ClinGen Variant Curation Interface: a variant classification platform for the application of evidence criteria from ACMG/AMP guidelines.

BACKGROUND: Identification of clinically significant genetic alterations involved in human disease has been dramatically accelerated by developments in next-generation sequencing technologies. However, the infrastructure and accessible comprehensive curation tools necessary for analyzing an individual patient genome and interpreting genetic variants to inform healthcare management have been lacking. RESULTS: Here we present the ClinGen Variant Curation Interface (VCI), a global open-source variant classification platform for supporting the application of evidence criteria and classification of variants based on the ACMG/AMP variant classification guidelines. The VCI is among a suite of tools developed by the NIH-funded Clinical Genome Resource (ClinGen) Consortium and supports an FDA-recognized human variant curation process. Essential to this is the ability to enable collaboration and peer review across ClinGen Expert Panels supporting users in comprehensively identifying, annotating, and sharing relevant evidence while making variant pathogenicity assertions. To facilitate evidence-based improvements in human variant classification, the VCI is publicly available to the genomics community. Navigation workflows support users providing guidance to comprehensively apply the ACMG/AMP evidence criteria and document provenance for asserting variant classifications. CONCLUSIONS: The VCI offers a central platform for clinical variant classification that fills a gap in the learning healthcare system, facilitates widespread adoption of standards for clinical curation, and is available at https://curation.clinicalgenome.org.

The Clinical Genome Resource (ClinGen) Familial Hypercholesterolemia Variant Curation Expert Panel consensus guidelines for LDLR variant classification.

PURPOSE: In 2015, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) published consensus standardized guidelines for sequence-level variant classification in Mendelian disorders. To increase accuracy and consistency, the Clinical Genome Resource Familial Hypercholesterolemia (FH) Variant Curation Expert Panel was tasked with optimizing the existing ACMG/AMP framework for disease-specific classification in FH. In this study, we provide consensus recommendations for the most common FH-associated gene, LDLR, where >2300 unique FH-associated variants have been identified. METHODS: The multidisciplinary FH Variant Curation Expert Panel met in person and through frequent emails and conference calls to develop LDLR-specific modifications of ACMG/AMP guidelines. Through iteration, pilot testing, debate, and commentary, consensus among experts was reached. RESULTS: The consensus LDLR variant modifications to existing ACMG/AMP guidelines include (1) alteration of population frequency thresholds, (2) delineation of loss-of-function variant types, (3) functional study criteria specifications, (4) cosegregation criteria specifications, and (5) specific use and thresholds for in silico prediction tools, among others. CONCLUSION: Establishment of these guidelines as the new standard in the clinical laboratory setting will result in a more evidence-based, harmonized method for LDLR variant classification worldwide, thereby improving the care of patients with FH.

Improving reporting standards for polygenic scores in risk prediction studies.

Polygenic risk scores (PRSs), which often aggregate results from genome-wide association studies, can bridge the gap between initial discovery efforts and clinical applications for the estimation of disease risk using genetics. However, there is notable heterogeneity in the application and reporting of these risk scores, which hinders the translation of PRSs into clinical care. Here, in a collaboration between the Clinical Genome Resource (ClinGen) Complex Disease Working Group and the Polygenic Score (PGS) Catalog, we present the Polygenic Risk Score Reporting Standards (PRS-RS), in which we update the Genetic Risk Prediction Studies (GRIPS) Statement to reflect the present state of the field. Drawing on the input of experts in epidemiology, statistics, disease-specific applications, implementation and policy, this comprehensive reporting framework defines the minimal information that is needed to interpret and evaluate PRSs, especially with respect to downstream clinical applications. Items span detailed descriptions of study populations, statistical methods for the development and validation of PRSs and considerations for the potential limitations of these scores. In addition, we emphasize the need for data availability and transparency, and we encourage researchers to deposit and share PRSs through the PGS Catalog to facilitate reproducibility and comparative benchmarking. By providing these criteria in a structured format that builds on existing standards and ontologies, the use of this framework in publishing PRSs will facilitate translation into clinical care and progress towards defining best practice.

