Evaluation of Oral Health and its Modifying Factors: Oral Microbiome, Genetic Markers, and Patient Knowledge in Phenylketonuria and Type 1 Diabetes

Background: Phenylketonuria (PKU) is an autosomal recessive inherited disorder of phenylalanine metabolism caused by a deficiency of the enzyme phenylalanine hydroxylase. Individuals with PKU must adhere to a lifelong low-protein diet and consume a specialised amino acid mixture daily. However, these amino acid supplements are often acidic, creating an unfavourable oral environment. The resulting low pH promotes plaque accumulation, which can lead to gingivitis and periodontitis, while also increasing the risk of dental caries due to the persistently acidic conditions in the mouth. Type 1 diabetes (T1D) is a metabolic disorder caused by defective insulin secretion and/or action and diet regimen is often suggested. In T1D chronic hyperglycaemia and impaired immune response increase the risk of periodontal disease, while altered salivary composition contributes to a higher susceptibility to dental caries and oral infections. Advancements in insulin therapy have simplified the disease management, allowing many patients with T1D to maintain a diet that is comparable to that of healthy individuals. The aim of this study was to evaluate oral health in patients with PKU and T1D compared to individuals in a control group from the Latvian population, and to analyse factors influencing it, such as oral hygiene habits, the oral microbiome, and genetic predisposition, as well as to develop specially tailored oral hygiene recommendations for patients with PKU. Material and methods: Our study included three groups – individuals with PKU, T1D and a control group without reported chronic diseases and dietary restrictions, all at least 12 years old. Oral health assessment was performed for all participants, followed by collection of saliva for microbial community analysis using 16S rRNA gene V3-V4 region sequencing and for genotyping of genetic variants in the IL1, DEFB1, TAS1R2, TAS2R38, CD36 genes. In this study, two questionnaires on oral hygiene habits and knowledge were prepared and administered by trained medical personnel, with a one-month interval between pre- and post-implementation of oral hygiene recommendations. These recommendations were based on previous oral health examinations and a literature review. The questionnaires were completed by all diagnosed patients with PKU in Latvia who were not lost to follow-up and who were older than two years. Results: Oral examination data was obtained from 45 patients with PKU (four were excluded due to severe intellectual disability), 31 T1D patients, and 70 control subjects. Clinical evaluation revealed that patients with PKU had the poorest oral hygiene index scores, with statistically significant differences in all variables compared to the T1D and control groups, except for the number of filled teeth. Patients with PKU also reported the lowest consistency in tooth brushing and flossing habits. Based on the questionnaires among patients with PKU under 18 years of age, 27 % cleaned their teeth once a day, while 6 % did not engage in regular tooth brushing at all. Among those aged 18 years and older, 26 % reported brushing only once a day. Additionally, a mere 39 % of patients with PKU aged ? 18 years and 17 % aged < 18 years incorporated flossing in their oral hygiene routine. In total, 45 % of patients with PKU aged ? 18 years and 44 % aged < 18 years visited a dentist once a year, but 40 % aged ? 18 years and 39 % aged < 18 years reported visiting a dentist less frequently than once per year. After reading the recommendations, a remarkable improvement in daily oral hygiene was observed among patients with PKU under 18 years of age: 25 % improved their toothbrushing habits and 23 % began flossing. Oral microbiome analysis revealed that the lowest number of shared genera was between the PKU and T1D groups. The highest number of differentially abundant genera was detected in the PKU group (n = 20), with significant enrichment of Actinomyces (padj = 4.17×10-22), Capnocytophaga (padj = 8.53×10-8) and Porphyromonas (padj = 1.18×10-5) compared to the control group. In contrast, the genus Leptotrichia (padj = 5.79×10-7) was enriched in T1D group, whereas the abundance of genera Fusobacterium (padj = 2.05×10-4) and Alloprevotella (padj = 7.24×10-4) was decreased compared to the control group. Analysis of genetic variant associations with oral health characteristics revealed that the DEFB1 rs11362 variant was associated with higher Silness-Löe plaque and Greene-Vermillion index scores in patients with PKU (p = 0.011 and p = 0.043, respectively). The IL1B rs1143634 variant was associated with a lower necessity for calculus removal in T1D patients (p = 0.030). Conclusions: PKU and T1D patients have a higher prevalence of carious teeth, and an increased risk of periodontal disease compared to the control group. Differences in the oral microbiome among PKU, T1D patients and controls may contribute to variations in oral health. In addition to metabolic disease and diet, genetic predisposition may also influence oral health outcomes in the PKU, T1D and control groups. Results of the survey on oral hygiene habits before and after receiving targeted recommendations suggest that a tailored approach may be needed to improve oral hygiene practices, particularly among adult patients with PKU.