Optimization of an allelic discrimination real-time RT-PCR assay for detection of H275Y oseltamivir resistance gene mutation among influenza A(H1N1)pdm09 patients from 2020 to 2022.

Influenza virus is known to cause mild to severe respiratory infections and is also prone to genetic mutations. Of all the mutations, neuraminidase (NA) gene mutations are a matter of concern, as most approved antivirals target this protein. During the 2020 influenza season, an emergence of mutation in the NA gene, affecting the binding of the World Health Organization (WHO)-recommended probes to the specific site of the NA gene, was reported by our group. As a result of this mutation, the WHO-recommended allelic discrimination real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was unable to detect wild-type (H275) or mutant oseltamivir-resistant (Y275) strains of influenza A(H1N1)pmd09 viruses. In the current study, the WHO-recommended probes were redesigned according to the mutation in the probe binding site. Fifty undetermined samples (2020-2021) from the previous study were retested with the newly designed probes and found to be positive for H275 and/or Y275. The results obtained were similar to the Sanger sequencing results from the previous study, suggesting that the redesigned probes were efficient in discriminating between wild-type and mutant-type viruses. Furthermore, 133 samples from 2022, making a total of 183 samples (2020-2022), were tested using improved allelic discrimination real-time RT-PCR, and the overall prevalence rate of oseltamivir resistance in 2020-2022 was found to be 0.54%.

Detection of H275Y oseltamivir resistance gene mutation among Influenza A(H1N1)pdm09 patients by allelic discrimination real-time RT-PCR.

Influenza viruses can mutate genetically and cause a range of respiratory ailments. The H275Y mutation in the neuraminidase (NA) gene reduces the effectiveness of oseltamivir, a widely used drug for the treatment of Influenza A and B virus infection. The World Health Organization (WHO) recommends single-nucleotide polymorphism assays to detect this mutation. The present study aims to estimate the prevalence of H275Y mutation conferring oseltamivir resistance in Influenza A(H1N1)pdm09 virus among hospitalized patients from June 2014 to December 2021. Following the WHO protocol, allelic discrimination real-time RT-PCR was performed for 752 samples. Out of the 752 samples, 1 tested positive for Y275 gene mutation by allelic discrimination real-time RT-PCR. In samples of years 2020 and 2021, neither the H275 nor Y275 genotype was detected. Sequencing of the NA gene of all negative samples showed a mismatch between the NA sequence and the probes used in the allelic discrimination assay. Also, Y275 mutation was detected in only 1 sample from 2020. The prevalence of oseltamivir resistance was estimated as 0.27% among the Influenza A(H1N1)pdm09 patients during 2014-2021. The study highlights that the WHO-recommended probes for detecting H275Y mutation may not be useful to detect 2020 and 2021 circulating strains of Influenza A(H1N1)pdm09, emphasizing the need for continuous monitoring of mutations in the influenza virus.

TaqMan real time PCR for the Detection of the Gilbert's Syndrome Markers UGT1A1*28; UGT1A1*36 and UGT1A1*37.

