Gene polymorphisms and gene

therapy

–

potential applications in dentistry

Gábor Varga

Department of Oral Biology, Semmelweis University

Budapest

2016

Examples of disorders in relation to

molecular diagnostics

• Cystic fibrosis

• Osteogenesis imperfecta

• Dentinogenesis imperfecta

• Amelogenesis imperfecta

• Hypohydrotic ectodermal dysplasia

• Head and neck cancers

• Gene therapy

• Human genome project

Genetic inheritance

All of our cells function

according to the

program stored in their

genetic material.

The genetic material is

located in the

chromosomes in the

nucleus of the cells.

The DNA is the most

important part of the

chromosomes.

cell

nucleus chromosome

The genetic

code of amino

acids –

base triplet

The genetic code of amino acids

Fir

st

Lett

er

Second Letter T

hird

Lette

r

Predictions from 1992-es about DNA sequencing:

1$/bp, 100,000 bp/year

Human genome 3x109bp:

30,000 év, 3 billion dollár ?

New methodological

developments

•Development of automated DNS sequencing

•Polymerase chain reaction (PCR)

•Development of data handling/informatics

(Bioinformatics)

Polymerase chain reaction (PCR) • What for? –PCR is suitable to

multiply a chosen DNA section from

the compley mixture of DNA molecules

– also for cDNA analysis (reversed

from RNA by reverse transcription).

• What is it good for? – Examples for

the fields of application:

detection of leukemias, lymphomas, chromosome-

translocations, causative agents, monoclonality

in justice (VNTR PCR: murder, rape, father trials)

Also for evaluating expressional profile

• How is it possible? –PCR uses some

features of DNA replication, and of

DNA-polymerases.

PCR- RFLP

Restriction fragment lenght polymorphism

Example: DRD4 promoter –521CT SNP 1. Multiplication of investigated area: PCR

–521CT

2.Restriction digestion: II. type restriction endonucleses: 6–8

bp-os palindrome sequence recognition

Spot of cutting

+

index

An example of sequencing results:

Genomics

The genome:

Out of a diploid cell the, haploid DNA content + mitochondrial DNA

Genomics:

Examination of the function, structure and interaction of the genome

and the used methods. The addition of nucleotides examination

includes examination of the proteins, bioinformatics, "Systems

Biology" and so on.

Genomics for example

• Structural genomics

• Comparative genomics

• Functional genomics

• Human genomics

• Pharmacogenomics

• Medical genomics

Characteristics of the human genome

(a few examples)

2003 April: the sequencing of the human genome was

completed

Only 1,2%-a of euchromatic regions code proteins, (exon)

At SNP-level only 1/1000 is the difference between two

human individuals (99,9% identity)

Average exonnumber: 12.2 exon/gene

Most usual intron size: 87 bp; exon size: 145 bp

Most exons in titin gene: 309 pieces

Characteristics of the human genome

(a few examples)

• 2003 April: the sequencing of the human genom was completed

• Only 1,2%-a of euchromatic regions code proteins, exon)

• At SNP-level only 1/1000 is the difference between two human individuals (99,9% identity)

• Average exonnumber: 12.2 exon/gene;

• Most usual intron size: 87 bp; exon size: 145 bp

• Most exons in titin gene: 309 db

What is SNP (Single Nucleotide

Polymorphism)?

ATGGTAAGCCTGAGCTGACTTAGCGT

ATGGTAAACCTGAGTTGACTTAGCGT

snp snp

SNP-s appear as replication error or DNA damage.

Difference in a single nucleotide basis between two individuals of the same species,

frequency >1 % .

