Estudio genómico de la incongruencia de género mediante la tecnología de microarrays

Programa Oficial de Doutoramento en Química Ambiental e Fundamental. 5031V01[Resumo] A incongruencia de xénero defínese no CIE-11 (World Health Organization, 2018) como unha marcada e persistente discordancia entre o xénero experimentado e o sexo asignado ao nacer. A súa orixe é complexa e multifactorial. Obxectivo: O obxectivo principal desta investigación centrouse na análise de diferentes polimorfismos xenéticos nunha poboación transxénero en contraste cunha poboación cisxénero con similares características. Na primeira parte analizáronse catro polimorfismos localizados no promotor do xene do receptor de estróxenos alfa (ESR1). Na segunda parte analizáronse 247 polimorfismos situados nos cofactores NCoA-1, NCoA-2, NCoA-3, NCoA-4, NCoA-5 e p300-CREBBP dada a íntima relación existente entre os esteroides e os coactivadores de esteroides. Finalmente, na terceira parte desta investigación analizáronse 242 polimorfismos situados no ADN mitocondrial dada a ausencia total de investigación relacionada ca incongruencia de xénero. Material e métodos: En todos os polimorfismos analizáronse as frecuencias alélicas e xenotípicas mediante o test de χ2, comparando as poboacións cis- e transxénero respecto a o seu sexo xenético. A forza de asociación de cada polimorfismo coa incongruencia de xénero mediuse mediante regresión loxística binaria. Respecto ao único polimorfismo de repetición (C2), analizouse o número de repeticións en cada poboación coa U de Mann- Whitney. O desequilibrio de ligamento e a estimación das frecuencias haplotípicas foron tamén analizados. Resultados: Respecto ao xene ESR1 atopouse que a media de repeticións para o polimorfismo C2 era máis baixa na poboación de homes transxénero que na poboación cisxénero. Os xenotipos S/S e S/L atopábanse sobrerrepresentados na poboación de homes transxénero (P<0,012 e P<0,003 respectivamente). Tamén se atopou unha sobrerrepresentación do xenotipo A/A para o polimorfismo C4 na poboación de homes transxénero (P<0,017), mentres que o xenotipo A/G estaba sobrerrepresentado na poboación cisxénero (P<0,009]. En canto ao estudo dos cofactores, atopáronse diferenzas significativas en once polimorfismos localizados en NCoA-1, NCoA-2, e p300-CREBBP, sendo os coactivadores NCoA-2 e p300-CREBBP, os que presentaron maior número de polimorfismos con significación estatística (5/64 e 2/9 respectivamente). Ademais, os polimorfismos P2 (localizado en NCoA-1), P9 e P10 (localizados en p300-CREBBP) mostraron diferente distribución xenotípica dependendo da covariable sexo. Respecto aos polimorfismos mitocondriales, atopáronse diferenzas significativas (P<0,05) en 26 polimorfismos. Só un deles pasou a corrección de Bonferroni (P<0,0002), estando ligado aos xenes MT-ND4 e MT-ND5 (OR=17,33). Conclusión: Os xenes ESR1, NCoA-1, NCoA-2, p300-CREBBP, MT-ND4 e MT-ND5 pódense considerar xenes implicados na base biolóxica da incongruencia de xénero.[Resumen] La marcada y persistente discordancia entre el género experimentado y el sexo asignado al nacer se define en el CIE-11 (World Health Organization, 2018) como incongruencia de género. Su origen es complejo y multifactorial. Objetivo: El objetivo principal de esta investigación se centró en el análisis de diferentes polimorfismos genéticos en una población transgénero en contraste con una población cisgénero de similares características. En la primera parte del estudio se analizaron cuatro polimorfismos localizados en el promotor del gen del receptor de estrógenos alfa (ESR1). En la segunda parte se analizaron 247 polimorfismos situados en las moléculas coactivadoras de esteroides NCoA-1, NCoA-2, NCoA-3, NCoA-4, NCoA-5 y p300- CREBBP dada la íntima relación existente entre los esteroides y los coactivadores de esteroides. Finalmente se analizaron 242 polimorfismos situados en el ADN mitocondrial, dada la ausencia total de investigación relacionada con la incongruencia de género. Material y métodos: En todos los polimorfismos se analizaron las frecuencias alélicas y genotípicas mediante el test de χ2, comparando las poblaciones cis- y transgénero según el sexo genético. La fuerza de asociación de cada polimorfismo con la incongruencia de género se midió mediante regresión logística binaria. Respecto al único polimorfismo de repetición (C2), se