Fine characterisation of a recombination hotspot at the DPY19L2 locus and resolution of the paradoxical excess of duplications over deletions in the general population

Charles Coutton, Farid Abada, Thomas Karaouzene, Damien Sanlaville, Véronique Satre, Joël Lunardi, Pierre-Simon Jouk, Christophe Arnoult, Nicolas Thierry-Mieg, Pierre F Ray, Charles Coutton, Farid Abada, Thomas Karaouzene, Damien Sanlaville, Véronique Satre, Joël Lunardi, Pierre-Simon Jouk, Christophe Arnoult, Nicolas Thierry-Mieg, Pierre F Ray

Abstract

We 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.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1. Strategy and validation of the…
Figure 1. Strategy and validation of the detection of DPY19L2 recombined alleles by PCR.
(A) Schematic representation of NAHR at the DPY19L2 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 DPY19L2 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 DPY19L2 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)).
Figure 2. Rate of de novo deletion…
Figure 2. Rate of de novo deletion and duplication events occurring at the DPY19L2 NAHR hotspot determined by digital PCR on sperm from 3 control donors.
(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 de novo deletion and duplication events in three control sperms. Error bars represent 95% CIs.
Figure 3. Details of the DPY19L2 LCR1…
Figure 3. Details of the DPY19L2 LCR1 and 2 and of the NAHR hotspot.
(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 Table S2. 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 , . Highly conserved nucleotides are red. For each locus the number of nucleotides identical to the consensus sequence is indicated on the right.
Figure 4. Distribution of deleted and duplicated…
Figure 4. Distribution of deleted and duplicated breakpoints observed from somatic DNA (left two panels) and sperm DNA (right two panels).
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.

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