遗传性胆红素代谢障碍与胆汁淤积NGS方法的建立及应用

doi:10.19586/j.2095-2341.2022.0128

生物技术进展 ›› 2022, Vol. 12 ›› Issue (4): 591-599.DOI: 10.19586/j.2095-2341.2022.0128

遗传性胆红素代谢障碍与胆汁淤积NGS方法的建立及应用

宋燚¹(), 贾思雨², 武丽娜³, 张伟³, 欧晓娟³, 黄坚¹()

^1.首都医科大学附属北京友谊医院临床医学研究所，北京 100050
^2.北京大学人民医院麻醉科，北京 100044
^3.首都医科大学附属北京友谊医院肝病中心，北京 100050

收稿日期:2022-06-28 接受日期:2022-07-08 出版日期:2022-07-25 发布日期:2022-08-10
通讯作者: 黄坚
作者简介:宋燚 E-mail: songmiaoxi@126.com;
基金资助:
国家自然科学基金项目(82000543);北京市医院管理中心消化内科学科协同发展中心项目(XXX0101)

Establishment and Application of NGS Method for Inherited Disorders of Hyperbilirubinemia and Cholestasis

Yi SONG¹(), Siyu JIA², Lina WU³, Wei ZHANG³, Xiaojuan OU³, Jian HUANG¹()

^1.Institute of Clinical Medicine，Beijing Friendship Hospital，Capital Medical University，Beijing 100050，China
^2.Department of Anesthesiology，Peking University People's Hospital，Beijing 100044，China
^3.Liver Research Center，Beijing Friendship Hospital，Capital Medical University，Beijing 100050，China

Received:2022-06-28 Accepted:2022-07-08 Online:2022-07-25 Published:2022-08-10
Contact: Jian HUANG

摘要/Abstract

摘要：

为快速准确、低成本、高通量地检测我国人群常见的遗传性胆红素代谢障碍及胆汁淤积综合征，选择了10个易感基因的全部外显子及内含子剪切区的SNP/CNV，建立了基于二代测序技术（next generation sequencing, NGS）的靶向捕获测序方法。通过6例已知突变位点的样本对该方法的准确性进行验证，准确率为100%。收集首都医科大学附属北京友谊医院遗传性胆红素代谢障碍及胆汁淤积综合征患者39例进行检测，共检测到58种突变。检测结果与HGMD、ClinVar、OMIM突变数据库比较，未报道的突变通过千人基因组数据集对比并按照哈温平衡检验（HWE_P＞0.05）和χ^２ 检验确定新突变19种。检测到的不同突变类型有效地揭示了该类疾病的遗传多样性。NGS方法的建立及应用为临床诊断提供了新的技术手段。

关键词: 遗传性胆红素代谢障碍, 胆汁淤积, 二代测序技术, 靶向捕获

Abstract:

In order to detect the common inherited disorders of hyperbilirubinemia and cholestasis syndrome in Chinese population more quickly， accurately， at lower cost and in higher throughput， the SNPs/CNVs of all exons and introns of ten susceptible genes were selected， and a targeted capture sequencing method based on next-generation sequencing （NGS） technology was established. The accuracy of this method was verified by six samples of known mutation sites， and the accuracy rate was 100%. The 39 patients with inherited disorders of hyperbilirubinemia and cholestasis syndrome were collected from Beijing Friendship Hospital， Capital Medical University. A total of 58 mutations were detected. The test results were compared with HGMD， ClinVar and OMIM mutation databases. The unreported mutations were compared using the international genome sample resource and according to the Hardy-Weinberg equilibrium （HWE_P>0.05） and chi-square test. 19 new mutations were identified. The different mutation types detected effectively reveal the genetic diversity of this disease. The establishment and application of NGS method provides a new technical means for clinical diagnosis.

Key words: inherited disorders of hyperbilirubinemia, cholestasis, next-generation sequencing technology, targeted capture

中图分类号:

Q789

宋燚, 贾思雨, 武丽娜, 张伟, 欧晓娟, 黄坚. 遗传性胆红素代谢障碍与胆汁淤积NGS方法的建立及应用[J]. 生物技术进展, 2022, 12(4): 591-599.

Yi SONG, Siyu JIA, Lina WU, Wei ZHANG, Xiaojuan OU, Jian HUANG. Establishment and Application of NGS Method for Inherited Disorders of Hyperbilirubinemia and Cholestasis[J]. Current Biotechnology, 2022, 12(4): 591-599.

