Allele-counting based non-invasive prenatal paternity testing for trisomy 21 fetuses:Methodology and case-based...

Prenatal paternity testing in fetuses with chromosomal aneuploidy poses interpretative challenges for traditional short tandem repeat (STR) analysis, whereas non-invasive SNP-based approaches may offer advantages. This study compared STR and non-invasive SNP testing for paternity determination in two fetuses with Down syndrome (trisomy 21). Amniotic fluid and parental peripheral blood samples were collected. STR typing was performed using the STRtyper-21G system. Non-invasive prenatal paternity testing (NIPPT) was conducted on maternal plasma cell-free fetal DNA using a custom capture panel of 5180 autosomal SNPs, followed by high-throughput sequencing and statistical analysis with the Prenatal Paternity Test Analysis System (PTAS). STR profiling revealed tri-allelic patterns and allelic dosage imbalance at chromosome 21 loci D21S11 and Penta D, forcing manual exclusion of these two loci for Combined Paternity Index (CPI) calculation. This reduced the final CPI by 2-fold (Case 1) and 21-fold (Case 2), yielding CPIs of 3.2 × 10⁹ and 5.7 × 10(Gao et al., 2023 [10]). In contrast, NIPPT produced CPIs of 2.40 × 10¹⁸³ and 7.18 × 10¹⁵⁴ with Cumulative Probability of Exclusion >99.999999% for both cases, requiring no adjustment for aneuploidy. SNP numbers and distribution were consistent between trisomy 21 and euploid controls. These findings demonstrate that while STR analysis is significantly confounded by chromosomal copy number variation in trisomy 21 pregnancies, SNP-based NIPPT - relying on allele counting, genome-wide locus redundancy, and a cumulative statistical model - provides robust, objective, and highly reliable paternity evidence without requiring exclusion of aneuploid loci. This approach represents a superior alternative for prenatal paternity testing in the context of fetal chromosomal aneuploidy.