Mutations in the stromal antigen 3 (STAG3) gene cause male infertility due to meiotic arrest.

Nils van der Bijl, Albrecht Röpke, Uddipta Biswas, Marius Wöste, Rolf Jessberger, Sabine Kliesch, Corinna Friedrich, Frank Tüttelmann, 04.11.2019

Abstract

STUDY QUESTION:
Are sequence variants in the stromal antigen 3 (STAG3) gene a cause for non-obstructive azoospermia (NOA) in infertile human males?

SUMMARY ANSWER:
Sequence variants affecting protein function of STAG3 cause male infertility due to meiotic arrest.

WHAT IS KNOWN ALREADY:
In both women and men, STAG3 encodes for a meiosis-specific protein that is crucial for the functionality of meiotic cohesin complexes. Sequence variants in STAG3 have been reported to cause meiotic arrest in male and female mice and premature ovarian failure in human females, but not in infertile human males so far.

STUDY DESIGN, SIZE, DURATION:
The full coding region of STAG3 was sequenced directly in a cohort of 28 men with NOA due to meiotic arrest. In addition, a larger group of 275 infertile men that underwent whole-exome sequencing (WES) was screened for potential STAG3 sequence variants. Furthermore, meiotic spreads, immunohistochemistry, WES and population sampling probability (PSAP) have been conducted in the index case.

PARTICIPANTS/MATERIALS, SETTING, METHODS:
This study included 28 infertile but otherwise healthy human males who underwent Sanger sequencing of the full coding region of STAG3. Additionally, WES data of 275 infertile human males with different infertility phenotypes have been screened for relevant STAG3 variants. All participants underwent karyotype analysis and azoospermia factor (AZF) screening in advance. In the index patient, segregation analysis, WES data, PSAP, lab parameters, testis histology and nuclear spreads have been added to suplort the findings.

MAIN RESULTS AND THE ROLE OF CHANCE:
Two compound-heterozygous variants in STAG3 (c.[1262T>G];[1312C>T], p.[(Leu421Arg)];[(Arg438Ter)]) have been found to cause male infertility due to complete bilateral meiotic arrest in an otherwise healthy human male. Compound heterozygosity was confirmed by Sanger sequencing of the parents and the patients brother. Other variants which may affect spermatogenesis have been ruled out through analysis of the patients WES data and application of the PSAP pipeline. As expected from Stag3 knockout-mice meiotic spreads, germ cells did not develop further than zygotene and showed drastic chromosome aberrations. No rare variants in STAG3 were found in the 275 infertile males with other phenotypes. Our results indicate that STAG3 variants that negatively affect its protein function are a rare cause of NOA (<1% of cases).

LIMITATIONS, REASONS FOR CAUTION:
We identified only one patient with compound-heterozygous variants in STAG3 causing NOA due to meiotic arrest. Future studies should evaluate STAG3 variants in larger cohorts to support this finding.
WIDER IMPLICATIONS OF THE FINDINGS:
Identification of STAG3 sequence variants in infertile human males should improve genetic counselling as well as diagnostics and treatment. Especially before testicular sperm extraction (TESE) for ICSI, STAG3 variants should be ruled out to prevent unnecessary interventions with frustrating outcomes for both patients and clinicians.

STUDY FUNDING/COMPETING INTEREST(S):
This work was carried out within the frame of the German Research Foundation (DFG) Clinical Research Unit Male Germ Cells: from Genes to Function (CRU326). Work in the laboratory of R.J. is supported by a grant of the European Union H2020 program GermAge. The authors declare no conflicts of interest.
TRIAL REGISTRATION NUMBER:
Not applicable.

VAN DER BIJL, N., et al. Mutations in the stromal antigen 3 (STAG3) gene cause male infertility due to meiotic arrest. Human Reproduction, 2019, 34. Jg., Nr. 11, S. 2112-2119.

Publication: https://doi.org/10.1093/humrep/dez204

Disclaimer

The publication Mutations in the stromal antigen 3 (STAG3) gene cause male infertility due to meiotic arrest. by Nils van der Bijl, Albrecht Röpke, Uddipta Biswas, Marius Wöste, Rolf Jessberger, Sabine Kliesch, Corinna Friedrich, Frank Tüttelmann is published under an open access license: http://creativecommons.org/licenses/by-nc/4.0/. Permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited.

Curation by the MFGA team
Relevant data sets presented in the publication have been identified. If possible, annotations (title, general information, conditions, processed tissue types and processed cell types) have been added based on information from the publication. Data tables and images that provide a good overview on the publication's findings on the data set have been extracted from the publication and/or supplement. If not stated otherwise, images are depicted with title and description exactly as in the publication. Tables have been adjusted to the MFGA table format. Conducted adjustments are explained in the detailed view of the tables. However, titles and descriptions have been adopted from the publication.

