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Background: Despite clinically demonstrated accuracy in next generation sequencing (NGS) data, many clinical laboratories continue to confirm variants with Sanger sequencing, which increases cost of testing and turnaround time. Several studies have assessed the accuracy of NGS in detecting single nucleotide variants; however, less has been reported about insertion, deletion, and deletion-insertion variants (indels)., Methods: We performed a retrospective analysis from 2015-2022 of indel results from a subset of NGS targeted gene panel tests offered through the Mayo Clinic Genomics Laboratories. We compared results from NGS and Sanger sequencing of indels observed in clinical runs and during the intra-assay validation of the tests., Results: Results demonstrated 100% concordance between NGS and Sanger sequencing for over 490 indels (217 unique), ranging in size from 1 to 68 basepairs (bp). The majority of indels were deletions (77%) and 1 to 5 bp in length (90%). Variant frequencies ranged from 11.4% to 67.4% and 85.1% to 100% for heterozygous and homozygous variants, respectively, with a median depth of coverage of 2562×. A subset of indels (7%) were located in complex regions of the genome, and these were accurately detected by NGS. We also demonstrated 100% reproducibility of indel detection (n = 179) during intra-assay validation., Conclusions: Together this data demonstrates that reportable indel variants up to 68 bp can be accurately assessed using NGS, even when they occur in complex regions. Depending on the complexity of the region or variant, Sanger sequence confirmation of indels is usually not necessary if the variants meet appropriate coverage and allele frequency thresholds., (© American Association for Clinical Chemistry 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Pathogenic variants in COL1A1 and COL1A2 are involved in osteogenesis imperfecta (OI) and, rarely, Ehlers-Danlos syndrome (EDS) subtypes and OI-EDS overlap syndromes (OIEDS1 and OIEDS2, respectively). Here we describe a cohort of 34 individuals with likely pathogenic and pathogenic variants in COL1A1 and COL1A2, 15 of whom have potential OIEDS1 (n = 5) or OIEDS2 (n = 10). A predominant OI phenotype and COL1A1 frameshift variants are present in 4/5 cases with potential OIEDS1. On the other hand, 9/10 potential OIEDS2 cases have a predominant EDS phenotype, including four with an initial diagnosis of hypermobile EDS (hEDS). An additional case with a predominant EDS phenotype had a COL1A1 arginine-to-cysteine variant that was originally misclassified as a variant of uncertain significance despite this type of variant being associated with classical EDS with vascular fragility. Vascular/arterial fragility was observed in 4/15 individuals (including one individual with an original diagnosis of hEDS), which underscores the unique clinical surveillance and management needs in these patients. In comparison to previously described OIEDS1/2, we observed differentiating features that should be considered to refine currently proposed criteria for genetic testing in OIEDS, which will be beneficial for diagnosis and management. Additionally, these results highlight the importance of gene-specific knowledge for informed variant classification and point to a potential genetic resolution (COL1A2) for some cases of clinically diagnosed hEDS., (© 2023 Wiley Periodicals LLC.)

CYP2D6 duplication has important pharmacogenomic implications. Reflex testing with long-range PCR (LR-PCR) can resolve the genotype when a duplication and alleles with differing activity scores are detected. We evaluated whether visual inspection of plots from real-time-PCR-based targeted genotyping with copy number variation (CNV) detection could reliably determine the duplicated CYP2D6 allele. Six reviewers evaluated QuantStudio OpenArray CYP2D6 genotyping results and the TaqMan Genotyper plots for seventy-three well-characterized cases with three copies of CYP2D6 and two different alleles. Reviewers blinded to the final genotype visually assessed the plots to determine the duplicated allele or opt for reflex sequencing. Reviewers achieved 100% accuracy for cases with three CYP2D6 copies that they opted to report. Reviewers did not request reflex sequencing in 49-67 (67-92%) cases (and correctly identified the duplicated allele in each case); all remaining cases (6-24) were marked by at least one reviewer for reflex sequencing. In most cases with three copies of CYP2D6 , the duplicated allele can be determined using a combination of targeted genotyping using real-time PCR with CNV detection without need for reflex sequencing. In ambiguous cases and those with >3 copies, LR-PCR and Sanger sequencing may still be necessary for determination of the duplicated allele.

