PATL 2 is a key actor of oocyte maturation whose invalidation causes infertility in women and mice
2018; Springer Nature; Volume: 10; Issue: 5 Linguagem: Inglês
10.15252/emmm.201708515
ISSN1757-4684
AutoresMarie Christou‐Kent, Zine‐Eddine Kherraf, Amir Amiri‐Yekta, Emilie Le Blévec, Thomas Karaouzène, Béatrice Conne, Jessica Escoffier, Saïd Assou, Audrey Guttin, Emeline Lambert, Guillaume Martinez, Magalie Boguenet, Sélima Fourati Ben Mustapha, Isabelle Cedrin Durnerin, Lazhar Halouani, Ouafi Marrakchi, Mounir Makni, Habib Latrous, Mahmoud Kharouf, Charles Coutton, Nicolas Thierry‐Mieg, Serge Nef, Serge P. Bottari, Raoudha Zouari, Jean Paul Issartel, Pierre F. Ray, Christophe Arnoult,
Tópico(s)Epigenetics and DNA Methylation
ResumoResearch Article16 April 2018Open Access Transparent process PATL2 is a key actor of oocyte maturation whose invalidation causes infertility in women and mice Marie Christou-Kent Genetics, Epigenetics 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 Genetics, Epigenetics 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 Genetics, Epigenetics and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France UM GI-DPI, CHU de Grenoble, Grenoble, France Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran Search for more papers by this author Emilie Le Blévec Genetics, Epigenetics 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 Karaouzène Genetics, Epigenetics 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 Béatrice Conne Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland Search for more papers by this author Jessica Escoffier Genetics, Epigenetics 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 Said Assou IRMB, INSERM U1183, CHRU Montpellier, Université Montpellier, Montpellier, France Search for more papers by this author Audrey Guttin Grenoble Neuroscience Institute, INSERM 1216, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Emeline Lambert Genetics, Epigenetics 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 Guillaume Martinez Genetics, Epigenetics and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France UM GI-DPI, CHU de Grenoble, Grenoble, France UM de Génétique Chromosomique, CHU de Grenoble, Grenoble, France Search for more papers by this author Magalie Boguenet Genetics, Epigenetics 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 Selima Fourati Ben Mustapha Polyclinique les Jasmins, Centre d'Aide Médicale à la Procréation, Centre Urbain Nord, Tunis, Tunisia Search for more papers by this author Isabelle Cedrin Durnerin Service de Médecine de la Reproduction, Centre Hospitalier Universitaire Jean Verdier, Assistance Publique - Hôpitaux de Paris, Bondy, France Search for more papers by this author Lazhar Halouani Polyclinique les Jasmins, Centre d'Aide Médicale à la Procréation, Centre Urbain Nord, Tunis, Tunisia Search for more papers by this author Ouafi Marrakchi Polyclinique les Jasmins, Centre d'Aide Médicale à la Procréation, Centre Urbain Nord, Tunis, Tunisia Search for more papers by this author Mounir Makni Polyclinique les Jasmins, Centre d'Aide Médicale à la Procréation, Centre Urbain Nord, Tunis, Tunisia Search for more papers by this author Habib Latrous Polyclinique les Jasmins, Centre d'Aide Médicale à la Procréation, Centre Urbain Nord, Tunis, Tunisia Search for more papers by this author Mahmoud Kharouf Polyclinique les Jasmins, Centre d'Aide Médicale à la Procréation, Centre Urbain Nord, Tunis, Tunisia Search for more papers by this author Charles Coutton Genetics, Epigenetics and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France UM GI-DPI, CHU de Grenoble, Grenoble, France UM de Génétique Chromosomique, CHU de Grenoble, Grenoble, France Search for more papers by this author Nicolas Thierry-Mieg Univ. Grenoble Alpes/CNRS, TIMC-IMAG, CNRS UMR 5525, Grenoble, France Search for more papers by this author Serge Nef Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland Search for more papers by this author Serge P Bottari Genetics, Epigenetics 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 Raoudha Zouari Polyclinique les Jasmins, Centre d'Aide Médicale à la Procréation, Centre Urbain Nord, Tunis, Tunisia Search for more papers by this author Jean Paul Issartel Grenoble Neuroscience Institute, INSERM 1216, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Pierre F Ray orcid.org/0000-0003-1544-7449 