## 2 Case Description
### Biochemical and Genetic Analyses  
Biochemical parameters included measuring circulating neurotransmitter levels. The potential involvement of the main catecholamines (DA and NA), as well as the role of glutamate and gamma-aminobutyric acid (GABA) neurotransmitters and related molecules were studied in blood/urine at different time points (see Table 1 for details and Supplementary Table S1 for complete data). In addition, a comprehensive panel of single-nucleotide polymorphisms (SNPs) (November 2021) (McMind company: [[https://shop.mcmind.de/](https://shop.mcmind.de/)]([https://shop.mcmind.de/](https://shop.mcmind.de/)), last accessed on 20 June 2024) showed the presence of some SNPs with potential relevance for the patient's risk of developing CFS (Table 2; for complete data set, please see Supplementary Table S2). Some implied a direct impact on the regulation of the monoamines and neurotransmitters (COMT and MAO), and others indicated a direct involvement in the response to stress (e.g., NR3C1, 24) and methylenetetrahydrofolate reductase (MTHFR) [24] (Table 2). The change in wt \"G\" by an \"A\" in the SNP rs46080 of the COMT gene (Val158 Met) is associated with lower COMT activity, which may lead to higher DA levels in the brain cortex, a reduced pain threshold, and an enhanced vulnerability to stress, which could align with the upper limit blood DA levels found in the patient (Table 1). Additional missense variants in heterozygosity: Ala222 Val of the MTHFR leads to reduced processing of folic acid, and Val16 Ala of the superoxide dismutase (SOD) gene translates into oxidative stress alterations. Importantly, some of these genetic conditions have previously been associated with CFS patients [25]. Further SNP analyses from Ancestry ([[https://www.ancestry.com/](https://www.ancestry.com/)]([https://www.ancestry.com/](https://www.ancestry.com/)) last accessed on 20 June 2024) and 23 andMe ([[https://www.23](https://www.23) andme.com/gen101/snps/]([https://www.23](https://www.23) andme.com/gen101/snps/)(2023) last accessed on 20 June 2024) were individually studied and classified for their role in cognitive processes, stress, motor disturbances (including physical fatigue), and inflammation, and the immune system (Supplementary Table S2). In addition, the HPA axis and immune system composition and function were studied by measuring glucocorticoidoid and cytokine levels, as well as the presence of autoantibodies. The patient presented with elevated proinflammatory cytokine levels (interleukin-6 (IL6), IL10, and interferon gamma (IFN\({}_{Y}\)) (2021-2022), indicative of neuroinflammation and a reduction in NK cells, suggesting a compromised immune system (Supplementary Table S1). In addition, the presence of Epstein-Barr virus (EBV) antibodies (Supplementary \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Gene Target** & **Locus (GRCh38)** & **rs ID** & **Genotype** & **Gene Region** \\ \hline COMT & 22q11.21 & rs4680 & A/A homozygote & coding \\ \hline MAOA & Xp11.3 & VNTR30 bp & High copy number & promoter \\ \hline MAOB & Xp11.3 & rs1799836 & T/T homozygote & intron \\ \hline FKBP5 & 6p21.31 & rs1360780 & C/T heterozygote & intron \\ \hline FKBP5 & 6p21.31 & rs9470080 & C/T heterozygote & intron \\ \hline FKBP5 & 6p21.31 & rs4713916 & G/A heterozygote & intron \\ \hline FKBP5 & 6p21.31 & rs9296158 & G/A heterozygote & intron \\ \hline NR3C1 & 5q31.3 & rs6198 & G/A heterozygote & 3’ UTR \\ \hline MTHFR & 1p36.22 & rs1801133 & C/T heterozygote & coding \\ \hline SOD2 & 6q25.3 & rs4880 & C/T heterozygote & coding \\ \hline \hline \end{tabular} Abbreviations: COMT (catechol-O-methyltransferaseerase); MAOA (monoamine oxidase A); MAOB (monoamine oxidase B); FKBP5 (FKBP probyl isomerase 5); NR3C1 (nuclear receptor subfamily 3 group C member 1); MTHFR (methylene-trabhydrofolate reductase); SOD2 (superoxide dismutase 2); GRCh38 (Genome Reference Consortium Human Build 38); rs (reference SNP cluster ID); VNTR (variable number of tandem repeat); UTR (untranslated region).  
\end{table}