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Novel causative mutations in patients with Nance-Horan syndrome and altered localization of the mutant NHS-A protein isoform.

Sharma S, Burdon KP, Dave A, Jamieson RV, Yaron Y, Billson F, Van Maldergem L, Lorenz B, Gécz J, Craig JE - Mol. Vis. (2008)

Bottom Line: Truncating mutations were found in 6 out of 10 unrelated patients from four countries.No mutation was found in the gene in four patients.Two disease-causing mutations (R134fs and R901X) and an artificial mutation (T1357fs) resulted in premature truncation of the NHS-A protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Flinders University, Bedford Park, South Australia, Australia. shiwani.sharma@flinders.edu.au

ABSTRACT

Purpose: Nance-Horan syndrome is typically characterized by severe bilateral congenital cataracts and dental abnormalities. Truncating mutations in the Nance-Horan syndrome (NHS) gene cause this X-linked genetic disorder. NHS encodes two isoforms, NHS-A and NHS-1A. The ocular lens expresses NHS-A, the epithelial and neuronal cell specific isoform. The NHS-A protein localizes in the lens epithelium at the cellular periphery. The data to date suggest a role for this isoform at cell-cell junctions in epithelial cells. This study aimed to identify the causative mutations in new patients diagnosed with Nance-Horan syndrome and to investigate the effect of mutations on subcellular localization of the NHS-A protein.

Methods: All coding exons of NHS were screened for mutations by polymerase chain reaction (PCR) and sequencing. PCR-based mutagenesis was performed to introduce three independent mutations in the NHS-A cDNA. Expression and localization of the mutant proteins was determined in mammalian epithelial cells.

Results: Truncating mutations were found in 6 out of 10 unrelated patients from four countries. Each of four patients carried a novel mutation (R248X, P264fs, K1198fs, and I1302fs), and each of the two other patients carried two previously reported mutations (R373X and R879X). No mutation was found in the gene in four patients. Two disease-causing mutations (R134fs and R901X) and an artificial mutation (T1357fs) resulted in premature truncation of the NHS-A protein. All three mutant proteins failed to localize to the cellular periphery in epithelial cells and instead were found in the cytoplasm.

Conclusions: This study brings the total number of mutations identified in NHS to 18. The mislocalization of the mutant NHS-A protein, revealed by mutation analysis, is expected to adversely affect cell-cell junctions in epithelial cells such as the lens epithelium, which may explain cataractogenesis in Nance-Horan syndrome patients. Mutation analysis also shed light on the significance of NHS-A regions for its localization and, hence, its function at epithelial cell junctions.

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Localization of GFP-NHS-A400delC mutant in MDCK cells. Cells were transfected with GFP-NHS-A and GFP-NHS-A400delC fusion constructs and pEGFP-C1 control. Transiently expressed fusion proteins were visualized by confocal microscopy. GFP-NHS-A wild type protein primarily localized to the cellular periphery whereas GFP-NHS-A400delC mutant protein localized in the cytoplasm and nucleus. Apparent peripheral distribution of GFP is an experimental artifact seen only between some adjoining transfected cells. Images were taken with a 60X objective.
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f3: Localization of GFP-NHS-A400delC mutant in MDCK cells. Cells were transfected with GFP-NHS-A and GFP-NHS-A400delC fusion constructs and pEGFP-C1 control. Transiently expressed fusion proteins were visualized by confocal microscopy. GFP-NHS-A wild type protein primarily localized to the cellular periphery whereas GFP-NHS-A400delC mutant protein localized in the cytoplasm and nucleus. Apparent peripheral distribution of GFP is an experimental artifact seen only between some adjoining transfected cells. Images were taken with a 60X objective.

Mentions: To study the subcellular distribution of the mutant proteins, GFP-NHS-A400delC was transiently expressed and FLAG-tagged C2701T and 4071del299bp mutants stably expressed in MDCK epithelial cells. Localization of the mutant proteins was compared with that of the wild type NHS-A protein. Unlike its localization to the peripheral cell membrane in lens epithelium in vivo, the NHS-A protein does not associate with the cell membrane in lens epithelial cells ex vivo because the latter do not form a polarized epithelium in culture [12]. Therefore, localization studies were performed in MDCK cells. As previously reported, transiently expressed wild type GFP-NHS-A primarily localized at the cellular periphery in a punctate fashion in MDCK cells (Figure 3) [12]. Cytoplasmic localization of the protein was seen in some cells. GFP-NHS-A400delC mutant protein mainly distributed in the cytoplasm and nucleus but was not found to associate with the peripheral cell membrane (Figure 3). GFP, expressed as a control, distributed both in the cytoplasm and nucleus. Upon immunolabeling the FLAG-NHS-A stable transfectants with an anti-FLAG tag antibody, intense immunoreactivity was observed at the cell boundary in the majority of cells (Figure 4). In some cells, immunoreactivity was also observed in the cytoplasm. In stable transfectants of FLAG-tagged C2701T and 4071del299bp mutants, FLAG tag immunolabeling was confined to the cytoplasm (Figure 4). No immunoreactivity was observed at the cellular periphery in cells expressing these mutants.


