern The main feature of modern knowledge about ASDknowledge about ASD – – its uncertainty its uncertainty..

Many parallel approaches are Many parallel approaches are necessary to understand genetic necessary to understand genetic factors which underlie ASDfactors which underlie ASD::1.1.Studies of the whole genomeStudies of the whole genome;;

2.2.Associative studiesAssociative studies;;

3.3.Revealing mutationRevealing mutation;;

4.4.Expansion of clinical genetic examination Expansion of clinical genetic examination of probands and their relativesof probands and their relatives

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Established genetic basis of autismEstablished genetic basis of autism::

1.1.Increase of the number of publications confirming Increase of the number of publications confirming that mutation and structural changes in any of that mutation and structural changes in any of several genes can significantly increase the risk of several genes can significantly increase the risk of this disease.this disease.

2.2.If the diagnosis of autism is established in a child, If the diagnosis of autism is established in a child, the risk for the family will be 25 tomes higherthe risk for the family will be 25 tomes higher..

3.3.Cognitive behavior features, which are similar to Cognitive behavior features, which are similar to those observed in probands, are more likely those observed in probands, are more likely observed in sibs and parents of an ill child than in observed in sibs and parents of an ill child than in controls controls ..

4.4.Independent studies of twins show concordance Independent studies of twins show concordance for for monozygoticmonozygotic twins twins 70-90 %, 70-90 %, for for dizygoticdizygotic twins twins fromfrom 0 0 toto 10%. 10%.

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

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Many studie indicate Many studie indicate system coursesystem course of disorders in ASD developmentof disorders in ASD development.. Different influence of maternal and Different influence of maternal and paternal paternal 1515q11q11 in ASDin ASD is an important is an important confirmation of cytogenetic disorders.confirmation of cytogenetic disorders. It is supposed that different It is supposed that different molecular events are at the level of molecular events are at the level of systems.systems.

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In recent years greater number of associated with

autism syndromes are found (Tab. 1).

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Table Table 1: 1: Syndromes associated withSyndromes associated with ASDASD№ Syndromes Genes which

are associated with syndromes

Proportions of patients with syndromes followed by ASD

Proportions of patients with ASD, who have these syndromes

1 15q dupAngelman syndrome

UBI3A(and other)

>40 1,2%

2 16p11 del Gene is unknown

high -1%

3 22q del SHANK3 high -1%

4 Syndrome of cortical dysplasia of focal epilepsy

CNTNAP2 ~ 70% rare

5 Fragile X-chromosome FMR1 25% of men;6% of women

1-2%

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Table Table 11 (continuation) (continuation): : Syndromes associated with ASDSyndromes associated with ASD

№ Syndromes Genes associated with syndromes

Proportions of patients with syndromes followed by ASD

Proportions of patients with ASD, who have these syndromes

6 Hobart syndrome GOUBIRT,Many loci

25% rare

7 Potocki-Lupski syndrome Chromosomes17 р 11

~ 90% unknown

8 Smith-Lemli-Opitz syndrome

DHSA7 50% rare

9 Rett syndrome MISP2 All individuals who have Rett syndrome

~ 0,5%

10 Timothy syndrome SASNAIS 60-80% unknown

11 Tuberous sclerosis TSC1, TSC2 20% ~1%

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•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.

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Name of enzyme GENE COENZYME

MUTATIONS DISEASES

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.B12 A2756G Thromboembolia

Colorectal cancer

Malignant lymphoma

Methionine synthase reductase

MTRR A66G Cardiovascular

Сystathionine-β-synthase CBS Vit.B6 Ile to Thr

Gly to SerHomocystinuria

Cystathionine-γ-lyase CSE / CBL Congenital cystathionineuria

Methionine

adenosyltransferase

MAT I / III Hypermethioninemia

Glycine N-methyltransferase deficiency

GNMT Liver pathology

S-Adenosyl-homocysteine hydrolase

SAHH Psychomotor development delay, neurological abnormalities, hepatitis, myopathy

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

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Polymorphisms of genes of folate and methionine cycle

Hyperhomocysteinemia was found in every third from Hyperhomocysteinemia was found in every third from examined patients with IHD and preeclampsiaexamined patients with IHD and preeclampsia

