Allele-specific PCR Primers Research Articles

Resolving Discrepancies in Idylla BRAF Mutational Assay Results Using Targeted Next-Generation Sequencing.

BRAF mutation identification is important for the diagnosis and treatment of several tumor types, both solid and hematologic. Rapid identification of BRAF mutations is required to determine eligibility for targeted BRAF inhibitor therapy. The Idylla BRAF mutation assay is a rapid, multiplex allele-specific PCR test designed to detect the most common oncogenic BRAF V600 mutations in formalin-fixed paraffin-embedded (FFPE) tissue samples. Here, we describe the validation of the Idylla BRAF mutation assay in our laboratory. During routine clinical practice, we noticed cases in which BRAF V600 mutations were identified with unusual amplification curves, with three cases displaying a delayed amplification within a double amplification pattern and two false-positive calls. We therefore initiated a quality improvement effort to systematically and retrospectively evaluate next-generation sequencing (NGS)-tested cases with BRAF mutations identified within five amino acids of BRAF codon V600 and did not identify additional false-positive cases. We hypothesize that late amplification in a double amplification pattern may represent non-specific amplification, whereas cases displaying single delayed amplification curves may stem from the presence of either non-V600 variants, very low-level V600 variants, cytosine deamination artifacts, and/or non-specific amplification by an allele-specific PCR primer. Regardless, we recommend that Idylla BRAF cases with non-classical amplification curves undergo reflex NGS testing. These findings are likely relevant for other Idylla assays interrogating hotspot mutations in genes such as EGFR, IDH1/2, KRAS, and NRAS.

Multiplicity of infection of Plasmodium knowlesi in Malaysia: an application of Pkmsp-1 block IV.

In Malaysia presently, the main cause of human malaria is by the zoonotic monkey parasite Plasmodium knowlesi. A previous study has suggested that the P. knowlesi merozoite surface protein 1 (Pkmsp-1) block IV to be a suitable multiplicity of infection (MOI) genotyping marker for knowlesimalaria. This study therefore aimed to investigate the usefulness of Pkmsp-1 block IV in assessing the MOI of P. knowlesi in clinical isolates from Malaysia. Two allele-specific PCR primer pairs targeting the two allelic families of block IV (T1 and T2) were designed, and used to genotype P. knowlesi in 200 blood samples (100 from Peninsular Malaysia and 100 from Malaysian Borneo). Results showed that the mean MOI in Malaysian Borneo was slightly higher as compared to Peninsular Malaysia (1.58 and 1.40, respectively). Almost half of the total blood samples from Malaysian Borneo (52%) had polyclonal infections (i.e., more than one allele of any family type) as compared to Peninsular Malaysia (33%) samples. The T1 allelic family was more prevalent in Peninsular Malaysia (n=75) than in Malaysian Borneo (n=60). The T2 allelic family, however, was more prevalent in the Malaysian Borneo (n=87 vs n=53 respectively). This study shows that the single locus Pkmsp-1 block IV can serve as a simple alternative genetic marker for estimating knowlesi malaria MOI in a population. Future MOI studies should focus on macaque populations as macaques are the natural host of P. knowlesi.

P-723 Pre-fertilization genetic testing to select healthy gametes in a mammalian Marfan syndrome model

