Pedigree of Hemophilia in One Family

Pedigree of Hemophilia in One Family

female normal male hemophilic male

Hemophilia 

Hemophilia - A sex linked genetic disorder in which blood clotting is deficient

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Hemophilia A - lack of antihemophilic globulin Most common type (80% of cases).

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Hemophilia B - defect in thromboplastic component - a milder form of the disease. Sex linked - trait found on X chromosome.

Chromosomes

22.3 22.2 22.1

p 21.2

21.3

21.1 11.4 11.3 11.23 11.22 11.21 11.1 11.1 11.2 12

X Chromosome growth control factor, X-linked Xg blood roup ocular albinism sensorineural deafness anemia, sideroblastic, with Spinocerebellar ataxia

13 21.2

22.2

21.1

cleft palate

21.3

lymphoproliferative syndrome

22.1 22.3 23 24 25

q

26 27 28

Simpson dysmorphia syndrome coagualation factor IX, hemophilia B blue-monochr. color blindness coagulation factor VIIIc,hemophiliaA homosexuality, male

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Tahun 50-an : DNA double stranded yang membentuk Helix (Watson and Crick),DNA Polymerase (Kornberg) Tahun 60-an : DNA extrachromosome (Plasmid), fungsi mRNA, Codon (Triplet Nucleotide) Tahun 70-an : Reverse Transcriptase, Restriction Endonuclease, DNA Ligase, Recombinant DNA (Berg), Cloning DNA (BIOTEKNOLOGI)

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Tahun 80-an : Transgenic mouse, Penerapan rekayasa genetika dalam bidang kedokteran, pertanian dan industri. Tahun 90-an : Gen therapy, Cloning dan Sequencing DNA (HGP), Diagnostic dll. Tahun 2000-an : HGP selesai, Pathogenese penyakit diketahui dari fungsi molekul.

BODY PROTEIN  Enzyme  Receptor  Hormone  Growth Factor  Immunoglobulin  Interferon, Interleukin  Adhesions molecules  HLA/MHC

α-1         

α-1 Acid Glycoprotein α-1 T Glycoprotein α-1 Antitrypsin Transcortin α-1 Antichymotrypsin α-1 B glycoprotein 9,5-s α-1 Glycoprotein Vitamin-D binding protein α-1 Lipoproteins

α-2          

Retinol binding protein α-2 HS Glycoprotein Histidine-rich 3,8 S α2 Glycoprotein Haptoglobin Pregnancy zone protein α2 Macrogobulin Prothrombin Antihemophilic factor C1 inactivator C1s

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STRUKTUR PROTEIN SIFAT PROTEIN FUNGSI PROTEIN PEMBENTUKAN PROTEIN DISTRIBUSI PROTEIN PEMERIKSAAN PROTEIN

STRUKTUR PROTEIN :    

Struktur Primer : Sequensi asam amino Struktur Sekunder : -helix, lipatan  Struktur Tertier : sub-unit protein (tiga dimensi) Struktur Kwaterner : gabungan bbrp struktur tertier

SIFAT PROTEIN : 

Ditentukan oleh sifat asam amino

FUNGSI PROTEIN : 

Sangat bervariasi

PEMBENTUKAN PROTEIN :  

Berdasarkan gen / DNA di inti sel Berlangsung di Organella (Ribosome)

Proteins are composed of subunits called amino acids

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Biokimia : DNA adalah Polymer dari Desoxyribonucleotide (Basa, zat Gula dan 1 atau lebih gugus Phosphat) Zat Gula : -D-2 Desoxyribose (Ribose) Ikatan N-Glykosida antara Desoxyribose (C1) dengan Pyrimidin (N1) atau Purin (N9)

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Sanger dan Gilbert (1975) : methode sequensi Basa Nukleotida (A, T, C, G) Nukleotida : 2,9 milyar (990 mm) di Chromosome (inti sel) Telah selesai disequensi pada Juli 2000 Gen : Sepotong DNA (Intron atau Exon) A-T G-C Satuan DNA : bp (base pair)

DNA Base Pairing A G C G A T C T G G T C G C T A G A C C Double helix consists of 2 complimentary strands of DNA.