Polymorphism

neutral ??? Risk factors

More than 1% Frequency Less than 1%

effect disease

Mutation

Single nucleotide variations/ SNP (“snips”)

•90% of known differencies/variations

• in most SNPs only two allelic variation exists

Length polymorphisms

• STR (short tandem repeats)

• VNTR (variable number of tandem repeats)

Mutation and polymorphism

Human Genetic Varibility

CCCCAGCCTCCTTGCCAACGCCCCCTTTCCCTCTCCCCCTCCCGCTCGGCGCTGACC

CCCCATCCCCACCCCCGTGGGAACACTGGGAGCCTGCACTCCACAGACCCTCTCCTT

GCCTCTTCCCTCACCTCAGCCTCCGCTCCCCGCCCTCTTCCCGGCCCAGGGCGCCG

GCCCACCCTTCCCTCCGCCGCCCCCCGGCCGCGGGGAGGACATGGCCGCGCACAG

GCCGGTGGAATGGGTCCAGGCCGTGGTCAGCCGCTTCGACGAGCAGCTTCCAATAA

AAACAGGACAGCAGAACACACATACCAAAGTCAGTACTGAGCACAACAAGGAATGTC

TAATCAATATTTCCAAATACAAGTTTTCTTTGGTTATAAGCGGCCTCACTACTATTTTAA

AGAATGTTAACAATATGAGAATATTTGGAGAAGCTGCTGAAAAAAATTTATATCTCTCT

CAGTTGATTATATTGGATACACTGGAAAAATGTCTTGCTGGGCAACCAAAGGACACAA

TGAGATTAGATGAAACGATGCTGGTCAAACAGTTGCTGCCAGAAATCTGCCATTTTCT

TCACACCTGTCGTGAAGGAAACCAGCATGCAGCTGAACTTCGGAATTCTGCCTCTGG

GGTTTTATTTTCTCTCAGCTGCAACAACTTCAATGCAGTCTTTAGTCGCATTTCTACCA

GGTTACAGGAATTAACTGTTTGTTCAGAAGACAATGTTGATGTTCATGATATAGAATTG

TTACAGTATATCAATGTGGATTGTGCAAAATTAAAACGACTCCTGAAGGAAACAGCAT

TTAAATTTAAAGCCCTAAAGAAGGTTGCGCAGTTAGCAGTTATAAATAGCCTGGAAAA

GGCATTTTGGAACTGGGTAGAAAATTATCCAGATGAATTTACAAAACTGTACCAGATC

CCACAGACTGATATGGCTGAATGTGCAGAAAAGCTATTTGACTTGGTGGATGGTTTTG

CTGAAAGCACCAAACGTAAAGCAGCAGTTTGGCCACTACAAATCATTCTCCTTATCTT

GTGTCCAGAAATAATCCAGGATATATCCAAAGACGTGGTTGATGAAAACAACATGAAT

AAGAAGTTATTTCTGGACAGTCTACGAAAAGCTCTTGCTGGCCATGGAGGAAGTAGG

CAGCTGACAGAAAGTGCTGCAATTGCCTGTGTCAAACTGTGTAAAGCAAGTACTTACA

TCAATTGGGAAGATAACTCTGTCATTTTCCTACTTGTTCAGTCCATGGTGGTTGATCTT

AAGAACCTGCTTTTTAATCCAAGTAAGCCATTCTCAAGAGGCAGTCAGCCTGCAGATG

TGGATCTAATGATTGACTGCCTTGTTTCTTGCTTTCGTATAAGCCCTCACAACAACCAA

CACTTTAAGATCTGCCTGGCTCAGAATTCACCTTCTACATTTCACTATGTGCTGGTAAA

TTCACTCCATCGAATCATCACCAATTCCGCATTGGATTGGTGGCCTAAGATTGATGCT

GTGTATTGTCACTCGGTTGAACTTCGAAATATGTTTGGTGAAACACTTCATAAAGCAG

TGCAAGGTTGTGGAGCACACCCAGCAATACGAATGGCACCGAGTCTTACATTTAAAG

AAAAAGTAACAAGCCTTAAATTTAAAGAAAAACCTACAGACCTGGAGACAAGAAGCTA

TAAGTATCTTCTCTTGTCCATGGTGAAACTAATTCATGCAGATCCAAAGCTCTTGCTTT

GTAATCCAAGAAAACAGGGGCCCGAAACCCAAGGCAGTACAGCAGAATTAATTACAG

GGCTCGTCCAACTGGTCCCTCAGTCACACATGCCAGAGATTGCTCAGGAAGCAATGG

AGGCTCTGCTGGTTCTTCATCAGTTAGATAGCATTGATTTGTGGAATCCTGATGCTCC

