Limits...
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

The expression pattern of ADAMTS10 and ADAMTS10-L14Q in HEK 293E cells. HEK 293 E cells were transfected with ADAMTS10-Myc-His6 and ADAMTS10_L14Q-Myc-His6 constructs. The polypeptides from supernatants (a) and cell lysates (b) were immunoblotted, and ADAMTS10 and ADAMTS10_L14Q protein detection in the cell lysate and the supernatant was performed using a polyclonal anti-His6 antibody. The ADAMTS10_L14Q protein was detected in the cell lysate as well as in the supernatant, whereas the ADAMTS10_L14Q polypeptide was only observed in the cell lysate, showing a mobility shift.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4538198&req=5

fig3: The expression pattern of ADAMTS10 and ADAMTS10-L14Q in HEK 293E cells. HEK 293 E cells were transfected with ADAMTS10-Myc-His6 and ADAMTS10_L14Q-Myc-His6 constructs. The polypeptides from supernatants (a) and cell lysates (b) were immunoblotted, and ADAMTS10 and ADAMTS10_L14Q protein detection in the cell lysate and the supernatant was performed using a polyclonal anti-His6 antibody. The ADAMTS10_L14Q protein was detected in the cell lysate as well as in the supernatant, whereas the ADAMTS10_L14Q polypeptide was only observed in the cell lysate, showing a mobility shift.

Mentions: To investigate whether the ADAMTS10 c.41T>A exchange leads to intracellular mis-localisation of the protein, plasmids containing eGFP fused to wild-type and ADAMTS10 c.41T>A cDNAs were transiently transfected in HEK 293E cells and examined by immunocytochemistry and fluorescence microscopy. In cells transfected with ADAMTS10-d2eGFP, a considerable amount of the protein was present in the ER, as shown by co-visualisation with the ER marker ERp72 (Figure 2c). In contrast, ADAMTS10_L14Q-d2eGFP is localised to the cytosol in a uniform diffuse pattern (Figure 2f). This finding confirms the in silico prediction and suggests that the leucine to glutamine exchange in the leader peptide leads to mis-targeting of the mutant protein to the cytoplasm. Next, we investigated the size of ADAMTS10 polypeptides by immunoblotting the media and cell lysates from HEK 293E cells transiently transfected with ADAMTS10-Myc-His6 and ADAMTS10_L14Q-Myc-His6 constructs (Figure 3). In the cell lysates and supernatants of ADAMTS10-Myc-His6-transfected cells, we observed an anti-His6 reactive band at the expected size (approximately 140 kDa). In cell lysates from ADAMTS10_LQ14-Myc-His6-transfected cells, a band at approximately 130 kDa was observed, but no reactive band was present in the serum-free medium.


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)

The expression pattern of ADAMTS10 and ADAMTS10-L14Q in HEK 293E cells. HEK 293 E cells were transfected with ADAMTS10-Myc-His6 and ADAMTS10_L14Q-Myc-His6 constructs. The polypeptides from supernatants (a) and cell lysates (b) were immunoblotted, and ADAMTS10 and ADAMTS10_L14Q protein detection in the cell lysate and the supernatant was performed using a polyclonal anti-His6 antibody. The ADAMTS10_L14Q protein was detected in the cell lysate as well as in the supernatant, whereas the ADAMTS10_L14Q polypeptide was only observed in the cell lysate, showing a mobility shift.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: The expression pattern of ADAMTS10 and ADAMTS10-L14Q in HEK 293E cells. HEK 293 E cells were transfected with ADAMTS10-Myc-His6 and ADAMTS10_L14Q-Myc-His6 constructs. The polypeptides from supernatants (a) and cell lysates (b) were immunoblotted, and ADAMTS10 and ADAMTS10_L14Q protein detection in the cell lysate and the supernatant was performed using a polyclonal anti-His6 antibody. The ADAMTS10_L14Q protein was detected in the cell lysate as well as in the supernatant, whereas the ADAMTS10_L14Q polypeptide was only observed in the cell lysate, showing a mobility shift.
Mentions: To investigate whether the ADAMTS10 c.41T>A exchange leads to intracellular mis-localisation of the protein, plasmids containing eGFP fused to wild-type and ADAMTS10 c.41T>A cDNAs were transiently transfected in HEK 293E cells and examined by immunocytochemistry and fluorescence microscopy. In cells transfected with ADAMTS10-d2eGFP, a considerable amount of the protein was present in the ER, as shown by co-visualisation with the ER marker ERp72 (Figure 2c). In contrast, ADAMTS10_L14Q-d2eGFP is localised to the cytosol in a uniform diffuse pattern (Figure 2f). This finding confirms the in silico prediction and suggests that the leucine to glutamine exchange in the leader peptide leads to mis-targeting of the mutant protein to the cytoplasm. Next, we investigated the size of ADAMTS10 polypeptides by immunoblotting the media and cell lysates from HEK 293E cells transiently transfected with ADAMTS10-Myc-His6 and ADAMTS10_L14Q-Myc-His6 constructs (Figure 3). In the cell lysates and supernatants of ADAMTS10-Myc-His6-transfected cells, we observed an anti-His6 reactive band at the expected size (approximately 140 kDa). In cell lysates from ADAMTS10_LQ14-Myc-His6-transfected cells, a band at approximately 130 kDa was observed, but no reactive band was present in the serum-free medium.

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