CAJ | 학술논문

目的阐明多重连接依赖的探针扩增技术(multiplex ligation-dependent probe amplification,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/Becker musculardystrophy,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MLPA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 MLPA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.
목적 천명다중련접의뢰적탐침확증기술(multiplex ligation-dependent probe amplification,MLPA)재Dystrophin기인외현자고패수이상검측중적우세화국한,탐토MLPA단외현자이상결과적처리방법.방법 응용MLPA대70례학진적Duchenne/Becker기영양불량(Duchenne/Becker musculardystrophy,DMD/BMD)환자진행Dystrophin기인외현자결실급확증적검측.대우단외현자이상결과통과PCR、측서화형광정량PCR진행진일보험증.결과 재70례양본중,MLPA검출외현자결실42례(60％),기중12례위단일외현자결실,1례위단일외현자가의결실；검출외현자확증7례(10％),기중2례위단일외현자확증；21례양본미검출외현자고패수이상(30％).재12례MLPA제시적단일외현자결실중,11례PCR험증결과여MLPA일치,1례위미결실돌변(c.4470-4471delAA)；재1례MLPA제시단외현자의사결실,경측서학정위기왕미보도적미결실돌변(c.4746-4747delCT)；재2례MLPA제시단일외현자확증적양본형광정량PCR결과여MLPA일치.결론 MLPA기술응성위Dystrophin기인외현자고패수이상적수선검측방법;MLPA제시적단외현자이상결과필수결합기타검측방법진행진일보험증.
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 70patients,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％).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.