Polymerase Chain Reaction With Sequence-specific Primers Research Articles

A genomic study on distribution of human leukocyte antigen (HLA)-A and HLA-B alleles in Lak population of Iran

Anthropological studies based on the highly polymorphic gene, human leukocyte antigen (HLA), provide useful information for bone marrow donor registry, forensic medicine, disease association studies, as well as infertility treatment, designing peptide vaccines against tumors, and infectious or autoimmune diseases. The aim of this study was to determine HLA-A and HLA-B allele frequencies in 100 unrelated Lak/lᴂk/individuals from Lorestan province of Iran. Finally, we compared the results with that previously described in Iranian population. Commercial HLA-Type kits from BAG (Lich, Germany) company were used for determination of the HLA-A and HLA-B allele frequencies in genomic DNA, based on polymerase chain reaction with sequence specific primer (PCR-SSP) assay. The differences between the populations in distribution of HLA-A and HLA-B alleles were estimated by chi-squared test with Yate's correction. The most frequent HLA-A alleles were *24 (20%), *02 (18%), *03 (12%) and *11 (10%), and the most frequent HLA-B alleles were *35 (24%), *51 (16%), *18 (6%) and *38 (6%) in Lak population. HLA-A*66 (1%), *74(1%) and HLA-B*48 (1%), *55(1%) were the least observed frequencies in Lak population. Our results based on HLA-A and HLA-B allele frequencies showed that Lak population possesses the previously reported general features of Iranians but still with unique.

Distribution of DI*A and DI*B Allele Frequencies and Comparisons among Central Thai and Other Populations.

Alloantibodies to the Diego (DI) blood group system, anti-Dia and anti-Dib are clinically significant in causing hemolytic transfusion reactions (HTRs) and hemolytic disease of the fetus and newborn (HDFN), especially in Asian populations with Mongolian ancestry. This study aimed to report the frequency of the DI*A and DI*B alleles in a Central Thai population and to compare them with those of other populations previously published. Altogether, 1,011 blood samples from unrelated healthy blood donors at the National Blood Centre, Thai Red Cross Society, Bangkok were included. Only 391 samples were tested with anti-Dia by conventional tube technique. All samples were genotyped for DI*A and DI*B alleles using an in-house polymerase chain reaction with sequence-specific primer (PCR-SSP) technique. The DI phenotyping and genotyping results were in 100% concordance. The DI*A and DI*B allele frequencies among 1,011 Central Thais were 0.0183 (37/2,022) and 0.9817 (1,985/2,022), respectively. Allele frequencies were compared between Central Thai and other populations. Our data shows that DI*A and DI*B allele frequencies are similar to Southeast Asian, Brazilian, Southern Brazilian and American Native populations; whereas, these frequencies significantly differ from those reported in East Asian, Italian, Alaska Native/Aleut, Hawaiian/Pacific Islander and Filipino populations (P<0.05), corresponding to the results of a matrix of geometric genetic distances. This study confirms that the prevalence of DI*A and DI*B alleles among Central Thais is similar to Southeast Asians and different to others populations of the world. A PCR-based identification of DI genotyping should overcome some of the serological limitations in transfusion medicine and provides a complementary tool for further population-genetic studies.

Killer Cell Immunoglobulin-Like Receptor Genes and their HLA-C Ligands in Hashimoto Thyroiditis in a Chinese Population

