Tetra-primer Amplification Refractory Mutation system-PCR Research Articles

Analysis of Clinical and Genetic Factors of Obesity and Psoriasis Concomitance-The Influence of Body Mass Composition, Prevalence of Mood Disorders, Environmental Factors and FTO Gene Polymorphisms (rs9939609, rs1558902).

The study enrolled 30 overweight or obese adult patients with chronic plaque psoriasis and 30 overweight or obese volunteers (northern Poland region, Caucasian population). Mood disorders, body mass composition by using bioelectrical impedance analysis (BIA) and FTO gene polymorphisms (rs9939609, rs1558902) by tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) were assessed. Results revealed significantly higher visceral adipose tissue levels in psoriatic patients (5.23 ± 2.29 [L] vs. 3.41 ± 1.86 [L]), p = 0.001), especially among men, along with elevated rates of moderate and severe depression (26.67% vs. 6.67% and 13.33% vs. 3.33%, p = 0.048 respectively). Additionally, FTO gene polymorphisms correlated with waist-hip ratio differences in both groups. The study highlights the importance of evaluating body composition beyond body mass index, recognizing its influence on psoriasis and associated conditions like depression. The FTO gene may serve as a potential genetic link between psoriasis and obesity, warranting further research for validation. Adiposity emerges as a key and modifiable risk factor, underscoring the clinical implications of body composition complexities in psoriasis management.

A novel tetra-primer ARMS-PCR for genotyping of the OPRM1 gene rs1799971 variant associated with opioid use disorders

ObjectivesA SNV is a single nucleotide change that can occur at any point in the genome. SNVs are the most common genetic variants that occur in the human genome, and a number of SNVs have been found to be associated with human traits and disease. Researchers genotype SNVs using TaqMan technology, DNA microarray, MALDI-TOF mass spectrometry, and automated sequencing, which are expensive and time-consuming. The OPRM1 gene rs1799971 (A118G) has been identified for its association with Opioid use disorder (OUD). The present study focused on developing a single step identification test using Tetra-Primer Amplification Refractory Mutation System-PCR (T-ARMS-PCR) to detect the presence of SNV OPRM1 rs1799971 (A118G). This study was performed to optimize the protocol for the designed four primers and validate it using a total of 52 buccal samples from volunteers who are currently under rehabilitation for the drug abuse disorder.ResultsUtilizing 52 DNA samples, a novel T-ARMS-PCR assay was successfully developed, tested, and validated. The products of the T-ARMS PCR for rs1799971 contained 395 bp as the control band, 186 bp as G allele (variant) and 257 bp as A allele (wild type), which were observed in the gel image. The genotype frequencies for the OPRM1 gene rs1799971 (A118G) were 44% (22/52) of homozygous variant type (GG), 28.9% (15/52) of homozygous wild type (AA) and 28.9% (15/22) of heterozygous (AG). The G allele frequency was 56.7% and A allele frequency was 43.3%.

FRI031 A Novel Strategy To Detect Leptin Receptor (Lepr, fa) Mutation In Obesity And Diabetic Animal Model (Zucker Rats)

Abstract Disclosure: X. Xu: None. X. Hu: None. G. Ma: None. T. Wang: None. J. Wang: None. G. Chen: None. L. Zhao: None. J. Chen: None. Globally, diabetes is among the leading causes of premature morbidity and mortality. Early intervention is crucial to achieve optimal outcomes and minimize the burden of diabetes. Animal models are indispensable tools to investigate etiology and explore novel strategies for diabetes treatment and prevention. Zucker strain and its derivatives, Zucker Fatty (ZF) and Zucker Diabetic Fatty (ZDF) rats, are one of the most used rodent models to study diabetes and diabetes-associated metabolic disorders. Homozygous Zucker rats with leptin receptor gene mutation (Lepr, fa-/-) spontaneously develop obesity, hypercholesterolemia, and hyperlipidemia. The wildtype (fa+/+) and heterozygous (fa+/-) littermates phenotypically are indistinguishable. Furthermore, early stages of diabetes are asymptomatic, and the obese phenotype is not appreciable in homogenous (fa-/-) Zucker rats until week 4 of age. Therefore, a rapid, cost-effective, and reliable genotyping method is essential to differentiate homozygous (fa-/-) rats from either wildtype (fa+/+) or heterozygotes (fa+/-) in early interventional studies. We reported a tetra-primer Amplification-Refractory Mutation System (ARMS)—PCR approach to detect leptin receptor fa mutation using genome DNA collected from ear and tail tissues of Zucker rats. Three different types of amplicon patterns are easily identified by agarose gel electrophoresis. Wildtype (fa+/+) genotype displays a 401 bp and a 201 bp amplicon band; heterozygous (fa+/-) consists of a 401 bp, a 263 bp, and a 201 bp amplicon band on the gel; homozygous (fa-/-) comes up with two bands of 401 bp and 263 bp. The genotypes identified by our tetra-primer ARMS—PCR method are further confirmed by sequence analysis. This method requires neither DNA purification nor restriction enzyme digestion, which is suitable for research exploring gene-environment interaction, as well as implementing appropriate intervention approaches in the research areas of diabetes and metabolic diseases. Presentation: Friday, June 16, 2023

