title: MS-MLPA analysis for FMR1 gene: evaluation in a routine diagnostic setting.
abstract: BACKGROUND
  Fragile X Syndrome (FXS), the most common cause of familiar mental retardation, is associated in over 99% of cases to an expansion over 200 repeats of a CGG sequence in the 5' UTR of the FMR1 gene (Xq27.3), leading to the hypermethylation of the promoter. Molecular diagnosis of FXS have been so far based on the use of the Southern Blot (SB) analysis, a low throughput and time consuming technique. In order to update the diagnostic approach for FXS, we evaluated the usefulness of the Methylation-Specific Multiplex-Ligation-dependent Probe Amplification assay (MS-MLPA).
  
  
  METHODS
  The study was carried out by retrospectively analysing 44 male patients, 10 Chorionic Villus Sampling (CVS) samples and 10 females previously analyzed by SB. In addition, a prospective study on 98 male subjects, 20 females and 1 CVS sample was carried out for assessing the feasibility and the impact of MS-MLPA in a routine lab work.
  
  
  RESULTS
  Results provided by both the retrospective and the prospective parts of this study strongly demonstrate the robustness and reproducibility of the MS-MLPA assay, able to correctly detect the methylation status in all normal and full mutation male samples analyzed, including CVS male samples. On the other hand, MS-MLPA analysis on females samples produced unreliable results.
  
  
  CONCLUSIONS
  Based on our results, we suggest the necessity of a separate workflow for male and female patients with suspected FXS in the routine diagnostic setting. MS-MLPA, in combination with CGG repeat sizing using a single-tube primed FMR1 PCR, represents a reliable diagnostic protocol in the molecular diagnosis of FXS male patients.
authors.affiliation: Laboratory of Molecular Genetics, Department of Psychological, Humanities and Territorial Sciences, School of Medicine and Health Sciences, G, d'Annunzio University, via dei Vestini 31, Chieti, 66013, Italy. v.gatta@unich.it
authors.forename: Valentina
authors.initials: V
authors.lastname: Gatta
authors.forename: Elena
authors.initials: E
authors.lastname: Gennaro
authors.forename: Sara
authors.initials: S
authors.lastname: Franchi
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Cecconi
authors.forename: Ivana
authors.initials: I
authors.lastname: Antonucci
authors.forename: Marco
authors.initials: M
authors.lastname: Tommasi
authors.forename: Giandomenico
authors.initials: G
authors.lastname: Palka
authors.forename: Domenico
authors.initials: D
authors.lastname: Coviello
authors.forename: Liborio
authors.initials: L
authors.lastname: Stuppia
authors.forename: Marina
authors.initials: M
authors.lastname: Grasso
mesh_terms: D015139:Blotting, Southern / Q000379:methods; D015193:Chorionic Villi Sampling / Q000379:methods; D019175:DNA Methylation; D005260:Female; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics*; D005600:Fragile X Syndrome / Q000175:diagnosis* / Q000235:genetics*; D006801:Humans; D008297:Male; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D011247:Pregnancy; D011401:Promoter Regions, Genetic; D011446:Prospective Studies; D015203:Reproducibility of Results; D012189:Retrospective Studies
chemical_list: D051860:Fragile X Messenger Ribonucleoprotein 1; C497683:FMR1 protein, human
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