Table_1_Differentially Methylated Regions in Desmoid-Type Fibromatosis: A Comparison Between CTNNB1 S45F and T41A Tumors.xlsx

IntroductionThe majority of desmoid-type fibromatosis (DTF) tumors harbor a β-catenin mutation, affecting specific codons in CTNNB1 exon 3. S45F tumors are reported to have a higher chance of recurrence after surgery and more resistance to systemic treatments compared to wild-type (WT) and T41A tumors. The aim of this pilot study was to examine the genome-wide DNA methylation profiles of S45F and T41A mutated DTF, to explain the observed differences in clinical behavior between these DTF subtypes.
Material and MethodsGenome-wide analysis of DNA methylation was performed using MeD-seq on formalin-fixed, paraffin-embedded primary DTF samples harboring a S45F (n = 14) or a T41A (n = 15) mutation. Differentially methylated regions (DMRs) between S45F and T41A DTF were identified and used for a supervised hierarchical cluster analysis. DMRs with a fold-change ≥1.5 were considered to be differentially methylated and differences between S45F and T41A tumors were quantitatively assessed. The effect of DMRs on the expression of associated genes was assessed using an independent mRNA expression dataset. Protein-protein interactions between WT β-catenin and mutant variants and DNA methyltransferase 1 (DNMT1) were examined by immunoprecipitation experiments.
ResultsMeD-seq analyses indicated 354 regions that displayed differential methylation. Cluster analysis yielded no distinct clusters based on mutation, sex, tumor site or tumor size. A supervised clustering based on DMRs between small (≤34 mm) and large (>87 mm) DTF distinguished the two groups. Only ten DMRs displayed a fold change of ≥1.5 and six of them were found associated with the following genes: NLRP4, FOXK2, PERM1, CCDC6, NOC4L, and DUX4L6. The effects of DMRs on gene expression yielded a significant difference (p < 0.05) in the expression between S45F and T41A for CCDC6 and FOXK2 but not for all Affymetrix probe-sets used to detect these genes. Immunoprecipitations did not reveal an association of WT β-catenin or mutant variants with DNMT1.
ConclusionThis study demonstrated that S45F and T41A DTF tumors did not exhibit gross differences in DNA methylation patterns. This implies that distinct DNA methylation profiles are not the sole determinant for the divergent clinical behavior of these different DTF mutant subtypes.

引言
绝大多数韧带样型纤维瘤病（desmoid-type fibromatosis, DTF）肿瘤携带β-连环蛋白（β-catenin）突变，该突变累及CTNNB1基因第3外显子的特定密码子。相较于野生型（wild-type, WT）及T41A型肿瘤，S45F型肿瘤术后复发风险更高，且对系统性治疗的耐药性更强。本预实验旨在分析携带S45F与T41A突变的DTF的全基因组DNA甲基化谱，以阐释不同DTF亚型临床行为的差异。

材料与方法
本研究针对携带S45F（n=14）或T41A（n=15）突变的原发性福尔马林固定石蜡包埋DTF样本，采用甲基化DNA免疫沉淀测序（MeD-seq）开展全基因组DNA甲基化分析。鉴定S45F与T41A型DTF间的差异甲基化区域（differentially methylated regions, DMRs），并将其用于监督分层聚类分析。将折叠变化≥1.5的DMRs认定为存在差异甲基化，随后对S45F与T41A型肿瘤的甲基化差异进行定量评估。利用独立的mRNA表达数据集，评估DMRs对相关基因表达的影响。采用免疫共沉淀实验，检测野生型β-连环蛋白及其突变体与DNA甲基转移酶1（DNA methyltransferase 1, DNMT1）之间的蛋白质相互作用。

结果
MeD-seq分析共鉴定出354个存在甲基化差异的区域。聚类分析未发现基于突变类型、性别、肿瘤部位或肿瘤大小的显著聚类分组。基于肿瘤大小≤34mm与>87mm的DTF样本间的DMRs进行监督聚类，可有效区分这两组样本。仅10个DMRs的折叠变化≥1.5，其中6个与下述基因相关：NLRP4、FOXK2、PERM1、CCDC6、NOC4L及DUX4L6。DMRs对基因表达的影响分析显示，CCDC6与FOXK2的表达在S45F与T41A型肿瘤间存在显著差异（p<0.05），但并非所有用于检测这两个基因的Affymetrix探针集均体现出该差异。免疫共沉淀实验未发现野生型β-连环蛋白及其突变体与DNMT1存在相互作用。

结论
本研究证实，S45F与T41A型DTF肿瘤的DNA甲基化模式无显著整体差异。这提示，独特的DNA甲基化谱并非决定不同DTF突变亚型临床行为差异的唯一因素。