Novel POLE mutations identified in patients with IMAGE-I syndrome cause aberrant subcellular localisation and protein degradation in the nucleus

Abstract

Background: DNA replisome is a molecular complex that plays indispensable roles in normal DNA replication. IMAGE-I syndrome is a DNA replisome-associated genetic disease caused by biallelic mutations in the gene encoding DNA polymerase epsilon catalytic subunit 1 (POLE). However, the underlying molecular mechanisms remain largely unresolved.

Methods: The clinical manifestations in two patients with IMAGE-I syndrome were characterised. Whole-exome sequencing was performed and altered mRNA splicing and protein levels of POLE were determined. Subcellular localisation, cell cycle analysis and DNA replication stress were assessed using fibroblasts and peripheral blood from the patients and transfected cell lines to determine the functional significance of POLE mutations.

Results: Both patients presented with growth retardation, adrenal insufficiency, immunodeficiency and complicated diffuse large B-cell lymphoma. We identified three novel POLE mutations: namely, a deep intronic mutation, c.1226+234G>A, common in both patients, and missense (c.2593T>G) and in-frame deletion (c.711_713del) mutations in each patient. The unique deep intronic mutation produced aberrantly spliced mRNAs. All mutants showed significantly reduced, but not null, protein levels. Notably, the mutants showed severely diminished nuclear localisation, which was rescued by proteasome inhibitor treatment. Functional analysis revealed impairment of cell cycle progression and increase in the expression of phospho-H2A histone family member X in both patients.

Conclusion: These findings provide new insights regarding the mechanism via which POLE mutants are highly susceptible to proteasome-dependent degradation in the nucleus, resulting in impaired DNA replication and cell cycle progression, a characteristic of DNA replisome-associated diseases.

Keywords: DNA replication; immune system diseases; mutation.

Figure 1

Novel POLE mutations and clinical manifestations in two patients with IMAGE-I syndrome. (A) Pedigrees of two unrelated kindreds, showing allelic segregations. ‘E?’ indicates an unknown genotype. The scheme below shows the location of the mutations in POLE. (B) Facial appearances of Patient 1 (P1) at 11 years of age and Patient 2 (P2) at 31 years of age. (C) Growth curve of P1. The upper curve shows the body height for ages, and the lower curve shows the body weight for ages. These were described using the clinical tool provided by the Japanese Society for Pediatric Endocrinology (http://jspe.umin.jp/medical/chart_dl.html). Exo, exonuclease domain; Pol, polymerase domain; POLE, gene encoding DNA polymerase epsilon catalytic subunit 1.

Figure 2

A deep intronic mutation of c.1226+234G>A causes aberrant splicing of POLE (gene encoding DNA polymerase epsilon catalytic subunit 1). (A) Electrophoresis of RT-PCR products between exons 12 and 13. Patient 1 (P1) and Patient 2 (P2) have the same aberrant splicing product. DNA ladder marker was shown in left. (B) Prediction of c.1226+234G>A mutation using the SpliceAI lookup platform. Δ score ranged from 0 to 1 and provided 0.2 (high recall), 0.5 (recommended) and 0.8 (high precision) as the cut-off values. Aberrant isoform 1 cannot be predicted using the predictive method. (C) Electrophoresis of RT-PCR products in minigene assay. The c.1226+234G>A mutant produced the two aberrant splicing products with 309-bp (aberrant isoform 1) and 106-bp insertions (aberrant isoform 2). DNA ladder marker was shown in left. (D) Scheme of minigene vector and aberrant splicing products. Both aberrant splicing products were cloned and confirmed using Sanger sequencing. PB, peripheral blood; EV, empty vector; Mut, c.1226+234G>A mutant; WT, wild type.

Figure 3

POLE levels in patient samples. Immunoblotting of POLE protein extracted from two normal controls (C1 and C2) and P1 primary fibroblasts (A), and from C1, C2 and P2 PBMCs (B). The experiment was performed in triplicate and the means were calculated. The error bars indicate ±SE of the mean. The p-value was calculated using one-way ANOVA, and the Dunnett test was used for post hoc analysis. ANOVA, analysis of variance; C1, control 1; C2, control 2; P1, patient 1; P2. patient 2; PBMCs, peripheral blood mononuclear cells; POLE, gene encoding DNA polymerase epsilon catalytic subunit 1.

Figure 4

Impaired cell cycle progression and replication stress response in two patients with IMAGE-I syndrome. (A), (B) FACS plot (left panels) and quantification of mid-S-phase cells (right panels). In FACS plots, each P4, P5, P6 gate shows G1/G0 phase, S phase, G2/M phase, respectively. P1 primary fibroblast (A) or P2 PBMCs (B) and three normal controls were treated with EdU for 1 hour and harvested at 0, 4, 8 and 12 hours. We performed experiments in triplicate and calculated the means. The error bars show means±SEM. The p-value was calculated using two-way ANOVA, and the Holm test was used for post hoc analysis. (C) Immunofluorescence staining of phospho-H2AX in P1 primary fibroblast and two normal controls is shown in red, and merged images with DAPI shown in blue (upper panels). Dots plots show quantification of means per cell (lower panel). The blue bars indicate the means. The p-value was calculated using the Kruskal-Wallis test, and the Mann-Whitney U test was used for post hoc test analysis. ANOVA, analysis of variance; EdU, 5-ethynyl-2-deoxyuridine; HU, hydroxyurea; P1, patient 1; P2. patient 2; PBMCs, peripheral blood mononuclear cells.

Figure 5

Reduced nuclear localisation of POLE mutants due to proteasome-dependent degradation. (A) Immunofluorescence staining of transfected COS-7 cells treated with vehicle (upper panels) and with CHX and MG132 (lower panels). FLAG-tagged POLE protein stained with anti-FLAG antibody is shown in red. DAPI is shown in blue, and polymerised actin stained with phalloidin is shown in green. Scale bar, 20 µm. (B) Immunoblotting of POLE in cytosolic and nuclear fraction extracted from COS-7 cells transfected with each plasmid. Introduced FLAG-tagged POLE protein was detected using anti-FLAG antibody. Lamin A/C is the marker of nuclear fraction, and α-tubulin of the cytosol fraction. *Denotes non-specific bands. (C) Immunoblotting of POLE in cytosolic and nuclear fractions extracted from P1 and two control fibroblasts. *Denotes non-specific bands. C1, control 1; C2, control 2; CHX, cycloheximide; P1, patient 1; POLE, gene encoding DNA polymerase epsilon catalytic subunit 1; WT, wild type.