Establishment of a Conditionally Immortalized Wilms Tumor Cell Line with a Homozygous <i>WT1</i> Deletion within a Heterozygous 11p13 Deletion and UPD Limited to 11p15

<div><p>We describe a stromal predominant Wilms tumor with focal anaplasia and a complex, tumor specific chromosome 11 aberration: a homozygous deletion of the entire <i>WT1</i> gene within a heterozygous 11p13 deletion and an additional region of uniparental disomy (UPD) limited to 11p15.5-p15.2 including the <i>IGF2</i> gene. The tumor carried a heterozygous p.T41A mutation in <i>CTNNB1</i>. Cells established from the tumor carried the same chromosome 11 aberration, but a different, homozygous p.S45Δ <i>CTNNB1</i> mutation. Uniparental disomy (UPD) 3p21.3pter lead to the homozygous <i>CTNNB1</i> mutation. The tumor cell line was immortalized using the catalytic subunit of human telomerase (h<i>TERT</i>) in conjunction with a novel thermolabile mutant (U19dl89-97tsA58) of SV40 large T antigen (LT). This cell line is cytogenetically stable and can be grown indefinitely representing a valuable tool to study the effect of a complete lack of <i>WT1</i> in tumor cells. The origin/fate of Wilms tumors with <i>WT1</i> mutations is currently poorly defined. Here we studied the expression of several genes expressed in early kidney development, e.g. <i>FOXD1</i>, <i>PAX3</i>, <i>SIX1</i>, <i>OSR1</i>, <i>OSR2</i> and <i>MEIS1</i> and show that these are expressed at similar levels in the parental and the immortalized Wilms10 cells. In addition the limited potential for muscle/ osteogenic/ adipogenic differentiation similar to all other <i>WT1</i> mutant cell lines is also observed in the Wilms10 tumor cell line and this is retained in the immortalized cells. In summary these Wilms10 cells are a valuable model system for functional studies of <i>WT1</i> mutant cells.</p></div

Comparison of <i>CTNNB1</i> mutations and UPD region on chromosome 3 in the primary Wilms10 tumor and tumor-derived cells.

<p>(A) DNA sequencing reveals a heterozygous A>G <i>CTNNB1</i> mutation at position c.121/p.T41A in the primary Wilms10 tumor. The position of the three deleted nucleoties in the tumor cells culture are boxed. (B) identification of a homozygous deletion c.133_135del TCT (p.S45Δ) in the <i>CTNNB1</i> gene in the Wilms10 tumor-derived cell line. The position of this deletion is indicated above the DNA sequence. (C) UPD region on chromosome 3p as shown with the cytogenomics workbench program.</p

Quantitative RT-PCR analysis of the <i>WT1</i> expression in various WT cell lines.

<p>Total RNA from the <i>WT1</i> mutant cell lines Wilms1, Wilms2, Wilms3, Wilms8 and Wilms10 was analyzed by Q-RT-PCR. The genetic alterations present in these cell lines are found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155561#pone.0155561.s016" target="_blank">S1 Table</a>. The expression level was normalized versus <i>RER1</i>, a gene with the lowest variation between the cell lines in our gene expression studies. The analysis was performed in triplicates. The error bars correspond to 95% confidence intervals. The relative expression is shown versus Wilms3.</p

Similar expression level of selected kidney marker genes in imWilms10 and Wilms10 cells.

<p>The selection of these genes was based on studies of gene expression in various compartments during kidney development. All of these genes are also expressed in the other <i>WT1</i> mutant cell lines that we have previously established. The data are derived from microarray analyses of two biological replicates. The expression level is indicated from microarray intensity and the bar represents standard error.</p

The top pathway down-regulated in imWilms10 versus non immortalized Wilms10 cells.

