耳聋基因筛查阴性和仅携带GJB2、SLC26A4单一杂合变异耳聋患儿测序分析

doi:10.3969/j.issn.1673-8640.2024.12.012

摘要/Abstract

摘要：

目的了解江西地区耳聋患儿4个致聋基因9个常规筛查变异位点（GJB2基因的35delG、235delC、176del16bp、299-300delAT，SLC26A4基因的919-2A>G、2168 A>G，线粒体12S rRNA基因的1494C>T、1555A>G，GJB3基因的538C>T）以外的变异携带情况。方法选取2018年1月1日—2021年9月30日江西省儿童医院耳聋基因筛查结果为阴性或仅携带GJB2和/或SLC26A4单一杂合变异的耳聋患儿118例。采用多重聚合酶链反应（PCR）扩增捕获技术检测耳聋基因数据库中GJB2、SLC26A4、12S rRNA基因所有已知的致病性位点，并分析变异位点的规律。结果 118例耳聋患儿中共检出其他变异位点19个，其中GJB2基因2个、SLC26A4基因16个、线粒体12S rRNA基因1个。有32例患儿检出GJB2基因c.109G>A位点变异，其中纯合子20例；检出GJB2 c.139G>T杂合突变1例。在检出的SLC26A4基因16个其他致病性变异位点中，c.1229C>T检出3次，c.916dup、c.1226G>A、c.1905G>A各检出2次，c.2T>G、c.84C>A、c.349del、c.757A>G、c.1079C>T、c.1174A>T、c.1286C>A、c.1692dup、c.1707+5G>A、c.1983C>A、c.2000T>C和c.2162C>T各检出1次。检出线粒体12S rRNA基因m.1027A>G变异位点携带者1例。结论江西省耳聋患儿除9个常规筛查变异位点以外，以GJB2基因c.109 G>A变异位点检出率最高；SLC26A4基因其他变异位点检出数量多，且分布分散，c.1229C>T、c.1226G>A、c.916dup、c.1905G>A的检出率较高。多重PCR扩增捕获技术能有效提高耳聋患儿基因变异位点检出率。

关键词: 耳聋基因, GJB2, SLC26A4, 12S rRNA, 多重聚合酶链反应扩增捕获技术

Abstract:

Objective To investigate the carrier status of hearing loss gene mutations beyond the 9 commonly screened variants (GJB2: 35delG,235delC, 176del16bp, 299-300delAT; SLC26A4: 919-2A>G,2168A>G;12S rRNA:1494C>T,1555A>G; GJB3: 538C>T) among pediatric hearing loss patients in Jiangxi, China. Methods A total of 118 pediatric patients diagnosed with hearing impairment at Jiangxi Provincial Children's Hospital between January 1, 2018 and September 30, 2021 were screened. Patients who tested negative for the 9 variants or carried a single heterozygous mutation in GJB2 and/or SLC26A4 were included. Multiplex polymerase chain reaction (PCR) amplification and capture techniques were employed to detect all known pathogenic variants in GJB2, SLC26A4 and 12S rRNA genes. Variant patterns were analyzed. Results Totally, 19 additional variants were detected among 118 patients, including 2 in the GJB2 gene, 16 in the SLC26A4 gene, and 1 in the 12S rRNA gene. Among these patients, 32 cases exhibited the GJB2 c.109G>A variant, with 20 in homozygous form. Additionally, one presented with the GJB2 c.139G>T heterozygous variant. Among the 16 other pathogenic variants detected in the SLC26A4 gene, the c.1229C>T variant appeared 3 times, while c.916dup, c.1226G>A and c.1905G>A were detected twice. The remaining variants (c.2T>G, c.84C>A, c.349del, c.757A>G, c.1079C>T, c.1174A>T, c.1286C>A, c.1692dup, c.1707+5G>A, c.1983C>A, c.2000T>C and c.2162C>T) were identified once. One case carried the m.1027A>G variant in the mitochondrial 12S rRNA gene. Conclusions In addition to the 9 commonly screened mutations, the GJB2 c.109G>A variant shows the highest detection rate in pediatric hearing impairment patients in Jiangxi. A diverse range of additional SLC26A4 gene variants is identified, with c.1229C>T, c.1226G>A, c.916dup and c.1905G>A occurring at relatively high frequencies and warranting further attention. The use of multiplex PCR amplification and capture techniques significantly improves the detection rate of gene variants in pediatric hearing impairment patients.

