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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