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Highly variable response shown by individuals against mosquito-borne infections suggests that host genetic factors play an important role in determining mosquito-borne disease onset. Therefore, it is necessary to determine the genetic risk of these diseases in specific populations. The current study aimed to determine the percentage of individuals in the general population carrying mosquito-borne disease susceptibility and protection-related variants. This study initially aggregated mosquito-borne disease susceptibility and protection-related variants from all publically available data and literature. Afterward, the allele frequency was calculated 1009 genetic variants of 366 genes associated with susceptibility and protection to estimate the global prevalence in multiple ethnicities (Middle Eastern, Ashkenazi Jewish, European (Non-Finnish), Latino/Admixed American, South Asian, East Asian, European (Finnish), North Asian, Southeast Asian, African American, and Swedish population). Furthermore, the cumulative allele frequency of all susceptibility and protection-related variants was calculated in diverse ethnic groups and the relationship with mosquito-borne disease-associated morbidity and mortality was examined to determine whether results are consistent with founder effect in these populations. Two prioritized genetic variants of IL-10 (rs1800871) and FcγRIIA (rs1801274) were examined in the Tehsil Haripur population to assess the genetic risks linked to susceptibility and protection against mosquito-borne diseases. The findings of this study revealed overlapping genes most implicated in mosquito-borne disease linked with susceptibility and protection across different ethnic ancestries. In the available sample size, the percentage of TC and TT genotypes in IL-10 genetic variant (rs1800871) was 12% and 88%, respectively and GA and GG genotypes in FcγRIIA(rs1801274) genetic variant were 6% and 94% respectively. Based on statistical analysis, the percentage allele frequency of IL-10 (rs1800871) variant was 0.2112% and the FcγRIIA (rs1801274) variant is 0.1128% in the current study. Additionally, this study reflects that screening of genetic variants associated with susceptibility and protection in a population gives better insights into organizing public health awareness campaigns to control diseases. [ABSTRACT FROM AUTHOR]

Abstract Background Hepatocellular carcinoma (HCC) is a global health concern. Due to late diagnosis and limited therapeutic strategies, HCC based mortality rate is exponentially increasing globally. Genetic predisposition is a non-avoidable intrinsic factor that could alter the genome sequence, ultimately leading to HCC. Protein kinase C eta (PKCη) is involved in key physiological roles, hence alteration in PKCη could aid in cancer progression. Research indicates association between non-synonymous (ns) SNPs and HCC onset. However, effect of nsSNP variants of PKCη on HCC development has not been explored yet. Hence, this study aimed to investigate the association between pathogenic nsSNPs of PKCη with HCC. Methods Non-synonymous (missense) variants of PKCη were obtained from Ensembl genome browser. These variants were filtered out to obtain pathogenic nsSNPs of PKCη. Genotyping of nsSNPs was done through Tetra ARMS PCR. For that, blood samples of 348 HCC patients and 337 controls were collected. The clinical factors that influence HCC were studied. Relative risk (RR) and Odds Ratio (OR) with 95% confidence interval was calculated by Chi-square test and P-value

In order to achieve its nucleotide polymorphism (SNP) accurate typing, this paper establishes a molecular diagnostic technique based on fluorescent quantitative PCR for accurate genotyping of the nucleotide polymorphism (SNP) at the rs755555 locus of the SLC39A13 gene. Firstly, Taqman fluorescent ARMS-PCR detection primers and probes were designed for the rs755555 locus and the internal reference gene peptidylprolyl isomerase A (PPIA). Secondly, positive control plasmids were constructed. Finally, based on genotyping accuracy, the primer and probe combinations were optimized, and the PCR reaction system and conditions for the detection reagents were optimized. The optimal primer and probe combination for the wild-type was: WF1, R1, FP1, PIRF5, PIRR5, PIRP5; the optimal combination for the mutant type was: FMF3, R1, FP1, PIRF5, PIRR5, PIRP5. The optimal reaction system for detecting samples was: 0.1 μL each of upstream and downstream primers and probes for the SLC39A13 gene, 0.1 μL each of upstream and downstream primers and probes for the internal standard, 10 μL PerfectStart Ⅱ Probe qPCR SuperMix UDG, 5.4 μL purified water, and 4 μL sample genome. The feasibility of this detection system was confirmed through reproducibility experiments and the detection of 70 samples. This provides a technical foundation for the development of a detection kit for the polymorphism at the rs755555 locus of the SLC39A13 gene.

Background: Hepatocellular carcinoma (HCC) is a global health concern. Due to late diagnosis and limited therapeutic strategies, HCC based mortality rate is exponentially increasing globally. Genetic predisposition is a non-avoidable intrinsic factor that could alter the genome sequence, ultimately leading to HCC. Protein kinase C eta (PKCη) is involved in key physiological roles, hence alteration in PKCη could aid in cancer progression. Research indicates association between non-synonymous (ns) SNPs and HCC onset. However, effect of nsSNP variants of PKCη on HCC development has not been explored yet. Hence, this study aimed to investigate the association between pathogenic nsSNPs of PKCη with HCC. Methods: Non-synonymous (missense) variants of PKCη were obtained from Ensembl genome browser. These variants were filtered out to obtain pathogenic nsSNPs of PKCη. Genotyping of nsSNPs was done through Tetra ARMS PCR. For that, blood samples of 348 HCC patients and 337 controls were collected. The clinical factors that influence HCC were studied. Relative risk (RR) and Odds Ratio (OR) with 95% confidence interval was calculated by Chi-square test and P-value < 0.05 was deemed significant. Results: Five nsSNP variants of PKCη including rs1162102190 (T/C), rs868127012 (G/T), rs750830348 (G/T), rs768619375 (T/C), and rs752329416 (T/C) were identified. The retrieved nsSNPs were frequently identified in HCC patients. However, rs752329416 T/C was significantly prevalent in patients having HCC family history. Moreover, all the variants were found in HCC patients manifesting the stage II than the advance stages of HCC. Conclusion: This study can be utilized to identify potential genetic markers for early screening of HCC. Moreover, consideration of further clinical factors, and mechanistic approach would enhance the understanding that how alteration in nsSNPs could impact the HCC onset. [ABSTRACT FROM AUTHOR]