A duplex tetra-primer ARMS-PCR assay to discriminate three species of the Schistosoma haematobium group: Schistosoma curassoni, S. bovis, S. haematobium and their hybrids

BackgroundThe use of applications involving single nucleotide polymorphisms (SNPs) has greatly increased since the beginning of the 2000s, with the number of associated techniques expanding rapidly in the field of molecular research. Tetra-primer amplification refractory mutation system—PCR (T-ARMS-PCR) is one such technique involving SNP genotyping. It has the advantage of amplifying multiple alleles in a single reaction with the inclusion of an internal molecular control. We report here the development of a rapid, reliable and cost-effective duplex T-ARMS-PCR assay to distinguish between three Schistosoma species, namely Schistosoma haematobium (human parasite), Schistosoma bovis and Schistosoma curassoni (animal parasites), and their hybrids. This technique will facilitate studies of population genetics and the evolution of introgression events.MethodsDuring the development of the technique we focused on one of the five inter-species internal transcribed spacer (ITS) SNPs and one of the inter-species 18S SNPs which, when combined, discriminate between all three Schistosoma species and their hybrid forms. We designed T-ARMS-PCR primers to amplify amplicons of specific lengths for each species, which in turn can then be visualized on an electrophoresis gel. This was further tested using laboratory and field-collected adult worms and field-collected larval stages (miracidia) from Spain, Egypt, Mali, Senegal and Ivory Coast. The combined duplex T-ARMS-PCR and ITS + 18S primer set was then used to differentiate the three species in a single reaction.ResultsThe T-ARMS-PCR assay was able to detect DNA from both species being analysed at the maximum and minimum levels in the DNA ratios (95/5) tested. The duplex T-ARMS-PCR assay was also able to detect all hybrids tested and was validated by sequencing the ITS and the 18S amplicons of 148 of the field samples included in the study.ConclusionsThe duplex tetra-primer ARMS-PCR assay described here can be applied to differentiate between Schistosoma species and their hybrid forms that infect humans and animals, thereby providing a method to investigate the epidemiology of these species in endemic areas. The addition of several markers in a single reaction saves considerable time and is of long-standing interest for investigating genetic populations.Graphical

Association of DROSHA rs6877842, rs642321 and rs10719 polymorphisms with increased susceptibility to breast cancer: A case-control study with genotype and haplotype analysis.

Multiple lines of evidence suggest that single nucleotide polymorphisms (SNPs) in genes encoding components of the microRNA processing machinery may underlie susceptibility to various human diseases, including cancer. The present study aimed to investigate whether rs6877842, rs642321 and rs10719 SNPs of DROSHA, a key component of the miRNA biogenesis pathway, are associated with increased risk of breast cancer. A total of 100 patients diagnosed with breast cancer and 100 healthy women were included. Following extraction of DNA, genotyping was performed by tetra primer- amplification refractory mutation system-PCR (T-ARMS-PCR) technique. Under the co-dominant, dominant and recessive inheritance models, the association between DROSHA SNPs and breast cancer risk was determined by logistic regression analysis. The association of DROSHA SNPs with patients' clinicopathological parameters was assessed. Also, haplotype analysis was performed to evaluate the combined effect of DROSHA SNPs on breast cancer risk. We observed a statistically significant association between DROSHA rs642321 polymorphism and breast cancer susceptibility (P <0.05). Under the dominant inheritance model, DROSHA rs642321 polymorphism was significantly associated with increased risk of breast cancer (OR: 6.091; 95% CI: 3.291-11.26; P =0.0001). Our findings demonstrated that DROSHA rs642321 T allele can contribute to the development of breast cancer (OR: 3.125; 95% CI: 1.984-4.923; P =0.0001). We also found that GTC and GTT haplotypes conferred significant risk for breast cancer (OR: 2.367; 95% CI: 1.453-3.856; P =0.0001 and OR: 7.944; 95% CI: 2.073-30.43; P =0.0001, respectively). These results provide the first evidence that DROSHA rs642321 polymorphism is associated with increased risk of breast cancer. However, further studies are needed to firmly validate these findings.

