A novel AMH variant at the prehelix loop impairs the binding to AMHR2 and causes persistent Müllerian duct syndrome.

Dear Editor, Persistent Müllerian duct syndrome (PMDS; Online Mendelian Inheritance in Man [OMIM]: #261550) is a rare autosomal recessive inherent difference in sex development in males with Müllerian duct derivatives, including the uterus, fallopian tubes, and upper part of the vagina.1 Approximately 88% of PMDS patients were found to have biallelic variants in the anti-Müllerian hormone (AMH; OMIM: *600957) or AMH type 2 receptor (AMHR2; OMIM: *600956) genes, and the rest were referred as idiopathic.1 The serum AMH levels are usually very low or undetectable in PMDS patients with AMH gene mutations but normal or elevated in those with AMHR2 gene mutations. Here, an adult Chinese patient with PMDS carrying biallelic variants of the AMH gene was reported to have unexpectedly normal serum AMH level. Please refer to Supplementary Information of Patient and Methods for methodologies. A 32-year-old male who was referred to the Center of Reproductive Medicine of Shengjing Hospital of China Medical University (Shenyang, China) on March 18th, 2021, was enrolled in this study. Written informed consent was obtained and all protocols were approved by the Ethics Committee of Shengjing Hospital of China Medical University (Approval No. 2021PS523K). He was born to nonconsanguineous parents, with a typical sister who delivered a healthy boy (Figure 1a). He had experienced 8 years of infertility, with occasional hematospermia. He had transverse testicular ectopia and underwent cryptorchidectomy at 1 year old after a failed attempt to return the left testis. Physical examination revealed an empty right scrotum and inguinal hernia on the left side. No gynecomastia or other abnormalities were observed. Semen analysis revealed oligoasthenozoospermia with low sperm volume, low normal morphology, low survival rate, high sperm nucleoprotein immaturity, low DNA fractionation index, and no progressive motility. Hormone analyses revealed slightly elevated levels of prolactin and estradiol. Serum AMH (3.18 ng ml−1) was within the normal range (Supplementary Table 1). Ultrasonography showed a single testis of 6.3 cm × 2.1 cm × 2.5 cm in the left scrotum. Magnetic resonance scanning showed 10.0 cm × 2.1 cm × 2.5 cm of tissue with a fluid-filled cavity (Figure 1b).Figure 1: (a) Pedigree of the patient with persistent Müllerian duct syndrome. The affected proband is indicated by a black arrow. (b) Pelvic magnetic resonance scanning. The Müllerian structure is indicated by red arrows. The left testis is indicated by blue arrows. (c) Sanger sequencing of the heterozygous c.1165G>T (p.E389X) and c.1507T>C (p.Y503H) variants (indicated by red arrows) of the AMH gene. (d) Evaluation of the amino acid conservation by Ugene. Each residue box in alignment is assigned a color according to ClustalX color scheme. The Y503 of AMH is indicated in a black box. (e) Three-dimension molecular docking of AMH (green molecule) with AMHR2 (red molecule). The cluster was constructed by Haddock2.410 and visualized by PyMOL. The interface of wild-type (left) and Y503H mutant (right) AMH with the extracellular domain of AMHR2 showed differences in both bonds and surface. (f) ELISA of the secreted AMH in the transfected HEK293 cell culture media. The kit was specific to the C-terminal bioactive domain. *P< 0.01 in the post hoc Tukey analysis. (g) The binding of AMH with AMHR2 evaluated by IP and IB. The Myc-tagged AMHR2-transfected cells were incubating with AMH-transfected culture media. The interactive bands (IP: Myc; IB: AMH) were normalized to that of internal control (IP: Myc; IB: Myc), with the wild-type taken as “1”. *P< 0.01 in the Student’s t-test. (h) H&E staining of the Müllerian remnant. Immunohistochemistry for (i) ER and (j) PR. Scale bars = 20 µm. AMH: anti-Müllerian hormone; ELISA: enzyme-linked immunosorbent assay; AMHR2: AMH type 2 receptor; IP: immunoprecipitation; IB: immunoblotting; H&E: hematoxylin and eosin; ER: estrogen receptor; PR: progesterone receptor.Supplementary Table 1: Sperm and hormone parameters in the patient with persistent Müllerian duct syndromeCytogenetic analysis revealed a typical 46,XY karyotype. Compound heterozygous variants of c.1165G>T (p.E389X) and c.1507T>C (p.Y503H) in the AMH gene were identified by whole-exome sequencing and confirmed by Sanger sequencing (Figure 1c). The nonsense variant has been reported in Chinese patients.2–4 The novel missense variant occurs at a highly conserved amino acid across various species (Figure 1d). In silico analyses by Sorting Intolerant From Tolerant (SIFT; http://sift.jcvi.org), ProVean (http://provean.jcvi.org/index.php), Polyphen2 (http://genetics.bwh.harvard.edu/pph2), and MutationTaster (http://www.mutationtaster.org), all predicted the variant as “deleterious”. Considering the unexpectedly normal serum AMH level, we evaluated three-dimensional (3D) molecular docking to the receptor AMHR2 using Haddock2.4 and visualized the top cluster using PyMOL (https://pymol.org/; Figure 1e). The Y503 at the prehelix loop of the AMH palm generated a hydrogen