Methylation of CYP1A1 and VKORC1 promoter associated with stable dosage of warfarin in Chinese patients

Objective To investigate the association between DNA methylation and the stable warfarin dose through genome-wide DNA methylation analysis and pyrosequencing assay. Method This study included 161 patients and genome-wide DNA methylation analysis was used to screen potential warfarin dose-associated CpGs through Illumina Infinium HumanMethylation 450 K BeadChip; then, the pyrosequencing assay was used to further validate the association between the stable warfarin dose and alterations in the methylation of the screened CpGs. GenomeStudio Software and R were used to analyze the differentially methylated CpGs. Results The methylation levels of CpGs surrounding the xenobiotic response element (XRE) within the CYP1A1 promoter, differed significantly between the different dose groups (P < 0.05), and these CpGs presented a positive correlation (r> 0, P < 0.05) with an increase in the stable dose of warfarin. At the VKORC1 promoter, two CpGs methylation levels were significantly different between the differential dose groups (P < 0.05), and one CpG (Chr16: 31106793) presented a significant negative correlation (r < 0, P < 0.05) among different dose (low, medium, and high) groups. Conclusion This is a novel report of the methylation levels of six CpGs surrounding the XRE within the CYP1A1 promoter and one differential CpG at the VKORC1 promoter associated with stable warfarin dosage; these methylation levels might be applied as molecular signatures for warfarin.

Genotyping of the Galactosemia GALT 591 A>G Mutation by Pyrosequencing

Introduction/Objective This project aimed to design a pyrosequencing assay capable of genetically analyzing the presence of GALT 591 A&gt;G mutation for the diagnosis of galactosemia. Galactosemia is an autosomal recessive disorder that affects enzyme activity of galactose-1-phosphate uridylytransferase (GALT). This enzyme is responsible for proper processing of galactose to glucose; if not broken down the accumulation of galactose in the body results in developmental delays, clouding of the eyes, speech difficulties, and intellectual disabilities. Pyrosequencing offers a valuable platform in assessing whether patients are wild type unaffected (A:A), heterozygous carrier (A:G), or homozygous affected (G:G) at the alleles within the GALT 591 locus by measuring percentage of the wild type A allele and the mutant G allele. Methods PCR primers were designed for the mutation locus, and PCR amplification was optimized for the target 77 bp product, with verification by gel electrophoresis. The pyrosequencing assay was designed and validated on the Qiagen PyroMark Q24 instrument. Control samples included commercially purchased DNA from the Coriell Cell Repository. Results One hundred and thirty-three reactions were utilized to establish the following assay validation parameters: accuracy (100% for G:G, 95% for A:G and A:A), precision (determined by mean and standard deviation with a standard deviation of 0.6 for G:G, 4.55 for A:G and 4.85 for A:A), and limit of detection (10 ng DNA pre-PCR and 2 µL of PCR product loaded). Twenty-six blinded samples were utilized to test assay clinical performance, as compared against a high resolution melt curve assay. Conclusion It was determined that detection of GALT 591 A&gt;G via pyrosequencing is highly sensitive and specific with a clinical sensitivity of 100% and a clinical specificity of 95.83%. It was concluded that this assay could be applied in a clinical environment for GALT 591 A&gt;G genotyping to aid in galactosemia diagnosis.

Teladorsagia circumcincta beta tubulin: the presence of the E198L polymorphism on its own is associated with benzimidazole resistance

Background Benzimidazole resistance is associated with isotype-1 β-tubulin gene F200Y, E198A and F167Y SNPs. In this study, the recently described polymorphism E198L was reported and analysed in Teladorsagia circumcincta. Methods The benzimidazole phenotypic resistance was measured by the faecal egg count reduction test (FECRT) and the egg hatch test (EHT) using a discriminating dose (DD) in 39 sheep flocks. Around 1000 larvae collected before and after treatment were used for DNA extraction. The resistant species identified in all flocks was T. circumcincta. The resistance alleles frequencies were measured for F200Y and E198A. A 371-bp fragment of the isotype-1 β-tubulin gene was analysed, including the three codons of interest, and a new pyrosequencing assay was designed for testing E198L. Results The percentage of resistant flocks was 35% by FECRT or 26% by EHT; however, F200Y and E198A SNPs were absent in T. circumcincta. The amplification of a 371-bp fragment confirmed the absence of F167Y and F200Y in 6 resistant flocks. Regarding codon 198, all samples after treatment carried a leucine (CTA). A pyrosequencing assay analysed the allele frequencies for the first two bases at codon 198 independently, G/C and A/T. The correlation between C and T frequencies was almost 1 (r = 0.929, P < 0.0001) and the mean value of both was calculated to measure the leucine frequency; this value ranged between 10.4–80.7% before treatment, and 82.3–92.8% after treatment. High and similar correlations were reported between the genotypic variables (C frequency, T frequency or mean of both frequencies) and phenotypic resistance (r > 0.720, P < 0.0001), although negatively associated with the FECRT and positively with the EHT. According to multivariate linear regression analysis, the T frequency was the most significant variable influencing the phenotypic resistance (FECRT or EHT; P < 0.0001). In the EHT, 67.1% of the phenotypic variability is associated with the T frequency but in the FECRT only 33.4%; therefore, the EHT using a DD seems to detect the genotypic resistance more accurately than the FECRT. Conclusions The E198L polymorphism can confer BZ resistance on its own in T. circumcincta.

