Download HUMAN MOLECULAR GENETICS

Molecular Basis of Genetic Diseases
and Tools of Human Molecular
Genetics
Ömer Faruk Bayrak
Genetic Diseases in Humans
Role of Genes in Human Disease
•
Most diseases -> phenotypes result from
the interaction between genes and the
environment
•
Some phenotypes are primarily
genetically determined
–
100%
Environmental
Achondroplasia (-> dwarfism)
Struck by lightning
Infection
Weight
•
Other phenotypes require genetic and
environmental factors
–
•
Mental retardation in persons with PKU
(polyketonuria)
Some phenotypes result primarily from
the environment or chance
–
Cancer
Lead poisoning
Diabetes
Height
100%
Genetic
Down syndrome, achondroplasia
Principles of molecular disease
 Molecular reason of a genetic disease is a mutation.
This mutation either inherited or acquired.
 The biochemical genetic is study of phenotype at the
level of proteins, biochemistry and metabolism.
Principles of molecular disease
Effects of Mutations,
1. Loss of function (α thalasemia)
2. Gain a function (Achondroplasia)
3. Acquision of a novel property by mutant protein.
(Sickle Cell Anemia)
4. Expression of a gene at the wrong time or wrong
place. (most of cancers)
Genetic Diseases in Humans
Types of Genetic Disorders:
-> Chromosomes and chromosome abnormalities (Down
Syndrome)
-> Single gene disorders (Haemophilia, sickle cell anaemia)
-> Polygenic Disorders (Cancer)
Genetic Diseases in Humans
Chromosomal disorders
•
Addition or deletion of entire chromosomes or parts of chromosomes
•
Typically more than 1 gene involved
•
1% of paediatric admissions and 2.5% of childhood deaths
•
Classic example is trisomy 21 - Down syndrome
KARYOTYPE
Genetic Diseases in Humans
Single gene disorders
• Single mutant gene has a large effect on the patient
• Transmitted in a Mendelian fashion
• Autosomal dominant, autosomal recessive, X-linked, Y-linked
• Osteogenesis imperfecta - autosomal dominant
• Sickle cell anaemia - autosomal recessive
• Haemophilia - X-linked
Genetic Diseases in Humans
Single gene disorders
Neonatal fractures
typical of osteogenesis
imperfecta, an
autosomal dominant
disease caused
by rare mutations
in the type I collagen
genes COL1A1 and
COL1A2
A famous carrier
of haemophilia A,
an X-linked disease
caused by mutation
in the factor VIII gene
Sickle cell anaemia,
an autosomal recessive
disease caused by
mutation in the
β-globin gene
Genetic Diseases in Humans
Polygenic disorders
• The most common yet still the least understood of human
genetic diseases
• Result from an interaction of multiple genes, each with a minor
effect
• The susceptibility alleles are common
• Type I and type II diabetes, autism, osteoarthritis, cancer
Monogenic Disorders
• Involve single mutant genes
• Classification:
(1) autosomal dominant - clinically evident if one
chromosome affected (heterozygote)
• e.g., Familial hypercholesterolemia
(2) autosomal recessive - both chromosomes
must be affected (homozygous)
• e.g., Sickle cell anemia
(3) X-linked - mutation present on X chromosome
• females may be either heterozygous or homozygous
for affected gene
• males affected if they inherit mutant gene
• e.g., Duchenne muscular dystrophy
Multifactorial Disorders
• Interplay of number of genes and environmental
factors
– pattern of inheritance does not conform to classic
Mendelian genetic principles
– due to complex genetics, harder to identify
affected genes; thus, less is known about this
category of disease
– e.g., Essential hypertension
The Various Types of Heterogeneity
Associated with Genetic Disease
Type of Heterogeneity
Genetic heterogeneity
Allelic heterogeneity
Locus heterogeneity
Clinical or phenotypic
heterogeneity
Definition
the phenomenon in which
different mutations at the
same locus causes a similar
phenotype
Examples
β-Thalassemia mutations in βglobin
Phenylalanine hydroxylase
mutations in PKU
Perinatal lethal osteogenesis
imperfecta (type II), from
mutations in the α1 collagen
gene
The association of more than Biopterin metabolism defects
one locus with a specific
causing hyperphenylalaninemia
clinical phenotype
The association of more than Phenylalanine hydroxylase
one phenotype with mutations mutations causing PKU, variant
at a single locus
PKU, or non-PKU
hyperphenylalaninemia
α-L-Iduronidase mutations
causing Hurler syndrome or
Scheie syndrome
Some examples of Classes of Proteins
Associated with Monogenic Diseases
• 1-Transport and storage
• İnterorgan Hemoglobin (thalassemias)(AR)
• İntracellular transport (copper transport prot.