Gilbert's syndrome is characterized by mild unconjugated hyperbilirubinemia. The key of this disease is a diminished activity of UDP-glucuronosyltransferase 1A1 (UGT1A1). TA insertion into the TATA box promoter region of the UGT1A1 gene on chromosome 2 is the genetic basis of Gilbert's syndrome (UGT1A1*28). An extra TA insert leads to eight (TA)8 repeats (UGT1A1*37) resulting in a further reduction of glucuronidation activity. A variant lacking one TA repeat (TA)5 (UGT1A1*36) has been identified. (TA)8 repeats (UGT1A1*37) and (TA)5 (UGT1A1*36) have been detected in Africans (frequency up to 0.07 and 0.08 respectively). We present a real time PCR method for genotyping the UGT1A1 (TA)n polymorphism (UGT1A1*28, UGT1A1*36, UGT1A1*37) using Taqman PCR on 7500 and cfx96 Real-Time PCR System. We present a real time PCR method for genotyping the UGT1A1 (TA)n polymorphism (UGT1A1*28, UGT1A1*36, UGT1A1*37) using Taqman PCR. About clinical validation, all 53 samples collected from patients referred for suspected Gilbert's syndrome were analyzed. We found 21 on the 53 patients (39.6%) were homozygotes (UGT1A1-TATA (TA)6) and referred as wild-type, 13 on the 53 patients (24.5%) were homozygotes (UGT1A1-TATA (TA)7) and referred as mutated, 1 on the 53 patients (1.9%) were homozygotes (UGT1A1-TATA (TA)8) and referred as mutated, 1 on the 53 patients (1.9%) were heterozygotes (UGT1A1-TATA (TA)7/8) and referred as mutated, 1 on the 53 patients (1.9%) were heterozygotes (UGT1A1-TATA (TA)5/6) and referred as mutated, and 16 on the 53 patients (30.2%) were heterozygotes (UGT1A1-TATA (TA)6/7). None were homozygotes UGT1A1-TATA (TA)5, homozygotes UGT1A1-TATA (TA)8, or heterozygotes with (TA)5 or (TA)8 alleles. The newly described technique represents a valid alternative method to sequencing, mainly due to its rapidity, easiness, and minor costs.

Functional Polymorphism Located in the microRNA Binding Site of the Insulin Receptor (INSR) Gene Confers Risk for Type 2 Diabetes Mellitus in the Bangladeshi Population.

Bangladesh has the second largest number of adults with diabetes in South Asia. Compelling evidence suggest that miRNAs contribute to the etiology of Type 2 diabetes mellitus (T2DM) by regulating many aspects of glucose homeostasis. Hence, we hypothesized that genetic polymorphisms in the diabetes-related miRNA target-binding sites could be associated with the risk of T2DM in Bangladesh. The reference Single nucleotide polymorphism (SNP) data from the Insulin Receptor (INSR) gene were downloaded from the ENSEMBL genome browser release 88 and further analyzed in silico for identifying SNPs with deleterious effect and clinical relationships. Further, case-control study using the microRNA-binding site polymorphism rs1366600 (T > C) located at the 3' UTR of the INSR gene was carried out in 217 T2DM patients and 237 healthy controls from Bangladesh. Genotyping was performed using the real time PCR based allele discrimination method. The results showed that the minor allele 'C' is associated with increased risk of T2DM [Odds ratio (OR) 1.87; 95% confidence intervals (CI) 1.28-2.74; P = 0.0010]. When we dissected our analysis to include the dominant model (CC + TC genotype against the TT genotype), we found that the CC and TC genotypes were associated with increased risk of T2DM in Bangladeshi population (OR 2.01; 95% CI 1.31-3.07; P = 0.0012). However, in recessive model (CC vs TT + TC); the effect was not statistically significant (OR 2.23; 95% CI 0.66-7.51; P = 0.1848). Stratification of our data based on the gender of the cases and controls showed similar degree of risk association with respect to different genotypes and alleles. Our study showed that the miRNA binding site polymorphism rs1366600 located at the 3'-UTR region of the INSR gene is associated with increased risk of T2DM in Bangladeshi individuals.

Toll-like receptor 7 gene single nucleotide polymorphisms and the risk for systemic lupus erythematosus: a case-control study.

BACKGROUND: Toll-like receptors (TLRs) are a family of pattern-recognition receptors which play a role in eliciting innate/adaptive immune responses and developing chronic inflammation. So, the aim of this study was to analyze the effect of TLR7 gene single nucleotide polymorphisms (SNPs) rs3853839 and rs179019 on systemic lupus erythematosus (SLE) susceptibility and to assess their relations with various clinical and laboratory data of the patients. METHODS: This is a case-control study including 50 SLE female patients and 50 healthy controls. TLR7 rs3853839 and rs179019 genotyping was performed using real-time polymerase chain reaction (PCR) TaqMan-based allelic discrimination assay. RESULTS: Regarding rs3853839, there was a statistically significant difference in the distribution of the genotypes between SLE patients and the control group in our study (P = 0.009). A significant association was detected between TLR7 genotypes (rs385389) and lupus nephritis (p = 0.021). Regarding rs179019, there was no statistically significant difference in the distribution of the genotypes between SLE patients and the control group in our study (P = 0.271) CONCLUSION: This study revealed the plausible role of TLR7 rs3853839 SNP in SLE in Egyptian women.

