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

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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.

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Acknowledgements

We wish to thank the participants and clinical staff for making this research study possible. We would also like to recognize Dr. Andrea Berman at the University of Pittsburgh, who provided expert assistance with GCNA 3D modeling. Additionally, we would like to acknowledge Magee-Womens Research Institute scientific editor Bruce Campbell for carefully proofreading the article.

Funding

This study was supported by The Eunice Kennedy Shriver NICHD Grant HD080755 (ANY), the Magee-Womens Research Institute University of Pittsburgh Start Up Fund (ANY), PA DoH Grant SAP4100085736 (ANY), NIH P50 Specialized Center Grant HD096723 (KO, ANY, DC, PNS, KH, and MBE), German Research Foundation Clinical Research Unit ‘Male Germ Cells’ grant DFG CRU326 (FT), National Science Centre in Poland, grants no.: 2017/26/D/NZ5/00789 (AM) and 2015/17/B/NZ2/01157; NCN 2020/37/B/NZ5/00549 (MK), Magee-Womens Research Institute University of Pittsburgh, Faculty Fellowship Award and NICHD T32 HD087194 (JH), GM125812 (MB), GM127569 (MB, JLY, and ANY), NIH R00H090289 (MABE), National Health and Medical Research Council Project grant APP1120356 (MKOB, JAV, and DC), UUKi Rutherford Fund Fellowship (BJH), Estonian Research Council, grants IUT34-12 and PRG1021 (ML), and The Netherlands Organization for Scientific Research grant no.: 918-15-667 as well as an Investigator Award in Science from the Wellcome Trust grant no.: 209451 (JAV). Computational analysis was supported in part by the University of Pittsburgh Center for Research Computing through the resources provided.

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Correspondence to Maciej Kurpisz, Frank Tüttelmann or Alexander N. Yatsenko.

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All study participants provided informed consent and were enrolled under respective IRB protocols, PRO10030036 (Pittsburgh), OKB-5-2/15, 772/15, and 1003/18 (Poznan), 2010-578-f-S (Münster), 0102004794 (New York) (GEMINI Consortium), 254/M-17, 21.12.2015 (Tartu) (GEMINI Consortium), or approval by the human ethics committees of Monash Surgical Private Hospital (Clayton), Monash Medical Centre and Monash University (Melbourne).

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Membership of the GEMINI Consortium is provided in the Appendix.

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Supplementary file1 (DOCX 17 kb)

439_2021_2287_MOESM2_ESM.docx

Supplementary file2 Motifs were derived from Regular Expressions (regex) of chemically conserved regions in GCNA N-terminal IDRs. The regex for motifs with identified GCNA variants are:

439_2021_2287_MOESM3_ESM.tif

Supplementary file3 Supplemental Figure 1. IGV imaging and Sanger confirmation of GCNA variants found in men with SPGF. Integrative Genomics Viewer (IGV; Broad Institute) imaging (left) and Sanger sequencing (right) confirm presence of identified variants in patients with SPGF (see also Table 2). Affected GCNA domains are given in parentheses above chromatogram next to variant position. Affected base is enclosed in black box on chromatograms. (TIF 14057 kb)

Supplementary file4 (TIF 17097 kb)

Supplementary file5 (TIF 1052 kb)

439_2021_2287_MOESM6_ESM.tif

Supplementary file6 Supplemental Figure 2. Human Testis scRNA-seq expression of GCNA and UTF1. (A) UMAP plot of human testicular cells in distinct clusters representing undifferentiated spermatogonia, differentiating spermatogonia, spermatocytes, spermatids, endothelial cells, Leydig cells, macrophages, myoid cells, and pericytes. (B) UMAP plot showing GCNA RNA expression and (C) spermatogonia marker UTF1 RNA expression in human testicular cells. Cells demonstrating positive expression shown in red, with no or little expression in blue. (TIF 10181 kb)

439_2021_2287_MOESM7_ESM.tif

Supplementary file7 Supplemental Figure 3. Spermatogonia scRNA-seq expression of selected markers. (A) UMAP plot of re-clustered human spermatogonia in distinct clusters representing undifferentiated (clusters 1 and 2) and differentiating spermatogonia (clusters 3 and 4), based on expression of spermatogonia markers (B-I) GFRA1, HORMAD1, KIT, ID4, MKI67, STRA8, TEX11, and UCHL1. Cells demonstrating positive expression shown in red, with no or little expression in blue. (TIF 7043 kb)