menkes syndr. (AR)
• Epitel membr. Cystic fibrosis (CFRT AR)
2-Enzyme defects
• Amino acids - PKU(phenyl alanine hydroxilase AR)
• Complex lipid-Tay sachs(Hexosaminidase A AR)
• Purines-immundeficiency( Adenosine deaminidase AR)
• Carbohydrates Galactose 1 phosphate uridyl
transferase
Some examples of Classes of Proteins
Associated with Monogenic Diseases
3-Structure of cells and organs
• Duschene muscular distrophy(dystrophin XR)
4-Control of growth and differantiation
• Tumor suppressors,
• RB gene products (AR),
• oncogenes(AD).
5-Intracellular metabolism and comunication
• Growth gormon(dwarfizm,AR),insulin(AD)
• Familial hypercholesterolemia(LDL receptor).
Tools of Human Molecular Genetics
PCR
• PCR was first conceived in 1983 by Kary Mullis, a molecular biologist
who received a Nobel Prize for the discovery 10 years later
• A PCR (Polymerase Chain Reaction) is performed in order to make a
large number of copies of a gene. Otherwise, the quantity of DNA is
insufficient and cannot be used for other methods such as
sequencing.
• A PCR is performed on an automated cycler, which heats and cools
the tubes with the reaction mixture in a very short time.
• Performed for 30-40 cycles, in three major steps: 1)denaturation,
2)annealing, and 3)extension.
1)
Denaturation at 94°C :
 During the denaturation, the double strand melts open to single stranded DNA, all
enzymatic reactions halt.
2)
Annealing at 50-60°C :
 The primers are freely moving due to Brownian motion. Ionic bonds are constantly
formed and broken between the single stranded primer and the single stranded
template.
 Primers that fit exactly will have stable bonds that last longer. The polymerase
attaches onto a piece of double stranded DNA (which is template and primer), and
starts copying the template. Once there are a few bases built in, the ionic bond is
so strong between the template and the primer, that it does not break anymore.
3) Extension at 72°C :
 This temperature is ideal for the polymerase. The primers, which have a few bases
built in, already have a stronger ionic attraction to the template than the forces
breaking these attractions.
 Primers that are on positions with no exact match, loosen their bonds again
(because of the higher temperature) and do not extend the fragment.
The bases (complementary to the template) are coupled to the primer on the 3' side
(the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side,
bases are added complementary to the template)
The ladder is a mixture of fragments with known size to compare with the
PCR fragments. Notice that the distance between the different fragments of
the ladder is logarithmic. Lane 1 : PCR fragment is approximately 1850 bases
long. Lane 2 and 4 : the fragments are approximately 800 bases long. Lane 3 :
no product is formed, so the PCR failed. Lane 5 : multiple bands are formed
because one of the primers fits on different places.
• Applications of PCR:
• 1) Diagnosis of Disease: Linkage analysis, detection of mutant
alleles, diagnosing infectious agents, epidemiological studies
• 2) Forensics: paternity testing, DNA typing for identification,
criminal investigations.
• 3)Recombinat DNA engineering
• 4) DNA sequence determination
• 5) new gene isolation
• 6) Anthropological studies: population genetics, migration studies.
• 7) Evolution studies
• If you need to look at 100 genes is PCR a good approach?