bond (3.7 Å) to AMHR2 C559 at the terminus of finger 4, creating a connective molecular surface. The substitution by H503 disrupted the H-bond and the connective surface, prolonging the distance to T47 of AMHR2 from 4.9 Å to 5.6 Å. Secretion and receptor binding were investigated in transfected HEK293 cells. In the enzyme-linked immunosorbent assay (ELISA) specific for C-terminal mature AMH (Figure 1f), the E389X mutant AMH was almost undetectable in the culture media. In contrast, the Y503H mutant AMH mildly decreased to 72.8% of the wild-type control. Receptor binding was assayed by incubating AMHR2-transfected cells in the AMH-transfected culture media. After immunoprecipitation with Myc-tagged AMHR2, the proteins were immunoblotted to the Y503H mutant AMH with a significantly decreased level (36.3% of the wild-type) but not to the E389X mutant AMH (Figure 1g). These data indicate that the pathogenic mechanism of E389X AMH is impaired biosynthesis, and Y503H AMH is impaired receptor binding. The patient underwent testicular sperm extraction and intracytoplasmic sperm injection (ICSI) and got a healthy child. An informed choice for subtotal hysterectomy was made. The resected Müllerian remnants included a seemingly well-developed uterus filled with 30 ml of bloody liquid and a fallopian tube; the cervix was preserved. Pathological analysis confirmed the uterine structure (Figure 1h). Immunohistochemistry revealed positive staining in estrogen receptor (Figure 1i) and progesterone receptor (Figure 1j). The left inguinal hernia was repaired without complications. Most cases of PMDS were incidentally identified during evaluation or corrective surgery for cryptorchidism or inguinal hernia in children. Overlooking adult patients were rare. The patients in our present and previous studies demonstrated asthenozoospermia with no progressive motility,2 which was supported by other adult patients with azoospermia, oligozoospermia, or asthenozoospermia.4–6 Infertility is a major concern in PMDS, and we have added successful experience in testicular sperm retrieval and ICSI. The development of the Müllerian remnant varies, and interventions remain controversial. The risk of Müllerian neoplastic degeneration (4%) is less than that of undescended testes (33%).7,8 Surgical removal may unavoidably damage the vas deferens or the deferential artery to some degree. However, the extended uterus may induce urinary retention by mechanical obstruction, and highly differentiated Müllerian derivatives may cause excess estrogen, inducing gynecomastia and hematospermia. Considering our patient’s large uterus and occasional hematospermia, subtotal hysterectomy was suggested after successful assisted reproduction. AMH comprises a large N-terminal pro region crucial for proper folding and stability and a smaller C-terminal mature domain responsible for receptor binding.1 The p.E389X variant at the N-terminal domain created a premature termination codon with a long distance to the poly(A) binding protein, which might activate nonsense-induced mRNA decay. Cells transfected with p.E389X expression vectors demonstrated null expression using C-terminal-specific antibodies.2 Indeed, almost all patients with AMH mutations showed very low or undetectable serum AMH levels. Our patient is an exception, with similar observations in two patients with homozygous p.Q496H and p.R502L variants.8,9 Together with the novel p.Y503H variant in our patient, all three resided in the highly conserved prehelix loop of the C-terminal domain, which is important for receptor interaction. The 3D molecular docking analysis of Y503H AMH demonstrated disruption in the H-bond and connective surface to AMHR2, which was confirmed by a co-immunoprecipitation experiment. Therefore, the Y503H variant may be destructive in tethering parallel fingers to maintain a stable interface for receptor binding. The decrease to 72.8% of wild-type AMH secretion might not exceed the wide reference range if set in human serum, indicating a minor pathogenic effect. In conclusion, we report an adult patient with PMDS carrying biallelic loss-of-function AMH variants with unexpectedly normal serum AMH level. The pathogenic mechanism of the novel p.Y503H variant in the prehelix loop of the palm was found to be defective in receptor binding. AUTHOR CONTRIBUTIONS QD evaluated the clinical data and drafted the manuscript. CQ performed the cellular and molecular experiment. YYZ performed the genetic analysis. XJS performed the bioinformatic analysis and reviewed the literature. XLL conceived and supervised the study. All the authors took part in revising the manuscript, and read and approved the final manuscript. COMPETING INTERESTS All authors declare no competing interests. ACKNOWLEDGMENTS This study was supported by grant from the Natural Science Foundation of Liaoning Province (No. 2022-MS-208), the 345 Talent of Shengjing Hospital of China Medical University (No. M1395), and the General Project of Shengjing Hospital of China Medical University (No. 2023-M1643). Supplementary Information is linked to the online version of the paper on the Asian Journal of Andrology website.