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Coutton C.,French National Center for Scientific Research | Coutton C.,Joseph Fourier University | Coutton C.,Grenoble University Hospital Center | Zouari R.,Clinique de la Reproduction les Jasmins | And 32 more authors.
Human Reproduction | Year: 2012

summary answer: Two DPY19L2 heterozygous deletions and three point mutations were identified, thus further confirming that genetic alterations of the DPY19L2 gene are the main cause of globozoospermia and indicating that DPY19L2 molecular diagnostics should not be stopped in the absence of a homozygous gene deletion. what is known already: Globozoospermia is a rare phenotype of primary male infertility characterized by the production of a majority of round-headed spermatozoa without acrosome.We demonstrated previously that most cases in man were caused by a recurrent homozygous deletion of the totality of the DPY19L2 gene, preventing sperm head elongation and acrosome formation. In mammals, DPY19L2 has three paralogs of yet unknown function and one highly homologous pseudogene showing .95% sequence identity with DPY19L2. Specific amplification and sequencing of DPY19L2 have so far been hampered by the presence of this pseudogene which has greatly complicated specific amplification and sequencing. study design, size, duration: In this cohort study, 34 patients presenting with globozoospermia were recruited during routine infertility treatment in infertility centers in France and Tunisia between January 2008 and December 2011. The molecular variants identified in patients were screened in 200 individuals from the general population to exclude frequent non-pathological polymorphisms. participants/materials, setting, methods: We developed a Multiplex Ligation-dependent Probe Amplification test to detect the presence of heterozygous deletions and identified the conditions to specifically amplify and sequence the 22 exons and intronic boundaries of the DPY19L2 gene. The pathogenicity of the identified mutations and their action on the protein were evaluated in silico. main results and the role of chance: There were 23 patients who were homozygous for the DPY19L2 deletion (67.6%). Only eight of the eleven non-homozygously deleted patients could be sequenced due to poor DNA quality of three patients. Two patients were compound heterozygous carrying one DPY19L2 deleted allele associated respectively with a nonsense (p.Q342) and a missense mutation (p.R290H). One patient was homozygous for p.M358K, another missense mutation affecting a highly conserved amino acid. Due to & The Author 2012. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com Human Reproduction, Vol.27, No.8 pp. 2549-2558, 2012 Advanced Access publication on May 24, 2012 doi:10.1093/humrep/des160 Downloaded from http://humrep.oxfordjournals.org/ at SCOPUS EFLOWI on November 23, 2012 the localization of this mutation and the physicochemical properties of the substituted amino acids, we believe that this variant is likely to disrupt one of the protein transmembrane domains and destabilize the protein. Overall, 84% of the fully analysed patients (n = 31) had a molecular alteration of DPY19L2. There was no clear phenotypic difference between the homozygous deleted individual, patients carrying a point mutation and undiagnosed patients. limitations, reasons for caution: Globally poor fertilization rates are observed after intracytoplasmic sperm injection of round spermatozoa. Further work is needed to assess whether DPY19L2 mutated patients present a better or worse prognostic than the non-diagnosed patients. Evaluation of the potential benefit of treatment with a calcium ionophore, described to improve fertilization, should be evaluated in these two groups. wider implications of the findings: In previous work, deletions of DPY19L2 had only been identified in North African patients. Here we have identified DPY19L2 deletions and point mutations in European patients, indicating that globozoospemia caused by a molecular defect of DPY19L2 can be expected in individuals from any ethnic background. study funding/competing interest(s): None of the authors have any competing interest. This work is part of the project 'Identification and Characterization of Genes Involved in Infertility (ICG2I)' funded by the program GENOPAT 2009 from the French Research Agency (ANR). © The Author 2012. Source

Ben Khelifa M.,French National Center for Scientific Research | Ben Khelifa M.,Joseph Fourier University | Ben Khelifa M.,Institute Pasteur Of Tunis | Coutton C.,French National Center for Scientific Research | And 36 more authors.
Human Reproduction | Year: 2012