Genetics, Epigenetics and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France UM GI-DPI, CHU de Grenoble, Grenoble, France Search for more papers by this author Christophe Arnoult Corresponding Author [email protected] orcid.org/0000-0002-3753-5901 Genetics, Epigenetics 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 Genetics, Epigenetics 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 Genetics, Epigenetics 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 Genetics, Epigenetics and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France UM GI-DPI, CHU de Grenoble, Grenoble, France Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran Search for more papers by this author Emilie Le Blévec Genetics, Epigenetics 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 Karaouzène Genetics, Epigenetics 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 Béatrice Conne Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland Search for more papers by this author Jessica Escoffier Genetics, Epigenetics 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 Said Assou IRMB, INSERM U1183, CHRU Montpellier, Université Montpellier, Montpellier, France Search for more papers by this author Audrey Guttin Grenoble Neuroscience Institute, INSERM 1216, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Emeline Lambert Genetics, Epigenetics 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 Guillaume Martinez Genetics, Epigenetics and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France UM GI-DPI, CHU de Grenoble, Grenoble, France UM de Génétique Chromosomique, CHU de Grenoble, Grenoble, France Search for more papers by this author Magalie Boguenet Genetics, Epigenetics 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 Selima Fourati Ben Mustapha Polyclinique les Jasmins, Centre d'Aide Médicale à la Procréation, Centre Urbain Nord, Tunis, Tunisia Search for more papers by this author Isabelle Cedrin Durnerin Service de Médecine de la Reproduction, Centre Hospitalier Universitaire Jean Verdier, Assistance Publique - Hôpitaux de Paris, Bondy, France Search for more papers by this author Lazhar Halouani Polyclinique les Jasmins, Centre d'Aide Médicale à la Procréation, Centre Urbain Nord, Tunis, Tunisia Search for more papers by this author Ouafi Marrakchi Polyclinique les Jasmins, Centre d'Aide Médicale à la Procréation, Centre Urbain Nord, Tunis, Tunisia Search for more papers by this author Mounir Makni Polyclinique les Jasmins, Centre d'Aide Médicale à la Procréation, Centre Urbain Nord, Tunis, Tunisia Search for more papers by this author Habib Latrous Polyclinique les Jasmins, Centre d'Aide Médicale à la Procréation, Centre Urbain Nord, Tunis, Tunisia Search for more papers by this author Mahmoud Kharouf Polyclinique les Jasmins, Centre d'Aide Médicale à la Procréation, Centre Urbain Nord, Tunis, Tunisia Search for more papers by this author Charles Coutton Genetics, Epigenetics and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France UM GI-DPI, CHU de Grenoble, Grenoble, France UM de Génétique Chromosomique, CHU de Grenoble, Grenoble, France Search for more papers by this author Nicolas Thierry-Mieg Univ. Grenoble Alpes/CNRS, TIMC-IMAG, CNRS UMR 5525, Grenoble, France Search for more papers by this author Serge Nef Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland Search for more papers by this author Serge P Bottari Genetics, Epigenetics 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 Raoudha Zouari Polyclinique les Jasmins, Centre d'Aide Médicale à la Procréation, Centre Urbain Nord, Tunis, Tunisia Search for more papers by this author Jean Paul Issartel Grenoble Neuroscience Institute, INSERM 1216, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Pierre F Ray orcid.org/0000-0003-1544-7449 Genetics, Epigenetics and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France UM GI-DPI, CHU de Grenoble, Grenoble, France Search for more papers by this author Christophe Arnoult Corresponding Author [email protected] orcid.org/0000-0002-3753-5901 Genetics, Epigenetics 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 Marie Christou-Kent1, Zine-Eddine Kherraf1, Amir Amiri-Yekta1,2,3, Emilie Le Blévec1, Thomas Karaouzène1, Béatrice Conne4, Jessica Escoffier1, Said Assou5, Audrey Guttin6, Emeline Lambert1, Guillaume Martinez1,2,7, Magalie Boguenet1, Selima Fourati Ben