Tetra-primer ARMS-PCR Technique Research Articles

Novel and known minor alleles of CNTNAP2 gene variants are associated with comorbidity of intellectual disability and epilepsy phenotypes: a case-control association study reveals potential biomarkers.

Neurodevelopmental disorders are heterogeneous due to underlying multiple shared genetic pathways and risk factors. Intellectual disability, epilepsy and autism spectrum disorder phenotypes overlap which indicates the diverse effects of common genes. Recent studies suggested the probable contribution of CNTNAP2 gene polymorphisms to the comorbidity of these neurological conditions. This study was conducted to investigate the role of CNTNAP2 polymorphisms rs147815978 (G>T) and rs2710102 (A>G) as a risk factor for comorbidity of intellectual disability and epilepsy in a group of 345 individuals including 170 patients and 175 healthy controls recruited from various ethnic groups of Pakistani population. Our case-control study group was genotyped by tetra primer ARMS-PCR technique and results were analysed to know the effects of CNTNAP2 rs147815978 (G>T) and rs2710102 (A>G) polymorphisms in the group. The frequency of risk allele T (rs147815978) and risk allele G (rs2710102) for homozygous recessive genotypes (TT/GG) in our study group was 36.47% while odds ratios for risk allele T (rs147815978) was 5.45 (3.90-7.61: 95% CI, P = 0.000) and that for risk allele G (rs2710102) was 2.39 (1.76-3.24: 95% CI, P = 0.0001). Homozygous recessive genotypes (TT/GG) appeared only in cases and not in control group which indicated these as suspected risk genotypes and the significant association (p < 0.05%) of CNTNAP2 gene polymorphisms rs147815978 (G>T) and rs2710102 (A>G) with co-occurrence of intellectual disability and epilepsy phenotypes in our study group which is in HWE (χ2 = 174, P < 0.0001). Logistic regression analysis shows additive (p < 0.0001) and multiplicative (p < 0.001) models which confirms significant association of both the polymorphisms in our data, which are closely located on same haplotype (D' = -0.168). We propose that CNTNAP2 rs147815978 (G>T) and rs2710102 (A>G) polymorphisms are possible risk loci for overlapping neurodevelopmental disorders in Pakistani population. We propose the role of a previously reported common SNP rs2710102 (A>G) with a rarely reported novel SNP rs147815978 (G>T) for CNTNAP2 gene association with neurodevelopmental disorders in our data. Our study has expanded the knowledge of CNTNAP2 gene polymorphisms as probable biomarkers for susceptibility of co-occurrence of intellectual disability and epilepsy phenotypes in Pakistani population. We hope that our study will open new horizons of CNTNAP2 gene variants research to cure the neurological conditions in Pakistani population where consanguinity is a tradition and prevalence of neurodevelopmental disorders has increased from 1 to 2% during last 5 years.

Allelic and Genotypic Analysis of LncRNA ANRIL rs4977574 A/G Mutations in Oral Squamous Cell Carcinoma Patients: Insights into Tumor Characteristics and Genotypic Correlations.

Long noncoding RNAs (lncRNA) ANRIL and its genetic polymorphisms are shown to be associated with the risk of several cancers. However, the single nucleotide polymorphisms (SNPs) of lncRNA ANRIL are not thoroughly assessed in oral squamous cell carcinoma (OSCC) which is the most prevalent cancer in the head and neck area. Thus, this study aimed to assess the association of SNP of lncRNA ANRIL rs4977574 in patients with OSCC. 106 blood samples from the patients with OSCC were obtained with a gender- and age-matched control group to evaluate the SNP of rs4977574 of lncRNA ANRIL. The DNA was extracted using the salt-out technique and DNA genotyping was undertaken using specific primer pairs in the tetra-primer ARMS-PCR technique. Eventually, the frequency of wild-type (A) and the mutated allele (G), as well as the genotypes were estimated between the groups of patients with OSCC and healthy individuals. The results of our study indicated no statistically significant difference in the frequency of rs4977574 A/G of lncRNA ANRIL among the patients with OSCC and healthy individuals (p > 0.05). Likewise, no significant difference was found in the genotypes' frequencies (p > 0.05). Nevertheless, the marked association of GG with smaller tumor size and the high level of differentiation of OSCC cells in the presence of AA or AG genotypes were interesting outcomes of this study (p < 0.05). Similarly, all the genotypes AA, AG, and GG were correlated with the site of the occurrence of OSCC. Furthermore, the association of the genotypes with the lymph node metastasis and the tumors stage was not found to be significant (p > 0.05). The results of our study indicate that rs4977574 A/G and its genotypes do not have any direct correlation with the presence of OSCC; however, its association with the smaller tumor size and the level of the cancer cells differentiation could imply its possible indirect role.

