Purine metabolites in cell lysates of non-transfected (non TR) and wt transfected (TR) CR-cells (n = 3).

<p>Purine metabolites in cell lysates of non-transfected (non TR) and wt transfected (TR) CR-cells (n = 3).</p

Immunodetection of PPAT and GART in CR-ATIC cells transfected with constructs encoding wt-ATIC proteins.

<p>In cells transfected with constructs encoding wt-ATIC protein, the endogenous proteins PPAT (A) and GART (B) were observed as fine granules with their fluorescent signals showing a high degree of overlap in purine-depleted medium (C), whereas in purine-rich medium, the proteins PPAT (G) and GART (H) remained diffuse and did not colocalize (I). The same behaviour was observed in the control HeLa cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201432#pone.0201432.g009" target="_blank">Fig 9</a>). When the cells were not transfected, the endogenous proteins remained diffuse regardless of the level of purines in the media (D, E, J, K) and did not colocalize (F, L). The values of the fluorescent signal overlaps are shown in pseudocolour, and the scale is shown at the lower right in the corresponding LUT.</p

Immunochemical labelling of the endogenous proteins PPAT and PAICS in CR-GART cells transfected with constructs encoding wt-GART proteins.

<p>In cells transfected with constructs encoding the wt-GART protein, the endogenous proteins PPAT (A) and PAICS (B) were observed in the form of fine granules with their fluorescent signals showing a high degree of overlap in purine-depleted medium (C), whereas in purine-rich medium, the proteins PPAT (G) and PAICS (H) remained diffuse and did not colocalize (I). The same behaviour was observed in the control HeLa cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201432#pone.0201432.g009" target="_blank">Fig 9</a>). Endogenous proteins in the non-transfected cells remained diffuse regardless of the level of purines in the media (D, E, J, K) and did not colocalize (F, L). The values of the fluorescent signal overlaps are shown in pseudocolour, and the scale is shown at the lower right in the corresponding LUT.</p

Scheme of <i>de novo</i> purine synthesis (DNPS), the salvage pathway, the degradation pathway and the purinosome.

<p>The initial substrate in DNPS is phosphoribosyl pyrophosphate (PRPP). Six enzymes are involved in DNPS and the purinosome multienzyme complex: phosphoribosyl pyrophosphate amidotransferase (PPAT), the trifunctional enzyme GART (glycinamide ribonucleotide synthetase/glycinamide ribonucleotide transformylase/aminoimidazole ribonucleotide synthetase), phosphoribosylformylglycinamidine synthetase (PFAS), the bifunctional enzyme PAICS (phosphoribosylaminoimidazole carboxylase/phosphoribosylaminoimidazolesuccinocarboxamide synthetase), adenylosuccinate lyase (ADSL), and the bifunctional enzyme ATIC (5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase). The final product is inosine monophosphate (IMP). IMP is converted into adenosine monophosphate (AMP) and guanosine monophosphate (GMP) and is also degraded to uric acid via the degradation pathway. The hypoxanthine intermediate can be recycled by the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) into IMP or GMP.</p

Immunodetection of PPAT and GART in CR-ADSL cells transfected with constructs encoding wt-ADSL proteins.

<p>In cells transfected with constructs encoding wt-ADSL protein, the endogenous proteins PPAT (A) and GART (B) were found in the form of fine granules with their fluorescent signals showing a high degree of overlap in purine-depleted medium (C), whereas in purine-rich medium, the proteins PPAT (G) and GART (H) remained diffuse and did not colocalize (I). The same behaviour was observed in the control HeLa cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201432#pone.0201432.g009" target="_blank">Fig 9</a>). When the cells were not transfected, the endogenous proteins remained diffuse regardless of the level of purines in the media (D, E, J, K) and did not colocalize (F, L). The values of the fluorescent signal overlaps are shown in pseudocolour, and the scale is shown at the lower right in the corresponding LUT.</p

Immunochemical labelling of the endogenous proteins GART and PPAT in CR-ADSL cells transfected with a construct encoding a mutant inactive protein, p.Y114H ADSL.

<p>CR-ADSL cells were transfected with a vector encoding the BFP-labelled inactive protein p.Y114H ADSL (A, E) and seeded in purine-depleted medium (A, B, C, D) or purine-rich medium (E, F, G, H). The endogenous proteins PPAT (B, F) and GART (C, G) remained diffuse, and the signals did not colocalize, regardless of the amount of purines in the growth media (D, H). The values of the fluorescent signal overlaps are shown in pseudocolour, and the scale is shown at the lower right in the corresponding LUT.</p

Immunodetection of PPAT, GART and PAICS in control HeLa cells.