Abstract Study question Is it possible to identify disease-free gametes before insemination to prevent vertical transmission of an autosomal dominant disease? Summary answer In a mammalian Marfan syndrome model, we successfully generated and identified healthy androgenotes to be utilized as male gametes to produce unaffected conceptuses. What is known already In patients with autosomal dominant genetic disorders such as Marfan syndrome, germline heterozygosity poses a risk to offspring health. For these patients, preimplantation genetic testing for monogenic disorders (PGT-M) allows us to select unaffected conceptuses for embryo replacement. However, PGT-M is inherently inefficient and can lead to embryo wastage. This has sparked interest in replicating the genome of individual gametes for (1) pre-fertilization genetic testing and (2) subsequent use as genotyped gamete substitutes during IVF. Study design, size, duration For this study, we selected 4 male B6 mice that were heterozygous for the Fbn1tm1Hcd mutation and displayed classic manifestations of Marfan syndrome. Spermatozoa from these mice were used to inseminate enucleated wildtype mouse oocytes and generate haploid androgenetic embryos for (1) Fbn1tm1Hcd genotyping and (2) use as unaffected male gamete substitutes. Participants/materials, setting, methods Using tail clippings and embryo biopsy specimens, a Fbn1tm1Hcd allele-specific PCR primer was developed and validated. Haploid androgenetic embryos were allowed to progress to the 8-cell stage. Subsequently, a single-haploid pseudo-blastomere was isolated for genotyping, while the remainder were individually grafted into activated recipient wildtype oocytes and fused using inactivated Sendai virus to generate biparental conceptuses. Resulting diploid conceptuses were cultured in a time-lapse incubator and then genotyped to confirm the healthy paternal haplotype. Main results and the role of chance A total of 175 wildtype B6 oocytes were enucleated yielding 169 ooplasts (96.6% survival rate). The ooplasts were inseminated with spermatozoa from mutant Fbn1tm1Hcd mice, yielding 144 (85.2%) monopronucleated haploid androgenetic embryos. Of those, 100 (69.4%) progressed to the 8-cell stage. In 47 (47.0%) of them, genetic analysis by PGT-M on an individual pseudo-blastomere characterized the embryo as unaffected because it harbored the wildtype Fbn1 allele. The remaining 7 haploid pseudo-blastomeres from 5 Fbn1 wildtype androgenetic embryos were individually isolated and grafted into 35 recipient oocytes to generate 31 (88.6%) biparental conceptuses with the desired paternal haplotype. These achieved a 61.3% (19/31) blastocyst development rate after 96 hours, displaying normal embryo morphokinesis. A blastocyst biopsy was carried out (n = 19), confirming the unaffected paternal haplotype void of the Fbn1tm1Hcd mutation. Limitations, reasons for caution Although this technique is feasible for pre-fertilization genetic testing, it needs to be reproduced with the same efficiency in humans. A major concern is related to the dynamics and roles of the sperm centrosome in the first mitotic division of human zygotes, which may be missing in the pseudo-gametes. Wider implications of the findings This approach to pre-fertilization genetic testing will benefit patients with heritable genetic conditions. In addition, the inherited decondensation of the male genome by the ooplasm may enable genome editing experiments to be performed. Trial registration number Not applicable

Development and Validation of an Allele-Specific Marker for Resistance to Bacterial Halo Blight in Coffea arabica

Bacterial halo blight (BHB) is a bacterial disease, caused by Pseudomonas syringae pv. garcae, which has been gaining prominence in the main coffee-producing regions. Chemical control of this disease increases production costs and is environmentally undesirable. In this scenario, the development of new cultivars resistant to BHB is the most economical and sustainable alternative. Marker-Assisted Selection (MAS) is an appropriate strategy to assist breeding programs for resistant genotype selection. In a previous Genome-Wide Association Study (GWAS) for C. arabica and P. syringae pv. garcae interaction, we identified a locus, probably linked to qualitative resistance to the pathogen. In this work, we developed and validated a pair of Allele-Specific-Polymerase Chain Reaction (AS-PCR) primers for this locus in C. arabica breeding populations. This pair of AS-PCR primers, called Psg_QL1, was tested both in a backcross (BC) (n = 38) and in an F2 population (n = 138) segregating for resistance to BHB. The linkage between the Psg_QL1 marker and qualitative resistance showed an accuracy of 93.75%. Our results demonstrated that the Psg_QL1 marker can be applied in MAS in a robust, simple, fast, and low-cost way.

