A Novel Technique for Validating Copy Number Variants and Its Application in Familial Exudative Vitreoretinopathy

Abstract

Inherited diseases have a strong genetic basis, with copy number variants (CNVs) playing a crucial role in their pathogenesis. However, the detection and validation of CNVs remain challenging. In this study, we introduce target enrichment polymerase chain reaction (tecPCR), a novel technique that integrates multiplex polymerase chain reaction (PCR) with next-generation sequencing (NGS) for enhanced CNV validation, and applied it in familial exudative vitreoretinopathy (FEVR). Using NGS, 97 variants linked to FEVR were identified, including 91 SNVs and 6 CNVs. Of the CNVs, 66.67% (4/6) were validated both by quantitative PCR and tecPCR, yielding a positive predictive value of 100% (95% confidence intervals: 39.8%–100%) and negative predictive value of 100% (95% confidence intervals: 15.8%–100%) for tecPCR in this pilot validation. The validated FEVR genetic test showed a positivity rate of 53.07% (95/179). Moreover, the demographic and ocular features of the FEVR patients with CNVs were comparable to those with SNVs. This study broadens the mutation spectrum of FEVR and demonstrates that tecPCR provides exceptional specificity and sensitivity, making it a promising tool for genetic diagnostics.