Association of plasma biomarkers with cognition, cognitive decline, and daily function across and within neurodegenerative diseases: Results from the Ontario Neurodegenerative Disease Research Initiative.

INTRODUCTION: We investigated whether novel plasma biomarkers are associated with cognition, cognitive decline, and functional independence in activities of daily living across and within neurodegenerative diseases. METHODS: Glial fibrillary acidic protein (GFAP), neurofilament light chain (NfL), phosphorylated tau (p-tau)181 and amyloid beta (Aß)42/40 were measured using ultra-sensitive Simoa immunoassays in 44 healthy controls and 480 participants diagnosed with Alzheimer's disease/mild cognitive impairment (AD/MCI), Parkinson's disease (PD), frontotemporal dementia (FTD) spectrum disorders, or cerebrovascular disease (CVD). RESULTS: GFAP, NfL, and/or p-tau181 were elevated among all diseases compared to controls, and were broadly associated with worse baseline cognitive performance, greater cognitive decline, and/or lower functional independence. While GFAP, NfL, and p-tau181 were highly predictive across diseases, p-tau181 was more specific to the AD/MCI cohort. Sparse associations were found in the FTD and CVD cohorts and for Aß42/40 . DISCUSSION: GFAP, NfL, and p-tau181 are valuable predictors of cognition and function across common neurodegenerative diseases, and may be useful in specialized clinics and clinical trials.

Cost-effectiveness of a gene sequencing test for Alzheimer's disease in Ontario.

Alzheimer's f disease (AD) affects approximately 250,000 Ontarians, a number that is expected to double by 2040. The Ontario Neurodegenerative Disease Research Initiative has developed an in-province genetic test (ONDRISeq), which currently runs in Ontario in an experimental capacity. The aim of this study is to estimate the costs and health outcomes associated with ONDRISeq to diagnose AD relative to out-of-country (OOC) testing (status quo). A cost-utility analysis was developed for a hypothetical cohort of 65-year-olds at risk of AD in Ontario over a 25-year time horizon. Costs and health outcomes (quality-adjusted life years (QALYs)) were assessed from a healthcare payer perspective. Cost-effectiveness was assessed with a $50,000 cost-effectiveness threshold. Probabilistic sensitivity analyses were conducted to evaluate parameter uncertainty. ONDRISeq saved $54 per patient relative to OOC testing and led to a small QALY gain in the base case (0.0014 per patient). Results were most sensitive to testing costs, uptake rates, and treatment efficacy. ONDRISeq represented better value for money relative to OOC testing throughout 75% of 10,000 probabilistic iterations. Using ONDRISeq is expected to provide health system cost savings. Switching to ONDRISeq for AD genetic testing in Ontario would be dependent on the ability to accommodate the expected testing volumes.

Characteristics of the Ontario Neurodegenerative Disease Research Initiative cohort.

INTRODUCTION: Understanding synergies between neurodegenerative and cerebrovascular pathologies that modify dementia presentation represents an important knowledge gap. METHODS: This multi-site, longitudinal, observational cohort study recruited participants across prevalent neurodegenerative diseases and cerebrovascular disease and assessed participants comprehensively across modalities. We describe univariate and multivariate baseline features of the cohort and summarize recruitment, data collection, and curation processes. RESULTS: We enrolled 520 participants across five neurodegenerative and cerebrovascular diseases. Median age was 69 years, median Montreal Cognitive Assessment score was 25, median independence in activities of daily living was 100% for basic and 93% for instrumental activities. Spousal study partners predominated; participants were often male, White, and more educated. Milder disease stages predominated, yet cohorts reflect clinical presentation. DISCUSSION: Data will be shared with the global scientific community. Within-disease and disease-agnostic approaches are expected to identify markers of severity, progression, and therapy targets. Sampling characteristics also provide guidance for future study design.

Association of apolipoprotein E variation with cognitive impairment across multiple neurodegenerative diagnoses.

