The need for standardized biobanks in Kazakhstan

Biobanks are an important tool for clinical and research studies conducted on biomarkers of genetic therapy, diagnostic tests and new drugs; however, most biobanks remain incomplete and are often used without uniform standards and criteria. There is also a a lack of high-quality biological samples and many bioethical problems are often overlooked.Currently, Kazakhstan has no standard requirements and protocols for biomedical organizations. However, .an analysis of published data shows that possibly hundreds of samples are analyzed. Therefore, an establishment of biobank with standardized requirements could create better quality research.The National Center for Biotechnology has already started a biobank with more than 1,500 blood samples, with the ultimate goal of creating a biobank including around 10,000 blood samples of healthy volunteers, the same number of samples obtained from individuals with cardiovascular and endocrine diseases with samples stored under special conditions. The database contains demographic characteristics of donor’s medical history. Informed consent for research received from all donors. This biobank can be considered as a national resource for scientific research

Pharmacogenetic research in Kazakhstan

Introduction: Pharmacogenomics is an emerging field of medicine that combines genetics and pharmacology. Pharmacogenomic research is relatively new in Kazahkstan, but, in recent years, significant progress has been made in this field. The National Scientific Laboratory for Biotechnology has launched several government-funded research projects focused on finding genetic markers that determine susceptibility to various drugs. Another goal of pharmacogenetic research in the laboratory is to find the pharmacogenomic markers that target cardiovascular diseases, accounting for allelic frequencies in selected genes in the Kazakh population. In addition, pharmacogenomic testing kits allow patients to choose the drug dosage. For example, the drug Warfarin has been developed within the framework of the "Technology Commercialization Project,” funded jointly by the Ministry of Education and Science of the Republic of Kazakhstan and the World Bank.Material and methods: The pharmacogenomic studies were conducted using the real-time PCR and direct DNA sequencing. DNA was isolated from venous blood or buccal cells, collected from patients.Results: To date, we have identified the most promising areas of research in the field of pharmacogenomics in Kazakhstan. The allelic frequencies of a number of polymorphisms in the Kazakh population have been calculated (CYP2C9, CYP2C19, CYP3A4, VKORC1, CYP4F2, GGCX, CYP2D6, CYP1A2, NAT2, GSTP1, SLC47A1). A unique repository of DNA samples was established and is being replenished during the implementation of aforementioned projects. Development of the testing kit for individual selection of Warfarin dosage is nearing completion. A patent, named "Method of Selection Based Dose Warfarin Genotyping for the Kazakh Population" has been recently obtained. An application for another patent, titled "Express Method of Correction of Warfarin Dosing, Based on Real-time PCR" has received positive evaluation. The results of domestic pharmacogenomic studies will allow a more rational selection of drugs and their dosage regimens specific to the Kazakh population

Frequency of NAT2 and GSTP1 polymorphisms in the Kazakh population

Introduction: Phase II xenobiotic biotransformation enzymes perform detoxification of hydrophilic and often toxic Phase I products by glutathionetransferase (GST), UDP-glucuronosyltransferase (UDF), N-acetyltransferase (NAT) families and other enzymes. GST protein family metabolizes a large number of electrophilic xenobiotics, including drugs, by conjugating them with glutathione. Arylamine-N-acetyltransferase (NAT) catalyzes the acetylation of the aromatic and heterocyclic amines.Materials and methods: The current study has assessed the frequencies of NAT2 and GSTP1 genes polymorphisms in 326 healthy individuals from different regions of Kazakhstan by using Real-Time PCR and direct sequencing methods.Results: Allele frequencies were derived for NAT2*5 (0.54) and GSTP1 (0.27). GSTP1 alleles were in Hardy – Weinberg equilibrium (p > 0.05), while NAT2*5 (p = 0.00) were not.  The population differences between North, Northeast and South Kazakhstan regions were determined. Statistically significant differences in the frequency of genotypes were not found.Conclusion: Allelic polymorphisms of NAT2*5 and GSTP1 genes vary widely in different populations. Kazakh population was significantly different from Asian, Caucasoid, African-American and Hispanic ones by NAT2*5 and GSTP1 genes. Allelic variants of the NAT2*5 were detected with a low frequency in Asian populations. Allelic frequency in other world populations varies from 30 to 50%. The differences between Kazakh (0.54) and the world population were statistically significant (p < 0.05). The frequency of GSTP1 (rs1695) in the African American population is 42%. The frequency of GSTP1 in Asian populations varies from 11% to 23%, in Caucasoid populations it is about 30%. The differences between Kazakh population (0.27) and other populations selected from the literature were statistically significant (p < 0.05).The study of mutations in GSTP1 and NAT2 genes is necessary to assess the risk of the development of various diseases, such as cancer. Information on allelic polymorphisms also might be useful for personalized drug prescription for such drugs as cyclophosphamide, cisplatin, methotrexate, isoniazid, pyrazinamide, and rifampin

