Unveiling genetic variants: Tetra-primer ARMS-PCR diagnosis and structural insights into BLAD, BC, and DUMPS in Pakistani cattle herds.

BACKGROUND: Bovine leukocyte adhesion deficiency (BLAD), bovine citrullinemia (BC), and deficiency of Uridine monophosphate synthetase (DUMPS) are the common autosomal recessive disorders affecting the global dairy industry. BLAD leads to poor wound healing and recurrent infections. In BC, ammonia builds up leading to neurological disorders and death. DUMPS results in developmental abnormalities. METHODOLOGY: In this study, tetra-primer amplification refractory mutation system polymerase chain reaction (ARMS PCR) based diagnostic tests were optimized for BLAD, BC, and DUMPS. A total of 250 animals (58 indigenous and 192 Holstein Friesian (HF)) were screened from all across Pakistan. In addition to validation of ARMS-PCR results through Sanger sequencing, the protein modeling provided structural insights of the disease-associated reported SNPs. Pathway analysis illustrated gene functions under normal and mutated conditions. Furthermore, haplotype and phylogenetic analysis of ASS1 (Argininosuccinate synthetase) gene were performed on study samples and NCBI retrieved sequences. RESULTS: The study's focus was to screen the herds for prevalence of carriers of genetic disorders, as they are the main source of disease dissemination. One animal was found carrier for BC, whereas no carriers were found for BLAD and DUMPS. The protein models corroborated the reported amino acid change in BLAD, and protein truncation in both BC and DUMPS proteins. SNPs found in NCBI retrieved sequences were either silent or missense and had no effect on protein structure. DNA network presented graphical illustration of haplotype interactions and phylogenetic analysis conferred evolutionary landscape of ASS1 gene. The combination of these approaches produced an in-depth genetic picture of BC in Pakistani cattle. CONCLUSION: The development of diagnostic tests and identification of the heterozygous BC sample underscores the significance of constant monitoring to avoid the unwanted dissemination of mutant alleles among Pakistani cattle, thereby promoting the general well-being and sustainability of the dairy sector.

Genotype Analysis of the JR Blood Group in an East Asian Population Using Tetra-Primer ARMS-PCR.

OBJECTIVE: The JR blood group system, officially designated ISBT JR 032, consists of a single antigen called Jra. This is a high frequency antigen in most populations. The Jr(a-) phenotype is more prevalent in Japanese and Asian populations. Individuals with the Jr(a-) blood type can be recognized incidentally by the production of anti-Jr(a) antibodies and verified by the existence of two null ABCG2 alleles. METHODS: We used direct sequencing to analyze the genotype frequency of the ABCG2 null allele (c.376C>T, rs72552713) and compared it with East Asian genomic databases. We developed tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR), which is a simple, precise method for determining an individual's genotype and suitable for clinical use, and analyzed a cohort of 300 healthy Koreans. RESULTS: Using direct sequencing, we found that 14 individuals in the cohort carried a heterozygous ABCG2 null allele. We optimized the ARMS-PCR technique to detect and identify this null allele precisely. We identified the presence of this null allele in a heterozygous state using ARMS-PCR. CONCLUSION: The minor allele frequency of the ABCG2 null allele in the Korean cohort was 2.3%. The estimated genotype frequencies of homozygotes and heterozygotes for this null allele are 0.05% and 4.56%, respectively. The newly developed ARMS-PCR assay would be useful for determining the Jr(a-) antigen status in patients who produce anti-Jr(a) antibodies as well as for selecting Jr(a-) blood donors.

Comparing PCR With High-resolution Melting Analysis for Apolipoprotein E Genotyping in Alzheimer's: A Case-control Study.

