ABO Blood Group Genotyping by Real-time PCR in Kazakh Population

Introduction. ABO blood group genotyping is a new technology in hematology that helps prevent adverse transfusion reactions in patients. Identification of antigens on the surface of red blood cells is based on serology; however, genotyping employs a different strategy and is aimed directly at genes that determine the surface proteins. ABO blood group genotyping by real-time PCR has several crucial advantages over other PCR-based techniques, such as high rapidity and reliability of analysis. The purpose of this study was to examine nucleotide substitutions differences by blood types using a PCR-based method on Kazakh blood donors.Methods. The study was approved by the Ethics Committee of the National Center for Biotechnology. Venous blood samples from 369 healthy Kazakh blood donors, whose blood types had been determined by serological methods, were collected after obtaining informed consent. The phenotypes of the samples included blood group A (n = 99), B (n = 93), O (n = 132), and AB (n = 45). Genomic DNA was extracted using a salting-out method. PCR products of ABO gene were sequenced on an ABI 3730xl DNA analyzer (Applied Biosystems). The resulting nucleotide sequences were compared and aligned against reference sequence NM_020469.2. Real-time PCR analysis was performed on CFX96 Touch™ Real-Time PCR Detection System (BioRad).Results. Direct sequencing of ABO gene in 369 samples revealed that the vast majority of nucleotide substitutions that change the ABO phenotype were limited to exons 6 and 7 of the ABO gene at positions 261, 467, 657, 796, 803, 930 and 1,060. However, genotyping of only three of them (261, 796 and 803) resulted in identification of major ABO genotypes in the Kazakh population. As a result, TaqMan probe based real-time PCR assay for the specific detection of genotypes 261, 796 and 803 was developed. The assay did not take into account several other mutations that may affect the determination of blood group, because they have a low occurrence rate and therefore have not been found in the population sample.Conclusion. Real-time PCR based method for fast and reliable ABO genotyping was developed. This assay may be used as a complement to classic serological blood typing

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

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

Collagen Type I alpha1 (COL1A1) Gene Polymorphism and Bone Mineral Density in Postmenopausal Kazakh Women

Introduction: Single nucleotide polymorphism (SNP) at the collagen type I alpha 1 gene (COL1A1) rs1800012 has been widely studied and has shown an association with bone mineral density (BMD) and fractures. A minor allele TT of this SNP was found to be greatly overrepresented in individuals with fractures compared to controls, thus becoming a good predictor of  increased fracture risk. The aim of this investigation was to evaluate potential association between COL1A1 gene polymorphism and osteoporosis in Kazakh postmenopausal women.Methods: The study population included 103 postmenopausal women recruited from Pavlodar and Almaty clinics. BMD was measured using DEXA. Genomic DNA was extracted from peripheral venous blood of study participants with Wizard® Genomic DNA Purification Kit (Promega, USA). Detection of COL1A1 +1245G/T (Sp1) polymorphism was done by the TaqMan® SNP Genotyping Assay of real-time PCR.Results: Densitometry results revealed 36 osteoporotic, 42 osteopenic, and 25 normal postmenopausal women. Data analysis of 1245G>T polymorphism in COL1A1 gene in the group of women with osteopenia and osteoporosis revealed deviation from Hardy-Weinberg equilibrium. The mutant TT genotype was prevalent compared to the heterozygous genotype GT in both groups. Distributions were 83% GG, 3% GT, and 14% TT in the group with osteopenia and 80% GG, 6% GT, and 14% TT in the group with osteoporosis. The distribution of genotypes frequency in the group of normal postmenopausal women was 76% GG, 16% GT, and 8% TT.Conclusion: These results suggest that TT genotype of COL1A1 +1245G/T (Sp1) polymorphism is associated with risk of postmenopausal osteoporosis in Kazakh women. Further studies involving a larger number of women are needed to clarify the relationship of this polymorphism with risk of osteoporosis.

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

Polymorphisms in genes involved in the absorption, distribution, metabolism, and excretion of drugs in the Kazakhs of Kazakhstan

A list of SNPs that were not found in heterozygous or homozygous variants. (DOC 83 kb

Mitochondrial and Y-chromosomal profile of the Kazakh population from East Kazakhstan

Aim To study the genetic relationship of Kazakhs from East Kazakhstan to other Eurasian populations by examining paternal and maternal DNA lineages. Methods Whole blood samples were collected in 2010 from 160 unrelated healthy Kazakhs residing in East Kazakhstan. Genomic DNA was extracted with Wizard® genomic DNA Purification Kit. Nucleotide sequence of hypervariable segment I of mitochondrial DNA (mtDNA) was determined and analyzed. Seventeen Y-short tandem repeat (STR) loci were studied in 67 samples with the Amp- FiSTR Y-filer PCR Amplification Kit. In addition, mtDNA data for 2701 individuals and Y-STR data for 677 individuals were retrieved from the literature for comparison. Results There was a high degree of genetic differentiation on the level of mitochondrial DNA. The majority of maternal lineages belonged to haplogroups common in Central Asia. In contrast, Y-STR data showed very low genetic diversity, with the relative frequency of the predominant haplotype of 0.612. Conclusion The results revealed different migration patterns in the population sample, showing there had been more migration among women. mtDNA genetic diversity in this population was equivalent to that in other Central Asian populations. Genetic evidence suggests the existence of a single paternal founder lineage in the population of East Kazakhstan, which is consistent with verbal genealogical data of the local tribes

