Phenotypic Spectrum and Molecular Characteristics of Variants of Uncertain Significance in Copy Number Variations Associated with Fetal Cardiac Malformations: A Case Series of 9 Patients

Qingsong Wang^1*Jun Yin¹Ting Lan¹Huimin Ou²Xiaoqin Zhou¹Lingli Zhong¹Hongmei Cao¹Le Zhang¹Yan Luo¹

• ¹The First People's Hospital of Jintang County/ Jintang Hospital, West China Hospital, Sichuan University, Chengdu, Sichuan, China
• ²Sichuan Provincial Women's and Children's Hospital / The Affiliated Women's and Children's Hospital of Chengdu Medical College, Chengdu, China

Background: Copy number variants of uncertain significance (VUS-CNVs) pose significant challenges in prenatal counseling for congenital heart malformations (CHMs). Their clinical relevance remains poorly defined, particularly in the absence of postnatal validation. Objective: To characterize the phenotypic and molecular features of VUS-CNVs in CHM fetuses and evaluate their clinical implications. Methods: From a cohort of 644 fetuses with CHMs undergoing chromosomal microarray analysis (CMA), we identified 9 VUS-CNV cases. All reportable CNVs (≥200 kb or in known pathogenic regions such as 22q11.21) were validated by TaqMan qPCR. Detailed prenatal echocardiography, trio CMA (n=7), and postnatal follow-up were integrated for comprehensive assessment. Results: VUS-CNVs were identified in 9 of 644 (1.40%) CMA-tested fetuses with CHMs. These included 3 in isolated CHM, 1 in complex CHM, 3 in CHM with extracardiac structural anomalies, and 2 in CHM with isolated soft markers. VUS-CNVs in non-isolated cases were larger (median: 2.5 Mb vs. 0.72 Mb in isolated CHM) and encompassed more OMIM-listed genes (mean: 3.8 vs. 1.5 in isolated CHM) than those in isolated CHM. Trio analysis reclassified 3 of 7 tested VUS-CNVs (2 as likely benign, 1 as likely pathogenic). Six pregnancies continued to term—3 with isolated and 3 with non-isolated CHM—and all live-born infants showed no major postnatal abnormalities. The three terminations involved non-isolated phenotypes; decisions were primarily driven by structural anomaly severity, though the VUS amplified prognostic uncertainty. Conclusion: Although VUS-CNVs are uncommon in fetuses with CHMs, they occur more frequently in non-isolated phenotypes and carry the potential for future reclassification—posing significant challenges in prenatal genetic counseling. These two aspects underscore the necessity of trio-based genomic testing, cautious interpretation of results, and longitudinal follow-up. Given the limited sample size of this study, no causal relationship between VUS-CNVs and clinical phenotypes can be inferred; however, our findings support a risk-stratified management framework that integrates detailed fetal phenotyping, trio analysis, and postnatal follow-up to enable accurate variant interpretation and informed decision-making.