Resequencing Genomic DNA of Patients With Severe Hypertriglyceridemia (MIM 144650)

【摘要】  Objective— The genetic determinants of severe hypertriglyceridemia (HTG; MIM 144650 ) in adults are poorly defined. We therefore resequenced 3 candidate genes, namely LPL, APOC2, and APOA5, to search for accumulation of missense mutations in patients with severe HTG compared with normolipidemic subjects.

Methods and Results— 2 million base pairs of genomic DNA from 110 nondiabetic patients with severe HTG and determined the prevalence of coding sequence variants compared with 472 age- and sex-matched normolipidemic controls. We found: (1) heterozygous mutations ( LPL 11X, p.D25H, p.W86R, p.G188E, p.I194T and p.P207L; APOC2 A) in 10.0% of severe HTG patients compared with 0.2% of controls (carrier odds ratio 52, 95% confidence interval 8.6 to 319); and (2) an association of the APOA5 p.S19W missense variant with severe HTG (carrier OR 5.5 95% CI 3.3 to 9.1). Furthermore, either rare mutations or the APOA5 p.S19W variant were found in 41.8% of HTG subjects compared with 8.9% of controls (carrier OR 7.4, 95% CI 4.5 to 12.0). Also, heterozygotes for rare mutations had a significantly reduced plasma triglyceride response to fibrate monotherapy.

Conclusions— Both common and rare DNA variants in candidate genes were found in a substantial proportion of severe HTG patients. The findings underscore the value of candidate gene resequencing to understand the genetic contribution in complex lipoprotein and metabolic disorders.

Severe hypertriglyceridemia has been presumed to have a genetic basis, but this has never been quantified. With resequencing, we now document a range of DNA variants in LPL, APOC2, and APOA5 genes which together were found in 41.8% of patients with severe HTG compared with 8.9% of control subjects ( P <10 –13 ).

【关键词】  complex trait metabolism atherosclerosis pancreatitis mutation

Introduction

Hypertriglyceridemia (HTG) is a commonly encountered phenotype that is a defining component of the metabolic syndrome 1 and is associated with numerous comorbidities, including increased coronary heart disease (CHD) risk. 2 Furthermore, plasma triglyceride 10 mmol/L—a level that defines adult patients with Frederickson type 5 hyperlipoproteinemia (MIM 144650 )—are associated with increased risk of acute pancreatitis. 3,4 10 mmol/L is seen in 1 in 600 adult North Americans. 5 Although both genetic and lifestyle factors determine plasma TG concentration, the genetic component remains incompletely defined. 6

Complex quantitative traits, such as plasma TG, do not conform to Mendelian inheritance patterns; instead their genetic basis represents the cumulative contribution of multiple DNA variants. 7 A promising new strategy in human genetics to understand common complex traits is called the "missense accumulation approach", which aims to detect enrichment of rare, deleterious missense DNA variants in cases taken from one extreme of the distribution of a quantitative trait versus a control group. 7 The cumulative frequency of missense mutations rather than their individual frequencies is then compared between cases and controls. 7 This method has proven to be successful for investigation of common disease traits that have a very heterogeneous spectrum of predisposing alleles. 7 For instance, the missense accumulation approach has been successfully used to evaluate the MC4R gene 8 and several other genes in obesity, 9 and the tyrosine phosphatome in colorectal cancers. 10

However, the most successful application of the missense accumulation strategy has been in lipoprotein metabolism, as evidenced by the pioneering work of Hobbs and Cohen. 11–14 They have found enrichment of missense mutations in individuals at the extremes of several plasma lipoprotein traits, including: (1) increased missense mutations in LCAT, APOA1, and ABCA1 among individuals with depressed high-density lipoprotein (HDL) cholesterol 13; (2) increased PCSK9 missense or nonsense mutations among individuals with depressed low-density lipoprotein (LDL) cholesterol 14; (3) increased missense mutations in NPC1L1 in individuals with reduced sterol absorption and low plasma LDL cholesterol 12; and (4) increased missense mutations in ANGPTL4 in individuals with depressed triglyceride (TG) and increased HDL cholesterol. 11 In these studies, the statistical association of the accumulation of rare coding sequence variants implicated the gene as contributing to the traits under study, whereas no direct experimental evidence of dysfunction for the mutations was provided. 11–14 Furthermore, most rare missense variants that accumulate in patients clustered at the extremes of a quantitative trait are dysfunctional. 7