midió el número de repeticiones en cada población y se analizó mediante la U de Mann-Whitney. También se realizaron análisis de desequilibrio de ligamiento y estimación de las frecuencias haplotípicas. Resultados: Respecto al gen ESR1 se encontró que la media de repeticiones para el polimorfismo C2 era más baja en la población de hombres transgénero que en la población cisgénero. Los genotipos S/S y S/L estaban sobrerrepresentados en la población de hombres transgénero (P<0,012 y P<0,003 respectivamente). También se encontró una sobrerrepresentación del genotipo A/A para el polimorfismo C4 en la población de hombres transgénero (P<0,017), mientras que el genotipo A/G estaba sobrerrepresentado en la población cisgénero (P<0,009]. En cuanto al estudio de los cofactores, se encontraron diferencias significativas en once polimorfismos localizados en NCoA-1, NCoA-2, y p300-CREBBP. Siendo los coactivadores NCoA-2 y p300-CREBBP los que presentaron mayor número de polimorfismos con significación estadística (5/64 y 2/9 respectivamente). Además, los polimorfismos P2 (localizado en NCoA-1), P9 y P10 (localizados en p300-CREBBP) mostraron diferente distribución genotípica dependiente de la covariable sexo. Respecto a los polimorfismos mitocondriales, se encontraron diferencias significativas (P<0,05) en 26 polimorfismos; sólo uno de ellos pasó la corrección de Bonferroni (P<0,0002), estando ligado a los genes MT-ND4 y MT-ND5 (OR=17,33). Conclusión: Los genes ESR1, NCoA-1, NCoA-2, p300-CREBBP, MT-ND4 y MT-ND5 se pueden considerar genes implicados en la incongruencia de género.[Abstract] Gender incongruence is defined in the ICD-11 (World Health Organization, 2018) as a marked and persistent disagreement between the experienced gender and the assigned natal sex. Its origin is complex and multifactorial. Objective: The main objective was focused on the analysis of different DNA polymorphisms in a transgender population in contrast to a cisgender population. The first part of the investigation involved the analysis of four polymorphisms that are encoded in the promoter of the estrogen receptor alpha (ESR1) gene. The second part focused on the study of 247 polymorphisms situated in the coactivator molecules NCoA-1, NCoA-2, NCoA-3, NCoA-4, NCoA-5 and p300-CREBBP given the close relationship between steroids and coactivators of steroids. Finally, 242 polymorphisms located in the mitochondrial DNA and their relationship with gender incongruence were analyzed, given the total absence of research related to this topic. Material and methods: In all polymorphisms, allele and genotypic frequencies were analyzed using the χ2 test, comparing the cis- and transgender populations with respect to their natal sex. The strength of association of each polymorphism with gender incongruence was measured using binary logistic regression. Regarding the only repeated polymorphism analyzed (C2), the number of repetitions was analyzed with the Mann- Whitney U test. Linkage disequilibrium and haplotype frequency analyses were also performed. Results: Regarding the ESR1 gene, it was found that the mean of repetitions for the C2 polymorphism was smaller in the transgender men population than in the cisgender population. The S/S and S/L genotypes were overrepresented in the transgender men population (P<0.012 and P<0.003 respectively). An overrepresentation of the A/A genotype was also found for the C4 polymorphism in the transgender men population (P<0.017), while the A/G was overrepresented in the cisgender population (P<0.009]. Regarding the study of cofactor molecules, significant differences were found in eleven polymorphisms located in NCoA-1, NCoA-2, and p300-CREBBP. Coactivators NCoA-2 and p300-CREBBP being those with the highest number of polymorphisms with statistical significance (5/64 and 2/9 respectively). Furthermore, only polymorphisms P2 (located in NCoA-1), P9 and P10 (located in p300-CREBBP) showed different genotypic distribution depending on the covariate “sex”. Regarding mitochondrial polymorphisms, significant differences (P<0.05) were found in 26 polymorphisms; only one of them passed the Bonferroni correction (P<0.0002), being linked to the MT-ND4 and MT-ND5 genes, with an OR=17.33. Conclusion: The ESR1, NCoA-1, NCoA-2, p300-CREBBP, MT-ND4 and MT-ND5 genes can be considered to be involved in gender incongruenc

