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.