Six years' experience with LipidSeq: clinical and research learnings from a hybrid, targeted sequencing panel for dyslipidemias.

BACKGROUND: In 2013, our laboratory designed a targeted sequencing panel, "LipidSeq", to study the genetic determinants of dyslipidemia and metabolic disorders. Over the last 6 years, we have analyzed 3262 patient samples obtained from our own Lipid Genetics Clinic and international colleagues. Here, we highlight our findings and discuss research benefits and clinical implications of our panel. METHODS: LipidSeq targets 69 genes and 185 single-nucleotide polymorphisms (SNPs) either causally related or associated with dyslipidemia and metabolic disorders. This design allows us to simultaneously evaluate monogenic-caused by rare single-nucleotide variants (SNVs) or copy-number variants (CNVs)-and polygenic forms of dyslipidemia. Polygenic determinants were assessed using three polygenic scores, one each for low-density lipoprotein cholesterol, triglyceride, and high-density lipoprotein cholesterol. RESULTS: Among 3262 patient samples evaluated, the majority had hypertriglyceridemia (40.1%) and familial hypercholesterolemia (28.3%). Across all samples, we identified 24,931 unique SNVs, including 2205 rare variants predicted disruptive to protein function, and 77 unique CNVs. Considering our own 1466 clinic patients, LipidSeq results have helped in diagnosis and improving treatment options. CONCLUSIONS: Our LipidSeq design based on ontology of lipid disorders has enabled robust detection of variants underlying monogenic and polygenic dyslipidemias. In more than 50 publications related to LipidSeq, we have described novel variants, the polygenic nature of many dyslipidemias-some previously thought to be primarily monogenic-and have uncovered novel mechanisms of disease. We further demonstrate several tangible clinical benefits of its use.

Progress in finding pathogenic DNA copy number variations in dyslipidemia.

PURPOSE OF REVIEW: DNA copy number variations (CNVs) are large-scale mutations that include deletions and duplications larger than 50 bp in size. In the era when single-nucleotide variations were the major focus of genetic technology and research, CNVs were largely overlooked. However, CNVs clearly underlie a substantial proportion of clinical disorders. Here, we update recent progress in identifying CNVs in dyslipidemias. RECENT FINDINGS: Until last year, only the LDLR and LPA genes were appreciated as loci within which clinically relevant CNVs contributed to familial hypercholesterolemia and variation in Lp(a) levels, respectively. Since 2017, next-generation sequencing panels have identified pathogenic CNVs in at least five more genes underlying dyslipidemias, including a PCSK9 whole-gene duplication in familial hypercholesterolemia; LPL, GPIHBP1, and APOC2 deletions in hypertriglyceridemia; and ABCA1 deletions in hypoalphalipoproteinemia. SUMMARY: CNVs are an important class of mutation that contribute to the molecular genetic heterogeneity underlying dyslipidemias. Clinical applications of next-generation sequencing technologies need to consider CNVs concurrently with familiar small-scale genetic variation, given the likely implications for improved diagnosis and treatment.

Severe hypertriglyceridemia is primarily polygenic.