ZNF804A intronic variant rs1344706 and NGRN rs12807809 genetic polymorphisms and risk of schizophrenia in Bangladesh

BackgroundGenetic polymorphism exerts an undeniable impact on schizophrenia (SCZ) pathogenesis. Two polymorphisms, including rs1344806 of ZNF804A and rs12807809 of NRGN gene have been identified as susceptible candidates bearing the risk of SCZ. The target of this experimental study is to investigate the role of these polymorphisms with SCZ in native Bangladeshi people. MethodsFor this study, 220 schizophrenic patients and 204 non-schizophrenic controls were recruited to perform this investigation. For genotyping, tetra-primer ARMS-PCR technique was applied. The Agarose gel electrophoresis system visualized the final genomic products of the desired size. ResultsFrom the genotypic analysis of ZNF804A rs1344706 polymorphism, it was revealed that the minor allele frequency was noticeably increased in the patients than in the healthy population (42.27% vs. 29.17%). Five genetic models showed significantly increased risk of SCZ, including codominant model 1 (1.56-times), codominant model 2 (3.02-times), dominant model (1.86-times), recessive model (2.43-times), and allele model (1.78-times) (p < 0.05). The over-dominant model also had 1.18 times higher risk for SCZ, although the result could not meet the significance level (p > 0.05). Genotyping of rs12807809 unveiled the presence of a comparatively higher genotypic frequency of the minor allele among the patients than in controls (39.09% vs. 17.65%). Every genetic model under investigation showed significantly increased risk for SCZ– codominant model 1 (2.85-times), codominant model 2 (6.45-times), dominant model (3.44-times), recessive model (4.31-times), over-dominant model (2.10-times), and allele model (3.00-times) (p < 0.05). ConclusionThis experimental study concluded that rs1344806 of ZNF804A and rs12807809 of NRGN SNPs are significantly associated with SCZ in the Bangladeshi population.

Establishment of tetra-primer ARMS-PCR technique for detecting exo-E415G SNP in Plasmodium falciparum isolatesfrom Central Highlands of Vietnam

Drug resistance in Plasmodium falciparum parasites is associated with the single nucleotide polymorphism (SNP) exo-E415G, which is considered as a marker to reduce the susceptibility to artimisinin and leads to treatment failure. This study aims to establish a procedure based on tetra-primer ARMS PCR technique to detect the SNP exo-E415G in malaria patients who experienced treatment failure with artemisinin. DNA sample from P. falciparum 3D7 isolate was used to optimise the procedure based on tetra-primer ARMS PCR technique to detect exo-E415G SNP. The method was then applied to screen for exo-E415G SNP on 123 blood samples from patients who experienced treatment failure with artemisinin in 3 provinces (Gia Lai, Dak Nong, Dak Lak) from Central Highlands of Vietnam. A DNA fragment (749 bp) containing the exo-E415GSNP was amplified using primer pairs E415G-Fc and E415G-Rc. Specific primers for the wildtype alen (A alen) and the mutant alen (G alen) amplified fragments with the length of 316 and 474 bp, respectively. The genotyping results were consistent with Sanger sequencing. The tetra-primer ARMS PCR technique was used for genotyping exo-E415G SNP on 123 patients who failed with artemisinin treatment, the frequency of exo-E415G alen was observed at 78% (96/123). In conclusion, the simple tetra-primer ARMS PCR technique was established to detect the SNP Exo-E415G. The prevalence of the mutant alenExo-E415G was 78% in the Central Highlands of Vietnam.

Genetic analysis of lncRNA H19 (rs217727) and MIAT (rs1894720) polymorphisms in patients with salivary gland tumors

