A novel PKD1 variant demonstrates a disease-modifying role in trans with a truncating PKD1 mutation in patients with Autosomal Dominant Polycystic Kidney Disease

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common form of Polycystic Kidney Disease (PKD) and occurs at a frequency of 1/800 to 1/1000 affecting all ethnic groups worldwide. ADPKD shows significant intrafamilial phenotypic variability in the rate of disease progression and extra-renal manifestations, which suggests the involvement of heritable modifier genes. Here we show that the PKD1 gene can act as a disease causing and a disease modifier gene in ADPKD patients

A novel PKD1 variant demonstrates a disease-modifying role in trans with a truncating PKD1 mutation in patients with Autosomal Dominant Polycystic Kidney Disease

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common form of Polycystic Kidney Disease (PKD) and occurs at a frequency of 1/800 to 1/1000 affecting all ethnic groups worldwide. ADPKD shows significant intrafamilial phenotypic variability in the rate of disease progression and extra-renal manifestations, which suggests the involvement of heritable modifier genes. Here we show that the PKD1 gene can act as a disease causing and a disease modifier gene in ADPKD patients

Separation of plasmacytoid dendritic cells from B-cell-biased lymphoid progenitor (BLP) and Pre-pro B cells using PDCA-1.

B-cell-biased lymphoid progenitors (BLPs) and Pre-pro B cells lie at a critical juncture between B cell specification and commitment. However, both of these populations are heterogenous, which hampers investigation into the molecular changes that occur as lymphoid progenitors commit to the B cell lineage. Here, we demonstrate that there are PDCA-1(+)Siglec H(+) plasmacytoid dendritic cells (pDCs) that co-purify with BLPs and Pre-pro B cells, which express little or no CD11c or Ly6C. Removal of PDCA-1(+) pDCs separates B cell progenitors that express high levels of a Rag1-GFP reporter from Rag1-GFP(low/neg) pDCs within the BLP and Pre-pro B populations. Analysis of Flt3-ligand knockout and IL-7Rα knockout mice revealed that there is a block in B cell development at the all-lymphoid progenitor (ALP) stage, as the majority of cells within the BLP or Pre-pro B gates were PDCA-1(+) pDCs. Thus, removal of PDCA-1(+) pDCs is critical for analysis of BLP and Pre-pro B cell populations. Analysis of B cell potential within the B220(+)CD19(-) fraction demonstrated that AA4.1(+)Ly6D(+)PDCA-1(-) Pre-pro B cells gave rise to CD19(+) B cells at high frequency, while PDCA-1(+) pDCs in this fraction did not. Interestingly, the presence of PDCA-1(+) pDCs within CLPs may help to explain the conflicting results regarding the origin of these cells

Neither Ly-6C nor CD11c eliminates PDCA-1+ pDCs from Pre-pro B cells.

<p>(A) Cells within the Pre-pro B gate were analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078408#pone-0078408-g003" target="_blank">Figure 3A</a>. (B) Cells within the Pre-pro B gate were analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078408#pone-0078408-g003" target="_blank">Figure 3A</a>, except that Ly-6C was also included. In the upper panels, cells were first gated to exclude Ly-6C. Exclusion of Ly-6C+ cells did not remove PDCA-1+ cells from Pre-pro B cells. In the lower panel, cells were first gated to exclude PDCA-1+. Exclusion of PDCA-1+ cells removed all Ly-6C+ cells from Pre-pro B cells. (C) Cells within the Pre-pro B fraction were analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078408#pone-0078408-g003" target="_blank">Figure 3A</a>, except that CD11c was also included. In the upper panels, cells were first gated to exclude CD11c. Exclusion of CD11c+ cells did not remove PDCA-1+ cells from Pre-pro B cells. In the lower panel, cells were first gated to exclude PDCA-1+. Exclusion of PDCA-1+ cells removed all CD11c+ cells from Pre-pro B cells.</p

Severe decrease in BLPs in IL-7Rα and Flt3-ligand deficient mice revealed upon exclusion of pDCs.

<p>(A) WT and Flt3-ligand (FL) knockout (KO) mice were examined for ALPs and BLPs using the gating strategy described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078408#pone-0078408-g001" target="_blank">Figure 1A</a>, except that Ly6C was analyzed separately from lineage (using a lineage cocktail as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078408#pone-0078408-g001" target="_blank">Figure 1A</a> except lacking Ly6C). Data shown is representative of 4 WT and 4 FL KO mice from 4 independent experiments. (B) WT and IL-7Rα KO mice were examined for ALPs and BLPs using the gating strategy as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078408#pone-0078408-g002" target="_blank">Figure 2A</a>, except that IL-7Rα expression was not used to define CLPs. Data shown is representative of 4 WT and 4 FL KO mice from 4 independent experiments. (C) The absolute number of PDCA-1⁻ BLPs, total Ly6D⁺ CLPs and PDCA-1⁺ pDCs within the CLP population per femur was calculated in WT, FL KO and IL-7Rα KO mice. In the left panel, PDCA-1⁻ BLPs were analyzed after exclusion of PDCA-1⁺ pDCs from the Ly-6D⁺ CLP gate. In the middle panel, total Ly6D⁺ CLPs were analyzed without exclusion of PDCA-1⁺ pDCs from the Ly6D⁺ CLPs gate. In the right panel, total PDCA-1⁺ pDCs within the CLP population were examined. Statistical analysis was performed using an unpaired two-tailed t test (GraphPad Prism).</p

Identification of a plasmacytoid dendritic cell subset within the common lymphoid progenitor population.