Custom pyrosequencing assay to detect short BRAF deletions in Langerhans cell histiocytic lesions

Langerhans cell histiocytosis (LCH) is a rare inflammatory myeloid neoplastic disease driven by activating mutations in the mitogen-activating protein kinase signalling pathway, including the BRAFV600E mutation and BRAF deletions (BRAFdel). Next-generation sequencing and whole exome sequencing (WES) are valuable and powerful approaches for BRAFdel identification, but these techniques are costly and time consuming. Pyrosequencing is an alternative method that has the potential to rapidly and reliably identify gene deletions. We developed a custom pyrosequencing assay to detect the exon-12 BRAFdel in 18 biopsies from adult patients with LCH, which were all genotyped in parallel using Sanger sequencing and WES. A BRAFdel was detected in 7/18 (39%), 6/18 (33%) and 3/18 (17%) LCH lesions using WES, pyrosequencing and Sanger, respectively, with good concordance between the WES and pyrosequencing results (Kappa-coefficient=0.88). Therefore, our pyrosequencing assay is reliable and useful for detecting BRAFdel, particularly in BRAFV600E-negative LCH lesions, for which targeted treatment is indicated.

Teladorsagia circumcincta beta tubulin: the presence of the E198L polymorphism on its own is associated with benzimidazole resistance

Background: Benzimidazole resistance is associated with isotype-1 β-tubulin gene F200Y, E198A and F167Y SNPs. In this study, the recently described polymorphism E198L was reported and analysed in Teladorsagia circumcincta.Methods: The benzimidazole phenotypic resistance was measured by the faecal egg count reduction test (FECRT) and the egg hatch test (EHT) using a discriminating dose (DD) in 39 sheep flocks. Around 1000 larvae collected before and after treatment were used for DNA extraction. The resistant species identified in all flocks was T. circumcincta. The resistance alleles frequencies were measured for F200Y and E198A. A 371-bp fragment of the isotype-1 β-tubulin gene was analysed, including the three codons of interest, and a new pyrosequencing assay was designed for testing E198L.Results: The percentage of resistant flocks was 35% by FECRT or 26% by EHT; however, F200Y and E198A SNPs were absent in T. circumcincta. The amplification of a 371-bp fragment confirmed the absence of F167Y and F200Y in 6 resistant flocks. Regarding codon 198, all samples after treatment carried a leucine (CTA). A pyrosequencing assay analysed the allele frequencies for the first two bases at codon 198 independently, G/C and A/T. The correlation between C and T frequencies was almost 1 (r = 0.929, P < 0.0001) and the mean value of both was calculated to measure the leucine frequency; this value ranged between 10.4–80.7% before treatment, and 82.3–92.8% after treatment. High and similar correlations were reported between the genotypic variables (C frequency, T frequency or mean of both frequencies) and phenotypic resistance (r > 0.720, P < 0.0001), although negatively associated with the FECRT and positively with the EHT. According to multivariate linear regression analysis, the T frequency was the most significant variable influencing the phenotypic resistance (FECRT or EHT; P < 0.0001). In the EHT, 67.1% of the phenotypic variability is associated with the T frequency but in the FECRT only 33.4%; therefore, the EHT using a DD seems to detect the genotypic resistance more accurately than the FECRT.Conclusions: The E198L polymorphism can confer BZ resistance on its own in T. circumcincta.

Teladorsagia circumcincta beta tubulin: the presence of the E198L polymorphism on its own is associated with benzimidazole resistance

Background: Benzimidazole resistance is associated with isotype-1 β-tubulin gene F200Y, E198A and F167Y SNPs. In this study, the recently described polymorphism E198L was reported and analysed in Teladorsagia circumcincta.Methods: The benzimidazole phenotypic resistance was measured by the Faecal Egg Count Reduction Test (FECRT) and the Egg Hatch Test (EHT) using a discriminating dose (DD) in 39 sheep flocks. Around 1,000 larvae collected before and after treatment were used for DNA extraction. The resistant species identified in all flocks was T. circumcincta. The resistance alleles frequencies were measured for F200Y and E198A. A 371 bp fragment of the isotype-1 β-tubulin gene was analysed, including the three codons of interest, and a new pyrosequencing assay was designed for testing E198L.Results: The percentage of resistant flocks was 35% by FECRT or 26% by EHT, however F200Y and E198A SNPs were absent in T. circumcincta. The amplification of a 371 bp fragment confirmed the absence of F167Y and F200Y in 6 resistant flocks. Regarding codon 198, all samples after treatment carried a leucine (CTA). A pyrosequencing assay analysed the allele frequencies for the first two bases at codon 198 independently, G/C and A/T. The correlation between C and T frequencies was almost 1 (r = 0.929; p < 0.0001) and the mean value of both was calculated to measure the leucine frequency; this value ranged between 10.4 and 80.7% before treatment, and from 82.3 to 92.8% after treatment. High and similar correlations were reported between the genotypic variables (C frequency, T frequency or mean of both frequencies) and phenotypic resistance (r > 0.720; p < 0.0001), although negatively associated with the FECRT and positively with the EHT. According to multivariate linear regression analysis, the T frequency was the most significant variable influencing the phenotypic resistance (FECRT or EHT; p < 0.0001). In the EHT, 67.1% of the phenotypic variability is associated with the T frequency but in the FECRT only 33.4%; therefore, the EHT using a DD seems to detect the genotypic resistance more accurately than the FECRT.Conclusions: The E198L polymorphism can confer BZ resistance on its own in T. circumcincta.

