## 3 Results and Discussion
### Phylogenetic Clustering and Population-Level Analysis of Novel Mutations in SE-Z-DNA Loci
The most conserved locus was chr10:119394546, where an alternative allele was observed in only 15% of the genomes. A short insertion at this locus led to the formation of a new motif, GGGGGTGTCC. The clustering analysis allowed to suggest potential functional relevance of the novel mutations basing on co-segregation with known pathogenic variants retrieved from ClinVar database, suggesting potential roles in multiallelic neurodegenerative diseases or associated with neurodevelopmental diseases etiology or regulatory mechanisms. Further experimental validation is required to confirm the mechanistic impact of these variants on transcriptional regulation and disease pathogenesis. Figure 5: Phylogenetic tree of individuals based on novel mutations in SE-Z-DNA loci. Red bars represent the Cumulative Allele Frequency Score (CAFS), while green bars indicate the allele frequencies in the cohort of 100 individuals. Therefore, tree structure, combined with allele frequency and calculated CAFs for gnomAD, considers population-wide variations with corresponding functional impact. The clustering of individuals into distinct clades supports the idea of frequency-specific genetic architecture across the whole non-Finnish European population. These findings highlight specific genomic loci enriched for Z-DNA and associated regulatory mutations that represent promising targets for future experimental studies. Specifically, these regions could be investigated using CRISPR-based functional assays or chromatin conformation capture techniques to elucidate how modulating Z-DNA formation affects transcription factor binding, chromatin accessibility, and downstream transcriptional networks implicated in disease pathology. While the mechanism of Z-DNA co-regulation within super-enhancers remains incompletely understood, its potential applications may primarily lie in the diagnosis of complex diseases, including cancer and autoimmune disorders. Z-DNA in super-enhancer regions could serve as a potential biomarker, indicating elevated expression levels of the target genes within these loci. However, further experimental validation is required to substantiate this hypothesis. Although direct evidence remains limited, the ability of super-enhancers to adopt Z-DNA flipons may serve as a potential biomarker for identifying active regulatory regions, particularly in super-enhancers associated with genes governing cell cycle progression, apoptosis, or key signaling pathways (e.g., JAK/STAT, NF-kB, MAPK/ERK, PI3K/AKT/mTOR, Wnt/\(\beta\)-catenin). The stable formation of Z-DNA within such super-enhancers could be linked to the hyperactivation of oncogenic programs, promoting uncontrolled proliferation, suppression of apoptosis, therapy resistance, epithelial-to-mesenchymal transition (EMT), and ultimately, cancer progression. While this study provides robust computational evidence supporting the enrichment and potential functional relevance of Z-DNA-forming sequences within enhancer and super-enhancer regions, it is acknowledged that the presented findings are primarily based on computational predictions. Experimental validation, such as targeted functional assays or CRISPR-based genome editing, would further elucidate the mechanistic roles of these predicted Z-DNA loci in transcriptional regulation, enhancer-promoter interactions, and chromatin dynamics. These experiments represent important next steps to build upon and extend the insights generated by our computational analyses.