Lung cancer is a serious health and life issue, with the fastest-growing incidence and fatality rates worldwide. It is now clear that inflammation is a key factor involved in all aspects of carcinogenesis, notably lung cancer development. Genetic changes, including polymorphisms in inflammatory genes, are supposed to be a significant cause of increased lung cancer risk. The main idea of this research was to disclose the linkage between both IL-6 rs1800795 and IL-1β rs16944 variants and susceptibility to non-small-cell lung cancer (NSCLC) in Egyptians. This case–control design was composed of 127 cases and 138 controls, which were genotyped using the ARMS-PCR technique. To examine the NSCLC susceptibility under various genetic models, the odds ratio (OR) and 95% confidence intervals (CIs) were determined by logistic regression. Rs1800795 of the IL-6 gene was linked to higher odds of NSCLC under the allele model (adjusted, OR 2.28; 95% CI 1.2–4.33; p = 0.011). In the genetic models, IL-6 rs1800795 elevated the odds of NSCLC, while IL-1β rs16944 decreased the odds of NSCLC. Stratification analysis showed that IL-6 rs1800795 greatly increased the NSCLC risk in females and adenocarcinoma subtypes, whereas IL-1β rs16944 largely decreased the NSCLC risk for males, patients aged < 55, and nonsmokers. Regarding clinical data, the IL-6 variant was remarkably correlated with tumor size. This work primarily established that IL-6 and IL-1β variants have a great impact on NSCLC development in the Egyptian population; thus, it may be a supportive guide for earlier NSCLC prevention. [ABSTRACT FROM AUTHOR]

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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. [ABSTRACT FROM AUTHOR]

Bactrocera tryoni and Bactrocera neohumeralis are morphologically similar sibling pest fruit fly species that possess different biological attributes, geographic distributions, and host ranges. The need to differentiate between the two species is critical for accurate pest status assessment, management, biosecurity, and maintenance of reference colonies. While morphologically similar, adults may be separated based on subtle characters; however, some characters exhibit intraspecific variability, creating overlap between the two species. Additionally, there is currently no single molecular marker or rapid diagnostic assay that can reliably distinguish between B. neohumeralis and B. tryoni; therefore, ambiguous samples remain undiagnosed. Here we report the first molecular marker that can consistently distinguish between B. tryoni and B. neohumeralis. Our diagnostic region consists of two adjacent single nucleotide polymorphisms (SNPs) within the pangolin (pan) gene region. We confirmed the genotypes of each species are consistent across their distributional range, then developed a tetra‐primer amplification refractory mutation system (ARMS) PCR assay for rapid diagnosis of the species. The assay utilizes four primers in multiplex, with two outer universal primers, and two internal primers: one designed to target two adjacent SNPs (AA) present in B. tryoni and the other targeting adjacent SNPs present in B. neohumeralis (GG). The assay accurately discriminates between the two species, but their SNP genotypes are shared with other nontarget tephritid fruit fly species. Therefore, this assay is most suited to adult diagnostics where species confirmation is necessary in determining ambiguous surveillance trap catches; maintaining pure colony lines; and in Sterile Insect Technique management responses. [ABSTRACT FROM AUTHOR]

Abstract 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

Abstract 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

HBV is a continuous major global health concern. Genetic factors of hosts are known to play a role in HBV infection outcomes. This study aimed to reveal the association of IL-12b 3′ UTR variant rs3212227 in HBV patients. Genotyping was performed using ARMS-PCR to detect IL-12b rs3212227 polymorphism. The patients were categorized into groups based on their response to the antiviral therapy. Group I: non-sustained virological response (NSR); Group II: sustained virological responders (SVR); and Group III: HBV-positive fresh cases. ALT levels were measured to evaluate liver function, and viral load was determined to evaluate viral infectivity among the study groups. The variant genotype CC was found to be significantly associated with the non-sustained virological response to the antiviral therapy (with a p-value of 0.0117; OR = 2.914; RR = 1.556). It was also determined that the genotype CC was the most prevalent genotype among both genders in the NSR group. Viral load was found to be 6-fold higher in Group III compared to Group I and Group II. The results suggest that genotype CC is the most prevalent genotype in the NSR groups, and it is associated with a poor response to antiviral therapy in Pakistani patients with HBV infection.

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. [ABSTRACT FROM AUTHOR]

Polymorphisms of CYP3A4*1G and CYP3A5*3 affect the pharmacokinetic profile of various drugs, e.g., fentanyl, tacrolimus, diltiazem, simvastatin. Tetra-primer amplification refractory mutation system-polymerase chain (ARMS-PCR) is a simple and economical method for SNP determination. The polymorphisms in the CYP3A4*1G and CYP3A5*3 genes have not yet been examined using this method in Javanese Indonesian. Our aim was to determine the frequency of polymorphisms in the CYP3A4*1G and CYP3A5*3 genes in Indonesian Javanese using the ARMSPCR method. Eighty-six patients at the Kalasan Community Health Centre in Yogyakarta, Indonesia, were chosen based on the inclusion criteria, which is Javanese ancestry. They gave their informed consent to blood collection by completing a form. Genetic variants were detected using Tetra-primer amplification refractory mutation system-polymerase chain (ARMS-PCR). The chi-square test was used to determine genotype deviations from Hardy-Weinberg equilibrium, with a significant threshold of 0.05. For homozygous wild types, CYP3A4 *1/*1 dominated overall among study participants (73.35%), whereas for CYP3A5*3/*3, homozygous mutants were more prevalent (83.72%). Hardy-Weinberg equilibrium is consistent with genotype frequencies (p > 0.005). One participant carried a homozygous mutation for both CYP3A4*1G and CYP3A5*3, while the other 49 subjects were heterozygous for CYP3A4*1G and homozygous mutant for CYP3A5*3, which is the highest number of SNP combinations. The findings of the current investigation demonstrate that the population has the highest proportion of homozygous CYP3A4*1G wild-types (CYP3A4*1/*1) and homozygous mutants for CYP3A5*3 (CYP3A5*3/*3). [ABSTRACT FROM AUTHOR]

Malay populations are sub-groups of the Malayo-Polynesian, with various sub-ethnic groups believed to have different ancestral origins based on their migration centuries ago. The variability in the genetic pattern within the Malay population might impose different risk and disease probabilities or certain phenotypes. This study aimed to analyze single nucleotide polymorphisms (SNPs) in Malayo-Polynesian sub-groups of the Malay population in Southeast Asia. SNPs were genotyped through T-ARMS PCR in 52 unrelated individuals from three Malay sub-groups: Champa (n=16), Kelantan (n=25) and Bugis (n=11). Most (60%) of the SNP genotypes showed a similarity with all Malay sub-ethnic groups. The PCA plot showed that all Malay sub-ethnic groups were slightly separated but clustered together with Asian populations compared with population groups from other geographical regions. Overall, the SNP genotyping generated from this study provides essential knowledge of the genetic relationships within Malay sub-ethnic groups in Southeast Asia and other global populations. Additionally, these findings may be used for future illness research, drug response estimation, and the development of preventive and therapeutic management strategies toward more personalized or precision medicine. [ABSTRACT FROM AUTHOR]

Abstract 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) 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.

