多重连接依赖的探针扩增技术在Dystrophin基因外显子拷贝数异常临床检测中的研究  

Clinical application of multiplex ligation-dependent probe amplification for the detection exonic copy number alterations in the Dystrophin gene

作　　者：龙飞[1] 孙维[1] 季星[1] 李西华[1] 刘晓青[1] 蒋雯婷[1] 陶炯[1] 

机构地区：[1]上海交通大学医学院附属新华医院上海市儿科医学研究所,200092

出　　处：《中华医学遗传学杂志》2011年第6期699-704,共6页Chinese Journal of Medical Genetics

基　　金：上海市自然科学基金（09ZRl425600）;上海市卫生局联合攻关重大项目（2008ZD004）

摘　　要：目的阐明多重连接依赖的探针扩增技术（multiplexligation—dependentprobeamplification，MLPA）在Dystrophin基因外显子拷贝数异常检测中的优势和局限，探讨MLPA单外显子异常结果的处理方法。方法应用MLPA对70例确诊的Duchenne／Becker肌营养不良（Duchenne／Beckermusculardystrophy，DMD／BMD）患者进行Dystrophin基因外显子缺失及扩增的检测。对于单外显子异常结果通过PCR、测序和荧光定量PCR进行进一步验证。结果在70例样本中，MLPA检出外显子缺失42例（60％），其中12例为单一外显子缺失，1例为单一外显子可疑缺失；检出外显子扩增7例（10％），其中2例为单一外显子扩增；21例样本未检出外显子拷贝数异常（30％）。在12例MLPA提示的单一外显子缺失中，11例PCR验证结果与MLPA一致，1例为微缺失突变（c．4470—4471delAA）；在1例MI。PA提示单外显子疑似缺失，经测序确定为既往未报道的微缺失突变（c．4746—4747delCT）；在2例MLPA提示单一外显子扩增的样本荧光定量PCR结果与MLPA一致。结论MI。PA技术应成为Dystrophin基因外显子拷贝数异常的首选检测方法；MLPA提示的单外显子异常结果必须结合其他检测方法进行进一步验证。Objective To clarify advantages and disadvantages of using multiplex ligation-dependent probe amplification （MLPA） for detecting exonic deletions and duplications of the Dystrophin gene, and to explore the appropriate management for single-exon abnormality detected by MLPA. Methods MLPA were performed to detect exonic copy number changes in 70 Duchenne/Becker muscular dystrophy （DMD/BMD） patients diagnosed by clinical and histological findings. PCR, DNA sequencing and real-time PCR were applied to the samples in which MLPA indicated single-exon deletion or duplication. Results Of all 70 patients, MLPA detected exonic deletions in 42 （60 % ）, including 12 with single-exon deletion and one with ambiguous single-exon deletion. Exon duplications were found in 7 patients （10~）, among which two were single-exon duplication. 21 patients showed normal results （30~/oo）. For the 12 patients with single-exon deletion, MLPA results were confirmed by PCR in 11. In one patient, a deletion of two nucleotides （c. 4470-4471delAA） was found by sequencing. A novel two-nucleotide deletion （c. 4746-4747delCT） was identified in the patient with the ambiguous single-exon deletion. For the two patients showed single-exon duplication, MLPA results were confirmed by real-time PCR. Conclusion MLPA should be the first choice in detecting Dystrophin gene exon deletions and duplications. Single-exon deletion/duplication resulted from MLPA should be further evaluated by other methods.

关 键 词：Duchenne/Becker肌营养不良 多重连接依赖的探针扩增技术 DYSTROPHIN基因 

分 类 号：R746.2[医药卫生—神经病学与精神病学]