Refractory Mutation System-polymerase Chain Reaction Research Articles

Effect of IL-23 Receptor Gene Polymorphism (Rs1884444) on the Prevalence of Oral Fungal Infection in Patients with Type 2 Diabetes Mellitus: A Case-Control Study in Iraqi Patients.

Type 2 diabetes mellitus (T2DM) is a complex metabolic disorder linked to several genetic disorders. Over the last decade, advancements in genetic association studies have resulted in the identification of at least 75 distinct genetic loci associated with T2DM, allowing for a better understanding of the genetic architecture of this disease. Recently, there has been a positive association between the prevalence of oral fungal infection and T2DM. The current study aimed to assess the effect of single nucleotide polymorphism in IL23R (rs1884444) on oral fungal infection and the distribution of alleles in T2DM patients compared to healthy controls. A total of 150 specimens, including oral swabs and whole blood samples, were collected from the Endocrinology and Diabetes Center in Baghdad. Oral swabs were collected via AIMS transport media. Routine tests and the Vitek 2 system carried out fungal identification; moreover, the tetra-primeramplification refractory mutation system-polymerase chain reaction (ARMS-PCR) was used for molecular detection. The findings revealed that the O blood group was positively associated with T2DM and oral fungal infection. Moreover, the TT genotype for IL23R SNP (rs1884444G/T) increased significantly in patients, as compared to that in healthy control. Furthermore, the T allele was increased in patients suffering from T2DM (P<0.001). The GT and TT were more frequent in oral fungal infection in patients with T2DM. The TT and T alleles were positively associated with the risk of developing T2DM. Moreover, GT and TT were associated with oral fungal infection and T2DM.

Comparison of Nonsequencing Techniques for Identification of NPM1 Mutations and Associated Blast Morphology in Patients With Acute Myeloid Leukemia.

Nucleophosmin 1 (NPM1) mutations affect 20% to 30% of all acute myeloid leukemia (AML) patients; several methods are employed to analyze NPM1 mutations, each of them with its advantages and limitations. To compare 3 nonsequencing protocols capable of detecting the main NPM1 mutations and to evaluate nuclear morphometric analysis (NMA) as an alternative to cuplike blast detection. We selected multiparameter flow cytometry (MFC), amplification refractory mutation system-polymerase chain reaction (ARMS-PCR), and a quantitative PCR (qPCR) kit to identify NPM1 mutations in AML patients at diagnosis. We also evaluated the presence of cuplike blasts and assessed nuclear morphometry using NMA. MFC appears as a screening method for NPM1 mutations because of its lower specificity. ARMS-PCR demonstrated specificity similar to that of the qPCR kit, although it was more laborious. qPCR testing, conversely, is relatively fast and easy to standardize. Of these methods, qPCR was the only one capable of identifying the type of NPM1 mutation. With regard to morphology, NMA could be used as an alternative for the evaluation of cuplike blasts in AML smears. qPCR appears to be the best option to identify NPM1 mutations, with ARMS-PCR representing a cheaper alternative. MFC may be used as a screening method, in which results falling within and above the gray zone should be confirmed by molecular testing.

P071 Isolate profiling and antifungal susceptibility determination for terbinafine and itraconazole among dermatophytes in a tertiary care hospital of western rajasthan