Background: Genetic-guided P2Y 12 inhibitor selection has been proposed to reduce ischemic events by identifying CYP2C19 loss-of-function (LOF) carriers at increased risk with clopidogrel treatment after percutaneous coronary intervention (PCI). A prespecified analysis of TAILOR-PCI (Tailored Antiplatelet Therapy Following PCI) evaluated the effect of genetic-guided P2Y 12 inhibitor therapy on cumulative ischemic and bleeding events., Objectives: Here, the authors detail a prespecified analysis of cumulative endpoints. The primary endpoint was cumulative incidence rate of ischemic events at 12 months. Cumulative incidence of major and minor bleeding was a secondary endpoint. Cox proportional hazards models as adapted by Wei, Lin, and Weissfeld were used to estimate the effect of this strategy on all observed events., Methods: The TAILOR-PCI trial was a prospective trial including 5,302 post-PCI patients with acute and stable coronary artery disease (CAD) who were randomized to genetic-guided P2Y 12 inhibitor or conventional clopidogrel therapy. In the genetic-guided group, LOF carriers were prescribed ticagrelor, whereas noncarriers received clopidogrel. TAILOR-PCI's primary analysis was time to first event in LOF carriers., Results: Among 5,276 patients (median age 62 years; 25% women; 82% acute CAD; 18% stable CAD), 1,849 were LOF carriers (903 genetic-guided; 946 conventional therapy). The cumulative primary endpoint was significantly reduced in the genetic-guided group compared with the conventional therapy (HR: 0.61; 95% CI: 0.41-0.89; P = 0.011) with no significant difference in cumulative incidence of major or minor bleeding (HR: 1.36; 95% CI: 0.67-2.76; P = 0.39)., Conclusions: Among CYP2C19 LOF carriers undergoing PCI, a genetic-guided strategy resulted in a statistically significant reduction in cumulative ischemic events without a significant difference in bleeding. (Tailored Antiplatelet Therapy Following PCI [TAILOR-PCI]; NCT01742117)., Competing Interests: Funding Support and Author Disclosures Funding for this research was provided by the National Institutes of Health (NIH) grants U01HL128606 to Drs Pereira and Farkouh and U01HL128626 to Dr Bailey. The content of this manuscript is solely the responsibility of the authors and does not necessarily reflect the views of the National Heart, Lung, and Blood Institute (NHLBI) or the National Institutes of Health. Dr Farkouh has received grants from NHLBI, Amgen, Novartis, and Novo Nordisk. Mr Lennon has received grants from the National Institutes of Health (NIH)/NHLBI and receiving nonfinancial support from Spartan Biosciences. Dr So has received grants from Eli Lilly Canada, Spartan Biosciences, Roche Diagnostics, and Aggredyne Inc; and has received personal fees from AstraZeneca Canada, Bayer Canada, and Servier Canada. Dr Goodman has received research grant support (eg, steering committee or data and safety monitoring committee) and/or speaker/consulting honoraria (eg, advisory boards) from Amgen, Anthos Therapeutics, AstraZeneca, Bayer, Boehringer Ingelheim, Bristol Myers Squibb, CSL Behring, Daiichi Sankyo/American Regent, Eli Lilly, Esperion, Ferring Pharmaceuticals, HLS Therapeutics, JAMP Pharma, Merck, Novartis, Novo Nordisk A/C, Pendopharm/Pharmascience, Pfizer, Regeneron, Sanofi, Servier, Tolmar Pharmaceuticals, and Valeo Pharma; and has received salary support/honoraria from the Heart and Stroke Foundation of Ontario/University of Toronto (Polo) Chair, Canadian Heart Research Centre and MD Primer, Canadian VIGOUR Centre, Cleveland Clinic Coordinating Centre for Clinical Research, Duke Clinical Research Institute, New York University Clinical Coordinating Centre, PERFUSE Research Institute, and the TIMI Study Group (Brigham Health). Dr Lerman has received personal fees from Itamar Medical, Phillips/Volcano, Shahal, and Wei Jian RC Inc. Dr Tanguay has received personal fees from Mayo Clinic, AstraZeneca, Bayer, Daiichi Sankyo, Servier, Novartis, and BMS-Pfizer Alliance. Dr Welsh has received grants and/or personal fees from AstraZeneca, Pfizer, Bayer, Canadian Cardiac Society, Mayo Clinic, Boehringer Ingelheim, and Novartis. Dr Bailey has received grants from NIH. Dr Pereira has received grants from the NHLBI. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2023. Published by Elsevier Inc.)

Loss-of-function CYP2C19 variants are associated with increased cumulative ischemic outcomes warranting CYP2C19 genotyping prior to clopidogrel administration. TAILOR-PCI was an international, multicenter (40 sites), prospective, randomized trial comparing rapid point of care (POC) genotype-guided vs. conventional anti-platelet therapy. The performance of buccal-based rapid CYP2C19 genotyping performed by non-laboratory-trained staff in TAILOR-PCI was assessed. Pre-trial training and evaluation involved rapid genotyping of 373 oral samples, with 99.5% (371/373) concordance with Sanger sequencing. During TAILOR-PCI, 5302 patients undergoing PCI were randomized to POC rapid CYP2C19 *2, *3, and *17 genotyping versus no genotyping. At 12 months post-PCI, TaqMan genotyping determined 99.1% (2,364/2,385) concordance with the POC results, with 90.7-98.8% sensitivity and 99.2-99.6% specificity. In conclusion, non-laboratory personnel can be successfully trained for on-site instrument operation and POC rapid genotyping with analytical accuracy and precision across multiple international centers, thereby supporting POC genotyping in patient-care settings, such as the cardiac catheterization laboratory.Clinical Trial Registration: https://www.clinicalTrials.gov (Identifier: NCT01742117)., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Rationale & Objective: The etiology of kidney disease remains unknown in many individuals with chronic kidney disease (CKD). We created the Mayo Clinic Nephrology Genomics Clinic to improve our ability to integrate genomic and clinical data to identify the etiology of unexplained CKD., Study Design: Retrospective study., Setting & Participants: An essential component of our program is the Nephrology Genomics Board which consists of nephrologists, geneticists, pathologists, translational omics scientists, and trainees who interpret the patient's clinical and genetic data. Since September 2016, the Board has reviewed 163 cases (15 cystic, 100 glomerular, 6 congenital anomalies of kidney and urinary tract (CAKUT), 20 stones, 15 tubulointerstitial, and 13 other)., Analytical Approach: Testing was performed with targeted panels, single gene analysis, or analysis of kidney-related genes from exome sequencing. Variant classification was obtained based on the 2015 American College of Medical Genetics and Genomics and the Association for Molecular Pathology guidelines., Results: A definitive genetic diagnosis was achieved for 50 families (30.7%). The highest diagnostic yield was obtained in individuals with tubulointerstitial diseases (53.3%), followed by congenital anomalies of the kidney and urological tract (33.3%), glomerular (31%), cysts (26.7%), stones (25%), and others (15.4%). A further 20 (12.3%) patients had variants of interest, and variant segregation, and research activities (exome, genome, or transcriptome sequencing) are ongoing for 44 (40%) unresolved families., Limitations: Possible overestimation of diagnostic rate due to inclusion of individuals with variants with evidence of pathogenicity but classified as of uncertain significance by the clinical laboratory., Conclusions: Integration of genomic and research testing and multidisciplinary evaluation in a nephrology cohort with CKD of unknown etiology or suspected monogenic disease provided a diagnosis in a third of families. These diagnoses had prognostic implications, and often changes in management were implemented., (© 2021 The Authors.)