Long noncoding RNAs (lncRNAs) represent a novel pharmacological application in cancer. Salivary gland tumors (SGTs) are essential to both maxillofacial and oral pathology. This study evaluated the H19 (rs217727) and Myocardial infarction-associated transcript (MIAT) (rs1894720) polymorphisms' connection to SGT susceptibility.We studied 104 Iranian patients with clinically definite SGTs and 114 healthy individuals. Samples collected from the participants were prepared, followed by DNA extraction. Following a tetra-primer ARMS-PCR technique employing specific primer pairs, DNA genotyping was carried out.The multivariate logistic regression analysis after adjusting the age revealed that the risk of SGTs was significantly reduced to a 0.17-fold in patients with TT genotype of rs217727 in the co-dominant model relative to CC genotype, while the risk of SGTs increased to 69-fold in patients with GT genotype of rs1894720 in the co-dominant model relative to GG genotype. In the dominant model, the risk of SGTs significantly were increased to a 41.8-fold in patients with GT + TT genotypes of rs1894720 relative to GG genotype. Also, in the recessive model, we found significant decrease in the risk of SGTs in patients with TT genotype of rs217727 compared to CT + CC and also in patients with TT genotype of rs1894720 relative to GT + GG genotype. In over-dominant model, the GT genotypes of rs1894720 significantly were associated to a 69-fold increase the risk of SGTs.Finally, the current study is the first to show that rs217727 and rs1894720 polymorphisms could be related to SGT risk in the Iranian population.

Assessment of the relationship of transcription factor 7-like 2 rs4506565 (T/A) variant with type 2 diabetes in Iraqi Arab patients

Researchers have consistently linked some TCF7L2 gene variations, such as rs7903146, rs12255372, and rs11255372, to Type 2 diabetes mellitus (T2DM) in numerous populations and ethnic groups. The goal of this study is to show if the TCF7L2 variations rs4506565 T/A is linked to T2DM in Iraqi Arab patients. For this purpose, a case-control study was conducted with 100 T2DM patients and 100 controls. The Tetra-Primer ARMS-PCR Technique was used to genotype TCF7L2 rs4506565 T/A, making use of newly-designed primers. The study concluded that the TCF7L2 gene polymorphism rs4506565 T/A shows no significant difference in genotypes in any of the codominant, dominant, over dominant, recessive, and additive models. The dominant model has a significance with age but no significance with other parameters.

Four Variations Detected in 3 Genes Related to Thrombophilia by Multiplex ARMS – PCR

