Truncating Mutations in the Adhesion G Protein-Coupled Receptor G2 Gene ADGRG2 Cause an X-Linked Congenital Bilateral Absence of Vas Deferens

Abstract

In 80% of infertile men with obstructive azoospermia caused by a congenital bilateral absence of the vas deferens (CBAVD), mutations are identified in the cystic fibrosis transmembrane conductance regulator gene (CFTR). For the remaining 20%, the origin of the CBAVD is unknown. A large cohort of azoospermic men with CBAVD was retrospectively reassessed with more stringent selection criteria based on consistent clinical data, complete description of semen and reproductive excurrent ducts, extensive CFTR testing, and kidney ultrasound examination. To maximize the phenotypic prioritization, men with CBAVD and with unilateral renal agenesis were considered ineligible for the present study. We performed whole-exome sequencing on 12 CFTR-negative men with CBAVD and targeted sequencing on 14 additional individuals. We identified three protein-truncating hemizygous mutations, c.1545dupT (p.Glu516Ter), c.2845delT (p.Cys949AlafsTer81), and c.2002_2006delinsAGA (p.Leu668ArgfsTer21), in ADGRG2, encoding the epididymal- and efferent-ducts-specific adhesion G protein-coupled receptor G2, in four subjects, including two related individuals with X-linked transmission of their infertility. Previous studies have demonstrated that Adgrg2-knockout male mice develop obstructive infertility. Our study confirms the crucial role of ADGRG2 in human male fertility and brings new insight into congenital obstructive azoospermia pathogenesis. In men with CBAVD who are CFTR-negative, ADGRG2 testing could allow for appropriate genetic counseling with regard to the X-linked transmission of the molecular defect.

Figure 1

Study Flow Chart and Genealogic Tree of the Pedigree with Two Related Individuals with CBAVD (A) Study flow chart showing the procedure for the identification of new male fertility genes from a cohort of azoospermic men with CBAVD. LoF, loss of function. (B) Genealogic tree of the two related CBAVD-affected individuals included in the cohort (II-3 and III-12, arrow). ?, probable infertility.

Figure 2

Schematic Representation of ADGRG2 Structure with the Position of the Amino Acid Changes Resulting from the Identified Truncating Mutations Like the other members of the adhesion-GPCR family, ADGRG2 is composed of a seven-transmembrane domain and a highly conserved cystein-rich motif, named the GAIN domain, containing GPS, which is the site of autoproteolytic cleavage. The ADGRG2 extracellular domain contains a serine-threonine-proline-rich region (STP). Positions of C-terminal and N-terminal epitopes recognized by the populations of polyclonal antibodies used for immunohistochemistry are indicated by yellow stars.

Figure 3

Immunohistochemical Staining of ADGRG2 in Sections of Epididymal and Efferent Ducts Obtained from a Control Individual with Non-obstructive Azoospermia and from the CBAVD-Affected Proband Carrying the p.Leu668Argfs^∗21 ADGRG2 Truncating Variant With the antibody directed against the N-terminal portion of ADGRG2, subapical staining (arrows) was observed on the control epididymal (A) and efferent (C) ducts. The same staining was observed on sections from the proband, respectively (B and D). In the control subject, staining of stereocilia and apical membrane is observed on the epididymis section (A) (arrowhead), whereas stereocilia are not stained in the proband (B) (arrowhead). With the antibody directed against the C-terminal portion of ADGRG2, positive staining was observed on the control subject at the apical level and stereocilia of cells from the epididymis (E) (arrows) or at the apical level of efferent ducts, especially in crypt-like grooves (G) (arrows). In contrast, in the CBAVD-affected proband whose mutation is predicted to delete the ADGRG2 C-terminal region, such staining was not observed on the epididymal (F) or efferent (H) ducts. Scale bars indicate 50 μm.