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM ObjectiveTo determine the species distribution of causative agents causing dermatophytosis, and their antifungal susceptibility pattern for terbinafine and itraconazole in trichophyton spp among samples collected in patients with dermatophytosis clinical suspicion during the period of December 1, 2020 to January 31, 2022.Materials and MethodsThis is a prospective study conducted in the Department of Microbiology of a tertiary care super specialty and referral Centre of western Rajasthan from December 1, 2020 to January 31, 2022.Skin scraping, nail clipping, and hair pluckings were collected in mycology lab from clinically suspected cases of dermatophytosis presenting to the department of dermatology for conventional identification, and antifungal susceptibility testing.The specimens were subjected to direct KOH and calcofluor white microscopy and conventional fungal culture on SDA at 25 ͦ C and 37 ͦ C.The cultures positive for dermatophytes were speciated by microslide culture lactophenol cotton blue mount, hair perforation test, and urease test.The isolates identified as Trichophyton spp were taken up for antifungal susceptibility testing against terbinafine and itraconazole by microbroth dilution according to CLSI-M38 A2 guidelines. Further terbinafine resistance gene evaluation for detection of C1191A and T1189C single nucleotide polymorphism in Squalene epoxide by Amplified Refractory Mutation System-Polymerase chain Reaction (ARMS-PCR) is undergoing for trichophyton spp.ResultsOver the 14-month study period, the laboratory processed total of 174 specimens: 134 skin scraping, 36 nail clipping, and 4 hair pluckings. Of them, 106 (61.62%) specimens were microscopy positive and 111 (63.79%) were culture positive. Out of the 111 culture-positive agents isolated, 94 (84.68%) were found to be dermatophytes. On isolate profiling of 94 dermatophytes T. mentagrophyte was found to be most common 45 (48%) followed by T. rubrum 27 (29%), T. tonsurans 20 (21%), T. verrucosum 1 (1.1%), and Microsporum spp 1(1.1%). Antifungal susceptibility of 93 Trichophyton spp against terbinafine showed resistance among 58.06% isolates with 83.33% isolates among terbinafine resistant cases showing ≥4 μg/ml minimum inhibitory concentration. There was no resistance detected for itraconazole with microbroth dilution.ConclusionA total of (54.02%) skin, hair, nail infections were found to be caused by dermatophytes.On isolate profiling, T. mentagrophyte, T. rubrum, and T. tonsurans were found to be predominant species among our isolates showing altered trend of local isolates from T. tonsurans being second most common spp isolated in past.On antifungal susceptibility >55% isolates showed resistance for Terbinafine with >80% having higher MIC of ≥4 μg/ml on the contrary there was no observed resistance for itraconazole.There is a need for encouraging dermatologists for prescribing routine fungal microscopy, culture, and AFST for dermatophytes in Western Rajasthan, to reduce the indiscriminate use of antifungals.

Detection of Benzimidazole Resistance-Associated Single-Nucleotide Polymorphisms in the Beta-Tubulin Gene in Trichuris trichiura from Brazilian Populations.

Preventive chemotherapy is recommended by the WHO as the main strategy for controlling infections caused by nematodes in humans, aiming to eliminate the morbidity associated with these infections. This strategy consists of routine periodic administration of benzimidazoles, among other drugs. Although these drugs decrease the intensity of infections, they have the potential to exert selection pressure for genotypes bearing mutations associated with drug resistance, which may result in the establishment of resistant worm populations. There is evidence in the literature of resistance to these drugs in nematodes that infect humans, including in the species Trichuris trichiura. Single-nucleotide polymorphisms (SNPs) in the beta-tubulin gene located at codons 167, 198, and 200 are associated with the mechanism of resistance to benzimidazoles in nematodes. Here, we standardized a molecular technique based on an amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) to analyze codons 167, 198, and 200 of T. trichiura. The ARMS-PCR methodology was successfully established to evaluate the codons of interest. A total of 420 samples of individual eggs were analyzed from populations obtained from five Brazilian states. A mutation in codon 198 was observed at a frequency of 4.8% (20/420), while for the other two codons, no polymorphism was observed. This is the first report of the presence of this mutation in populations of T. trichiura in Brazil. This fact and the emergence of the problem already observed in other species reinforces the need for regular monitoring of SNPs related to benzimidazole resistance using techniques that are highly sensitive and specific.

The impact of deiodinase type II gene on the therapeutic response to levothyroxine in a sample of Iraqi hypothyroidism patients

BackgroundThere is a clinical observation in the local population of hypothyroid patients that many patients still have symptoms and disease-complaints even after they treated with levothyroxine as a replacement therapy. Deiodinase type II enzyme play a central role in the conversion of thyroxin to the active form triiodothyronine. This study searches the effect of rs225014; 274 T>C single nucleotide polymorphism (SNP) of deiodinase type II (DIO2) gene on the clinical response to levothyroxine replacement therapy in Iraqi hypothyroidism patients. MethodologyIn this cross –sectional study, one hundred fifty Iraqi female patients with primary hypothyroidism of the age of 40 and above, who were treated with levothyroxine were recruited. Thyroid hormones were assessed and the genetic analysis to detect rs225014 SNP was done using the tetra primers amplification refractory mutation system-polymerase chain reaction technique. ResultsThe genotypes distribution of rs225014; 274 T>C SNP was 22(14.66 %), 19 (12.66 %) and 72(72.66 %) as TT, TC and CC, respectively. Total T4 was significantly higher in TC carriers than in CC carriers, while there were no significant differences in the levels of TSH, total T3, free T3 and free T4 in TT, TC and CC groups of patients. The TC group also had significantly higher fasting serum insulin and homeostatic model assessment for insulin resistance (HOMA-IR) than the carriers of CC genotype. ConclusionsSince the rs225014 SNP of DIO2 gene is not associated with the most of the thyroid hormones levels, it could not affect the response to levothyroxine treatment in our sample of Iraqi hypothyroidism patients. Although this SNP cannot be excluded from being related to the hypothyroidism because the homozygous mutant type (CC) of it is the most frequent genotype in our sample of Iraqi hypothyroidism patients. Since TC genotype is associated with elevated levels of fasting serum insulin and HOMA-IR, it could have an impact in developing insulin resistance and type 2 diabetes mellitus.

