Elias, Marjanu Hikmah
(2010)
Exploration Of Real Time PCR As A New Diagnostic Method Of Fragile X Syndrome.
Masters thesis, Universiti Sains Malaysia.
Abstract
Fragile X Syndrome (FXS) is the most common form of inherited mental retardation in
males caused by FMR1 gene abnormality associated with CGG repeats expansion and
hypermethylation status of its promoter. Methylated alleles usually lead to
transcriptional inhibition and consequently loss of Fragile X Mental Retardation Protein
(FMRP) production. Chemical modification of cytosine to uracil by sodium bisulfite
treatment has provided an additional method for the laboratory diagnosis of FXS, thus
avoiding the use of the laborious Southern blot analysis, which is the gold standard test
for FXS diagnosis. Thus, a study was done to explore a rapid, easy, reliable and cheap
method for FXS diagnosis that can replace the laborious, time consuming and expensive
Southern blot method. Genomic DNA was extracted from peripheral blood of 45
clinically diagnosed FXS patients. Samples were treated with sodium bisulfite followed
by amplification using real-time multiplex methylation specific PCR (RT-M-MSPCR)
with XIST gene as an internal control, followed by melting curve analysis. Our results
showed all control samples with known methylation status were correctly diagnosed
using Real Time Multiplex Methylation Specific PCR. For method validation purpose,
the methylation status of other 45 male patients sample were also successfully diagnosed
using our RT-M-MSPCR method and were concordance with the results of the Southern
blot.
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