Detection of D151G/N mutations in the neuraminidase gene of influenza A (H3N2) viruses by real-time RT-PCR allelic discrimination assay.

Single nucleotide polymorphisms (SNPs) at D151 position of neuraminidase (NA) gene of influenza A (H3N2) virus has been associated with drug resistance and increased binding affinity. NA-D151G/N-substitutions of influenza A (H3N2) viruses are frequently induced and selected by culturing in Madin-Darby canine kidney (MDCK) cell lines. It is important to consider and exclude D151G/N mutants after isolation of influenza virus in MDCK cell line; since, the substitutions can highly influence the results of experimental research. The study aims to develop an allelic discrimination real-time reverse transcriptase polymerase chain reaction (RT-PCR) for the screening of D151G/N mutants. Thirty-six influenza A (H3N2) virus isolates were included and screened for D151G/N mutants using allelic discrimination assay. Out of the 36 isolates, 11 isolates (30.5%) were detected as heterozygous for D and G/N substitutions. Twenty-one (58.3%) isolates were identified as homozygous wild type and four isolates (11.1%) were undetermined. Isolates with substitutions at D151 position were sequenced by Sanger sequencing method. The present study demonstrates a rapid and convenient method for primary screening of the mutation after culturing of the influenza virus in MDCK cell lines in order to avoid potential misinterpretations of results and improve the quality of experimental research.

Association Between AIRE Polymorphisms rs870881(C>T), rs1003854(T>C) and Rheumatoid Arthritis Risk: A Hungarian Case-control Study.

BACKGROUND/AIM: Autoimmune regulator (AIRE) is a transcription factor that plays pivotal role in controlling autoimmunity. In the thymus, it supports the presentation of peripheral tissue antigens to developing T cells, where recognition of these self-antigens negatively selects the autoimmune naïve T-cells by central tolerance. Studies demonstrated that single-nucleotide polymorphisms (SNPs) in AIRE alter transcription and propagate clonal survival of autoimmune T cells, therefore increase susceptibility to autoimmune diseases. This study intended to identify SNPs in exon and intron sequences that determine AIRE transcription, where their genotypes are associated with rheumatoid arthritis (RA) risk and clinical parameters. PATIENTS AND METHODS: After a thorough in silico research, we enrolled 100 patients with RA and 100 healthy controls to analyze the association of SNP rs870881(C>T) and rs1003854(T>C) in AIRE coding sequence with RA risk by using five different genetic models and selected clinical parameters. Multiplex quantitative polymerase chain reaction was used to determine allelic discrimination of SNPs. RA risk was assessed by odds ratios (ORs) and confidence intervals (CIs). RESULTS: In a recessive model of rs878081, minor allele TT homozygotes were associated with RA (p=0.032, OR=5.44, 95%CI=1.16-25.52); in a recessive model of rs1003854, minor allele CC homozygotes were associated with RA (p=0.047, OR=4.84, 95%CI=1.02-23.02). Higher C-reactive protein (CRP) levels in patients with RA were significantly associated with minor allele homozygotes in recessive and codominant genetic models (p=0.029 and p=0.043, respectively) of rs1003854. CONCLUSION: Genotypes for minor alleles of rs878081 and rs1003854 might be involved in RA pathogenesis and risk prediction.

Angio-Long Noncoding RNA MALAT1 (rs3200401) and MIAT (rs1061540) Gene Variants in Ovarian Cancer.

