Rstudio version 4.2.3

For continuous variables, the median and IQR were reported, and absolute and relative frequencies were assessed as summary measures of categorical variables. According to the nature of variables, χ² tests and Kruskal-Wallis tests were performed to investigate the associations between germinal variant status and characteristics of patients and tumors. The Mann-Whitney U test was used to compare ages between probands and family members. Disease-free survival was estimated using the Kaplan-Meier method, and survival distributions were compared using the log-rank test. A multivariable Cox proportional hazards regression model was used to determine the independent association of germinal variant status with cancer progression or death, adjusting for age. The significance level was set at a global 2-tailed P < .05 for all analyses. The statistical analyses were performed with RStudio, version 4.2.3 (RStudio).

Statistics was done in R (RStudio Version 4.2.3, 2023 RStudio, Inc., www.posit.co) using a p-value ≤ 0.05. One-way Anova with Tukey’s HSD post-hoc test was used to determine significant differences in concentrations.

The daily intake of BTs was estimated based on urinary concentrations of ∑8 BTs and a simple steady-state kinetic model. To calculate ∑8 BTs, imputed and SG-adjusted values were used for analytes with a detection frequency higher than 50%, while for the rest of the BTs, concentrations below MLOQ were substituted with the value MLOQ/square root of 2. The values of all 8 BTs were then summed for each participant. The estimation of daily intake was undertaken using the following formula derived from an equation used in previous studies (Katsikantami et al. 2019 (link); Šulc et al. 2022 (link)): $$EDI\left(\mu g/kg/day\right)=\frac{c_U\left(\mu g/L\right)\times V_{\mathit{UO}}(L/day)}{F_{\mathit{UE}}\times b.w.(kg)}$$ where EDI is Estimated Daily Intake, c_U is the SG-adjusted concentration of BTs in urine, V_UO is the urine output volume (1.7 L/day (Perucca et al. 2007 (link))), F_UE is the urinary excretion factor, and b.w. is the body weight of the participant. However, the pharmacokinetics of BTs is not well known; hence, 3 theoretical excretion scenarios with different F_UE were considered: A) a worst-case scenario, in which only 10% of daily intake is excreted through urine (F_UE = 0.10); B) a medium scenario, in which 50% of daily intake is excreted via urine (F_UE = 0.50); and lastly C) a best-case scenario, in which 90% of daily intake is excreted via urine (F_UE = 0.90). All statistical analyses were performed using Rstudio version 4.2.3 (RStudio Team 2020 ).