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Analysis of RNA splicing defects in PITX2 mutants supports a gene dosage model of Axenfeld-Rieger syndrome.

ABSTRACT: Axenfeld-Rieger syndrome (ARS) is associated with mutations in the PITX2 gene that encodes a homeobox transcription factor. Several intronic PITX2 mutations have been reported in Axenfeld-Rieger patients but their effects on gene expression have not been tested.We present two new families with recurrent PITX2 intronic mutations and use PITX2c minigenes and transfected cells to address the hypothesis that intronic mutations effect RNA splicing. Three PITX2 mutations have been analyzed: a G>T mutation within the AG 3' splice site (ss) junction associated with exon 4 (IVS4-1G>T), a G>C mutation at position +5 of the 5' (ss) of exon 4 (IVS4+5G>C), and a previously reported A>G substitution at position -11 of 3'ss of exon 5 (IVS5-11A>G).Mutation IVS4+5G>C showed 71% retention of the intron between exons 4 and 5, and poorly expressed protein. Wild-type protein levels were proportionally expressed from correctly spliced mRNA. The G>T mutation within the exon 4 AG 3'ss junction shifted splicing exclusively to a new AG and resulted in a severely truncated, poorly expressed protein. Finally, the A>G substitution at position -11 of the 3'ss of exon 5 shifted splicing exclusively to a newly created upstream AG and resulted in generation of a protein with a truncated homeodomain.This is the first direct evidence to support aberrant RNA splicing as the mechanism underlying the disorder in some patients and suggests that the magnitude of the splicing defect may contribute to the variability of ARS phenotypes, in support of a gene dosage model of Axenfeld-Rieger syndrome.

SUBMITTER: Maciolek NL

PROVIDER: S-EPMC1553432 | biostudies-literature | 2006 Jul

REPOSITORIES: biostudies-literature

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Analysis of RNA splicing defects in PITX2 mutants supports a gene dosage model of Axenfeld-Rieger syndrome.

Maciolek Nicole L NL Alward Wallace L M WL Murray Jeffrey C JC Semina Elena V EV McNally Mark T MT

BMC medical genetics 20060711

<h4>Background</h4>Axenfeld-Rieger syndrome (ARS) is associated with mutations in the PITX2 gene that encodes a homeobox transcription factor. Several intronic PITX2 mutations have been reported in Axenfeld-Rieger patients but their effects on gene expression have not been tested.<h4>Methods</h4>We present two new families with recurrent PITX2 intronic mutations and use PITX2c minigenes and transfected cells to address the hypothesis that intronic mutations effect RNA splicing. Three PITX2 mutat ...[more]

PMID: 16834779

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Project description:Purpose:To identify the genetic cause in a four-generation Chinese family with Axenfeld-Rieger syndrome (ARS). Methods:The family members received clinical examinations of the eye, tooth, periumbilical skin, and heart. Sanger sequencing and whole-exome sequencing (WES) were performed to screen potential mutations. The genomic deletion region around the PITX2 gene was estimated from single nucleotide polymorphism (SNP) data from WES and then confirmed with "quantitative PCR (qPCR) using a set of primers. The DNA breakpoint was further identified with long-range PCR and Sanger sequencing. Results:Symptoms including anterior segment dysplasia of the eye (iris dysplasia, multiple pupils, and posterior embryotoxon), dental dysplasia, and periumbilical skin redundancy were present in all of the affected individuals. Three of them had glaucoma. Corneal abnormalities (inferior sclerocornea, corneal endothelial dystrophy, and central corneal scar) were seen in most of the affected individuals. Cataract, limited eye movement, electrocardiographic abnormalities, intellectual disability, and recurrent miscarriages were observed in some of the affected individuals. No mutations in the coding and exon-intron adjacent regions of the PITX2 and FOXC1 genes were identified with Sanger sequencing. According to the SNP data from WES, we suspected that there might be a deletion region (at most 1.6 Mb) around the PITX2 gene. With the use of qPCR and long-range PCR, we identified a 53,840 bp deletion (chr4: 111,535,454-111,588,933) spanning PITX2 and PANCR. The genomic deletion cosegregated with the major ARS symptoms observed in the family members. Conclusions:With the help of WES, qPCR, and long-range PCR, we identified a genomic deletion encompassing PITX2 and the adjacent noncoding gene PANCR in a Chinese family with ARS. The clinical features of the affected individuals are reported. This work may broaden understanding of the phenotypic and mutational spectrums related to ARS.

Project description:PurposeThe aims of this study were to examine novel mutations in PITX2 and FOXC1 in Chinese patients with anterior segment dysgenesis (ASD) and to compare the clinical presentations of these mutations with previously reported associated phenotypes.MethodsTwenty-six unrelated patients with different forms of ASD were enrolled from our paediatric and genetic eye clinic. The ocular manifestations of both eyes of each patient were recorded. Genomic DNA was prepared from venous leukocytes. All coding exons of PITX2 and FOXC1 were amplified by polymerase chain reaction (PCR) from genomic DNA and subjected to direct DNA sequencing. Analysis of mutations in control subjects was performed by heteroduplex single-strand conformation polymorphism (SSCP) analysis.ResultsSequence analysis of the PITX2 gene revealed four mutations, including c.475_476delCT (P.L159VfsX39), c.64C > T (P.Q22X), c.296delG (P.R99PfsX56), and c.206G > A (P.R69H). The first three mutations were found to be novel. The c.475_476delCT (P.L159VfsX39) mutation, located at the 3' end of the PITX2-coding region, was identified in a Chinese Axenfeld-Rieger syndrome (ARS) patient who presented with an unusual severe phenotype of bilateral aniridia. The clinical characteristics, including the severity and manifestations of the patient's phenotype, were compared with reported PITX2-associated aniridia phenotypes of ARS in the literature.ConclusionsThese results expand the mutation spectrum of the PITX2 gene in patients with ARS. The PITX2 gene may be responsible for a significant portion of ARS with additional systemic defects in the Chinese population. This is the first reported case of a mutation at the 3' end of the PITX2-coding region extending the phenotypic consequences to bilateral aniridia. The traits of ARS could display tremendous variability in severity and manifestations due to the dominant-negative effect of PITX2. Our results further emphasize the importance of careful clinical and genetic analysis in determining mutation-disease associations and may lead to a better understanding of the role of PITX2 in ocular development.

Dataset Information

Analysis of RNA splicing defects in PITX2 mutants supports a gene dosage model of Axenfeld-Rieger syndrome.

Publications

Analysis of RNA splicing defects in PITX2 mutants supports a gene dosage model of Axenfeld-Rieger syndrome.

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