DNA-Methylation Patterns in Trisomy 21 Using Cells from Monozygotic Twins

DNA methylation is essential in mammalian development. We have hypothesized that methylation differences induced by trisomy 21 (T21) contribute to the phenotypic characteristics and heterogeneity in Down syndrome (DS). In order to determine the methylation differences in T21 without interference of the interindividual genomic variation, we have used fetal skin fibroblasts from monozygotic (MZ) twins discordant for T21. We also used skin fibroblasts from MZ twins concordant for T21, normal MZ twins without T21, and unrelated normal and T21 individuals. Reduced Representation Bisulfite Sequencing (RRBS) revealed 35 differentially methylated promoter regions (DMRs) (Absolute methylation differences = 25%, FDR < 0.001) in MZ twins discordant for T21 that have also been observed in comparison between unrelated normal and T21 individuals. The identified DMRs are enriched for genes involved in embryonic organ morphogenesis (FDR = 1.60 e -03) and include genes of the HOXB and HOXD clusters. These DMRs are maintained in iPS cells generated from this twin pair and are correlated with the gene expression changes. We have also observed an increase in DNA methylation level in the T21 methylome compared to the normal euploid methylome. This observation is concordant with the up regulation of DNA methyltransferase enzymes (DNMT3B and DNMT3L) and down regulation of DNA demethylation enzymes (TET2 and TET3) observed in the iPSC of the T21 versus normal twin. Altogether, the results of this study highlight the epigenetic effects of the extra chromosome 21 in T21 on loci outside of this chromosome that are relevant to DS associated phenotypes

DPY19L2, its mutation in about half globozoospermia

International audienceIntroduction: Testicular sperm extraction (TESE) combined with intracytoplasmic sperm injection is a promising assessment in reproductive practice particularly for patients with non obstructive azoospermia (NOA). There was evidence that impaired spermatogenesis could be related to an imbalance in the intratesticular oestradiol/testosterone ratio. We carried out a prospective observational study in order to evaluate the putative variation of the expression of genes implicated in the estrogen synthesis (aromatase) and mediation of estrogen action (estrogen receptors and GRP30 for the respective initiation of genomic and non-genomic pathways) in human testicular biopsies and to understand the mechanisms involved in different testicular disorders in relation to NOA such as hypospermatogenesis (Hsg), germ cell arrest (GCA) and Sertoli Cell Only (SCO) syndrome. Material and Methods: Histological evaluation, sperm recovery and ARN extraction followed by the measurement of relative mRNA level of cyp19, Esr1, Esr2 and gpr30 using real time polymerase chain reaction were realized in testicular bilateral fragments (n = 98) providing from 49 azoospermic patients. Taking into account the existence of potential discordant patterns in bilateral biopsies, the high prevalence of mixed patterns in a same testes and the fact that histological evaluation was always performed in a testicular biopsy different from this studied, we have reported the expression of specific genes considered as cells markers (Prm1 for round spermatids, H1t for pachytene spermatocytes and vimentin for mature Sertoli cells) for the selection of pure and homogeneous NOA forms. Then the expression of genes encoding for aromatase, estrogen receptors and GPR 30 has been evaluated in obstructive azoospermia group (0A) used as control and NOA groups (Hsg,GCA and SCO). Results are expressed as means + S.E.M. Statistical analysis was performed using ANOVA (Graphpad Instat 3, GraphPad Software, San Diego, CA, USA) and means are compared using Tukey-Kramer multiple comparisons test. Statistical significance was accepted at p < 0.05. Results: We have at first described specific patterns of pure forms of Hsg, GCA, SCO and OA with the helpful of cell markers. A pure form of SCO could be defined as a relative expression of vimentin transcript higher than 2 associated with an absence of Prm 1 or H1t transcripts. The level of Prm1 transcripts and the ratio Prm1 mRNA/H1t mRNA are significantly correlated with the number of spermatozoa recovered by TESE. A reduced expression of GRP30 is observed in all groups but seems more elevated in GCA group. Levels of the two isoforms ERalpha and ERbeta transcripts are significantly increased in OA and Hsg groups. But only the ERalpha level is strongly correlated with that of Prm1 and sperm recovery. Aromatase expression doest not differ significantly in the four groups studied. However we have found an intensive expression of aromatase and ERalpha in the SCO group associated with Leydig cell hyperplasia. Conclusions: Studying the putative variation of transcripts implicated in the estrogen synthesis and mediation of estrogen action in testicular biopsies could represent a helpful for the understanding of mechanisms involved in the pathogenesis of NOA forms and bring new insights about the role of estrogens during spermatogenesis. GRP30 expression seems to be restricted to testicular cells implicated in the first steps of spermatogenesis. The relative important expression of the two isoforms ERalpha and ERbeta in post-meiotic cells suggests their role during spermiogenesis. But an enhanced expression of ERalpha in Leydig cell hyperplasia and a tight correlation between ERalpha and Prm1 expression could argue the case for a differential role of the two ER isoforms during spermatogenesis