Megan E. Grove

Clinical Interpretation and Implications of Whole-Genome Sequencing

IMPORTANCE Whole-genome sequencing (WGS) is increasingly applied in clinical medicine and is expected to uncover clinically significant findings regardless of sequencing indication. OBJECTIVES To examine coverage and concordance of clinically relevant genetic variation provided by WGS technologies; to quantitate inherited disease risk and pharmacogenomic findings in WGS data and resources required for their discovery and interpretation; and to evaluate clinical action prompted by WGS findings. DESIGN, SETTING, AND PARTICIPANTS An exploratory study of 12 adult participants recruited at Stanford University Medical Center who underwent WGS between November 2011 and March 2012. A multidisciplinary team reviewed all potentially reportable genetic findings. Five physicians proposed initial clinical follow-up based on the genetic findings. MAIN OUTCOMES AND MEASURES Genome coverage and sequencing platform concordance in different categories of genetic disease risk, person-hours spent curating candidate disease-risk variants, interpretation agreement between trained curators and disease genetics databases, burden of inherited disease risk and pharmacogenomic findings, and burden and interrater agreement of proposed clinical follow-up. RESULTS Depending on sequencing platform, 10% to 19% of inherited disease genes were not covered to accepted standards for single nucleotide variant discovery. Genotype concordance was high for previously described single nucleotide genetic variants (99%-100%) but low for small insertion/deletion variants (53%-59%). Curation of 90 to 127 genetic variants in each participant required a median of 54 minutes (range, 5-223 minutes) per genetic variant, resulted in moderate classification agreement between professionals (Gross κ, 0.52; 95% CI, 0.40-0.64), and reclassified 69% of genetic variants cataloged as disease causing in mutation databases to variants of uncertain or lesser significance. Two to 6 personal disease-risk findings were discovered in each participant, including 1 frameshift deletion in the BRCA1 gene implicated in hereditary breast and ovarian cancer. Physician review of sequencing findings prompted consideration of a median of 1 to 3 initial diagnostic tests and referrals per participant, with fair interrater agreement about the suitability of WGS findings for clinical follow-up (Fleiss κ, 0.24; P < 001). CONCLUSIONS AND RELEVANCE In this exploratory study of 12 volunteer adults, the use of WGS was associated with incomplete coverage of inherited disease genes, low reproducibility of detection of genetic variation with the highest potential clinical effects, and uncertainty about clinically reportable findings. In certain cases, WGS will identify clinically actionable genetic variants warranting early medical intervention. These issues should be considered when determining the role of WGS in clinical medicine.

Views of Genetics Health Professionals on the Return of Genomic Results

As exome and whole genome sequencing become clinically available, the potential to receive a large number of clinically relevant but incidental results is a significant challenge in the provision of genomic counseling. We conducted three focus groups of a total of 35 individuals who were members of ASHG and/or NSGC, assessing views towards the return of genomic results. Participants stressed that patient autonomy was primary. There was consensus that a mechanism to return results to the healthcare provider, rather than patient, and to streamline integration into the electronic health record would ensure these results had the maximal impact on patient management. All three focus groups agreed that pharmacogenomic results were reasonable to return and that they were not felt to be stigmatizing. With regard to the return of medically relevant results, there was much debate. Participants had difficulty in consistently assigning specific diseases to ‘bins’ that were considered obligatory versus optional for disclosure. Consensus was reached regarding the importance of informed consent and pretest counseling visits to clarify what the return of results process would entail. Evidence based professional guidelines should continue to be developed and regularly revised to assist in consistently and appropriately providing genomic results to patients.