Diagnostic laboratories gather phenotypic data through requisition forms, but there is no consensus as to which data are essential for variant interpretation. The ClinGen Cardiomyopathy Variant Curation Expert Panel defined a phenotypic data set for hypertrophic cardiomyopathy (HCM) variant interpretation, with the goal of standardizing requisition forms. Phenotypic data elements listed on requisition forms from nine leading cardiomyopathy testing laboratories were compiled to assess divergence in data collection. A pilot of 50 HCM cases was implemented to determine the feasibility of harmonizing data collection. Laboratory directors were surveyed to gauge potential for adoption of a minimal data set. Wide divergence was observed in the phenotypic data fields in requisition forms. The 50-case pilot showed that although demographics and assertion of a clinical diagnosis of HCM had 86% to 98% completion, specific phenotypic features, such as degree of left ventricular hypertrophy, ejection fraction, and suspected syndromic disease, were completed only 24% to 44% of the time. Nine data elements were deemed essential for variant classification by the expert panel. Participating laboratories unanimously expressed a willingness to adopt these data elements in their requisition forms. This study demonstrates the value of comparing and sharing best practices through an expert group, such as the ClinGen Program, to enhance variant interpretation, providing a foundation for leveraging cumulative case-level data in public databases and ultimately improving patient care., (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Gene-specific knowledge can enhance genetic variant classification, but may not be routinely incorporated into clinical laboratory practice. For example, FBN1 variants associated with Marfan syndrome may be variably classified depending on knowledge of FBN1-specific critical regions. In order to assess variability in classification of FBN1 variants, 674 FBN1 missense variants from 18 ClinVar submitters were compared and reanalyzed using FBN1-specific criteria and ACMG/AMP 2015 guidelines for variant interpretation. Conflicting variant classifications occurred in 30.7% of the missense variants that had multiple submitters. There were 451 classifications of 361 critical residue missense variants, with 80.0% (361/451) classified as likely pathogenic or pathogenic [(L)P]. Non-cysteine critical residue variants were less likely to be classified as (L)P [55.3% (78/141)] than cysteine variants [91.3% (283/310)] and were more likely to lack evidence citing the functional significance of the amino acid impacted. Application of FBN1-specific knowledge allowed for reclassification or discrepancy resolution in 65/361 (18.0%) critical residue variants. There were 522 classifications of 313 unique missense variants not known to impact a critical residue. Of these, 31.6% (165/522) were likely overclassified as either (L)P or uncertain significance (VUS), especially when minor allele frequency (MAF) was taken into account, and we reclassified or resolved classification discrepancies in 128/313 (40.9%) of these variants. Our results provide a refined framework and resource for FBN1 variant classification, and further supports the more global implications of combining gene-based knowledge with ACMG/AMP criteria and appropriate MAF cutoffs for variant classification that extend beyond FBN1.

Objective: To evaluate perceptions toward pharmacogenetic testing of patients undergoing percutaneous coronary intervention (PCI) who are prescribed dual antiplatelet therapy (DAPT) and whether geographical differences in these perceptions exist., Participants and Methods: TAILOR-PCI is the largest genotype-based cardiovascular clinical trial randomizing participants to conventional DAPT or prospective genotyping-guided DAPT. Enrolled patients completed surveys before and 6 months after randomization., Results: A total of 1327 patients completed baseline surveys of whom 28, 29, and 43% were from Korea, Canada and the USA, respectively. Most patients (77%) valued identifying pharmacogenetic variants; however, fewer Koreans (44%) as compared with Canadians (91%) and USA (89%) patients identified pharmacogenetics as being important (P<0.001). After adjusting for age, sex, and country, those who were confident in their ability to understand genetic information were significantly more likely to value identifying pharmacogenetic variants (odds ratio: 30.0; 95% confidence interval: 20.5-43.8). Only 21% of Koreans, as opposed to 86 and 77% of patients in Canada and USA, respectively, were confident in their ability to understand genetic information (P<0.001)., Conclusion: Although genetically mediated clopidogrel resistance is more prevalent amongst Asians, Koreans undergoing PCI identified pharmacogenetic variants as less important to their healthcare, likely related to their lack of confidence in their ability to understand genetic information. To enable successful implementation of pharmacogenetic testing on a global scale, the possibility of international population differences in perceptions should be considered.

Common genetic variation in CYP2C19 (cytochrome P450, family 2, subfamily C, polypeptide 19) *2 and *3 alleles leads to a loss of functional protein, and carriers of these loss-of-function alleles when treated with clopidogrel have significantly reduced clopidogrel active metabolite levels and high on-treatment platelet reactivity resulting in increased risk of major adverse cardiovascular events, especially after percutaneous coronary intervention. The Food and Drug Administration has issued a black box warning advising practitioners to consider alternative treatment in CYP2C19 poor metabolizers who might receive clopidogrel and to identify such patients by genotyping. However, routine clinical use of genotyping for CYP2C19 loss-of-function alleles in patients undergoing percutaneous coronary intervention is not recommended by clinical guidelines because of lack of prospective evidence. To address this critical gap, TAILOR-PCI (Tailored Antiplatelet Initiation to Lessen Outcomes due to Decreased Clopidogrel Response After Percutaneous Coronary Intervention) is a large, pragmatic, randomized trial comparing point-of-care genotype-guided antiplatelet therapy with routine care to determine whether identifying CYP2C19 loss-of-function allele patients prospectively and prescribing alternative antiplatelet therapy is beneficial.

Objective- Ceramides are sphingolipids involved with cellular signaling. Synthesis of ceramides occurs in all tissues. Ceramides accumulate within tissues and the blood plasma during metabolic dysfunction, dyslipidemia, and inflammation. Elevations of ceramides are predictive of cardiovascular mortality. We sought to verify the utility of plasma concentrations of 4 ceramides: N-palmitoyl-sphingosine [Cer(16:0)], N-stearoyl-sphingosine [Cer(18:0)], N-nervonoyl-sphingosine [Cer(24:1)], and N-lignoceroyl-sphingosine [Cer(24:0)] in predicting major adverse cardiovascular events in a diverse patient population referred for coronary angiography. Approach and Results- Plasma ceramides were measured in 495 participants before nonurgent coronary angiography. Coronary artery disease, defined as >50% stenosis in ≥1 coronary artery, was identified 265 (54%) cases. Ceramides were not significantly associated with coronary artery disease. Patients were followed for a combined primary end point of myocardial infarction, percutaneous intervention, coronary artery bypass, stroke, or death within 4 years. Ceramides were significantly predictive of outcomes after adjusting for age, sex, body mass index, hypertension, smoking, LDL (low-density lipoprotein) cholesterol, HDL (high-density lipoprotein) cholesterol, triglycerides, serum glucose, and family history of coronary artery disease. The fully adjusted per SD hazard ratios (95% confidence interval) were 1.50 (1.16-1.93) for Cer(16:0), 1.42 (1.11-1.83) for Cer(18:0), 1.43 (1.08-1.89) for Cer(24:1), and 1.58 (1.22-2.04) for the ceramide risk score. Conclusions- Elevated plasma concentrations of ceramides are independently associated with major adverse cardiovascular events in patients with and without coronary artery disease.