BACKGROUND: Hypertriglyceridemia (HTG) is a complex trait defined by elevated plasma triglyceride levels. Genetic determinants of HTG have so far been examined in a piecemeal manner; understanding of its molecular basis, both monogenic and polygenic, is thus incomplete. OBJECTIVE: The objective of this study was to characterize genetic profiles of patients with severe HTG, and quantify the genetic determinants and molecular contributors. METHODS: We concurrently assessed rare and common variants in two independent cohorts of 251 and 312 Caucasian patients with severe HTG. DNA was subjected to targeted next-generation sequencing of 73 genes and 185 SNPs associated with dyslipidemia. LPL, APOC2, GPIHBP1, APOA5, and LMF1 genes were screened for rare variants, and a polygenic risk score was used to assess the accumulation of common variants. RESULTS: As there were no significant differences in the prevalence of genetic determinants between cohorts, data were combined for all 563 patients: 1.1% had biallelic (homozygous or compound heterozygous) rare variants, 14.4% had heterozygous rare variants, 32.0% had an extreme accumulation of common variants (ie, high polygenic risk), and 52.6% remained genetically undefined. Patients with HTG were 5.77 times (95% CI [4.26-7.82]; P < .0001) more likely to carry one of these types of genetic susceptibility compared with controls. CONCLUSIONS: We report the most in-depth, systematic evaluation of genetic determinants of severe HTG to date. The predominant feature was an extreme accumulation of common variants (high polygenic risk score), whereas a substantial proportion of patients also carried heterozygous rare variants. Overall, 46.3% of patients had polygenic HTG, whereas only 1.1% had biallelic or homozygous monogenic HTG.

Efficacy of Evolocumab in Monogenic vs Polygenic Hypercholesterolemia.

BACKGROUND: Inhibitors of proprotein convertase subtilisin kexin 9 are indicated in Canada for treatment of patients with familial hypercholesterolemia (FH). Classically, FH is considered to be a monogenic condition caused by rare pathogenic mutations; however, some patients have hypercholesterolemia on a polygenic basis. Whether the effect of proprotein convertase subtilisin kexin 9 inhibitor treatment differs between patients with monogenic hypercholesterolemia and patients with polygenic hypercholesterolemia is unclear. METHODS: We performed retrospective chart reviews on patients treated with evolocumab 140 mg subcutaneously biweekly from the Lipid Genetics Clinic, London Health Sciences Centre. Evolocumab-treated patients with hypercholesterolemia were grouped into monogenic or polygenic categories on the basis of their genotype determined by targeted next-generation sequencing. Absolute and relative changes in low-density lipoprotein cholesterol (LDL-C) levels before and after evolocumab treatment were studied. RESULTS: In 32 patients with monogenic heterozygous FH and 7 patients with polygenic hypercholesterolemia treated with evolocumab, absolute incremental reductions in LDL-C were 2.94 ± 1.22 mmol/L and 3.15 ± 0.90 mmol/L, respectively (P = not significant), whereas percent reductions in LDL-C were 63.9% ± 16.0% and 67.7% ± 20.7%, respectively (P = not significant). CONCLUSION: Although the sample size is small, the findings suggest comparable biochemical responsiveness to evolocumab in both monogenic (heterozygous) and polygenic hypercholesterolemia.

INTRODUCTION: Au Canada, les inhibiteurs de la proprotéine convertase subtilisine/kexine de type 9 sont indiqués dans le traitement des patients atteints d'hypercholestérolémie familiale (HF). Traditionnellement, la HF est considérée comme une maladie monogénique causée par des mutations pathogènes rares. Toutefois, certains patients ont une hypercholestérolémie de forme polygénique. On ignore si les effets du traitement par inhibiteurs de la proprotéine convertase subtilisine/kexine de type 9 diffèrent entre les patients ayant une hypercholestérolémie monogénique et les patients ayant une hypercholestérolémie polygénique. MÉTHODES: Nous avons réalisé une revue rétrospective de dossiers de patients traités par évolocumab à raison de 140 mg par voie sous-cutanée 2 fois par semaine à la Lipid Genetics Clinic du London Health Sciences Centre. Les patients atteints d'hypercholestérolémie qui étaient traités par évolocumab ont été regroupés dans la catégorie de la forme monogénique et la catégorie de la forme polygénique en fonction de leur génotype déterminé par le séquençage ciblé de nouvelle génération. Nous avons étudié les changements absolus et relatifs des concentrations de cholestérol à lipoprotéines de faible densité (LDL-C) avant et après le traitement par évolocumab. RÉSULTATS: Chez 32 patients ayant une HF hétérozygote monogénique et 7 patients ayant une hypercholestérolémie polygénique traités par évolocumab, les réductions progressives absolues du LDL-C étaient respectivement de 2,94 ± 1,22 mmol/l et de 3,15 ± 0,90 mmol/l (P = non significatif), alors que les réductions du LDL-C en pourcentage étaient respectivement de 63,9 % ± 16,0 % et de 67,7 % ± 20,7 % (P = non significatif). CONCLUSION: Bien que la taille de l'échantillon soit petite, les résultats montrent une capacité de réponse biochimique comparable à l'évolocumab lors d'hypercholestérolémie (hétérozygote) monogénique et d'hypercholestérolémie polygénique.

