Polymerase δ deficiency causes syndromic immunodeficiency with replicative stress
Cecilia Domínguez Conde, … , Mirjam van der Burg, Kaan Boztug
Cecilia Domínguez Conde, … , Mirjam van der Burg, Kaan Boztug
Published August 26, 2019
Citation Information: J Clin Invest. 2019;129(10):4194-4206. https://doi.org/10.1172/JCI128903.
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Research Article Genetics Immunology

Polymerase δ deficiency causes syndromic immunodeficiency with replicative stress

Abstract

Polymerase δ is essential for eukaryotic genome duplication and synthesizes DNA at both the leading and lagging strands. The polymerase δ complex is a heterotetramer comprising the catalytic subunit POLD1 and the accessory subunits POLD2, POLD3, and POLD4. Beyond DNA replication, the polymerase δ complex has emerged as a central element in genome maintenance. The essentiality of polymerase δ has constrained the generation of polymerase δ–knockout cell lines or model organisms and, therefore, the understanding of the complexity of its activity and the function of its accessory subunits. To our knowledge, no germline biallelic mutations affecting this complex have been reported in humans. In patients from 2 independent pedigrees, we have identified what we believe to be a novel syndrome with reduced functionality of the polymerase δ complex caused by germline biallelic mutations in POLD1 or POLD2 as the underlying etiology of a previously unknown autosomal-recessive syndrome that combines replicative stress, neurodevelopmental abnormalities, and immunodeficiency. Patients’ cells showed impaired cell-cycle progression and replication-associated DNA lesions that were reversible upon overexpression of polymerase δ. The mutations affected the stability and interactions within the polymerase δ complex or its intrinsic polymerase activity. We believe our discovery of human polymerase δ deficiency identifies the central role of this complex in the prevention of replication-related DNA lesions, with particular relevance to adaptive immunity.

Authors

Cecilia Domínguez Conde, Özlem Yüce Petronczki, Safa Baris, Katharina L. Willmann, Enrico Girardi, Elisabeth Salzer, Stefan Weitzer, Rico Chandra Ardy, Ana Krolo, Hanna Ijspeert, Ayca Kiykim, Elif Karakoc-Aydiner, Elisabeth Förster-Waldl, Leo Kager, Winfried F. Pickl, Giulio Superti-Furga, Javier Martínez, Joanna I. Loizou, Ahmet Ozen, Mirjam van der Burg, Kaan Boztug

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Figure 3

Polymerase δ –deficient cells show reduced proliferation, cell-cycle delay, and disturbed replication fork dynamics.

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Polymerase δ –deficient cells show reduced proliferation, cell-cycle del...
(A) T cell proliferation as measured by 48-hour thymidine incorporation upon stimulation of PBMCs with anti-CD3, PMA, PMA-ionomycin, and PHA. The hash marks separate independent experiments performed in P1 and P2. (B) T cell proliferation as measured by VPD450 three days after stimulation of PBMCs with anti-CD3 and anti-CD28. Results are representative of 2 independent experiments. (C) Cell-cycle analysis of patient and healthy control T cells as measured by BrdU incorporation. Data are representative of 2 independent experiments. (D) T cell proliferation as measured by VPD450 at the indicated time points after anti-CD3 and anti-CD28 stimulation of in vitro–expanded T cells. (E) Cell-cycle analysis of patient and healthy control fibroblasts upon double-thymidine block and release. (F) Scheme of dual-pulse labeling in DNA fiber analysis and representative images of the fiber analysis experiment performed using patient and HD fibroblasts. Original magnification, ×40. Representative green tracks are marked with an asterisk. Fiber data are representative of 2 independent experiments. Number of fibers counted: 1859 in HDs and 1213 in P1. (G) Replication fork speed in P1 (average of 3.899 μm/min) and HD (average of 2.89 μm/min) fibroblasts as measured by the length of IdU tracks. Statistical significance calculated by Wilcoxon rank sum test. (H) Analysis of initiation events in P1 (12.45%) and HD (18.93%) fibroblasts as measured by the frequency of only IdU-labeled tracks.