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1、Dynamic mutations are an important mutation class. Describe the full range of methods and strategies that are currently being used to diagnose 3 inherited diseases of your choice. Choose examples which maximise the number of methods and strategies you describe. Sarah Waller,Key words: Trinucleotide
2、repeat; Huntington disease; polyglutamine; myotonic dystrophy; Fragile X syndrome; Southern blot; triplet-primed PCR; exclusion testing. Useful reference: La Spada & Taylor, Nat Rev Genet 11:247 (2010),Dynamic mutations,A dynamic mutation is an unstable heritable element, where the initial change to
3、 the DNA sequence alters the chance of further changes to it. These mutations typically occur in areas of tandem repeats, and are exemplified in the trinucleotide repeat disorders. Triplet expansion is caused by slippage during DNA replication. Loop out structures may form during DNA replication due
4、 to the repetitive nature of these DNA sequences; if this occurs on the daughter strand the number of repeats will be increased. If the loop out structure occurs on the parent strand the number of repeats can decrease and the mutation can revert to a normal or premutation carrier state.,Trinucleotid
5、e repeat disorders,The length of the repeat determines the probability and extent of its pathogenicity. Anticipation the tendency of the age of onset to decrease and the severity of symptoms to increase through successive generations due to continued repeat expansion. More common in paternal transmi
6、ssion due to instability of the CAG repeat in spermatogenesis. Most of these diseases have neurological symptoms. Methods & strategies currently in use to diagnose: Huntington disease Myotonic dystrophy Fragile X syndrome,Huntington disease (HD),HD is a progressive neuronal degenerative disease caus
7、ed by expansion of a CAG repeat in exon 1 of the HTT gene, resulting in production of a toxic protein containing a polyglutamine repeat. Pathogenicity of HD allele is determined by its size: 26 or fewer repeats normal 27-35 intermediate; due to repeat instability the allele may expand to a HD-causin
8、g allele in subsequent generations 36-39 reduced penetrance HD-causing allele; patient is at risk but may not develop HD 40 or more full penetrance HD-causing allele There is a significant correlation between CAG repeat size and the age of onset and rate of deterioration of motor & cognitive functio
9、n CAG tracts interrupted with CGG are more stable Accurate sizing of HD allele crucial for proper diagnosis & genetic counselling,PCR of HD alleles,HD alleles can be accurately sized by PCR followed by capillary gel electrophoresis: Sequence analysis can be performed on gel purified products of inte
10、rmediate or reduced penetrance alleles to determine if there are any stabilising CGG interruptions.,Triplet-primed PCR (TP-PCR),3 primers used: forward (different to that used in genomic PCR), triplet repeat primer, consisting of GTC repeats linked to annealing site for 3rd primer. Triplet repeat pr
11、imer anneals randomly along CAG tract, generating ladder of products.,TP-PCR can also be used to detect the presence of CAG repeats:,Exclusion testing,Exclusion testing can be offered to couples who wish to have prenatal testing but who do not want to know their own HD status. Analysis of polymorphi
12、c markers is carried out to determine if the fetus has inherited the HD locus from the affected grandparent:,Diagnosis of HD using PCR & TP-PCR,Both of these methods are: cheap quick and straightforward to perform have very high specificity and sensitivity,Myotonic dystrophy,DM1 caused by CTG repeat
13、 expansion in 3 UTR of DMPK gene, which encodes a myosin kinase. The predominant symptom in classic DM1 is distal muscle weakness. Expanded RNA sequesters muscleblind 1 (MBNL1) protein, which leads to down-regulation of mRNAs which normally interact with MBLN1, eg genes involved in Ca signalling. Al
14、ternative splicing of certain genes is also disrupted. As with HD, pathogenicity is determined by allele size:,- 37 or fewer repeats: normal - 38-49 repeats: premutation, patients asymptomatic but children at risk of inheriting larger allele with pathologically expanded repeat - 50-4000 repeats: ful
15、l penetrance,Myotonic dystrophy,Larger repeat expansions correlate with earlier age of onset and more severe disease phenotype, particularly if less than 400 repeats. Alleles with greater than 1000 repeats give rise to congenital DM1, a severe disease often presenting before birth. Anticipation usua
16、lly occurs through maternal inheritance very large alleles believed to be toxic to sperm. However smaller alleles inherited from father can also expand to full mutation. Sizing of alleles performed by PCR; if only one allele detected then Southern blotting is performed since large expansions are beyond detection limit of PCR & capillary electrophoresis. This will distinguish patients with homozygous normal alleles and those with one normal and one expanded allele.,Southern bl