MET overexpression in EGFR L858R mutant treatment-naïve advanced lung adenocarcinoma correlated with poor prognosis: a real-world retrospective study.

Mesenchymal epithelial transition (MET) overexpression has been reported in approximately 50-60% of epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancers. However, the prognostic significance of MET overexpression has not been established in advanced lung adenocarcinoma (ADC) patients with EGFR-sensitive mutations. A retrospective study was performed on a total of 406 treatment-naïve advanced ADC patients with EGFR mutation detection and MET expression information. EGFR mutations were detected by next-generation sequencing or amplification refractory mutation system-polymerase chain reaction. Immuno-histochemistry staining of MET expression was evaluated by H-score and overexpression was defined as an H-score ≥ 200. Overall survival (OS) and progression-free survival (PFS) were analyzed according to MET expression. Among the 406 patients, 208 patients had EGFR mutations, including 102 exon 19_del mutations, 94 L858R mutations and 12 other types of mutations. Of 110 patients with concomitant EGFR mutations and MET overexpression, 61 (59.8%) patients had 19_del mutations, 44 (46.8%) patients had L858R mutations and five (41.7%) patients had others. Patients with MET overexpression had a markedly shorter PFS and OS than patients with MET H-score < 200 in the EGFR L858R mutation subgroup (median PFS: 12 versus 26months, p = 0.001; median OS: 24 versus 32months, p = 0.038), whereas no significant difference was observed in 19_del mutation subgroup. Multivariate Cox analysis showed that MET overexpression was an independent poor prognostic factor for PFS and OS in patients with the L858R mutations (HR = 3.064, 95% CI 1.705-5.507, p < 0.001; HR = 2.043, 95% CI 1.000-4.172; p = 0.049), rather than 19_del. MET overexpression is a poor prognostic factor for advanced ADC patients with the EGFR L858R mutation.

Association of rs3212220 G/T single nucleotide polymorphism of IL-12B with tuberculosis in the population of Punjab, India

Many single nucleotide polymorphisms (SNPs) in the cytokine genes are reported to be associated with altered host susceptibility to tuberculosis (TB). Interleukin (IL)-12 has crucial role in immunity against Mycobacterium tuberculosis (MTB), the causative organism of TB. IL-12 is a key cytokine required for the release of IFN-γ by NK and Th1 cells, which is regarded as pivotal cytokine against TB. IL-12 has two subunits IL-p35 and IL-12p40 encoded by IL-12A and IL-12B gene respectively. Present study was designed for evaluating the association of rs3212220 G/T SNP of IL-12B and to check plasma levels of IL-12p40 in TB patients from population of Punjab, India. This case-control study was conducted on 300 TB patients and 150 normal healthy controls (NHCs). Genotyping of selected SNP of IL-12B was done by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) method. Plasma levels of IL-12p40 were measured by enzyme linked immunosorbent assay (ELISA). Statistical significance level was calculated at 0.05. No statistically significant difference was found between genotypic and allelic frequencies of rs3212220 G/T SNP of IL-12B among TB patients and NHCs. IL-12p40 levels were found to be significantly elevated in TB patients when compared with NHCs (p < 0.0001). These levels declined with the course of anti-tuberculosis therapy (ATT). GG and GT genotypes were found to be associated with increased level of IL-12p40 in TB patients when compared to NHCs.