The etiology of statin intolerance is hypothesized to be due to genetic variants that impact statin disposition and clearance. We sought to determine whether genetic variants were associated to statin intolerance. The studied cohort consisted of hyperlipidemic participants (n = 90) clinically diagnosed with statin intolerance by a cardiologist and matched controls without statin intolerance. Creatine kinase activity, lipid profiles and genetic analyses were performed on genes involved in statin metabolism and included UGT1A1 and UGT1A3 sequencing and targeted analyses of CYP3A4*22, CYP3A5*3, SLCO1B1*5 and *1b, ABCB1 c.3435C>T, ABCG2 c.421C>A and GATM rs9806699. Although lipids were higher in cases, genetic variant minor allele frequencies were similar between cases and controls, except for UGT1A1*28, which was less prevalent in cases than controls.

Background: Postmortem genetic testing for heritable cardiovascular (CV) disorders is often lacking because ideal specimens (ie, whole blood) are not retained routinely at autopsy. Formalin-fixed paraffin-embedded tissue (FFPET) is ubiquitously collected at autopsy, but DNA quality hampers its use with traditional sequencing methods. Targeted next-generation sequencing may offer the ability to circumvent such limitations, but a method has not been previously described. The primary aim of this study was to develop and evaluate the use of FFPET for heritable CV disorders via next-generation sequencing., Methods and Results: Nineteen FFPET (heart) and blood (whole blood or dried blood spot) specimens underwent targeted next-generation sequencing using a custom panel of 101 CV-associated genes. Nucleic acid yield and quality metrics were evaluated in relation to FFPET specimen age (6 months to 15 years; n=14) and specimen type (FFPET versus whole blood and dried blood spot; n=12). Four FFPET cases with a clinical phenotype of heritable CV disorder were analyzed. Accuracy and precision were 100% concordant between all sample types, with read depths >100× for most regions tested. Lower read depth, as low as 40×, was occasionally observed with FFPET and dried blood spot. High-quality DNA was obtained from FFPET samples as old as 15 years. Genomic analysis of FFPET from the 4 phenotype-positive/genotype unknown cases all revealed putative disease-causing variants., Conclusions: Similar performance characteristics were observed for next-generation sequencing of FFPET, whole blood, and dried blood spot in the evaluation of inherited CV disorders. Although blood is preferable for genetic analyses, this study offers an alternative when only FFPET is available., (© 2017 American Heart Association, Inc.)

Background: Neonatal hyperbilirubinemia can be severe or prolonged and warrant exploration into the underlying etiology, which may include genetic assessment of UGT1A1 for inherited disorders (i.e. Crigler-Najjar syndrome or Gilbert syndrome)., Methods: In our reference laboratory, we performed UGT1A1 gene sequencing analysis on 346 pediatric patients referred for a clinical indication of hyperbilirubinemia., Results: Males (n = 241) had significantly higher mean total bilirubin concentration compared to females (n = 105) (9.7 and 7.3 mg/dL, respectively, p = 0.042); however, no sex-based difference was observed in frequency of known or suspected reduced function UGT1A1 variants. The presence of two UGT1A1 variants (consistent with Gilbert or Crigler-Najjar syndrome) occurred less frequently in neonates (aged ≤28 days) than older children (aged 1-18 years) (31.3% in neonates vs. 85.1%, p < 0.0001), and among neonates there was no significant difference in mean total bilirubin between those with two UGT1A1 variants and those without (p = 0.47). Three novel variants, including c.337T>G (p.Y113D), c.1037C>A (p.A346E), and c.1469A>C (p.D490A) were identified. Among older children, the most common reason for referral was Gilbert syndrome (83.8%) and UGT1A1 genetic analysis confirmed a diagnosis of Gilbert syndrome in 79.0% of those children., Conclusions: Among neonates, a population in which hyperbilirubinemia is common and often of multifactorial etiology, UGT1A1 genetic testing served as a useful clinical tool in ruling in or ruling out inherited hyperbilirubinemia. Here we describe our experience as a reference laboratory in clinical UGT1A1 full gene sequencing. Our results highlight the challenges in predicting the contribution of genetic variation in UGT1A1 to hyperbilirubinemia based on clinical parameters alone, particularly in neonates, and the utility of UGT1A1 full gene sequencing in the evaluation of neonatal and pediatric hyperbilirubinemia.

Objective: The objective of our study was to measure the effect of genetic variants of these two enzymes, UGT1A1 and SLCO1B1, in the bilirubin metabolic pathway on the degree of hyperbilirubinemia in a cohort of African-American (AA) infants from our well-baby nursery. In addition, a second objective was to document the types and frequencies of genetic variations of these enzymes in our cohort., Study Design: A prospective study of 180 AA infants from the Well Baby Nursery of an inner city community hospital, all of whose mothers were type O pos. Sixty infants were ABO-incompatible direct antiglobulin test (DAT) pos, 60 were ABO-incompatible DAT neg and 60 were type O + . Blood for carboxyhemoglobin (COHb) and variants of the enzymes uridine diphosphoglucuronosyltransferase 1A1 and hepatic solute carrier organic anion transporter 1B1 (SLCO1B1) was drawn at the time of the infants' initial bilirubin, and the infants' precise percentile on the Bhutani nomogram was calculated., Results: Variants in the two enzymes studied were quite common. In total, 21.1% were positive for a Gilbert phenotype, whereas an additional 42.4% were heterozygous for the *28 or *37 variant of UGT1A1. In total, 67.2% were homozygous for the *60 variant of the phenobarbital responsive enhancer module. In total, 41.1% were homozygous for the *1b variant of SLCO1B1, whereas an additional 12.7% were positive for the *4 variant of this gene. In total, 20.6% of infants had variations in both genes. Using logistic regression when COHbc was assessed with each of the different variants, only COHbc (P<0.0001 to 0.0004) was significantly associated with the level of hyperbilirubinemia as defined by the Bhutani nomogram., Conclusion: Although we have found quite a large number of genetic variants of the UGT1A1 and SLCO1B1 genes in the AA population, it does not appear that they have a significant impact on the incidence of hyperbilirubinemia among this group of infants.

