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Somatic mutations associated with oncological diseases, including Ph-myeloproliferative neoplasms (Ph-MPN), are very diverse, occur with different frequencies and different allelic burden levels. Therefore, at the initial stage of performing molecular-genetic diagnostic procedures, it is desirable to be able to conduct screening tests in the laboratory. This is especially important when analyzing rare and diverse mutations. Analysis of high resolution melting curves (HRM analysis), which has high sensitivity and is suitable for screening all types of mutations, in a number of studies is proposed for the analysis of Ph-MPN associated mutations in the JAK2 and CALR genes. For analysis of somatic mutations in the majority of literature sources that we reviewed, the authors use the LightCycler (Roche) thermocycler and much rarely the CFX96 (Bio-Rad), which is often presented in Russian scientific and practical and medical organizations. The aim of the study was to screen the somatic JAK2 and CALR mutations by HRM analysis using the CFX96 thermocycler and the Precision Melt Analysis software (Bio-Rad, USA) for patients with Ph-MPN. In the present research, HRM analysis was conducted on the DNA samples from patients with mutations in the JAK2 or in the CALR gene. The Precision Melt Analysis software identified all variants of the analyzed mutations, both a single nucleotide substitution in the JAK2 gene (with allelic burden level in the range of 5-40%), and various indel mutations in the CALR gene (with allelic burden level in the range of 40-50%) Therefore, the HRM analysis that was conducted on the CFX96 allows screening of highly specific mutation for the diagnosis of Ph-MPN in the exon 14 of the JAK2 gene and in the exon 9 of the CALR gene. The inclusion of this screening research in the laboratory testing algorithm improves the efficiency and accessibility of molecular genetic technologies in the diagnosis of Ph-MPN.

Aim: The purpose of our study was to analyze the predictive ability of the multiplicative model of genetic risk of nonlacunar ischemic stroke (IS) for independent samples from Russia. Patients & methods: A total of 181 patients and 360 healthy controls were included in this study. The discriminative accuracy of model was evaluated by the area under the receiver operating characteristic curve (AUC). Results: Classification model based on 15 single-nucleotide polymorphisms (SNPs), which are associated with a cardioembolic subtype of IS, had an AUC of 0.62 in patients with corresponding subtypes and an AUC of 0.58 for all patients. Conclusion: Risk calculation approach based on IS-associated SNPs had satisfactory performance in predicting the predisposition to the disease.

The possibilities of early detection of chronic myelo-proliferative tumors (MPT) are determined by sensitivity of techniques implemented for finding somatic mutation V617F in gene JAK2. The mutation V617F can also be found in individuals without unfolded picture of hematological diseases. The detection of mutation even in low concentrations is associated with increasing of risk of cerebral stroke and thrombosis of arterial and venous vessels. The study was carried out to develop techniques based on COLD polymerase chain reaction and allele-specific polymerase chain reaction targeted to increasing of sensitivity of finding mutation V617F detected using pyro-sequencing. The analytical sensitivity of techniques was evaluated by control samples with different ratio of alleles. For allele-specific polymerase chain reaction analytical sensitivity amounted to 0.25% of mutant allele at concentration of analyzed control sample 10 copies of DNA per mkl. For COLD polymerase chain reaction sensitivity amounted to 0.5% at concentration 10 copies of DNA per mkl. The comparative approbation of techniques was implemented using clinical material obtained from 106 patients with suspicion on MPT. The analysis of clinical samples using COLD polymerase chain reaction revealed 13 (14%) and using technique of allele-specific polymerase chain reaction - 15 (16%) positive samples. In all 15 cases of detection of mutation clinical confirmations of diagnosis of MPT was received. The proposed techniques permit increasing efficiency of amplification of mutant DNA in analyzed samples and hence to increase sensitivity of subsequent analysis of products of amplification using technique of pyro-sequencing. Therefore, the mentioned techniques can be recommended to be applied for confirmation of diagnosis of MPT and early identification of individuals with increased risk of development of venous and arterial thromboses.