<p>(A) Flow cytometry analysis was used to examine CLP populations from Rag1-GFP mice. Cells were first gated according to expression of IL-7Rα and low/negative of expression of lineage markers using a lineage cocktail containing antibodies against CD3ε, TCRβ, TCRγδ, B220, CD19, Ly6C, Ter119, CD11c, CD11b, Gr-1, NK1.1 and CD8α (left panel). These IL-7Rα⁺lineage^low cells were examined for expression of c-kit and Flt3 (middle panel). CLPs were identifed as IL-7Rα⁺lineage^low c-Kit^loFlt3⁺. CLPs were separated into three populations based on expression of Ly6D and PDCA-1 (right panel): ALP (Ly6D⁻PDCA-1⁻, green gate), BLP (Ly6D⁺PDCA-1⁻, red gate), and PDCA-1⁺ CLPs (Ly6D⁺PDCA-1⁺, blue gate). Each population was examined for expression of Rag1-GFP reporter, Flt3, c-kit and IL-7Rα. The analysis shown is representative of 4 mice analyzed in 3 independent experiments. (B) Analysis of expression of Siglec H and DX5 in ALP (green gate), BLP (red gate) or PDCA-1⁺ CLPs (blue gate) populations, gated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078408#pone-0078408-g001" target="_blank">figure 1(A)</a>. (C) Quantitative PCR was performed on ALPs, BLPs and PDCA-1⁺ CLPs (color coded as in the gating shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078408#pone-0078408-g001" target="_blank">figure 1(A)</a>). Each population was independently isolated by FACS sorting using the gating strategy in (A). Relative expression of EBF1, Pax5, Runx2 and Pacsin1 is shown. Data shown is the average of 4 independently sorted samples from 4 WT mice, and error bars reflect the standard error of the mean. Data was normalized to the expression of each gene in one of the four WT BLP samples. Significance was quantified using unpaired two-tailed t test (GraphPad Prism).</p

Severe decrease in Pre-pro B cells in IL-7Rα and Flt3-ligand deficient mice revealed upon exclusion of pDCs.

<p>A. WT, IL-7Rα knockout, and Flt3 ligand knockout mice were analyzed for the presence of Pre-pro B cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078408#pone-0078408-g003" target="_blank">Figure 3</a>. Data shown is from a representative experiment that was replicated independently four times. B. Absolute numbers of Pre-pro B cells from WT, IL-7Rα knockout, and Flt3 ligand knockout mice. On the left is the analysis of Pre-pro B cells (B220⁺CD19⁻AA4.1⁺Ly6D⁺) after elimination of PDCA-1+ pDCs. On the right is the analysis of Pre-pro B cells without elimination of PDCA-1+ pDCs. Error bars represent SEM from 4 mice in each group analyzed in 4 independent experiments. Statistical analysis was performed using an unpaired two-tailed t test (GraphPad Prism).</p

pDCs also contaminate the Pre-pro B population.

<p>(A) Flow cytometry analysis was performed on bone marrow cells to subfractionate B220⁺CD19⁻ Fraction A. Using AA4.1, Ly6D and PDCA-1, four populations were distinguished within the B220⁺CD19⁻ population. Each of the gated populations defined were examined for expression of Rag1-GFP mice. The black histogram is the negative control, showing lack of GFP expression in WT mice, with each population color coded to the gating of each population highlighted in A (Purple is B220⁺CD19⁻AA4.1⁻; Red is B220⁺CD19⁻AA4.1⁺Ly6D⁺PDCA-1⁻; Blue is B220⁺CD19⁻AA4.1⁺Ly6D⁺PDCA-1⁺; and Green is B220⁺CD19⁻AA4.1⁺Ly6D⁻PDCA-1⁺). Shown are representative plots of 4 mice from 3 independent experiments. (B) Examination of cell surface markers to distinguish the two populations of B220⁺CD19⁻AA4.1⁺Ly6D⁺ cells. Histograms are color coded as in A and B, with red representing expression in B220⁺CD19⁻AA4.1⁺Ly6D⁺PDCA-1⁻ cells and blue representing expression in B220⁺CD19⁻AA4.1⁺Ly6D⁺PDCA-1⁺ cells. Shown are representative plots from four mice analyzed in two independent experiments. (C) B lineage potential of B220⁺CD19⁻AA4.1⁺Ly6D⁺PDCA-1⁻ cells (red gate in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078408#pone-0078408-g003" target="_blank">figure 3A</a>) and B220⁺CD19⁻AA4.1⁺Ly6D⁺PDCA-1⁺ cells (blue gate in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078408#pone-0078408-g003" target="_blank">figure 3A</a>) was examined by sorting each population onto OP9 stromal cells in media containing IL-7, Stem cell factor (SCF) and Flt3 ligand. After 4 days, cells were stained with CD19 and the total number of CD19⁺ cells generated compared to the number of input sorted cells per well was calculated (frequency of generation of CD19⁺ cells). The frequency was calcuated from 7 independent wells per sample from 3 independent sorts. Statistical analysis was performed using an unpaired two-tailed t test (GraphPad Prism).</p