Analysis of Four Polymorphisms Located at the Promoter of the Estrogen Receptor Alpha ESR1 Gene in a Population With Gender Incongruence

[Abstract] Introduction: Gender incongruence defines a state in which individuals feel discrepancy between the sex assigned at birth and their gender. Some of these people make a social transition from male to female (transwomen) or from female to male (trans men). By contrast, the word cisgender describes a person whose gender identity is consistent with their sex assigned at birth. Aim: To analyze the implication of the estrogen receptor a gene (ESR1) in the genetic basis of gender incongruence. Main Outcome Measures: Polymorphisms rs9478245, rs3138774, rs2234693, rs9340799. Method: We carried out the analysis of 4 polymorphisms located at the promoter of the ESR1 gene (C1 ¼ rs9478245, C2 ¼ rs3138774, C3 ¼ rs2234693, and C4 ¼ rs9340799) in a population of 273 trans women, 226 trans men, and 537 cis gender controls. For SNP polymorphisms, the allele and genotype frequencies were analyzed by c2 test. The strength of the SNP associations with gender incongruence was measured by binary logistic regression. For the STR polymorphism, the mean number of repeats were analyzed by the ManneWhitney U test. Measurement of linkage disequilibrium and haplotype frequencies were also performed. Results: The C2 median repeats were shorter in the trans men population. Genotypes S/S and S/L for the C2 polymorphism were overrepresented in the trans men group (P ¼ .012 and P ¼ .003 respectively). We also found overtransmission of the A/A genotype (C4) in the trans men population (P ¼ .017), while the A/G genotype (C4) was subrepresented (P ¼ .009]. The analyzed polymorphisms were in linkage disequilibrium. In the trans men population, the T(C1)-L(C2)-C(C3)-A(C4) haplotype was overrepresented (P ¼ .019) while the T(C1)-L(C2)-C(C3)-G(C4) was subrepresented (P ¼ .005). Conclusion: The ESR1 is associated with gender incongruence in the trans men populationThis work was supported by grants: ED431B 019/02 (EP), PGC2018-094919-B-C21 (AG), PGC2018-094919-B-C22 (RF), and FPU 15/02558 (JCC)Xunta de Galicia; ED431B 019/0

Implications of the Estrogen Receptor Coactivators SRC1 and SRC2 in the Biological Basis of Gender Incongruence

[Abstract] Introduction Brain sexual differentiation results from the effects of sex steroids on the developing brain. The presumptive route for brain masculinization is the direct induction of gene expression via activation of the estrogen receptors α and β and the androgen receptor through their binding to ligands and to coactivators, regulating the transcription of multiple genes in a cascade effect. Aim To analyze the implication of the estrogen receptor coactivators SRC-1, SRC-2, and SRC-3 in the genetic basis of gender incongruence. Main Outcome Measures Analysis of 157 polymorphisms located at the estrogen receptor coactivators SRC-1, SRC-2, and SRC-3, in 94 transgender versus 94 cisgender individuals. Method Using SNPStats software, the allele and genotype frequencies were analyzed by χ2, the strength of the association was measured by binary logistic regression, estimating the odds ratio for each genotype. Measurements of linkage disequilibrium and haplotype frequencies were also performed. Results We found significant differences at level P < .05 in 8 polymorphisms that correspond to 5.09% of the total. Three were located in SRC-1 and 5 in SRC-2. The odds ratio analysis showed significant differences at level P < .05 for multiple patterns of inheritance. The polymorphisms analyzed were in linkage disequilibrium. The SRC-1 haplotypes CGA and CGG (global haplotype association P < .009) and the SRC-2 haplotypes GGTAA and GGTAG (global haplotype association P < .005) were overrepresented in the transgender population. Conclusion The coactivators SRC-1 and SRC-2 could be considered as candidates for increasing the list of potential genes for gender incongruence. Ramírez KDV, Fernández R, Delgado-Zayas E, et al. Implications of the Estrogen Receptor Coactivators SRC1 and SRC2 in the Biological Basis of Gender Incongruence. Sex Med 2021;9:100368.This work was supported by grants: ED431B 019/02 (EP), PGC2018-094919-B-C21 (AG), PGC2018-094919-B-C22 (RF and EP