RT-PCR
 An RT-PCR (Reverse transcriptase-polymerase chain reaction) is a
highly sensitive technique for the detection and quantitation of mRNA
(messenger RNA).
 The technique consists of two parts:
1) The synthesis of cDNA (complementary DNA) from RNA by
reverse transcription (RT)
2) The amplification of a specific cDNA by PCR.
Compared to Northern blot analysis and RNase protection assay used
to quantify mRNA, RT-PCR can be used from much smaller samples. It
is sensitive enough to enable quantitation of RNA from a single cell.
 Real-time RT-PCR is the method of choice for quantitating changes in
gene expression. Furthermore, real-time RT-PCR is the preferred
method for validating results obtained from array analyses and other
techniques that evaluate gene expression changes.
Real-time PCR advantages
* not influenced by non-specific amplification
* amplification can be monitored real-time
* no post-PCR processing of products
(high throughput, low contamination risk)
* ultra-rapid cycling (30 minutes to 2 hours)
* wider dynamic range of up to 1010-fold
* requirement of 1000-fold less RNA than conventional assays
(3 picogram = one genome equivalent)
* detection is capable down to a 2-fold change
* confirmation of specific amplification by melting curve analysis
* most specific, sensitive and reproducible
* not much more expensive than conventional PCR
(except equipment cost)
Microarray
 DNA microarrays allow researchers to analyze the expression of
thousands of genes simultaneously.
 DNA microarrays contain thousands of individual gene sequences in
microscopic spots of ≈1-kb DNA sequences representing thousands of
genes bound to the surface of glass microscope slides.
 Provide a means for analyzing gene expression patterns on a genomic
scale.
 Provides a medium for matching known and unknown DNA samples based
on base-pairing rules and automating the process of identification.
Microarray
Microarray
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•
•
•
•
•
•
•
Applications
Gene discovery
Disease diagnosis
Drugs and toxicological research : The goal of pharmacogenomics is
to find correlations between therapeutic responses to drugs and the
genetic profiles of patients.
Expression screening. The focus of most current microarray-based
studies is the monitoring of RNA expression levels which can be done
by using either cDNA clone microarrays or gene-specific
oligonucleotide microarray
Screening of DNA variation. There is also huge potential for
assaying in drug development and patient susceptibility, as well as for
mutations in known disease genes such as cardiovascular disease and
cancer as seen in the case of the breast cancer susceptibility gene,
BRCA1.
In addition, there have been vigorous efforts to identify and catalog
human single nucleotide polymorphism (SNP) markers.
Gene Expression Analysis Technologies
>10,000
Number of samples
Real-time PCR
100
Low-density
Arrays
10
High-density
Arrays
SAGE
RPA
Northern
1
2-10
100-1000
>10,000
Number of genes
To consider: Ratio samples / genes, needs for accuracy
Comparison of Quantitative Assays (RNA/DNA):
Sensitivity
100
101
102
103
104
105
106
107
108
Dynamic Range
Real-Time PCR
Amplicor/TMA
NASBA
bDNA
XPLORE
Microarrays
RPA
Northern
108
107
106
105
104
103
102
101
100
NASBA: nucleic acid seq based amplificationTMA: transcription mediated amplification
bDNA: branched DNA assay
RPA: RNAse protection assay
Xplore: based on Invader technology
Advantage: Real-Time PCR
Southern Blot
• Southern Blotting (named after Ed Southern, the inventor) is the
detection of specific sequences of DNA on a gel by hybridisation
with a labelled DNA probe.
• DNA is first transferred out of a gel by capilliarity (the "blot") to
a thin membrane which can be incubated with a probe and washed.
• By hybridising at different temperatures, and washing to different
ionic strengths ("stringencies") it is possible to tune the process to
pick up sequences that are either similar, or exactly identical, to
the probe.
Southern Blot
• Applications:
• 1) To confirm the presence of a gene, often in conjunction with
PCR.
• 2) To test for the presence of a specific allele of a gene (i.e.
human disease genetics).
