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Patient-Oriented and Epidemiological Research| Volume 59, ISSUE 12, P2421-2435, December 2018

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Genetic and secondary causes of severe HDL deficiency and cardiovascular disease1

Open AccessPublished:October 17, 2018DOI:https://doi.org/10.1194/jlr.M088203
      We assessed secondary and genetic causes of severe HDL deficiency in 258,252 subjects, of whom 370 men (0.33%) and 144 women (0.099%) had HDL cholesterol levels <20 mg/dl. We excluded 206 subjects (40.1%) with significant elevations of triglycerides, C-reactive protein, glycosylated hemoglobin, myeloperoxidase, or liver enzymes and men receiving testosterone. We sequenced 23 lipid-related genes in 201 (65.3%) of 308 eligible subjects. Mutations (23 novel) and selected variants were found at the following gene loci: 1) ABCA1 (26.9%): 2 homozygotes, 7 compound or double heterozygotes, 30 heterozygotes, and 2 homozygotes and 13 heterozygotes with variants rs9282541/p.R230C or rs111292742/c.-279C>G; 2) LCAT (12.4%): 1 homozygote, 3 compound heterozygotes, 13 heterozygotes, and 8 heterozygotes with variant rs4986970/p.S232T; 3) APOA1 (5.0%): 1 homozygote and 9 heterozygotes; and 4) LPL (4.5%): 1 heterozygote and 8 heterozygotes with variant rs268/p.N318S. In addition, 4.5% had other mutations, and 46.8% had no mutations. Atherosclerotic cardiovascular disease (ASCVD) prevalence rates in the ABCA1,LCAT,APOA1, LPL, and mutation-negative groups were 37.0%, 4.0%, 40.0%, 11.1%, and 6.4%, respectively. Severe HDL deficiency is uncommon, with 40.1% having secondary causes and 48.8% of the subjects sequenced having ABCA1,LCAT,APOA1, or LPL mutations or variants, with the highest ASCVD prevalence rates being observed in the ABCA1 and APOA1 groups.
      It has been concluded that genetic HDL deficiency is not causative for atherosclerotic cardiovascular disease (ASCVD), in contrast to genetic hypercholesterolemia associated with elevated levels of LDL cholesterol (LDL-C) or genetic hypertriglyceridemia (
      • Voight B.F.
      • Peloso G.M.
      • Orho-Melander M.
      • Frikke-Schmidt R.
      • Barbalic M.
      • Jensen M.K.
      • Hindy G.
      • Hólm H.
      • Ding E.L.
      • Johnson T.
      • et al.
      Plasma HDL cholesterol and risk of myocardial infarction: a Mendelian randomization study.
      ,
      • Do R.
      • Willer C.J.
      • Schmidt E.M.
      • Sengupta S.
      • Gao C.
      • Peloso G.M.
      • Gustafsson S.
      • Kanoni S.
      • Ganna A.
      • Chen J.
      • et al.
      Common variants associated with plasma triglycerides and risk for coronary artery disease.
      ). This assessment has been reported in high-impact journals by a very large number of authors, representing some of the most prominent and well-known scientists in our field. The data generated stem from a very large number of subjects studied with and without ASCVD. The analyses, however, were based entirely on SNPs, mainly in intronic DNA regions, and excluded the four most important genes that regulate HDL cholesterol (HDL-C) levels: ABCA1,LCAT, APOA1, and LPL. The exclusions were justified by study findings that 1) genetic variations at the ABCA1,LCAT, APOA1, and LPL gene loci also affect TG and/or LDL-C levels and 2) an LIPC variant was not associated with ASCVD (
      • Voight B.F.
      • Peloso G.M.
      • Orho-Melander M.
      • Frikke-Schmidt R.
      • Barbalic M.
      • Jensen M.K.
      • Hindy G.
      • Hólm H.
      • Ding E.L.
      • Johnson T.
      • et al.
      Plasma HDL cholesterol and risk of myocardial infarction: a Mendelian randomization study.
      ,
      • Do R.
      • Willer C.J.
      • Schmidt E.M.
      • Sengupta S.
      • Gao C.
      • Peloso G.M.
      • Gustafsson S.
      • Kanoni S.
      • Ganna A.
      • Chen J.
      • et al.
      Common variants associated with plasma triglycerides and risk for coronary artery disease.
      ). In our view this justification is flawed. Our recent review of severe HDL deficiency has documented that patients with HDL-C levels <20 mg/dl in the absence of secondary causes may have premature ASCVD, especially if such patients are homozygous or compound heterozygous for mutations in the APOA1 or ABCA1 genes (
      • Schaefer E.J.
      • Anthanont P.
      • Diffenderfer M.R.
      • Polisecki E.
      • Asztalos B.F.
      Diagnosis and treatment of high density lipoprotein deficiency.
      ).
      Although many studies have examined potential genetic causes of low HDL-C, there is no prior study to our knowledge that has carefully examined both secondary and genetic causes of severe HDL-C deficiency in a population of more than 250,000 subjects and related such findings to ASCVD (
      • Schaefer E.J.
      • Anthanont P.
      • Diffenderfer M.R.
      • Polisecki E.
      • Asztalos B.F.
      Diagnosis and treatment of high density lipoprotein deficiency.
      ). Our purpose in this investigation was to assess the prevalence of severe HDL deficiency in a large reference laboratory population and to determine potential underlying secondary and genetic causes by direct DNA sequencing of genes that are germane for lipoprotein metabolism, including APOA1,ABCA1,LCAT, and LPL, as well as to ascertain the prevalence of ASCVD in the various genetic HDL deficiency states.
      Decreased levels of serum HDL-C (<40 mg/dl in men and <50 mg/dl in women) have been found to be a very significant independent risk factor for ASCVD and in this regard were superior to serum apoA-I levels in the Framingham Offspring Study (
      • Goff D.C.
      • Lloyd-Jones D.M.
      • Bennett G.
      • Coady S.
      • D'Agostino R.B.
      • Gibbons R.
      • Greenland P.
      • Lackland D.T.
      • Levy D.
      • O'Donnell C.J.
      • et al.
      2013 ACC/AHA guideline on the assessment of cardiovascular risk: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines.
      ,
      • Ingelsson E.
      • Schaefer E.J.
      • Contois J.H.
      • McNamara J.R.
      • Sullivan L.
      • Keyes M.J.
      • Pencina M.J.
      • Schoonmaker C.
      • Wilson P.W.
      • D'Agostino R.B.
      • et al.
      Clinical utility of different lipid measures for prediction of coronary heart disease in men and women.
      ). An even better marker of ASCVD risk may be decreased HDL function as assessed by cellular cholesterol efflux (
      • Khera A.V.
      • Cuchel M.
      • de la Llera-Moya M.
      • Rodrigues A.
      • Burke M.F.
      • Jafri K.
      • French B.C.
      • Phillips J.A.
      • Mucksavage M.L.
      • Wilensky R.L.
      • et al.
      Cholesterol efflux capacity, high-density lipoprotein function, and atherosclerosis.
      ,
      • Rohatgi A.
      • Khera A.
      • Berry J.D.
      • Givens E.G.
      • Ayers C.R.
      • Wedin K.E.
      • Neeland I.J.
      • Yuhanna I.S.
      • Rader D.R.
      • de Lemos J.A.
      • et al.
      HDL cholesterol efflux capacity and incident cardiovascular events.
      ). Pre-β1 HDL, the smallest HDL subparticle separated by two-dimensional gel electrophoresis, serves as the acceptor of cellular free cholesterol and phospholipids via the ATP binding cassette transporter 1 (ABCA1) and, in the process, is converted to larger α HDL particles, while α1 HDL serves as both a donor and acceptor of cholesterol in its interaction with liver cells via scavenger receptor B1 (
      • Asztalos B.F.
      • De la Llera-Moya M.
      • Dallal G.E.
      • Horvath K.V.
      • Schaefer E.J.
      • Rothblat G.H.
      Differential effects of HDL subpopulations on cellular ABCA1- and SRB1-mediated cholesterol efflux.
      ,
      • Asztalos B.F.
      • Horvath K.V.
      • Mehan M.
      • Yokota Y.
      • Schaefer E.J.
      Influence of HDL particles on cell-cholesterol efflux under various pathological conditions.
      ,
      • Asztalos B.F.
      • Horvath K.V.
      • Schaefer E.J.
      High-density lipoprotein particles, cell-cholesterol efflux, and coronary heart disease risk.
      ). In patients with low HDL and premature ASCVD, compared with control subjects, there are significantly lower apoA-I levels in α1 HDL and higher apoA-I levels in pre-β1 HDL than in other HDL particles (
      • Asztalos B.F.
      • Cupples L.A.
      • Demissie S.
      • Horvath K.V.
      • Cox C.E.
      • Batista M.C.
      • Schaefer E.J.
      High-density lipoprotein subpopulation profile and coronary heart disease prevalence in male participants of the Framingham Offspring Study.
      ,
      • Lamon-Fava S.
      • Herrington D.M.
      • Reboussin D.M.
      • Sherman M.
      • Horvath K.V.
      • Cupples L.A.
      • White C.
      • Demissie S.
      • Schaefer E.J.
      • Asztalos B.F.
      Plasma levels of HDL subpopulations and remnant lipoproteins predict the extent of angiographically defined disease in post-menopausal women.
      ,
      • Asztalos B.F.
      • Collins D.
      • Cupples L.A.
      • Demissie S.
      • Horvath K.V.
      • Bloomfield H.E.
      • Robins S.J.
      • Schaefer E.J.
      Value of high density lipoprotein (HDL) subpopulations in predicting recurrent cardiovascular events in the Veterans Affairs HDL Intervention Trial.
      ,
      • Guey L.T.
      • Pullinger C.R.
      • Ishida B.Y.
      • O'Connor P.M.
      • Zellner C.
      • Francone O.L.
      • Laramie J.M.
      • Naya-Vigne J.M.
      • Siradze K.A.
      • Deedwania P.
      • et al.
      Relation of increased prebeta-1 high-density lipoprotein levels to risk of coronary heart disease.
      ). Hypertriglyceridemia is also associated with increased levels of pre-β1 HDL (
      • Stock EO
      • Ferrara C.T.
      • O'Connor P.M.
      • Naya-Vigne J.M.
      • Frost P.H.
      • Malloy M.J.
      • Kane J.P.
      • Pullinger C.R.
      Levels of prebeta-1 high-density lipoprotein are elevated in 3 phenotypes of dyslipidemia.
      ). Increasing apoA-I levels in α1 HDL by means of treatment with the simvastatin/niacin combination has been associated with decreased coronary atherosclerosis (
      • Asztalos B.F.
      • Batista M.
      • Horvath K.V.
      • Cox C.E.
      • Dallal G.E.
      • Morse J.S.
      • Brown G.B.
      • Schaefer E.J.
      Change in alpha 1 HDL concentration predicts progression in coronary artery stenosis.
      ).
      In prior studies of families with premature ASCVD, we reported that 19% had lipoprotein (a) excess, 15% had familial dyslipidemia (high TGs and low HDL-C), 14% had familial combined hyperlipidemia (high LDL-C, high TGs, and usually low HDL-C), 4% had isolated low HDL (hypoalphalipoproteinemia), and 1% had familial hypercholesterolemia, according to the 10th and 90th percentile values from the Framingham Offspring Study (
      • Genest J.J.
      • Martin-Munley S.S.
      • McNamara J.R.
      • Ordovas J.M.
      • Jenner J.
      • Meyers R.H.
      • Silberman S.R.
      • Wilson P.W.F.
      • Salem D.N.
      • Schaefer E.J.
      Familial lipoprotein disorders in patients with premature coronary artery disease.
      ,
      • Genest J.
      • Bard J.M.
      • Fruchart J.C.
      • Ordovas J.M.
      • Schaefer E.J.
      Familial hypoalphalipoproteinemia in premature coronary artery disease.
      ). Our data indicate that low HDL-C in families with premature ASCVD is frequently associated with either elevated TGs or elevations of both TGs and LDL-C. In the general population, we have also documented that low HDL-C levels are significantly associated with hypertriglyceridemia, obesity, diabetes, men, sedentary lifestyle, and cigarette smoking (
      • Schaefer E.J.
      • Lamon-Fava S.
      • Ordovas J.M.
      • Cohn S.D.
      • Schaefer M.M.
      • Castelli W.P.
      • Wilson P.W.F.
      Factors associated with low and elevated plasma high density lipoprotein cholesterol and apolipoprotein A-I levels in the Framingham Offspring Study.
      ). Patients with markedly low plasma concentrations of HDL-C (<20 mg/dl) in the absence of marked hypertriglyceridemia, marked inflammation, diabetes, liver disease, or the use of anabolic steroids are therefore uncommon (
      • Schaefer E.J.
      • Anthanont P.
      • Diffenderfer M.R.
      • Polisecki E.
      • Asztalos B.F.
      Diagnosis and treatment of high density lipoprotein deficiency.
      ) and are most likely to have homozygous, compound heterozygous, or heterozygous defects involving the APOA1, ABCA1, or LCAT genes (
      • Voight B.F.
      • Peloso G.M.
      • Orho-Melander M.
      • Frikke-Schmidt R.
      • Barbalic M.
      • Jensen M.K.
      • Hindy G.
      • Hólm H.
      • Ding E.L.
      • Johnson T.
      • et al.
      Plasma HDL cholesterol and risk of myocardial infarction: a Mendelian randomization study.
      ).
      The overall data presented in this investigation are consistent with the concept that approximately 40% of patients with severe HDL deficiency have secondary causes, including severe hypertriglyceridemia, increased inflammation, and uncontrolled diabetes. In the absence of such secondary causes, about half of patients with markedly low HDL-C have mutations or variants at the ABCA1, LCAT, or APOA1 gene loci as determined by DNA sequencing. Moreover, compared with nonaffected control subjects, patients with ABCA1 or APOA1 mutations or variants have an increased prevalence of ASCVD. In contrast to the conclusions of some investigators, the data indicate that genetic causes of severe HDL deficiency can, in turn, cause premature ASCVD (
      • Voight B.F.
      • Peloso G.M.
      • Orho-Melander M.
      • Frikke-Schmidt R.
      • Barbalic M.
      • Jensen M.K.
      • Hindy G.
      • Hólm H.
      • Ding E.L.
      • Johnson T.
      • et al.
      Plasma HDL cholesterol and risk of myocardial infarction: a Mendelian randomization study.
      ,
      • Do R.
      • Willer C.J.
      • Schmidt E.M.
      • Sengupta S.
      • Gao C.
      • Peloso G.M.
      • Gustafsson S.
      • Kanoni S.
      • Ganna A.
      • Chen J.
      • et al.
      Common variants associated with plasma triglycerides and risk for coronary artery disease.
      ,
      • Schaefer E.J.
      • Anthanont P.
      • Diffenderfer M.R.
      • Polisecki E.
      • Asztalos B.F.
      Diagnosis and treatment of high density lipoprotein deficiency.
      ).