图/表 7

参考文献 20

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易感基因	疾病信息	目标区域类型	起始位置	终止位置	目标区域探针个数
ATP8B1	良性复发型肝内胆汁淤积，进行性家族性肝内胆汁淤积症1型	CDS+splice region	55315698	55399065	136
ABCB11	进行性家族性肝内胆汁淤积症2型	CDS+splice region	169780116	169874654	124
ABCB4	进行性家族性肝内胆汁淤积症 3型	CDS+splice region	87031398	87104818	149
UGT1A1	Gilbert综合征和Crigler-Najjar综合征	CDS+splice region	234668914	234681236	54
JAG1	Alagille综合征	CDS+splice region	10620125	10654270	148
ABCC2	Dubin-Johnson综合征	CDS+splice region	101542567	101611439	165
SLCO1B1	Rotor综合征	CDS+splice region	21294434	21392178	87
SLCO1B3	Rotor综合征	CDS+splice region	20968592	21069206	95
SLC25A13	Citrin 缺乏症	CDS+splice region	95750479	95951328	82
SERPINA1	α1-抗胰蛋白酶缺乏症	CDS+splice region	94844765	94849630	44

易感基因	疾病信息	目标区域类型	起始位置	终止位置	目标区域探针个数
ATP8B1	良性复发型肝内胆汁淤积，进行性家族性肝内胆汁淤积症1型	CDS+splice region	55315698	55399065	136
ABCB11	进行性家族性肝内胆汁淤积症2型	CDS+splice region	169780116	169874654	124
ABCB4	进行性家族性肝内胆汁淤积症 3型	CDS+splice region	87031398	87104818	149
UGT1A1	Gilbert综合征和Crigler-Najjar综合征	CDS+splice region	234668914	234681236	54
JAG1	Alagille综合征	CDS+splice region	10620125	10654270	148
ABCC2	Dubin-Johnson综合征	CDS+splice region	101542567	101611439	165
SLCO1B1	Rotor综合征	CDS+splice region	21294434	21392178	87
SLCO1B3	Rotor综合征	CDS+splice region	20968592	21069206	95
SLC25A13	Citrin 缺乏症	CDS+splice region	95750479	95951328	82
SERPINA1	α1-抗胰蛋白酶缺乏症	CDS+splice region	94844765	94849630	44

项目	测量值
平均总得率/Mb	208.12
平均总读长/bp	149
平均碱基质量	36.4
平均 Q30 碱基数百分比/%	96.07
平均 Q20 碱基数百分比/%	98.47
平均总基因组比对后碱基数/Mb	198.52
平均基因组比对率/%	99.63
比对到目标区域上的碱基数/Mb	181.8
比对到目标上的平均比例/%	99.31
平均30X覆盖度	100
平均碱基捕获率	86.34
目标区域平均深度（以总目标区域为分母）	184.2

项目	测量值
平均总得率/Mb	208.12
平均总读长/bp	149
平均碱基质量	36.4
平均 Q30 碱基数百分比/%	96.07
平均 Q20 碱基数百分比/%	98.47
平均总基因组比对后碱基数/Mb	198.52
平均基因组比对率/%	99.63
比对到目标区域上的碱基数/Mb	181.8
比对到目标上的平均比例/%	99.31
平均30X覆盖度	100
平均碱基捕获率	86.34
目标区域平均深度（以总目标区域为分母）	184.2