Homozygous mutations in candidate genes for plasma TG metabolism, namely LPL encoding the main plasma hydrolytic enzyme lipoprotein lipase (LPL), and APOC2 encoding its circulating cofactor apolipoprotein (apo) C-II, are found in patients with Frederickson type 1 hyperlipoproteinemia 15–17 (MIM 238600 ), a disorder that affects 1 in 1 million people. 13,15 Also, homozygous nonsense mutations in APOA5 encoding apo A-V, a protein that promotes LPL activity, 18 have been found in probands with late-onset chylomicronemia. 19 Because the prevalence of coding sequence variants in adults with severe HTG is unknown, 2 million base pairs of genomic DNA from nondiabetic patients with severe HTG and used the missense accumulation approach to determine the association of variants in LPL, APOC2, and APOA5 with severe hypertriglyceridemia.

Methods

Subjects

We studied 110 nondiabetic patients of European geographic ancestry 10 mmol/L documented on 2 occasions, from a single tertiary referral lipid clinic. Patients underwent a complete medical history and examination; basic clinical, biochemical, and demographic variables were collected. Normolipidemic nondiabetic adult controls were taken from the European subgroup of the Study of Health Assessment and Risk in Ethnic groups (SHARE), a survey of cardiovascular risk factors in Canadian subpopulations 20 together with healthy population-based controls from the same region of Canada. No control had ischemic heart disease and there was no use of medications among these healthy control subjects. All patients provided informed consent for DNA analysis.

DNA Analysis

DNA was extracted as described. 21 Coding regions and intron-exon boundaries of LPL (10 exons), APOC2 (4 exons), and APOA5 (4 exons) were amplified, purified, and then directly sequenced in 5'- and 3'- directions in an ABI 3730 DNA Analyzer (Applied Biosystems) using reagents shown in supplemental Table I. DNA sequences were analyzed using Sequence Navigator software (Applied Biosystems). DNA variants were confirmed in an independent sample on another day. Screening of controls for sequence variants was performed using allele-specific methods such as restriction endonuclease analysis or a method called SNaPshot (Applied Biosystems), as summarized in supplemental Table II. Blinded between-day 99.9% concordance. DNA variants with a minor allele frequency (MAF) <1% in controls were analyzed separately from variants with 1%.

Bioinformatic Studies

We used both the PANTHER (www.pantherdb.org) 22,23 and PolyPhen 24 algorithms to impute dysfunction of sequence variants. Predictions of dysfunction from both programs are well correlated with in vitro functional assessment. 24,25 The scores from each program were grouped into 3 categories: "probably deleterious", "possibly deleterious", and "benign". The majority of biochemically-proven functional mutations have scores of either "probably" or "possibly" deleterious for both programs. 24,25

Statistical Analysis

Analyses were performed using SAS version 9.1 (SAS Institute). Between-group differences in discrete and quantitative traits were determined using chi-square analysis and unpaired Student t tests, respectively. Odds ratios (OR) were calculated using the "case-control" method in the FREQ procedure in SAS. Log transformed TG was used for parametric analyses, but untransformed values are shown in the tables and figure. The nominal level for significance was P <0.05.

Results

Clinical and Biochemical Features

Baseline attributes of the study sample are shown in Table 1. 110 nondiabetic severe HTG cases were each matched with up to 4 controls based on age within 5 years and sex. By definition, severe HTG patients had markedly higher plasma TG and total cholesterol and significantly lower HDL cholesterol. Plasma TG concentration in severe HTG patients ranged from 10.1 to 180 mmol/L. In addition, 32/110 severe HTG patients (29.0%) had been hospitalized on 1 occasion with pancreatitis.

