Download Molecular medicine

Applications of genome sequencing projects
1) Molecular Medicine
2) Energy sources and environmental applications
3) Risk assessment
4) Bioarchaeology, anthropology, human evolution,
human migration
5) DNA forensics
6) Agriculture, livestock breeding, and
bioprocessing
http://www.ornl.gov/hgmis/project/benefits.html
Molecular medicine
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improved diagnosis of disease
earlier detection of genetic predisposition to
disease
Rational drug design
Gene therapy and control systems for drugs
pharmacogenomics "custom drugs"
Definitions
DNA polymorphism: A DNA sequence that occurs in two or more variant
forms
Alleles: any variations in genes at a particular location (locus)
Haplotype: combination of alleles at multiple, tightly-linked loci that are
transmitted together over many generations
Anonymous locus : position on genome with no known function
DNA marker: polymorphic locus useful for mapping studies
RFLP Variation in the length of a restriction fragment due to nucleotide
changes at a restriction site, detected by a particular probe / PCR.
SNP: presence of two different nucleotides at the same loci in genomic
DNA from different individuals
DNA fingerprinting: Detection of genotype at a number of unlinked highly
polymorphic loci using one probe
Genetic testing: Testing for a pathogenic mutation in a certain gene in an
individual that indicate a person’s risk of developing or transmitting a
disease
The spectrum of human diseases
Cystic fibrosis
thalassemia
<5%
cancer
Huntington’s
‘Mendelian’ diseases (<5%)
Autosomal dominant inheritance:
e.g huntington’s disease
Autosomal codominant inheritance
e.g Hb-S sickle cell disease
Autosomal recessive inheritance:
e.g cystic fibrosis, a & b thalassemias
X-linked inheritance:
e.g Duchenne muscular dystrophy (DMD)
How to identify disease genes
• Identify pathology
• Find families in which the disease is
segregating
• Find ‘candidate gene’
• Screen for mutations in segregating
families
How to map candidate genes
2 broad strategies have been used
A. Position independent approach (based
on knowledge of gene function)
1) biochemical approach
2) animal model approach
B. Position dependent approach (based on
mapped position)
Position independent approach
1) Biochemical: when the causative protein has
been identified E.g. Factor VIII haemophilia
Blood-clotting
cascade in
which vessel
damage causes a
cascade of
inactive factors
to be converted
to active
factors
Blood tests determine if active
form of each factor in the cascade
is present
Fig. 11.16 c
Techniques used to purify Factor
VIII and clone the gene
Fig.
Fig.11.16
11.16d d Hartwell
2) Animal model approach
compares animal mutant models for a phenotypically similar human
disease.
E.g. Identification of the SOX10 gene in human Waardenburg
syndrome4 (WS4)
Dom (dominant megacolon)
mutant mice shared phenotypic
traits similar to human patient
with WS4 (Hirschsprung
disease, hearing loss, pigment
abnormalities)
Waardenburg
WS4 patients screened for
SOX10 mutations
confirmed the role of this gene
in WS4.
Dom mouse
Hirschsprung
B) Positional dependent approach
Positional cloning
identifies a disease
gene based on only
approximate
chromosomal location.
It is used when nature
of gene product /
candidate genes is
unknown.
Candidate genes can be
identified by a
combination of their
map position and
expression, function or
homology
B) Positional Cloning Steps
Step 1 – Collect a large number of affected
families as possible
Step 2 - Identify a candidate region based
on genetic mapping (~ 10Mb or more)
Step 3 - Establish a transcript map,
cataloguing all the genes in the region
Step 4- Identify potential candidate genes
Step 5 – confirm a candidate gene and
screen for mutations in affected families
Step 2 - Identifying a candidate region
Genetic map of <1Mb
Genetic markers: RFLPs,
SSLPs, SNPs
Linkage association:
Lod scores (log of the odds):
ratio of the odds that 2 loci are
linked or not linked
need a lod of 3 to prove linkage
and a lod of -2 against linkage
Chromosmal
abnormalities
Halpotype association
HapMap published in Oct27 2005 Nature
DNA markers/polymorphisms
RFLPs (restriction fragment length polymorphisms)
- Size changes in fragments due to the loss or gain of a
restriction site
SSLPs (simple sequence length polymorphisms) or
microsatellite repeats. Copies of bi, tri or tetra
nucleotide repeats of differing lengths e.g. 25 copies
of a CA repeat can be detected using PCR analysis.