      METHODS

      The subject population consisted of 112,776 men and 145,476 women referred to our reference laboratory over a 3-year period (2014–2016) that had the following parameters assessed in serum or plasma after an overnight fast using methods as previously described (
      • Schaefer E.J.
      • Tsunoda F.
      • Diffenderfer M.
      • Polisecki E.
      • Thai N.
      • Asztalos B.
      The measurement of lipids, lipoproteins, apolipoproteins, fatty acids, and sterols, and next generation sequencing for the diagnosis and treatment of lipid disorders.
      ): total cholesterol, TGs, direct LDL-C, small dense LDL-C (sdLDL-C), HDL-C, apoA-I, apoB, HDL particle analysis, glucose, insulin, adiponectin, high-sensitivity C-reactive protein (hsCRP), fibrinogen, myeloperoxidase (MPO), and liver transaminases. We utilized results from only the first laboratory sample if a subject was sampled more than once. The median age of this population, the gender percentage, and the fasting lipid concentrations were quite similar to values previously obtained from subjects participating in cycle 6 of the Framingham Offspring Study (
      • Asztalos B.F.
      • Cupples L.A.
      • Demissie S.
      • Horvath K.V.
      • Cox C.E.
      • Batista M.C.
      • Schaefer E.J.
      High-density lipoprotein subpopulation profile and coronary heart disease prevalence in male participants of the Framingham Offspring Study.
      ). However, because our subjects were not randomly selected but were rather selected by their healthcare providers to have their serum and plasma biochemistries characterized by a reference laboratory, they may not be representative of middle-aged and elderly subjects in the general US population.
      The lipid assays were standardized by the Lipid Standardization Program of the Centers for Disease Control (Atlanta, GA), and the apolipoprotein assays were standardized by the Northwest Lipid Research Clinics Program of the University of Washington (Seattle, WA). All assays except the HDL particle analyses and the MPO assays were performed using automated enzymatic analyses on Roche COBAS automated analyzers with reagents obtained from Roche Diagnostics (Indianapolis, IN). The within- and between-run coefficients of variation for these assays were <4%. The MPO measurements were performed using a chemiluminescence assay obtained from Siemens (Malvern, PA) on a Siemens Dimension 200 EXL platform, with within- and between-run coefficients of variation of <5%. The apoA-I content of HDL particles was analyzed by two-dimensional gel electrophoresis followed by immunoblotting as previously described, with within- and between-run coefficients of variation of <10.0% for all particles except for pre-β1 HDL, which was <15.0% (
      • Asztalos B.F.
      • Cupples L.A.
      • Demissie S.
      • Horvath K.V.
      • Cox C.E.
      • Batista M.C.
      • Schaefer E.J.
      High-density lipoprotein subpopulation profile and coronary heart disease prevalence in male participants of the Framingham Offspring Study.
      ).
      In this population, 0.33% of men (n = 370) and 0.099% of women (n = 144) had fasting HDL-C concentrations <20 mg/dl. From this population, we excluded all subjects with hsCRP concentrations >10.0 mg/l, fasting TG concentrations >600 mg/dl, glycosylated hemoglobin (HbA1c) levels >8.0%, and liver transaminase levels >120 U/l, as well as men receiving testosterone replacement. These cutoffs for TGs, MPO, and liver enzymes were chosen because they represent values greater than the 99th percentile of the values in our population, while the remaining cutoffs were chosen because they represent significant inflammation, uncontrolled diabetes mellitus, and/or hormonal effects that are known to lower HDL-C levels (
      • Thirumalai A.
      • Rubinow K.B.
      • Page S.T.
      An update on testosterone, HDL and cardiovascular risk in men.
      ).
      Under a protocol approved by the Schulman Investigational Review Board (Cincinnati, OH), a total of 201 of 308 (65.3%) eligible subjects were studied, provided that their healthcare providers consented to an anonymized genetic analysis at no cost to either the patient or the healthcare provider. The remaining 107 subjects were not tested because 1) we never received a sample for DNA analysis (the main reason), 2) the healthcare provider did not give consent because he/she was no longer following the patient or did not want to participate in the study, or 3) the healthcare provider did not respond to our letter or phone calls. Data on these 107 subjects are provided in the Results section.
      Clinical information from the study subjects was obtained from the healthcare provider by telephone interview using a questionnaire and was placed in an anonymized database. The questionnaire asked about the subject's history of angina pectoris, myocardial infarction, coronary artery bypass grafting or angioplasty, stroke, transient ischemic attacks, carotid or peripheral artery disease, and/or surgery for these latter conditions. If any of these responses was positive, the subject was deemed to have ASCVD. The questionnaire also asked about the subject's history of any form of kidney disease, decreased kidney function, proteinuria, neuropathy or neurological disease, seizures, hepatosplenomegaly, and/or corneal opacification or visual impairment.
      DNA was isolated from whole blood of the 201 eligible subjects and analyzed by next-generation sequencing at 23 gene loci (APOA1, ABCA1, LCAT, LPL, LIPC, LDLR, APOB, PCSK9, LDLRAP1, STAP1, ABCG5, ABCG8, APOE, LIPA,GPIHBP1, APOA5, APOC2, APOC3, CETP, SCARB1, CYP27A1,DHCR24, and LIPG) on an Illumina MiSeqDx platform using 2 × 150 paired-end reads as previously described (
      • Johansen C.T.
      • Dubé J.B.
      • Loyzer M.N.
      • MacDonald A.
      • Carter D.E.
      • McIntyre A.D.
      • Cao H.
      • Wang J.
      • Robinson J.F.
      • Hegele R.A.
      LipidSeq: a next-generation clinical resequencing panel for monogenic dyslipidemias.
      ). With the exception of CYP27A1, these genes were selected by Johansen et al. (
      • Johansen C.T.
      • Dubé J.B.
      • Loyzer M.N.
      • MacDonald A.
      • Carter D.E.
      • McIntyre A.D.
      • Cao H.
      • Wang J.
      • Robinson J.F.
      • Hegele R.A.
      LipidSeq: a next-generation clinical resequencing panel for monogenic dyslipidemias.
      ) because of their known role in causing monogenic lipid disorders. We added the gene CYP27A1 because of its role in causing cerebrotendinous xanthomatosis. For each gene, sequencing was performed on all known exons plus an additional 25 base pair padding at each intron-exon border. The median read depth was 517×, with 100% sensitivity and specificity for SNPs and 100% sensitivity and 91% specificity for insertions/deletions.
      FASTQ files were processed using a custom workflow in CLC Biomedical Genomics Workbench version 3.2 (Qiagen, Valencia, CA). All mapping, variant calling and primary annotation, and quality control were performed using validated protocols. Variant call format files containing all identified variants in targeted regions were annotated and further analyzed using Ingenuity Variant Analysis (Qiagen) with a customized filtering cascade designed to identify potentially disease-causing variants. The analysis incorporated the classification guidelines described by the American College of Medical Genetics and Genomics (
      • Richards S.
      • Aziz N.
      • Bale S.
      • Bick D.
      • Das S.
      • Gastier-Foster J.
      • Grody W.W.
      • Hegde M.
      • Lyon E.
      • Spector E.
      • et al.
      Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
      ). In addition, copy number variations and large structural variants were assessed using built-in tools from Biomedical Genomics Workbench (Qiagen) and VarSeq copy number variant analysis (Golden Helix, Bozeman, MT). Bioinformatic analysis of the sequencing results was confirmed by Dron et al. (
      • Dron J.S.
      • Wang J.
      • Low-Kam C.
      • Khetarpal S.A.
      • Robinson J.F.
      • McIntyre A.D.
      • Ban M.R.
      • Cao H.
      • Vhainds D.
      • Dubé M-P.
      • et al.
      Polygenic determinants in extremes of high-density lipoprotein cholesterol.
      ) as previously described. Our genetic analysis focused on three gene loci directly involved in HDL particle metabolism (APOA1, ABCA1, and LCAT). We also selected four common SNPs (ABCA1 rs111292742, ABCA1 rs9282541, LCAT rs4986970, and LPL rs268) known to be associated with HDL-C and TG plasma concentrations (discussed below) and analyzed their prevalence and minor allele frequency in our study population of 201 subjects versus the reported global population (
      • Lek M.
      • Karczewski K.J.
      • Minikel E.V.
      • Samocha K.E.
      • Banks E.
      • Fennell T.
      • O'Donnell-Luria A.H.
      • Ware J.S.
      • Hill A.J.
      • Cummings B.B.
      • et al.
      Analysis of protein-coding genetic variation in 60,706 humans.
      ). Results of the analysis were given to the healthcare provider if requested.
      Further analysis was required for variants that were novel or of uncertain significance to ascertain the potential pathogenicity. Prediction scores used for this assessment included 1) Genomic Evolutionary Rate Profiling scores to evaluate conservation; 2) DANN pathogenicity scores, which use computational deep-learning methodologies to classify variants; and 3) dbNSFP, which aggregates several informatics prediction algorithms and uses a proportion of these algorithms to indicate the number of scores predicting the mutation to be deleterious versus the number predicting low functional impact. These scores were assessed using Varsome (Saphetor, Lausanne, Switzerland) and other computational and literature-based tools in Ingenuity Variant Analysis.