Mustapha8, Isabelle Cedrin Durnerin9, Lazhar Halouani8, Ouafi Marrakchi8, Mounir Makni8, Habib Latrous8, Mahmoud Kharouf8, Charles Coutton1,2,7, Nicolas Thierry-Mieg10, Serge Nef4, Serge P Bottari1, Raoudha Zouari8, Jean Paul Issartel6, Pierre F Ray1,2,‡ and Christophe Arnoult *,1,‡ 1Genetics, Epigenetics and Therapies of Infertility, Institute for Advanced Biosciences, Inserm U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France 2UM GI-DPI, CHU de Grenoble, 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, Switzerland 5IRMB, INSERM U1183, CHRU Montpellier, Université Montpellier, Montpellier, France 6Grenoble Neuroscience Institute, INSERM 1216, Université Grenoble Alpes, Grenoble, France 7UM de Génétique Chromosomique, CHU de Grenoble, Grenoble, France 8Polyclinique les Jasmins, Centre d'Aide Médicale à la Procréation, Centre Urbain Nord, Tunis, Tunisia 9Service de Médecine de la Reproduction, Centre Hospitalier Universitaire Jean Verdier, Assistance Publique - Hôpitaux de Paris, Bondy, France 10Univ. Grenoble Alpes/CNRS, TIMC-IMAG, CNRS UMR 5525, Grenoble, France ‡These authors contributed equally to this work as senior authors *Corresponding author. Tel: +33 476 637 408; E-mail: [email protected] EMBO Mol Med (2018)10:e8515https://doi.org/10.15252/emmm.201708515 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 The genetic causes of oocyte meiotic deficiency (OMD), a form of primary infertility characterised by the production of immature oocytes, remain largely unexplored. Using whole exome sequencing, we found that 26% of a cohort of 23 subjects with OMD harboured the same homozygous nonsense pathogenic mutation in PATL2, a gene encoding a putative RNA-binding protein. Using Patl2 knockout mice, we confirmed that PATL2 deficiency disturbs oocyte maturation, since oocytes and zygotes exhibit morphological and developmental defects, respectively. PATL2's amphibian orthologue is involved in the regulation of oocyte mRNA as a partner of CPEB. However, Patl2's expression profile throughout oocyte development in mice, alongside colocalisation experiments with Cpeb1, Msy2 and Ddx6 (three oocyte RNA regulators) suggest an original role for Patl2 in mammals. Accordingly, transcriptomic analysis of oocytes from WT and Patl2−/− animals demonstrated that in the absence of Patl2, expression levels of a select number of highly relevant genes involved in oocyte maturation and early embryonic development are deregulated. In conclusion, PATL2 is a novel actor of mammalian oocyte maturation whose invalidation causes OMD in humans. Synopsis A novel mutation in the gene PATL2 causes oocyte maturation arrest at the germinal vesicle (GV) stage. In mice, Patl2 deficiency during oocyte growth modifies the global transcriptional landscape of GV oocytes, causing dramatic defects and hampering normal maturation. In a cohort of 23 infertile women from North Africa with oocyte meiotic deficiency (OMD), a truncating mutation in the gene PATL2, encoding an RNA-binding protein, was identified in 26% of patients. Patl2 knockout female mice presented severe subfertility, confirming the human diagnostic. Patl2 knockout mouse oocytes could progress to the MII stage, however with numerous morphological defects hampering normal fertilisation and development. Patl2 is not detectable in primordial follicle oocytes, but is strongly expressed during oocyte growth and remains detectable at least until the MII stage. Patl2 has a unique expression pattern from primordial follicle-stage to MII-stage oocytes and did not colocalise with Cpeb1, Msy2 or Ddx6 known to stabilise mRNA during oocyte growth. Patl2 deficiency leads to a down or up-regulation of a subset of mRNAs encoding proteins which are crucial for oocyte meiotic progression and early embryonic development, with no effect on the GV-MII transition. Introduction In humans, oocyte production is a lengthy process that begins during embryonic development and is characterised by a long diapause lasting over a decade until resumption of maturation at puberty. The quiescent oocytes, contained within primordial follicles, are arrested in the prophase of meiosis I. Periodically, a group of primordial follicles are recruited to the pool of growing follicles. The germinal vesicle (GV) oocyte and surrounding follicular cells develop in tight coordination to produce a fully grown GV oocyte