title: Pitfalls of Multiple Ligation-Dependent Probe Amplifications in Detecting DMD Exon Deletions or Duplications.
abstract: Multiple ligation-dependent probe amplifications (MLPAs) are a key technology for the molecular diagnosis of Duchenne/Becker muscular dystrophy, which is mainly caused by large gene arrangements. However, little is known about the false-positive rates of MLPA for this disease. Here, we review MLPA analysis results from 398 patients suspected to have Duchenne/Becker muscular dystrophy. MLPA assay was used for screening the entire coding region. If these amplifications produced normal results, direct sequencing was performed to search for sequence variations and to determine single-exon deletions, duplications, or indeterminate results. Using MLPA, 290 cases (72.9%) showed exon deletion or duplication results. Among those, 75 cases (25.9%) resulted in a deletion or duplication of a single exon. Direct sequencing revealed that 11 single-exon deletion cases resulted in false-positives due to sequence variations within the patient population interfering with probe binding at the probe-hybridization sites. Abnormal MLPA results were closely related to the type of sequence change and the position within the probe-hybridization locus. The most common type was C-T transition (n = 19, 55.9%). Abnormal MLPA results correlated with CA mismatch and low melting temperature (≤75°C). False-positive events for large gene rearrangements involving a single exon in DMD accounted for approximately 15% (11/75). Therefore, careful design of MLPA probes is required to avoid false-positive results.
authors.affiliation: Department of Laboratory Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Man Jin
authors.initials: MJ
authors.lastname: Kim
authors.affiliation: Department of Laboratory Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Sung Im
authors.initials: SI
authors.lastname: Cho
authors.affiliation: Department of Pediatrics, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Jong-Hee
authors.initials: JH
authors.lastname: Chae
authors.affiliation: Department of Pediatrics, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Byung Chan
authors.initials: BC
authors.lastname: Lim
authors.affiliation: Department of Laboratory Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Jee-Soo
authors.initials: JS
authors.lastname: Lee
authors.affiliation: Department of Laboratory Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Seung Jun
authors.initials: SJ
authors.lastname: Lee
authors.affiliation: Department of Laboratory Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Soo Hyun
authors.initials: SH
authors.lastname: Seo
authors.affiliation: Department of Laboratory Medicine, Gyeongsang National University Hospital, Jinju, Republic of Korea.
authors.forename: Hyunwoong
authors.initials: H
authors.lastname: Park
authors.affiliation: Department of Pediatrics, Ewha Womans University School of Medicine, Seoul, Republic of Korea.
authors.forename: Anna
authors.initials: A
authors.lastname: Cho
authors.affiliation: Department of Laboratory Medicine, National Medical Center, Seoul, Republic of Korea.
authors.forename: So Yeon
authors.initials: SY
authors.lastname: Kim
authors.affiliation: Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea.
authors.forename: Ji Yeon
authors.initials: JY
authors.lastname: Kim
authors.affiliation: Department of Laboratory Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Sung Sup
authors.initials: SS
authors.lastname: Park
authors.affiliation: Department of Laboratory Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea. Electronic address: mwseong@snu.ac.kr.
authors.forename: Moon-Woo
authors.initials: MW
authors.lastname: Seong
mesh_terms: D015342:DNA Probes / Q000235:genetics; D016189:Dystrophin / Q000235:genetics; D005091:Exons; D020440:Gene Duplication; D015321:Gene Rearrangement; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D020388:Muscular Dystrophy, Duchenne / Q000175:diagnosis / Q000235:genetics*; D017384:Sequence Deletion
chemical_list: C484258:DMD protein, human; D015342:DNA Probes; D016189:Dystrophin