      RESULTS

      Prevalence of severe HDL deficiency

      Data on our population are presented in Tables 1 and 2. Of the men in our population, 74.3% had normal HDL-C levels (≥40 mg/dl), 25.4% had low HDL-C levels (20–39 mg/dl), and 0.3% had severe HDL deficiency (<20 mg/dl) (Table 1). Men with severe HDL deficiency, by definition, had significantly (P < 0.001) lower levels of HDL-C (−66.7%), as well as significantly (P < 0.001) lower levels of total apoA-I (−47.0%) and apoA-I in very large α1 HDL (−77.2%), large α2 HDL (−51.3%), medium α3 HDL (−17.9%), small α4 HDL (−28.1%), and very small pre-β1 HDL (−36.9%) compared with men with normal HDL-C levels. They had 37% lower median HDL-C/apoA-I ratios at 0.214 versus 0.341 in controls.
      TABLE 1Men in the Boston Heart Diagnostics population grouped by HDL-C levels (n = 112,776)
      ParameterNormalLowSevere
      P < 0.001 for trend across HDL-C groups for all parameters, as determined by the nonparametric Kruskal-Wallis test.
      % Difference: Severe vs. Normal
      HDL-C ≥40 mg/dl (n = 83,812; 74.32%)HDL-C 20–39 mg/dl (n = 28,594; 25.35%)HDL-C <20 mg/dl (n = 370; 0.33%)
      Age (years)56 (21)54 (20)50 (21)−10.7
      BMI (kg/m2)28 (7)31 (7)32 (9)+14.3
      Weight (lbs.)195 (48)218 (56)215 (60)+10.3
      Lipids and apolipoproteins (mg/dl)
      HDL-C
      HDL-C/apoA-I ratio medians in men for the normal HDL-C, low HDL-C, and severely deficient HDL-C groups are 0.341, 0.288, and 0.214, respectively.
      51 (16)34 (5)17 (4)−66.7
      apoA-I
      HDL-C/apoA-I ratio medians in men for the normal HDL-C, low HDL-C, and severely deficient HDL-C groups are 0.341, 0.288, and 0.214, respectively.
      149.5 (29.8)118.1 (17.7)79.3 (31.0)−47.0
      TGs101 (67)167 (124)245 (348)+242.5
      LDL-C116 (54)113 (54)86 (60)−25.9
      sdLDL-C26 (19)34 (27)38 (29)+46.2
      apoB95 (38)100 (40)97 (45)+2.1
      HDL subpopulations (mg/dl)
      α1 apoA-I26.3 (13.6)14.1 (5.9)6.0 (4.9)−77.2
      α2 apoA-I59.8 (13.7)45.5 (8.6)29.1 (10.0)−51.3
      α3 apoA-I22.9 (6.0)23.2 (6.1)18.8 (7.1)−17.9
      α4 apoA-I19.2 (5.7)18.2 (5.2)13.8 (10.0)−28.1
      Pre-β1 apoA-I8.4 (5.4)6.9 (4.6)5.3 (1.7)−36.9
      Glucose metabolism
      Glucose (mg/dl)98 (16)102 (27)105 (43)+7.1
      HbA1c (%)5.6 (0.6)5.8 (1.0)5.8 (1.7)+9.4
      Insulin (µU/ml)10.0 (10)16.0 (16.0)18.0 (18.0)+80.0
      Adiponectin (µg/dl)9.9 (6.6)6.8 (4.1)6.0 (4.2)−39.4
      HOMA-IR2.4 (2.6)4.4 (4.9)5.0 (4.9)+108.0
      HOMA-β98.2 (90.8)140.5 (139.3)140.0 (167.2)+42.6
      Inflammation
      hsCRP (mg/l)1.0 (1.9)1.9 (3.2)2.6 (6.2)+160.0
      Fibrinogen (mg/dl)355 (105)422 (143)483 (341)+36.1
      MPO (pmol/l)259 (139)287 (160)324 (222)+25.1
      Data are expressed as median (interquartile range).
      a P < 0.001 for trend across HDL-C groups for all parameters, as determined by the nonparametric Kruskal-Wallis test.
      b HDL-C/apoA-I ratio medians in men for the normal HDL-C, low HDL-C, and severely deficient HDL-C groups are 0.341, 0.288, and 0.214, respectively.
      TABLE 2Women in the Boston Heart Diagnostics population grouped by HDL-C levels (n = 145,476)
      ParameterNormalLowSevere
      P < 0.001 for trend across HDL cholesterol groups for all parameters, as determined by the nonparametric Kruskal-Wallis test.
      % Difference: Severe vs. Normal
      HDL-C ≥50 mg/dl (n = 110,774; 76.15%)HDL-C 20–49 mg/dl (n = 34,558; 23.76%)HDL-C <20 mg/dl (n = 144; 0.099%)
      Age (years)55 (20)53 (23)56 (21)+1.8
      BMI (kg/m2)26 (8)32 (10)27 (6)+3.8
      Weight (lbs.)154 (47)185 (62)173 (46)+12.3
      Lipids and apolipoproteins (mg/dl)
      HDL-C
      HDL-C/apoA-I ratio medians in women for the normal HDL-C, low HDL-C, and severely deficient HDL-C groups are 0.374, 0.310, and 0.197, respectively.
      66.0 (21)42.0 (8)17.0 (5)−74.2
      apoA-I
      HDL-C/apoA-I ratio medians in women for the normal HDL-C, low HDL-C, and severely deficient HDL-C groups are 0.374, 0.310, and 0.197, respectively.
      176.3 (37.2)135.3 (21.5)86.3 (32.3)−51.0
      TGs88 (57)148 (99)289 (338)+228.4
      LDL-C118 (50)121 (53)73 (50)−25.9
      sdLDL-C24 (14)30 (24)31 (30)+29.2
      apoB94 (34)103 (40)90 (42)+2.1
      HDL subpopulations (mg/dl)
      α1 apoA-I39.1 (18.0)20.7 (8.1)7.3 (5.0)−81.3
      α2 apoA-I71.5 (16.4)54.7 (11.0)31.8 (13.9)−55.5
      α3 apoA-I22.8 (5.9)23.0 (6.2)19.2 (9.3)−15.8
      α4 apoA-I19.0 (6.7)18.4 (6.1)14.4 (8.7)−24.2
      Pre-β1 apoA-I8.7 (6.0)7.1 (5.2)6.4 (6.2)−26.4
      Glucose metabolism
      Glucose (mg/dl)92 (14)98 (21)112 (62)+21.7
      HbA1c (%)5.5 (0.5)5.7 (0.7)5.8 (1.6)+5.5
      Insulin (µU/ml)8.0 (8.0)15.0 (14.0)20.0 (22.2)+150.0
      Adiponectin (µg/dl)15.3 (9.7)9.2 (5.7)6.8 (8.5)−55.6
      HOMA-IR1.9 (1.9)3.9 (4.2)7.2 (10.0)+278.9
      HOMA-β98.2 (83.7)152.1 (137.3)138.8 (142.0)+41.3
      Inflammation
      hsCRP (mg/l)1.3 (2.8)3.1 (5.1)3.3 (7.8)+153.8
      Fibrinogen (mg/dl)357 (105)415 (125)471 (230)+31.9
      MPO (pmol/l)273 (151)315 (175)333 (313)+22.0
      Data are expressed as median (interquartile range).
      a P < 0.001 for trend across HDL cholesterol groups for all parameters, as determined by the nonparametric Kruskal-Wallis test.
      b HDL-C/apoA-I ratio medians in women for the normal HDL-C, low HDL-C, and severely deficient HDL-C groups are 0.374, 0.310, and 0.197, respectively.
      Men with severe HDL deficiency were also significantly (P < 0.001) younger (−10.7%) and had significantly (P < 0.001) higher BMI (+14.3%), body weight (+10.3%), sdLDL-C (+46.2%), TGs (+242.5%), HbA1c (+9.4%), glucose (+7.1%), insulin (+80.0%), calculated homeostasis model assessment of insulin resistance (HOMA-IR) (+108%), calculated homeostasis model of insulin production (HOMA-β) (+42.6%), hsCRP (+160.0%), fibrinogen (+36.1%), and MPO (+25.1%) than controls. They also had significantly (P < 0.001) lower levels of direct LDL-C (−25.9%). Men with low HDL-C (20–39 mg/dl) generally had values for all of these parameters that were intermediate between the men with normal HDL-C and those with severe HDL deficiency (Table 1). The largest percentage difference between men with severe HDL deficiency and normal men was for elevated TG and hsCRP levels.
      Of the women in our population (Table 2), 76.1% had normal HDL-C concentrations (≥50 mg/dl), 23.8% had low HDL-C concentrations (20–49 mg/dl), and 0.1% had severe HDL deficiency (<20 mg/dl). Women with severe HDL deficiency, by definition, had significantly (P < 0.001) lower levels of HDL-C (−74.2%), as well as significantly (P < 0.001) lower levels of total apoA-I (−51.0%) and apoA-I in very large α1 HDL (−81.3%), large α2 HDL (−55.5%), medium α3 HDL (−15.8%), small α4 HDL (−24.2%), and very small pre-β1 HDL (−26.4%) compared with women with normal HDL-C levels. They also had 47% lower median HDL-C/apoA-I ratios at 0.197 versus 0.374 in controls.
      In addition, women with severe HDL deficiency had significantly (P < 0.001) higher sdLDL-C (+29.2%), TGs (+228.4%), HbA1c (+5.5%), glucose (+21.7%), insulin (+150.0%), HOMA-IR (+278.9%) and HOMA-β (+41.3%) scores, hsCRP (+153.8%), fibrinogen (+31.9%), and MPO (+22.0%). They also had significantly (P < 0.001) lower levels of LDL-C (−25.9%) and adiponectin (−55.6%). Women with low HDL-C (20–49 mg/dl) in general had values for all of these parameters that were intermediate between the women with normal HDL-C and those with severe HDL deficiency (Table 2). As with men, there were large percentage differences between women with severe HDL deficiency and normal women for elevated TG and hsCRP levels.