Polymorphisms of CYP3A4*1G and CYP3A5*3 affect the pharmacokinetic profile of various drugs, e.g., fentanyl, tacrolimus, diltiazem, simvastatin. Tetra-primer amplification refractory mutation system-polymerase chain (ARMS-PCR) is a simple and economical method for SNP determination. The polymorphisms in the CYP3A4*1G and CYP3A5*3 genes have not yet been examined using this method in Javanese Indonesian. Our aim was to determine the frequency of polymorphisms in the CYP3A4*1G and CYP3A5*3 genes in Indonesian Javanese using the ARMS-PCR method. Eighty-six patients at the Kalasan Community Health Centre in Yogyakarta, Indonesia, were chosen based on the inclusion criteria, which is Javanese ancestry. They gave their informed consent to blood collection by completing a form. Genetic variants were detected using Tetra-primer amplification refractory mutation system-polymerase chain (ARMS-PCR). The chi-square test was used to determine genotype deviations from Hardy-Weinberg equilibrium, with a significant threshold of 0.05. For homozygous wild types, CYP3A4 *1/*1 dominated overall among study participants (73.35%), whereas for CYP3A5*3/*3, homozygous mutants were more prevalent (83.72%). Hardy-Weinberg equilibrium is consistent with genotype frequencies (p > 0.005). One participant carried a homozygous mutation for both CYP3A4*1G and CYP3A5*3, while the other 49 subjects were heterozygous for CYP3A4*1G and homozygous mutant for CYP3A5*3, which is the highest number of SNP combinations. The findings of the current investigation demonstrate that the population has the highest proportion of homozygous CYP3A4*1G wild-types (CYP3A4*1/*1) and homozygous mutants for CYP3A5*3 (CYP3A5*3/*3)

تُرمِّز الجين PIP4K2A لإحدى أنزيمات الكيناز التي تنتمي إلى الصف الثاني لعائلة من عائلات الكينازات تعرف بالفوسفوتيديل إنوزيتول 5 فوسفات 4 كيناز (PIP4K)، والتي تضم فضلاً عن PIP4K2A كل من PIP4K2B وPIP4K2C. يمتلك الأنزيم PIP4K2A أعلى فعالية أنزيمية من بين أفراد هذه العائلة. قد أشارت العديد مِنَ الدِّراسات إلى أهمية الجين PIP4K2A كإحدى مواقع الاختطار Risk Loci التي تؤهب جينياً Genetic Predisposition للإصابة بابيضاض الدم اللمفاوي الحاد ALL وبيَّنت دراسات أخرى أن التعبير عن PIP4K2A يتأثر بشدة بالأنماط الجينية Genotypes للـتعددات الشكلية مفرد النكليوتيد SNPs المجاورة لموقع هذه الجين، مما يجعل هناك ارتباطاً بين SNPs هذه الجين وخاصة rs7088318 وحادثة تشكل الابيضاضات Leukemogenesis من خلال تحريض زيادة التعبير Overexpression عن الجين. وفي نفس الوقت، أظهرت الأدلة الحالية ارتباطاً بين قابلية الإصابة بـ ALL والجين PIP4K2A خاص بالعرق Ethnicity-Specific وخاص بتحت النمط Subtype-Specific المناعي للابيضاض. هدفت هذه الدراسة إلى استعراف تواتر أليلا التعدد الشكل rs7088318 لدى جمهرة من مرضى سوريين مصابين بابيضاض لمفاوي حاد واستقصاء العلاقة بين النمط الجيني وكلاّ من جنس المصاب وعمره ونمطه المناعي. تضمّنت دراستنا الوصفية المقطعية 114 مريضاً مصاباً بـALL من الجنسين ومن أطفالٍ وبالغين ومن أنماط مناعية مختلفة، استعرف نمطهم الجيني عن طريق تفاعل البوليميراز السلسلي من نمط (ARMS-PCR). حُلّلت البيانات اعتماداً على البرمجية الإحصائية SPSS بإصدارها 23. بَلغ تواتر الأليل المتغاير T نسبة قدرها 80% في جمهرة الدراسة، ولم يكن هناك فوارق يعتد بها إحصائياً بين تواتر هذا الأليل وتحت المجموعات المرضية مما يظهر عدم وجود علاقة ارتباط بين التعدد الشكلي rs7088318 وكلٌ من الجنس والعمر والنمط المناعي للمرضى السوريين المصابين بابيضاض الدم اللمفاوي الحاد. تُلقي هذه الدراسة الضوء على الحاجة لإجراء مزيد من الاستقصاءات لاستعراف تواترات الألائل في المواقع الجينية الأخرى عند المرضى السوريين المصابين بابيضاض الدم اللمفاوي الحاد وتقييم تأثيرها على القابلية لإحداث المرض، والاستجابة للعلاج الكيميائي، والتنبؤ بحدوث النكس عند المرضى.

The Pacific white shrimp (Litopenaeus vannamei) is Indonesia's main export commodity, but its production is constrained by the white spot syndrome virus (WSSV). Selective breeding of disease-resistant broodstock based on single nucleotide polymorphism (SNP) in the anti-lipopolysaccharide factor (ALF) gene is an alternative strategy for solving the disease problem. This study aimed to detect the SNP g.455 A>G in the anti-lipopolysaccharide factor (ALF) shrimp gene, evaluate the correlation of SNP with WSSV-resistance trait, analyze the expression level of immunity genes and genotype frequencies of the WSSV-resistance population shrimp and analyze the SNP inheritance in the first generation of selected shrimp. A total of 120 individuals from 4 families were used to detect the SNP marker using tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). The correlation of the SNP marker with survival rate (SR) was analyzed using a general linear model (GLM) between genotype frequencies and SR. Genotypic similarities between broodstock and pedigree were analyzed using Chi-square. SNP g.455 A>G was successfully detected using the ARMS-PCR method and had a strong correlation between the marker and SR (p-value of AA = 0.012; AG = 0.359, and GG = 0.001). The resistant population has significantly higher ALF and SOD gene expression levels and AA genotype frequency. The SNP marker was inherited, so the broodstock and pedigree have the same genotype frequencies according to chi-square analysis (χ² = 0.46 and p-value = 0.497). These results suggested that the g.455 genotype AA could be selected to produce WSSV-resistant Pacific white shrimp. [ABSTRACT FROM AUTHOR]

