Post on 12-Dec-2020
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