屈光手术患者<italic style="font-style: italic">TGFBI</italic>突变多重qPCR检测体系的建立

顾云峰; 毛丽萍; 李小玲; 孙康轩; 江秋若; 吴文辉; 沈洋洋; 陈世豪; 郑美琴; 徐一

doi:10.1631/jzus.B2500747

Your Location：

Home >

Browse articles >

屈光手术患者TGFBI突变多重qPCR检测体系的建立

Scan QR Code

文章被引用时，请邮件提醒。

Submit

Research Article | Updated：2026-06-23

屈光手术患者TGFBI突变多重qPCR检测体系的建立
Integrative analytical and statistical framework for optimization of multiplex qPCR detection of TGFBI mutations in refractive surgery candidates
浙江大学学报（英文版）（B辑：生物医学和生物技术） 2026年27卷第6期页码：645-655
Affiliations：

1.National Clinical Research Center for Ocular Diseases, Eye Hospital, Wenzhou Medical University, Wenzhou 325027, China
2.Institute of PSI Genomics, Wenzhou Global Eye & Vision Innovation Center, Wenzhou 325024, China
3.Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325035, China
Funds：

the Wenzhou Science and Technology Plan Project, China(ZY2022018)
DOI：10.1631/jzus.B2500747
中图分类号：
CSTR：
收稿：2025-11-19，

修回：2026-03-24，

纸质出版：2026-06-15
Accepted：

顾云峰,毛丽萍,李小玲等.屈光手术患者,TGFBI突变多重qPCR检测体系的建立[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2026,27(06):645-655.

Yunfeng GU, Liping MAO, Xiaoling LI, et al. Integrative analytical and statistical framework for optimization of multiplex qPCR detection of TGFBI mutations in refractive surgery candidates[J]. Journal of Zhejiang University-SCIENCE B, 2026, 27(6): 645-655. DOI: 10.1631/jzus.B2500747.
顾云峰,毛丽萍,李小玲等.屈光手术患者,TGFBI突变多重qPCR检测体系的建立[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2026,27(06):645-655. DOI： 10.1631/jzus.B2500747.

Yunfeng GU, Liping MAO, Xiaoling LI, et al. Integrative analytical and statistical framework for optimization of multiplex qPCR detection of TGFBI mutations in refractive surgery candidates[J]. Journal of Zhejiang University-SCIENCE B, 2026, 27(6): 645-655. DOI: 10.1631/jzus.B2500747. DOI：

摘要

TGFBI

基因突变是导致角膜营养不良的重要病因，也是屈光手术术前必须排查的遗传风险因素。然而，目前仍缺乏稳定、灵敏且可标准化的多重检测体系。本研究构建了一套整合分析与统计框架，用于优化针对

TGFBI

高频突变位点的多重等位基因特异性定量聚合酶链反应（qPCR）检测方法，并系统评估其临床应用价值。通过条件优化、阈值确定、重复性与特异性验证，建立了可同时检测R124C/L/H、R555W/Q等关键突变的多重qPCR体系。统计分析结果表明，该方法扩增效率良好、重复性高且检出限低，在梯度稀释样本中表现出稳定的定量能力，且无明显非特异性扩增。受试者工作特征曲线（ROC）与概率单位（Probit）分析进一步验证了体系的准确性与可靠性，能够有效检出微量模板中突变序列。将该体系应用于大规模屈光手术候选者术前筛查，成功检出了低频

TGFBI

突变携带者，且结果与Sanger测序完全一致。上述结果证实，基于整合分析与统计优化的多重qPCR体系，能够高效、可靠地用于

TGFBI

突变筛查，为屈光手术提供标准化的术前遗传风险评估工具，也为其他遗传病位点的多重检测优化提供了可借鉴的统计与方法学框架。

Abstract

Refractive surgery can unmask or accelerate transforming growth factor-β-induced (

