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

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

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

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

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

A Recessive Founder Mutation in Regulator of Telomere Elongation Helicase 1, <i>RTEL1</i>, Underlies Severe Immunodeficiency and Features of Hoyeraal Hreidarsson Syndrome

<div><p>Dyskeratosis congenita (DC) is a heterogeneous inherited bone marrow failure and cancer predisposition syndrome in which germline mutations in telomere biology genes account for approximately one-half of known families. Hoyeraal Hreidarsson syndrome (HH) is a clinically severe variant of DC in which patients also have cerebellar hypoplasia and may present with severe immunodeficiency and enteropathy. We discovered a germline autosomal recessive mutation in <i>RTEL1</i>, a helicase with critical telomeric functions, in two unrelated families of Ashkenazi Jewish (AJ) ancestry. The affected individuals in these families are homozygous for the same mutation, R1264H, which affects three isoforms of <i>RTEL1</i>. Each parent was a heterozygous carrier of one mutant allele. Patient-derived cell lines revealed evidence of telomere dysfunction, including significantly decreased telomere length, telomere length heterogeneity, and the presence of extra-chromosomal circular telomeric DNA. In addition, <i>RTEL1</i> mutant cells exhibited enhanced sensitivity to the interstrand cross-linking agent mitomycin C. The molecular data and the patterns of inheritance are consistent with a hypomorphic mutation in <i>RTEL1</i> as the underlying basis of the clinical and cellular phenotypes. This study further implicates <i>RTEL1</i> in the etiology of DC/HH and immunodeficiency, and identifies the first known homozygous autosomal recessive disease-associated mutation in <i>RTEL1</i>.</p></div

Inhibiting DNA replication blocks T-circle formation in MSK-41 <i>RTEL1^R1264H</i> cells.

<p>(A) Phi29-dependent T-circles in BJ hTERT and MSK-41. (B) Phi29-dependent T-circles in RTEL1 floxed/- MEFs ± Cre, BJ hTERT and MSK-41. (C) Phi29-dependent T-circles in BJ hTERT and MSK-41 ± aphidicolin (APD; 5 µM). (D) Dot blot of the Phi29-dependent T-circles in BJ hTERT and MSK-41 ± aphidicolin (APD; 5 µM). (E) Quantification of the fold increase in intensity of Phi29-dependent T-circles in the different cell lines subjected to the indicated treatments. Intensity mean and standard deviation were calculated over two independent experiments; statistical analysis (one-way ANOVA) was calculated with Prism (GraphPad).</p

Telomere length is altered in individuals with <i>RTEL1^R1264H</i>.

<p>(A) Primary lymphocyte telomeres in family NCI-318 were measured by flow cytometry with fluorescent <i>in situ</i> hybridization (FISH) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003695#pgen.1003695-Alter2" target="_blank">[3]</a>. The proband is indicated by a triangle, the mother by a circle, and the father by a square. (B) Telomere FISH analysis of MSK-41 hTERT-immortalized fibroblasts revealed extreme telomere length heterogeneity. Quantitation of chromatids lacking detectable telomeric signal is shown. BJ hTERT, a normal hTERT-immortalized fibroblast line, and SaOS-2, an osteosarcoma cell line that relies on recombination-based telomere maintenance (ALT), are presented for comparison. (C) Representative metaphase spreads of MSK-41 and BJ hTERT are shown.</p

<i>RTEL1^R1264H</i> affects a putative conserved C4C4 domain.

<p>As displayed on the schematic (representing ENSP00000353332), the RTEL1 mutation is at the C-terminus of the protein, distal to the helicase domain. The affected amino acid is in a putative C4C4 domain. All eight key cysteines and R1264 are conserved in human, orangutan, cattle, and mouse sequences. Higher percent identity at a given amino acid position is indicated by a deeper purple color.</p