Knowledge of variant pathogenicity is key to implementing genomic medicine. We describe variability between expert reviewers in assigning pathogenicity to sequence variants in LDLR, the causal gene in the majority of cases of familial hypercholesterolemia. LDLR was sequenced on the Illumina HiSeq platform (average read depth >200 × ) in 1013 Mayo Biobank participants recruited from 2012 to 2013. Variants with a minor allele frequency (MAF) <5% predicted to be functional or referenced in HGMD (Human Gene Mutation Database) or NCBI-ClinVar databases were reviewed. To assign pathogenicity, variant frequency in population data sets, computational predictions, reported observations and patient-level data including electronic health record-based post hoc phenotyping were leveraged. Of 178 LDLR variants passing quality control, 25 were selected for independent review using either an in-house protocol or a disease/gene-specific semi-quantitative framework based on the American College of Medical Genetics and Genomics-recommended lines of evidence. NCBI-ClinVar included interpretations for all queried variants with 74% (14/19) of variants with >1 submitter showing inconsistency in classification and 26% (5/19) appearing with conflicting clinical actionability. The discordance rate (one-step level of disagreement out of five classes in variant interpretation) between the reviewers was 40% (10/25). Two LDLR variants were independently deemed clinically actionable and returnable. Interpretation of LDLR variants was often discordant among ClinVar submitters and between expert reviewers. A quantitative approach based on strength of each predefined criterion in the context of specific genes and phenotypes may yield greater consistency between different reviewers.

Significant barriers, such as lack of professional guidelines, specialized training for interpretation of pharmacogenomics (PGx) data, and insufficient evidence to support clinical utility, prevent preemptive PGx testing from being widely clinically implemented. The current study, as a pilot project for the Right Drug, Right Dose, Right Time-Using Genomic Data to Individualize Treatment Protocol, was designed to evaluate the impact of preemptive PGx and to optimize the workflow in the clinic setting. We used an 84-gene next-generation sequencing panel that included SLCO1B1, CYP2C19, CYP2C9, and VKORC1 together with a custom-designed CYP2D6 testing cascade to genotype the 1013 subjects in laboratories approved by the Clinical Laboratory Improvement Act. Actionable PGx variants were placed in patient's electronic medical records where integrated clinical decision support rules alert providers when a relevant medication is ordered. The fraction of this cohort carrying actionable PGx variant(s) in individual genes ranged from 30% (SLCO1B1) to 79% (CYP2D6). When considering all five genes together, 99% of the subjects carried an actionable PGx variant(s) in at least one gene. Our study provides evidence in favor of preemptive PGx testing by identifying the risk of a variant being present in the population we studied., (Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Current clinical laboratory practice guidelines for next-generation sequencing (NGS) do not provide definitive guidance on confirming NGS variants. Sanger confirmation of NGS results can be inefficient, redundant, and expensive. We evaluated the accuracy of NGS-detected single-nucleotide variants (SNVs) and insertion/deletion variants (indels) and the necessity of NGS variant confirmation using four NGS target-capture gene panels covering 117 genes, 568 Kbp, and 77 patient DNA samples. Unique NGS-detected variants (1080 SNVs and 124 indels) underwent Sanger confirmation and/or were compared to data from the 1000 Genomes Project (1000G). Recurrent variants in unrelated samples resulted in 919 comparisons between NGS and Sanger, with 100% concordance. In a second comparison, 762 unique NGS results (736 SNVs, 26 indels) from seven 1000G samples were found to have 97.1% concordance with 1000G phase 1 data. Sanger sequencing and 1000G phase 3 data confirmed the accuracy of the NGS results for all 1000G phase 1 discrepancies. In all samples, the depth of coverage exceeded 100× in >99.7% of bases in the target regions. In conclusion, confirmatory analysis by Sanger sequencing of SNVs detected via capture-based NGS testing that meets appropriate quality thresholds is unnecessarily redundant. In contrast, Sanger sequencing for indels may be required for defining the correct genomic location, and Sanger may be used for quality-assurance purposes., (Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

The diagnosis of Marfan syndrome (MFS) remains challenging despite the 2010 revision to Ghent nosology criteria, and there is a lack of published information regarding FBN1 genotype associations in patients since the update in Ghent criteria. Applying revised Ghent criteria, we reviewed consecutive proband cases (n=292) submitted for FBN1 sequencing. Testing yielded 207 pathogenic or likely pathogenic FBN1 variants, with 114/207 (55%) missense, 67/207 (32%) non-sense or frameshift, and 28/207 (13%) splicing. There were 130 novel FBN1 variants predicted as pathogenic or likely pathogenic (n=109) or variant of undetermined significance (n=21). Of the 104 patients who met 2010 revised Ghent criteria, 87/104 (82%) had a pathogenic or likely pathogenic variant. There was a significantly lower frequency of missense variants (41 vs 89%; P<0.0001) observed in the Ghent-positive (vs Ghent-negative) patients, and this association held true in age-based groupings. Previously described genotype associations with ectopia lentis and early onset/'neonatal' MFS were confirmed in our cohort. Overall, our study points to the imperfect nature of relying solely on clinical criteria to diagnose MFS as well as the potential importance of truncating/splicing variants in Ghent-positive cases. Furthermore, the description of numerous novel variants and associated clinical findings may be useful for future clinical interpretation of FBN1 genotype in patients with suspected MFS.

