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W2617453476 abstract "Research Article29 May 2017Open Access Transparent process SPINK2 deficiency causes infertility by inducing sperm defects in heterozygotes and azoospermia in homozygotes Zine-Eddine Kherraf Zine-Eddine Kherraf Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Marie Christou-Kent Marie Christou-Kent Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Thomas Karaouzene Thomas Karaouzene Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Amir Amiri-Yekta Amir Amiri-Yekta Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France CHU de Grenoble, UF de Biochimie Génétique et Moléculaire, Grenoble, France Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran Search for more papers by this author Guillaume Martinez Guillaume Martinez Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Alexandra S Vargas Alexandra S Vargas Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Emeline Lambert Emeline Lambert Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Christelle Borel Christelle Borel Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva 4, Switzerland Search for more papers by this author Béatrice Dorphin Béatrice Dorphin Laboratoire d'Aide Médicale à la Procréation, Centre AMP 74, Contamine-sur-Arve, France Search for more papers by this author Isabelle Aknin-Seifer Isabelle Aknin-Seifer Laboratoire de Biologie de la Reproduction, Hôpital Nord, Saint Etienne, France Search for more papers by this author Michael J Mitchell Michael J Mitchell Aix Marseille Univ, INSERM, GMGF, Marseille, France Search for more papers by this author Catherine Metzler-Guillemain Catherine Metzler-Guillemain Aix Marseille Univ, INSERM, GMGF, Marseille, France Search for more papers by this author Jessica Escoffier Jessica Escoffier Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Serge Nef Serge Nef Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva 4, Switzerland Search for more papers by this author Mariane Grepillat Mariane Grepillat Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Nicolas Thierry-Mieg Nicolas Thierry-Mieg Univ. Grenoble Alpes / CNRS, TIMC-IMAG, Grenoble, France Search for more papers by this author Véronique Satre Véronique Satre Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France CHU de Grenoble, UF de Génétique Chromosomique, Grenoble, France Search for more papers by this author Marc Bailly Marc Bailly Department of Reproductive Biology and Gynaecology, Poissy General Hospital, Poissy, France EA 7404 GIG, Université de Versailles Saint Quentin, Montigny le Bretonneux, France Search for more papers by this author Florence Boitrelle Florence Boitrelle Department of Reproductive Biology and Gynaecology, Poissy General Hospital, Poissy, France EA 7404 GIG, Université de Versailles Saint Quentin, Montigny le Bretonneux, France Search for more papers by this author Karin Pernet-Gallay Karin Pernet-Gallay Grenoble Neuroscience Institute, INSERM 1216, Grenoble, France Search for more papers by this author Sylviane Hennebicq Sylviane Hennebicq Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France CHU de Grenoble, UF de Biologie de la procréation, Grenoble, France Search for more papers by this author Julien Fauré Julien Fauré CHU de Grenoble, UF de Biochimie Génétique et Moléculaire, Grenoble, France Grenoble Neuroscience Institute, INSERM 1216, Grenoble, France Search for more papers by this author Serge P Bottari Serge P Bottari Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France CHU de Grenoble, UF de Radioanalyses, Grenoble, France Search for more papers by this author Charles Coutton Charles Coutton Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France CHU de Grenoble, UF de Génétique Chromosomique, Grenoble, France Search for more papers by this author Pierre F Ray Corresponding Author Pierre F Ray [email protected] orcid.org/0000-0003-1544-7449 Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France CHU de Grenoble, UF de Biochimie Génétique et Moléculaire, Grenoble, France Search for more papers by this author Christophe Arnoult Christophe Arnoult Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Zine-Eddine Kherraf Zine-Eddine Kherraf Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Marie Christou-Kent Marie Christou-Kent Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Thomas Karaouzene Thomas Karaouzene Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Amir Amiri-Yekta Amir Amiri-Yekta Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France CHU de Grenoble, UF de