Diagnostic evaluation and clinical utility of C-MoKa for prenatal genetic testing

C-MoKa (Chromosome Conformation-based Karyotyping) is a novel 3D genome mapping platform, that enables simultaneous detection of structural variations (SVs), aneuploidies, copy number variations (CNVs), and uniparental disomy (UPD) in a single test. However, its performance in routine prenatal diagnosis is still unexplored. This study aimed to comprehensively evaluate the diagnostic performance and clinical utility of C-MoKa in prenatal diagnosis, and to assess its technical concordance with standard of care (SOC) testings in prenatal diagnosis.

A two-phase study was designed. In phase 1, 56 retrospective participants with known chromosomal abnormalities (CAs) were recruited, and C-MoKa was performed on their cultured amniotic fluid (AF) samples. Concordance between C-MoKa and known CAs was analyzed. In phase 2, a prospective cohort of 208 participants for prenatal diagnosis were recruited. Samples underwent C-MoKa and karyotyping (KT), with parallel chromosomal microarray analysis (CMA) or improved whole-exome sequencing (iWES). In our study, supplementary copy number probes were enhanced in iWES detection, which could provide ~ 100 kb resolution across the genome, thus either CMA or iWES platform employed was used to assess the CNV detection. Diagnostic yields and concordance were evaluated, and the discordance of SVs and CNVs were further validated by fluorescence in situ hybridization (FISH) and CNV-seq, respectively.

In the retrospective cohort with known CAs, C-MoKa achieved a 94.6% (53/56) diagnostic yield and 92.8% (52/56) concordance with KT + CNV. In the prospective cohort, its diagnostic yield was 21.2% (44/208), higher than KT (12.5%, 26/208) and CNV (18.3%, 38/208). The diagnostic yield (22.6%, 47/208) was achieved when CNV platform and C-MoKa were used together. C-MoKa exhibited concordance rates of 89.4% (186/208) with KT, 89.9% (187/208) with CNV, and 90.4% (188/208) with KT + CNV. Two SVs identified by C-MoKa but missed by KT were successfully determined by FISH, and five samples with additional CNVs identified by C-MoKa were consistently detected by CNV-seq.

Our findings demonstrate that C-MoKa is a highly effective and reliable method for prenatal diagnosis, exhibiting high concordance with SOC techniques. Compared to the conventional application of "KT + CNV" in prenatal setting, the combined use of CNV and C-MoKa appears to maximize the diagnostic yield.