      Prevalence of secondary causes of severe HDL deficiency

      As shown in Table 3, among men with severe HDL deficiency, 19.1% had fasting TG values >600 mg/dl versus 0.1% of normal controls; 18.9% had hsCRP values >10.0 mg/l versus 3.7%; 14.2% had HbA1c values >8.0% versus 3.1%; 4.1% had MPO values >1,000 pmol/l versus 0.6%; and 3.3% had elevated liver transaminase levels >120 U/l versus 0.5% (all P < 0.001). These cutoffs for TGs, MPO, and liver enzymes were chosen because they represent values greater than the 99th percentile of the values in our population, while the remaining cutoffs were chosen because they represent significant inflammation and/or uncontrolled diabetes mellitus. Each of these parameters is known to be associated with HDL deficiency (
      • Schaefer E.J.
      • Anthanont P.
      • Diffenderfer M.R.
      • Polisecki E.
      • Asztalos B.F.
      Diagnosis and treatment of high density lipoprotein deficiency.
      ). Men with low HDL-C (20–39 mg/dl) generally had prevalence rates for these abnormalities that were intermediate between men with severe HDL deficiency and normal controls. All men with these abnormalities were excluded from the genetic studies, as were 38 men (10.2%) with an HDL-C value <20 mg/dl who were receiving testosterone replacement, which is known to lower HDL-C levels significantly (
      • Thirumalai A.
      • Rubinow K.B.
      • Page S.T.
      An update on testosterone, HDL and cardiovascular risk in men.
      ).
      TABLE 3Prevalence of abnormal parameters used for excluding subjects from genetic analysis
      ParameterNormalLowSevere
      P < 0.001 for trend across HDL-C groups for all parameters except AST in women (P = 0.002), as determined by a chi-squared test.
      % Difference: Severe vs. Normal
      HDL-C ≥40 mg/dl (n = 83,812; 74.32%)HDL-C 20–39 mg/dl (n = 28,594; 25.35%)HDL-C <20 mg/dl (n = 370; 0.33%)
      Men (n = 112,776)
      TG >600 mg/dl84 (0.1)772 (2.7)71 (19.1)+19,000.0
      HbA1c >8%2,598 (3.1)2,430 (8.5)53 (14.2)+358.0
      hsCRP >10 mg/dl3,101 (3.7)1,973 (6.9)70 (18.9)+410.8
      MPO >1,000 pmol/l503 (0.6)286 (1.0)15 (4.1)+583.3
      ALT >120 U/l19 (0.5)229 (0.8)12 (3.3)+560.0
      AST >120 U/l251 (0.3)114 (0.4)12 (3.3)+1,000.0
      ParameterNormalLowSevere
      P < 0.001 for trend across HDL-C groups for all parameters except AST in women (P = 0.002), as determined by a chi-squared test.
      % Difference: Severe vs. Normal
      HDL-C ≥50 mg/dl (n = 110,774; 76.15%)HDL-C 20–49 mg/dl (n = 34,558; 23.76%)HDL-C <20 mg/dl (n = 144; 0.099%)
      Women (n = 145,476)
      TG >600 mg/dl0 (0.0)276 (0.8)32 (21.9)NC
      HbA1c >8%1,772 (1.6)2,143 (6.2)16 (11.2)+600.0
      hsCRP >10 mg/dl5,982 (5.4)4,527 (13.1)35 (24.5)+353.7
      MPO >1,000 pmol/l775 (0.7)415 (1.2)7 (5.1)+628.6
      ALT >120 U/l332 (0.3)207 (0.6)2 (1.6)+433.3
      AST >120 U/l222 (0.2)104 (0.3)0 (0.0)−100.0
      Data are expressed as the number (%) of subjects in the HDL-C group. ALT, alanine aminotransferase; AST, aspartate aminotransferase; NC, not calculated.
      a P < 0.001 for trend across HDL-C groups for all parameters except AST in women (P = 0.002), as determined by a chi-squared test.
      Table 3 also shows that among women with severe HDL deficiency, 24.5% had hsCRP values >10.0 mg/l versus 5.4% of controls, 21.9% had fasting TG values >600 mg/dl versus 0.0%, 11.2% had HbA1c values >8.0% versus 1.6%, 5.1% had MPO values >1,000 pmol/l versus 0.7%, and 1.6% had elevated liver transaminase levels >120 U/l versus 0.3% (all P < 0.001). Women with low HDL-C (20–49 mg/dl) generally had prevalence rates for these abnormalities that were intermediate between women with severe HDL deficiency and those with normal HDL-C. All women with these abnormalities were excluded from the genetic studies. In both men and women the major reasons for exclusion were because of severe hypertriglyceridemia, markedly increased inflammation (markedly elevated hsCRP and/or MPO levels), and/or uncontrolled diabetes mellitus.

