Loss-of-Function CREB3L3 Variants in Patients With Severe Hypertriglyceridemia.

OBJECTIVE: Genetic determinants of severe hypertriglyceridemia include both common variants with small effects (assessed using polygenic risk scores) plus heterozygous and homozygous rare variants in canonical genes directly affecting triglyceride metabolism. Here, we broadened our scope to detect associations with rare loss-of-function variants in genes affecting noncanonical pathways, including those known to affect triglyceride metabolism indirectly. Approach and Results: From targeted next-generation sequencing of 69 metabolism-related genes in 265 patients of European descent with severe hypertriglyceridemia (≥10 mmol/L or ≥885 mg/dL) and 477 normolipidemic controls, we focused on the association of rare heterozygous loss-of-function variants in individual genes. We observed that compared with controls, severe hypertriglyceridemia patients were 20.2× (95% CI, 1.11-366.1; P=0.03) more likely than controls to carry a rare loss-of-function variant in CREB3L3, which encodes a transcription factor that regulates several target genes with roles in triglyceride metabolism. CONCLUSIONS: Our findings indicate that rare variants in a noncanonical gene for triglyceride metabolism, namely CREB3L3, contribute significantly to severe hypertriglyceridemia. Secondary genes and pathways should be considered when evaluating the genetic architecture of this complex trait.

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.

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.

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.

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.

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.

Polygenic determinants in extremes of high-density lipoprotein cholesterol.

HDL cholesterol (HDL-C) remains a superior biochemical predictor of CVD risk, but its genetic basis is incompletely defined. In patients with extreme HDL-C concentrations, we concurrently evaluated the contributions of multiple large- and small-effect genetic variants. In a discovery cohort of 255 unrelated lipid clinic patients with extreme HDL-C levels, we used a targeted next-generation sequencing panel to evaluate rare variants in known HDL metabolism genes, simultaneously with common variants bundled into a polygenic trait score. Two additional cohorts were used for validation and included 1,746 individuals from the Montréal Heart Institute Biobank and 1,048 individuals from the University of Pennsylvania. Findings were consistent between cohorts: we found rare heterozygous large-effect variants in 18.7% and 10.9% of low- and high-HDL-C patients, respectively. We also found common variant accumulation, indicated by extreme polygenic trait scores, in an additional 12.8% and 19.3% of overall cases of low- and high-HDL-C extremes, respectively. Thus, the genetic basis of extreme HDL-C concentrations encountered clinically is frequently polygenic, with contributions from both rare large-effect and common small-effect variants. Multiple types of genetic variants should be considered as contributing factors in patients with extreme dyslipidemia.

Polygenic Versus Monogenic Causes of Hypercholesterolemia Ascertained Clinically.

OBJECTIVE: Next-generation sequencing technology is transforming our understanding of heterozygous familial hypercholesterolemia, including revision of prevalence estimates and attribution of polygenic effects. Here, we examined the contributions of monogenic and polygenic factors in patients with severe hypercholesterolemia referred to a specialty clinic. APPROACH AND RESULTS: We applied targeted next-generation sequencing with custom annotation, coupled with evaluation of large-scale copy number variation and polygenic scores for raised low-density lipoprotein cholesterol in a cohort of 313 individuals with severe hypercholesterolemia, defined as low-density lipoprotein cholesterol >5.0 mmol/L (>194 mg/dL). We found that (1) monogenic familial hypercholesterolemia-causing mutations detected by targeted next-generation sequencing were present in 47.3% of individuals; (2) the percentage of individuals with monogenic mutations increased to 53.7% when copy number variations were included; (3) the percentage further increased to 67.1% when individuals with extreme polygenic scores were included; and (4) the percentage of individuals with an identified genetic component increased from 57.0% to 92.0% as low-density lipoprotein cholesterol level increased from 5.0 to >8.0 mmol/L (194 to >310 mg/dL). CONCLUSIONS: In a clinically ascertained sample with severe hypercholesterolemia, we found that most patients had a discrete genetic basis detected using a comprehensive screening approach that includes targeted next-generation sequencing, an assay for copy number variations, and polygenic trait scores.

Targeted next-generation sequencing in monogenic dyslipidemias.

