Penelitian biologi molekular

THE APPROACH OF RESEARCH IN
MOLECULAR & GENETIC DISEASES
dr. Retno Sutomo, Sp.A(K), Ph.D
Cluster of Molecular & Genetic Diseases
Cluster of Child Health
Department of Child Health, School of Medicine, UGM
rsutomo@yahoo.com, rsutomo@ugm.ac.id
Basic concept
chromosome
DNA
gene
RNA
codon
amino acid
exon
Intron
coding region
non-coding
region
promoter
allele
Molecular-based study
• Direct testing
– Is there any mutation in a certain gene of interest?
– Sample: DNA, RNA, protein, etc
– Individual test
• Gene tracking
– Does one inherit high-risk chromosome from a heterozygous
parent?
– Test of a family  segregation of a chromosomal segment in
the family
– Use of linked markers
Direct test
• To test a one’s gene or gene product directly –
sequence --- normal or mutant?
• Pre-requisite
– Known target gene
– Known the relevant ‘normal’ (wild-type) sequence
• Mostly apply PCR-based test
Sources of sample
•
•
•
•
•
•
•
Blood samples
Mouthwashes or buccal scrapes
Chorionic villous biopsy samples
One or two cells removed from eight-cell stage embryos
Hair, semen, etc
Archived pathological specimens
Guthrie cards
Research in
molecular & genetic diseases
Level of study
• DNA level
• RNA level
• Protein level
DNA or RNA?
• DNA  easier to obtain and handle (relatively stable)
• RNA has advantages over DNA, but is more difficult to
obtain and handle
• RT-PCR is more suitable for gene with many exons
• Only RT-PCR can reliably detect aberrant splicing
– Sometimes hard to predict from a DNA sequence change
– May be caused by activation of a cryptic splice site deep
within an intron
DNA or RNA?
• RNA is much less convenient to obtain and work with
– Samples must be handled with extreme care and processed rapidly
to avoid degrading mRNA
– The gene of interest may not be expressed in readily accessible
tissues
– Many mutations are unstable  RT-PCR product from a
heterozygous person may show only the normal allele
Nonsense mutations
Frame-shift mutation
Premature
termination codon
Unstable RNA
Truncated protein
Exon skipping
Functional assay of protein
• The essential question in most genetic diagnoses 
mutated protein -- functional or not?
• Protein-based functional assay -- functional or nonfunctional protein
• Functional protein assays -- specific to a particular protein
• By contrast, DNA technology is generic
Is a DNA mutation pathogenic?
MUTATION
• Deletions : ranging from 1 bp to megabases
• Insertions (including duplications)
• Single base substitutions -- SNP (single nucleotide
polymorphism)
– Missense mutation
– Nonsense mutation
– Splice site mutation  create or destroy signals for exon-intron splicing
• Frame-shifts mutation  deletions, insertions, or splicing errors
• Dynamic mutations (tandem repeats that often change size on
Mutation in highly conserved region
• Region conserved among different species
• Reflects the important role of the region
• House-keeping region : survival, critical
function, etc
Frame-shift mutation
• Mutation alters the reading frame
of RNA transcript
• Ex: dystrophinopathy
o Duchenne muscular dystrophy (DMD)
o Frame-shift mutation
o Becker muscular dystrophy (BMD)
o Non frame-shit mutation
Class of amino acids
Mutation resulting in change
of AA class is more likely to
be pathogenic
How to prove that a mutation
associated with the disease?
• Expression study  expression cloning
• Check the presence/absence of the
mutation in control individuals
– 50 unrelated individual (100 alleles) would
represent population
Design of molecular genetic study
• Observational study
– Mostly, case-control study
• Experimental study
– Therapeutic study (animal or human-based)
Type of study
• Diagnostic study
• Association study
– Genotype-phenotype correlation
– Genetic risk factor
• Prognostic study
• Therapy
Tools for molecular biology research
• Polymerase chain reaction (PCR)
– Pure amplification
– Specific-designed PCR: allele specific PCR, multiplex
PCR, etc
• Restriction enzyme digestion analysis
• Sequencing analysis
• Expression cloning
Research question
Scenario 1:
• A mutation in a certain gene has been reported
to be associated with the development of a
disease in some population
• Question: Does that mutation associate with
the disease in Indonesia as well?
Research question
Scenario 2:
• A mutation has been reported to be associated with the
development of some disease in some country/countries
• A study/studies in Indonesia revealed no such association
• Questions:
– Is/are there other responsible mutation/mutations in the same
gene?