      Genetic causes of severe HDL deficiency

      Of a total of 308 eligible subjects, there were 107 (72% men) for whom no DNA was available. Their mean age was 55 years, and their mean lipid and lipoprotein concentrations were as follows: non-HDL-C, 154 mg/dl; direct LDL-C, 107 mg/dl; TGs, 198 mg/dl; HDL-C, 18 mg/dl; and apoA-I, 87 mg/dl, with an HDL-C/apoA-I ratio of 0.207. Of these subjects, 5 (4.7%; all men) were reported by their healthcare provider to have established ASCVD, with a mean age of onset of 61 years. There were 201 subjects (65.3%) with HDL-C levels <20 mg/dl and without known secondary causes of low HDL-C who underwent DNA analysis. Of these subjects, 172 (85.6%) were Caucasian, 16 (8.0%) were Hispanic, 10 (5.0%) were African American, and 3 (1.5%) were Asian. Of 94 of the 201 subjects sequenced (46.8%; 72 men and 22 women), no ABCA1, LCAT, or APOA1 mutations, selected variants (ABCA1 rs111292742, ABCA1 rs9282541, LCAT rs4986970, LPL rs268), or other mutations were found (mutation-negative group). The data on these subjects were similar to the data on the subjects for whom no DNA was available for sequencing. The mean age of the mutation-negative group was 53 years; the mean lipid and lipoprotein concentrations were as follows: non-HDL-C, 142 mg/dl; direct LDL-C, 99 mg/dl; TGs, 237 mg/dl; HDL-C, 16 mg/dl; and apoA-I, 81 mg/dl, with an HDL-C/apoA-I ratio of 0.198. Of these 94 subjects, 6 (6.4%; 4 men and 2 women) were reported to have established ASCVD by their healthcare provider, with a mean age of disease onset of 64 years.

