Alida Harahap

Dept. Clinical Pathology

FKUI/RSCM
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Need to know:

Structure and function of

nucleic acids (DNA and RNA)
 replication
 transcription
 translation
 codon
 etc

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Early 90’s: Molecular biology approach
used as a diagnostic tool

Genetic diseases Infectious diseases

Molecular diagnosis
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MOLECULAR DIAGNOSTIC AREAS

Malignancies
Inherited diseases
Identity assesment
Pharmacogenetics

Infectious diseases
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Mutation detection:

Methods:
 PCR
 PCR-RFLP
 oligo specific probe
 ARMS.
 real time PCR for quantitation

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Mutation detection:

Specimens:
• peripheral blood/cord blood lymphocytes
• villi choriales
• amniotic fluid
• circulating nucleic acid
• others

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Mutation detection at DNA level in inherited diseases

fertilization
Sperm

Mature oocyte
Fertilization
placenta
differentiation
Trophoblast

Embryoblast

fetus

Blastocyst
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CIRCULATING NUCLEIC ACIDS
cell free DNA/RNA

Cancer DNA

 Oncogene mutation
 Viral nucleic acid
 etc

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CIRCULATING NUCLEIC ACIDS
cell free DNA/RNA

Fetal DNA
in maternal circulation
 b-thalassemia
 Achondroplasia
 Congenital adrenal hyperplasia

Problem: most of the DNA in maternal plasma

is still of maternal origin.
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CIRCULATING NUCLEIC ACIDS
cell free DNA/RNA

Others

 pulmonary embolism
 myocardial infarction
 stroke
 organ transplantation

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 Molecular Diagnosis
in Inherited Diseases

DNA Mutation

Nuclear Mitochondrial

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Specimens:
• peripheral blood/cord blood lymphocytes
• villi choriales
• amniotic fluid
• circulating nucleic acid

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Mitochondrial genome

Maternal inheritance
Multiple copies in each mitochondrion
High rate of mutation
Heteroplasmy, homoplasmy

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 Mitochondrial DNA mutation

Germline Somatic
Associated with
Neurodegenerative
 aging
and/or myopathic
 cancer
diseases

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 Mitochondrial DNA Neuro-
degenerative/
germline mutation (s) Myopathy

MELAS LHON
Myopathy Leber’s
Encephalopathy Hereditary
Lactic Acidosis Optic
Stroke like Neuropathy
episode
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 Application of
Molecular Diagnosis:

1. Differential diagnosis
2. Carrier detection in the family
3. Carrier detection in the population
4. Prenatal diagnosis
5. Presymptomatic diagnosis
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1. Differential diagnostic testing

X-linked muscular dystrophies

Other neurological disorders

Fragile X syndrome

Other cause of mental retardation

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2. Carrier detection within family

Congenital Adrenal Hyperplasia

(adrenogenital syndrome)
 21-hydroxylase defficiency

Consequences of the salt-losing

defect in early infancy.
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3. Carrier detection within population
For autosomal recessive disorders
Cystic Fibrosis:
Multiple organ – lung, pancreas
Thick mucous secretion
- blockage of airways
- secondary infection
CFTR (CF Transmembrane Conductance
Regulator) – chloride channel
Not efficient for hemoglobinopathies
- too many types of mutation
- use: hematology parameters 22
4. Prenatal Diagnosis
Severe childhood onset diseases
with a poor prognosis
and no effective treatment

5. Presymptomatic diagnosis
For adult onset disorders

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Time limitation: (Examples)

a-thalassemia
Carrier detection in pregnancy

Prenatal diagnosis

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Mutation detection:

Methods:
• PCR
• PCR-RFLP
(restriction fragment length polymorphism)
• Oligo specific probe
• ARMS
(amplification refractory mutation system)
• etc
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PCR

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Sickle-cell disease

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Sickle-cell disease

1300
1100
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Heteroduplex

Homoduplex

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Heteroduplex

Homoduplex

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 PHENYLKETONURIA (PKU)

Inborn errors of metabolism

Mutation in gene encoding phenylalanine hydroxylase

Phenylalanine  Tyrosine
A cause of mental retardation --- can be corrected

Newborn screening
 assay of blood phenylalanine levels

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 TREATMENT OF GENETIC DISEASE

CONVENTIONAL GENE THERAPY

APPROACHES

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Dietary restriction PKU: removal phenylalanine
from the diet

Hormon replacement
Cortison: Congenital Adrenal Hyperplasia
Thyroxine: Congenital Hypothyroidism

Protein replacement
Factor VIII: Hemophilia A
Blood transfusion: thalassemia
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Drug treatment
To lower cholesterol level:
in familial hypercholesterolemia

Avoid drug
Sulphonamide, etc: G6PD deficiency

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AIM
to correct a genetic defect

restore normal gene expression.

To deliver genes to appropriate target cells

To obtain optimal expressions
of introduced genes

To produce a product that the patient lacks

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 GENE THERAPY

Germ line Somatic cell

Genetic changes in Genetic changes in
somatis and germ cells somatic cells only

Transmitted to
next generations

prohibited
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SOMATIC CELL GENE THERAPY

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Technical aspects need to be considered

1. Gene characterization
Gene should have been cloned,
Include the control and regulator region.

2. Target cells/tissue/organ
Must be identified and accessible.

3. Vector system
Gene to be introduced
has to be efficient and safe 39
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EXAMPLES

Hemophilia A Factor VIII

Familial hypercholesterolemia
Low-density lipoprotein Receptor

Cystic Fibrosis
Cystic Fibrosis Trasmembrane
conductance Regulator (CFTR)

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Emery’s Elements of Medical Genetics
Robert F. Mueller, Ian D. Young
Churchill Livingstone

Molecular Diagnosis of Genetic Diseases

Rob Elles
Human Press

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