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Identification and molecular characterisation of a homozygous missense mutation in the ADAMTS10 gene in a patient with Weill-Marchesani syndrome.

Steinkellner H, Etzler J, Gogoll L, Neesen J, Stifter E, Brandau O, Laccone F - Eur. J. Hum. Genet. (2014)

Bottom Line: The ADAMTS10 missense mutation was analysed in silico, with conflicting results as to its effects on protein function, but it was predicted to affect the leader sequence.Molecular characterisation in HEK293 Ebna cells revealed an intracellular mis-targeting of the ADAMTS10 protein with a reduced concentration of the polypeptide in the endoplasmic reticulum.A large reduction in glycosylation of the cytoplasmic fraction of the mutant ADAMTS10 protein versus the wild-type protein and a lack of secretion of the mutant protein are also evident in our results.In conclusion, we identified a novel missense mutation of the ADAMTS10 gene and confirmed the functional consequences suggested by the in silico analysis by conducting molecular studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, Medical University of Vienna, Vienna, Austria.

ABSTRACT
Weill-Marchesani syndrome is a rare disorder of the connective tissue. Functional variants in ADAMTS10 are associated with Weill-Marchesani syndrome-1. We identified a homozygous missense mutation, c.41T>A, of the ADAMTS10 gene in a 19-year-old female with typical symptoms of WMS1: proportionate short stature, brachydactyly, joint stiffness, and microspherophakia. The ADAMTS10 missense mutation was analysed in silico, with conflicting results as to its effects on protein function, but it was predicted to affect the leader sequence. Molecular characterisation in HEK293 Ebna cells revealed an intracellular mis-targeting of the ADAMTS10 protein with a reduced concentration of the polypeptide in the endoplasmic reticulum. A large reduction in glycosylation of the cytoplasmic fraction of the mutant ADAMTS10 protein versus the wild-type protein and a lack of secretion of the mutant protein are also evident in our results.In conclusion, we identified a novel missense mutation of the ADAMTS10 gene and confirmed the functional consequences suggested by the in silico analysis by conducting molecular studies.

No MeSH data available.


Related in: MedlinePlus

N-linked deglycosylation assay of HEK 293E cells transfected with ADAMTS10-Myc-His6 or ADAMTS10_L14Q-Myc-His6. Whole cell extracts of HEK 293E cells transiently transfected with ADAMTS10-Myc-His6 (WT) and ADAMTS10_L14Q-Myc-His6 (MUT) or empty vector (Ctrl) were exposed to the PNGase F (+) enzyme. The protein extracts were size-fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and characterised by western blotting analysis using an anti-c-myc antibody. The position of specific bands is indicated by a molecular mass marker (55–170 kDa), and β-tubulin detection was used to confirm uniform loading of the total protein.Whereas deglycosylation of the ADAMTS10_L14Q-Myc-His6 protein with PNGaseF does not alter protein mobility, PNGase treatment of ADAMTS10-Myc-His6 results in a mobility shift with an electrophoretic mobility corresponding to the ADAMTS10_L14Q-Myc-His6 protein.
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fig4: N-linked deglycosylation assay of HEK 293E cells transfected with ADAMTS10-Myc-His6 or ADAMTS10_L14Q-Myc-His6. Whole cell extracts of HEK 293E cells transiently transfected with ADAMTS10-Myc-His6 (WT) and ADAMTS10_L14Q-Myc-His6 (MUT) or empty vector (Ctrl) were exposed to the PNGase F (+) enzyme. The protein extracts were size-fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and characterised by western blotting analysis using an anti-c-myc antibody. The position of specific bands is indicated by a molecular mass marker (55–170 kDa), and β-tubulin detection was used to confirm uniform loading of the total protein.Whereas deglycosylation of the ADAMTS10_L14Q-Myc-His6 protein with PNGaseF does not alter protein mobility, PNGase treatment of ADAMTS10-Myc-His6 results in a mobility shift with an electrophoretic mobility corresponding to the ADAMTS10_L14Q-Myc-His6 protein.

Mentions: The different kDa values for the expressed ADAMTS10 fusion proteins prompted us to investigate whether the mobility shift is due to posttranslational modification. Protein extracts of HEK 293E cells transfected with the ADAMTS10-Myc-His6 and ADAMTS10_L14Q-Myc-His6 plasmid DNA were treated with endoglycosidase PNGase F (Figure 4). Endoglycosidase PNGase F cleaves N-acetylglucosamine side chains from asparagine residues and thus removes all asparagine-associated glycans.18 Treatment with PNGase F markedly increased the electrophoretic mobility of wild-type ADAMTS10 but had no effect on mutant ADAMTS10_L14Q, indicating that the mobility shift observed in untreated protein extracts is caused by a diminished N-glycosylation of the mutated ADAMTS10 protein.