Novel causative mutations in patients with Nance-Horan syndrome and altered localization of the mutant NHS-A protein isoform.

Sharma S, Burdon KP, Dave A, Jamieson RV, Yaron Y, Billson F, Van Maldergem L, Lorenz B, Gécz J, Craig JE - Mol. Vis. (2008)

Localization of GFP-NHS-A400delC mutant in MDCK cells. Cells were transfected with GFP-NHS-A and GFP-NHS-A400delC fusion constructs and pEGFP-C1 control. Transiently expressed fusion proteins were visualized by confocal microscopy. GFP-NHS-A wild type protein primarily localized to the cellular periphery whereas GFP-NHS-A400delC mutant protein localized in the cytoplasm and nucleus. Apparent peripheral distribution of GFP is an experimental artifact seen only between some adjoining transfected cells. Images were taken with a 60X objective.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2571945&req=5

f3: Localization of GFP-NHS-A400delC mutant in MDCK cells. Cells were transfected with GFP-NHS-A and GFP-NHS-A400delC fusion constructs and pEGFP-C1 control. Transiently expressed fusion proteins were visualized by confocal microscopy. GFP-NHS-A wild type protein primarily localized to the cellular periphery whereas GFP-NHS-A400delC mutant protein localized in the cytoplasm and nucleus. Apparent peripheral distribution of GFP is an experimental artifact seen only between some adjoining transfected cells. Images were taken with a 60X objective.
Mentions: To study the subcellular distribution of the mutant proteins, GFP-NHS-A400delC was transiently expressed and FLAG-tagged C2701T and 4071del299bp mutants stably expressed in MDCK epithelial cells. Localization of the mutant proteins was compared with that of the wild type NHS-A protein. Unlike its localization to the peripheral cell membrane in lens epithelium in vivo, the NHS-A protein does not associate with the cell membrane in lens epithelial cells ex vivo because the latter do not form a polarized epithelium in culture [12]. Therefore, localization studies were performed in MDCK cells. As previously reported, transiently expressed wild type GFP-NHS-A primarily localized at the cellular periphery in a punctate fashion in MDCK cells (Figure 3) [12]. Cytoplasmic localization of the protein was seen in some cells. GFP-NHS-A400delC mutant protein mainly distributed in the cytoplasm and nucleus but was not found to associate with the peripheral cell membrane (Figure 3). GFP, expressed as a control, distributed both in the cytoplasm and nucleus. Upon immunolabeling the FLAG-NHS-A stable transfectants with an anti-FLAG tag antibody, intense immunoreactivity was observed at the cell boundary in the majority of cells (Figure 4). In some cells, immunoreactivity was also observed in the cytoplasm. In stable transfectants of FLAG-tagged C2701T and 4071del299bp mutants, FLAG tag immunolabeling was confined to the cytoplasm (Figure 4). No immunoreactivity was observed at the cellular periphery in cells expressing these mutants.

Bottom Line: Truncating mutations were found in 6 out of 10 unrelated patients from four countries.No mutation was found in the gene in four patients.Two disease-causing mutations (R134fs and R901X) and an artificial mutation (T1357fs) resulted in premature truncation of the NHS-A protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Flinders University, Bedford Park, South Australia, Australia. shiwani.sharma@flinders.edu.au

ABSTRACT

Purpose: Nance-Horan syndrome is typically characterized by severe bilateral congenital cataracts and dental abnormalities. Truncating mutations in the Nance-Horan syndrome (NHS) gene cause this X-linked genetic disorder. NHS encodes two isoforms, NHS-A and NHS-1A. The ocular lens expresses NHS-A, the epithelial and neuronal cell specific isoform. The NHS-A protein localizes in the lens epithelium at the cellular periphery. The data to date suggest a role for this isoform at cell-cell junctions in epithelial cells. This study aimed to identify the causative mutations in new patients diagnosed with Nance-Horan syndrome and to investigate the effect of mutations on subcellular localization of the NHS-A protein.

Methods: All coding exons of NHS were screened for mutations by polymerase chain reaction (PCR) and sequencing. PCR-based mutagenesis was performed to introduce three independent mutations in the NHS-A cDNA. Expression and localization of the mutant proteins was determined in mammalian epithelial cells.

Results: Truncating mutations were found in 6 out of 10 unrelated patients from four countries. Each of four patients carried a novel mutation (R248X, P264fs, K1198fs, and I1302fs), and each of the two other patients carried two previously reported mutations (R373X and R879X). No mutation was found in the gene in four patients. Two disease-causing mutations (R134fs and R901X) and an artificial mutation (T1357fs) resulted in premature truncation of the NHS-A protein. All three mutant proteins failed to localize to the cellular periphery in epithelial cells and instead were found in the cytoplasm.

Conclusions: This study brings the total number of mutations identified in NHS to 18. The mislocalization of the mutant NHS-A protein, revealed by mutation analysis, is expected to adversely affect cell-cell junctions in epithelial cells such as the lens epithelium, which may explain cataractogenesis in Nance-Horan syndrome patients. Mutation analysis also shed light on the significance of NHS-A regions for its localization and, hence, its function at epithelial cell junctions.

Show MeSH
Related in: MedlinePlus