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A novel, non-stop mutation in FOXE3 causes an autosomal dominant form of variable anterior segment dysgenesis including Peters anomaly.

ABSTRACT: Anterior segment dysgenesis (ASD) is a spectrum of disorders that affect the anterior ocular chamber. Clinical studies on a Newfoundland family over the past 30 years show that 11 relatives have a variable ocular phenotype ranging from microcornea to Peters anomaly, segregating as an autosomal dominant trait. To determine the molecular etiology of the variable ASD in this family, we sequenced nine functional candidate genes and identified 44 variants. A point mutation in FOXE3, which codes for a transcription factor involved in the formation of the lens and surrounding structures, co-segregated with the variable ocular phenotype. This novel mutation (c.959G>T) substitutes the stop codon for a leucine residue, predicting the addition of 72 amino acids to the C-terminus of FOXE3. Two recent reports have also identified non-stop mutations in FOXE3 in patients with variable ocular phenotypes and predict an extended protein. Although FOXE3 is a lens-specific gene, we successfully isolated complementary DNA from lymphoblasts of an affected family member, and our sequencing results show that the c.959T allele is absent, suggesting that it may be degraded at the RNA level. Though preliminary, our results challenge the notion that an extended FOXE3 protein causes ASD, and instead suggests a mechanism of haploinsufficiency in the case of non-stop mutations. This study adds to several reports that suggest that autosomal-dominant mutations within FOXE3 cause ASD and has important clinical utility, especially for the diagnosis of mildly affected patients.

SUBMITTER: Doucette L

PROVIDER: S-EPMC3062009 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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A novel, non-stop mutation in FOXE3 causes an autosomal dominant form of variable anterior segment dysgenesis including Peters anomaly.

Doucette Lance L Green Jane J Fernandez Bridget B Johnson Gordon J GJ Parfrey Patrick P Young Terry-Lynn TL

European journal of human genetics : EJHG 20101208 3

Anterior segment dysgenesis (ASD) is a spectrum of disorders that affect the anterior ocular chamber. Clinical studies on a Newfoundland family over the past 30 years show that 11 relatives have a variable ocular phenotype ranging from microcornea to Peters anomaly, segregating as an autosomal dominant trait. To determine the molecular etiology of the variable ASD in this family, we sequenced nine functional candidate genes and identified 44 variants. A point mutation in FOXE3, which codes for a ...[more]

PMID: 21150893

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Project description:FOXE3 is a lens specific transcription factor that has been associated with anterior segment ocular dysgenesis. To determine the transcriptional target(s) of FOXE3 that are indispensable for the anterior segment development, we examined the transcriptome and the proteome of cells expressing truncated FOXE3 responsible for Peters anomaly identified through linkage-coupled next-generation whole exome sequencing. We found that DNAJB1, an autophagy-associated protein, was the only candidate exhibiting differential expression in both screens. We confirmed the candidacy of DNAJB1 through chromatin immunoprecipitation and luciferase assays while knockdown of DNAJB1 in human lens epithelial cells resulted in mitotic arrest. Subsequently, we targeted dnajb1a in zebrafish through injection of a splice-blocking morpholino. The dnajb1a morphants exhibited underdeveloped cataractous lenses with persistent apoptotic nuclei. In conclusion, we have identified DNAJB1 as a transcriptional target of FOXE3 in a novel pathway that is crucial for development of the anterior segment of the eye. Human Embryonic Kidney (HEK293FT) cells were transfected with the expression vector (pT-RexTM-DEST30) harboring either the wild type or the mutant (C240*) FOXE3 ORF (open reading frame). The experimental design included a total of eight biological replicates of cells expressing the wild type and eight replicates of mutant FOXE3 along with eight non-transfected controls. Cells were harvested 24-hour post-transfection and subjected to total RNA isolation for the preparation of whole transcriptome next-generation sequencing libraries. Initially, we examined the quality of transcriptome libraries on a MiSeq genome analyzer. Subsequent to confirmation of the quality, all libraries were paired-end sequenced (2 x 100 bp) using Illumina TruSeq Cluster V3 flow cell at a concentration of 13.0 pM in two separate lanes (12 bar-coded mRNA pooled libraries in each lane) on a HiSeq 2000 genome analyzer.

