Molecular Diagnostic In Reproductive Endocrinology.

Tri H Achmad

Molecular diagnostic
in
reproductive endocrinology
Tri Hanggono Achmad
Department of Biochemistry
Medical school – Universitas Padjadjaran
Kursus Pencitraan Laboratorium Imunoneuroendokrin Biomolekuler Endokrinologi Reproduksi
Pertemuan Ilmiah Tahunan
Perkumpulan Obstetri & Ginekologi Indonesia XIV
Bandung, 11 – 15 Juli 2004

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Clinical genetic science has moved
beyond classical mendelian principles
Nontraditional genetic processes :
- germline mocaism
- uniparental disomy
- mitochondrial inheritance
Require detail inherited disease mechanism
When to recognize that developmental abnormality
primarily genetic or not
How to recognize

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Germline mocaism
- the presence of two or more cell lines w/ differ genotype
- due to mutation occurs in a cell of the developing organism
- after fertilization
- only somatic manifestation or affect gonad
Uniparental disomy
- child possesses two copies of one parent’s chromosome

- child affected if allele causes recessive condition
- eq. Cystic fibrosis
- possible to be detected by DNA analysis
Mitochondrial inheritance
- mtDNA (DNA extra chromosomal)
- contains 13 genes
- matrilineally inherited
- eq. NIDDM, LOHN etc

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Functional
cloning

Positional
cloning
Clinical
phenotype
Mappinglinkage to a
chromosomal

region

Biochemical
abnormality

Abnormal gene
product (protein)

Identify
candidate gene
Gene
cloning

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Disease w/ genetic component
Map
Time

Clone gene

Diagnostics
Gene th/
Preventive
medicine

Understand basic
biologic defect

Drug th/

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DNA “chip” micro array technology

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Chromosome painting

Fluorescent
In
Situ
Hybridization
(FISH)

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Kini ilmu kedokteran
lebih dari sekedar intuisi dan “common sense”.
Ilmu kedokteran adalah ketepatan
yang didasarkan pada
perbaikan pemahaman tentang penyakit
dalam terminologi yang spesifik
yang berkembang lebih dari satu abad

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Kita kini berada pada

era kedokteran biofisik-molekuler,
suatu pengaruh
yang meleburkan dan menyatukan
bagian-bagian tradisi dari kedokteran.
Apakah seseorang berbicara tentang
gangguan metabolisme bawaan,
neurotransmitter, sitokin, onkogen, atau regulasi hormon,
semua dibicarakan secara terperinci,
jelas dan komprehensif pada tingkat molekuler

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Organisms use just a few of
evolutionary conserved mechani sms
to detect extracellular signals
and transduce them into intracellular changes

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Steps in Signal Communication

1. Synthesis
2. Release
3. Transport to target cell
4. Signal detection by specific receptor
5. Change in cellular metabolism
6. Signal removal
termination cellular response

HYPOTHALAMUS

GnRH

Anterior pituitary

Gonadotropic
cell
FSH

LH
Ovulation

Folicle

CORPUS
LUTEUM

Inhibin
ESTRADIOL

PROGESTERONE

Uterus, mammary glands,
Secondary sex characteristics

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Membrane
Events

Intracellular
metabolism
Lysosome

Cholesterol
esters: LDL

Cholesterol
source

Membrane
events

Lypid stores
choleterol esters
esterase

r ot peceR

ACTH

et al ynedA

esal cyc

sdi pil ohpsohP

Choleterol

Ca2+

Choleterol
Denovo
Cholesterol synthesis

ATP
cAMP

Protein
kinase
HMG-CoA Reductase
Glycogenolysis
glucose shunt

NADPH

O2
CYT.
NAD2+
P-450
NAD2Choleterol
Pregnenolone

21 CH3
18 =O
17
12
16
11
13
1 19
15
10 9 8 14

Lipoprotein
Acetate
2
HO

CH3
=O
OH

Cholesterol

HO 3
5

pathway

4

5

7

5

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pathway

6
Pregnenolone

CH3
=O

O
HO

O

17-OH-pregnenolone

Progesterone

=O

O
OH Dehyroepiandrosterone
(DHEA)

Androstanediol

CH2OH

17-OH-progesterone

O

OH
O

Deoxycorticosterone (DOC)

11-deoxycortisol
4-androstenedione

Corticosterone

O Testosterone

HO

OH

H

Dihydrotestosterone

Esterone

CH2OH

OH

=O
OH

O

HO
Estradiol

HO

O
Cortisol

CH2OH
CH =O

O

CH3
=O

O
Aldosterone

Progesterone

Pathways of syntheis of the major classes of steroid hormones, Cholesterol is devided from acetate by sybthesis or from lipoprotein
partcles. The numbering of the steroid molecule is shown for pregnenolone. The major pathways thought to be used are shown.

