DNA Polymerase Epsilon Deficiency Causes IMAGe Syndrome with Variable Immunodeficiency

Abstract

During genome replication, polymerase epsilon (Pol ε) acts as the major leading-strand DNA polymerase. Here we report the identification of biallelic mutations in POLE, encoding the Pol ε catalytic subunit POLE1, in 15 individuals from 12 families. Phenotypically, these individuals had clinical features closely resembling IMAGe syndrome (intrauterine growth restriction [IUGR], metaphyseal dysplasia, adrenal hypoplasia congenita, and genitourinary anomalies in males), a disorder previously associated with gain-of-function mutations in CDKN1C. POLE1-deficient individuals also exhibited distinctive facial features and variable immune dysfunction with evidence of lymphocyte deficiency. All subjects shared the same intronic variant (c.1686+32C>G) as part of a common haplotype, in combination with different loss-of-function variants in trans. The intronic variant alters splicing, and together the biallelic mutations lead to cellular deficiency of Pol ε and delayed S-phase progression. In summary, we establish POLE as a second gene in which mutations cause IMAGe syndrome. These findings add to a growing list of disorders due to mutations in DNA replication genes that manifest growth restriction alongside adrenal dysfunction and/or immunodeficiency, consolidating these as replisome phenotypes and highlighting a need for future studies to understand the tissue-specific development roles of the encoded proteins.

Keywords: DNA replication; IMAGe syndrome; adrenal failure; cell cycle; growth; immunodeficiency; microcephaly; polymerase epsilon.

Figure 1

Mutations Causing POLE-Associated IMAGe Syndrome Are Distinct from Mutations Conferring a Non-syndromic Susceptibility to Cancer Schematic of the POLE gene, which encodes POLE1, the catalytic subunit of DNA polymerase epsilon. Domains: Pol, polymerase; Exo, exonuclease. Mutations identified in POLE subjects indicated above gene and protein (green). Recurrent intronic mutation underlined. For comparison, heterozygous germline missense mutations located in the exonuclease domain predisposing to colorectal cancer and other malignancies highlighted below (red).

Figure 2

Individuals with Biallelic POLE Mutations Have Severely Impaired Pre- and Post-natal Growth and a Recognizable Facial Gestalt (A) Photographs of POLE-deficient subjects demonstrating facial similarities. Written consent obtained from all families for photography. (B and C) Severe pre-natal onset growth restriction occurs in POLE-deficient individuals. (B) Adult POLE-deficient subject next to a control individual of average stature. (C) Growth is severely impaired pre- and postnatally. Z-scores (standard deviations from population mean for age and sex) for birth weight and postnatal height and head circumference (OFC). Dashed lines 95% confidence interval for general population. Circles, individual subject data points; red bars, mean values.

Figure 3

Common Intronic Variant Identified Causes Aberrant Splicing and POLE-Deficient Cells Show Deficiency of Polymerase Epsilon and Slowed S-phase Progression (A) The c.1686+32C>G mutation causes aberrant splicing of intron 15 in subject cells. RT-PCR of POLE transcripts from primary fibroblasts. Primers indicated by arrows in schematic. P1, P3, POLE-deficient subjects; C1, C2, control subjects. (B) Minigene assay demonstrating that aberrant splicing is a direct consequence of the c.1686+32C>G mutation. +ve control, point mutation in splice donor site, c.1686+1G>A. 5′ & 3′ indicate artificial vector-associated exons. (C) POLE1 levels are markedly reduced in subject fibroblasts. Immunoblot of total cell extracts. POLE1 antibody raised against AA1-176. Vinculin, loading control. ^∗ non-specific band. (D and E) Fibroblast cells from affected individuals exhibit delayed S phase progression. Schematic, experimental set-up. (D) Representative FACS plots. (E) Quantification of n = 3 affected and n = 3 control cell lines from representative experiment (of n = 3 expts with n ≥ 2 biological replicates per group). Mid-S-phase mean (±SEM) BrdU-labeled cells, normalized to t = 0 time point are plotted for each group. p value, two-way ANOVA.

Figure 4

POLE1 Deficiency Links CDKN1C-IMAGe Syndrome with Other Replisome-Associated Disorders Schematic of replication initiation (adapted by permission from Gaillard et al. copyright 2015 Macmillan Publishers), highlighting the sequential action of replisome-associated proteins, mutation of which causes MGS (blue text) and those that are associated with dwarfism with adrenal insufficiency and/or immune deficiency, including IMAGe syndrome (red text). During replication licensing, MCM helicases (MCM2-7) are loaded at replication origins by the ORC complex (ORC1-6) with CDC6 and CDT1 to form the pre-replicative complex (pre-RC). Subsequently, loading of additional replisome protein occurs, regulated by DDK and CDK kinases, to form the pre-initiation complex (pre-IC), that contains the CMG (CDC45, MCMs, GINS) complex. CDKN1C inhibits CDK activity. In the active replisome, Primase-Pol α initiates DNA synthesis with strands extended by the PCNA-associated DNA polymerases δ and ε. POLE1 and POLE2 are part of the Pol ε holoenzyme.