Project description:BackgroundCongenital cataracts are clinically and genetically heterogeneous with more than 45 known loci and 38 identified genes. They can occur as isolated defects or in association with anterior segment developmental anomalies. One of the disease genes for congenital cataract with or without anterior segment dysgenesis (ASD) is PITX3, encoding a transcription factor with a crucial role in lens and anterior segment development. Only five unique PITX3 mutations have been described, of which the 17-bp duplication c.640_656dup, p.(Gly220Profs*95), is the most common one and the only one known to cause cataract with ASD. The aim of this study was to perform a genetic study of the PITX3 gene in five probands with autosomal dominant congenital cataract (ADCC) and ASD, to compare their clinical presentations to previously reported PITX3-associated phenotypes and to functionally evaluate the PITX3 mutations found.MethodsSanger sequencing of the coding region and targeted exons of PITX3 was performed in probands and family members respectively. Transactivation, DNA-binding and subcellular localization assays were performed for the PITX3 mutations found. Ophthalmological examinations included visual acuity measurement, slit-lamp biomicroscopy, tonometry and fundoscopy.ResultsIn four Belgian families with ADCC and ASD the recurrent 17-bp duplication c.640_656dup, p.(Gly220Profs*95), was found in a heterozygous state. A novel PITX3 mutation c.573del, p.(Ser192Alafs*117), was identified in heterozygous state in a Belgo-Romanian family with a similar phenotype. Functional assays showed that this novel mutation retains its nuclear localization but results in decreased DNA-binding and transactivation activity, similar to the recurrent duplication.ConclusionsOur study identified a second PITX3 mutation leading to congenital cataract with ASD. The similarity in phenotypic expression was substantiated by our in vitro functional studies which demonstrated comparable molecular consequences for the novel p.(Ser192Alafs*117) and the recurrent p.(Gly220Profs*95) mutations.

Project description:Purpose: Peters anomaly (PA) is a heterogeneous developmental disorder characterized by central corneal opacity and iridocorneal or corneolenticular adhesions. Although many causative genes have been identified, most screened patients do not have mutations in the known genes. We aimed to identify the genetic cause of Peters anomaly in a pedigree with three affected individuals.Methods: Slit-lamp biomicroscopy and ultrasound biomicroscopy were performed for definitive diagnosis. Exome sequencing was conducted on the DNA of all three patients. After identification of a candidate causative gene, expression of the gene was assessed with real-time PCR in various ocular tissues of three human embryos and three adults.Results: The patients were affected with isolated PA. The parents of the patients were related to one another. Inheritance of PA was autosomal recessive. After appropriate filtering of the exome data, a homozygous variation in DOP1B remained as the only candidate genetic cause of PA in the pedigree. The variant segregated with disease status in the pedigree and was absent among 800 control Iranians. The variant has been reported in various databases at frequencies of 0.006 or less only in the heterozygous state in some cohorts of African origin. The p.Val1660 amino acid affected by the mutation is completely conserved in mammals and birds during evolution. Expression of DOP1B was shown in all adult and embryonic lens, iris, cornea, sclera, and retina tissues that were tested.Conclusions: DOP1B that encodes DOP1 leucine zipper like protein B was identified as the putative PA-causing gene in pedigree PA-101. As DOP1B is positioned within the Down syndrome chromosomal region on chromosome 21, until now this gene has mostly been studied with respect to brain functions. However, members of the Dopey gene family have been shown to have roles in development in other organisms. Evidence of the expression of DOP1B in various PA-relevant eye tissues, which, to the best of our knowledge, is shown here for the first time, is to be noted. However, this finding does not necessarily implicate a specific role for DOP1B in eye development as the gene is expressed in many tissues. Ultimately, definitive assessment of the contribution of DOP1B to PA pathology awaits identification of mutations in the gene in unrelated patients with PA and functional studies.

Dataset Information

A novel, non-stop mutation in FOXE3 causes an autosomal dominant form of variable anterior segment dysgenesis including Peters anomaly.

Publications

A novel, non-stop mutation in FOXE3 causes an autosomal dominant form of variable anterior segment dysgenesis including Peters anomaly.

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