Variants in GCNA, X-linked germ-cell genome integrity gene, identified in men with primary spermatogenic failure

Abstract

Male infertility impacts millions of couples yet, the etiology of primary infertility remains largely unknown. A critical element of successful spermatogenesis is maintenance of genome integrity. Here, we present a genomic study of spermatogenic failure (SPGF). Our initial analysis (n = 176) did not reveal known gene-candidates but identified a potentially significant single-nucleotide variant (SNV) in X-linked germ-cell nuclear antigen (GCNA). Together with a larger follow-up study (n = 2049), 7 likely clinically relevant GCNA variants were identified. GCNA is critical for genome integrity in male meiosis and knockout models exhibit impaired spermatogenesis and infertility. Single-cell RNA-seq and immunohistochemistry confirm human GCNA expression from spermatogonia to elongated spermatids. Five identified SNVs were located in key functional regions, including N-terminal SUMO-interacting motif and C-terminal Spartan-like protease domain. Notably, variant p.Ala115ProfsTer7 results in an early frameshift, while Spartan-like domain missense variants p.Ser659Trp and p.Arg664Cys change conserved residues, likely affecting 3D structure. For variants within GCNA's intrinsically disordered region, we performed computational modeling for consensus motifs. Two SNVs were predicted to impact the structure of these consensus motifs. All identified variants have an extremely low minor allele frequency in the general population and 6 of 7 were not detected in > 5000 biological fathers. Considering evidence from animal models, germ-cell-specific expression, 3D modeling, and computational predictions for SNVs, we propose that identified GCNA variants disrupt structure and function of the respective protein domains, ultimately arresting germ-cell division. To our knowledge, this is the first study implicating GCNA, a key genome integrity factor, in human male infertility.

Fig. 1

Single-cell transcriptome profiling of adult human spermatogonia. a Violin plot showing expression level of GCNA mRNA across the spermatogonia clusters. b UMAP plot of re-clustered human spermatogonia in distinct clusters representing undifferentiated (clusters 1 and 2) and differentiating spermatogonia (clusters 3 and 4) (see Supplemental Materials for single-cell transcriptome clustering of all testicular cells and expression patterns of common markers utilized to distinguish spermatogonia). Cells demonstrating positive expression shown in red with no or little expression in blue. c UMAP plot showing GCNA RNA expression predominantly in clusters representing differentiating spermatogonia. d UMAP plot showing known RNA marker UTF1 expression primarily in clusters representing undifferentiated spermatogonia

Fig. 2

GCNA protein expression in normal adult human seminiferous tubules. Immunofluorescent detection of GCNA and VASA (DDX4) in Bouin-fixed adult human testis confirms GCNA is present in all spermatogenesis cell types, including spermatogonia (SG), spermatocytes (SC), and spermatids (ST), but absent in visible mature spermatozoa (SZ)

Fig. 3

Diagram of human GCNA protein domains and variant locations. Schematic depiction of human GCNA protein with known domains (intrinsically disordered region, Spartan like, and zinc finger) and amino acid coordinates. Potentially disease-causing variants observed in 7 patients with SPGF are labeled and correspond to the IDR and SprT-like domains (see also Table 2). Repeat 1 = 2 exon repeat, Repeat 2 = DD(N/S)DDS(E/D)(A/V)P 21x, Repeat 3 = DD(N/S)DDS(E/D)(A/V)P 21x (Carmell, Dokshin et al. 2016)

Fig. 4

3D Modeling and conservation of GCNA variant positions in SprT-like domain residues. Three dimensional models depict a highly conserved aa residue Ser659 in H. sapiens (Hs) and C. elegans (Ce, left) and H. sapiens and D. melanogaster (Dm, right) which was altered in a Sertoli cell only individual with variant p.Ser659Trp and b moderately conserved aa residue Arg664 in H. sapiens and C. elegans which was altered in an azoospermic individual with variant p.Arg664Cys. Human catalytic histidines (H592, H596 and H609) are represented by sticks. c Partial amino acid sequences for multiple species of the conserved SprT-like protease domain also demonstrate conservation of affected amino acids (see also Table 2)

Fig. 5

Histological and immunohistochemical imaging of human seminiferous tubules with normal and likely altered GCNA expression. Appearance of human seminiferous tubule in controls (left) and Sertoli cell-only phenotype (right). PAS staining shows presence of all testicular germ cell stages in control and presence of only Sertoli cells in individual harboring GCNA variant p.Ser659Trp in the Spartan-like domain (row 1). GCNA and VASA colocalize in normal spermatogonia, spermatocytes, and spermatids but are not seen in Sertoli cells confirming germ cell specific expression (row 2). Both germ cells stained with DAPI and somatic Sertoli cells labelled with SOX9 are present in individuals with normal GCNA but only Sertoli cells are seen in the seminiferous tubules of the patient with variant p.Ser659Trp in the Spartan-like domain (row 3)