Biochimie Génétique et Moléculaire, Grenoble, France Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran Search for more papers by this author Guillaume Martinez Guillaume Martinez Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Alexandra S Vargas Alexandra S Vargas Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Emeline Lambert Emeline Lambert Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Christelle Borel Christelle Borel Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva 4, Switzerland Search for more papers by this author Béatrice Dorphin Béatrice Dorphin Laboratoire d'Aide Médicale à la Procréation, Centre AMP 74, Contamine-sur-Arve, France Search for more papers by this author Isabelle Aknin-Seifer Isabelle Aknin-Seifer Laboratoire de Biologie de la Reproduction, Hôpital Nord, Saint Etienne, France Search for more papers by this author Michael J Mitchell Michael J Mitchell Aix Marseille Univ, INSERM, GMGF, Marseille, France Search for more papers by this author Catherine Metzler-Guillemain Catherine Metzler-Guillemain Aix Marseille Univ, INSERM, GMGF, Marseille, France Search for more papers by this author Jessica Escoffier Jessica Escoffier Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Serge Nef Serge Nef Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva 4, Switzerland Search for more papers by this author Mariane Grepillat Mariane Grepillat Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Nicolas Thierry-Mieg Nicolas Thierry-Mieg Univ. Grenoble Alpes / CNRS, TIMC-IMAG, Grenoble, France Search for more papers by this author Véronique Satre Véronique Satre Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France CHU de Grenoble, UF de Génétique Chromosomique, Grenoble, France Search for more papers by this author Marc Bailly Marc Bailly Department of Reproductive Biology and Gynaecology, Poissy General Hospital, Poissy, France EA 7404 GIG, Université de Versailles Saint Quentin, Montigny le Bretonneux, France Search for more papers by this author Florence Boitrelle Florence Boitrelle Department of Reproductive Biology and Gynaecology, Poissy General Hospital, Poissy, France EA 7404 GIG, Université de Versailles Saint Quentin, Montigny le Bretonneux, France Search for more papers by this author Karin Pernet-Gallay Karin Pernet-Gallay Grenoble Neuroscience Institute, INSERM 1216, Grenoble, France Search for more papers by this author Sylviane Hennebicq Sylviane Hennebicq Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France CHU de Grenoble, UF de Biologie de la procréation, Grenoble, France Search for more papers by this author Julien Fauré Julien Fauré CHU de Grenoble, UF de Biochimie Génétique et Moléculaire, Grenoble, France Grenoble Neuroscience Institute, INSERM 1216, Grenoble, France Search for more papers by this author Serge P Bottari Serge P Bottari Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France CHU de Grenoble, UF de Radioanalyses, Grenoble, France Search for more papers by this author Charles Coutton Charles Coutton Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France CHU de Grenoble, UF de Génétique Chromosomique, Grenoble, France Search for more papers by this author Pierre F Ray Corresponding Author Pierre F Ray [email protected] orcid.org/0000-0003-1544-7449 Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France CHU de Grenoble, UF de Biochimie Génétique et Moléculaire, Grenoble, France Search for more papers by this author Christophe Arnoult Christophe Arnoult Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Author Information Zine-Eddine Kherraf1,‡, Marie Christou-Kent1,‡, Thomas Karaouzene1, Amir Amiri-Yekta1,2,3, Guillaume Martinez1, Alexandra S Vargas1, Emeline Lambert1, Christelle Borel4, Béatrice Dorphin5, Isabelle Aknin-Seifer6, Michael J Mitchell7, Catherine Metzler-Guillemain7, Jessica Escoffier1, Serge Nef4, Mariane Grepillat1, Nicolas Thierry-Mieg8, Véronique Satre1,9, Marc Bailly10,11, Florence Boitrelle10,11, Karin Pernet-Gallay12, Sylviane Hennebicq1,13, Julien Fauré2,12, Serge P Bottari1,14, Charles Coutton1,9, Pierre F Ray *,1,2,‡ and Christophe Arnoult1,‡ 1Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France 2CHU de Grenoble, UF de Biochimie Génétique et Moléculaire, Grenoble, France 3Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran 4Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva 4, Switzerland 5Laboratoire d'Aide Médicale à la Procréation, Centre AMP 74, Contamine-sur-Arve, France 6Laboratoire de Biologie de la Reproduction, Hôpital Nord, Saint Etienne, France 7Aix Marseille