Molecular Diagnosis of Long-QT syndrome at 10 Days of Life by Rapid Whole Genome Sequencing

BACKGROUND The advent of clinical next generation sequencing is rapidly changing the landscape of rare disease medicine. Molecular diagnosis of long QT syndrome (LQTS) can affect clinical management, including risk stratification and selection of pharmacotherapy on the basis of the type of ion channel affected, but results from the current gene panel testing requires 4-16 weeks before return to clinicians. OBJECTIVE A term female infant presented with 2:1 atrioventricular block and ventricular arrhythmias consistent with perinatal LQTS, requiring aggressive treatment including epicardial pacemaker and cardioverter-defibrillator implantation and sympathectomy on day of life 2. We sought to provide a rapid molecular diagnosis for the optimization of treatment strategies. METHODS We performed Clinical Laboratory Improvement Amendments-certified rapid whole genome sequencing (WGS) with a speed-optimized bioinformatics platform to achieve molecular diagnosis at 10 days of life. RESULTS We detected a known pathogenic variant in KCNH2 that was demonstrated to be paternally inherited by follow-up genotyping. The unbiased assessment of the entire catalog of human genes provided by WGS revealed a maternally inherited variant of unknown significance in a novel gene. CONCLUSION Rapid clinical WGS provides faster and more comprehensive diagnostic information at 10 days of life than does standard gene panel testing. In selected clinical scenarios such as perinatal LQTS, rapid WGS can provide more timely and clinically actionable information than can a standard commercial test.

Early somatic mosaicism is a rare cause of long-QT syndrome

Significance Most genetic studies and clinical genetic testing do not look for the possibility of mosaic variation. The genetic form of long-QT syndrome (LQTS) can result in life-threatening arrhythmias, but 30% of patients do not have a genetic diagnosis. We performed deep characterization of a mosaic variant in an infant with perinatal LQTS and developed a computational model showing how abnormal cellular repolarization in only 8% of heart cells may cause arrhythmia. Finally we looked at the prevalence of mosaicism among patients with LQTS; in a population of 7,500 individuals we found evidence of pathogenic early somatic mosaicism in approximately 0.17% of LQTS patients without a genetic diagnosis. Together these data establish an unreported mechanism for LQTS and other genetic arrhythmias. Somatic mosaicism, the occurrence and propagation of genetic variation in cell lineages after fertilization, is increasingly recognized to play a causal role in a variety of human diseases. We investigated the case of life-threatening arrhythmia in a 10-day-old infant with long QT syndrome (LQTS). Rapid genome sequencing suggested a variant in the sodium channel NaV1.5 encoded by SCN5A, NM_000335:c.5284G > T predicting p.(V1762L), but read depth was insufficient to be diagnostic. Exome sequencing of the trio confirmed read ratios inconsistent with Mendelian inheritance only in the proband. Genotyping of single circulating leukocytes demonstrated the mutation in the genomes of 8% of patient cells, and RNA sequencing of cardiac tissue from the infant confirmed the expression of the mutant allele at mosaic ratios. Heterologous expression of the mutant channel revealed significantly delayed sodium current with a dominant negative effect. To investigate the mechanism by which mosaicism might cause arrhythmia, we built a finite element simulation model incorporating Purkinje fiber activation. This model confirmed the pathogenic consequences of cardiac cellular mosaicism and, under the presenting conditions of this case, recapitulated 2:1 AV block and arrhythmia. To investigate the extent to which mosaicism might explain undiagnosed arrhythmia, we studied 7,500 affected probands undergoing commercial gene-panel testing. Four individuals with pathogenic variants arising from early somatic mutation events were found. Here we establish cardiac mosaicism as a causal mechanism for LQTS and present methods by which the general phenomenon, likely to be relevant for all genetic diseases, can be detected through single-cell analysis and next-generation sequencing.

