Abstract Background: Despite outstanding advances in understanding the genetic background of uveal melanoma (UM) development and prognosis, the role of DNA methylation reprogramming remains elusive. This study aims to clarify the extent of DNA methylation deregulation in the context of gene expression changes and its utility as a reliable prognostic biomarker. Methods: Transcriptomic and DNA methylation landscapes in 25 high- and low-risk UMs were interrogated by Agilent SurePrint G3 Human Gene Expression 8×60K v2 Microarray and Human Infinium Methylation EPIC Bead Chip array, respectively. DNA methylation and gene expression of the nine top discriminatory genes, selected by the integrative analysis, were validated by pyrosequencing and qPCR in 58 tissues. Results: Among 2,262 differentially expressed genes discovered in UM samples differing in metastatic risk, 60 were epigenetic regulators, mostly histone modifiers and chromatin remodelers. 44,398 CpGs were differentially methylated, 27,810 hypomethylated, and 16,588 hypermethylated in high-risk tumors, with Δβ values ranging between -0.78 and 0.79. By integrative analysis, 944 differentially expressed DNA methylation-regulated genes were revealed, 635 hypomethylated/upregulated, and 309 hypermethylated/downregulated. Aberrant DNA methylation in high-risk tumors was associated with the deregulation of key oncogenic pathways such as EGFR tyrosine kinase inhibitor resistance, focal adhesion, proteoglycans in cancer, PI3K-Akt signaling, or ECM-receptor interaction. Notably, DNA methylation values of nine genes, HTR2B , AHNAK2, CALHM2, SLC25A38, EDNRB, TLR1, RNF43, IL12RB2 , and MEGF10, validated by pyrosequencing, demonstrated excellent risk group prediction accuracies (AUC ranging between 0.870 and 0.956). Moreover, CALHM2 hypomethylation and MEGF10, TLR1 hypermethylation, as well as two three-gene methylation signatures, Signature 1 combining A HNAK2, CALHM2, and IL12RB and Signature 2 A HNAK2, CALHM2, and SLC25A38 genes, correlated with shorter overall survival (HR = 4.38, 95% CI 1.30-16.41, HR = 5.59, 95% CI 1.30-16.41; HR = 3.43, 95% CI 1.30-16.41, HR = 4.61, 95% CI 1.30-16.41 and HR = 4.95, 95% CI 1.39-17.58, respectively). Conclusions: Our results demonstrate a significant role of DNA methylation aberrancy in UM progression. The advantages of DNA as a biological material and excellent prediction accuracies of methylation markers open the perspective for their more extensive clinical use.
Phenylketonuria (PKU) and hyperphenylalaninemia (HPA) are a group of genetic disorders predominantly caused by mutations in the phenylalanine hydroxylase (PAH) gene.To date, more than 950 variants have been identified, however the pathogenic mechanism of many variants remains unknown.In this study, in silico prediction and in vitro prokaryotic and eukaryotic expression systems were used to functionally characterise five PAH missense variants (p.F233I, p.R270I, p.F331S, p.S350Y, and p.L358F) previously identified in Slovak and Czech patients.p.F233I, p.R270I, and p.S350Y were classified as deleterious mutations since they showed no specific activity in functional assay and no response to chaperone co-expression.Protein levels of these PAH variants were very low when expressed in HepG2 cells, and only p.S350Y responded to BH4 precursor overload by significant increase in PAH monomer, probably due to reduced rate of protein degradation as the result of proper protein folding.Variants p.F331S and p.L358F exerted residual enzymatic activity in vitro.While the first can be classified as probably pathogenic due to its very low protein levels in HepG2 cells, the latter is considered to be mild mutation with protein levels of approximately 17.85% compared to wt PAH.Our findings contribute to better understanding of structure and function of PAH mutated enzymes and optimal treatment of PKU patients carrying these mutations using BH4 supplementation.
