MS-MLPA analysis for FMR1 gene: evaluation in a routine diagnostic setting

Valentina Gatta,Liborio Stuppia,Sara Franchi,Elena Gennaro,Marina Grasso,Ivana Antonucci,Massimiliano Cecconi,Marco Tommasi,Domenico Coviello,Giandomenico Palka

doi:10.1186/1471-2350-14-79

Abstract

BackgroundFragile 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).MethodsThe 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.ResultResults 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.ConclusionBased 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.

Highlights

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
Recent epidemiological studies indicate that FXS is responsible for moderate to severe mental retardation in 1:4000–6000 males of European descent; the same condition is responsible for mild-to-mo
Since the distinction between a premutation and a full mutation is related to the methylation status rather than to the exact size of the repeat, in the present study we evaluated the usefulness of the Methylation-Specific Multiplex-Ligation-dependent Probe Amplification (MS-MLPA) assay to assess the methylation status of the promoter of the FMR1 gene for the molecular diagnosis of FXS

Summary

Introduction

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

Results

Discussion

Conclusion