For many years there has been uncertainty regarding how apolipoprotein E (APOE) E2 and E4 variants may influence overlapping features of neurodegeneration, such as cognitive impairment. We aimed to identify whether the APOE variants are associated with cognitive function across various neurodegenerative and cerebrovascular diagnoses (n = 513). Utilizing a comprehensive neuropsychology battery, multivariate multiple regression was used to assess the influence of APOE carrier status and disease cohort on performance across five cognitive domains. Irrespective of disease cohort, E4 carriers had significantly lower performance in verbal memory and visuospatial domains than those with E3/3, while E2 carriers' cognitive performance was not significantly different. However, E2 carriers with frontotemporal dementia (FTD) performed significantly worse than those with E3/3 in the attention/working memory, executive function, and visuospatial domains. Our results highlight that the influence of APOE variation on cognition is complex, in some cases varying based on diagnosis and possibly underlying disease pathology.

Enrichment of loss-of-function and copy number variants in ventricular cardiomyopathy genes in 'lone' atrial fibrillation.

AIMS: Atrial fibrillation (AF) is a complex heritable disease whose genetic underpinnings remain largely unexplained, though recent work has suggested that the arrhythmia may develop secondary to an underlying atrial cardiomyopathy. We sought to evaluate for enrichment of loss-of-function (LOF) and copy number variants (CNVs) in genes implicated in ventricular cardiomyopathy in 'lone' AF. METHODS AND RESULTS: Whole-exome sequencing was performed in 255 early onset 'lone' AF cases, defined as arrhythmia onset prior to 60 years of age in the absence of known clinical risk factors. Subsequent evaluations were restricted to 195 cases of European genetic ancestry, as defined by principal component analysis, and focused on a pre-defined set of 43 genes previously implicated in ventricular cardiomyopathy. Bioinformatic analysis identified 6 LOF variants (3.1%), including 3 within the TTN gene, among cases in comparison with 4 of 503 (0.80%) controls [odds ratio: 3.96; 95% confidence interval (CI): 1.11-14.2; P = 0.033]. Further, two AF cases possessed a novel heterozygous 8521 base pair TTN deletion, confirmed with Sanger sequencing and breakpoint validation, which was absent from 4958 controls (P = 0.0014). Subsequent cascade screening in two families revealed evidence of co-segregation of a LOF variant with 'lone' AF. CONCLUSION: 'Lone' AF cases are enriched in rare LOF variants from cardiomyopathy genes, findings primarily driven by TTN, and a novel TTN deletion, providing additional evidence to implicate atrial cardiomyopathy as an AF genetic sub-phenotype. Our results also highlight that AF may develop in the context of these variants in the absence of a discernable ventricular cardiomyopathy.

Simplifying Detection of Copy-Number Variations in Maturity-Onset Diabetes of the Young.

OBJECTIVES: Copy-number variations (CNVs) are large-scale deletions or duplications of DNA that have required specialized detection methods, such as microarray-based genomic hybridization or multiplex ligation probe amplification. However, recent advances in bioinformatics have made it possible to detect CNVs from next-generation DNA sequencing (NGS) data. Maturity-onset diabetes of the young (MODY) 5 is a subtype of autosomal-dominant diabetes that is often caused by heterozygous deletions involving the HNF1B gene on chromosome 17q12. We evaluated the utility of bioinformatic processing of raw NGS data to detect chromosome 17q12 deletions in MODY5 patients. METHODS: NGS data from 57 patients clinically suspected to have MODY but who were negative for pathogenic mutations using a targeted panel were re-examined using a CNV calling tool (CNV Caller, VarSeq version 1.4.3). Potential CNVs for MODY5 were then confirmed using whole-exome sequencing, cytogenetic analysis and breakpoint analysis when possible. RESULTS: Whole-gene deletions in HNF1B, ranging from 1.46 to 1.85 million basepairs in size, were detected in 3 individuals with features of MODY5. These were confirmed by independent methods to be part of a more extensive 17q12 deletion syndrome. Two additional patients carrying a 17q12 deletion were subsequently diagnosed using this method. CONCLUSIONS: Large-scale deletions are the most common cause of MODY5 and can be detected directly from NGS data, without the need for additional methods.