Association of Single Nucleotide Polymorphisms in the PYGO2 and PRDM9 Genes with Idiopathic Azoospermia in Iranian Infertile Male Patients.

Azoospermia is a risk factor for infertility affecting approximately 1% of the male population. Genetic factors are associated with non-obstructive azoospermia (NOA). Pygo2 and PRDM9 genes are involved in the spermatogenesis process. The present study aimed to assess the association of single nucleotide polymorphism (SNP) in the Pygo2 (rs61758740 and rs61758741) and PRDM9 (rs2973631 and rs1874165) genes with idiopathic azoospermia (IA). A cross-sectional study was conducted from October 2018 to August 2019 at Rooya Infertility Centre (Qom, Iran). A total of 100 infertile patients with NOA and 100 men with normal fertility were enrolled in the study. Tetra-primer amplification refractory mutation system-PCR method was used to detect SNPs rs61758740, rs61758741, and rs2973631. The restriction fragment length polymorphism method was used for SNP rs1874165. In addition, luteinizing, follicle-stimulating, and testosterone hormone levels were measured. The results showed a significant increase in luteinizing and follicle-stimulating hormone levels in the patient group (P<0.001), but a non-significant difference in testosterone levels in both groups. SNP rs61758740 (T>C) was associated with the increased risk of azoospermia (OR: 2.359, 95% Cl: 1.192-4.666, P=0.012). SNP rs2973631 showed a significant difference in genotype frequency between the patient and control groups in the dominant, recessive, and codominant models. However, in the case of SNP rs1874165, the difference was significant in the dominant, codominant, and overdominant models. There is an association between azoospermia and SNPs in Pygo2 and PRDM9 genes in Iranian infertile male patients with IA. SNPs can be considered a risk factor for male infertility. It should be noted that this article was published in preprint form on the website of europepmc (https://europepmc.org/article/ppr/ppr416800).

Designing and implementation of a T-ARMS-PCR assay to genotype genetic variants associated with retinoblastoma in a cohort of Sri Lankan population

Retinoblastoma (RB) is the most common intraocular malignancy, characterized by high mortality if not detected early and treated promptly. The rare childhood malignancy retinoblastoma serves one of the most important models in modern cancer genetics. Since the study of its familial and sporadic occurrence has led to the identification of the first tumor suppressor gene RB1. Mutations screening is important for risk assessment in future siblings and offspring of RB patients.The aim of this study was to design and implement a novel genetic assay to identify genetic variants associated with retinoblastoma in a cohort of Sri Lankan patients.Materials and methods: A prospective descriptive study was carried out with 59 patients referred to the Eye Unit of the Lady Ridgeway Hospital. Genomic DNA of 59 patients were genotyped using primers designed for Tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR).Results: The median age at diagnosis was 2 years and 7 months. Female to male ratio was 3:2. Out of which, 63% had unilateral retinoblastoma and 36% had bilateral retinoblastoma. Family history of RB was seen in 6.78% patients. Most cases were advanced group D at presentation. As the final result, all patients tested homozygous for the ancestral allele for both rs587776789 and rs121913305 variants of the RB1 gene.Discussion and conclusion: The discovery of germ-line mutations in unilateral patients is valuable since they can be segregated based on their mutational status and this would impact the genetic counselling given to them as they age. In conclusion, this assay can be introduced as a sensitive, specific and simple diagnostic technique for screening related genetic variants for retinoblastoma in the Sri Lankan population.