Table 1. Baseline Attributes of Study Subjects

Rare Mutations in Candidate Genes

Mutations, defined as DNA sequence variants with MAF <1% in controls or known functional disease-causing mutations that were found in the genomic DNA of severe HTG patients, with their highest recorded plasma TG concentrations, are summarized in Table 2. In the severe HTG patients, we found 12 occurrences of heterozygous candidate gene mutations. Carriers were heterozygous for 1 of 9, mostly known disease-causing, mutations: 6 in LPL, 2 in APOC2 and 1 in APOA5. For instance, in the homozygous state, LPL p.W86R, p.G188E, p.I194T, and p.P207L each cause HLP type 1 and each is dysfunctional in vitro, with significantly impaired or absent hydrolytic capacity of the mutant gene product. 26–29 Among the novel heterozygous mutations observed in this study, LPL 11X was a frameshift mutation with a very prematurely truncated product, whereas LPL p.D25H was predicted to be deleterious in both PANTHER and PolyPhen. The known APOC2 p.K19T variant was previously associated with dyslipidemia. 30,31 The novel APOC2 A variant potentially affects RNA splicing. APOA5 p.A315V 32 was predicted to be possibly deleterious; however, in the absence of more definitive demonstration of dysfunction, we treated the single carrier of this mutation as a noncarrier in subsequent analyses.

Table 2. Rare DNA Sequence Mutations in LPL, APOC2, and APOA5 in Patients With Severe Hypertriglyceridemia

Common DNA Sequence Variants

By resequencing, we found 5 reported candidate gene single nucleotide 1% in controls: 3 in LPL, namely p.D9N, p.N291S, and p.S447X and 2 in APOA5, namely p.S19W and p.V153 mol/L ( Table 3 ). The LPL SNPs were previously functionally assessed: p.D9N had compromised LDL uptake but not impaired hydrolysis, 33 whereas p.N291S 34 and p.S447X 35 had 50% decreased and 30% increased hydrolytic capacity, respectively. APOA5 p.S19W is defectively secreted in vitro. 36 The APOA5 p.S19W allele was significantly more prevalent in severe HTG cases compared with controls ( Table 3 ).

Table 3. Common DNA Sequence Polymorphisms in LPL, APOC2 and APOA5 in Patients With Severe Hypertriglyceridemia

Table 4. Carrier Frequencies for DNA Variants Found in This Study

Differences in Distribution of DNA Variants Between Cases and Controls

Frequencies of carriers of rare mutations in severe HTG patients and normotriglyceridemic controls are shown in Table 4. Heterozygous LPL mutations p.Q-12E-11X (once), p.D25H (once), p.W86R (once), p.G188E (twice), p.I194T (once), and p.P207L (once) were present cumulatively in 7/110 (6.4%) of severe HTG patients compared with 0/472 controls; the carrier odds ratio (OR) was infinite ( P <0.00001). When heterozygotes for either APOC2 p.K19T or A were included, 10.0% of severe HTG patients compared with 0.2% of controls were carriers of mutations (carrier OR 52, 95% confidence interval 8.6 to 319; P <10 –7 ).

The APOA5 p.S19W loss-of-function allele was strongly associated with severe HTG: 34.6% of HTG subjects were carriers versus 8.8% of controls (OR 5.5, 95% CI 3.3 to 9.1; P <10 –9 ). The LPL p.D9N variant was modestly associated with severe HTG: 10.9% of cases were carriers versus 3.6% of controls (OR 3.2, 95% CI 1.5 to 7.0; P =0.0017). The LPL p.S447X variant had a borderline associated with protection from severe HTG: 6.4% of HTG subjects were carriers versus 10.8% of controls (OR 0.44, 95% CI 0.20 to 0.99; P =0.043). To quantify the total genetic contribution of the most significantly associated variants, we determined carrier OR for subjects with 1 copy of either the heterozygous rare mutations or 1 copy of the APOA5 p.S19W allele. We found that 41.8% of HTG subjects were carriers compared with 8.9% of controls (OR 7.4, 95% CI 4.5 to 12.0; P <10 –13 ).

