CASE REVIEW

Tata, 2 YO, Boy

CC: Looked Very Pale
HT PE LE
Looked very pale had
for 6 months
This similar health
happens to both
family members of
father and mother
They receive blood
transfusion reguraly
Immunization (+)
Sick (-)
Nutritional problem (-)
Chronic bleeding (-)
Screened
abnormalities (-)
Looks pale (+)
Conjunctiva anemis
(+)
Sclera yellowish (+)
Abdomen
hepatosplenomegaly
(+)

Haemoglobin
(normal)
His parents are
carriers (from
screening) -
thalassemia
Mutation in -globin
chain (+)
Diagnosis: -Thalassemia
Treatment:
Pharmacology: -
Non-Pharma: Routine blood transfussion
FINAL CONCEPT


















, 2 YO
Mutation In Gene Expression
Parents Carrier
Gene Exp &
Gene Tech
Mutation
Failure In mRNA Splicing
Nucleic Acid,
DNA, RNA, &
Protein Syntesys
Mutation -Globin Chains
Imbalance Of -Globin Chains
-Thalassemia Mayor
Basic Diagnosys
and Clinicall Signs
Free -Chains
Agregat -Chains
Erythroblast Destroyed
(Ineffective
Erytrhopoesis)
Free Precipitates to
Damage RBC
Abnormal Structure
Intravascular
Hemolysys
Erytrocyte
Hb
RBC Life Spen
Spleen Destroys RBC
RBC
RBC Production
Erythropoetic Tissues (In
Liver & Spleen) / Work Hard
Pale Anemia Heme



















Hepatosplenomegaly
Biliverdin
Unconjugat
ed Bilirubin
Icteric
Sclera
Treatment BHP & PHOP
-THALASSEMIA MAYOR
1. Explain the structure, function, resources and utility of nucleic
acid
2. Explain the DNA organization, replication and repair
3. Explain the RNA structure, synthesis and processing
4. Explain protein synthesis and genetic code
5. Explain the regulation of gene expression
6. Explain the gene technology and genetic examination for
diagnosis
7. Explain the definition of gene, chromosom, and mutation
8. BHP: Ethical issue in frequent blood transfusion
9. PHOP: Premarital screening










NUCLEIC ACID
1. Structure of Nucleic Acid


Each nucleotide is composed of:
Pentose sugar
o Deoxyribose (DNA) without oxygen atom on carbon-2
o Ribose (RNA)

Nitrogen Bases
o Purin (Body : Adenine, Guanine ; Free : Xanthine, Hypoxanthine)
o Pyrimidine (Cytocine, Thymine (DNA), Uracil (RNA))

Phosphate group
o Provided by H3PO4, able to support segments of nucleic acid


2. Function / Utility
Form genetic code
important component of protein synthesis
Energy source for protein synthesis
Gene therapy and gene technology
Metabolic process

3. Source :
- Amino acids
- Sugar
- Food rich in nucleotides (brain, spinach, anchovy, caviar)
- Nucleotide released in intracellular metabolism


















DNA
Structure
Nucleotide : Consist of 3 major component : pentose sugar, phosphate
group, and nitrogen base
DNAs structure is double helix that coiled around common axis. The chains are
paired in an antiparallel manner, that is, the 5end of one strand is paired with
the 3- end of the other strand
Complementary chain structure of DNA show that the amount of purine and
pyrimidine are the same (A + G = C + T). Adenine will be paired with
thymine by 2 hydrogen bond, while Guanine will be paired with Cytosine by 3
hydrogen bond
Backbone of DNA consist of phosphate-sugar group. Pentose sugar of DNA is 2-
deoxyribose
2 nucleotide joined by phosphodiesther bond on 3rd or 5th C atom
Nucleosome : consist of DNA segment that warp around histone octamer.
Nucleosome + H1 histone (histone protein tightly bond to chromatin)
called chromatosome.
Histone protein : protein with + charge that contain may arginine and lysine that
could interact with charge and phosphate group p(ionic bond). Kind of histone
protein
H1 joined in the solenoid
H2A
H2B
H3
H4
There are three major structural forms of DNA: the B form, described by Watson
and Crick in 1953, the A form, and the Z form. The B form is a right-handed helix
with ten residues per 360 turn of the helix, and with the planes of the bases
perpendicular to the helical axis. The A form is produced by moderately
dehydrating the B form. It is also a right-handed helix, but there are eleven base
pairs per turn. The Z-DNA is a left handed helix that contains about twelve base
pairs per turn.

