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

Direct Visualization of the Highly Polymorphic <i>RNU2</i> Locus in Proximity to the <i>BRCA1</i> Gene

<div><p>Although the breast cancer susceptibility gene <i>BRCA1</i> is one of the most extensively characterized genetic loci, much less is known about its upstream variable number tandem repeat element, the <i>RNU2</i> locus. <i>RNU2</i> encodes the U2 small nuclear RNA, an essential splicing element, but this locus is missing from the human genome assembly due to the inherent difficulty in the assembly of repetitive sequences. To fill the gap between <i>RNU2</i> and <i>BRCA1</i>, we have reconstructed the physical map of this region by re-examining genomic clone sequences of public databases, which allowed us to precisely localize the <i>RNU2</i> array 124 kb telomeric to <i>BRCA1</i>. We measured by performing FISH analyses on combed DNA for the first time the exact number of repeats carried by each of the two alleles in 41 individuals and found a range of 6-82 copies and a level of heterozygosity of 98%. The precise localisation of the <i>RNU2</i> locus in the genome reference assembly and the implementation of a new technical tool to study it will make the detailed exploration of this locus possible. This recently neglected macrosatellite could be valuable for evaluating the potential role of structural variations in disease due to its location next to a major cancer susceptibility gene.</p></div

Description of the <i>RNU2</i> array alleles identified in 46 unrelated chromosomes.

<p>Description of the <i>RNU2</i> array alleles identified in 46 unrelated chromosomes.</p

Visualization by molecular combing of the 17q21 region around <i>BRCA1</i>.

<p>(A) Schematization of the genomic morse code used. The <i>BRCA1</i> Genomic Morse Code (GMC) depicted (v4.0) is an improvement of the published code (v1.0) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076054#pone.0076054-Cheeseman1" target="_blank">[23]</a>. It covers a genomic region of 200 kb and consist in 17 signals of a distinct color (green, red or blue), each composed of 1 to 3 small horizontal bars corresponding to a single DNA probe. The signals for the flanking probes FP1-4 are each composed of 2 green or blue horizontal bars, while the signal for the <i>RNU2</i> array repeat unit is composed of 1 red horizontal bar. Of note, the probe for the <i>RNU2</i> array cross-reacts with <i>RNU2-4P</i>. (B) Fourteen fibres displaying different numbers of <i>RNU2</i> signals are shown. The first six fibres display the entire bar code from the <i>BRCA1</i> GMC to <i>RNU2-4P</i>, while the followings miss either the beginning of the <i>BRCA1</i> GMC or <i>RNU2-4P</i>.</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

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

Schematic representation of the chromosome 17q21 region around the <i>BRCA1</i> gene.

<p>(A) Gene locations and physical map distances as reported in the literature <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076054#pone.0076054-Liu1" target="_blank">[16]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076054#pone.0076054-Pavelitz2" target="_blank">[19]</a>. (B) Gene locations within a 300 Kb window as shown in the UCSC Genome Browser. Arrows indicate transcription direction. BRCA1P1: <i>BRCA1</i> pseudogene.</p

Localisation of the <i>RNU2</i> macrosatellite within the chromosome 17 sequence assemblies from NCBI Build 37.p10.

<p>(A) Schema of the region surrounding the <i>RNU2</i> array. The location of the portion of the <i>RNU2</i> repeat unit (not comprising the <i>RNU2</i> gene) and of the right junction found in the assemblies are depicted, as well as the probes used in molecular combing experiments that flank the <i>RNU2</i> array (FP1-4), and the <i>NBR1</i> and <i>TMEM106A</i> genes. (B) Clones covering the region. The reference sequence assemblies is based upon the complete sequence of 3 overlapping BACs, RP11-242D8, CTD-3014M21 and RP11-100E5 (AC060780.18, AC109326.11 and AC087650.12 respectively), represented by brown arrows. The complete sequence of the WI2-3095P13 fosmid (AC160862.2, green arrow) matches the reference sequence. The sequence of the ABC10-44487500M2 fosmid (AC231386.2, green arrow) matches the reference sequence up to its centromeric extremity where it contains several <i>RNU2</i> repeat units (depicted in a dotted curl). The five unassembled contigs of the working draft sequence of the RP11-570A16 BAC clone (AC087365.3) showing homology with the reference sequence are represented by a purple arrow. Contig 15 has been mis-assembled, as it contains several <i>RNU2</i> repeat units (depicted in dotted curls) at both its extremities.</p