Development of Tetra-primer Amplification Refractory Mutation System (ARMS) PCR for Detection of CHRNA3 rs8040868

BACKGROUND: Single nucleotide variations (SNV) have been mapped to be associated with several human conditions and diseases. To validate the association between SNV to certain human traits or diseases, a large number of subjects must be included. Thus, in need of a fast, relatively economic, and reliable genotyping method. This can be achieved through the use of tetra-primer amplification refractory mutation system polymerase chain reaction (Tetra-primer ARMS PCR). This study reports strategy to develop Tetra-primer ARMS PCR-based genotyping of CHRNA3 rs8040868.METHODS: The optimization of Tetra-primer ARMS PCR was done through these steps: identification of gene sequence and position of single mutation; designing outer and inner PCR primers; amplification of target gene fragments through PCR by using outer primer; confirming genotype of the PCR product by using sequencing; determining an optimum ratio of outer and inner primer; and determining optimum annealing temperature and cycles for the PCR program. The PCR products were run in 2% gel agarose electrophoresis and visualized under UV illumination.RESULTS: Outer and inner primer ratio of 1:3 with annealing temperature of 64.4°C and 40x cycles was found to be the most optimum condition. Tetra-primer ARMS PCR was able to confirm the results of the DNA sequence of 2 samples, confirming wild-type variants (TT allele) and the heterozygous mutant (CT allele).CONCLUSION: Tetra-primer ARMS PCR was able to genotype rs8040868 of the CHRNA3 gene.KEYWORDS: tetra-primer ARMS PCR, CHRNA3, rs8040868, genotyping

Novel manifestations of Warburg micro syndrome type 1 caused by a new splicing variant of RAB3GAP1: a case report

BackgroundThe present study aimed to determine the underlying genetic factors causing the possible Warburg micro syndrome (WARBM) phenotype in two Iranian patients.Case presentationA 5-year-old female and a 4.5-year-old male were referred due to microcephaly, global developmental delay, and dysmorphic features. After doing neuroimaging and clinical examinations, due to the heterogeneity of neurodevelopmental disorders, we subjected 7 family members to whole-exome sequencing. Three candidate variants were confirmed by Sanger sequencing and allele frequency of each variant was also determined in 300 healthy ethnically matched people using the tetra-primer amplification refractory mutation system-PCR and PCR-restriction fragment length polymorphism. To show the splicing effects, reverse transcription-PCR (RT-PCR) and RT-qPCR were performed, followed by Sanger sequencing. A novel homozygous variant—NM_012233.2: c.151-5 T > G; p.(Gly51IlefsTer15)—in the RAB3GAP1 gene was identified as the most likely disease-causing variant. RT-PCR/RT-qPCR showed that this variant can activate a cryptic site of splicing in intron 3, changing the splicing and gene expression processes. We also identified some novel manifestations in association with WARBM type 1 to touch upon abnormal philtrum, prominent antitragus, downturned corners of the mouth, malaligned teeth, scrotal hypoplasia, low anterior hairline, hypertrichosis of upper back, spastic diplegia to quadriplegia, and cerebral white matter signal changes.ConclusionsDue to the common phenotypes between WARBMs and Martsolf syndrome (MIM: 212720), we suggest using the “RABopathies” term that can in turn cover a broad range of manifestations. This study can per se increase the genotype-phenotype spectrum of WARBM type 1.

Rapid detection of ERG11 polymorphism associated azole resistance in Candida tropicalis.

Increasing reports of azole resistance in Candida tropicalis, highlight the development of rapid resistance detection techniques. Nonsynonymous mutations in the lanosterol C14 alpha-demethylase (ERG11) gene is one of the predominant mechanisms of azole resistance in C. tropicalis. We evaluated the tetra primer-amplification refractory mutation system-PCR (T-ARMS-PCR), restriction site mutation (RSM), and high-resolution melt (HRM) analysis methods for rapid resistance detection based on ERG11 polymorphism in C. tropicalis. Twelve azole-resistant and 19 susceptible isolates of C. tropicalis were included. DNA sequencing of the isolates was performed to check the ERG11 polymorphism status among resistant and susceptible isolates. Three approaches T-ARMS-PCR, RSM, and HRM were evaluated and validated for the rapid detection of ERG11 mutation. The fluconazole MICs for the 12 resistant and 19 susceptible isolates were 32-256 mg/L and 0.5-1 mg/L, respectively. The resistant isolates showed A339T and C461T mutations in the ERG11 gene. The T-ARMS-PCR and RSM approaches discriminated all the resistant and susceptible isolates, whereas HRM analysis differentiated all except one susceptible isolate. The sensitivity, specificity, analytical sensitivity, time, and cost of analysis suggests that these three methods can be utilized for the rapid detection of ERG11 mutations in C. tropicalis. Additionally, an excellent concordance with DNA sequencing was noted for all three methods. The rapid, sensitive, and inexpensive T-ARMS-PCR, RSM, and HRM approaches are suitable for the detection of azole resistance based on ERG11 polymorphism in C. tropicalis and can be implemented in clinical setups for batter patient management.