replication
Type of DNA replication accepted by universal is semiconservative type. The
process are :
Separation of the two complementary DNA strands: DNA strain must first
separate (or melt), at least in a small region. In prokaryote, it begins at a single,
unique nucleotide sequencea site called the origin of replication. In eukaryote,
replication begins at multiple sites along the DNA helix. Helicase is the enzyme
responsible for separating the double chain on the replication forks on the
recognizable sequence of DNA.
Single strain DNA protein is responsible to prevent the two separated DNA chain
back together
Because of the separation of DNA chain in one side, the other part of DNA starts
getting tight. Topoisomerase is responsible for unwinding tight parts of DNA.
After the DNA chain is separated into 2 part, the synthesis begins. DNA
Polymerase 3 starts to synthesis new-born DNA. The synthesis begins from the 3
end. Because DNA consist of 2 anti-parallel backbone, that means they move in
different direction. So, there are 2 sites :
Leading strain : continuously synthesize from 5 to 3 direction without
distraction
Lagging strain : Because DNA synthesize need 3-OH end, the synthesize
must be initiated by RNA-primase.
From that, DNA polymerase 3 start to synthesize newborn DNA. After finished
synthesizing 1 DNA fragment, the Okazaki fragment, RNA primase are removed
by DNA polymerase 1. Then, they will be joined together by ligase enzyme






DNA repair

Mismatch repair

Base excision repair

Nucleotide excision repair

Double strand break








RNA

A. Definition
RNA stands for ribonucleic acid, which is a polymeric molecule made up of one or more
nucleotides. A strand of RNA can be thought of as a chain with a nucleotide at each
chain link. Each nucleotide is made up of a base (adenine, cytosine, guanine, or uracil), a
ribose sugar, and a phosphate.

RNA is divided into four types:
a. rRNA
b. mRNA
c. tRNA
d. snRNA

B. Structure



A, G, C, U nitrogenous bases
pentose sugar (ribose sugar)
P phosphate group











C. Function

D. RNA Synthesis
RNA synthesis is the process of transcribing DNA nucleotide sequence information into
RNA sequence information. It is catalyzed by a large enzyme called RNA polymerase.

In eukaryotic cells it occurs in the cell nucleus, while in prokaryotic cells, it occurs in the
cytoplasm.

There are three steps to this process, which are initiation, elongation, and termination.
Illustration to these steps is shown in the picture on the next page.



E. RNA Processing














PROTEIN SYNTHESIS AND GENETIC CODE
Protein synthesis is translating genetic code to make amino acids which will bind each other to
form polypeptides.
The genetic code is a dictionary that identifies the correspondence between a sequence of nucleotide
bases and a sequence of amino acids. Each individual word in the code is composed of three nucleotide bases.
These genetic words are called codons. Codons are presented in the mRNA language of adenine (A), guanine (G),
cytosine (C), and uracil (U). The four nucleotide bases are used to produce the three-base codons.
Characteristics of genetic code:
1. Specificity (unambigous), a particular codon always codes for the same amino acid. Example: UUU always
codes for phenylalanine

2. Universality, the genetic codes virtually universal, same encoding will occur in every synthesis that
happens. An exception occurs in mitochondria, wich a few codons have different meanings.
3. Degeneracy, a given amino acid may have more than one triplet coding for it. For example, arginine
is specified by six different codons. Only Met and Trp have just one coding triplet.
4. Nonoverlapping and commaless, the code is read from a fixed starting point as a continuous
sequence of bases, taken three at a time. For example, AGCUGGAUACAU is read as
AGC/UGG/AUA/CAU without any punctuation between the codons.













PROTEIN SYNTHESIS
Components required for translation:
1. Amino acid
2. tRNA
3. aminoacyl-tRNA synthetases
4. mRNA
5. ribosome
6. protein factor
7. ATP and GTP(Guanosine Triphosphate)
8. EIF(Eukaryotic Initiation Factor) = certain compound which become factors that help protein
synthesis
STEPS
1. INITIATION
1) Dissociation of ribosome into its 40s(small ribosome) and 60s units(large ribosome)
2) Binding of ternary complex (met-tRNA/codon start, GTP, eIF-2) to the 40s ribosomal
subunit form preinitiation complex
3) Binding of mRNA to the 40s preinitiation complex to form initiation complex
4) Combination of initiation complex with the 60s ribosomal subunit to form 80s initiation
complex
2. ELONGATION
Catalyzed by proteins called elongation factors
1) Binding of aminoacyl-tRNA to the A site
2) Peptide bond formation
Attachment of growing peptide chain to the tRNA in the A site
3) Translocation
Displace the peptidyl tRNA(tRNA that carries amino acid) from A site to P site. Ready for
new aminoacyl-tRNA attachment and continuity of elongation process.
3. TERMINATION
1) After multiple cycle of elongation, the stop or terminating codon of mRNA (UAA, UAG,
UGA) apperas in A site.
2) Complex contain releasing factor with bound GTP will recognizes stop codon appears in
A site.
3) The protein and tRNA will be released from the P site because of hydrolysis
4) When hydrolisis and release, 80s ribosome dissociate into 40s and 60s subunit, which
are then recycled
5) mRNA is released from ribosome
6) another recycle can be repeated
7)
