Gender-Affirming Hormone Therapy Modifies the CpG Methylation Pattern of the ESR1 Gene Promoter After Six Months of Treatment in Transmen

[Abstract] Background Brain sexual differentiation is a process that results from the effects of sex steroids on the developing brain. Evidence shows that epigenetics plays a main role in the formation of enduring brain sex differences and that the estrogen receptor α (ESR1) is one of the implicated genes. Aim To analyze whether the methylation of region III (RIII) of the ESR1 promoter is involved in the biological basis of gender dysphoria. Methods We carried out a prospective study of the CpG methylation profile of RIII (−1,188 to −790 bp) of the ESR1 promoter using bisulfite genomic sequencing in a cisgender population (10 men and 10 women) and in a transgender population (10 trans men and 10 trans women), before and after 6 months of gender-affirming hormone treatment. Cisgender and transgender populations were matched by geographical origin, age, and sex. DNAs were treated with bisulfite, amplified, cloned, and sequenced. At least 10 clones per individual from independent polymerase chain reactions were sequenced. The analysis of 671 bisulfite sequences was carried out with the QUMA (QUantification tool for Methylation Analysis) program. Outcomes The main outcome of this study was RIII analysis using bisulfite genomic sequencing. Results We found sex differences in RIII methylation profiles in cisgender and transgender populations. Cismen showed a higher methylation degree than ciswomen at CpG sites 297, 306, 509, and at the total fragment (P ≤ .003, P ≤ .026, P ≤ .001, P ≤ .006). Transmen showed a lower methylation level than trans women at sites 306, 372, and at the total fragment (P ≤ .0001, P ≤ .018, P ≤ .0107). Before the hormone treatment, transmen showed the lowest methylation level with respect to cisgender and transgender populations, whereas transwomen reached an intermediate methylation level between both the cisgender groups. After the hormone treatment, transmen showed a statistically significant methylation increase, whereas transwomen showed a non-significant methylation decrease. After the hormone treatment, the RIII methylation differences between transmen and transwomen disappeared, and both transgender groups reached an intermediate methylation level between both the cisgender groups. Clinical Implications Clinical implications in the hormonal treatment of trans people. Strengths & Limitations Increasing the number of regions analyzed in the ESR1 promoter and increasing the number of tissues analyzed would provide a better understanding of the variation in the methylation pattern. Conclusions Our data showed sex differences in RIII methylation patterns in cisgender and transgender populations before the hormone treatment. Furthermore, before the hormone treatment, transwomen and transmen showed a characteristic methylation profile, different from both the cisgender groups. But the hormonal treatment modified RIII methylation in trans populations, which are now more similar to their gender. Therefore, our results suggest that the methylation of RIII could be involved in gender dysphoria.This work was supported by grants: Xunta de Galicia ED431 B 019/02 (EP), PGC2018-094919-B-C21 (AG), Ministerio de ciencia, innovación y Universidades PGC2018-094919-B-C22 (RF, EP). J. Cortés-Cortés was supported by a doctoral fellowship FPU 15/02558Xunta de Galicia ED431 B 019/0

Epigenetics Is Implicated in the Basis of Gender Incongruence: An Epigenome-Wide Association Analysis

