荧光PCR熔解曲线法联合Sanger测序技术在新生儿耳聋基因筛查中的应用

潘澍青; 潘婕文; 鲍幼维; 潘小莉; 庄丹燕

doi:10.16766/j.cnki.issn.1674-4152.003965

荧光PCR熔解曲线法联合Sanger测序技术在新生儿耳聋基因筛查中的应用

doi: 10.16766/j.cnki.issn.1674-4152.003965

宁波大学附属妇女儿童医院出生缺陷综合防治中心宁波市胚胎源性疾病防治重点实验室宁波市基因组医学与出生缺陷防治重点实验室，浙江宁波 315012

基金项目:

浙江省医药卫生科技计划项目 2023KY1121

宁波市医学重点扶植学科儿童保健学 2022-F26

详细信息

通讯作者:
庄丹燕，E-mail：nbsclab@126.com

中图分类号: R764.43 R446.7
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出版历程
- 收稿日期: 2024-06-11
- 网络出版日期: 2025-06-30

Application of the real-time fluorescence PCR melting curve method in gene screening for newborn hearing loss

The Central Laboratory of Birth Defects Prevention and Control, Women and Children' s Hospital Affiliated to Ningbo University, Ningbo Key Laboratory for the Prevention and Treatment of Embryogenic Diseases, Ningbo Key Laboratory of Genomic Medicine and Birth Defects Prevention, Ningbo, Zhejiang 315012, China

摘要

摘要: 目的分析荧光PCR熔解曲线法在新生儿耳聋基因筛查中的准确性，了解本地区常见耳聋基因的变异类型及频率，为今后耳聋基因筛查和耳聋防控提供参考。方法回顾性选取2022年11月—2023年10月于宁波大学附属妇女儿童医院分娩，并进行耳聋基因检测的5 563例新生儿。采用荧光PCR熔解曲线法检测4个常见耳聋基因(GJB2、SLC26A4、线粒体12S rRNA和GJB3)的15个变异位点，再采用Sanger测序验证阳性样本。结果 5 563例新生儿中，检出耳聋基因变异256例，总检出率为4.60%，其中GJB2基因变异156例，检出率为2.80%；SLC26A4基因变异73例，检出率为1.31%；线粒体12S rRNA基因变异10例，检出率为0.18%；GJB3基因变异16例，检出率为0.29%；同时检出1例复合杂合变异，检出率为0.02%。阳性样本经Sanger测序验证，发现9例结果不相符，因此，荧光PCR熔解曲线法的准确率为96.48%。结论本地区新生儿耳聋基因变异以GJB2和SLC26A4基因为主；荧光PCR熔解曲线法联合Sanger测序法可准确检出耳聋基因变异情况，有助于及早发现先天性、药物性和迟发性耳聋，对早期预防和早期治疗耳聋具有重要意义。
- 耳聋基因 /
- 基因变异 /
- Sanger测序 /
- 新生儿
Abstract: Objective To analyze the accuracy of the fluorescence PCR melting curve method in screening for newborn deafness genes, and to understand the variation types and frequencies of common deafness genes in the local population. It provides reference for gene screening and prevention of deafness in the future. Methods A retrospective analysis was conducted on 5 563 newborns and screened for deafness genes at Ningbo Women and Children' s Hospital Affiliated to Ningbo University, from November 2022 to October 2023. Firstly, the fluorescent PCR melting curve method was used to detect 15 mutation sites in four deafness genes (GJB2, SLC26A4, mitochondrial 12S rRNA, and GJB3), followed by Sanger sequencing to verify the positive samples. Results Among the 5 563 newborns, 256 cases of deafness gene mutation were detected, yielding a total detection rate of 4.60%. Mutations in the GJB2 gene were found in 156 cases (2.80%), in the SLC26A4 gene in 73 cases (1.31%), in the mitochondrial 12S rRNA gene in 10 cases (0.18%), and in the GJB3 gene in 16 cases (0.29%). Additionally, one case of complex heterozygous mutations was detected (0.02%). The positive samples were verified by Sanger sequencing, revealing 9 cases with inconsistent results. Therefore, the accuracy of the fluorescence PCR melting curve method is 96.48%. Conclusion The GJB2 gene and SLC26A4 gene are the main mutations associated with deafness in newborns in this region. Fluorescence PCR fusion curve method combined with Sanger sequencing method accurately detects genetic mutations related to deafness. This mothed is helpful for early detection of congenital, drug-induced, and delayed-onset deafness, playing an important role in the early prevention and treatment of deafness.
- Deafness gene /
- Gene mutation /
- Sanger sequencing /
- Newborn

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图 1 9例不同结果的Sanger测序图

Figure 1. Sanger sequencing images from 9 cases with varied results

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表 1 荧光PCR熔解曲线法检测结果分析

Table 1. Analysis of the detection results using fluorescence PCR melting curve method

