Download Slide 1

POSITIONAL CLONING
TWO EXAMPLES
Huntington's disease
•
•
•
•
•
•
•
Inheritance pattern - dominant autosomal
Entirely penetrant and fatal
Frequency - about 1/10,000 live births
Late onset - age 35 to 45
No biochemical defect known, until very recently
No methods of treatment
Because of late onset, many have had children
before symptoms appear
Huntington’s disease
results from nerve cell
degeneration in the
basal ganglia
Influence of the Wexler family
Families with a history of
Huntington's disease
• Indiana University maintains a National
Research Roster for Huntington's Patients
• Large family with a history of Huntington's
disease discovered living on shore of lake
Maracaibo in Venezuela
For both families with a history of
Huntington's disease:
• Blood samples taken from each member
• Permanent cell lines established
• Each family member analyzed by a
neurologist for disease symptoms
• Paternity verified
• Objective: Finding a probe that uncovered an
RFLP that was linked to Huntington's disease.
• 1981 - Gusella's group started with a group of
anonymous probes that uncovered RFLPs very few available.
• They were incredibly lucky - the 12th probe
they tried -called G8 - indicated linkage.
I
II
III
IV
V
AD AA AA AA
AA AA AD
AB AA
AA
AA AC
AC
AB
AB AC AC BC AC
AB AB AB AA AA AA CD
BC
AC
AC CD
BC CC AC CC AC AC CC CC CC CC AA
AA
AA
AC AC AC AC AC AC
AC
AA AB
AC CD AC AC AB AC AC AC AA AC
AB BC BC
• Disease associated with the A haplotype
in the American family and the C
haplotype in the Venezuelan family.
LOD Scores
0.0
A 1.81
HD – G8 V 6.72
T 8.53
HD-GC
G8-GC
0.05
1.59
5.96
7.55
-2.27
-2.73
0.1
1.36
5.16
6.52
-1.20
-1.17
0.2
0.90
3.46
4.36
-0.32
-0.08
0.3
0.48
1.71
2.19
0.00
0.14
0.4
0.16
0.33
0.49
0.07
0.08
1983 - G8 (also called D4S10)
mapped approximately 4 cM from
the HD locus.
It took 10 more years to clone
the gene. Why?
1986-87 DNA markers were used
and D4S10 was localized by in situ
hybridization and somatic cell
genetics to chromosome region
4p16.3
Further linkage studies for isolating
HD
Further linkage studies for isolating HD
Identification of Putative Coding
Sequences
Exon Trapping
Use trapped exons to identify
candidate genes from cDNAs
Four transcripts were analyzed.
IT15 - Huntingtin
Repeat Sequence Diseases
Implications
• No cure yet
• Testing is possible
• Ethical issues arise
Cystic fibrosis
•
•
•
•
Autosomal recessive
Not late onset
Average life expectancy 27 years
Treatment but no cure
The most common fatal genetic disease in the US today. It is an
autosomal recessive, and it is found mostly in people of Northern
European background.
Affects multiple organs, all of which are involved with secretion. Mucus
is very thick and sticky. Variable symptoms.
Lungs: thick mucus harbors microorganisms, often leading to
pneumonia. Also asthma, bronchitis, and other lung problems.
Pancreas: decreased secretion of digestive enzymes lead to
malnutrition, intestinal blockage, and other digestive problems
male infertility
abnormally salty sweat: often detectable in newborns. A sweat
test is the standard method of diagnosis for CF.
About 1/20 to 1/25 Caucasians are
carriers of the defective gene (two
carriers have to mate to produce an
affected individual). Why is the
prevalence of this defect so high?
Biochemical work done before the gene was cloned pointed to
salty skin as a primary clue. Problem in chloride ion transport.
Chloride can't get out of the cell, so water stays in to dilute it-can't come out to dilute the mucus.
Work on cloning the gene started with a search in lots of
families with many RFLP probes.
Pedigree Analysis
• A loosely linked RFLP
marker was found.
• By in situ hybridization,
this marker and
therefore the CF gene
were mapped to
chromosome 7.
Further RFLP markers known to be located on
chromosome 7 were then tested for linkage
to CF.
Two markers were found that flanked CF, met
and D7S8, but they were 1600 kb apart.
Eventually two more closely linked markers
were found that narrowed the region to
about 500 kb.
In the case of the CF gene, 7 jumps
of 50 to 75 kb were made with each
arrival point then serving as a new
origin for chromosome walking.
• The map position agreed with the linkage
data
• Zoo blot
• The probe had a CpG island at the start of
the coding sequence
• The gene was detected in Northern blots
using RNA extracted from the "correct"
tissue, in this case cultured epithelial
(sweat gland) cells
Diagnostic tests are available, but
extensive testing is necessary
because there are so many different
mutations.
Since the function of the gene is
known, treatment strategies can be
devised.
Conventional Therapy:
• Antibiotics
• Inhalers to reduce mucus blockage
in lungs
• Medicines to reduce inflammation
in lungs (e.g. corticosteroids, ibuprofen)
• Pancreatic enzymes
• Vitamins
• Extra calories
Gene therapy
• Insert CFTR gene in adenovirus vector.
• Virus is used to infect CF patient by inhalation.
• Virus inserts itself into lung cells, where gene
functions normally.
Why is disease so prevalent?
• Selective advantage of heterozygotes?
• Big gene, many mutations