Análisis del perfil de metilación CpG del ADN genómico de una población con incongruencia de género

Abstract

Gender incongruence in the ICD-11 (International Classification of Diseases-11) is characterized by “a persistent incongruence between the individual's experience of gender and the sex assigned at birth”. The origin of gender incongruence is complex and multifactorial. Aim: The first part of the research focused on the prospective CpG (cytosine-phosphate-guanine) analysis of the fragment III (RIII) of the estrogen receptor α promoter, in a population with gender incongruence before vs after six months of gender affirming hormone treatment (GAHT). The second part focused on the prospective global CpG methylation using the Illumina© Infinium Human Methylation 850k BeadChip. Material and Methods: The analysis of the CpG profile of the RIII of the estrogen receptor α promoter was carried out using bisulfite genomic sequencing in 20 cis gender and 20 trans gender people before vs after six months of GAHT. DNAs were bisulfited, amplified, cloned, and sequenced. The statistical analysis was carried out with the QUMA (QUantification tool for Methylation Analysis) program. The global CpG analysis was performed on blood from 16 trans gender people before vs after GAHT, in comparison to 16 cis gender people using the Illumina© Infinium Human Methylation 850k BeadChip, after bisulfite conversion. Global methylation profiles were analyzed with the Partek® Genomics Suite® program by a 3-way ANOVA test comparing populations by group, sex and treatment. An enrichment analysis was performed with the Partek® Pathway® program and the WebGestalt. Results: The first part of this study showed (i) that in cis and trans populations, men and women had different RIII methylation patterns; (ii) that before the GAHT, both transgender groups showed a characteristic RIII methylation profile, which did not match any of the cisgender groups; (iii) that GAHT modified the RIII methylation pattern towards more similar methylation profiles to the corresponding gender. The main result of the global methylation study is that the cis and trans populations differ in the degree of methylation before GAHT treatment. In the male population (according to the assignment at birth), 22 CpG islands were found that passed the statistic criteria (FDR p <0.05; fold change ≥ ± 2). The most significant CpGs were related to the genes WDR45, SLC6A20, NHLH1, PLEKHA5, UBALD1, SLC37A1, ARL6IP1, GRASP and NCOA6. Regarding the female population (according to the assignment at birth), 2 CpGs were found that passed the statistical criteria, but none of them were located in islands. One of these CpGs, the one related to the MPPED2 gene, is shared by both trans men and trans women. Enrichment analysis showed that these genes are involved in important functions such as negative regulation of gene expression (GO: 0010629), development of the central nervous system (GO: 0007417), brain development (GO: 0007420), ribonucleotide binding (GO: 0032553) and RNA binding (GO: 0003723). Furthermore, six months of GAHT treatment modified the methyloma in both trans populations. The before vs after methylome study revealed a significant decrease in methylation in both trans populations. Trans men showed variations in 95 CpG islands, of which 72.63% decreased the degree of methylation after treatment. Regarding trans women, six months of GAHT modified the methylation in 78 CpGs islands, of which 85.9% were hypomethylated after treatment. Thus, in both trans populations, GAHT induced a significant reduction in methylation.