Functional Analysis of POLD1 P.Ser605del Variant: the Aging Phenotype of MDPL Syndrome Is Associated with an Impaired DNA Repair Capacity
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www.aging-us.com AGING 2021, Vol. 13, No. 4 Research Paper Functional analysis of POLD1 p.ser605del variant: the aging phenotype of MDPL syndrome is associated with an impaired DNA repair capacity Michela Murdocca1, Paola Spitalieri1, Claudia De Masi1, Ion Udroiu2, Jessica Marinaccio2, Massimo Sanchez3, Rosa Valentina Talarico1, Chiara Fiorillo4, Monica D’Adamo5, Paolo Sbraccia5, Maria Rosaria D’Apice6, Giuseppe Novelli1,6, Antonella Sgura2, Federica Sangiuolo1,6 1Department of Biomedicine and Prevention, Tor Vergata University, Rome 00133, Italy 2Department of Science, “Roma Tre” University, Rome 00154, Italy 3Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome 00161, Italy 4Paediatric Neurology and Neuromuscular Disorders, University of Genoa and Istituto Gaslini, Genoa 16147, Italy 5Department of Systems Medicine, Tor Vergata University, Rome 00133, Italy 6Laboratory of Medical Genetics, Tor Vergata Hospital, Rome 00133, Italy Correspondence to: Maria Rosaria D’Apice, Antonella Sgura; email: [email protected], [email protected] Keywords: MDPL syndrome, POLD1 gene, age-related disease, DNA repair, telomere damage Received: October 29, 2020 Accepted: January 4, 2021 Published: February 22, 2021 Copyright: © 2021 Murdocca et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Mandibular hypoplasia, Deafness and Progeroid features with concomitant Lipodystrophy define a rare systemic disorder, named MDPL Syndrome, due to almost always a de novo variant in POLD1 gene, encoding the DNA polymerase δ. We report a MDPL female heterozygote for the recurrent p.Ser605del variant. In order to deepen the functional role of the in frame deletion affecting the polymerase catalytic site of the protein, cellular phenotype has been characterised. MDPL fibroblasts exhibit in vitro nuclear envelope anomalies, accumulation of prelamin A and presence of micronuclei. A decline of cell growth, cellular senescence and a blockage of proliferation in G0/G1 phase complete the aged cellular picture. The evaluation of the genomic instability reveals a delayed recovery from DNA induced-damage. Moreover, the rate of telomere shortening was greater in pathological cells, suggesting the telomere dysfunction as an emerging key feature in MDPL. Our results suggest an alteration in DNA replication/repair function of POLD1 as a primary pathogenetic cause of MDPL. The understanding of the mechanisms linking these cellular characteristics to the accelerated aging and to the wide spectrum of affected tissues and clinical symptoms in the MDPL patients may provide opportunities to develop therapeutic treatments for progeroid syndromes. INTRODUCTION seven subjects showing a clinical phenotype overlapping with mandibuloacral dysplasia syndromes (MADA and Mandibular hypoplasia, Deafness and Progeroid features MADB) such as mandibular hypoplasia, prominent eyes, with concomitant Lipodystrophy, represent a rare stiff joints, beaked nose, and lipodystrophy, but also systemic disorder, named MDPL syndrome (MDPL; specific additional clinical hallmarks, including OMIM #615381) with a prevalence <1/1,000,000. MDPL sensorineural hearing loss, hypogonadism and absent was described for the first time in 2010 [1], reporting clavicular hypoplasia/acroosteolyses. www.aging-us.com 4926 AGING MAD and MDPL belong to the group of diseases holoenzyme can result in an increase in stalled or characterized by premature aging, which can be caused collapsed forks and the inability to quickly repair these by inheritable nuclear envelope and/or DNA repair breaks leads to genomic instability and apoptosis [12, defects [2]. 13]. Most importantly Polδ is involved in telomere break repair: in particular for the synthesis of both C- To date, only 26 patients with MDPL Syndrome have and G-rich telomere strands [14, 15]. been described and all of them reported variants in POLD1 gene (NM_002691.3) [3], encoding for the Moreover, an inverse correlation between POLD1 gene evolutionarily conserved p125 subunit of DNA expression and age has been described both in vitro and polymerase delta (Polδ). It provides the essential in vivo [16], suggesting that POLD1 may be associated catalytic activities of the enzyme, mediated by 5′–3′ with aging, but the functional link still remains unclear. DNA polymerase and 3′–5′ exonuclease moieties [4]. p125 subunit forms a heterotetramer with three smaller We report the in vitro characterisation of a known in- accessory subunits encoded by the POLD2 (p50), frame deletion p.Ser605del identified in a 22 years old POLD3 (p66), and POLD4 (p12) genes which, together Italian girl with clinical features of MDPL syndrome. In with Replication Factor C and Proliferating Nuclear order to elucidate the functional role of this deletion, Cell Antigen, constitute the polymerase holoenzyme MDPL cellular phenotype has been characterised in [5]. Both exonuclease and polymerase activities of Polδ terms of nuclear morphology, cellular proliferation, are fundamental to the nuclear function of the enzyme. senescence, and cell cycle progression. In particular, we Most recently, a cytoplasmic function of Polδ has also shed light on the capacity of MDPL cells to respond and been reported residing on the Golgi complex, where the repair DNA induced-damage, especially at telomeric Polδ controls microtubule growth [6]. Other studies level. The understanding of the pathogenic mechanism have evidenced that Polδ also acts as a lying at the basis of the MDPL syndrome allows us to nucleocytoplasmic shuttling protein transported into and explore the link existing among DNA repair and age- out of the nucleus in a controlled manner [7]. related diseases. POLD1 gene transcription is regulated throughout the RESULTS cell cycle, where relatively small increases in mRNA levels occur in late G1/S phase, accompanied by Clinical phenotype corresponding modest increases in p125 protein levels [8]. Song and colleagues also demonstrated that POLD1 MDPL clinical diagnosis was done at 21 years old, downregulation is able to block the cell cycle at G1 and although the mother reported growth retardation since G2/M phases and results in reduced DNA synthesis [9], the proband was 3 years old (Figure 1A). At the time of demonstrating the potential role of POLD1 in the the diagnosis, the patient was normotensive with a BMI regulation of cell cycle progression. of 19.5. Although fasting plasma glucose concentration was normal, an oral glucose challenge revealed type 2 Furthermore, evidence about Polδ activity has diabetes with marked hyperinsulinemia compatible with highlighted its fundamental involvement in DNA extreme insulin resistance. Skin scleroderma, replication process, cooperating with a DNA helicase, telangiectasia, and subcutaneous lipoatrophy were WRN, to maintain genome stability [10]. Also the clinically evident throughout the body concurrently with downregulation of p125 subunit is sufficient to induce reduced limb muscle mass (Figure 1A). A DEXA scan genomic instability, culminating in DNA replication detected percentages of fat at both the upper and lower errors [11]. Polδ function is in fact essential for limb <10%, whereas bone mineral density at lumbar replication, with a primary role as the replicase for the and femoral sites was in the osteopenic range (data not lagging strand, but it also has an important proofreading shown). MRI of the abdomen showed an over- ability conferred by the exonuclease activity, which is representation of mesenteric fat, as reported in other critical for ensuring replicative fidelity. Polδ serves to cases of MDPL (Figure 1B). Finally, she had primary repair DNA lesions arising as a result of exposure to amenorrhea. Clinical signs of MDPL in the mother were mutagens, acting in multiple forms of DNA repair, very mild. including nucleotide excision repair, double strand break repair, base excision repair, and mismatch repair MDPL syndrome: a case of germline POLD1 variant [5]. During double strand breaks, cells can choose, as repair mechanism, between non homologous end DNA sequencing of the proband by Next Generation joining (NHEJ) or homologous recombination (HR) Sequencing (NGS) reveals the heterozygous deletion process, depending on the type of lesion and the timing c.1812_1814delCTC, p.Ser605del in POLD1 gene, of the cell cycle. Sometimes, the instability of the segregating from the mother (Figure 1C, 1D), while www.aging-us.com 4927 AGING healthy siblings and father are negative. The mother than in WT-HDFs (7.3% respect to 1.5%; P<0.01) shows a mosaic pattern of the MDPL-causing variant in (Supplementary Figure 1A–1C). In order to analyze the peripheral lymphocytes, hair, urine and saliva (20%, nuclear envelope proteins, immunofluorescence data not shown). analyses were conducted both for the detection of prelamin and mature lamin A. Prelamin A showed an Nuclear envelope characterization and micronuclei abnormal accumulation in about 30% of MDPL nuclei, assessment of MDPL-HDFs mostly located at the nuclear rim, within membrane invaginations and occasionally in intranuclear Once established primary cell lines from dermal structures. As expected, prelamin A was rarely detected fibroblasts (HDFs), cells were first analysed for nuclear (4,3%) in WT cells which did not show any nuclear architecture instability, a sign that