Key words: Hearing loss gene, GJB2, SLC26A4, 12S rRNA, Multiplex polymerase chain reaction amplification and capture technique

中图分类号:

R446.5

刘发娣, 乐萍, 吴英龙, 刘会华, 易婷, 付晶晶, 柯江维. 耳聋基因筛查阴性和仅携带GJB2、SLC26A4单一杂合变异耳聋患儿测序分析[J]. 检验医学, 2024, 39(12): 1202-1207.

LIU Fadi, LE Ping, WU Yinglong, LIU Huihua, YI Ting, FU Jingjing, KE Jiangwei. Sequencing analysis of hearing-impaired children with negative hearing loss gene screening results and carrying only a single heterozygous variant in GJB2 or SLC26A4[J]. Laboratory Medicine, 2024, 39(12): 1202-1207.

图/表 2

参考文献 21

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基因名称	正向引物序列（5'~3'）	反向引物序列（5'~3'）	引物大小/bp
GJB2
T1P1	TCTTAATCTAACAACTGGGCAATGC	CTTCTATGTCATGTACGACGGCT	249
T1P2	CGGACCTTCTGGGTTTTGATCTC	GATGAGCAGGCCGACTTTGT	245
T1P3	CAAGGACGTGTGTTGGTCCA	AGAGACCCCAACGCCGA	183
SLC26A4
T1P4	GTCCTCCCTCCTCGCTGT	GCTTCTCTCTACGCAGGCC	232
T1P5	CTGCATACTGTAACTTTGGTTTGTGA	TGCACCAACCTAATAGAGGTATAATGC	255
T1P6	GGACTTTCTGCATACTGTAACTTTGG	TAGCCAAAACACTTTAAACATGAGCA	227
T1P7	TGGTTTGTGAATGTAATCACTTTGCA	GTGAAAACCGCAATTACTTTTGCAC	259
T1P8	TGCAGCTAGAGATACAGCTAGAGT	GTGAGCCTTAATAAGTGGGGTCTT	258
T1P9	TGCGTGTAGCAGCAGGAAG	CCTTGTTTGTCAACCAAATAATGCTG	251
T1P10	GATGGGGAAAAAGGATGGTGGT	TCCTGTTTCCAGCCCTATAAAACC	256
T1P11	ACAAGGGAGAAGGACGAATCCT	AGAGAGACAGAGAAGGCTGTGT	260
T1P12	GGTAGTTATCACATGATGGTACCTGA	GGTATAAGGAAGCTCAGAGTGTGTT	237
T1P13	TGACTCCTTGCTAAGTAGCCAGA	TGGACCCCAGTAAATACTTGTAGAATG	258
T1P14	CAAATCTTTGACAATTAAGTTGACAGTGT	TCCAACAACGTCCAGGAAAGATAT	255
T1P15	CTCCTGAGCAAGTAACTGAATGC	GACATAATGTGACCACAGTCCCA	221
T1P16	GACAGATGAGAAGCACCAGGAA	ACCTCATCCTGGACATCAAGTTC	184
12S rRNA
T1P17	TGTTTAGACGGGCTCACATCAC	GGTTAATCACTGCTGTTTCCCG	214
T1P18	CAATAGAAGCCGGCGTAAAGAGT	TCTGGCGAGCAGTTTTGTTGA	218
T1P19	CTAGAGGAGCCTGTTCTGTAATCG	TCCACCTTCGACCCTTAAGTTTC	230
T1P20	TTAGTTGAACAGGGCCCTGAAG	TGGTAGTAAGGTGGAGTGGGTT	258
T2P15	AGCTCAAAACGCTTAGCCTAGC	TTGGGGAGGGGGTGATCTAAAA	192
T2P16	GCTTAGCCCTAAACCTCAACAGT	GTAGCCTTCATCAGGGTTTGCT	202
T2P17	GGGCTACATTTTCTACCCCAGA	CTACACTCTGGTTCGTCCAAGT	252
GJB2
T2P1	CACAAAGCAGTCCACAGTGTTG	CTACCGGAGACATGAGAAGAAGAG	259
T2P2	GAAGTAGTGATCGTAGCACACGT	GTGTGCATTCGTCTTTTCCAGAG	250
SLC26A4
T2P3	TCCTGATAGGATCGGTTGGGAA	CACTTAAGAGTCCCATCCGCAA	255
T2P4	GCAAATTGGTTGTGACTGAGATTGG	TGCTGGAGACCAGAACTCTCAA	235
T2P5	AACCCTATGCAGACACATTGAAC	ACCTGTATAATTCCAACCAGCAGA	257
T2P6	GAGAGTAGGTTTCTATCTCAGGCAA	CATCCCTGGAGCAAGAAGCAA	252
T2P7	CAGCATTATTTGGTTGACAAACAAGG	AGGAGTATCAGTGAAATGAAGCTTGT	260
T2P8	GGATCGTTGTCATCCAGTCTCTTC	CTCTGTTGCCATTCCTCGACTT	203
T2P9	CCCTGAATAACACAGCCTTCTCTG	CTCTGGAGTTCCCAAAGCACAA	251
T2P10	TGATGGGCTCTTTAGTAGCTGTTG	AGCTGCTGAAACTTCAGGCTAG	222
T2P11	AAGCTTTAGGTGCCAGGCAT	ACTGCTCTCATCAGGGAAAGGA	203
T2P12	GGATTGGAACTCTGAGCTTCCA	GTTGCAATACTGGACAACCCAC	199
T2P13	CCTAGGAACTAACAAAACATTGTGTCT	CATCTGTAGAAAGGTTGAATATTTACCG	210
T2P14	TGTTTTCCCCTTGCTTCCACA	AAGCCTAGAAGCAGTCTTAGTGC	188

