Fine Characterisation of a Recombination Hotspot at the DPY19L2 Locus and Resolution of the Paradoxical Excess of Duplications over Deletions in the General Population.

International audienceWe demonstrated previously that 75% of infertile men with round, acrosomeless spermatozoa (globozoospermia) had a homozygous 200-Kb deletion removing the totality of DPY19L2. We showed that this deletion occurred by Non-Allelic Homologous Recombination (NAHR) between two homologous 28-Kb Low Copy Repeats (LCRs) located on each side of the gene. The accepted NAHR model predicts that inter-chromatid and inter-chromosome NAHR create a deleted and a duplicated recombined allele, while intra-chromatid events only generate deletions. Therefore more deletions are expected to be produced de novo. Surprisingly, array CGH data show that, in the general population, DPY19L2 duplicated alleles are approximately three times as frequent as deleted alleles. In order to shed light on this paradox, we developed a sperm-based assay to measure the de novo rates of deletions and duplications at this locus. As predicted by the NAHR model, we identified an excess of de novo deletions over duplications. We calculated that the excess of de novo deletion was compensated by evolutionary loss, whereas duplications, not subjected to selection, increased gradually. Purifying selection against sterile, homozygous deleted men may be sufficient for this compensation, but heterozygously deleted men might also suffer a small fitness penalty. The recombined alleles were sequenced to pinpoint the localisation of the breakpoints. We analysed a total of 15 homozygous deleted patients and 17 heterozygous individuals carrying either a deletion (n = 4) or a duplication (n = 13). All but two alleles fell within a 1.2-Kb region central to the 28-Kb LCR, indicating that >90% of the NAHR took place in that region. We showed that a PRDM9 13-mer recognition sequence is located right in the centre of that region. Our results therefore strengthen the link between this consensus sequence and the occurrence of NAHR

Details of the <i>DPY19L2</i> LCR1 and 2 and of the NAHR hotspot.

<p>(A) Detailed scaled representation of the 28.2 Kb LCR 1 (orange) and 27 Kb LCR2 (yellow). Pale blue rectangles correspond to sequences specific to one of the LCRs facing a gap in the other LCR. The presence of a 13 bp consensus PRDM9 recognition site (CCNCCNTNNCCNC) on LCR1 or LCR2 is indicated by a green circle when identified on the forward DNA strand and by a red circle when identified on the reverse strand (GTGGNNAGGGTGG). The LCR arrows point toward the chromosome 12 telomere. (B) The analysed recombination region is represented in grey. The positions of LCR-specific markers (diamonds and bold numbering) and variable nucleotides (crossed circles) are represented. Details of the markers' sequences and localisations are indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003363#pgen.1003363.s003" target="_blank">Table S2</a>. The five identified breakpoints (BP1–BP5) are shown as double arrows. One PRDM9 consensus sequence is localised in the centre of BP2, the central and most frequent breakpoint. (C) The central nucleotide from the consensus sequence corresponds to one of the identified SNPs (snp 20). A perfect match for the consensus sequence is present on LCR1, while the central thimine is replaced by a cytidine in LCR2. The 39 nt surrounding the 5 matches to the PRDM9 consensus sequence identified in LCR1 and 2 (sites a–e) are compared with the consensus sequence described in Myers et al <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003363#pgen.1003363-Paigen1" target="_blank">[8]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003363#pgen.1003363-Hayashi1" target="_blank">[11]</a>. Highly conserved nucleotides are red. For each locus the number of nucleotides identical to the consensus sequence is indicated on the right.</p

Rate of <i>de novo</i> deletion and duplication events occurring at the <i>DPY19L2</i> NAHR hotspot determined by digital PCR on sperm from 3 control donors.

<p>(A) Illustration of PCR results obtained by real time PCR. The left plots show amplification profiles obtained with primers specific to the recombined deleted LCR, the right plots show profiles obtained with the duplication-specific primers. No amplification was observed with either pairs of primers from 200 ng of somatic (blood) DNA, indicating that the NAHR did not occur during mitosis. Sperm DNA was diluted in order to obtain a positive amplification in approximately 25% of the wells. (B) The number of positive wells allowed estimating the frequency of <i>de novo</i> deletion and duplication events in three control sperms. Error bars represent 95% CIs.</p

Strategy and validation of the detection of <i>DPY19L2</i> recombined alleles by PCR.

<p>(A) Schematic representation of NAHR at the <i>DPY19L2</i> locus. 1) LCR1 and LCR2 correspond to the centromeric and telomeric LCRs respectively. The two LCRs are separated by approximately 200 Kb and each measures 28 Kb. 2) NAHR can occur following the mis-alignment of Low Copy Repeats 1 and 2 located either on 1) the same chromatid and results in the production of a) a deleted allele with a recombined 1-2 LCR, and b) a small circular molecule with a recombined 2-1 LCR and the <i>DPY19L2</i> gene. This small molecule will not survive through the cell cycle. 3) NAHR can occur following the mis-alignment from two distinct chromatids (whether sister-chromatids or chromatids from homologous chromosomes). This results in the production of a) a deleted allele with a 1-2 recombined LCR, and b) a complementary duplicated allele with a 2-1 recombined LCR. (B) Illustration of the specificity of the LCR-specific amplification when amplifying DNA from <i>DPY19L2</i> homozygously deleted globozoospermic patients (G) and control individuals (C). 1) Primers specific to the deleted 1-2 LCR yield a 2088 nt fragment in globozoospermic patients only. 2,3) Specific amplification of LCR 1 and 2 is only obtained from non-deleted controls. 4) Co-amplification of a control locus (bottom band) with a deleted 1-2 LCR-specific sequence. 5) Co-amplification of a control locus (bottom band) with a duplicated 2-1 LCR-specific sequence. A duplicated allele is identified in one control individual (first lane after the molecular weight markers (mw)).</p

Frequency of deleted and duplicated alleles in sperm from three control donors.

<p>Frequency of deleted and duplicated alleles in sperm from three control donors.</p

Distribution of deleted and duplicated breakpoints observed from somatic DNA (left two panels) and sperm DNA (right two panels).

<p>Somatic deletions were identified from sequence analysis of 15 homozygous deleted patients and two heterozygous deleted control individuals. Somatic duplications were identified from 12 positive control individuals. Data from sperm were pooled from three control donors.</p

Identification of a new recurrent Aurora kinase C mutation in both European and African men with macrozoospermia.

International audienceSTUDY 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 CHANCE: We 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)

A Recurrent Deletion of DPY19L2 Causes Infertility in Man by Blocking Sperm Head Elongation and Acrosome Formation

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