Data set 1: Screening of infertile men with meiotic arrest for relevant STAG3 variants

Genome: Sanger Sequencing

Species

Species
Human

Conditions

Human phenotype ontology	Participants	Comment
HP:0011961: Non-obstructive azoospermia	28	Complete bilateral meiotic arrest

Images

Figure 1: Identification of compound heterozygous variants in the stromal antigen 3 (STAG3) gene in an infertile patient (M870) leading to meiotic arrest and azoospermia

(A) Pedigree of the index patient with meiotic arrest (arrow). Squares indicate men, circles women. The index patient carries two variants in Exon 13 of the STAG3 gene (c.[1262T>G];[1312C>T], p.[(Leu421Arg)];[(Arg428Ter)], depicted in grey and black, respectively) in a compound heterozygous state. Electropherograms indicate that c.1262T>G is inherited from the father while c.1312C>T is inherited from the mother. The unaffected brother only carries the variant c.1262T>G leading to the missense variant p.(Leu421Arg).</br>(B) Orthologous alignment shows that both mutated amino acids are highly conserved, which underlines the likely pathogenic impact of the variants.</br>(C) Three-dimensional structure of the STAG3 protein predicted by Phyre-2 based on the template c4pjwA covering 74% of the protein (904 residues) with a confidence of 100%. Identified variants are highlighted in red.</br>(D) Exon–intron structure of STAG3 gene obtained by ENSEMBL (reference transcript ENST00000317296.9) and the corresponding protein domain structure. Both variants lie within Exon 13 affecting the armadillo (ARM)-type domain, which is predicted to interact with a nucleic acid or another protein. In case of the stop variant (p.(Arg428Ter)) this domain would be interrupted.

Source: VAN DER BIJL, N., et al. Mutations in the stromal antigen 3 (STAG3) gene cause male infertility due to meiotic arrest. Human Reproduction, 2019, 34. Jg., Nr. 11, S. 2112-2119.

Licensed under: http://creativecommons.org/licenses/by-nc/4.0/

Data set 3: Meiotic spreads and immunofluorescence staining of patient M870 with STAG3 variant and meiotic arrest

Other: Other

Species

Species
Human

Conditions

Human phenotype ontology	Participants	Comment
HP:0011961: Non-obstructive azoospermia	1	Patient M870; Complete bilateral meiotic arrest

Images

Figure 2: Histological staining of the left (A/C) and right (B/D) testes obtained by microsurgical testicular sperm extraction (mTESE) from patient M870 with azoospermia

Spermatogenesis arrests at the stage of primary spermatocytes, indicating bilateral meiotic arrest being the underlying cause of non-obstructive azoospermia of the affected patient. Overviews (A and B) were imaged at ×20, single tubuli (C and D, labelled by asterisks) at ×60 magnification. Scale bars as indicated.

Source: VAN DER BIJL, N., et al. Mutations in the stromal antigen 3 (STAG3) gene cause male infertility due to meiotic arrest. Human Reproduction, 2019, 34. Jg., Nr. 11, S. 2112-2119.

Licensed under: http://creativecommons.org/licenses/by-nc/4.0/

Figure 3: Analysis of chromosome features of spermatocytes from patient M870 and a control

(A) Immunofluorescence staining of chromosome spreads from control and patient samples with antibodies against synaptonemal complex protein 3 (SYCP3, red), STAG3 (green; n = 52 cells) and structural maintenance of chromosomes protein 3 (SMC3, green; n = 50). (B) Immunofluorescence staining of chromosome spreads from control (n = 46) and patient (n = 40) samples with antibodies against phospho-Histone H2A.X (H2AX) (red) and STAG3 (green). (C) Immunofluorescence staining of centromeres by anti centromeric antibody (ACA, red) and axis STAG3 (green) from control and patient samples, extended exposure. (D) Graphical representation of centromere signals in control and patient samples. Average ACA signals (with SD) in control: 28 ± 6.0 (n = 28) and in patient samples: 40 ± 12 (n = 36). Unpaired t test was performed, and the P value was <0.0001. Scale bars: 10 μm.

Source: VAN DER BIJL, N., et al. Mutations in the stromal antigen 3 (STAG3) gene cause male infertility due to meiotic arrest. Human Reproduction, 2019, 34. Jg., Nr. 11, S. 2112-2119.

Licensed under: http://creativecommons.org/licenses/by-nc/4.0/