Bulletin of RSMU

ISSN Print 2500–1094 ISSN Online 2542–1204

Bulletin of RSMU

BIOMEDICAL JOURNAL OF PIROGOV UNIVERSITY (MOSCOW, RUSSIA)

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Kolomeichuk SN, Alekseev RV, Putilov AA, Meigal AYu

This work aims to study distribution of allele frequencies of the ACE and BDKRB2 genes coding for the angiotensin-converting enzyme and the bradykinin receptor β2, respectively, in athletes specializing in different sports and to establish the associations between the studied genotypes and heart rate variability. The study included 75 male athletes. Polymorphisms of ACE and BDKRB2 (I/D and +9/−9, respectively) were studied by PCR. A significant difference was revealed in the −9/−9 genotype frequency between the studied groups of athletes. Parasympathetic nerve activity prevailed in the athletes with the I allele of the ACE gene. Time-domain parameters of heart rate variability had low values in the carriers of the D/D genotype. In the athletes with the ACE I/I genotype the time-domain parameters differed from those typical for the I/D and D/D genotype carriers. Participants homozygous for −9 BDKRB2 had the lowest heart rate in the studied sample, implying an increased contribution of parasympathetic activity to heart rate regulation. The −9 allele of BDKRB2 was found to be associated with the minimal R — R interval between consecutive hear beats. We conclude that polymorphisms I/D of ACE and +9/−9 of BDKRB2 can indicate individual patterns of heart rate regulation in athletes from the Republic of Karelia.

Received: 2017-08-25

Published online: 2017-10-30

DOI: 10.24075/brsmu.2017-04-08

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VIEWS 4810

Nikiforova AI, Abramov DD, Kadochnikova VV, Zobkova GU, Ogurtsova KA, Brjuhanova NO, Shestopalova EA, Kochetkova TO, Shubina ES, Donnikov AE, Trofimov DYu

The present study aimed to determine frequencies of mutations in the phenylalanine hydroxylase gene (PAH) in unrelated children (n = 71) diagnosed with phenylketonuria, who presented to Morozovskaya Children’s City Clinical hospital (Moscow) over the period from 2015 to 2016. The patients were tested for the most common PAH mutations using the original real- time PCR-based technique for the identification of nucleotide variants; additionally, next generation sequencing (NGS) was performed on the unidentified genotypes. The original PCR-based technique allowed us to effectively identify 83 % of the pathogenic allelic variants in the sample. Using the combination approach (real-time PCR + NGS), we found mutations in both alleles of PAH in 66 of total 71 patients. Altogether, 26 pathogenic PAH mutations were identified, the most common being p.R408W (47.9 %) and p.R261Q (9.9 %). Frequencies of mutations common for the Russian population, such as IVS10nt546, IVS12+1G>A, p.R158Q, p.Y414C, and IVS4+5G>T, ranged from 4.2 to 2.8 %. Half of the identified variants accounted for the total frequency of < 10 %. Sequencing of PAH revealed a few functional mutations previously unreported for Moscow region residents, including p.D222Terfs, p.R111Ter, p.F161S, p.G188D, p.R270K, p.L311P, p.F55L, p.F55Leufs, IVS1+5G>T, and IVS8-7A>G. It could be reasonable to include mutations p.D222Terfs and p.R111Ter (carrier frequency of 2.1 %) in PCR testing panels. The data obtained in our study can also be used in the development of genetic tests for phenylketonuria.

Received: 2017-08-07

Published online: 2017-10-30

DOI: 10.24075/brsmu.2017-04-07

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VIEWS 4996

Levchenko OA, Zinchenko RA, Lavrov AV

Cohen syndrome is a rare autosomal-recessive disorder characterized by intellectual disability, myopia, hypotonia, and skeletal malformations. Its clinical diagnosis is impeded by marked inter- and intrafamilial phenotypic variability. Gene VPS13B that carries disease-associated mutations has 62 exons, making Sanger sequencing of the entire gene unsuitable for routine clinical use due to high costs. In this work we report a case of Cohen syndrome in a brother and sister born to a mixed Abazin-Circassian marriage and diagnosed with moderate mental retardation. Both patients had psychomotor retardation, were unable to study at school, and never learned to read, write and count. Although the patients shared a few nonspecific phenotypic characteristics, phenotypic differences made it impossible to arrive at a clear diagnosis. Therefore, whole exome sequencing was performed revealing the single nucleotide variant с.7603C>T that results in the premature stop codon R2535* in VPS13B. This mutation was found in the mother, the affected sibs and one of the two other healthy sibs. The second mutation remained undetected. Considering the identified mutation and the analyzed phenotypic traits, we concluded Cohen syndrome in both patients.

