Abstract
Non-obstructive azoospermia (NOA), the lack of spermatozoa in semen due to impaired spermatogenesis affects nearly 1% of men. In about half of cases, an underlying cause for NOA cannot be identified. This study aimed to identify novel variants associated with idiopathic NOA. We identified a nonconsanguineous family in which multiple sons displayed the NOA phenotype. We performed whole-exome sequencing in three affected brothers with NOA, their two unaffected brothers and their father, and identified compound heterozygous frameshift variants (one novel and one extremely rare) in Telomere Repeat Binding Bouquet Formation Protein 2 (TERB2) that segregated perfectly with NOA. TERB2 interacts with TERB1 and Membrane Anchored Junction Protein (MAJIN) to form the tripartite meiotic telomere complex (MTC), which has been shown in mouse models to be necessary for the completion of meiosis and both male and female fertility. Given our novel findings of TERB2 variants in NOA men, along with the integral role of the three MTC proteins in spermatogenesis, we subsequently explored exome sequence data from 1495 NOA men to investigate the role of MTC gene variants in spermatogenic impairment. Remarkably, we identified two NOA patients with likely damaging rare homozygous stop and missense variants in TERB1 and one NOA patient with a rare homozygous missense variant in MAJIN. Available testis histology data from three of the NOA patients indicate germ cell maturation arrest, consistent with mouse phenotypes. These findings suggest that variants in MTC genes may be an important cause of NOA in both consanguineous and outbred populations.
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06 February 2021
A Correction to this paper has been published: https://doi.org/10.1007/s00439-020-02244-1
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Acknowledgements
We thank all the participants and their families for their enthusiastic collaboration. Variant calling and interpretation for Family 1 were performed at the Utah Center for Genetic Discovery Core, part of the Health Sciences Center Cores at University of Utah. We are grateful to Jochen Wistuba for help with the CREM staining of case M1646, and we thank Nadja Rotte for her help with testicular histology in controls. This work was supported in part by funding from the National Institutes of Health (R01HD078641) and the German Research Foundation (Clinical Research Unit ‘Male Germ Cells: from Genes to Function’, DFG CRU326). GEMINI Consortium members: Donald F. Conrad, Kenneth I. Aston, Douglas T. Carrell, James M. Hotaling, Liina Nagirnaja, Timothy G. Jenkins, Moira K. O’Bryan, Rob McLachlan, Peter N. Schlegel, Michael L. Eisenberg, Jay I. Sandlow, James F. Smith, Puneet Kamal, Carole Ober, Mark Sigman, Kathleen Hwang, Emily S. Jungheim, Kenan R. Omurtag, Alexandra M. Lopes, Filipa Carvalho, Susana Fernandes, Alberto Barros, João Gonçalves, Maris Laan, Margus Punab, Ewa Rajpert-De Meyts, Niels Jørgensen, Kristian Almstrup, Csilla G. Krausz, Keith A. Jarvi, Davor Jezek.
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All authors contributed to the study conception and design. Web resources: Gene Cards, https://www.genecards.org/. UCSC Genome Browser, https://genome.ucsc.edu/. Genome Aggregation Database, https://gnomad.broadinstitute.org/. Greater Middle East Variome, https://igm.ucsd.edu/gme/. OMIM, https://www.omim.org/. Protein Atlas, https://www.proteinatlas.org/. PubMed, https://www.ncbi.nlm.nih.gov/PubMed/. UCSC In-Silico PCR, https://genome.ucsc.edu/cgi-bin/hgPcr. UniProt, https://www.uniprot.org/.
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439_2020_2236_MOESM1_ESM.tif
Fig. S1. Protein, genetic context, validation and alignment of the TERB2 variants detected in Family 1. a) Position of TERB2 on chromosome 15. b) Sequence length and exon structure for TERB2. c) Corresponding protein domain structure. d) Primers for the region of interest in exon 6, wild type sequence and validation of exon 6 TERB2 variant using Sanger sequencing in all samples analyzed. e) Primers for the region of interest in exon 7, wild type sequence and validation of exon 7 TERB2 variant using Sanger sequencing in all the analyzed samples. (TIF 1015 KB)
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Fig. S2. Protein sequence conservation of the exon 6 TERB2 variant (p.Thr153fs*17) detected in Family 1 was assessed across 100 vertebrate species using Multiz alignment in the UCSC Genome Browser. * Coelacanth was excluded from the species count because its sequence is truncated. (TIF 183 KB)
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Fig. S3. Protein sequence conservation of the exon 7 TERB2 variant (p.Met182fs*31) detected in Family 1 was assessed across 100 vertebrate species using Multiz alignment in the UCSC Genome Browser. * Coelacanth was excluded from the species count because its sequence is truncated. (TIF 231 KB)
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Fig. S4. Protein, genetic context, validation and alignment of the TERB1 variants detected in Individuals 2 and 3. a) Position of TERB1 on chromosome 16. b) Sequence length and exon structure for TERB1. c) Corresponding protein domain structure. d) Primers for the region of interest and validation of exon 11 TERB1 variant using Sanger sequencing in individual 2. e) Primers for the region of interest and validation of exon 18 TERB1 variant using Sanger sequencing in individual 3 (M2073). (TIF 591 KB)
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Fig. S5. Protein sequence conservation of the TERB1 variant (c.977A>G, p.Glu326Gly) detected in Individual 2 was assessed across 100 vertebrate species using Multiz alignment in the UCSC Genome Browser. (TIF 74 KB)
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Fig. S6. Protein, genetic context, validation and alignment of the MAJIN variant detected in Individual 4 (M1646). a) Position of MAJIN on chromosome 11. b) Sequence length and exon structure for MAJIN gene. c) Corresponding protein domain structure. d) Primers for the region of interest and validation of exon 5 MAJIN variant using Sanger sequencing in the analyzed sample. (TIF 453 KB)
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Fig. S7. Protein sequence conservation of the MAJIN variant (c.158G>A, p.Arg53His) detected in Individual 4 (M1646) was assessed across 100 vertebrate species using Multiz alignment in the UCSC Genome Browser. (TIF 73 KB)
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Fig. S8. RNA expression of TERB2, TERB1 and MAJIN genes with NX data (Consensus Normalized eXpression) levels for 55 tissue types and 6 blood cell types, showing strong enrichment in the testis, created by combining the data from the three transcriptomic datasets (HPA, GTEx and FANTOM5). (TIF 653 KB)
439_2020_2236_MOESM9_ESM.xlsx
Table S1. List of 170 candidate genes that were previously reported to be associated with impaired spermatogenesis according to a systematic review by Oud et al. 2019 and eight recently published genes associated with non-obstructive azoospermia (Krausz et al. 2020) (XLSX 13 KB)
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able S3 Filtered PSAP output for three patients with TERB1 and MAJIN variants based on the following criteria: 1) minor allele frequency [MAF] <0.01 in gnomAD, 1000 Genomes, NHLBI Exome Sequencing Project and ExAC databases, 2) CADD-score ≥20) and 3) PSAP p-value ≤ 0.001. Tabs 1-3 represent the highest-ranked variants for Individual 2, M2073 and M1646, respectively, and tab 4 includes the legend to define column headers in PSAP tables (XLSX 37 KB)
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Salas-Huetos, A., Tüttelmann, F., Wyrwoll, M.J. et al. Disruption of human meiotic telomere complex genes TERB1, TERB2 and MAJIN in men with non-obstructive azoospermia. Hum Genet 140, 217–227 (2021). https://doi.org/10.1007/s00439-020-02236-1
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DOI: https://doi.org/10.1007/s00439-020-02236-1