Cut-off values for diagnosis of G6PD deficiency by flow cytometry in Thai population

In heterozygous females, X-inactivation causes a change in glucose-6-phosphate dehydrogenase (G6PD) activity from normal to deficient. Most G6PD screening tests are used to accurately diagnose hemizygous males, but they are less reliable for diagnosing heterozygous females. This study established flow cytometric cut-off values for screening of G6PD deficiency in hemizygous males and heterozygous or homozygous females. We studied 205 (125 females, 80 males) leftover blood samples from quantitative methemoglobin reduction (MR) screening. G6PD gene mutations determined by multiplex amplification refractory mutation system-polymerase chain reaction and direct DNA sequencing were used as the gold standard reference. Accuracy of the test, including the sensitivity, specificity, and positive and negative predictive values, was analyzed using MedCalc software. The optimal cut-off values for classification of %red blood cells with normal G6PD activity or %bright cells into homozygous normal, heterozygous, and homozygous deficiency in females were 85.4–100%, 6.3–85.3%, and 0–6.2%, respectively (sensitivity 93.2%, specificity 100%). The cut-offs for classification into hemizygous normal and hemizygous deficiency in males were 76.5–100% and 0–76.4%, respectively (sensitivity 100%, specificity 96.5%). Flow cytometry can be used to differentiate heterozygous females with intermediate phenotype from homozygous females, but cannot distinguish between heterozygous females with extreme phenotype and homozygous females. By flow cytometry, heterozygous and homozygous deficiency was detected in 29.6% and 3.2% of females, respectively. Among males, hemizygous deficiency was found in 31.3%. Flow cytometry can be used to screen patients with G6PD deficiency, and reliably and efficiently identify heterozygous and homozygous females, and hemizygous males based on cellular G6PD activity.

Genomics Analysis and Nomogram Risk Predictionof Occult Lymph Node Metastasis in Non-Predominant Micropapillary Component of Lung Adenocarcinoma Measuring ≤ 3 cm.

BackgroundThe efficacy of sublobar resection and selective lymph node dissection is gradually being accepted by thoracic surgeons for patients within early-stage non-small cell lung cancer (NSCLC). Nevertheless, there are still some NSCLC patients develop lymphatic metastasis at clinical T1 stage. Lung adenocarcinoma with a micropapillary (MP) component poses a higher risk of lymph node metastasis and recurrence even when the MP component is not predominant. Our study aimed to explore the genetic features and occult lymph node metastasis (OLNM) risk factors in patients with a non-predominant micropapillary component (NP-MPC) in a large of patient’s cohort with surgically resected lung adenocarcinoma.MethodsBetween January 2019 and December 2021, 6418 patients who underwent complete resection for primary lung adenocarcinoma at the Qilu Hospital of Shandong University. In our study, 442 patients diagnosed with lung adenocarcinoma with NP-MPC with a tumor size ≤3 cm were included. Genetic alterations were analyzed using amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). Abnormal protein expression of gene mutations was validated using immunohistochemistry. A nomogram risk model based on clinicopathological parameters was developed to predict OLNM. This model was invalidated using the calibration plot and concordance index.ResultsIn our retrospective cohort, the incidence rate of the micropapillary component was 11.17%, and OLNM was observed in 20.13% of the patients in our study. ARMS-PCR suggested that EGFR exon 19 del was the most frequent alteration in NP-MCP patients compared with other gene mutations (frequency: 21.2%, P<0.001). Patients harboring exon 19 del showed significantly higher risk of OLNM (P< 0.001). A nomogram was developed based on five risk parameters, which showed good calibration and reliable discrimination ability (C-index = 0.84) for evaluating OLNM risk.Conclusions.Intense expression of EGFR exon 19 del characterizes lung adenocarcinoma in patients with NP-MCP and it’s a potential risk factor for OLNM. We firstly established a nomogram based on age, CYFRA21-1 level, tumor size, micropapillary and solid composition, that was effective in predicting OLNM among NP-MCP of lung adenocarcinoma measuring ≤ 3 cm.

Impact of interferon-induced transmembrane protein 3 gene rs12252 polymorphism on COVID-19 mortality

Background and aimsInterferon-induced transmembrane protein 3 (IFITM3) plays a critical role in the adaptive and innate immune response by preventing membrane hemifusion between the host and viral cell cytoplasm. This study aimed to evaluate whether IFITM3 rs12252 polymorphism is related to an increased mortality rate of coronavirus disease 2019 (COVID-19). MethodsThe IFITM3 rs12252 polymorphism was genotyped using the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) in 548 dead and 630 improved patients positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ResultsIn the present study, the minor allele frequency of IFITM3 rs12252 (C) was significantly more frequent in dead patients than in improved cases. The results of the multivariate logistic regression analysis indicated that the lower lipid profiles, PCR Ct value, 25-hydroxyvitamin D, and uric acid and higher levels of erythrocyte sedimentation rate (ESR), liver enzymes, and creatinine, and IFITM3 rs12252 CC genotypes were related to the COVID-19 infection mortality. ConclusionsIn summary, our findings suggested a possible link between the mortality of COVID-19 infection, the CC genotypes of IFITM3 rs12252, and clinical parameters. Further investigations are required worldwide to prove the link relationship of COVID-19 mortality with host genetic factors.

