AMH mutations with reduced in vitro bioactivity are related to premature ovarian insufficiency.

STUDY QUESTION: Could anti-Müllerian hormone (AMH) mutations be implicated in the development of idiopathic premature ovarian insufficiency (POI)?

SUMMARY ANSWER: Three rare or unknown missense variants of the AMH gene were identified in a cohort of 55 POI patients; all three variants showed a drastically reduced in vitro bioactivity.

WHAT IS KNOWN ALREADY: Genetic factors are implicated in 5-15% of cases of POI. However, only a few genes have been shown to be involved in its development. AMH inhibits the recruitment of primordial follicles in the ovary and defective or absent AMH leads to premature depletion of the primordial follicle pool in AMH null mice.

STUDY DESIGN, SIZE, DURATION: The whole coding sequence and the exon-intron junction of the AMH gene was sequenced in a cohort of 55 POI patients recruited over a period of 8 years. The studied variants were also sequenced in 197 ethnically matched controls.

PARTICIPANTS/MATERIALS, SETTING, METHODS: POI was defined as amenorrhea of more than 4 months with increased FSH before the age of 40. Patients with POI resulting from radio- or chemotherapy, surgery, chromosomal anomalies or FMR1 gene pre-mutation were excluded from the study. Recombinant human wild-type (wt) and mutated AMH proteins were produced in HEK293 T cells. KK-1 cells transfected with the AMH receptor type 2 (AMHR2) and a BMP responsive element coupled to a luciferase reporter vector were stimulated with different concentrations of wt AMH and the three tested variants.

MAIN RESULTS AND THE ROLE OF CHANCE: The whole coding sequence of the AMH gene could be performed and analyzed for 50 POI patients: 16 variants were found, including 6 missense variants from which 1 was unknown (R444H) and 2 were very rare (G264R and D288E). The variant D288E was also found in one of the patient's mother who also underwent POI at 32 years old. The stimulation of the AMHR2 assessed by the luciferase activity was drastically reduced for the three variants when compared with the wt AMH.

LIMITATIONS, REASONS FOR CAUTION: The study is limited by a relatively small number of patients in the POI cohort.

WIDER IMPLICATIONS OF THE FINDINGS: This is the first time that the bioactivity of AMH variants related to POI patients is tested in vitro. The functional study showed a drastic reduction of the protein activity for the three variants, supporting their contribution to the development of the ovarian insufficiency. The familial segregation further supports the implication of AMH in the development of POI.

STUDY FUNDING/COMPETING INTERESTS: The study was performed thanks to funding from the 'Fondation Erasme'. No conflicts of interest are declared.