Natural killer (NK) cells serve as primary immune surveillance and are partially regulated by combinations of killer immunoglobulin-like receptors (KIR) and their human leukocyte antigen-C (HLA-C) ligands. Alterations in NK cell activity have been associated with Hashimoto thyroiditis (HT). The aim of this study was to determine whether certain KIR/HLA-C genotype combinations play a role in HT pathogenesis. The present study enrolled 107 unrelated HT patients and 108 random healthy individuals in a case-control study. Blood was collected for DNA extraction; typing of KIR genes and HLA-C alleles was performed by polymerase chain reaction with sequence specific primers (PCR-SSP), followed by electrophoresis on agarose gels. Among a panel of KIR2D/HLA-C genotype combinations, the frequency of KIR2DS2/HLA-C1 was significantly increased in HT patients compared to controls (33.64% vs. 12.96%, P<.001). To further analyze the precise genotype, we investigated inhibitory or activating KIR/HLA-C gene pairs when their corresponding activating or inhibitory KIR genes were absent in the 2 groups. Only the frequency of KIR2DS2(-)2DL2/3(+)HLA-C1(+) was significantly decreased in HT patients compared to controls (48.60% vs. 70.37%, P = .001). Our data suggest that KIR2DS2/HLA-C1 may correlate with HT pathogenesis. On the contrary, the predominance of KIR2DL2/3/HLA-C1 in the absence of KIR2DS2 suggests a potential inhibitory role in HT pathogenesis. In conclusion, our findings may further elucidate the mechanisms underlying the pathogenesis of HT and other autoimmune diseases. HLA-C = human leukocyte antigen-C HT = Hashimoto thyroiditis KIR = killer immunoglobulin-like receptor NK = natural killer PCR = polymerase chain reaction.

Ankylosing Spondylitis Patients with HLA-B*2704 have More Uveitis than Patients with HLA-B*2705 in a North Chinese Population

ABSTRACTPurpose: To investigate the correlation between clinical features of ankylosing spondylitis (AS) and different human leukocyte antigen (HLA)-B27 subtypes.Methods: We conducted a cross-sectional study of 216 patients with AS. HLA-B27 and its subtypes were detected by polymerase chain reaction with sequence specific primer (PCR-SSP). Clinical features were compared between the different HLA-B27 subtypes. A meta-analysis on uveitis frequencies in AS patients with HLA-B*2705 vs 2704 was performed.Results: The most prevalent subtypes of HLA-B27 were HLA-B*2704 (66.1%) and HLA-B*2705 (32.2%). There were 57 HLA-B27-positive AS patients with the history of uveitis; 45 were B*2704 and 12 were B*2705. Patients with B*2704 had more uveitis than B*2705 (p = 0.021). After meta-analysis, there was no significant difference in the presence of uveitis between HLA-B*2704 and HLA-B*2705.Conclusions: AS patients with B*2704 have a higher risk of uveitis than AS with B*2705 in a north Chinese people.

Polymorphism of Platelet Specific Antigens (HPA-1-5, 15) in Han Donors in the North Area of Henan Province

To study the gene polymorphism distribution characteristics of human platelet HPA-1-5 and 15 blood group antigens and construct a certain scale of platelet HPA database in the north area of Henan Province so as to provide platelet apheresis for clinical departments. Using polymerase chain reaction with sequence-specific primers (PCR-SSP), the genotyping of HPA-1-5 and 15 system was carried out; the periperal blood of 500 healthy Han donors in north area of Henan Province was collected randomly, the gene and genotype frequencies were detected by direct counting method, and the population distribution frequncy of HPA genes was analyzed by Hardy-Weinberg balance test, and compared with other regions and ethnics by using χ(2) test. There was statistically significant (P < 0.05) of increase HPA-3b and HPA-5a in North area of Henan Province, compared with Chinese Han population; the HPA-3b and 5a increase and HPA-2a decrease were statistically significant (P < 0.05), compared with Ethnic minority of China. There was partly increase of HPA-1a, 2a, 3a and 5a, compared with different regions and ethnic in abroad. HPA allele genes of 500 Han donors in the North area of Henan Province were as follows: 0.985 and 0.015 for 1a and 1b; 0.924 and 0.076 for 2a and 2b; 0.469 and 0.531 for 3a and 3b; 1.000 and 1.000 for 4a and 5a; 0.532 and 0.468 for 15a and 15b, respectively. HPA allele gene frequencies were 1aa0.970, 1ab0.030; 2aa0.848, 2ab0.152; 3aa0.222, 3ab0.494, 3bb0.284; 4aa1.000; 5aa1.000; 15aa0.282, 15ab0.500, 15bb0.218. Compared with other regions and ethnic, HPA gene frequencies partly had statistical significance. Distribution of HPA allele frequencies in the North area of Henan province is in accordence with the Hardy-Weinberg law. There are race and regional differences in HPA allele gene frequencies, compared with other regions and countries. And the HPA systems HPA-3 and 15 display the genetic polymorphisms, which provides a theoretical basis for the relevant research of the same type platelet infusion and alloimmune thrombocytopenia.

