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Distinct mutations in IRAK-4 confer hyporesponsiveness to lipopolysaccharide and interleukin-1 in a patient with recurrent bacterial infections.

Medvedev AE, Lentschat A, Kuhns DB, Blanco JC, Salkowski C, Zhang S, Arditi M, Gallin JI, Vogel SN - J. Exp. Med. (2003)

Bottom Line: A lack of IL-18 responsiveness, coupled with diminished LPS and/or IL-1-induced nuclear factor-kappaB and activator protein-1 translocation, p38 phosphorylation, gene expression, and dysregulated IL-1R-associated kinase (IRAK)-1 activity in vitro support the hypothesis that the defect lies within the signaling pathway common to toll-like receptor 4, IL-1R, and IL-18R.When overexpressed in HEK293T cells, neither truncated form augmented endogenous IRAK-1 kinase activity, and both inhibited endogenous IRAK-1 activity modestly.Thus, IRAK-4 is pivotal in the development of a normal inflammatory response initiated by bacterial or nonbacterial insults.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Maryland, Baltimore, Baltimore, MD 20101, USA.

ABSTRACT
We identified previously a patient with recurrent bacterial infections who failed to respond to gram-negative LPS in vivo, and whose leukocytes were profoundly hyporesponsive to LPS and IL-1 in vitro. We now demonstrate that this patient also exhibits deficient responses in a skin blister model of aseptic inflammation. A lack of IL-18 responsiveness, coupled with diminished LPS and/or IL-1-induced nuclear factor-kappaB and activator protein-1 translocation, p38 phosphorylation, gene expression, and dysregulated IL-1R-associated kinase (IRAK)-1 activity in vitro support the hypothesis that the defect lies within the signaling pathway common to toll-like receptor 4, IL-1R, and IL-18R. This patient expresses a "compound heterozygous" genotype, with a point mutation (C877T in cDNA) and a two-nucleotide, AC deletion (620-621del in cDNA) encoded by distinct alleles of the IRAK-4 gene (GenBank/EMBL/DDBJ accession nos. AF445802 and AY186092). Both mutations encode proteins with an intact death domain, but a truncated kinase domain, thereby precluding expression of full-length IRAK-4 (i.e., a recessive phenotype). When overexpressed in HEK293T cells, neither truncated form augmented endogenous IRAK-1 kinase activity, and both inhibited endogenous IRAK-1 activity modestly. Thus, IRAK-4 is pivotal in the development of a normal inflammatory response initiated by bacterial or nonbacterial insults.

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Identification and functionality of deletion mutation in patient's IRAK-4. (A) Illustration of effect of AC deletion at nucleotides 620-621 in the patient (620-621del), resulting in a truncated form of IRAK-4 (M #2). Arrow indicates approximate location of truncation in IRAK-4 protein. (B) Vectors encoding the WT (N) or mutated (M #2) forms of IRAK-4 were expressed in HEK293T cells (10 μg vector/transfection), and cell lysates subjected to Western blot analysis using anti-Flag Ab as described in Fig. 8. (C) Overexpression of WT (N), but not the 620-621 deleted mutant (M #2), IRAK-4 expression vectors (10 μg/transfection) differentially modulate IRAK-1 kinase activity (see Fig. 8 B; n = 3).
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fig9: Identification and functionality of deletion mutation in patient's IRAK-4. (A) Illustration of effect of AC deletion at nucleotides 620-621 in the patient (620-621del), resulting in a truncated form of IRAK-4 (M #2). Arrow indicates approximate location of truncation in IRAK-4 protein. (B) Vectors encoding the WT (N) or mutated (M #2) forms of IRAK-4 were expressed in HEK293T cells (10 μg vector/transfection), and cell lysates subjected to Western blot analysis using anti-Flag Ab as described in Fig. 8. (C) Overexpression of WT (N), but not the 620-621 deleted mutant (M #2), IRAK-4 expression vectors (10 μg/transfection) differentially modulate IRAK-1 kinase activity (see Fig. 8 B; n = 3).