Prevalence of CYP2C19 and ITGB3 polymorphisms among Bangladeshi patients who underwent percutaneous coronary intervention.

Introduction:Antithrombotic agents are the basic therapeutic option for patients with arterial thrombosis who underwent percutaneous coronary intervention (PCI). In Bangladesh, aspirin and clopidogrel are frequently prescribed as antithrombotics or platelet inhibitors. Studies reported the genetic polymorphisms of CYP2C19*2, CYP2C19*17, and ITGB3 cause an alteration of the pharmacodynamic and pharmacokinetic profile of aspirin and clopidogrel. Therefore, we aimed to assess the prevalence of CYP2C19*2, CYP2C19*17, and ITGB3 polymorphisms among Bangladeshi patients with cardiovascular disease (CVD) who underwent PCI.Methods:Here we assessed a total of 1,000 CVD patients (male 782 and female 218) who underwent PCI and were treated with clopidogrel and/or aspirin. We performed genotyping of patients treated with clopidogrel and aspirin by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) and tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) methods. The PCR products of clopidogrel-treated patients were screened with agarose gel electrophoresis and then digested with SmaI and NsiI-HF for CYP2C19*2 and CYP2C19*17, respectively. We genotyped aspirin-treated patients with T-ARMS-PCR for missense rs5918 (PlA1/A1) polymorphism of the ITGB3 gene. Then we ran the digested PCR products on 2% agarose gel electrophoresis to detect the mentioned polymorphisms.Results:Among the clopidogrel-treated patients, we observed 64.1% polymorphism (hetero + mutant) of CYP2C19*2 (loss-of-function allele) and 22.7% (hetero + mutant) of CYP2C19*17 (gain-of-function allele). On the other hand, among the aspirin-treated patients, polymorphisms of ITGB3 were 84.1% homozygous (PlA1/A1), 15.6% heterozygous (PlA1/A2), and 0.3% mutant homozygous.Conclusion:In the present study, we observed a high prevalence of genetic polymorphisms of CYP2C19 and ITGB3 genes. Therefore, we recommend genotyping of CVD patients before prescribing clopidogrel or aspirin to prevent coagulation. Based on the genotyping study, the adjustment of doses or alternative generics might require to avoid therapeutic failure or toxicity in some cases.

Prognosis of vascular endothelial growth factor gene polymorphism in Egyptian patients with acute myeloid leukemia

BackgroundAngiogenesis is an important process in hematological malignancies especially leukemia. Its role in acute leukemia has been discussed since the cloning of the vascular endothelial growth factor (VEGF) gene from acute myelogenous leukemia (AML). HL-60 is a human promyelocytic leukemia cell line. VEGF, a polymorphic gene, plays a key role in angiogenesis and tumor growth. Its different genotypes are associated with many human diseases. Increased angiogenesis, mediated by VEGF, was associated with poor prognosis in AML patients. AML that arises from neoplastic transformation of hematopoietic stem and progenitor cells remains one of the greater challenges in treating this AML. In adults, leukemia is included in the top 15 of the most common forms of cancer. Single-nucleotide polymorphism of the VEGF gene, 699947 is found in peripheral blood mononuclear cells of Iranian, Australian, Caucasian, and African patients with AML. The main target of this study is to investigate the relationship between VEGF gene polymorphism (rs699947) with AML.Patients and methodsThis a controlled study which included 100 and 76 individuals in each group, respectively. Group 1: a healthy control group and group 2: AML diagnosed patients. Blood samples were genotyped using the tetra-primer amplification-refractory mutation system-PCR and the results were confirmed by PCR-restriction fragment length polymorphism method.ResultsOur results suggest that there is a correlation between rs699947 single-nucleotide polymorphism and AML development and prognosis. However, patients with −2578 AA/CC genotypes were associated with significant better complete remission in comparison to patients with −2578 CA genotype (P = 0.0001). We found that certain polymorphisms are associated significantly with remissions while others with worse response to induction chemotherapy.