The complete chloroplast genome sequence of Cyathula officinalis and comparative analysis with four related species

Cyathula officinalis is a medicinal and edible herb, which can remove blood stasis, stimulate menstrual flow, and ease joint movement. In this study, the complete chloroplast genome of Cyathula officinalis was sequenced, assembled, and analyzed. Compared with the chloroplast genomes of Cyathula capitata, Achyranthes bidentata, Achyranthes longifolianine and Achyranthes aspera, the basic characteristics, codon usage bias, repeat sequences, simple sequence repeats, and phylogenetic tree were analyzed. In addition, according to nucleotide diversity analysis and sequence alignment, DNA barcoding and allele-specific PCR primers were designed to identify and distinguish Cyathula officinalis from its fake drugs, which has effectively practical significance for the authentication of “Chuan Niuxi” crude drug in the market.

Association of MDR1 C1236T Polymorphisms and B Cell Non-Hodgkin Lymphoma.

Background: Multidrug resistance gene 1 (MDR-1) encodes for P-glycoprotein (P-gp) recognized for removing cytostatic drugs from tumor cells. MDR1 gene polymorphisms change function of P-gp. In this study, we are interested in investigating whether MDR1 C1236T single nucleotide polymorphisms (SNPs) affect the susceptibility and treatment-related toxicities in B-cell non-Hodgkin lymphoma (B-NHL) in the population of eastern China.Materials and methods: A group of 107 B-NHL patients and 150 healthy donors, unrelated ethnic Han Chinese and residents of eastern China, were included in this study. The MDR1 C1236T polymorphisms were determined using polymerase chain reaction-allele specific primers after extraction of genomic DNA. Analyses were performed using SPSS and Arlequin software.Results: MDR1 C1236T polymorphisms were not significantly related to the risk and treatment-related toxicities of B-NHL. A significant association between extranodal sites and C1236T allele was observed (C vs T: P=0.01).Conclusion: Our findings could expand our understanding of MDR1 in B-NHL and provide references for further research in multidrug resistance.

Correlation Analysis of Th17/Treg Cells and PTPN22 Gene Polymorphism in HT Patients with Different Iodine Nutritional Status

Background: The incidence of Hashimoto's thyroiditis (HT) may be related to environmental, immune, and genetic factors. Different iodine nutritional status can affect thyroid function. Th17/Treg cell imbalance is involved in the pathogenesis of autoimmune thyroid diseases, and PTPN22 is an important susceptibility gene related to autoimmune diseases. To study the relationship between Th17/Treg cells and cytokines and PTPN22 gene polymorphism in HT patients with different iodine nutritional status is to determine the role of immune and genetic factors in the pathogenesis of HT. Objective: To investigate the correlation between Th17/Treg cells and factors protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene-1123G>C site (rs2488457) polymorphism in peripheral blood mononuclear cells (PBMC) of Hashimoto's thyroiditis (HT) patients with different iodine nutritional status. Methods: 100 HT patients and 60 healthy subjects in Cang Zhou are selected from 2019 to 2020. Flow cytometry and real-time fluorescent quantitative PCR are used to detecting the proportion and ratio of Th17 and Treg cells in PBMC, as well as retinoic acid-related orphan receptor-γt (ROR-γt) and fork head/winged spiral transcription factor 3 (Foxp3) mRNA expression levels. Single allele-specific primer-polymerase chain reaction (SASP-PCR) technology is taken to sequence PTPN gene promoter-1123SNP leading to calculate the gene Type and allele frequency. Results: Th17 and Treg cells and factors in the PBMC of HT patients have a certain correlation with the-1123G>C locus of PTPN22 gene (rs2488457). The distribution frequency of rs2488457 genotype is consistent with Hardy Weinberg's law of genetic equilibrium. The distribution frequencies of various genotypes and alleles are significantly different between HT patients and the control group (P<0.05). PTPN22 gene-1123G>C site C/G, C/C genotype can increase of the risk of HT, while G/G genotype can decrease of the risk of HT. In Conclusion: The polymorphism of rs2488457 is related to susceptibility to HT. Carrying C/G and C/C genotypes may be risk factors for HT, and G/G genotypes may be protective factors for HT, but it has nothing to do with iodine nutrition status.