基因	突变位点	检出人数(%)
GJB2	c.176-191del16杂合突变	6(0.11)
	c.299-300delAT杂合突变	29(0.52)
	c.235delC纯合突变	1(0.02)
	c.235delC杂合突变	119(2.14)
	c.35delG杂合突变	1(0.02)
GJB3	c.538C＞T	16(0.29)
SLC26A4	c.919-2A＞G纯合突变	1(0.02)
	c.919-2A＞G杂合突变	47(0.84)
	c.2168A＞G杂合突变	8(0.14)
	c.1174A＞T杂合突变	2(0.04)
	c.1226G＞A杂合突变	3(0.05)
	c.1229C＞T杂合突变	6(0.11)
	c.1707+5G＞A杂合突变	4(0.07)
	c.1975G＞C杂合突变	1(0.02)
	c.2027T＞A杂合突变	1(0.02)
线粒体12S rRNA	m.1494C＞T同质性突变	1(0.02)
	m.1555A＞G同质性突变	6(0.11)
	m.1555A＞G异质性突变	3(0.05)
GJB2/线粒体12S rRNA	c.235delC/m.1494C＞T同质性突变	1(0.02)
合计		256(4.60)

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表 2 Sanger测序结果分析

Table 2. Analysis of Sanger sequencing results

基因	荧光PCR熔解曲线法	Sanger测序
GJB2	c.235delC杂合突变	c.232G＞A杂合突变
	c.35delG杂合突变	c.35G＞T杂合突变
SLC26A4	c.1229C＞T杂合突变	c.1233C＞T杂合突变
	c.1229C＞T杂合突变	c.1233C＞T杂合突变
	c.1707+5G＞A	c.1707A＞G杂合突变
	c.1707+5G＞A	c.1707A＞G杂合突变
	c.1707+5G＞A	c.1707A＞G杂合突变
	c.919-2A＞G杂合突变	c.921G＞A杂合突变
	c.919-2A＞G杂合突变	c.919-2A＞G杂合突变；c.1983C＞A杂合突变

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参考文献(18)