Genotyping of blood groups in alloimmunized patients with β-thalassemia major by T-ARMS-PCR and multiplex-aso-pcr

IntroductionBeta-thalassemia major is a severe hemolytic anemia requiring life-long blood transfusion. Planned random donor blood transfusion is associated with alloimmunization against incompatible antigens. Determination of the minor blood group systems phenotype or genotype, and administration of the compatible blood components can significantly reduce the rate of alloimmunization.The present study aimed to determine the prevalence of alloimmunization, and genotype/phenotype characteristics of the minor blood groups systems in patients with β-thalassemia major. Material and methodsThis study was conducted on 1147 β-thalassemia major patients. Initially, antibody screening and antibody identification were performed. Then, phenotyping and genotyping for the Rh, Kell, Kidd, and Duffy blood groups were done in alloimmunized patients using monoclonal antibodies and Multiplex-Allele Specific Oligonucleotide-Polymerase Chain Reaction (Multiplex-ASO-PCR) and Tetra-primer amplification refractory mutation system–PCR (T-ARMS-PCR), respectively. Any phenotype/genotype discrepancy was assessed by direct sequencing. ResultsNinety-seven (8.5 %) out of 1147 patients had alloantibodies against the minor blood group antigens (44 males, 45.4 %, and 53 female, 54.6 %). The most common alloantibodies were against the RH (n: 47, 48.5 %), and the Kell (n: 23, 23.7 %) blood groups systems. Twenty-three (2.1 %) genotype/phenotype discrepancies out of 1067 tests, including 9 in the Rh (9.3 %), 8 in Duffy (34.8 %), and 6 in Kidd (26.1 %) blood groups were detected. No discrepancy was found in the Kell blood group system. Direct sequencing revealed that the results of molecular methods were correct. ConclusionMultiplex-ASO-PCR and T-ARMS-PCR molecular methods are fast, reliable and cost-benefit molecular methods for the minor blood group genotyping in multi-transfused β-thalassemia major patients.

Polymorphism study of BMP-15 (FecXR) and GDF-9 gene related with twinning in cattle

Natural mutations in prolificacy genes in some species especially sheep has been shown transforming growth factor beta (TGF-β) super family ligands such as growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) and their type I receptor (bone morphogenetic protein receptor, BMPR1B) crucial for ovulation and as well as for increasing litter size. Mutations in any of these genes increased prolificacy in carrier. The present investigation was carried out for identification of fecundity genes in cattle, with comparison of DNA fragment between dams bearing twin and singleton and to see inheritance pattern of these fragments in their twin progenies. The animals belonged to six breeds viz. pure (Sahiwal and Hariana) and three crossbreeds (Karan Swiss, Karan Fires, Holstein Friesian) besides other cross breeds of unknown lineage selected from field. The study involved analysis of genome related to twinning using molecular markers (PCR) on BMP-15 (FecXR) gene and A competitive technique called tetra-primer amplification refractory mutation system-PCR was adapted to type G1 locus mutation GDF-9 fecundity gene. PCR analysis of the FecXR allele of the BMP-15 gene detected the presence of wild-type allele in the dams and their progenies. An allele specific primer used for detection of GDF-9 gene mutation, identified only wild-type and control fragment which revealed the absence of the mutant fragment for this gene locus. The study revealed monomorphic pattern of the two fecundity genes BMP-15 and GDF-9 genes, which has no effect on twinning in cattle.

Premarital Screening Program for Congenital Factor XIII Deficiency in Iran.

Factor XIII deficiency is one of the most severe congenital bleeding disorders with high rate of life-threatening bleeding including central nervous system bleeding, umbilical cord bleeding, and recurrent miscarriages. Due to the highest global incidence of the disorder in Iran, this study aimed to design a premarital screening program in Iran. This descriptive study was performed on 30 couples with a positive family history of factor XIII deficiency. Underling F13A gene mutation was determined in the family members, and all the selected couples underwent molecular testing mutations by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), tetra primer-amplification refractory mutation system-PCR (T-ARMS-PCR), and sequencing. The probability of affected childbirth existed for ten couples. Three couples decided not to marry, while seven got married and three of them decided to have a baby. The genotypes of the fetuses were determined and revealed that none of them was a homozygote for the F13A gene mutation. Because of the importance of factor XIII deficiency diagnosis, it can be helpful to control the incidence of factor XIII deficiency by implementing preventive programs such as premarital screening.

Polymorphisms within the Boule Gene Detected by Tetra-Primer Amplification Refractory Mutation System PCR (T-ARMS-PCR) are Significantly Associated with Goat Litter Size.