SNPs (single nucleotide polymorphisms)- presence of two
different nucleotides at the same loci in genomic DNA
from different individuals
RFLPs
- Amplify fragment
- Expose to
restriction enzyme
- Gel
electrophoresis
e.g., sickle-cell
genotyping with a
PCR based
protocol
Fig. 11.7 – genetics/ Hartwell
SSLPs
Similar principles used in detection of RFLPs
However, no change in restriction sites
Changes in length of repeats
SNPs (single nucleotide polymorphisms)
presence of two different nucleotides at the same loci in
genomic DNA from different individuals
SNP detection using allele-specific oligonucleotides
(ASOs)
Very short probes (<21 bp) specific which hybridize to one
allele or other
ASOs can determine genotype at any SNP locus
Fig. 11.8
Fig. 11.9 a-c
Hybridized and
labeled with ASO
for allele 1
Hybridized and labeled
with ASO for allele 2
Fig. 11.9 d, e
Step 2 – identifying candidate regions
Chromosomal abnormalities: Rare patients who
show chromosomal abnormalities linked to an
unexplained phenotype. E.g DMD
Boy’BB’ with a single large Xp21 deletion who had
- Duschenne’s muscular dystrophy (DMD gene)
- Chronic granulomatoses disease (CYBB gene)
- retinitis pigmentosa (RPGR gene)
- McLeod phenotype (XK gene)
Step 3 – transcript map which defines
all genes within the candidate region
Search browsers e.g. Ensembl
Computational analysis
– Usually about 17 genes per 1000 kb fragment
– Identify coding regions, conserved sequences
between species, exon-like sequences by looking for
codon usage, ORFs, and splice sites etc
Experimental checks – double check sequences,
clones, alignments etc
Direct searches – cDNA library screen
Step 4 – identifying candidate genes
Expression: Gene expression patterns can pinpoint
candidate genes
RNA expression by Northern blot or
RT-PCR or microarrays
Look for misexpression (no
expression, underexpression,
overexpression)
CFTR gene
Northern blot analysis reveals only one of candidate genes is
expressed in lungs and pancreas
Step 4 – identifying candidate genes
Function: Look for obvious function or most likely
function based on sequence analysis
e.g. retinitis pigmentosa
Candidate gene RHO part of
phototransduction pathway
Linkage analysis mapped disease gene
on 3q (close to RHO)
Patient-specific mutations identified in
a year
Step 4 – identifying candidate genes
Homology: look for homolog (paralog or ortholog)
Beals syndrome
fibrillin gene FBN2
Both mapped to 5q
Marfan syndrome
fibrillin gene FBN1
Step 4 – identifying candidate genes
Animal models: look for homologous genes in
animal models especially mouse
e.g. Waardenburg syndrome type 1
Linkage analysis localised
WS1 to 2q
Splotch mouse mutant
showed similar phenotype
Splotch mouse
WS type1
Could sp and WS1 be
orthologous genes?
Pax-3 mapped to sp locus
Homologous to HuP2
Step 5 – confirm a candidate gene
Mutation screening
Sequence differences
- Missense mutations identified by
sequencing coding region of candidate gene
from normal and abnormal individuals
Transgenic model
- Knockout / knockin the mutant gene into a
model organism
Modification of phenotype
Transgenic analysis can prove
candidate gene is disease locus
Fig. 11.21
Reading
HMG3 by T Strachan & AP Read : Chapter 14
AND/OR
Genetics by Hartwell (2e) chapter 11
Optional Reading on Molecular medicine
Nature (May2004) Vol 429 Insight series
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human genomics and medicine pp439 (editorial)
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predicting disease using medicine by John Bell pp
453-456.