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. 2017 Dec 4:8:1683.
doi: 10.3389/fimmu.2017.01683. eCollection 2017.

Clinical and Biological Manifestation of RNF168 Deficiency in Two Polish Siblings

Barbara Pietrucha  1 , Edyta Heropolitańska-Pliszka  1 , Robert Geffers  2 , Julia Enßen  3 , Britta Wieland  3 , Natalia Valerijevna Bogdanova  3   4 , Thilo Dörk  3
Affiliations

Clinical and Biological Manifestation of RNF168 Deficiency in Two Polish Siblings

Barbara Pietrucha et al. Front Immunol. .

Abstract

Germline mutations in the RING finger protein gene RNF168 have been identified in a combined immunodeficiency disorder called RIDDLE syndrome. Since only two patients have been described with somewhat different phenotypes, there is need to identify further patients. Here, we report on two Polish siblings with RNF168 deficiency due to homozygosity for a novel frameshift mutation, c.295delG, that was identified through exome sequencing. Both patients presented with immunoglobulin deficiency, telangiectasia, cellular radiosensitivity, and increased alpha-fetoprotein (AFP) levels. The younger sibling had a more pronounced neurological and morphological phenotype, and she also carried an ATM gene mutation in the heterozygous state. Immunoblot analyses showed absence of RNF168 protein, whereas ATM levels and function were proficient in lymphoblastoid cells from both patients. Consistent with the absence of RNF168 protein, 53BP1 recruitment to DNA double-strand breaks (DSBs) after irradiation was undetectable in lymphoblasts or primary fibroblasts from either of the two patients. γH2AX foci accumulated normally but they disappeared with significant delay, indicating a severe defect in DSB repair. A comparison with the two previously identified patients indicates immunoglobulin deficiency, cellular radiosensitivity, and increased AFP levels as hallmarks of RNF168 deficiency. The variability in its clinical expression despite similar cellular phenotypes suggests that some manifestations of RNF168 deficiency may be modified by additional genetic or epidemiological factors.

Keywords: DNA repair; chromosome instability; double-strand break repair; immunodeficiency syndrome; radiosensitivity.

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Figures

Figure 1
Figure 1
Assessment of ATM mutation (A), ATM protein level (B), and ATM kinase activity (C). (A) Direct sequencing of ATM exon 12 reveals heterozygosity for the novel frameshift mutation c.1402_1403delAA in genomic DNA from the sister (HA591) but not the brother (HA592). (B) Immunoblotting detects ATM protein in lymphoblastoid cells from both patients (HA591, HA592). Lymphoblastoid cell lines (LCLs) from a healthy individual were used as an ATM-proficient control (HA325), and LCLs from a patient with classical ataxia–telangiectasia were used as an ATM-deficient control (HA56). DNA-dependent protein kinase, catalytic subunit, served as the loading control (DNA-PKcs). (C) Immunoblotting of cells after irradiation reveals radiation-induced phosphorylation of the ATM substrate KAP1 at Ser824. Cells were irradiated with 0, 1.5, or 6 Gy, respectively, and proteins were extracted at 30 min after irradiation. LCLs from a healthy individual were used as an ATM-proficient control (HA325), and LCLs from a patient with classical ataxia–telangiectasia were used as an ATM-deficient control (HA56). β-actin served as the loading control (ACTB).
Figure 2
Figure 2
Assessment of RNF168 mutation (A) and RNF168 protein level (B). (A) Direct sequencing of RNF168 exon 12 reveals homozygosity for the novel frameshift mutation c.295delG in genomic DNA from either of both patients (HA591, HA592). (B) RFLP analysis of PCR products on 2% agarose gel electrophoresis. S, size marker; 1, undigested PCR product; 2–6, PCR products cleaved with MnlI: 2, wild-type control, 3, paternal sample, 4. maternal sample, 5, patient HA591, 6, patient HA592. (C) Western blot analysis reveals strongly reduced immunoreactivity for RNF168 protein in lymphoblastoid cells from either of both patients (HA591, HA592). Lymphoblastoid cell lines (LCLs) from a healthy individual were used as an RNF168-proficient control (HA325), and LCLs from a patient with classical ataxia–telangiectasia were used for comparison (HA56). β-actin served as the loading control (ACTB).
Figure 3
Figure 3
Immunocytochemical analysis of irradiation-induced repair foci in RNF168-deficient fibroblasts and lymphoblastoid cell lines. Detection of γH2AX foci (second column) and 53BP1 foci (third column) in reference ADP fibroblasts (upper panel) compared to patient fibroblasts F591 and F592 (middle and bottom panel) at 1 h after 6 Gy irradiation. DAPI staining and merged pictures are shown in the outer columns as controls for intracellular localization.
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