HBV is a continuous major global health concern. Genetic factors of hosts are known to play a role in HBV infection outcomes. This study aimed to reveal the association of IL-12b 3′ UTR variant rs3212227 in HBV patients. Genotyping was performed using ARMS-PCR to detect IL-12b rs3212227 polymorphism. The patients were categorized into groups based on their response to the antiviral therapy. Group I: non-sustained virological response (NSR); Group II: sustained virological responders (SVR); and Group III: HBV-positive fresh cases. ALT levels were measured to evaluate liver function, and viral load was determined to evaluate viral infectivity among the study groups. The variant genotype CC was found to be significantly associated with the non-sustained virological response to the antiviral therapy (with a p-value of 0.0117; OR = 2.914; RR = 1.556). It was also determined that the genotype CC was the most prevalent genotype among both genders in the NSR group. Viral load was found to be 6-fold higher in Group III compared to Group I and Group II. The results suggest that genotype CC is the most prevalent genotype in the NSR groups, and it is associated with a poor response to antiviral therapy in Pakistani patients with HBV infection. [ABSTRACT FROM AUTHOR]

Highlight Research • A molecular marker for shrimp selection in disease resistance. • Single nucleotide polymorphisms (SNP) in the ALF gene strongly correlate with shrimp resistance to WSSV infection. • Higher ALF gene expression in survivor shrimp. • SNP as molecular marker inherited in the first generation (G1) shrimp. • ARMS-PCR method successfully detect SNP in the shrimp ALF gene. Abstract The Pacific white shrimp (Litopenaeus vannamei) is Indonesia's main export commodity, but its production is constrained by the white spot syndrome virus (WSSV). Selective breeding of disease-resistant broodstock based on single nucleotide polymorphism (SNP) in the anti-lipopolysaccharide factor (ALF) gene is an alternative strategy for solving the disease problem. This study aimed to detect the SNP g.455 A>G in the anti-lipopolysaccharide factor (ALF) shrimp gene, evaluate the correlation of SNP with WSSV-resistance trait, analyze the expression level of immunity genes and genotype frequencies of the WSSV-resistance population shrimp and analyze the SNP inheritance in the first generation of selected shrimp. A total of 120 individuals from 4 families were used to detect the SNP marker using tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). The correlation of the SNP marker with survival rate (SR) was analyzed using a general linear model (GLM) between genotype frequencies and SR. Genotypic similarities between broodstock and pedigree were analyzed using Chi-square. SNP g.455 A>G was successfully detected using the ARMS-PCR method and had a strong correlation between the marker and SR (p-value of AA = 0.012; AG = 0.359, and GG = 0.001). The resistant population has significantly higher ALF and SOD gene expression levels and AA genotype frequency. The SNP marker was inherited, so the broodstock and pedigree have the same genotype frequencies according to chi-square analysis (χ2 = 0.46 and p-value = 0.497). These results suggested that the g.455 genotype AA could be selected to produce WSSV-resistant Pacific white shrimp.

Aspergillus fumigatus is a common human fungal pathogen that can cause a range of diseases. Triazoles are used to treat A. fumigatus infections, but resistance is increasing due to mutations in genes such as cyp51A, hmg1 and overexpression of efflux pumps. Verifying the importance of these mutations is time-consuming, and although the use of CRISPR-Cas9 methods has shortened this process, it still relies on the construction of repair templates containing a selectable marker. Here, employing in vitro-assembled CRISPR-Cas9 along with a recyclable selectable marker, we devised a quick and easy way to effectively and seamlessly introduce mutations conferring triazole resistance in A. fumigatus. We used it to introduce, alone and in combination, triazole resistance-conferring mutations in cyp51A, cyp51B and hmg1. With the potential to seamlessly introduce genes imparting resistance to additional existing and novel antifungals, toxic metals, and environmental stressors, this technique can considerably improve the ability to introduce dominant mutations in A. fumigatus. [ABSTRACT FROM AUTHOR]

ABSTRACT: The 2 major subvariants of β-casein (A1 and A2), coded by CSN2 gene, have received great interest in the last decade both from the scientific community and the dairy sector due to their influence on milk quality. The consumption of the A1 variant, compared with the A2 variant, has a potential negative effect on human health after its digestion but, at the same time, its presence improves the milk technological properties. The aim of the present study was to compare the best method in terms of time required, costs, and technical engagement for the identification of β-casein A1 and A2 variants (homozygous and heterozygous animals) in milk to offer a reliable service for large-scale screening studies. Two allele-specific PCR procedures, namely RFLP-PCR and amplification refractory mutation system (ARMS-PCR), and one biochemical technique (HPLC) were evaluated and validated through sequencing. Manual and automated DNA extraction protocols from milk somatic cells were also compared. Automated DNA extraction provided better yield and purity. Chromatographic analysis was the most informative and the cheapest method but unsuitable for large-scale studies due to lengthy procedures (45 min per sample). Both allele-specific PCR techniques proved to be fast and reliable for differentiating between A1 and A2 variants but more expensive than HPLC analysis. Specifically, RFLP-PCR was the most expensive and labor-demanding among the evaluated techniques, whereas ARMS-PCR was the fastest while also requiring less technical expertise. Overall, automated extraction of DNA from milk matrix combined with ARMS-PCR is the most suitable technique to provide genetic characterization of the CSN2 gene on a large scale.