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R

Hsp90
Hsp90
S

R
S

S
S

R* R*

S

DNA
Response
S

S

R*

S

R*

GRE

Protein

CBG
mRNA
(Editing)
Cytoplasm

PremRNA

Transcription
Machinery
(RNA polymerase, etc

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Bound steroid
Inhibitor protein hsp 90
Hormone
Binding
domain

NH2
Gene regulatory domain

COOH

Hormone
Binding site
DNA – binding domain
Steroid
hormone

Hinge region

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COOH
H2N

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Signal transductions

Play movie

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Steroid/thyroid
hormone
retinoic acid

Peptide or
peptidergic

second-messenger
regulated kinase or
receptor kinase

Transcription
factor(TF)

steroid/thyroid hormone/retinoic
acid receptor

PO4-TF
nucleus

Gene A
HREs
mRNA A

mRNA A

cytoplasm
Protein A

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Nuclear
localication signal
Transcription
activation subdomain

Zinc
Fingers

Transcription
activation
subdomain

Heat shock
Proteinbinding
site

GR

N-

VARIABLE
(IMMUNOGENIC)

DNA

HOMOLOGIES : 60 - 95%

STEROID

65 - 75%

30 - 60%

-C

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Hormone Response
Element (HRE)

Promoter
Element (PE)

5’

3’

1’
Termination
Transcription
site
initiation site

Regulatory DNA Region

Structural
DNA Region

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G
C
T
A

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Definitions

Bases
Thymine
Adenine (A)

Guanine (G)

Cytosine (C)

The combination of a deoxyribose and a base constitutes a
deoxynucleoside .

Deoxyadenosine

Deoxyguanosine

Deoxycytidine

Deoxythymidine

The combination of a phosphate, a deoxyribose and a
base constitutes a deoxynucleotide.

Deoxyadenylate

Deoxyguanylate

Deoxycytidylate

Deoxythymidylate

The rule A+C=T+G

(T)

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Definitions

Bases

Uracyl (U)
Adenine (A)

Guanine (G)

Cytosine (C)

The combination of a ribose and a base constitutes a nucleoside .

Adenosine

Guanosine

Cytidine

Uridine

The combination of a phosphate, a ribose and a base constitutes a nucleotide.

Adenylate

Guanylate

Cytidylate

Uridylate

The rule A+C=U+G CAN'T BE APPLIED HERE

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Biosintesa Protein
mRNA

hn RNA

DNA
Transkripsi

Splicing

Translasi
Protein
Penyusunan
bentuk 3
dimensi
Fungsi Protein

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Gene
TRANSCRIPTION

Primary
transcript
NUCLEUS

Degradation

MODIFICATION / PROCESSING

mRNA

Degradation
Transport

mRNA
CYTOPLASM

Active
degradation

TRANSLATION

Protein

Degradation

inactive

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Hubungan penyakit dengan kelainan molekul :
1.

Kelainan struktur biomolekul dapat mengganggu fungsi.
Kurang atau tidak berfungsinya biomolekul tertentu akan
mengganggu fungsi sel
 organ  penyakit

2.

Gangguan produksi biomolekul normal
- hiperfungsi
- hipofungsi  panyaikit

3.

Kelainan struktur dan jumlah biomplekul
- gangguan berat
- gangguan ringan

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Hubungan penyakit dengan kelainan molekul :
4. Keberadaan suatu biomolekul ditentukan oleh
gena
5. Kelainan suatu biomolekul dapat menyebabkan
kelainan organel  sel  organ
6. Gangguan pada berbagai macam biomolekul
dapat menyebabkan gejala klinik dan
laboratorium yang sama

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Penyakit genetik :
1.