Gene frequency of human platelet alloantigens‐1 to ‐6 and ‐15 in Saudi blood donors

Human platelet antigens (HPAs) are involved in the pathogenesis of several clinical conditions, such as platelet transfusion purpura (PTP), refractoriness to platelet transfusion and neonatal alloimmune thrombocytopenia (NAITP). Typing of HPA (1-6 and 15) has not been carried on the Saudi population. This is the first study of all the seven HPA systems on Arabs. The aim of this prospective study was to determine the frequency of HPA (1-6 and 15) in Saudis. A total of 100 randomly selected Saudi blood donor samples were genotyped using the polymerase chain reaction with sequence-specific primers (PCR-SSP). The most common HPA genotypes among Saudis were HPA-1 a + b- (75%), HPA-2 a + b- (62%), HPA-3 a + b- (51·5%), HPA-4 a + b- (99%), HPA-5 a + b- (76·5%), HPA-6 a + b- (100%) and HPA-15 a + b + (50%). The prevalent allele among the HPA systems was (a), except in the HPA-15 system where the (b) allele was found in 52% of the subjects. Comparisons with other ethnic populations uncovered marked differences in the distribution of HPA alleles. Studying the prevalence of HPA antigens in Saudi population will help in the understanding of its role in platelet-related disorders. It will also enable the blood bank to establish an HPA-based donor registry that will be a valuable source of compatible platelet-therapeutic products to alloimmunised patients. This will also enhance the safety and efficacy of platelet transfusion. This data obtained will form an addition to the existing body of literature in transfusion research.

Development of a Multiplex PCR for Human Neutrophil Antigens-1, -3, -4, and -5 Genotyping Among Thai Blood Donors.

Different polymerase chain reaction (PCR) techniques for human neutrophil antigens (HNA) genotyping have been implemented to diagnose the clinical conditions of patients with alloimmune neutropenia, febrile non-hemolytic transfusion reactions, and transfusion-related acute lung injury and to provide effective HNAmatched granulocyte transfusions. The present study aimed to develop an in-house multiplex-PCR for HNA-1, -3, -4, and -5 genotyping in the Thai population. Altogether, 500 DNA samples obtained from unrelated, healthy Thai blood donors at the National Blood Centre, Thai Red Cross Society, Bangkok, Thailand were included. Three hundred DNA samples of known HNA genotyping based on PCR with sequence specific primers (PCR-SSP), as previously described, were tested with the newly developed multiplex PCR. Additionally, 200 DNA samples of unknown HNA genotyped donors were tested for HNA-1, -3, -4, and -5 genotyping using multiplex-PCR. Validity of HNA-1, -3, -4, and -5 genotyping by multiplex PCR using known DNA controls and the comparison of the genotyping results between PCR-SSP and multiplex PCR were in agreement. Interestingly, the rare genotype HNA-4b4b was not found in this study, similar to previous studies in Thai and other populations. Moreover, 30 samples were randomly tested twice for HNA genotyping using the multiplex-PCR and demonstrated reproducible results and were confirmed by DNA sequencing. This study shows that the newly developed multiplex-PCR is cost effective and less time consuming compared with PCR-SSP. The multiplex PCR can be used as an alternative technique for HNA-1, -3, -4, and -5 genotyping for routine testing, especially in other developing countries due to its simplicity and accuracy.