      ABCA1 mutations and variants

      Of the 201 subjects sequenced, 54 (26.9%; 38 men and 16 women) were noted to have ABCA1 mutations (Tables 4 and 5) or SNPs (Table 6); 18 of the mutations were determined to be novel. Among the 39 subjects (19.4%) having ABCA1 mutations, 2 men, subjects ABCA1-01 and ABCA1-02, were homozygous for the novel mutations ABCA1 p.G851R and p.P1412R, respectively. Their plasma concentrations of HDL-C, apoA-I, nonHDL-C, LDL-C, and TGs were 5 and 6 mg/dl, 10 and 24 mg/dl, 35 and 91 mg/dl, 5 and 77 mg/dl, and 312 and 67 mg/dl, respectively. ABCA1-01 was also heterozygous for LCAT rs4986970 (p.S232T) (see below) and at the age of 56 years had evidence of splenomegaly, anemia, and peripheral neuropathy (numbness and tingling of his right arm) but no evidence of ASCVD. At the age of 47 years, he had undergone coronary angiography at the National Institutes of Health that showed no evidence of coronary atherosclerosis. It should be noted that his direct LDL-C value was very low (5 mg/dl). HDL particle analysis indicated that almost all of his apoA-I was in pre-β1 HDL. ABCA1-02 was recently reported, after we supplied the healthcare provider with the genetic information; unfortunately, in the published report the mutation was mislabeled as ABCA1 p.P1312R (
      • Carcora Y.
      • Brook R.D.
      • Farhat L.
      • Willer C.J.
      • Rubenfire M.
      • Kim D.S.
      A novel homozygous ABCA1 variant in an asymptomatic man with profound hypoalphalipoproteinemia.
      ). At the age of 38 years, this patient was asymptomatic with no evidence of ASCVD and had a marked decrease in apoA-I in all HDL particles, especially in very large α1 HDL, which was approximately 2% of normal.
      TABLE 4Subjects with homozygous, compound heterozygous, and double heterozygous mutations in ABCA1 (n = 9)
      CaseGenderAge (years)HDL-C (mg/dl)apoA-I (mg/dl)HDL-C/apoA-I RatioNon-HDL-C (mg/dl)LDL-C (mg/dl)TGs (mg/dl)CVDABCA1 Mutation
      GeneProtein
      Homozygous
      ABCA1-01
      Subject was also heterozygous for the LCAT rs4986970 (p.S232T) SNP.
      Male565100.50355312c.2551G>Ap.G851R
      ABCA1-02Male386240.25917767c.4235C>Gp.P1412R
      Compound heterozygous
      ABCA1-03Male4919181.069857375+c.1770G>Cp.W590C
      c.1769G>Tp.W590L
      ABCA1-04Female425300.16192146291c.720+6T>C
      c.4465-1G>C
      ABCA1-05Female495300.17989173c.4430G>Tp.C1477F
      c.1447A>C
      Novel mutation (n = 5).
      p.T483P
      ABCA1-06Female6010530.19967692+c.6204G>Cp.L2068L
      c.5604_5605insG
      Novel mutation (n = 5).
      p.L1869fs*22
      ABCA1-07Male305170.2910610267c.4175+1G>T
      c.4037G>Ap.G1346E
      Mean2 males/ 3 females469300.37118941802/5
      Double heterozygous
      ABCA1-08Male3516790.20164136111ABCA1 c.2311G>CABCA1 p.V771L
      LCAT c.475C>TLCAT p.R159W
      ABCA1-09Male3817950.1811675260ABCA1 c.2328G>CABCA1 p.K776N
      APOA1 c.327G>AAPOA1 p.E109E
      a Subject was also heterozygous for the LCAT rs4986970 (p.S232T) SNP.
      b Novel mutation (n = 5).
      TABLE 5Subjects with heterozygous mutations in ABCA1 (n = 30)
      CaseGenderAge (years)HDL-C (mg/dl)apoA-I (mg/dl)HDL-C/apoA-I RatioNon-HDL-C (mg/dl)LDL-C (mg/dl)TGs (mg/dl)CVDABCA1 Mutation
      GeneProtein
      ABCA1-10Female5617690.25165126259+c.1510-1G>T
      ABCA1-11Female3911510.22836570c.811G>Tp.E271*
      ABCA1-12
      Subject was also heterozygous for the LPL rs268 (p.N318S) SNP.
      Female6618620.2915210469+c.4492C>Tp.Q1498*
      ABCA1-13Female5916750.21137111113+c.5577delAp.V1861fs*9
      ABCA1-14Female3413560.23138119125c.2338-3_2338-2delCA
      ABCA1-15Male3413610.21156118255+c.2568delGp.W856fs*40
      ABCA1-16Female615210.2411270225+c.4465-1G>C
      ABCA1-17Male7529960.30116102102+c.4175+1G>T
      ABCA1-18Male3917760.22129110189c.1529T>Gp.L510R
      ABCA1-19
      Subject was also heterozygous for a previously reported ABCG8 c.550C>T (p.R184C) mutation.
      Male3616750.217557176c.1699A>Tp.N567Y
      ABCA1-20
      Subject was also heterozygous for the LCAT rs4986970 (p.S232T) SNP.
      Male6412670.1810670272+c.916C>Tp.R306C
      ABCA1-21Female58171000.1710158263c.5477A>Tp.Q1826L
      ABCA1-22Male5214640.22167131209c.1862A>Gp.Q621R
      ABCA1-23Male7618820.226749159+c.3098T>Cp.L1033P
      ABCA1-24Female4419970.2010783202c.1759C>Tp.R587W
      ABCA1-25
      Subject was also heterozygous for the LCAT rs4986970 (p.S232T) SNP.
      Male555360.14161103320c.1774G>Tp.G592C
      ABCA1-26Female3616730.2212289282c.6218C>Tp.T2073I
      ABCA1-27Female265160.31151133143c.1769G>Tp.W590L
      ABCA1-28Male446430.1413389297c.5376C>Tp.T1792T
      ABCA1-29
      Subject was also heterozygous for the LPL rs268 (p.N318S) SNP.
      Male4718940.19177110587+c.254C>Tp.P85L
      ABCA1-30Male49191140.17184140288+c.5398A>Cp.N1800H
      ABCA1-31Male4316710.238563115+c.360C>Tp.T120T
      ABCA1-32Male456190.32400317388c.474G>Cp.L158L
      ABCA1-33Male4019890.21241188424c.720+6T>C
      ABCA1-34Male78141040.1314860500+c.4037G>Ap.G1346E
      ABCA1-35Male4310340.29193166130c.254C>Tp.P85L
      ABCA1-36Male7718750.24132105102+c.4518G>Ap.S1506S
      ABCA1-37Male7319840.2313390303+c.720+6T>C
      ABCA1-38Male6318780.239068101c.5376C>Tp.T1792T
      ABCA1-39Male6515620.24125103143+c.32T>Cp.L11P
      Mean20 males/ 10 females5315680.2214310722715/30
      a Novel mutation (n = 13).
      b Subject was also heterozygous for the LPL rs268 (p.N318S) SNP.
      c Subject was also heterozygous for a previously reported ABCG8 c.550C>T (p.R184C) mutation.
      d Subject was also heterozygous for the LCAT rs4986970 (p.S232T) SNP.
      TABLE 6Subjects with selected SNPs at the ABCA1, LCAT, and/or LPL gene loci (n = 31)
      CaseGenderAge (years)HDL-C (mg/dl)apoA-I (mg/dl)HDL-C/apoA-I RatioNon-HDL-C (mg/dl)LDL-C (mg/dl)TGs (mg/dl)CVDSNP
      a Global minor allele frequencies for the identified SNPs are as follows: ABCA1 rs9282541 (p.R230C), 1.10%; ABCA1 rs111292742 (c.-279C>G), 2.46%; LCAT rs4986970 (p.S232T), 1.76%; and LPL rs268 (p.N318S), 1.29%. In our study population, including subjects with ABCA1, LCAT, and APOA1 mutations, the corresponding minor allele frequencies were 1.74%, 3.48%, 2.74%, and 2.99%, respectively.
      ABCA1
      SNP-01Male807440.1610858155+rs9282541/p.R230C
      SNP-02
      c Subject was also heterozygous for a novel APOA5 c.1028G>C (p.R343P) mutation.
      Female3419750.2512091204rs9282541/p.R230C
      SNP-03Female5319950.2012686287rs9282541/p.R230C
      SNP-04Female4618660.27151120144rs9282541/p.R230C
      SNP-05Male197380.18170108302rs9282541/p.R230C
      SNP-06Male6817970.189932394+rs111292742/c.-279C>G
      SNP-07Male36191150.17176113341rs111292742/c.-279C>G
      SNP-08Male4113600.22179144161rs111292742/c.-279C>G
      SNP-09Male54181160.16161111342rs111292742/c.-279C>G
      SNP-10Male64191100.1715091451+rs111292742/c.-279C>G
      SNP-11Male6119860.226648125rs111292742/c.-279C>G
      SNP-12Male3118920.2014071462rs111292742/c.-279C>G
