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.

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.

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.

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.

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.

Impact of IL-6 and IL-1ß Gene Variants on Non-small-cell Lung Cancer Risk in Egyptian Patients.

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-1ß 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-1ß 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-1ß 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-1ß variants have a great impact on NSCLC development in the Egyptian population; thus, it may be a supportive guide for earlier NSCLC prevention.

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.

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.

Association of CDKAL1 gene polymorphism (rs10946398) with gestational diabetes mellitus in Pakistani population.

BACKGROUND: CDK5 regulatory subunit associated protein 1 like 1 (CDKAL1) encodes a tRNA modifying enzyme involved in the proper protein translation and regulation of insulin production encoded by the CDKL gene. Sequence variations in the CDKAL1 gene lead to the misreading of the Lys codon in proinsulin, resulting in decreased glucose-stimulated proinsulin production. Various polymorphic sequence variants of the CDKAL1 gene such as rs7754840, rs7756992, rs9465871, and rs10946398 are reported to be associated with type 2 diabetes mellitus and gestational diabetes mellitus (GDM) incidence. One of these single nucleotide polymorphisms i.e., rs10946398 has been reported to impact the risk of GDM and its outcomes in pregnant women of different ethnicities i.e., Egypt, Chinese, Korean, Indian, Arab, and Malaysian. Numerous findings have shown that rs10946398 overturns the regulation of CDKAL1 expression, resulting in decreased insulin production and elevated risk of GDM. However, there is no data regarding rs10946398 genotype association with GDM incidence in our population. METHODOLOGY: In this study, 47 GDM patients and 40 age-matched controls were genotyped for rs10946398 CDKAL1 variant using Tetra primer Amplification Refractory Mutation System Polymerase Chain Reaction (Tetra ARMS-PCR). RESULTS: Analysis of the results showed the significant association of the C allele of CDKAL1 SNP rs10946398 (χ2 = 0.02 p = 0.001) with the risk of GDM development. Conclusively, the results support the role of SNP i.e., rs10946398 of CDKAL1 gene in GDM development in Pakistani female patients. However, future large-scale studies are needed to functionally authenticate the role of variant genotypes in the disease pathogenesis and progression.

Association of single nucleotide polymorphisms in heat stress protein 70 (HSP70) and 90 (HSP90) with the susceptibility of Pakistani sheep breeds to hemoparasitic infections.

The present study was designed to report the genotypic and allelic frequency of single nucleotide polymorphism (SNP) at 222 G > A in HSP70 and at ex6-7390T22G in the HSP90 gene of 204 sheep (Baluchi = 11, Kajli = 29, Latti = 06 and Mundri = 158) enrolled from District Rajanpur in Punjab and to report the susceptibility of these sheep to the blood-borne parasitic infection. The tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) approach revealed a significant variation (p < 0.001) in the genotype frequency of four enrolled sheep breeds at SNP 222 G > A in the HSP70 gene while the allelic frequency remained unaffected (p = 0.08). In all sheep breeds, GG (wild) genotype was most common. T-ARMS-PCR analysis revealed a similar trend for ex6-7390T22G in the HSP90 gene and it was observed that sheep had significantly higher wild-type (GG) (p < 0.05) at the studied SNPs. Studied epidemiological factors (sex and sampling sites) were not found associated with both SNPs. Chi-square test revealed that no specific genotype and allelic frequency at 222 G > A in HSP70 and at ex6-7390T22G in the HSP90 gene of the enrolled sheep breed was associated with the susceptibility to blood-borne parasitic infection (p > 0.05). In conclusion, we are reporting that Pakistan is blessed to have majority of sheep, from all breeds, having wild genotype at analyzed SNPs in heat stress genes. We highly recommend the genotypic screening of sheep before their selection as breeders to reduce the possibility of having sheep with polymorphic genotypes at 222 G > A in HSP70 and at 7390T22G in HSP90 genes that will improve the profitability and sustainability of animal production systems in Pakistan.