GENE EXPRESSION

Influenced by
1. Hormones
2. Heavy metals
3. Chemicals

Regulation
a. Positive: Expression of genetic information increased by the presence of specific regulatory
element (activator)
b. Negative: Expression of genetic information diminished by the presence of specific
regulatory element (repressor)
Regulation response
1. Type A (prokaryotes): An increased extent of gene expression that is dependent upon the
continued presence of the inducing signal. When the signal is removed, the amount of gene
expression diminishes to its basal level.
2. Type B (during development of organism): Increased amount of gene that is transient even
in the continued presence of the regulatory signal.
2. Type C (during development of differenciated function in a tissue or organ): In response to
the regulatory signal, an increased extent of gene expression that persists indefinitely after
termination of the signal (the signal acts as a trigger)

Regulation of gene expression
Pada prokariot, sebagian besar DNA cetakan dapat dilakukan transkripsi namun untuk
eukariot hrus dilakukan remodeling kromatin (modifikasi kromatin) terlebih dahulu guna
ditranskripsi. Khusus pada prokariot, DNA cetakan itu berupa operon. APA ITU OPERON?
Operon: Gugus gugus cetakan DNA yang diawali dengan promoter, operator dan beberapa
gen yang bersebelahan(operon lac). Guna promoter adalah tempat terdapatnya RNA
polymerase dan operator berfungsi untuk menyalakan dan mematikan(repressor) dalam
proses regulasi.Operator pada prokariot berfungsi sama dengan metilasi DNA pada eukariot.

1. Modifikasi kromatin
Gen dalam kromatin sangat terpadatkan biasanya tidak ditranskripsikan.
Asetilasi histon tampaknya melonggarkan struktur kromatin dan meningkatkan transkripsi.
Metilasi DNA umumnya mengurangi transkripsi.
2. Transkripsi
Regulasi inisiasi transkripsi: unsur kontrol DNA mengikat faktor transkripsi spesifik.
Penekukan DNA memungkinkan aktivator menyentuh protein di promoter, sehingga
menginisiasi transkripsi.
Regulasi terkoordinasi.
3. Pemrosesan mRNA
Penyambungan RNA alternatif
4. Translasi
Inisiasi dari translasi dapat dikontrol melalui regulasi dari faktor inisiasi.
5. Pemrosesan dan degradasi protein.
Oleh proteasom
6. Degradasi mRNA.
Setiap mRNA memiliki panjang usia yang khas, ditentukan sebagian oleh sekuens pada UTR 5
dan 3








GENE EXAMINATION

RFLP (Restriction Fragment Length Polymorphism)
RFLP analysis is used to identify changes in genetic sequences which occur at a site where the
restriction enzyme cuts.
RFLP is based on the chance of comparing each DNA profile resulted from the cutting of
targeted DNA with the restriction enzyme.
Steps of RFLP:
1. DNA Isolation
The process of isolating the DNA fragments with extraction and lytic method. Usually the
process is done with the help of homogenization and usage of additional extraction buffer
to prevent DNA from damaging.
Separation of DNA from other cell components is done using the centrifuge.
2. Cutting of DNA using the restriction enzyme and electrophoresis gel
The result of DNA isolation will then cut using certain restriction enzyme which selected
carefully. Every restriction enzyme which place in a suitable environment will recognize and
slice the DNA to form the fragments. The fragments will then undergo electrophoresis using
the Aragosa gel.

Electrophoresis
The standard lab procedure for separating DNA by size (e.g. length in base pairs) for visualization
and purification.
The principle of the basic categories of analyses used to characterize DNA and RNA:
Electrophoretic Separation
Movement of DNA or RNA in response to an electric field will be proportional to the molecular
weight or length of the molecule. This property is used to characterize the size of nucleic acid
fragments by electrophoretic separation.

Hybridization
The process of combining two complementary single-stranded DNA or RNA molecules and
allowing them to form a double-stranded molecule through base pairing.
Hybridization is defined as the interaction between two single-stranded nucleic acid molecules
to form a duplex (double-stranded) molecule.
Principle: based on the complementary base pairing of their respective sequences.
High-stringency conditions high temperature (close to Tm ), low salt, and high formamide
will only allow the most perfectly matched duplex structures to remain in a stable helix
conformation.


