Senogles 6

Senogles 6 Huntington’s Disease
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Huntington’s is a trinucleotide repeat expansion disease, but has unique characteristics. Dementia is due to severe effects on basal ganglia of brain. 2 forms: adult onset and juvenile form. Juvenile form gave insights into inheritance
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Emphasis on juvenile is because of insights.
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RFLP marker assigned to chromosome 4 in 1983. One of 1st testable markers for a disease.
Marker led to cloning of disease gene. Linkage disequilibrium- indicates ancestor gene close to marker.
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Huge pedigrees back to 1800s from families around Lake Maracaibo in Venezuela.
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Assigned to end of chromosome 4- 5 million base pair fragment.
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Segregation with HD: Markers land close to disease gene. Diamonds indicate blind study- did not want to give identifying information about individuals.
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Restriction map and theoretical locus: Knew that disease mapped to this region.Wanted to clone gene.
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Problems: big region. Instead of chromosome walking and jumping and cros-hybridization, they used exon amplification.
Make family of fragments, generate probes, screen library.
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Take vector with known sequences on either side of multiple cloning site (commonly use a beta globulin). Clone in DNA. Get family of fragments. Transfect cell. Isolate RNA, amplify by PCR using primers for known sequence. PCR product is exon flanked by known sequence. If it has an exon, will be transcribed and can be identified. Idea of trapping- if vector has exonic sequence- RNA will result. Can sequence PCR product. Now have probe to screen CDNA library and Northern blots.
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Using strategy, cloned a number of CDNAs. Composite is 340 KB protein. Found that in giant clone had large ORF with 2 repeats in middle of coding sequence. Poly Q, and poly P. Poly P is not related to disease. Poly Q is.
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Repeat size is larger, but not horrendously larger. This is more subtle. Disease state centers around 45 repeats.
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Juvenile Huntington’s: imprinting phenomenon. Repeat size and age of onset correlate.
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Huntington’s is autosomal dominant, so affected male has an allele in the normal range, and an expanded one. For autosomal dominant disease, one parent must be affected, shows up in every generation.
Some individuals have larger repeats than parent . 8 and 10 do. Age of onset correlates to size of expanded allele.
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In Venezuela you can get huntington’s disease homozygous. About 25% of kids are homozygous dominant. Some are heterozygous. Age onset homozygous is 6-7 yrs.
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Sporadic mutation occurs. PCR shown. Huntington’s can be caused by sporadic increase in repeats.
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No evidence of Huntington’s until mutation. Expansions in fragile X were in UTR. These are in code for a protein. Changes in poly Q yield changes in protein function.
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Genetic anticipation- bigger expanded allele, earlier child diagnosed with disease. Not Mendelian phenomenon. Through mother- repeat sizes do not change all that much. Changes dramatically in gene passed down from father.
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Scattergram of juvenile age onset vs number of repeats.
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Age of onset is a function of repeat length- no correlation with maternal descent- is correlated with paternal in juvenile Huntington’s.
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Large repeats have paternal origin, in contrast to Fragile X.
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Expansion may occur during male gametogenesis. Somatic mosaicism- different numbers of repeats in different somatic cells from same patient. Different tissues are different.
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Protein product- you get a functional protein and can observe multiple species. Proteins are made with larger repeat. Heterogeneity detectable at protein level.
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Lots of expression in basal ganglia.
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From a given individual, samples from cortex and cerebellum are different. Individuals cannot efficiently replicate repeats. Different size proteins are reflected.
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Result:
Basis of Huntington’s is undefined. Cellular inclusions form in cells and cause cell death. This may be a gain-of-function mutation- as protein expands, interacts with new things and causes bad effects.
Transgenic mice- can get Huntington’s phenotype.