Introduction: The apolipoprotein E (APOE) genotype has a heterogeneous distribution throughout the world. The present study aimed to characterize the APOE genotype (rs429358, rs7412) in healthy individuals compared with Alzheimer cases in Kerman, southeastern Iran, by two standard mutation scanning methods. Methods: In this case-control study, 90 Alzheimer patients as a case group and 90 healthy individuals as a control group were examined. APOE genotyping was carried out using high-resolution melting (HRM) analysis assay and multiplex tetra-primer amplification-refractory mutation system polymerase chain reaction (T-ARMS PCR) techniques. Results: In contrast to Multiplex T-ARMS PCR, HRM analysis was not efficient in rs7412 genotyping. The results of multiplex T-ARMS showed that ɛ2ɛ3 genotype (P=0.006, odd ratio [OR]=0.119) and ɛ2 allele (P=0.004, OR=0.129) were more prevalent in the control group compared with the case ones, whereas ɛ4 allele was associated with borderline risk of Alzheimer disease (P=0.099, OR=1.76). Conclusion: We concluded that Multiplex T-ARMS PCR could be considered as a better option than HRM analysis for APOE genotyping in terms of speed, accuracy, simplicity, and cheapness in large-scale use. Also, the present study revealed that ɛ2 ɛ3 genotype and ɛ2 allele are protective against Alzheimer whereas the ɛ4 allele cannot be strongly considered as Alzheimer genetic risk factor in Kerman, Iran. The results may help to choose a better technique for APOE genotyping.

Exploring the Association of Biochemical Characterization and Genetic Determinants of TNF-α, CXCR2, and CCR5 Delta 32 Mutation with Predisposition to Polycystic Ovary Syndrome.

PCOS is a heterogeneous, multifactorial endocrine disorder with a complex pathophysiology. It is a globally rising infertility disorder that affects a large percentage of women of reproductive age, with a relatively high prevalence of 8-13%. Genome-wide association studies have revealed associations of genetic variations with many diseases, including PCOS. The cellular activity of IL8 is mediated by the receptor CXCR2, and transcription of IL8 is controlled by TNF-α. Therefore, this study aimed to investigate the association of TNF-α, CCR5-delta32, and CXCR2 gene variations with PCOS. METHODOLOGY: In this case control study, we used amplification-refractory mutation system (ARMS)-PCR to detect and determine the presence of the polymorphic variants TNF-α, CCR5-delta32, and CXCR2 in the study subjects. These gene polymorphs may serve as critical candidate gene variants in PCOS pathogenesis and therapeutics. RESULTS: The case-control study's findings revealed that the majority of the biochemical and endocrine serum biomarkers examined in the investigation-including lipids (LDL, HDL, and cholesterol), T2DM markers (fasting glucose, free insulin, and HOMA-IR), and hormones (FSH, LH, testosterone, and progesterone)-exhibited statistically significant changes in PCOS patients. The distributions of TNF-α (rs1800629), CCR5-delta32, and CXCR2 (rs2230054) genotypes analyzed within PCOS patients and healthy controls in the considered population were significant (p < 0.05). The heterozygosity of CXCR2-CA, TNF-α GA, and CCR5(WT+Δ32*) genotypes was significantly associated with PCOS susceptibility, with high OR and p < 0.05 in the codominant model. Similarly, the A allele of the TNF-α and CXCR2 genes, along with the CCR5Δ32*(mutant) allele, was significantly associated with PCOS susceptibility, with high OR and p < 0.05. Likewise, the CXCR2 (CA+AA) vs CC genotype was associated with increased susceptibility to PCOS, with OR 2.25, p < 0.032. CONCLUSIONS: Our study concludes that TNF-α rs1800629G>A, CXCR2-rs2230054C>T, and CCR5-Delta32 rs333 are potential loci for developing PCOS in the Tabuk population. These findings might eventually be useful in identifying and classifying those who are at risk for PCOS. To validate these results, it is advised that further longitudinal studies be conducted in diverse ethnic populations and with larger sample sizes.

Genotyping single point mutations in rd1 and rd8 mice using melting curve analysis of qPCR fragments.

PCR is tolerant to single nucleotide mismatches. Therefore, genotyping of point mutations by PCR requires special conditions for the amplification of allele-specific PCR fragments. MS-PCR (mutagenically separated PCR) is an improved version of ARMS (amplification refractory mutation system) in which additional nucleotide mismatches near the mutation site are used to separate the wt fragments from the mutant fragments in a single-tube PCR. In the originally described procedure, the resulting fragments are resolved on agarose gels according to differences in size introduced by different lengths of the allele-specific primers. In order to evaluate the PCR fragments by melting curve analysis, we enlarged the difference in the melting temperatures of the fragments of the two alleles by increasing the GC content of the longer allele-specific primer resulting in a higher melting temperature of the corresponding fragment. Using the murine retinal degeneration mutations rd1 and rd8 as an example, we show that such primers result in an easy to handle genotyping procedure: qPCR followed by melting curve analysis. In summary, MS-PCR is a simple and easy-to-use method for detecting single nucleotide variants.

