Citation: Timperio, A.M.; Gevi, F.; Cucinotta, F.; Ricciardello, A.; Turriziani, L.; Scattoni, M.L.; Persico, A.M. Urinary Untargeted Metabolic Profile Differentiates Children with Autism from Their Unaffected Siblings. Metabolites 2022, 12, 797. https://doi.org/10.3390/ metabo12090797 Academic Editor: J.J.M. (Judith) Jans Received: 8 July 2022 Accepted: 22 August 2022 Published: 26 August 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). metabolites H OH OH Article Urinary Untargeted Metabolic Profile Differentiates Children with Autism from Their Unaffected Siblings Anna Maria Timperio 1, * ,† , Federica Gevi 1,† , Francesca Cucinotta 2,3 , Arianna Ricciardello 2,4 , Laura Turriziani 2 , Maria Luisa Scattoni 5 and Antonio M. Persico 6, * 1 Department of Ecological and Biological Sciences, University of Tuscia, 01100 Viterbo, Italy 2 Interdepartmental Program “Autism 0-90”, “G. Martino” University Hospital, 98124 Messina, Italy 3 IRCCS Centro Neurolesi “Bonino-Pulejo”, 98124 Messina, Italy 4 Villa Miralago, 21050 Cuasso al Monte, Italy 5 Research Coordination and Support Service, Istituto Superiore di Sanità, 00161 Rome, Italy 6 Child & Adolescent Neuropsychiatry Program, Modena University Hospital & Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, 41121 Modena, Italy * Correspondence: timperio@unitus.it (A.M.T.); antonio.persico@unimore.it (A.M.P.) † These authors contributed equally to this work. Abstract: Autism Spectrum Disorder (ASD) encompasses a clinical spectrum of neurodevelopmental conditions that display significant heterogeneity in etiology, symptomatology, and severity. We previously compared 30 young children with idiopathic ASD and 30 unrelated typically-developing controls, detecting an imbalance in several compounds belonging mainly to the metabolism of purines, tryptophan and other amino acids, as well as compounds derived from the intestinal flora, and reduced levels of vitamins B6, B12 and folic acid. The present study describes significant uri- nary metabolomic differences within 14 pairs, including one child with idiopathic ASD and his/her typically-developing sibling, tightly matched by sex and age to minimize confounding factors, al- lowing a more reliable identification of the metabolic fingerprint related to ASD. By using a highly sensitive, accurate and unbiased approach, suitable for ensuring broad metabolite detection coverage on human urine, and by applying multivariate statistical analysis, we largely replicate our previous results, demonstrating a significant perturbation of the purine and tryptophan pathways, and further highlight abnormalities in the “phenylalanine, tyrosine and tryptophan” pathway, essentially involv- ing increased phenylalanine and decreased tyrosine levels, as well as enhanced concentrations of bacterial degradation products, including phenylpyruvic acid, phenylacetic acid and 4-ethylphenyl- sulfate. The outcome of these within-family contrasts consolidates and extends our previous results obtained from unrelated individuals, adding further evidence that these metabolic imbalances may be linked to ASD rather than to environmental differences between cases and controls. It further under- scores the excess of some gut microbiota-derived compounds in ASD, which could have diagnostic value in a network model differentiating the metabolome of autistic and unaffected siblings. Finally, it points toward the existence of a “metabolic autism spectrum” distributed as an endophenotype, with unaffected siblings possibly displaying a metabolic profile intermediate between their autistic siblings and unrelated typically-developing controls. Keywords: metabolomics; autism; mass spectrometry; sibling 1. Introduction Autism Spectrum Disorder (ASD) spans a wide range of neurodevelopmental condi- tions characterized by deficits in social interaction and communication, repetitive behaviors, restricted and unusual interests, rigid adherence to routines, and abnormal sensory pro- cessing [1]. Its prevalence rates range from 1/54 children and 1/45 adults in the United States [2,3] to 1/87 children in Italy and 1/102 adults in England [4,5]. ASD displays significant heterogeneity in terms of etiology, symptomatology, developmental trajectory, Metabolites 2022, 12, 797. https://doi.org/10.3390/metabo12090797 https://www.mdpi.com/journal/metabolites