HLA class II alleles and risk for peripheral neuropathy in type 2 diabetes patients.

The potential impact of human leukocyte antigen (HLA) genotype variations on development of diabetic peripheral neuropathy (DPN) is not well determined. This study aimed to identify the association of HLA class II alleles with DPN in type 2 diabetes (T2D) patients. Totally 106 T2D patients, 49 with DPN and 57 without DPN, and 100 ethnic-matched healthy controls were analyzed. Both groups of the patients were matched based on sex, age, body mass index (BMI) and duration of T2D. Polyneuropathy was diagnosed using electrodiagnostic methods. HLA-DRB1 and DQB1 genotyping was performed in all subjects by the polymerase chain reaction with sequence-specific primers (PCR-SSP) method. T2D patients with DPN showed higher frequencies of HLA-DRB1*10 and DRB1*12 alleles compared to control group (P = 0.04). HLA-DQB1*02 allele and HLA-DRB1*07-DQB1*02 haplotype were associated with a decreased risk for developing DPN in T2D patients (P = 0.02 and P = 0.05 respectively). Also, patients with severe neuropathy showed higher frequencies of DRB1*07 (P = 0.003) and DQB1*02 (P = 0.02) alleles than those with mild-to-moderate form of neuropathy. The distribution of DRB1 and DQB1 alleles and haplotypes were not statistically different between all patients and healthy controls. Our findings implicate a possible protective role of HLA-DQB1*02 allele and HLA-DRB1*07-DQB1*02 haplotype against development of peripheral neuropathy in T2D patients. Therefore, variations in HLA genotypes might be used as genetic markers for prediction and potentially management of neuropathy in T2D patients.

Genotype frequency of human neutrophil antigen-3 polymorphisms in the Yi, Han, and Tibetan populations of China.

Human neutrophil antigen-3 (HNA-3) alloantibodies can cause fatal transfusion-related acute lung injury (TRALI). Most frequencies of SLC44A2 alleles encoding the HNA-3a/b antigens have been established in Han individuals by polymerase chain reaction with sequence-specific priming (PCR-SSP). We sequenced SLC44A2 gene fragments and determined allele frequencies in three ethnicities of China. Genomic DNA was extracted from 448 samples of 100 blood donors of Yi ethnicity in Xichang, Liangshan; 248 Han in Nanjing, Jiangsu; and 100 Tibetan in Lhasa, Tibet. A PCR-SSP was applied to determine the phase of two single-nucleotide polymorphisms (SNPs); SLC44A2 haplotypes were constructed. In the 567 nucleotides of the SLC44A2 gene covered by our sequencing approach in Han individuals, we detected the known 331-44G>A (rs12972963) and 461G>A (rs2288904) polymorphisms. In the 243 nucleotides sequenced in Yi and Tibetan populations, we detected the known 461G>A and 503-15T>C (rs1560711) polymorphisms. A PCR-SSP for the common HNA-3a/b SNP was 100% concordant. The frequencies of the HNA-3a allele were 0.58, 0.66, and 0.69 in Yi, Han (Nanjing), and Tibetan, respectively (0.42, 0.34, and 0.31 for HNA-3b). The Yi population of China had the highest frequency of blood donors at risk of harboring anti-HNA-3a compared to any population studied so far. We confirmed that the underlying SLC44A2*2 allele is more common in China than in any European or African populations.

IL-1A rs1800587, IL-1B rs1143634 and IL-1R1 rs2234650 polymorphisms in Iranian patients with systemic sclerosis.