Simple SummaryMutations of the Boule gene, a gene that contributes to spermatogenesis, are a main cause of mammal infertility in reproduction. As a conserved gene, only some single nucleotide polymorphisms (SNPs) in the introns within it have been reported in humans with meiosis block, but not in goats, and especially its fundamental roles in female reproduction are still unknown. Therefore, this study detected the potential polymorphisms of the Boule gene in goats via the tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR). Herein, one predicted SNP locus (rs661484476: g.7254T>C) of the Boule gene was first detected and displayed moderate polymorphism among all the studied groups. Notably, the polymorphisms of the goat Boule gene were significantly associated with the goat litter size in different groups, and female goats with the heterozygous genotype (CT) had more possibilities to produce multiple lambs than others, indicating that the Boule gene might underlie female reproductive capacities and that g.7254T>C could be a potential marker in the marker-assisted selection process for litter size in goats.As a gene contributing to spermatogenesis, the Boule gene (also called Boll), whose mutations result in azoospermia and sterility of flies and mice, was conserved in reductional maturation divisions. However, in goats, the polymorphisms of Boule, especially with regard to their fundamental roles in female reproduction traits, are still unknown. Therefore, the aims of this study were to detect a potential mutation (rs661484476: g.7254T>C) located in intron 2 of the Boule gene by tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) and to explore its potential association with the litter size of Shaanbei White-Cashmere goats (SBWGs). In this study, g.7254T>C was firstly detected. The TT genotype was the dominant genotype in the single-lamb group, and T was also the dominant allele in all tested groups. Additionally, the detected locus displayed moderate polymorphism with polymorphism information content (PIC) values among all studied goats ranging from 0.303 to 0.344. Notably, according to the χ2 test, the distribution differences for the genotypic frequencies between the single- and multi-lamb groups was significant (p = 0.014). Furthermore, the polymorphisms of the goat Boule gene were significantly associated with the goat litter size in SBWGs (p < 0.05), which indicated that g.7254T>C could be a potential marker in the marker-assisted selection process for potential litter size in goats. These results also indicated that the Boule gene might exercise important functions in female goat reproduction, which provided new insight for female goat breeding and could accelerate the process of goat breeding.

Effect of variation in miRNA-binding site (rs8176318) of the BRCA1 gene in breast cancer patients

Background/aimA variation in the 3 prime untranslated regions (3′-UTRs) affects the binding of microRNA (miRNA) to the breast cancer (BC) susceptibility gene 1 (BRCA1) gene, and thus regulate its expression. In this study, the consequences of a variation in the miRNA-binding site (rs8176318G>T) in the 3′-UTRs of BRCA1 and its association with the risk of BC were investigated.Materials and methodsThe selected variation (rs8176318G>T) was genotyped in BC patients (n = 300) and healthy controls (n = 300) using allele-specific polymerase chain reaction (PCR) [tetra-primer amplification refractory mutation system-PCR (T-ARMS-PCR)]. The results of the T-ARMS-PCR were further confirmed by Sanger sequencing through a random selection of 10% previously analyzed samples by T-ARMS-PCR. Association of this variation with BC was tested by calculating the odds ratio (OR) (at 95% CI) and χ2-value using 4 different genetic models (codominant, dominant, recessive, and additive models).ResultsUsing Fisher’s exact test, a significant association between variant rs8176318 (G>T) and BC was found in codominant [χ2-value = 15.68, df: 2 P < 0.0004], dominant [OR = 1.557 (1.082–2.241), P <0.0213], recessive [OR = 0.474 (0.3204–0.7017), P = 0.0002] and additive models [OR = 1.609 (1.282–2.018), P < 0.0001].ConclusionIt was therefore concluded that there is a significant association between rs8176318 and BC risk in a case-control study in a Pakistani population. Furthermore, an association study using a large sample size is required to further verify these findings.