基因名称	正向引物序列（5'~3'）	反向引物序列（5'~3'）	引物大小/bp
GJB2
T1P1	TCTTAATCTAACAACTGGGCAATGC	CTTCTATGTCATGTACGACGGCT	249
T1P2	CGGACCTTCTGGGTTTTGATCTC	GATGAGCAGGCCGACTTTGT	245
T1P3	CAAGGACGTGTGTTGGTCCA	AGAGACCCCAACGCCGA	183
SLC26A4
T1P4	GTCCTCCCTCCTCGCTGT	GCTTCTCTCTACGCAGGCC	232
T1P5	CTGCATACTGTAACTTTGGTTTGTGA	TGCACCAACCTAATAGAGGTATAATGC	255
T1P6	GGACTTTCTGCATACTGTAACTTTGG	TAGCCAAAACACTTTAAACATGAGCA	227
T1P7	TGGTTTGTGAATGTAATCACTTTGCA	GTGAAAACCGCAATTACTTTTGCAC	259
T1P8	TGCAGCTAGAGATACAGCTAGAGT	GTGAGCCTTAATAAGTGGGGTCTT	258
T1P9	TGCGTGTAGCAGCAGGAAG	CCTTGTTTGTCAACCAAATAATGCTG	251
T1P10	GATGGGGAAAAAGGATGGTGGT	TCCTGTTTCCAGCCCTATAAAACC	256
T1P11	ACAAGGGAGAAGGACGAATCCT	AGAGAGACAGAGAAGGCTGTGT	260
T1P12	GGTAGTTATCACATGATGGTACCTGA	GGTATAAGGAAGCTCAGAGTGTGTT	237
T1P13	TGACTCCTTGCTAAGTAGCCAGA	TGGACCCCAGTAAATACTTGTAGAATG	258
T1P14	CAAATCTTTGACAATTAAGTTGACAGTGT	TCCAACAACGTCCAGGAAAGATAT	255
T1P15	CTCCTGAGCAAGTAACTGAATGC	GACATAATGTGACCACAGTCCCA	221
T1P16	GACAGATGAGAAGCACCAGGAA	ACCTCATCCTGGACATCAAGTTC	184
12S rRNA
T1P17	TGTTTAGACGGGCTCACATCAC	GGTTAATCACTGCTGTTTCCCG	214
T1P18	CAATAGAAGCCGGCGTAAAGAGT	TCTGGCGAGCAGTTTTGTTGA	218
T1P19	CTAGAGGAGCCTGTTCTGTAATCG	TCCACCTTCGACCCTTAAGTTTC	230
T1P20	TTAGTTGAACAGGGCCCTGAAG	TGGTAGTAAGGTGGAGTGGGTT	258
T2P15	AGCTCAAAACGCTTAGCCTAGC	TTGGGGAGGGGGTGATCTAAAA	192
T2P16	GCTTAGCCCTAAACCTCAACAGT	GTAGCCTTCATCAGGGTTTGCT	202
T2P17	GGGCTACATTTTCTACCCCAGA	CTACACTCTGGTTCGTCCAAGT	252
GJB2
T2P1	CACAAAGCAGTCCACAGTGTTG	CTACCGGAGACATGAGAAGAAGAG	259
T2P2	GAAGTAGTGATCGTAGCACACGT	GTGTGCATTCGTCTTTTCCAGAG	250
SLC26A4
T2P3	TCCTGATAGGATCGGTTGGGAA	CACTTAAGAGTCCCATCCGCAA	255
T2P4	GCAAATTGGTTGTGACTGAGATTGG	TGCTGGAGACCAGAACTCTCAA	235
T2P5	AACCCTATGCAGACACATTGAAC	ACCTGTATAATTCCAACCAGCAGA	257
T2P6	GAGAGTAGGTTTCTATCTCAGGCAA	CATCCCTGGAGCAAGAAGCAA	252
T2P7	CAGCATTATTTGGTTGACAAACAAGG	AGGAGTATCAGTGAAATGAAGCTTGT	260
T2P8	GGATCGTTGTCATCCAGTCTCTTC	CTCTGTTGCCATTCCTCGACTT	203
T2P9	CCCTGAATAACACAGCCTTCTCTG	CTCTGGAGTTCCCAAAGCACAA	251
T2P10	TGATGGGCTCTTTAGTAGCTGTTG	AGCTGCTGAAACTTCAGGCTAG	222
T2P11	AAGCTTTAGGTGCCAGGCAT	ACTGCTCTCATCAGGGAAAGGA	203
T2P12	GGATTGGAACTCTGAGCTTCCA	GTTGCAATACTGGACAACCCAC	199
T2P13	CCTAGGAACTAACAAAACATTGTGTCT	CATCTGTAGAAAGGTTGAATATTTACCG	210
T2P14	TGTTTTCCCCTTGCTTCCACA	AAGCCTAGAAGCAGTCTTAGTGC	188