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.

Genetic and epigenetic study of an Alzheimer's disease family with monozygotic triplets.

Age at onset of Alzheimer's disease is highly variable, and its modifiers (genetic or environmental) could act through epigenetic changes, such as DNA methylation at CpG sites. DNA methylation is also linked to ageing-the strongest Alzheimer's disease risk factor. DNA methylation age can be calculated using age-related CpGs and might reflect biological ageing. We conducted a clinical, genetic and epigenetic investigation of a unique Ashkenazi Jewish family with monozygotic triplets, two of whom developed Alzheimer's disease at ages 73 and 76, while the third at age 85 has no cognitive complaints or deficits in daily activities. One of their offspring developed Alzheimer's disease at age 50. Targeted sequencing of 80 genes associated with neurodegeneration revealed that the triplets and the affected offspring are heterozygous carriers of the risk APOE ε4 allele, as well as rare substitutions in APP (p.S198P), NOTCH3 (p.H1235L) and SORL1 (p.W1563C). In addition, we catalogued 52 possibly damaging rare variants detected by NeuroX array in affected individuals. Analysis of family members on a genome-wide DNA methylation chip revealed that the DNA methylation age of the triplets was 6-10 years younger than chronological age, while it was 9 years older in the offspring with early-onset Alzheimer's disease, suggesting accelerated ageing.

Partial LPL deletions: rare copy-number variants contributing towards severe hypertriglyceridemia.

Severe hypertriglyceridemia (HTG) is a relatively common form of dyslipidemia with a complex pathophysiology and serious health complications. HTG can develop in the presence of rare genetic factors disrupting genes involved in the triglyceride (TG) metabolic pathway, including large-scale copy-number variants (CNVs). Improvements in next-generation sequencing technologies and bioinformatic analyses have better allowed assessment of CNVs as possible causes of or contributors to severe HTG. We screened targeted sequencing data of 632 patients with severe HTG and identified partial deletions of the LPL gene, encoding the central enzyme involved in the metabolism of TG-rich lipoproteins, in four individuals (0.63%). We confirmed the genomic breakpoints in each patient with Sanger sequencing. Three patients carried an identical heterozygous deletion spanning the 5' untranslated region (UTR) to LPL exon 2, and one patient carried a heterozygous deletion spanning the 5'UTR to LPL exon 1. All four heterozygous CNV carriers were determined to have multifactorial severe HTG. The predicted null nature of our identified LPL deletions may contribute to relatively higher TG levels and a more severe clinical phenotype than other forms of genetic variation associated with the disease, particularly in the polygenic state. The identification of novel CNVs in patients with severe HTG suggests that methods for CNV detection should be included in the diagnostic workup and molecular genetic evaluation of patients with high TG levels.

Targeted sequencing reveals expanded genetic diversity of human transfer RNAs.

Transfer RNAs are required to translate genetic information into proteins as well as regulate other cellular processes. Nucleotide changes in tRNAs can result in loss or gain of function that impact the composition and fidelity of the proteome. Despite links between tRNA variation and disease, the importance of cytoplasmic tRNA variation has been overlooked. Using a custom capture panel, we sequenced 605 human tRNA-encoding genes from 84 individuals. We developed a bioinformatic pipeline that allows more accurate tRNA read mapping and identifies multiple polymorphisms occurring within the same variant. Our analysis identified 522 unique tRNA-encoding sequences that differed from the reference genome from 84 individuals. Each individual had ~66 tRNA variants including nine variants found in less than 5% of our sample group. Variants were identified throughout the tRNA structure with 17% predicted to enhance function. Eighteen anticodon mutants were identified including potentially mistranslating tRNAs; e.g., a tRNASer that decodes Phe codons. Similar engineered tRNA variants were previously shown to inhibit cell growth, increase apoptosis and induce the unfolded protein response in mammalian cell cultures and chick embryos. Our analysis shows that human tRNA variation has been underestimated. We conclude that the large number of tRNA genes provides a buffer enabling the emergence of variants, some of which could contribute to disease.

