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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 1 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. 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 2 mapped by systematic screening of polymorphic markers for linkage Fig. 9.25 Gelehrter RFLP test G8 marker (12th tested) closely associated with disease RFLP marker 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 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 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 3 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 4