应用多重竞争性PCR联合毛细管电泳技术进行脊髓性肌萎缩症携带者筛查

doi:10.16288/j.yczz.22-042

遗传 ›› 2022, Vol. 44 ›› Issue (7): 618-628.doi: 10.16288/j.yczz.22-042

• 技术与方法 • 上一篇

应用多重竞争性PCR联合毛细管电泳技术进行脊髓性肌萎缩症携带者筛查

李烨荣(), 吕娟, 王玉国, 谭建新, 邵彬彬, 张菁菁()

南京医科大学附属妇产医院(南京市妇幼保健院)遗传医学中心, 南京 210004

收稿日期:2022-03-29 修回日期:2022-05-13 出版日期:2022-07-20 发布日期:2022-06-01
通讯作者: 张菁菁 E-mail:lyr15835364548@163.com;18913384682@163.com
作者简介:李烨荣,在读硕士研究生,专业方向：妇产科学,优生学。E-mail: lyr15835364548@163.com
基金资助:
国家自然科学基金项目资助编号(82001612)

Application of multiplex competitive PCR combined with capillary electrophoresis in carrier screening of spinal muscular atrophy

Yerong Li(), Juan Lv, Yuguo Wang, Jianxin Tan, Binbin Shao, Jingjing Zhang()

Department of Medical Genetics, Women's Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital, Nanjing 210004, China

Received:2022-03-29 Revised:2022-05-13 Online:2022-07-20 Published:2022-06-01
Contact: Zhang Jingjing E-mail:lyr15835364548@163.com;18913384682@163.com
Supported by:
Supported by the National Natural Science Foundation of China No(82001612)

摘要/Abstract

摘要：

脊髓性肌萎缩症(spinal muscular atrophy, SMA)是一种常染色体隐性遗传、儿童致死性神经系统疾病。SMA致病基因为运动神经元存活基因(survival motor neuron1, SMN1)。虽然检测SMN1基因拷贝数的方法众多,但目前适于大规模人群筛查的技术较少。为寻求一种快速准确的实验技术可以用于人群中SMA携带者的大规模筛查,了解区域人群携带情况及常见变异的分布,本研究应用多重竞争性PCR联合毛细管电泳技术检测12例SMA患者及其父母SMN1基因拷贝数,同时对江苏地区151例健康孕妇人群SMN1基因进行拷贝数检测,并通过多重连接依赖探针扩增(multiplex ligation-dependent probe amplification, MLPA)技术验证检测结果。多重竞争性PCR联合毛细管电泳技术结果与MLPA结果一致,显示12例SMA患者均为SMN1基因零拷贝,其父母的SMN1基因拷贝数均为单拷贝,151例健康人群中检测出SMN1基因单拷贝3例(即SMA携带者),占2.0%;SMN1基因双拷贝134例,占88.7%;SMN1基因大于双拷贝14例,占9.3%。因此,多重竞争性PCR联合毛细管电泳技术作为一种快速、简便和准确的检测技术具有着应用于人群中SMA携带者的大规模筛查的潜力。

关键词: 脊髓性肌萎缩症, 多重竞争性PCR联合毛细管电泳技术, SMN1基因, 携带者筛查

Abstract:

Spinal muscular atrophy (SMA) is an autosomal recessive, fatal neurological disorder in children. The pathogenic gene of SMA is survival motor neuron1 (SMN1). There are many methods to detect SMN1 gene copy number, but few techniques are suitable for large-scale population screening. In order to find a rapid and accurate experimental technique for mass screening of SMA carriers in the population, the SMN1 gene copy number of 12 SMA patients and their parents was analyzed by multiplex competitive PCR combined with capillary electrophoresis. Meanwhile, the copy number of SMN1 gene in 151 healthy pregnant women in Jiangsu was screened with the MLPA technology to confirm their copy number of the SMN genes. The results showed that the 12 SMA patients had 0 copy of SMN1 gene, and all their parents had 1 copy of SMN1 gene only. Among 151 healthy subjects, 3 cases (2.0%) had 1 copy of SMN1 gene, and hence designated as SMA carriers. One hundred and thirty-four cases (88.7%) had 2 copies of the SMN1 gene. There were 14 cases (9.3%) with more than 2 copies of the SMN1 gene. Therefore, multiplex competitive PCR combined with capillary electrophoresis is a rapid, simple and accurate method for the detection of SMA carriers; and potentially applicable to mass screening of SMA carriers in the population.

