Génétique de l'infertilité masculine

Performing a genome wide scan by SNP microarray on a Jordanian consanguineous family where five brothers were diagnosed with complete globozoospermia, we show in a first study that the four out of five analysed infertile brothers carried a homozygous deletion of 200 kb on chromosome 12 encompassing only DPY19L2. The gene encodes for a transmembrane protein and is surrounded by two low copy repeats (LCRs). Very similar deletions were found in three additional unrelated patients. Later, we have pursued our patient screen by recruiting a largest cohort of patients. Out of a total of 54 patients analysed, 36 (66.7%) showed a mutation in DPY19L2. Out of 36 mutated patients, 20 are homozygous deleted, 7 heterozygous composite and 4 showed a homozygous point mutation. We characterized a total of nine breakpoints that clustered in two recombination hotspots, both containing direct repeat elements. These findings confirm that the deletion is due to a nonallelic homologous recombination (NAHR) between the two LCRs. Thus, Globozoospermia can be considered as a new genomic disorder. This study confirms that DPY19L2 is the major gene responsible for globozoospermia and enlarges the spectrum of possible mutations in the gene.Le génotypage d’une famille jordanienne consanguine constituée de 5 frères globozoospermiques et de 3 frères fertiles sur puce Affymetrix, a permis d’identifier un nouveau gène responsable de la globozoospermie situé dans un intervalle de 6.4Mb en 12q14.2. Au regard de son expression prédominante dans le testicule et l’implication de son orthologue, chez C. elegans, dans la polarisation cellulaire, le gène DPY19L2 est un gène candidat parfait. Le gène, codant pour une protéine transmembranaire, est flanqué par deux séquences répétées (LCRs) qui partagent 96,5% d’identité. Dans une première étude, une délétion de 200Kb englobant l’ensemble du gène a été mise en évidence chez les 4 frères infertiles de cette famille jordanienne ainsi que chez 3 autres patients non apparentés. Nous avons ensuite recruté une plus grande cohorte de 54 patients. Parmi ces patients, 20 sont homozygotes pour la délétion de DPY19L2 et 7 sont hétérozygotes composites associant la délétion hétérozygote et une mutation ponctuelle. En outre, nous avons identifié, 4 patients avec des mutations ponctuelles homozygotes. Par conséquent, la fréquence d’implication de DPY19L2 s’élève à 66.7%. En tout, 9 points de cassures, regroupés en deux hotspots au sein des LCRs, ont pu être mis en évidence. Ceci confirme que le mécanisme sous-jacent de la délétion est une recombinaison homologue non allélique (NAHR) entre les LCRs. En conclusion, nous confirmons que DPY19L2 est le principal gène de la globozoospermie et nous élargissons le spectre des mutations possible dans ce gène

Human genetics of male infertility

Le génotypage d’une famille jordanienne consanguine constituée de 5 frères globozoospermiques et de 3 frères fertiles sur puce Affymetrix, a permis d’identifier un nouveau gène responsable de la globozoospermie situé dans un intervalle de 6.4Mb en 12q14.2. Au regard de son expression prédominante dans le testicule et l’implication de son orthologue, chez C. elegans, dans la polarisation cellulaire, le gène DPY19L2 est un gène candidat parfait. Le gène, codant pour une protéine transmembranaire, est flanqué par deux séquences répétées (LCRs) qui partagent 96,5% d’identité. Dans une première étude, une délétion de 200Kb englobant l’ensemble du gène a été mise en évidence chez les 4 frères infertiles de cette famille jordanienne ainsi que chez 3 autres patients non apparentés. Nous avons ensuite recruté une plus grande cohorte de 54 patients. Parmi ces patients, 20 sont homozygotes pour la délétion de DPY19L2 et 7 sont hétérozygotes composites associant la délétion hétérozygote et une mutation ponctuelle. En outre, nous avons identifié, 4 patients avec des mutations ponctuelles homozygotes. Par conséquent, la fréquence d’implication de DPY19L2 s’élève à 66.7%. En tout, 9 points de cassures, regroupés en deux hotspots au sein des LCRs, ont pu être mis en évidence. Ceci confirme que le mécanisme sous-jacent de la délétion est une recombinaison homologue non allélique (NAHR) entre les LCRs. En conclusion, nous confirmons que DPY19L2 est le principal gène de la globozoospermie et nous élargissons le spectre des mutations possible dans ce gène.Performing a genome wide scan by SNP microarray on a Jordanian consanguineous family where five brothers were diagnosed with complete globozoospermia, we show in a first study that the four out of five analysed infertile brothers carried a homozygous deletion of 200 kb on chromosome 12 encompassing only DPY19L2. The gene encodes for a transmembrane protein and is surrounded by two low copy repeats (LCRs). Very similar deletions were found in three additional unrelated patients. Later, we have pursued our patient screen by recruiting a largest cohort of patients. Out of a total of 54 patients analysed, 36 (66.7%) showed a mutation in DPY19L2. Out of 36 mutated patients, 20 are homozygous deleted, 7 heterozygous composite and 4 showed a homozygous point mutation. We characterized a total of nine breakpoints that clustered in two recombination hotspots, both containing direct repeat elements. These findings confirm that the deletion is due to a nonallelic homologous recombination (NAHR) between the two LCRs. Thus, Globozoospermia can be considered as a new genomic disorder. This study confirms that DPY19L2 is the major gene responsible for globozoospermia and enlarges the spectrum of possible mutations in the gene