      SNP-13Male43191030.1810666372rs111292742/c.-279C>G
      SNP-14Male509450.20153125127rs111292742/c.-279C>G
      Mean11 males/3 females4916820.20136902763/14
      LCAT
      SNP-15Male5314960.1514586361rs4986970/p.S232T
      SNP-16Female6420950.2111274220rs4986970/p.S232T
      SNP-17Male2917770.2212390174rs4986970/p.S232T
      SNP-18Male2810420.24262215184rs4986970/p.S232T
      SNP-19Female666370.16164100318rs4986970/p.S232T
      SNP-20Male2410400.25232210169rs4986970/p.S232T
      SNP-21Female4413730.186940162rs4986970/p.S232T
      SNP-22Female765240.21190102338+rs4986970/p.S232T
      Mean4 males/4 females4812610.201621152411/8
      LPL
      SNP-23Male53171010.17190135440rs268/p.N318S
      SNP-24Male4916670.24311269327rs268/p.N318S
      SNP-25Female7810680.158129169+rs268/p.N318S
      SNP-26Male7116940.176939218rs268/p.N318S
      SNP-27Male3317670.25176130239rs268/p.N318S
      SNP-28Male3319770.25201152296rs268/p.N318S
      SNP-29Male43181050.1713051504rs268/p.N318S
      SNP-30Male5517970.187951198rs268/p.N318S
      Mean7 males/1 females5216850.201551072991/8
      Double
      SNP-31Male636350.16199116407ABCA1 rs111292742
      LPL rs268
      a a Global minor allele frequencies for the identified SNPs are as follows: ABCA1 rs9282541 (p.R230C), 1.10%; ABCA1 rs111292742 (c.-279C>G), 2.46%; LCAT rs4986970 (p.S232T), 1.76%; and LPL rs268 (p.N318S), 1.29%. In our study population, including subjects with ABCA1, LCAT, and APOA1 mutations, the corresponding minor allele frequencies were 1.74%, 3.48%, 2.74%, and 2.99%, respectively.
      b b Homozygous SNP.
      c c Subject was also heterozygous for a novel APOA5 c.1028G>C (p.R343P) mutation.
      As shown in Table 4, there were 5 ABCA1 compound heterozygotes (2 men, 3 women) with a mean age of 46 years and mean lipid and lipoprotein concentrations as follows: HDL-C, 9 mg/dl; apoA-I, 30 mg/dl; non-HDL-C, 118 mg/dl; LDL-C, 94 mg/dl; and TG, 180 mg/dl. Three of the mutations found in these subjects were novel. Subjects ABCA1-03 and ABCA1-06 had established ASCVD (with the age of disease onset of 46 and 58 years, respectively), with non-HDL-C concentrations >80 mg/dl. When analyzed together, the 7 subjects (4 men, 3 women; mean age: 46 years) who were either homozygous or compound heterozygous for mutations in ABCA1 (Table 4) had mean plasma lipid values as follows: HDL-C, 8 mg/dl; apoA-I, 26 mg/dl; nonHDL-C, 102 mg/dl; direct LDL-C, 79 mg/dl; and TG, 182 mg/dl, with an HDL-C/apoA-I ratio of 0.37. Of these subjects, 29% had evidence of ASCVD.
      Subjects ABCA1-08 and ABCA1-09 were double heterozygous, having one mutation in ABCA1 and the other in LCAT and APOA1, respectively (Table 4). They were men, aged 35 and 38 years, with no evidence of ASCVD and with the following respective values: HDL-C, 16 and 17 mg/dl; apoA-I, 79 and 95 mg/dl; non-HDL-C, 164 and 116 mg/dl; direct LDL-C, 136 and 75 mg/dl; and TG, 111 and 260 mg/dl. Their HDL-C/apoA-I ratios were 0.20 and 0.18, respectively. All 9 ABCA1 homozygotes, compound heterozygotes, and double heterozygotes had a marked deficiency of apoA-I in very large α1 and large α2 HDL (<10% of normal; data not shown).
      Table 5 shows that there were 20 men and 10 women heterozygous for ABCA1 (includes 13 novel mutations) with a mean age of 53 years and mean values as follows: HDL-C, 15 mg/dl; apoA-I, 68 mg/dl; non-HDL-C, 143 mg/dl; direct LDL-C, 107 mg/dl; and TG, 227 mg/dl, with an HDL-C/apoA-I ratio of 0.22. Among these 30 subjects, 15 (50.0%) had evidence of ASCVD (11 men, 4 women; mean age of disease onset: 58 years); none had evidence of neuropathy, splenomegaly, or anemia. ABCA1-20 and ABCA1-25 were also found to be heterozygous for LCAT rs4986970 (see below), while ABCA1-12 and ABCA1-29 were also heterozygous for LPL rs268 (see below).
      Data regarding the study subjects having only ABCA1 vari­ant rs9282541 (p.R230C) and/or rs111292742 (c.-279C>G) and no ABCA1, APOA1, or LCAT mutations are provided in Table 6. An 80-year-old Hispanic man (SNP-01) and a 34-year-old unrelated Hispanic woman (SNP-02) were found to be homozygous for the ABCA1 rs9282541 variant, which has been reported in the Mexican population to be associated with low HDL-C levels (
      • Aguilar-Salinas C.A.
      • Canizales-Quinteros S.
      • Rojas-Martínez R.
      • Mehta R.
      • Rodriguez-Guillén R.
      • Ordoñez-Sanchez M.L.
      • Riba L.
      • Tusié-Luna M.T.
      The non-synonymous Arg230Cys variant (R230C) of the ATP-binding cassette transporter A1 is associated with low HDL cholesterol concentrations in Mexican adults: a population based nationwide study.
      ). Their respective values were as follows: HDL-C, 7 and 19 mg/dl; apoA-I, 44 and 75 mg/dl; non-HDL-C, 108 and 120 mg/dl; direct LDL-C, 58 and 91 mg/dl; and TG, 155 and 204 mg/dl. SNP-01 also had evidence of ASCVD (postmyocardial infarction at the age of 71 years). SNP-03, SNP-04, and SNP-05 were heterozygous for the ABCA1 rs9282541 variant. None of these subjects had ASCVD. Their mean values were as follows: HDL-C, 15 mg/dl; apoA-I, 66 mg/dl; non-HDL-C, 149 mg/dl; direct LDL-C, 105 mg/dl; and TG, 244 mg/dl. This allele was found in 1.74% of our subjects versus 1.10% in the global population (not significant).
      The ABCA1 rs111292742 (ABCA1 c.-279C>G) variant was found in 10 subjects, one of whom (SNP-31) was also heterozygous for LPL rs268. ABCA1 rs111292742 has previously been associated with low HDL-C and hypertension in a Japanese population, while LPL rs268 has previously been reported to be associated with hypertriglyceridemia and low HDL-C levels (
      • Yamada Y.
      • Kato K.
      • Yoshida T.
      • Yokoi K.
      • Matsuo H.
      • Watanabe S.
      • Ichihara S.
      • Metoki N.
      • Yoshida H.
      • Satoh K.
      • et al.
      Association of polymorphisms of ABCA1 and ROS1 with hypertension in Japanese individuals.
      ,
      • Wright W.T.
      • Young I.S.
      • Nicholls D.P.
      • Graham C.A.
      Genetic screening of the LPL gene in hypertriglyceridaemic patients.
      ). These 10 subjects (all men; mean age: 51 years) had the following mean values: HDL-C, 16 mg/dl; apoA-I, 86 mg/dl; non-HDL-C, 143 mg/dl; direct LDL-C, 92 mg/dl; and TG, 318 mg/dl, with an HDL-C/apoA-I ratio of 0.19. The ABCA1 rs111292742 allele was found in 3.48% of our subjects versus 2.46% in the global population (not significant). Evidence of ASCVD was noted in 3 of the 15 subjects with ABCA1 variants (SNP-01, SNP-06, and SNP-10). Overall, 20 of the 54 subjects (37.0%) with ABCA1 mutations or variants had evidence of ASCVD (mean age of disease onset: 59 years), significantly higher than the prevalence (6.4%) observed in our mutation-negative group (P < 0.05). Three of these subjects had evidence of neuropathy: ABCA1-01 with right-arm neuropathy, and ABCA1 SNP-01 and SNP-10 both with numbness and tingling in their lower extremities. SNP-01 and SNP-10 also had diabetes mellitus, which is well-known to cause neuropathy.