Identification of Bupleurum (Apiaceae) seeds by allele-specific PCR based on ITS sequences.

The traditional Chinese medicine Bupleuri radix (chaihu) is the dried roots of Bupleurum chinense and Bupleurum scorzonerifolium and many adulterants exist because of the differences in traditional understanding, medication habits and seed resources. Therefore, rapid and accurate identification methods for Bupleurum (Apiaceae) seeds are required. We analyzed the internal transcribed spacer (ITS) sequences of five common Bupleurum species to detect variations in them, including B. chinense, B. scorzonerifolium, B. marginatum var. stenophyllum, B. falcatum and B. smithii var.parvifolium. Based on single nucleotide polymorphisms (SNPs) in the ITS region, we designed five specific primer pairs and used these primers in an allele-specific PCR technique to establish a robust molecular identification method. The neighbor-joining (NJ) tree of ITS sequences showed that five Bupleurum species formed their own monophyly. Five specific primer pairs were designed and integrated into a specific PCR master mix and cycling conditions. The primer pair of BCF/R8 for B. chinense could amplify a specific identification band of 429bp and the minimum detection limit of the 5g mixture was about 5%; BSF/R11 for B. scorzonerifolium could amplify a specific 464bp band and the minimum detection limit was about 5%; BMSF/R1 for B. marginatum var. stenophyllum could amplify a specific 344bp band and the minimum detection limit was about 1%; BFF/R7 for B. falcatum could amplify a specific 137bp band and the minimum detection limit was about 1%; BSPF/R1 for B. smithii var. parvifolium could amplify a specific 390bp band. Subsequent analysis proved the reliable accuracy and good practicability of the five specific identification primers, indicating that the allele-specific primer PCR identification method can quickly identify Bupleurum seeds. The method elaborated in this study has the advantages of simple operation, good accuracy and high efficiency.

Molecular S-genotyping of sweet cherry (Prunus avium L.) genetic resources

Sweet cherries are self-incompatible, which is determined by a gametophytic self-incompatibility system (GSI). The self-incompatibility is controlled by a multi-allelic S-locus. Knowledge about the S-allele constitution of the cultivars is essential for fruit growers and breeders. Recently, molecular PCR-based methods have been developed to distinguish all S-alleles in sweet cherries. In our work, we analysed S-locus genotypes by 13 universal and allele-specific PCR primer combinations within 117 registered, old and local sweet cherry cultivars from the Czech genetic resources of the Research and Breeding Institute of Pomology in Holovousy, the Czech Republic. We confirmed the previous S-genotyping for 66 accessions except for Drogans Gelbe, Hedelfinger, Erika, Meckenheimer Frühe, Badeborner, Bing, Alfa, Gamma, Huldra, Rivan, Valerij Tschkalov, Viola and Winkler’s Frühe. It could be due to either mislabelling or mistakes in the previous analyses. Newly, S-genotyping was determined for 51 accessions in which we found 4 new S-loci combinations. We detected the S-locus combinations in 19 incompatibility groups. The most frequent incompatibility groups were III (S&lt;sub&gt;3&lt;/sub&gt;S&lt;sub&gt;4&lt;/sub&gt;), II (S&lt;sub&gt;1&lt;/sub&gt;S&lt;sub&gt;3&lt;/sub&gt;), IV (S&lt;sub&gt;2&lt;/sub&gt;S&lt;sub&gt;3&lt;/sub&gt;), and VI (S&lt;sub&gt;3&lt;/sub&gt;S&lt;sub&gt;6&lt;/sub&gt;) with 22, 20, 12 and 12 genotypes, respectively.

A Re-Evaluation of Olive Fruit Fly Organophosphate-Resistant Ace Alleles in Iberia, and Field-Testing Population Effects after in-Practice Dimethoate Use.