The genotyping of long non-coding RNA (lncRNA)-related single-nucleotide polymorphisms (SNPs) could be associated with cancer risk and/or progression. This study aimed to analyze the angiogenesis-related lncRNAs MALAT1 (rs3200401) and MIAT (rs1061540) variants in patients with ovarian cancer (OC) using "Real-Time allelic discrimination polymerase chain reaction" in 182 formalin-fixed paraffin-embedded (FFPE) samples of benign, borderline, and primary malignant ovarian tissues. Differences in the genotype frequencies between low-grade ovarian epithelial tumors (benign/borderline) and malignant tumors and between high-grade malignant epithelial tumors and malignant epithelial tumors other than high-grade serous carcinomas were compared. Odds ratios (ORs)/95% confidence intervals were calculated as measures of the association strength. Additionally, associations of the genotypes with the available pathological data were analyzed. The heterozygosity of MALAT1 rs3200401 was the most common genotype (47.8%), followed by C/C (36.3%). Comparing the study groups, no significant differences were observed regarding this variant. In contrast, the malignant epithelial tumors had a higher frequency of the MIAT rs1061540 C/C genotype compared to the low-grade epithelial tumor cohorts (56.7% vs. 37.6, p = 0.031). The same genotype was significantly higher in high-grade serous carcinoma than its counterparts (69.4% vs. 43.8%, p = 0.038). Multivariate Cox regression analysis showed that the age at diagnosis was significantly associated with the risk of OC development. In contrast, the MIAT T/T genotype was associated with a low risk of malignant epithelial tumors under the homozygote comparison model (OR = 0.37 (0.16-0.83), p = 0.017). Also, MIAT T allele carriers were less likely to develop high-grade serous carcinoma under heterozygote (CT vs. CC; OR = 0.33 (0.12-0.88), p = 0.027) and homozygote (TT vs. CC; OR = 0.26 (0.07-0.90), p = 0.034) comparison models. In conclusion, our data provide novel evidence for a potential association between the lncRNA MIAT rs1061540 and the malignant condition of ovarian cancer, suggesting the involvement of such lncRNAs in OC development.

Are GES carbapenemases underdiagnosed? An allelic discrimination assay for their accurate detection and differentiation.

GES (Guiana Extended Spectrum) carbapenemases belong to "minor class A carbapenemases" and its prevalence could be underestimated due to the lack of specific tests. The aim of this study was to develop an easy PCR method to differentiate between GES ß-lactamases with or without carbapenemase activity, based on an allelic discrimination system of SNPs that encode E104K and G170S mutations, without need of sequencing. Two pair of primers and Affinity Plus probes, labeled with different fluorophores; FAM/IBFQ and YAK/IBFQ, were designed for each one of the SNPs. This allelic discrimination assay allows to detect in real time the presence of all type of GES- ß-lactamases, being able to differentiate between carbapenemases and extended-spectrum ß-lactamase (ESBL), through a quick PCR test that avoid costly sequencing approaches and could help to decrease the current underdiagnosis of minor carbapenemases that scape of phenotypic screenings.

Allelic Discrimination of Vitamin D Receptor Polymorphisms and Risk of Type 2 Diabetes Mellitus: A Case-Controlled Study.

(1) Background: Type 2 diabetes mellitus (T2DM) is one of the rapidly growing healthcare problems, and several vitamin D receptor (VDR) polymorphisms seem to modulate the risk of T2DM. Our research was designed to investigate the allelic discrimination of VDR polymorphisms and T2DM occurrence risk. (2) Methods: This case-control research included 156 patients with T2DM and 145 healthy control subjects. Most of the study population were males 56.6% vs. 62.8% in the case and control groups, respectively. Genotyping for VDR single nucleotide polymorphisms (SNPs), rs228570 (Fok1), rs7975232 (Apa1), and rs1544410 (Bsm1) was compared between both groups. (3) Results: There was a negative link between vitamin D levels and insulin sensitivity. A significant difference was noted in the allelic discrimination of VDR polymorphism rs228570 and rs1544410 between the study groups (p < 0.001). No difference was observed in the allelic discrimination of VDR polymorphism rs7975232 between the groups (p = 0.063). Moreover, T2DM patients had significantly higher levels of fasting blood sugar (FBS), glycated hemoglobin HbA1c, 2-h post-prandial blood sugar (PP), serum glutamic oxaloacetic transaminase (SGOT), serum glutamic-pyruvic transaminase (SGPT), total cholesterol, and triglycerides (p < 0.001), while High-Density Lipoprotein (HDL) Cholesterol (HDL-C) was significantly decreased (p = 0.006). (4) Conclusions: VDR polymorphisms had a positive association with T2DM risk among the Egyptian population. Further large-scale research using deep sequencing of samples is strongly urged to investigate different vitamin D gene variants and interactions, as well as the influence of vitamin D on T2DM.