Background: Toxoplasma gondii (T. gondii) causes toxoplasmosis, a dangerous and prevalent disease. Toll-like receptors (TLRs) are the best-studied pattern recognition receptors in mammals. Objective: The current study was conducted at Al-Diwaniya Maternity and Children Teaching Hospital, Diwaniyah city, Iraq, from December 2020 to August 2021. Methods: Blood samples and placenta tissue pieces were collected prospectively from 30 patients newly diagnosed with toxoplasmosis and 64 healthy controls. Women in both groups underwent a spontaneous abortion. A human Toll-Like Receptor 4 (TLR4) ELISA kit was used to measure TLR4 levels and blood DNA extraction. ARMS-PCR was adopted to analyze the polymorphism for TLR4 Asp299Gly, and SAG3 marker was used to analyze the genotype of Toxoplasma strains. Results: The frequency distribution of the A allele and G allele was statistically non-significant between the patients and the control group. The polymorphism analysis of the Asp299Gly SNP in the TLR4 gene showed abortion had no significant association with toxoplasma infection (P> 0.05). Genotype II was more prevalent in aborted Iraqi women (22/30) than in control. The obtained result revealed that the concentration of TLR4 in serum women infected with type I Toxoplasma was significantly higher (P< 0.05) in the AA genotype of TLR4 Asp299Gly (mean 1328 pg/ml, SD: 266.1pg/ml), compared to AG genotype (mean; 923.6 pg/ml, SD: 27.2 pg/ml). Discussion: These findings highlighted the significance of TLR molecules in Toxoplasma gondii infection and the need for close collaboration between practitioners and to lessen the illness burden of toxoplasmosis. [ABSTRACT FROM AUTHOR]

Abstract Background and aim Primary Ovarian Insufficiency (POI) is defined by the occurrence of menopause before the age of 40 years. It is often associated with cardiovascular disease (CVD). The purpose of this study was to explore the relationship between POI-associated genotypes cardiometabolic disorder risk factors. Methods One hundred seventeen women with POI and one hundred eighty-three healthy women without POI were recruited in this study. DNA was extracted and analyzed using ASO-PCR or Tetra ARMS-PCR. Lipid profiles were also assessed. Results Multivariate logistic regression analysis showed that individuals with GG vs. TT genotype of the rs1046089 SNP were more likely to have a higher serum LDL (p = 0.03) compared to the control group. There was also a significant association between low serum HDL and rs2303369 and rs4806660 SNP genotypes in the POI group. In the POI group, the percentage of those with high total cholesterol was lower in those with a CC genotype compared to those with a TT genotype (p = 0.03). Conclusion Some SNPs reported to be associated with POI appear to be independently associated with dyslipidemia. These results may be helpful to identify subjects with POI who may be susceptible to CVD.

Single nucleotide polymorphism (SNP) C4685T located in 14. intron of bovine CAPN1 gene have shown significant association with a higher lean share in valuable cuts for mutant genotype TT. The work was oriented to developed a sensitive single tube tetra-primer amplification refractory mutation system PCR (ARMS-PCR) method for detection of C4685T polymorphism in CAPNI gene and analysis of genotype structure in population of 130 animals of Slovak Pinzgau cattle. The genomic DNA was isolated from samples of blood and hairs of cattle. Design of primers for ARMS-PCR was realized by using program Tetra-Primer ARMS-PCR. The presence of wild allele C and mutant allele T on agarose gel was detected by one control 439 bp fragment for both alleles and one specific fragment for each allele C - 204 bp and T - 290 bp. For the checking of correct genotyping was used PCR-RFLP method with restriction endonuclease BseGI. In the population of Slovak Pinzgau cattle we detected all genotypes. There were detected homozygote genotype CC with frequency 0.3308, heterozygote genotype CT with frequency 0.4 and homozygote genotype TT with frequency 0.2692. Frequency of alleles C and T for SNP C4685T of gene CAPN1 were 0.5308 and 0.4692.

Introduction: Thalassemia is a common disease caused by mutations in the beta globins gene. Today, this disease is high frequency due to some factors including lack of control, the increase of population growth, and lack of implementation of appropriate methods of family planning. In present study, the molecular ß-thalassemia has been investigated in patients with thalassemia major in Ardabil, Iran. Methods: Blood samples of patients were collected over the province and in the process of collecting; sampling of venous blood was performed under blood expert guidance and with the consent of 50 patients with ß-thalassemia major. The samples were stored at -70 °C in the freezer and DNA after extraction was amplified by amplification refractory mutation system - polymerase chain reaction (ARMS-PCR) method. Results: The results showed that the frequency of mutation was 16%, 14% and 4% for IVSI-110, IVSII-1 and IVSI-5 mutations, respectively. Conclusion: It can be concluded that the incidence of ß-thalassemia major in Ardabil IVSI-110 has the highest ratio of the disease, IVSII-1 is in the second place and IVSI-5 has a bit effect on the creation of thalassemia major patients. It is recommended to create a database of mutations in ß-thalassemia patients to find appropriate therapeutic solutions. [ABSTRACT FROM AUTHOR]

The increasing antibiotic resistance of Helicobacter pylori infection is a globally urging problem. To investigate the H. pylori resistance situation in Nanjing, China, we enrolled patients in Nanjing First Hospital from January 2018 to May 2021. H. pylori strains were isolated from patients who had at least one positive 13C-urea breath or rapid urease result. Subsequently, we performed antibiotic susceptibility tests on the isolated strains to clarithromycin, metronidazole, levofloxacin, amoxicillin, furazolidone and tetracycline. ARMS-PCR was conducted to determine H. pylori clarithromycin resistance gene mutation. Our results demonstrated that the primary resistance rates of metronidazole, clarithromycin, levofloxacin, amoxicillin, furazolidone and tetracycline were 67.19% (1417/2109), 35.99% (759/2109), 24.23% (511/2109), 0.76% (16/2109), 0.28% (6/2109) and 0.09% (2/2109), respectively. The resistance rates of metronidazole, clarithromycin and levofloxacin elevated significantly after treatment and the three antibiotics composed the majority of multi-resistance patterns. However, the resistance rates of amoxicillin, furazolidone and tetracycline were still in low levels after treatment. ARMS-PCR showed a rather good consistency with antibiotic susceptibility test in detecting clarithromycin resistance, with a kappa value of 0.79. Overall, this study revealed the latest complex situation of antibiotic resistance of H. pylori infection in Nanjing and offered suggestions on clinical medication for curing H. pylori. [ABSTRACT FROM AUTHOR]

• Associations of common genetic risk variants with GDM risk in the north Indian population were investigated. • Relative risk, population penetrance and attributable risk for risk allele variants was higher in GDM mother. • Four variants FTO, PPARG2, SLC30A8, and TCF7L2 were significantly associated with BMI, HbA1c and insulin. • Four variants FTO, PPARG2, SLC30A8, and TCF7L2 were significantly associated with GDM in North Indian population. 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. [ABSTRACT FROM AUTHOR]