Genetic variants related to insulin metabolism are associated with gestational diabetes mellitus.

The current study sought to investigate the associations of common genetic risk variants with gestational diabetes mellitus (GDM) risk in the north Indian population and to evaluate their utility in identifying GDM cases. A case-control study, including 300 pregnant women, was included, and clinical and pathological information was collected. The amplification-refractory mutation system (ARMS) was used for genotyping four single nucleotide polymorphisms (SNPs), namely FTO (rs9939609), PPARG2 (rs1801282), SLC30A8 (rs13266634), and TCF7L2 (rs12255372). The odds ratio and confidence interval were determined for each SNP in different genetic models. Further, attributable risk, population penetrance, and relative risk were also calculated. The risk allele A of FTO (rs9939609) poses a two times higher risk of GDM (p = 0.02, OR = 2.5). The CG and GG genotypes of PPARG2 (rs1801282) have half a lower risk of GDM. In SLC30A8 (rs13266634), the recessive model analysis showed a two times higher risk of having GDM, while the recessive model (TT vs. GG + GT) analysis in TCF7L2 (rs12255372) indicates a lower risk of GDM. Finally, the relative risk, population penetrance, and attributable risk for risk allele in all four variants was higher in GDM mothers. All four polymorphisms were found to be significantly associated with BMI, HbA1c, and insulin. Our study first time confirmed a significant association with GDM for four variants, FTO, PPARG2, SLC30A8, and TCF7L2, in the North Indian population.

Where do obesity and male infertility collide?

The parallel rise in obesity and male infertility in modern societies necessitates the identification of susceptibility genes underlying these interconnected health issues. In our study, we conducted a comprehensive search in the OMIM database to identify genes commonly associated with male infertility and obesity. Subsequently, we performed an insilico analysis using the REVEL algorithm to detect pathogenic single nucleotide polymorphisms (SNPs) in the coding region of these candidate genes. To validate our findings in vivo, we conducted a comprehensive analysis of SNPs and gene expression of candidate genes in 200 obese infertile subjects and 240 obese fertile individuals using ARMS-PCR. Additionally, we analyzed 20 fertile and 22 infertile obese individuals using Realtime-qPCR. By removing duplicated queries, we obtained 197 obesity-related genes and 102 male infertility-related genes from the OMIM database. Interestingly, the APOB gene was found in common between the two datasets. REVEL identified the rs13306194 variant as potentially pathogenic with a calculated score of 0.524. The study identified a significant association between the AA (P value = 0.001) genotype and A allele (P value = 0.003) of the APOB rs13306194 variant and infertility in obese men. APOB expression levels were significantly lower in obese infertile men compared to obese fertile controls (p < 0.01). Moreover, the AA genotype of rs13306194 APOB was associated with a significant decrease in APOB gene expression in obese infertile men (p = 0.05). There is a significant association between the Waist-to-Hip Ratio (WHR) and LH with infertility in the obese infertile group. These results are likely to contribute to a better understanding of the causes of male infertility and its association with obesity.

Determination of IL-23 receptor expression and gene polymorphism (rs1884444) in Iranian patients with ankylosing spondylitis.