Rapid and accurate identification of SARS-CoV-2 Omicron variants using droplet digital PCR (RT-ddPCR).

BACKGROUND: Some mutations in the receptor binding domain of the SARS-CoV-2 Spike protein are associated with increased transmission or substantial reductions in vaccine efficacy, including in recently described Omicron subvariants. The changing frequencies of these mutations combined with their differing susceptibility to available therapies have posed significant problems for clinicians and public health professionals. OBJECTIVE: To develop an assay capable of rapidly and accurately identifying variants including Omicron in clinical specimens to enable case tracking and/or selection of appropriate clinical treatment. STUDY DESIGN: Using three duplex RT-ddPCR reactions targeting four amino acids, we tested 419 positive clinical specimens from February to December 2021 during a period of rapidly shifting variant prevalences and compared genotyping results to genome sequences for each sample, determining the sensitivity and specificity of the assay for each variant. RESULTS: Mutation determinations for 99.7% of detected samples agree with NGS data for those samples, and are accurate despite wide variation in RNA concentration and potential confounding factors like transport medium, presence of additional respiratory viruses, and additional mutations in primer and probe sequences. The assay accurately identified the first 15 Omicron variants in our laboratory including the first Omicron in Washington State and discriminated against S-gene dropout Delta specimen. CONCLUSION: We describe an accurate, precise, and specific RT-ddPCR assay for variant detection that remains robust despite being designed prior the emergence of Delta and Omicron variants. The assay can quickly identify mutations in current and past SARS-CoV-2 variants, and can be adapted to future mutations.

Reliable and Fast Genotyping Protocol for Galactosylceramidase (Galc) in the Twitcher (Twi) Mouse.

Twitcher (Twi) is a neurological Krabbe disease (KD, or globoid cell leukodystrophy) spontaneous mutant line in mice. The genome of the Twi mouse presents a single nucleotide polymorphism (SNP), leading to an enzymatically inactive galactosylceramidase (Galc) protein that causes KD. In this context, mouse Twi genotyping is an essential step in KD research. To date, the genotyping method used is labor-intensive and often has ambiguous results. Here, we evaluated a novel protocol for the genotype determination of Galc mutation status in Twi mice based on the allele-discrimination real-time polymerase chain reaction (PCR). Here, DNA is extracted from Twi mice (n = 20, pilot study; n = 120, verification study) and control group (n = 10, pilot study; n = 30 verification study) and assessed by allele-discrimination real-time PCR to detect SNP c.355G>A. Using the allele-discrimination PCR, all of the samples are identified correctly with the genotype GG (wild-type, WT), GA (heterozygote, HET), or AA (homozygote, HOM) using the first analysis and no animals are not genotyped. We demonstrated that this novel method can be used to distinguish KD timely, accurately, and without ambiguity in HOM, WT, and HET animals. This protocol represents a great opportunity to increase accuracy and speed in KD research.

Development of Breeder-Friendly KASP Markers from Genome-Wide Association Studies Results.

Array-based SNP markers are commonly used in genome-wide association studies (GWAS) to identify genomic regions involved in important agronomical traits. However, conversion of these SNP markers into breeder-friendly kompetitive allele-specific PCR (KASP) markers for use in marker-assisted selection is often challenging. In this chapter we describe general considerations and successfully applied protocols for the conversion of Illumina array SNP markers into locus-specific KASP markers with a special emphasis and examples on how to overcome difficulties in polyploid wheat.

Pharmacogenomics of Methotrexate Pathway in Rheumatoid Arthritis Patients: Approach toward Personalized Medicine.

