Identification of candidate genes by a combination of

their map position and expression, function or homology

Positional cloning information

Identification of most disease genes

Positional independent strategies

BUT WHY BOTH ARE REQUIRED??

BECAUSE
Purely positionindependent approach
Rarely suceed as molecular pathology is too complicated Predictions of the biochemical function of an unknown disease gene are often proved wrong once the gene is isolated predicting the phenotypic effect of mutations in a known gene works only in general terms.

Purely positional approach

inefficient because candidate regions identified by positional cloning usually contains dozens of genes. Very time consuming and laboriousto identify every transcript from the region and to screen them all for mutation. Before mutation screening to be started, exon-intron structure must be determined.

Candidate gene
Any gene thought likely to cause a disease. The gene may be a candidate because it is located in a particular chromosome region suspected of being involved in the disease or its protein product may suggest that it could be the disease gene in question.

Criteria for selecting a candidate gene

Appropriate expression and function

Homology to a relevant human gene or EST

Homology to a relevant gene in a model organism

1. Appropriate expression pattern

gene should have an expression pattern consistent with the disease phenotype. Expression need not be restricted to the affected tissue but candidate should at least be expressed at the time and in the place where the pathology is seen. Testing the expression of candidate genes by i. RT-PCR ii. Northern blotting, iii. in situ hybridization against mRNA in tissue sections (best method)

2. Appropriate function
May be suggested on the basis of a close functional relationship to a gene known to be involved in a similar disease. could be related by encoding a receptor and its ligand, or other interacting components in the same metabolic or developmental pathway.

Examples of positional candidate genes identified.. i. Rhodopsin inRetinitis Pigmentosa (RP) ii. Fibrillin in Marfan syndrome (MFS)

3. Homology to a relevant human gene or EST

Preliminary identification of transcripts - matching genomic sequence generated from the candidate region against unmapped ESTs in the databases. Finding a match suggests the presence of an exon in the genomic DNA, and may provide leads to identifying more of the gene or to guessing its function. Sometimes a gene in the candidate region turns out to be closely related to a known disease gene. If the diseases are similar, the new gene becomes a compelling candidate. Example: Fibrillin gene mutated in MFS and second fibrillin gene (FBN2) mapped on 5q, mutated in Congenital contractural Arachnodactyly.

4. Homology to a relevant gene in a model organism

More successful as extensive homologies can be detected between human genes and genes in zebrafish, Drosophila, the nematode worm Caenorhabditis elegans and even yeast. Even pathways are often highly conserved. Example: transfection of human apterous homolog Lhx2
gene in wingless Drosophila mutant-Apterous, makes flies to grow wings.

 Using clues from the mouse

genetically well explored organisms much the closest to humans in evolutionary terms orthologous gene mutations are more likely to produce similar phenotypes.(may differ) Mouse phenotypic information often translates readily into positional candidate information Backcross mapping allows quick and accurate mapping. Once a chromosomal location for a gene of interest is known in mouse or humans possible to predict the likely location of that gene in the other species (not always) . Exon sequences are usually well conserved between orthologous human and mouse genes. Once gene is isolated, probes or primers can be designed to screen DNA libraries from the other species in order to identify the orthologous gene.

 Sequence homologies with lower organisms

genomic or cDNA sequences generated in the exploration of a candidate disease region are routinely checked against data from lower organisms. A match of genomic sequence presence of a gene, if the match is to a known gene, suggests the nature of the human gene. Example:
using Drosophila phenotypic information systematically to identify positional candidates for human diseases is the DRES database. dbEST human EST database was searched for matches to Drosophila genes with known mutant phenotypes. Sixty six novel matches were detected