Received: 2017-08-02

Published online: 2017-10-30

DOI: 10.24075/brsmu.2017-04-06

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VIEWS 4791

Shirshova AN, Shamovskaya DA, Boyarskikh UA, Apalko SV, Leskov LS, Sokolov AV, Kovalenko SA, Scherbak SG, Pikalov IV, Kel AE, Filipenko ML

Colorectal cancer (CRC) is one of the most common cancer types in the world. Timely diagnosis of CRC and adenomatous polyps aided by effective screening techniques can considerably reduce mortality from this disease. MicroRNAs constitute a new class of promising biomarkers for a range of human diseases including cancer. The following article assesses the diagnostic significance of miR-146a concentrations in the blood plasma of patients with colorectal cancer. The main group included patients with stages I to III colorectal cancer (n = 102); the control group comprised patients with chronic colitis, nonspecific ulcerative colitis and Crohn’s disease (n = 58) and healthy individuals (n = 42). MicroRNA levels were quantified by reverse- transcription real-time PCR, revealing significantly higher miR-146a concentrations in the samples of patients with CRC than in the controls (p < 0.0001). The optimal diagnostic sensitivity determined by ROC analysis was 47.3 %, specificity was 91.5 %, with AUC = 0.79 ± 0.018. Our findings demonstrate that the studied approach does not have sufficient specificity, but still suggest that miR-146a can be included into screening tests based on quantification of other microRNAs with improved specificity.

Received: 2017-08-25

Published online: 2017-10-30

DOI: 10.24075/brsmu.2017-04-05

Comments 0

VIEWS 4864

Tyschik EA, Kometova VV, Rodionov VV, Rebrikov DV

The present study aims to estimate and compare the levels of cell-free circulating RNAs of three interleukins IL-6, IL-8, and IL-18 and three splice variants of the vascular endothelial growth factor (VEGF), namely 121, 165 and 189, in blood plasma of patients with stage I / II breast cancer and healthy controls. The study reveals that patients with breast cancer have significantly elevated levels of circulating VEGF121 and VEGF165 RNAs, so far unreported in the literature. We also confirm that levels of circulating IL-8 and IL-18 RNAs are considerably increased in breast cancer patients.

Received: 2017-08-15

Published online: 2017-10-30

DOI: 10.24075/brsmu.2017-04-04

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VIEWS 4768

Sokolova EA, Khlistun IV, Kushlinsky DN

There is evidence that size distribution of cell-free DNA (cfDNA) fragments can be diagnostically relevant. The present work describes a multiplex quantitative real-time polymerase chain reaction technique modified and validated by the authors to study the degree of cfDNA fragmentation in blood plasma. Based on the detection of Alu and hLINE-1 repeats, this technique employs fluorescent probes. We selected suitable primers and probes, optimized PCR conditions and estimated the dynamic range and sensitivity threshold of the assay. The modified PCR had a dynamic range of 6 logs, its efficiency being over 90 %. We demonstrated that cfDNA fragmentation index did not differ significantly between healthy women (n = 16) and women with stage III–IV ovarian cancer (n = 14). Therefore, further research on a larger sample is needed using electrophoretic cfDNA fractionation.

Received: 2017-08-25

Published online: 2017-10-29

DOI: 10.24075/brsmu.2017-04-03

Comments 0

VIEWS 4888

Telysheva EN, Snigireva GP

Screening for cell-free DNA usually referred to as liquid biopsy holds great promise in cancer diagnosis and treatment. This article presents the results of the analysis of somatic tumor-specific mutations in circulating free DNA (cfDNA) isolated from the blood plasma of patients with stages I–IV colorectal cancer, based on the use of wild-type blocking allele-specific real-time polymerase chain reaction. This technique was specially designed for the analysis of biological specimens containing small amounts of mutant circulating tumor DNA. The study included 46 patients (18 female and 28 male participants) between 48 and 86 years of age (mean age was 67.1 ± 8.8 years). All patients underwent surgical treatment (radical surgery was performed on 85 % of the participants). Besides the molecular genetic analysis of cfDNA isolated from the blood plasma, standard histological staining was performed. Patients’ blood samples were collected before the surgery and on day 5 after it to test for KRAS and BRAF mutations. The applied PCR technique proved to be effective in detecting mutations in the RAS genes in stages II–IV of the disease, its sensitivity threshold being 0.1 %. Analysis of cfDNA before and after surgery may provide additional information on the surgical treatment outcome, development of new metastases, or presence of those previously overlooked. Wild-type blocking allele-specific real-time PCR is awaiting further validation in different clinical situations.

Received: 2017-08-08

Published online: 2017-10-29

DOI: 10.24075/brsmu.2017-04-02

Comments 0

VIEWS 4522

Cell-free DNA (cfDNA) was discovered in human blood plasma as early as the middle of the 20th century, but it was not until a few decades ago that knowledge of human genome and epigenome in health and pathology became sufficient and methods of nucleic acid analysis became more advanced to encourage active research of the diagnostic potential of cfDNA. The use of cfDNA as a diagnostic biomarker is conventionally referred to as liquid biopsy. The following review tells a story of cfDNA discovery, summarizes contemporary views on cfDNA sources inside the body and touches upon possible prognostic and diagnostic applications of cfDNA analysis in medicine, specifically in cancer and prenatal screening, prediction of implant failure and sepsis development.

Received: 2017-08-25

Published online: 2017-10-29

DOI: 10.24075/brsmu.2017-04-01

Comments 0

VIEWS 5212