Study Question Can we identify new sequence variants in the aurora kinase C gene (AURKC) of patients with macrozoospermia and establish a genotype-phenotype correlation? Summary Answer We identified a new non-sense mutation, p.Y248 (*), that represents 13 of all mutant alleles. There was no difference in the phenotype of individuals carrying this new mutation versus the initially described and main mutation c.144delC. What is Known Already The absence of a functional AURKC gene causes primary infertility in men by blocking the first meiotic division and leading to the production of tetraploid large-headed spermatozoa. We previously demonstrated that most affected men were of North African origin and carried a homozygous truncating mutation (c.144delC). Study Design, Size, Duration This is a retrospective study carried out on patients consulting for infertility and described as having >5 large-headed spermatozoa. A total of 87 patients are presented here, 43 patients were published previously and 44 are new patients recruited between January 2008 and December 2011. Participants/Materials, Setting , Methods All patients consulted for primary infertility in fertility clinics in France (n 44), Tunisia (n 30), Morocco (n 9) or Algeria (n 4). Sperm analysis was carried out in the recruiting fertility clinics and all molecular analyses were performed at Grenoble teaching hospital. DNA was extracted from blood or saliva and the seven AURKC exons were sequenced. RT-PCR was carried out on transcripts extracted from leukocytes from one patient homozygous for p.Y248 (*). Microsatellite analysis was performed on all p.Y248 (*) patients to evaluate the age of this new mutation. Main Results and The Role of ChanceWe identified a new non-sense mutation, p.Y248 (*), in 10 unrelated individuals of European (n 4) and North African origin (n 6). We show that this new variant represents 13 of all mutant alleles and that the initially described c.144delC variant accounts for almost all of the remaining mutated alleles (85.5). No mutated transcripts could be detected by RT-PCR suggesting a specific degradation of the mutant transcripts by non-sense mediated mRNA decay. A rare variant located in the 3′ untranslated region was found to strictly co-segregate with p.Y248 (*), demonstrating a founding effect. Microsatellite analysis confirmed this linkage and allowed us to estimate a mutational age of between 925 and 1325 years, predating the c.144delC variant predicted by the same method to have arisen 250-650 years ago. Patients with no identified AURKC mutation (n 15) have significantly improved parameters in terms of vitality and concentration of normal spermatozoa, and a decreased rate of spermatozoa with a large head and multiple flagella (P < 0.001). Limitations, Reasons for Caution Despite adherence to the World Health Organization guidelines, large variations in most characteristic sperm parameters were observed, even for patients with the same homozygous mutation. We believe that is mainly related to inter-laboratory variability in sperm parameter scoring. This prevented us from establishing clear-cut values to indicate a need for molecular analysis of patients with macrozoospermia. Wider Implications of The Findings This study confirms yet again the importance of AURKC mutations in the aetiology of macrozoospermia. Although a large majority of patients are of North African origin, we have now identified European patients carrying a new non-sense mutation indicating that a diagnosis of absence of a functional AURKC gene should not be ruled out for non-Magrebian individuals. Indirect evidence indicates that AURKC might be playing a role in the meiotic spindle assembly checkpoint (SAC) during meiosis. We postulate that heterozygous men might have a more relaxed SAC leading to a more abundant sperm production and a reproductive advantage. This could be the reason for the rapid accumulation of the two AURKC mutations we observe in North African individuals. Study Funding/Competing Interest (S)None of the authors have any competing interest. This work is part of the project 'Identification and Characterization of Genes Involved in Infertility (ICG2I)' funded by the programme GENOPAT 2009 from the French Research Agency (ANR). © 2012 The Author. Source

Harbuz R.,Joseph Fourier University | Harbuz R.,French National Center for Scientific Research | Harbuz R.,Grenoble University Hospital Center | Zouari R.,Clinique de la Reproduction les Jasmins | And 41 more authors.
American Journal of Human Genetics | Year: 2011

An increasing number of couples require medical assistance to achieve a pregnancy, and more than 2% of the births in Western countries now result from assisted reproductive technologies. To identify genetic variants responsible for male infertility, we performed a whole-genome SNP scan on patients presenting with total globozoospermia, a primary infertility phenotype characterized by the presence of 100% round acrosomeless spermatozoa in the ejaculate. This strategy allowed us to identify in most patients (15/20) a 200 kb homozygous deletion encompassing only DPY19L2, which is highly expressed in the testis. Although there was no known function for DPY19L2 in humans, previous work indicated that its ortholog in C. elegans is involved in cell polarity. In man, the DPY19L2 region has been described as a copy-number variant (CNV) found to be duplicated and heterozygously deleted in healthy individuals. We show here that the breakpoints of the deletions are located on a highly homologous 28 kb low copy repeat (LCR) sequence present on each side of DPY19L2, indicating that the identified deletions were probably produced by nonallelic homologous recombination (NAHR) between these two regions. We demonstrate that patients with globozoospermia have a homozygous deletion of DPY19L2, thus indicating that DPY19L2 is necessary in men for sperm head elongation and acrosome formation. A molecular diagnosis can now be proposed to affected men; the presence of the deletion confirms the diagnosis of globozoospermia and assigns a poor prognosis for the success of in vitro fertilization. © 2011 The American Society of Human Genetics. Source

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