Systemic Sclerosis (SSc) is a systemic autoimmune disorder, with ambiguous pathogenesis. Genetic and environmental factors were proved to be correlated with SSc aetiology. Single nucleotide polymorphisms (SNPs) in cytokine genes can alter the structure and function of the cytokines and consequently may increase the susceptibility to a specific disease. In this study, we investigated SNPs of the IL-1 gene cluster in Iranian SSc patients. We obtained blood samples from 170 SSc patients and 213 healthy individuals. Cytokine genotyping results were obtained by polymerase chain reaction with sequence-specific primers (PCR-SSP). IL-1A rs1800587, IL-1B rs1143634 and IL-1R1 rs2234650 were evaluated for SNP study. The frequency of the IL-1B rs1143634 CT genotype was significantly lower in SSc patients compared to the controls (OR=0.584; 95% CI=0.385-0.886; P-value=0.023), so we propose that CT genotype of this allele might be protective. According to our haplotype analysis, CCC haplotype frequency is higher in the control group compared to SSc patients (OR=1.575; 95% CI=1.176-2.111; P-value=0.008) and in contrast, CTC haplotype frequency is lower in the control group compared to SSc patients (OR=0.152; 95% CI=0.047-0.484; P-value=0.002), so they might decrease and increase the susceptibility of having SSc, respectively. In addition, we reported two significant diplotypes frequency differences among SSc patients and healthy individuals. It is highly important that there is not much resemblance between the IL-1 gene cluster polymorphism in different populations, so we can indicate that SNPs may play critical roles when they are combined with other genetic and environmental factors.

Association study of interleukin-1 family and interleukin-6 gene single nucleotide polymorphisms in recurrent aphthous stomatitis.

Recurrent aphthous stomatitis (RAS) is a common painful, ulcerative oral inflammatory disorder with unknown aetiology. Immune system and aberrant cytokine cascade deemed to be critical in outbreaks of RAS ulcers. Interleukin-1 (IL-1) and IL-6 are the most potent pro-inflammatory cytokines. Single nucleotide polymorphisms (SNPs) of IL-1 and IL-6 genes can affect the secretion of these cytokines. The aim of this study was to investigate the association between RAS and IL-6 and IL-1 in Iranian subjects with minor RAS. Genomic DNA was obtained from 64 Iranian patients with RAS. IL-1α C -889 T, IL-1β C -511 T, IL-1β C +3962 T, IL-1R C pst-I 1970 T, IL-1Ra C Mspa-I11100 T, IL-6 C -174 G and IL-6 A nt +565 G polymorphisms were determined using polymerase chain reaction with sequence-specific primers (PCR-SSP). The frequency of C -174 C genotype in the patients group was significantly different from the healthy control. No other significant differences were found in genotype and alleles frequencies between the two groups. These results indicate that certain SNPs of IL-6 gene at position -174 which located in promoter have association with predisposition of individuals to RAS.

Duffy blood group genotyping in Thai blood donors.

BackgroundDuffy (FY) blood group genotyping is important in transfusion medicine because Duffy alloantibodies are associated with delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. In this study, FY allele frequencies in Thai blood donors were determined by in-house PCR with sequence-specific primers (PCR-SSP), and the probability of obtaining compatible blood for alloimmunized patients was assessed.MethodsFive hundred blood samples from Thai blood donors of the National Blood Centre, Thai Red Cross Society, were included. Only 200 samples were tested with anti-Fya and anti-Fyb using the gel technique. All 500 samples and four samples from a Guinea family with the Fy(a-b-) phenotype were genotyped by using PCR-SSP. Additionally, the probability of obtaining antigen-negative red blood cells (RBCs) for alloimmunized patients was calculated according to the estimated FY allele frequencies.ResultsThe FY phenotyping and genotyping results were in 100% concordance. The allele frequencies of FY*A and FY*B in 500 central Thais were 0.962 (962/1,000) and 0.038 (38/1,000), respectively. Although the Fy(a-b-) phenotype was not observed in this study, FY*BES/FY*BES was identified by PCR-SSP in the Guinea family and was confirmed by DNA sequencing.ConclusionsOur results confirm the high frequency of the FY*A allele in the Thai population, similar to that of Asian populations. At least 500 Thai blood donors are needed to obtain two units of antigen-negative RBCs for the Fy(a-b+) phenotype.

