14 research outputs found
Immune deficiency caused by impaired expression of nuclear factor-kappaB essential modifier (NEMO) because of a mutation in the 5' untranslated region of the NEMO gene.
BACKGROUND: Nuclear factor-ÎșB (NF-ÎșB) is a key transcription factor that regulates both innate and adaptive immunity as well as ectodermal development. Mutations in the coding region of the IÎșB kinase Îł/NF-ÎșB essential modifier (NEMO) gene cause X-linked ectodermal dysplasia with immunodeficiency. OBJECTIVE: To determine the genetic cause of recurrent sinopulmonary infections and dysgammaglobulinemia in a patient with a normal NEMO coding sequence and his affected brother. METHODS: TNF-α and IFN-α production in response to Toll-like receptor (TLR) stimulation was analyzed by ELISA, NEMO mRNA levels were measured by quantitative PCR, and NEMO protein expression was measured by Western blotting. NF-ÎșB activation was assessed by nuclear translocation of p65 and luciferase reporter gene assays. RESULTS: TLR-induced TNF-α and IFN-α production by PBMCs was impaired in the patient and his brother. Sequencing of the patientâs NEMO gene revealed a novel mutation in the 5âČ untranslated region, which was also present in the brother, resulting in abnormally spliced transcripts and a 4-fold reduction in mRNA levels. NEMO protein levels in EBV transformed B cells and fibroblasts from the index patient were 8-fold lower than normal controls. NF-ÎșB p65 nuclear translocation in the patientâs EBV B cells after TLR7 ligation was defective. NF-ÎșBâdependent luciferase gene expression in IL-1âstimulated fibroblasts from the patient was impaired. CONCLUSION: This is the first description of immune deficiency resulting from low expression of a normal NEMO protein
A 23-Year Follow-Up of a Patient with Gain-of-Function IkB-Alpha Mutation and Stable Full Chimerism After Hematopoietic Stem Cell Transplantation
Compound Heterozygous Mutation of Rag1 Leading to Omenn Syndrome
Omenn syndrome is a primary immunodeficiency disorder, featuring susceptibility to infections and autoreactive T cells and resulting from defective genomic rearrangement of genes for the T cell and B cell receptors. The most frequent etiologies are hypomorphic mutations in ânon-coreâ regions of the Rag1 or Rag2 genes, the protein products of which are critical members of the cellular apparatus for V(D)J recombination. In this report, we describe an infant with Omenn syndrome with a previously unreported termination mutation (p.R142*) in Rag1 on one allele and a partially characterized substitution mutation (p.V779M) in a âcoreâ region of the other Rag1 allele. Using a cellular recombination assay, we found that while the p.R142* mutation completely abolished V(D)J recombination activity, the p.V779M mutation conferred a severe, but not total, loss of V(D)J recombination activity. The recombination defect of the V779 mutant was not due to overall misfolding of Rag1, however, as this mutant supported wild-type levels of V(D)J cleavage. These findings provide insight into the role of this poorly understood region of Rag1 and support the role of Rag1 in a post-cleavage stage of recombination
p.R142* maternal and p.V779M paternal mutations.
<p>P1 harbors a maternally inherited c.424C>T mutation, resulting in a premature stop codon. <b>A.</b> Sequencing chromatogram demonstrating the presence of a heterozygous c.424C>T mutation. <b>B.</b> Alignment of the wildtype and mutant Rag1 cDNA and protein sequences. c.424C>T creates a premature stop codon at position 142 of the protein. <b>C.</b> P1 harbors a paternally inherited c.2335G>A mutation, resulting in the non-synonymous coding mutation p.V779M. Sequencing chromatogram demonstrating the presence of a heterozygous c.2335G>A mutation. <b>D.</b> Alignment of the wildtype and mutant Rag1 cDNA and protein sequences. c.2335G>A creates a missense p.V779M mutation in the Rag1 protein.</p
Rag1<sub>R142*</sub> is a null mutant and Rag1<sub>V779M</sub> is a hypomorphic mutant.
<p>A. Western analysis of Flag-tagged full-length Rag1 proteins expressed in Br3neo human fibroblast cells confirms that the wild-type (Rag1) and mutant (Rag1<sub>V779M</sub>) proteins are expressed at comparable levels <i>in vivo</i>. B. Representative recombination data from using the indicated constructs for transient V(D)J recombination assays in Br3neo cells. C. Absolute recombination activity using wild-type Rag1 (hatched) or the p.V779M mutant (shaded) with signal-joint substrates (left) or coding-joint substrates (right). Results represent the mean ±s.d. of six independent experiments. D. Normalized recombination activity of the p.V779M mutant. Recombination activity of the p.V779M mutant on each substrate was normalized to the activity of wild-type Rag1. Results represent the mean ± s.d. of six independent experiments.</p
Overall domain structure of the human RAG1 protein.
<p>The human RAG1 protein is 1043 amino acid long and consists of a core region (aa 387â1011; yellow) and non-core regions (aa 1â386 and 1012â1043; white). There are two potential domains within the core region of Rag1: the central domain (aa 531â763; purple bar); and the C-terminal domain (aa 764â983; orange bar). Rag1 contains four basic regions (BI: aa 142â147; BII: aa 219â237; BIII: aa 244â252; BIV: aa 829â843; gray), a RING finger (aa 293â331; red), two zinc fingers (ZFA: aa 356â379; ZFB: aa 728â753; blue), a nonamer DNA-binding region (NBR: 387â457; dark yellow), a nuclear localization signal (NLS: aa 972â976; green), an Asp-Asp-Glu active site motif (D603, D711, E965), and two C-terminal zinc-binding sites (C905/C907 and H940/H945). The four basic regions serve as binding sites for the nuclear transport proteins Srp1 and Rch1. The RING finger functions as an E3 ubiquitin ligase and, together with zinc finger A, mediates Rag1 multimerization. Zinc finger B is thought to function as a Rag2 binding site. The positions of R142 and V779 are indicated.</p
Rag1<sub>V779M</sub> has wild-type V(D)J cleavage activity.
<p><b>A</b>, Wild-type and mutant Rag1 proteins express and purify equally well. Coomassie stained gel of wild-type (Rag1) and mutant (Rag1<sub>V779M</sub>) recombinant core Rag1 proteins purified from E. coli. A serial dilution series (5-fold dilutions between lanes) is shown for each protein. <b>B</b>, The p.V779M mutant protein catalyzes wild-type V(D)J cleavage <i>in vitro</i>. Cleavage reactions were performed with recombinant wild-type core Rag1 and core Rag1<sub>V779M</sub> in the presence of recombinant full-length Rag2 and resolved by denaturing polyacrylamide gel electrophoresis. The positions of the substrate (S) and cleavage products (hairpin (H) and nick (N)) are indicated. <b>C</b>, Absolute V(D)J cleavage activity of wild-type Rag1 (left) and the p.V779M mutant (right). Results represent the mean ± s.d. of four independent experiments.</p
Peripheral blood analysis of P1 at time of initial presentation (age 4Âœ months) is consistent with Omennâs Syndrome.
<p>Normal values from the Childrenâs Hospital Laboratory, the Cincinnati Childrenâs Hospital Laboratory, or from reference [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121489#pone.0121489.ref030" target="_blank">30</a>].</p><p>Peripheral blood analysis of P1 at time of initial presentation (age 4Âœ months) is consistent with Omennâs Syndrome.</p
Collapsed T cell repertoire in Omenn Syndrome Patient.
<p>TCR VÎČ spectratype analysis of CDR3 reveals profound oligoclonality and monoclonality, consistent with Omenn Syndrome.</p