Ankyrin-B dysfunction predisposes to arrhythmogenic cardiomyopathy and is amenable to therapy.

Arrhythmogenic cardiomyopathy (ACM) is an inherited arrhythmia syndrome characterized by severe structural and electrical cardiac phenotypes, including myocardial fibrofatty replacement and sudden cardiac death. Clinical management of ACM is largely palliative, owing to an absence of therapies that target its underlying pathophysiology, which stems partially from our limited insight into the condition. Following identification of deceased ACM probands possessing ANK2 rare variants and evidence of ankyrin-B loss of function on cardiac tissue analysis, an ANK2 mouse model was found to develop dramatic structural abnormalities reflective of human ACM, including biventricular dilation, reduced ejection fraction, cardiac fibrosis, and premature death. Desmosomal structure and function appeared preserved in diseased human and murine specimens in the presence of markedly abnormal ß-catenin expression and patterning, leading to identification of a previously unknown interaction between ankyrin-B and ß-catenin. A pharmacological activator of the WNT/ß-catenin pathway, SB-216763, successfully prevented and partially reversed the murine ACM phenotypes. Our findings introduce what we believe to be a new pathway for ACM, a role of ankyrin-B in cardiac structure and signaling, a molecular link between ankyrin-B and ß-catenin, and evidence for targeted activation of the WNT/ß-catenin pathway as a potential treatment for this disease.

Targeted next generation sequencing as a tool for precision medicine.

BACKGROUND: Targeted next-generation sequencing (NGS) enables rapid identification of common and rare genetic variation. The detection of variants contributing to therapeutic drug response or adverse effects is essential for implementation of individualized pharmacotherapy. Successful application of short-read based NGS to pharmacogenes with high sequence homology, nearby pseudogenes and complex structure has been previously shown despite anticipated technical challenges. However, little is known regarding the utility of such panels to detect copy number variation (CNV) in the highly polymorphic cytochrome P450 (CYP) 2D6 gene, or to identify the promoter (TA)7 TAA repeat polymorphism UDP glucuronosyltransferase (UGT) 1A1*28. Here we developed and validated PGxSeq, a targeted exome panel for pharmacogenes pertinent to drug disposition and/or response. METHODS: A panel of capture probes was generated to assess 422 kb of total coding region in 100 pharmacogenes. NGS was carried out in 235 subjects, and sequencing performance and accuracy of variant discovery validated in clinically relevant pharmacogenes. CYP2D6 CNV was determined using the bioinformatics tool CNV caller (VarSeq). Identified SNVs were assessed in terms of population allele frequency and predicted functional effects through in silico algorithms. RESULTS: Adequate performance of the PGxSeq panel was demonstrated with a depth-of-coverage (DOC) ≥ 20× for at least 94% of the target sequence. We showed accurate detection of 39 clinically relevant gene variants compared to standard genotyping techniques (99.9% concordance), including CYP2D6 CNV and UGT1A1*28. Allele frequency of rare or novel variants and predicted function in 235 subjects mirrored findings from large genomic datasets. A large proportion of patients (78%, 183 out of 235) were identified as homozygous carriers of at least one variant necessitating altered pharmacotherapy. CONCLUSIONS: PGxSeq can serve as a comprehensive, rapid, and reliable approach for the detection of common and novel SNVs in pharmacogenes benefiting the emerging field of precision medicine.

Genetic Variation in the Ontario Neurodegenerative Disease Research Initiative.