[1]	中国耳聋基因筛查与诊断临床多中心研究协作组, 中华耳鼻咽喉头颈外科杂志编辑委员会, 中华医学会耳鼻咽喉头颈外科学分会. 中国耳聋基因诊断与遗传咨询临床实践指南(2023)[J]. 中华耳鼻咽喉头颈外科杂志, 2023, 58(1): 3-14. Chinese Multi-Center Clinical Research Collaboration Group on Genetic Screening and Diagnosis of Deafness, Editorial Board of Chinese Journal of Otolaryngology Head and Neck Surgery, Chinese Medical Association Otolaryngology Head and Neck Surgery. Clinical practice guidelines for the genetic diagnosis and counseling of hearing loss in China (2023)[J]. Chinese Journal of otolaryngology Head and Neck Surgery, 2023, 58(1): 3-14.
[2]	何红琴, 苏力, 徐佳, 等. 山西运城地区新生儿听力与耳聋基因联合筛查结果分析[J]. 中华医学遗传学杂志, 2023, 40(7): 815-820. HE H Q, SU L, XU J, et al. Analysis of combined screening of newborn hearing and deafness genes in Yuncheng area of Shanxi Province[J]. Chinese Journal of Medical Genetics, 2023, 40(7): 815-820.
[3]	FENG J, ZENG Z, LUO S, et al. Carrier frequencies, trends, and geographical distribution of hearing loss variants in China: the pooled analysis of 2 161 984 newborns[J]. Heliyon, 2024, 10(3): e24850. DOI: 10.1016/j.heliyon.2024.e24850.
[4]	董学妍, 余道军, 王贤军, 等. 遗传性耳聋基因筛查中2种耳聋基因检测方法的应用比较[J]. 中华全科医学, 2021, 19(1): 93-98. doi: 10.16766/j.cnki.issn.1674-4152.001740 DONG X Y, YU D J, WANG X J, et al. Comparison of two deafness genes detection methods in genetic hearing loss gene screening[J]. Chinese Journal of General Practice, 2021, 19(1): 93-98. doi: 10.16766/j.cnki.issn.1674-4152.001740
[5]	ZHANG C R, SHI Y P, ZHANG C X, et al. Mutation screening and functional study of SLC26A4 in Chinese patients with congenital hypothyroidism[J]. J Clin Res Pediatr Endocrinol, 2022, 14(1): 46-55. doi: 10.4274/jcrpe.galenos.2021.2021.0122
[6]	侯小娟, 杨丽, 丁伟, 等. 301例重度及极重度非综合征型耳聋患儿耳聋基因结果分析[J]. 医学研究杂志, 2024, 53(1): 157-161. HOU X J, YANG L, DING W, et al. Analysis of the results of deafness gene screening in 301 severe and very severe non syndromic deafness children[J]. Journal of Medical Research, 2024, 53(1): 157-161.
[7]	文铖, 黄丽辉, 解舒婷, 等. 中国部分地区新生儿耳聋基因筛查现况调查[J]. 临床耳鼻咽喉头颈外科杂志, 2020, 34(11): 972-977. WEN C, HUANG L H, XIE S T, et al. Current status of newborn deafness gene screeningin parts of China[J]. Journal of Clinical Otorhinolaryngology Head and Neck Surgery, 2020, 34(11): 972-977.
[8]	WEN C, YANG X, CHENG X, et al. Optimized concurrent hearing and genetic screening in Beijing, China: a cross-sectional study[J]. Biosci Trends, 2023, 17(2): 148-159. http://www.jstage.jst.go.jp/article/bst/17/2/17_2023.01051/_article/-char/en
[9]	MAO L, WANG Y, AN L, et al. Molecular mechanisms and clinical phenotypes of GJB2 missense variants[J]. Biology (Basel), 2023, 12(4): 505. DOI: 10.3390/biology12040505.
[10]	LIANG S, LI W, CHEN Z, et al. Analysis of GJB2 gene mutations spectrum and the characteristics of individuals with c. 109G>A in western Guangdong[J]. Mol Genet Genomic Med, 2023, 11(8): e2185. DOI: 10.1002/mgg3.2185.
[11]	耿国兴, 林彩娟, 黄小桃, 等. 42 708例新生儿耳聋基因筛查结果分析[J]. 中华全科医学, 2020, 18(10): 1688-1690. GENG G X, LIN C J, HUANG X T, et al. Analysis of gene screening results for 42 708 newborns with deafness[J]. 2020, 18(10): 1688-1690.
[12]	YUAN L, WANG X, LIU X, et al. Genotypic and allelic frequencies of GJB2 variants and features of hearing phenotypes in the Chinese population of the dongfeng-tongji cohort[J]. genes (Basel), 2023, 14(11): 2007. DOI: 10.3390/genes14112007.
[13]	黄丽辉, 赵雪雷, 程晓华, 等. 850例耳聋基因筛查SLC26A4单杂合突变新生儿的基因型及合并第二突变位点者表型分析[J]. 中华耳鼻咽喉头颈外科杂志, 2023, 58(2): 117-125. HUANG L H, ZHAO X L, CHENG X H, et al. Analysis of genotypes on 850 newborns with SLC26A4 single-allele mutation and the phenotypes of those with second variant[J]. Chinese Journal of Otorhinolaryngology Head and Neck Surgery, 2023, 58(2): 117-125.
[14]	肖艳荣, 管明凤, 陆鸿略. 苏州地区新生儿听力与耳聋基因联合筛查结果分析[J]. 中国听力语言康复科学杂志, 2023, 21(4): 390-393. XIAO Y R, GUAN M F, LU H L. Analysis of combined screening results of hearing and deafness gene in Suzhou area[J]. Chinese Scientific Journal of Hearing and Speech Rehabilitation, 2023, 21(4): 390-393.
[15]	邹凌, 蔡娟, 杨馨婷, 等. 成都地区471 994例新生儿耳聋基因筛查突变情况分析[J]. 中华耳科学杂志, 2021, 19(4): 608-615. ZOU L, CAI J, YANG X T, et al. Deafness gene mutation identified through screening among 471 994 newborns in Chengdu[J]. Chinese Journal of Otology, 2021, 19(4): 608-615.
[16]	邱小兵, 黄俊高, 陈俊坤, 等. 122, 635例赣南地区新生儿耳聋基因筛查及确诊者结果回溯分析[J]. 中华耳科学杂志, 2022, 20(3): 402-408. QIU X B, HUANG J G, CHEN J K, et al. A retrospective study of deafness gene screening and confirmed deafness among 122635 newborns in southern Jiangxi[J]. Chinese Journal of Otology, 2022, 20(3): 402-408.
[17]	王智慧, 吴洁丽, 项延包. 温州地区3 620例孕妇耳聋基因产前筛查及临床意义分析[J]. 浙江医学, 2023, 45(5): 460-463, 469. WANG Z H, WU J L, XIANG Y B. Prenatal screening of deafness genes in pregnant women in Wenzhou area[J]. Zhejiang Medical Journal, 2023, 45(5): 460-463, 469.
[18]	ADADEY S M, WONKAM-TINGANG E, TWUMASI ABOAGYE E, et al. Connexin genes variants associated with non-syndromic hearing impairment: a systematic review of the global burden[J]. Life(Basel), 2020, 10(11): 258. DOI: 10.3390/life10110258.