High frequency of BRCA1, but not CHEK2 or NBS1 (NBN), founder mutations in Russian ovarian cancer patients

<p>Abstract</p> <p>Background</p> <p>A significant portion of ovarian cancer (OC) cases is caused by germ-line mutations in BRCA1 or BRCA2 genes. BRCA testing is cheap in populations with founder effect and therefore recommended for all patients with OC diagnosis. Recurrent mutations constitute the vast majority of BRCA defects in Russia, however their impact in OC morbidity has not been yet systematically studied. Furthermore, Russian population is characterized by a relatively high frequency of CHEK2 and NBS1 (NBN) heterozygotes, but it remains unclear whether these two genes contribute to the OC risk.</p> <p>Methods</p> <p>The study included 354 OC patients from 2 distinct, geographically remote regions (290 from North-Western Russia (St.-Petersburg) and 64 from the south of the country (Krasnodar)). DNA samples were tested by allele-specific PCR for the presence of 8 founder mutations (BRCA1 5382insC, BRCA1 4153delA, BRCA1 185delAG, BRCA1 300T>G, BRCA2 6174delT, CHEK2 1100delC, CHEK2 IVS2+1G>A, NBS1 657del5). In addition, literature data on the occurrence of BRCA1, BRCA2, CHEK2 and NBS1 mutations in non-selected ovarian cancer patients were reviewed.</p> <p>Results</p> <p>BRCA1 5382insC allele was detected in 28/290 (9.7%) OC cases from the North-West and 11/64 (17.2%) OC patients from the South of Russia. In addition, 4 BRCA1 185delAG, 2 BRCA1 4153delA, 1 BRCA2 6174delT, 2 CHEK2 1100delC and 1 NBS1 657del5 mutation were detected. 1 patient from Krasnodar was heterozygous for both BRCA1 5382insC and NBS1 657del5 variants.</p> <p>Conclusion</p> <p>Founder BRCA1 mutations, especially BRCA1 5382insC variant, are responsible for substantial share of OC morbidity in Russia, therefore DNA testing has to be considered for every OC patient of Russian origin. Taken together with literature data, this study does not support the contribution of CHEK2 in OC risk, while the role of NBS1 heterozygosity may require further clarification.</p

Tissue Specificity of Human Angiotensin I-Converting Enzyme.

Angiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, as well as in reproductive functions, is expressed as a type-1 membrane glycoprotein on the surface of endothelial and epithelial cells. ACE also presents as a soluble form in biological fluids, among which seminal fluid being the richest in ACE content - 50-fold more than that in blood.We performed conformational fingerprinting of lung and seminal fluid ACEs using a set of monoclonal antibodies (mAbs) to 17 epitopes of human ACE and determined the effects of potential ACE-binding partners on mAbs binding to these two different ACEs. Patterns of mAbs binding to ACEs from lung and from seminal fluid dramatically differed, which reflects difference in the local conformations of these ACEs, likely due to different patterns of ACE glycosylation in the lung endothelial cells and epithelial cells of epididymis/prostate (source of seminal fluid ACE), confirmed by mass-spectrometry of ACEs tryptic digests.Dramatic differences in the local conformations of seminal fluid and lung ACEs, as well as the effects of ACE-binding partners on mAbs binding to these ACEs, suggest different regulation of ACE functions and shedding from epithelial cells in epididymis and prostate and endothelial cells of lung capillaries. The differences in local conformation of ACE could be the base for the generation of mAbs distingushing tissue-specific ACEs

The structures of N and C domains of ACE with potential glycosylation sites and epitopes for mAbs.

<p>Human N domain structure was based on PDB P2C6N and C domain structure—based on PDB 1O86. The epitopes were marked on the N and C domains according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.ref027" target="_blank">27</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.ref031" target="_blank">31</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.ref041" target="_blank">41</a>]. The positions of the epitopes for some mAbs (12 out of 17) are shown by circles on both sides of domain globule. The potential sites of N-glycosylation, 9 on the N domain and 6 on the C domain, are marked by green; Asn494 on the N domain is not seen while Asn1196 is not present in structure of the C domain. The glycosylation sites which might be differently glycosylated in seminal fluid ACE and lung ACE are shown by arrows. Some amino acid residues are shown by numbers according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.ref038" target="_blank">38</a>] for orientation.</p

N-glycosylated peptides and glycan structures found by the GlycoMod tool in ACE from lung and seminal fluid using MS data.