Human genetics of male infertility

Le génotypage d’une famille jordanienne consanguine constituée de 5 frères globozoospermiques et de 3 frères fertiles sur puce Affymetrix, a permis d’identifier un nouveau gène responsable de la globozoospermie situé dans un intervalle de 6.4Mb en 12q14.2. Au regard de son expression prédominante dans le testicule et l’implication de son orthologue, chez C. elegans, dans la polarisation cellulaire, le gène DPY19L2 est un gène candidat parfait. Le gène, codant pour une protéine transmembranaire, est flanqué par deux séquences répétées (LCRs) qui partagent 96,5% d’identité. Dans une première étude, une délétion de 200Kb englobant l’ensemble du gène a été mise en évidence chez les 4 frères infertiles de cette famille jordanienne ainsi que chez 3 autres patients non apparentés. Nous avons ensuite recruté une plus grande cohorte de 54 patients. Parmi ces patients, 20 sont homozygotes pour la délétion de DPY19L2 et 7 sont hétérozygotes composites associant la délétion hétérozygote et une mutation ponctuelle. En outre, nous avons identifié, 4 patients avec des mutations ponctuelles homozygotes. Par conséquent, la fréquence d’implication de DPY19L2 s’élève à 66.7%. En tout, 9 points de cassures, regroupés en deux hotspots au sein des LCRs, ont pu être mis en évidence. Ceci confirme que le mécanisme sous-jacent de la délétion est une recombinaison homologue non allélique (NAHR) entre les LCRs. En conclusion, nous confirmons que DPY19L2 est le principal gène de la globozoospermie et nous élargissons le spectre des mutations possible dans ce gène.Performing a genome wide scan by SNP microarray on a Jordanian consanguineous family where five brothers were diagnosed with complete globozoospermia, we show in a first study that the four out of five analysed infertile brothers carried a homozygous deletion of 200 kb on chromosome 12 encompassing only DPY19L2. The gene encodes for a transmembrane protein and is surrounded by two low copy repeats (LCRs). Very similar deletions were found in three additional unrelated patients. Later, we have pursued our patient screen by recruiting a largest cohort of patients. Out of a total of 54 patients analysed, 36 (66.7%) showed a mutation in DPY19L2. Out of 36 mutated patients, 20 are homozygous deleted, 7 heterozygous composite and 4 showed a homozygous point mutation. We characterized a total of nine breakpoints that clustered in two recombination hotspots, both containing direct repeat elements. These findings confirm that the deletion is due to a nonallelic homologous recombination (NAHR) between the two LCRs. Thus, Globozoospermia can be considered as a new genomic disorder. This study confirms that DPY19L2 is the major gene responsible for globozoospermia and enlarges the spectrum of possible mutations in the gene