<p>Control HeLa cells were immunolabelled with PPAT and GART (A, B, C, G, H, I) or with PPAT and PAICS (D, E, F, J, K, L). Both combinations of the endogenous proteins formed granules (A, B, D, E) with high signal colocalization in the purine-depleted medium (C, F), whereas in the purine-rich medium, the proteins remained diffuse (G, H, J, K) with no colocalization (I, L). The values of the fluorescent signal overlaps are shown in pseudocolour, and the scale is shown at the lower right in the corresponding LUT.</p

Characterization of the HGPRT knockout cells.

<p>(A): Illustration of the sgRNA targeting sequence in exon 4 of the <i>HPRT1</i> gene in control and CR-HGPRT cells and the protein sequences of wild-type and mutated HGPRT. The 20-bp target sgRNA sequence is indicated in the blue box, adjacent to the NGG (TGG) PAM motif sequence (red coloured). The probable Cas9 cut site is indicated by the red flash-shaped object. CR-HGPRT is shown with the appropriate c.373-378delTTAACT mutation. In the HGPRT protein sequence, the affected amino acids are coloured red; the CR-HGPRT protein shows a p. 125-126del deletion. (B): The activity of the HGPRT enzyme was determined in the control and CR-HGPRT cells. The activity of the enzyme in the CR-HGPRT cells was 0% of the activity in the control cells (n = 3). (C): Growth curves of CR-HGPRT cells and control HeLa cells. The cells were grown in normal growth medium and in growth medium containing 0.03 mM thioguanine (TG). The CR-HGPRT cells grew in the normal growth medium (light red line) and more slowly in the growth medium containing TG (dark red line). All the control cells in the medium containing TG died within 72 h (dark blue line), while the control cells in the normal medium showed ten times more growth at the same time point (light blue line).</p

Immunodetection of PPAT and GART in CR-PAICS cells transfected with constructs encoding wt-PAICS proteins.

<p>In cells transfected with constructs encoding wt-PAICS protein, the endogenous proteins PPAT (A) and GART (B) were observed in the form of fine granules with their fluorescent signals showing a high degree of overlap in purine-depleted medium (C), whereas in purine-rich medium, the proteins PPAT (G) and GART (H) remained diffuse and did not colocalize (I). The same behaviour was observed in the control HeLa cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201432#pone.0201432.g009" target="_blank">Fig 9</a>). Endogenous proteins in the non-transfected cells remained diffuse regardless of the level of purines in the media (D, E, J, K) and did not colocalize (F, L). The values of the fluorescent signal overlaps are shown in pseudocolour, and the scale is shown at the lower right in the corresponding LUT.</p

Validation of CZECANCA (CZEch CAncer paNel for Clinical Application) for targeted NGS-based analysis of hereditary cancer syndromes

<div><p>Background</p><p>Carriers of mutations in hereditary cancer predisposition genes represent a small but clinically important subgroup of oncology patients. The identification of causal germline mutations determines follow-up management, treatment options and genetic counselling in patients’ families. Targeted next-generation sequencing-based analyses using cancer-specific panels in high-risk individuals have been rapidly adopted by diagnostic laboratories. While the use of diagnosis-specific panels is straightforward in typical cases, individuals with unusual phenotypes from families with overlapping criteria require multiple panel testing. Moreover, narrow gene panels are limited by our currently incomplete knowledge about possible genetic dispositions.</p><p>Methods</p><p>We have designed a multi-gene panel called CZECANCA (CZEch CAncer paNel for Clinical Application) for a sequencing analysis of 219 cancer-susceptibility and candidate predisposition genes associated with frequent hereditary cancers.</p><p>Results</p><p>The bioanalytical and bioinformatics pipeline was validated on a set of internal and commercially available DNA controls showing high coverage uniformity, sensitivity, specificity and accuracy. The panel demonstrates a reliable detection of both single nucleotide and copy number variants. Inter-laboratory, intra- and inter-run replicates confirmed the robustness of our approach.</p><p>Conclusion</p><p>The objective of CZECANCA is a nationwide consolidation of cancer-predisposition genetic testing across various clinical indications with savings in costs, human labor and turnaround time. Moreover, the unified diagnostics will enable the integration and analysis of genotypes with associated phenotypes in a national database improving the clinical interpretation of variants.</p></div