Mutation of POLB causes lupus in mice

Abstract

A replication study of a previous genome-wide association study (GWAS) suggested that a SNP linked to the POLB gene is associated with systemic lupus erythematosus (SLE). This SNP is correlated with decreased expression of Pol β, a key enzyme in the base excision repair (BER) pathway. To determine whether decreased Pol β activity results in SLE, we constructed a mouse model of POLB that encodes an enzyme with slow DNA polymerase activity. We show that mice expressing this hypomorphic POLB allele develop an autoimmune pathology that strongly resembles SLE. Of note, the mutant mice have shorter immunoglobulin heavy-chain junctions and somatic hypermutation is dramatically increased. These results demonstrate that decreased Pol β activity during the generation of immune diversity leads to lupus-like disease in mice, and suggest that decreased expression of Pol β in humans is an underlying cause of SLE.

Figure 1. The POL B Y265^c/c and Y265^c/+ mice exhibit multi-organ symptoms of SLE

A. The levels of lupus-like dermatitis are increased significantly in the POL B Y265^c/c and Y265^c/+ mice compare to WT mice as the mice age. Shown are curves for each genotype (POL B^{+/+, c/+, and c/c}) representing the percentage of mice with lupus-like dermatitis. B. The POLB^Y265c/c and POLB^Y265c/+ mice have higher levels of ANA than WT mice. Shown above the graph is an example of immunofluorescence intensity in 12M old mutant mice compared to the WT littermates and the MRL/lpr mice. Note that the fluorescence pattern observed in the MRL/lpr mice differs from that of the POLB^Y265c/c and POLB^Y265c/+ mice. In the graph, levels of ANA were quantified from several mice (WT, blue squares; c/+, red circles; c/c, green triangles) at various ages. Briefly, ANA was tested by immunofluorescence using human epithelial (HEp-2) cells in 12-well slides (Diasorin Inc). For each run 1:50 diluted sera were used at a screening investigation. Samples from mice with high and low ANA scores were used on the same slide to confirm the sensitivity and specificity of the test during each scoring. C. The POLB^Y265c/c and POLB^Y265c/+ mice exhibit increased severity of glomerular nephritis compared to WT mice. The severity of kidney lesions was scored from 0 to 3 for normal, mild, moderate, or severe. For each mouse, more than 10 glomerular, tubular, or interstitial areas were evaluated and scored for glomerular cellularity, infiltrating leukocytes, severity of tubular lesions, mesangial matrix expansion, crescent formation, interstitial mononuclear cell infiltrates in the medulla and cortex. Lesion scores for each mouse were calculated as the mean of the summed individual scores from each evaluated factors. D. Examples of renal disease in the POLB^Y265c/c mice compared to WT. The black arrow points to mesangial hypercellularity. The white arrow denotes basement membrane thickening and white arrowheads are glomerular crescents. The gray arrow denotes deposits of antibodies and shrunken glomeruli in the outer cortex denoted by the black arrow. E. The kidneys of the POLB^Y265c/c exhibit staining of IgG that is of significantly higher intensity than what is observed in WT mice. F. The POLB^Y265c/c exhibit significantly increased levels of cervical lymphadenopathy compared to WT mice. G. Examples of enlarged cervical lymph nodes in the POLB^{Y265c/c and c/+} mice compared to the normal nodes shown in the WT mouse. Arrows point to the cervical lymph nodes.

Figure 2. VDJ recombination is aberrant in the POLB^Y265c/c mice

A. Polymerase chain reaction (PCR)-amplified products from B220⁺ IgM⁻ bone marrow and splenic cell genomic DNA of 3–4 three week-old POLB^{+/+ and C/C} mice were cloned and sequenced. The lengths of the CDR3 sequences as a function of the percent of sequences with particular lengths are plotted in the graph. Note that the majority of CDR3 sequences from the POLB^Y265c/c mice have a length between 31–35 bases whereas the CDR3 region of WT mice is 41–45 bases in length. At least three mice of each genotype were characterized. B. The table shows the ranges of lengths of the CDR3 regions from spleen and bone marrow found in WT and POLB^Y265c/c mice. C. The percentage of D regions in the POLB^C/C mice that were unidentifiable were significantly greater than WT controls. We identified D-regions on the basis of six contiguous nucleotides. D. The average length of N/P-nucleotide addition was reduced in the POLB^C/C mice. The allocation of N/P nucleotides was based on the known sequences of the germline elements. The average length of the N/P region between the rearranged V and D junction (N1) was significantly shorter in POLB^C/C mice compared to WT controls. The N/P region between the rearranged D and J junction (N2) was not significantly different between the POLB^{+/+ and C/C} mice.

Figure 3. CSR is normal in the POLB^Y265c/c mice

Naïve splenic B cells were isolated from spleens of 3–5 POLB^Y265C/C and WT control mice and induced in vitro with either LPS to induce switching to IgG1, LPS with IL-4 to induce switching to IgG3, LPS with INFγ to induce switching to IgG2a, or LPS with TGFβ to induce switching to IgG2b. No significant change in CSR in the B cells was observed between the POL B^Y265C/C and WT mice.

