Rag1_R142* is a null mutant and Rag1_V779M 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_V779M) 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

Collapsed T cell repertoire in Omenn Syndrome Patient.

<p>TCR Vβ spectratype analysis of CDR3 reveals profound oligoclonality and monoclonality, consistent with Omenn Syndrome.</p

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_V779M 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_V779M) 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_V779M 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 4½ months) is consistent with Omenn’s Syndrome.

<p>Normal values from the Children’s Hospital Laboratory, the Cincinnati Children’s 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 4½ months) is consistent with Omenn’s Syndrome.</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 387–1011; yellow) and non-core regions (aa 1–386 and 1012–1043; white). There are two potential domains within the core region of Rag1: the central domain (aa 531–763; purple bar); and the C-terminal domain (aa 764–983; orange bar). Rag1 contains four basic regions (BI: aa 142–147; BII: aa 219–237; BIII: aa 244–252; BIV: aa 829–843; gray), a RING finger (aa 293–331; red), two zinc fingers (ZFA: aa 356–379; ZFB: aa 728–753; blue), a nonamer DNA-binding region (NBR: 387–457; dark yellow), a nuclear localization signal (NLS: aa 972–976; 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