The risk of relapse in breast cancer patients is associated with MMP-9 gene polymorphism: a prospective study in a sample of the Iranian population

We investigated the association of MMP-9 rs1056628 and rs17576 polymorphisms with breast cancer (BC) relapse in a cohort of prospectively observed BC patients. The polymorphisms were genotyped in 200 BC subjects with(case) and without(control) relapse by Tetra-primer Amplification Refractory Mutation System-Polymerase Chain Reaction (T-ARMS-PCR) method. A significant association was found between the rs1056628C allele and increased risk of BC relapse (OR = 1.8, P = 0.006). Also, rs17576 allele and genotypes were shown protective effects against BC relapse. Increased risk of relapse was observed in haplotype CG (OR = 2.1, P = 0.001). Our results suggest for the first time that rs1056628 and rs17576 polymorphisms may influence BC relapse.

The STAT4 SNP (rs7574865) and systemic lupus erythematosus

Introduction: Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease affecting several systems and organs in the body. The association of STAT4 transcription factor with SLE risk remains unclear. Objectives: The aim of this study was to investigate the association of STAT4 gene polymorphism (rs7574865) with the incidence of SLE. Patients and Methods: One hundred and sixty participants (80 patients with SLE and 80 healthy individuals) were included in this study. Gene analysis was conducted by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) in peripheral blood samples. Results: Fifty-seven percent (n=45) of patients with SLE had SLE disease activity index (SLEDAI) above six and had active disease. In the SLE group, the frequency of G and T alleles were 81% and 19%, respectively. Moreover, 72.50% (n=58) of patients carried the GG genotype, 17.5% (n=14) had the GT genotype and 10.1% (n=8) carried the TT genotype. There was no significant difference between allele frequency and genotypic distribution for rs7574865 polymorphism (P&gt;0.05) between SLE and control groups. Significant differences were observed between the distribution of genotypes and clinical manifestations including leukopenia (P=0.04), pulmonary (P=0.01) and ophthalmic (P=0.049) problems. The T allele with an odd ratio of 1.47 and confidence interval of 0.80 to 2.6 could increase the risk of SLE; however, it was not statistically significant (P=0.20). Conclusion: The T allele and TT genotype of the STAT4 rs7574865 polymorphism could increase the risk of lupus; however, these observations were not statistically significant.

IL-17 gene polymorphism (rs763780) in kidney recipients with post-transplant diabetes

Introduction: New-onset diabetes mellitus after transplantation (NODAT) is a common complication of organ transplantation, leading to allograft dysfunction. Genetic alterations of inflammatory cytokines have been reported to be associated with glucose homeostasis and diabetes. Objectives: This study evaluated the rs763780 polymorphism of IL-17F gene in transplant recipients with and without NODAT. Patients and Methods: The present retrospective study was conducted on ninety-one patients who have had a kidney transplant for at least three months. Patients were divided into two subgroups; recipients with NODAT (n=32) and kidney recipients without NODAT (n=59). After DNA extraction from patients’ blood samples, amplification and evaluation of specific polymorphism of the gene were performed using amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). Clinical and demographic data of patients were collected. Results: The NODAT was detected in 81.3% (n=26) of TT genotype carriers, 12.5% of TC genotype carriers and 6.3% of CC genotype carriers. No statistically significant differences between the studied groups in the frequency of C and T alleles and the distribution of the abovementioned genotypes were detected (P≥0.721). In the NODAT group, graft rejection and age of patients were higher significantly (P≤0.017). Conclusion: No significant correlation between the incidence of diabetes and rs763780 polymorphism of IL-17F gene was observed.

A study of association between presence or absence of GSTT1 and GSTM1 and/or single nucleotide polymorphism in FABP2 and GSTP1 with incidence of diabetes type 2: A case-control study.