The detection of somatic mutations in the 9 exon of the calreticulin gene (CALR) is regulated by the clinical recommendations as a diagnostic criterion for chronic Ph-negative myeloproliferative neoplasms (MPN). Some methods of nucleic acids testing are used to identify CALR gene mutations with different requirements for special skills of personnel and expensive equipment. The purpose of this work is to compare the results of the detection of CALR gene mutations in venous blood samples by allele-specific RT-PCR with subsequent electrophoresis, fragment analysis and Sanger- or pyro- sequencing. We used 1284 blood samples of patients with suspected MPN and 20 blood donor samples. Mutations in the CALR gene of the I and II type were identified using PCR-RT with the original primers and TaqMan probes. Also, all samples were tested for mutations in the CALR gene by electrophoretic detection of PCR results in an agarose gel. The use of allele-specific RT-PCR followed by electrophoretic detection made it possible to determine clinically significant mutations in the CALR gene in 81 venous blood samples of JAK2- and MPL-negative patients, including 42 cases of type I mutation, 33 cases of type II mutation and 8 rare CALR mutations. Mutations in the 9 exon of the CALR gene were not detected in any of the 20 blood donor samples or in 121 blood samples of patients with polycythemia vera. In randomly selected 20 negative samples, CALR gene mutations were also not detected using Sanger sequencing. All positive samples were confirmed by fragment analysis, as well as with Sanger- sequencing and pyro- sequencing. The described combined approach to detect mutations of the CALR gene in peripheral blood samples can be used in clinical diagnostic laboratories that have a standard set of equipment for electrophoresis of nucleic acids and a PCR-RT. We also propose a confirmatory test based on the pyrosequencing of DNA using the system of genetic analysis "PyroMark Q24".

One of the prevalent genetic causes of idiopathic male sterility is related to micro-deletions in AZF locus located in Y-chromosome. In total population, rate of such micro-deletions makes up to 1:4000. however, in infertile males their rate varies from 2% to 10%. In AZF locus three subregions are distinguished: AZFa, AZFb and AZFc. The loss of one or several subregions can result in disorder of spermatogenesis of various degree - from decreasing of its activity to Sertoli-cell syndrome manifested by azoospermia or oligospermia of severe degree. Therefore, implementation of genetic testing for presence of micro-deletions in AZF locus is a necessary test in case of prognosis of male sterility and its treatment. The purpose of study is to develop and test a diagnostic system of detection of micro-deletions in subregions of AZF locus using multiplex polymerase chain reaction in real-time. As a reference method a technique was implemented described in guidelines of the European Academy of Andrology conjointly with European Molecular Genetics Quality Network. The technique testing specified analysis of 33 samples of DNA separated from blood of males with azoospermia and oligospermia of severe degree. No discordant results were received as compared with reference method. In 27 DNA samples the deletions were detected in AZF locus: 4 AZFa deletions (15%), 2 AZFb deletions (7%), 17 AZFc deletions (63%) and 6 combined deletions of AZFb+candи AZFa+b+с (22%). The proposed technique permits detect micro-deletions of subregions of AZF locus.

To study genetic characteristics of the population of the Moscow region and analyze the association of rs1801133 and rs1801131 of MTHFR with the risk of ischemic stroke (IS).A sample of 170 and 115 patients with atherothrombotic and cardioembolic subtypes of IS and 360 residents of the Moscow region without IS were examined. MTHFR alleles were determined by a multiplex real-time polymerase chain reaction.No association between the frequencies of MTHFR alleles and the risk of ischemic stroke was found. The comparison of allele frequencies with those in Caucasian populations published in the dbSNP (NCBI) and 1000 Genomes Project databases revealed significant differences for rs1801133 from the EUR 1000 Genomes Project. The allele frequency data for MTHFR could increase the accuracy and reliability of the individual risk calculation for multifactorial diseases in the Russian population.Цель исследования. Изучение генетических особенностей населения московского региона и анализ влияния аллелей и генотипов полиморфных локусов rs1801133 и rs1801131 гена MTHFR на риск развития ишемического инсульта (ИИ). Материал и методы. Обследовано 170 и 115 пациентов с атеротромботическим и кардиоэмболическим подтипами ИИ соответственно и 360 жителей московского региона без признаков острого нарушения мозгового кровообращения на момент участия в исследовании. Аллели гена MTHFR определяли методом мультиплексной ПЦР в реальном времени. Результаты и заключение. В исследованной выборке не обнаружено ассоциации полиморфных локусов гена MTHFR с риском развития ишемического инсульта. При сопоставлении частот аллелей в популяционном контроле с частотами, представленными для популяции европеоидов в базах данных dbSNP и 1000 Genomes Project, выявлены статистически значимые различия для rs1801133 в сравнении с выборкой EUR 1000 Genomes Project. Описанные генетические особенности могут быть использованы для повышения точности и надежности расчета индивидуального генетического риска развития других патологических состояний, связанных с нарушением обмена фолатов в наблюдаемой популяции.

