Department of Physiological Sciences, Faculty of Medicine, Pontificia Universidad Javeriana, Bogotá, Colombia.
Human Genetics Institute, Faculty of Medicine, Pontificia Universidad Javeriana, Bogotá, Colombia.
J Appl Lab Med. 2023 May 4;8(3):469-478. doi: 10.1093/jalm/jfac136.
The MLPA (multiplex ligation dependent probe amplification) technique is currently the test most widely used to detect mutations in the Duchenne/Becker muscular dystrophy (DMD) gene in the initial assessment. However, several studies have suggested that MLPA results require implementing other detection methods due to false duplication. Our aim was to evaluate variables that could alter the peak ratio in MLPA in individuals with Duchenne/Becker muscular dystrophy (DMD/BMD) who present sequence variants at the probe hybridization site, such as the location of sequence variants (SVs), melting temperature of the probe, and the type of variant.
We analyzed patients with clinical suspicion of DMD/BMD through the MLPA technique. The DMD gene was sequenced in patients with normal results in MLPA.
Of 111 patients, 72 had an abnormal MLPA result, of which 10 had a single exon abnormal peak, and 39 had a normal peak ratio. Out of 10 patients, 4 (40%) with a single exon abnormal peak ratio had SV at the hybridization site of the probe. In the other 6, the deletion was confirmed. Out of 39 patients with a normal peak ratio, 11 presented SVs at the hybridization site of the probe, and DMD/BMD was confirmed.
In cases of abnormal peak ratio results of MLPA in a single exon, it would be valuable to sequence the DMD gene to assess whether variants in the probe hybridization site might result in a false positive that could be interpreted as an exon deletion.
MLPA（多重连接依赖探针扩增）技术是目前在初始评估中检测杜氏/贝克肌营养不良症（DMD）基因突变最广泛使用的测试方法。然而，几项研究表明，由于假阳性重复，MLPA 结果需要实施其他检测方法。我们的目的是评估在 DMD/BMD 患者中，位于探针杂交位点的序列变异（SVs）、探针的熔点和变异类型等因素可能改变 MLPA 中峰比的变量。
我们通过 MLPA 技术分析了具有 DMD/BMD 临床疑似症状的患者。在 MLPA 结果正常的患者中对 DMD 基因进行了测序。
在 111 名患者中，72 名患者的 MLPA 结果异常，其中 10 名患者的单个外显子异常峰，39 名患者的峰比正常。在 10 名患者中，4 名（40%）具有单个外显子异常峰比的患者在探针杂交位点存在 SV。在另外 6 名患者中，确认存在缺失。在 39 名峰比正常的患者中，11 名患者在探针杂交位点存在 SV，且确认了 DMD/BMD。
在 MLPA 单个外显子异常峰比结果的情况下，对 DMD 基因进行测序以评估探针杂交位点的变异是否可能导致假阳性，从而被解释为外显子缺失，这将是有价值的。
