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Recombination activity of human recombination-activating gene 2 (RAG2) mutations and correlation with clinical phenotype.

ABSTRACT: BACKGROUND:Mutations in recombination-activating gene (RAG) 1 and RAG2 are associated with a broad range of clinical and immunologic phenotypes in human subjects. OBJECTIVE:Using a flow cytometry-based assay, we aimed to measure the recombinase activity of naturally occurring RAG2 mutant proteins and to correlate our results with the severity of the clinical and immunologic phenotype. METHODS:Abelson virus-transformed Rag2-/- pro-B cells engineered to contain an inverted green fluorescent protein (GFP) cassette flanked by recombination signal sequences were transduced with retroviruses encoding either wild-type or 41 naturally occurring RAG2 variants. Bicistronic vectors were used to introduce compound heterozygous RAG2 variants. The percentage of GFP-expressing cells was evaluated by using flow cytometry, and high-throughput sequencing was used to analyze rearrangements at the endogenous immunoglobulin heavy chain (Igh) locus. RESULTS:The RAG2 variants showed a wide range of recombination activity. Mutations associated with severe combined immunodeficiency and Omenn syndrome had significantly lower activity than those detected in patients with less severe clinical presentations. Four variants (P253R, F386L, N474S, and M502V) previously thought to be pathogenic were found to have wild-type levels of activity. Use of bicistronic vectors permitted us to assess more carefully the effect of compound heterozygous mutations, with good correlation between GFP expression and the number and diversity of Igh rearrangements. CONCLUSIONS:Our data support genotype-phenotype correlation in the setting of RAG2 deficiency. The assay described can be used to define the possible disease-causing role of novel RAG2 variants and might help predict the severity of the clinical phenotype.

SUBMITTER: Tirosh I

PROVIDER: S-EPMC6295349 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Recombination activity of human recombination-activating gene 2 (RAG2) mutations and correlation with clinical phenotype.

Tirosh Irit I Yamazaki Yasuhiro Y Frugoni Francesco F Ververs Francesca A FA Allenspach Eric J EJ Zhang Yu Y Burns Siobhan S Al-Herz Waleed W Noroski Lenora L Walter Jolan E JE Gennery Andrew R AR van der Burg Mirjam M Notarangelo Luigi D LD Lee Yu Nee YN

The Journal of allergy and clinical immunology 20180618 2

<h4>Background</h4>Mutations in recombination-activating gene (RAG) 1 and RAG2 are associated with a broad range of clinical and immunologic phenotypes in human subjects.<h4>Objective</h4>Using a flow cytometry-based assay, we aimed to measure the recombinase activity of naturally occurring RAG2 mutant proteins and to correlate our results with the severity of the clinical and immunologic phenotype.<h4>Methods</h4>Abelson virus-transformed Rag2<sup>-/-</sup> pro-B cells engineered to contain an ...[more]

PMID: 29772310

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Project description:Holoprosencephaly (HPE), which results from failed or incomplete midline forebrain division early in gestation, is the most common forebrain malformation. The etiology of HPE is complex and multifactorial. To date, at least 12 HPE-associated genes have been identified, including TGIF (transforming growth factor beta-induced factor), located on chromosome 18p11.3. TGIF encodes a transcriptional repressor of retinoid responses involved in TGF-? signaling regulation, including Nodal signaling. TGIF mutations are reported in approximately 1-2% of patients with non-syndromic, non-chromosomal HPE. We combined data from our comprehensive studies of HPE with a literature search for all individuals with HPE and evidence of mutations affecting TGIF in order to establish the genotypic and phenotypic range. We describe 2 groups of patients: 34 with intragenic mutations and 21 with deletions of TGIF. These individuals, which were ascertained from our research group, in collaboration with other centers, and through a literature search, include 38 probands and 17 mutation-positive relatives. The majority of intragenic mutations occur in the TGIF homeodomain. Patients with mutations affecting TGIFrecapitulate the entire phenotypic spectrum observed in non-chromosomal, non-syndromic HPE. We identified a statistically significant difference between the 2 groups with respect to inheritance, as TGIF deletions were more likely to be de novo in comparison to TGIF mutations (?(2) ((2)) = 6.97, p(permutated) = 0.0356). In addition, patients with TGIF deletions were also found to more commonly present with manifestations beyond the craniofacial and neuroanatomical features associated with HPE (p = 0.0030). These findings highlight differences in patients with intragenic mutations versus deletions affecting TGIF, and draw attention to the homeodomain region, which appears to be particularly relevant to HPE. These results may be useful for genetic counseling of affected patients.

Dataset Information

Recombination activity of human recombination-activating gene 2 (RAG2) mutations and correlation with clinical phenotype.

Publications

Recombination activity of human recombination-activating gene 2 (RAG2) mutations and correlation with clinical phenotype.

Similar Datasets

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