The somatic mutation V617F in gen JAK2 is a frequent cause of chronic myeloprolific diseases not conditioned by BCR/ABL mutation. The quantitative testing of relative percentage of mutant allele can be used in establishing severity of disease and its prognosis and in prescription of remedy inhibiting activity of JAK2. To quantitatively test mutation the pyrosequencing technique was applied. The developed technique permits detecting and quantitatively, testing percentage of mutation fraction since 7%. The "gray zone" is presented by samples with percentage of mutant allele from 4% to 7%. The dependence of expected percentage of mutant fraction in analyzed sample from observed value of signal is described by equation of line with regression coefficients y = - 0.97, x = -1.32 and at that measurement uncertainty consists ± 0.7. The developed technique is approved officially on clinical material from 192 patients with main forms of myeloprolific diseases not conditioned by BCR/ABL mutation. It was detected 64 samples with mautant fraction percentage from 13% to 91%. The developed technique permits implementing monitoring of therapy of myeloprolific diseases and facilitates to optimize tactics of treatment.

Development and testing of a real-time PCR method for detection of Neisseria meningitiis serogroup A, B, C and W DNA.Reference strains and 187 samples of cerebrospinal fluid (CSF) from meningocci meningitis patients were used in the study. Multiplex PCR was carried out in an instrument with 5 channels of fluorescent detection:Analysis of specific serogroup loci of the genome and design of oligonucleotides for the detection of DNA of all the capsule meningococci and 4 serogroups in particular was carried out. PCR conditions were optimized; specificity was shown and analytical sensitivity was evaluated using reference strains. DNA of the following serogroups was detected during study of clinical CSF samples: A--in 103 samples (55%), B--in 45 (24%), C--in 30 (16%), W--in 5 (3%). Only DNA of meningococci capstle gene ctrA was found in 4 samples; presumably, they contained DNA of other serogroups. Multilocus sequence-typing and detection of antigenic determinants of PorA and FetA genes for 27 DNA samples of group A menincococci as well as DNA of 5 group W meningococci and 4 ungroupable was carried out.The method proposed allows to carry out serogrouping of no less than 95% of strains or DNA samples isolated from CSF of meningococci infection patients. Combined with other recommended non-cultural methods of genotyping, it may be useful for complex characteristics of pathogenic meningococci.

To develop a method of the complex assessment of genetic risk for ischemic stroke (IS) and evaluate its effectiveness.Genotyping of 182 patients with atherothrombotic and cardioembolic subtypes of IS and 360 healthy individuals of 48 single nucleotide polymorphic loci (SNP) associated with the risk of II and its subtypes was performed.In each group of SNPs, composite indicators of genetic risk of IS in groups of patients and healthy controls were identified. Differences between the calculated values of the genetic risk in these groups were significant (p0,05). The quality of the binary classification validated by ROC-analysis confirmed the predictive potential of the proposed method of risk calculation for determining the genetic predisposition to the development of IS.Цель исследования. Разработка методики комплексной оценки генетически обусловленного риска развития ишемического инсульта (ИИ) и оценка ее эффективности. Материал и методы. Проведено генотипирование 182 больных с атеротромботическим и кардиоэмболическим подтипами ИИ (группа больнах) и 360 здоровых (контрольная группа) по 48 однонуклеотидным полиморфным локусам (SNP), ассоциированным с риском развития ИИ и его подтипов. Результаты и заключение. Для каждой группы SNP в соответствии с предложенной моделью определены комплексные показатели генетического риска развития ИИ в группе больных и контрольной группе. Различия между группой больных и контрольной группой по показателям генетического риска определены как статистически значимые (p0,05). Качество бинарной классификации, проверенное ROC-анализом, подтверждает прогностический потенциал использования предложенной модели расчета риска для определения генетической предрасположенности к развитию ИИ.