Human genetics of male infertility

Le génotypage d une famille jordanienne consanguine constituée de 5 frères globozoospermiques et de 3 frères fertiles sur puce Affymetrix, a permis d identifier un nouveau gène responsable de la globozoospermie situé dans un intervalle de 6.4Mb en 12q14.2. Au regard de son expression prédominante dans le testicule et l implication de son orthologue, chez C. elegans, dans la polarisation cellulaire, le gène DPY19L2 est un gène candidat parfait. Le gène, codant pour une protéine transmembranaire, est flanqué par deux séquences répétées (LCRs) qui partagent 96,5% d identité. Dans une première étude, une délétion de 200Kb englobant l ensemble du gène a été mise en évidence chez les 4 frères infertiles de cette famille jordanienne ainsi que chez 3 autres patients non apparentés. Nous avons ensuite recruté une plus grande cohorte de 54 patients. Parmi ces patients, 20 sont homozygotes pour la délétion de DPY19L2 et 7 sont hétérozygotes composites associant la délétion hétérozygote et une mutation ponctuelle. En outre, nous avons identifié, 4 patients avec des mutations ponctuelles homozygotes. Par conséquent, la fréquence d implication de DPY19L2 s élève à 66.7%. En tout, 9 points de cassures, regroupés en deux hotspots au sein des LCRs, ont pu être mis en évidence. Ceci confirme que le mécanisme sous-jacent de la délétion est une recombinaison homologue non allélique (NAHR) entre les LCRs. En conclusion, nous confirmons que DPY19L2 est le principal gène de la globozoospermie et nous élargissons le spectre des mutations possible dans ce gène.Performing a genome wide scan by SNP microarray on a Jordanian consanguineous family where five brothers were diagnosed with complete globozoospermia, we show in a first study that the four out of five analysed infertile brothers carried a homozygous deletion of 200 kb on chromosome 12 encompassing only DPY19L2. The gene encodes for a transmembrane protein and is surrounded by two low copy repeats (LCRs). Very similar deletions were found in three additional unrelated patients. Later, we have pursued our patient screen by recruiting a largest cohort of patients. Out of a total of 54 patients analysed, 36 (66.7%) showed a mutation in DPY19L2. Out of 36 mutated patients, 20 are homozygous deleted, 7 heterozygous composite and 4 showed a homozygous point mutation. We characterized a total of nine breakpoints that clustered in two recombination hotspots, both containing direct repeat elements. These findings confirm that the deletion is due to a nonallelic homologous recombination (NAHR) between the two LCRs. Thus, Globozoospermia can be considered as a new genomic disorder. This study confirms that DPY19L2 is the major gene responsible for globozoospermia and enlarges the spectrum of possible mutations in the gene.STRASBOURG-Bib.electronique 063 (674829902) / SudocSudocFranceF

Histone H2AFX Links Meiotic Chromosome Asynapsis to Prophase I Oocyte Loss in Mammals.

Chromosome abnormalities are common in the human population, causing germ cell loss at meiotic prophase I and infertility. The mechanisms driving this loss are unknown, but persistent meiotic DNA damage and asynapsis may be triggers. Here we investigate the contribution of these lesions to oocyte elimination in mice with chromosome abnormalities, e.g. Turner syndrome (XO) and translocations. We show that asynapsed chromosomes trigger oocyte elimination at diplonema, which is linked to the presence of phosphorylated H2AFX (γH2AFX). We find that DNA double-strand break (DSB) foci disappear on asynapsed chromosomes during pachynema, excluding persistent DNA damage as a likely cause, and demonstrating the existence in mammalian oocytes of a repair pathway for asynapsis-associated DNA DSBs. Importantly, deletion or point mutation of H2afx restores oocyte numbers in XO females to wild type (XX) levels. Unexpectedly, we find that asynapsed supernumerary chromosomes do not elicit prophase I loss, despite being enriched for γH2AFX and other checkpoint proteins. These results suggest that oocyte loss cannot be explained simply by asynapsis checkpoint models, but is related to the gene content of asynapsed chromosomes. A similar mechanistic basis for oocyte loss may operate in humans with chromosome abnormalities

DNA damage response protein TOPBP1 regulates X chromosome silencing in the mammalian germ line

Meiotic synapsis and recombination between homologs permits the formation of cross-overs that are essential for generating chromo-somally balanced sperm and eggs. In mammals, surveillance mechanisms eliminate meiotic cells with defective synapsis, thereby minimizing transmission of aneuploidy. One such surveillance mechanism is meiotic silencing, the inactivation of genes located on asynapsed chromosomes, via ATR-dependent serine-139 phosphorylation of histone H2AFX (γH2AFX). Stimulation of ATR activity requires direct interaction with an ATR activation domain (AAD)containing partner. However, which partner facilitates the meiotic silencing properties of ATR is unknown. Focusing on the best-characterized example of meiotic silencing, meiotic sex chromosome inactivation, we reveal this AAD-containing partner to be the DNA damage and checkpoint protein TOPBP1. Conditional TOPBP1 deletion during pachynema causes germ cell elimination associated with defective X chromosome gene silencing and sex chromosome condensation. TOPBP1 is essential for localization to the X chromosome of silencing “sensors,” including BRCA1, and effectors, including ATR, γH2AFX, and canonical repressive histone marks. We present evidence that persistent DNA double-strand breaks act as silencing initiation sites. Our study identifies TOPBP1 as a critical factor in meiotic sex chromosome silencing

DNA damage response protein TOPBP1 regulates X chromosome silencing in the mammalian germ line