Thrombophilia is a condition that may cause venous obstruction, leading to serious illnesses such as stroke, respiratory failure, and even death. This condition is hereditary or related to the immune system. Inherited thrombophilia is screened by analysing genetic variants (allele). In this study, we analyse 4 variations in 3 genes, which occur at high variable frequency, namely Methytetrahydrofolate Reductase (MTHFR C677T, MTHFR A1298C), Factor V Leiden (FVL C1691T) and Plasminogen Activator Inhibitor 1 (PAI-1 5G/4G), using allele-specific polymerase chain reaction (ARMS-PCR). The primers were designed using FastPCR program and PCR conditions were optimized to identify 8 alleles of 4 variants through 3 multiplex ARMS - PCR reactions. The accuracy of the ARMS - PCR results was confirmed by Sanger sequencing and the real time PCR commercial kit (SNP). Mastering the allele specific PCR technique contributes to the survey of genetic variations in the Vietnamese population in order to screen and treat thrombophilia-related diseases.&#x0D; Keywords&#x0D; Thrombophilia, allele-specific polymerase chain reaction (ARMS-PCR), variants MTHFR C677T, MTHFR A1298C, FVL C1691T, PAI-1 4G/5G.&#x0D; References&#x0D; [1] A. Dautaj, G. Krasi, V. Bushati et al., Hereditary Thrombophilia, Acta Biomedica, Vol. 90, No. 10S, 2019, pp. 44-46, https://doi.org/10.23750/abm.v90i10-S.8758.[2] M. G. Beckman, W. C. Hooper, S. E. Critchley, T. L. Ortel, Venous Thromboembolism: A Public Health Concern, American Journal of Preventive Medicine, Vol. 38, No. 4S, 2010, pp. S495-S501, https://doi.org/10.1016/j.amepre.2009.12.017.[3] G. E. Raskob, R. Silverstein, D. W. Bratzler et al., Surveillance for Deep Vein Thrombosis and Pulmonary Embolism: Recommendations from A National Workshop, American Journal Preventive Medicine, Vol. 38, No. 4S, 2010, pp. S502-S509, https://doi.org/10.1016/j.amepre.2010.01.010.[4] J. A. Heit, M. D. Silverstein, D. N. Mohr et al., The Epidemiology of Venous Thromboembolism in the Community, Thrombosis and Haemostasis, Vol. 86, No. 1, 2001, pp. 452-463, https://doi.org/10.1055/s-0037-1616243.[5] M. Cushman, A. W. Tsai, R. H. White et al., Deep Vein Thrombosis and Pulmonary Embolism in Two Cohorts: The Longitudinal Investigation of Thromboembolism Etiology, The American Journal of Medicine, Vol. 117, No. 1, 2004, pp. 19-25, https://doi.org/10.1016/j.amjmed.2004.01.018.[6] J. A. Heit, The Epidemiology of Venous Thromboembolism in the Community: Implications for Prevention and Management, Journal of Thrombosis and Thrombolysis, Vol. 21, No. 1, 2006, pp. 23-29, https://doi.org/10.1007/s11239-006-5572-y.[7] R. H. White, The Epidemiology of Venous Thromboembolism, Circulation, Vol. 107, No. 23 S1, 2003, pp. I4-I8, https://doi.org/10.1161/01.cir. 0000078468.11849.66.[8] A. Wahed, A. Dasgupta, Thrombophilias and Their Detection, Hematology and Coagulation: A Comprehensive Review for Board Preparation, Certification and Clinical Practice, Elsevier, United States of America, 2015, pp. 263-275.[9] SNP Biotechnology, SNP Detection Real-Time PCR Kits, http://www.snp.com.tr/EN,37/snp-detection-real-time-pcr-kits.html/, 2021 (accessed on: May 1st, 2021).[10] V. S. Le, K. T. Tran, H. T. P. Bui et al., A Vietnamese Human Genetic Variation Database, Human Mutation, Vol. 40, No. 10, 2019,pp. 1664-1675, https://doi.org/10.1002/humu.23835.[11] dbSNP – NCBI, Reference SNP (rs) Report (rs6025, rs1799762), https://www.ncbi.nlm.nih.gov/snp/,02021 2021 (accessed on: May 1st, 2021).[12] D. C. Rees, M. Cox, J. B. Clegg, World Distribution of Factor V Leiden, The Lancet, Vol. 346, No. 8983, 1995, pp. 1133-1134, https://doi.org/10.1016/s0140-6736(95)91803-5. A. Fateh, M. Aghasadeghi, S. D. Siadat et al., Comparison of Three Different Methods for Detection of IL28 rs12979860 Polymorphisms as A Predictor of Treatment Outcome in Patients with Hepatitis C Virus, Osong Public Health and Research Perspectives, Vol. 7, No. 2, 2016, pp. 83-89, https://doi.org/10.1016/j.phrp.2015.11.004. [13] T. Huang, J. Zhuge, W. W. Zhang, Sensitive Detection of BRAF V600E Mutation by Amplification Refractory Mutation System (ARMS)-PCR, Biomarker Research, Vol. 1, No. 3, 2013, https://doi.org/10.1186/2050-7771-1-3.[14] R. F. V. Medrano, C. A. D. Oliveira, Guidelines for the Tetra-primer ARMS-PCR Technique Development, Molecular Biotechnology, Vol. 56, 2014, pp. 599-608, https://doi.org/10.1007/s12033-014-9734-4.[15] C. Runcharoen, K. Fukunaga, I. Sensorn et al., Prevalence of Pharmacogenomic Variants in 100 Pharmacogenes Among Southeast Asian Populations under Tarmacogenomics Research Network (SEAPharm), Human Genome Variation, Vol. 8, No. 7, 2021, https://doi.org/10.1038/s41439-021-00135-z.

Genetic Polymorphism of miR-218-2 (rs11134527) in Cervical Cancer: A Case-Control Study on the Bangladeshi Women.

The prevalence of Cervical Cancer (CC) is disproportionately higher in developing countries. It is the second most frequent cancer type among Bangladeshi women and the major cause of morbidity and mortality. However, no previous data reported the association of miR-218-2 gene polymorphisms in Bangladeshi cervical cancer patients. This case-control study was designed to find the link between the rs11134527 polymorphism in miR-218-2 and CC. A total of 488 subjects were recruited, comprising 256 cervical cancer patients and 232 healthy females. Genotyping was conducted with the tetra-primer ARMS-PCR technique to detect the association. The results of genotype data showed that rs11134527 was in the Hardy-Weinberg equilibrium in both CC cases and controls (P >0.05). Overall, the polymorphism was found to be significantly associated with an increased risk of cervical cancer with AG genotype (AG vs. GG: OR = 2.26, 95% Cl = 1.40-3.66, P = 0.0008), AA genotype (AA vs. GG: OR = 3.64, 95% Cl = 2.17-6.10, P <0.0001), dominant model (AG+AA vs. GG: OR = 2.75, 95% Cl = 1.75-4.31, P <0.0001), recessive model (AA vs. GG+AG: OR = 2.08, 95% Cl = 1.41-3.08, P = 0.0002), and A allele (A vs. G: OR = 1.94, 95% Cl = 1.51-2.51, P <0.0001). All of these correlations remained statistically significant after performing Bonferroni correction (P <0.008). Our study suggests that the rs11134527 polymorphism in the miR-218-2 gene contributes to the susceptibility of CC in Bangladeshi women.