Analysis of association between RAGE polymorphisms and stroke risk

Receptor for advanced glycation end products (RAGE) is a kind of immunoglobulins that interacts with several ligands and participates in regulation of immune responses. Variants within RAGE gene have been associated with risk of stroke in different populations. In the present study, we have assessed associations between two single nucleotide polymorphisms (SNPs) in this gene and risk of ischemic stroke in Iranian persons by using tetra primer-amplification refractory mutation system-PCR approach. Totally, 184 patients with history of ischemic stroke and 192 age- and sex-matched controls were recruited. After correction for multiple comparisons, there were no significant difference in allele frequencies of rs184003 and rs1800625 between cases and controls (P values of 0.38 and 0.06 respectively). A significant association was found between rs1800625 genotypes and risk of stroke in recessive and additive models (OR (95% CI) = 0.2 (0.04–0.93), adjusted P value = .04; OR (95% CI) = 1.52 (1.04–2.21), P value = .03 respectively) which implies that CC genotype has a protective role against stroke in the assessed population when compared with TT + CT genotypes. The estimated haplotypes distributed similarly between cases and controls. Our data propose a role for RAGE variants in modulating the risk of stroke in Iranian population. However, our data need to be appraised in larger cohorts of patients.

Detection of Bovine TMEM95 p.Cys161X Mutation in 13 Chinese Indigenous Cattle Breeds.

Simple SummaryChinese indigenous cattle are economically important cattle breeds for animal husbandry development. The promotion and development of Chinese cattle breeds is essential. A previous study found that a nonsense mutation (rs378652941, c.483C > A, p.Cys161X) in bovine transmembrane protein 95 gene (TMEM95) seriously reduced reproductive performance in male Fleckvieh cattle; therefore, this locus was considered a candidate genetic marker in bovine marker-assisted selection (MAS) breeding. Until now, no study has identified this mutation in Chinese cattle breeds. Herein, we detected this c.483C > A mutation in 13 Chinese cattle breeds. Importantly, we found that this mutation did not exist at this locus in our analyzed breeds. Interestingly, we first identified a frameshift insertion/deletion (indel) mutation (NC_037346.1: g.27056998_27057000delCT) in the bovine TMEM95 gene in 11 cattle breeds. Together, the results of this study suggest that the mutation c.483C > A cannot serve as a genetic marker for molecular breeding among Chinese indigenous cattle breeds.Chinese indigenous cattle breeds have abundant genetic resources, which are valuable for the molecular breeding of cattle around the world. Thus, identifying important candidate genes and their genetic markers is very important for cattle molecular breeding. A previous study found that a nonsense mutation (rs378652941, c.483C > A, p.Cys161X) in the bovine transmembrane protein 95 gene (TMEM95) seriously reduced the reproductive performance in bulls, but few studies have detected this mutation in Chinese indigenous cattle breeds. Since the mutation c.483C > A may serve as a potential genetic marker for selecting higher fertility bulls, in the present study, using tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR), forced PCR restriction fragment length polymorphism (forced PCR-RFLP), and DNA sequencing methods, the mutation c.483C > A was detected in 765 individuals from 13 Chinese cattle breeds. However, the results showed that this mutation did not exist at this locus in our analyzed breeds. Interestingly, we identified a newly frameshift insertion/deletion (indel) mutation (NC_037346.1: g.27056998_27057000delCT) in the bovine TMEM95 gene in 11 cattle breeds, which changed the location of the termination codon and changed the 16 amino acids in the C-terminal to 21 amino acids. Combined with previous studies, our study provides evidence that in Chinese cattle breeds the mutation c.483C > A cannot be used as a genetic marker in molecular breeding.

Association of Fc gamma-receptor genes polymorphisms with chronic periodontitis and Peri-Implantitis.

This study was conducted on 87 patients with chronic periodontitis (CP), 50 patients with peri-implantitis and 90 periodontally healthy individuals referring to the Department of Periodontics for evaluating the association between Fc gamma-receptor genes polymorphisms with CP and peri-implantitis. After obtaining consent, venous blood samples (5cc) were obtained from patients and DNA was extracted using Miller's salting-out method. Polymerase chain reaction (PCR)-restriction fragment length polymorphism and tetra-primer amplification refractory mutation system-PCR methods were used to assess the polymorphisms of FcγRs IIa, IIIa, and IIIb genes. Analyzing showed a significant association between specific genotypes with increasing CP and peri-implantitis risks in codominant and dominant models. For FcγR IIIa, analyzing revealed a significant association between specific genotypes with increasing CP and peri-implantitis risks in codominant, dominant, and recessive models. For FcγR IIIb, we also detected a significant association between specific genotypes with increasing CP and peri-implantitis risks in codominant, dominant, and recessive models ( P < 0.05). According to the results of this study, the FCGRIIa (rs1801274), FCGRIIIa (rs396991), and FCGRIIIb (rs1050501) polymorphisms were significantly associated with CP and peri-implantitis and may have a role in the pathogenesis of these diseases.