within an antral follicle. This process takes around 290 days Williams & Erickson, 2012; Li & Albertini, 2013). At this stage, the oocyte is sensitive to hormonal stimulation, which causes meiosis to resume, as revealed by GV breakdown (GVBD), and extrusion of the first polar body before arresting again at the metaphase 2 (MII) stage of meiosis II. The second meiosis is completed, with exclusion of the second polar body, upon fertilisation. Several reports have been published describing cases of infertile women whose ovaries repeatedly produce mostly/only immature oocytes. This poorly defined syndrome is known as "oocyte factor infertility" or "bad eggs syndrome" (Hartshorne et al, 1999; Levran et al, 2002; Beall et al, 2010; Hourvitz et al, 2010). We studied a cohort of patients who had all had at least one in vitro fertilisation (IVF) cycle yielding only GV, MI or atretic oocytes, and named this phenotype oocyte meiotic deficiency (OMD). Generation of knockout mouse models has allowed the identification of several genetic variants linked to oocyte meiotic arrest at various stages. For instance, mice deficient in Cdc25b, a gene involved in cyclic AMP control, show GV arrest (Lincoln et al, 2002; Vaccari et al, 2008). Similarly, deletion of H1foo, a transcription factor for Mei1 (required for normal meiotic chromosome synapsis) and Ubb (a ubiquitin controlling the destruction of key cell cycle regulators), resulted in MI arrest (Libby et al, 2002; Furuya et al, 2007; Ryu et al, 2008). Finally, invalidation of Smc1b, a meiosis-specific component of the cohesin complex, causes MII arrest (Takabayashi et al, 2009) while deletion of Mlh3, which maintains homologous chromosome pairing at meiosis, induces mixed arrests (Lipkin et al, 2002). While it is tempting to suggest that mutation of any of the above-mentioned genes could cause OMD in women, none has so far been associated with this disease. Recently, heterozygous mis-sense mutations in TUBB8, an oocyte-specific tubulin required to form the meiotic spindle, were identified in a cohort of Chinese patients with OMD (Feng et al, 2016). Thus, TUBB8 was established as the first human gene linked to OMD. Here, we analysed 23 unrelated OMD patients from North Africa and found that six (26%) had the same homozygous truncating mutation in the PATL2 gene, encoding a putative oocyte-specific RNA-binding protein. The role of this protein has yet to be characterised in mammals. A TUBB8 variant was only found in a single patient in our cohort, indicating that absence of PATL2 is the main cause of OMD in this region. Results A homozygous truncating mutation in PATL2 identified by whole exome and Sanger sequencing in 26% of tested subjects We analysed a cohort of 23 infertile women presenting with OMD (Table 1). These patients responded normally to ovarian stimulation, and the number of follicles and oocytes harvested was similar to numbers for control patients. However, examination of the oocytes revealed only either GV or MI-arrested or atretic cells (identified by an irregular shape with a dark ooplasm), and a complete absence of MII oocytes. Table 1. Medical history, laboratory investigations and oocyte collection outcomes for patients presenting with OMD Origin Age (years) Number of oocytes collected FSH U/L LH U/L TSH U/L Prolactin μg/L Menst. Comments GV MI MII At. Tot. Patients with PATL2 mutation P1 Tunisia 35 4 0 0 1 5 1.36 34 2 Medical records not available 34 8 Medical records not available P2 Tunisia 28 9 0 0 11 20 YES 28.9 15 0 0 4 19 P3 Tunisia 24 11 0 0 5 16 10.31 3.54 22.28 1 GV maturated to M1 in vitro P4 Tunisia 34.28 8 0 0 2 10 1.07 23 YES P5 Arab 41 2 0 0 2 4 9.39 6.1 2.3 12.9 YES 42 3 1 0 1 5 P6 Mauritania 36 2 10 0 4 16 3.01 3.38 2.88 25 YES Cytoplasmic vacuoles in MI oocyte 36.8 0 0 0 5 5 Patients without PATL2 mutation P7 Algeria 37 2 0 0 2 4 10.1 9.15 2.3 YES P8 Algeria 32 0 4 0 0 4 32 0 2 0 0 2 P9 Tunisia 32 0 0 0 8 8 3.73 First cousin couple P10 Tunisia 37 0 2 0 3 5 37 2 3 0 3 8 P11 Tunisia 38.9 0 3 2 2 7 8.49 3.42 1.17 17.05 P12 Libya 28 2 15 0 0 17 1.8 9.6 P13 Arab 33 0 3 2 7 12 37 3 1 0 11 15 P14 Tunisia 26 0 0 0 7 7 YES P15 Arab 38 0 0 0 4 4 39 0 0 0 5 5 P16 Arab 33 0 5 2 2 9 4.65 2.71 14.25 Heterozygous mutation in TUBB8 P17 Arab 34 0 0 0 0 0 YES P18 Arab-FR 27 0 8 0 4 12 P19 Arab-FR 24 0 3 0 0 3 P20 29 0 0 0 7 7 P21 Tunisia 29 0 0 0 13 13 YES P22 