This document discusses genetic polymorphisms and their role in disease pathogenesis and complications. It begins with definitions of health and disease, then discusses various disease causative agents including environmental, genetic, and microbial factors. It describes the organization of the human genome and genes. The document outlines DNA structure and organization, including DNA packaging into chromosomes. It discusses polymorphisms, mutations, and how they can be analyzed using restriction endonucleases and techniques like PCR and gel electrophoresis. Specific examples are provided of how genetic variations can impact disease by changing enzymes, receptors, transport proteins, and other molecular factors.

1. Genetic Polymorphisms in the Disease
Pathogenesis and its Complications
Muhammad Saiedullah
Prof Zahid Hassan
Dept of Physiology & Molecular Biology
Bangladesh University of Heath Sciences
70th NITUB Workshop; 14-19 July 2018; BUHS

2. Definition: Health and Disease
 Health
 Health is a state of complete physical, mental and social well-
being and not merely the absence of disease or infirmity.
 Disease
 A condition of the living animal or plant body or of one of its
parts that impairs normal functioning and is typically manifested
by distinguishing signs and symptoms.
 A disordered or incorrectly functioning organ, part, structure, or
system of the body resulting from the effect of genetic or
developmental errors, infection, poisons, nutritional deficiency or
imbalance, toxicity, or unfavorable environmental factors
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3. Disease causative agents
 Environmental factors
 Physical agent
 Chemical agent
 Microbial agents
 Bacteria
 virus
 Genetic factors
 Monogenic
 Polygenic
 Multifactorial
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4. From DNA duplex to metaphase chromosome. The figure shows human chromosome 17, as seen
in a G-banded, 400 band preparation. The estimated packaging ratios (the degree of compaction
of the linear DNA duplex) for human chromosomes are 1:6 for nucleosomes, 1:36 for the 30 nm
fiber and >1 :10 000 for the metaphase chromosome. Presently, it is uncertain whether the DNA
at the centromere of the metaphase chromosome has been delayed in its replication unlike the
rest of the chromatid, or whether full DNA replication has occurred in the S phase and the
constriction at the centromere is due to some other cause.
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5. DNA Macrostructure Strands of DNA are wrapped around
supporting histones. These proteins are increasingly
bundled and condensed into chromatin, which is packed
tightly into chromosomes when the cell is ready to divide.
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6. Organization of the human genome
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7. Gene
 DNA sequence that encodes protein and RNA.
 Dispersed throughout the genome- nuclear and
mitochondrial DNA.
 Protein coding DNA sequence ‘exone’ in a gene is
interrupted by noncoding ‘intron’
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8. Restriction endonuclease
 Recognition sequence
 TaaI
 5’- A C N^G T-3’
 3́ - T G^N C A-5’
 RarI
 5’-CTCTTC(N)^-3’
 3’-GAGAAG(N)^-5;
 EcoR1
 5’-G^ATTC-3’
 5’-CTAA^G-3’
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9. Polymorphism
 Variation in the DNA sequence in the population.
 Involves at least 1–2% variations in the
population.
 Variations cause creation or abolishment of
endonuclease recognition site.
 Allow researchers to analyze variations.
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10. DNA mutation
 Heritable change in the DNA and/ or chromosome.
 Mutation abolishes or create restriction enzyme
(endonuclease) recognition site.
 Changes length variations in the DNA sequence.
 Mutation(s) often associated with pathogenesis of
disease, however, may remain silent ie no effect
 Mutation can be analyzed by DNA amplification and
followed by restriction enzyme digestion.
 Enzyme digestion products containing variable DNA
fragment lengths analyzed by agarose or polyacrylamide
gel electrophoresis
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11. Molecular Basis
Of Diseases
Environment
And genes
Changes an Enzyme
e.g. Phenylalanine hydroxylase
Splice site mutation of PAH gene leading to reduced amount
phenylalanine hydroxylase Causing phenylketonuria
Changes an Enzyme inhibitor
e.g. 1-Antitrypsin: Missense mutation of SERPINA1
gene that impair secretion from liver to serum causing
Emphysema and Liver disease
Changes a receptor
e.g. Low density lipoprotein receptor
Deletion or point mutation of LDLR gene that
reduce synthesis, Or transport to the cell surface
or binding to low density lipoprotein
Causing Familial hypercholesterolemia
Change a transport or carrier protein
1.e.g. Haemoglobin
Mutations in splice sites (commonest) leading to
Reduced -globin.causing -Thalassemia in
-Thalassemia the -globin gene is usually deleted
2.e.g. Cystic fibrosis transmembrane conductance
Regulator. Deletions or point mutation causing
Cystic fibrosis.
Changes in Hemostasis
e.g. Factor VIII deletions, insertions, nonsense
Mutation reduce synthesis or abnormal factor VIII
Causing Hemophilia A.
Changes in structural
Proteins
1.e.g. collagen: Mutations in
the COL1A1, COL1A2, CRTAP,
and P3H1 genes - Deletions or point
mutation that produce reduced amount
of normal collagen or Normal
amounts of mutant Collagen. Causing
Osteogenesis imperfecta.
2.e.g cell membrane Fibrillin:
Missense mutations in the FBN1 gene
cause Marfan syndrome.
Or deletion of dystrophin ( DMD )
gene Causing Duchene (☺dushen)
muscular Dystrophy
Growth regulation
e.g.RB1 gene mutation causing
Retinoblastoma etc