The management of the olive fruit fly (Bactrocera oleae) is traditionally based upon the use of organophosphate insecticides, mainly dimethoate. In this evolutionary arms race between man and pest, the flies have adapted a pesticide resistance, implying two point-mutations of the Ace gene -I214V and G488S- and a 9bp deletion -Δ3Q. We revisited 11 Iberian locations to evaluate this adaptation of organophosphate (OP)-resistant alleles through amplicon sequencing. Screening for populations where the wild type is prevalent allows an identification of hotspots for targeted mitigation measures; we have hence refined the scale to the region with the lowest OP-resistant alleles frequency 71 locations were sampled and individuals checked using a fast and low-cost allele-specific-primer polymerase chain reaction (ASP-PCR) method]. An increase in Ace gene point-mutations was observed, and the Δ3Q mutation remains undetected. The lowest frequencies of the OP-resistant alleles remain in the west, underlining the hypothesis of an introduction of resistance from eastern Mediterranean areas. A field test was performed by sampling the fly population before and after in-practice dimethoate application. A clear reduction in olive fruit fly numbers was observed, with no relevant changes in the genotypic frequencies of the resistance alleles. The findings are discussed in frame of the type and intensity of the selection pressure that has led to the adaptation to resistance and its consequences from the producer perspective.

CD44 Gene rs8193 C Allele Is Significantly Enriched in Gastric Cancer Patients.

Objective Gastric cancer is a multifactorial disease. In addition to environmental factors, many genes are involved in this malignancy. One of the genes associated with gastric cancer is CD44 gene and its polymorphisms. CD44 gene plays role in regulating cell survival, growth and mobility. The single nucleotide polymorphism (SNP) rs8193, located in the CD44 gene, has not been studied in gastric cancer patients of the Iranian population. The present study aims to study this polymorphism in 86 gastric cancer patients and 96 healthy individuals. Materials and Methods In this cross-sectional case-control study, rs8193 polymorphism was genotyped by allele specific primer polymerase chain reaction (ASP-PCR) technique. The obtained data were statistically analyzed. To find the potential mechanism of action, rs8193 was bioinformatically investigated. Results rs8193 C allele played a risk factor role for gastric cancer. Patients carrying this allele were more susceptible to have gastric cancer, with lymph node spread. On the other hand, rs8193 T allele, a protective factor, was associated with a higher chance of accumulation in the lower stages of cancer. C allele might impose its effect via destabilizing CD44 and miR-570 interaction. Conclusionrs8193 is statistically associated with the risk of malignancy, lymph node spread and stage of gastric cancer in Iranian population.

Rapid screening mutations of first-line-drug-resistant genes in Mycobacterium tuberculosis strains by allele-specific real-time quantitative PCR.

Tuberculosis (TB) is a worldwide health, economic, and social burden, especially in developing countries. Drug-resistant TB is the most serious type of this burden. Thus, it is necessary to screen drug-resistant mutations by using a simple and rapid detection method. A total of 32 pairs of allele-specific PCR (AS-PCR) primers were designed to screen mutation and/or wild-type alleles of 16 variations in four first-line drug-resistant genes (katG, rpoB, rpsL, and embB) of TB strains. A pair of primers was designed to amplify 16S rRNA gene and to verify successful amplification. Subsequently, we tested the specificity and sensitivity of these AS-PCR primers. The optimized condition of these AS-PCR primers was first confirmed. All mutations could be screened in general AS-PCR, but only 13 of 16 variations were intuitively investigated by using real-time quantitative PCR (qPCR) and AS-PCR primers. The results of specificity assay suggested that the AS-PCR primers with mutation and/or wildtype alleles could successfully amplify the corresponding allele under optimized PCR conditions. The sensitivity of nine pairs of primers was 500 copy numbers, and the other seven pairs of primers could successfully amplify correct fragments with a template comprising 103 or 104 copy numbers template. An optimized AS-qPCR was established to screen drug-resistant mutations in TB strains with high specificity and sensitivity.