[Abstract] Introduction: The main objective was to carry out a global DNA methylation analysis in a population with gender incongruence before gender-affirming hormone treatment (GAHT), in comparison to a cisgender population. Methods: A global CpG (cytosine-phosphate-guanine) methylation analysis was performed on blood from 16 transgender people before GAHT vs. 16 cisgender people using the Illumina© Infinium Human Methylation 850k BeadChip, after bisulfite conversion. Changes in the DNA methylome in cisgender vs. transgender populations were analyzed with the Partek® Genomics Suite program by a 2-way ANOVA test comparing populations by group and their sex assigned at birth. Results: The principal components analysis (PCA) showed that both populations (cis and trans) differ in the degree of global CpG methylation prior to GAHT. The 2-way ANOVA test showed 71,515 CpGs that passed the criterion FDR p < 0.05. Subsequently, in male assigned at birth population we found 87 CpGs that passed both criteria (FDR p < 0.05; fold change ≥ ± 2) of which 22 were located in islands. The most significant CpGs were related to genes: WDR45B, SLC6A20, NHLH1, PLEKHA5, UBALD1, SLC37A1, ARL6IP1, GRASP, and NCOA6. Regarding the female assigned at birth populations, we found 2 CpGs that passed both criteria (FDR p < 0.05; fold change ≥ ± 2), but none were located in islands. One of these CpGs, related to the MPPED2 gene, is shared by both, trans men and trans women. The enrichment analysis showed that these genes are involved in functions such as negative regulation of gene expression (GO:0010629), central nervous system development (GO:0007417), brain development (GO:0007420), ribonucleotide binding (GO:0032553), and RNA binding (GO:0003723), among others. Strengths and Limitations: It is the first time that a global CpG methylation analysis has been carried out in a population with gender incongruence before GAHT. A prospective study before/during GAHT would provide a better understanding of the influence of epigenetics in this process. Conclusion: The main finding of this study is that the cis and trans populations have different global CpG methylation profiles prior to GAHT. Therefore, our results suggest that epigenetics may be involved in the etiology of gender incongruence.Xunta de Galicia; ED431 B 019/02 (EP) Ministerio de Ciencia, Innovación y Universidades; PGC2018-094919-B-C21 (AG) e PGC2018-094919-B-C22 (RF and EP) Ghent University.; BOF interdisciplinary project (IOP003-18

The Biological Basis of Gender Incongruence

Gender incongruence (GI) is defined as an individual’s discontent with their assigned gender at birth and their identification with a gender other than that associated with their sex based on physical sex characteristics. The origin of GI appears to be multifactorial. From the extensive research that has been conducted over the past few years, four main factors have been identified as key mechanisms: genes, hormones, epigenetics, and the environment. One of the current hypotheses suggests that GI could be related to a different sexual differentiation of the brain as a result of changes in the DNA sequence of the estrogen receptors ERs and androgen receptor AR genes. These changes in the DNA sequence would imply a variability in the sensitivity of the hormone receptors, causing a genetic vulnerability

Implications of the Estrogen Receptor Coactivators SRC1 and SRC2 in the Biological Basis of Gender Incongruence

ABSTRACT: Introduction: Brain sexual differentiation results from the effects of sex steroids on the developing brain. The presumptive route for brain masculinization is the direct induction of gene expression via activation of the estrogen receptors α and β and the androgen receptor through their binding to ligands and to coactivators, regulating the transcription of multiple genes in a cascade effect. Aim: To analyze the implication of the estrogen receptor coactivators SRC-1, SRC-2, and SRC-3 in the genetic basis of gender incongruence. Main Outcome Measures: Analysis of 157 polymorphisms located at the estrogen receptor coactivators SRC-1, SRC-2, and SRC-3, in 94 transgender versus 94 cisgender individuals. Method: Using SNPStats software, the allele and genotype frequencies were analyzed by χ2, the strength of the association was measured by binary logistic regression, estimating the odds ratio for each genotype. Measurements of linkage disequilibrium and haplotype frequencies were also performed. Results: We found significant differences at level P < .05 in 8 polymorphisms that correspond to 5.09% of the total. Three were located in SRC-1 and 5 in SRC-2. The odds ratio analysis showed significant differences at level P < .05 for multiple patterns of inheritance. The polymorphisms analyzed were in linkage disequilibrium. The SRC-1 haplotypes CGA and CGG (global haplotype association P < .009) and the SRC-2 haplotypes GGTAA and GGTAG (global haplotype association P < .005) were overrepresented in the transgender population. Conclusion: The coactivators SRC-1 and SRC-2 could be considered as candidates for increasing the list of potential genes for gender incongruence. Ramírez KDV, Fernández R, Delgado-Zayas E, et al. Implications of the Estrogen Receptor Coactivators SRC1 and SRC2 in the Biological Basis of Gender Incongruence. Sex Med 2021;9:100368