IntroductionCo-inheritance of hereditary hemochromatosis (HFE) gene variants p. C282Y and p.H63D worsen iron overload in transfusion-dependent thalassemia. Data on the HFE gene variants in Sri Lankan patients with thalassemia have not been extensively studied. This study aimed to analyze the p.C282Y and p.H63D variants in transfusion-dependent beta (β) and HbE/β-thalassemia patients and establish an association between these variants and their serum ferritin levels.Materials and MethodsA total of 125 transfusion-dependent β-thalassemia major and HbE/β thalassemia patients were tested for the c.845G>A (p.C282Y) and c.187C>G (p.H63D) HFE gene variants using the multiplex Amplification Refractory Mutation System Polymerase Chain Reaction method. For phenotype-genotype correlation, serum ferritin levels, the erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) levels were measured. The standard descriptive statistics were used for data analysis.ResultsThe study cohort consisted of transfusion-dependent 123 β-thalassemia and 2 HbE/β-thalassemia patients. The p.C282Y variant was not detected in any patient; allele frequency for the wild type (c.845GG) was 100%. Twenty-three patients were heterozygous for the p.H63D variant allele, and the allele frequencies were c.187CC 91.8%, c.187CG 9.2%, and c.187GG 0%. The mean serum ferritin level was relatively higher (mean level 4,987 ng/ml) in the p.H63D heterozygous (c.187CG) group compared to the wild type (c.187CC) group (mean level 4,571 ng/ml), but the difference was statistically not significant (p = 0.865). Among the total study population, CRP, ESR, and serum glutamine aspartate transaminase (SGPT) were elevated in 9 (7.2%), 65 (52%), and 82 (65.6%) patients, respectively. Among the p.H63D c.187CG group, elevated CRP, ESR, and SGPT were present in 5 (5%), 15 (12%), and 18 (14.4%) patients, respectively. The detected sample number was low to correlate with the confounding effect of inflammatory disorders and liver damage on the serum ferritin levels.ConclusionsThe HFE gene variant p.C282Y is unlikely to cause iron overload in the Asian β-thalassemia patients; the rarity of this variant in the study cohort replicates the findings of other South Asian population studies of this variant. The presence of the p.H63D variant could be a potential risk factor for iron overload in the β-thalassemia patients. A more extensive cohort study is required to validate this finding.

The presence of single nucleotide variations in the coding region of micro-RNA (miRNA)-encoding genes plays a significant role in the expression and function of these molecules in oncogenesis and cancer. The association of rs2682818 in miR-618 with increased risk of breast cancer was investigated in the Iranian population. rs2682818/miR-618 was genotyped using amplification-refractory mutation system PCR (ARMS-PCR) in 200 healthy individuals and patients with breast cancer. The data revealed the presence of Hardy-Weinberg equilibrium (HWE) for this marker. The frequency of alleles C and A was 70% and 30%, respectively, in healthy individuals; the frequency of alleles C and A was 44% and 56%, respectively, in patients with breast cancer. Analysis of odd ratios showed that the rs2682818/miR-618 polymorphism is associated with increased probability of breast cancer and is statistically significant (OR=2.97, P=0.0003). The data suggest that rs2682818/miR-618 could be considered a novel marker of increased risk of breast cancer.

Asthma (AS) is one of the most important health problems that affect people worldwide. IL-33, one of IL-1 cytokine family members plays and important role in asthma throughout affecting many pathways. The aim of the study was to estimate and genotyped asthma patients according to IL-33. Serum IL-33 and IL-33 (rs1342326T/G) were estimated in asthma patient vs healthy controls using ELISA and ARMS-PCR, respectively. The majority of AS patients were in their fourth decade of their life with the dominancy of females over males. AS patients have a significant high level of IgE and a non-significant level of IL-33. The heterozygote genotype (TG) of (IL-33 rs1342326) was significantly associated with asthma and AS patient with this genotype have a significant high level of IL-33. In conclusion, AS patients with IL-33 TG genotype have the highest level of IL-33 suggesting have more sever asthmatic symptoms making it a good candidate for therapeutic purposes. [ABSTRACT FROM AUTHOR]

This study dealt with IL-13 1024 (C/T) gene genotyping among patients with Thyroid goiter in Iraq. Forty blood samples from patients with Thyroid goiter were collected and compared with 30 healthy persons as controls. The genotyping results of IL-13 1024 (C/T) gene using ARMS-PCR revealed presence TT, CC and CT genotypes beside T and C alleles. The T allele and TT genotype frequency were higher in Thyroid goiter patients compared to the same genotype and allele in healthy persons (P = 0.060). These increasing results were related with increasing risk factor of Thyroid goiter (odds ratio [OR] 2.15; 95% confidence interval [CI] 0.99-71.4). No significant differences between genotypes for Thyroid goiter patients and controls were revealed by using Hardy-Weinberg distribution. In conclusion, Thyroid goiter increasing risk was related with the TT and TC genotypes and T allele and these are showed as etiological fraction (EF) with risk having Thyroid goiter, while the CC genotype ratio percentage in healthy persons was higher in comparisons with Thyroid goiter patients suggesting the CC genotype have preventive fraction (PF). [ABSTRACT FROM AUTHOR]

Single nucleotide polymorphisms (SNPs) are one of the most common and abundant class of molecular markers present in the genome of many organisms. The current study represents the first attempt to investigate the natural variations in the RK-MTases genes; Ribosomal N-lysine methyltransferase 1 (RKM1) and Ribosomal N-lysine methyltransferase4 (RKM4) in Sordariafimicola using SNP markers. A total seven SNPs in the RKM1 gene and nine in RK.X14 gene were identified. A subset of SNPs were unique in SFS strains and others were fixed in the NFS strains of S.fimicola. These polymorphisms might be adaptive in stressful environmental conditions. Genotyping of eight SNPs of RK-MTases genes of S.fimicola was accomplished by designing allele specific primers via amplification refractory mutation system-PCR (ARMS-PCR) yielding amplicons of different sizes. This study concluded that SNP markers are an efficient and informative marker system in S.fimicola. Most of the studied SNPs are non-synonymous substitutions, which might underpin functional differences in their protein products. [ABSTRACT FROM AUTHOR]

This study aims to reveal the genotypic structure of four cattle breeds; Holstein (HS), Turkish Grey Steppe (TGS), Anatolian Black (AB) and East Anatolian Red (EAR), raised in Turkey in terms of CD14, MBL, ITGB6, SLC11A1 and TLR2 genes and to evaluate their usefulness in Marker Assisted Selection (MAS). It also assesses whether the loci associated with resistance to diseases are suitable for phylogenetic analysis. Desired alleles and/or genotypes were detected in native Turkish cattle breeds at different frequencies in terms of polymorphisms of CD14, MBL, ITGB6, SLC11A1 and TLR2 genes which were previously reported to be associated with mastitis, foot-and-mouth disease and tuberculosis. These variations offer opportunities to improve selection strategies against diseases in the future. These results preliminary indicate that associated studies between these variations and disease resistance in native Turkish cattle breeds should be conducted. On the other hand, phylogenetic tree constructed based on genetic distance clearly separated native Turkish cattle breeds from HS breed. The gene regions related to diseases can be used to distinguish native cattle breeds from exotic ones. [ABSTRACT FROM AUTHOR]