The effect of insulin-like growth factor 1 receptor gene single nucleotide polymorphism on growth and milk production traits in two native Indian tropical goat breeds.

Insulin like growth factor1(IGF-1) is an essential growth factor that mediates the growth-promoting functions of pituitary growth hormone. Insulin like growth factor 1 receptor (IGF1R) is a tyrosine kinase receptor that mediates the actions of IGF1. Therefore, IGF1R is a candidate gene for examining SNPs linked with growth and production traits. The objective of this study was to detect the c.546 + 179170A > T transversion in intron 2 of the gene encoding IGF1R in two goat breeds, Attappady Black and Malabari, and examine the association of this polymorphism with growth and milk production. For the identification of the SNP, the T-ARMS-PCR was utilized. All three genotypes were present in the two investigated breeds. The polymorphism was found to be significantly (p < 0.05) linked with growth traits. At birth, 3 and 6 months of age, Attappady goats with the AT genotype had significantly (p < 0.05) higher body weights than those with the AA and TT genotypes. Malabari goats with the AT genotype had significantly (p < 0.05) higher body weights at birth and at 3 months of age. The genotypes of the IGF1R gene had no effect on total or peak milk production. Therefore, this SNP could be used as a molecular marker in selection of meat-producing goat breeds.HIGHLIGHTSc.546 + 179170A > T IGF1R transversion was detected using T-ARMS-PCR in two indigenous goat breeds from Kerala.Attappady Black and Malabari goat breeds of Kerala possessed all the three genotypesIn these breeds, there was a significant correlation between this SNP, c.546 + 179170A > T IGF1R, and body weight.In both the breeds, there was no association between this SNP and milk production traits.

Association of genotypes at rs438228855 in bovine SLC35A3 receptor gene of Pakistani cattle with the susceptibility to develop complex vertebral malformation.

The aim of this study was to report the genotype and allelic frequency at rs438228855 (G > T) in SLC35A3 receptor gene and its association with a complex vertebral malformation (CMV) in the enrolled Pakistani cattle. Our results indicated that allelic and genotype frequency at rs438228855 varied non-significantly (p > .05) among the three enrolled cattle breeds. GT (heterozygous) genotype was most abundant (0.54) followed by GG (wild type) genotype (0.45) while the mutant genotype (TT) was not observed among the enrolled cattle. It was observed that the Holstein Friesian breed had more GG (wild) than GT (heterozygous) genotypes while Sahiwal and cross cattle breed had more heterozygous (GT) combination at rs438228855 than the wild (GG) genotype. Significant variations in white blood cell count, % lymphocytes, red blood cell count, % monocytes, haemoglobin, mean corpuscular volume and mean corpuscular haemoglobin concentration were observed when compared between the enrolled cattle breeds. Most of the studied haematological parameters showed no association with the genotype at rs438228855. In conclusion, the heterozygosity at rs438228855 is not limited to the Holstein Friesian breed as local Sahiwal and crossbred cattle had also higher heterozygosity at rs438228855. We recommend that animals must be genotyped for rs438228855 before their selection as breeders to prevent economic losses.

Impact of cytokines genes polymorphisms and their serum levels on childhood asthma in Egyptian population.