Long-read genome sequencing identifies causal structural variation in a Mendelian disease

PurposeCurrent clinical genomics assays primarily utilize short-read sequencing (SRS), but SRS has limited ability to evaluate repetitive regions and structural variants. Long-read sequencing (LRS) has complementary strengths, and we aimed to determine whether LRS could offer a means to identify overlooked genetic variation in patients undiagnosed by SRS.MethodsWe performed low-coverage genome LRS to identify structural variants in a patient who presented with multiple neoplasia and cardiac myxomata, in whom the results of targeted clinical testing and genome SRS were negative.ResultsThis LRS approach yielded 6,971 deletions and 6,821 insertions > 50 bp. Filtering for variants that are absent in an unrelated control and overlap a disease gene coding exon identified three deletions and three insertions. One of these, a heterozygous 2,184 bp deletion, overlaps the first coding exon of PRKAR1A, which is implicated in autosomal dominant Carney complex. RNA sequencing demonstrated decreased PRKAR1A expression. The deletion was classified as pathogenic based on guidelines for interpretation of sequence variants.ConclusionThis first successful application of genome LRS to identify a pathogenic variant in a patient suggests that LRS has significant potential for the identification of disease-causing structural variation. Larger studies will ultimately be required to evaluate the potential clinical utility of LRS.

Sequence to Medical Phenotypes: A Framework for Interpretation of Human Whole Genome DNA Sequence Data

Abstract High throughput sequencing has facilitated a precipitous drop in the cost of genomic sequencing, prompting predictions of a revolution in medicine via genetic personalization of diagnostic and therapeutic strategies. There are significant barriers to realizing this goal that are related to the difficult task of interpreting personal genetic variation. A comprehensive, widely accessible application for interpretation of whole genome sequence data is needed. Here, we present a series of methods for identification of genetic variants and genotypes with clinical associations, phasing genetic data and using Mendelian inheritance for quality control, and providing predictive genetic information about risk for rare disease phenotypes and response to pharmacological therapy in single individuals and father-mother-child trios. We demonstrate application of these methods for disease and drug response prognostication in whole genome sequence data from twelve unrelated adults, and for disease gene discovery in one father-mother-child trio with apparently simplex congenital ventricular arrhythmia. In doing so we identify clinically actionable inherited disease risk and drug response genotypes in pre-symptomatic individuals. We also nominate a new candidate gene in congenital arrhythmia, ATP2B4, and provide experimental evidence of a regulatory role for variants discovered using this framework.

Clinically impactful differences in variant interpretation between clinicians and testing laboratories: a single-center experience

PurposeTo describe the frequency and nature of differences in variant classifications between clinicians and genetic testing laboratories.MethodsRetrospective review of variants identified through genetic testing ordered in routine clinical care by clinicians in the Stanford Center for Inherited Cardiovascular Disease. We compared classifications made by clinicians, the testing laboratory, and other laboratories in ClinVar.ResultsOf 688 laboratory classifications, 124 (18%) differed from the clinicians’ classifications. Most differences in classification would probably affect clinical care of the patient and/or family (83%, 103/124). The frequency of discordant classifications differed depending on the testing laboratory (P < 0.0001) and the testing laboratory’s classification (P < 0.00001). For the majority (82/124, 66%) of discordant classifications, clinicians were more conservative (less likely to classify a variant pathogenic or likely pathogenic). The clinicians’ classification was discordant with one or more submitter in ClinVar in 49.1% (28/57) of cases, while the testing laboratory’s classification was discordant with a ClinVar submitter in 82.5% of cases (47/57, P = 0.0002).ConclusionThe clinical team disagreed with the laboratory’s classification at a rate similar to that of reported disagreements between laboratories. Most of this discordance was clinically significant, with clinicians tending to be more conservative than laboratories in their classifications.