BACKGROUND/OBJECTIVE: Apolipoprotein E (APOE) E4 is the main genetic risk factor for Alzheimer's disease (AD). Due to the consistent association, there is interest as to whether E4 influences the risk of other neurodegenerative diseases. Further, there is a constant search for other genetic biomarkers contributing to these phenotypes, such as microtubule-associated protein tau (MAPT) haplotypes. Here, participants from the Ontario Neurodegenerative Disease Research Initiative were genotyped to investigate whether the APOE E4 allele or MAPT H1 haplotype are associated with five neurodegenerative diseases: (1) AD and mild cognitive impairment (MCI), (2) amyotrophic lateral sclerosis, (3) frontotemporal dementia (FTD), (4) Parkinson's disease, and (5) vascular cognitive impairment. METHODS: Genotypes were defined for their respective APOE allele and MAPT haplotype calls for each participant, and logistic regression analyses were performed to identify the associations with the presentations of neurodegenerative diseases. RESULTS: Our work confirmed the association of the E4 allele with a dose-dependent increased presentation of AD, and an association between the E4 allele alone and MCI; however, the other four diseases were not associated with E4. Further, the APOE E2 allele was associated with decreased presentation of both AD and MCI. No associations were identified between MAPT haplotype and the neurodegenerative disease cohorts; but following subtyping of the FTD cohort, the H1 haplotype was significantly associated with progressive supranuclear palsy. CONCLUSION: This is the first study to concurrently analyze the association of APOE isoforms and MAPT haplotypes with five neurodegenerative diseases using consistent enrollment criteria and broad phenotypic analysis.

Étude de variance génétique dans le cadre de l'initiative de recherche sur les maladies neurodégénératives en Ontario. Contexte/Objectif : L'apolipoprotéine E4 (ApoE4) constitue le principal facteur de risque génétique de la maladie d'Alzheimer. En raison de cette association systématique, il existe un intérêt certain à savoir dans quelle mesure cette classe d'apolipoprotéines peut influencer le risque d'autres maladies neurodégénératives. En outre, le milieu de la recherche n'a de cesse d'identifier d'autres biomarqueurs génétiques, par exemple les haplotypes H1 de la protéine tau associée aux microtubules, qui contribuent à certains phénotypes, Dans le cadre de cette étude, des participants à l'initiative de recherche sur les maladies neurodégénératives en Ontario ont été « génotypés ¼ afin de déterminer si l'ApoE4 ou l'haplotype H1 mentionné ci-dessus peuvent être associés à cinq maladies neurodégénératives : 1) la maladie d'Alzheimer et d'autres troubles cognitifs légers ; 2) la sclérose latérale amyotrophique ; 3) la démence fronto-temporale ; 4) la maladie de Parkinson ; 5) et finalement les déficits cognitifs d'origine vasculaire. Méthodes : Pour chaque participant, la cartographie des génotypes a été établie en fonction de leur ApoE4 respectif et de la présence d'haplotypes H1 de la protéine tau associée aux microtubules. Des analyses de régression logistique ont été ensuite effectuées dans le but d'identifier de possibles liens avec ces maladies neurodégénératives. Résultats : Nos travaux ont confirmé l'association entre l'ApoE4 et une plus grande occurrence de cas d'Alzheimer, et ce, en tenant compte de l'effet d'une dose de médicament. Ils ont aussi montré une association entre la seule ApoE4 et des troubles cognitifs légers. Cela dit, il convient de préciser que les quatre autres maladies n'ont pas été associées à cet allèle. Plus encore, nous avons trouvé que l'allèle E2 de l'apolipoprotéine était associé à une occurrence plus faible de cas d'Alzheimer et de troubles cognitifs légers. Fait à souligner, aucune association n'a été détectée entre l'haplotype H1 de la protéine tau associée aux microtubules et nos cohortes atteintes de maladies neurodégénératives. Toutefois, à la suite du sous-typage de la cohorte de participants atteints de démence fronto-temporale, il s'est avéré que l'haplotype H1 était associé de façon notable à la paralysie supra-nucléaire progressive. Conclusion : Il s'agit de la première étude à analyser simultanément, au moyen de critères de participation cohérents et d'une analyse phénotypique élargie, les associations entre les isoformes de l'ApoE, l'haplotype H1 de la protéine tau associée aux microtubules et cinq maladies neurodégénératives.

Copy Number Variation in GCK in Patients With Maturity-Onset Diabetes of the Young.