Mutational Analysis of Clarithromycin and Levofloxacin Resistance in Helicobacter pylori from Gastric Biopsy Specimens in a Tertiary Care Hospital in Dhaka, Bangladesh

Background: Clarithromycin and Levofloxacin are most frequently included in the standard triple therapies for H. pylori eradication in our country. Resistance to clarithromycin and fluoroquinolones are particularly related with treatment failure.&#x0D; Objectives: The objective of this study was to detect, clarithromycin and levofloxacin resistance associated with gene mutations in H. pylori directly from gastric biopsies using an allele specific primer-PCR (ASP-PCR) assay.&#x0D; Materials and Methods: Gastric biopsy specimens were collected from 143 adult dyspeptic patients, from Department of Gastroenterology, BSMMU and Dhaka Medical College Hospital (DMCH), during the period of March, 2018 to February, 2019. H. pylori was identified by rapid urease test, ureC gene by PCR, histological staining and culture. ASP-PCR was used to identify 23S rRNA gene and gyrA gene mutation predictive of clarithromycin and levofloxacin resistant H. pylori respectively.&#x0D; Results: H. pylori positive cases were 32.9% based on the case definition used in the study. Among 42 ureC positive H. pylori cases, point mutations in 23Sr RNA gene for clarithromycin resistance were detected only at A2142G position in 9 (21.4%) cases and gyrA gene mutations for levofloxacin resistance were detected in 16 (38.1%) cases. Only 1 (2.4%) case had mutation both in 23Sr RNA and gyrA gene.&#x0D; Conclusion: Those findings may guide toward the therapeutic choices in our country. PCR based diagnostic assays can be the alternative approach for rapid detection of antibiotic resistances of H. pylori directly from gastric biopsies, where culture and susceptibility tests are not routinely performed.&#x0D; Bangladesh J Med Microbiol 2019; 13 (1): 12-19

KIR and HLA-C Genetic Polymorphisms Influence Plasma IP-10 Concentration in Antiretroviral Therapy-Naive HIV-Infected Adult Zimbabweans.

Past studies on the relationship between Killer cell Immunoglobulin-like Receptor (KIR) and Human Leukocyte Antigen (HLA) genetic variation and chronic immune activation (CIA) in HIV infection are not uniformly consistent. Moreover, interferon-γ-induced protein 10 (IP-10) is a soluble biomarker of immune activation, with high plasma concentrations predicting accelerated disease progression in HIV infection. Thus, we investigated the association of KIR and HLA-C genetic polymorphisms with plasma IP-10 concentration in 183 treatment-naive chronically HIV-infected adults of Bantu origin from Zimbabwe. KIR genetic variation was determined using allele-specific primer PCR while HLA-C typing was characterized by sequencing. Plasma IP-10 was quantified using enzyme-linked immunosorbent assay. The KIR2DL3 gene was significantly associated with CIA as observed from IP-10 concentrations among KIR2DL3 carriers (265.20 pg/mL, IQR: 179.99-385.19) compared with KIR2DL3 noncarriers (183.56 pg/mL; IQR: 110.98-230.81; p = 0.001) and among KIR2DL3+HLA-C2 carriers (226.23 pg/mL, IQR: 187.96-394.73) compared with KIR2DL3+HLA-C2 noncarriers (212.86 pg/mL, IQR: 160.15-344.99; p = 0.017), respectively. Similarly, IP-10 concentrations were significantly higher (p = 0.030) in the KIR3DS1 carriers (313.86 pg/mL, IQR: 230.05-469.20) compared with KIR3DS1 noncarriers (246.01 pg/mL, IQR: 169.58-373.32). Thus, KIR and HLA-C could be playing important roles in HIV-associated immune activation. The elevation of IP-10 in KIR2DL3 and KIR2DL3+C2 could potentially be explained by increased IFN-γ secretion from activated NK cell activation due to the absence of KIR2DL3's cognate C1 ligand. To the best of our knowledge, this is the first study on a potential link between KIR and HLA-C genetic determinants and plasma IP-10 concentration in this population sample. Future studies are called for in other world populations for biomarkers of disease progression and mechanisms of IP-10 variability in HIV infection.