Mentions: Both the patient's parents responded vigorously to LPS (Fig. 5 A) and neither exhibited a history of recurrent infection. Therefore, we hypothesized that a second mutation on the patient's alternate IRAK-4 allele must be present to account for such a profound phenotype. A two-nucleotide deletion was identified in the cDNA at nucleotides 620–621, which corresponds to genomic DNA nucleotides 15978–15979. This second mutation was not seen in any clones that carried the point mutation, supporting the hypothesis that it was derived from the alternate allele. The predicted effect of this frameshift mutation would be to create a stop codon 36–39 nucleotides downstream in the IRAK-4 CDS, resulting in a second truncated form with a predicted size of 218 amino acids or ∼22 kD (Fig. 9 A) and would be predicted to express the entire DD, but a smaller region of the kinase domain than M #1. As in the case of the point mutation, the presence of the mutation was confirmed by sequencing PCR products amplified from genomic DNA that showed a C/A double peak at nucleotide 623 with a scrambled sequence thereafter (unpublished data). In addition, sequencing of cloned PCR products confirmed that approximately half expressed the deletion. Using site-directed mutagenesis, we introduced the deletion into the pRK7-FlagIRAK4 (N) expression vector to obtain the mutant vector, pRK7-IRAK4 (M #2) and overexpressed both in HEK293T cells to compare them functionally. Fig. 9 B shows that pRK7-IRAK4 (M #2) led to expression of two truncated Flag-tagged proteins with molecular weights of ∼26 and 30 kD, suggesting posttranscriptional modification, and/or possibly “read through” of the first stop codon. Fig. 9 C demonstrates that, unlike WT IRAK-4, this more-truncated form of IRAK-4 also failed to augment endogenous IRAK-1 kinase activity and inhibited endogenous basal and IL-1–induced IRAK-1 activity, although less than observed upon overexpression of pRK7-IRAK4 (M #1).


Distinct mutations in IRAK-4 confer hyporesponsiveness to lipopolysaccharide and interleukin-1 in a patient with recurrent bacterial infections.

Medvedev AE, Lentschat A, Kuhns DB, Blanco JC, Salkowski C, Zhang S, Arditi M, Gallin JI, Vogel SN - J. Exp. Med. (2003)

Identification and functionality of deletion mutation in patient's IRAK-4. (A) Illustration of effect of AC deletion at nucleotides 620-621 in the patient (620-621del), resulting in a truncated form of IRAK-4 (M #2). Arrow indicates approximate location of truncation in IRAK-4 protein. (B) Vectors encoding the WT (N) or mutated (M #2) forms of IRAK-4 were expressed in HEK293T cells (10 μg vector/transfection), and cell lysates subjected to Western blot analysis using anti-Flag Ab as described in Fig. 8. (C) Overexpression of WT (N), but not the 620-621 deleted mutant (M #2), IRAK-4 expression vectors (10 μg/transfection) differentially modulate IRAK-1 kinase activity (see Fig. 8 B; n = 3).
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Related In: Results  -  Collection