TGFBI

)-related corneal dystrophies that are undetectable by routine slit-lamp examination

creating a clear need for a rapid

standardized

preoperative genetic screening. We developed a multiplex

allele-specific real-time quantitative polymerase chain reaction (qPCR) panel

targeting five high-frequency

TGFBI

hotspots (R124C/L/H

R555W/Q) and built a statistics-driven analytical framework to optimize assay decisions. Receiver operating characteristic (ROC) analysis defined locus-specific cycle threshold (

) cut-offs that were harmonized to a single decision threshold (

=36) to simplify deployment. Analytical sensitivity was established by Probit modeling of serial two-fold dilutions

and confirmed by ≥20 replicates per level. In a 158-sample validation set (38 mutation-positive; 120 negative)

qPCR agreed perfectly with Sanger sequencing (Cohen’s kappa coefficient (

)=1.0). Probit analysis yielded locus-specific limit of detection (LoD) values ranging from 0.035 to 0.200 ng/µL; at 0.200 ng/µL

the detection rate was over 95%. Repeatability and intermediate precision were high (

coefficient of variation (CV) 0.34%–1.21%). No cross-reactivity was observed against non-target

TGFBI

variants or other ophthalmic genes

and interference from blood

oral flora/rinse

or toothpaste produced small

bounded shifts (approximately -7.8% to +2.8%). Calibration with serial dilutions demonstrated linear

–log(copy) relationships suitable for routine quality control. Prospective screening of 10 055 refractive surgery candidates identified six

TGFBI

carriers (0.06%) harboring R124H (including one homozygote)

R124L

R124C

or R555W mutation

all confirmed by Sanger sequencing. This study established a clinically applicable

statistically optimized multiplex qPCR platform that integrated ROC-derived cut-offs and Probit-defined LoD with rigorous evaluations of precision

specificity

and robustness

enabling large-scale population implementation. Positive screening results guide clinical decision-making through a standardized post-screening workflow

and the targeted hotspot screening strategy serves as a cost-effective

first-tier high-throughput approach for preoperative risk assessment. The framework provides a transparent

reproducible path to standardize preoperative

TGFBI

screening and reduce iatrogenic risk in refractive surgery candidates.

关键词

Keywords

references

Banning CS , Kim WC , Randleman JB , et al. , 2006 . Exacerbation of Avellino corneal dystrophy after LASIK in North America . Cornea , 25 ( 4 ): 482 - 484 . https://doi.org/10.1097/01.ico.0000195949.93695.37 https://doi.org/10.1097/01.ico.0000195949.93695.37

Bostan C , Randleman JB , 2024 . Unilateral granular type 2 corneal dystrophy with exacerbation after LASIK . Cornea , 43 ( 5 ): 648 - 651 . https://doi.org/10.1097/ICO.0000000000003490 https://doi.org/10.1097/ICO.0000000000003490

Chao-Shern C , DeDionisio LA , Jang JH , et al. , 2019 . Evaluation of TGFBI corneal dystrophy and molecular diagnostic testi ng . Eye , 33 ( 6 ): 874 - 881 . https://doi.org/10.1038/s41433-019-0346-x https://doi.org/10.1038/s41433-019-0346-x

Cho EH , Lee M , Ki CS , et al. , 2025 . Genetic epidemiology of epithelial-stromal TGFBI dystrophies in a large Korean population . Sci Rep , 15 : 25360 . https://doi.org/10.1038/s41598-025-08189-7 https://doi.org/10.1038/s41598-025-08189-7

Han KE , Choi SI , Kim TI , et al. , 2016 . Pathogenesis and treatments of TGFBI corneal dystrophies . Prog Retin Eye Res , 50 : 67 - 88 . https://doi.org/10.1016/j.preteyeres.2015.11.002 https://doi.org/10.1016/j.preteyeres.2015.11.002