Genetic polymorphism in MAP1B gene associated with conception rate in Holstein Friesian crossbred breeding bulls

The microtubule associated protein 1 B (MAP1B) gene is one of the candidate genes which was found to be strongly associated with the sire conception rate. In the present study, identification of single nucleotide polymorphisms (SNPs) in the targeted region of MAP1B gene and their effect on conception rate (CR) in Karan Fries (KF) breeding bulls were sought. A tetra-primer ARMS PCR technique was adapted to genotype an SNP, g. 3,066A&gt;G located on intron 1 of MAP1B gene. The relationship between the CR of KF breeding bulls and polymorphic locus of MAP1B gene was assessed. Genotyping of the targeted region revealed three kinds of genotype (AA, AG and GG) with respect to g. 3,066A&gt;G SNP locus. The identified SNP had highly significant (P=0.0009) influence on CR. The KF bulls with AA and AG genotype had comparatively higher CR than the bulls with GG genotype. The identified SNP was significantly associated with CR, thus it may be utilized as a molecular marker for developing marker-assisted selection strategy to enhance the CR in KF cattle population.

The LncRNA H19 rs217727 Polymorphism Is Associated with Oral Squamous Cell Carcinoma Susceptibility in Iranian Population.

Lack of protein-coding capacity is a main characteristic of long noncoding RNAs (lncRNAs) which, as molecular biomarkers, have found a novel pharmacological application in cancer and are reported to be important regulators of gene expression. H19 is reportedly involved in cancer progression and tumorigenesis. One of the most common types of head and neck cancers is oral squamous cell carcinoma (OSCC). The main objective of the present study was to evaluate the correlation of OSCC susceptibility with H19 gene in an Iranian population. This research was performed on 400 subjects of both sexes referred to the Namazi Hospital affiliated with the Shiraz University of Medical Sciences (SUMS). Individuals aged 15-88 years were divided into two groups: pathologically diagnosed patients with new-onset OSCC and healthy controls. After written and informed consent was obtained from the individuals, genomic DNA was extracted. The tetra-primer ARMS-PCR technique was performed for DNA genotyping by the use of specific primer pairs. The susceptibility of OSCC and H19 gene polymorphism sites was further analyzed (rs217727 and rs2107425). The allele and genotype frequencies of H19 rs2107425 polymorphism were similar between OSCC cases and controls. The H19 rs217727T allele frequency was significantly higher in OSCC cases (P = 0.002), and the polymorphism of H19 rs217727 was associated with OSCC susceptibility in the codominant (OR = 6.04, 95%CI = 1.70 − 21.42, P = 0.001 for TT genotype), dominant (OR = 1.62, 95%CI = 1.08 − 2.43, P = 0.01), and recessive (OR = 5.32, 95%CI = 1.51 − 18.69, P = 0.003) models. This study showed that rs217727 and OSCC susceptibility were statistically correlated in the Iranian population.

Detection of Single-Nucleotide Polymorphism in AGPAT6 Gene, Associated with Milk Fat Content, using Tetra-Primer ARMS PCR-Based Assay, in Karan Fries Breeding Bulls.

Background: The bovine AGPAT6 gene is one of the potential candidate genes governing milk fat synthesis.Objectives: Identification of single nucleotide polymorphisms (SNP) in the targeted region of AGPAT6 gene and their effect on expected breeding values (EBV) of first lactation milk production traits viz. fat %, fat yield and 305 days milk yield in Karan Fries (KF) breeding bulls were sought.Materials and Methods: A tetra-primer ARMS PCR technique was adapted to genotype an SNP, g.36,175,805C>T located on 5’ flanking region of AGPAT6 gene. The relationship between EBV of milk production traits and polymorphic locus of AGPAT6 gene was assessed. Results: Three kinds of genotype (CC, CT, and TT) with respect to g.36,175,805C>T SNP locus were observed. The identified SNP had significant (P<0.05) influence on EBV of fat % (EBV-FP). The KF bulls with CC and CT genotype had comparatively higher EBV-FP than the bulls with the TT genotype. The substitution of “C” allele by “T” allele led to a decrease of 0.0045 % in the EBV-FP.Conclusion: The identified SNP was significantly associated with EBV-FP, thus it may be utilized as a molecular marker for developing marker-assisted selection strategy to enhance the milk fat content in KF cattle population.