Identification and molecular characterisation of a homozygous missense mutation in the ADAMTS10 gene in a patient with Weill-Marchesani syndrome.

Steinkellner H, Etzler J, Gogoll L, Neesen J, Stifter E, Brandau O, Laccone F - Eur. J. Hum. Genet. (2014)

N-linked deglycosylation assay of HEK 293E cells transfected with ADAMTS10-Myc-His6 or ADAMTS10_L14Q-Myc-His6. Whole cell extracts of HEK 293E cells transiently transfected with ADAMTS10-Myc-His6 (WT) and ADAMTS10_L14Q-Myc-His6 (MUT) or empty vector (Ctrl) were exposed to the PNGase F (+) enzyme. The protein extracts were size-fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and characterised by western blotting analysis using an anti-c-myc antibody. The position of specific bands is indicated by a molecular mass marker (55–170 kDa), and β-tubulin detection was used to confirm uniform loading of the total protein.Whereas deglycosylation of the ADAMTS10_L14Q-Myc-His6 protein with PNGaseF does not alter protein mobility, PNGase treatment of ADAMTS10-Myc-His6 results in a mobility shift with an electrophoretic mobility corresponding to the ADAMTS10_L14Q-Myc-His6 protein.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4538198&req=5

fig4: N-linked deglycosylation assay of HEK 293E cells transfected with ADAMTS10-Myc-His6 or ADAMTS10_L14Q-Myc-His6. Whole cell extracts of HEK 293E cells transiently transfected with ADAMTS10-Myc-His6 (WT) and ADAMTS10_L14Q-Myc-His6 (MUT) or empty vector (Ctrl) were exposed to the PNGase F (+) enzyme. The protein extracts were size-fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and characterised by western blotting analysis using an anti-c-myc antibody. The position of specific bands is indicated by a molecular mass marker (55–170 kDa), and β-tubulin detection was used to confirm uniform loading of the total protein.Whereas deglycosylation of the ADAMTS10_L14Q-Myc-His6 protein with PNGaseF does not alter protein mobility, PNGase treatment of ADAMTS10-Myc-His6 results in a mobility shift with an electrophoretic mobility corresponding to the ADAMTS10_L14Q-Myc-His6 protein.
Mentions: The different kDa values for the expressed ADAMTS10 fusion proteins prompted us to investigate whether the mobility shift is due to posttranslational modification. Protein extracts of HEK 293E cells transfected with the ADAMTS10-Myc-His6 and ADAMTS10_L14Q-Myc-His6 plasmid DNA were treated with endoglycosidase PNGase F (Figure 4). Endoglycosidase PNGase F cleaves N-acetylglucosamine side chains from asparagine residues and thus removes all asparagine-associated glycans.18 Treatment with PNGase F markedly increased the electrophoretic mobility of wild-type ADAMTS10 but had no effect on mutant ADAMTS10_L14Q, indicating that the mobility shift observed in untreated protein extracts is caused by a diminished N-glycosylation of the mutated ADAMTS10 protein.

Bottom Line: The ADAMTS10 missense mutation was analysed in silico, with conflicting results as to its effects on protein function, but it was predicted to affect the leader sequence.Molecular characterisation in HEK293 Ebna cells revealed an intracellular mis-targeting of the ADAMTS10 protein with a reduced concentration of the polypeptide in the endoplasmic reticulum.A large reduction in glycosylation of the cytoplasmic fraction of the mutant ADAMTS10 protein versus the wild-type protein and a lack of secretion of the mutant protein are also evident in our results.In conclusion, we identified a novel missense mutation of the ADAMTS10 gene and confirmed the functional consequences suggested by the in silico analysis by conducting molecular studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, Medical University of Vienna, Vienna, Austria.

ABSTRACT
Weill-Marchesani syndrome is a rare disorder of the connective tissue. Functional variants in ADAMTS10 are associated with Weill-Marchesani syndrome-1. We identified a homozygous missense mutation, c.41T>A, of the ADAMTS10 gene in a 19-year-old female with typical symptoms of WMS1: proportionate short stature, brachydactyly, joint stiffness, and microspherophakia. The ADAMTS10 missense mutation was analysed in silico, with conflicting results as to its effects on protein function, but it was predicted to affect the leader sequence. Molecular characterisation in HEK293 Ebna cells revealed an intracellular mis-targeting of the ADAMTS10 protein with a reduced concentration of the polypeptide in the endoplasmic reticulum. A large reduction in glycosylation of the cytoplasmic fraction of the mutant ADAMTS10 protein versus the wild-type protein and a lack of secretion of the mutant protein are also evident in our results.In conclusion, we identified a novel missense mutation of the ADAMTS10 gene and confirmed the functional consequences suggested by the in silico analysis by conducting molecular studies.

No MeSH data available.


Related in: MedlinePlus