Univ, INSERM, GMGF, Marseille, France 8Univ. Grenoble Alpes / CNRS, TIMC-IMAG, Grenoble, France 9CHU de Grenoble, UF de Génétique Chromosomique, Grenoble, France 10Department of Reproductive Biology and Gynaecology, Poissy General Hospital, Poissy, France 11EA 7404 GIG, Université de Versailles Saint Quentin, Montigny le Bretonneux, France 12Grenoble Neuroscience Institute, INSERM 1216, Grenoble, France 13CHU de Grenoble, UF de Biologie de la procréation, Grenoble, France 14CHU de Grenoble, UF de Radioanalyses, Grenoble, France ‡These authors contributed equally to this work ‡These authors contributed equally to this work as senior authors *Corresponding author. Tel: +33 4 76 76 55 73; E-mail: [email protected] EMBO Mol Med (2017)9:1132-1149https://doi.org/10.15252/emmm.201607461 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Azoospermia, characterized by the absence of spermatozoa in the ejaculate, is a common cause of male infertility with a poorly characterized etiology. Exome sequencing analysis of two azoospermic brothers allowed the identification of a homozygous splice mutation in SPINK2, encoding a serine protease inhibitor believed to target acrosin, the main sperm acrosomal protease. In accord with these findings, we observed that homozygous Spink2 KO male mice had azoospermia. Moreover, despite normal fertility, heterozygous male mice had a high rate of morphologically abnormal spermatozoa and a reduced sperm motility. Further analysis demonstrated that in the absence of Spink2, protease-induced stress initiates Golgi fragmentation and prevents acrosome biogenesis leading to spermatid differentiation arrest. We also observed a deleterious effect of acrosin overexpression in HEK cells, effect that was alleviated by SPINK2 coexpression confirming its role as acrosin inhibitor. These results demonstrate that SPINK2 is necessary to neutralize proteases during their cellular transit toward the acrosome and that its deficiency induces a pathological continuum ranging from oligoasthenoteratozoospermia in heterozygotes to azoospermia in homozygotes. Synopsis SPINK2, a serine protease inhibitor, is believed to target the acrosin, the main sperm acrosomal protease. This study confirms SPINK2 in that role and finds it essential for spermiogenesis as SPINK2 deficiency induces a post meiotic block at the round spermatid stage leading to azoospermia in mice and men. In round spermatids, SPINK2 is necessary to inactivate the acrosin during its transit through the endoplasmic reticulum and the Golgi apparatus. In the absence of SPINK2, acrosin can auto-activate, disorganize the Golgi apparatus, prevent the production of the acrosome and induce a block at the round spermatid stage. A reduced amount of SPINK2 in heterozygotes is also deleterious, inducing a milder phenotype of oligozoospermia and/or teratozoospermia without a systematic infertility. Introduction The World Health Organization estimates that 50 million couples worldwide are confronted with infertility. Assisted reproduction technologies (ART) initiated 35 years ago by Nobel Prize Winner Robert Edwards have revolutionized the practice of reproductive medicine, and it is now estimated that approximately 15% of couples in Western countries seek assistance from reproductive clinics for infertility or subfertility. Despite technological breakthroughs and advances, approximately half of the couples concerned still fail to achieve a successful pregnancy even after repeated treatment cycles. Alternative treatment strategies should therefore be envisaged to improve ART success rate, especially for patients impervious to usual assisted reproductive technologies. Improvement in treatment efficiency essentially depends upon an accurate diagnosis and the characterization of the molecular etiology of the defect. These efforts to better characterize infertility subtypes should first be concentrated on the most severe defects since they generally have a poor prognosis and affected patients would benefit the most from new treatments. Moreover, the most severe phenotypes are more likely to be caused by monogenic defects which are easier to identify. As such, the genetic exploration of non-obstructive azoospermia (NOA), the absence of spermatozoa in the ejaculate due to a defect in spermatogenesis, should be considered a priority. NOA is a common cause of infertility found in approximately 10% of the couples assessed for infertility. Although a genetic etiology is likely to be present in most cases of azoospermia, only a few defective genes have so far