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<p>Refractive surgery can unmask or accelerate transforming growth factor-β-induced (<i>TGFBI</i>)-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 <i>TGFBI</i> 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 (<i>C</i><sub>T</sub>) cut-offs that were harmonized to a single decision threshold (<i>C</i><sub>T</sub>=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 (<i>κ</i>)=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 (<i>C</i><sub>T</sub> coefficient of variation (CV) 0.34%–1.21%). No cross-reactivity was observed against non-target <i>TGFBI</i> 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 <i>C</i><sub>T</sub>−log(copy) relationships suitable for routine quality control. Prospective screening of 10 055 refractive surgery candidates identified six <i>TGFBI</i> 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 <i>TGFBI</i> screening and reduce iatrogenic risk in refractive surgery candidates.</p>

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

  • 期刊论文

  • 发表日期：

    2026年6月27日

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  • Yunfeng Gu,
  • Liping Mao,
  • Xiaoling Li,
  • Kangxuan Sun,
  • Qiuruo Jiang,
  • Wenhui Wu,
  • Yangyang Shen,
  • Shihao Chen,
  • Meiqin Zheng,
  • Yi Xu

摘要

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 (CT) cut-offs that were harmonized to a single decision threshold (CT=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 (κ)=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 (CT 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 CT−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.