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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

SignalP V4.1 graphic output for wild-type and mutant ADAMTS10 signal peptides. The c-score (raw cleavage site score) predicts the first amino-acid residue of the mature protein. The s-score (signal peptide score) calculates the probability of amino-acid residues to be part of a functioning signal peptide. The y-score combines the c-score and s-score, resulting in better cleavage site prediction. The d-score (discrimination score) is a weighted average of the mean s-score and the maximum y-score. (a) Analysis of the wild-type protein predicts a cleavage site between amino-acid positions 25 and 26 and yields a d-score value of 0.759, well above the default cutoff for signal peptides. (b) The presence of a polar glutamine in exchange for leucine is predicted to disrupt the hydrophobic core of the ADAMTS10 signal peptide, thus reducing the probability that the amino-acid sequence acts as a signal peptide. The d-score value drops below the cutoff of 0.450.
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fig1: SignalP V4.1 graphic output for wild-type and mutant ADAMTS10 signal peptides. The c-score (raw cleavage site score) predicts the first amino-acid residue of the mature protein. The s-score (signal peptide score) calculates the probability of amino-acid residues to be part of a functioning signal peptide. The y-score combines the c-score and s-score, resulting in better cleavage site prediction. The d-score (discrimination score) is a weighted average of the mean s-score and the maximum y-score. (a) Analysis of the wild-type protein predicts a cleavage site between amino-acid positions 25 and 26 and yields a d-score value of 0.759, well above the default cutoff for signal peptides. (b) The presence of a polar glutamine in exchange for leucine is predicted to disrupt the hydrophobic core of the ADAMTS10 signal peptide, thus reducing the probability that the amino-acid sequence acts as a signal peptide. The d-score value drops below the cutoff of 0.450.

Mentions: ADAMTS10 enters the secretory pathway during posttranslational modification.17 The base pair exchange described here affects the leucine at position 14, which is in the hydrophobic region of the leader peptide, resulting in an exchange to glutamine (p.(Leu14Gln)). Insight into the possible effect of an amino-acid exchange within a signal sequence can be gained with the SignalP signal peptide prediction program (http://www.cbs.dtu.dk/services/SignalP/).15 To assess whether the amino-acid exchange might result in protein mis-localisation because of a disrupted signal peptide, the N-termini of the wild-type and mutant ADAMTS10 proteins were submitted to the SignalP V4.1 signal peptide prediction program. Analysis of the 60 N-terminal amino acids of the ADAMTS10 wild-type protein predicted a cleavage site between amino-acid positions 25 and 26, with a d-score value of 0.759 for amino acids 1–25, considerably exceeding the default d-score cutoff value of 0.450 (Figure 1a). In silico analysis of the c.41T>A mutant, however, yielded a d-score value of only 0.446 for the first 25 amino acids, which are thereby no longer predicted to function as a signal peptide (Figure 1b).


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)

SignalP V4.1 graphic output for wild-type and mutant ADAMTS10 signal peptides. The c-score (raw cleavage site score) predicts the first amino-acid residue of the mature protein. The s-score (signal peptide score) calculates the probability of amino-acid residues to be part of a functioning signal peptide. The y-score combines the c-score and s-score, resulting in better cleavage site prediction. The d-score (discrimination score) is a weighted average of the mean s-score and the maximum y-score. (a) Analysis of the wild-type protein predicts a cleavage site between amino-acid positions 25 and 26 and yields a d-score value of 0.759, well above the default cutoff for signal peptides. (b) The presence of a polar glutamine in exchange for leucine is predicted to disrupt the hydrophobic core of the ADAMTS10 signal peptide, thus reducing the probability that the amino-acid sequence acts as a signal peptide. The d-score value drops below the cutoff of 0.450.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4538198&req=5

fig1: SignalP V4.1 graphic output for wild-type and mutant ADAMTS10 signal peptides. The c-score (raw cleavage site score) predicts the first amino-acid residue of the mature protein. The s-score (signal peptide score) calculates the probability of amino-acid residues to be part of a functioning signal peptide. The y-score combines the c-score and s-score, resulting in better cleavage site prediction. The d-score (discrimination score) is a weighted average of the mean s-score and the maximum y-score. (a) Analysis of the wild-type protein predicts a cleavage site between amino-acid positions 25 and 26 and yields a d-score value of 0.759, well above the default cutoff for signal peptides. (b) The presence of a polar glutamine in exchange for leucine is predicted to disrupt the hydrophobic core of the ADAMTS10 signal peptide, thus reducing the probability that the amino-acid sequence acts as a signal peptide. The d-score value drops below the cutoff of 0.450.
Mentions: ADAMTS10 enters the secretory pathway during posttranslational modification.17 The base pair exchange described here affects the leucine at position 14, which is in the hydrophobic region of the leader peptide, resulting in an exchange to glutamine (p.(Leu14Gln)). Insight into the possible effect of an amino-acid exchange within a signal sequence can be gained with the SignalP signal peptide prediction program (http://www.cbs.dtu.dk/services/SignalP/).15 To assess whether the amino-acid exchange might result in protein mis-localisation because of a disrupted signal peptide, the N-termini of the wild-type and mutant ADAMTS10 proteins were submitted to the SignalP V4.1 signal peptide prediction program. Analysis of the 60 N-terminal amino acids of the ADAMTS10 wild-type protein predicted a cleavage site between amino-acid positions 25 and 26, with a d-score value of 0.759 for amino acids 1–25, considerably exceeding the default d-score cutoff value of 0.450 (Figure 1a). In silico analysis of the c.41T>A mutant, however, yielded a d-score value of only 0.446 for the first 25 amino acids, which are thereby no longer predicted to function as a signal peptide (Figure 1b).

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