SF1 transactivation of the AMH gene through binding to a specific site of the AMH proximal promoter is essential for Mullerian duct regression in humans

NATHALIE DI CLEMENTE; JOSSO NATHALIE; VALERI CLARA; MARSHALL I; REY RODOLFO A.; SCHTEINGART HELENA; PICARD JEAN-YVES

Copenhagen

Simposio; 6th International DSD Symposium; 2017

International Federation Of Infant and Juvenile Gynecology

Regression of Müllerian ducts, the anlagen of the uterus and Fallopian tubes, in the male fetus depends on the action of anti-Müllerian hormone (AMH) during a short developmental window, after which Müllerian ducts become insensitive to AMH. Therefore, AMH expression needs to be tightly regulated. Failure of AMH action during the specific window in the 46,XY fetus underlies the Persistent Müllerian Duct Syndrome (PMDS), characterised by the presence of uterus and Fallopian tubes in a newborn with normally virilized external genitalia. SOX9, SF1, GATA4, WT1 and DAX1 are major modulators of AMH expression in early fetal life in the mouse. However, their relevance in humans is less clear owing to species differences. For instance, SF1 knockout in mice results in a complete lack of testis development and feminisation of the XY fetus, whereas in humans SF1 mutations lead to variable phenotypes.Two SF1 consensus sites are present in the human AMH proximal promoter: one 228-222 bp and one 102-96 bp upstream of the AMH transcription start site. In vitro studies using luciferase reporter assays showed that when the -228 or the -102 SF1 elements was modified by site-directed mutagenesis, the transcriptional activity of a 0.4-kb AMH promoter was significantly impaired (0.66 ± 0.05 RLU for the -228 mutation and 0.41 ± 0.17 RLU for the -102 mutations, as compared to 1.03 ± 0.07 RLU for the wild-type (WT) promoter, ANOVA + Bonferroni test, n=9). However, in a longer AMH promoter context (3 kb), only the mutation of the -228 site significantly impaired the promoter activity (0.35 ± 0.06 RLU for -228, 0.84 ± 0.06 for -102, and 1.02 ± 0.07 for WT, ANOVA + Bonferroni test, n=9). These results indicate that in experimental in vitro conditions, the SF1 element at -228 of the human AMH promoter is relevant for AMH expression.Recently, the diagnosis of PMDS due to AMH deficiency was made in a non-dysmorphic newborn who presented with a normal sized penis and non-palpable gonads. Laboratory work-up showed normal serum testosterone (358 ng/dl), extremely low serum AMH (7.8 pmol/l) and a 46,XY karyotype. A sonogram and MRI showed a uterus measuring 5 x 1.4 x 1.9 cm; VCUG showed a normal male urethra without a urogenital sinus. Unexpectedly, DNA sequencing detected no mutations in the AMH gene coding sequences. However, a homozygous single-base deletion (c.-225delA) was identified, coinciding with one of the putative SF1 response elements of the AMH promoter. The c.-225delA variant was experimentally created by site-directed mutagenesis, and analysed in luciferase assays, showing a decrease to a similar extent (0.54 ± 0.08 RLU in the 0.4-kb promoter and 0.59 ± 0.04 in the 3-kb promoter context) of what was observed when the -228 SF1 site was completely disrupted, as shown above. Electro-mobility shift assays (EMSA) showed that the interaction between SF1 and its -228 binding site was lost when the oligonucleotide carried c.-225delA or the fully disrupted -228 site.In conclusion, the single base deletion c.-225delA within the SF1 site of the AMH gene promoter impaired SF1 binding to and transactivation of the AMH promoter, resulting in extremely decreased AMH production, leading to PMDS in this patient. This is the first description of an AMH promoter mutation leading to PMDS.