Genotypes with high predisposition to homocysteine-Genotypes with high predisposition to homocysteine-associated thrombophilia in the case of their associated thrombophilia in the case of their assessment by four polymorphic loci MTHFRassessment by four polymorphic loci MTHFR СС677677TT__AA1298C1298C / / MTRMTR 2756 2756 AGAG / / MTRRMTRR 66 66 AGAG:: CT_AA/AA/GG, CT_AC/AA/GG , CС_AA/AA/GG , CT_AA/AA/GG, CT_AC/AA/GG , CС_AA/AA/GG , CT_AC/AA/AA , CС_AC/GG/GG, CC_AC/AA/AG, CT_AC/AA/AA , CС_AC/GG/GG, CC_AC/AA/AG, CC_AA/AA/AGCC_AA/AA/AG were foundwere found..

The risk of hyperhomocysteinemia is likely associated The risk of hyperhomocysteinemia is likely associated with with AAAA carrier of carrier of MTR MTR genotype genotype andand GG GG of of MTRRMTRR genotypegenotype

Carriers of four and more mutant alleles (MTHFRCarriers of four and more mutant alleles (MTHFR,, MTR, MTR, MTRRMTRR) need screening for homocysteine content in ) need screening for homocysteine content in blood plasmablood plasma..

There is necessity of hyperhomocysteinemia There is necessity of hyperhomocysteinemia verification using methionine loading testverification using methionine loading test..

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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)

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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.

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•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.

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

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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

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Comparative characteristics of IDDComparative characteristics of IDD by screening by screening

data on Kharkiv region for data on Kharkiv region for 2000-20082000-2008

Diseases

2000 2001 2002 2003 2004 2005 2006 2007 2008

Ab. amount

In. value

Ab. amount

In. value

Ab. amount

In. value

Ab. amount

In. value

Ab. amount

In. value

Ab. amount

In. value

Ab. amount

In. value

Ab. amount

In. value

Ab. amount

In. value

1.Anencephaly 2 0,993 1,53 3 1,45 3 1,38 1 0,44 2 0,90 1 0,41 1 0,39 1 0,36

2.Myelocele 7 3,495 2,55 6 2,90 9 4,16 10 4,40 11 4,75 5 2,07 12 4,72 8 2,9

3.Meningocele - -1 0,53 1 0,48 1 0,46 - - - - - - 3 1,18 2 0,72

4.Hydrocephaly 3 1,499 4,59 14 6,78 10 4,63 13 5,72 7 3,56 13 5,38 12 4,72 8 2,9

5.Microcephaly 5 2,493 1,53 - - 4 1,85 5 2,20 5 2.97 9 3,72 5 1,96 5 1,8

6.Anotia - -- - - - 2 0,92 1 0,44 2 0,90 4 1,64 5 1,96 3 1,09

7.Anophthalmia 1 0,49- - 1 0,48 - - - - - - - - 1 0,39 - -

8.Microphthalmia 2 0,991 0,53 1 0,48 - - 1 0,44 4 1,79 4 1,64 1 0,39 2 0,72

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Polymorphisms Genotypes and alleles

Population selection n=200, %

Patient selectionn=4586,%

Expected genotype

frequency, %

С677ТMTHFR

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

А66GMTRR

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

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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.

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DNA methylationDNA methylationCytosine Guanine

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Methylation of biologically active substances

3636Betaine 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

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Hyperhomocysteinemia and methylation disorder(G.R. Akopyan)

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О2

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

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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.

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METHYLATION FUNCTIONMETHYLATION FUNCTION::

1.1. DNA methylation is necessary for DNA methylation is necessary for support of differential expression of support of differential expression of paternal and maternal gene copy paternal and maternal gene copy susceptible to the genome susceptible to the genome imprinting imprinting ..