TGTAGAAACATTTTGGGAGATTAGCTCACAAATGCTTTTTTACATCTGCAAGAAATTAA

CTAGTCATCAAATGCTTAGTAGCACAGAAATTCTCAAGTGGTTGCGGGAAATATTGAT

CTGCAGGAATAAATTTCTTCTTAAAAATAAGCAGGCAGATAGAAGTTCCTGTCACTTTC

CCCCAGCCTCCTTGCCAACGCCCCCTTTCCCTCTCCCCCTCCCGCTCGGCGCTGACC

CCCCATCCCCACCCCCGTGGGAACACTGGGAGCCTGCACTCCACAGACCCTCTCCTT

GCCTCTTCCCTCACCTCAGCCTCCGCTCCCCGCCCTCTTCCCGGCCCAGGGCGCCG

GCCCACCCTTCCCTCCGCCGCCCCCCGGCCGCGGGGAGGACATGGCCGCGCACAG

GCCGGTGGAATGGGTCCAGGCCGTGGTCAGCCGCTTCGACGAGCAGCTTCCAATAA

AAACAGGACAGCAGAACACACATACCAAAGTCAGTACTGAGCACAACAAGGAATGTC

TAATCAATATTTCCAAATACAAGTTTTCTTTGGTTATAAGCGGCCTCACTACTATTTTAA

AGAATGTTAACTATATGAGAATATTTGGAGAAGCTGCTGAAAAAAATTTATATCTCTCT

CAGTTGATTATATTGGATACACTGGAAAAATGTCTTGCTGGGCAACCAAAGGACACAA

TGAGATTAGATGAAACGATGCTGGTCAAACAGTTGCTGCCAGAAATCTGCCATTTTCT

TCACACCTGTCGTGAAGGAAACCAGCATGCAGCTGAACTTCGGAATTCTGCCTCTGG

GGTTTTATTTTCTCTCAGCTGCAACAACTTCAATGCAGTCTTTAGTCGCATTTCTACCA

GGTTACAGGAATTAACTGTTTGTTCAGAAGACAATGTTGATGTTCATGATATAGAATTG

TTACAGTATATCAATGTGGATTGTGCAAAATTAAAACGACTCCTGAAGGAAACAGCAT

TTAAATTTAAAGCCCTAAAGAAGGTTGCGCAGTTAGCAGTTATAAATAGCCTGGAAAA

GGCATTTTGGAACTGGGTAGAAAATTATCCAGATGAATTTACAAAACTGTACCAGATC

CCACAGACTGATATGGCTGAATGTGCAGAAAAGCTATTTGACTTGGTGGATGGTTTTG

CTGAAAGCACCAAACGTAAAGCAGCAGTTTGGCCACTACAAATCATTCTCCTTATCTT

GTGTCCAGAAATAATCCAGGATATATCCAAAGACGTGGTTGATGAAAACAACATGAAT

AAGAAGTTATTTCTGGACAGTCTACGAAAAGCTCTTGCTGGCCATGGAGGAAGTAGG

CAGCTGACAGAAAGTGCTGCAATTGCCTGTGTCAAACTGTGTAAAGCAAGTACTTACA

TCAATTGGGAAGATAACTCTGTCATTTTCCTACTTGTTCAGTCCATGGTGGTTGATCTT

AAGAACCTGCTTTTTAATCCAAGTAAGCCATTCTCAAGAGGCAGTCAGCCTGCAGATG

TGGATCTAATGATTGACTGCCTTGTTTCTTGCTTTCGTATAAGCCCTCACAACAACCAA

CACTTTAAGATCTGCCTGGCTCAGAATTCACCTTCTACATTTCACTATGTGCTGGTAAA

TTCACTCCATCGAATCATCACCAATTCCGCATTGGATTGGTGGCCTAAGATTGATGCT

GTGTATTGTCACTCGGTTGAACTTCGAAATATGTTTGGTGAAACACTTCATAAAGCAG

TGCAAGGTTGTGGAGCACACCCAGCAATACGAATGGCACCGAGTCTTACATTTAAAG

AAAAAGTAACAAGCCTTAAATTTAAAGAAAAACCTACAGACCTGGAGACAAGAAGCTA

TAAGTATCTTCTCTTGTCCATGGTGAAACTAATTCATGCAGCTCCAAAGCTCTTGCTTT

GTAATCCAAGAAAACAGGGGCCCGAAACCCAAGGCAGTACAGCAGAATTAATTACAG

GGCTCGTCCAACTGGTCCCTCAGTCACACATGCCAGAGATTGCTCAGGAAGCAATGG

AGGCTCTGCTGGTTCTTCATCAGTTAGATAGCATTGATTTGTGGAATCCTGATGCTCC

TGTAGAAACATTTTGGGAGATTAGCTCACAAATGCTTTTTTACATCTGCAAGAAATTAA

CTAGTCATCAAATGCTTAGTAGCACAGAAATTCTCAAGTGGTTGCGGGAAATATTGAT

CTGCAGGAATAAATTTCTTCTTAAAAATAAGCAGGCAGATAGAAGTTCCTGTCACTTTC

At SNP level the difference between two people is approximately