Hieda O , Kobayashi A , Sotozono C , et al. , 2023 . Corneal electrolysis for granular corneal dystrophy type 2 (Avellino corneal dystrophy) exacerbation after LASIK . J Refract Surg , 39 ( 1 ): 61 - 65 . https://doi.org/10.3928/1081597X-20221129-01 https://doi.org/10.3928/1081597X-20221129-01

Holden BA , Fricke TR , Wilson DA , et al. , 2016 . Global prevalence of myopia and high myopia and temporal trends from 2000 through 2050 . Ophthalmology , 123 ( 5 ): 1036 - 1042 . https://doi.org/10.1016/j.ophtha.2016.01.006 https://doi.org/10.1016/j.ophtha.2016.01.006

International Myopia Institute , 2021 . IMI impact of myopia . Gao JH , Liu K, Chen Z, translators, Chin J Exp Ophthalmol, 39 ( 12 ): 1091 - 1103 (in Chinese) . https://doi.org/10.3760/cma.j.cn115989-20210623-00369 https://doi.org/10.3760/cma.j.cn115989-20210623-00369

Jiang X , Zhang H , 2021 . Deterioration of Avellino corneal dystrophy in a Chinese family after LASIK . Int J Ophthalmol , 14 ( 6 ): 795 - 799 . https://doi.org/10.18240/ijo.2021.06.02 https://doi.org/10.18240/ijo.2021.06.02

Kwak JJ , Yoon SH , Seo KY , et al. , 2021 . Exacerbation of granular corneal dystrophy type 2 after small incision lenticule extraction . Cornea , 40 ( 4 ): 519 - 524 . https://doi.org/10.1097/ICO.0000000000002655 https://doi.org/10.1097/ICO.0000000000002655

Li W , Qu N , Li JK , et al. , 2021 . Evaluation of the genetic variation spectrum related to corneal dystrophy in a large cohort . Front Cell Dev Biol , 9 : 632946 . https://doi.org/10.3389/fcell.2021.632946 https://doi.org/10.3389/fcell.2021.632946

Liskova P , Skalicka P , Dudakova L , et al. , 2025 . Genotype-phenotype correlations in corneal dystrophies: advances in molecular genetics and therapeutic insights . Clin Exp Ophthalmol , 53 ( 3 ): 232 - 245 . https://doi.org/10.1111/ceo.14516 https://doi.org/10.1111/ceo.14516

Munier FL , Korvatska E , Djemaï A , et al. , 1997 . Kerato-epithelin mutations in four 5q31-linked corneal dystrophies . Nat Genet , 15 ( 3 ): 247 - 251 . https://doi.org/10.1038/ng0397-247 https://doi.org/10.1038/ng0397-247

Poulsen ET , Nielsen NS , Jensen MM , et al. , 2016 . LASIK surgery of granular corneal dystrophy type 2 patients leads to accumulation and differential proteolytic processing of transforming growth factor beta-induced protein (TGFBIp) . Proteomics , 16 ( 3 ): 539 - 543 . https://doi.org/10.1002/pmic.201500287 https://doi.org/10.1002/pmic.201500287

Rocha-de-Lossada C , Rachwani-Anil R , Colmenero-Reina E , et al. , 2021 . Laser refractive surgery in corneal dystrophies . J Cataract Refract Surg , 47 ( 5 ): 662 - 670 . https://doi.org/10.1097/j.jcrs.0000000000000468 https://doi.org/10.1097/j.jcrs.0000000000000468

Skonier J , Neubauer M , Madisen L , et al. , 1992 . cDNA cloning and sequence analysis of βig-h3, a novel gene induced in a human adenocarcinoma cell line after treatment with transforming growth factor-β . DNA Cell Biol , 11 ( 7 ): 511 - 522 . https://doi.org/10.1089/dna.1992.11.511 https://doi.org/10.1089/dna.1992.11.511

