Copy number analysis of NIPBL in a cohort of 510 patients reveals rare copy number variants and a mosaic deletion

Abstract

Cornelia de Lange syndrome (CdLS) is a genetically heterogeneous disorder characterized by growth retardation, intellectual disability, upper limb abnormalities, hirsutism, and characteristic facial features. In this study we explored the occurrence of intragenic NIPBL copy number variations (CNVs) in a cohort of 510 NIPBL sequence-negative patients with suspected CdLS. Copy number analysis was performed by custom exon-targeted oligonucleotide array-comparative genomic hybridization and/or MLPA. Whole-genome SNP array was used to further characterize rearrangements extending beyond the NIPBL gene. We identified NIPBL CNVs in 13 patients (2.5%) including one intragenic duplication and a deletion in mosaic state. Breakpoint sequences in two patients provided further evidence of a microhomology-mediated replicative mechanism as a potential predominant contributor to CNVs in NIPBL. Patients for whom clinical information was available share classical CdLS features including craniofacial and limb defects. Our experience in studying the frequency of NIBPL CNVs in the largest series of patients to date widens the mutational spectrum of NIPBL and emphasizes the clinical utility of performing NIPBL deletion/duplication analysis in patients with CdLS.

Keywords: Array-CGH; Cornelia de Lange; MLPA; NIPBL; copy number variation.

Figure 1

Characterization of deletions involving NIPBL by whole-genome microarray analysis in Patients 5, 11, and 13. In Patient 5 (Pt_5), a 24.4 kb deletion extending from the distal end of NIPBL into the intergenic region was observed. In Patient 11 (Pt_11), a 193.4 kb deletion was detected and extended to C5orf42. In Patient 13 (Pt_13), the deletion spanned 5.1 Mb and included NIPBL and 22 other genes. The copy number state segments (red for deletion) and copy number state data are shown for each patient. Genes involved are indicated. Not drawn to scale.

Figure 2

Break-point analysis of the mosaic deletion of NIPBL exons 12–14 in Patient 7. (A) Array-CGH profile (top) with the magnified region of interest (bottom) showing low-level reduction in the Cy5/Cy3 fluorescence log 2 ratio of oligonucleotide probes spanning exons 11–16; (B) MLPA histogram: NIPBL exons arrowed in red (12–14) showing reduced height ratio in comparison with control probes (∼0.7 vs. 1); (C) nucleotide sequence of the break points. Proximal reference sequence and patient break-point sequence that match with the proximal reference sequence are shown in green, whereas the distal reference sequence and patient break-point sequence that match with the distal reference sequence are shown in red. Dash boxed sequence corresponds to a region of microhomology and reveals the break-point junction.

Figure 3

Breakpoint analysis of the duplication of NIPBL exons 23–27 in Patient 8. (A) Array-CGH results revealed a duplication of NIPBL exons 23–27; (B) MLPA histogram: NIPBL exons arrowed in red (23–27) showing increased height ratio (∼1.4 vs. 1); (C) schematic diagram of the duplicated region with dashed square boxes representing the exons duplicated in tandem. Arrow heads indicate the location of PCR primers used to amplify the breakpoint junction of the duplication; (D) Nucleotide sequence of the breakpoint revealing insertion (purple) of seven nucleotides (ATATAAT) and 1bp-microhomology (dash box) at the breakpoint junction; (E) Photo of Patient 8 taken at two weeks of age.

Figure 4

Schematic diagram of the NIPBL gene displaying exonic deletions and duplications in 13 patients. On top: graphic view of 47 exons (vertical blue bars) of NIPBL: solid horizontal bars represent NIPBL genomic regions deleted (red) or duplicated (green) and approximate sizes. The deletion in mosaic state in Patient 7 is indicated by a striped red bar. Narrow dotted bars indicate rearrangements extending beyond the gene in Patients 4, 5, 11, and 13. The graphical data for each patient were obtained by inputting the most distal and proximal oligonucleotide genomic probe coordinates into the custom track at the University of California, Santa Cruz website: http://genome.ucsc.edu/cgi-bin/hgGateway.