Development and application of real-time PCR (RT-PCR) procedure for determination of Streptococcus pneumoniae serotypes.S. pneumoniae cps-locus wzx, wzy, wzz, wcwV and galU genes were chosen as PCR targets to select serotype-specific oligonucleotide primers and fluorescent labeled probes. 89 samples of cerebrospinal fluid (CSF) obtained in 2007 - 2010 from patients with pneumococcal meningitis diagnosis undergoing therapy in the Infectious Clinical Hospital No. 2, Moscow, were studied with the aim of testing the possibility of practical use of RT-PCR.Primers and probes were selected for the determination of 16 vaccine and/or frequently encountered serotypes distributed among 4 reaction mixtures also including a pair of primers and a probe for cpsA gene detection that is present in all the capsule pneumococci (internal control). The procedure was tested on a collection of 108 pneumococci strains gathered in Research Institute of Antimicrobial Therapy and serotyped earlier by specific PCR with electrophoretic detection and serologically by using Pneumotest-Latex kit. The sensitivity and specificity of the RT-PCR was 100%. RT-PCR procedure allowed to determine pneumococcus serotype in 79% of CSF clinical samples containing S. pneumoniae DNA. Serotype 3 and 23F were detected most frequently (13%, each).RT-PCR application does not assume causative agent seeding stage, significantly reduces analysis execution time and increases sensitivity of the study. The developed procedure will allow to begin addressing the important problem--clarification of spectra and frequency of occurrence of pneumococci serotypes circulating on the territory of Russia.

The technique to detect all possible variants of mutations in 12, 13 and 15 codons of gene KRAS was developed on the basis of the pyrosequencing technology. The analytical characteristics of the developed technique were identified. The limit of detection for mutations G34T, G35A and G38A detected on the cloned control samples consisted 3%. The limit of blank for various mutations consisted from 0.3% to 4.1%. The system was tested on clinical samples. The 7 different types of mutations were identified and detected in quantitative format. No discrepancy of pyrosequencing data with results of sequencing according Sanger was established.

Genetic characterization of 37 strains and CSF samples containing DNA of Haemophilus influenzae type b isolated in Moscow during 2007-2009.Multilocus sequence-typing method was used and also variant of method for capsule type determination was approbated.Ten sequence types, of which 7 were described in previous studies and 3 were revealed for the first time during this work, were detected in studied sample. ST-6 and ST-92 were the most frequently detected--9 strains (24%) of Hib belonging to each sequence type were revealed. All detected sequence types, except one, belong to clonal complex "ST-6" ("A1/A2"). Obtained data were compared with results of typing of Hib strains isolated in Moscow in 1999-2001. Genetic changes in studied population of Hib are characterized by decreased proportion of Hib belonging to ST-6 (from 54% to 24%) and increased number of sequence types belonging to clonal complex "ST-6" differing from ST-6 on more than one locus of allelic profile (from 2 types [2 strains, 5.4%] to 5 types [9 strains, 24%]).In 2007-2009, number of Hib strains with sequence type ST-95 (7 strains, 19%), which is typical for strains circulating in Russia, is markedly increased. Capsule type I was detected in 32 (86.5%) of studied strains, whereas capsule type II--in 5 (13.5%) of studied strains. Capsule type II was detected only in Hib strains with ST-80 sequence type.

Genotyping of Hib strains isolated in regions of Russia as well as characterization of genetic relations of typed strains with strains isolated in other areas.Genetic characterization of 31 strains of Hib isolated in Russian regions during 2005-2008 was performed by multilocus sequence typing.Studied strains belonged to 11 variants of sequence types, 6 of which were described in previous studies, whereas other 5 were isolated for the first time during this study. The most common isolated strains were ST-92 (13 strains or 42%) and ST-6 (6 strains or 19%). Typed strains were distributed to two clonal complexes. Clonal complex "A1/A2" ("ST-6") incorporates all typed strains except ST-93 strain belonging to clonal complex "B1b" ("ST-93"). The majority of studied strains (19 or 61%) had difference from "central" sequence type of clonal complex, A1/A2 ("ST-6") on not more than one allele.Clonal structure of isolated strains is analogous to the one observed in Moscow and foreign strains.

To perform advanced antigenic characterization of meningococci belonging to serogroups A and B and circulating in Moscow according to modern nomenclature of Neisseria meningitidis strains.Method of typing of "VR" fragment of FetA protein together with methods of genetic subtyping and multilocus sequence typing was used.Detailed information about studied strains was inputed in Internet database--http://pubmlst.org/neisseria/. Typing of serogroup B strains did not allow to define dominating variant of "VR, fragment of FetA protein which is in accordance with subtyping data obtained previously. Serogroup A strains were notable for less variability of "VR" fragment variants: 6 variants were detected. For the majority of serogroup A strains, it was possible to trace connection between belonging of the strain to particular genetic subgroup and its revealed antigenic profile. For strains from genetic subgroup VI, antigenic profile P1.5-2, 10; F1-5 detected in 14(18%) strains was typical, whereas antigenic profile P1.5-2, 10; F3-5 was typical for genetic subgroup X and was detected in 50 (63%) strains. Antigenic profile P1.5-2, 10-67; F3-5 was detected in 5 (6%) strains, and other 10 antigenic profiles were revealed in one strain each.Prevalence of strains with antigenic profile P1.5-2, 10; F3-5 is explained by change of predominant genetic subgroup from subgroup VI to subgroup X in Moscow population serogroup A meningococci observed after 2003.