PURPOSE OF REVIEW: To evaluate the potential clinical translation of high-throughput next-generation sequencing (NGS) methods in diagnosis and management of dyslipidemia. RECENT FINDINGS: Recent NGS experiments indicate that most causative genes for monogenic dyslipidemias are already known. Thus, monogenic dyslipidemias can now be diagnosed using targeted NGS. Targeting of dyslipidemia genes can be achieved by either: designing custom reagents for a dyslipidemia-specific NGS panel; or performing genome-wide NGS and focusing on genes of interest. Advantages of the former approach are lower cost and limited potential to detect incidental pathogenic variants unrelated to dyslipidemia. However, the latter approach is more flexible because masking criteria can be altered as knowledge advances, with no need for re-design of reagents or follow-up sequencing runs. Also, the cost of genome-wide analysis is decreasing and ethical concerns can likely be mitigated. DNA-based diagnosis is already part of the clinical diagnostic algorithms for familial hypercholesterolemia. Furthermore, DNA-based diagnosis is supplanting traditional biochemical methods to diagnose chylomicronemia caused by deficiency of lipoprotein lipase or its co-factors. SUMMARY: The increasing availability and decreasing cost of clinical NGS for dyslipidemia means that its potential benefits can now be evaluated on a larger scale.

LipidSeq: a next-generation clinical resequencing panel for monogenic dyslipidemias.

We report the design of a targeted resequencing panel for monogenic dyslipidemias, LipidSeq, for the purpose of replacing Sanger sequencing in the clinical detection of dyslipidemia-causing variants. We also evaluate the performance of the LipidSeq approach versus Sanger sequencing in 84 patients with a range of phenotypes including extreme blood lipid concentrations as well as additional dyslipidemias and related metabolic disorders. The panel performs well, with high concordance (95.2%) in samples with known mutations based on Sanger sequencing and a high detection rate (57.9%) of mutations likely to be causative for disease in samples not previously sequenced. Clinical implementation of LipidSeq has the potential to aid in the molecular diagnosis of patients with monogenic dyslipidemias with a high degree of speed and accuracy and at lower cost than either Sanger sequencing or whole exome sequencing. Furthermore, LipidSeq will help to provide a more focused picture of monogenic and polygenic contributors that underlie dyslipidemia while excluding the discovery of incidental pathogenic clinically actionable variants in nonmetabolism-related genes, such as oncogenes, that would otherwise be identified by a whole exome approach, thus minimizing potential ethical issues.

An increased burden of common and rare lipid-associated risk alleles contributes to the phenotypic spectrum of hypertriglyceridemia.

OBJECTIVE: Earlier studies have suggested that a common genetic architecture underlies the clinically heterogeneous polygenic Fredrickson hyperlipoproteinemia (HLP) phenotypes defined by hypertriglyceridemia (HTG). Here, we comprehensively analyzed 504 HLP-HTG patients and 1213 normotriglyceridemic controls and confirmed that a spectrum of common and rare lipid-associated variants underlies this heterogeneity. METHODS AND RESULTS: First, we demonstrated that genetic determinants of plasma lipids and lipoproteins, including common variants associated with plasma triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) from the Global Lipids Genetics Consortium were associated with multiple HLP-HTG phenotypes. Second, we demonstrated that weighted risk scores composed of common TG-associated variants were distinctly increased across all HLP-HTG phenotypes compared with controls; weighted HDL-C and LDL-C risk scores were also increased, although to a less pronounced degree with some HLP-HTG phenotypes. Interestingly, decomposition of HDL-C and LDL-C risk scores revealed that pleiotropic variants (those jointly associated with TG) accounted for the greatest difference in HDL-C and LDL-C risk scores. The APOE E2/E2 genotype was significantly overrepresented in HLP type 3 versus other phenotypes. Finally, rare variants in 4 genes accumulated equally across HLP-HTG phenotypes. CONCLUSIONS: HTG susceptibility and phenotypic heterogeneity are both influenced by accumulation of common and rare TG-associated variants.

A polygenic basis for four classical Fredrickson hyperlipoproteinemia phenotypes that are characterized by hypertriglyceridemia.

