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Background: Blood of pregnant women contains cell-free fetal DNA (cffDNA), which is widely used in non-invasive prenatal diagnosis. The modern laboratory equipment market provides huge variety of commercial kits for isolation of circulating nucleic acids, but unfortunately none of them are standardized for isolation of cffDNA, which is a crucial step for success of subsequent analysis., Aim: To compare DSPVK and CNAK in terms of cffDNA, cell-free total DNA (cftDNA) yield and resulting cffDNA fraction, as well as to try to explain the possible difference between the efficacy of these kits., Methods: Peripheral blood samples were collected from 18 healthy pregnant women (6 th -14th week of pregnancy) and from 12 healthy unpregnant subjects. cftDNA was isolated using QIAamp Circulating Nucleic Acid Kit (CNAK) (Qiagen, Germany) and QIAamp DSP Virus Kit (DSPVK) (Qiagen, Germany) from 1 ml of plasma of each sample. Methylation-sensitive restriction was carried out to isolate cffDNA. Yield of cffDNA and cftDNA was quantified using digital PCR. To explain the difference in resulting efficacy of these two kits PCR inhibitors analysis was performed, as well as the optimal plasma input for DSPVK was investigated., Results: Yield of cffDNA using CNAK was statistically significantly higher than using DSPVK (167.62 (125.34-192.47) vs 52.88 (35.48-125.42) GEq/mL, p < 0.001). The same applies to cftDNA yield, CNAK appears to be statistically significantly superior to DSPVK (743.42 (455.02-898.33) vs 371.07 (294.37-509.89) GEq/mL, p < 0.001). cffDNA fraction using CNAK was also higher than using DSVPK (24.75 (14.5-31.53) vs 14.20 (6.88-25.83) %, p = 0.586), although the difference was not statistically significant due to inconsistency of DSPVK results from sample to sample. PCR inhibitors analysis uncovered increased amount of PCR inhibitors in CNAK cftDNA solution, compared to DSPVK (p = 0.002). Usage of 0.5 mL of plasma for cftDNA extraction with DSPVK over 1 mL demonstrates almost 1.8 times higher cftDNA output (p = 0.028), which suggests that this kit is not so viable for volumes of plasma larger than 0.5 mL., Conclusions: We recommend CNAK over DSPVK for quantitative analysis of cffDNA. Nevertheless, DSPVK is definitely suitable for qualitative analysis as well as for research with limited budget, since it is almost 3 times cheaper than CNAK., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Gaucher disease is characterized by the activation of splenic and hepatic macrophages, accompanied by dramatically increased levels of angiotensin-converting enzyme (ACE). To evaluate the source of the elevated blood ACE, we performed complete ACE phenotyping using blood, spleen and liver samples from patients with Gaucher disease and controls., Methods: ACE phenotyping included 1) immunohistochemical staining for ACE; 2) measuring ACE activity with two substrates (HHL and ZPHL); 3) calculating the ratio of the rates of substrate hydrolysis (ZPHL/HHL ratio); 4) assessing the conformational fingerprint of ACE by evaluating the pattern of binding of monoclonal antibodies to 16 different ACE epitopes., Results: We show that in patients with Gaucher disease, the dramatically increased levels of ACE originate from activated splenic and/or hepatic macrophages (Gaucher cells), and that both its conformational fingerprint and kinetic characteristics (ZPHL/HHL ratio) differ from controls and from patients with sarcoid granulomas. Furthermore, normal spleen was found to produce high levels of endogenous ACE inhibitors and a novel, tightly-bound 10-30 kDa ACE effector which is deficient in Gaucher spleen., Conclusions: The conformation of ACE is tissue-specific. In Gaucher disease, ACE produced by activated splenic macrophages differs from that in hepatic macrophages, as well as from macrophages and dendritic cells in sarcoid granulomas. The observed differences are likely due to altered ACE glycosylation or sialylation in these diseased organs. The conformational differences in ACE may serve as a specific biomarker for Gaucher disease., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Hereditary hemochromatosis (HH) is a common cause of primary iron overload induced by genetic impairment of iron metabolism. More than 80% of HH patients in populations of European origin are homozygotes for a single mutation C282Y, or compound heterozygotes for C282Y and H63D mutations in the HFE gene. However, in the majority of Asian, African, Australasian, and Amerindian populations, frequencies of C282Y are close to zero. Data on the prevalence of HFE mutations in Russian population and in Russian patients with HH are very limited. In this work, we determined frequencies of C282Y and H63D in ethnical Russians living in the Central European region of Russia. Furthermore, we tested whether homozygocity for C282Y is the major cause of HH in Russians. We found that, in the Russian population, the frequency of C282Y mutation in the HFE gene is relatively high and corresponds to mean European levels. However, in contrast to the majority of European populations, homozygocity for C282Y is found only in a small proportion (5%) of patients with biochemical and clinical signs of HH. These data suggest that either the penetrance of C282Y in Russia is lower than in Western countries, or that a more frequent non-HFE dependent mechanism of primary iron overload dominates in Russian population.