Kelainan khromosom
Adanya mutasi pada satu gene
- autosomal dominan atau resesif
- X-linked

2.

Monogenik
Adanya mutasi pada satu gene
- autosomal dominan atau resesif
- X-linked

3.

Multifaktorial
Kelainan pada
lingkungan

beberapa

gena

disertai

pengaruh

Penyakit genetik disebabkan oleh
kelainan pada materi genetik.
Kelainan pada materi genetik sebagai akibat mutasi DNA
1. DNA  RNA

 Struktur
mutant
protein
normal

 Fungsi
protein
normal

2. DNA  RNA
 Struktur
mutant mutant
protein
berubah

 Fungsi
protein
normal

mutant

3. DNA  RNA
 Struktur  Fungsi
mutant
mutant
protein
terganggu
berubah
ringan
4. DNA  RNA
 Struktur  Fungsi
mutant
mutant
protein
terganggu
berubah
sedang/berat

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Dengan mengetahui dasar-dasar molekuler
suatu penyakit akan dapat dilakukan:
1. proses diagnosis secara rasional
2. melakukan terapi secara tepat (rasional & efektif)
3. mencegah terjadinya penyakit atau terjadinya
kekambuhan maupun memburuknya penyakit

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Apakah mutasi DNA akan selalu
mengganggu fungsi protein?
Tidak, karena DNA pembentuk protein
hanya kurang dari lima persen dari
seluruh DNA pembentuk gena dalam
kromosom

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Proses pengaturan sintesa protein

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POLYMERASE CHAIN REACTION (PCR)
(Karl B. Mullis)
Teknik Amplifikasi sekuen DNA yang spesifik sehingga dapat
dianalisis lebih lanjut
PRINSIP: ~ Proses Replikasi DNA
- Templat DNA
- Primer ( 20 - 25 nukleotida)
- Enzim polimerase (Taq Polimerase)
- Substrat (dNTP)
Perbedaan : Pada PCR pemisahan DNA dengan pengaruh fisik
(suhu tinggi)
Pada Proses Replikasi memerlukan enzim helikase

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3 TAHAP PENTING DALAM PROSES PCR:
1. Denaturasi
Terjadi penguraian rantai ganda DNA menjadi rantai
tunggal dengan bantuan suhu tinggi (90-940C)
2. Annealling
Terjadi penempelan primer pada templat.
Diperlukan suhu yang sesuai dengan primer yang dipakai
(3-50C dibawah melting temperatur;Tm)
Tm = 4(G+C) + 2(A+T)
3. Ekstensi
Terjadi proses pemanjangan untaian nukleotida membentuk
fragmen berupa komplemen dari DNA templat
Suhu yang digunakan 720C merupakan suhu optimal untuk
enzim Taq polimerase

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Wayne W. Grody :
Molecular techniques have already revolutionized
laboratory diagnostics in many areas
and have vastly expanded the horizons
of both academic and practice
The revolution is as global and profound
as the last major advance in all field of practice,
because molecular techniques are applicable
to all sections of the laboratory
While perhaps intimidating to some classically laboratory practitioners,
the advent of this new technology should be welcomed
for its inherent scientific excitement and its promise
to rejuvenate traditional laboratory practice

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This new molecular tests are not likely
to replace traditional testing in the immediate future.
The cost and complexity of this technology
tends to restrict its initial applications to special diagnostic situations
where the information obtained cannot be provided
by any other method
Increased automation and commercially designed methods
will bring cost down,
reduce the level of technical expertise required to perform the tests,
and result in integration of molecular technology
into the mainstream of laboratory testing
Molecular analyses have the potential to greatly expand
the role of the laboratory in areas beyond disease diagnosis

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Work in small size
You never really “see”
Laboratory techniques and procedure will be the “eyes”

General laboratory safety guidelines :
1. Contact lenses should never be worn
2. Never work alone
3. Be familiar w./ all materials used
4. Eating, drinking & smoking are strictly prohibited
5. Unauthorized experiments are not allowed
6. Do not use mouth suction
7. Be familiar w/. Location & standard safety features

Laboratory notebook

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