Browsing by Author "Kliesch, Sabine"
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- Bi-allelic Mutations in M1AP Are a Frequent Cause of Meiotic Arrest and Severely Impaired Spermatogenesis Leading to Male InfertilityPublication . Wyrwoll, Margot J.; Temel, Şehime G.; Nagirnaja, Liina; Oud, Manon S.; Lopes, Alexandra M.; van der Heijden, Godfried W.; Heald, James S.; Rotte, Nadja; Wistuba, Joachim; Wöste, Marius; Ledig, Susanne; Krenz, Henrike; Smits, Roos M.; Carvalho, Filipa; Gonçalves, João; Fietz, Daniela; Türkgenç, Burcu; Ergören, Mahmut C.; Çetinkaya, Murat; Başar, Murad; Kahraman, Semra; McEleny, Kevin; Xavier, Miguel J.; Turner, Helen; Pilatz, Adrian; Röpke, Albrecht; Dugas, Martin; Kliesch, Sabine; Neuhaus, Nina; Aston, Kenneth I.; Conrad, Donald F.; Veltman, Joris A.; Friedrich, Corinna; Tüttelmann, FrankMale infertility affects ∼7% of men, but its causes remain poorly understood. The most severe form is non-obstructive azoospermia (NOA), which is, in part, caused by an arrest at meiosis. So far, only a few validated disease-associated genes have been reported. To address this gap, we performed whole-exome sequencing in 58 men with unexplained meiotic arrest and identified the same homozygous frameshift variant c.676dup (p.Trp226LeufsTer4) in M1AP, encoding meiosis 1 associated protein, in three unrelated men. This variant most likely results in a truncated protein as shown in vitro by heterologous expression of mutant M1AP. Next, we screened four large cohorts of infertile men and identified three additional individuals carrying homozygous c.676dup and three carrying combinations of this and other likely causal variants in M1AP. Moreover, a homozygous missense variant, c.1166C>T (p.Pro389Leu), segregated with infertility in five men from a consanguineous Turkish family. The common phenotype between all affected men was NOA, but occasionally spermatids and rarely a few spermatozoa in the semen were observed. A similar phenotype has been described for mice with disruption of M1ap. Collectively, these findings demonstrate that mutations in M1AP are a relatively frequent cause of autosomal recessive severe spermatogenic failure and male infertility with strong clinical validity.
- Disruption of human meiotic telomere complex genes TERB1, TERB2 and MAJIN in men with non-obstructive azoospermiaPublication . Salas-Huetos, Albert; Tüttelmann, Frank; Wyrwoll, Margot J.; Kliesch, Sabine; Lopes, Alexandra M.; Gonçalves, João; Boyden, Steven E.; Wöste, Marius; Hotaling, James M.; GEMINI Consortium; Nagirnaja, Liina; Conrad, Donald F.; Carrell, Douglas T.; Aston, Kenneth I.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.
- Diverse monogenic subforms of human spermatogenic failurePublication . Nagirnaja, Liina; Lopes, Alexandra M.; Charng, Wu-Lin; Miller, Brian; Stakaitis, Rytis; Golubickaite, Ieva; Stendahl, Alexandra; Luan, Tianpengcheng; Friedrich, Corinna; Mahyari, Eisa; Fadial, Eloise; Kasak, Laura; Vigh-Conrad, Katinka; Oud, Manon S.; Xavier, Miguel J.; Cheers, Samuel R.; James, Emma R.; Guo, Jingtao; Jenkins, Timothy G.; Riera-Escamilla, Antoni; Barros, Alberto; Carvalho, Filipa; Fernandes, Susana; Gonçalves, João; Gurnett, Christina A.; Jørgensen, Niels; Jezek, Davor; Jungheim, Emily S.; Kliesch, Sabine; McLachlan, Robert I.; Omurtag, Kenan R.; Pilatz, Adrian; Sandlow, Jay I.; Smith, James; Eisenberg, Michael L.; Hotaling, James M.; Jarvi, Keith A.; Punab, Margus; Rajpert-De Meyts, Ewa; Carrell, Douglas T.; Krausz, Csilla; Laan, Maris; O’Bryan, Moira K.; Schlegel, Peter N.; Tüttelmann, Frank; Veltman, Joris A.; Almstrup, Kristian; Aston, Kenneth I.; Conrad, Donald F.Non-obstructive azoospermia (NOA) is the most severe form of male infertility and typically incurable. Defining the genetic basis of NOA has proven chal lenging, and the most advanced classification of NOA subforms is not based on genetics, but simple description of testis histology. In this study, we exome sequenced over 1000 clinically diagnosed NOA cases and identified a plausible recessive Mendelian cause in 20%. We find further support for 21 genes in a 2-stage burden test with 2072 cases and 11,587 fertile controls. The disrupted genes are primarily on the autosomes, enriched for undescribed human “knockouts”, and, for the most part, have yet to be linked to a Mendelian trait. Integration with single-cell RNA sequencing data shows that azoospermia genes can be grouped into molecular subforms with synchronized expression patterns, and analogs of these subforms exist in mice. This analysis framework identifies groups of genes with known roles in spermatogenesis but also reveals unrecognized subforms, such as a set of genes expressed across mitotic divisions of differentiating spermatogonia. Our findings highlight NOA as an understudied Mendelian disorder and provide a conceptual structure for organizing the complex genetics of male infertility, which may provide a rational basis for disease classification
