Massive parallel sequencing in steroid-resistant nephrotic syndrome (SRNS)

<p><strong>ABSTRACT</strong></p> <p><strong>Introduction</strong>. To date abnormalities in more than 20 genes have been associated with SRNS. Sequencing of all SRNS genes requires ~ 600 PCR amplicons, rendering conventional mutation testing unfeasible due to financial and time constraints. Hence, current screening algorithms usually include only the most common disease genes and/or use preselection according to additional phenotypic criteria. This practice typically allows for mutation detection in ~15% of patients. We have evaluated targeted NGS screening of SRNS patients enrolled in the PodoNet registry who were found negative for mutations in the first-line SRNS-associated genes.</p> <p><strong>Material and Methods</strong>. Molecular analysis of 31 known or plausible SDNS disease genes was performed by NGS using a custom-designed multiplex PCR kit (MASTR FSGS, Multiplicom). The pilot group consisted of 22 patients with 4.7 years median age at disease onset (range 0.5-20 years), positive family history in 68%, parental consanguinity in 18% and chronic renal insufficiency at last observation in 59%.</p> <p><strong>Results.</strong> Mean coverage was 1269x (median 1286, range 929-1826). 18/22 runs had at least 15x read depth covering 99% of the target sequences. One patient was diagnosed with hereditary SRNS due to a previousy described homozygous pathogenic mutation in SMARCAL1 gene. In addition, three novel sequence variants in the genes PLCE1 (homozygous), LAMB2 (homozygous) and WT1 (heterozygous) were detected. In silico studies support their classification as pathogenic, even though the patients do not present the characteristic clinical and/or histopathological features typically reported for patients with mutations in these genes.</p> <p><strong>Conclusions</strong>. Our detection of pathogenic mutations in 4 out of 22 SRNS patients screened negative by conventional selective screening approaches support targeted NGS testing in all SRNS patients, regardless of age at diagnosis, absence of extrarenal manifestations or histological subtype. We anticipate that systematic NGS screening of the SRNS cohorts collected in EURenOmics will allow re-evaluation of mutation incidence rates in SRNS and become the new standard of genetic diagnostics in this condition.</p

Plasma ACE activity of the members of the affected family.

<p>ACE activity in heparinized plasma (1/5 dilution in PBS) of members of the affected family, the father (F), mother (M), and brother (B) as well as in 5 healthy individuals (marked by initials) that served as controls was determined by fluorimetric assay of 40 µl diluted plasma with 200 µl each of substrates Hip-His-Leu (HHL) and Z-Phe-His-Leu(ZPHL) during 1 hour of incubation. <b>A.</b> ACE activity with HHL: mean (± SD) of three independent determinations. <b>B.</b> Ratio of hydrolysis of the two substrates (<b>ZPHL/HHL ratio</b>) in the tested samples, which is used to characterize for each catalytic domain of ACE in somatic two-domain ACE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010438#pone.0010438-Danilov3" target="_blank">[28]</a>. ND-not determined. * p<0.05 vs. control samples.</p

Chaperone-like effect of anti-ACE mAbs on mutant ACE.

<p>Membrane-bound (<b>A</b>) and soluble (<b>B</b>) mutant ACE (Q1069R) were pre-incubated with N-domain specific mAb 9B9 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010438#pone.0010438-Danilov4" target="_blank">[30]</a> or C-domain specific mAbs 1B3 and 1B8 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010438#pone.0010438-Naperova1" target="_blank">[16]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010438#pone.0010438-Balyasnikova6" target="_blank">[36]</a> at the indicated concentrations for 2 hrs at room temperature and then residual activity was determined with 5 mM HHL and 2 mM ZPHL as substrates. Experimental conditions are as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010438#pone-0010438-g005" target="_blank">Fig. 5</a>. Data presented as ZPHL/HHL ratio of ACE activity in the presence mAb. Results shown are the mean ± SD of 3–4 experiments.</p

Mutant ACE protein and mRNA quantification.

<p>CHO cells were transiently transfected with plasmids coding for wild-type (WT) and mutant (Q1069R) ACE (4 µg of plasmid DNA per 35 mm dish). The lysates of these cells (normalized by equal protein loading of 10 µg per lane) were subjected to SDS-PAGE (4–15% gradient gel) in reducing conditions for Western blotting (<b>A</b>) or ACE mRNA quantification (<b>C</b>). <b>A</b>. Western blotting was performed with rat mAb (4G6) that recognizes the denatured epitope on the N-domain of human ACE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010438#pone.0010438-Balyasnikova8" target="_blank">[38]</a>. Proteins transferred on PVDF-Plus membrane were revealed with 2 µg/ml of indicated mAb. Molecular weight markers are shown by arrows on the left of panel A, which is a representative experiment. <b>B</b>. The relative amount of WT and mutant ACE revealed by Western blotting with mAb 4G6 (<b>A</b>) was quantified by the image analysis (densitometry) using ImageJ software (NIH). Data are expressed as mean ± SD of 3 independent experiments. <b>C</b>. Relative mRNA concentrations were calculated from the takeoff point of reactions using manufacturer's software and normalized to α-tubulin. Data are expressed as mean ± SD of 3 independent experiments.</p

Effect of temperature, chaperones, and proteolytic inhibitors on ACE activity.