Numerous single nucleotide polymorphisms (SNPs) have been found in recent genome wide association studies (GWAS) to be associated with subtle plasma triglyceride (TG) variation in normolipidemic subjects. However, since these GWAS did not specifically evaluate patients with rare disorders of lipoprotein metabolism--'hyperlipoproteinemia' (HLP)--it remains largely unresolved whether any of these SNP determinants of modest physiological changes in TG are necessarily also determinants of most HLP phenotypes. To address this question, we evaluated 28 TG-associated SNPs from GWAS in 386 unrelated adult patients with one of five Fredrickson phenotypes (HLP types 2A, 2B, 3, 4 and 5) and 242 matched normolipidemic controls. We found that several SNPs associated with TG in normolipidemic samples, including APOA5 p.S19W and -1131T>C, TRIB1 rs17321515, TBL2 rs17145738, GCKR rs780094, GALNT2 rs4846914 and ANGPTL3 rs12130333, were significantly associated with HLP types 2B, 3, 4 and 5. The findings indicate that: (i) the TG-associated Fredrickson HLP types 2B, 3, 4 and 5 are polygenic traits; (ii) these Fredrickson HLP types share numerous genetic determinants among themselves; and (iii) genetic determinants of modest TG variation in normolipidemic population samples also underlie--to an apparently even greater degree--susceptibility to these rare HLP phenotypes. Thus, the TG-associated Fredrickson HLP types 2B, 3, 4 and 5, although historically considered to be distinct are actually complex traits sharing among them several common genetic determinants seen in GWAS of normolipidemic population samples.

HNF1A G319S variant, active cigarette smoking and incident type 2 diabetes in Aboriginal Canadians: a population-based epidemiological study.

BACKGROUND: In a recent report of large-scale association analysis, a type 2 diabetes susceptibility locus near HNF1A was identified in predominantly European descent populations. A population-specific G319S polymorphism in HNF1A was previously identified in Aboriginal Canadians who have a high prevalence of type 2 diabetes. We aimed to investigate the association of the HNF1A G319S polymorphism with incident type 2 diabetes and to assess whether clinical risk variables for type 2 diabetes influence the association in an Aboriginal population. METHODS: Of 606 participants who were free of diabetes at baseline in 1993-1995, 540 (89.1%) participated in 10-year follow-up assessments in 2003-2005. Fasting glucose and a 75-g oral glucose tolerance test were obtained to determine incident type 2 diabetes. Participants were genotyped for the HNF1A G319S polymorphism. Interviewers administered questionnaires on smoking behavior. RESULTS: The incidence rates of type 2 diabetes were 14.2% (55/388) in major allele homozygotes and 31.2% (29/93) in minor allele carriers (p < 0.001). The HNF1A G319S carrier status was associated with incident type 2 diabetes (odds ratio [OR] 3.78 [95% CI 2.13-6.69]) after adjustment for age, sex, hypertension, triglyceride, HDL cholesterol, and waist circumference. A statistical interaction was observed between HNF1A G319S and baseline active cigarette smoking on the development of type 2 diabetes with similar adjustment (p = 0.006). When participants were stratified by baseline smoking status, HNF1A G319S carriers who were active smokers had increased risk of developing diabetes (OR 6.91 [95% CI 3.38-14.12]), while the association was attenuated to non-significance among non-smokers (1.11 [0.40-3.08]). CONCLUSIONS: The HNF1A G319S variant is associated with incident type 2 diabetes in Aboriginal Canadians. Furthermore, cigarette smoking appears to amplify incident diabetes risk in carriers of HNF1A G319S.

Combined hyperlipidemia is genetically similar to isolated hypertriglyceridemia.