Single nucleotide polymorphism (SNP) is hereditary change in a DNA sequence that occurs when a single nucleotide in a genome is modified; SNPs are usually considered to be point mutations that have been evolutionarily successful enough to recur in a significant proportion of the population of a species. The ability of certain individuals to respond appropriately to Toll-like receptor (TLR) ligands may be impaired by single-nucleotide polymorphisms (SNPs) within TLR genes, terminating in a modified susceptibility to infectious or in?ammatory disease that might contribute to the pathogenesis of complex diseases. Out of the 400 clinical samples of blood collected from calves and lambs suffering from diarrhea, the samples originated from Babylon Veterinary Teaching Hospital and Veterinary Clinics during the period from October 2018 to January 2019, 200 samples were suffering from diarrhea (100 samples of calves and 100 samples of lambs), and 200 samples were the control groups. Genotyping of TLR4 polymorphisms was carried out by using amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) technique. For calves' TLR4 (rs8193046) gene polymorphisms, the results showed there were significant differences in genotypes and alleles frequencies between the diarrheic cases and control groups (p < 0.05). GG homozygous genotype was overrepresented among diarrheic calves. The frequencies of G allele was higher in diarrheic calves compared to control groups. The calves' GG homozygous genotype was significantly associated with increased susceptibility to Colibacillosis in calves. For lamb-TLR4 (rs160202325) gene polymorphism, AG heterozygous genotype was overrepresented among the cases as of 20 (52.63%), whereas AA was obviously more presented among control individuals as of 6 (60%). There were no difference between diarrheic cases and control groups for allele frequencies.

APAF1 is an autosomal recessive inherited mutation, associated with Holstein haplotype 1 (HH1) and characterized by a substitution of cytosine for a thymine (c.1741C>T) in chromosome 5. The mutation causes fetal and embryonic loss, between 60 and 200 days of gestation, and reduced conception rate. The ARMS-PCR is considered a simple and low-cost method to determine single nucleotide polymorphism (SNP) with no need for genetic sequencing of the animal genome. This study aimed to verify the allelic frequency of APAF1 mutation in Brazilian Holstein cattle. A total of 248 Holstein DNA samples (210 cows and 38 bulls) were analyzed, and synthetic genes were manufactured to validate the primers developed by the authors. All animals assessed in this study were classified as wild-type for APAF1 mutation. The primers and protocol developed for the ARMS-PCR technique work with 100% specificity and efficiency since the amplicon formations are as expected according to the genotypes. In conclusion, the mutation responsible for APAF1 was not detected in the Brazilian Holstein cattle population assessed in this prevalence study, although it is not possible to affirm that APAF1 does not occur in Brazilian Holstein animals. The tetra-primer ARMS-PCR protocol for APAF1 mutation that has been validated here may be a relatively simple and economical method to determine the animals' genotype.

Background and Aim: Primary Ovarian Insufficiency (POI) is defined by the occurrence of menopause before the age of 40 years. It is often associated with cardiovascular disease (CVD). The purpose of this study was to explore the relationship between POI-associated genotypes cardiometabolic disorder risk factors.Methods: One hundred seventeen women with POI and one hundred eighty-three healthy women without POI were recruited in this study. DNA was extracted and analyzed using ASO-PCR or Tetra ARMS-PCR. Lipid profiles were also assessed.Results: Multivariate logistic regression analysis showed that individuals with GG vs. TT genotype of the rs1046089 SNP were more likely to have a higher serum LDL (p = 0.03) compared to the control group. There was also a significant association between low serum HDL and rs2303369 and rs4806660 SNP genotypes in the POI group. In the POI group, the percentage of those with high total cholesterol was lower in those with a CC genotype compared to those with a TT genotype (p = 0.03).Conclusion: Some SNPs reported to be associated with POI appear to be independently associated with dyslipidemia. These results may be helpful to identify subjects with POI who may be susceptible to CVD. [ABSTRACT FROM AUTHOR]

The main purpose of this study was to work out the relationship between different polymorphisms of PAI (Plasminogen Activator Inhibitor type 1) 4G/5G and XIII Val34Leu genes in women with first-semester recurrent miscarriage (RM) syndrome. Recurrent miscarriage (RM) is an obstetric challenge. Polymorphisms of factor XIII (FXIII)and plasminogen activator inhibitor-1 (PAI-1) may cause an imbalance between coagulation and fibrinolysis that can end in RM. This case-control study enclosed 48 women with at least two or three abortions and 50 women with at least one pregnancy as a control.DNA molecule was extracted from peripheral blood samples by the phenolchloroform methodology. Different variants of the two genes were amplified by Amplification Refractory Mutation System - Polymerase chain reaction (ARMS-PCR) method. Finally, we analyzed allele frequencies and genotypes, Odd Ratios, Chi-square, Fisher's and Students T-test for the data. During this study, it has been found that 50% of the case population have the normal genotype for the PAI-1 (rs1799762) gene, 31.25% had a heterozygous genotype and 18.75% had a mutant homozygote. The frequency of the mutated allele in the patient population compared to the controls have p-values <0.001 and it is statistically significant for this allele (OR = 0.068, p-values <0.001, CI = 0.014-0.322). In contrast, no significant results were observed for the factor XIII (rs5985) gene and this variant was considered in this population as an ineffective polymorphism on recurrent miscarriage syndrome (p-values = 0.238). Finally, it is suggested that other variants of the given gene should be examined in Golestan. [ABSTRACT FROM AUTHOR]

