PITX2 and FOXC1 spectrum of mutations in ocular syndromes

Abstract

Anterior segment dysgenesis (ASD) encompasses a broad spectrum of developmental conditions affecting anterior ocular structures and associated with an increased risk for glaucoma. Various systemic anomalies are often observed in ASD conditions such as Axenfeld-Rieger syndrome (ARS) and De Hauwere syndrome. We report DNA sequencing and copy number analysis of PITX2 and FOXC1 in 76 patients with syndromic or isolated ASD and related conditions. PITX2 mutations and deletions were found in 24 patients with dental and/or umbilical anomalies seen in all. Seven PITX2-mutant alleles were novel including c.708_730del, the most C-terminal mutation reported to date. A second case of deletion of the distant upstream but not coding region of PITX2 was identified, highlighting the importance of this recently discovered mechanism for ARS. FOXC1 deletions were observed in four cases, three of which demonstrated hearing and/or heart defects, including a patient with De Hauwere syndrome; no nucleotide mutations in FOXC1 were identified. Review of the literature identified several other patients with 6p25 deletions and features of De Hauwere syndrome. The 1.3-Mb deletion of 6p25 presented here defines the critical region for this phenotype and includes the FOXC1, FOXF2, and FOXQ1 genes. In summary, PITX2 or FOXC1 disruptions explained 63% of ARS and 6% of other ASD in our cohort; all affected patients demonstrated additional systemic defects with PITX2 mutations showing a strong association with dental and/or umbilical anomalies and FOXC1 with heart and hearing defects. FOXC1 deletion was also found to be associated with De Hauwere syndrome.

Figure 1

Photograph of a patient (case 11) with ARS, demonstrating the characteristic facial features (a, b), dental defects (c, d), and umbilical anomaly (e). The color reproduction of this figure is available at the European Journal of Human Genetics online.

Figure 2

Summary of PITX2 mutations identified in this study. (a) Schematic drawing of PITX2 gene and upstream region. Identified point mutations are shown with red numbered arrows (1–18) whereas identified deletions are shown as red lines (19–24); the solid line shows the minimum deletion size, with the dashed line representing the maximum extent of the deletions. PITX2 exons are shown as numbered boxes, regions corresponding to the homeobox and 14-amino-acid-conserved domains are indicated as black and light gray boxes, respectively. Initiation codons corresponding to the PITX2 isoforms are shown with black arrows. The stop codon is indicated with a black arrow and asterisk. The positions of the TaqMan probes are shown and labeled P1–P6; for upstream probes P3B and P4–P6, the distance (in base pairs) from the beginning of the gene is indicated in parenthesis. The positions of probes that demonstrated haploid state are shown as black diamonds, whereas probes that showed diploid state are shown as gray diamonds. (b) DNA sequencing chromatograms for normal and mutants alleles; mutation position is indicated with an arrow. (c) Copy number analysis (TaqMan assay and Affymetrix array) data. For PITX2 TaqMan assays, probes P1–P3 correspond to PITX2 exons and probes P3B and P4–P6 correspond to the upstream region, patient sample values were compared with control (C) results for every assay. Probes that showed half of the normal value are noted in red. For Affymetrix array analysis, Genotyping Console representation of chromosome 4 marker data is shown with heterozygous deletions indicated with arrows. The color reproduction of this figure is available at the European Journal of Human Genetics online.

Figure 3

Summary of FOXC1 deletions identified in this study. (a) Schematic drawing of the 6p25 region. All known human protein-coding and non-protein-coding genes from the NCBI RNA reference sequences collection (RefSeq) for the selected region are shown as seen in the UCSC Genome Browser (http://www.genome/ucsc.edu); GRCh37/hg19 assembly was utilized. Identified deletions are shown as red lines (25–28); the solid line shows the minimum deletion size, with the dashed line representing the maximum extent of the deletions. The positions of the TaqMan probes that demonstrated haploid state are shown as black diamonds; the positions of the Affymetrix probes that exhibited haploid state are shown as black circles and probes that displayed diploid state as gray circles. (b) Affymetrix array identification of FOXC1/6p25 deletions. Genotyping Console representation of chromosome 6 marker data for cases 25, 26 and 28 is shown with FOXC1 position within the heterozygous deletions indicated with arrows. (c) Copy number analysis using TaqMan assays showing deletion of FOXC1 and GMDS for cases 25, 26 and 28. Patient sample values were compared with control (C) results for every assay. The color reproduction of this figure is available at the European Journal of Human Genetics online.