Triplet Repeat Disease
Describe examples of triplet/trinucleotide repeat disease
Review clinical characteristics & genetic basis of Huntington's Disease & Fragile X Syndrome
Describe the use of QF-PCR & Southern blotting methods for detecting triplet repeats & methylated DNA
Trinucleotide Repeat Disorders
Diseases resulting from an abnormal expansion of repetitive sequences
Threshold number of repeats must be reached - clinical symptoms
Huntington's Disease (HD)
Fragile X Syndrome (FXS)
HD
FXS
Progressive neurodegenerative disorder
Adult onset, 30 - 40s
Depression, poor coordination
HTT gene
Chromosome 4p16.3
Expansion of CAGn repeat in exon1 of the HTT gene
Extended poly Q tract in huntingtin protein
CAG repeat size (at >40 repeats)
Clinical characteristics
Delayed speech
Mild to moderate mental retardation
Distinctive physical & behavioural traits
Molecular basis
∼1% heterogeneous mutations: large deletions, missense, nonsense, small in/del frame shift
∼99% (CGG)n expansion in the 5'UTR of FMR1
FMR1 gene becomes silenced following repeat expansion
Methylation sensitive Southern blotting RFLP analysis
FX results in methylation of high copy number repeat sequences
Detect using RFLP
EcoRI cuts independently of methylation status of genomic DNA
Eag1 is methylation sensitive (recognition site methylated - not cut)
Southern Blot
Restriction Digest Electrophoresis
Transfer to membrane
Anneal probe
Detect
Use a FMR1 radioactive DNA probe to detect alleles
Methylated (silent, condition associated) cut only with EcoR1 - larger frags
Unmethylated (express FMR1 protein) - cut with EcoR1 + Eag1
Coding
Non-coding