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Functional analysis of naturally occurring DCLRE1C mutations and correlation with the clinical phenotype of ARTEMIS deficiency.

ABSTRACT: BACKGROUND:The endonuclease ARTEMIS, which is encoded by the DCLRE1C gene, is a component of the nonhomologous end-joining pathway and participates in hairpin opening during the V(D)J recombination process and repair of a subset of DNA double-strand breaks. Patients with ARTEMIS deficiency usually present with severe combined immunodeficiency (SCID) and cellular radiosensitivity, but hypomorphic mutations can cause milder phenotypes (leaky SCID). OBJECTIVE:We sought to correlate the functional effect of human DCLRE1C mutations on phenotypic presentation in patients with ARTEMIS deficiency. METHODS:We studied the recombination and DNA repair activity of 41 human DCLRE1C mutations in Dclre1c(-/-) v-abl kinase-transformed pro-B cells retrovirally engineered with a construct that allows quantification of recombination activity by means of flow cytometry. For assessment of DNA repair efficacy, resolution of ?H2AX accumulation was studied after ionizing radiation. RESULTS:Low or absent activity was detected for mutations causing a typical SCID phenotype. Most of the patients with leaky SCID were compound heterozygous for 1 loss-of-function and 1 hypomorphic allele, with significant residual levels of recombination and DNA repair activity. Deletions disrupting the C-terminus result in truncated but partially functional proteins and are often associated with leaky SCID. Overexpression of hypomorphic mutants might improve the functional defect. CONCLUSIONS:Correlation between the nature and location of DCLRE1C mutations, functional activity, and the clinical phenotype has been observed. Hypomorphic variants that have been reported in the general population can be disease causing if combined in trans with a loss-of-function allele. Therapeutic strategies aimed at inducing overexpression of hypomorphic alleles might be beneficial.

SUBMITTER: Felgentreff K

PROVIDER: S-EPMC4494888 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Functional analysis of naturally occurring DCLRE1C mutations and correlation with the clinical phenotype of ARTEMIS deficiency.

Felgentreff Kerstin K Lee Yu Nee YN Frugoni Francesco F Du Likun L van der Burg Mirjam M Giliani Silvia S Tezcan Ilhan I Reisli Ismail I Mejstrikova Ester E de Villartay Jean-Pierre JP Sleckman Barry P BP Manis John J Notarangelo Luigi D LD

The Journal of allergy and clinical immunology 20150425 1

<h4>Background</h4>The endonuclease ARTEMIS, which is encoded by the DCLRE1C gene, is a component of the nonhomologous end-joining pathway and participates in hairpin opening during the V(D)J recombination process and repair of a subset of DNA double-strand breaks. Patients with ARTEMIS deficiency usually present with severe combined immunodeficiency (SCID) and cellular radiosensitivity, but hypomorphic mutations can cause milder phenotypes (leaky SCID).<h4>Objective</h4>We sought to correlate t ...[more]

PMID: 25917813

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Project description:BackgroundGlucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common X-linked inherited erythroenzymopathy in Thailand. The clinical and hematological manifestations of G6PD deficiency are variable.ObjectiveThis study aimed to characterize the genotype-phenotype correlation of G6PD mutations in Thai pediatric patients who were followed-up in Phramongkutklao Hospital, a tertiary center in central Thailand. Material and Method. A total of 102 children including 73 males (71.6%) and 29 females (28.4%) were included in our study. Mutation analysis was performed by direct DNA sequencing of all coding exons of the G6PD gene. Ninety-one patients (89.2%) were presented with neonatal hyperbilirubinemia and 11 patients (10.8%) were presented with acute hemolytic anemia beyond the neonatal period.ResultsMolecular analysis of the G6PD gene in 102 G6PD-deficient Thai children identified 12 different mutations. G6PD Viangchan (871G > A) and G6PD Canton (1376G > T) were the first (46.2%) and the second (15.4%) most common identified mutations among both male and female G6PD-deficient individuals, respectively. All affected males were hemizygous for G6PD mutations and had an average G6PD level of 16.7 ± 11.5 (3-76) IU/ml.RBC. Majority of female patients (27 in 29, 93.1%) were heterozygous for G6PD mutations and had an average G6PD level of 133.6 ± 43.4 (9-195) IU/ml.RBC. Two female patients (6.9%) were either homozygous or compound heterozygous for the mutations and had G6PD level in the affected male range (35 and 10 IU/ml.RBC). Only 1 in 27 heterozygous females (3.7%) had G6PD level in the affected male range (9 IU/ml.RBC) which is possibly explained by nonrandom X-chromosome inactivation. The correlation of genotypes, G6PD levels, and clinical phenotypes was not demonstrated in our study in which all of the included G6PD-deficient patients were presented with neonatal hyperbilirubinemia and acute hemolytic anemia, since the genotype-phenotype correlation is normally demonstrated in chronic nonspherocytic hemolytic anemia (CNSHA) G6PD-deficient individuals.ConclusionThis study characterizes the molecular heterogeneity of G6PD variants causing G6PD deficiency in Thai children. Our study demonstrated the efficiency of direct DNA sequencing which can identify 12 missense mutations in Thai children.

Project description:Sequencing of Ebola virus (EBOV) genomes during the 2014-2016 epidemic identified several naturally occurring, dominant mutations potentially impacting virulence or tropism. In this study, we characterized EBOV variants carrying one of the following substitutions: A82V in the glycoprotein (GP), R111C in the nucleoprotein (NP), or D759G in the RNA-dependent RNA polymerase (L). Compared with the wild-type (WT) EBOV C07 isolate, NP and L mutants conferred a replication advantage in monkey Vero E6, human A549, and insectivorous bat Tb1.Lu cells, while L mutants displayed a disadvantage in human Huh7 cells. The replication of the GP mutant was significantly delayed in Tb1.Lu cells and similar to that of the WT in other cells. The L mutant was less virulent, as evidenced by increased survival for mice and a significantly delayed time to death for ferrets, but increased lengths of the period of EBOV shedding may have contributed to the prolonged epidemic. Our results show that single substitutions can have observable impacts on EBOV pathogenicity and provide a framework for the study of other mutations.IMPORTANCE During the Ebola virus (EBOV) disease outbreak in West Africa in 2014-2016, it was discovered that several mutations in the virus emerged and became prevalent in the human population. This suggests that these mutations may play a role impacting viral fitness. We investigated three of these previously identified mutations (in the glycoprotein [GP], nucleoprotein [NP], or RNA-dependent RNA polymerase [L]) in cell culture, as well as in mice and ferrets, by generating recombinant viruses (based on an early West African EBOV strain) each carrying one of these mutations. The NP and L mutations appear to decrease virulence, whereas the GP mutation slightly increases virulence but mainly impacts viral tropism. Our results show that these single mutations can impact EBOV virulence in animals and have implications for the rational design of efficacious antiviral therapies against these infections.

Dataset Information

Functional analysis of naturally occurring DCLRE1C mutations and correlation with the clinical phenotype of ARTEMIS deficiency.

Publications

Functional analysis of naturally occurring DCLRE1C mutations and correlation with the clinical phenotype of ARTEMIS deficiency.

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