SIRT1 Gene SNP rs932658 Is Associated With Medication-Related Osteonecrosis of the Jaw

Medication-related osteonecrosis of the jaw (MRONJ) is a rare but serious adverse drug reaction. Our previous whole-exome sequencing study found SIRT1 intronic region single-nucleotide polymorphism (SNP) rs7896005 to be associated with MRONJ in cancer patients treated with intravenous (iv) bisphosphonates (BPs). This study aimed to identify causal variants for this association. In silico analyses identified three SNPs (rs3758391, rs932658, and rs2394443) in the SIRT1 promoter region that are in high linkage disequilibrium (r² > 0.8) with rs7896005. To validate the association between these SNPs and MRONJ, we genotyped these three SNPs on the germline DNA from 104 cancer patients of European ancestry treated with iv BPs (46 cases and 58 controls). Multivariable logistic regression analysis showed the minor alleles of these three SNPs were associated with lower odds for MRONJ. The odds ratios (95% confidence interval) and p values were 0.351 (0.164–0.751; p = 0.007) for rs3758391, 0.351 (0.164–0.751; p = 0.007) for rs932658, and 0.331 (0.157–0.697; p = 0.0036) for rs2394443, respectively. In the reporter gene assays, constructs containing rs932658 with variant allele A had higher luciferase activity than the reference allele, whereas constructs containing SNP rs3758391 and/or rs2394443 did not significantly affect activity. These results indicate that the promoter SNP rs932658 regulates the expression of SIRT1 and presumably lowers the risk of MRONJ by increasing SIRT1 expression. © 2020 American Society for Bone and Mineral Research (ASBMR).     

Comparison of cytochrome P450 2C9 genotyping methods and implications for the clinical laboratory

STUDY OBJECTIVE: To compare the accuracy, speed, and cost of two methodologies used for genotyping known variants in the cytochrome P450 (CYP) 2C9 metabolizing enzyme gene. DESIGN: Comparative study. SETTING: University research center. SAMPLES: Fifteen-milliliter mouthwash samples collected from 253 subjects participating in a warfarin pharmacogenomic study. INTERVENTION: Genotyping for the isoleucine-to-leucine change at codon 359 (Ile359Leu [*3] polymorphism) was performed by using the Pyrosequencing and polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) methods in all 253 samples. Genotyping for the arginine-to-cysteine change at codon 144 (Arg144Cys [*2] polymorphism) was performed by using Pyrosequencing in all samples and by PCR-RFLP in a random subset of 136 samples. MEASUREMENTS AND MAIN RESULTS: Comparisons of genotyping success rates, time efficiency, and cost analyses were conducted for Pyrosequencing and PCR-RFLP at each variant site. Pyrosequencing and PCR-RFLP produced similar success rates on the first genotyping attempt for the Arg144Cys variant (93.3% vs 90.4%, respectively) and the Ile359Leu variant (83.8% vs 79.1%, respectively). With Pyrosequencing, genotyping 96 samples for either polymorphism could be performed in 1 hour. In contrast, genotyping 96 samples by RFLP took 10 hours for the Arg144Cys variant and 20 hours for the Ile359Leu variant. Total cost/sample for Arg144Cys genotyping was dollars 1.90 with PCR-Pyrosequencing and dollars 3.14 with PCR-RFLP. Total cost/sample for Ile359Leu genotyping was dollars 1.88 with PCR-Pyrosequencing and dollars 10.18 with PCR-RFLP CONCLUSION: Compared with RFLP, genotype determination by Pyrosequencing is a more time-efficient, cost-effective, and robust method for CYP2C9 genotyping. Because of its wide applicability and ease of use, Pyrosequencing is a promising technology for future pharmacogenomic investigations.     