Chromosomes Long strands of DNA packaged and compressed very tightly  Everyone has 2 sets (1 pair) of chromosomes 

1 pair of each of the 22 ‘autosomes’ plus XX for a female (46XX)  or XY for a male (46XY) 

1 is inherited from mum, 1 from dad  You pass 1 of each pair onto each child 

The Human Genome 

The haploid human genome is made up 9 of 3 x 10 base pairs of DNA

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This contains 50,00050,000- 100,000 genes arranged on 46 chromosomes

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Packaged within the nucleus of the cell

DNA Replication 

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Each of the 2 DNA strands is copied by machinery in the cell Each new ‘daughter’ strand has a sequence complimentary to the original ‘template’ strand Replication essential to allow cell division (Mitosis) where 1 cell becomes 2

DNA Replication

C

C A T

A

T

T

A G

T T A A G

G A T C

semi-conservative 2 daughter cells

DNA Replication

DNA Replication   

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Replication fork : leading strand and lagging strand DNA synthesized in the 5’ – 3’ The 5’-3’ synthesis of the leading strand is continuous. The lagging strand is also synthesized in the 5’-3’ direction but in small segments This segments referred to as Okazaki fragments Okazaki fragments has 100 – 200 nucleotides DNA ligase joined the Okazaki fragments. 5 DNA Polymerase : α, β, δ, ε and γ

The DNA Replication Fork

DNA Replication in Meiosis 

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During the replication of chromosomes, there is a cross-over of portions of one DNA strand to another (of the same chromosome). This cross-over, along with randomization assures that offspring differ from the parents.

meiosis

+

Genes Segments of DNA code for proteins (or parts of proteins)  Each coding segment is called a gene  One gene codes one protein (or part of)  Genes contain the information which makes us what we are 

Gene Structure Every three bases of DNA is called a ‘codon’  Each codon specifies an amino acid which join together to form the protein eg ATG = methionine = START TAA = STOP TAG = STOP TGA = STOP 

Gene Structure Introns Promoter

Exons ATG start

TAA TAG ‘stop’ TGA

Exon = coding sequence Intron= intervening sequence (non-coding)

Protein Synthesis transcription

DNA

RNA

Protein

translation

Transcription  

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3 Nuclear RNA Polymerase : mRNA transcribed by RNA Polymerase II The initiation of transcription involves binding RNA Polymerase to a specific DNA sequence called a Promoter Many promoters for RNA Polymerase II contain consensus sequences, referred to as the TATA box ( T A T A A/T A A/T A/G) which occur about 25-35 bp upstream from the transcription initiation site. The activity of many promoters is affected by Enhancers (regulatory sequences that may occur thousands of base pairs upstream or downstream of the gene they affect.

Protein Synthesis - Transcription  

Each gene codes for a protein DNA sense strand acts as template and is ‘transcribed’ into messenger RNA (mirror image of the DNA but Uracil instead of Thymine)

DNA mRNA

AT C G G UAG CC

Protein Synthesis- Translation   

Introns are spliced out of the mRNA mRNA leaves the nucleus In the cytoplasm, ribosomes attach to the mRNA ensuring the correct amino acid, for each codon, is added to a growing chain of amino acids which forms the resulting protein.

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rRNA : 40s particle (sebuah 18S RNA dan 55 % protein) ; 60S particle (28S; 5,8S; 5S rRNA dan protein)(-ribosom:RNA dan protein. Setiap tRNA membawa 1 protein) Translation: 1. Initiation(-mencari AUG) 2. Elongation 3 Termination Translational initiation signal : AUG mRNA become translated through 5’ → 3’ direction Elongation : Peptidyl transferase.(-enzim) Termination : Stop Codon (UAG, UAA, UGA) Amino acid will be activated and linked to the tRNA by Aminoacyl-tRNA synthetase.