Abstract Background: Despite outstanding advances in understanding the genetic background of uveal melanoma (UM) development and prognosis, the role of DNA methylation reprogramming remains elusive. This study aims to clarify the extent of DNA methylation deregulation in the context of gene expression changes and its utility as a reliable prognostic biomarker. Methods: Transcriptomic and DNA methylation landscapes in 25 high- and low-risk UMs were interrogated by Agilent SurePrint G3 Human Gene Expression 8×60K v2 Microarray and Human Infinium Methylation EPIC Bead Chip array, respectively. DNA methylation and gene expression of the nine top discriminatory genes, selected by the integrative analysis, were validated by pyrosequencing and qPCR in 58 tissues. Results: Among 2,262 differentially expressed genes discovered in UM samples differing in metastatic risk, 60 were epigenetic regulators, mostly histone modifiers and chromatin remodelers. A total of 44,398 CpGs were differentially methylated, 27,810 hypomethylated, and 16,588 hypermethylated in high-risk tumors, with Δβ values ranging between -0.78 and 0.79. By integrative analysis, 944 differentially expressed DNA methylation-regulated genes were revealed, 635 hypomethylated/upregulated, and 309 hypermethylated/downregulated. Aberrant DNA methylation in high-risk tumors was associated with the deregulation of key oncogenic pathways such as EGFR tyrosine kinase inhibitor resistance, focal adhesion, proteoglycans in cancer, PI3K-Akt signaling, or ECM-receptor interaction. Notably, the DNA methylation values of nine genes, HTR2B , AHNAK2, CALHM2, SLC25A38, EDNRB, TLR1, RNF43, IL12RB2 , and MEGF10, validated by pyrosequencing, demonstrated excellent risk group prediction accuracies (AUCs ranging between 0.870 and 0.956). Moreover, CALHM2 hypomethylation and MEGF10, TLR1 hypermethylation, as well as two three-gene methylation signatures, Signature 1 combining A HNAK2, CALHM2, and IL12RB and Signature 2 A HNAK2, CALHM2, and SLC25A38 genes, correlated with shorter overall survival (HR = 4.38, 95% CI 1.30-16.41, HR = 5.59, 95% CI 1.30-16.41; HR = 3.43, 95% CI 1.30-16.41, HR = 4.61, 95% CI 1.30-16.41 and HR = 4.95, 95% CI 1.39-17.58, respectively). Conclusions: Our results demonstrate a significant role of DNA methylation aberrancy in UM progression. The advantages of DNA as a biological material and the excellent prediction accuracies of methylation markers open the perspective for their more extensive clinical use.
Abstract Rationale: Periventricular nodular heterotopia-7 (PVNH7) is a neurodevelopmental disorder associated with improper neuronal migration during neurogenesis in cortex development caused by pathogenic variants in the NEDD4L gene. Patient concerns: We report the case of a polystigmatized 2-year-old boy having significant symptomatologic overlap with PVNH7, such as delayed psychomotor and mental development, seizures and infantile spasms, periventricular nodular heterotopia, polymicrogyria, cleft palate, 2 to 3 toe syndactyly, hypotonia, microretrognathia, strabismus, and absent speech and walking. The patient showed also distinct symptoms falling outside PVNH7 symptomatology, also present in the proband's older brother, such as blue sclerae, hydronephrosis, transversal palmar crease (found also in their father), and bilateral talipes equinovarus . In addition, the patient suffered from many other symptoms. Diagnoses: The boy, his brother and their parents were subjected to whole-exome sequencing. Because of uncertainties in symptomatology and inheritance pattern, the top-down approach was hard to apply. Using the bottom-up approach, we identified a known pathogenic variant, NM_001144967.2(NEDD4L):c.2677G>A:p.Glu893Lys, in the proband's genome that absented in any other analyzed family member, suggesting its de novo origin. Interventions and outcomes: The patient was treated with Convulex 300 mg/mL for the successful seizure control and Euthyrox 25mg for the treatment of thyroid malfunction. He also took various supplements for the metabolism support and digestion regulation. Moreover, the patient underwent the corrective surgeries of cleft palate and talipes equinovarus . Lessons: We successfully identified the causative mutation NM_001144967.2(NEDD4L):c.2677G>A:p.Glu893Lys explaining symptoms overlapping those reported for PVNH7. Symptoms shared with the brother were not explained by this variant, since he was not a carrier of the pathogenic NEDD4L variant. These are most likely not extended phenotypes of PVNH7, rather an independent clinical entity caused by a yet unidentified genetic factor in the family, highlighting thus the importance of thorough evaluation of symptomatology and genomic findings in affected and unaffected family members, when such data are available.
