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Clinical and biochemical effects of the E139K missense mutation in the TIMP3 gene, associated with Sorsby fundus dystrophy.

Saihan Z, Li Z, Rice J, Rana NA, Ramsden S, Schlottmann PG, Jenkins SA, Blyth C, Black GC, McKie N, Webster AR - Mol. Vis. (2009)

Bottom Line: The TIMP3 p.E139K mutation is another cause of SFD.It is the second TIMP3 sequence variant reported that does not affect the number of cysteine residues in the mutant protein yet dimerizes in vitro.The clinical presentation of this family is in keeping with previous clinical reports of this disorder.

View Article: PubMed Central - PubMed

Affiliation: Moorfields Eye Hospital, London, UK. z.saihan@ucl.ac.uk

ABSTRACT

Purpose: To determine the phenotypic and biochemical characteristics of the p.E139K missense variant in tissue inhibitor of metalloproteinase 3 (TIMP3) associated with Sorsby fundus dystrophy (SFD).

Methods: The coding regions and adjacent intronic sequence of TIMP3 were amplified by polymerase chain reaction and then analyzed by bidirectional sequencing. Allele-specific PCR was used to determine the minimum allele frequency of the mutant allele in ethnically matched controls. Clinical examination and imaging of affected individuals with color fundus photography, scanning laser ophthalmoscope (fundal autofluorescence), and optical coherence tomography was performed. A mutant construct of the TIMP3 protein was created and expressed in human retinal pigment epithelium (ARPE19) cells, which were then assayed for oligomerization and intrinsic matrix metalloproteinase (MMP) inhibitory activity.

Results: Three affected individuals from a family of Welsh origin each harbored one allele of the TIMP3 missense variant c.415 G>A, (p.E139K), which was not identified in 534 ethnically matched control chromosomes and thus presumed pathogenic. The mutant protein was shown to dimerize in culture cells and retain its MMP inhibitory activity. Retinal examination was variable between eyes of affected individuals and between family members. Drusen-like deposits were common to all three affected individuals and yellow subretinal deposits, exudative maculopathy, and geographic atrophy were also observed. Optical coherence tomography (OCT) images of affected individuals demonstrated hyperreflectivity of the RPE-photoreceptor-choroid complex.

Conclusions: The TIMP3 p.E139K mutation is another cause of SFD. It is the second TIMP3 sequence variant reported that does not affect the number of cysteine residues in the mutant protein yet dimerizes in vitro. The clinical presentation of this family is in keeping with previous clinical reports of this disorder.

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Related in: MedlinePlus

Western Blot of E139K TIMP-3 mutant. TIMP3 immunoblot (western blot) of the extracellular matrix sample from ARPE-19 cells transfected with E139K TIMP-3 expression plasmid, in nonreduced form demonstrating dimerization.
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f8: Western Blot of E139K TIMP-3 mutant. TIMP3 immunoblot (western blot) of the extracellular matrix sample from ARPE-19 cells transfected with E139K TIMP-3 expression plasmid, in nonreduced form demonstrating dimerization.

Mentions: Western blotting of the ECM extracts revealed a monomeric TIMP3 form in addition to a dimeric form at circa 48 kDa (Figure 8). The inhibitory activity of p.E139K TIMP3 mutant protein was tested by reverse zymography, using recombinant MMP-2 as an in-gel MMP activity. The dimerized TIMP3 p.E139K mutant could be seen as a high band with the size of 48 kDa in the gel with the presence of p.E139K TIMP3 monomer in the size of 24 kDa. Results of reverse zymography demonstrated that p.E139K TIMP3 mutant retained its MMP inhibitory activity and also could form a dimer within the ECM (Figure 9).


Clinical and biochemical effects of the E139K missense mutation in the TIMP3 gene, associated with Sorsby fundus dystrophy.

Saihan Z, Li Z, Rice J, Rana NA, Ramsden S, Schlottmann PG, Jenkins SA, Blyth C, Black GC, McKie N, Webster AR - Mol. Vis. (2009)

Western Blot of E139K TIMP-3 mutant. TIMP3 immunoblot (western blot) of the extracellular matrix sample from ARPE-19 cells transfected with E139K TIMP-3 expression plasmid, in nonreduced form demonstrating dimerization.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2697491&req=5

f8: Western Blot of E139K TIMP-3 mutant. TIMP3 immunoblot (western blot) of the extracellular matrix sample from ARPE-19 cells transfected with E139K TIMP-3 expression plasmid, in nonreduced form demonstrating dimerization.
Mentions: Western blotting of the ECM extracts revealed a monomeric TIMP3 form in addition to a dimeric form at circa 48 kDa (Figure 8). The inhibitory activity of p.E139K TIMP3 mutant protein was tested by reverse zymography, using recombinant MMP-2 as an in-gel MMP activity. The dimerized TIMP3 p.E139K mutant could be seen as a high band with the size of 48 kDa in the gel with the presence of p.E139K TIMP3 monomer in the size of 24 kDa. Results of reverse zymography demonstrated that p.E139K TIMP3 mutant retained its MMP inhibitory activity and also could form a dimer within the ECM (Figure 9).

Bottom Line: The TIMP3 p.E139K mutation is another cause of SFD.It is the second TIMP3 sequence variant reported that does not affect the number of cysteine residues in the mutant protein yet dimerizes in vitro.The clinical presentation of this family is in keeping with previous clinical reports of this disorder.

View Article: PubMed Central - PubMed

Affiliation: Moorfields Eye Hospital, London, UK. z.saihan@ucl.ac.uk

ABSTRACT

Purpose: To determine the phenotypic and biochemical characteristics of the p.E139K missense variant in tissue inhibitor of metalloproteinase 3 (TIMP3) associated with Sorsby fundus dystrophy (SFD).

Methods: The coding regions and adjacent intronic sequence of TIMP3 were amplified by polymerase chain reaction and then analyzed by bidirectional sequencing. Allele-specific PCR was used to determine the minimum allele frequency of the mutant allele in ethnically matched controls. Clinical examination and imaging of affected individuals with color fundus photography, scanning laser ophthalmoscope (fundal autofluorescence), and optical coherence tomography was performed. A mutant construct of the TIMP3 protein was created and expressed in human retinal pigment epithelium (ARPE19) cells, which were then assayed for oligomerization and intrinsic matrix metalloproteinase (MMP) inhibitory activity.

Results: Three affected individuals from a family of Welsh origin each harbored one allele of the TIMP3 missense variant c.415 G>A, (p.E139K), which was not identified in 534 ethnically matched control chromosomes and thus presumed pathogenic. The mutant protein was shown to dimerize in culture cells and retain its MMP inhibitory activity. Retinal examination was variable between eyes of affected individuals and between family members. Drusen-like deposits were common to all three affected individuals and yellow subretinal deposits, exudative maculopathy, and geographic atrophy were also observed. Optical coherence tomography (OCT) images of affected individuals demonstrated hyperreflectivity of the RPE-photoreceptor-choroid complex.

Conclusions: The TIMP3 p.E139K mutation is another cause of SFD. It is the second TIMP3 sequence variant reported that does not affect the number of cysteine residues in the mutant protein yet dimerizes in vitro. The clinical presentation of this family is in keeping with previous clinical reports of this disorder.

Show MeSH
Related in: MedlinePlus