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A large novel deletion downstream of PAX6 gene in a Chinese family with coloboma of iris

ABSTRACT: The paired box gene 6 (PAX6) is an essential transcription factor for eye formation. Genetic alterations in PAX6 can lead to various ocular malformations including aniridia. The purpose of this study was to identify genetic defects as the underlying cause of familial coloboma of iris in a large Chinese family. After linkage analysis was carried out in this family, all exons of PAX6 in the proband were sequenced by the Sanger sequencing technique. Then the genome of the proband was evaluated by a microarray-based comparative genomic hybridization (aCGH). Quantitative real-time PCR was applied to verify the abnormal aCGH findings. All patients presented bilateral partial coloboma of iris, severe congenital nystagmus, hyperpresbyopia and congenital posterior polar cataracts. Two-point linkage analysis in the autosomal dominant family showed loss of heterozygosity at the D11S914 locus. There was no pathogenic mutation in the exons of PAX6. The aCGH analysis revealed a 681 kb heterozygous deletion on chromosome 11p13. Quantitative real-time PCR verified the deletion in the patients and further confirmed this deletion cosegregation with the coloboma of iris phenotype in the family. The 681 kb large deletion of chromosome 11p13 downstream of PAX6 is the genetic cause of the familial coloboma of ocular in this large Chinese family. aCGH should be applied if there is a negative result for the mutation detection of PAX6 in patients with aniridia.

ORGANISM(S): Homo sapiens

PROVIDER: GSE50577 | GEO | 2013/09/05

SECONDARY ACCESSION(S): PRJNA217974

REPOSITORIES: GEO

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Project description:PAX6-related Aniridia is a sight-threatening disease due to progression of secondary glaucoma and aniridia associated keratopathy (AAK). Changes or loss of limbal epithelial progenitors causes the epithelial surfaces defects. We analyzed how PAX6 contribute to this with a two-step approach. 1) mRNA Sequencing of limbal epithelial cells isolated from controls and aniridia patients. 2) confirming the bioinformatical and literature-based result list on a siRNA based primary aniridia cell model (PAX6- knockdown). With this approach genes which are directly influenced by PAX6 should be distinguishable from genes secondary affected by AAK disease state. Therefore epithelial cells were isolated from the limbus region of two patients with aniridia and cultured in KSFM medium. Normal cells were obtained from limbus region of cadaveric control patients. For the siRNA based anridia cell model cells were transfected with lipofectamine and 5 nM siRNA against PAX6 or scrambled control. All cells were lysed to obtain DNA, RNA and protein. Reduction of PAX6 protein was controlled by Western Blot. Aniridia and Control Poly-A enriched RNA-librarys were subjected to Next Generation Sequencing. The differential analysis was a combination of quantification with RSEM and differential tests with edgeR. Gene lists were filtered by comparing to NCBI GEO Datasets, annotation with DAVID and manually annotation using literature search. For the resulting filtered gene list qPCR primer were ordered and postulated changes were verified with qPCR on siRNA based aniridia- cell model (Namely: TP63, ABCG2, ADH7, ALDH1A1, PITX1, DKK1, DSG1, K12, K3, K13, SPINK6, SPINK7, CTSV, SPERPINB1). We could identify genes which might be regulated by PAX6. We could show that SPINK7 mRNA coding for a protease inhibitor is downregulated in patients as well as in our primary aniridia cell model. ALDH1A1 and AHD7 mRNAs were reduced in limbal epithelial cells of aniridia patients (AN-LEC). Both transcripts were downregulated upon PAX6 knockdown in our cell model.Our siRNA-based aniridia cell model is a tool to proof identified PAX6 affected genes which might drive AAK pathogenesis. We could demonstrate, that this is the fact for transcripts coding for retinol converting enzymes. This provides evidence that PAX6 might drive corneal epithelial differentiation process by direct or indirect controlling retinoic acid signaling processes.

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A large novel deletion downstream of PAX6 gene in a Chinese family with coloboma of iris

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