BACKGROUND: Combined hyperlipidemia (CHL) is a common disorder defined by concurrently elevated low-density lipoprotein cholesterol (LDL-C) and triglyceride (TG) levels. Despite decades of study, the genetic basis of CHL remains unclear. OBJECTIVE: To characterize the genetic profiles of patients with CHL and compare them to those in patients with isolated hypercholesterolemia and isolated hypertriglyceridemia (HTG). METHODS: DNA from 259, 379 and 124 patients with CHL, isolated hypercholesterolemia and isolated HTG, respectively, underwent targeted sequencing. We assessed: 1) rare variants disrupting canonical LDL-C or TG metabolism genes; and 2) two polygenic scores-for elevated LDL-C and TG-calculated using common trait-associated single-nucleotide polymorphisms (SNPs). Genetic profiles were compared against 1000 Genomes Project controls. RESULTS: Both CHL and isolated HTG patients had significantly increased odds of a high polygenic score for TG: 2.50 (95% confidence interval [CI] 1.61-3.88; P < 0.001) and 3.72 (95% CI 2.24-6.19; P < 0.001), respectively. CHL patients had neither a significant accumulation of rare variants for LDL-C or TG, nor a high polygenic score for LDL-C. In contrast, patients with isolated hypercholesterolemia had a 3.03-fold increased odds (95% CI 2.22-4.13; P < 0.001) of carrying rare variants associated with familial hypercholesterolemia, while patients with isolated HTG had a 2.78-fold increased odds (95% CI 1.27-6.10; P = 0.0136) of carrying rare variants associated with severe HTG. CONCLUSION: CHL is genetically similar to isolated HTG, a known polygenic trait. Both cohorts had a significant accumulation of common TG-raising variants. Elevated LDL-C levels in CHL are not associated with common or rare LDL-C-related genetic variants.

Ancestry-specific profiles of genetic determinants of severe hypertriglyceridemia.

BACKGROUND: Susceptibility to severe hypertriglyceridemia (HTG), defined as plasma triglyceride (TG) levels ≥10 mmol/L (880 mg/dL), is conferred by both heterozygous rare variants in five genes involved in TG metabolism and numerous common single-nucleotide polymorphisms (SNPs) associated with TG levels. OBJECTIVE: To date, these genetic susceptibility factors have been comprehensively assessed primarily in severe HTG patients of European ancestry. Here, we expand our analysis to HTG patients of East Asian and Hispanic ancestry. METHODS: The genomic DNA of 336, 63 and 199 severe HTG patients of European, East Asian and Hispanic ancestry, respectively, was evaluated using a targeted next-generation sequencing panel to screen for: 1) rare variants in LPL, APOA5, APOC2, GPIHBP1 and LMF1; 2) common, small-to-moderate effect SNPs, quantified using a polygenic score; and 3) common, large-effect polymorphisms, APOA5 p.G185C and p.S19W. RESULTS: While the proportion of individuals with high polygenic scores was similar, frequency of rare variant carriers varied across ancestries. Compared with ancestry-matched controls, Hispanic patients were the most likely to have a rare variant (OR = 5.02; 95% CI 3.07-8.21; p < 0.001), while European patients were the least likely (OR = 2.56; 95% CI 1.58-4.13; p < 0.001). The APOA5 p.G185C polymorphism, exclusive to East Asians, was significantly enriched in patients compared with controls (OR = 10.1; 95% CI 5.6-18.3; p < 0.001), showing the highest enrichment among the measured genetic factors. CONCLUSION: While TG-associated rare variants and common SNPs are both found in statistical excess in severe HTG patients of different ancestral backgrounds, the overall genetic profiles of each ancestry group were distinct.

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.

APOC1 T45S polymorphism is associated with reduced obesity indices and lower plasma concentrations of leptin and apolipoprotein C-I in aboriginal Canadians.

Apolipoprotein (apo) C-I is a constituent of chylomicrons, very low density lipoprotein, and high density lipoprotein. The role of apo C-I in human metabolism is incompletely defined. We took advantage of a naturally occurring amino acid polymorphism that is present in aboriginal North Americans, namely apo C-I T45S. We assessed the hypothesis that metabolic traits, including obesity-related and lipoprotein-related traits, would differ between carriers and noncarriers of apo C-I T45S. A genotyping assay was developed for APOC1 T45S and genotypes were determined in a sample of 410 Canadian Oji-Cree subjects. The allele frequency of the apo C-I S45 allele was approximately 8% in this sample. We observed the apo C-I S45 allele was significantly associated with 1) lower percent body fat (P < 0.05), 2) lower waist circumference (P = 0.058), 3) lower serum leptin levels (P < 0.05), and 4) lower plasma apo C-I levels (P < 0.0001), using a newly developed ELISA-based method. Taken together, these results suggest that at the whole human phenotype level, apo C-I is associated with the complex metabolic trait of obesity as well as with serum leptin levels.