Genetic Determinants of Myocardial Infarction Risk in Familial Hypercholesterolemia.

BACKGROUND: Familial hypercholesterolemia (FH) is an inherited condition of elevated serum low-density lipoprotein (LDL) cholesterol leading to premature coronary heart disease. We evaluated whether FH mutations are independently associated with the development of myocardial infarction (MI), after adjusting for LDL cholesterol level and clinical risk factors. METHODS: In 182 unrelated patients from different families referred with clinically suspected FH, targeted next-generation DNA sequencing was performed on 73 lipid-related genes and 178 single nucleotide polymorphisms, at 300-times mean read depth, to identify monogenic mutations and high-risk single nucleotide polymorphisms. RESULTS: Pathogenic FH mutations were identified in 27% of patients. Patients with mutations, compared with those without, were 12 years younger when referred to the lipid clinic (P < 0.001) and had higher baseline and post-treatment LDL cholesterol by 1.11 mmol/L (P < 0.001) and 0.62 mmol/L (P = 0.01), respectively. The hazard ratio for premature MI with respect to having an FH mutation, controlling for sex, hypertension, body mass index, diabetes, LDL cholesterol, and smoking, was 4.51 (P = 0.002). CONCLUSION: FH is a genetically diverse condition. FH mutations are independently associated with higher risk of premature MI in patients referred for hypercholesterolemia. Therefore, genotyping could guide cardiovascular risk stratification in the personalized treatment of FH.

INTRODUCTION: L'hypercholestérolémie familiale (HF) est une maladie héréditaire caractérisée par des concentrations élevées de lipoprotéines de faible densité (LDL) qui entraîne de manière précoce la maladie coronarienne. Nous avons évalué si les mutations propres à la HF sont indépendamment associées au développement de l'infarctus du myocarde (IM) après l'ajustement des concentrations de cholestérol LDL et des facteurs de risque cliniques. MÉTHODES: Chez les 182 patients non apparentés de différentes familles envoyés en consultation en raison d'une suspicion clinique de HF, nous avons réalisé le séquençage ciblé de l'ADN de nouvelle génération de 73 gènes du métabolisme des lipides et 178 polymorphismes mononucléotidiques, à une profondeur de lecture moyenne de 300 fois, pour déterminer les mutations monogènes et les polymorphismes mononucléotidiques à haut risque. RÉSULTATS: Nous avons déterminé les mutations pathogènes à l'origine de la HF de 27 % des patients. Comparativement aux patients qui ne présentaient pas de mutations, les patients qui en présentaient étaient 12 ans plus jeunes lorsqu'ils étaient envoyés en consultation à la clinique des lipides (P < 0,001) et avaient des concentrations respectives plus élevées de cholestérol LDL de 1,11 mmol/l (P < 0,001) et de 0,62 mmol/l (P = 0,01), au début et après le traitement. Le rapport de risque d'IM prématuré par rapport à la présence d'une mutation à l'origine de la HF, qui tient compte du sexe, de l'hypertension, de l'indice de masse corporelle, du diabète, du cholestérol LDL et de la consommation du tabac, était de 4,51 (P = 0,002). CONCLUSION: La HF est une maladie diversifiée sur le plan génétique. Les mutations à l'origine de la HF sont indépendamment associées à un risque plus élevé d'IM prématuré chez les patients envoyés en consultation en raison d'une hypercholestérolémie. Par conséquent, le génotypage pourrait être utile à la stratification du risque cardiovasculaire dans le traitement personnalisé de la HF.

