applications of genome sequencing projects 4) bioarchaeology, anthropology, human evolution, human...
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Applications of genome sequencing projects
http://www.ornl.gov/hgmis/project/benefits.html
4) Bioarchaeology, anthropology, human evolution, human migration 5) DNA forensics 6) Agriculture, livestock breeding, and bioprocessing
1) Molecular Medicine 2) Energy sources and environmental applications 3) Risk assessment
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"
The spectrum of human diseases
Cystic fibrosis thalassemia
Huntington’s
cancer
<5%
‘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, 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 approach1) 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. 11.16 dFig. 11.16 d Hartwell
2) Animal model approachcompares 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)
WS4 patients screened for SOX10 mutations
confirmed the role of this gene in WS4.
Dom mouse
Hirschsprung
Waardenburg
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 StepsStep 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 regionGenetic 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 linkedneed 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
Northern blot analysis reveals only one of candidate genes is expressed in lungs and pancreas
RNA expression by Northern blot or RT-PCR or microarrays
Look for misexpression (no expression, underexpression, overexpression)
CFTR gene
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)
Both mapped to 5q
Beals syndromefibrillin gene FBN2
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
Could sp and WS1 be orthologous genes?
Pax-3 mapped to sp locusHomologous to HuP2
Splotch mouse WS type1
Step 5 – confirm a candidate gene
Mutation screeningSequence differences
Missense mutations identified by sequencing coding region of candidate gene from normal and abnormal individualsTransgenic modelKnockout / knockin the mutant gene into
a model organismModification of phenotype
Transgenic analysis can prove candidate gene is disease locus
Fig. 11.21
Figure 1 | Genetic and chemical-genetic approaches identify genes and proteins, respectively, that regulate biological processes.
a | Forward genetics entails introducing random mutations into cells, screening mutant cells for a phenotype of interest and identifying mutated genes in affected cells. In the example shown, yeast cells are randomly mutated, cells showing a large-bud phenotype are selected, and genes mutated in these cells are identified. Reverse genetics entails introducing a mutation into a specific gene of interest and studying the phenotypic consequences of the mutation in a cellular or organismal context. In the example shown, a single mutated gene is introduced into yeast cells and a large-bud phenotype is observed.
b | Forward chemical-genetics entails screening exogenous ligands in cells, selecting a ligand that induces a phenotype of interest, and identifying the protein target of this ligand. In the example shown, one compound that induces a large-bud phenotype is selected and the protein target of this ligand is subsequently identified. Reverse chemical-genetics entails overexpressing a protein of interest, screening for a ligand for the protein, and using the ligand to determine the phenotypic consequences of altering the function of this protein in a cellular context. In the example shown, a ligand for a specific protein is found to induce a large-bud phenotype.
ReadingHMG3 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.