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fig9: Identification and functionality of deletion mutation in patient's IRAK-4. (A) Illustration of effect of AC deletion at nucleotides 620-621 in the patient (620-621del), resulting in a truncated form of IRAK-4 (M #2). Arrow indicates approximate location of truncation in IRAK-4 protein. (B) Vectors encoding the WT (N) or mutated (M #2) forms of IRAK-4 were expressed in HEK293T cells (10 μg vector/transfection), and cell lysates subjected to Western blot analysis using anti-Flag Ab as described in Fig. 8. (C) Overexpression of WT (N), but not the 620-621 deleted mutant (M #2), IRAK-4 expression vectors (10 μg/transfection) differentially modulate IRAK-1 kinase activity (see Fig. 8 B; n = 3).
Mentions: Both the patient's parents responded vigorously to LPS (Fig. 5 A) and neither exhibited a history of recurrent infection. Therefore, we hypothesized that a second mutation on the patient's alternate IRAK-4 allele must be present to account for such a profound phenotype. A two-nucleotide deletion was identified in the cDNA at nucleotides 620–621, which corresponds to genomic DNA nucleotides 15978–15979. This second mutation was not seen in any clones that carried the point mutation, supporting the hypothesis that it was derived from the alternate allele. The predicted effect of this frameshift mutation would be to create a stop codon 36–39 nucleotides downstream in the IRAK-4 CDS, resulting in a second truncated form with a predicted size of 218 amino acids or ∼22 kD (Fig. 9 A) and would be predicted to express the entire DD, but a smaller region of the kinase domain than M #1. As in the case of the point mutation, the presence of the mutation was confirmed by sequencing PCR products amplified from genomic DNA that showed a C/A double peak at nucleotide 623 with a scrambled sequence thereafter (unpublished data). In addition, sequencing of cloned PCR products confirmed that approximately half expressed the deletion. Using site-directed mutagenesis, we introduced the deletion into the pRK7-FlagIRAK4 (N) expression vector to obtain the mutant vector, pRK7-IRAK4 (M #2) and overexpressed both in HEK293T cells to compare them functionally. Fig. 9 B shows that pRK7-IRAK4 (M #2) led to expression of two truncated Flag-tagged proteins with molecular weights of ∼26 and 30 kD, suggesting posttranscriptional modification, and/or possibly “read through” of the first stop codon. Fig. 9 C demonstrates that, unlike WT IRAK-4, this more-truncated form of IRAK-4 also failed to augment endogenous IRAK-1 kinase activity and inhibited endogenous basal and IL-1–induced IRAK-1 activity, although less than observed upon overexpression of pRK7-IRAK4 (M #1).

Bottom Line: A lack of IL-18 responsiveness, coupled with diminished LPS and/or IL-1-induced nuclear factor-kappaB and activator protein-1 translocation, p38 phosphorylation, gene expression, and dysregulated IL-1R-associated kinase (IRAK)-1 activity in vitro support the hypothesis that the defect lies within the signaling pathway common to toll-like receptor 4, IL-1R, and IL-18R.When overexpressed in HEK293T cells, neither truncated form augmented endogenous IRAK-1 kinase activity, and both inhibited endogenous IRAK-1 activity modestly.Thus, IRAK-4 is pivotal in the development of a normal inflammatory response initiated by bacterial or nonbacterial insults.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Maryland, Baltimore, Baltimore, MD 20101, USA.

ABSTRACT
We identified previously a patient with recurrent bacterial infections who failed to respond to gram-negative LPS in vivo, and whose leukocytes were profoundly hyporesponsive to LPS and IL-1 in vitro. We now demonstrate that this patient also exhibits deficient responses in a skin blister model of aseptic inflammation. A lack of IL-18 responsiveness, coupled with diminished LPS and/or IL-1-induced nuclear factor-kappaB and activator protein-1 translocation, p38 phosphorylation, gene expression, and dysregulated IL-1R-associated kinase (IRAK)-1 activity in vitro support the hypothesis that the defect lies within the signaling pathway common to toll-like receptor 4, IL-1R, and IL-18R. This patient expresses a "compound heterozygous" genotype, with a point mutation (C877T in cDNA) and a two-nucleotide, AC deletion (620-621del in cDNA) encoded by distinct alleles of the IRAK-4 gene (GenBank/EMBL/DDBJ accession nos. AF445802 and AY186092). Both mutations encode proteins with an intact death domain, but a truncated kinase domain, thereby precluding expression of full-length IRAK-4 (i.e., a recessive phenotype). When overexpressed in HEK293T cells, neither truncated form augmented endogenous IRAK-1 kinase activity, and both inhibited endogenous IRAK-1 activity modestly. Thus, IRAK-4 is pivotal in the development of a normal inflammatory response initiated by bacterial or nonbacterial insults.

Show MeSH
Related in: MedlinePlus