only 0,1%

sensitive population to a given disease

Thousands of SNP-s for the whole population

ATGATTATAG ATGTTTATAG

In all resistant individuals A a is present

at position 4 at gene X, hhile in the

sensitive population T is present at the same location

Gene X

Resistant population

Genomics for personalized medicine

Inherited disease or state

Mapping

Cloning of gene

Diagnostics Understanding the

biologcal basis of

disease

Preventive

medicine

Pharmaco-

genomics

Gene therapy

Drug

therapy

Time

Human

Genome

Project

speeded it

up

Hypodontia

The etiology of tooth deficiency is complex, and is in

relationship with both genetic and environmental factors

Genetic polymorphism and animal studies show that

tooth development is influenced by various genes

Although the weight of some factors are not known, the

complexity of tooth development is shown through the

molecular study of the odontogenesis of mice, that

includes the involvement of more than 200 gene

Oligodontia (more than 6 missing teeth)

Hypodontia

(tooth development)

MSX1 PAX9

Hypodontia

(tooth development)

MSX1 PAX9

MSX1 is highly expressed in the

healthy mesenchyme, but is absent

from the dental epithelium during

the bud formation.Presumable the

mutation of MSX1 coding regions

cause the deficience of various

teeth, mainly premolars.

Pax9 is also highly expressed in

the maxillar and mandibular

mesenchyme, and has a role in the

development of palatine and teeth.

In humans the mutation or deletion

of Pax9 coding sequence causes

the deficiency of molars mainly.

The results show that there is a link between tooth deficiency and

MSX1, PAX9 and the TGF-α gene markers regulated by them, but to

explore these connections in detail, more, high case number studies

are needed.

Periodontitis

50% periodontitis

20% destructive periodontitis (100% = the whole population)

In the 60s, the classical epidemiological and clinical studies parodontitis was explained only with the bad oral hygene and dental plaque. They thought there is no other etiological factor causing periodontical diseases.

For today it became clear that the chronical destructive periodontitis is a multifactorial disease and the dental bacterial biofilm is necessary but not the only cause for the development of the destructive periodontitis.

