molecular diagnostic in reproductive...
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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
Transcript of molecular diagnostic in reproductive...
Tri H Achmad
Molecular diagnosticin
reproductive endocrinology
Tri Hanggono Achmad
Department of BiochemistryMedical school – Universitas Padjadjaran
Kursus Pencitraan Laboratorium Imunoneuroendokrin Biomolekuler Endokrinologi ReproduksiPertemuan Ilmiah Tahunan
Perkumpulan Obstetri & Ginekologi Indonesia XIVBandung, 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 abnormalityprimarily 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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Functionalcloning
Positionalcloning
Clinicalphenotype
Biochemicalabnormality
Abnormal geneproduct (protein)
Genecloning
Identifycandidate gene
Mapping-linkage to a
chromosomalregion
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Disease w/ genetic component
Map
Clone gene
Diagnostics
Preventivemedicine
Gene th/
Drug th/
Understand basicbiologic defect
Time
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DNA “chip” micro array technology
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FluorescentIn
SituHybridization
(FISH)
Chromosome painting
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Kini ilmu kedokteranlebih dari sekedar intuisi dan “common sense”.
Ilmu kedokteran adalah ketepatanyang didasarkan pada
perbaikan pemahaman tentang penyakitdalam terminologi yang spesifik
yang berkembang lebih dari satu abad
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Kita kini berada padaera kedokteran biofisik-molekuler,
suatu pengaruhyang 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 ofevolutionary conserved mechanisms
to detect extracellular signalsand transduce them into intracellular changes
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Steps in Signal Communication
1. Synthesis2. Release3. Transport to target cell4. Signal detection by specific receptor5. Change in cellular metabolism6. Signal removal termination cellular response
Tri H AchmadHYPOTHALAMUS
ESTRADIOL
CORPUSLUTEUM
PROGESTERONE
Ovulation
FSH LH
Anterior pituitary
GnRH
Gonadotropiccell
Folicle
Inhibin
Uterus, mammary glands,Secondary sex characteristics
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Membrane Events
Intracellular metabolism
Cholesterol source
Membrane events
Cholesterol esters: LDL
Lysosome Lypid stores choleterol esters
CYT.P-450
Choleterol Choleterol
esterase
DenovoCholesterol synthesis
HMG-CoA Reductase
Protein kinase
Glycogenolysisglucose shunt NADPH
cAMP
ATP
Choleterol
Pregnenolone
O2Receptor
PhospholipidsAdenylate
cyclase
Ca2+
ACTH
NAD2+
NAD2-
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Lipoprotein
CholesterolHO
Acetate
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.
DihydrotestosteroneHO H
OH OH
HO O
=OOH
CH2OH
O
CH2OH=OCH
O
HO
CH3
=O
O
Estradiol Cortisol Aldosterone Progesterone
12
34
56
78910
1112
13
14 15
161718
19
21 CH3=O
HOCH3=O
OH
HO 17-OH-pregnenolone
O
Dehyroepiandrosterone(DHEA)
OH
OHAndrostanediol
O Testosterone
5 pathway Pregnenolone
5 pathway=O
CH3
O Progesterone CH2OH=O
Deoxycorticosterone (DOC)O
Corticosterone
17-OH-progesterone
11-deoxycortisol
O
O4-androstenedione
Esterone
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R
CBG
SS
S
SS
S
S
S
DNA
GRE
TranscriptionMachinery(RNA poly-merase, etc
Pre-mRNA(Editing)
mRNA
Protein
Response
Cytoplasm
R Hsp90Hsp90
R* R*
R*R*
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COOH
Bound steroidInhibitor protein hsp 90
HormoneBindingdomain
Hinge region
Steroidhormone
HormoneBinding site
DNA – binding domain
Gene regulatory domain
NH2
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COOH
H2N
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Signal transductions
Play movie
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Protein A
nucleus
mRNA A
HREs
steroid/thyroid hormone/retinoic acid receptor
Steroid/thyroid hormone
retinoic acidPeptide or peptidergic
Gene A
mRNA A
Transcription factor(TF)
PO4-TF
second-messengerregulated kinase orreceptor kinase
cytoplasm
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HOMOLOGIES : 60 - 95% 65 - 75% 30 - 60%
Transcription activation subdomain
Zinc Fingers
Nuclear localication signal
Transcription activation subdomain
-CVARIABLE(IMMUNOGENIC)
N-GR
Heat shock Proteinbindingsite
DNA STEROID
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3’5’
Termination site
1’Transcription initiation site
Structural DNA Region
Regulatory DNA Region
PromoterElement (PE)
Hormone ResponseElement (HRE)
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A
G
CT
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DeoxythymidylateDeoxycytidylateDeoxyguanylateDeoxyadenylateThe combination of a phosphate, a deoxyribose and a base constitutes a deoxynucleotide.