BACKGROUND: Through investigating genetic variations, it has been demonstrated that single nucleotide polymorphisms (SNPs) in the IL-23 receptor (IL23R) gene have a critical role in the pathophysiology of ankylosing spondylitis (AS). Here, we investigated whether the IL23R variant (rs1884444) is associated with AS in the Iranian population. METHODS AND MATERIAL: In this research, we analyzed rs1884444 in a group of 425 patients with AS and 400 matched controls. For DNA extraction, the phenol/chloroform technique was utilized. Peripheral blood mononuclear cells (PBMCs) were obtained from the whole blood of 39 patients and 43 healthy controls and total RNA was extracted. Genotyping was performed by amplification-refractory mutation system (ARMS)-PCR method. Afterward, the expression level of IL23R was analyzed by the real-time quantitative (Q)-PCR method. RESULTS: We observed no significant association between the distribution of alleles and genotypes of rs1884444 and susceptibility to AS. In addition, the expression level of IL23R did not differ between PBMCs from AS patients compared to the control group (P = 0.167). Furthermore, the relative expression level of IL23R was positively correlated with the BASDAI (P < 0.01) and BASFI (P < 0.05) scores of the patients. CONCLUSION: It appears that IL23R polymorphism (rs1884444) and the level of gene expression might not contribute to the susceptibility to AS in the Iranian population. The correlation of IL23R expression with the level of BASDAI and BASFI scores in patients may be due to the role of the IL-23/IL-23R signaling cascade in inflammation and exert a critical role in the development of AS.

Multiplex fluorescent amplification-refractory mutation system PCR method for the detection of 10 genetic defects in Holstein cattle and its comparison with the KASP genotyping assay.

The common deleterious genetic defects in Holstein cattle include haplotypes 1-6 (HH1-HH6), haplotypes for cholesterol deficiency (HCD), bovine leukocyte adhesion deficiency (BLAD), complex vertebral malformation (CVM) and brachyspina syndrome (BS). Recessive inheritance patterns of these genetic defects permit the carriers to function normally, but homozygous recessive genotypes cause embryo loss or neonatal death. Therefore, rapid detection of the carriers is essential to manage these genetic defects. This study was conducted to develop a single-tube multiplex fluorescent amplification-refractory mutation system (mf-ARMS) PCR method for efficient genotyping of these 10 genetic defects and to compare its efficiency with the kompetitive allele specific PCR (KASP) genotyping assay. The mf-ARMS PCR method introduced 10 sets of tri-primers optimized with additional mismatches in the 3' end of wild and mutant-specific primers, size differentiation between wild and mutant-specific primers, fluorescent labeling of universal primers, adjustment of annealing temperatures and optimization of primer concentrations. The genotyping of 484 Holstein cows resulted in 16.12% carriers with at least one genetic defect, while no homozygous recessive genotype was detected. This study found carrier frequencies ranging from 0.0% (HH6) to 3.72% (HH3) for individual defects. The mf-ARMS PCR method demonstrated improved detection, time and cost efficiency compared with the KASP method for these defects. Therefore, the application of mf-ARMS PCR for genotyping Holstein cattle is anticipated to decrease the frequency of lethal alleles and limit the transmission of these genetic defects.

Spectrum of Rare and Novel Indel Mutations Responsible for ß Thalassemia in Eastern India.

There is limited data available regarding the clinical utility of routine molecular diagnosis of ß Thalassaemia in addition to HPLC-based screening in low resource settings. The current study highlights the caveats of an HPLC-based screening compared to the inclusion of genetic confirmation as a second-tier test and its implications in terms of genotype-phenotype correlation. A prospective, institution-based, observational study was conducted at the Department of Paediatric Medicine, including 103 children aged up to 12 years. Five common mutations for ß Thalassemia and the HbE mutation in the HBB gene were tested by a two-tiered approach using multiplex ARMS PCR and PCR RFLP methods respectively. Sanger sequencing of all three exons of the HBB gene was performed in all negative cases. Sequencing revealed many rare pathogenic mutations like c.316-106 C > G (dbSNP: 34,690,599); Hb Kairouan (c.92G > C); c.33 C > A (dbSNP rs35799536); c.47G > A (dbSNP rs63750783); c.51delC (HbVar ID 799); c.[93-2 A > C] and c.118 C > T (HbVar ID 845). We detected a novel Pathogenic M_000518.5(HBB):c.164_168delinsGGCATCA (p.Val55fs) mutation in a heterozygous state which was reported in the ClinVar database with accession ID VCV000590977.2. We also encountered several cases of silent carrier on HPLC and de novo occurrence of mutation. We conclude that the multiplex touchdown ARMS PCR methodology employed in the present study provides a low-cost solution for molecular diagnostics of Β Thalassaemia. The problem of silent carriers in HPLC is significant enough to rethink if we need supplemental genetic testing in the couple when one of the partners is a carrier. Supplementary Information: The online version contains supplementary material available at 10.1007/s12291-022-01098-w.