易感基因	染色体定位	突变形式	突变类型	该突变/总例数	正常人中频率
ATP8B1	chr18:55342156	c.1729A>G(p.I577V)	错义突变	1/39（0.026）	0.005
	chr18:55336626	c.2021T>C(p.M674T)	错义突变	1/39（0.026）	0.005
	chr18:55362421	c.922G>A(p.G308S)	错义突变	1/39（0.026）	4.061e-06
	chr18:55317591	c.3531+8G>T	剪接突变	3/39（0.0769）	4.0e-06
ABCB11	chr2:169830328	c.1331T>C.(p.V444A)	错义突变	16/39（0.410）	0.569
	chr2:169801131	c.2594C>T(p.A865V)	错义突变	2/39（0.051）	0.001
	chr2:169851971	c.499G>A(p.A167T)	错义突变	1/39（0.026）	8.186e-06
	chr2:169814596	c.2221G>A(p.V741I)	错义突变	1/39（0.026）	8.173e-06
	chr2:169820817	c.2077G>C(p.A693P)	错义突变	1/39（0.026）	1.792e-05
ABCB4	chr7:87069639	c.1436C>T(p.P479L)	错义突变	1/39（0.026）	8.13e-06
	chr7:87056178	c.1952C>A(p.T651N)	错义突变	1/39（0.026）	0.001
	chr7:87056176	c.1954A>G(p.R652G)	错义突变	1/39（0.026）	0.099
UGT1A1	chr2:234669144	c.211G>A(p.G71R)	错义突变	15/39（0.385）	0.022
	chr2:234669268	c.335C>G(p.T112R)	错义突变	1/39（0.026）	1.625e-05
	chr2:234676979	c.1198A>C(p.N400H)	错义突变	1/39（0.026）	4.102e-06
	chr2:234681059	c.1456T>G(p.Y486D)	错义突变	4/39（0.103）	0.000 2
	chr2:234676872	c.1082C>T(p.P364L)	错义突变	4/39（0.103）	0.001
JAG1	chr20:10639284	c.526G>A(p.V176I)	错义突变	2/39（0.0513）	0.000 4
JAG1	chr20:10622501	c.2612C>G(p.P871R)	错义突变	1/39（0.026）	0.064
ABCC2	chr10:101572833	c.2026G>C(p.G676R)	错义突变	1/39（0.026）	1.5e-05
	chr10:101571331	c.1939G>T(p.E647X)	翻译终止	1/39（0.026）	4.067e-06
	chr10:101578849	c.2443C>T(p.R815X)	翻译终止	2/39（0.051）	3.657e-05
	chr10:101603639	c.3825C>G(p.Y1275X)	翻译终止	1/39（0.026）	6.497e-05
SLCO1B1	chr12:21375289	c.1738C>T(p.R580X)	翻译终止	4/39（0.103）	0.002
	chr12:21329738	c.388A>G(p.N130D)	错义突变	17/39（0.436）	0.472
	chr12:21331549	c.521T>C(p.V174A)	错义突变	8/39（0.205）	0.133
	chr12:21329802	c.452A>G(p.N151S)	错义突变	1/39（0.026）	0.000 3
	chr12:21327652	c.359+9_359+10insA	剪接突变	4/39（0.103）	4.0e-06
SLCO1B3	chr12:21011480	c.334T>G(p.S84A)	错义突变	18/39（0.462）	0.809 4
	chr12:20968666-chr12:20968669	c.-7_-4del	剪接突变	1/39（0.026）	2.1e-04
	chr12:21015760	c.699G>A(p.M205I)	错义突变	18/39（0.462）	0.809
	chr12:21015372-21015373	c.508_509del(p.M142fs)	移码突变	1/39（0.026）	3.659e-05
	chr12:21011481	c.335C>A(p.S84Y)	错义突变	4/39（0.103）	0.001
	chr12:21013948	c.360-3C>T	剪接突变	6/39（0.1538）	0.002 3
SERPINA1	chr14:94844843	c.1200A>C(p.E400D)	错义突变	10/39（0.256）	0.278 0
	chr14:94849201	c.374G>A(p.R125H)	错义突变	4/39（0.103）	0.160
	chr14:94847415	c.710T>C(p.V237A)	错义突变	1/39（0.026）	0.209
	chr14:94844968	c.1075A>G(p.K359E)	错义突变	1/39（0.026）	0.0001
SLC25A13	chr7:95864108	c.328+6A>G	剪接突变	7/39（0.179）	0.258

遗传性胆红素代谢障碍与胆汁淤积NGS方法的建立及应用

Establishment and Application of NGS Method for Inherited Disorders of Hyperbilirubinemia and Cholestasis

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易感基因	染色体定位	突变形式	突变类型
ATP8B1	chr18:55328438	c.2675G>A(p.G892E)	错义突变
ABCB11	chr2:169825967	c.1904T>A(p.V635E)	错义突变
ABCB11	chr2:169788962	c.3138A>G(p.I1046M)	错义突变
ABCB4	chr7:87069062	c.1652C>T(p.P551L)	错义突变
ABCB4	chr7:87046787	c.2523C>A(p.N841K)	错义突变
UGT1A1	chr2:234669301	c.368T>G(p.L123R)	错义突变
	chr2:234680996	c.1393_1394insTGA:p.V465delinsVM	非移码插入
	chr2:234676937	c.1156G>T(p.V386F)	错义突变
ABCC2	chr10:101571317	c.1926del:p.(Ala642Alafs*16)	移码突变
	chr10:101578706	c.2433del:p.(Lys811Lysfs*22)	移码突变
	chr10:101595969	c.3536del:p.(Gln1179Argfs*5)	移码突变
	chr10:101591889-101591889	c.3258+1G>A	剪接突变
	chr10:101557089	c.867+1_867+2ins	剪接突变
	chr10:101572870	c.2063T>C(p.M688T)	错义突变
	chr10:101578592	c.2317A>G(p.R773G)	错义突变
SLCO1B1	chr12:21032460	c.1226A>G:p.(Lys409Arg)	错义突变
SLCO1B3	chr12:21033881	c.1424G>A(p.G475E)	错义突变
SLC25A13	chr7:95822344	c.615+5G>A	剪接突变
SLC25A13	chr7:95818885	c.848+8G>T	剪接突变

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