MS-MLPA is a specific and sensitive technique for detecting all chromosome 11p15.5 imprinting defects of BWS and SRS in a single-tube experiment

Abstract

Human chromosome 11p15.5 harbours a large cluster of imprinted genes. Different epigenetic defects at this locus have been associated with both Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS). Multiple techniques (Southern blotting, COBRA and microsatellite analysis) have been used so far to detect various DNA methylation abnormalities, uniparental disomies and copy number variations, which are characteristics of these two diseases. We have now evaluated a methylation-specific multiplex-ligation-dependent probe amplification assay (MS-MLPA) for the molecular diagnosis of BWS and SRS. Seventy-three samples derived from BWS-and SRS-affected individuals and 20 controls were analysed by conventional tests and MS-MLPA in blind. All cases that were found positive with conventional methods were also identified by MS-MLPA. These included cases with paternal UPD11, hyper-or hypomethylation at the Imprinting Centre 1 or Imprinting Centre 2 and rare 11p15.5 duplications. In summary, this MS-MLPA assay can detect both copy number variations and methylation defects of the 11p15.5 critical region within one single experiment and represents an easy, low cost and reliable system for the molecular diagnostics of BWS and SRS.

Novel deletion of the E3A ubiquitin protein ligase gene detected by multiplex ligation-dependent probe amplification in a patient with Angelman syndrome

“…In line with hundreds previous MLPA studies performed in various syndromes and diseases (Ainsworth et al, 2004;Madrigal et al, 2007;Priolo et al, 2008), we confirm that MLPA is an useful method to detect UBE3A exon deletions which otherwise would be missed using conventional gene-scanning methods. However, at the same time we need to claim about some limitations in the performance of MLPA for quantification analysis possibly leading to false positives as documented in our previous study (Calì et al, 2010).…”

(Epi)genotype–phenotype correlations in Beckwith–Wiedemann syndrome

“…Methylation analysis of the 11p15.5 chromosomal regions containing IC1 and IC2 was carried out in all patients and performed either by Southern blotting (n = 170), COBRA (n = 45) 15 or Methylation-Sensitive Multiple Ligation Probe Amplification (MS-MLPA MRC-HOLLAND kit) (n = 103). 16 The results obtained by these techniques have been shown to be comparable. 16,17 In patients with suspected UPD, confirmation was obtained by microsatellite analysis of probands and parents, as described.…”

“…16 The results obtained by these techniques have been shown to be comparable. 16,17 In patients with suspected UPD, confirmation was obtained by microsatellite analysis of probands and parents, as described. 18 The presence of genome-wide UPD was tested in 28 UPD patients by microsatellite analysis and single-nucleotide polymorphism array.…”

Addition of H19 ‘Loss of Methylation Testing’ for Beckwith-Wiedemann Syndrome (BWS) Increases the Diagnostic Yield

“…2,17,73 Notwithstanding these advantages, MS-MLPA-detection rates are similar to former standards and ϳ78% in well-characterized patients with BWS. 125 Therefore, none of the current methods, including those applied here, are able to replace a multimodal approach including detection of translocations/inversion via karyotyping and CDKN1C alterations by DNA sequencing. 2 Cytogenetic abnormalities are observed in ϳ1% (FISH: ϳ2%) and CDKN1C mutations (occurring independent of H19/LIT1 22,38,46,52,69,126 ) have been described in 1-3% of sporadic and 5-10% of familial BWS.…”