To assess the association of single nucleotide polymorphisms in fatty acid binding protein-2 (rs1799883) and glutathione S-transferase pi (rs1695) genes with presence/absence of glutathione S-transferase mu and glutathione S-transferase theta genes in type 2 diabetes. The cross-sectional case-control study was conducted at Institute of Molecular Biology and Biotechnology during March till September 2019 and comprised type 2 diabetes patients and non-diabetic controls from two districts in southern Punjab. Polymerase chain reaction, polymerase chain reaction-restriction fragment length polymorphism and tetra-primer amplification refractory mutation system-polymerase chain reaction were applied to investigate glutathione S-transferase theta, mu and pi genes as well as fatty acid binding protein-2, as appropriate. The association of single nucleotide polymorphisms in all genes with the disease were studied either individually or in various combinations. Data was analysed using Minitab 18. Of the 448 subjects, 248(55.4%) were patients and 200(44.6%) were controls. Overall there were 213(47.5%) males and 235(52.5) were females, and 141(31.5%) were aged 30-46 years. The presence of rs1799883 in fatty acid binding protein-2 (p=0.03) and the absence of glutathione S-transferase mu gene (p<0.001) had significant association with type 2 diabetes, while the presence of glutathione S-transferase theta and rs1695 in glutathione S-transferase pi genes were not associated with the disease. Individuals with glutathione S-transferase mu gene null and Ileu/Ileu or Ileu/Val genotype of rs1695 in glutathione S-transferase pi gene have potential to develop type 2 diabetes in their lifetime (p<0.05). The presence of rs1799883 in fatty acid binding protein-2 and the absence of glutathione S-transferase mu gene were found to play significantly in the development of type 2 diabetes.

Association of CATSPER1, SPATA16 and TEX11 genes polymorphism with idiopathic azoospermia and oligospermia risk in Iranian population

BackgroundMale infertility is a heterogeneous disease which can occur due to spermatogenesis defects. The idiopathic azoospermia and oligospermia are the common cause of male infertility with unknown underlying molecular mechanisms. The aim of this study was to investigate association of idiopathic azoospermia and oligospermia with single-nucleotide polymorphisms of CATSPER1, SPATA16 and TEX11 genes in Iranian-Azeri men.MethodsIn this case–control study, we recruited 100 infertile men (case group) and 100 fertile men (control group) from Azeri population in north western provinces, Iran, population. The genomic DNA was extracted using a proteinase K method from peripheral blood leukocytes. The genotypes analysis was conducted using tetra-primer amplification refractory mutation system-polymerase chain reaction method. The obtained data were analyzed by statistical software.ResultsWe found a significant difference in the frequencies of heterozygote AB and mutant homozygote BB genotypes in the CATSPER1 (rs2845570) gene polymorphism between patients and healthy controls (p < 0.05). Moreover, we observed a significant difference in the frequencies of heterozygote BA genotype in the SPATA16 (rs1515442) gene polymorphism between patients and healthy controls (p < 0.05). However, no significant difference was found in genotypes distribution of case and control groups in the TEX11 (rs143246552) gene polymorphism.ConclusionOur finding showed that the CATSPER1 (rs2845570) and SPATA16 (rs1515442) genes polymorphism may play an important role in idiopathic azoospermia and oligospermia in Iranian Azeri population. However, more extensive studies with larger sample sizes from different ethnic origins are essential for access more accurate results.

Effect of deletions in the α-globin gene on the phenotype severity of β-thalassemia

Thalassemia is the most common inherited hemoglobinopathy worldwide. Variation of clinical symptoms in this hemoglobinopathy entails differences in disease-onset and transfusion requirements. The aim of this study was to investigate the role of α-globin gene deletions in modulating the clinical heterogeneity of β-thalassemia (β-thal) syndromes. A total number 270 β-thal subjects were enrolled. Hematological parameters were recorded. β-Globin mutations were determined by amplified refractory mutation system-polymerase chain reaction (ARMS-PCR), gap-PCR and Sanger sequencing. α-Globin gene deletions were determined by multiplex PCR. Out of 270 β-thal subjects, 19 carried β+/β+, 74 had β0/β0 and 177 had the β0/β+ genotype. When we determined the severity of the different β-thal subjects in coinherited with the α gene deletion, it was revealed that, 84.2% β+/β+ subjects carried a non severe phenotype and did not have an α gene deletion. Of the β0/β0 individuals, 95.9% presented a severe phenotype, irrespective of α-globin gene deletions. In cases with the β0/β+ genotype, 19.2% subjects also carried a deletion on the α gene. Of these, 61.8% presented a non severe phenotype and 38.2% were severely affected. Only in the β0/β+ category did α gene deletions make a significant contribution (p < 0.001) toward alleviation of clinical severity. Therefore, it can be stated that α-globin gene deletions play a role in ameliorating the phenotype in patients with a β+/β0 genotype.