BACKGROUND: Asthma is chronic immune-mediated airway inflammation, and it is affected by a complex network of interacting cytokines. To date, the exact role of each cytokine and its genetic polymorphisms in childhood asthma development and its severity has remained poorly understood. The purpose of this study was to explore potential roles of four cytokine genes polymorphism and serum levels l [(T helper-2 (Th2) cytokine); Interleukin-4 (IL-4) 590, (Th3 cytokine); and transforming growth factor ß1 (TGF-ß1) 509T; (Th17) including tumor necrosis factor-alpha (TNF-α), and IL17A rs8193036] in childhood asthma risk and control in Egyptian children, for the 1st time. MATERIALS AND METHODS: This case-control study included two children subgroups; Group1 included 216 non-asthmatic controls and (Group 2) 216 cases diagnosed with asthma (clinically and spirometry-based) were classified as controlled, partly controlled, and uncontrolled. Polymorphisms of TGF-ß1-509, IL-4 590, and TNF-α-308 genes were detected using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). IL-17 was genotyped using tetra-primer amplification refractory mutation system polymerase chain reaction (ARMS-PCR). Serum cytokines levels were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Serum total IgE, TGF-ß1, IL4, TNF-α, and IL17A levels were significantly higher in asthmatic compared to controls. Also, significant increases in serum total IgE, IL-4, TGF-ß1, and TNF-α levels are combined with poor asthma control, while no significant IL17A changes. There were significant changes of IL-4-590, TNF-α-308, and IL17A genotypes and allele distributions between asthmatic and controls groups as well as different asthma control levels; while no impact of TGF-ß1 SNP on asthma risk and control level. Four cytokines SNPs affected their serum levels among asthmatic patients. CONCLUSION: There are impacts of cytokine gene polymorphisms (IL-4-590, TNF-α-308, and IL17A); but not TGF-ß1 on asthma susceptibility and poor asthma control in Egyptian children.

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

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

Polymorphisms in IL-17A Gene and Susceptibility of Colorectal Cancer in Bangladeshi Population: A Case-Control Analysis.

OBJECTIVE: Interleukin-17A (IL-17A) genetic polymorphisms are associated with multiple cancer types, including colorectal cancer (CRC). However, no previous study was performed in the Bangladeshi population to evaluate the association. Our study aimed to find the association between two IL-17A variants (rs10484879 C/A and rs3748067 G/A) and susceptibility of CRC. METHODS AND MATERIALS: This retrospective case-control study comprised 292 CRC patients and 288 age, sex, and BMI matched healthy volunteers. Genotyping of both variants was done by the tetra-primer ARMS-PCR method, and the results were analyzed by the SPSS software package (version-25.0). RESULTS: Logistic regression analysis indicated that in case of IL-17A rs10484879 polymorphism, AC and AA genotype carriers showed 2.44- and 3.27-times significantly increased risk for CRC development (OR = 2.44, P = .0008 and OR = 3.27, P = .0133, individually). A significant association was also observed for AC + AA genotype (OR = 2.58, P = .0001). Again, over-dominant and allelic model revealed statistically significant link to CRC risk (OR = 2.13, P = .0035 and OR = 2.22, P = .001). For rs3748067 polymorphism, AG and AA genotype carriers showed 2.30- and 2.45-times enhanced risk for CRC (OR = 2.30, P = .005 and OR = 2.45, P = .031). A statistically significant association was also observed for AG + AA genotype (OR = 2.35, P = .001), over-dominant model (OR = 2.05, P = .014), and allelic model (OR = 2.11, P = .0004). CONCLUSION: This study highlights that IL-17A rs10484879 and rs3748067 polymorphisms may be associated with CRC development. However, further functional research with larger samples may reveal more statistically significant outcomes.

The relationship between genetic variants associated with primary ovarian insufficiency and lipid profile in women recruited from MASHAD cohort study.

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.

Association between TBXT rs2305089 polymorphism and chordoma in Iranian patients identified by a developed T-ARMS-PCR assay.

BACKGROUND: Chordoma is a locally aggressive bone tumor with a high capability of recurrence. Because chordoma often occurs at critical locations next to neurovascular structures, there is an urgent need to introduce validated biomarkers. T-box transcription factor T (TBXT; OMIM: 601397) plays an important role in the pathogenesis and survival of chordoma cells. METHODS: Herein, we aimed to show whether rs2305089 polymorphism is correlated with chordoma in the Iranian population. In order to detect rs2305089, tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) was used. In total, 19 chordoma patients and 108 normal healthy individuals were recruited and screened using T-ARMS-PCR. The results were subsequently validated by Sanger sequencing. RESULTS: The genotype distributions and allele frequencies were significantly different among the patient and healthy groups (p-value <0.05). The A allele of rs2305089 showed a significant positive association with chordoma risk (p-value <0.05). DNA sequencing verified the T-ARMS-PCR results as well. This study demonstrated the association between TBXT rs2305089 and chordoma in an Iranian population using a simple, accurate, and cost-effective T-ARMS-PCR assay. CONCLUSIONS: Our results were in line with those of previous studies showing that TBXT rs2305089 is associated with chordoma development. We also developed an efficient T-ARMS-PCR assay to determine the genotype of rs2305089.