<p>N-glycosylated peptides and glycan structures found by the GlycoMod tool in ACE from lung and seminal fluid using MS data.</p

Observed [M+H]⁺ ions of unglycosylated peptides in the mass spectra of human ACE tryptic digests.

<p>^a Acrylamide adduct on cysteine.</p><p>^b Oxidized methionine.</p><p>^c Contains one or two missed cleavage(s) by trypsin.</p><p>Peptides that contain potential N-glycosylation sites are shown in bold.</p><p>Observed [M+H]⁺ ions of unglycosylated peptides in the mass spectra of human ACE tryptic digests.</p

Effect of different additives on mAbs binding to seminal fluid and lung ACEs.

<p>ACE activity immunoprecipitated by 17 mAbs to ACE (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.g001" target="_blank">Fig 1</a>) was presented as a normalized value (“binding ratio”) to highlight differences in immunoprecipitation pattern (“conformational fingerprint”) after adding of tested compounds to purified seminal fluid and lung ACEs with that without additives. <b>(A)</b> Effect of 20% of human heat-inactivated plasma. <b>(B</b>) Effect of 80% 3 kDa filtrate of human citrated plasma. <b>C</b>-<b>D</b>. Effect of bilirubin (150 ug/ml) in the absence (<b>C</b>) or presence (<b>D</b>) of human albumin at 8 mg/ml concentration (which correspond to its concentration in 20% serum). Data are presented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.g001" target="_blank">Fig 1</a>.</p

Effect of human plasma, seminal fluid and albumins on mAbs binding to ACEs.

<p>ACE activity immunoprecipitated by 17 mAbs to ACE (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.g001" target="_blank">Fig 1</a>) was presented as a normalized value (“binding ratio”) to highlight differences in immunoprecipitation pattern (“conformational fingerprint”) after adding heat-inactivated human citrated plasma, heat-inactivated seminal fluid, as well as human and bovine albumins to purified seminal fluid and lung ACEs with that without additives. <b>A-B</b>. Effect of 20% of heat-inactivated human plasma (<b>A</b>) and heat-inactivated seminal fluid (<b>B</b>); <b>C-D</b>. Effect of human (<b>C</b>) and bovine (<b>D</b>) albumins at concentrations of 8 mg/ml (similar to albumin concentration in 20% serum). Data are presented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.g001" target="_blank">Fig 1</a>.</p

Primary immune response in mice to pure somatic ACE from seminal fluid.

<p>Culture fluids from 670 post-fusion cell populations grown in 96-well plates (primary screening) were anylyzed for the presence of antibodies to seminal fluid ACE and lung ACE in parallel in plate precipitation assay (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.g001" target="_blank">Fig 1</a>). Presence of antibodies to seminal fluid and/or lung ACE was detected in 91 wells by precipitated ACE activity and the data are presented as the ratio of ACE activity precipitation from seminal fluid ACE to that from lung ACE (SF/Lung ratio). Discrimination of these two ACEs by antibodies from these positive wells was observed in a wide range (A). Besides expected antibodies, recognizing both ACEs (C) with SF/Lung ratio in the region from 0.5 to 1.5, we identified significant proportions of antibodies which preferentially recognized seminal fluid ACE (B) with SF/Lung ratio more than 1.5, and antibodies which preferentially recognized lung ACE (D) with SF/Lung ratio less than 0.5, correspondingly.</p

Effect of anti-catalytic mAbs on the activity of pure seminal fluid and lung ACEs.

<p>Pure seminal fluid and lung ACEs (5 mU/ml with ZPHL as a substrate) were incubated with mAbs (10 ug/ml), which are anti-catalytic for the N-domain active center, i2H5 and 3A5 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.ref027" target="_blank">27</a>], and for the C domain active center, 1E10 and 4E3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.ref030" target="_blank">30</a>], of ACE. Residual ACE activity was determined with substrates HHL (<b>A</b>) and ZPHL (<b>B</b>) and is presented as the ratio of ACE activity in the presence of mAbs to that without mAbs. Data are also presented as ZPHL/HHL ratio of ACE activity in the presence of mAbs to that without mAbs (<b>C</b>). Results are the mean ± SD of 2–4 experiments, made in duplicates.</p

Amino acid sequences of the lung and seminal fluid ACEs.

<p>Peptides identified by MALDI TOF MS are shown in bold; potential sites of trypsin cleavage are underlined; potential glycosylation sites are marked by green; zinc-recognizing motives are marked by red; putative glycopeptides are shaded.</p