2.2. For stable gene silencing on For stable gene silencing on inactive X-chromosome.inactive X-chromosome.

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3.3. Stable transcriptional repression of provirus Stable transcriptional repression of provirus genomes and endogen retrotransposons depends genomes and endogen retrotransposons depends on DNA methylationon DNA methylation

DNA methylation takes part in management and DNA methylation takes part in management and support tissue-specific patterns of gene support tissue-specific patterns of gene expression in developmentexpression in development

Absence of DNA methylationAbsence of DNA methylation decreases reliability decreases reliability of support of chromosomes number that leads to of support of chromosomes number that leads to chromosomal aberrationschromosomal aberrations

DNA hypomethylation in consequence of DNA hypomethylation in consequence of influence of DNA-methyltransferase inhibitors influence of DNA-methyltransferase inhibitors leads to elimination of tumorsleads to elimination of tumors

Formation of other types of tumors increases in Formation of other types of tumors increases in DNA hypomethylationDNA hypomethylation

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Entirety of methylation systems Entirety of methylation systems determines genome and it means psychic, determines genome and it means psychic, physic, reproductive health.physic, reproductive health.

Studies, which explain how environmental Studies, which explain how environmental factors can induce epigenetic changes and factors can induce epigenetic changes and biologic effects, have appearedbiologic effects, have appeared..

En Li, Adrian Bira En Li, Adrian Bira (2010)(2010)

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DNA methylation and DNA methylation and chromosomal instabilitychromosomal instability

Ehrlich, 2003Ehrlich, 2003;; Dobge et al, 2005 Dobge et al, 2005 established that DNMT3B mutations in established that DNMT3B mutations in patients with ICF syndromepatients with ICF syndrome or Dnmt 3bor Dnmt 3b

inactivation in mice lead to various inactivation in mice lead to various chromosomal aberrations (structural and chromosomal aberrations (structural and quantitative)quantitative)

There is the hypothesis that DNA There is the hypothesis that DNA methylation contributes exact methylation contributes exact chromosomal disjunction and in its chromosomal disjunction and in its absence more often there is leading to absence more often there is leading to chromosomal disorderschromosomal disorders nondisjunction nondisjunction (hypomethylation, demethylation) (hypomethylation, demethylation)

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

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Identification of folate cycle Identification of folate cycle disorders includedisorders include::

1.1. Inherent malabsorption of folic acid Inherent malabsorption of folic acid caused by mutations in the gene caused by mutations in the gene which encodes folic acid which encodes folic acid transportertransporter..

2.2. Deficiency of formiminotransferase Deficiency of formiminotransferase caused by the mutation in FTCD caused by the mutation in FTCD gene.gene.

3.3. Deficiency of Deficiency of methylentetrahydrofolate reductase methylentetrahydrofolate reductase caused by the mutation in MTHFR caused by the mutation in MTHFR gene.gene.

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4-5.4-5. Deficiency of functional methionine Deficiency of functional methionine synthase as a result of mutations in MTR synthase as a result of mutations in MTR gene affecting methionine synthase gene affecting methionine synthase ((cblG) cblG) or mutations affecting methionine or mutations affecting methionine synthase reductase synthase reductase ((cblE due to the cblE due to the mutation in MTRmutation in MTRРР gene gene). ).

6.6. Cerebral deficiency of folic acid caused by Cerebral deficiency of folic acid caused by mutations in FOLR1 genemutations in FOLR1 gene..

7.7. Deficiency of thrifunctional enzyme Deficiency of thrifunctional enzyme containing methylenetetragydrofolate containing methylenetetragydrofolate cyclohydrogenase and cyclohydrogenase and formyltetrahydrofolate synthase caused formyltetrahydrofolate synthase caused by mutations in MTHFD1 gene by mutations in MTHFD1 gene ((Mac Gill, Mac Gill, Rosenblatt et al.Rosenblatt et al.). ).

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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

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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

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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)

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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.

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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.

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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.

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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 testingVarious 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.

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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.

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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.

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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.

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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.

18.04.2318.04.23

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.

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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

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Factors which lead to switching on epigenesis:

- Nutrition;- Infection;- Smoking;- Stress;- Trauma;- Operation;- Alcohol.

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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.

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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.

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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.

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Effects of epigenetics

Genome imprinting (and its disorders)Cellular differentiationTransgenerative epigenetic effectsMutation processBlastemasOrganism ageingConservatism of genetic information

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Mechanisms of epigeneticsDNA methylationChromatine remodelingRNA-mediated modificationsProtein preionizationX-chromosome inactivation

67

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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).

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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).