Background: Methotrexate (MTX) is one of the most common medications used for rheumatoid arthritis (RA) treatment. Single-nucleotide polymorphisms (SNPs) could potentially predict variability in therapeutic outcomes. Aim: This study aims to assess the impact of SNPs in genes encoding for the MTX pathway for predicting clinical and therapeutic responses to MTX in a cohort of Egyptian patients with RA. Subjects and Methods: Data from 107 Egyptian RA patients (aged 44.4 ± 11.4 years) treated with MTX monotherapy, for a duration of 3.7 ± 3.3 years, were collected. Genotypes of 10 SNPs from four different genes were analyzed using the allelic discrimination PCR technique. Results: The ATIC rs3821353 G/T (p = 0.034) and the C/T and C/C of SLC19A1 rs7279445 (p = 0.0018) were associated with a non-response to MTX, while DHFR rs10072026 C/T and C/C were associated with a good response (p < 0.001). Carriers of the ATIC rs382135 3 G (p = 0.001) and ATIC rs4673990 G (p < 0.001) alleles were more likely to develop RA, while the SLC19A1 rs11702425 T (p < 0.001) and GGH rs12681874 T (p = 0.003) allele carriers were more likely to be protected against RA. Carriers of the ATIC rs4673990 A/G genotype (p < 0.001) were at risk of developing RA, while carriers of the following genotypes were mostly protected against RA: ATIC rs3821353 T/T (p < 0.001), ATIC rs3821353 G/G (p = 0.004), SLC19A1 rs11702425 T/T (p = 0.001), SLC19A1 rs11702425 C/T (p = 0.003), GGH rs12681874 C/T (p = 0.004) and GGH rs12681874 T/T (0.002). Conclusion: The genotyping of genes involved in the MTX pathway may be helpful to predict which RA patients will/will not benefit from MTX, and thus, may help to apply a personalized medicine approach in RA.

Development of a TaqMan® Allelic Discrimination qPCR Assay for Rapid Detection of Equine CXCL16 Allelic Variants Associated With the Establishment of Long-Term Equine Arteritis Virus Carrier State in Stallions.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of equids. Following natural infection, up to 70% of the infected stallions can remain persistently infected over 1 year (long-term persistent infection [LTPI]) and shed EAV in their semen. Thus, the LTP-infected stallions play a pivotal role in maintaining and perpetuating EAV in the equine population. Previous studies identified equine C-X-C motif chemokine ligand 16 (CXCL16) as a critical host cell factor determining LTPI in the stallion's reproductive tract. Two alleles (CXCL16 S and CXCL16 r ) were identified in the equine population and correlated with the susceptibility or resistance of a CD3+ T cell subpopulation in peripheral blood to in vitro EAV infection, respectively. Interestingly, CXCL16 S has been linked to the establishment of LTPI in stallions, and thus, genotyping stallions based on CXCL16 S/r would allow identification of those at the highest risk of establishing LTPI. Thus, we developed a TaqMan® allelic discrimination qPCR assay for the genotyping of the equine CXCL16 gene based on the identification of a single nucleotide polymorphism in position 1,073 based on NCBI gene ID: 100061442 (or position 527 based on Ensembl: ENSECAG00000018406.2) located in exon 2. One hundred and sixty horses from four breeds were screened for the CD3+ T cell susceptibility phenotype to EAV infection by flow cytometry and subsequently sequenced to determine CXCL16 allelic composition. Genotyping by Sanger sequencing determined that all horses with the resistant CD3+ T cell phenotype were homozygous for CXCL16 r while horses with the susceptible CD3+ T cell phenotype carried at least one CXCL16 S allele or homozygous for CXCL16 S . In addition, genotypification with the TaqMan® allelic discrimination qPCR assay showed perfect agreement with Sanger sequencing and flow cytometric analysis. In conclusion, the new TaqMan® allelic discrimination genotyping qPCR assay can be used to screen prepubertal colts for the presence of the CXCL16 genotype. It is highly recommended that colts that carry the susceptible genotype (CXCL16  S/S or CXCL16 S/r ) are vaccinated against EAV after 6 months of age to prevent the establishment of LTPI carriers following possible natural infection with EAV.

