Implementation of preemptive DNA sequence-based pharmacogenomics testing across a large academic medical center: The Mayo-Baylor RIGHT 10K Study.

PURPOSE: The Mayo-Baylor RIGHT 10K Study enabled preemptive, sequence-based pharmacogenomics (PGx)-driven drug prescribing practices in routine clinical care within a large cohort. We also generated the tools and resources necessary for clinical PGx implementation and identified challenges that need to be overcome. Furthermore, we measured the frequency of both common genetic variation for which clinical guidelines already exist and rare variation that could be detected by DNA sequencing, rather than genotyping. METHODS: Targeted oligonucleotide-capture sequencing of 77 pharmacogenes was performed using DNA from 10,077 consented Mayo Clinic Biobank volunteers. The resulting predicted drug response-related phenotypes for 13 genes, including CYP2D6 and HLA, affecting 21 drug-gene pairs, were deposited preemptively in the Mayo electronic health record. RESULTS: For the 13 pharmacogenes of interest, the genomes of 79% of participants carried clinically actionable variants in 3 or more genes, and DNA sequencing identified an average of 3.3 additional conservatively predicted deleterious variants that would not have been evident using genotyping. CONCLUSION: Implementation of preemptive rather than reactive and sequence-based rather than genotype-based PGx prescribing revealed nearly universal patient applicability and required integrated institution-wide resources to fully realize individualized drug therapy and to show more efficient use of health care resources.

The Genetic Counselor's Role in Managing Ethical Dilemmas Arising in the Laboratory Setting.

Ethical dilemmas are encountered commonly in the setting of the clinical genetic testing laboratory due to the complexity of genetic testing and the number of relevant stakeholders involved in the genetic testing process. Based on their clinical training and role within the laboratory, genetic counselors are uniquely equipped to identify and facilitate management of ethical dilemmas. This paper reviews the historical context of ethical theory and its application to the field of genetic counseling. Theoretical and applied ethics are explored in the context of dilemmas arising in the laboratory setting, with a focus on the role of the laboratory genetic counselor in managing ethical dilemmas. Two illustrative case examples are provided.

Increased frequency of FBN1 truncating and splicing variants in Marfan syndrome patients with aortic events.

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.

Decreased frequency of FBN1 missense variants in Ghent criteria-positive Marfan syndrome and characterization of novel FBN1 variants.

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.

Genetic and biochemical analyses in dyslipidemic patients undergoing LDL apheresis.

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.

Looking back and moving forward: an historical perspective from laboratory genetic counselors.

Despite a consistent increase in genetic counselors who report working in laboratory positions, there is a relative dearth of literature on laboratory genetic counseling. Semi-structured interviews were completed with nine laboratory genetic counselors to document how positions were created and have changed with time. Interview transcriptions were analyzed for emerging themes. Several common themes were identified, including that early positions were often part-time, laboratory-initiated and had a lack of job definition. Laboratory genetic counselors commented on their evolving roles and responsibilities, with their positions becoming more technical and specialized over time and many taking on managerial and supervisory roles. All genetic counselors surveyed reported using core genetic counseling skills in their positions. The expansion of diagnostic testing and quickly evolving technology were common themes in regards to the future of laboratory genetic counselors, and participants commented on laboratory genetic counselors having expanding roles with data management, result interpretation and reporting, and guidance of other healthcare providers. Other comments included the impact of competition among laboratories and how training programs can better prepare genetic counseling students for a career in the laboratory setting. This study describes the emergence, and subsequent evolution, of laboratory genetic counseling positions as a significant subspecialty of genetic counseling.

UGT1A1 genetic analysis as a diagnostic aid for individuals with unconjugated hyperbilirubinemia.

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.

Targeted Genotyping in Clinical Pharmacogenomics: What Is Missing?

Clinical pharmacogenomic testing typically uses targeted genotyping, which only detects variants included in the test design and may vary among laboratories. To evaluate the potential patient impact of genotyping compared with sequencing, which can detect common and rare variants, an in silico targeted genotyping panel was developed based on the variants most commonly included in clinical tests and applied to a cohort of 10,030 participants who underwent sequencing for CYP1A2, CYP2C19, CYP2C9, CYP2D6, CYP3A4, CYP3A5, DPYD, SLCO1B1, TPMT, UGT1A1, and VKORC1. The results of in silico targeted genotyping were compared with the clinically reported sequencing results. Of the 10,030 participants, 2780 (28%) had at least one potentially clinically relevant variant/allele identified by sequencing that would not have been detected in a standard targeted genotyping panel. The genes with the largest number of participants with variants only detected by sequencing were SLCO1B1, DPYD, and CYP2D6, which affected 13%, 6.3%, and 3.5% of participants, respectively. DPYD (112 variants) and CYP2D6 (103 variants) had the largest number of unique variants detected only by sequencing. Although targeted genotyping detects most clinically significant pharmacogenomic variants, sequencing-based approaches are necessary to detect rare variants that collectively affect many patients. However, efforts to establish pharmacogenomic variant classification systems and nomenclature to accommodate rare variants will be required to adopt sequencing-based pharmacogenomics.

Variability in gene-based knowledge impacts variant classification: an analysis of FBN1 missense variants in ClinVar.

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.

Clinical UGT1A1 Genetic Analysis in Pediatric Patients: Experience of a Reference Laboratory.

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.

Technical Advances for the Clinical Genomic Evaluation of Sudden Cardiac Death: Verification of Next-Generation Sequencing Panels for Hereditary Cardiovascular Conditions Using Formalin-Fixed Paraffin-Embedded Tissues and Dried Blood Spots.

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.

Genetic testing utilization and the role of the laboratory genetic counselor.

Laboratory genetic counselors within hospital laboratories and genetic testing laboratories have an important role in increasing the appropriate utilization of genetic tests. This service is becoming more important as genetic testing becomes more complex and the demand for genetic testing in healthcare increases. Additionally genetic tests are among the most expensive assays in the clinical laboratory test catalog. Laboratory genetic counselors are able to increase genetic test utilization through review and assessment of the appropriateness of the ordered testing, developing protocols, and by increasing communication with ordering providers.