Factor V Leiden, MTHFR, and FXIIIVal34Leu gene polymorphisms and their association with clinical features and risk of diabetic retinopathy in patients with type 2 diabetes.

Background: Diabetic retinopathy (DR) is expanding to epidemic levels globally due to the progressing prevalence of diabetes mellitus (DM). In this study, the association between factor V Leiden (FVL), MTHFRC677T, and FXIIIVal34Leu polymorphisms and diabetic retinopathy was investigated in Eastern Iran. Methods: This case-control study enlisted the participation of 300 people (diabetic patients=100, diabetic retinopathy patients=100, healthy controls=100), and polymorphisms were examined by Tetra primer ARMS-PCR. Results: The frequency of FVL (p=0.294) and FXIIIVal34Leu (P=0.349) polymorphism showed no significant results between the genotype frequency in the mentioned groups. In contrast, MTHFRC677T SNP was significantly different in diabetic patients and controls (P=0.008). The MTHFRC677T polymorphism was found to be connected with increased systolic blood pressure in patients who had the TT genotype (130.96±11.92mm/Hg; P=0.011). Conclusion: Our study recommended that the MTHFRC677T polymorphism may offer to DR development. Studies with larger sample sizes and a wider spectrum of populations are authorized to verify this finding.

Targeted next-generation sequencing of 491 lung cancers in clinical practice: Implications for future detection strategy and targeted therapy.

Although lung cancer remains the most common cause of global cancer-related mortality, the identification of oncogenic driver alterations and the development of targeted drugs has dramatically altered the therapeutic landscape. In this retrospective study, we found that 97.7% samples carried at least one mutation in the 25 genes tested in our cohort. 53.6% samples were positive for EGFR mutations, followed by TP53 (41.1%), KRAS (11.8%), ERBB2 (4.3%). EGFR mutations were mainly found in female adenocarcinomas, while TP53 was mainly found in male non-adenocarcinomas. Significant differences can be found in the mutation rate of EGFR (60.9% vs 11.9%), KRAS (12.2% vs 25.0%), STK11 (1.5% vs 11.9%), FGFR3 (2.4% vs 0.0%) and ERBB4 (1.2% vs 6.1%) between adenocarcinoma in our cohort and TCGA-LUAD data (all p < 0.001). What's more, we found that the mutation of EGFR increased significantly from adenocarcinomas in situ (AIS, 21.4%) to microinvasive adenocarcinomas (MIA, 52.4%) and invasive adenocarcinomas (IA, 61.1%), while the mutation of ERBB2 dropped markedly from AIS (21.4%) to MIA (9.5%) and IA (4.1%). At last, comparations between targeted NGS and ARMS-based single gene test in the detection of EGFR showed a 94.6% consistence. In conclusion, targeted NGS can provide a comprehensive mutational profile of lung cancer. Considering the high mutation rate of EGFR in NSCLC of Asian populations, a specialized detection strategy should be conducted.

The Association of miRNA-146a Gene Variation and Multiple Sclerosis in The Iranian Population.

Background: Multiple sclerosis (MS) is a complex human autoimmune-type inflammatory disease of the central nervous system (CNS). MicroRNA-146a (miR-146a) belongs to an endogenous and non-coding RNA family with 18-22 nucleotides long, which modulates the innate and adaptive immune response. Methods: Our study aimed to investigate a possible association between rs2910164 and rs2431697 polymorphisms of the miR-146a gene and multiple sclerosis in the Iranian population. A total of 60 MS cases and 100 controls were recruited. Single nucleotide polymorphism (SNP) rs2431697 was genotyped by utilizing polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and SNP rs2910164 was genotyped by using Tetra-primer ARMS-PCR. Statistical Analysis conducted by the chi-squared test utilizing SPSS version 21.0 Software. The Hardy-Weinberg equilibrium assumption was evaluated. Results: The results of the present study suggest the miR-146a gene rs2431697 polymorphism is not associated with multiple sclerosis. However, there is a significant relationship between polymorphism rs2910164 of the miR-146a gene and multiple sclerosis in the population studied (P = 0.012). Conclusion: Our data provide evidence that the miR-146a gene may be involved in creating the susceptibility to MS in the Iranian population.

