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Involvement of the same TNFR1 residue in mendelian and multifactorial inflammatory disorders.

Jéru I, Charmion S, Cochet E, Copin B, Duquesnoy P, Garcia MT, Le Borgne G, Cathebras P, Gaillat J, Karabina S, Dodé C, Lohse P, Hentgen V, Amselem S - PLoS ONE (2013)

Bottom Line: Most TNFRSF1A mutations are missense changes and, apart from those affecting conserved cysteines, their deleterious effect remains often questionable.Similar results were obtained with R92P, another mutation previously identified in a very small familial form with incomplete penetrance and variable expressivity.Combined with previous reports on arginine 92 mutations, this study discloses an unusual situation in which different amino acid substitutions at the same position in the protein are associated with a clinical spectrum bridging Mendelian to multifactorial conditions.

View Article: PubMed Central - PubMed

Affiliation: UMR_S933, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France. isabelle.jeru@trs.aphp.fr

ABSTRACT

Objectives: TNFRSF1A is involved in an autosomal dominant autoinflammatory disorder called TNFR-associated periodic syndrome (TRAPS). Most TNFRSF1A mutations are missense changes and, apart from those affecting conserved cysteines, their deleterious effect remains often questionable. This is especially true for the frequent R92Q mutation, which might not be responsible for TRAPS per se but represents a susceptibility factor to multifactorial inflammatory disorders. This study investigates TRAPS pathophysiology in a family exceptional by its size (13 members) and compares the consequences of several mutations affecting arginine 92.

Methods: TNFRSF1A screening was performed by PCR-sequencing. Comparison of the 3-dimensional structure and electrostatic properties of wild-type and mutated TNFR1 proteins was performed by in silico homology modeling. TNFR1 expression was assessed by FACS analysis, western blotting and ELISA in lysates and supernatants of HEK293T cells transiently expressing wild-type and mutated TNFR1.

Results: A TNFRSF1A heterozygous missense mutation, R92W (c.361C>T), was shown to perfectly segregate with typical TRAPS manifestations within the family investigated (p<5.10(-4)). It was associated with very high disease penetrance (0.9). Prediction of its impact on the protein structure revealed local conformational changes and alterations of the receptor electrostatic properties. R92W also impairs the TNFR1 expression at the cell surface and the levels of soluble receptor. Similar results were obtained with R92P, another mutation previously identified in a very small familial form with incomplete penetrance and variable expressivity. In contrast, TNFR1-R92Q behaves like the wild-type receptor.

Conclusions: These data demonstrate the pathogenicity of a mutation affecting arginine 92, a residue whose involvement in inflammatory disorders is deeply debated. Combined with previous reports on arginine 92 mutations, this study discloses an unusual situation in which different amino acid substitutions at the same position in the protein are associated with a clinical spectrum bridging Mendelian to multifactorial conditions.

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Trafficking of WT and mutated forms of TNFR1 at the cell surface.FACS analysis was performed in HEK293T cells transiently expressing the WT and mutated forms of the receptor. Cells were incubated either with a PE-conjugated monoclonal anti-TNFR1 antibody or with a goat polyclonal anti-TNFR1 antibody followed by an Alexa fluor-conjugated anti-goat antibody. A, Percentage of cells expressing TNFR1 at their surface. B, Percentage of cells expressing TNFR1 after permeabilization.
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pone-0069757-g004: Trafficking of WT and mutated forms of TNFR1 at the cell surface.FACS analysis was performed in HEK293T cells transiently expressing the WT and mutated forms of the receptor. Cells were incubated either with a PE-conjugated monoclonal anti-TNFR1 antibody or with a goat polyclonal anti-TNFR1 antibody followed by an Alexa fluor-conjugated anti-goat antibody. A, Percentage of cells expressing TNFR1 at their surface. B, Percentage of cells expressing TNFR1 after permeabilization.

Mentions: We then assessed the effect of mutations affecting arginine 92 on TNFR1 expression. To this purpose, we transfected plasmids encoding WT and mutated forms of the receptor into HEK293T cells. As controls, we generated expression vectors for several TNFRSF1A unambiguous mutations (T50M, C70S, C73R) as well as for P46L, which is considered as a polymorphism or as a mutation with very mild pro-inflammatory effects [6]. As shown by western blotting in cell lysates (Figure 3A), TNFR1 expression was similar for TNFR1-WT and all mutated forms of the receptor. Nevertheless, western blot and ELISA assays performed on cell supernatants revealed that the soluble form of the receptor (sTNFR1) was detectable only for cells expressing TNFR1-WT, TNFR1-P46L and TNFR1-R92Q (Figure 3A and 3B). These data demonstrate that R92W and R92P result in a dramatic diminution of the pool of sTNFR1, like unambiguous mutations. In contrast, R92Q behaves like the WT protein. We also evaluated the membrane expression of TNFR1 by FACS analysis. We first used a PE-conjugated monoclonal anti-TNFR1 antibody (R&D Systems) used previously by other teams [7], [22]–[23]. Consistent with a previous observation [22], the receptor carrying the R92P mutation was not recognized by this antibody (Figure 4), so that we could not conclude on its subcellular localization. The subsequent use of a polyclonal antibody (R&D Systems) showed that TNFR1-R92P is indeed expressed in the cytoplasm, whereas very low levels are detectable at the cell surface. As shown in Figure 4A, the percentage of cells expressing the receptor at their surface is higher in the presence of TNFR1-WT, TNFR1-P46L and TNFR1-R92Q than in the presence of TNFR1-T50M, TNFR1-C70S, TNFR1-C73R, TNFR1-R92P and TNFR1-R92W. The same experiment performed after cell permeabilization did not reveal a different intracellular expression of the different forms of the receptor (Figure 4B).