Scanning RNA-Seq and RAD-Seq approach to develop SNP markers closely linked to MALE STERILITY 1 (MS1) in Cryptomeria japonica D. Don.

Cryptomeria japonica is a major forestry tree species in Japan. Male sterility of the species is caused by a recessive gene, which shows dysfunction of pollen development and results in no dispersed pollen. Because the pollen of C. japonica induces pollinosis, breeding of pollen-free C. japonica is desired. In this study, single nucleotide polymorphism (SNP) markers located at 1.78 and 0.58 cM to a male sterility locus (MS1) were identified from an analysis of RNA-Seq and RAD-Seq, respectively. SNPs closely linked to MS1 were first scanned by a method similar to MutMap, where a type of index was calculated to measure the strength of the linkage between a marker sequence and MS1. Linkage analysis of selected SNP markers confirmed a higher efficiency of the current method to construct a partial map around MS1. Allele-specific PCR primer pair for the most closely linked SNP with MS1 was developed as a codominant marker, and visualization of the PCR products on an agarose gel enabled rapid screening of male sterile C. japonica. The allele-specific primers developed in this study would be useful for establishing the selection of male sterile C. japonica.

Simple, rapid and inexpensive typing of common HLA class I alleles for immunological studies

Current HLA-typing methods are typically designed to provide exquisitely-detailed identification of multiple HLA-alleles to satisfy the requirements for organ and bone marrow transplantation or genetic studies. Many human immunological studies, on the other hand, focus around only a small number of HLA alleles that are abundant or of relevance to specific diseases. Consequently, for such studies, many HLA typing approaches are not cost-effective and are potentially complicated, slow and not easily performed in-house. Work-flow would be streamlined by a simple, inexpensive and rapid typing method able to be performed in-house. We outline a straightforward approach that provides appropriate data for much immunological research. In a predominantly Caucasian population, flow cytometry using anti-HLA-A2, -B8 and -B7 antibodies consistently and accurately screened for samples carrying the highly-abundant HLA class I alleles HLA-A*02:01, -B*08:01 and -B*07:02 that form the focus of immunological studies. Next, we describe a straightforward and simple strategy for design and use of allele-specific PCR primers to identify, at high-resolution, alleles of interest. When combined with a simple gDNA extraction technique this provides reliable, simple and inexpensive in-house HLA typing demonstrated here for highly-abundant HLA class I alleles.

Risk factor effect of rs1044165 and rs3745453 as neighboring variants of miR-223, miR-24, miR-23a and miR-27a on the onset of MS disease in Isfahan/Iran

Multiple sclerosis (MS) is a multifactorial, chronic and inflammatory disease due to the myelin sheath damage of the central nervous system (CNS). Genome-wide association studies reveal that progression of MS is associated with single nucleotide polymorphisms (SNP). To elucidate the frequency of two SNPs (rs3745453 and rs1044165) with the incidence of MS, a population composed of 310 MS patients and 280 healthy volunteers were selected in Isfahan as a high prevalent location in the heart of Iran. After genomic extraction of blood samples, Allele specific primer PCR (ASP-PCR) was implemented for genotyping of the samples. Data indicated that frequency of carriage of CC genotype in rs3745453 in MS patients compared to healthy individuals and frequency of carriage of rs1044165 TT genotype increased in female MS patients compared to the controls. Logistic regression analyses indicated a risk factor role of rs1044165 and rs3745453 in the pathogenesis of MS.

TP53 rs1625895 is Related to Breast Cancer Incidence and Early Death in Iranian Population.