Rapid Tetra-Primer Amplification Refractory Mutation System-Polymerase Chain Reaction Protocol for Detection of Y132F Mutation in Fluconazole Resistant Candida parapsilosis.

There is an emerging fluconazole resistance in Candida parapsilosis in recent years. The leading mechanism causing azole resistance in C. parapsilosis is the Y132F codon alteration in the ERG11 gene which encodes the target enzyme of azole drugs. In this study, we evaluated the sensitivity, compatibility, and specificity of a novel tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) method for rapid detection of the Y132F mutation in fluconazole nonsusceptible C. parapsilosis. Antifungal susceptibility tests for detection of fluconazole resistance were performed by broth microdilution according to the CLSI guidelines. All susceptible and nonsusceptible C. parapsilosis isolates were analyzed for ERG11 mutations with Sanger sequencing. T-ARMS-PCR was fully concordant with the Sanger sequencing (100% of sensitivity and specificity) for detection of Y132F mutations. T-ARMS-PCR method could be a rapid, simple, accurate, and economical assay in the early detection of the most common cause of fluconazole resistance in C. parapsilosis isolates. In routine laboratories with high C. parapsilosis isolation rates, performing the T-ARMS-PCR for early detection of the most common reason of fluconazole resistance in C. parapsilosis, could be a life-saving approach for directing antifungal therapy before obtaining the definitive antifungal susceptibility tests results.

Integrated approach for detection of SARS-CoV-2 and its variant by utilizing LAMP and ARMS-PCR.

Global impact of COVID-19 pandemic has heightened the urgency for efficient virus detection and identification of variants such as the Q57H mutation. Early and efficient detection of SARS-CoV-2 among densely populated developing countries is paramount objective. Although RT-PCR assays offer accuracy, however, dependence on expansive kits and availability of allied health resources pose an immense challenge for developing countries. In the current study, RT-LAMP based detection of SARS-Cov-2 with subsequent confirmation of Q57H variant through ARMS-PCR was performed. Among the 212 collected samples, 134 yielded positive results, while 78 tested negative using RT-LAMP. Oropharyngeal swabs of suspected individuals were collected and processed for viral RNA isolation. Isolated viral RNA was processed further by using either commercially available WarmStart Master Mix or our in house developed LAMP master mix separately. Subsequently, the end results of each specimen were evaluated by colorimetry. For LAMP assays, primers targeting three genes (ORF1ab, N and S) were designed using PrimerExplorer software. Interestingly, pooling of these three genes in single reaction tube increased sensitivity (95.5%) and specificity (93.5%) of LAMP assay. SARS-CoV-2 positive specimens were screened further for Q57H mutation using ARMS-PCR. Based on amplicon size variation, later confirmed by sequencing, our data showed 18.5% samples positive for Q57H mutation. Hence, these findings strongly advocate use of RT-LAMP-based assay for SARS-CoV-2 screening within suspected general population. Furthermore, ARMS-PCR also provides an efficient mean to detect prevalent mutations against SARS-Cov-2.

Investigating the impact of the genetic variant CXCR1 (rs2234671) in individuals with urinary tract infections.