基因变异位点	携带者例数	等位基因频率/%	MAF^①	氨基酸改变^①	变异类型^①	致病性
基因变异位点	携带者例数	等位基因频率/%	MAF^①	氨基酸改变^①	变异类型^①	耳聋基因变异数据库	ClinVar数据库
GJB2
c.109G>A	32	22.46	0.015 380	p.Val37Ile	错义突变	致病	致病
c.139G>T	1	0.42	0.000 128	p.Glu47Ter	无义突变	致病	致病
SLC26A4
c.1229C>T	3	1.27	0.000 043	p.Thr410Met	错义突变	致病	致病
c.916dup	2	0.85	0.000 012	p.Val306fs	框移突变	致病	致病/可能致病
c.1226G>A	2	0.85	0.000 124	p.Arg409His	错义突变	致病	致病/可能致病
c.1905G>A	2	0.85	0.000 111	p.Glu635=	同义突变	致病	有争议
c.2T>G	1	0.42	0.000 006	p.Met1Arg	起始密码突变	致病	致病/可能致病
c.84C>A	1	0.42	0.000 014	p.Ser28Arg	错义突变	致病	致病/可能致病
c.349del	1	0.42	0.000 007	p.Leu117fs	框移突变	致病
c.757A>G	1	0.42	0.000 014	p.Ile253Val	错义突变	致病
c.1079C>T	1	0.42	0.000 007	p.Ala360Val	错义突变	致病	致病/可能致病
c.1174A>T	1	0.42	0.000 004	p.Asn392Tyr	错义突变	致病	致病
c.1286C>A	1	0.42	0.000 048	p.Ala429Glu	错义突变	致病	临床意义未明
c.1692dup	1	0.42	0.000 004	p.Cys565fs	框移突变	致病	致病
c.1707+5G>A	1	0.42	0.000 008		剪接区域变异	致病	致病
c.1983C>A	1	0.42	0.000 021	p.Asp661Glu	错义突变	致病	有争议
c.2000T>C	1	0.42	0.000 001	p.Phe667Ser	错义突变	致病	临床意义未明
c.2162C>T	1	0.42	0.000 029	p.Thr721Met	错义突变	致病	致病
12S rRNA
m.1027A>G	1				线粒体突变	可能致病	可能致病