Amino acid assembly during translation occurs on ribosomes; tRNA serves as the crucial adaptor molecule(-antibiotik m’blok ribosom menjadi tidak bekerja)

Nukleotida 1. (5’) U

Nukleotida 2.

Nukleotida 3.

C

A

G

U

Phe

Ser

Tyr

Cys

U

U

Phe

Ser

Tyr

Cys

C

U

Leu

Ser

STOP

STOP

A

U

Leu

Ser

STOP

Trp

G

C

Leu

Pro

His

Arg

U

C

Leu

Pro

His

Arg

C

C

Leu

Pro

Gln

Arg

A

C

Leu

Pro

Gln

Arg

G

(3’)

U

C

A

G

A

Ile

Thr

Asn

Ser

U

A

Ile

Thr

Asn

Ser

C

A

Ile

Thr

Lys

Arg

A

A

Met

Thr

Lys

Arg

G

G

Val

Ala

Asp

Gly

U

G

Val

Ala

Asp

Gly

C

G

Val

Ala

Glu

Gly

A

G

Val

Ala

Glu

Gly

G

Perbedaan Sandi Nukleotida 

Nukleotida : UGA AUA AGA AGG

Chr. :

Mit. :

Stop Ile Arg Arg

Trp Met Stop Stop

MITOCHONDRIAL ENERGY TRANSDUCTION

Human body synthesizes body weight of ATP per day • motoric functions • biosynthetic activities • heat maintenance ATP

ADP + Pi

ATP synthase Proton Motive Force

NADH

I coQ

Succinate

II

III

Cytc

IV

O2 H2O

MITOCHONDRIAL RESPIRATORY ENZYME COMPLEXES

Cytosolic side ΔμH+ ADP

c I

Q

NADH NAD + H2

Matrix side

III Q II

IV

H++½O2

UCP

H2O

ATP

V

Succinate ADP+Pi

ANT

ATP

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Gen Mitochondria Gen yang berbentuk sirkuler, terdiri dari 16569 bp Diturunkan secara maternal, mudah bermutasi Menyandi : 7 sub unit kompleks I (NADH Q-Reduktase), 3 sub unit kompleks IV (Sitokrom Oksidase), 2 sub unit ATP Synthase dan 1 sub unit kompleks III (Apositokrom B) Mutasi noktah (point mutation) pada gen mitochondria : A3243G(di jepang) G3316A A3260G T3394C A3256G A3252G luas dijumpai : T16189C(di indonesia)

(jumlah ATP yang dibentuk selama 24 jam adalah sebanyak berat badan stiap org)

MITOCHONDRIAL BIOLOGY AND GENETICS  Semi-autonomous Outer Inner organelles, contain multiple Compartment Membrane copies of mtDNA  Double membrane structure, cristae containing respiratory chain enzymes  Most mitochondrial proteins encoded by nuclear genome Inner Outer Matrix Cristae Compartment Membrane and imported into mitochondria  Functions in cellular metabolism and the regulation of cell death 

(mitokondria tidak sibentuk baru oleh sel, tetapi melalui pembelahan)

MITOCHONDRIAL PROTEINS – mtDNA

• Circular DNA - 16,569 bp • Encodes  13 polypeptides - for OXPHOS  22 tRNA  2 rRNA • D-loop - initiation of replication and transciption • Evolves at higher rate than nDNA • Maternally inherited

Pathogenese NIDDM 

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Patophysiologi secara genetik yang berkorelasi dengan metabolisme energi Timbul oleh karena perobahan cara hidup dengan cepat (terutama dalam hal nutrisi) Sel  Pankreas berfungsi untuk mensekresikan Insulin bergantung pada energi yang dibentuk di Mt. Phosphorilasi oksidatif pada rantai respirasi Mt ATP ATP dependent Potassium Channel tertutup Calcium Channel terbuka sekresi Insulin Mutasi MtDNA penurunan ATP (fungsi insulin:mengaktifkan GLUT 4, kalau insulin sudah ckp byk, reseptor tdk dimunculkan/recycling, jadi reseptor masuk melalui jalur degradasi = down regulation)