Purpose: Marfan syndrome is a systemic disorder that typically involves FBN1 mutations and cardiovascular manifestations. We investigated FBN1 genotype-phenotype correlations with aortic events (aortic dissection and prophylactic aortic surgery) in patients with Marfan syndrome., Methods: Genotype and phenotype information from probands (n = 179) with an FBN1 pathogenic or likely pathogenic variant were assessed., Results: A higher frequency of truncating or splicing FBN1 variants was observed in Ghent criteria-positive patients with an aortic event (n = 34) as compared with all other probands (n = 145) without a reported aortic event (79 vs. 39%; P < 0.0001), as well as Ghent criteria-positive probands (n = 54) without an aortic event (79 vs. 48%; P = 0.0039). Most probands with an early aortic event had a truncating or splicing variant (100% (n = 12) and 95% (n = 21) of patients younger than 30 and 40 years old, respectively). Aortic events occurred at a younger median age in patients with truncating/splicing variants (29 years) as compared with those with missense variants (51 years). A trend toward a higher frequency of truncating/splicing variants in patients with aortic dissection (n = 21) versus prophylactic surgery (n = 13) (85.7 vs. 69.3%; not significant) was observed., Conclusion: These aortic event- and age-associated findings may have important implications for the management of Marfan syndrome patients with FBN1 truncating and splicing variants.Genet Med 17 3, 177-187.

Recent reports have suggested an association between variation in the serotonin transporter and primary pulmonary hypertension and myocardial infarction. We set out to determine whether these associations were present in a population of patients who underwent SLC6A4 genotyping and to explore whether genetic variation in the serotonin transporter might be also associated with other cardiovascular functional and structural abnormalities. Included were 3473 patients who were genotyped for the SLC6A4 5HTTLPR polymorphism and a subset for rs25531 (n=816) and STin2 (n=819). An association was observed between 5HTTLPR and primary pulmonary hypertension (p=0.0130), anomalies of the cerebrovascular system (p<0.0001), and other anomalies of great veins (p=0.0359). The combined 5HTTLPR and rs25531 genotype was associated with tachycardia (p=0.0123). There was an association of the STin2 genotype with abnormal electrocardiogram (ECG) (p=0.0366) and abnormal cardiac study (0.0311). Overall, these results represent a step toward the understanding of the impact of SLC6A4 variation on cardiovascular pathology.

Objective: Familial hypercholesterolemia (FH) can be due to mutations in LDLR, PCSK9, and APOB. In phenotypically defined patients, a subset remains unresponsive to lipid-lowering therapies and requires low density-lipoprotein (LDL) apheresis treatment. In this pilot study, we examined the genotype/phenotype relationship in patients with dyslipidemia undergoing routine LDL apheresis., Design: LDLR, APOB, and PCKS9 were analyzed for disease-causing mutations in seven patients undergoing routine LDL apheresis. Plasma and serum specimens were collected pre- and post-apheresis and analyzed for lipid concentrations, Lp(a) cholesterol, and lipoprotein particle concentrations (via NMR)., Results: We found that four patients harbored LDLR mutations and of these, three presented with xanthomas. While similar reductions in LDL-cholesterol (LDL-C), apolipoprotein B, and LDL particles (LDL-P) were observed following apheresis in all patients, lipid profile analysis revealed the LDLR mutation-positive cohort had a more pro-atherogenic profile (higher LDL-C, apolipoprotein B, LDL-P, and small LDL-P) pre-apheresis., Conclusion: Our data show that not all clinically diagnosed FH patients who require routine apheresis have genetically defined disease. In our small cohort, those with LDLR mutations had a more proatherogenic phenotype than those without identifiable mutations. This pilot cohort suggests that patients receiving the maximum lipid lowering therapy could be further stratified, based on genetic make-up, to optimize treatment., (© 2013 Wiley Periodicals, Inc.)

Objective: To report the design and implementation of the Right Drug, Right Dose, Right Time-Using Genomic Data to Individualize Treatment protocol that was developed to test the concept that prescribers can deliver genome-guided therapy at the point of care by using preemptive pharmacogenomics (PGx) data and clinical decision support (CDS) integrated into the electronic medical record (EMR)., Patients and Methods: We used a multivariate prediction model to identify patients with a high risk of initiating statin therapy within 3 years. The model was used to target a study cohort most likely to benefit from preemptive PGx testing among the Mayo Clinic Biobank participants, with a recruitment goal of 1000 patients. We used a Cox proportional hazards model with variables selected through the Lasso shrinkage method. An operational CDS model was adapted to implement PGx rules within the EMR., Results: The prediction model included age, sex, race, and 6 chronic diseases categorized by the Clinical Classifications Software for International Classification of Diseases, Ninth Revision codes (dyslipidemia, diabetes, peripheral atherosclerosis, disease of the blood-forming organs, coronary atherosclerosis and other heart diseases, and hypertension). Of the 2000 Biobank participants invited, 1013 (51%) provided blood samples, 256 (13%) declined participation, 555 (28%) did not respond, and 176 (9%) consented but did not provide a blood sample within the recruitment window (October 4, 2012, through March 20, 2013). Preemptive PGx testing included CYP2D6 genotyping and targeted sequencing of 84 PGx genes. Synchronous real-time CDS was integrated into the EMR and flagged potential patient-specific drug-gene interactions and provided therapeutic guidance., Conclusion: This translational project provides an opportunity to begin to evaluate the impact of preemptive sequencing and EMR-driven genome-guided therapy. These interventions will improve understanding and implementation of genomic data in clinical practice., (Copyright © 2014 Mayo Foundation for Medical Education and Research. Published by Elsevier Inc. All rights reserved.)