An Improved Method With High Specificity for KIR2DL1 Functional Allele Typing.

As new killer-cell immunoglobulin-like receptor (KIR) alleles are discovered, a challenge in KIR typing is maintaining sensitivity and specificity. A single nucleotide polymorphism assay can be used to type functional KIR2DL1 alleles. We improved recently on the earlier method by using a higher-specificity assay. The major modifications include the development of sequence-specific primers to selectively amplify the transmembrane domain of all known KIR2DL1 alleles via polymerase chain reaction with sequence-specific primers (PCR-SSP), and using the PCR products as the template for a revised KIR2DL1 functional allele-typing assay. This modified method allows high-throughput typing with high specificity.

Association between polymorphisms of killer cell immunoglobulin-like receptor gene and the risk of essential hypertension: a case-control study

To assess the association between killer cell immunoglobulin-like receptor (KIR) gene polymorphisms and the risk of hypertension in autoimmune mechanism. We conducted a case-control study including 205 hypertensives and 205 controls matched with sex and age, from a community-based population. KIR genes of all subjects were genotyped by polymerase chain reaction with sequence-specific primers (PCR-SSP). Conditional logistic regression model and generalized multifactor dimensionality reduction (GMDR) method were used to estimate the association among KIR gene polymorphisms and the risk of hypertension. The genotypic frequencies of KIRs were not significantly different between the hypertensives and the control groups (P > 0.05). Among all the models of GMDR concerning the association between interactions of KIR genes and essential hypertension, the testing accuracy of the interaction between KIR2DS2 and KIR2DS3 was the highest (55.13%), with cross-validation consistency as 10/10 (P = 0.054). Results from the conditional logistic regression showed that individuals with KIR2DS2+: KIR2DS3- were significantly associated with an increased risk on hypertension (OR = 2.555, 95% CI: 1.203-5.429, P = 0.015). However, individuals with KIR2DS2+: KIR2DS3+ were significantly associated with a reduced risk of hypertension (OR = 0.268, 95% CI: 0.088-0.815, P = 0.020). Individuals with KIR2DS2- KIR2DS3+ did not seem to be associated with the risk of hypertension (OR = 1.602, 95% CI: 0.785-3.266, P = 0.195), when compared to the KIR2DS2- KIR2DS3- group. Interactions between KIR2DS2 and KIR2DS3 were significantly associated with the risk of hypertension, after adjusted for BMI, smoking, drinking and family history of hypertension (OR = 0.065, 95% CI: 0.013-0.317, P = 0.001). Individuals with KIR2DS2 and no KIR2DS3 were associated with the increased risk of hypertension. KIR2DS2 that coexisted with KIR2DS3 were associated with the reduced risk of hypertension. Antagonism between KIR2DS2 and KIR2DS3 might serve as a protect factor for hypertension.

New associations: INFG and TGFB1 genes and the inhibitor development in severe haemophilia A.

The development of factor VIII (FVIII) inhibitor is the main complication of replacement therapy in patients with haemophilia A (HA). A ratio of 5-7% of individuals HA develops antibodies (inhibitors) against the FVIII infused during the treatment, thereby reducing their pro-coagulant activity. The immunomodulatory cytokine genes have been related to the risk of development of alloantibodies in several studies, mainly in HA with severe form. We investigated the polymorphisms in regulatory regions of cytokine genes (IL1A, IL1B, IL1R, IL1RA, IL4RA, IL12, INFG, TGFB1, TNF, IL2, IL4, IL6, IL10) that could influence the risk of developing inhibitors in patients with severe HA. The genotyping of cytokine genes of 117 patients with HA was performed by polymerase chain reaction with sequence-specific primers (PCR-SSP) using the protocol recommended by the manufacturer (Invitrogen kit Cytokines(®) , Canoga Park, USA) RESULTS: From the cohort of 117 patients with severe HA, 35 developed inhibitors. There was a higher frequency of +874 T allele in INFG and of +869 TT and TG/TG in TGFB1 genes on patients with inhibitors. This suggests that polymorphisms in INFG and in TGFB1 genes are related to risk of developing inhibitor, and could contribute to a genetic profile of the individual HA for the risk of inhibitors development to FVIII.