Risk factors in periodontitis

Bad oral hygiene Diseases weakening the

immune system

Local plaque retention

factors Nutritional problems

Occlusal overload Osteoporosis

Smoking Earlier periodontitis

Emotional stress Genetic factors

Age and gender Ethnical group

Periodontitis

Patomechanism of development – importance of cytokines and cytokine

receptors

IL-1 TNF

The role of interleukin-1 (IL-1) genes

and the tumor necrosis factor- (TNF-)

have been studied most as a

predisposing genetic factor.

The combinated polymorphism of IL-1

genes (IL-1A -889, IL-1B +3953) show

strong correlation with the seriousness

of periodontitis.

According to the references available

today, the polymorphism of IL-1 gene

predisposes periodontitis by a different

degree in different ethnical groups.

The parodontal effect of the

polymorphism of the gene of TNF-a

shows discrepancy. Some studies states

that the polymorphism of TNF-a is not

predisposing factor, while others

consider the alleles of IL-1 and TNF-a

genes together as the genetic markers

of complex inflammatory diseases.

The combinated genotype of the

polymorphisms of TNF-a and TNF-b

genes increases the tendency for

developing parodontitis in a given

population.

Beside these the polymorphism of numerous citokin- and non-citokin-

coding genes can affect the development of periodontitis.

Periodontitis

Patomechanism of development – importance of cytokines and cytokine

receptors

IL-1 TNF

Potential application of molecular genomics in

dentistry

– Tooth development

– Caries formation

– Tooth erosion

– Gingivitis

– Periodontitis

– Genomics of pathogenic bacteria

– Salivary gland diseases

Naked DNA Target

Cell

Therapeutic

Protein

AAV

Retrovirus/Lentivirus

Adenovirus

Nucleus

Gene Therapy Principles

Naked DNA Target

Cell Therapeutic

Protein

AAV

Retrovirus/Lentivirus

Adenovirus

Nucleus

Gene Therapy Principles

ADA/gen therapy – the first one

Ashanti de Silva: the first patient who ever

received gene therapy (in 1990).

Adenosin-desaminase-deficiency (ADA)

results in disturbance of T-lymphocyte

function and this led to immunodeficieny.

Ex vivo gene therapy: T-lymphocytes were

infected by ADA cDNA containing

retrovirus, then reinfused to the patient. In

parallel, enzyme substitution therapy was

applied.

(a) (b) (c)

Gene therapy – SCID example

(a) (b) (c)

.

Gene therapy – SCID example

Acinus Ductus Primer secretion

- isotonic Secondary modification

- hypotonic

H2O

K+

HCO3ˉ

H2O

Na+

Cl-

Cl-

Na+

HCO3ˉ

K+

Saliva

Structure of salivary glands – acinus and ductus

Ductus Acinus Vector

Salivary glands as targets of genetherapy

constitutive

regulated

SALIVA

apical

basal

tight junction

BLOODSTREAM

Genetherapeutic treatment of systemic and oral diseases

Salivary glands as targets of gene therapy

constitutive

regulated

Reconstitution of salivary function–ductal-acinar transzdifferention

apical

basal

TJ

circulation

Salivary glands as targets of gene therapy 1

Gene therapeutic treatment of systemic and oral diseases

constitutive

regulated

SALIVA – ie. Antimicrobial substances, antiinflammatory cytokines

apical

basal

TJ

circulation

Salivary glands as targets of gene therapy 2

Gene therapeutic treatment of systemic and oral diseases

constitutive

regulated

NYÁL

apikális

bazális

TJ

Blood circulation – ie. eritopoietin, growth hormon, insulin, antitumor agents etc

Salivary glands as targets of gene therapy 3

Szisztémás és szájüregi betegségek génterápiás kezelése

Treatment of

neck cancer

by use of

Onyx-015

Treatment of oral-head-neck cancers by

application of Onyx-015

Cancer therapy – future perspectives?

Targeted oncolitic virus

+

Anti-angiogenesis transgene

Thank you for your attention