PURPOSE: Next generation sequencing (NGS) methods to diagnose maturity-onset diabetes of the young (MODY), a monogenic autosomal dominant cause of diabetes, do not typically detect large-scale copy number variations (CNVs). New techniques may allow assessment for CNVs using output data from targeted NGS, without requiring additional sequencing. Using this technique, two kindreds of patients presenting with features of MODY were found to bear the same heterozygous large-scale deletion in GCK. METHODS: Patients suspected of having MODY but with negative targeted NGS pathogenic variant calling were reanalyzed using the CNV caller tool (VarSeq v1.4.3). Two patients were identified as having a possible heterozygous whole exon deletion affecting exon 1 of GCK. For confirmation and determination of the exact breakpoints, whole exome sequencing followed by Sanger sequencing were used. Familial samples from both affected and nonaffected first-degree relatives were then analyzed for each proband. RESULTS: A heterozygous whole-exon deletion spanning 4763 bp affecting the entire exon 1 of GCK was detected in two apparently unrelated patients with clinical features of MODY. This deletion showed segregation concordance across generations in affected and nonaffected family members. CONCLUSIONS: Our findings confirm the utility of applying the CNV caller tool to screen for CNVs in GCK from NGS data. In so doing, we identified a deletion of exon 1 of GCK as likely causal for MODY. Our data indicate that incorporating CNV analysis routinely when assessing for MODY via targeted NGS may increase diagnostic yield and reduce false negative genetic testing rates.

Clinical Utility and Practical Considerations of a Coronary Artery Disease Genetic Risk Score.

BACKGROUND: Coronary artery disease (CAD) risk traditionally has been assessed using clinical risk factors. We evaluated whether molecular genetic markers for CAD risk could add information to traditional variables. METHODS: We developed a false discovery rate 267-marker genetic risk score (FDR267) from markers that were significantly associated with CAD in the UK Biobank cohort meta-analysis. FDR267 was tested in the Atherosclerosis Risk in Communities cohort using logistic regression and Cox proportional hazards analyses in the European and African American groups. RESULTS: Our genetic risk score (FDR267) was associated with a 1.45 (95% confidence interval, 1.39-1.51) increase in odds ratio and a 1.32 (95% confidence interval, 1.26-1.38) increase in hazard ratio per standard deviation of the score. The score modestly improved the area under the curve (AUC) statistic when added to a clinical model (ΔAUC = 0.0112, P = 0.0002). FDR267 predicted incident CAD (C-index = 0.60), although it did not improve on clinical risk factors (ΔAUC = 0.0159, P = 0.0965). Individuals in the top quintile of FDR267 genetic risk were at approximately 2-fold increased risk compared with the bottom quintile, which is comparable to risk associated with self-reported family history. The performance of FDR267 was less robust in the African American sample. CONCLUSIONS: FDR267 is significantly associated with CAD in the European sample, with an effect size comparable to self-reported family history. FDR267 discriminated between individuals with and without CAD, but did not improve CAD risk prediction over clinical variables. FDR267 was less predictive of CAD risk in African Americans.

INTRODUCTION: L'évaluation du risque de maladie coronarienne (MC) a traditionnellement reposé sur les facteurs de risque cliniques. Nous avons évalué si les marqueurs génétiques moléculaires de risque de MC pourraient servir de complément aux variables traditionnelles. MÉTHODES: Nous avons élaboré un taux de fausses découvertes (FDR pour false discovery rate) du score de risque génétique du marqueur 267 (FDR267) provenant des marqueurs qui étaient associés de manière significative à la MC dans la méta-analyse de cohortes de la UK Biobank. Le FDR267 a été testé dans la cohorte du Atherosclerosis Risk in Communities à l'aide de la régression logistique et des analyses selon le modèle à risques proportionnels de Cox dans des groupes européens et afro-américains. RÉSULTATS: Notre score de risque génétique (FDR267) a été associé à une augmentation de 1,45 (intervalle de confiance [IC] à 95 %, 1,39-1,51) du rapport des cotes et d'une augmentation de 1,32 (IC à 95 %, 1,26-1,38) du risque relatif par l'écart-type des scores. Le score a modestement amélioré l'aire sous la courbe (ASC) lorsqu'il a été ajouté à un modèle clinique (ΔASC = 0,0112, P = 0,0002). Le FDR267 a prédit les nouveaux cas de MC (C-index [indice de concordance] = 0,60), mais il n'a pas amélioré les facteurs de risque cliniques (ΔASC = 0,0159, P = 0,0965). Les individus dans le quintile supérieur du risque génétique du FDR267 ont montré un risque accru d'environ 2 fois par rapport au quintile inférieur, soit un risque comparable au risque associé aux antécédents familiaux auto-rapportés. La performance du FDR267 s'est révélée moins robuste chez les Afro-Américains. CONCLUSIONS: Le FDR267 est associé de manière significative à la MC dans l'échantillon d'Européens et a une taille de l'effet comparable aux antécédents familiaux auto-rapportés. Le FDR267 a fait la discrimination entre les individus atteints ou non atteints de MC, mais n'a pas amélioré la prédiction du risque de MC par rapport aux variables cliniques. Le FDR267 a moins bien prédit le risque de MC chez les Afro-Américains.