The molecular genetics of well-characterized inherited diseases, such as phenylketonuria (PKU) and hyperphenylalaninemia (HPA) predominantly caused by mutations in the phenylalanine hydroxylase (PAH) gene, is often complicated by the identification of many novel variants, often with no obvious impact on the associated disorder. To date, more than 1100 PAH variants have been identified of which a substantial portion have unknown clinical significance. In this work, we study the functionality of seven yet uncharacterized PAH missense variants p.Asn167Tyr, p.Thr200Asn, p.Asp229Gly, p.Gly239Ala, p.Phe263Ser, p.Ala342Pro, and p.Ile406Met first identified in the Czech PKU/HPA patients. From all tested variants, three of them, namely p.Asn167Tyr, p.Thr200Asn, and p.Ile406Met, exerted residual enzymatic activity in vitro similar to wild type (WT) PAH, however, when expressed in HepG2 cells, their protein level reached a maximum of 72.1% ± 4.9%, 11.2% ± 4.2%, and 36.6% ± 7.3% compared to WT PAH, respectively. Remaining variants were null with no enzyme activity and decreased protein levels in HepG2 cells. The chaperone-like effect of applied BH4 precursor increased protein level significantly for p.Asn167Tyr, p.Asp229Gly, p.Ala342Pro, and p.Ile406Met. Taken together, our results of functional characterization in combination with in silico prediction suggest that while p.Asn167Tyr, p.Thr200Asn, and p.Ile406Met PAH variants have a mild impact on the protein, p.Asp229Gly, p.Gly239Ala, p.Phe263Ser, and p.Ala342Pro severely affect protein structure and function.
DEAR EDITOR Uveal melanoma (UM) is a rare, aggressive cancer with limited treatment options. Despite significant advancements in understanding its genetic background,1, 2 the precise contribution of epigenomic alterations to the pathogenesis and progression of the disease remain elusive. In this study, we utilized a carefully curated set of UM samples to define the epigenomic and transcriptomic landscapes of high-risk tumours and identify novel, clinically relevant methylation markers and therapeutic targets. We stratified UM patients into risk groups based on UM-specific chromosomal rearrangements, particularly monosomy 3 (M3) and BAP1 mutations in tumour tissues (Figure 1A). Please refer to the Supporting information for a detailed description of patient clinical characteristics and methods (Additional file 1: Tables S1-S3). Specifically, we identified 25 low-risk and 33 high-risk patients, of which 21 (63.6%) carried the BAP1 mutation (Figure 1B, C). We observed extensive gene expression reprogramming in high-risk UMs, resulting in 2262 differentially expressed genes (DEGs) including 60 epigenetic regulators, histone modifiers and chromatin remodelers. Furthermore, we identified 44 398 differentially methylated CpGs, with hypermethylation more frequent in TSS1500 and CpG shores (Supporting Information Additional file 1: Figures S1, S2; Additional file 2: Tables S1-4). Integrative analysis revealed 635 hypomethylated upregulated and 309 hypermethylated downregulated genes in high-risk tumours (Figure 2A,B). A significant proportion of methylation-regulated DEGs belong to specific functional groups, including epigenetic modifiers, transcription factors, tumour suppressor genes and oncogenes (Figure 2C), demonstrating the critical role of DNA methylation in controlling cell fate. The median β values of differentially methylated CpGs, were lower in the high-risk UMs, suggesting that epigenetic gene activation can be more common than repression (Figure 2D). BAP1 expression negatively correlated with cg01493712 DNA methylation β value (r = −.496; p = .014), implying epigenetic control of BAP1 itself (Figure 2E). Aberrant DNA methylation, distributed relatively uniformly across the entire genome, was associated with the dysregulation of key oncogenic pathways such as EGFR tyrosine kinase inhibitor resistance, focal adhesion, proteoglycans in cancer, PI3K-Akt signalling, or ECM-receptor interaction (Figure 2F,G; Supporting Information Additional file 2: Table S5). These findings highlight the critical role of DNA methylation aberrancy in driving transcriptomic changes associated with poor prognosis. Based on integrative analysis findings, we selected nine candidate genes, three upregulated and six downregulated. The selection was guided by fold change (FC), Δβ values and the number of CpGs inversely correlated with gene expression. The upregulated genes were HTR2B (FC = 191.4), AHNAK2 (FC = 12.6) and CALHM2 (FC = 7.8), while the downregulated genes were SLC25A38 (FC = −4.6), EDNRB (FC = −4.7), TLR1 (FC = −8.6), RNF43 (FC = −10.8), IL12RB2 (FC = −18.1) and MEGF10 (FC = −25.2). Their expression (Supporting Information Additional file 1: Figure S3) was significantly associated with UM overall survival (OS) data, available in The Cancer Genome Atlas dataset (Supporting Information Additional file 1: Figure