Estrogen Receptor Gene (ESR1) PVUII and XBAI Polymorphisms and Bone Mineral Density in Kazakh Women

Introduction: Osteoporosis is a common age-related disease that is strongly influenced by genetics. Polymorphisms of the estrogen receptor gene alpha (ESR1) are consistently been associated with bone mineral density (BMD) and fracture.The purpose of this investigation was to evaluate potential association of single nucleotide polymorphism (SNP) variants of the ESR1 gene and bone mineral density (BMD) of the lumbar spine in Kazakh women.Methods: 140 female participants in Pavlodar clinics with varying measures of BMD. We are examined the potential association of BMD with 2 SNPs from the ESR1 gene (rs2234693 [PvuII] and rs9340799 [XbaI]). Genotyping of the PvuII and XbaI polymorphisms was performed by direct sequencing of the gene fragments containing restriction sites with the identification of genotypes PP, Pp, pp and XX, Xx, xx respectively.Results: Unadjusted mean BMD values ranged from 1.14±0.14 g/cm2 in Caucasian women and 1.03±0.11 g/cm2 in Asian women. The association between PvuII polymorphism and BMD at the lumbar spine (p= 0.04 for PP=Pp=pp) was statistically significant in all women. The XbaI polymorphism was not associated with BMD at lumbar spine. The relative risk for low BMD was higher for the marker PvuII (RR=1.51) than for the marker XbaI (RR=1.35).Conclusion: The PvuII polymorphism had a weak association with lumbar spine BMD.  XbaI polymorphism was unlikely to be a predictor of lumbar spine BMD in Kazakh women. These conclusions could help to determine the genetic risk factors for osteoporosis; however, further studies on the association between gene polymorphisms and BMD are needed including larger numbers of participants and genes to clarify genetic risks

Vitamin D Receptor Gene Polymorphisms and Breast Cancer Risk in Kazakhstan

Introduction: The steroid hormone 1,25-dihydroxyvitamin D3 is thought to protect against breast cancer. The activity of 1,25-dihydroxyvitamin D3 is mediated via the vitamin D receptor (VDR), and a number of polymorphisms in the VDR gene have been identified. These result in distinct genotypes, some of which may alter susceptibility to breast cancer. Two common single nucleotide polymorphisms (SNP) in the VDR gene (VDR), rs1544410 (BsmI) and rs2228570 (FokI), have been inconsistently associated with breast cancer risk. Increased risk has been reported for the FokI ff genotype, which encodes a less transcriptionally active isoform of VDR. A reduced risk has been reported for the BsmI BB genotype which may influence VDR mRNA stability.Aim: We have investigated whether specific VDR gene polymorphisms are associated with breast cancer risk in Kazakhstan women.Material and Methods: In a case–control study, female breast cancer patients (315) and a female control group (n=604) were tested for two VDR polymorphisms. Statistical analysis was conducted using SPSS19.0.Results: The VDR rs2228570 (FokI) polymorphism was associated with an increased occurence of BC [rs2228570 (folk) ff vs. FF genotype: OR=1.71; 95% CI=1.21-2.43]. No association was noted between rs1544410 (BsmI) BB and breast cancer risk [OR=0.68; 95% CI=0.49-0.95].Conclusion: Although the factors that increase breast cancer susceptibility remain uncertain, future large studies should integrate genetic variation in VDR with biomarkers of vitamin D status. Additional testing on the effect of varying genotypes on the functional mechanisms of the VDR could help to improve future testing and treatment of woman at risk for breast cancer

BRCA1 and BRCA2 Gene Mutations Screening In Sporadic Breast Cancer Patients In Kazakhstan.