AMELX Gene Association with Dental Caries in Iranian Adults.

Dental decay is a disease that is greatly affected by environmental components, but recently there have been an increasing number of documents supporting a genetic factor in the development of caries. The purpose of this study was to examine the association between dental caries and single-nucleotide polymorphisms in the AMELX gene. This research was carried out on 360 individuals of both sexes, who were referred to the dental school at the Shiraz University of Medical Sciences. In this research, individuals aged 20–65 years were divided into two groups: controls (decayed, missed, or filled teeth (DMFT) ≤ 5; n = 180) and cases (DMFT ≥ 14; n = 180). The tetra-primer ARMS-PCR technique was performed for genotyping the DNA extracted from blood cells. Analysis of the AMELX rs946252 polymorphism showed that the T allele of rs946252 was a significant protective factor against dental caries in Iranian adults (T vs. C: OR = 0.70, 95% CI: 0.49–0.98, P = 0.04). We demonstrated the significant differences in the genotype frequencies under two genetic models: overdominant (TC vs. TT + CC: OR 0.35, 95% CI 0.19–0.64, P = 0.0006) and recessive (CC vs. TC + TT: OR 2.57, 95% CI 1.39–4.76, P = 0.002). Our results show that the SNPs of the AMELX gene may be related with susceptibility to dental caries in Iranian adults.

UGT1A1 gene linkage analysis: application of polymorphic markers rs4148326/rs4124874 in the Iranian population.

Objective(s):Mutations in the UGT1A1 gene are responsible for hyperbilirubinemia syndromes including Crigler-Najjar type 1 and 2 and Gilbert syndrome. In view of the genetic heterogeneity and involvement of large numbers of the disease causing mutations, the application of polymorphic markers in the UGTA1 gene could be useful in molecular diagnosis of the disease.Materials and Methods:In the present study, two polymorphic markers including rs4148326 and rs4124874 in the UGT1A1 gene region were characterized. The markers were selected using bioinformatics analysis of the UGT1A1 gene region and genotyped in 212 unrelated healthy individuals and 13 family trios in the Iranian population using Tetra-Primer ARMS PCR technique. The allele frequency and population status of the alleles were estimated using GENEPOP, FBAT, PowerMarker and Arlequin software.Results:The results indicated that in the case of rs4148326 marker, allele frequency for T and C allele was 66.04% and 33.96%, respectively. For rs4124874 marker, allele frequency for G and T alleles was 39.4% and 60.6%, respectively. The values of heterozygosity index for the markers examined were 64.1 for rs4148326 and 72.1 for rs4124874, respectively. The haplotype estimation analysis of the markers resulted in three informative haplotypes with frequencies ≥0.05. Moreover, the results suggested the presence of linkage disequilibrium between two markers.Conclusion:Altogether, the data suggested that rs4148326 and rs4124874 could be introduced as informative markers for molecular diagnosis of Crigler-Najjar type 1 and 2 and Gilbert syndrome in the Iranian population.

Allele Frequency and Genotyping of rs2288258 Polymorphism in HEXA Gene Region in the Isfahan Population

Introduction: Tay-Sachs is a genetic disorder with autosomal recessive inheritance affecting the central nervous system. The disorder mainly results from mutations in the HEXA gene on chromosome 15. Sequencing is used to detect mutations and sequence variations in the HEXA gene, which is expensive and time consuming. Alternatively, linkage analysis of polymorphic markers such as single nucleotide polymorphism (SNP) could be used in heterozygous carrier detection and prenatal diagnosis of the disease in families with an affected individual. Materials & methods: A large number of SNP markers have been introduced for the HEXA gene in the electronic databases. In the present study the genotype and informative situation of rs2288258 genetic marker in HEXA gene region was investigated using Tetra-primer ARMS PCR technique with newly designed primers in Isfahan population. Estimation of allelic frequency, genotype frequency, presence of Hardy Weinberg Equilibrium (HWE) and the amount of polymorphism information content (PIC) was computed by Power Marker software. Findings: The results indicated 9.12% minor allele frequency (MAF) and PIC=0.1520 for rs2288258 marker in the Isfahan population. Also, analysis of Hardy-Weinberg Equilibrium showed the presence of equilibrium for this marker in the mentioned population. Discussion & conclusions: The results indicated 9.12% minor allele frequency (MAF) and PIC=0.1520 for rs2288258 marker in the Isfahan population. Also, analysis of Hardy-Weinberg Equilibrium showed the presence of equilibrium for this marker in the mentioned population. Totally, according to the results of this study rs2288258 genetic marker could be introduced as an SNP marker for linkage analysis in carrier detection and prenatal diagnosis of HEXA mutations in Isfahan population.