Background: Variability of response to statins has been related to polymorphisms in genes involved in cholesterol homeostasis and statin metabolism, such as CYP3A4 and CYP3A5. We investigated the effects of atorvastatin on CYP3A4 and CYP3A5 mRNA expression in mononuclear cells and on CYP3A activity and their interactions with common variants., Methods: Unrelated individuals (n=121) with hypercholesterolemia (HC) were treated with atorvastatin (10 mg/day/4 weeks). Ninety-two normolipidemic (NL) subjects were selected as a control group. Genotype analysis of CYP3A4*1B (rs2740574), CYP3A4*22 (rs35599367), CYP3A5*3C (rs776746), and CYP3A5*1D (rs15524) and mRNA levels in peripheral blood mononuclear cells (PBMCs) were estimated. CYP3A activity was phenotyped by the urinary cortisol to 6-beta-hydroxy-cortisol ratio., Results: LDL cholesterol reduction in response to atorvastatin was positively correlated with change in CYP3A4 (R(2)=0.039, p=0.037) and CYP3A5 (R(2)=0.047, p=0.019) mRNA levels and negatively correlated with CYP3A activity (R(2)=0.071, p=0.022). CYP3A5*3C (AGT haplotype) was associated to lower basal CYP3A5 mRNA expression in HC (p<0.045), however none of the haplotype groups impacted treatment., Conclusion: It is likely that cholesterolemia status changes promoted by atorvastatin play a role in regulating CYP3A4 and CYP3A5 mRNA expression in PBMCs, as well as CYP3A activity. CYP3A5*3C (AGT haplotype) also contributes for the variability of CYP3A5 mRNA levels in PBMCs., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Objective: To assess the clinical utility of UGT1A1 genetic testing and describe the spectrum and prevalence of UGT1A1 variations identified in pediatric unconjugated hyperbilirubinemia (UCH), and to characterize specific genotype-phenotype relationships in suspected Gilbert and Crigler-Najjar syndromes., Study Design: A retrospective study was conducted to review clinical information and UGT1A1 genotyping data from 181 pediatric patients referred for UCH. In silico analyses were performed to aid in the assessment of novel UGT1A1 variants., Results: Overall, 146/181 pediatric patients had at least one heterozygous UGT1A1 functional variant. Identified UGT1A1 variants included 17 novel variants, 7 rare star alleles, and 1 rare variant. There were 129 individuals who possessed the TA7 (*28) promoter repeat and 15 individuals who possessed the *6 (c.211G > A) variation. Out of the 104 individuals with accompanying bilirubin levels, 41 individuals did not have identifiable UGT1A1 variants that explained their UCH, although glucose-6-phosphate dehydrogenase deficiency and other causes of UCH could not be ruled out., Conclusion: Much of the observed UCH could be attributed to variation at the UGT1A1 locus, and UGT1A1 testing helped to substantiate a genetic diagnosis, thereby aiding in individual and family disease management. Although UGT1A1 variation plays a large role in UCH, genetic assessment of UGT1A1 alone may not be comprehensive. Assessment of additional genes may also be useful to evaluate genetic causes for UCH., (Copyright © 2013 Mosby, Inc. All rights reserved.)

Technological applications and novel biomarkers in the field of molecular diagnostics have never been evolving at a more rapid pace. These novel applications have the promise to change the face of clinical care as we move into the era of personalized medicine. While some of these technologies and biomarkers have been adopted by some clinical laboratories, most laboratories face a steep learning curve in bringing these dramatically new and different molecular diagnostic applications on board. Furthermore, interpreting the vast amounts and new types of data produced by these novel applications brings forth challenges for laboratorians and clinicians alike. In this article, we discuss how some of these emerging novel molecular diagnostic technologies and analytes, such as next-generation sequencing, chromosomal microarray, microRNAs and circulating fetal nucleic acids are revolutionizing patient care and personalized medicine.

Long QT syndrome (LQTS) is a cardiac channelopathy associated with syncope, seizures, and sudden death. Approximately 75% of LQTS is due to mutations in genes encoding for 3 cardiac ion channel α-subunits (LQT1 to LQT3). However, traditional mutational analyses have limited detection capabilities for atypical mutations such as large gene rearrangements. We set out to determine the prevalence and spectrum of large deletions/duplications in the major LQTS-susceptibility genes in unrelated patients who were mutation negative after point mutation analysis of LQT1- to LQT12-susceptibility genes. Forty-two unrelated, clinically strong LQTS patients were analyzed using multiplex ligation-dependent probe amplification, a quantitative fluorescent technique for detecting multiple exon deletions and duplications. The SALSA multiplex ligation-dependent probe amplification LQTS kit from MRC-Holland was used to analyze the 3 major LQTS-associated genes, KCNQ1, KCNH2, and SCN5A, and the 2 minor genes, KCNE1 and KCNE2. Overall, 2 gene rearrangements were found in 2 of 42 unrelated patients (4.8%, confidence interval 1.7 to 11). A deletion of KCNQ1 exon 3 was identified in a 10-year-old Caucasian boy with a corrected QT duration of 660 ms, a personal history of exercise-induced syncope, and a family history of syncope. A deletion of KCNQ1 exon 7 was identified in a 17-year-old Caucasian girl with a corrected QT duration of 480 ms, a personal history of exercise-induced syncope, and a family history of sudden cardiac death. In conclusion, because nearly 5% of patients with genetically elusive LQTS had large genomic rearrangements involving the canonical LQTS-susceptibility genes, reflex genetic testing to investigate genomic rearrangements may be of clinical value., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The response to beta blockers in patients with heart failure could be associated with the genotype of drug-metabolizing enzymes and/or drug targets. The purpose of the present study was to determine whether specific genetic polymorphisms in ADRB1 (encoding the beta1-adrenergic receptor), CYP2D6, and UGT1A1 correlated with dose of, or response to, metoprolol or carvedilol treatment in patients with heart failure. A cohort of patients with heart failure (n = 93), characterized as responders or nonresponders to metoprolol (n = 19) or carvedilol (n = 74) therapy, was retrospectively identified. Individual genotyping was performed for a panel of polymorphisms in the ADRB1, CYP2D6, and UGT1A1 genes. Univariate and multivariate analyses were performed to compare the genotype to the metoprolol or carvedilol response status and dose. A nonresponse was identified in 10 of 19 patients taking metoprolol and 32 of 74 patients taking carvedilol. None of the polymorphisms in ADRB1, CYP2D6, and UGT1A1 were associated with a response or nonresponse. However, a significant relation between the carvedilol (but not metoprolol) dose and the ADRB1 and CYP2D6 genotype was observed. Patients homozygous for the ADRB1 389Gly variant or who were CYP2D6 poor metabolizers achieved a significantly higher dose of carvedilol (p = 0.01 and p = 0.02, respectively). In conclusion, polymorphisms in ADRB1, CYP2D6, and UGT1A1 were not associated with a response to metoprolol or carvedilol therapy in our cohort of patients with heart failure. The ADRB1 and CYP2D6 genotype, alone and in haplotype, were significantly associated with the dose of carvedilol., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