Risk estimation of HNA-3 incompatibility and alloimmunization in Thai populations.

Severe transfusion-related acute lung injury (TRALI) is often due to antibodies in blood components directed against human neutrophil antigen (HNA)-3a. This study aimed to report the genotype frequencies of the HNA-3 system and to estimate the potential risk of HNA-3 incompatibility and alloimmunization in two Thai populations. Eight hundred DNA samples obtained from 500 unrelated healthy blood donors at the National Blood Centre, Thai Red Cross Society, Bangkok and 300 samples from the Blood Bank, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand were included. HNA-3 genotyping was performed using an in-house polymerase chain reaction with sequence-specific primer (PCR-SSP) technique. The observed frequencies of the HNA-3a/3a, HNA-3a/3b, and HNA-3b/3b genotypes were 0.528, 0.380, and 0.092 in central Thais and 0.600, 0.350, and 0.050 in northern Thais, respectively. The frequencies were used to estimate HNA-3 incompatibility and risk of HNA-3a alloimmunization. The HNA-3 incompatibility in central Thais (33.28%) was higher than northern Thais (28.75%), corresponding to a significantly higher probability of HNA-3a alloimmunization (P<0.05) similar to Japanese and Chinese populations. This study showed the high risk of HNA-3 incompatibility and alloimmunization, especially in central Thai blood donors. A molecular-based identification of the HNA-3 genotype of female donors is suggested to reduce the risk of TRALI following plasma and whole blood allogeneic transfusion.

PCR with sequence-specific primers for typing of diallelic blood groups.

PCR with sequence-specific primers (PCR-SSP) is a cost-effective and robust method for the analysis of single nucleotide polymorphisms (SNPs). Many blood group antigens and the antithetic antigens are based on a diallelic SNP in the coding region of the corresponding blood group gene. Here, we describe PCR-SSP protocols for genotyping 24 blood group antigens based on 12 diallelic SNPs. We also provide protocols for molecular determination of the rare blood group phenotypes Yk(a-) and Vel-.

Variations in Immediate-early Genes Encoding c-Fos, c-Jun and IER5 Transcription Factors are Associated with Ischemic Stroke

Transcription factors make a great contribution in the regulation of mechanisms, which are involved in the preand post-stroke events. The main goal of the current study was to evaluate the potential association of single nucleotide polymorphisms FOS (rs7101, rs1063169), JUN (rs11688) and IER5 (rs6425663) with ischemic stroke in Armenian population. A total 161 patients with ischemic stroke and 165 controls were involved in this study. Transcription factors were genotyped using polymerase chain reaction with sequence-specific primers (PCR-SSP). Association of genotype, allelic and carriage frequencies with ischemic stroke was assessed using Pearson's Chisquare test. Multiple test corrected p values less than 0.05 were considered significant. The data obtained demonstrated a significant negative association between the FOS rs1063169*T, JUN rs11688*A and IER5 rs6425663*T minor alleles with ischemic stroke. On the other hand, the frequency distribution of rs7101*T minor allele of FOS gene and its carriage rate in the group of patients were significantly higher than in controls. Our results indicate that the minor alleles of FOS rs1063169, JUN rs11688 and IER5 rs6425663 SNPs can be considered as protective against ischemic stroke, whereas the minor allele of FOS rs7101 SNP represents a risk factor for this disease.