Standardization and application of the tetraprimer ARMS-PCR technique for screening of the E198A SNP in the β-tubulin gene of hookworm populations in Brazil

The tetraprimer ARMS-PCR technique is efficient for SNP detection and can be used to search for polymorphisms associated with drug resistance. However, the establishment of this methodology is not always straightforward because of the constraints on primer design due to the restrictions of the polymorphic regions. Here, we describe the standardization of the tetraprimer ARMS-PCR methodology for the detection of a SNP at codon 198 of the Ancylostoma caninum β-tubulin gene. This SNP is associated with resistance to albendazole in various nematodes. The methodology was used to screen 327 individuals from 6 different locations. No mutation was found in any of the samples. This methodology will be useful for screening for the E198A SNP in the β-tubulin gene of canine hookworms in a broader population to determine whether this SNP is associated with benzimidazole resistance in this species. The method could also be adapted for the analysis of other SNPs in other nematode species.

Relationship between phosphoinositide-3-kinase genetic polymorphism and schizophrenia

Schizophrenia, with incidence of 1% worldwide, is a common mental disorder. Phosphoinositide-3-kinases (PI3Ks) are a family of enzymes involved in cellular functions such as cell growth, proliferation, differentiation, motility, intracellular trafficking, and survival. These enzymes play an important role in the PI3K/AKT signalling pathway. The PIK3CA gene encodes the alpha catalytic subunit of the PI3K enzyme. The present study analysed the role of three SNPs of the PIK3CA gene (rs6443624 (A/C), rs7640662(C/G) and rs7621329(C/T)) in the development of schizophrenia. Methods: In this case-controlled study, DNA was extracted from blood samples from 108 patients with schizophrenia and 108 healthy patients as controls. Genotypic analyses of PIK3CA SNPs rs6443624 (A/C), rs7640662(C/G) and rs7621329(C/T) were made using the tetra primer ARMS-PCR technique. Results: The outcome shows significant difference between CT and the combined genotype (CT + TT) of rs7621329 and the risk of schizophrenia (OR = 6.4, 95% CI = 3.023-14.23, p < 0.0001). Outcome showed no significant difference for were for analyses of the rs6443624 and rs7640662 genotypes. Conclusions: These results indicate an association between PIK3CA gene polymorphism on the rs7621329(C/T) site and the risk of schizophrenia. Further study of the genetic population using a larger sample size is necessary in order to validate these present findings.

Role of XmnIgG Polymorphism in Hydroxyurea Treatment and Fetal Hemoglobin Level at Isfahanian Intermediate β-Thalassemia Patients.

Background:β-thalassemia is the most common monogenic disorder in human. The (CT) polymorphism at -158 upstream region of the γG-globin gene and pharmacological factors such as hydroxyurea have been reported to influence γ-globin gene expression and the severity of clinical symptoms of β-thalassemia. Methods:In the present study, 51 β-thalassemia intermediate patients were studied. Xmn1γG polymorphism genotype was determined using Tetra-Primer ARMS-PCR technique. Hemoglobin (Hb) and fetal hemoglobin (HbF) levels were determined by gel electrophoresis. Results:Of 51 patients, 35 (68.6%) patients were heterozygous (CT) and 16 (31.4%) patients were homozygous (CC). Of 30 patients under treatment by hydroxyurea, 20 (66.7%) patients were heterozygous (CT) and 10 (33.3%) patients were homozygous (CC). Our results demonstrated that in the heterozygous (CT) genotype, the Hb (9.58 ± 1.25 gm/dl) and HbF (89.30 ± 21.87) levels were significantly higher in comparison with homozygous (CC) genotype (7.94 ± 1.34 gm/dl and 70.32 ± 40.56, respectively). Furthermore, we observed that after drug usage, the Hb and HbF levels in patients with heterozygous (CT) genotype (0.7 ± 1.26 gm/dl and 5.95±14.8, respectively) raised more in comparison with homozygous (CC) genotype (0.26 ± 1.43 gm/dl and 0.8 ± 1.31, respectively). Conclusion:Hb and HbF levels in the patients carrying T allele are increased significantly, and they also response to hydroxyurea treatment.