Tunisia 39 0 10 0 0 10 P23 Tunisia-FR No data available No fertilisation Mean P1-P23 33.42 2.17 2.50 0.21 3.90 8.45 Mean P1-P6 34.00 6.00 1.38 0 3.89 10.00 Mean P7-P23 33.1 0.45 2.95 0.3 3.9 7.6 Control cohort values (n = 238) 34.4 2.2 1.8 6 2.3 9.1 < 10.2 < 16.9 0.5–5 2–20 Normal values correspond to couples where the male suffers from azoospermia or teratozoospermia (n = 234). Arab-FR = French of Arab origin, At. = atretic, Tot. = total, Menst. = menstruation. Given that most of the patients were Tunisian and that 20–30% of marriages are consanguineous in this country, we hypothesised that infertility could be transmitted through recessive inheritance and we therefore focused on homozygous mutations. Exome analysis was performed first on samples from 15 patients. After exclusion of common variants and application of technical and biological filters (Coutton et al, 2018), three genes were found to be homozygously mutated in at least two subjects. Only one gene carried a homozygous variant scored as "high" and was predicted to induce loss of function by the "Variant Effect Predictor" tool (Ensembl). Interestingly, the same variant, p.Arg160Ter, c.478C>T in PATL2 transcript ENST00000434130, was detected in five different patients. Since the orthologue of PATL2 in Xenopus is described as an important factor in Xenopus oocyte maturation (Nakamura et al, 2010), it was possible that this variant could be the cause of these subjects' infertility. The variant identified is expected to lead to either the production of a truncated protein (Fig 1A) or a complete absence of expression due to possible nonsense-mediated mRNA decay. The truncated protein would contain less than one-third of the complete amino acid sequence, in particular lacking the topoisomerase II-associated protein PAT1 domain. This domain, common to all Pat1 proteins, has been shown to be necessary for its paralogue, PATL1, to function through interaction with its partners (Braun et al, 2010) (Fig 1A). Because we did not have access to the relevant biological material (patients' ovaries), it was impossible to assess RNA decay in the presence of this mutation. Figure 1. Identification of a truncating mutation in PATL2 Location of the PATL2 mutation in the intron–exon structure and in a representation of the corresponding amino acid sequence. The variant identified, homozygous in the six patients, is located in exon 6 and creates a STOP codon, ending translation and producing a truncated 158-amino acid (aa) protein instead of the full-length 543 aa, and lacking the essential PAT1 (topoisomerase II-associated protein PAT1) domain. Electropherograms of Sanger sequencing for patients harbouring PATL2 mutations compared to reference sequence. Download figure Download PowerPoint The presence of the genetic variant was confirmed by Sanger sequencing for the five mutated patients (Fig 1B). This variant was also identified in a heterozygous state in five out of 148,732 alleles (rs548527219) in the Genome Aggregation Database (gnomAD). This rate corresponds to a very low frequency of 0.003362%, compatible with recessive transmission of a genetic disease. Sanger sequencing of PATL2 coding sequences was then performed on another eight OMD subjects. An additional patient was identified with the same homozygous mutation, increasing the final number to six out of 23 subjects analysed (26%) carrying the PATL2 p.Arg160Ter variant. To complete the analysis of the cohort, WES was performed on the newly recruited patients (n = 8) except for the subject harbouring the PATL2 mutation. WES analysis was therefore performed on a total of 22 subjects. From these data, we also sought TUBB8 heterozygous mutations, which have also been described to induce OMD (Feng et al, 2016). One deleterious heterozygous variant (ENST00000309812.4:c.363_366del, ENSP00000311042.4:p.Lys122ArgfsTer13) was identified in patient P16 (Table 1), which could be the reason for this patient's infertility. Overall, in this cohort, six out of 23 subjects analysed (26%) were observed to carry the PATL2 p.Arg160Ter mutation, and one patient presented a new TUBB8 variant (4.5%, 1/22), the pathogenicity of which remains to be confirmed. In our cohort, we compared patient characteristics between subjects with a PATL2 mutation or presenting no PATL2 mutation (Fig EV1). Although both groups were of similar ages at the time