XPG protein is a crucial component of the nucleotide excision repair pathway. Various single nucleotide polymorphisms of XPG gene have been reported to modulate colorectal cancer susceptibility. Therefore, this case control study evaluated the association of XPG (rs1 047 768 T>C) polymorphism with risk of colorectal cancer. In this study total 175 individuals comprising of age matched one hundred pathologically confirmed colorectal cancer cases and seventy-five controls were genotyped for XPG (rs1 047 768 T>C) polymorphism. Genotyping was accomplished with "Tetra amplification-refractory mutation system (ARMS) PCR" and PCR products were electrophoretically resolved on agarose gel. To validate the results 10% samples were re-analysed for XPG genotyping. Demographic factors were represented by mean ± SD. Multivariate logistic regression analysis was applied to compute odds ratio and confidence interval considering association between genotypes, demographic factors and colorectal cancer risk. Results were computed by MedCalc and SPSS version 24. Results showed significant association of XPG rs1 047 768—T>C genotype (OR: 7.51, CI: 1.63–35.02) and increased risk of colorectal cancer. Additionally, smoking and family history were significant contributors in colorectal cancer development. In summary, XPG (rs1 047 768 T>C) polymorphism is a low susceptibility penetrance gene for colorectal cancer and has never been screened before in Pakistani population. [ABSTRACT FROM AUTHOR]

Enteritis caused by CPV-2 antigenic variants (CPV-2a, 2b, and 2c) is frequently reported in dogs worldwide leading to significant morbidity and mortality. Here, we describe about a simple, single-step, ARMS-PCR strategy targeting the mutant 426 amino acid of VP2 to differentiate CPV-2 antigenic types. A total of 150 fecal samples were subjected to ARMS-PCR of which 18 were typed as CPV-2a, 79 were typed as CPV-2b, and 6 were typed as CPV-2c. The ARMS-PCR results were validated by randomly sequencing partial VP2 gene of 14 samples. Phylogenetic analysis of partial VP2 gene sequencing of each of the CPV-2 variants revealed that CPV-2a and CPV-2b isolates formed a separate clade of Indian lineage, while CPV-2c shared common evolutionary origin with Asian lineage. The developed technique is first of its kind, one-step, rapid, sequencing independent method for typing of CPV-2 antigenic variants. [ABSTRACT FROM AUTHOR]

• The Super-ARMS method has better sensitivity and specificity than ARMS-PCR. • Super-ARMS can meet the clinical needs of ESR1 mutation detection. • ESR1 mutations are associated with AIs use and tumor metastasis. • The frequency of ESR1 mutation increased with the increase of metastatic sites and the line of endocrine therapy. ESR1 mutation is an important mechanism of drug resistance and recurrence in hormone receptor-positive breast cancer patients during AI treatment. Patient could still benefit from treatment with fulvestrant after ESR1 mutated. At present, there is still no suitable method to detect ESR1 mutation in plasma as clinical promotion method. We aim to improve from ARMS-PCR to get a method with higher sensitivity but no additional cost is incurred. We designed new primers for ESR1. Then positive and negative standard sample was used for sensitivity and specificity tests. Lastly, we collected patient peripheral blood sample and analyzed the performance of Super-ARMS in plasma ctDNA samples. A total of 207 patients were enrolled in this study, including 142 prime breast cancer (PBC) patients and 65 metastasis breast cancer(MBC) patients. The mutation rate was as high as 27.9%(12/43) in MBC patients with AI treatment. But only 2.97%(3/101) in PBC patients with AI and 0% in both MBC or PBC patient without AI. There was no significant difference in Super-ARMS results compared with DDPCR method. Super-ARMS is a method that has sensitivity close to DDPCR and has the convenience and low price of ARMS-PCR for plasma ctDNA ESR1 mutation detection. It has obvious advantages compared with other method such NGS and DDPCR as clinical promotion method. [ABSTRACT FROM AUTHOR]

Minor deviations in the script of human life (the genetic code) are known to have major consequences on human health and well-being. Even substitution of a single nucleotide (single nucleotide polymorphism — SNP) in the gene sequences can cause diseases. SNP genotyping refers to a variety of techniques that allow simultaneous detection of such changes that can be associated with the pathophysiology of different diseases. However, most of the current, cutting-edge SNP genotyping technologies are complex, expensive, and require post-PCR manipulation. The Tetra-Amplification-Refractory Mutation System-Polymerase Chain Reaction (T-ARMS-PCR) method is a simple tool that meets modern diagnostic laboratories' expectations. It is a fast, reliable, and economical method of genotyping for genetic diseases such as β-thalassemia. [ABSTRACT FROM AUTHOR]

Abstract The goal of this study was to evaluate the feasibility of detecting Helicobacter pylori clarithromycin resistance in gastric mucosa using the amplification refractory mutation system combined with quantitative real‐time PCR (ARMS‐PCR). Gastric mucosal specimens (150) were collected from patients who were unsuccessfully treated for H. pylori eradication. Each specimen was divided into 2 samples. One sample was used to extract genomic DNA and detect any gene mutations of H. pylori produced by ARMS‐ PCR. Sequencing was used to assess the accuracy of this method. The other sample was used to culture H. pylori. The E‐test minimum inhibitory concentration (MIC) was used to assess clarithromycin resistance. The results were compared with a paired chi‐square test to validate the coincidence rate among the 3 methods. The coincidence rate between the sequencing and ARMS‐PCR results was 98.7%, thus verifying the accuracy of ARMS‐PCR. E‐tests detected 144 clarithromycin resistance cases, including 45 sensitivity cases; the resistance rate was 70%. The coincidence rate between the results of the E‐test and ARMS‐PCR was 97.1%, and no significant difference between the 2 methods was observed. ARMS‐PCR is a simple and fast method that has high sensitivity and specificity and can be used to detect the clarithromycin resistance of H. pylori in gastric mucosa. ARMS‐PCR is expected to be used to study drug resistance mechanisms and use in assays of individual therapies for H. pylori eradication.

Purpose: The present study was designed to screen the genetic polymorphisms and expression profiling of CEP-152 and CEP-63 genes in brain tumor patients. Methods: The amplification refractory mutation system PCR technique (ARMS-PCR) was used for mutation analysis using 300 blood samples of brain tumor patients and 300 overtly healthy controls. For expression analysis, 150 brain tumor tissue samples along with adjacent uninvolved/normal tissues (controls) were collected. Results: A significantly higher frequency of the mutant genotype of the CEP-152 single nucleotide polymorphism (rs2169757) and CEP-63 single nucleotide polymorphisms (rs9809619 and rs13060247) was observed in patients versus overtly healthy controls. The authors' results showed highly significant deregulation of CEP-152 (p < 0.0001) and CEP-63 (p < 0.0001) in glioma/meningioma tumor tissues versus adjacent normal tissue. Conclusion: The present study showed that variations in CEP-152 and CEP-63 genes were associated with an increased risk of brain tumor. [ABSTRACT FROM AUTHOR]