Phenylketonuria (PKU) is a common inborn error of amino acid metabolism in which the enzyme phenylalanine hydroxylase, which converts phenylalanine to tyrosine, is functionally impaired due to pathogenic variants in the PAH gene. Thirty-four Brazilian patients with a biochemical diagnosis of PKU, from 33 unrelated families, were analyzed through next-generation sequencing in the Ion Torrent PGM™ platform. Phenotype–genotype correlations were made based on the BioPKU database. Three patients required additional Sanger sequencing analyses. Twenty-six different pathogenic variants were identified. The most frequent variants were c.1315+1G>A (n = 8/66), c.473G>A (n = 6/66), and c.1162G>A (n = 6/66). One novel variant, c.524C>G (p.Pro175Arg), was found in one allele and was predicted as likely pathogenic by the American College of Medical Genetics and Genomics (ACMG) criteria. The molecular modeling of p.Pro175Arg indicated that this substitution can affect monomers binding in the PAH tetramer, which could lead to a change in the stability and activity of this enzyme. Next-generation sequencing was a fast and effective method for diagnosing PKU and is useful for patient phenotype prediction and genetic counseling.

Although next-generation sequencing has emerged as a powerful tool for diagnosing rare diseases (RD), many cases of inherited metabolic diseases (IMD) remain unsolved, hindering the diagnosis, clinical and therapeutic management of the patients. The primary aim of this study is to address the most elusive cases by applying long-read sequencing (LRS) targeted to the gene of interest on seven patients ( FARS2 , GYS2 , PEX1 , SLC2A1 , AGL , ACAT1 , and ACADM ), identifying six novel pathogenic variants including two intronic variants, a structural variant and three transposable elements (TE) insertions. In addition, we have demonstrated the effect on splicing of an exonic variant previously reported as missense. Functional genetic tests specific for the expected effect of each variant of uncertain significance were designed, such as minigenes analysis or chromatin conformation capture assay. From the TE insertions, two were located in the genomic region of GYS2 or PEX1 , causing a reduction in their mRNA expression. The third was located 7.6 kb downstream of SLC2A1 ; it alters the interaction between the SLC2A1 promoter and its distal regulatory element via the establishment of a loop with the 3’ border of the native topologically associating domain. This study shows that the combination of LRS and functional genetic assays confers a powerful approach for expanding the mutational spectrum of IMD, adding data to improve the diagnosis of this large group of RD.

Patients with phenylketonuria (PKU) who retain residual phenylalanine hydroxylase (PAH) activity may benefit from sapropterin dihydrochloride (sapropterin) administration. Objective: To characterize sapropterin responsiveness in patients with PKU and investigate the impact of natural fluctuations in phenylalanine (PHE) levels and variations in PHE intake on sapropterin responsiveness. Methods: Retrospective chart review study. Patients with PKU who underwent the 24 h responsiveness test, including correction for natural PHE fluctuations, were included. Responders were defined as those who exhibited a >30% reduction in PHE levels within 8 h and/or 24 h after intake of 20 mg/kg sapropterin, correcting for the natural fluctuation in plasma PHE levels on day 1. Patients with a 28–30% reduction in PHE were considered sapropterin-responsive only if they had a concordant genotype. Results: Fifteen patients completed the test; however, three were excluded due to non-compliance. Additionally, one patient with mild PKU exhibited a borderline response, but the genotype agreement could not be assessed, rendering the test inconclusive. The rate of responsiveness could be assessed for eleven patients (six mild and five classical PKU). Among the patients with mild PKU, four were classified as responders: three at both 8 h and 24 h (reduction in plasma PHE: −75.9 ± 20.2% at 8 h and −75.7 ± 37.0% at 24 h) and one at 8 h only (reduction in plasma PHE: −28.7%). All patients with classic PKU (n = 5) were non-responders. Conclusions: A 24 h sapropterin responsiveness test incorporating correction for natural fluctuations in PHE levels identified a proportion of sapropterin-responsive patients with mild PKU similar to that described in the literature. PHE consumption should be objectively controlled during the protocol to avoid bias in determining responsiveness.

Phenylketonuria (PKU), one of the most common inherited metabolic disorders, is caused by biallelic loss-of-function variants in the phenylalanine hydroxylase ( PAH ) gene. More than 1000 pathogenic variants have been described in this gene. Although genotype-phenotype correlations are imperfect, PAH genotypes can influence treatment decision-making. To better characterize the genetic landscape of PKU in Brazil, we compiled a dataset of PAH genotypes from 742 patients by combining data from the literature ( n = 486), public databases ( n = 117), and direct physician reports ( n = 139). Most patients were classified as having classical PKU ( n = 455/742; 61.3%). The most prevalent variants were c.1162G > A (16.6%), c.1066-11G > A (13.9%), and c.782G > A (11.8%), which are also the predominant ones in Portugal, Spain, and Latin America. This study confirms the allelic heterogeneity of PKU in the Brazilian population and provide relevant insights to support public health policies for this disorder.

Phenylketonuria (PKU), one of the most common inherited metabolic disorders, is caused by biallelic loss-of-function variants in the phenylalanine hydroxylase ( PAH ) gene. More than 1000 pathogenic variants have been described in this gene. Although genotype-phenotype correlations are imperfect, PAH genotypes can influence treatment decision-making. To better characterize the genetic landscape of PKU in Brazil, we compiled a dataset of PAH genotypes from 742 patients by combining data from the literature (n = 486), public databases (n = 117), and direct physician reports (n = 139). Most patients were classified as having classical PKU (n = 455/742; 61.3%). The most prevalent variants were c.1162G > A (16.6%), c.1066-11G > A (13.9%), and c.782G > A (11.8%), which are also the predominant ones in Portugal, Spain, and Latin America. This study confirms the allelic heterogeneity of PKU in the Brazilian population and provide relevant insights to support public health policies for this disorder.