Figure 4. The rate of SHM is increased in the POLB^Y265c/c mice

A. Products were amplified using PCR and genomic DNA isolated from B220⁺ PNA^high Peyer’s patches of 3–5 month-old POLB^{+/+ and C/C} mice. These products were cloned and sequenced. Somatic hypermutation results showing that the overall frequencies are increased in the POLB^Y265c/c mice compared to WT. B. For SHM the 344 nucleotides downstream of the J_H4 gene region of rearranged VDJ segments on the heavy chain locus of the VHJ558 gene was PCR-amplified and sequenced. The numbers of mutations versus the total length of DNA sequences and the mutation frequency were analyzed in three mutant and three age-matched WT animals, using SHMTool, which is a webserver for comparative analysis of somatic hypermutation datasets. At the center of the pie chart is shown the numbers of clones analyzed for each genotype. Segments show the percentage of each clone that contained a defined number of mutations and these are indicated with different colors. C. All types of mutations are increased (p=3.7 × 10⁻¹⁸) in the J_H4 intron from the POLB^Y265c/c, but the types of mutations that are most significantly increased are transversions at GC base pairs (p= 2.8 × 10⁻⁸), followed by transitions at GC (p = 7.0 × 10⁻⁷), transversions at AT (p= 3 × 10⁻⁵), and transitions at AT base pairs (p = 0.0029). At least three mice of each genotype were used for these experiments. D. Amino acid motifs where many of the SHM mutations occur in the POLB^Y265c/c mice compared to WT. Mutations arise predominantly in the GYW (p = 7.4 × 10⁻⁵), RGYW (p=0.0003), and DGYW (p = 7.4 × 10⁻⁵), which are the motifs in which deamination catalyzed by activation-induced cytidine deaminase (AID) occur.

Figure 5. The POLB^Y265c/c mice have increased numbers of germinal centers compared to WT mice

A. Spleens of 3–4 five month-old mice were frozen with OCT compound, prepared as a frozen section and stained overnight at 4°C for confocal analysis. PNA-positive germinal centers were counted per field (25×). The graph compares the numbers of germinal centers in POLB^Y265c/c and WT mice and the image depicts an example of multiple germinal centers in the spleen of a POLB^Y265c/c mouse. B. Examples of FACs sorted splenic cells. For flow cytometry, splenic cells from 3–5 five month-old mice were processed and stained with anti-mouse antibodies (eBioscience) as described in Supplementary Methods. Top. Germinal center B-cells from a 5 month-old mouse were CD19⁺IgD-lo gated followed by gating for CD95 Gl7 double-positive cells (WT (+/+) is on the left and c/c is on the right). Bottom. Follicular help T-cells (T_FH) were CD4+ CD44-lo gated followed by gating for CXCR5 PD-1 double-positive cells. C. Graph showing percent TUNEL positive cells from the POLB^Y265c/c and WT mice spleen, which is representative of at least three mice of each genotype. D. Spleen frozen sections were stained overnight at 4°C for c onfocal analysis. The image represents an example of germinal centers in the spleen of POLB^Y265c/c and WT mice. PNA-positive germinal centers are shown green (FITC), the blue (αCD4-Cy5) shows the CD4 positive helper T cells, and the TUNEL positive cells are shown in red. E. Graph showing percent TUNEL positive cells in the germinal center of POLB^Y265c/c and WT mice, which is representative of 8–10 germinal centers of 2–3 mice of each genotype.

Figure 6. Model of aberrant V(D)J and SHM in the POLB^Y265c/c mice

A. Y265C Pol β promotes deletion during joining of the V and D fragments. This cartoon depicts joining of the V to the D fragment. After the Rag proteins cleave the DNA, hairpins form. Artemis nicks the hairpins which sometime results in a substrate with DNA gaps. WT Pol β or other × family polymerases fill the gaps. However, in the presence of the slow Y265C Pol β polymerase, gaps are filled inefficiently, which can result in nuclease activity leading to deletions and shortening of the CDR3 junction. B. Y265C permits highly error-prone SHM resulting in autoimmunity. After deamination of cytosine by AID, replication can occur resulting in transitions. Alternatively, the mismatch repair pathway can bind to the U:G mismatch and recruit Exo 1, resulting in mutations at A:T base pairs. A third possibility is that APE1 incises the DNA at the AP site, resulting in a single nucleotide gap that is normally filled in by Pol β. However, in the presence of Y265C Pol β the gap is filled inefficiently or not at all, leading to translesion synthesis and increased mutagenesis.

Figure 6. Model of aberrant V(D)J and SHM in the POLB^Y265c/c mice

A. Y265C Pol β promotes deletion during joining of the V and D fragments. This cartoon depicts joining of the V to the D fragment. After the Rag proteins cleave the DNA, hairpins form. Artemis nicks the hairpins which sometime results in a substrate with DNA gaps. WT Pol β or other × family polymerases fill the gaps. However, in the presence of the slow Y265C Pol β polymerase, gaps are filled inefficiently, which can result in nuclease activity leading to deletions and shortening of the CDR3 junction. B. Y265C permits highly error-prone SHM resulting in autoimmunity. After deamination of cytosine by AID, replication can occur resulting in transitions. Alternatively, the mismatch repair pathway can bind to the U:G mismatch and recruit Exo 1, resulting in mutations at A:T base pairs. A third possibility is that APE1 incises the DNA at the AP site, resulting in a single nucleotide gap that is normally filled in by Pol β. However, in the presence of Y265C Pol β the gap is filled inefficiently or not at all, leading to translesion synthesis and increased mutagenesis.