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Nascer e Crescer

versão impressa ISSN 0872-0754

Nascer e Crescer vol.23  supl.1 Porto mar. 2014

 

POSTER ABSTRACTS / RESUMOS DE POSTERS

 

P-02

Next generation fragile-X testing: getting away from southern blots

 

 

Nuno MaiaI; Isabel MarquesI; Paula JorgeI,II; Rosário SantosI

IUnidade de Genética Molecular, Centro de Genética Médica Doutor Jacinto Magalhães, Centro Hospitalar do Porto E.P.E., Porto, Portugal
IIUnidade Multidisciplinar de Investigação Biomédica, ICBAS-UP

nuno.maia@chporto.min-saude.pt

 

 

Fragile X syndrome (FXS)  is  the  most  common  form of intellectual disability  in  the general population, usually caused by an expansion of a trinucleotide CGG repeat in the 5’ untranslated region of the FMR1 gene. In most cases, expansions over 200 repeats, termed full mutations, cause silencing of FMR1 gene due to methylation of its promoter, and consequently loss of protein product. Expansions with 55-199 CGG repeats called pre-mutations or others even smaller (45-54 CGG repeats)  named  intermediate, do not cause FXS, but are frequently associated with lateonset neurological and/or reproductive disorders (FXTAS/ FXPOI). Southern Blot (SB) is still considered the gold standard for molecular diagnosis of FXS, because it is able to clearly characterize size and methylation status of full and pre-mutated FMR1 alleles (following DNA digestion with  methylation  sensitive   enzymes).   Nevertheless,   SB is a very time-consuming technique and requires a large amount of intact and high-molecular weight DNA. As such, several methodologies have been developed to replace SB and overcome its disadvantages. The aim of this work was to test different techniques which can substitute totally or partially the SB, by comparing (1) their ability to quantify or discriminate normal, pre-mutated and fully mutated alleles; (2) the maximum number of CGG repeats detected; (3) their capacity to determine the DNA methylation state; (4) their power to discriminate size and methylation mosaics; and (5) the amount of DNA required for each technique. The tested techniques were High Resolution Melting Curve Analysis (currently in experimental process with prototype reagents), the FragilEase™ assay from PerkinElmer®, the Amplidex® FMR1 mPCR Protocol from Asuragen® and a multiplex assay developed by our  group,  for  the  simultaneous  screening of 3 genes FMR1, AFF2 e ARX. For this work we tested 7 DNA samples from patients previously characterized at the molecular level in our laboratory: 6 from females and 1 obtained from a chorionic villus sample of a male fetus. These samples were previously characterized as normal (n=2), fully mutated (n=3), pre-mutated (n=1) and a size mosaic (n=1). Although using a very small number of samples, this work aims to describe and compare four different methodologies in an attempt to establish if they can adequately replace SB.

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