Download 19. Positional cloning

BIOL 311 Human Genetics
Fall 2006
Lecture: Positional Cloning
Reading: Chap. 14, Selections from Principles in Medical Genetics, Collins et al.
Lecture Outline:
1. Cloning strategies
2. Muscular dystrophy
3. Huntington's disease
4. Cystic fibrosis
Lecture:
1. Strategies for identifying disease genes
Fig. 14-1
Position-independent:
Requires knowledge of protein product
Work backward from protein DNA
Positional cloning
No knowledge of protein needed
Isolate clone based on approximate
chromosomal location
Synthesize degenerate oligonucleotide PCR Work from candidate region clones
or screen for cDNA use to isolate
possible genes test genes for mutations
genomic clone
in affected people
Identify gene in mouse and use to clone
human gene
Genome-wide screening for triplet repeats
has identified new disease genes
Limitations of positional cloning:
Not enough DNA markers mapped
Extensive amount of work to isolate gene
Unless region is well pinpointed
Limit of resolution ~1 cM approximately = 1 Megabase
"Clues" used in positional cloning
 autozygosity mapping
 inspect pattern of transmission of group of markers (Chap. 13-9)
 chromosomal rearrangements in individuals with disease
 translocations
 inversions
 deletions
2. Duchenne Muscular Dystrophy
 X-linked recessive trait
 Mainly affects boys
 Boys lose muscle tone/wheelchair bound by age 10, death by age 20
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Mapping of disease gene locus to Xp21
A few girls with MD had translocation breakpoint at Xp21.
Why do these girls have MD despite having a normal X chromosome? X-inactivation
normal allele is inactivated.
A boy with no family history of genetic disease had 4 X-linked traits:
 DMD
 Chronic granulomatous disease
 2 other linked traits
traced to small deletion of Xp21.2
Positional cloning
2 groups used different approaches to clone MD
 one used X-chromosome translocation breakpoint--cloned region adjacent to
some rRNA genes
 one used small deletion from boy with 4 X-linked diseases; then carried out
subtractive hybridization vs. normal DNA
Which DNA fragments are missing?
Southern blot of boys DNAs having deletion in region of ~50 kb.
Fig. 9-20 Gehlerter et al.
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Found five with same small deletion, not detectable cytologically.
Later detected transcript in normal mRNA, missing in MD patients cloned
cDNA identify intron/exon structure
Duchenne's muscular dystrophy and Becker's muscular dystrophy are both due to
mutations in dystrophin gene.
 In DMD, mutations result in no expression of dystrophin (427 kDa protein) or
production of truncated or non-functional protein.
 In Becker's, mutations and deletions maintain reading frame for dystrophin
protein.
3. Huntington's Disease (HD)
 Autosomal dominant
 1/20,000 individuals affected
 average age of onset 37
 ~100% penetrance by age 80
 symptoms: personality changes, memory loss, motor problems
 autopsy: dramatic neural loss in basal ganglia of brain
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 mapped by systematic screening of polymorphic markers for linkage
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Fig. 9.25 Gelehrter RFLP test
G8 marker (12th tested) closely associated with disease RFLP marker
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LOD analysis of many families throughout the world gave score of +80
high association of G8 with HD gene on chromosome 4
"C" haplotype for G8 closely associated with disease
genetic distance 3 cM
gene was cloned in 1993
disease associated with CAG triplet repeat expansion in amino terminus of HD
protein (huntingtin)
disease allele can be detected by PCR (Gelehrter Fig. 9-27)
10-30 copies in normal chromosomes
36-121 copies in HD chromosomes
correlation between length of repeats and age of onset  longer repeats is
correlated with earlier age of onset = "anticipation"
ethical issues such as chosing to learn ones status by DNA testing when no
treatment is available
4. Cystic fibrosis
 One of the most common autosomal recessive diseases of white populations
 Affects 1/2500 newborns
 Carrier frequency 1/25
 Elevated Cl- levels in sweat
 Viscous secretion of pancreas and lungs
 Respiratory therapy required
 Males exhibit fertility problems
 Average survival ~30 years
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Linkage to chromosomal markers demonstrated in 1985
As gene was approached, observed increased "linkage disequilibrium" with
neighboring markers
Associated disease with particular haplotype
Linkage disequilibrium
 Identify shared ancestral chromosome segments on CF chromosomes from
unrelated patients. Same haplotype or marker alleles.
 Key tool for identifying susceptibility genes for complex diseases
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500,000 bp candidate interval
no major rearrangements commonly associated with disease
three incorrect genes were identified before the correct gene was cloned
identified transcript and cloned cDNA of a fourth gene
250,000 bp gene
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26 exons
mRNA 6129 bp encodes 1480 amino acid protein
CFTR=cystic fibrosis transmembrane conductance regulator
Chloride ion channel protein
Identified mutations that correlate with disease
Exon 10: 3 bp deletion results in deletion of phenylalanine at amino acid 508
Carrier screening possible
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