The antithrombotic benefits of warfarin are countered by a narrow therapeutic index that contributes to excessive bleeding or cerebrovascular clotting and stroke in some patients. This article reviews the current literature describing warfarin sensitivity genotyping and compares the results of that review to the findings of our study in 189 patients at Mayo Clinic conducted between June 2001 and April 2003. For the review of the literature, we identified relevant peer-reviewed articles by searching the Web of Knowledge using key word warfarin-related adverse event. For the 189 Mayo Clinic patients initiating warfarin therapy to achieve a target international normalized ratio (INR) in the range of 2.0 to 3.5, we analyzed the CYP2C9 (cytochrome P450 2C9) and VKORC1 (vitamin K epoxide reductase complex, subunit 1) genetic loci to study the relationship among the initial warfarin dose, steady-state dose, time to achieve steady-state dose, variations in INR, and allelic variance. Results were compared with those previously reported in the literature for 637 patients. The relationships between allelic variants and warfarin sensitivity found in our study of Mayo Clinic patients are fundamentally the same as in those reported by others. The Mayo Clinic population is predominantly white and shows considerable allelic variability in CYP2C9 and VKORC1. Certain of these alleles are associated with increased sensitivity to warfarin. Polymorphisms in CYP2C9 and VKORC1 have a considerable effect on warfarin dose in white people. A correlation between steady-state warfarin dose and allelic variants of CYP2C9 and VKORC1 has been demonstrated by many previous reports and is reconfirmed in this report. The allelic variants found to most affect warfarin sensitivity are CYP2C9*1*1-VKORC1BB (less warfarin sensitivity than typical); CYP2C9*1*1-VKORC1AA (considerable variance in INR throughout initiation); CYP2C9*1*2-VKORC1AB (more sensitivity to warfarin than typical); CYP2C9*1*3-VKORC1AB (much more sensitivity to warfarin than typical); CYP2C9*1*2-VKORC1AB (much more sensitivity to warfarin than typical); CYP2C9*1*3-VKORC1AA (much more sensitivity to warfarin than typical); and CYP2C9*2*2-VKORC1AB (much more sensitivity to warfarin than typical). Although we were unable to show an association between allelic variants and initial warfarin dose or dose escalation, an association was seen between allelic variant and steady-state warfarin dose. White people show considerable variance in CYP2C9 allele types, whereas people of Asian or African descent infrequently carry CYP2C9 allelic variants. The VKORC1AA allele associated with high warfarin sensitivity predominates in those of Asian descent, whereas white people and those of African descent show diversity, carrying either the VKORC1BB, an allele associated with low warfarin sensitivity, or VKORC1AB or VKORC1AA, alleles associated with moderate and high warfarin sensitivity, respectively.

With the advent of the genome-wide association (GWA) study, a promising new avenue for identifying genetic markers for complex diseases like coronary artery disease (CAD) has been opened. This avenue, however, is not without challenges and limitations, including the need for carefully designed and executed studies and the risk of false positive associations. Nonetheless, new markers have been identified through such studies that could potentially revolutionize the ways that individuals with CAD are identified and managed.

Pharmacologic therapies are essential in the management of patients with hemostatic and thrombotic diseases because these therapies are able to modify components of the coagulation pathway and platelet response. Nevertheless, responses to different drugs vary significantly, and the best clinical outcome frequently involves a delicate risk/benefit balance. The recent exponential growth of pharmacogenomics has led to the emergence of individualized medicine that has revolutionized modern medical practice, allowing for a deeper understanding of pathophysiology, increased diagnostic specificity, and better markers for risk stratification and an enhanced potential for gene therapy. Management of drugs prescribed to treat thrombotic and hemostatic abnormalities may benefit from pharmacogenetics, and our focus in this review will be on the pharmacogenetics related to some of the more common drugs that fall into this category.

Familial hypercholesterolemia (FH) is the most common form of autosomal-dominant hypercholesterolemia, and is caused by mutations in the low-density lipoprotein receptor (LDLR) gene. Heterozygous FH is characterized by elevated low-density lipoprotein (LDL) cholesterol and early-onset cardiovascular disease, whereas homozygous FH results in more severe LDL cholesterol elevation with death by 20 years of age. We present here the case of an African-American female FH patient presenting with a myocardial infarction at the age of 48, recurrent angina pectoris and numerous coronary artery stents. Her pretreated LDL cholesterol levels were more typical of a homozygous FH pattern and she was resistant to conventional lipid-lowering treatment, yet her other clinical parameters were not necessarily consistent with homozygous FH. Genetic testing revealed two LDLR variants on the same chromosome: one a novel missense mutation in exon 14 (Cys681Gly) and the other a promoter variant (IVS1-217C>T) previously shown to result in increased LDLR transcription. Disease-associated PCSK9 or APOB mutations were not identified in this individual. Overall, her genetic and clinical profile suggests that enhanced expression of the mutant LDLR allele resulted in a severe phenotype with characteristics of both heterozygous and homozygous FH.

MUTYH adenomatous polyposis (MAP) can mimic both the familial adenomatous polyposis (FAP) and hereditary nonpolyposis colon cancer (HNPCC) phenotypes. As a result of MAP's phenotypic overlap with FAP, some DNA diagnostic laboratories perform MUTYH testing in conjunction with APC testing in patients with suspected FAP or attenuated FAP (AFAP). In addition to testing FAP/AFAP samples for MUTYH mutations, we were interested whether there would also be value in testing samples referred for HNPCC testing. To determine this, we tested a consecutive series of 229 samples referred for HNPCC testing for the two most common MUTYH mutations in the Caucasian population. To enrich our study population with MAP cases, we only included samples from patients with early onset colorectal cancer (CRC diagnosed <50 years old) in whom HNPCC had been excluded by microsatellite instability testing (microsatellite stable or low microsatellite instability). Four biallelic (2%) and six monoallelic (3%) MUTYH mutation carriers were identified. No clinical factors predicted MUTYH mutation status. Specifically, a family history of vertical transmission of CRC or having few polyps (<15) did not rule out the possibility of biallelic MUTYH mutations. Thus, MUTYH mutation testing may be a reasonable cascade test in early onset CRC found to have proficient DNA mismatch repair, regardless of pattern of family history or number of polyps.