Download Identifying human disease genes

Peter John
M.Phil, PhD
Atta-ur-Rahman School of Applied Biosciences (ASAB)
National University of Sciences & Technology (NUST)
Position Dependent Strategies
Functional complementation in
transgenic mice
 A mouse gene has been identified by constructing transgenic
mice
 Using non mutant BAC clones from a candidate region,
crossing them to mice carrying the mutation
Human MYO15 gene
 DFNB3 had been mapped to a location that corresponded in the
mouse to the location of the deafness gene shaker-2
 Transgenic mice were constructed using BACs from the shaker-2
candidate region, and a BAC that corrected the shaker-2
phenotype was identified.
Human MYO15 gene
 This led to identifying the shaker-2 gene
 Human MYO15 gene was then isolated based on its close
homology to the mouse gene,
 Its position within the DFNB3 candidate region confirmed,
 Mutations demonstrated in DFNB3 affected people.
Functional Complementation in Transgenic Mice
Functional Complementation in Transgenic
Mice
 The shaker-2 mouse mutation was identified by finding a
wild-type clone that corrected the defect.
Functional complementation in
mammalian cell lines
 Mammalian cell lines have been generated that are
deficient in DNA repair
 They show abnormal responses following exposure to
UV irradiation or chemical mutagens.
 These mutant cells, can be transformed by fragments
of normal human DNA or human chromosomes in
order to produce a repair-competent phenotype.
Functional complementation in
mammalian cell lines
 The ability of transferred chromosomes or clones to
correct the uncontrolled growth of tumor cell lines has
been used to help locate and then identify tumor
suppressor genes
Positional cloning
 First step in positional cloning is to define the
candidate region
 Initial localization from genetic mapping defines a
candidate region of 10 Mb or more.
 The next step is to collect as many families as possible
and establish a dense cover of polymorphic markers
across the region.
Positional cloning
 Suitable markers may be found by database searching.
 Otherwise YACs, BACs and cosmids must be isolated
from the candidate
polymorphisms.
region
and
screened
for
 Pairs of closely spaced markers define the positions of
the closest recombinations on either side of the disease
locus.
Positional cloning
 This is decided by inspecting individual haplotypes.
 Linkage disequilibrium may allow very high resolution
mapping
Candidate Region Search
 cDNA library screening, using as probes genomic
clones from the candidate region
 CpG island identification, to seek the regions of
under-methylated DNA which often lie close to gene
Chromosomal aberrations
 chromosomal break can cause a loss-of-function
phenotype if it disrupts the coding sequence of a gene,
or separates it from a nearby regulatory region.
 The breakpoint provides a valuable clue to the exact
physical location of the disease gene.
Deletions and duplications
 Chromosomal deletions cause abnormalities due to
loss of genes
 Microdeletions can be identified by several methods.
 FISH mapping.
 Hybridization-based restriction mapping.
Positional candidate strategies
 Identify candidate genes by a combination of their
 Map position
 Expression Pattern
 Function or homology
Positional candidate strategies
 Predictions of the biochemical function of an
unknown disease gene are often proved wrong once
the gene is isolated.
 candidate regions identified by positional cloning
usually contain dozens of genes.
 It can be very time-consuming to identify every
transcript from the region, and excessively laborious to
screen them all for mutations.
Criteria for candidate gene
 Appropriate expression pattern
 Candidate gene should have an expression pattern
consistent with the disease phenotype.
 Expression of candidate genes can be tested by RT-
PCR or Northern blotting, but the best method for
revealing the exact expression pattern is in situ
hybridization against mRNA in tissue sections
Appropriate function
 Candidate genes may also be suggested on the basis of
a close functional relationship to a gene known to be
involved in a similar disease.
 The genes could be related by encoding a receptor and
its ligand, or other interacting components in the
same metabolic or developmental pathway.
Homology to a relevant human gene
or EST
 Selecting candidate disease genes by homology is often
more successful using model organisms
 Identification
of
transcripts
often
comes
from
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
Homology to a relevant gene in a
Model Organism
 Powerful means of selecting good candidates from
among a set of human genes is therefore to search the
databases for evidence of homologous genes in these
well-studied model organisms
 Such data might include the pattern of expression and
the phenotype of mutants
Confirming a candidate gene
 Mutation screening
 Restoration of normal phenotype in vitro by using cell
lines
 Production of a mouse model of the disease, a
transgenic mouse model can be constructed.
Functional Analysis
 Once a candidate gene is confirmed, the next step is to
understand its function
 Understanding the molecular pathology may also lead
to insight into related diseases, and hopefully
eventually to more effective treatment including
perhaps gene therapy
Thanks