P02-014 - Consequences of Arginine 92 mutations in TNFR1

International audienc

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

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

Modeling of structural and electrostatic changes induced by mutations affecting arginine 92 on the TNFR1 ectodomain.

<p><b>A,</b> 3D structures of wild-type and mutated TNFR1 proteins. Protein models were generated by homology modeling. Carbon, oxygen, nitrogen and sulphur atoms are shown in white, red, blue and yellow, respectively. Residue 92 is represented with balls and other amino acids with sticks. <b>B,</b> Electrostatic properties of wild-type and mutated TNFR1 proteins. Electrostatic potentials are mapped on the surface of the TNFR1 3D structure. Blue color indicates regions of positive potential, whereas red depicts negative potential values. The position of the residue at position 92 is indicated by a white arrow.</p

Clinical presentation of the patients carrying the R92W mutation.

*<p>nd: not determined.</p

Comparison of sTNFR1 levels for the WT and mutated forms of the receptor.

<p><b>A</b>, Detection of TNFR1 by western blotting in supernatants and lysates from HEK293T cells transiently expressing the WT and mutated forms of the receptor. The data presented are representative of those obtained in three independent experiments. <b>B</b>, Measurement of soluble TNFR1 (sTNFR1) in supernatants from the same cells. Results from ELISA are presented as means ± SD of three independent experiments performed in duplicate. Data were normalized to the concentration measured in the mock condition.</p

Trafficking of WT and mutated forms of TNFR1 at the cell surface.

<p>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. <b>A,</b> Percentage of cells expressing TNFR1 at their surface. <b>B,</b> Percentage of cells expressing TNFR1 after permeabilization.</p

Genealogical tree and mutation analysis.

<p>Filled symbols represent patients with TRAPS; open squares and circles indicate healthy individuals. No clinical information was available for individuals from generations I and II. A sequencing chromatogram showing the mutation identified in family members III.2, III.3, III.4, III.6, III.7, III.8, III.9, III.11, IV.2, and IV.3 is presented below. The sequence in healthy relatives (III.1, III.5, III.10, IV.1) is shown as a control. The heterozygous transition generating the missense mutation is encircled.</p

Involvement of the Same TNFR1 Residue in Mendelian and Multifactorial Inflammatory Disorders

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