Alu-mediated deletion of SOX10 regulatory elements in Waardenburg syndrome type 4

Abstract

Waardenburg syndrome type 4 (WS4) is a rare neural crest disorder defined by the combination of Waardenburg syndrome (sensorineural hearing loss and pigmentation defects) and Hirschsprung disease (intestinal aganglionosis). Three genes are known to be involved in this syndrome, that is, EDN3 (endothelin-3), EDNRB (endothelin receptor type B), and SOX10. However, 15-35% of WS4 remains unexplained at the molecular level, suggesting that other genes could be involved and/or that mutations within known genes may have escaped previous screenings. Here, we searched for deletions within recently identified SOX10 regulatory sequences and describe the first characterization of a WS4 patient presenting with a large deletion encompassing three of these enhancers. Analysis of the breakpoint region suggests a complex rearrangement involving three Alu sequences that could be mediated by a FosTes/MMBIR replication mechanism. Taken together with recent reports, our results demonstrate that the disruption of highly conserved non-coding elements located within or at a long distance from the coding sequences of key genes can result in several neurocristopathies. This opens up new routes to the molecular dissection of neural crest disorders.

Figure 1

Characterisation of SOX10 regulatory sequences deletion identified by QMF-PCR and CGH array. (a) Schematic representation from centromere to telomere of the deletion identified by QMF-PCR. The scheme indicates the orientation of the SOX10 and PICK1 genes, with the localisation of the enhancers (U1–5 and D6+7). Segments analysed by the initial QMF-PCR are indicated by black arrowheads, whereas additional primers used to delineate the deleted regions are indicated by grey arrowheads (see Supplementary Table 1 for primer sequences). QMF-PCR results are shown below: ‘+'=not deleted or ‘−'=deleted. Localisation of Alu1 and 2 sequences described in Figure 2 are indicated. (b) Graphical overview of the CGH-array results encompassing the chromosome 22 deletion. The results are represented using the Genomic Workbench software (V5.0.14, Agilent technologies). Dots show the relative intensities as deviation from the horizontal line of log2Ratio=0. The deleted region is highlighted. The genes are represented and the genomic locations are indicated in the horizontal scale.

Figure 2

Sequence analysis of the breakpoint region (a) Chromatogram of the junction fragment corresponding to the mutant allele amplified from the propositus (b) Alignment of mutant and Alu1, 2 and 3 sequences. The mutant sequence observed in the propositus is shown on the first line. Dots indicate conserved nucleotides. Non-conserved nucleotides are indicated in normal upper case. The patient's mutant allele and the respective Alu1, 2, and 3 sequence fragments maintained during the deletion event are indicated in bold and highlighted in grey. The 38 bp region homologous to part of the Alu3 sequence is boxed in a and b. (c) Top: schematic representation of Alu 1, 2, and 3 from centromere to telomere. Bottom: deletion detected in the propositus. Fragments of Alu1 and 2 maintained during the deletion event are indicated in grey. The 38-bp insertion corresponding to the Alu3 sequence (Genbank accession number NT_011520.12) is indicated in black (and ^*).

Figure 3

Proposed model to explain the rearrangement observed. Schematic representation from centromere to telomere of the region analysed. Respective orientations of SOX10, and Alu1, 2, and 3 sequences are indicated by arrows. Numbers 1 and 2 indicate the localisation of proposed FoSTeS/MMBIR events. Doted lines indicate deleted regions.