ClinVar database of global familial hypercholesterolemia-associated DNA variants.

Accurate and consistent variant classification is imperative for incorporation of rapidly developing sequencing technologies into genomic medicine for improved patient care. An essential requirement for achieving standardized and reliable variant interpretation is data sharing, facilitated by a centralized open-source database. Familial hypercholesterolemia (FH) is an exemplar of the utility of such a resource: it has a high incidence, a favorable prognosis with early intervention and treatment, and cascade screening can be offered to families if a causative variant is identified. ClinVar, an NCBI-funded resource, has become the primary repository for clinically relevant variants in Mendelian disease, including FH. Here, we present the concerted efforts made by the Clinical Genome Resource, through the FH Variant Curation Expert Panel and global FH community, to increase submission of FH-associated variants into ClinVar. Variant-level data was categorized by submitter, variant characteristics, classification method, and available supporting data. To further reform interpretation of FH-associated variants, areas for improvement in variant submissions were identified; these include a need for more detailed submissions and submission of supporting variant-level data, both retrospectively and prospectively. Collaborating to provide thorough, reliable evidence-based variant interpretation will ultimately improve the care of FH patients.

Whole-Gene Duplication of PCSK9 as a Novel Genetic Mechanism for Severe Familial Hypercholesterolemia.

BACKGROUND: Familial hypercholesterolemia (FH) is a common genetic disorder of severely elevated low-density lipoprotein (LDL) cholesterol, characterized by premature atherosclerotic cardiovascular disease. Although copy number variations (CNVs) are a large-scale mutation-type capable of explaining FH cases, they have been, to date, assessed only in the LDLR gene. Here, we performed novel CNV screening in additional FH-associated genes using a next-generation sequencing-based approach. METHODS: In 704 patients with FH, we sequenced FH-associated genes APOB, PCSK9, LDLRAP1, APOE, STAP1, LIPA, and ABCG5/8 using our LipidSeq targeted next-generation sequencing panel. Bioinformatic tools were applied to LipidSeq data for CNV screening, and identified CNVs were validated using whole-exome sequencing and microarray-based copy number analyses. RESULTS: We identified a whole-gene duplication of PCSK9 in 2 unrelated Canadian FH index cases; this PCSK9 CNV was also found to cosegregate with affected status in family members. Features in affected individuals included severely elevated LDL cholesterol levels that were refractory to intensive statin therapy, pronounced clinical stigmata, premature cardiovascular events, and a plasma PCSK9 of approximately 5000 ng/mL in 1 index case. We found no CNVs in APOB, LDLRAP1, APOE, STAP1, LIPA, and ABCG5/8 in our cohort of 704 FH individuals. CONCLUSIONS: Here, we report the first description of a CNV affecting the PCSK9 gene in FH. This finding is associated with a profound FH phenotype and the highest known plasma PCSK9 level reported in a human. This finding also has therapeutic relevance, as elevated PCSK9 levels may limit the efficacy of high-dose statin therapy and also PCSK9 inhibition.

Large-scale deletions of the ABCA1 gene in patients with hypoalphalipoproteinemia.

Copy-number variations (CNVs) have been studied in the context of familial hypercholesterolemia but have not yet been evaluated in patients with extreme levels of HDL cholesterol. We evaluated targeted, next-generation sequencing data from patients with very low levels of HDL cholesterol (i.e., hypoalphalipoproteinemia) with the VarSeq-CNV® caller algorithm to screen for CNVs that disrupted the ABCA1, LCAT, or APOA1 genes. In four individuals, we found three unique deletions in ABCA1: a heterozygous deletion of exon 4, a heterozygous deletion that spanned exons 8 to 31, and a heterozygous deletion of the entire ABCA1 gene. Breakpoints were identified with Sanger sequencing, and the full-gene deletion was confirmed by using exome sequencing and the Affymetrix CytoScan HD array. Previously, large-scale deletions in candidate HDL genes had not been associated with hypoalphalipoproteinemia; our findings indicate that CNVs in ABCA1 may be a previously unappreciated genetic determinant of low levels of HDL cholesterol. By coupling bioinformatic analyses with next-generation sequencing data, we can successfully assess the spectrum of genetic determinants of many dyslipidemias, including hypoalphalipoproteinemia.

