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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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Expression of mutant NHS-A proteins in mammalian cells. A: Lysates of HEK 293A cells transiently transfected with GFP-NHS-A400delC and pEGFP-C1 constructs and untransfected cells were analyzed by western blotting with anti-GFP antibody. B: Lysates of HEK 293A cells transiently transfected with FLAG-NHS-A, FLAG-NHS-AC2701T and FLAG-NHS-A4071del299bp in pCMV-Tag 2A and untransfected cells were analyzed by western blotting with anti-FLAG tag antibody. A protein band of greater than 150 kDa seen in all the lanes is due to non-specific binding of the anti-FLAG tag antibody (indicated with an asterisk). A very faint protein band of approximately 130 kDa in the NHS-A and 4071Δ299bp lanes is most likely due to protein degradation. The molecular masses of proteins standards are indicated. UT=untransfected cells.
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f2: Expression of mutant NHS-A proteins in mammalian cells. A: Lysates of HEK 293A cells transiently transfected with GFP-NHS-A400delC and pEGFP-C1 constructs and untransfected cells were analyzed by western blotting with anti-GFP antibody. B: Lysates of HEK 293A cells transiently transfected with FLAG-NHS-A, FLAG-NHS-AC2701T and FLAG-NHS-A4071del299bp in pCMV-Tag 2A and untransfected cells were analyzed by western blotting with anti-FLAG tag antibody. A protein band of greater than 150 kDa seen in all the lanes is due to non-specific binding of the anti-FLAG tag antibody (indicated with an asterisk). A very faint protein band of approximately 130 kDa in the NHS-A and 4071Δ299bp lanes is most likely due to protein degradation. The molecular masses of proteins standards are indicated. UT=untransfected cells.

Mentions: To understand the effect of disease-causing mutations on the NHS-A protein, two previously reported mutations, 400delC (R134fs) in exon 1 and C2635T (R879X) in exon 6 (named C2701T [R901X] in this study because the mutant cDNA included the alternatively spliced exon 3a) [3,8,10], and an artificial mutation, 4071del299bp (T1357fs), also in exon 6 were created in the wild type cDNA by PCR-based mutagenesis. The 400delC mutation was reported in a family with typical features of NHS and is predicted to prematurely truncate only the NHS-A protein isoform. The family with the C2635T mutation displayed most features of the syndrome, and the mutation would abolish the COOH-terminal half of the NHS-A protein. The 4071del299bp mutant was obtained as a cloning artifact while attempting to create the S1484X artificial mutation in exon 8. While cloning the PCR-amplified fragment carrying this mutation to replace the corresponding wild type fragment, a 299 bp deletion was incorporated due to star activity of ScaI, one of the restriction enzymes used for cloning. The resulting 4071del299bp mutation is expected to give rise to a truncated NHS-A protein very similar to that predicted to result from 3908del11bp mutation identified here in patient 122 and was therefore included in the study. The 400delC mutant was fused to GFP at the NH2-terminus, and the C2701T and 4071del299bp mutants were fused with a FLAG epitope tag. To confirm that a mutant cDNA expressed the truncated protein, it was transiently expressed in HEK 293A cells and protein expression determined by western blotting. A truncated protein of approximately 65 kDa was detected with an anti-GFP antibody in cells expressing the 400delC mutant (Figure 2A). Similarly, around 140 and 225 kDa truncated proteins were revealed with an anti-FLAG tag antibody in cells respectively expressing the C2701T and 4071del299bp mutants (Figure 2B). These protein sizes are greater than those expected for each of the mutant proteins (Table 3). The wild type NHS-A has a predicted size of 181 kDa but migrates slower than its expected size probably due to post-translational modification (Figure 2B) [12]. The greater than expected size of each mutant protein observed here is therefore not unusual and is also likely to result from post-translational modification.


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)

Expression of mutant NHS-A proteins in mammalian cells. A: Lysates of HEK 293A cells transiently transfected with GFP-NHS-A400delC and pEGFP-C1 constructs and untransfected cells were analyzed by western blotting with anti-GFP antibody. B: Lysates of HEK 293A cells transiently transfected with FLAG-NHS-A, FLAG-NHS-AC2701T and FLAG-NHS-A4071del299bp in pCMV-Tag 2A and untransfected cells were analyzed by western blotting with anti-FLAG tag antibody. A protein band of greater than 150 kDa seen in all the lanes is due to non-specific binding of the anti-FLAG tag antibody (indicated with an asterisk). A very faint protein band of approximately 130 kDa in the NHS-A and 4071Δ299bp lanes is most likely due to protein degradation. The molecular masses of proteins standards are indicated. UT=untransfected cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f2: Expression of mutant NHS-A proteins in mammalian cells. A: Lysates of HEK 293A cells transiently transfected with GFP-NHS-A400delC and pEGFP-C1 constructs and untransfected cells were analyzed by western blotting with anti-GFP antibody. B: Lysates of HEK 293A cells transiently transfected with FLAG-NHS-A, FLAG-NHS-AC2701T and FLAG-NHS-A4071del299bp in pCMV-Tag 2A and untransfected cells were analyzed by western blotting with anti-FLAG tag antibody. A protein band of greater than 150 kDa seen in all the lanes is due to non-specific binding of the anti-FLAG tag antibody (indicated with an asterisk). A very faint protein band of approximately 130 kDa in the NHS-A and 4071Δ299bp lanes is most likely due to protein degradation. The molecular masses of proteins standards are indicated. UT=untransfected cells.
Mentions: To understand the effect of disease-causing mutations on the NHS-A protein, two previously reported mutations, 400delC (R134fs) in exon 1 and C2635T (R879X) in exon 6 (named C2701T [R901X] in this study because the mutant cDNA included the alternatively spliced exon 3a) [3,8,10], and an artificial mutation, 4071del299bp (T1357fs), also in exon 6 were created in the wild type cDNA by PCR-based mutagenesis. The 400delC mutation was reported in a family with typical features of NHS and is predicted to prematurely truncate only the NHS-A protein isoform. The family with the C2635T mutation displayed most features of the syndrome, and the mutation would abolish the COOH-terminal half of the NHS-A protein. The 4071del299bp mutant was obtained as a cloning artifact while attempting to create the S1484X artificial mutation in exon 8. While cloning the PCR-amplified fragment carrying this mutation to replace the corresponding wild type fragment, a 299 bp deletion was incorporated due to star activity of ScaI, one of the restriction enzymes used for cloning. The resulting 4071del299bp mutation is expected to give rise to a truncated NHS-A protein very similar to that predicted to result from 3908del11bp mutation identified here in patient 122 and was therefore included in the study. The 400delC mutant was fused to GFP at the NH2-terminus, and the C2701T and 4071del299bp mutants were fused with a FLAG epitope tag. To confirm that a mutant cDNA expressed the truncated protein, it was transiently expressed in HEK 293A cells and protein expression determined by western blotting. A truncated protein of approximately 65 kDa was detected with an anti-GFP antibody in cells expressing the 400delC mutant (Figure 2A). Similarly, around 140 and 225 kDa truncated proteins were revealed with an anti-FLAG tag antibody in cells respectively expressing the C2701T and 4071del299bp mutants (Figure 2B). These protein sizes are greater than those expected for each of the mutant proteins (Table 3). The wild type NHS-A has a predicted size of 181 kDa but migrates slower than its expected size probably due to post-translational modification (Figure 2B) [12]. The greater than expected size of each mutant protein observed here is therefore not unusual and is also likely to result from post-translational modification.

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