Large novel deletions detected in Chinese families with aniridia: correlation between genotype and phenotype

Abstract

Purpose: To describe the clinical and genetic findings in two Chinese families with aniridia and other ocular abnormalities.

Methods: Two unrelated families were examined clinically. After informed consent was obtained, genomic DNA was extracted from the venous blood of all participants. Mutation screening of all exons of the PAX6 (paired box gene 6) gene was performed by direct sequencing of PCR-amplified DNA fragments. Multiplex ligation-dependent probe amplification (MLPA) was performed to detect large deletions. Linkage analysis was used to validate the large deletions revealed by MLPA in all available family members.

Results: Clinical examination and pedigree analysis revealed one four-generation family (85) and one three- generation family (86) with total aniridia, congenital cataracts, foveal hypoplasia, and glaucoma. No mutation in PAX6 was identified after PCR-sequencing. Through MLPA analysis, a large deletion including the whole PAX6 gene, DKFZp686k1684 (hypothetical LOC440034), and the RCN1 (reticulocalbin 1) gene was detected in family 85; a 3' deletion to the PAX6 gene including the ELP4 (elongator complex protein 4) and the DCDC1 (doublecortin domain containing 1) gene was identified in family 86.The two large deletions were confirmed with linkage analysis and the "loss of heterozygous" in the different PAX6 regions were co-segregated with the phenotype of the two families, respectively.

Conclusions: Patients with the PAX6 contiguous gene deletion, including the RCN1 gene, presented more severe vision impairments than those carrying the PAX6 3' deletion. Large deletions may account for several Chinese families and sporadic cases with aniridia and screening for these kinds of alterations should be included in aniridia patients' analyses.

Figure 1

Schematic representation of WT1/ RCN1/ DKFZ p686k1684/PAX6/ ELP4/ DCDC1 on chromosome 11p13 and the relative positions of the microsatellite markers and single nucleotide polymorphism (SNP) used in linkage and real-time quantitative PCR analysis. D presents DKFZ p686k1684. Brown arrows below the each gene indicated the transcription direction for each gene. The region between the two blue vertical lines presents the deleted region (407 Kb) of family 85, the region between the two purple vertical lines presents the deleted region (527 Kb) of family 86.

Figure 2

Family structure and haplotype analysis of two Chinese families with aniridia. Pedigree and haplotype analysis of family 85 and 86 with aniridia showed “loss of heterozygous” segregation with the microsatellite marker PAX6.CA/GT (family 85), D11S995, and D11S2001 (family 86), respectively. All markers are on chromosome 11, listed in descending order from the centromeric end. Squares indicate males; circles indicate females; slashed symbols indicate deceased; solid symbols indicate affected; open symbols indicate unaffected.

Figure 3

Ophthalmological findings in patients from the two families. A: Photograph of anterior segment of patient III-4 of 85 with complete absence of iris and the progressing dense congenital cataract. B: Fundus images of patient III-4 showed late-stage glaucomatous cupping of the optic disc. C: Complete hypoplasia of the iris and congenital cataract were observed in patient IV-7 of family 85. D: Fundus images of patient IV-7 showing foveal hypoplasia. E: Photograph of anterior segment of patient II-4 of family 86 with complete absence of iris and congenital cataract. F: Fundus image of patient II-4 showing a tessellated appearance. G: Photograph of the anterior segment of patient III-3 of family 86 with complete absence of iris and mild cataract. H: Fundus image of patient III-3 showing a normal foveal reflex.

Figure 4

The normalized MLPA results of the probands of the two families. A: The normalized MLPA result of III-4 of family 85. B: The result of II-4 of family 86. The height of the columns represents of the dosage of the respective segments in the genomic DNA with two alleles. The light blue columns represent chromosome 11p13 specific probes. The orange columns represent the deleted probes. The dark blue columns represent the control probes. The allele dosage of the deleted probes was found in the range of about 0.5–0.7 of normal control, which corresponds to one allele.

Figure 5

Analysis of deletions in the PAX6 gene region by real-time quantitative PCR. A: Fluorescence amplification plots of the real-time quantitative PCR for exon 8 of the PAX6 gene, detected deletion by MLPA. B: Fluorescence amplification plots of the real-time PCR for rs7104670. Arrows in A and B indicated triplicate signals obtained for a control subject (Normal) and the patient III-1 of family 85 (Patient). C: Histogram indicated the relative quantity (RQ) between the exon8 of PAX6 or rs7104670 and GAPDH values. Bars represent mean values±standard deviations for the triplicate values.