Teladorsagia circumcincta beta tubulin: the presence of the E198L polymorphism on its own is associated with benzimidazole resistance

Background . Benzimidazole resistance is associated with isotype-1 β-tubulin gene F200Y, E198A and F167Y SNPs. In this study, the recently described polymorphism E198L was reported and analysed in Teladorsagia circumcincta . Methods . The benzimidazole phenotypic resistance was measured by the Faecal Egg Count Reduction Test (FECRT) and the Egg Hatch Test (EHT) using a discriminating dose (DD) in 39 sheep flocks. Around 1,000 larvae collected before and after treatment were used for DNA extraction. The resistant species identified in all flocks was T. circumcincta . The resistance alleles frequencies were measured for F200Y and E198A. A 371 bp fragment of the isotype-1 β-tubulin gene was analysed, including the three codons of interest, and a new pyrosequencing assay was designed for testing E198L. Results . The percentage of resistant flocks was 35% by FECRT or 26% by EHT, however F200Y and E198A SNPs were absent in T. circumcincta . The amplification of a 371 bp fragment confirmed the absence of F167Y and F200Y in 6 resistant flocks. Regarding codon 198, all samples after treatment carried a leucine (CTA). A pyrosequencing assay analysed the allele frequencies for the first two bases at codon 198 independently, G/C and A/T. The correlation between C and T frequencies was almost 1 (r = 0.929; p = 0.000) and the mean value of both was calculated to measure the leucine frequency; this value ranged between 10.4 and 80.7% before treatment, and from 82.3 to 92.8% after treatment. High and similar correlations were reported between the genotypic variables (C frequency, T frequency or mean of both frequencies) and phenotypic resistance (r > 0.720; p = 0.000), although negatively associated with the FECRT and positively with the EHT. According to multivariate linear regression analysis, the T frequency was the most significant variable influencing the phenotypic resistance (FECRT or EHT; p = 0.000). In the EHT, 67.1% of the phenotypic variability is associated with the T frequency but in the FECRT only 33.4%; therefore, the EHT using a DD seems to detect the genotypic resistance more accurately than the FECRT. Conclusions . The E198L polymorphism can confer BZ resistance on its own in T. circumcincta .

Teladorsagia Circumcincta Beta Tubulin: The Presence of the E198L Polymorphism on Its Own is Associated with Benzimidazole Resistance

Background: Benzimidazole resistance is associated with isotype-1 β-tubulin gene F200Y, E198A and F167Y SNPs. In this study, the recently described polymorphism E198L was reported and analysed in Teladorsagia circumcincta.Methods: The benzimidazole phenotypic resistance was measured by the Faecal Egg Count Reduction Test (FECRT) and the Egg Hatch Test (EHT) using a discriminating dose in 39 sheep flocks. Around 1,000 larvae collected before and after treatment were used for DNA extraction. The resistant species identified in all flocks was T. circumcincta . The resistance alleles frequencies were measured for F200Y and E198A. A 371 bp fragment of the isotype-1 β-tubulin gene was analysed, including the three codons of interest, and a new pyrosequencing assay was designed for testing E198L. Results: The percentage of resistant flocks was 32% by FECRT or 26% by EHT, however F200Y and E198A SNPs were absent in T. circumcincta . The amplification a 371 bp fragment confirmed the absence of F167Y and F200Y in 6 resistant flocks. Regarding codon 198, all samples after treatment carried a leucine (CTA). A pyrosequencing assay analysed the allele frequencies for the first two bases at codon 198 independently, G/C and A/T. The correlation between C and T frequencies was almost 1 (r = 0.929; p = 0.000) and the mean value of both was calculated to measure the leucine frequency; this value ranged between 10.4 and 80.7% before treatment, and from 82.3 to 92.8% after treatment. Very high and similar correlations were reported between the genotypic variables (C frequency, T frequency or mean of both frequencies) and phenotypic resistance (r > 0.720; p = 0.000), although negatively associated with the FECRT and positively with the EHT. According to multivariate linear regression analysis, the T frequency was the most significant variable influencing the phenotypic resistance (FECRT or EHT; p = 0.000). In the EHT, 67.1% of the phenotypic variability is associated with the T frequency but in the FECRT only 33.4%; therefore, the EHT using a DD seems to detect the genotypic resistance more accurately than the FECRT. Conclusions: The E198L polymorphism can confer BZ resistance on its own in T. circumcincta .