Background: A large number of distinct mutations in the BRCA1 and BRCA2 genes have been reported worldwide, but little is known regarding the role of these inherited susceptibility genes in breast cancer risk among Kazakhstan women.Aim: To evaluate the role of BRCA1/2 mutations in Kazakhstan women presenting with sporadic breast cancer.Methods: We investigated the distribution and nature of polymorphisms in BRCA1 and BRCA2 entire coding regions in 156 Kazakhstan sporadic breast cancer cases and 112 age-matched controls using automatic direct sequencing.Results: We identified 22 distinct variants, including 16 missense mutations and 6 polymorphisms in BRCA1/2 genes. In BRCA1, 9 missense mutations and 3 synonymous polymorphisms were observed. In BRCA2, 7 missense mutations and 3 polymorphisms were detected. There was a higher prevalence of observed mutations in Caucasian breast cancer cases compared to Asian cases (p<0.05); higher frequencies of sequence variants were observed in Asian controls. No recurrent or founder mutations were observed in BRCA1/2 genes. There were no statistically significant differences in age at diagnosis, tumor histology, size of tumor, and lymph node involvement between women with breast cancer with or without the BRCA sequence alterations.Conclusions:Considering the majority of breast cancer cases are sporadic, the present study will be helpful in the evaluation of the need for the genetic screening of BRCA1/2 mutations and reliable genetic counseling for Kazakhstan sporadic breast cancer patients. Evaluation of common polymorphisms and mutations and breast cancer risk in families with genetic predisposition to breast cancer is ongoing in another current investigation.

О стандартизации и оценке систем непрерывного мониторинга уровня глюкозы

   Continuous glucose monitoring (CGM) systems are often used to monitor blood glucose levels. Most commercially available CGM systems continuously measure glucose concentrations in the interstitial fluid of subcutaneous adipose tissue. However, there is currently no internationally accepted reference method for measuring interstitial fluid glucose, which is a prerequisite for metrological traceability of glucose measurements obtained using CGM. Since manufacturers do not provide information about the traceability chain and measurement uncertainty of their systems, CGM-derived glucose values cannot currently be adequately traced to standards or higher order reference measurement procedures. Additionally, the «mean absolute relative difference» (MARD) often used to describe the analytical performance of CGM systems is dependent on many factors. For example, the MARD can be significantly affected by the «lag time» between the change in blood glucose and interstitial glucose, especially at high rates of change in glucose. Finally, modern automated insulin delivery (ADI) systems with integrated CGM can automatically suspend or increase insulin infusion in response to current and/or predicted hypoglycemic and hyperglycemic phenomenon in children and adults with type 1 diabetes mellitus (T1DM).   The purpose of the review is justification of the necessity to establish metrological traceability of glucose measurements with CGM systems, as well as a discussion of the analytical and clinical characteristics of CGM systems proposed by various professional communities.   Based on the results of the review, it was concluded that it is necessary to 1) develop metrological support for glucose measurements performed using CGM systems, 2) solve the problems of ensuring the accessibility and usability of CGM systems by patients in real conditions.   Контроль уровня глюкозы в крови осуществляется с помощью систем непрерывного мониторинга глюкозы (НМГ). Среди всех коммерчески доступных систем НМГ превалируют системы, непрерывно измеряющие концентрацию глюкозы в интерстициальной жидкости подкожной жировой ткани. Однако сегодня не существует международно признанной референтной методики измерения глюкозы в интерстициальной жидкости, – значит, не соблюдается необходимое условие для обеспечения метрологической прослеживаемости результатов измерений глюкозы, полученных с применением НМГ. К тому же производители не предоставляют информацию о цепочке прослеживаемости и неопределенности измерений их систем, следовательно, полученные с помощью НМГ значения глюкозы не могут быть отслежены до эталонов или референтных методик измерений более высокого порядка. Кроме того, часто используемый для описания аналитической эффективности систем НМГ показатель – средняя абсолютная относительная разница (МАRD) – зависит от многих факторов. Например, на МАRD может существенно влиять «время задержки» между изменением уровня глюкозы в крови и интерстициальной глюкозой, особенно при высоких скоростях изменения уровня глюкозы. Наконец, современные системы автоматизированной доставки инсулина (АДИ) со встроенным НМГ могут автоматически приостанавливать или увеличивать инфузию инсулина в ответ на текущие и/или прогнозируемые гипогликемические и гипергликемические явления у детей и взрослых с сахарным диабетом 1 типа (СД1).   Целью обзора является обоснование необходимости установления метрологической прослеживаемости измерений глюкозы системами НМГ, а также обсуждение аналитических и клинических характеристик систем НМГ, предложенных различными профессиональными сообществами.   По результатам обзора сделаны выводы о необходимости, первое – развития метрологического обеспечения измерений глюкозы, выполняемых с применением систем НМГ, и второе – решения проблем обеспечения пациентам доступности и удобства пользования системами НМГ в реальных условиях