Song XD , Wu QY , Zhu PR , et al. , 2023 . Pathogenic mutation of TGFBI gene in a family with corneal dystrophy . Chin J Clin Lab Sci , 41 ( 3 ): 176 - 179 (in Chinese) . https://doi.org/10.13602/j.cnki.jcls.2023.03.04 https://doi.org/10.13602/j.cnki.jcls.2023.03.04

Song YZ , Sun MS , Wang NL , et al. , 2017 . Prevalence of transforming growth factor β–induced gene corneal dystrophies in Chinese refractive surgery candidates . J Cataract Refract Surg , 43 ( 12 ): 1489 - 1494 . https://doi.org/10.1016/j.jcrs.2017.07.038 https://doi.org/10.1016/j.jcrs.2017.07.038

Stenson PD , Mort M , Ball EV , et al. , 2020 . The Human Gene Mutation Datab ase (HGMD ® ): optimizing its use in a clinical diagnostic or research setting . Hum Genet , 139 ( 10 ): 1197 - 1207 . https://doi.org/10.1007/s00439-020-02199-3 https://doi.org/10.1007/s00439-020-02199-3

Valasek MA , Repa JJ , 2005 . The power of real-time PCR . Adv Physiol Educ , 29 ( 3 ): 151 - 159 . https://doi.org/10.1152/advan.00019.2005 https://doi.org/10.1152/advan.00019.2005

Wang XR , Zhou BT , Zheng QM , et al. , 2020 . A recognition survey of granular corneal dystrophy type 2 genetic detection in China . Int J Ophthalmol , 13 ( 12 ): 1976 - 1982 . https://doi.org/10.18240/ijo.2020.12.20 https://doi.org/10.18240/ijo.2020.12.20

Wang Y , Shi WY , Li Y , 2020 . The rapid development and changes of corneal refractive surgery in China . Chin J Ophthalmol , 56 ( 2 ): 81 - 85 (in Chinese) . https://doi.org/10.3760/cma.j.issn.0412-4081.2020.02.001 https://doi.org/10.3760/cma.j.issn.0412-4081.2020.02.001

Weiss JS , Rapuano CJ , Seitz B , et al. , 2024 . IC3D classification of corneal dystrophies—Edition 3 . Cornea , 43 ( 4 ): 466 - 527 . https://doi.org/10.1097/ico.0000000000003420 https://doi.org/10.1097/ico.0000000000003420

Zeng L , Zhao J , Chen Y , et al. , 2017 . TGFBI gene mutation analysis of clinically diagnosed granular corneal dystrophy patients prior to PTK: a pilot study from eastern China . Sci Rep , 7 : 596 . https://doi.org/10.1038/s41598-017-00716-5 https://doi.org/10.1038/s41598-017-00716-5

Zhang FJ , Song YZ , 2023 . Interpretation of the group standard “Technical specifications for laser corneal refractive surgery Part 1”. Chin J Ophthalmol , 59 ( 6 ): 505 - 508 (in Chinese) . https://doi.org/10.3760/cma.j.cn112142-20230117-00025 https://doi.org/10.3760/cma.j.cn112142-20230117-00025

浏览量

Downloads

CSCD

工具集

关联资源

表皮更新过程中的细胞增殖与分化

耐盐植物促生菌Enterobacter sp. Av16和Acinetobacter sp. Av23减轻盐胁迫对白麻（Apocynum pictum）种子萌发和幼苗光合作用的抑制

RNA甲基转移酶HENMT1在生物学和疾病中的作用

秦皮乙素可通过抑制降钙素基因相关肽（CGRP）和调节脑膜肥大细胞减轻大鼠偏头痛样疼痛

施耐德膜来源间充质干细胞在上颌窦底提升模型与颅骨缺损模型中的再生潜能

屈光手术患者TGFBI突变多重qPCR检测体系的建立

Integrative analytical and statistical framework for optimization of multiplex qPCR detection of TGFBI mutations in refractive surgery candidates

DOI：10.1631/jzus.B2500747

摘要

Abstract

关键词

Keywords

references