Development and clinical application of CAPAH: a long-read sequencing approach for accurate second-tier screening of phenylketonuria in newborns

Abstract

Phenylketonuria (PKU) is a common inherited metabolic disorder in newborns. Early diagnosis and timely intervention are essential to prevent neurodevelopmental impairment. Traditional newborn screening primarily relies on measuring blood phenylalanine (Phe) levels, which can lead to both false-positive and false-negative results. Long-read sequencing (LRS) offers high coverage and long-fragment resolution, enabling comprehensive detection of PAH gene variants and potentially improving PKU screening and diagnosis. We developed a comprehensive PAH gene sequencing technology (CAPAH) based on the HiFi LRS platform, and applied it to the second-tier screening for PKU in newborns. First, CAPAH was validated for consistency with traditional NGS, MLPA, and Sanger sequencing in 20 samples with known genotypes. Subsequently, a retrospective analysis of 200 newborns with positive or borderline primary screening results was performed to assess the impact of CAPAH on variant detection, genotype-phenotype associations, and second-tier screening performance. CAPAH achieved complete consistency (100%) with traditional methods in the 20 validation samples. It enabled direct determination of cis-trans configurations and precise localization of large deletion breakpoints. Among 200 newborns, CAPAH identified 103 newborns with biallelic variants, 47 carriers, and 50 negative individuals, reducing the recall rate from primary screening by 48.5% (97/200). Among 131 newborns with negative or excluded PKU based on rescreening blood Phe levels, CAPAH detected 40 newborns carrying compound heterozygous variants, preventing approximately 20% of potential missed detections. In the 103 newborns with biallelic variants, CAPAH identified 64 PAH variants, including 47 missense, eight splice-site, four nonsense, and five deletion variants. Genotype-phenotype analysis revealed that the proportion of severe biallelic PKU-related variants (classic and mild PKU) was significantly higher in the rescreen-positive group than in the rescreen-negative group (25.00% vs. 14.29%). Homozygosity for c.158G > A or its combination with PKU- or mild hyperphenylalaninemia (MHP)-related variants was associated with blood Phe levels within the MHP range (2–6 mg/dL). When c.158G > A was combined with PKU-related variants, rescreening Phe levels were significantly elevated ( p  < 0.01). CAPAH enables comprehensive detection of the PAH gene, including complex variants and cis–trans configurations, thereby improving the accuracy of second-tier PKU screening in newborns and reducing unnecessary recalls. This approach provides an efficient and practical molecular diagnostic tool for newborn screening of inherited metabolic disease.