The Association of KIR2DS4 and Its Variant KIR1D with CMV Infection after HLA-Matched Sibling Hematopoietic Stem Cell Transplantation

Objective To expolore the association of KIR2DS4 and its variant KIR1D with cytomegalovirus(CMV) infection after HLA-matched sibling hematopoietic stem cell transplantation.Methods Polymerase chain reaction with sequence-specific primers (PCR-SSP) method was used to genotype KIR genes in 267 donor-recipient pairs from Oct 2005 to Apr 2014. Posttransplant monitoring for CMV infection was performed by immune histochemically assays .165 donor-recipient pairs who belong to KIR gene haplotype AA were analyzed for the presence of KIR2DS4 and its variant KIR1D and then further subdivided into the following groups: 2DS4-/1D+ (homozygous for the deletion variant KIR1D), 2DS4+/1D+ (heterozygous), 2DS4+/1D- (two intact KIR2DS4 alleles). Furthermore, we investigated the influence of the KIR2DS4 variants on CMV infection of 165 patients receiving Sibling related HLA matched transplantation.Results There were no significant differences in frequency of KIR2DS4 or KIR1D between donors and recipients in the haplotype AA group. The ratio of 2DS4+ and KIR1D in haplotype AA group was 2:1.There was no difference on neutrophil engraftment and platelet recovery among the three groups after hematopoietic stem cell transplantation. The CMV infection rate was significantly higher in 2DS4+/1D- group compared with 2DS4+/1D+ group (44.0% vs 19.0%，P=0.002)．In 2DS4-/1D+ group ，the CMV infection rate was higer than that in 2DS4+KIR1D+ group (50.0% vs 19%，P=0.028). However，there was no difference in CMV infection rate between 2DS4+/1D-group and 2DS4-/1D+ group.Conclusion KIR2DS4 and its variant KIR1D are associated with CMV infection after HLA-matched sibling hematopoietic stem cell transplantation DisclosuresNo relevant conflicts of interest to declare.

Correlation Analysis Between Protein Phosphatase-1 Regulatory Inhibitor Subunit-11 Polymorphism and Hepatitis C Virus Spontaneous Clearance

Objective To explore the association of protein phosphatase-1 regulatory inhibitor subunit-11 (PPP1R11) polymorphism and hepatitis C virus (HCV) spontaneous clearance.Methods From January 2009 to May 2013,a total of 328 blood donors whose blood samples were anti-HCV positive with enzyme-linked immunosorbent assay (ELISA) were included into this study.According to follow-up results,they were divided into spontaneous clearance group whose HCV RNA were negative but anti-HCV recombinant immunoblot assay results were positive (n =134),and chronic infection group whose HCV RNA were positive (n=198).Polymerase chain reaction with sequence specific primer(PCR-SSP) was performed to determine the genotype of rs3807031 of PPP1R11.Statistical software SPSS was used to analyze the association between genotype of rs3807031 and HCV spontaneous clearance.Results The average age of donors in spontaneous clearance group was (28.8± 10.2) years old,which was significantly lower than that of chronic infection group whose average age was (31.0±9.0) years old (t=2.135,P =0.034).Higher percentage of female was observed in spontaneous clearance group compared to chronic infection group (35.8％ vs 21.6％ ; x2 =7.994,P=0.005).Importantly,rs3807031 A allele composed 30.6％ and 22.4％,while C allele composed 69.4％ and 77.6％ in spontaneous clearance and chronic infection group,respectively.The frequencies of rs3807031 alleles were statistically different between these two groups (x2 =5.538,P =0.023).Multivariate unconditional logistic regression analysis showed that gender (OR=1.957,95％CI:1.118-3.224,P=0.008) and genotype of rs3807031 (OR=0.650,95％CI:0.450-0.973,P =0.021) are independent factors associated with HCV spontaneous clearance.Conclusions PPP1R11 rs3807031 is associated with HCV spontaneous clearance,which has the potential to predict the spontaneous clearance of HCV infection.Our results provide information to the research on the mechanism and molecular marker of HCV spontaneous clearance. Key words: Hepatitis C, chronic; Hepacivirus; Genotype; Polymorphism, single nucleotide