BACKGROUND: Urinary tract infections (UTIs) are currently posing a worldwide health concern by affecting millions of people. The genetic variant rs2234671 in the CXCR1-interleukin-8 receptor is closely related to a raised UTI risk. OBJECTIVES: In this work, the impact of CXCR1 (rs2234671) on UTI individuals was examined. METHODS: The demographic features of 30 recurrent UTI patients and 20 controls were thoroughly investigated. Bacterial isolation and identification were performed by the implementation of cultural and biochemical methods. DNA extraction, purification of all samples from both patients and healthy people, and IL-8 rs2234671 (C/G) SNP genotyping using T-ARMS-PCR were performed. The significance of the results was evaluated by carrying out a statistical analysis. FINDINGS: The patient's average age was 34.63 ± 11.44 years, and controls averaged 30.30 ± 8.59 years (P= 0.156). No significant gender difference existed (P= 0.804). Escherichia coli (63.3%) was predominant, followed by Proteus mirabilis (26.7%), Enterococcus faecalis (23.3%), Klebsiella pneumoniae (10.0%), and Pseudomonas aeruginosa (20.0%). No significant association was found between bacterial species frequency, age, or sex. From the CXCR1 (rs2234671) frequency comparison, a higher GG genotype incidence in UTI patients than controls was extracted (26.7% vs. 15.0%), though not statistically significant. Risk analysis revealed that GG homozygous and C/G heterozygous genotypes were not UTI risk factors (OR = 2.47 and OR = 1.85, respectively). Moreover, the allele frequencies displayed no significant difference between the patients and controls (G allele: 66.7% vs. 66.7%; C allele: 33.3% vs. 33.3%). MAIN CONCLUSIONS: Although no significant association between CXCR1 (rs2234671) and UTI was found, the GG genotype may point to the increasing probability of UTI risk. Additional research is required to confirm and expand these conclusions.

The linkage between IL-6 rs1800797 variant and breast cancer susceptibility in Bangladeshi women: A case-control study.

Background and Aims: Breast cancer is one of the deadliest diseases affecting women in Bangladesh, and its prevalence is increasing year by year. Although several IL-6 single nucleotide polymorphisms have been implicated in BC susceptibility and prognosis in various studies, no research has been done to investigate the relationship between breast cancer and IL-6 in Bangladeshi women. This investigation aimed to explore the linkage between the rs1800797 variant of IL-6 and the susceptibility to breast carcinoma among women in Bangladesh. Methods: The IL-6 rs1800797 variant was genotyped in 218 subjects (110 cases and 108 controls) using the tetra-primer ARMS-PCR method. The statistical analysis was applied utilizing the SPSS software version 24.0. UALCAN database was used for IL-6 mRNA analysis, and genotype-based gene expression was retrieved from GTEx Portal. Results: This study found a significant link between IL-6 rs1800797 variants and increased chance of breast cancer across different genetic inheritance models, including additive model 1 (AG vs. GG: OR = 2.16, p = 0.035); dominant model (AG + AA vs. GG: OR = 2.26, p < 0.05); overdominant model (AG vs. GG + AA: OR = 2.08, p < 0.05); and allelic model (A vs. G: OR = 2.15, p < 0.05). However, an insignificant association of breast cancer was found in both additive model 2 (AA vs. GG: OR = 2.91, p > 0.05) and the recessive model (AA vs. GG + AG: OR = 2.52, p > 0.05). Under the analysis of the probability of false positive reports, no significant values were found in different models when the OR was 1.5, and the prior probability was 0.25. Conclusions: A significant relationship was found between the IL-6 rs1800797 genetic variant and the risk of breast cancer. However, the findings of the study should be further investigated with a larger sample size to validate the correlation.

Micronutrients intake and genetic variants associated with premature ovarian insufficiency; MASHAD cohort study.

BACKGROUND AND AIM: premature ovarian insufficiency (POI) is defined as the menopause before 40 years of age, and its prevalence is reported to be two-fold higher in Iranian women than the average for woman globally. POI is associated with several cardio/cerebrovascular complications as well as an increased overall mortality. Genetic factors, and serum levels of minerals and vitamin D, have been reported to be related to the prevalence of POI. We have investigated the association between some POI -related genotypes with the serum levels of some important micronutrients. METHODS: One hundred and seventeen women with POI and 183 controls without any renal, hepatic, and thyroid abnormalities were recruited as part of the MASHAD study. Demographic and anthropometric features were recorded and blood samples were collected and processed. DNA was extracted from the buffy coat of blood samples from all participants and 8 POI-related single nucleotide polymorphisms (SNPs) were determined using ASO-PCR or Tetra ARMS-PCR. Serum minerals and vitamin D concentrations were measured using routine methods. RESULTS: In women with POI, serum copper, phosphate, and calcium were significantly different for those with rs244715, rs16991615, and rs4806660 genotypes, respectively. In our control population, significant differences were also found in serum copper concentrations between different genotypes of rs4806660, rs7246479, rs1046089, and rs2303369. After adjusting for all confounding factors, the women with POI carrying TC genotype (rs4806660) had a lower risk to have serum copper levels < 80 (µg/dL) than those carrying a TT genotype. Furthermore, women with POI carrying GG genotype (rs244715) had a 6-fold higher risk to have serum copper levels > 155 than those carrying AA genotype. CONCLUSION: The C and G alleles of the rs4806660 and rs244715 polymorphisms respectively are independently associated with serum copper in women with POI. Further studies are necessary to investigate the association of serum copper and other micronutrients in women and other POI -related polymorphisms.

