title: A dominant negative mutation at the ATP binding domain of AMHR2 is associated with a defective anti-Müllerian hormone signaling pathway.
abstract: Does a heterozygous mutation in AMHR2, identified in whole-exome sequencings (WES) of patients with primary ovarian insufficiency (POI), cause a defect in anti-Müllerian hormone (AMH) signaling?
  
  
  
  The I209N mutation at the adenosine triphosphate binding domain of AMHR2 exerts dominant negative defects in the AMH signaling pathway.
  
  
  
  Previous studies have demonstrated the associations of several sequence variants in AMH or AMHR2 with POI, but no functional assay has been performed to verify whether there was any defect on AMH signaling.
  
  
  
  Ninety-six unrelated female Chinese Han patients were diagnosed with idiopathic POI and subjected to WES. In silico analysis was done for the sequence variants followed by molecular assays to examine the functional effects of the sequence variants in human granulosa cells. In silico analysis, immunostaining, Western analysis, genome-wide expression analysis, quantitatively polymerase chain reaction were applied to the characterization of the sequence variants.
  
  
  
  We identified one novel heterozygous missense variant, p.Ala17Glu (A17E), in AMHR2. Subsequently, A17E and two independently reported missense variants, p.Ile209Asn (I209N) and p.Leu354Phe (L354F), were evaluated for effects on the AMH signaling pathway. In silico analysis predicted that all three variants may be deleterious. However, only one variant, I209N, showed severe defects in transducing the AMH signal as well as impaired SMAD1/5/8 phosphorylation. Furthermore, using genome-wide gene expression analysis, we identified genes whose expression was affected by the mutation, these included genes previously reported to participate in AMH signaling as well as newly identified genes. They are EMILIN2, FAM155A, GATA2, HES5, ID1, ID2, RLTPR, SMAD7, CBL, MALAT1 and SMARCA2.
  
  
  
  None.
  
  
  
  Although the in vitro assays demonstrated the causative effect of I209N on AMH signaling, further studies need to validate its long-term effects on folliculogenesis and POI.
  
  
  
  These results will aid both researchers and clinicians in understanding the molecular pathology of AMH signaling and POI to develop diagnostic assays or therapeutics approaches.
  
  
  
  Research funding is provided by the Ministry of Science and Technology of China [2012CB944704; 2012CB966702], and the National Natural Science Foundation of China [Grant number: 31171429]. The authors declare no conflict of interest.
authors.affiliation: School of Life Sciences, Tsinghua University, Haidian District, Beijing, 100084, China Center for Stem Cell Biology and Regenerative Medicine, Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Haidian District, Beijing 100084, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Li
authors.affiliation: The Hong Kong Jockey Club Building for Interdisciplinary Research, The University of Hong Kong, 5 Sassoon Road, Pokfulam, Hong Kong.
authors.forename: Xueya
authors.initials: X
authors.lastname: Zhou
authors.affiliation: Center for Genetics, National Research Institute for Family Planning, No. 12 Da-Hui-Si Road, Haidian District, Beijing 100081, China Graduate School of Peking Union Medical College, Dongdan three 9, Dongcheng District, Beijing 100730, China.
authors.forename: Xi
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Medical Genetics and Developmental Biology, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, Youanmenwai, Fengtai District, Beijing 100069, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.affiliation: Center for Genetics, National Research Institute for Family Planning, No. 12 Da-Hui-Si Road, Haidian District, Beijing 100081, China Graduate School of Peking Union Medical College, Dongdan three 9, Dongcheng District, Beijing 100730, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Center for Genetics, National Research Institute for Family Planning, No. 12 Da-Hui-Si Road, Haidian District, Beijing 100081, China Graduate School of Peking Union Medical College, Dongdan three 9, Dongcheng District, Beijing 100730, China.
authors.forename: Binbin
authors.initials: B
authors.lastname: Wang
authors.affiliation: Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, Meishan Road, Shushan District, Hefei 230022, China Institute of Reproductive Genetics, Anhui Medical University, Meishan Road, Shushan District, Hefei 230022, China Anhui Provincial Engineering Technology Research Center for Biopreservation and Artificial Organs, Meishan Road, Shushan District, Hefei 230022, China kkee@mail.tsinghua.edu.cn caoyunxia_profr@126.com.
authors.forename: Yunxia
authors.initials: Y
authors.lastname: Cao
authors.affiliation: Center for Stem Cell Biology and Regenerative Medicine, Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Haidian District, Beijing 100084, China kkee@mail.tsinghua.edu.cn caoyunxia_profr@126.com.
authors.forename: Kehkooi
authors.initials: K
authors.lastname: Kee
mesh_terms: D054304:Anti-Mullerian Hormone / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D059472:Exome / Q000235:genetics; D005260:Female; D006801:Humans; D009154:Mutation / Q000235:genetics*; D016649:Primary Ovarian Insufficiency / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D000072417:Protein Domains; D018000:Receptors, Peptide / Q000235:genetics / Q000378:metabolism; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology
chemical_list: D018000:Receptors, Peptide; D018125:Receptors, Transforming Growth Factor beta; C084621:anti-Mullerian hormone receptor; D054304:Anti-Mullerian Hormone
keywords: AMHR2; anti-Müllerian hormone (AMH); dominant negative mutation; granulosa cell; ovarian follicle development; premature ovarian failure; primary ovarian insufficiency; signaling pathway; transforming growth factor-beta (TGF-β)