GENE TECHNOLOGY

DNA Cloning
DNA cloning is a process of producing the identical DNA from its parental DNA.
Vectors are the agent that carries DNA fragment into the living cell in order to increase the
amount of DNA fragments.
There are a few vectors used within the process:
1. Bacteria
a. Plasmid : small circular DNA molecule which have an ability to replicate itself
b. Phage : viruses that infected the bacteria
c. Cosmides : a larger fragments of DNA which resulted from the combination of
plasmid and phage
2. Viral
a. Adenovirus
b. Retrovirus
3. Artificial Chromosome

DNA Recombinant
DNA recombinant is the formation of new genetic
combination by inserting a DNA molecule into certain
vector, making it possible for them to integrate and
undergo a replication process within other organism.
Purpose of DNA recombinant:
1. Gene mapping: determine the specific location of
gene in particular chromosome.
2. Production of protein for research and diagnosis
purposes.
3. Examination of molecular analysis in disease.



Gene Therapy
Gene therapy is the use of DNA as a drug to treat disease by delivering therapeutic DNA into a patient's
cells. Mostly involves using DNA that encodes a functional, therapeutic gene to replace a mutated gene.
There are two types of gene therapy, yet only one of which has been used in humans:
1. Somatic Gene Therapy
2. Germline Gene Therapy
Principle of gene therapy
Inserting precursor cell to the location of the damaged cell so that the precursor cell will have the same
expression with damaged cells and able to replace the function of the damaged cells.
PCR (Polymerase Chain Reaction)
Technic used in certain DNA fragments amplification in an in vitro medium using a pair of
oligonucleotide.







Seperating the double-strand DNA, breaking the
hydrogen bond in order to form the single-
strand DNA. Temperature needed: 92-94
o
C.
Denaturation
Primer forward and reverse is searching for its
pair in the DNA strands.
Annealing
Taq polymerase replicate the DNA fragment.
Temperature needed: 72
0
C
Elongation
MUTATION
Definition
A mutation is defined as a permanent change in the DNA. Mutatations that affect germ
cells are transmitted to the progeny and can give rise to inherited disease. Mutations
that arise in somatic cells understandably do not cause hereditary diseases but are
important in the genesis of cancers and some congenital malformations. Human genetic
disorders can be broadly classified into three categories :
1. Disorders related to mutations in single genes with large effects: Highly penetrant,
they usually follow the classic Mendelian pattern of inheritance and are also
referred to as Mendelian disorders.
2. Chromosomal disorder: Uncommon, but with high penetrance
3. Complex multigenic disorders: Often expressed in polymorphisms, although it is often
of small effect and low penetrance unless in huge amounts of polymorphisms
expressed (in multigenic or polygenic conditions as it is termed) Penetrance = the
frequency of manifestation of a hereditary condition in individuals. Mutations may
result in partial or complete deletion of a gene or affect a single base.

Types of mutations
1. Point mutations
It may alter the code in a triplet of bases and lead to the replacement of one
amino acid by another in the gene product. There are several types of this
mutation
. Missense mutations: Replacement of normal amino acid with a very different
one that can cause some physiological or physical changes (often detrimental).
Eg. -thalassemia due to CTC changed in CAC.
. Nonsense mutations: May cause severe translation errors, causing often severe
physiological or physical effects. Eg. Beta-thalassemia major due to codon for
glutamine (CAG) creates stop codon (UAG), this creates premature termination
of betaglobin gene translation and causes severe form of anemia called o-
thalassemia.
. Silent mutation: No change in protein function can be observed due to the
mutation being silent (has no visible physiological or physical effects on the
mutant subject).
Caused due to base substitution, there are 2 types of base substitution :
. Transition: Changes purine to purine / pyrimidine to pyrimidine.
. Transversion: Changes purine to pyrimidine / pyrimidine to purine.
2. F rameshift mutations
It alters the reading frame of the DNA strand, it is usually detrimental for an
organisms fitness. They are caused by :
. Insertions: Addition of one or more DNA that alters the reading frame of the
DNA.

. Deletions: Deletion of one or more DNA that alters the reading frame of the
DNA.

3. Trinucleotide-repeat mutations
Special category of genetic anomaly. Characterized by amplification (gene
duplication) of a sequence of three nucleotides.
4. Chromosomal mutation
There are 4 types of chromosome according to the position of centromeres :
. Metacentric = at the centre of the chromosome
. Submetacentric = near the centre of the chromosome
. Acrocentric = near the end of the chromosome
. Telocentric = at the end of the chromosome
Chromosme mutation is usually more severe than point mutations, there are
several types of chromosmal mutations :
. Deletion
. Duplication
. Inversion












BHP & PHOP
BHP
Informing patient about the reason why the patient should get the transfusion blood.
Informing patient about the procedure of blood transfussion.
Informing patient about the risk, benefit, purpose, and requirement of blood transfusion to
the donature and the recepient.

PHOP
Preventive : Doing genetic counseling and genetic screening before married
(premartial screening )
Promotive : Educate and persuading people the importance of genetic counseling
before married to prevent the genetical disease happen
Currative : Giving the patient blood transfusion, stem cell transfusion, and bone
marrow transplant.
Rehabilitative : Doing routine check up to prevent any risk happen after blood
transfusion