Abstract

ABSTRACT: Phenylketonuria (PKU) is a rare autosomal recessive inborn error of phenylalanine (Phe) metabolism due to a deficiency of the hepatic-based enzyme phenylalanine hydroxylase. Dietary treatment is the cornerstone of therapy and its goal is to prevent excessive Phe accumulation in the blood.

The gut microbiota plays a major role in the maintenance of the host health, including intestinal health and function. It is influenced by several factors, including dietary pattern, antibiotic exposure, mode of delivery of a neonate, among others. However, dietary pattern is one of the most important, as it strongly influences the composition of gut microbiota. This may be particularly relevant in PKU patients, since their special Phe- restricted diet begins in the neonatal period and is maintained throughout life, possibly modifying gut microbiota composition and affecting gut homeostasis.

This study aims to i) characterize the gut microbiota of adult PKU patients; ii) compare gut microbiota of adult PKU patients with healthy adult population; and iii) understand the role of the Phe-restricted diet as a potential inducer of gut dysbiosis.

This will be an observational, cross-sectional study. A total of 24 participants will be recruited from the National Reference Centre in Hereditary Metabolic Disorders, Centro Hospitalar de Lisboa Norte. Anthropometric data of the population will be collected, and body composition will be assessed by bioimpedance, after overnight fasting. Faecal samples will be collected, as well as food records from the three days prior to faecal sample collection. Data from medical records will also be collected, including prescribed protein substitutes, natural and total daily protein intakes, median blood Phe concentrations in the previous year preceding the study, and clinical relevant data.

STATE OF THE ART

Phenylketonuria (PKU) is a rare autosomal recessive inborn error of phenylalanine (Phe) metabolism due to a deficiency of the hepatic-based enzyme phenylalanine hydroxylase (PAH). This leads to a complete or partial inability to convert the essential amino acid Phe to tyrosine (Tyr) (1, 2). PKU is identified by newborn screening and the estimated incidence in Europe is one in 10 000 newborn babies (1). Patients with PKU, when left untreated, have high Phe concentrations in blood and tissues and low to normal Tyr concentrations. Clinically, this leads to irreversible intellectual disability, microcephaly, seizures, aberrant behaviour, psychiatric symptoms, motor disturbances and eczematous rash (1). For this reason, treatment should be started as soon as possible. Dietary treatment is the cornerstone of therapy and its goal is to prevent excessive blood Phe accumulation. It consists of a dietary Phe restriction by 1) strict control of natural protein intake according to individual Phe tolerance; 2) administration of Phe-free L- amino acids supplements (L-AAs) usually with added vitamins and minerals in order to meet protein and non-protein requirements; 3) special low-protein foods (SLPF) to meet energy requirements (1, 2). Patients following this dietary therapy can have a successful outcome, even though poor dietary compliance and concomitant deteriorated blood Phe control is associated with lower intellectual quotient (2). Additionally, other treatment approaches are available such as administration of pharmacological doses of tetrahydrobiopterin (BH4) – cofactor of PAH – to BH4- responsive patients (3), use of large neutral amino acids (LNAA) and of glycomacropeptide supplemented with rate-limiting amino acids, vitamins and minerals (GMP-AAs) (4). The human microbiota is a collection of microbes living in the human body. The human gut has around 1013 to 1014 bacterial cells, outnumbering human cells by a factor of ten (5). The gut microbiota plays a major role in the maintenance of the host health, including intestinal health and function. In addition, it has the ability to hydrolyse protein, supplying free amino acids for both host and microbial metabolism functions (6) and to synthetize vitamins, neurotransmitters, short-chain fatty acids (SCFAs), among others (7, 8). In the gut, SCFAs act as an energetic substrate for the epithelial and peripheral tissues of the colon and as modulators of inflammation, providing health benefits to the host (7). Dysbiosis is a phenomenon where changes in diversity and composition of gut microbiota result in an increased colonization by potentially detrimental bacteria (7). This could influence gastrointestinal homeostasis, predispose to chronic inflammation and several inflammatory gastrointestinal diseases and modulate other metabolic functions through gut-liver axis and gut-brain axis (7, 9, 10). Several factors – endogenous and exogenous - influence the gut microbiota, including dietary pattern, antibiotic exposure, mode of delivery of a neonate, host genetic features, infections and environmental microbial exposures (7, 8). From these factors, dietary pattern is one of the most important, as it strongly influence the composition of gut microbiota (8, 10). In fact, different dietary patterns result in different substrates for microbial fermentation which leads to the enrichment or depletion of specific genera in gut microbiota. These changes in gut microbiota composition lead to distinct production of microbial metabolites, mostly SCFAs. For instance, acetate, butyrate, and propionate are derived from carbohydrate fermentation, while branched-chain fatty acids derive from proteins and amino acids fermentation by proteolytic bacteria (10). This influence of diet on gut microbiota composition may be particularly relevant in PKU patients, since their special Phe-restricted diet begins in the neonatal period and is maintained throughout life, possibly modifying gut microbiota composition and affecting gut homeostasis (10). When comparing gut microbiota of PKU children with healthy controls, Bacteroidetes and Verrucomicrobia were more prevalent and Firmicutes less prevalent in PKU patients’ samples (7). Conversely, Bassanini et al (9) found that the relative abundance of Bacteroidetes and Firmicutes was similar, with both phyla slightly diminished in the PKU group. In both studies, PKU children showed a diminished alpha diversity (7, 9) characteristic of gut dysbiosis (10). According to Pinheiro de Oliveira et al. the microbiome of PKU patients had less genes involved in starch and sucrose metabolism and in glycolysis/gluconeogenesis. This could affect SCFAs production, leading to lower acetate, propionate and butyrate production (7). In line with this, findings from Verduci and colleagues (10) showed a decrease in total SCFAs and butyrate production in PKU children. Up to date, only two studies aimed to characterize the gut microbiota of PKU patients and compare it to healthy controls or patients with mild hyperphenylalaninemia (7, 9). However, these studies have been performed in children. Thus, knowledge about gut microbiota of adult PKU patients is still lacking.