<p>ACE activity of membrane-bound form of WT and mutant ACE after culturing cells 24 hrs in different conditions listed below as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010438#s2" target="_blank">Materials and Methods</a>. ACE activity was determined by fluorimetric assay as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010438#pone-0010438-g005" target="_blank">Figure5</a> using Z-Phe-His-Leu (ZPHL) as substrate. The following compounds were tested: Sodium butyrate (5 mM); Enalaprilat (1 µM), MG132 (5 µM), Bortezomib (5 µM). Data are the mean values (± SD) of 3–4 independent experiments measured in duplicate expressed as the percentage of ACE activity in the lysate of WT or mutant ACE expressing cells cultured at 37°C.</p

Conformational fingerprinting of mutant ACE.

<p>Membrane-bound WT and mutant ACE lysates were normalized to achieve 5 mU/ml ACE activity with Z-Phe-His-Leu as substrate and incubated in microtiter plate wells covered with 16 mAbs to human ACE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010438#pone.0010438-Naperova1" target="_blank">[16]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010438#pone.0010438-Danilov4" target="_blank">[30]</a> via goat-anti-mouse IgG. Precipitated ACE activity was quantified by fluorimetric plate precipitation assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010438#pone.0010438-Danilov4" target="_blank">[30]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010438#pone.0010438-Danilov6" target="_blank">[54]</a>. Data (mean ± SD of 6–8 independent experiments in duplicate) are expressed as ratio of ACE activity precipitated by mAbs from mutant ACE to that of WT ACE. * p<0.05 vs. WT ACE.</p

Differential subcellular localization of wild type and mutant ACE.

<p>Confocal microscopy was used to determine the localization of WT- vs. Q1069R-ACE mutant expressed in CHO cells following immunostaining with anti-ACE mAb 9B9 and Alexa 488 goat anti-mouse secondary Ab. A-C: WT-ACE expressing cells; D-F: Q1069R-ACE expressing cells. A,B and D,E. Immunostaining of WT and Q1069R ACE in 0.3% paraformaldehye for 15 min at 4C (fixed only). B and E. Treatment with the chemical chaperone sodium butyrate and proteosome inhibitor MG132 for 24 hrs followed by immunostaining with 9B9 mAb. C and F. CHO cells fixed and permeabilized with 3% paraformaldehyde for 15 min at 37C and labeled as above show WT-ACE on the plasma membrane and to a lesser extent in the cytosol and Q1069R-ACE in perinuclear areas.</p

Localization of new mutation (Q1069R) in the C domain of somatic ACE.

<p>The localization of Gln1069 in the C-domain of human ACE which was mutated to Arg is shown using molecular surface (<b>A</b>) and ribbon (<b>B</b>) representations of the substrate-bound crystal structure of the C-domain fragment of human ACE, where the 36 amino acid residues unique to tACE were deleted <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010438#pone.0010438-Watermeyer2" target="_blank">[75]</a>. The surface and ribbon are light brown, and amino acid residues that were crucial for orientation are colored. Key amino acids referred to in the text are denoted using somatic ACE numbering. Gln1069 (brown) is located right under the bump created by Lys1067 and Tyr1068 (purple) and shown by arrow. Dark blue indicates the first N-terminal amino acid residue (Asp616) seen in this structure; orange-indicates the C terminal end of the C-domain (and the epitope for mAb 1B3; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010438#pone.0010438-Naperova1" target="_blank">[16]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010438#pone.0010438-Balyasnikova6" target="_blank">[36]</a>. Light blue (P730) and red colored amino acid residues indicate highly immunogenic bumps on the surface of the C-domain. Light green indicates Asp in putative glycosylation sites.</p

ACE activity of mutant ACE.

<p>ACE activity of the membrane-bound form of ACE in lysates of CHO cells expressing WT and mutant ACE was determined by fluorimetric assay of 40 µl aliquotes of cells with 200 µl each of substrates Hip-His-Leu (HHL) and Z-Phe-His-Leu (ZPHL) during 1 hr incubation. <b>A.</b> ACE activity with HHL and ZPHL (mU/ml), correspondingly, from a representative experiment performed in triplicate. B.<b> </b> Ratio of hydrolysis of the two substrates (ZPHL/HHL ratio) in the tested samples. The data are presented as mean (± SD) from three independent experiments. *p<0.05 vs. control samples.</p

Catalytic properties of mutant ACE bound to ACE mAbs.

<p>Membrane-bound WT (<b>A</b>) and mutant ACE (<b>B</b>) lysates were normalized to achieve 5 mU/ml ACE activity with Z-Phe-His-Leu as substrate and incubated in microtiter plates covered by 16 mAbs to human ACE as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010438#pone-0010438-g007" target="_blank">Fig. 7</a>. ACE activity precipitated by each mAb was quantified by fluorimetric assay with two substrates (Hip-His-Leu and Z-Phe-His-Leu) as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010438#pone-0010438-g005" target="_blank">Fig. 5</a>. Data are expressed as the ratio of ACE activity precipitated by each mAb determined with each substrate. Data are mean ± SD of 6–8 independent experiments in duplicate. * - p<0.05 vs. ZPHL/HHL ratio for corresponding values of WT and mutant ACE in solution (horizontal lines in A and B). The ratio for duplicate samples of WT ACE or mutant ACE was approximately 1.0 and the SD was less than 10%.</p