<p>(A) Using a gene set derived from the stringent parameters (FDR adjusted p-value of 0.1) the top down-regulated pathway in imWilms10 is Development_Hedgehog and PTH signalling pathways in bone and cartilage development". Down-regulated genes from this pathway are labelled with a thermometer. The height of the blue colour in the thermometer shows the fold down-regulation in the immortalized cells. (B) shown is the down-regulation of the four genes mapping to this pathway by expression intensity on the biological replicates on the arrays.</p

Differentiation potential of Wilms10 and imWilms10 cell lines.

<p>(A) Analysis of osteogenesis as measured by quantification of calcium production. Parental Wilms10 cells deposit some calcium in the absence of induction conditions. hMSC cells were used as controls and they show a high level of calcium production after induction of differentiation. The imWilms10 cells, demonstrate a modest increase of calcium production as compared to parental Wilms10 cells. (B) Analysis of muscle differentiation by immunofluorescence analysis using a Titin antibody. Left: Most Wilms10 cells show positive staining for Titin after 9 days of induction. Right: the same analysis was performed with imWilms10 cells. A lower percentage of cells showed a positive staining for Titin. (C) Quantitative analysis <i>PPARG</i> mRNA expression, as a marker for adipogenesis. Left: After 10 days of induction a significant increase is seen in Wiilms10 cells compared to the uninduced control. The expression was normalized versus <i>RER1</i> and the analysis was done in triplicates. The error bar corresponds to 95% confidence intervals and * corresponds to a significance level of p = 0.00001- Right: The same analysis was conducted with imWilms10 cells and a slightly lower induction was observed when compared to uninduced control cells. Expression analysis was done after 18 days of induction of imWilms10 cells. The error bar corresponds to 95% confidence intervals and * corresponds to a significance level of p = 0.00001.</p

Down-regulation of the two embryonal growth factors <i>IFG2</i> and <i>MEST</i>.

<p>(A) Down-regulation of <i>IGF2</i> in imWilms10 cells cultured at 33°C as seen in two biological replicates on the Agilent array (left). The down-regulation was confirmed by Q-RT-PCR and is seen when cells are cultured at 33° and 37°C (right). The error bars correspond to 95% confidence intervals and * corresponds to a significance level of p 0.000001. (B) Down-regulation of <i>MEST</i> RNA as seen in the two biological replicates (left) and the confirmation of the protein down-regulation by western blot analysis. The down-regulation is seen at all temperatures, even at 39°C, the nonpermissive temperature for the tsLT, indicating that it is due to h<i>TERT</i> expression and is independent on the functional LT.</p

Biallelic variants in YRDC cause a developmental disorder with progeroid features

The highly conserved YrdC domain-containing protein (YRDC) interacts with the well-described KEOPS complex, regulating specific tRNA modifications to ensure accurate protein synthesis. Previous studies have linked the KEOPS complex to a role in promoting telomere maintenance and controlling genome integrity. Here, we report on a newborn with a severe neonatal progeroid phenotype including generalized loss of subcutaneous fat, microcephaly, growth retardation, wrinkled skin, renal failure, and premature death at the age of 12 days. By trio whole-exome sequencing, we identified a novel homozygous missense mutation, c.662T > C, in YRDC affecting an evolutionary highly conserved amino acid (p.Ile221Thr). Functional characterization of patient-derived dermal fibroblasts revealed that this mutation impairs YRDC function and consequently results in reduced t(6)A modifications of tRNAs. Furthermore, we established and performed a novel and highly sensitive 3-D Q-FISH analysis based on single-telomere detection to investigate the impact of YRDC on telomere maintenance. This analysis revealed significant telomere shortening in YRDC-mutant cells. Moreover, single-cell RNA sequencing analysis of YRDC-mutant fibroblasts revealed significant transcriptome-wide changes in gene expression, specifically enriched for genes associated with processes involved in DNA repair. We next examined the DNA damage response of patient's dermal fibroblasts and detected an increased susceptibility to genotoxic agents and a global DNA double-strand break repair defect. Thus, our data suggest that YRDC may affect the maintenance of genomic stability. Together, our findings indicate that biallelic variants in YRDC result in a developmental disorder with progeroid features and might be linked to increased genomic instability and telomere shortening