Association of single nucleotide polymorphisms of interferon-γ with pulmonary tuberculosis in population of Himachal Pradesh, India

Interferon-gamma (IFN-γ) plays an integral role in the host immunity against tuberculosis (TB). The gene encoding IFN-γ is polymorphic and several studies have reported the association of its genetic polymorphisms with TB in different populations of the world. The present study investigated the association of rs2069705 (C/T), rs1861494 (C/T), rs1861493 (A/G) and rs2069718 (C/T) single nucleotide polymorphisms (SNPs) of IFN-γ with pulmonary TB in a population of Himachal Pradesh, India. For present study, 210 pulmonary TB patients and 205 healthy controls (HCs) were recruited. The selected SNPs of IFN-γ were genotyped by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and plasma IFN-γ levels were measured by ELISA. The ‘T’ allele of rs1861494 SNP was found to increase susceptibility to TB in the studied population (OR = 2.18, 95%CI = 1.57–3.03; p < 0.001). After stratifying the subjects on basis of sex, males with ‘T’ allele of rs2069718 SNP were found to be at higher risk to TB (OR = 1.55, 95%CI = 1.07–2.25; p = 0.02). We also found moderate linkage disequilibrium among the studied SNPs. The haplotypes C-T-A-T and T-T-G-T of rs2069705-rs1861494-rs1861493-rs2069718 were overrepresented in TB patients and found to increase susceptibility to TB (p = 0.012). The plasma IFN-γ levels in TB patients were around seven times higher in comparison to HCs (p < 0.0001). The HCs with genotype ‘AA’ of SNP rs1861493 were found with higher plasma IFN-γ levels than ‘AG/GG’ genotype (p = 0.023). In conclusion, the results suggest the association of rs1861494 (C/T) and rs2069718 (C/T) SNPs of IFN-γ with TB and genotype ‘AA’ of rs1861493 is associated with higher plasma IFN-γ levels in the population of Himachal Pradesh, India.

Cell HE staining smears and paired cell paraffin sections in detection of epithelial growth factor receptor gene of pleural fluid specimens.

The advanced non-small cell lung cancer (NSCLC) patients with pleural effusion have no opportunity for surgery treatment. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are the first-line drugs for these patients with EGFR-sensitive mutation. However, the disease progression and drug update during or after treatment of EGFR-TKIs bring more challenges and puzzles to clinical diagnosis and treatment, which inevitably requires archived pleural cell samples for EGFR re-examination or comparative study. Understanding the DNA quality of archived pleural fluid samples and effectively using archival data of pleural fluid cells are of great significance for tracing the origin of cases and basic medical research. This study aims to evaluate the consistency of EGFR mutant gene expression between the 2 methods, and to explore a reliable way for preserving cytological data and making full use of cytological archival data via cell HE staining smear and cell paraffin section. A total of 57 pleural fluid cytology cases in the Department of Pathology of China Aerospace Center Hospital from October 2014 to April 2021 were selected. Tumor cells were detected by cell HE staining smears and immunohistochemical staining for TTF-1 and Napsin A in the paired cell paraffin sections. There were more than 200 tumor cells in cell HE staining smear and the proportion of tumor cells were ≥70% in matched cell paraffin sections. Patients with 2 cell smears (one for cell data retention and the other for DNA extraction) were selected as the research subjects, and 57 pleural fluid samples were enrolled. EGFR gene mutation was detected by amplification refractory mutation system-polymerase chain reaction in 57 paired cell HE staining smears and cell paraffin sections. DNA concentration was 2 ng/μL. Cell HE smear was amplified side-by-side with DNA samples from paired cell paraffin sections. Result determination was according to the requirements of the reagent instructions. The external control cycle threshold (Ct) value of the No. 8 well of the samples to be tested was between 13 and 21, which was considered as successful and reliable samples. When the Ct value of EGFR gene mutation was <26, it was considered as positive; when the Ct value was between 26 and 29, it was critical positive; when the Ct value was equal or more than 29, it was negative. ΔCt value was the difference between mutant Ct value and externally controlled Ct value. The smaller the ΔCt value was, the better the quality of DNA of the detected sample was. Among the 57 pleural effusion samples, 42 patients were hospitalized with pleural effusion as the first symptom, accounting for 73.7% (42/57). EGFR mutation was detected in 37 samples [64.9% (37/57)]. The mutation rate for 19del was 37.8% (14/37) while for L858R was 48.6% (18/37). Females were 56.7% (21/37) of mutation cases. The mutation consistency rate of cell HE staining smear and matched cell paraffin sections was 100%. The ΔCt values of cell HE staining smears were less than those of matched cell paraffin sections. The mutation Ct values of 37 cytological samples were statistically analyzed according to the preservation periods of the years of 2014-2015, 2016-2017, 2018-2019, and 2020-2021. There were significant differences in cell paraffin section in the years of 2014-2015 and 2016-2017 compared with the years of 2018-2019 and 2020-2021, while no significant differences were found in cell HE staining smear. Statistical analysis of externally controlled Ct values of 57 cytological samples showed that there were significant differences between cell HE staining smears and cell paraffin section in the years of 2014-2015 and 2016-2017, compared with the years of 2018-2019 and 2020-2021. The mutational Ct values of 37 paired cell blocks and smears were all <26, and the externally controlled Ct values of 57 paired cell paraffin sections and HE staining smears were all between 13 and 21. The DNA quality of cell HE smears and matched cell paraffin section met the qualified requirements. Two methods possess show an excellent consistency in detecting EGFR mutation in NSCLC pleural fluid samples. The DNA quality of cell HE staining smear is better than that of cell paraffin sections, so cell HE staining smear can be used as important supplement of the gene test source. It should be noted that the limitation of cell HE staining smears is non-reproducibility, so multiple smears of pleural fluid are recommended to be prepared for multiple tests.