Breast cancer as the second most common cancer worldwide tend to be experienced by Iranian women 10years earlier with a peak incidence at the premenopausal stage. Genetic mutations of TP53 tumor suppressor gene has been shown to be related to early onset of breast cancer. It has been shown already that rs1625895 polymorphic site is related to glioma as well as lung cancer. In this study, we have investigated the role of rs1625895 polymorphism in breast cancer incidence in Iranian women. DNA extraction of 86 breast cancer patients and 96 control individuals have been used for allele-specific primer-PCR and genotyping of allele A and allele G of the TP53 rs1625895. Genotypes frequencies have been shown that GG homozygosis as the most frequent genotype is a significant association with increased risk of breast cancer development in Iranian women (odds ratio = 6, p = 0.002). On the other hand and in comparison to allele G, allele A could cause early death of breast cancer patients by threefolds significantly (p = 0.011). As a conclusion, we show that allele A is the minor allele in both breast cancer patients and also control individuals and major allele G, is related to the increased risk of breast cancer development in Iranian women.

MiR-581-Related Single Nucleotide Polymorphism, rs2641726, Located in MUC4 Gene, is Associated with Gastric Cancer Incidence.

MUC4 is aberrantly expressed in several carcinomas including breast, colon, ovarian, lung, prostate, stomach and pancreatic cancers. MUC4 can regulate cell apoptosis negatively and facilitate stomach tumorigenesis. In this research, we aimed to evaluate the possible association between rs2641726 (C > A) polymorphism of MUC4 and gastric cancer risk in the Iranian population. In this case-control study, we collected blood samples from 168 gastric cancer patients and 66 healthy subjects. Allele-specific primer polymerase chain reaction method was applied to genotype rs2641726 in the obtained DNA samples. This study demonstrated that rs2641726 C allele was significantly associated with the incidence of gastric cancer, odds ratio = 3.382, 95% confidence interval: 1.840-6.217 (P < 0.001). Furthermore, the distribution of this risk allele was highly enriched in the samples with stage III. In silico studies revealed that the C allele of rs2641726, located within MUC4 3'UTR, is potential to attenuate the interaction between miR-581 and MUC4 mRNA. This disturbing effect, which might result in higher expression of MUC4 oncoprotein, was proposed for the mechanism of action of the rs2641726 risk allele. rs2641726 C allele is significantly enriched in gastric cancer specimens. The attenuating effect of this allele on miR-581 and MUC4 interaction might be a potential mechanism of action by which C allele imposes its oncogenic impact.

MDR1 polymorphisms affect the outcome of Chinese multiple myeloma patients

ObjectiveTo illustrate the association of MDR1 (Multidrug Resistance 1) polymorphisms at loci 1236, 2677, 3435 and the prognosis of multiple myeloma (MM) in Jiangsu population. MethodsA total of 129 MM patients were recruited from Jiangsu Province, China. The DNA was extracted from white blood cells (WBC) of peripheral blood and was amplified by polymerase chain reaction-allele specific primers (PCR-ASP). MDR1 polymorphisms at 3 loci were analyzed by electrophoresis followed by photograph or DNA direct sequencing. The association between the MDR1 and clinical outcomes were calculated by Graphpad and SPSS. ResultsMDR1 alleles at locus C1236T with T had significant lower calcium level in MM patients compared with C. The genotype CT had a significantly prolonged progress free survival (PFS) compared genotype CC at locus C1236T (median time: 48 months vs. 28 months, respectively; p=0.0062; HR=0.21; 95%CI0.061–0.715) while patients carrying T allele (CT and TT) at locus C3435T had a longer PFS than patients without T allele (CC) (median time: 60 months vs. 29 months, respectively; p=0.038; HR=0.508; 95%CI 0.264–0.978). And a borderline significance was found in haplotype at loci 2677-3435 and PFS. No significant findings were revealed between OS and MDR1 polymorphisms. ConclusionMDR1 polymorphisms could affect the prognosis of multiple myeloma whereas more samples and a longer follow-up are also needed.