– Is it a mutation/mutation in other gene/genes responsible for that
disease?
Research question
Scenario 3:
• A novel mutation is found in patient with a
certain disease
• Question:
– Is it the causative mutation?
Research question
Scenario 4:
• Comparison of molecular-based diagnostic
tool
The longer TATA box
polymorphism is common and
associated with neonatal
jaundice in US and European
countries
However, in Japanese and
Korean populations TATA box
polymorphism is rare and not
associated with neonatal
jaundice
How about in Indonesian
and Malaysian population?
To detect the presence of the G71R mutation in the UGTA1A gene, a sequencing analysis has to be
applied since the simple detection method, such as restriction enzyme digestion is not applicable.
It results in expensive and time-consuming practice.
Does DHPLC technique have good accuracy in detecting the mutation?
• DNA-based diagnosis of spinal muscular
atrophy
• Restriction enzyme digestion has been
considered as a standard detection
method
• Is there other method available with
comparative advantage?
• DHPLC
– Faster
– More simple procedure
– Cheaper
Genotype-phenotype association study
Proving a novel W92S mutation in the SMN1 gene is pathogenic
- Located in a highly conserved region of Tudor domain
- Absent in 50 control individuals
- Disrupt the function of protein
The G71R mutation of the UGT1A1
gene is a genetic risk factor for the
development of hyperbilirubinemia
Does it impair the conjugation
function of the enzyme?
Genetic aspect of ADHD
• The presence of genetic association between ADHD
and polymorphisms in the dopamine transporter
(DAT1) and dopamine receptor (DRD2, DRD4, DRD5)
genes
• Many studies in many countries
• The common polymorphisms identified
• No data available in Indonesian population
Cornerstone of ADHD medication
Blocks reuptake of dopamine by DAT
Releases dopamine from vesicle stores
 Dopamine in synaptic cleft
Dopamine transporter gene
Dopamine receptor genes
DRD4 gene in Indonesian children with ADHD
• The DRD4 gene 7 repeat (7R) allele of the 48-bp VNTR is
widely considered as a genetic risk factor of ADHD
• The 2R allele has been suggested as a genetic protective
factor
• How about in Indonesian children?
500 bp
400 bp
300 bp
2R/4R
4R/4R
2R/2R
M1
4R/5R
DRD4-48 bp repeat polymorphism
M2
1078 bp
872 bp
603 bp
310 bp
281/271bp
Sutomo (2014), manuscript in preparation
DRD4-48bp VNTR
Genotype distribution
Patient
Control
2R/2R
2R/4R
4R/4R
4R/5R
2R/5R
7 (21.9%)
7 (14.0%)
11 (34.4%
22 (44.0%)
11(34.4%)
19 (38.0%)
3 (9.4%)
1 (2.0%)
0
1 (2.0%)
The known ADHD-related allele
(7R) not exist at all
Sutomo (2014), manuscript in preparation
DRD4-48bp VNTR
Allele distribution
2R-allele  known as protective allele in some studies
2R-allele
non 2R-allele
25 (39.1%)
37 (37.0%)
39 (60.9%)
63 (63.0%)
4R-allele
non 4R-allele
Patient
36 (56.25%)
28 (43.75%)
Control
61 (61.0%)
39 (39.0%)
Patient
Control
4R-allele
No difference in allele distribution among patients and controls
DRD4 gene in Indonesian children with ADHD
• The longer 120-bp duplication in the DRD4 gene has been
associated with the development of ADHD
• Is it the case in Indonesian children?
DRD4-120 bp duplication
L/L
S/L
549 bp
429 bp
S/S
M
500 bp
400 bp
300 bp
200 bp
S = short allele
L = long allele
Sutomo (2014), manuscript in preparation
DRD4-120bp duplication
Genotype distribution
Patient
S/S
1 (3.1%)
S/L
13 (40.6%)
L/L
18 (56.3%)
Σ
32
Control
5(10.0%)
19 (38.%)
26 (52.0%)
50
Allele distribution
Patient
S-allele
49 (76.6%)
L-allele
15 (23.4%)
Control
71 (71.0%)
29 (29.0%)
No difference in the genotype and allele distribution among patients and controls
Still…many spaces
• There remains large space for research in
molecular genetics field in Indonesia
• Recently, there is increasing interest in molecular
genetics research in Indonesia, including among
clinicians