Key words: spinal muscular atrophy, multiplex competitive PCR combined with capillary electrophoresis, SMN1 gene, carrier screening

李烨荣, 吕娟, 王玉国, 谭建新, 邵彬彬, 张菁菁. 应用多重竞争性PCR联合毛细管电泳技术进行脊髓性肌萎缩症携带者筛查[J]. 遗传, 2022, 44(7): 618-628.

Yerong Li, Juan Lv, Yuguo Wang, Jianxin Tan, Binbin Shao, Jingjing Zhang. Application of multiplex competitive PCR combined with capillary electrophoresis in carrier screening of spinal muscular atrophy[J]. Hereditas(Beijing), 2022, 44(7): 618-628.

图/表 8

图1

表1

PCR扩增的基因片段及引物序列"

基因片段	位置(GRCh37/hg19)	荧光引物(FAM, 5′→3′)	反向引物(5′→3′)
2p	Chr.2:84500611-84500685	TTGCTTGGAAGGCAGGCAAAC	GTTTCTTTGAGCCAAAAATTCAGAATACAAGGA
20q	Chr.20:35866080-35866163	TTTTCCCACCAGAGCTTCCCTTAGC	GTTTCATGCAGAACCACCAGGAAAAGG
10p	Chr.10:31120531-31120675	TTTTTAATGCACACCTCCAGGGAAAAC	GTTTCTTCACTGAGCCCCAGAGACCTGAC
16p	Chr.16:25258133-25258312	AAAGCTATCGAAAGGTGAGAAATGG	GTTTCCCACCAAGCTGATGTGTTCCTTAG
SMN-E2a	Chr.5:69359231-69359324 70234655-70234748	ACCCTTTCCAGAGCGATGATTCT	GTTTCTTTTTTTGCATTTCATACCTTAAATGAAGCCACA
SMN-E1-US	Chr.5:69344093-69344199 70219501-70219607	TGGTCAACATCATCCCATTCTCC	GTTTCTTAACTCCACGAAAGGAACTTTGAGC
SMN-E2b	Chr.5:69361736-69361853 70237160-70237277	ACTTTTATTCTGTGCACCACCCTGT	GTTTCTTCTTTTAGGTGTGGTTTTTGGTTTACCC
SMN-E4	Chr.5:69363077-69363209 70238501-70238633	GCAGATTTGGGCTTGATGTTATCTG	GTTTCACACCCTTATAACAAAAACCTGCAT
SMN-E6	Chr.5:69366476-69366612 70241901-70242037	CCCACCACCTCCCATATGTCC	GTTTCTTAATTGTCAGGAAAAGATGCTGAGTGA
SMN-E5	Chr.5:69365098-69365262 70240521-70240685	TCTGGCCCAAGGGATGTTCT	GTTTCTTCCACCACCACCACCCCACT
NAIP-E4-II	Chr.5:70308186-70308380	TCCAGATTGTGGGTTCCTTTTGAAC	GTTTCTTAAGCCAGCCTCTGAGAGCACA
NAIP-E4-I	Chr.5:70308414-70308621	AGGAGCAGAAGGAGCGAGCA	GTTTCTTGTGAGGCCGGCACCAAAG
NAIP-E5	Chr.5:70307103-70307328	GGCCAATGAGATCGGGAGTG	GTTTGTTGGGGAACCATTTGGCATGT
SMN-E7-SMN1	Chr.5:70247563-70247802	GCAGCCTAATAATTGTTTTCTTTGGG ATAACT	GTTTCTTTTTGTGAGCACCTTCCTTCTTTTTGATTTTGTATG
SMN-E8-SMN1	Chr.5:69374299-69374557 70249719-70249977	CCACCACACTACCTGGCTGGA	GTTAATGGCAACCCTGCAGAAAAGT
SMN-E8-SMN1	Chr.5:70248466-70248731	GCCACATACGCCTCACATACATTTTG	GTTTCGGACTCTATTTTGAAAAACCATCTGTAAAAGACAGG
SMN-E1-DS-FA	Chr.5:69345517-69345796 70220935-70221214	ATCAAGGAGCCCAAACTGCTCG	GTTTCTTCGATGAGCAGCGGCGGGAA
SMN-E1-DS-FG	Chr.5:69345517-69345796 70220935-70221214	ATCAAGGAGCCCAAACTGCTCG	GTTTCTTTTTCGATGAGCAGCGGCGGAAG
SMN-E7-SMN2	Chr.5:69372143-69372382	GCAGCCTAATAATTGTTTTCTTTGGG ATAACT	GTTGTGAGCACCTTCCTTCTTTTTGATTTTGTGTA
SMN-E8-SMN2	Chr.5:69373046-69373311	GCCACATACGCCTCACATACATTTTG	GTTTCTTTTTTTGGACTCTATTTTGAAAAACCATCTGTAAAAGACGGA