Involvement of the same TNFR1 residue in mendelian and multifactorial inflammatory disorders.

Jéru I, Charmion S, Cochet E, Copin B, Duquesnoy P, Garcia MT, Le Borgne G, Cathebras P, Gaillat J, Karabina S, Dodé C, Lohse P, Hentgen V, Amselem S - PLoS ONE (2013)

Trafficking of WT and mutated forms of TNFR1 at the cell surface.FACS analysis was performed in HEK293T cells transiently expressing the WT and mutated forms of the receptor. Cells were incubated either with a PE-conjugated monoclonal anti-TNFR1 antibody or with a goat polyclonal anti-TNFR1 antibody followed by an Alexa fluor-conjugated anti-goat antibody. A, Percentage of cells expressing TNFR1 at their surface. B, Percentage of cells expressing TNFR1 after permeabilization.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3722142&req=5

pone-0069757-g004: Trafficking of WT and mutated forms of TNFR1 at the cell surface.FACS analysis was performed in HEK293T cells transiently expressing the WT and mutated forms of the receptor. Cells were incubated either with a PE-conjugated monoclonal anti-TNFR1 antibody or with a goat polyclonal anti-TNFR1 antibody followed by an Alexa fluor-conjugated anti-goat antibody. A, Percentage of cells expressing TNFR1 at their surface. B, Percentage of cells expressing TNFR1 after permeabilization.
Mentions: We then assessed the effect of mutations affecting arginine 92 on TNFR1 expression. To this purpose, we transfected plasmids encoding WT and mutated forms of the receptor into HEK293T cells. As controls, we generated expression vectors for several TNFRSF1A unambiguous mutations (T50M, C70S, C73R) as well as for P46L, which is considered as a polymorphism or as a mutation with very mild pro-inflammatory effects [6]. As shown by western blotting in cell lysates (Figure 3A), TNFR1 expression was similar for TNFR1-WT and all mutated forms of the receptor. Nevertheless, western blot and ELISA assays performed on cell supernatants revealed that the soluble form of the receptor (sTNFR1) was detectable only for cells expressing TNFR1-WT, TNFR1-P46L and TNFR1-R92Q (Figure 3A and 3B). These data demonstrate that R92W and R92P result in a dramatic diminution of the pool of sTNFR1, like unambiguous mutations. In contrast, R92Q behaves like the WT protein. We also evaluated the membrane expression of TNFR1 by FACS analysis. We first used a PE-conjugated monoclonal anti-TNFR1 antibody (R&D Systems) used previously by other teams [7], [22]–[23]. Consistent with a previous observation [22], the receptor carrying the R92P mutation was not recognized by this antibody (Figure 4), so that we could not conclude on its subcellular localization. The subsequent use of a polyclonal antibody (R&D Systems) showed that TNFR1-R92P is indeed expressed in the cytoplasm, whereas very low levels are detectable at the cell surface. As shown in Figure 4A, the percentage of cells expressing the receptor at their surface is higher in the presence of TNFR1-WT, TNFR1-P46L and TNFR1-R92Q than in the presence of TNFR1-T50M, TNFR1-C70S, TNFR1-C73R, TNFR1-R92P and TNFR1-R92W. The same experiment performed after cell permeabilization did not reveal a different intracellular expression of the different forms of the receptor (Figure 4B).

Bottom Line: Most TNFRSF1A mutations are missense changes and, apart from those affecting conserved cysteines, their deleterious effect remains often questionable.Similar results were obtained with R92P, another mutation previously identified in a very small familial form with incomplete penetrance and variable expressivity.Combined with previous reports on arginine 92 mutations, this study discloses an unusual situation in which different amino acid substitutions at the same position in the protein are associated with a clinical spectrum bridging Mendelian to multifactorial conditions.

View Article: PubMed Central - PubMed

Affiliation: UMR_S933, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France. isabelle.jeru@trs.aphp.fr

ABSTRACT

Objectives: TNFRSF1A is involved in an autosomal dominant autoinflammatory disorder called TNFR-associated periodic syndrome (TRAPS). Most TNFRSF1A mutations are missense changes and, apart from those affecting conserved cysteines, their deleterious effect remains often questionable. This is especially true for the frequent R92Q mutation, which might not be responsible for TRAPS per se but represents a susceptibility factor to multifactorial inflammatory disorders. This study investigates TRAPS pathophysiology in a family exceptional by its size (13 members) and compares the consequences of several mutations affecting arginine 92.

Methods: TNFRSF1A screening was performed by PCR-sequencing. Comparison of the 3-dimensional structure and electrostatic properties of wild-type and mutated TNFR1 proteins was performed by in silico homology modeling. TNFR1 expression was assessed by FACS analysis, western blotting and ELISA in lysates and supernatants of HEK293T cells transiently expressing wild-type and mutated TNFR1.

Results: A TNFRSF1A heterozygous missense mutation, R92W (c.361C>T), was shown to perfectly segregate with typical TRAPS manifestations within the family investigated (p<5.10(-4)). It was associated with very high disease penetrance (0.9). Prediction of its impact on the protein structure revealed local conformational changes and alterations of the receptor electrostatic properties. R92W also impairs the TNFR1 expression at the cell surface and the levels of soluble receptor. Similar results were obtained with R92P, another mutation previously identified in a very small familial form with incomplete penetrance and variable expressivity. In contrast, TNFR1-R92Q behaves like the wild-type receptor.

Conclusions: These data demonstrate the pathogenicity of a mutation affecting arginine 92, a residue whose involvement in inflammatory disorders is deeply debated. Combined with previous reports on arginine 92 mutations, this study discloses an unusual situation in which different amino acid substitutions at the same position in the protein are associated with a clinical spectrum bridging Mendelian to multifactorial conditions.

Show MeSH
Related in: MedlinePlus