of analysis, and the numbers of oocytes retrieved were comparable, the two groups were clearly distinct in terms of the type of oocyte arrest. Oocytes from PATL2 patients were mainly arrested at the GV stage, whereas oocytes from non-PATL2 patients were generally arrested at the MI stage (Fig EV1). Click here to expand this figure. Figure EV1. Quality of oocytes collected from patients harbouring PATL2 mutation and control patients after ovarian stimulation The mean age of the six patients harbouring a PATL2 mutation at the time of 11 hormonal stimulations was compared to the non-PATL2 patients within the cohort and also to a control cohort corresponding to women from infertile couples of similar geographical origin where the male was diagnosed with a male infertility (mean ± SEM, n = 234). There was no significant (NS) age difference between the PATL2 patients, the non-PATL2 patients and the control cohort. Numbers of oocytes retrieved after hormonal stimulation (mean ± SEM) were similar in PATL2 patients, non-PATL2 patients and the control cohort. Collected oocytes were sorted according to their maturation stage. For patients harbouring PATL2 mutation, the mean numbers of GV and atretic oocytes were significantly increased and no MII oocytes were collected. Non-PATL2 patients from the same cohort showed a comparably larger proportion of MI-arrested oocytes. Data information: Statistical differences were assessed using unpaired two-tailed t-tests. Download figure Download PowerPoint During evaluation of our data, PATL2 gene mutations were also reported to be associated with OMD in two Asian studies based on cohorts from China and Saudi Arabia (Chen et al, 2017; Maddirevula et al, 2017). These findings support the causality of our PATL2 variant and indicate a wide global spread for PATL2-dependent OMD. Patl2 is not expressed in the hypothalamic–pituitary–gonadal axis in mice x-pat1a, the Xenopus orthologue of PATL2, has been reported to be specifically expressed in growing oocytes (Marnef et al, 2010; Nakamura et al, 2010), and analysis of publicly accessible data banks shows that PATL2 is also expressed at high levels in both human and mouse oocytes (Appendix Fig S1), indicating an important role for PATL2 in female gametogenesis. It should be noted that PATL2 expression is very low in human follicular cells (Appendix Fig S1), suggesting that the maturation defect is of oocyte rather than follicular origin. It also appears that PATL2 is expressed at low levels in a number of other tissues (Appendix Fig S1). We therefore wondered whether an element of the infertility phenotype could be caused by alteration of the hypothalamic–pituitary–gonadal axis. To address this question, we performed comparative Western blots on extracts from GV oocytes, hypothalamus and pituitary glands from Patl2-HA-tagged mice created using CrispR-cas9 technology. Whereas a clear and specific signal is observed for oocyte extracts from PATL2-HA females, no signal was observed in extracts from the hypothalamus or pituitary gland indicating that the direct control of the hypothalamus/pituitary gland on oocyte maturation is not altered in mice (Appendix Fig S2). Since our PATL2 patients exhibited normal hormone levels (when data were available, Table 1) and reported regular menstrual cycles, these results taken together suggest that the human infertility phenotype is purely due to an oocyte defect. Absence of Patl2 modifies the number of the primordial follicles at 26 dpp but not at 12 dpp To decipher the molecular pathogenesis of the phenotype observed in our PATL2 patients, we assessed the reproductive phenotype of Patl2-deficient mice (Patl2−/−). The gene was invalidated by insertion of a LacZ cassette and deletion of exon 7, inducing a downstream translational frameshift (Appendix Fig S3). The putative transcript produced from this construct would consist of the first 102 amino acids (out of 529) tethered to β-galactosidase. Even if a protein product was generated from the modified Patl2 gene, it would not contain the topoisomerase II-associated protein (PAT1) domain and would therefore not be functional. Initially, we performed a comparative histological study of control and Patl2−/− ovaries at 12 and 26 days postpartum (dpp). At 12 dpp, ovary sections from control and Patl2−/− female
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