The study, entitled “Genetic and in silico functional characterization of a novel structural variant in the PAH gene by long-reads sequencing and structural modeling” and published in Frontiers, used long-read sequencing (nanopore) with CRISPR/Cas9 enrichment (nCATS) and confirmed a tandem duplication of approximately 18 kb between exons 1 and 3 in an initial patient. Subsequently, using a simpler methodology—PCR—the same structure was confirmed in 7 additional patients. Through structural modeling, it was predicted that the duplication generates an additional segment at the N-terminal end of the PAH protein, which could alter the enzyme’s ability to metabolize phenylalanine.
This article is part of the Master’s thesis in Nutrition and Food Sciences, Human Nutrition track, at INTA, University of Chile, by Viviana Gallardo. “This variant produces a mild, but relevant, alteration in enzymatic function, and allows us to advance in the understanding of the genotype-phenotype relationship in PKU,” the authors explain in the text. The findings could contribute to the development of personalized therapeutic strategies and enrich both national and international databases of PKU variants.
Corresponding author Lorena Santa María, PhD in Sciences and Head of the Human Cytogenetics and Genomics Laboratory at INTA, states that “the characterization of this new variant enables a more precise genotype-phenotype correlation. Individuals carrying this duplication may present hyperphenylalaninemia or a mild to moderate PKU phenotype. Although the recommended treatment of a phenylalanine-restricted diet does not change, the presence of this variant suggests that these patients might tolerate a slightly higher intake of phenylalanine without compromising their health”.
Q & A with Viviana Gallardo and Professor Santa María on the study
– Could this finding be linked to the methodology used, a local genetic component, or some other factor?
The methodology allowed us to elucidate an alteration that could not be characterized in detail with the tools available up to that point. Since this had not been previously reported and was later detected in several Chilean patients, it is believed that this variant may have a founder effect.
– Did the patient present any special characteristics, or was this a finding from follow-up?
The patient had no special characteristics; the finding was part of a genetic study initiated by Valerie Hamilton in 2017.
– What type of personalized therapeutic strategies could be considered for PKU patients based on this study?
Since the alteration decreases but does not completely abolish enzymatic function, it may be susceptible to management with specific enzymatic cofactors, thereby improving phenylalanine tolerance.
– Are there implications for early diagnosis or clinical follow-up of patients with complex structural variants such as this one?
The use of next-generation methodologies not only allowed us to delineate the alteration precisely, but also to subsequently design a simple, fast, and inexpensive detection method for the same alteration. This would make it possible to study many patients without having to apply the original characterization methodology, which, although effective, remains costly.
Reference: Gallardo V, Gaete A, Maldonado J, Morales P, Peña A, Hamilton V, Faundes V and Santa María L (2025) Genetic and in silico functional characterization of a novel structural variant in the PAH gene by long-reads sequencing and structural modeling. Front. Genet. 16:1669007. doi: 10.3389/fgene.2025.1669007
Keywords: Phenylketonuria (PKU), PAH gene, CRISPR/Cas9, Oxford Nanopore sequencing (ONT), Exon duplication, Protein modeling