been associated with this pathology accounting for a minority of cases. At present, only chromosomal abnormalities (mainly 47XXY, Klinefelter syndrome identified in 14% of cases) and microdeletions of the Y chromosome are routinely diagnosed, resulting in a positive genetic diagnosis in < 20% of azoospermia cases (Tuttelmann et al, 2011). The evolution of sequencing technologies and the use of whole-exome or whole-genome sequence (WES/WGS) analysis paves the way to a great improvement in our ability to characterize the causes of genetically heterogeneous pathologies such as NOA. Spermatogenesis can be subdivided into three main steps: (i) multiplication of diploid germ cells; (ii) meiosis, with the shuffling of parental genes and production of haploid cells; and (iii) spermiogenesis, the conversion of round spermatids into one of the smallest and most specialized cells in the body, the spermatozoa. NOA is expected to be mainly caused by failures in steps 1 and 2, and it is indeed what has been observed in a majority of cases so far. Very recently, defects in six genes were linked to azoospermia in man. Most of these genes code for meiosis-controlling proteins such as TEX11, TEX15, SYCE1, or MCM8, and the absence of the functional proteins induces a blockage of meiosis (Tuttelmann et al, 2011; Maor-Sagie et al, 2015; Okutman et al, 2015; Yang et al, 2015; Yatsenko et al, 2015). Another WES analysis of two consanguineous families identified likely causal mutations in TAF4B and ZMYND15 (Ayhan et al, 2014). Study of Taf4b KO mice showed that homozygous mutant males are subfertile with extensive pre-meiotic germ cell loss due to altered differentiation and self-renewal of the spermatogonial stem cell pool, thus illustrating that pre-meiotic block induces NOA. More surprisingly, ZMYND15 codes for a spermatid-specific histone deacetylase-dependent transcriptional repressor and its absence in mice induced a significant depletion of late-stage spermatids (Yan et al, 2010) suggesting that NOA can also be induced by post-meiotic defects. Here, WES analysis of two brothers with NOA led to the identification of a homozygous truncating mutation in the SPINK2 gene coding for a Kazal family serine protease inhibitor. Studying KO mice, we observed that homozygous KO animals also suffered from azoospermia thus confirming the implication of SPINK2 in NOA. Furthermore, we observed that SPINK2 is expressed from the round-spermatid stage onwards thus confirming that post-meiotic anomalies can result in NOA. We suggest that SPINK2 is necessary to neutralize the action of acrosomal proteases shortly after their synthesis and before they can be safely stored in the acrosome where they normally remain dormant until their release during the acrosome reaction. We also show that in the absence of SPINK2, protease-induced stress initiates Golgi fragmentation contributing to the arrest of spermatid differentiation and their shedding from the seminiferous epithelium. The characterization of the molecular pathophysiology of this defect opens several novel therapeutic perspectives which may allow the restoration of a functional spermatogenesis. Results Medical assessment of two brothers with defective sperm production Two French brothers (Br1 and Br2), born from second cousin parents (Fig 1A), and their respective wives sought medical advice from infertility clinics in France (Chatellerault, Tours, Poissy, and Grenoble) between 2005 and 2014 after 2 years of unsuccessful attempts to spontaneously conceive. Analyses of their ejaculates (Fig 1B; n = 5) evidenced the absence of spermatozoa for the first brother (Br1) and a very low concentration (0–200,000/ml, mean 126,000/ml n = 5) for the second (Br2). Moreover, all spermatozoa were immotile and presented an abnormal morphology (pin-shaped head devoid of acrosome; detached flagella) and were not suitable for in vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI). Interestingly, ejaculates of both brothers presented a significant concentration of germ cells (8.6 × 106 ± 6.2 × 106/ml and 9.0 × 106 ± 7.0 × 106/ml for Br-1 and Br-2, respectively) likely corresponding to spermatids. As Br1 and Br2 both present a severe default of sperm production with a high number of spermatids in the ejaculate, we believe that they present the same phenotype, likely caused by the same genetic defect. A normal karyotype was observed for both brothers (46,XY), and no deletions of the Y chromosome were observed at the AZF loci. Testis sperm extraction was carried out twice for Br1 in 2008 and 2014. Each