- Immune and spermatogenesis-related loci are involved in the development of extreme patterns of male infertilityPublication . Cerván-Martín, Miriam; Tüttelmann, Frank; Lopes, Alexandra M.; Bossini-Castillo, Lara; Rivera-Egea, Rocío; Garrido, Nicolás; Lujan, Saturnino; Romeu, Gema; Santos-Ribeiro, Samuel; Castilla, José A.; Carmen Gonzalvo, M.; Clavero, Ana; Maldonado, Vicente; Vicente, F. Javier; González-Muñoz, Sara; Guzmán-Jiménez, Andrea; Burgos, Miguel; Jiménez, Rafael; Pacheco, Alberto; González, Cristina; Gómez, Susana; Amorós, David; Aguilar, Jesus; Quintana, Fernando; Calhaz-Jorge, Carlos; Aguiar, Ana; Nunes, Joaquim; Sousa, Sandra; Pereira, Isabel; Pinto, Maria Graça; Correia, Sónia; Sánchez-Curbelo, Josvany; López-Rodrigo, Olga; Martín, Javier; Pereira-Caetano, Iris; Marques, Patricia I.; Carvalho, Filipa; Barros, Alberto; Gromoll, Jörg; Bassas, Lluís; Seixas, Susana; Gonçalves, João; Larriba, Sara; Kliesch, Sabine; Palomino-Morales, Rogelio J.; Carmona, F. DavidWe conducted a genome-wide association study in a large population of infertile men due to unexplained spermatogenic failure (SPGF). More than seven million genetic variants were analysed in 1,274 SPGF cases and 1,951 unaffected controls from two independent European cohorts. Two genomic regions were associated with the most severe histological pattern of SPGF, defined by Sertoli cell-only (SCO) phenotype, namely the MHC class II gene HLA-DRB1 (rs1136759, P = 1.32E-08, OR = 1.80) and an upstream locus of VRK1 (rs115054029, P = 4.24E-08, OR = 3.14), which encodes a protein kinase involved in the regulation of spermatogenesis. The SCO-associated rs1136759 allele (G) determines a serine in the position 13 of the HLA-DRβ1 molecule located in the antigen-binding pocket. Overall, our data support the notion of unexplained SPGF as a complex trait influenced by common variation in the genome, with the SCO phenotype likely representing an immune-mediated condition.
- New insight into the genetic contribution of common variants to the development of extreme phenotypes of unexplained male infertility: a multicenter genome-wide association studyPublication . Cerván-Martín, Miriam; Tüttelmann, Frank; Lopes, Alexandra; Castillo, Lara B.; Garrido, Nicolás; Luján, Saturnino; Castilla, José A.; Azoonomic, S.G.; Gromoll, J.; Seixas, Susana; Gonçalves, João; Larriba, Sara; Kliesch, Sabine; Palomino-Morales, Rogelio; Carmona, Francisco D.Study question: What is the contribution of the common genetic variation to the development of unexplained male infertility due to severe spermatogenic failure (SPGF)? Summary answer: Genetic polymorphisms of key immune and spermatogenesis loci are involved in the etiology of the most severe SPGF cases, defined by Sertoli cell-only (SCO) phenotype. What is known already: Male infertility is a rising worldwide concern that affects millions of couples. Non-obstructive azoospermia (NOA) and severe oligospermia (SO) are two extreme manifestations characterized by SPGF. A genetic cause can be established in only around 20% of affected men, with the remaining cases being classified as otherwise unexplained. To date, the genomewide association study (GWAS) strategy, although already successfully applied in several other complex traits and diseases, was less fruitful in studies that attempted to decipher the genetic component of unexplained SPGF, mainly due to both a lack of well-powered samples in different ancestries and limitations in study design. Study design, size, duration: We designed a GWAS for unexplained male infertility due to SPGF including a total of 1,274 affected cases and 1,951 fertile controls from the Iberian Peninsula (Spain and Portugal) and Germany. Different biostatistics and bioinformatics approaches were used to evaluate the possible effect of single-nucleotide polymorphisms (SNPs) across the whole genome in the susceptibility to specific subtypes of unexplained SPGF. Participants/materials, setting, methods: The case cohort comprised 502 SO and 772 NOA patients, who were subdivided according to histological phenotypes (SCO, maturation arrest, and hypospermatogenesis) and the outcome of testicular sperm extraction techniques (TESE) from testis biopsies. Genotyping was performed with the GSA platform (Illumina). After quality-control and genotype imputation, 6,539,982 SNPs remained for the analysis, which was performed by logistic regression models. The datasets went through a meta-analysis by the inverse variance weighted method under fixed effects. Main results and the role of chance: Genetic associations with SCO at the genome-wide-level of significance were identified in the major histocompatibility (MHC) class II region (rs1136759, OR=1.80, P=1.32E-08) and in a regulatory region of chromosome 14 nearby the vaccinia-related kinase 1 (VRK1) gene (rs115054029, OR=3.14, P=4.37-08). VRK1 is a relevant proliferative factor for spermatogenesis that causes progressive loss of spermatogonia when disrupted in mouse models. The role of the MHC system in SCO susceptibility was comprehensively evaluated through a validated imputation method that infers classical MHC alleles and polymorphic amino acid positions. A serine at position 13 of the HLA-DRβ1 protein (defined by the risk allele of the lead variant rs1136759) explained most of the SCO association signals within the MHC class II region. This residue is located in the binding pocket of the HLA-DR molecule and interacts directly with the presented antigen. Interestingly, position 13 of HLADRβ1 is the most relevant risk amino acid position for a wide spectrum of immune-mediated disorders. The HLA-DRB1*13 haplotype (which includes the serine at position 13 and represents the strongest NOA-associated marker in Asians to date) was the strongest signal amongst the classical MHC alleles in our study cohort (OR=1.93, P=9.90E-07). Limitations, reasons for caution: Although the statistical power for the overall analysis was appropriate, the subphenotype analyses performed had considerably lower counts, which may influence the identification of genetic variants conferring low to moderate risk effects. Independent studies in larger SCO study cohorts should be performed to confirm our findings. Wider implications of the findings: The molecular mechanisms underlying unexplained SPGF are largely unknown. Our data suggest a relevant role of common genetic variation in the development of SCO, the most extreme histological phenotype of NOA. SCO is characterized by the loss of germ cells and, therefore, implies a considerably higher probability of unsuccessful TESE.
- Variants in GCNA, X-linked germ-cell genome integrity gene, identified in men with primary spermatogenic failurePublication . Hardy, Jimmaline J.; Wyrwoll, Margot J.; Mcfadden, William; Malcher, Agnieszka; Rotte, Nadja; Pollock, Nijole C.; Munyoki, Sarah; Veroli, Maria V.; Houston, Brendan J.; Xavier, Miguel J.; Kasak, Laura; Punab, Margus; Laan, Maris; Kliesch, Sabine; Schlegel, Peter; Jaffe, Thomas; Hwang, Kathleen; Vukina, Josip; Brieño-Enríquez, Miguel A.; Orwig, Kyle; Yanowitz, Judith; Buszczak, Michael; Veltman, Joris A.; Oud, Manon; Nagirnaja, Liina; Olszewska, Marta; O’Bryan, Moira K.; Conrad, Donald F.; Kurpisz, Maciej; Tüttelmann, Frank; Yatsenko, Alexander N.; Conrad, Donald F.; Nagirnaja, Liina; Aston, Kenneth I.; Carrell, Douglas T.; Hotaling, James M.; Jenkins, Timothy G.; McLachlan, Rob; O’Bryan, Moira K.; Schlegel, Peter N.; Eisenberg, Michael L.; Sandlow, Jay I.; Jungheim, Emily S.; Omurtag, Kenan R.; Lopes, Alexandra M.; Seixas, Susana; Carvalho, Filipa; Fernandes, Susana; Barros, Alberto; Gonçalves, João; Caetano, Iris; Pinto, Graça; Correia, Sónia; Laan, Maris; Punab, Margus; Meyts, Ewa Rajpert-De; Jørgensen, Niels; Almstrup, Kristian; Krausz, Csilla G.; Jarvi, Keith A.; on behalf of GEMINI ConsortiumMale 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 identifed a potentially signifcant 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 identifed. GCNA is critical for genome integrity in male meiosis and knockout models exhibit impaired spermatogenesis and infertility. Single-cell RNA-seq and immunohistochemistry confrm human GCNA expression from spermatogonia to elongated spermatids. Five identifed 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 afecting 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 identifed 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-specifc expression, 3D modeling, and computational predictions for SNVs, we propose that identifed GCNA variants disrupt structure and function of the respective protein domains, ultimately arresting germ-cell division. To our knowledge, this is the frst study implicating GCNA, a key genome integrity factor, in human male infertility.