β1‐receptor polymorphisms and junctional ectopic tachycardia in children after cardiac surgery

Junctional ectopic tachycardia (JET) is a potentially life‐threatening postoperative arrhythmia in children with specific congenital heart defects and can contribute significantly to postoperative morbidity for at‐risk populations. In adults, β1‐adrenergic receptor (ADRB1) and β2‐adrenergic receptor (ADRB2) genotypes have been associated with increased risk for arrhythmias. However, their association with arrhythmia risk in children is unknown. We aimed to test associations between ADRB1 and ADRB2 genotypes and postoperative JET in patients with congenital heart defects. Children who underwent cardiac surgery were genotyped for the ADRB1 p.Ser49Gly (rs1801252; c.145A>G), p.Arg389Gly (rs1801253; c.1165C>G), ADRB2 p.Arg16Gly (rs1042713; c.46A>G), and p.Glu27Gln (rs1042714; c.79G>C) polymorphisms. The occurrence of postoperative JET was assessed via cardiologist‐interpreted electrocardiograms. Genotype associations with JET were analyzed via logistic regression, adjusted for clinical variables associated with JET, with separate analysis in patients not on a β‐blocker. Of the 343 children included (median age 8 months, 53% boys, 69% European ancestry), 45 (13%) developed JET. The Arg389Arg genotype was not significantly associated with JET in the overall population (odds ratio [OR] = 1.96, 95% confidence interval [CI] = 0.96–4.03, p = 0.064), but was nominally associated in patients not taking a β‐blocker (n = 324, OR = 2.25, 95% CI = 1.05–4.80. p = 0.034). None of the other variants were associated with JET. These data suggest that the ADRB1 Arg389Arg genotype may predict risk for JET following cardiac surgery in pediatric patients in the absence of β‐blockade. Whether treatment with a β‐blocker ameliorates this association requires further research.

Study Highlights

• WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?

ADRB variants have been associated with arrhythmias in an adult population; however, the contribution of these variants to postoperative arrhythmia risk in children with congenital heart disease is unknown.

• WHAT QUESTION DID THIS STUDY ADDRESS?

Is there an association among ADRB1 Ser49Gly, ADRB1 Arg389Gly, ADRB2 Arg16Gly, or ADRB2 Gln27Glu genotypes and postoperative junctional ectopic tachycardia (JET) occurrence in children undergoing cardiac surgery?

• WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?

ADRB1 Arg389Arg genotype may help predict risk for JET following cardiac surgery in pediatric patients in the absence of β‐blockade.

• HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?

These findings serve as a foundation for further study of postoperative JET risk and ADRB1 Arg389Arg genotype and suggest a potential role of β‐blockade in reducing genotype‐mediated risk for JET in ADRB1 Arg389Arg genotype patients.     

Common laboratory methods in pharmacogenomics studies.

PURPOSE: Common laboratory methods used in pharmacogenomics studies are described. SUMMARY: The reliable and accurate determination of a person's genetic makeup at a particular locus in the DNA molecule, or genotype, is fundamental to pharmacogenomics. Whole blood cells and buccal cells are commonly collected to obtain a DNA sample. Once DNA is collected, the genomic DNA must be isolated from other cellular material. Next, a specific region of interest must be identified and amplified, performed via polymerase chain reaction (PCR). Gel electrophoresis is often performed after PCR to verify that PCR was successful and that the amplified target sequence is the correct size. Numerous methods are available to determine a person's genotype and differ based on allele discrimination and detection. PCR coupled with restriction fragment length polymorphism (RFLP) analysis, a conventional genotyping method, does not rely on automated technology and is practical for laboratories that genotype a limited number of samples. Pyrosequencing is an automated genotyping method in which the principal allele discrimination method is a primer extension reaction coupled with a luciferase-based enzyme reaction. TaqMan relies on the use of fluorescencelabeled probes, in addition to PCR primers, in the reaction mixture, enabling PCR amplification and allele discrimination in the same step. Mass spectrometry differentiates DNA molecules using a defined mass. Denaturing high-performance liquid chromatography (DHPLC) uses a reverse-phase ion-pair column to discriminate between variant and nonvariant alleles. CONCLUSION: An understanding of the common genotyping methods used in pharmacogenomics studies, including PCR-RFLP analysis, pyrosequencing, TaqMan, mass spectrometry, and DHPLC, will aid pharmacy practitioners and students when interpreting the methods sections of such studies.     

Isolation and Expression Profiling of Insecticidal Lectins from Wild Alliums Against Onion Thrips (Thrips tabaci Lindeman)

Proceedings of the National Academy of Sciences, India Section B: Biological Sciences - Lectins are important for plant defence against various insect and viral invasions. Allium species are rich...