time the recovery was unsuccessful (although a few spermatozoa were observed in fixed dilacerated testicular tissues) suggesting a diagnosis of post-meiotic NOA. Histological analysis of seminiferous tubules obtained from Br1 biopsies showed: (i) a disorganization of the structure of the tubules; (ii) that the lumen of the seminiferous tubules were filled with immature germ cells, an indication of intense desquamation of the germinal epithelium; and (iii) a reduced number of round spermatids, with an overrepresentation of early round spermatids (Fig 1C–F). Brother Br2 has only had spermograms for diagnostic purposes which did not show any ICSI-compatible spermatozoa and has not been able to attempt ART. Figure 1. Azoospermia in two consanguineous brothers A. Genetic tree of the studied family showing affected brothers Br1 and Br2 illustrating the consanguinity of the parents (P1 and P2). B. Comparisons of ejaculate volume (n = 5) and spermograms (n = 5) of brothers Br1 and Br2 with those of fertile controls (n = 35) evidence the absence of mature sperm and the presence of round cells in the ejaculates. Data represent mean ± SEM. P-values are P = 4 × 10−4 (a), P = 0.6 (b, non-significant), and P = 4 × 10−5 (c); statistical differences were assessed using t-test. C, D. Testis sections from a fertile control and (D) patient Br1 stained with periodic acid–Schiff (PAS). The lumen of tubules from the control is large and mature sperm are present (C), whereas the lumen of most of seminiferous tubules from patient Br1 is filled with non-condensed and early condensed round spermatids and no mature sperm are observed. Scales bars, 100 μm. E, F. In the fertile control (E) seminiferous tubule cross sections, spermatogonia (Sg), spermatocytes (Sc) and spermatids (RS) are regularly layered, whereas the different types of spermatogenic cells are disorganized in patient Br1 (F). Scales bars, 100 μm. Download figure Download PowerPoint Whole-exome sequencing identifies a homozygous truncating mutation in SPINK2 Since the brothers were married to unrelated women, we excluded the possibility of a contributing female factor and focused our research on the brothers. Given the familial history of consanguinity, we postulated that their infertility was likely caused by a common homozygous mutation. We proceeded with WES to identify a possible genetic defect(s) which could explain the observed azoospermia. After exclusion of common variants, both bothers carried a total of 121 identical missense heterozygous variants (none appearing as obvious candidate) and only five identical homozygous variants common to both brothers (Appendix Table S1). Among these different genes, only the Chr4:57686748G>C SPINK2 variant was described to be predominantly expressed in human testis (Appendix Fig S1A) as well as in mouse testis (Appendix Fig S1B). The mutation was validated by Sanger sequencing in both brothers (homozygous) and their parents (heterozygous) (Fig 2A). SPINK2 thus appeared as the best candidate to explain the human condition. The variant Chr4:57686748G>C was not present in > 121,000 alleles analyzed in the ExAC database (http://exac.broadinstitute.org) and could have an effect on RNA splicing. SPINK2 is located on chromosome 4 and contains four exons (Fig 2B). The gene codes for a Kazal type 2 serine protease inhibitor also known as an acrosin–trypsin inhibitor. The Ensembl expression database (www.ensembl.org) predicts the presence of four transcripts. We studied the expression of the different transcripts in human testis by RT–PCR, and only one band was present corresponding to NM_021114, ENST00000248701, which codes for a protein of 9.291 kDa consisting of 84 amino acids (Fig 2C). All nucleotide sequences herein refer to this transcript. The identified mutation, c.56-3C>G, is located three nucleotides before exon 2 and may create a new splice acceptor site, leading to a frameshift and premature stop codon in exon 2 and the generation of an abnormal transcript (T1) and/or to the skipping of exon 2 (44 nt) giving rise to an early stop codon at the beginning of exon 3 and the generation of another abnormal transcript (T2) (Fig 2B). To validate these hypotheses, RT–PCR was performed on testicular extract from Br1. Two bands were observed (Fig 2C) and sequenced after isolation of each band following gel electrophoresis. Sequence analysis demonstrated that the bands corresponded to T1 and T2, demonstrating that both abnormal transcripts were present in the patient's testis (Fig 2D). Since the protease inhibitor and binding