Results of microbiological monitoring for serogroup A Neisseria meningitidis circulated in Moscow from 2002 to 2006 are presented. Using multilocus sequence-typing, molecular and epidemiologic characteristics of 32 cultures isolated from cerebro-spinal fluid of patients with generalized forms of meningococcal infection. Typed isolates belonged to 4 sequence types: CT-3349 (detected in 24 cultures), CT-2 (detected in 5 cultures), CT-75 (detected in 2 cultures), and CT-5803 (detected in 1 culture). All sequence types (except CT-5803) were detected in Moscow in previous years. Using Internet database (http://pubmlst.org/neisseria) they were genetically characterized and compared with data on serogroup A meningococci circulated in Moscow before 2002., meningococci belonging to epidemically dangerous genetic subgroup III were not detected between characterized strains. Typed isolates were distributed between subgroups VI and X, which are typical for the area under surveillance. Genetic changes in Moscow population of Neisseria meningitidis serogroup A, which manifested by shift of dominating genetic subgroup after 2002-2003, were analyzed.

46 Haemophilus influenzae strains of serotype b, isolated on the territory of Moscow and compared with foreign strains, were characterized by the method of multilocus sequencing-typing. Among the strains circulating in Moscow 10 variants of sequence-types were observed; of these, only one variant (CT-6) had been described earlier. The analysis of the data revealed that the strains under study were distributed in two clonal complexes; the overwhelming majority of the strains belonged to sequence-type 6. The distribution of Moscow strains in clonal complexes repeated the clonal organization detected in foreign strains. The conclusion was made that lower morbidity rate in Hib meningitis in Moscow (in comparison with that in the countries of Europe and America before the introduction of prophylactic vaccination) was not due to the peculiar genetic features of the circulating strains.

The analysis of meningococcal strains of different serogroups, isolated from the liquor of patients in Moscow, which was carried out with the method of multilocus sequencing-typing (MLST), was presented. At the periods of epidemic morbidity rises in Moscow the prevalence of group A meningococcal strains, belonging to subgroups III with sequence-types 5 (in the 1970s) and 7 (in 1996), was noted, and at a period between epidemics strains of genetic subgroups VI and X were isolated. Meningococcal strains, groups B and C, isolated in 1995 - 2002, had, as a rule, unique sequence-types, differing both one from another and from N. meningitidis sequence-types detected in other countries. Among group B meningococci the prevalence of strains belonging to clonal complex ST-18 was noted, while for group C meningococcci strains belonging to clonal complex ST-41/44 were most typical. Such genetic variability of circulating meningococci was regarded as characteristic of the period between epidemics, observed in Moscow since the end of the 1980s.

Haemophilius influenzae, type b (Hib) bacteria, were genotyped by multilocus sequence typing (MLST) using 5 loci (adk, fucK, mdh, pgi, recA). 42 Moscow Hib strains (including 38 isolates form cerebrospinal fluid of children, who had purulent meningitis in 1999-2001, and 4 strains isolated from healthy carriers of Hib), as well as 2 strains from Yekaterinburg were studied. In MLST a strain is characterized, by alleles and their combinations (an allele profile) referred to also as sequence-type (ST). 9 Sts were identified within the Russian Hib bacteria: ST-1 was found in 25 strains (57%), ST-12 was found in 8 strains (18%), ST-11 was found in 4 strains (9%) and ST-15 was found in 2 strains (4.5%); all other STs strains (13, 14, 16, 17, 51) were found in isolated cases (2.3%). A comparison of allelic profiles and of nucleotide sequences showed that 93% of Russian isolates, i.e. strain with ST-1, 11, 12, 13, 15 and 17, belong to one and the same clonal complex. 2 isolates from Norway and Sweden from among 7 foreign Hib strains studied up to now can be described as belonging to the same clonal complex; 5 Hib strains were different from the Russian ones.