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POSITIONAL CLONING -

HUNTINGTON'S DISEASE
AS AN EXAMPLE
• 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 children
before symptoms appear
Influence of the Wexler family
Hereditary Disease
Foundation
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.
• Disease associated with the A
haplotype in the American family
and the C haplotype in the
Venezuelan family.
LOD Scores

0.0 0.05 0.1 0.2 0.3 0.4


A 1.81 1.59 1.36 0.90 0.48 0.16
HD – G8 V 6.72 5.96 5.16 3.46 1.71 0.33
T 8.53 7.55 6.52 4.36 2.19 0.49
HD-GC -2.27 -1.20 -0.32 0.00 0.07
G8-GC -2.73 -1.17 -0.08 0.14 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
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
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?
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.
Cloning the cystic fibrosis gene

D788
7q31
met
Step 1: Chromosome walking to make a contig

probe for 1st step

met
probe for 3rd step
probe for 2nd step
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.
Step 2: Identification of candidate genes: Zoo blot

A C
B D

H M D H M D H M D H M D

H = human genomic DNA


M = mouse genomic DNA
D = Drosophila genomic DNA
Step 2: Identification of candidate genes: Northern blots

L B T L B T L B T L B T

probe D probe F probe Q probe Z

L = lung mRNA
B = brain mRNA
T = testis mRNA
• The map position agreed with the
linkage data
• 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.
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

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