Evaluation of seven gene signature for predicting HCV recurrence post-liver transplantation.

BACKGROUND: Orthotropic liver transplantation (OLT) offers a therapeutic choice for hepatocellular carcinoma (HCC) patients. The poor outcome of liver transplantation is HCV recurrence. Several genome-wide associated studies (GWAS) have reported many genetic variants to be associated with HCV recurrence. Seven gene polymorphisms formed a cirrhosis risk score (CRS) signature that could be used to distinguish chronic HCV patients at high risk from those at low risk for cirrhosis in non-transplant patients. This study aims to examine the association of CRS score and other clinical parameters with the probability for HCC emergence and/or the rate of HCV recurrence following liver transplantation. RESULTS: Seven gene polymorphisms, forming the CRS, were genotyped by real-time PCR using allelic discrimination protocol in 199 end-stage liver disease patients (79 child A, 43 child B, and 77child C), comprising 106 patients who encountered liver transplantation. Recipient CRS scores were correlated with HCV recurrence (HCV-Rec) at the end of the third year after OLT. Around 81% (39) recipients with low steatosis (LS; < 3.5%) donor percentage revealed no HCV recurrence (non-Rec) (p<0.001). CRS score could distinguish between child A, child B, and child C only at the low-risk group. Among the HCV Rec group 27% (8/30), 40% (12/30), and 33% (10/30) fell into the high, moderate, and low CRS risk groups, respectively. Stepwise logistic regression evinced two features more likely to be seen in HCV-Rec patients: abnormal ALT [OR, 1.1; 95% CI, 1.02-1.2] and donor steatosis >3.5% [OR, 46.07; 95% CI, 1.5-1407.8]. CONCLUSIONS: Accordingly, the CRS score seems to be less useful to predict HCV recurrence after OLT. ALT and donor steatosis (exceed 3.5%) can significantly promote the HCV recurrence post-OLT. Moreover, the combination of MMF and CNI positively heightens HCV recurrence.

Detection of the ABCB11930_1931del TC Mutation in Two Suspected Ivermectin-Sensitive Cats and Their Relatives by a Novel TaqMan Allelic Discrimination Assay.

The multidrug resistance gene MDR1 (syn. ABCB1) encodes for the multidrug efflux transporter P-glycoprotein (P-gp), which is highly expressed at the blood-brain barrier and protects the brain from potentially neurotoxic compounds, such as ivermectin. MDR1 mutation in dogs is known to be linked to dramatically increased brain accumulation of ivermectin and life-threatening neurological toxicity. The present report describes two suspected ivermectin-sensitive Maine Coon cats, which exhibited neurological toxicity following subcutaneous application of therapeutic doses of ivermectin. Both cats showed a homozygous 2-bp deletion in the MDR1/ABCB1 coding sequence (ABCB11930_1931del TC, syn. MDR1 nt1930(del2)) that had previously been associated with a drug-sensitive phenotype in cats. For cat MDR1 genotyping, a novel TaqMan allelic discrimination assay was established and validated. This assay was used for ABCB11930_1931del TC genotyping of the drug-sensitive cats as well as of more than 50 relatives. About half of them had the heterozygous MDR1(+/-) genotype, while none of these related cats with former ivermectin treatment had a history of drug-sensitivity. In conclusion: The present study supports previous findings on drug-sensitivity in cats with homozygous ABCB11930_1931del TC mutation. The newly established TaqMan allelic discrimination assay provides a useful and reliable method for routine MDR1 genotyping in cats in order to identify drug-sensitive cats prior to treatment with established P-gp substrates such as ivermectin and other macrocyclic lactones and thus to improve therapeutic safety.

Digital PCR for Genotype Quantification: A Case Study in a Pasta Production Chain.