• 3) To estimate gene complexity, before you have the gene
sequence.
• 4) To detect Restriction Fragment Length Polymorphism (RFLP)
and Variable Number of Tandem Repeat Polymorphism
(VNTR). The latter is the basis of DNA fingerprinting.
Southern Blot
Techniques: Southern
Blot
Southern Blot
Northern Blots
 Northern blots are similar to Southern, except that RNA from
different tissues is run out on a gel, and probed with a DNA or
RNA probe corresponding to a particular gene.
 Northern blotting is used for detecting and quantitation of RNA
fragments, instead of DNA fragments. The technique is exactly
like Southern Blotting. It is called "Northern" simply because it
is similar to "Southern", not because it was invented by a person
named "Northern".
 RNA samples are first separated by size via electrophoresis in an
agarose gel under denaturing conditions. The RNA is then
transferred to a membrane, crosslinked and hybridized with a
labeled probe.
Western Blot
• Western blot analysis can detect one protein in a mixture of any
number of proteins while giving you information about the size of
the protein.
• Allows investigators to determine with a specific primary
antibody, the relative amounts of the protein present in different
samples.
 Western blots are analogous to Northern and Southern, except
that proteins are run out in an SDS polyacrylamide gel, and are
detected with specific antibodies.
 In clinical settings, Western Blotting is routinely used to confirm
serious diagnosis suggested by ELISA such as HIV seroconversion
Nucleic Acid Hybridization
• The Basic Process of Binding a Single Strand of Nucleic Acid (DNA
or RNA) to Its Complementary Strand Is Called Nucleic Acid
Hybridization.
• Double-stranded DNA Can Be Denatured by Agents Such As Heat
or High PH. When Denatured, the Two Strands Separate Into
Single Strands and Diffuse Away From Each Other. If Conditions
Are Then (Slowly) Reversed (Lower the Temperature or Return the
PH to Neutrality) Then the DNA Will renature.
• If the Temperature Is Slowly Decreased, Then Each Strand of
DNA Will Find Its Corresponding Mate: the Complementary
Strands of the DNA Will Anneal and Re-form the Double Strand
With Correct Watson-crick If a Radioactively Labeled Probe
Corresponding to a Part of the Sequence of One of the Fragment
Is Included in the renaturation Mixture, It Will Participate in the
renaturation, Finding and Annealing to Its Complementary Partner
Nucleic Acid Hybridization
Probe present
No probe
A
C
C
C
T
G
C
G
FISH
• Fluorescence In-Situ Hybridization is a method used to identify
specific parts of a chromosome. For example, if you know the
sequence of a certain gene, but you don't know on which chromosome
the gene is located, you can use FISH to identify the chromosome in
question and the exact location of the gene.
• If you suspect that there has been a translocation in a chromosome,
you can use a probe that spans the site of breakage/translocation. If
there has been no translocation at that point, you will see one signal,
since the probe hybridizes to one place on the chromosome. If,
however, there has been a translocation, you will see two signals, since
the probe can hybridize to both ends of the translocation point.
• To use FISH efficiently, you have to know what you're looking for, i.e.
you usually suspect a particular defect, based on the appearance of
certain chromosomes, etc.
FISH
Method:
• Make a probe complementary to the known sequence. When making
the probe, label it with a fluorescent marker, e.g. digoxigenin, by
incorporating nucleotides that have the marker attached to them.
• Put the chromosomes on a microscope slide and denature them.
• Denature the probe and add it to the microscope slide, letting the
probe hybridize to its complementary site.
• Wash off the excess probe and look at the chromosomes in a
fluorescence microscope. The probe will show as one or more
fluorescent signals in the microscope, depending on how many sites
it can hybridize to.
FISH
Applications
• Diagnosis in clinical and cancer cytogenetics.
• Interspecies studies of evolutionary divergence.
• Analysis of aberrations in animal models of human diseases.
Interphase/Metaphase FISH
Multicolour-FISH, chromosome paints
FISH
Thank you