S4).3 The correlation between DNA methylation percentage measured by pyrosequencing in 58 UM tumours and β values were highly significant (Supporting Information Additional file 1: Table S4). In addition, individual DNA methylation values showed minimal overlap between high- and low-risk tissues (p < .001) (Figure 3A), indicating the potential use of these markers for predicting risk groups with excellent diagnostic accuracy. AUC values ranged from .870 to .956 (p < .001) (Figure 3B; Supporting Information Additional file 1: Table S5). By combining methylation values of hypomethylated AHNAK2 and CALHM2 genes with values of hypermethylated IL12RB2 (Signature 1) or SLC25A38 (Signature 2) genes, we achieved AUC values of .999 and .994, respectively (p < .001), demonstrating the robustness and potential clinical utility of these epigenetic markers in UM risk stratification. Kaplan–Meier survival curves were generated with the log-rank test, and univariate Cox regression analysis was performed to confirm that DNA methylation of CALHM2 and MEGF10 genes and both methylation signatures were sufficient to stratify patients reasonably well as the standard risk groups based on chromosomal rearrangements and mutation profiling (Figure 3C, Table 1). The DNA methylation repatterning in UM was initially attributed to the loss of BAP1, a gene coding for a deubiquitinating hydrolase that exerts diverse functions such as cell cycle regulation, DNA damage repair, chromatin remodelling and gene expression control.4 Although UM is considered poorly immunogenic due to its immune-privileged site of origin, it has been proposed that BAP1 loss may promote the immunosuppressive tumour microenvironment (TME).5 UM is a unique tumour type in which a high density of tumour-infiltrating lymphocytes and tumour-associated macrophages paradoxically correlates with a worse prognosis, highlighting the complex interaction between the TME and the immune response. Epigenetic regulations play a critical role in shaping these intricate relations.6 Accordingly, six of the top nine methylation-regulated genes have been previously linked to immune functions. Specifically, EDNRB, IL12RB2, CALHM2 and RNF43, were identified among UM prognostic genes that interact with immune and stromal cells in the TME.7 IL-12Rβ2, a subunit of the IL-12 receptor, generates high-affinity binding sites for IL-12, one of the most potent antitumor cytokines.8 The prognostic significance of CALHM2 and RNF43 is further reinforced by their listing among the most important DEGs related to UM survival.3 Additionally, AHNAK2, shown to promote UM cell proliferation and migration,9 was found to correlate with infiltration of immune cell subpopulations such as CD8+ and CD4+.10 Our findings align with a recent report by Figueiredo et al.,5 which revealed that the downregulation of TLR1, a gene responsible for immune activation, is correlated with M3 status but not BAP1 expression, indicating epigenomic reprogramming independent of BAP1 mutations. These results highlight the importance of further exploring epigenetic regulation of the unique immune landscape of UM, which presents both challenges and opportunities for developing effective treatments for high-risk patients. Overall, our study provides compelling evidence for the substantial role of DNA methylation in UM progression by regulating the expression of genes involved in critical biological processes such as immune evasion, calcium homeostasis, adhesion and migration. Importantly, we demonstrate that the DNA methylation status of carefully selected CpG sites has the potential to serve as reliable prognostic biomarkers, underscoring the clinical relevance of DNA methylation analysis in UM. By leveraging the power of epigenetic profiling, we can gain a powerful tool for patient stratification, which can aid in personalized therapy and ultimately lead to improved outcomes. Foremost, we express our gratitude to all patients who consented to participate in this study. We would also like to thank Dr. Andrea Štanclova (Department of Molecular Biology and Genomics, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Bratislava, Slovakia) for her technical assistance and valuable contributions. Finally, we thank the Slovak Cancer Research Foundation for their enduring assistance and support. The authors declare that they have no competing interests. This research was funded by the Slovak Research and Development Agency Grant number APVV-17-0369, The Ministry of Education, Science, Research and Sport of the Slovak Republic, Grant number VEGA 1/0395/21 and LISPER (ITMS 313011V446: Integrative strategy in the development of personalized medicine of selected malignant tumors and its impact on quality of life. Operational program integrated infrastructure 2014−2020) projects. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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