MITOCHONDRIAL ENERGY METABOLISM AND INSULIN SECRETION

MODY2

Transmembrane Protein Synthesis

Mutations A change in the DNA sequence of the gene  All cells acquire mutations as they divide -6  rate of approx 10 per gene per cell  Mutations can alter protein product of DNA, stop gene working or activate gene 

Types of Mutation   

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Deletion - DNA missing Insertion - extra DNA inserted Expansion (Amplification) - DNA repeat size has increased Point Mutation - change in one base

Types of Mutation (in coding sequence)

AGC TTC GAC CCG AGC TCG ACC CG AGC TTC CGA CCC G AGC TTC TTC GAC CCG ATC TTC GAC CGG

Wild type Deletion Insertion Expansion Point mutation

(klo berubah as.amino = MISSENSE MUTATION Klo timbul as.amino = nonsense mutation Klo brubah pada protein = silence mutation)

POINT MUTATION UAA (Termination Codon) UCA (Codon for Serine) UCU (Codon for Serine) CCA (Codon for Proline)

Polimerase Chain Reaction (PCR) Tahun 1985, Kary Mullis, California  Metode untuk mengmeng-amplifikasi (melipat gandakan) fragment DNA (Gen)  Dibutuhkan : DNA atau RNA Oligonucleotidprimer (PRIMER) Enzym Taq Taq--Polimerase Campuran dari 4 Basa Nukleotida (d’NTPs) 10 x Reactions Buffer Larutan MgCl2 

Alat : Thermal Cycler Prinsip : perobahan temperatur secara otomatis dengan waktu yang telah ditentukan  Dapat diatur (Program)  Contoh : 95 °C------ Denaturasi 55 °C------ Hybridisasi (Annealing) 72 °C------ Synthese DNA (Extension)  Lama reaksi, bervariasi tergantung panjang fragment DNA (2 min. : < 1000 Nukleotida) 

DNA DNA didi-isolasi dari sel (darah atau jaringan)  DNA menjadi “template” atau “matrix” untuk proses amplifikasi  Sense : 5’5’- ATG(Start) -GGT GGT--TCT TCT--GTT GTT--GCT GCT-GCT--TGG GCT TGG--TAA(Stop) TAA(Stop)-- 3’  Antisense : 3’ - TAC TAC--CCA CCA--AGA AGA--CAA CAA--CGA CGA-CGA--ACC CGA ACC--ATT ATT-- 5 ‘  Exon dan/atau Intron dapat berfungsi sebagai Matrix untuk amplifikasi 

RNA

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Single strand (Uracil pengganti Thymin) Transkripsi dari DNA mRNA Mengandung informasi genetik dari Exon Dengan Enzym Reverse Transkriptase diperoleh DNA dari RNA cDNA Reaksi PCR nya disebut RTRT-PCR

Taq--Polimerase Taq

Klenow - DNA Polymerase dari E.Coli  1988 : TaqTaq-Polymerase dari Bakteri Thermus aquaticus  Hybridisasi dan Polimerisasi berlangsung pada temp. 5050-70 °C  Perhatikan : Buffer yang digunakan (10 x RB) dan diperlukan MgCl2 

Primer Sequence dari Nukleotida tertentu (Intron atau Exon) : 20 – 30 bp  Prinsip : merupakan complementare dari kedua strand DNA (Forward Primer dan Reverse Primer).  Dari kedua Primer ini disinthese DNA yang baru dan seterusnya berfungsi sebagai matrix untuk siklus berikutnya.  Penentu bagi fragment DNA yang akan diamplifikasi 

