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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
Lipoprotein Acetate 2 HO
CH3 =O OH
Cholesterol
HO 3 5
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21 CH3 18 =O 17 12 16 11 13 1 19 15 10 9 8 14
pathway
4
5
7
5
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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1-7
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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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