The Impact of Single Nucleotide Polymorphisms on the Pharmacokinetics of Tacrolimus in Kidney Allograft Recipients of Northern-West, Iran.

Purpose: Calcineurin inhibitors (CNIs) such as tacrolimus are a major immunosuppressive therapy after renal transplantation, which inhibit cytokine expression. The pharmacokinetics of such drugs is influenced by cytochrome P450 (CYP) enzymes, multi-drug resistance-1 (MDR-1), and C25385T pregnane X receptor (PXR). This study aimed to investigate the impact of single nucleotide polymorphisms (SNP) in these genes on the ratio of tacrolimus level per drug dosage (C/D ratio), acute graft rejection, and viral infections. Methods: Kidney transplantation recipients (n=65) under similar immunosuppressive treatment were included. Amplification refractory mutation systempolymerase chain reaction (ARMS-PCR) method was applied to amplify the loci containing the SNPs of interest. Results: Overall, 65 patients with a male/female ratio of 37/28 were included. The mean age was 38±1.75 years. The variant allele frequencies of CYP3A5*3, MDR-1 C3435T, and PXR C25385T were 95.38, 20.77, and 26.92%, respectively. No significant correlations were found between the studied SNPs and the tacrolimus C/D ratios. However, there was a significant difference in the C/D ratios at 2 and 8 weeks in homozygote CYP3A5 *3/*3 carriers (P=0.015). No significant association was found between the studied polymorphisms and viral infections and acute graft rejection (P>0.05). Conclusion: Homozygote CYP3A5 *3/*3 genotype could influence the tacrolimus metabolism rate (C/D ratio).

Detection of Tri-allelic Single Nucleotide Polymorphisms of ABCB1 and CRP Genes by Penta-Primer Amplification Refractory Mutation System-Polymerase Chain Reaction.

Objective: The standard methods for tri-allelic single nucleotide polymorphism (SNP) genotyping require special equipment and are costly to perform. The aim of this study was to establish a fast, simple, and low-cost method to differentiate among tri-allelic SNPs in general laboratories. Methods: Based on the tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) typing of bi-allelic SNPs, we developed a penta-primer amplification refractory mutation system-polymerase chain reaction (P-ARMS-PCR) method characterize tri-allelic SNPs. The two most studied tri-allelic SNPs, rs2032582 and rs3091244, were genotyped using P-ARMS-PCR in 110 volunteers, and the results were verified by direct DNA sequencing. Results: For rs2032582, there were 20 samples (18.18%) with the GG genotype, 3 (2.73%) with the AA genotype, 24 (21.82%) with the TT genotype, 43 (39.09%) with the GT genotype, 11 (10.00%) with the AG genotype, and 9 (8.18%) with the AT genotype. For rs3091244, there were 67 samples (60.91%) with the CC genotype, 1 (0.91%) with the AA genotype, 8 (7.27%) with the CT genotype, 31 (28.18%) with the CA genotype, and 3 (2.73%) with the AT genotype. The genotypic distributions of rs2032582 (p = 0.482) and rs3091244 (p = 0.492) were in Hardy-Weinberg equilibrium. The DNA sequencing results were entirely consistent with the results of P-ARMS-PCR. Conclusion: P-ARMS-PCR is an accurate, rapid, simple, and low-cost characterization method for tri-allelic SNP genotyping of rs2032582 and rs3091244.