表1

表2

表3

图2

图3

表4

表5

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反应体系	基因片段	扩增产物长度(bp) (竞争性DNA/待测样本DNA)	毛细管电泳参考位置(bp) (竞争性DNA/待测样本DNA)	扩增片段说明
Pane1	REF2p	79/81	76.72/78.7	参照基因片段
	REF10	150/152	153.96/155.95	参照基因片段
	REF16p	183/185	184.32/186.36	参照基因片段
	REF20q	91/93	88.76/90.64	参照基因片段
	SMN-US	111/114	108.04/111.1	SMN上游
	SMN-E2a	103/106	100.44/103.29	SMN外显子2a
	SMN-E2b	122/125	118.99/121.84	SMN外显子2b
	SMN-E4	133/136	131.33/134.22	SMN外显子4
	SMN-E5	169/172	169.19/172.3	SMN外显子5
	SMN-E6	141/144	140.12/143.58	SMN外显子6
	SMN1-E7C	249/252	249.91/252.82	SMN1外显子7(C)
	SMN-DS	159/262	260.18/262.99	SMN下游
	SMN-E8G	268/271	266.62/269.34	SMN1外显子8(G)
	NAIP-E4I	212/215	211.7/214.82	NAIP外显子4
	NAIP-E4II	199/202	198.5/201.48	NAIP外显子4
	NAIP-E5	229/232	229.21/232.29	NAIP外显子5
Panel2	REF2p	85/87	82.33/84.32	参照基因片段
	REF10p	157/159	160.54/162.5	参照基因片段
	REF16p	191/193	189.77/191.89	参照基因片段
	SMN2-E7T	249/252	242.83/245.97	SMN2外显子7(T)
	SMN-E8A	168/171	272.88/275.6	SMN2外显子8(A)

SMN 拷贝数	多重竞争性PCR
SMN 拷贝数	QC值范围	平均值±标准偏差	变异系数(%)
0	0.00~0.20	0.00±0.00	0
1	0.80~1.20	1.02±0.07	6.86
2	1.70~2.30	2.01±0.10	4.98
3	2.70~3.30	3.02±0.13	4.30
4	3.60~4.40	4.04±0.15	3.71

SMN1 拷贝数	SMN2拷贝数				合计
SMN1 拷贝数	0拷贝	1拷贝	2拷贝	3拷贝	合计
1拷贝	-	-	2	1	3(2.0%)
2拷贝	4	49	80	1	134(88.7%)
3拷贝	3	8	3	-	14(9.3%)

特点	MLPA	荧光定量PCR技术	多重竞争性PCR联合毛细管电泳技术
位点	对SMN1及SMN2等多重位点进行检测; 含多个参照位点	对SMN1单基因检测; 单参照位点	对SMN1及SMN2等多重位点进行检测;含3~4个参照位点
操作性	繁琐(操作步骤多,时间久)	中等	简单
检测周期	48 h(耗时、效率低)	3 h	4 h
准确性	高(CV约10%~20%) DNA质量不一, 波动更大	中(CV约10%~20%) 无其他位点辅助验证	中(CV约5%)有其他位点辅助验证
样本要求	高(同试剂同批次抽提、检测)	低	低
结果判读	软件判读,需要经验	标准化判读	计算公式直接得出准确拷贝数柱形图直观展示

应用多重竞争性PCR联合毛细管电泳技术进行脊髓性肌萎缩症携带者筛查

Application of multiplex competitive PCR combined with capillary electrophoresis in carrier screening of spinal muscular atrophy

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[1]	曹延延, 程苗苗, 宋昉, 瞿宇晋, 白晋丽, 金煜炜, 王红. 脊髓性肌萎缩症SMN1基因 2+0基因型携带者的家系研究[J]. 遗传, 2021, 43(2): 160-168.
[2]	陈万金, 张奇杰, 何瑾, 林翔, 王柠. 脊髓性肌萎缩症患者尿液细胞模型的建立[J]. 遗传, 2014, 36(11): 1168-1172.