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.

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease.

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The technique is highly efficient with millions of sequencing reads being produced in a short time span and at relatively low cost. Specifically, targeted NGS is able to focus investigations to genomic regions of particular interest based on the disease of study. Not only does this further reduce costs and increase the speed of the process, but it lessens the computational burden that often accompanies NGS. Although targeted NGS is restricted to certain regions of the genome, preventing identification of potential novel loci of interest, it can be an excellent technique when faced with a phenotypically and genetically heterogeneous disease, for which there are previously known genetic associations. Because of the complex nature of the sequencing technique, it is important to closely adhere to protocols and methodologies in order to achieve sequencing reads of high coverage and quality. Further, once sequencing reads are obtained, a sophisticated bioinformatics workflow is utilized to accurately map reads to a reference genome, to call variants, and to ensure the variants pass quality metrics. Variants must also be annotated and curated based on their clinical significance, which can be standardized by applying the American College of Medical Genetics and Genomics Pathogenicity Guidelines. The methods presented herein will display the steps involved in generating and analyzing NGS data from a targeted sequencing panel, using the ONDRISeq neurodegenerative disease panel as a model, to identify variants that may be of clinical significance.

Identification of a novel synaptic protein, TMTC3, involved in periventricular nodular heterotopia with intellectual disability and epilepsy.

Defects in neuronal migration cause brain malformations, which are associated with intellectual disability (ID) and epilepsy. Using exome sequencing, we identified compound heterozygous variants (p.Arg71His and p. Leu729ThrfsTer6) in TMTC3, encoding transmembrane and tetratricopeptide repeat containing 3, in four siblings with nocturnal seizures and ID. Three of the four siblings have periventricular nodular heterotopia (PVNH), a common brain malformation caused by failure of neurons to migrate from the ventricular zone to the cortex. Expression analysis using patient-derived cells confirmed reduced TMTC3 transcript levels and loss of the TMTC3 protein compared to parental and control cells. As TMTC3 function is currently unexplored in the brain, we gathered support for a neurobiological role for TMTC3 by generating flies with post-mitotic neuron-specific knockdown of the highly conserved Drosophila melanogaster TMTC3 ortholog, CG4050/tmtc3. Neuron-specific knockdown of tmtc3 in flies resulted in increased susceptibility to induced seizures. Importantly, this phenotype was rescued by neuron-specific expression of human TMTC3, suggesting a role for TMTC3 in seizure biology. In addition, we observed co-localization of TMTC3 in the rat brain with vesicular GABA transporter (VGAT), a presynaptic marker for inhibitory synapses. TMTC3 is localized at VGAT positive pre-synaptic terminals and boutons in the rat hypothalamus and piriform cortex, suggesting a role for TMTC3 in the regulation of GABAergic inhibitory synapses. TMTC3 did not co-localize with Vglut2, a presynaptic marker for excitatory neurons. Our data identified TMTC3 as a synaptic protein that is involved in PVNH with ID and epilepsy, in addition to its previously described association with cobblestone lissencephaly.

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.