DeoxythymidineDeoxycytidineDeoxyguanosineDeoxyadenosineThe combination of a deoxyribose and a base constitutes a deoxynucleoside .
Thymine (T)
Cytosine (C) Guanine (G) Adenine (A)
Bases Definitions
The rule A+C=T+G
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UridylateCytidylateGuanylateAdenylateThe combination of a phosphate, a ribose and a base constitutes a nucleotide.
UridineCytidineGuanosineAdenosine The combination of a ribose and a base constitutes a nucleoside .
Uracyl (U)
Cytosine (C) Guanine (G) Adenine (A)
Bases Definitions
The rule A+C=U+G CAN'T BE APPLIED HERE
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Transkripsi
Biosintesa Protein
hn RNADNA
Splicing
Penyusunanbentuk 3dimensi
Translasi
mRNA
Protein
Fungsi Protein
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Gene
Primary transcript
mRNA
mRNA
Protein
TRANSCRIPTION
Degradation
MODIFICATION / PROCESSING
Degradation
Degradation
Active inactivedegradation
Transport
TRANSLATION
NUCLEUS
CYTOPLASM
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Hubungan penyakit dengan kelainan molekul :
1. Kelainan struktur biomolekul dapat mengganggu fungsi.Kurang atau tidak berfungsinya biomolekul tertentu akanmengganggu 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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4. Keberadaan suatu biomolekul ditentukan olehgena
5. Kelainan suatu biomolekul dapat menyebabkankelainan organel sel organ
6. Gangguan pada berbagai macam biomolekuldapat menyebabkan gejala klinik danlaboratorium yang sama
Hubungan penyakit dengan kelainan molekul :
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Penyakit genetik :1. Kelainan khromosom
Adanya mutasi pada satu gene- autosomal dominan atau resesif- X-linked
2. MonogenikAdanya mutasi pada satu gene
- autosomal dominan atau resesif- X-linked
3. MultifaktorialKelainan pada beberapa gena disertai pengaruhlingkungan
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Penyakit genetik disebabkan olehkelainan pada materi genetik.Kelainan pada materi genetik sebagai akibat mutasi DNA1. DNA RNA Struktur Fungsi
mutant mutant protein proteinnormal normal
2. DNA RNA Struktur Fungsimutant mutant protein protein
berubah normal
3. DNA RNA Struktur Fungsimutant mutant protein terganggu
berubah ringan
4. DNA RNA Struktur Fungsimutant mutant protein terganggu
berubah sedang/berat
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Dengan mengetahui dasar-dasar molekulersuatu penyakit akan dapat dilakukan:
1. proses diagnosis secara rasional2. melakukan terapi secara tepat (rasional & efektif)3. mencegah terjadinya penyakit atau terjadinya
kekambuhan maupun memburuknya penyakit
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Apakah mutasi DNA akan selalumengganggu fungsi protein?
Tidak, karena DNA pembentuk protein hanya kurang dari lima persen dariseluruh DNA pembentuk gena dalamkromosom
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Proses pengaturan sintesa protein
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POLYMERASE CHAIN REACTION (PCR)(Karl B. Mullis)
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
Teknik Amplifikasi sekuen DNA yang spesifik sehingga dapat dianalisis lebih lanjut
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3 TAHAP PENTING DALAM PROSES PCR:
1. DenaturasiTerjadi penguraian rantai ganda DNA menjadi rantai tunggal dengan bantuan suhu tinggi (90-940C)
2. AnneallingTerjadi 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. EkstensiTerjadi proses pemanjangan untaian nukleotida membentuk fragmen berupa komplemen dari DNA templatSuhu yang digunakan 720C merupakan suhu optimal untuk enzim Taq polimerase
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Molecular techniques have already revolutionizedlaboratory diagnostics in many areasand have vastly expanded the horizons
of both academic and practice
The revolution is as global and profoundas the last major advance in all field of practice,
because molecular techniques are applicableto all sections of the laboratory
While perhaps intimidating to some classically laboratory practitioners,the advent of this new technology should be welcomedfor its inherent scientific excitement and its promise
to rejuvenate traditional laboratory practice
Wayne W. Grody :
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This new molecular tests are not likelyto replace traditional testing in the immediate future.
The cost and complexity of this technologytends to restrict its initial applications to special diagnostic situations
where the information obtained cannot be providedby any other method
Increased automation and commercially designed methodswill 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 expandthe role of the laboratory in areas beyond disease diagnosis
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Work in small sizeYou 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 alone3. Be familiar w./ all materials used4. Eating, drinking & smoking are strictly prohibited5. Unauthorized experiments are not allowed6. Do not use mouth suction7. Be familiar w/. Location & standard safety features
Laboratory notebook
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