12. Study of DNA
 Amplification
 Visualization
 Genotyping
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13.  PCR
 PCR-RFLP
 Sequencing
 Pyerosequencing
 Satellite microsatellite
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16. Restriction Fragment Length
Polymorphisms
The presence of RFLP is inferred from
changes in fragment sizes.
1 2
A B C
+ -
AGATCT ATATCT
TCTAGA TATAGA
1 2 Size Number
+ + A,B,C 3
+ - A, (B+C) 2
- + (A+B), C 2
- - (A+B+C) 1
+/+ +/- -/+ -/-
Polymorphism
Restriction site
Gel band
pattern
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vertical

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22.  PCR: After measuring the concentration of the DNA,
5-10 ng will be used for standard polymer chain
reaction (PCR) in 15µl reaction volume of the target
genes (ApoB EcoRI) using specific primer.
 Amplified PCR product will be digested with specific
restriction enzymes and fragments will be analyzed in
agarose gel (electrophoresis) and visualized by
ethidium bromide staining.
 Presence or absence of SNP will be determined by
different DNA fragments formed due to enzyme
digestion of PCR products resolved in agarose gel.

23. ApoB EcoRI SNP (12669G>A)
The 480 bp sequence
containing the EcoR1
restriction site will be
digested into fragments of
253 and 227 bp,
respectively.
In the absence of the EcoR1
restriction site, the 480 bp
sequence will be produced
only a single band on the
agarose gel.

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26. Gel electrophoresis
 Selection of types of gel depends on the size of the
target fragments.
 There are options in choosing gel.
 Agarose gel
 For DNA fragment size 100bp and more agarose gel is
conventionally used and run in horizontal gel system.
 Can discriminates 3% base pair difference.
 Polyacrylamide gel
 Incase of PCR product <100bp polyacrulamide gel is used
 Polyacrylamide gel run in vertical gel system
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27. Gel run
 Power supply needs to be uninterrupted and measure
voltage or ampere.
 Voltage applied depends on the length of gel.
 Room temperature needs to be controlled.
 During electrophoresis there is generation of heat which
softens gel.
 Soft gels are subjected to be distorted during handling.
In addition these gels are fragile.
 To avoid the situation run often done in relatively low
voltage.
 Gel run in low temperature is recommended when it is
planned to carryout the experiment in high voltage.
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28. Trouble shooting
 Failure to initiate electrophoresis- check the wire
connection and secure the power connection.
 No band- make sure the staining of the gel is done.
 Keep an eye the gel(s) are not over run.
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29. Thank
you
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