439_2021_2287_MOESM8_ESM.tif

Supplementary file8 Supplemental Figure 4. Consensus Sequences in GCNA IDR domains altered in patients with SPGF. Missense variants A) p.Val76Met and B) p.Ser295Pro affect consensus motifs in GCNA’s intrinsically disordered region C0xZx and 0-SPiCY where C0xZx corresponds to charged residue, (allowed 2 gaps), 2+ aliphatic residues, and 4+ Z-residues (Ser, Asp, or Glu) in 24 GCNA-IDR sequences and 0-SPi(C/Y) is composed of aliphatic residue, 6+ Z-residues, π-residue, (allowed 1 gap), and terminal amino acid Cys, Tyr, Glu, or Asp) in 23/24 GCNA-IDRs as calculated by CLC7. Number above column on graph corresponds to amino acid position where color matches human reference amino acid identity. Residue frequencies (y-axis) were calculated for each position (x-axis). Motifs with calculated residue frequencies were loaded into MEME suite (Grant, Bailey et al. 2011) and are represented here as logos. FIMO (Grant, Bailey et al. 2011) was used to determine locations of significant motif matches to human GCNA, and those containing the SNV are reported in Suppl. Table 2. Logo visualizations generated by Seq2Logo (Thomsen and Nielsen 2012). (TIF 147 kb)

Appendix

Appendix

GEMINI Consortium Members

Department of Genetics, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, OR, USA

Donald F. Conrad, Liina Nagirnaja

Andrology and IVF Laboratory, Department of Surgery (Urology), University of Utah School of Medicine, Salt Lake City, UT, USA

Kenneth I. Aston, Douglas T. Carrell, James M. Hotaling, Timothy G. Jenkins

(1) Hudson Institute of Medical Research and the Department of Obstetrics and Gynaecology, Monash University, Clayton, Victoria, Australia; (2) Monash IVF and the Hudson Institute of Medical Research, Clayton, Victoria, Australia

Rob McLachlan

School of Biological Sciences, Monash University, Clayton, Victoria, Australia

Moira K. O’Bryan

Department of Urology, Weill Cornell Medicine, New York, NY, USA

Peter N. Schlegel

Department of Urology, Stanford University School of Medicine, Stanford, CA 94,305, USA

Michael L. Eisenberg

Department of Urology, Medical College of Wisconsin, Milwaukee, WI, 53226, USA

Jay I. Sandlow

Washington University in St Louis, School of Medicine, St Louis, MO, USA

Emily S. Jungheim, Kenan R. Omurtag

(1) i3S—Instituto de Investigação e Inovação em Saúde, Universidade do University of Porto.

(2) IPATIMUP—Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Porto, Portugal.

(3) Serviço de Genética, Departamento de Patologia, Faculdade de Medicina da Universidade do Porto, Porto, Portugal

Alexandra M. Lopes 1,2 , Susana Seixas 1,2 , Filipa Carvalho 1,3 , Susana Fernandes 1,3 , Alberto Barros 1,3

(1) Departamento de Genética Humana, Instituto

Nacional de Saúde Dr Ricardo Jorge, Lisboa,

Portugal

(2) ToxOmics, Faculdade de Ciências Médicas,

Universidade Nova de Lisboa, Portugal

(3) Centro de Medicina Reprodutiva, Maternidade Dr.

Alfredo da Costa, Lisboa, Portugal

João Gonçalves 1,2 , Iris Caetano1, Graça Pinto 3 , Sónia Correia 3

Institute of Biomedicine and Translational Medicine, University of Tartu, 51010 Tartu, Estonia

Maris Laan

Andrology Center, Tartu University Hospital, 50406 Tartu, Estonia

Margus Punab

Department of Growth and Reproduction, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark

Ewa Rajpert-De Meyts, Niels Jørgensen, Kristian Almstrup

(1) Department of Experimental and Clinical Biomedical Sciences, University of Florence, Florence, Italy; (2) Andrology Department, Fundacio Puigvert, Instituto de Investigaciones Biomédicas Sant Pau (IIB-Sant Pau), Barcelona, Spain

Csilla G. Krausz

Division of Urology, Department of Surgery, Mount Sinai Hospital, University of Toronto, Toronto, ON, Canada

Keith A. Jarvi

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Hardy, J.J., Wyrwoll, M.J., Mcfadden, W. et al. Variants in GCNA, X-linked germ-cell genome integrity gene, identified in men with primary spermatogenic failure. Hum Genet 140, 1169–1182 (2021). https://doi.org/10.1007/s00439-021-02287-y

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