Transcript mol medicine 1

applications of genome
sequencing projects
1) Molecular Medicine
2) Energy sources and environmental
applications
3) Risk assessment
4) Bioarchaeology, anthropology, evolution,
and human migration
5) DNA forensics
6) Agriculture, livestock breeding, and
bioprocessing
http://www.ornl.gov/hgmis/project/benefits.html
Molecular medicine
improved diagnosis of disease
eearlier detection of genetic predisposition to
disease
rational drug design
gene therapy and control systems for drugs
ppharmacogenomics "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 detected by a
particular probe due to nucleotide changes at a restriction site
SNP: two different nucleotides appear at the same position 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
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 polymorphism) 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)-Sites
resulting from a single change in individual bp.
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)
Sites resulting from a single change in individual bp
SNP detection using allele-specific oligonucleotides
(ASOs)
• Very short probes (<21 bp) specific which
hybridize to one allele or other
• Such probes are called ASOs
Fig. 11.8
ASOs can
determine
genotype at
any SNP
locus
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
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 approach: when the disease
protein is known 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
2) Animal model approach
compares animal mutant models in 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)
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
Step 2 - Identifying a candidate region
Genetic map of <1Mb
Genetic markers:
RFLPs, SSLPs, SNPs
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
Halpotype maps
HapMap published in Oct27 2005 Nature
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,
CFTR gene
overexpression)
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
•
human genomics and medicine pp439 (editorial)
•
predicting disease using medicine by John Bell pp 453-456.