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

• earlier detection of genetic predisposition to disease

• Rational drug design• Gene therapy and control systems for drugs • pharmacogenomics "custom drugs"

DefinitionsDNA polymorphism: A DNA sequence that occurs in two or more

variant formsAlleles: 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 functionDNA marker: polymorphic locus useful for mapping studiesRFLP 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 individualsDNA fingerprinting: Detection of genotype at a number of unlinked

highly polymorphic loci using one probeGenetic 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

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 genes2 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 approachPosition independent approach1) 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. 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 approachB) Positional dependent approachPositional 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 genesStep 5 – confirm a candidate gene and

screen for mutations in affected families

Step 2 - Identifying a candidate regionGenetic 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 linkedneed 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

Fig. 11.7 – genetics/ Hartwell

- Amplify fragment- Expose to

restriction enzyme- Gel

electrophoresis

e.g., sickle-cell genotyping with a PCR based protocol

SSLPs Similar principles used in detection of RFLPs However, no change in restriction sitesChanges in length of repeats

SNPs (single nucleotide polymorphisms)

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

presence of two different nucleotides at the same loci in genomic DNA from different individuals

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

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 1Linkage 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 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

ReadingHMG3 by T Strachan & AP Read : Chapter 14

AND/ORGenetics 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.