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EPIGENETIC DISEASES INCLUDEEPIGENETIC DISEASES INCLUDE

(HUDS Y.ZOGHBI, ART(HUDS Y.ZOGHBI, ARTHUR LHUR L. . BEANDETBEANDET)):: 11..Genome imprinting disorderGenome imprinting disorder..

1.1.1.1. Sister syndromes Sister syndromes;; Prader-Willi syndrome Prader-Willi syndrome..1.2. 1.2. Beckwith-Wiedemann syndromeBeckwith-Wiedemann syndrome1.3. 1.3. Silver-Russell syndromeSilver-Russell syndrome1.4.1.4. Pseudohypoparathyroidism Pseudohypoparathyroidism..

2. 2. Disorders influencing on chromatin structure in Disorders influencing on chromatin structure in transconfigurationtransconfiguration::

2.1. 2.1. Rubinshtein-Taybi syndromeRubinshtein-Taybi syndrome2.2. 2.2. Rett syndromeRett syndrome2.3. 2.3. X-linked X-linked ά-ά-thalassemia followed by mental delay. The thalassemia followed by mental delay. The

syndrome of immunodeficiency, instability of the syndrome of immunodeficiency, instability of the centromeric region and facial anomalies centromeric region and facial anomalies

2.4.2.4. Spondyloepiphyseal dysplasia of Schimke Spondyloepiphyseal dysplasia of Schimke..2.52.5. . Methylenetetrahydrofolate reductase deficiencyMethylenetetrahydrofolate reductase deficiency..

3.3. Disorders influencing on chromatin structure in cis- Disorders influencing on chromatin structure in cis-configurationconfiguration..

3.1. άδβ-δβ-3.1. άδβ-δβ-thalassemiathalassemia3.2. 3.2. X-fragile syndromeX-fragile syndrome3.3. 3.3. Facioscapulohumeral muscular dysrtrophyFacioscapulohumeral muscular dysrtrophy

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Classification of epigenetic human diseases Classification of epigenetic human diseases ((SS.А..А. Nazarenko Nazarenko, 2004), 2004)

Epigenetic status disorder of separate Epigenetic status disorder of separate regions of the genome (locate effectregions of the genome (locate effect))

Disorder of epigenetic status of the Disorder of epigenetic status of the whole genome whole genome ((global effectglobal effect))

1. 1. Diseases caused by inherited mutations Diseases caused by inherited mutations disturbing monoallele gene expression – disturbing monoallele gene expression – diseases of genome imprinting diseases of genome imprinting ((Beckwith-Beckwith-Wiedemann syndromeWiedemann syndrome, , Prader-Willi Prader-Willi syndrome, Engelman syndromesyndrome, Engelman syndrome))

1. 1. Diseases caused by inherited mutations Diseases caused by inherited mutations of genes, products of which are involved of genes, products of which are involved in the support of DNA methylation level in the support of DNA methylation level or modification of methionine structure - or modification of methionine structure - ICFICF syndrome syndrome,, Rett syndrome Rett syndrome, ATR-X, ATR-X syndromesyndrome, , Rubinshtein-Taybi syndromeRubinshtein-Taybi syndrome, , Coffin-Lowry syndromeCoffin-Lowry syndrome

2. 2. Diseases caused by methylation status Diseases caused by methylation status disorder of separate genes in the result of disorder of separate genes in the result of de novo mutations in somatic cells - de novo mutations in somatic cells - aa))cancer areas connected with imprinting cancer areas connected with imprinting loss leading to inactive gene activation or loss leading to inactive gene activation or inhibition of active gene expressioninhibition of active gene expression; ; bb))cancer diseases caused by cancer diseases caused by hypermethylation of promoters of tumor hypermethylation of promoters of tumor suppressor gene suppressor gene

2. 2. Diseases caused by global disorders of Diseases caused by global disorders of genome methylation in the result of de genome methylation in the result of de novo mutations in somatic cells – cancer novo mutations in somatic cells – cancer disease connected with the global genome disease connected with the global genome hypomethylation leading to activation of hypomethylation leading to activation of oncogenes, retrotransposons and oncogenes, retrotransposons and chromosomal instabilitychromosomal instability

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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.

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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).