Digital polymerase chain reaction (dPCR) is a breakthrough technology based on the partitioning of the analytical sample and detection of individual end-point amplifications into the separate compartments. Among the numerous applications of this technology, its suitability in mutation detection is relevant and characterized by unprecedented levels of precision. The actual applicability of this analytical technique to quantify the presence of a specific plant genotype, in both raw materials and transformed products, by exploiting a point polymorphism has been evaluated. As proof of concept, an Italian premium pasta production chain was considered and a dPCR assay based on a durum wheat target variety private point mutation was designed and evaluated in supply-chain samples. From the results obtained, the assay can be applied to confirm the presence of a target variety and to quantify it in raw materials and transformed products, such as commercial grain lots and pasta. The performance, costs, and applicability of the assay has been compared to analytical alternatives, namely simple sequence repeats (SSRs) and genotype-by-sequencing based on Diversity Arrays Technology sequencing (DArTseqTM).

Genetic Association Between Growth Differentiation Factor 5 Single Nucleotide Polymorphism and Primary Knee Osteoarthritis in a Group of Egyptian Patients: A Pilot Study.

AIM: This study aimed to determine the genetic association between Growth Differentiation Factor 5 (GDF5) gene (rs143383 T/C) single nucleotide polymorphism (SNP) and primary knee osteoarthritis (OA) in a group of Egyptian patients. PATIENTS AND METHODS: The study included 47 patients with primary knee OA and 40 apparently healthy control subjects. The disease was assessed using Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score and Health Assessment Questionnaire (HAQ). Radiological assessment was done by Kellgren-Laurence (K/L) grading system. The genetic association of the SNP with primary knee OA was assessed by restriction fragment length polymorphism - polymerase chain reaction (RFLP-PCR). RESULTS: The mean total WOMAC index was significantly higher in patients with TT genotype as compared to patients with CC and CT genotypes (P<0.001). Similarly, the HAQ score was significantly higher among patients with TT genotype when compared to patients with CT and CC genotypes (P<0.001). There was a statistically significant association between different GDF5 genotypes and K/L radiological grading of knee OA among the studied patients (P=0.029). No statistically significant association was detected on comparing the frequency distribution of GDF5 alleles and genotypes frequencies of the SNP in patients and healthy controls. CONCLUSION: There is a possible genetic association between GDF5 (rs143383) SNP and severity of primary knee OA, which might facilitate the detection of patients with high risk for disease progression. The present study did not detect an association between the SNP and development of primary knee OA.

Comparison of three PCR-based assays for SNP genotyping in plants.

BACKGROUND: PCR allelic discrimination technologies have broad applications in the detection of single nucleotide polymorphisms (SNPs) in genetics and genomics. The use of fluorescence-tagged probes is the leading method for targeted SNP detection, but assay costs and error rates could be improved to increase genotyping efficiency. A new assay, rhAmp, based on RNase H2-dependent PCR (rhPCR) combined with a universal reporter system attempts to reduce error rates from primer/primer and primer/probe dimers while lowering costs compared to existing technologies. Before rhAmp can be widely adopted, more experimentation is required to validate its effectiveness versus established methods. RESULTS: The aim of this study was to compare the accuracy, sensitivity and costs of TaqMan, KASP, and rhAmp SNP genotyping methods in sugar beet (Beta vulgaris L.). For each approach, assays were designed to genotype 33 SNPs in a set of 96 sugar beet individuals obtained from 12 parental lines. The assay sensitivity was tested using a series of dilutions from 100 to 0.1 ng per PCR reaction. PCR was carried out on the QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific, USA). The call-rate, defined as the percentage of genotype calls relative to the possible number of calls, was 97.0, 97.6, and 98.1% for TaqMan, KASP, and rhAmp, respectively. For rhAmp SNP, 24 of the 33 SNPs demonstrated 100% concordance with other two technologies. The genotype concordance with either technologies for the other 9 targets was above 99% (99.34-99.89%). CONCLUSION: The sensitivity test demonstrated that TaqMan and rhAmp were able to successfully determine SNP genotypes using as little as 0.2 ng DNA per reaction, while the KASP was unable to ascertain SNP states below 0.9 ng of DNA per reaction. Comparative cost per reaction was also analyzed with rhAmp SNP offering the lowest cost per reaction. In conclusion, rhAmp produced more calls than either TaqMan or KASP, higher signal to NTC data while offering the lowest cost per reaction.