Mutational and Biochemical Analysis of Isoprenylcysteine Carboxyl Methyltransferase

Ninety percent of pancreatic cancers are attributed to mutations in the Ras protein, making it paramount to concentrate on Ras activity. This study focuses on Ras activity by targeting a post-translational modifying enzyme of Ras called Isoprenylcysteine carboxyl methyltransferase (Icmt). Elucidation of the binding site of Icmt will allow the development of therapeutics that effectively inhibit Icmt causing the mislocalization of Ras, and in turn, aid in the treatment of Ras driven cancers. Currently, the hydrophobic substrate binding site of Icmt is unknown. In order to characterize the substrate binding site of Icmt, site-directed mutagenesis was used to design mutations in the yeast homolog of Icmt, Ste14p, and these mutants were tested on substrate specificity. Residues L33, L34, L40, L176, L190, and L195 were mutated to alanine and residue F80 was mutated to tyrosine. When tested with a methyltransferase assay, all of the mutants lost activity as compared to wild type (WT). Noticeably, L190A had only 44% WT activity. This suggests that L190A is important for either substrate binding or the overall structural integrity of Ste14p. The results from trypsin digestion show that all mutants have cleavage patterns similar to WT. This indicates that the structural integrity of Ste14p remains intact regardless of these mutations. Substrate specificity and photolabeling experiments should be conducted in the future to elucidate if these residues are vital for the substrate binding site of Icmt. These results could be utilized to design more potent and effective drug therapies to minimize Ras signaling in cancer cells

Injectable Contraceptive, Depo-Provera, Produces Erratic Beating Patterns in Patient-Specific Induced Pluripotent Stem Cell-derived Cardiomyocytes with Type 2 Long QT Syndrome.

BACKGROUND Long QT syndrome type 2 (LQT2) is caused by pathogenic variants in KCNH2. LQT2 may manifest as QT prolongation on an ECG and present with arrhythmic syncope/seizures, sudden cardiac arrest/death. Oral progestin-based contraceptives may increase the risk of LQT2-triggered cardiac events in women. We previously reported on a LQT2 woman with recurrent cardiac events temporally related and attributed to the progestin-based contraceptive, medroxyprogesterone acetate ("Depo-Provera", Depo). OBJECTIVE To evaluate the arrhythmic-risk of Depo in a patient-specific induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model of LQT2. METHODS An iPSC-CM line was generated from a 40-year-old female with p.G1006Afs*49-KCNH2. A CRISPR/Cas9 gene-edited/variant-corrected, isogenic control (IC) iPSC-CM line was generated. FluoVolt was used to measure the action potential duration (APD) following treatment with 10 μM Depo. Erratic beating patterns characterized as alternating spike amplitudes, alternans, or early after depolarization-like phenomena were assessed using multi-electrode array (MEA) following 10 μM Depo, 1 μM isoproterenol (ISO), or combined Depo + ISO treatment. RESULTS Depo treatment shortened the APD-90 of the G1006Afs*49 iPSC-CMs from 394±10 ms to 303±10 ms (p<0.0001). Combined Depo and ISO treatment increased the percent of electrodes displaying erratic beating in G1006Afs*49 iPSC-CMs [baseline 18±5% vs. Depo + ISO 54±5% (p<0.0001)] but not in IC iPSC-CMs [baseline 0±0% vs. Depo + ISO 10±3% (p=0.9659)]. CONCLUSION This cell study provides a potential mechanism for the patient's clinically documented Depo-associated episodes of recurrent ventricular fibrillation. This in-vitro data should prompt a large-scale clinical assessment of Depo's potential pro-arrhythmic effect in women with LQT2

A phenotype-enhanced variant classification framework to decrease the burden of missense variants of uncertain significance in type 1 long QT syndrome

Background: Pathogenic/likely pathogenic (P/LP) variants in the KCNQ1-encoded Kv7.1 potassium channel cause type 1 long QT syndrome (LQT1). Despite the revamped 2015 American College of Medical Genetics (ACMG) variant interpretation guidelines, the burden of KCNQ1 variants of uncertain significance (VUS) in patients with LQTS remains ∼30%. Objective: The purpose of this study was to determine whether a phenotype-enhanced (PE) variant classification approach could reduce the VUS burden in LQTS genetic testing. Methods: Retrospective analysis was performed on 79 KCNQ1 missense variants in 356 patients from Mayo Clinic and an independent cohort of 42 variants in 225 patients from Amsterdam University Medical Center (UMC). Each variant was classified initially using the ACMG guidelines and then readjudicated using a PE-ACMG framework that incorporated the LQTS clinical diagnostic Schwartz score plus 4 “LQT1-defining features”: broad-based/slow upstroke T waves, syncope/seizure during exertion, swimming-associated events, and a maladaptive LQT1 treadmill stress test. Results: According to the ACMG guidelines, Mayo Clinic variants were classified as follows: 17 of 79 P variants (22%), 34 of 79 LP variants (43%), and 28 of 79 VUS (35%). Similarly, for Amsterdam UMC, the variant distribution was 9 of 42 P variants (22%), 14 of 42 LP variants (33%), and 19 of 42 variants VUS (45%). After PE-ACMG readjudication, the total VUS burden decreased significantly from 28 (35%) to 13 (16%) (P = .0007) for Mayo Clinic and from 19 (45%) to 12 (29%) (P = .02) for Amsterdam UMC. Conclusion: Phenotype-guided variant adjudication decreased significantly the VUS burden of LQT1 case–derived KCNQ1 missense variants in 2 independent cohorts. This study demonstrates the value of incorporating LQT1-specific phenotype/clinical data to aid in the interpretation of KCNQ1 missense variants identified during genetic testing for LQTS