747474

Metabolism

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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.

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Biological function of methionine

An essential acidA component of aminoacyl tRNA

biosyntaseA component of glycine metabolism,

serine and trianineA component of histidine metabolismA component of methionine metabolismA component of selenium amino acid

metabolismA component of tyrosine metabolism

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Enzymes of methionine metabolism are presented by

Methionine syntaseThyrosine amino transferaseS-adenosyl methionine synthetase isoform II

typeArsenit methyltransferaseIndomethylamine N-methyltransferaseS-adenosyl methionine synthetase isoform I

typeBetaine homocysteine S-methyltransferase I.Methionine-tRNA synthetase, cytoplasmicMethionine adenosyltranferase II beta

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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.

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Folate and methionine cycles

8080

Classification (N. Blau et al., 1996)

Disorder —Affected component

Tissue distribution Chromosome

localisation

№ MIM

10.1 Methionine adenosyltransferase (МАТ) of the liver

Liver 250850

10.2 Cystathionine beta-synthase (CBS)

Liver, brain, lymphoblasts, cultured fibroblasts, amniocytes and choroidal fibers

21q22.3 236200

10.3 Gamma-cystatathionase(СТН)

Liver, lymphoblasts 16 219500

10.4 Sulfitoxidase, isolated or molybdenum cofactor10.4.1. Type А10.4.2 Type В

Liver, kidneys, lungs, heart, lymphoblasts, choroidal fibers, cultured fibroblasts and amniocytes

272300

252150252160

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Classification (N. Blau et al., 1996)

Disorder —Affected component

Tissue distribution Chromosome

localization

№ MIM

10.5 5,10- Methylenetetrahydrofolate reductase (MTHFR)

Liver, lymphocytes, lymphoblasts, choroidal fibers, cultured fibroblasts

1р36.3 236250

10.6 Methionine synthase (methyl cobalamin) cblE, cblG

236270250940

10.7 Methylmalonyl-СоА-mutase(adenosylcobalamine) and methionine synthase methylcobalamin)

Liver, cultured fibroblasts, amniocytes

277400277410277380

Liver, cultured fibroblasts, amniocytes

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

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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/ Hmz

gN/

HtzgHmzg/

NHtzg/N

Deficiency of folate cycle enzymes (16%) 6 11 36 37 69 99 5 36

Deficiency of folate cycle enzymes 3 3 11 13 37 51 1 15

Homocysteine remethylation disorder 1 1 3 3

Spina bifida 1 2

HCU 1 3 7 10 8 19 2 8

HCU (in relatives) 1 1

Thromboembolias/thrombosis 1 1 4 2 3 7 6

Thromboembolias/thrombosis(in relatives ) 1 3 1 2 1

Varicose vein dilatation 1 6 6 11 11 1 5

Varicose vein dilatation (in relatives ) 1 2 4 3 4 4 1 2

8484

Spectrum of nosologies in combination of polymorphisms

С677Т MTHFR / А66G MTRR in patient selection (n=1938)

Nosology spectrumС677Т MTHFR / А66G MTRR

Hmzg/Hmzg

Hmzg/Htzg

Htzg/Htzg

Htzg/Hmzg

N/ Hmzg

N/Htzg Hmzg/N

Htzg/N

IMD (7.8%) 9 10 71 64 71 124 10 44

IMD 2 10 11 13 23 4 13

IMD (in relatives) 2 2 2 1

IMD of amino acids 1 1 2 1 2 2 3

IMD of sulfur-containing amino acids 4 8 5 9 17 1 3

IMD of sulfur-containing amino acids (in relatives) 2 1 1

IMD of fatty acids 3 1 2 4

IMD of methionine 2 7 8 8 3 1

CTD 1 3 21 19 13 33 2 11

CTD(in relatives)

DMD 1 1 3 1

Disorder of tryptophan metabolism 1

Disaccharidose deficiency 1

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Nosology spectrum


Hmzg/Hmzg

Hmzg/Htzg

Htzg/Htzg

Htzg/Hmzg

N/ Hmzg

N/Htzg

Hmzg/N

Htzg/N

Sulfite oxidase deficiency 2 1 3

Metabolism disorder in urea cycle 1Maple syrup disease 1Hyperprolinemia 2 1 1Aminoacidemia 1Aciduria 2 2 4 1Hypothyrodism 1Autism 2Mitochondrial diseases 1 1 8 9 10 19 1 7

