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      Comparison of Quantitative Analysis of Methylated Alleles Real-Time PCR and Methylation-Specific MLPA for Molecular Diagnosis of Beckwith-Wiedemann Syndrome

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          Background/Aims: Beckwith-Wiedemann syndrome (BWS) is a congenital overgrowth disorder predisposing to tumorigenesis caused by abnormal expression or function of imprinted genes of the chromosome 11p15.5 imprinting gene cluster. This real-time PCR-based assay determines the methylation status of a selected CpG island and has been proposed for use in high-throughput methylation analysis. Methods: Here, we use quantitative analysis of methylated alleles (QAMA) for the detection of methylation status of the KCNQ10T1 gene, in a region immediately upstream of the transcription initiation site, and the CTCF binding site 6, located approximately 2 kb upstream of the SmaI site currently used for clinical laboratory testing. We assayed a series of controls and patients diagnosed with BWS at two different loci at 11p15.5 to assess the diagnostic yield of QAMA PCR for clinical laboratory testing. Results: These results compare favorably with methylation-specific multiple ligation probe amplification (MS-MLPA) analysis at both differentially methylated region (DMR)1 and DMR2. There are several advantages of the QAMA PCR over MS-MLPA. The QAMA PCR is less labor-intensive and therefore more cost-effective and does not require dedicated analysis software. A second advantage is that the assay is amenable to high-throughput analysis. Conclusions: The small sample size reflects the rare nature of this epigenetic disorder, and the range of ages was quite wide, as was the degree of disease severity. Therefore, further validation with larger cohorts is warranted.

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          Most cited references 22

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          Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.

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            Beckwith-Wiedemann syndrome.

            Beckwith-Wiedemann syndrome (BWS) is a model disorder for the study of imprinting, growth dysregulation, and tumorigenesis. Unique observations in this disorder point to an important embryonic developmental window relevant to the observations of increased monozygotic twinning and an increased rate of epigenetic errors after subfertility/assisted reproduction.
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              Beckwith-Wiedemann syndrome.

              Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder characterized by overgrowth, tumor predisposition, and congenital malformations. Approximately 85% of reported BWS cases are sporadic, while the remaining 15% are familial. BWS is caused by epigenetic or genomic alterations which disrupt genes in one or both of the two imprinted domains on chromosome 11p15.5. In each domain, an imprinting center regulates the expression of imprinted genes in cis. Normally in domain 1, insulin-like growth factor 2 (IGF2) and the untranslated mRNA H19 are monoallelically expressed. In BWS, increased expression of IGF2 occurs via several mechanisms. In domain 2, CDKN1C, a growth repressor, and an untranslated RNA, KCNQ1OT1, are normally expressed monoallelically. In cases of BWS, several mechanisms result in reduced expression of CDKN1C. Recent reports of BWS cases have identified mutations outside the chromosome 11p15.5 critical region, thereby broadening the challenges in the diagnosis and genetic counseling of individuals and families with BWS.

                Author and article information

                S. Karger AG
                August 2019
                25 June 2019
                : 86
                : 4
                : 217-224
                aDepartment of Public Health and Pediatric Sciences, University of Turin Medical School, Turin, Italy
                bHuman Molecular Genetics Laboratory, IRCCS Istituto Auxologico Italiano, Milano, Italy
                Author notes
                *Massimiliano Bergallo, Department of Public Health and Pediatric Sciences, University of Turin Medical School, Piazza Polonia 94, IT–10136 Turin (Italy), E-Mail massimiliano.bergallo@unito.it
                500627 Pathobiology 2019;86:217–224
                © 2019 S. Karger AG, Basel

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                Page count
                Figures: 3, Tables: 3, Pages: 8
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/500627
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