基因变异位点	携带者例数	等位基因频率/%	MAF^①	氨基酸改变^①	变异类型^①	致病性
基因变异位点	携带者例数	等位基因频率/%	MAF^①	氨基酸改变^①	变异类型^①	耳聋基因变异数据库	ClinVar数据库
GJB2
c.109G>A	32	22.46	0.015 380	p.Val37Ile	错义突变	致病	致病
c.139G>T	1	0.42	0.000 128	p.Glu47Ter	无义突变	致病	致病
SLC26A4
c.1229C>T	3	1.27	0.000 043	p.Thr410Met	错义突变	致病	致病
c.916dup	2	0.85	0.000 012	p.Val306fs	框移突变	致病	致病/可能致病
c.1226G>A	2	0.85	0.000 124	p.Arg409His	错义突变	致病	致病/可能致病
c.1905G>A	2	0.85	0.000 111	p.Glu635=	同义突变	致病	有争议
c.2T>G	1	0.42	0.000 006	p.Met1Arg	起始密码突变	致病	致病/可能致病
c.84C>A	1	0.42	0.000 014	p.Ser28Arg	错义突变	致病	致病/可能致病
c.349del	1	0.42	0.000 007	p.Leu117fs	框移突变	致病
c.757A>G	1	0.42	0.000 014	p.Ile253Val	错义突变	致病
c.1079C>T	1	0.42	0.000 007	p.Ala360Val	错义突变	致病	致病/可能致病
c.1174A>T	1	0.42	0.000 004	p.Asn392Tyr	错义突变	致病	致病
c.1286C>A	1	0.42	0.000 048	p.Ala429Glu	错义突变	致病	临床意义未明
c.1692dup	1	0.42	0.000 004	p.Cys565fs	框移突变	致病	致病
c.1707+5G>A	1	0.42	0.000 008		剪接区域变异	致病	致病
c.1983C>A	1	0.42	0.000 021	p.Asp661Glu	错义突变	致病	有争议
c.2000T>C	1	0.42	0.000 001	p.Phe667Ser	错义突变	致病	临床意义未明
c.2162C>T	1	0.42	0.000 029	p.Thr721Met	错义突变	致病	致病
12S rRNA
m.1027A>G	1				线粒体突变	可能致病	可能致病