Mitochondrial diseases(in relatives) 1Kearns-Sayre syndrome 1Epilepsy 1 1 3 1 5 10 1 2

8686



Hmzg/Hmzg

Hmzg/Htzg

Htzg/Htzg

Htzg/Hmzg

N/ Hmzg

N/Htzg Hmzg/N

Htzg/N

Vascular pathology(8%) 2 5 13 13 46 43 2 22

Inborn heart defect 2 1 5 1

Inborn heart defect(in relatives) 1 3

Cardiopathy 1 1 2

Myocardial infarction 1 1 1

Myocardial infarction (in relatives) 1 1 2 2 3

Ischemic heart disease 1 1

Ischemic heart disease (in relatives) 1 1 1 1

Vascular pathology 1 11 8 8

Vascular pathology (in relatives) 1 9 6 2

Insults 2 1 3

Insults (in relatives) 2 3 4 13 7 4

8787



Hmzg/Hmzg

Hmzg/Htzg

Htzg/Htzg

Htzg/Hmzg

N/ Hmzg

N/Htzg

Hmzg/N

Htzg/N

Monogene pathology (5%) 2 4 11 11 20 21 4 8

Ehlers-Danlos syndrome 1 2 1 4 5 6 2

Prader-Willy syndrome 1 1 1 1

Louis-Bar syndrome 1 1

Klippel-Trenaunay syndrome 1 1

Tuberous sclerosis 2 2 1

Rendu-Osler syndrome 1 1

Silver-Russell syndrome 1

Rubinstein-Taybi syndrome 1

Hyperirritability syndrome 1 2 1

Arnold-Kiari syndrome 1

IDD syndrome 1

AGS 1

Anonichia-ectodactyly syndrome 1

McCune-Albright syndrome 1

Lesch-Nyhan syndrome 1

8888



Hmzg/Hmzg

Hmzg/Htzg

Htzg/Htzg

Htzg/Hmzg

N/ Hmzg

N/Htzg

Hmzg/N

Htzg/N

Hemorrhagic syndrome 1Erb-Rott myopathy 1Myopathy syndrome 1 1Duchenne muscular dystrophy 2PKU 1 1 1Cystic fibrosis 3 2 1 2Reiter syndrome 1Marfan syndrome 1 1 2Dandy-Walker syndrome 1Fridreich’s ataxia 1Polyneuropathy 1 1Pertas disease 1Gilbert’s syndrome 1 2DHD syndrome 1Hypophyseal nanism 1

8989


Nosology spectrum


Hmzg/Hmzg

Hmzg/Htzg

Htzg/Htzg

Htzg/Hmzg

N/ Hmzg

N/Htzg

Hmzg/N

Htzg/N

Chromosomal pathology(6%) 2 16 10 29 27 2 14

Down syndrome 1 5 1 5 14 1 2Down syndrome(in relatives) 6 3 14 6 1 6

Shereshevsky-Turner syndrome 2 5 4 4

Various chromosomal pathologies/polymorphisms 1 3 6 5 3 2

9090

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.

9191

Rendu-Osler diseaseRendu-Osler disease. .

Polymorphic variant of 677 C/T MTHFR gene in homozygotous

state

9292

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?).

9393

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

9494

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+.

9595

Saethre-Chotzen syndrome,

secondary mitochondriopathy, folate cycle deficiency

9696

McCune-Albright syndrome in mother.

Polymorphic variants of Polymorphic variants of 677ТТ677ТТ MTHFR/66A/G MTRR genesMTHFR/66A/G MTRR genes.

Healthy children

9797

Robinow syndromeRobinow syndrome. .

Multiple harmatose growth in the Multiple harmatose growth in the liver Polymorphic variants ofliver Polymorphic variants of 677ТТ677ТТ

MTHFR/66GG MTRR genesMTHFR/66GG MTRR genes

98

Ukrainian Institute of Clinical Genetics

KhNMUKharkiv-22, Pravdu avenue, 13

Е-mail: [email protected]

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