Role of DNA copy number variation in dyslipidemias.

PURPOSE OF REVIEW: DNA copy number variations (CNVs) are quantitative structural rearrangements that include deletions, duplications, and higher order amplifications. Because of technical limitations, the contribution of this common form of genetic variation to regulation of lipid metabolism and dyslipidemia has been underestimated. RECENT FINDINGS: Recent literature involving CNVs and dyslipidemias has focused mainly on rare CNVs causing familial hypercholesterolemia, and a common CNV polymorphism as the major determinant of lipoprotein(a) plasma concentrations. Additionally, there is tantalizing evidence of largely uninvestigated but plausible presence of CNVs underlying other dyslipidemias. We also discuss the future role of improved technologies in facilitating more economic, routine CNV assessment in dyslipidemias. SUMMARY: CNVs account for large proportion of human genetic variation and are already known to contribute to susceptibility of dyslipidemias, particularly in about 10% of familial hypercholesterolemia patients. Increasing availability of clinical next-generation sequencing and bioinformatics presents a cost-effective opportunity for novel CNV discoveries in dyslipidemias.

Use of next-generation sequencing to detect LDLR gene copy number variation in familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is a heritable condition of severely elevated LDL cholesterol, caused predominantly by autosomal codominant mutations in the LDL receptor gene (LDLR). In providing a molecular diagnosis for FH, the current procedure often includes targeted next-generation sequencing (NGS) panels for the detection of small-scale DNA variants, followed by multiplex ligation-dependent probe amplification (MLPA) in LDLR for the detection of whole-exon copy number variants (CNVs). The latter is essential because ∼10% of FH cases are attributed to CNVs in LDLR; accounting for them decreases false negative findings. Here, we determined the potential of replacing MLPA with bioinformatic analysis applied to NGS data, which uses depth-of-coverage analysis as its principal method to identify whole-exon CNV events. In analysis of 388 FH patient samples, there was 100% concordance in LDLR CNV detection between these two methods: 38 reported CNVs identified by MLPA were also successfully detected by our NGS method, while 350 samples negative for CNVs by MLPA were also negative by NGS. This result suggests that MLPA can be removed from the routine diagnostic screening for FH, significantly reducing associated costs, resources, and analysis time, while promoting more widespread assessment of this important class of mutations across diagnostic laboratories.

Recent advances in genetic testing for familial hypercholesterolemia.

INTRODUCTION: Familial hypercholesterolemia (FH) is a common genetic cause of premature coronary heart disease that is widely underdiagnosed and undertreated. To improve the identification of FH and initiate timely and appropriate treatment strategies, genetic testing is becoming increasingly offered worldwide as a central part of diagnosis. Areas covered: Recent advances have been propelled by an improved understanding of the genetic determinants of FH together with substantially reduced costs of appropriate screening strategies. Here we review the various methods available for obtaining a molecular diagnosis of FH, and highlight the particular advantages of targeted next-generation sequencing (NGS) platforms as the most robust approach. Furthermore, we note the importance of screening for copy number variants and common polymorphisms to aid in molecularly defining suspected FH cases. Expert commentary: The need for genetic analysis of FH will increase, both for diagnosis and reimbursement of new therapies. An effective molecular diagnostic method must detect: 1) molecular and gene locus heterogeneity; 2) a wide range of mutation types; and 3) the polygenic component of FH. As availability of genetic testing for FH expands, standardization of variant curation, maintenance of clinical databases and registries, and wider health care provider education all assume greater importance.