Polygenic determinants of severe hypertriglyceridemia.

Recent genome-wide association (GWA) studies have identified new genetic determinants of complex quantitative traits, including plasma triglyceride (TG). We hypothesized that common variants associated with mild TG variation identified in GWA studies would also be associated with severe hypertriglyceridemia (HTG). We studied 132 patients of European ancestry with severe HTG (fasting plasma TG > 10 mmol/l), who had no mutations found by resequencing of candidate genes, and 351 matched normolipidemic controls. We determined genotypes for: GALNT2 rs4846914, TBL2/MLXIPL rs17145738, TRIB1 rs17321515, ANGPTL3 rs12130333, GCKR rs780094, APOA5 rs3135506 (S19W), APOA5 rs662799 (-1131T > C), APOE (isoforms) and LPL rs328 (S447X). We found that: (i) genotypes, including those of APOA5 S19W, APOA5 -1131T > C, APOE, GCKR, TRIB1 and TBL2/MLXIPL, were significantly associated with severe HTG; (ii) odds ratios for these genetic variables were significant in both univariate and multivariate regression analyses, irrespective of the presence or absence of diabetes or obesity; (iii) a significant fraction-about one-quarter-of the explained variation in disease status was associated with these genotypes. Therefore, common SNPs (single nucleotide polymorphisms) that are associated with mild TG variation in GWA studies of normolipidemic subjects are also associated with severe HTG. Our findings are consistent with the emerging model of a complex genetic trait. At the extremes of a quantitative trait, such as severe HTG, are found the cumulative contributions of both multiple rare alleles with large genetic effects and common alleles with small effects.

Assessing the association of the HNF1A G319S variant with C-reactive protein in Aboriginal Canadians: a population-based epidemiological study.

BACKGROUND: C-reactive protein (CRP), a biomarker of inflammation, has been associated with increased risk of developing cardiovascular disease. Common variants of the hepatocyte nuclear factor 1A (HNF1A) gene encoding HNF-1alpha have been associated with plasma CRP in predominantly European Caucasian samples. HNF1A might therefore have an impact on vascular disease and diabetes risk that is mediated by CRP. In an Aboriginal Canadian population, a private polymorphism, HNF1A G319S, was associated with increased prevalence of type 2 diabetes. However, it has not been investigated whether this association is mediated by CRP. We aimed to investigate whether CRP was mediating the association between HNF1A G319S and type 2 diabetes in an Aboriginal Canadian population with a high prevalence of diabetes. METHODS: A total of 718 individuals who participated in a diabetes prevalence and risk factor survey were included in the current analysis. Participants were genotyped for HNF1A G319S. Fasting plasma samples were analyzed for CRP. Fasting plasma glucose and a 75-g oral glucose tolerance test were obtained to determine type 2 diabetes. RESULTS: The prevalence rate of type 2 diabetes was 17.4% (125/718) using the 1999 World Health Organization definition and was higher among S319 allele carriers compared to G/G homozygotes (p < 0.0001). Among participants without type 2 diabetes, CRP levels were higher among G/G homozygotes (1.64 [95% confidence interval 1.35-2.00] mg/l) than in S319 carriers (1.26 [1.04-1.54] mg/l) (p = 0.009) after adjustment for age, sex, 2-h post-load glucose, waist circumference, and serum amyloid A. CRP levels were elevated among those with diabetes after similar adjustment (4.39 [95% confidence interval 3.09-6.23] and 4.44 [3.13-6.30] mg/L, respectively), and no significant difference in CRP was observed between S319 carriers and non-carriers (p = 0.95). CONCLUSIONS: CRP levels were lower in S319 allele carriers of the HNF1A gene compared to non-carriers among individuals without diabetes, but this difference was not present among those with diabetes, who uniformly had elevated CRP levels. Therefore, while HNF1A appears to influence CRP concentrations in the non-diabetic state, chronic elevation of CRP is unlikely mediating the association between the HNF1A polymorphism and the high prevalence of type 2 diabetes in this Aboriginal population.