X-linked TEX11 mutations, meiotic arrest, and azoospermia in infertile men

Abstract

Background: The genetic basis of nonobstructive azoospermia is unknown in the majority of infertile men.

Methods: We performed array comparative genomic hybridization testing in blood samples obtained from 15 patients with azoospermia, and we performed mutation screening by means of direct Sanger sequencing of the testis-expressed 11 gene (TEX11) open reading frame in blood and semen samples obtained from 289 patients with azoospermia and 384 controls.

Results: We identified a 99-kb hemizygous loss on chromosome Xq13.2 that involved three TEX11 exons. This loss, which was identical in 2 patients with azoospermia, predicts a deletion of 79 amino acids within the meiosis-specific sporulation domain SPO22. Our subsequent mutation screening showed five novel TEX11 mutations: three splicing mutations and two missense mutations. These mutations, which occurred in 7 of 289 men with azoospermia (2.4%), were absent in 384 controls with normal sperm concentrations (P=0.003). Notably, five of those TEX11 mutations were detected in 33 patients (15%) with azoospermia who received a diagnosis of azoospermia with meiotic arrest. Meiotic arrest in these patients resembled the phenotype of Tex11-deficient male mice. Immunohistochemical analysis showed specific cytoplasmic TEX11 expression in late spermatocytes, as well as in round and elongated spermatids, in normal human testes. In contrast, testes of patients who had azoospermia with TEX11 mutations had meiotic arrest and lacked TEX11 expression.

Conclusions: In our study, hemizygous TEX11 mutations were a common cause of meiotic arrest and azoospermia in infertile men. (Funded by the National Institutes of Health and others.).

Figure 1. Hemizygous Deletion of TEX11 Exons 10 to 12 and Flanking Intronic Regions in Two Men with Azoospermia

Panel A shows the array comparative genomic hybridization (aCGH) plot of the X chromosome. On the left, an idiogram of the X chromosome shows a region of interest (blue line) at the Xq13.2 band. On the right, dots represent oligonucleotide DNA probes, arranged according to their physical map locations from the distal p arm (top) to the distal q arm (bottom) of the X chromosome. For each probe, the fluorescence intensity of the test signal relative to the reference signal is converted to a logarithmic (log₂) value and shown along the x axis. Probes with a log₂ ratio clustered around zero (black dots) indicate DNA segments with a normal copy number. A positive log₂ ratio (above +0.3 [red dots]) indicates a gain (extra copy) of the chromosomal region, whereas intervals with a negative log₂ ratio (below −0.5 [green dots]) represent a loss (deletion) in DNA copy number. Panel B shows a magnified view of the deleted region detected by 12 probes with the use of the 400K whole-genome array (gray shaded area). Genomic coordinates (GRCh37/hg19 assembly) of the deletion start at 69,956,055 and stop at 70,045,530 nucleotides. Panels C and D show that the aCGH plot of the 180K oligonucleotide X-chromosome high-resolution microarray more precisely delineates the deletion intervals in Patients 1 and 3. Chromosomal alterations suggestive of a homozygous or heterozygous deletion are indicated by brown and red dots, respectively. In a sample obtained from Patient 1 (Panel C), the deletion spans chrX:69,954,488 to 70,045,530. In a sample obtained from Patient 3 (Panel D), the deletion spans chrX:69,954,488 to 70,046,552 regions (pink shaded areas). On the right side of Panel D, Xq13.2 deletions in both patients encompass exons 10 to 12 (blue triangles) of TEX11.

Figure 2. TEX11 Mutations Detected in Men with Azoospermia

Panel A shows the genomic structure of TEX11, with mutations mapped to isoform 1 (GenBank accession number, NM 001003811.1). Gold rectangles represent noncoding exons, and blue rectangles represent coding exons. The coding sequence of the gene begins at nucleotides that encode a start codon in exon 3 and ends in exon 31 at a stop codon. Point mutations detected in men with azoospermia in the initial and follow-up studies are shown. The red bracket below exons 10 to 12 shows the approximately 99-kb deletion detected in two men with azoospermia. Panel B shows sequence chromatograms of five point mutations detected in TEX11. Reference and mutant DNA sequences are shown, with an arrowhead identifying the mutation. Panel C shows predicted TEX11 domains with multiple tetratricopeptide repeat (TPR)–containing regions (amino acid positions 37–188, 418–450, and 457–814) and a sporulation domain (SPO22) meiosis-specific motif (amino acid positions 189–417). Mutations in the coding region (black lines) and splicing changes (blue arrowheads) are located in predicted domains. The red bracket shows the deletion that encompasses 79 amino acids of the SPO22 domain (35% of the entire length of the SPO22 domain) and includes 52 conserved positions.

Figure 3. Immunohistochemical Detection of TEX11 in Testicular Tissue Sections Obtained from a Man with Normal Spermatogenesis, a Man with Mixed Testicular Atrophy, and a Man with Meiotic Arrest

In a man with normal spermatogenesis (Panels A and B), TEX11 was highly expressed in late spermatocytes (arrow), round spermatids (arrowhead), and elongated spermatids (asterisk). Testicular tissue stained with IgG antibody was used as a negative control (Panel C). TEX11 was also present in tubules with remaining spermatogenesis in a patient with mixed testicular atrophy. TEX11 expression was observed in late spermatocytes (arrow) and round spermatids (arrowhead) (Patient 6) (Panels D and E). Seminiferous tubules containing only less-differentiated germ cells and Sertoli cells show no TEX11 expression (Panel F). In a man with complete meiotic arrest (Patient 4), germ cells did not have TEX11 expression in the majority of tubules (Panels F, G, and H). However, TEX11 staining was observed in rare seminiferous tubules with remaining late spermatocytes (arrow) and round spermatids (arrowhead) (Panel I). Scale bars indicate 50 μm.