Ureaplasma antigenic variation beyond MBA phase variation: DNA inversions generating chimeric structures and switching in expression of the MBA N-terminal paralogue UU172

Phase variation of the major ureaplasma surface membrane protein, the multiple-banded antigen (MBA), with its counterpart, the UU376 protein, was recently discussed as a result of DNA inversion occurring at specific inverted repeats. Two similar inverted repeats to the ones within the mba locus were found in the genome of Ureaplasma parvum serovar 3; one within the MBA N-terminal paralogue UU172 and another in the adjacent intergenic spacer region. In this report, we demonstrate on both genomic and protein level that DNA inversion at these inverted repeats leads to alternating expression between UU172 and the neighbouring conserved hypothetical ORF UU171. Sequence analysis of this phase-variable ‘UU172 element’ from both U. parvum and U. urealyticum strains revealed that it is highly conserved among both species and that it also includes the orthologue of UU144. A third inverted repeat region in UU144 is proposed to serve as an additional potential inversion site from which chimeric genes can evolve. Our results indicate that site-specific recombination events in the genome of U. parvum serovar 3 are dynamic and frequent, leading to a broad spectrum of antigenic variation by which the organism may evade host immune responses

Mixed Infection with cagA Positive and cagA Negative Strains of Helicobacter pylori Lowers Disease Burden in The Gambia

BACKGROUND: The prevalence of Helicobacter pylori including strains with putatively virulent genotypes is high, whereas the H. pylori-associated disease burden is low, in Africa compared to developed countries. In this study, we investigated the prevalence of virulence-related H. pylori genotypes and their association with gastroduodenal diseases in The Gambia. METHODS AND FINDINGS: DNA extracted from biopsies and H. pylori cultures from 169 subjects with abdominal pain, dyspepsia or other gastroduodenal diseases were tested by PCR for H. pylori. The H. pylori positive samples were further tested for the cagA oncogene and vacA toxin gene. One hundred and twenty one subjects (71.6%) were H. pylori positive. The cagA gene and more toxigenic s1 and m1 alleles of the vacA gene were found in 61.2%, 76.9% and 45.5% respectively of Gambian patients harbouring H. pylori. There was a high prevalence of cagA positive strains in patients with overt gastric diseases than those with non-ulcerative dyspepsia (NUD) (p = 0.05); however, mixed infection by cagA positive and cagA negative strains was more common in patients with NUD compared to patients with gastric disease (24.5% versus 0%; p = 0.002). CONCLUSION: This study shows that the prevalence of H. pylori is high in dyspeptic patients in The Gambia and that many strains are of the putatively more virulent cagA+, vacAs1 and vacAm1 genotypes. This study has also shown significantly lower disease burden in Gambians infected with a mixture of cag-positive and cag-negative strains, relative to those containing only cag-positive or only cag-negative strains, which suggests that harbouring both cag-positive and cag-negative strains is protective

Comparative Genomics of Helicobacter pylori Strains of China Associated with Different Clinical Outcome

In this study, a whole-genome CombiMatrix Custom oligonucleotide tiling microarray with 90000 probes covering six sequenced Helicobacter pylori (H. pylori) genomes was designed. This microarray was used to compare the genomic profiles of eight unsequenced strains isolated from patients with different gastroduodenal diseases in Heilongjiang province of China. Since significant genomic variation was found among these strains, an additional 76 H. pylori strains associated with different clinical outcomes were isolated from various provinces of China. These strains were tested by polymerase chain reaction to demonstrate this distinction. We identified several highly variable regions in strains associated with gastritis, gastric ulceration, and gastric cancer. These regions are associated with genes involved in the bacterial type I, type II, and type III R-M systems. They were also associated with the virB gene, which lies on the well-studied cag pathogenic island. While previous studies have reported on the diverse genetic characterization of this pathogenic island, in this study, we find that it is conserved in all strains tested by microarray. Moreover, a number of genes involved in the type IV secretion system, which is related to horizontal DNA transfer between H. pylori strains, were identified in the comparative analysis of the strain-specific genes. These findings may provide insight into new biomarkers for the prediction of gastric diseases