D.A. and L.F.-H.; Investigation, M.V.-A., M.A.A.-O., A.G.-d.A., L.F.-H., S.G.-L., L.L.-M., R.I.C.-N. and L.B.-M.; Methodology, M.A.A.-O., A.G.-d.A., S.G.-L., L.L.-M. and R.I.C.-N.; Project administration, M.V.-A., M.A.A.-O. and C.F.-L.; Resources, M.V.-A., M.A.A.-O., I.I.-G. in addition to a.G.-d.A.; Software, M.A.A.-O., I.I.-G., L.F.-H. and C.F.-L.; Supervision, M.A.A.-O. and C.F.-L.; Validation, M.A.A.-O., I.I.-G., L.F.-H. and C.F.-L.; Visualization, M.A.A.-O., A.G.-d.A., L.F.-H. and C.F.-L.; Writing–original draft, M.V.-A., M.A.A.-O., I.I.-G. and C.F.-L.; Writing–review and editing, M.V.-A., M.A.A.-O., I.I.-G., A.G.-d.A., L.F.-H., S.G.-L., L.L.-M., R.I.C.-N., L.B.-M. and C.F.-L. All authors have read and agreed for the published version from the manuscript. Funding: This investigation was funded by the National Institute of Pediatrics (Recursos Fiscales 2018020, Programa E022 Investigaci y Desarrollo Tecnol ico en Salud, Ciudad de M ico, Mexico). Institutional Evaluation Board PF-07038124 Metabolic Enzyme/Protease Statement: This study was authorized prior to information collection by the investigation, biosecurity and ethics committees with the National Institute of Pediatrics (approval numbers 2010/30 and 2020/014).Genes 2021, 12,19 ofInformed Consent Statement: All participants provided written consent of their participation plus the publication of data in an anonymized kind. Data Availability Statement: The datasets analyzed throughout the present study are accessible from the corresponding author on affordable request. Acknowledgments: We thank the patients, their families, and the Asociaci Mexicana de Fenilcetonuria, A.C. (Laura CRANAD-2 supplier Patricia Camacho Chavar and Josde Jes Mu z Navarro) for their assistance and commitment. The authors gratefully acknowledge Chemist A a Jannet Hern dez Montiel, Luis Ricardo Morales Gonz ez and Jaime Torres Marcial for their technical help. Conflicts of Interest: The authors declare no conflict of interest. The funders had no part in the style of your study; in the collection, analyses, or interpretation of data; within the writing in the manuscript, or in the selection to publish the outcomes.Appendix A Overview of PCR and Sanger Sequencing Situations: All reactions have been carried out in 30 beneath the typical situations suggested for HotStarTaqDNA Polymerase (www.qiagen/HB-0452, QIAGEN GmbH, Hilden, Germany, accessed on ten October 2019), with 55 ng of genomic DNA per reaction and 0.1 of every single primer. PCR primers to cover the entire coding exon and its exon ntron or untranslated regions borders have been made together with the Primer BLAST designing tool (ncbi.nlm.nih.gov/tools/ primer-blast/, accessed on 10 October 2019) applying the NG_008690.2 PAH RefSeqGene. The utilized annealing temperature was 58 C (exon 12) or 64 C (rest of exons). All reactions had been carried out with a final concentration of 0.3M of betaine (MP Biomedicals, LLC., Fountain Parkway Solon, OH, USA). All PCR solutions were evaluated by agarose gel electrophoresis, subjected to further enzymatic purification (ExoSAP-ITPCR Solution Cleanup, Affymetrix, Inc., Santa Clara, CA, USA), and then unidirectionally sequenced using the universal M13F primer (five -GTAAAACGACGGCCAGT-3) and Significant DyeTerminator Cycle Sequencing chemistry (Life Technologies Corp.; performed at PSOMAGEN Inc., Rockville, MD, USA). Clinically relevant variants have been confirmed by performing oppositestrand sequencing with all the attached universal primer. Variant annotation compliant with Human Genome Variation Society nomenclature (https://varnomen.hgvs.org/, accessed on.
