An in-frame deletion at the polymerase active site of POLD1 causes a multisystem disorder with lipodystrophy

Abstract

DNA polymerase δ, whose catalytic subunit is encoded by POLD1, is responsible for lagging-strand DNA synthesis during DNA replication. It carries out this synthesis with high fidelity owing to its intrinsic 3'- to 5'-exonuclease activity, which confers proofreading ability. Missense mutations affecting the exonuclease domain of POLD1 have recently been shown to predispose to colorectal and endometrial cancers. Here we report a recurring heterozygous single-codon deletion in POLD1 affecting the polymerase active site that abolishes DNA polymerase activity but only mildly impairs 3'- to 5'-exonuclease activity. This mutation causes a distinct multisystem disorder that includes subcutaneous lipodystrophy, deafness, mandibular hypoplasia and hypogonadism in males. This discovery suggests that perturbing the function of the ubiquitously expressed POLD1 polymerase has unexpectedly tissue-specific effects in humans and argues for an important role for POLD1 function in adipose tissue homeostasis.

Figure 1. Clinical characteristics of patients (full details in Supplementary Table 1)

(a) Patient 1 (aged 15yrs), demonstrating prominent lipodystrophy, small nose, “pseudo”-proptosis, secondary to lack of subcutaneous periorbital fat, tight skin, mandibular hypoplasia, bilateral hearing aids and reduced limb muscles. Informed consent was obtained to use Patient 1’s photo (b and c) Abnormal fat distribution shown by abdominal MRI at level L3 from patient 1 (b) and an age, gender and BMI matched control (c) showing the striking reduction of subcutaneous fat and marked increase in intra-abdominal fat as demonstrated by an increase in high intensity signal. (d and e) Picrosirius red stain of an abdominal subcutaneous adipose tissue image of (d) patient 1 with S605del POLD1 mutation and (e) a representative comparative image of a BMI matched subject. The black scale bars = 500μm. Adipose tissue of the subject with POLD1 mutation shows a considerable amount of fibrosis (in red).

Figure 2. Gene Schematic and mutational modelling of POLD1 mutations

(a) Gene Schematic of POLD1. Mutations predisposing to cancer (P327L and S478N) affect residues within the exonuclease (proofreading) domain whereas the S605del (found in all our 4 patients) is within the polymerase active site. (b) Global structure of POLD1 and sites of relevant mutations. The structure of POLD1 (residues 77-983, modelled on template 3iayA) is shown in ribbon form, coloured by secondary structure succession (blue, N-terminal, to red, C-terminal). Backbone and sidechains are displayed in stick format for residues P327 and S478 (pink), D602 and D757 (yellow), S604 (dark green), S605 (red) and L606 (blue), as indicated. The trinucleotide substrate is shown in stick format (light blue), with hydrogen bonds to the polymerase catalytic site indicated by green broken lines. Template DNA has been omitted for clarity. (c and d) Detail of the polymerase catalytic site, shown from the same viewpoint as in (b); (c) wild type POLD1, and (d) mutant S605del. Note the predicted deformation of the catalytic domain and loss of hydrogen bonding to substrate in S605del compared to normal POLD1; in contrast, the predicted structure of the catalytic site, and bonding to the trinucleotide substrate, was identical to normal POLD1 for both P327L and S478N mutants (not shown).

Figure 3. Pol delta S605del has no detectable polymerase activity, but robust exonuclease activity

(a) Polymerase reactions using increasing concentrations of either wild type (WT) or S605del (Del) Pol delta reveal that the mutant is incapable of extending the primer band (23 nt) in a 2 minute reaction with 25 μM dNTPs. (b) Exonuclease reactions on the same 23/46 primed duplex DNA, in the absence of dNTPs, show that S605del exhibits a 2- to 3-fold decrease in exonuclease activity when compared to wild type. ^aValues for percent of primer band degraded (% deg.) reflect three independent experiments, and are derived from a comparison of the intensity of the primer band (P) and the exonuclease product bands (E), given by the following equation: % deg = 1-(P/(P+E)). ^bThe % degradation values of Pol delta-S605del at 90, 150, and 300 fmol were plotted against the line obtained from measurements with the wild type polymerase (y=0.0029+0.445; R²=0.96). The values derived indicate that ~1.7 to 2.5-fold more mutant enzyme is required to observe the same amount of primer degradation as the wild type enzyme under these experimental conditions.