This study aims to:

1. i)  characterize the gut microbiota of adult PKU patients;
2. ii)  compare gut microbiota of adult PKU patients with healthy adult population;
3. iii)  understand the role of the Phe-restricted diet as a potential inducer of gut dysbiosis.

METHODOLOGY

This will be an observational, cross-sectional study. A total of 24 participants will be recruited from the National Reference Centre in Hereditary Metabolic Disorders, Centro Hospitalar de Lisboa Norte. PKU patients will be asked to identify a friend or family member of similar age and living in the same geographical area to take part of the study - healthy individuals group - an approach previously used by our team (11). This study will be registered at clinicaltrials.gov and will be submitted to Ethical committees of Centro Hospitalar de Lisboa Norte and NOVA Medical School. The inclusion criteria for the PKU group includes the following: 1) PKU patients, identified by neonatal screening, who received early and continuous dietary treatment; 2) under diet therapy with protein substitute prescription, except GMP-AAs; 3) male or female; 4) 2 over 18 years old; and 5) body mass index (BMI) between 18.5 and 30.0 kg/m . Exclusion criteria are 1) clinically significant illness; 2) taking sapropterin dihydrochloride; 3) have received antibiotic, prebiotic or probiotics in the 3 months preceding the study or 4) pregnant or breastfeeding. The study design will be explained, and participants will sign an informed consent form. In order to assess their eligibility, a clinical interview will be performed. Participants will receive a kit for faecal sample collection. After overnight fasting, body composition of all participants will be assessed. Participants will deliver the faecal sample collection, to be analysed by 16S rRNA sequencing. They will be instructed to fill out a food record concerning the three days prior to sample collection. Data from medical records from the last appointment at Hospital Santa Maria, Centro Hospitalar Universitário de Lisboa Norte, National Reference Centre in Hereditary Metabolic Disorders will be collected – prescribed protein substitutes, natural and total daily protein intakes, median blood Phe concentrations in the previous year preceding the study, and clinical relevant data.

EXPECTED RESULTS

After this study, we expect to be able to characterize adults PKU patient’s gut microbiota. In addition, a comparison with an age, sex, BMI, matched healthy adult population. Moreover, this study’ results could help to understand the role of the Phe-restricted diet as a potential inducer of gut modulation. This will provide brand new knowledge, supporting clinicians’ therapeutic prescription of prebiotic or probiotics. Regarding future protein substitute formulations and SLPF, this study may be a contribution as it may lead to their reformulation and supplementation in prebiotic or probiotic. Thus, this project will generate promising results, giving pertinent answers to the scientific community. It will also have a major impact in PKU patients gut health, allowing the establishment of intervention models in order to reverse dysbiosis and promoting a beneficial shift in PKU patients gut microbiota.

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