Tetra-primers ARMS-PCR Based Association Analyses of Synonymous and Intronic Variants in the ADAM12 Gene with Susceptibility to Knee Osteoarthritis: A Case-Control Study.

Genetic variations in a disintegrin and metalloprotease 12 (ADAM12) gene may contribute to develop Osteoarthritis (OA) that is characterized by cartilage matrix degradation and osteophytes formation. Therefore, the aim of present study was to analyze the association between the ADAM12 gene variants and knee OA predisposition. Tetra-primers ARMS-PCR was employed, to genotype the ADAM12 gene polymorphisms (rs1044122 and rs1871054) in 400 knee OA patients and equal number of age-matched controls. The association between ADAM12 gene variants and OA susceptibility was estimated using the Chi-square, logistic regression, haplotypes and linkage analyses. A significant association of rs1044122 (genotype: χ2 = 18.94; P < 0.001, allele: χ2 = 19.10; P < 0.001) and rs1871054 (genotype: χ2 = 10.04; P = 0.007, allele: χ2 = 10.57; P = 0.001) was observed with increased OA susceptibility. The variant genotype of rs1044122 increased OA risk more than twice [odds ratio (OR) 2.20; P = 0.001] and the risk was higher in females (OR 2.43; P = 0.001). The variant genotype of rs1871054 was perceived to almost double the risk in females (OR 1.97; P = 0.003). Moreover, a significant association of rs1044122 and rs1871054 under the additive genetic model (P < 0.001 and P = 0.002, respectively) was observed. The targeted ADAM12 gene polymorphisms, showed significant association with knee OA susceptibility. Females harboring the polymorphisms might be at risk. Besides, the haplotype CC of rs1044122 and rs1871054 in the ADAM12 gene may double knee OA risk. These findings may help in determining the etiology of OA and recognizing the people at risk of developing knee OA.

Characterization of the genetic polymorphism linked to the ß-casein A1/A2 alleles using different molecular and biochemical methods.

The 2 major subvariants of ß-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 ß-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.

Association of vitamin D receptor polymorphisms with cardiometabolic conditions in Pakistani population.

Apart from bone related effects, vitamin D has roles in immune modulation, hypertension, diabetes and cardiovascular diseases. Metabolic functions of vitamin D are mediated after binding with vitamin D receptor (VDR). VDR polymorphisms affect its physiological functions. Several VDR single nucleotide polymorphisms (SNPs) are reported previously. However, VDR polymorphisms causing influence on cardiovascular and metabolic disorders have not been investigated in Pakistani population so far. Therefore, present study was conducted to evaluate the role of VDR polymorphisms (rs2228570 and rs7975232) in the pathobiology of cardiometabolic disorders. In all, 400 cardiometabolic patients and 226 healthy control human adults were enrolled from Faisalabad, Pakistan. Biochemical parameters (serum glucose, liver function test, renal function test and lipid profile) were analyzed by standard kit methods. Genetic analysis was done by ARMS-PCR assay. Data was analyzed in SPSS v20. Regression analysis revealed that GG and AG genotypes of rs2228570 A>G polymorphism significantly increased the risk of hypertension in cardiovascular patients by 5.29 and 5.94 times respectively (GG: OR=5.29, 95% CI=1.63-17.2, p=0.005; AG: OR=5.94, 95% CI=1.70-20.7, p=0.005). However, rs7975232 C>A polymorphism was not correlated with cardiometabolic conditions. In conclusion, GG and AG genotypes of VDR SNP rs2228570 significantly contribute for hypertension in cardiovascular disease patients.