Genetic variation of CYP2C9 gene and its correlation with cardiovascular disease risk factors.

BACKGROUND: The major enzyme that is responsible for Sulfonylureas (SUs) metabolism is hepatic cytochrome P-450 2C9 (CYP2C9). It is encoded by the polymorphic gene CYP2C9, which has many allelic variants, among those the CYP2C9*2 and CYP2C9*3 are the most common and clinically significant allelic variations. People with diabetes mellitus type 2 (T2DM) are more likely to develop cardiovascular disease (CVD), and their risk of dying from it is more than two times higher than that of people without the condition. The purpose of this study was to evaluate the association of genetic variations in the CYP2C9 gene with cardiovascular risk factors by investigating CYP2C9*1, *2, *3, *5, *11, and *13 allelic variants. METHODS AND RESULTS: A total of 226 participants were enrolled in the current case-control study. Allele-specific amplification- PCR (ASA-PCR) was used to determine the allele of different variations and the results were confirmed by sequencing. The findings of this study showed the presence of the CYP2C9*2 allele in the T2DM group does not differ from its percentage in the control group. Also, CYP2C9*3 allele frequencies identified by Hardy-Weinberg equilibrium (HWE) analysis law were not significant, p = 0.6593 and 0.5828 in T2DM and control groups. There is no statistically significant difference between the control and diabetes groups involving the distribution of CYP2C9 alleles and CYP2C9*5, *11, and *13 polymorphisms were absent in the Iraqi population. No carrier for the CYP2C9*3 homozygous state was found in both groups. CONCLUSIONS: According to these results T2DM patients with the CYP2C9*2 and *3 variants have an increased risk of developing hypertension.

The miRNA variants MIR196A2 (rs11614913) and MIR423 (rs6505162) contribute to an increase in the risk of myocardial infarction.

INTRODUCTION: MicroRNAs (miRNAs) are small, single-stranded RNA molecules that negatively regulate gene expression and play a key role in the pathogenesis of human diseases. Recent studies have suggested that miRNAs contribute to cardiovascular diseases (CVDs). However, the association between single-nucleotide polymorphisms (SNPs) in miRNAs and myocardial infarction (MI) remains in infancy. AIM: The current study was designed to find out the association of SNPs in MIR196A2 and MIR423 (rs11614913 and rs6505162, respectively). METHODS: Using Tetra-Primer Amplification Refractory Mutation System-Polymerase Chain Reaction (T-ARMS PCR) in 400 cases (MI patients) and 336 healthy controls. Using different inheritance models (co-dominant, homozygous dominant, homozygous recessive, and additive models), the association of these SNPs was genotyped with MI risk. RESULTS: For variant rs11614913, significant distribution of the genotypes among the cases and controls was determined by co-dominant [χ2 = 29.19, 2; p value < 0.0001], dominant (C/C vs. C/T + T/T) [OR = 0.45 (0.34 to 0.61); p < 0.0001], recessive (T/T vs. C/T + C/C) [OR = 1.009 (0.63 to 1.63); p-value p value > 0.999], and additive models [OR = 0.65 (0.52 to 0.80); p value = 0.0001]. Similarly, a significant association of rs6505162 was determined by co-dominant [χ2 = 24.29, 2; p value < 0.0001], dominant (C/C vs. A/C+ A/A) [OR = 0.44 (0.32 to 0.61); p value < 0.0001], recessive (A/A vs. A/C + C/C) [OR = 1.29 (0.85 to 1.98); p value = 0.28], and additive models [OR = 0.65 (0.52 to 0.81); p value = 0.0001]. CONCLUSION: Therefore, the current study showed that both variants rs11614913 and rs6505162 are significantly associated with MI in the Pakistani population.