Response to Fibrate Therapy According to Genotype

To determine a possible between-genotype difference in plasma TG response to oral fibrate treatment, we performed an exploratory post hoc analysis in the subgroup of 53 nondiabetic HTG patients whose treatment consisted only of dietary counseling and usual doses of fenofibrate, gemfibrozil, or bezafibrate as monotherapy. In this subgroup (50.1±12.9 years, 34% female), we recorded the maximal percent change from baseline plasma lipoproteins within 12 months of initiating treatment. The subgroup comprised 7, 18, and 28 HTG patients who had 1 copy of either the heterozygous rare mutations, 1 copy of the APOA5 p.S19W allele, and neither, respectively. We observed a significant difference in plasma TG response between genotypes: patients who had 1 copy of the rare mutations had a blunted maximal decrease in plasma TG compared with other subjects ( Figure ). We observed significant between-group differences in increased plasma HDL cholesterol but no difference in total cholesterol on treatment ( Figure ).

Plasma lipoprotein response (percent change from baseline) in nondiabetic patients with severe hypertriglyceridemia treated with fibrate monotherapy. Patients were stratified by genotype: 7 had 1 copy of rare heterozygous mutations (black), 18 had 1 copy of the APOA5 p.S19W allele (gray), and 28 had no genetic variant (white). Mean and standard error of maximal lipoprotein changes from baseline and significance levels for between-genotype differences are shown.

Discussion

By resequencing 3 candidate genes, we found an association of several genetic variants with severe HTG in Canadian patients of European ancestry. Specifically, we found that 41.8% of subjects 10 mmol/L have 1 copy of several rare coding sequence variants in candidate genes ( LPL or APOC2 ) or 1 copy of the common APOA5 p.S19W allele, whereas this assortment of genetic variants is present in only 8.9% of controls (OR 7.4, 95% CI 4.5 to 12.0; P <10 –13 ). This is among the most substantial genetic contribution yet detected for a dyslipoproteinemia phenotype. We also observed that carriers of 1 copy of several rare mutations in LPL, APOC2, or APOA5 genes had a smaller decrease in plasma TG in response to oral fibrate treatment than subjects with other genotypes.

Previous studies of patients with extreme lipoprotein phenotypes showed that rare candidate gene mutations are present in a significant minority of cases. For instance among patients with low HDL, 16% had candidate gene mutations compared with 2% of controls. 13 Our findings support a similar significant contribution of rare candidate gene mutations in a minority of patients with severe HTG: rare mutations were seen in 10.0% of severe HTG patients compared with 0.2% of controls were carriers of mutations (OR 52, 95% CI 8.6 to 319; P <10 –7 ). In addition, we also found a very strong association of severe HTG with the common dysfunctional APOA5 p.S19W variant: 34.6% of HTG subjects were carriers versus 8.8% of controls (OR 5.5, 95% CI 3.3 to 9.1; P <10 –9 ). Thus, the genetic component of this complex metabolic trait is comprised of both rare and common variants, which together account for a greater proportion of affected 40% in this sample) than rare mutations alone.

The present findings quantify the potential contribution of mutant LPL to type 5 hyperlipoproteinemia and solidify a key physiological role for apo A-V, which was only discovered 5 years ago using bioinformatic analysis. 37 The APOA5 p.S19W allele has been evaluated in several studies, 32 some of which have shown modest associations with mildly elevated plasma TG. Recently, plasma apo A-V concentrations and p.S19W allele frequency were shown to be elevated in patients with relatively mild TG elevation. 38 Among numerous SNPs at the APOA5 locus, p.S19W is unique because it: (1) alters the amino acid sequence and has proven dysfunction in vitro 36; (2) is relatively common, with an allele frequency of 7% to 11% in control samples of European ancestry 18,39; and (3) is the defining polymorphism of a unique haplotype associated with moderately elevated TG. 37,39 Together, the data would indicate that APOA5 p.S19W might be a clinically useful risk marker of this extreme phenotype.