Identification of novel risk loci, causal insights, and heritable risk for Parkinson's disease: a meta-analysis of genome-wide association studies

Background Genome-wide association studies (GWAS) in Parkinson's disease have increased the scope of biological knowledge about the disease over the past decade. We aimed to use the largest aggregate of GWAS data to identify novel risk loci and gain further insight into the causes of Parkinson's disease. Methods We did a meta-analysis of 17 datasets from Parkinson's disease GWAS available from European ancestry samples to nominate novel loci for disease risk. These datasets incorporated all available data. We then used these data to estimate heritable risk and develop predictive models of this heritability. We also used large gene expression and methylation resources to examine possible functional consequences as well as tissue, cell type, and biological pathway enrichments for the identified risk factors. Additionally, we examined shared genetic risk between Parkinson's disease and other phenotypes of interest via genetic correlations followed by Mendelian randomisation. Findings Between Oct 1, 2017, and Aug 9, 2018, we analysed 7·8 million single nucleotide polymorphisms in 37 688 cases, 18 618 UK Biobank proxy-cases (ie, individuals who do not have Parkinson's disease but have a first degree relative that does), and 1·4 million controls. We identified 90 independent genome-wide significant risk signals across 78 genomic regions, including 38 novel independent risk signals in 37 loci. These 90 variants explained 16–36% of the heritable risk of Parkinson's disease depending on prevalence. Integrating methylation and expression data within a Mendelian randomisation framework identified putatively associated genes at 70 risk signals underlying GWAS loci for follow-up functional studies. Tissue-specific expression enrichment analyses suggested Parkinson's disease loci were heavily brain-enriched, with specific neuronal cell types being implicated from single cell data. We found significant genetic correlations with brain volumes (false discovery rate-adjusted p=0·0035 for intracranial volume, p=0·024 for putamen volume), smoking status (p=0·024), and educational attainment (p=0·038). Mendelian randomisation between cognitive performance and Parkinson's disease risk showed a robust association (p=8·00 × 10−7). Interpretation These data provide the most comprehensive survey of genetic risk within Parkinson's disease to date, to the best of our knowledge, by revealing many additional Parkinson's disease risk loci, providing a biological context for these risk factors, and showing that a considerable genetic component of this disease remains unidentified. These associations derived from European ancestry datasets will need to be followed-up with more diverse data. Funding The National Institute on Aging at the National Institutes of Health (USA), The Michael J Fox Foundation, and The Parkinson's Foundation (see appendix for full list of funding sources)

ABO Blood Group Genotyping by Real-time PCR in Kazakh Population

Introduction. ABO blood group genotyping is a new technology in hematology that helps prevent adverse transfusion reactions in patients. Identification of antigens on the surface of red blood cells is based on serology; however, genotyping employs a different strategy and is aimed directly at genes that determine the surface proteins. ABO blood group genotyping by real-time PCR has several crucial advantages over other PCR-based techniques, such as high rapidity and reliability of analysis. The purpose of this study was to examine nucleotide substitutions differences by blood types using a PCR-based method on Kazakh blood donors. Methods. The study was approved by the Ethics Committee of the National Center for Biotechnology. Venous blood samples from 369 healthy Kazakh blood donors, whose blood types had been determined by serological methods, were collected after obtaining informed consent. The phenotypes of the samples included blood group A (n = 99), B (n = 93), O (n = 132), and AB (n = 45). Genomic DNA was extracted using a salting-out method. PCR products of ABO gene were sequenced on an ABI 3730xl DNA analyzer (Applied Biosystems). The resulting nucleotide sequences were compared and aligned against reference sequence NM_020469.2. Real-time PCR analysis was performed on CFX96 Touch™ Real-Time PCR Detection System (BioRad). Results. Direct sequencing of ABO gene in 369 samples revealed that the vast majority of nucleotide substitutions that change the ABO phenotype were limited to exons 6 and 7 of the ABO gene at positions 261, 467, 657, 796, 803, 930 and 1,060. However, genotyping of only three of them (261, 796 and 803) resulted in identification of major ABO genotypes in the Kazakh population. As a result, TaqMan probe based real-time PCR assay for the specific detection of genotypes 261, 796 and 803 was developed. The assay did not take into account several other mutations that may affect the determination of blood group, because they have a low occurrence rate and therefore have not been found in the population sample. Conclusion. Real-time PCR based method for fast and reliable ABO genotyping was developed. This assay may be used as a complement to classic serological blood typing

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