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Daejeon, South Korea

Lee B.H.,University of Ulsan | Kim G.-H.,University of Ulsan | Oh T.J.,Genomictree | Kim J.H.,University of Ulsan | And 8 more authors.
Journal of Human Genetics | Year: 2013

Methylation-specific (MS) multiplex ligation-dependent probe amplification (MLPA) at two differentially methylated regions (DMRs) at chromosome 11p15, H19-DMR and LIT1-DMR, and microsatellite analysis for uniparental disomy (UPD) at chromosome 7 or 11, have been recommended for the genetic diagnosis of the Beckwith-Wiedemann syndrome (BWS) and the Silver-Russell syndrome (SRS). In this study, the efficacy of the MS pyrosequencing method at H19-DMR and LIT1-DMR at 11p15 and SGCE-DMR at 7q21 was evaluated for the genetic diagnosis of BWS (n=18) and SRS (n=20) patients. Epigenetic alterations or UPD were detected in 83% of BWS and 50% of SRS individuals by MS-MLPA, but the detection rate increased to 95% of BWS and 70% of SRS by MS pyrosequencing. Thirteen BWS patients (72%) harbored loss-of-methylation (LOM) at LIT1-DMR and two patients (11%) harbored gain-of-methylation (GOM) at H19-DMR, whereas two patients (11%) had both LOM at LIT1-DMR and GOM at H19-DMR, reflecting paternal UPD 11. Thirteen SRS patients (65%) harbored LOM at H19-DMR, whereas one patient (5%) had GOM at SGCE-DMR, reflecting maternal UPD 7. Birth anthropometric profiles were significantly correlated to methylation scores at either H19-DMR or LIT1-DMR. In conclusion, MS pyrosequencing enhanced the detection rate of molecular defects in BWS and SRS. Moreover, it indicates that methylation status at 11p15.5 might have an important role in fetal growth. © 2013 The Japan Society of Human Genetics All rights reserved.

Oh M.,Genomictree | Hoehn B.D.,Genomictree | Moon Y.,Genomictree | Oh T.,Genomictree | And 2 more authors.
Journal of Molecular Diagnostics | Year: 2012

Herein, we describe a novel multiplex genotyping method, GTPlex-PyroSeq. This method consists of two phases: multiplex PCR followed by a single reaction of pyrosequencing. This study demonstrates how GTPlex-PyroSeq can be adapted for the determination of multiple human papillomavirus (HPV) genotypes. A biotinylated consensus primer, GP6+, and 15 high-risk HPV type-specific primers are used for multiplex PCR. Each type-specific primer has a 5′-tag unique ID sequence connected to a pyrosequencing primer binding region. The unique ID sequence is composed of three parts: i) a single nucleotide ID representing a specific genotype; ii) a sign post; and iii) an end mark. This design allows multiple genotype determination under an ID sequence-dependent nucleotide dispensation order during pyrosequencing. Following initial studies using HPV plasmids and cell lines, we evaluated the clinical utility and effectiveness by comparing our assay with direct sequencing and HPV DNA chip analysis of 80 samples from high-risk, HPV-positive patients. We found in single-type infections, 100% concordance with direct sequencing (70 of 80 perfect matches) and 97.5% concordance with HPV DNA chip data (50 of 80 perfect matches). Additionally, our system was superior to direct sequencing in detection of multiple infections (12 of 80), with a limit of detection of 100 copies. The scalability of this multiplex system, with its open-platform design and ability to use various sample types, makes the GTPlex applicable for use in multiple settings. Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

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