Faster REM sleep EEG and worse restedness in older insomniacs with HLA DQB1*0602

HLA DQB1*0602 is found in most individuals with hypocretin-deficient narcolepsy, a disorder characterized by a severe disruption of sleep and wake. Population studies indicate that DQB1*0602 may also be associated with normal phenotypic variation of rapid eye movement (REM) sleep. Disruption of REM sleep has been linked to specific symptoms of insomnia. We here examine the relationship of sleep and DQB1*0602 in older individuals (n=46) with primary insomnia, using objective (polysomnography, wrist actigraphy) and subjective (logs, scales) measures. DQB1*0602 positivity was similarly distributed in the older individuals with insomnia (24%) as in the general population (25%). Most sleep variables were statistically indistinguishable between DQB1*0602 positive and negative subjects except that those with the allele reported that they were significantly less well rested than those without it. When sleep efficiencies were lower than 70%, DQB1*0602 positive subjects reported being less well rested at the same sleep efficiency than those without the allele. Examination of EEG during REM sleep also revealed that DQB1*0602 positive subjects had EEG shifted towards faster frequencies compared with negative subjects. Thus, DQB1*0602 positivity is associated with both a shift in EEG power spectrum to faster frequencies during REM sleep and a diminution of restedness given the same sleep quantity.

Identification of rare-disease genes in diverse undiagnosed cases using whole blood transcriptome sequencing and large control cohorts

RNA sequencing (RNA-seq) is a complementary approach for Mendelian disease diagnosis for patients in whom exome-sequencing is not informative. For both rare neuromuscular and mitochondrial disorders, its application has improved diagnostic rates. However, the generalizability of this approach to diverse Mendelian diseases has yet to be evaluated. We sequenced whole blood RNA from 56 cases with undiagnosed rare diseases spanning 11 diverse disease categories to evaluate the general application of RNA-seq to Mendelian disease diagnosis. We developed a robust approach to compare rare disease cases to existing large sets of RNA-seq controls (N=1,594 external and N=31 family-based controls) and demonstrated the substantial impacts of gene and variant filtering strategies on disease gene identification when combined with RNA-seq. Across our cohort, we observed that RNA-seq yields a 8.5% diagnostic rate. These diagnoses included diseases where blood would not intuitively reflect evidence of disease. We identified RARS2 as an under-expression outlier containing compound heterozygous pathogenic variants for an individual exhibiting profound global developmental delay, seizures, microcephaly, hypotonia, and progressive scoliosis. We also identified a new splicing junction in KCTD7 for an individual with global developmental delay, loss of milestones, tremors and seizures. Our study provides a broad evaluation of blood RNA-seq for the diagnosis of rare disease.

Navigating Genetic and Phenotypic Uncertainty in Left Ventricular Noncompaction

Once called spongiform cardiomyopathy for its distinct lace-like morphology, left ventricular noncompaction (LVNC) describes a ventricular wall with a prominent noncompacted layer; the excessive trabeculations and deep intertrabecular recesses are separated by thin compacted myocardium.1,2 The diagnosis of LVNC is made primarily by imaging studies to index the thickness of the trabeculated layer to that of the compacted layer. With the advent of cardiac magnetic resonance allowing for high-resolution imaging of the myocardium, hypertrabeculation in the LV has become increasingly recognized in clinical practice. Significant individual variability exists in the extent of trabeculation, however, making its diagnosis extremely challenging. In addition, LVNC frequently occurs in association with other cardiomyopathies,2 congenital heart defects,3 neuromuscular disorders, and genetic syndromes. As a result, there has been ongoing debate as to whether LVNC is an independent disease entity, a clinical phenotype shared among various cardiomyopathies, or a mere bystander.2 Contributing to this debate is the little data on the genetic architecture underlying LVNC. Although the next-generation sequencing has facilitated comprehensive and cost-effective approaches to identify potentially causative genetic variation in LVNC, the ability to identify such variation has outpaced our capacity to clearly interpret its clinical significance. Thus, in this era of rapidly evolving clinical imaging and sequencing technologies, there is a pressing need to integrate the accumulating imaging and genetic data in a large cohort, to better understand the entity that is LVNC. See Article by Author In this issue of Circulation: Cardiovascular Genetics , Jefferies et al4 expand on the known genetic causes of LVNC and provide insights into the genetic landscape of LVNC and LV hypertrabeculation (LVHT) in the largest prospective cohort to date. By leveraging whole exome sequencing and cardiac magnetic resonance, the research team has assembled a deeply genotyped and phenotyped study …