MLPA技术在3种遗传性疾病基因诊断中的应用

Application of MLPA technology in molecular diagnosis of three inherited diseases

目的评估多重连接探针扩增技术（MLPA）在脊髓性肌肉萎缩症（SMA）、DiGeorge综合征和先天性肾上腺皮质增生症（CAH）基因诊断中的应用价值。方法采集2010年2月至2011年2月期间，上海儿童医学中心门诊21例确诊为SMA、DiGeorge综合征和CAH患者及家系成员（3例SMA先证者和家系成员6人、DiGeorge综合征4例、CAH患者8例）外周血并抽提基因组DNA。通过探针和靶序列DNA杂交、连接、PCR扩增，产物经毛细管电泳分离及GeneMarker~分析软件收集分析数据，建立MLPA技术，检测SMA、DiGeorge综合征和CAH患者及携带者致病基因拷贝数，并通过传统PCR酶切法和染色体芯片分析技术验证部分MLPA检测结果。结果用MLPA技术检测3个SMA家系3例先证者SMNl基因第7和8号外显子均为0拷贝（纯合缺失），其父母均为该区域杂合缺失携带者，用传统酶切分析SMN基因扩增产物证实MLPA结果准确可靠；4例DiGeorge综合征患者中，3例患者22q11．2区域为0．5拷贝左右（杂合缺失），缺失基因均包含Tbxl等，用染色体芯片技术分析其中1例患者全基因组拷贝数变异为22q11．2区域的1．3Mb缺失，结果与MLPA检出的拷贝数变异相符；8例CAH患者中，6例存在21-羟化酶的编码基因（CYP21A2）及相关基因CAB和CYP21A1P的不同程度杂合缺失。结论MLPA技术作为传统检测拷贝数变异技术的补充，可有效提高相对定量检测SMA、DiGeorge综合征和CAH患者及携带者致病基因拷贝数变异。

Objective To evaluate multiplex ligation-dependent probe amplification （MLPA） as a senetic diagnostic method to detect copy number variants （CNVs） in SMA, DiGeorge syndrome and CAH. Methods Genomic DNA was extracted from peripheral blood of 21 patients and their family members received in Shanghai Children＇s Medical Centre during February 2010 to February 2011. The procedure of MLPA includes hybridization of probe and target sequence, ligation, PCR amplification, capillary electrophoresis and data collection. MPLA data was analyzed by GeneMarker software. The MLPA results were compared with conventional PCR-enzyme method and chromosomal microarray analysis. Results All of the 3 probands in SMA pedigrees suffer homozygous loss of exon 7 and 8 of SMN1 gene （ copy number state was zero）, and their parents have heterozygous loss on exon 7 and exon 8, respectively. Heterozygous deletions encompassing TBX1 were detected in 3 DiGeorge syndrome patients （copy number state ~0. 5）. The result of MLPA was confirmed by genomie microarray analysis, and the variant was scaled as a 1.3 Mb deletion. Various deletions including pathogenetic gene CYP21A2, the pseudogene CYP21A1P and module gene C4B were detected in six of eight CAH patients. Conclusions As a complement and enhancement ofconventional methods for detecting CNVs. MLPA is effective in relative quantitative detection of copy number variations of pathogenic gene in SMA, DiGeorge syndrome and CAH patients and carriers.