PCR-REACTION

PCR-Reaction

Polymerase Chain Reaction

PCR Product (Amplifikat)    

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Gel-elektrophorese (Agarose) GelSouthern Blot (Hybridisasi dengan Sonde DNA spesifik) Dot - Blot (deteksi : Enhanced Chemie Luminescense = ECL) Denaturating Gradient Gel Electrophorese (DGGE) atau Pulse Field Gel Electrophorese (PFGE) Enzym Restriksi : Restriction Endonuclease Sequence analysis (DNA Sequencing)

cytoplasmic proteins

free ribosomes

RER

Protein Traffic

MOLECULARE MICROBIOLOGY Aplikasi teknologi DNA      

INFEKSI SALURAN CERNA: Membedakan jenis : pathogen – non pathogen (Eschericia coli) Untuk bakteri yang sulit dikultur oleh karena memerlukan syarat tertentu (Campylobacter) Membedakan jenis bakteri dari toxin yang diproduksinya (E. coli dan Shigela sp.) Subklas bakteri : Campylobacter, Helicobacter Mengidentifikasi jenis Rotavirus (A, B, C)

Aplikasi teknologi DNA       

INFEKSI SALURAN NAFAS: Mycobacterium tuberculosis : Membedakan jenis atypic, dengan mikroskop hal ini tidak mungkin Kultur : waktu yang lama dan bakteri harus banyak (terutama untuk sensitivity test) Diagnose cepat dibutuhkan, mis. pada penderita AIDS. Ditemui jenis yang multi drug resistant (MDR) Diagnosa dengan PCR dan Hybridisasi (contoh : dot-blot)

RESULT

MOLECULARE ONKOLOGY  PROTOONKOGEN : gen yang normal pada

Genom yang berperan penting dalam proliferasi dan differensiasi sel  ONKOGEN : protoonkogen yang oleh karena mutasi atau gangguan pada ekspresinya menyebabkan proliferasi sel yang neoplastis  TUMORSUPPRESSOR GEN : gen yang berperan pada proliferasi dan differensiasi sel, dimana bila gen ini di-inaktivasi atau tidak terdapat, akan terbentuk sel neoplastis

MOLECULARE ONKOLOGY  Contoh Neoplastic Transformation :  1. Gentranslocation : bcr-abl (chr. 9 dan 22)  2. Genamplification : N-myc gen 300 x pada

Neuroblastoma pada anak-anak  3. Point mutation : ras mengontrol GTP(aktif) → GDP (inaktif)  4. Insertion gen virus : virus Hepatitis B  5. Tumorsuppressorgen : p53 dan gen retinoblastoma : regulasi siklus sel (stop pada G1 untuk DNA - repair)

Second-Messenger Mechanism Adenosine 3’,5’-cyclic monophosphate (cAMP)

Hormone

Receptor Transducer G Protein (+,-) (+,-) Adenylate cyclase

ATP cAMP Protein Kinase A Membrane Enzymes Channels Structural Proteins

Produk Protoonkogen SIS ABL

SRC RAS

FMS

Inti Sel FOS MYC JUN

Orga nella

MOS

ERB-B1

FMS

Carcinogenesis (Colorectal Cancer)

Penerapan Teknologi Gen/DNA dalam Therapy

Produk dari gen untuk therapy dan prophylaxis :  Erythropoietin  Insulin  Hormon pertumbuhan  Faktor pembekuan darah VIII  Plasminogen aktivator  Vaksin Hepatitis B 

Aplikasi gen dalam Forensik 

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Sebelum teknologi DNA diterapkan (1978) biasanya digunakan protein, misalnya antigen gol.darah, HLA, dll. 1985 : DNA Polymorphismus. Nov.1987 : DNA sebagai barang bukti di pengadilan di Inggris. Sampai akhir 80-an : lebih dari 1000 perkara dibantu oleh bukti-bukti DNA Juga dapat menentukan Paternity Profil DNA tiap individu berbeda