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Biomedical research is a multifaceted discipline encompassing translational, pathophysiological, and molecular studies with particular emphasis on genetics and genomics. Its primary aim is to understand the cellular and molecular mechanisms underlying physiological function and disease development. To advance our understanding of disease mechanisms in two quite heterogeneous diseases, this thesis examined genetic alterations associated with Mayer-Rokitansky-Küster-Hauser syndrome (MRKH) and epigenetic changes in Huntington’s disease (HD), interpreting both within a broader biological context.
The first study focused on the genetic etiology of MRKH, a congenital disorder characterized by agenesis of Müllerian ductderived structures, such as the uterus and upper vagina, and, in some cases, kidney and skeletal malformations. Whole genome sequencing was conducted on blood and uterine tissue samples from a unique cohort of five monozygotic twin pairs discordant for the condition. Data analysis involved various filtering criteria and comparison strategies, followed by Sanger sequencing to validate novel variants.
Single-sample analysis of MRKH tissue identified two variants of unknown significance in PAX8 (ENST00000263334.9: c.1315G>A, p.Ala439Thr) and WNT9B (ENST00000290015.7:c.205C>T, p.Arg69Trp) and one pathogenic variant in GREB1L (ENST00000269218.10 c.4665T>A, p.Tyr1555*). Multi-sample analysis comparing blood and tissue DNA of the MRKH patients to their healthy twins, revealed a novel ACTR3B variant. Subsequent Sanger sequencing confirmed this variant as mosaic—present in uterine tissue but absent in blood.
To contextualize these findings, literature on embryological development of the genitourinary tract, MRKH pathogenesis, and the function of affected genes was reviewed. The PAX8 variant was classified as of uncertain significance due to its presence in unaffected relatives, likely attributable to reduced penetrance. Nevertheless, the gene’s role in mesenchymal-epithelial transition (MET) during uterine development and its known association with MRKH highlight PAX8 as a promising candidate for further investigation. Similarly, the WNT9B variant aligns with previous studies linking the gene to Müllerian duct anomalies. Its presence in unaffected individuals, however, also suggests a contributory rather than a solely causative role. The GREB1L variant was associated with renal anomalies in both twins and additional uterine malformations in one—findings consistent with literature linking the gene to both types of genitourinary anomalies. Observed maternal transmission and reduced penetrance point to a complex inheritance pattern and phenotypic variability. The mosaic variant in ACTR3B, a gene regulating processes essential to the genitourinary tract formation, like MET and cytoskeletal remodeling, provides a novel perspective on potential contributors to MRKH pathogenesis. However, ACTR3B’s weak expression in target tissues and lack of direct links to Müllerian anomalies limit its current interpretability. Taken together, these findings underscore the enigmatic nature of MRKH and the need to explore etiological factors beyond genetics alone.
The second investigation presented in this thesis explored CpG methylation changes in HD in response to enriched, normal, and stressed environmental conditions. By comparing epigenome-wide striatal DNA methylation data from BACHD and wildtype rats, the initial analysis indicated that genotype-dependent differentially methylated sites (DMSs) were the most prominent findings. Although fewer in number and effect strength, DMSs in animals exposed to different environmental stimuli could also be seen. Four candidate sites in genes including CDK19 (Site 1 and Site 3), TMEFF1 (Site 2), and ARHGAP20 (Site 4) were selected for pyrosequencing and successfully validated, highlighting a strong genotype-dependent effect. An additional environment-dependent effect was initially observed at Site 4, where stressed transgenic animals displayed significantly higher methylation than their non-stressed counterparts. However, this finding could not be reliably confirmed due to technical variability in the pyrosequencing results. To contextualize and interpret these findings within a broader biological context, we explored the genes’ physiological functions and association with HD.
CDK19 encodes a kinase component of the Mediator complex, which regulates transcription and modulates key signaling pathways, including JAK/STAT, NFκB, SMAD, estrogen receptor signaling, and Wnt/β-catenin—most of which are dysregulated in HD. The increased methylation at Sites 1 and 3, alongside reduced CDK19 expression, aligns with known disruptions in many of these pathways. However, in some cases, the methylation changes did not match previously reported alterations of related pathway components in HD patients. These contrasting observations raise the question of whether such epigenetic alterations act as pathogenic drivers or represent compensatory mechanisms aimed at restoring expression balance within the affected pathways.
TMEFF1 is a key regulator of BMP2 signaling, a pathway essential for neuronal differentiation and the regulation of brain-derived neurotrophic factor (BDNF) expression. Previous studies have reported decreased TMEFF1 expression in HD patient brains—findings consistent with our results, suggesting its potential involvement in HD pathophysiology, as a biomarker or therapeutic target.
ARHGAP20 participates in signal transduction during neurite outgrowth and has been linked to stress-induced methylation changes—an effect mirrored in our stressed BACHD rats. Although the environmental effect could not be conclusively validated, the initial methylation and expression data point to a potential gene-environment interaction relevant to early developmental vulnerability in HD.
Our work on epigenetic profiling in BACHD rats provided insights into how disease and environmental factors influence methylation and how they might contribute to the complex pathophysiology of HD.
By juxtaposing these two projects, we highlight a broader narrative in biomedical research: the shift from a sole focus on (epi)genetic underpinnings toward a more integrated understanding of disease that considers diverse biological processes and developmental factors. This approach emphasizes the need to interpret (epi)genetic changes within a broader biological context to shed light on their roles in disease mechanisms and evaluate their potential as future biomarkers or therapeutic targets. As research progresses, integrating insights from diverse disciplines will be key to developing holistic strategies for understanding—and ultimately treating—complex diseases like MRKH and HD. |
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