Association analysis of DISC1 gene polymorphisms with Attention-deficit/hyperactivity disorder in Iranian population

Background & Objectives:Attention deficit hyperactivity disorder (ADHD) is a common heritable psychiatric disorder with a worldwide prevalence of 5%. The etiology of ADHD is still incompletely understood, but several studies, consistently indicate the strong role of genetic factors on this disorder. The aim of this study was to determine the effect of three SNPs rs11122319, rs11122330 and rs6675281 in the etiology of ADHD in an Iranian childrenMethods:In this research work, for the first time, we investigated the association of three SNPs (rs11122330, rs6675281 and rs11122319) in the DISC1 gene with ADHD in Iranian population. Two hundred fourthy subjects composed of 120 patients and 120 healthy controls were included and tetra-primer ARMS PCR technique was used for genotyping all selected SNPs.Results:We found differences in genotype and allele distributions of rs 6675281 polymorphism between our patients and controls. The A, T and A alleles were the more frequent alleles in rs11122319, rs6675281 and rs11122330 polymorphisms in both case and control groups respectively. The TT genotype was more frequent in control group compared to patients. (P value = 0.008, OR= 1.5837, 95% CI= 1.1012 to 2.2776).Conclusion:Our findings strengthens the role of DISC1 gene as a susceptibility locus for ADHD and indicate that rs6675281 polymorphism is a susceptibility factor for ADHD for the first time in children reported in an Iranian population in this part of the world.

Investigation of sensitivity, specificity and accuracy of Tetra primer ARMS PCR method in comparison with conventional ARMS PCR, based on sequencing technique outcomes in IVS-II-I genotyping of beta thalassemia patients

PurposeBeta thalassemia is one of the most important hematic diseases all around the world and solving the problems caused by this abnormality is strongly dependent on precise detection and reliable screening of high-risk couples. The aim of our study was the investigation of sensitivity, specificity and accuracy of Tetra primer ARMS PCR method comparing with conventional ARMS PCR, based on sequencing technique outcomes for genotyping of IVS-II-I mutation in beta thalassemia patients. MethodsFifty seven samples including two homozygote, 49 heterozygote and 6 normal specimens were analyzed by Tetra primer ARMS PCR and conventional ARMS PCR methods. DNA was extracted by the standard method of salting out for leukocyte genomic DNA extraction of blood specimens and a high pure PCR template preparation kit was used for DNA purification of CVS samples. The results obtained by Tetra primer ARMS PCR and conventional ARMS PCR methods were compared with gold standard technique, i.e. sequencing. ResultsAll three parameters including specificity, sensitivity and accuracy were 100% for Tetra primer ARMS PCR method, while they were 100%, 92.45% and 92.7% for conventional ARMS PCR technique respectively. Comparing with Tetra primer ARMS PCR which represented 100% agreement with sequencing method, conventional ARMS PCR technique only showed 47.1% agreement, because of 4 discordant results. ConclusionTetra primer ARMS PCR method is an almost reliable, sensitive and accurate technique and it is suggested that it can be used as a complementary method for diagnostic cases instead of conventional ARMS PCR method. This suggestion originated with perfect rate of agreement between outcomes of sequencing method, as a gold standard method of detecting the mutations, and Tetra primer ARMS PCR technique comparing with conventional ARMS PCR method.

CAPN1 markers in three Argentinean cattle breeds: report of a new InDel polymorphism within intron 17

In this study, the genotype distribution and allelic frequencies of CAPN1 (Calcium activated neutral protease) single nucleotide polymorphisms (SNPs) were analyzed taking advantage of the different genetic backgrounds provided by Hereford, Brahman and Braford cattle. We report a new insertion/deletion (InDel) polymorphism, consisting of a change of seven nucleotides for only one nucleotide (TCTGGGT → C) within intron 17 of the CAPN1 gene. The segregation pattern of this polymorphism was analyzed together with the markers CAPN316, CAPN530 and CAPN4751 already described. The allele distribution of CAPN1 markers in the Braford crossbreed (3/8 Brahman 5/8 Hereford) is described for the first time. Four assays of allelic discrimination were designed: the tetra primer ARMS-PCR technique for genotyping the new InDel and the CAPN4751 marker, and a PCR-RFLP method for genotyping the markers CAPN316 and CAPN530. The genotypic and minor allele frequencies (MAFs) obtained showed that the InDel polymorphism does not provide redundant information to that already provided by the other CAPN1 markers and segregates differently between breeds, being a common SNP (MAF ≥ 0.05) in the herds with a high percentage of Bos indicus background. The high percentage of heterozygous individuals found in the Braford crossbreed for the markers assessed reveals enough genetic variation that could help to solve the tenderness problem of tropical-adapted cattle.