Meiotic synapsis and recombination between homologs permits the formation of cross-overs that are essential for generating chromo-somally balanced sperm and eggs. In mammals, surveillance mechanisms eliminate meiotic cells with defective synapsis, thereby minimizing transmission of aneuploidy. One such surveillance mechanism is meiotic silencing, the inactivation of genes located on asynapsed chromosomes, via ATR-dependent serine-139 phosphorylation of histone H2AFX (γH2AFX). Stimulation of ATR activity requires direct interaction with an ATR activation domain (AAD)containing partner. However, which partner facilitates the meiotic silencing properties of ATR is unknown. Focusing on the best-characterized example of meiotic silencing, meiotic sex chromosome inactivation, we reveal this AAD-containing partner to be the DNA damage and checkpoint protein TOPBP1. Conditional TOPBP1 deletion during pachynema causes germ cell elimination associated with defective X chromosome gene silencing and sex chromosome condensation. TOPBP1 is essential for localization to the X chromosome of silencing “sensors,” including BRCA1, and effectors, including ATR, γH2AFX, and canonical repressive histone marks. We present evidence that persistent DNA double-strand breaks act as silencing initiation sites. Our study identifies TOPBP1 as a critical factor in meiotic sex chromosome silencing

Oocytes in the Tc1 accessory chromosome mouse model do not exhibit diplotene elimination.

<p>(<b>A</b>) Pachytene Tc1 oocyte with an asynapsed h21 chromosome (arrow, inset), labelled with γH2AFX (red, arrow) and HORMAD1 (magenta, inset). (<b>B</b>) Pachytene Tc1 oocyte with self-synapsed h21 chromosome (arrow, inset), which is negative for γH2AFX and HORMAD1. Scale bar represents 10μm. (<b>C</b>) Mean percentage (± s.e.m.) of Tc1 oocytes with a γH2AFX-positive or γH2AFX-negative h21 chromosome between pachynema and late diplonema, demonstrating no significant elimination of Tc1 oocytes with asynapsed h21 chromosomes. n is the number of oocytes counted from three non-littermate mice at 18.5 d<i>pc</i>. (<b>D</b>) Comparison of γH2AFX domain integrated intensity between XO and Tc1 oocytes at diplonema, the stage when oocyte losses are observed in XO but not Tc1 females. n refers to the number of oocytes analyzed. (<b>E</b>) Schematic showing that the asynapsed h21 chromosome does not trigger oocyte losses at diplonema.</p

DNA damage induces a kinetochore-based ATM/ATR-independent SAC arrest unique to the first meiotic division in mouse oocytes

Mouse oocytes carrying DNA damage arrest in meiosis I, thereby preventing creation of embryos with deleterious mutations. The arrest is dependent on the spindle assembly checkpoint, which results in anaphase-promoting complex (APC) inhibition. However, little is understood about how this checkpoint is engaged following DNA damage. Here, we find that within minutes DNA damage assembles checkpoint proteins at the kinetochore, not at damage sites along chromosome arms, such that the APC is fully inhibited within 30 min. Despite this robust response, there is no measurable loss in k-fibres, or tension across the bivalent. Through pharmacological inhibition we observed the response is dependent on Mps1 kinase, Aurora kinase, and haspin. Using oocyte specific knockouts we find the response does not require the DNA damage response kinases ATM or ATR. Furthermore, checkpoint activation does not occur in response to DNA damage in fully mature eggs during meiosis II, despite the divisions being separated by just a few hours. Therefore, mouse oocytes have a unique ability to sense DNA damage rapidly by activating the checkpoint at their kinetochores

Silencing of X-Linked MicroRNAs by Meiotic Sex Chromosome Inactivation

<div><p>During the pachytene stage of meiosis in male mammals, the X and Y chromosomes are transcriptionally silenced by Meiotic Sex Chromosome Inactivation (MSCI). MSCI is conserved in therian mammals and is essential for normal male fertility. Transcriptomics approaches have demonstrated that in mice, most or all protein-coding genes on the X chromosome are subject to MSCI. However, it is unclear whether X-linked non-coding RNAs behave in a similar manner. The X chromosome is enriched in microRNA (miRNA) genes, with many exhibiting testis-biased expression. Importantly, high expression levels of X-linked miRNAs (X-miRNAs) have been reported in pachytene spermatocytes, indicating that these genes may escape MSCI, and perhaps play a role in the XY-silencing process. Here we use RNA FISH to examine X-miRNA expression in the male germ line. We find that, like protein-coding X-genes, X-miRNAs are expressed prior to prophase I and are thereafter silenced during pachynema. X-miRNA silencing does not occur in mouse models with defective MSCI. Furthermore, X-miRNAs are expressed at pachynema when present as autosomally integrated transgenes. Thus, we conclude that silencing of X-miRNAs during pachynema in wild type males is MSCI-dependent. Importantly, misexpression of X-miRNAs during pachynema causes spermatogenic defects. We propose that MSCI represents a chromosomal mechanism by which X-miRNAs, and other potential X-encoded repressors, can be silenced, thereby regulating genes with critical late spermatogenic functions.</p></div