Improving molecular diagnosis of aniridia and WAGR syndrome using customized targeted array-based CGH

Abstract

Chromosomal deletions at 11p13 are a frequent cause of congenital Aniridia, a rare pan-ocular genetic disease, and of WAGR syndrome, accounting up to 30% of cases. First-tier genetic testing for newborn with aniridia, to detect 11p13 rearrangements, includes Multiplex Ligation-dependent Probe Amplification (MLPA) and karyotyping. However, neither of these approaches allow obtaining a complete picture of the high complexity of chromosomal deletions and breakpoints in aniridia. Here, we report the development and validation of a customized targeted array-based comparative genomic hybridization, so called WAGR-array, for comprehensive high-resolution analysis of CNV in the WAGR locus. Our approach increased the detection rate in a Spanish cohort of 38 patients with aniridia, WAGR syndrome and other related ocular malformations, allowing to characterize four undiagnosed aniridia cases, and to confirm MLPA findings in four additional patients. For all patients, breakpoints were accurately established and a contiguous deletion syndrome, involving a large number of genes, was identified in three patients. Moreover, we identified novel microdeletions affecting 3' PAX6 regulatory regions in three families with isolated aniridia. This tool represents a good strategy for the genetic diagnosis of aniridia and associated syndromes, allowing for a more accurate CNVs detection, as well as a better delineation of breakpoints. Our results underline the clinical importance of performing exhaustive and accurate analysis of chromosomal rearrangements for patients with aniridia, especially newborns and those without defects in PAX6 after diagnostic screening.

Fig 1. Identification of intragenic PAX6 deletion in patients with isolated aniridia.

Targeted array-based comparative genomic hybridization (aCGH) analysis identified two deletions involving partial PAX6 deletions in two patients. Colored bars represent the genomic positions of the deletions. Schematic representation of the complete intron-exon structure of PAX6 is shown. Exons are indicated by colored rectangles that are wider for the coding regions. CGH array data for both individuals is shown. The patient versus reference log2-ratio for the relative hybridization intensities of probes is plotted. Dots with log2-ratio around -1 indicate a heterozygous deletion (green dots), log2-ratio 0 indicates a normal pattern, and +0.6 indicates a heterozygous amplification (red dots). Shaded areas indicate deletions. Genomic coordinates are shown in the x-axis and are based on the Human Genome Assembly hg19. The red bar indicates a ~63 kb deletion encompassing from exon 5a to exon 13 of PAX6 found in patient ANIRIDIA-052 (chr11:31,760,458–31,823,847). The grey bar represents a ~3.3 kb deletion encompassing from exon 5a to exon 7 of PAX6 gene found in patient ANIRIDIA-039 (chr11:31,820,789–31,824,052).

Fig 2. Identification of 3’ regulatory deletions of PAX6 in patients with isolated aniridia.

Targeted array-based comparative genomic hybridization (aCGH) analysis identified deletions involving telomeric deletions to PAX6 in two patients ANIRIDIA-008 and ANIRIDIA-021. Patient ANIRIDIA-067 was used as positive control for validation purposes. The colored bars represent the genomic positions of the deletions. The red asterisks indicate a cluster of PAX6 regulatory regions located in intronic positions of ELP4, as reviewed by Bathia, et al, 2013. Exons are indicated by colored rectangles that are wider for the coding regions. CGH array data for the two patients with previously unknown 3' telomeric PAX6 deletions are shown. The patient versus reference log2-ratio for the relative hybridization intensities of probes is plotted. Dots with log2-ratio around -1 indicate a heterozygous deletion (green dots), log2-ratio 0 indicates a normal pattern and +0.6 indicates a heterozygous amplification (red dots). Shaded areas indicate deletions. Genomic coordinates are shown in the x-axis and are based on the Human Genome Assembly hg19. The grey bar indicates a ~567 kb deletion in patient ANIRIDIA-008 (chr11:31,147,306–31,714,853). The orange bar indicates a ~511 kb deletion in patient ANIRIDIA-021 (chr11:31,186,493–31,698,208).

Fig 3. Identification of chromosomal rearrangements in WAGR locus in syndromic and non-syndromic patients with aniridia.

Targeted array-based comparative genomic hybridization (aCGH) analysis identified deletions of different sizes ranging from3.3 Kb to 13.4 Mb. The red bars show intragenic PAX6 deletions in two patients with isolated aniridia, ANIRIDIA-039 (chr11:31,820,789–31,824,052) and ANIRIDIA-052 (chr11:31,760,458–31,823,847). The green bars show 3’ upstream deletions affecting 3' regulatory regions of PAX6, identified in three families with isolated aniridia: ANIRIDIA-008 (chr11:31,147,306–31,714,853), ANIRIDIA-021 (chr11:31,186,493–31,698,208) and ANIRIDIA-067 (chr11:31,083,877–31,704,548). Purple bars show large deletions affecting several contiguous genes, in two patients with WAGR (ANIRIDIA-020, chr11:29,750,813–32,752,091), and WAGRO (ANIRIDIA-070, chr11:21,586,131–33,168,232) syndromes. Genes delineating both syndromes are highlighted in red. The blue bar represents a novel gene contiguous deletion syndrome involving PAX6 and 45 upstream genes in a syndromic patient with aniridia (ANIRIDIA-064, chr11:18,536,224–31,923,308). Genomic coordinates are shown in the x-axis and are based on the Human Genome Assembly hg19.