High‐Resolution Haplotyping of the PAH Gene Enables Early Gestation Noninvasive Prenatal Diagnosis of Phenylketonuria and Evolution Analysis of Recurrent Pathogenic Variations

ABSTRACT Background The clinical performance of RHDO‐based NIPD for PKU during early gestation remains under‐evaluated. Furthermore, studies focused on SNP loci obtained by next‐generation sequencing to analyze the genetic evolution of pathogenic variations in PKU is limited. Methods Maternal peripheral blood, along with proband and paternal samples, was collected between 7 and 12 weeks of gestation. The PAH gene and surrounding high heterozygosity SNPs were targeted for enrichment and sequencing. Fetal genotypes were inferred using RHDO‐based NIPD. High‐resolution PAH haplotypes were used for the analysis of two common pathogenic variants in the Chinese population: c.728G>A and c.1238G>C. Results Sixty one PKU families participated with an average fetal fraction of 6.08%. The median gestational age was 8 +6 weeks. RHDO‐based NIPD successfully identified fetal genotypes in 59 cases (96.72%, 59/62). Two cases failed because of insufficient informative SNPs. In addition, a recombination event was assessed in one fetus of 59 cases. Six, and three haplotypes were identified for c.728G>A(p.Arg243Gln) and c.1238G>C(p.Arg413Pro), respectively. Hap_3 and hap_8 were identified as the ancestral haplotypes for these pathogenic variants, with other haplotypes arising from mutations or recombination based on these ancestral haplotypes. Conclusions This study validates the feasibility of an RHDO‐based assay for NIPD of PKU in early pregnancy and introduces its application in the demonstration of founder effects in recurrent pathogenic variations, offering new insights into the evolutionary analysis of PAH variations.