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.

Phenylalanine ammonia-lyase (PAL) has various applications in fine chemical manufacturing and the pharmaceutical industry. In particular, PAL derived from Anabaena variabilis (AvPAL) is used as a therapeutic agent to the treat phenylketonuria in clinical settings. In this study, we aligned the amino acid sequences of AvPAL and PAL derived from Nostoc punctiforme (NpPAL) to obtain several mutants with enhanced activity, expression yield, and thermal stability via amino acid substitution and saturation mutagenesis at the N-terminal position. Enzyme kinetic experiments revealed that the kcat values of NpPAL-N2K, NpPAL-I3T, and NpPAL-T4L mutants were increased to 3.2-, 2.8-, and 3.3-fold that of the wild-type, respectively. Saturation mutagenesis of the fourth amino acid in AvPAL revealed that the kcat values of AvPAL-L4N, AvPAL-L4P, AvPAL-L4Q and AvPAL-L4S increased to 4.0-, 3.7-, 3.6-, and 3.2-fold, respectively. Additionally, the soluble protein yield of AvPAL-L4K increased to approximately 14 mg/L, which is approximately 3.5-fold that of AvPAL. Molecular dynamics studies further revealed that maintaining the attacking state of the reaction and N-terminal structure increased the rate of catalytic reaction and improved the solubility of proteins. These findings provide new insights for the rational design of PAL in the future.

Phenylketonuria (PKU) is the most common inherited metabolic disease in humans. Clinical screening of newborn heel blood samples for PKU is costly and time-consuming because it requires multiple procedures, like isotope labeling and derivatization, and PKU subtype identification requires an additional urine sample. Delayed diagnosis of PKU, or subtype identification can result in mental disability. Here, plasmonic silver nanoshells are used for laser desorption/ionization mass spectrometry (MS) detection of PKU with label-free assay by recognizing metabolic profile in dried blood spot (DBS) samples. A total of 1100 subjects are recruited and each DBS sample can be processed in seconds. This platform achieves PKU screening with a sensitivity of 0.985 and specificity of 0.995, which is comparable to existing clinical liquid chromatography MS (LC-MS) methods. This method can process 360 samples per hour, compared with the LC-MS method which processes only 30 samples per hour. Moreover, this assay enables precise identification of PKU subtypes without the need for a urine sample. It is demonstrated that this platform enables high-performance and fast, low-cost PKU screening and subtype identification. This approach might be suitable for the detection of other clinically relevant biomarkers in blood or other clinical samples.

Phenylalanine hydroxylase deficiency (PAHD) is an autosomal recessive disorder affecting phenylalanine (Phe) metabolism caused by mutations in the phenylalanine hydroxylase (PAH) gene. It has a complex phenotype with many variants and genotypes in various populations. This study sets out to analyze the screening results of children with phenylketonuria (PKU) in Yinchuan City and characterize the mutation variants of the PAH gene.
Phenylketonuria screening results were retrospectively analyzed in 398,605 neonates (207,361 males and 191,244 females) born in different maternity hospitals in Yinchuan City between January 2017 and December 2021. Screening for genetic metabolic diseases was performed with parental consent at their own expense. A comprehensive diagnosis was performed by integrating tandem mass spectrometry (MS/MS) findings with clinical presentations. High-throughput sequencing (HTS) was used to detect genetic and metabolic disease-associated genes in children with PKU who were clinically diagnosed and voluntarily tested. The identified loci were validated through Sanger sequencing and parental verification.
Among the screened newborns, 45 (11.3/100,000) PKU cases were diagnosed. In the 38 cases that underwent self-financed PAH sequencing, 56 mutations were detected in 76 chromosomes, with an overall detection rate of 73.7%. All patients harbored mutant genes, and the 56 mutations detected identified represented 14 variants, including 8 missense mutations, 2 splicing mutations, 2 nonsense mutations, and 2 silent mutations. The mutations were primarily distributed in exons 2, 3, 6, 7, 9, 11, and intron 4, with the highest frequency observed in exon 7 (25 [44.7%]), followed by exon 11 (15 [26.7%]). The most prevalent mutations were exon 7-p.R252W (10 [17.9%]) and exon 7-p.R261Q (8 [14.3%]).
The PAH gene mutations in children with PKU in Yinchuan City are predominantly concentrated in exons 6, 7, and 11, with the highest detection rates observed for p.R252W and p.R261Q mutations.