Molecular Diagnostic In Reproductive Endocrinology.

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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

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Tri H Achmad

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

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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

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Tri H Achmad

Functional

cloning Positional_cloning Clinical phenotype Biochemical abnormality Abnormal gene product (protein) Gene cloning Identify candidate gene Mapping-linkage to a chromosomal

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Disease w/ genetic component

Map

Clone gene

Diagnostics

Preventive medicine

Gene th/

Drug th/ Understand basic biologic defect

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Tri H Achmad

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

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Tri H Achmad

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,

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Organisms use just a few of

evolutionary conserved mechani sms

to detect extracellular signals

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Tri H Achmad

Steps in Signal Communication

1. Synthesis 2. Release

3. Transport to target cell

4. Signal detection by specific receptor 5. Change in cellular metabolism

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ESTRADIOL CORPUS LUTEUM PROGESTERONE Ovulation FSH LH Anterior pituitary GnRH Gonadotropic cell Folicle Inhibin

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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 Denovo Cholesterol synthesis HMG-CoA Reductase Protein kinase Glycogenolysis

glucose shunt NADPH

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-Tri H Achmad Lipoprotein

Cholesterol HO

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.

Dihydrotestosterone HO H OH OH HO O =O OH CH₂OH

O

CH₂OH =O CH O HO CH₃ =O O

Estradiol Cortisol Aldosterone Progesterone 1

2

3

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

21 CH₃ =O HO CH₃ =O OH HO

17-OH-pregnenolone O Dehyroepiandrosterone (DHEA) OH OH Androstanediol O Testosterone

5pathway Pregnenolone

5_pathway

=O CH₃

O _Progesterone _CH

2OH

=O

Deoxycorticosterone (DOC) O

Corticosterone 17-OH-progesterone

11-deoxycortisol O

O

4_{-androstenedione}

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Tri H Achmad

COOH

Bound steroid Inhibitor protein hsp 90

Hormone Binding domain Hinge region Steroid hormone Hormone Binding site

DNA – binding domain

Gene regulatory domain

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COOH

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Signal transductions

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Protein A nucleus mRNA A HREs steroid/thyroid hormone/retinoic acid receptor Steroid/thyroid hormone retinoic acid Peptide or peptidergic Gene A mRNA A Transcription factor(TF) PO4-TF second-messenger regulated kinase or receptor kinase

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Tri H Achmad

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3’ 5’ Termination site 1’ Transcription initiation site Structural DNA Region Regulatory DNA Region

Promoter Element (PE) Hormone Response

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A

G C

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Deoxythymidylate Deoxycytidylate

Deoxyguanylate Deoxyadenylate

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

Deoxythymidine Deoxycytidine

Deoxyguanosine Deoxyadenosine

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

Thymine (T) Cytosine (C)

Guanine (G) Adenine (A)

Bases Definitions

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Uridylate Cytidylate

Guanylate Adenylate

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

Uridine Cytidine

Guanosine Adenosine

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

Uracyl (U) Cytosine (C) Guanine (G) Adenine (A) Bases Definitions

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Gene Primary transcript mRNA mRNA Protein TRANSCRIPTION Degradation

MODIFICATION / PROCESSING

Degradation

Active inactive degradation

Transport

TRANSLATION

NUCLEUS

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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

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Tri H Achmad

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

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Tri H Achmad

Penyakit genetik disebabkan oleh

kelainan pada materi genetik.

Kelainan pada materi genetik sebagai akibat mutasi DNA

1. DNA  RNA  Struktur  Fungsi mutant mutant protein protein

normal normal

2. DNA  RNA  Struktur  Fungsi mutant mutant protein protein

berubah normal

3. DNA  RNA  Struktur  Fungsi mutant mutant protein terganggu

berubah ringan

4. DNA  RNA  Struktur  Fungsi mutant mutant protein terganggu

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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

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Tri H Achmad

Apakah mutasi DNA akan selalu

mengganggu fungsi protein?

Tidak, karena DNA pembentuk protein

hanya kurang dari lima persen dari

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Tri H Achmad

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)

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3 TAHAP PENTING DALAM PROSES PCR:

1. Denaturasi

Terjadi penguraian rantai ganda DNA menjadi rantai tunggal dengan bantuan suhu tinggi (90-940_C)

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 720_{C merupakan suhu optimal untuk}

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Tri H Achmad

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

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Tri H Achmad

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

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