sites of the protein are coded mostly by exon 3, it is expected that the truncated proteins corresponding to T1 and T2 transcripts are not functional (Appendix Fig S2). Sequencing of Br1's transcripts therefore confirms that the identified splice variant abrogates the production of a full-length protein thereby confirming its role as a deleterious mutation. Figure 2. Identification of a SPINK2 variant (c.56-3C>G) by exome sequencing and its consequences on splicing and translation The identified variant, homozygous in patients 1 and 2 and heterozygous in their parents, is located three nucleotides before exon 2 and creates an AG that immediately precedes the original AG splice acceptor site. If recognized during splicing, this new acceptor site is expected to add two nucleotides (AG) at the beginning of exon 2, inducing a frameshift leading to a stop codon 3 amino acids later (transcript 1). The non-recognition of the abnormal acceptor site is expected to induce the skipping of exon 2 (transcript 2). The first stop codon can be observed 15 codons after the mis-inserted exon 3. RT–PCR of mRNA extracts from fertile control (Ctrl) and the brother Br1. Results show one band for Ctrl. The sequencing of this band showed that it corresponds to transcript NM_021114. For Br1, two bands were present, named T1 and T2. Bottom gel shows T1 and T2 after gel isolation. Transcripts T1 and T2 were collected and sequenced: T1 showed the insertion of an additional AG (red-dashed rectangle) leading to a premature stop codon (black box), whereas transcript T2 showed that exon 2 had been excised; these two transcripts correspond to the expected transcripts 1 and 2 from panel (B). Stop codons are shown in black boxes. Download figure Download PowerPoint Importance of SPINK2 variants as a cause for human infertility: sequence analysis of a cohort of infertile men with an altered spermatogenesis We sequenced SPINK2 whole coding sequences of 611 patients affected by azoo- or oligozoospermia (210 patients with azoospermia, 393 subjects with oligozoospermia and 8 with unspecified cause). Only one variant, identified in patient 105 (P105), was not described in ExAC and was likely deleterious (Appendix Table S2). This variant, c.1A>T (Fig EV1A), abrogates the SPINK2 start codon and was present heterozygously in P105, a man with oligozoospermia. An alternate start site could potentially be use" @default.
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W2617453476 title "<scp>SPINK</scp> 2 deficiency causes infertility by inducing sperm defects in heterozygotes and azoospermia in homozygotes" @default.
W2617453476 cites W1508970569 @default.
W2617453476 cites W1563800735 @default.
W2617453476 cites W1973609206 @default.
W2617453476 cites W1973632178 @default.
W2617453476 cites W1974427864 @default.
W2617453476 cites W1975534833 @default.
W2617453476 cites W1978695171 @default.
W2617453476 cites W1979715747 @default.
W2617453476 cites W1980996405 @default.
W2617453476 cites W1986499227 @default.
W2617453476 cites W1989609207 @default.
W2617453476 cites W2028748494 @default.
W2617453476 cites W2029036137 @default.
W2617453476 cites W2031724207 @default.
W2617453476 cites W2035557358 @default.
W2617453476 cites W2037485665 @default.
W2617453476 cites W2044088661 @default.
W2617453476 cites W2064397275 @default.
W2617453476 cites W2066449582 @default.
W2617453476 cites W2071646318 @default.
W2617453476 cites W2083174192 @default.
W2617453476 cites W2083445028 @default.
W2617453476 cites W2089334040 @default.
W2617453476 cites W2089436328 @default.
W2617453476 cites W2094809870 @default.
W2617453476 cites W2098346462 @default.
W2617453476 cites W2103665463 @default.
W2617453476 cites W2104628102 @default.
W2617453476 cites W2117086890 @default.
W2617453476 cites W2127911142 @default.
W2617453476 cites W2132658004 @default.
W2617453476 cites W2140766436 @default.
W2617453476 cites W2156400902 @default.
W2617453476 cites W2157938250 @default.
W2617453476 cites W2158162999 @default.
W2617453476 cites W2158899309 @default.
W2617453476 cites W2160577038 @default.
W2617453476 cites W2160903957 @default.
W2617453476 cites W2162190911 @default.
W2617453476 cites W2162395803 @default.
W2617453476 cites W2166011264 @default.
W2617453476 cites W2230734953 @default.
W2617453476 cites W327137974 @default.
W2617453476 cites W4240778206 @default.
W2617453476 cites W4376848309 @default.
W2617453476 cites W3145949091 @default.
W2617453476 doi "https://doi.org/10.15252/emmm.201607461" @default.
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