We selected LPL, APOC2, or APOA5 as candidate genes for association with type 5 hyperlipoproteinemia because homozygous mutations in each cause severe HTG with chylomicronemia and especially type 1 hyperlipoproteinemia (MIM 238600 ), a disorder whose genetic basis is well understood. 3,15 Most LPL mutations that we found in severe HTG patients were already proven to be disease-causing in the homozygous state, with documented functional impairment. 26–29 The novel mutations were by and large imputed to have functional impact through their statistical association with severe HTG, their virtual absence from the control sample and also through bioinformatic analysis. Although the findings clearly link heterozygosity for these rare mutations with severe HTG, other factors must be important—both in severe HTG patients with and without the heterozygous mutations.

In the premolecular era, analysis of pedigrees of probands with familial chylomicronemia and biochemical deficiency of LPL suggested that obligate heterozygote carriers of presumed LPL mutations had an increased risk of combined hypercholesterolemia and hypertriglyceridemia. 40 Similarly, analysis of pedigrees of probands with familial chylomicronemia attributable to mutant apoC-II isoforms suggested that obligate heterozygote carriers of APOC2 mutations had elevated plasma TG and high TG content in lipoprotein subfractions. 41 Finally, heterozygous relatives of probands homozygous for truncating mutations in APOA5 and severe HTG were variably found to have moderately elevated plasma TG. 42,43 Thus, evidence from the pre- and postgenomic eras, including findings from this study, indicates that heterozygosity for rare dysfunctional coding sequence mutations is strongly associated with severe HTG.

Besides APOA5 p.S19W, among the other coding variants with MAF 1%, LPL p.D9N, and p.S447X were found to be associated with susceptibility and protection from HTG, respectively. We noted that the presence of the so-called "gain-of-function" LPL p.S447X variant 35 was not "protective" for 7 carriers among the severe HTG patients. Such anecdotal cases may be important considering that p.S447X variant is being proposed as the central component of a gene therapy strategy designed to treat patients with severe HTG. 35

Thus, our findings are consistent with the emerging model that the cumulative contributions of multiple rare alleles with large genetic effects are found among individuals at the extremes of a complex genetic trait. 13,44 However, in contrast to findings from studies of patients at extremes of very low HDL and LDL 5%, 12–14 our findings indicate that a variety of low frequency mutations together with the common APOA5 p.S19W polymorphism, underlie an increased risk in a large segment of patients with extremely high plasma TG. We do not suggest that the heterozygous mutations identified here are directly causative because hyperlipoproteinemia type 5 is a complex trait with no single simple genetic cause and other factors, both genetic and nongenetic are likely to be very important. We also showed a difference in plasma TG response to fibrates, with carriers of 1 copy of several rare coding sequence variants in either LPL, APOC2 or APOA5 having a significantly less favorable response compared with other subjects. The findings further confirm that the genetic contribution to severe HTG is complex and suggest that other genes may still have an important role to play.

Acknowledgments

Rebecca Provost, Rachel Rollings, and Valerie Orr each provided outstanding technical assistance.

Sources of Funding

Dr Hegele is a Career Investigator of the Heart and Stroke Foundation of Ontario and holds the Edith Schulich Vinet Canada Research Chair (Tier I) in Human Genetics and the Jacob J. Wolfe Distinguished Medical Research Chair.

This work was supported by operating grants from the Canadian Institutes of Health Research (MT14030 and MOP-79533), the Heart and Stroke Foundation of Ontario, and Genome Canada through the Ontario Genomics Institute.

Disclosures

None.

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作者单位：Vascular Biology Research Group (J.W., H.C., M.R.B., B.A.K., S.Z., R.L.P., R.A.H.), Robarts Research Institute and Schulich School of Medicine and Dentistry, London, Ontario, Canada; and Population Health Research Institute (S.A., S.Y.), McMaster University, Hamilton Health Sciences, Hamilton, Ontar