      LCAT mutations and variants

      LCAT mutations or selected variants were found in 25 of the subjects (12.4%); 4 of the mutations were novel (Table 7). Subject LCAT-01, a 52-year-old man, was found to have a homozygous LCAT c.950T>G (p.M317R) mutation with the following values: HDL-C, 7 mg/dl; apoA-I, 44 mg/dl; non-HDL-C 80 mg/dl; direct LDL-C, <5 mg/dl; and TGs, 145 mg/dl, with an HDL-C/apoA-I ratio of 0.16. The direct LDL-C value of <5 mg/dl was below the detectable limits of the direct LDL-C assay, presumably due to the lack of a cholesteryl ester core in the LDL particles. As reported previously, this subject had marked corneal opacification and renal insufficiency, subsequently requiring hemodialysis (
      • Roshan B.
      • Ganda O.P.
      • Desilva R.
      • Ganim R.B.
      • Ward E.
      • Haessler S.D.
      • Polisecki E.Y.
      • Asztalos B.F.
      • Schaefer E.J.
      Homozygous lecithin:cholesterol acyltransferase (LCAT) deficiency due to a new loss of function mutation and review of the literature.
      ). He was the first lecithin-cholesterol acyltransferase (LCAT)-deficient patient to receive LCAT enzyme replacement therapy, which transiently stabilized his renal disease over approximately a 4-month period, until such therapy was no longer available (
      • Shamburek R.D.
      • Bakker-Arkema R.
      • Auerbach B.J.
      • Krause B.R.
      • Homan R.
      • Amar M.J.
      • Freeman L.A.
      • Remaley A.T.
      Familial lecithin:cholesterol acyltransferase deficiency: first-in-human treatment with enzyme replacement.
      ). HDL particle analysis indicated that his apoA-I was mainly in very small pre-β1 and small α4 HDL, with irregularly sized and poorly lipidated large HDL particles (
      • Roshan B.
      • Ganda O.P.
      • Desilva R.
      • Ganim R.B.
      • Ward E.
      • Haessler S.D.
      • Polisecki E.Y.
      • Asztalos B.F.
      • Schaefer E.J.
      Homozygous lecithin:cholesterol acyltransferase (LCAT) deficiency due to a new loss of function mutation and review of the literature.
      ). After enzyme replacement, his apoA-I-containing particle distribution was transiently normalized (
      • Shamburek R.D.
      • Bakker-Arkema R.
      • Auerbach B.J.
      • Krause B.R.
      • Homan R.
      • Amar M.J.
      • Freeman L.A.
      • Remaley A.T.
      Familial lecithin:cholesterol acyltransferase deficiency: first-in-human treatment with enzyme replacement.
      ). He had no evidence of ASCVD.
      TABLE 7Subjects with mutations in LCAT (n = 17)
      CaseGenderAge (years)HDL-C (mg/dl)apoA-I (mg/dl)HDL-C/apoA-I RatioNon-HDL-C (mg/dl)LDL-C (mg/dl)TGs (mg/dl)CVDLCAT Mutation
      GeneProtein
      Homozygous
      LCAT-01Male527440.1680<5145c.950T>Gp.M317R
      Compound heterozygous
      LCAT-02Female236360.17190157158c.110C>Tp.T37M
      c.101C>Tp.P34L
      LCAT-03Male263140.21214156290c.110C>Tp.T37M
      c.101C>Tp.P34L
      LCAT-04Male6913790.169654216c.491G>Ap.R164H
      c.382G>Ap.G128S
      Mean2 males/1 female397430.181671222210/3
      Heterozygous
      LCAT-05Female40191040.1810879176c.491G>Ap.R164H
      LCAT-06Male5315800.19162114270c.397delGp.V133fs*131
      LCAT-07Male7515770.199957236c.911G>Ap.R304H
      LCAT-08
      Subject was also homozygous for the ABCA1 rs111293742 (c.-279C>G) SNP.
      Male7218990.18191137316c.154+5G>A
      LCAT-09Male3416720.22267249103c.110C>Tp.T37M
      LCAT-10Female1719900.21133100244c.301G>Ap.D101N
      LCAT-11Female47191130.17167117304c.1112C>Tp.T371M
      LCAT-12Male4017780.22153115168c.1123C>Tp.R375C
      LCAT-13Male4417850.20143107226c.101dupCp.H35fs*7
      LCAT-14Female7518660.2711492112c.1139G>Ap.C380Y
      LCAT-15
      Subject was also heterozygous for a previously reported LPL c.-241G>C mutation.
      Male3213780.17151108292c.512G>Ap.R171Q
      LCAT-16Male6010690.14987790c.101C>Tp.P34L
      LCAT-17Male4919970.20159116340c.169G>Cp.G57R
      Mean9 males/4 females4917850.201501132210/13
      a Novel mutation (n = 4).
      b Subject was also homozygous for the ABCA1 rs111293742 (c.-279C>G) SNP.
      c Subject was also heterozygous for a previously reported LPL c.-241G>C mutation.
      Three subjects were found to be LCAT compound heterozygotes. LCAT-02 and LCAT-03, with heterozygous LCAT c.110C>T, p.T37M and LCAT c.101C>T, p.P34L mutations, respectively, were siblings and have been previously reported (
      • Dimick S.M.
      • Sallee B.
      • Asztalos B.F.
      • Pritchard P.H.
      • Frohlich J.
      • Schaefer E.J.
      A kindred with fish eye disease, corneal opacities, marked high-density lipoprotein deficiency, and statin therapy.
      ). They were aged 23 and 26 years with the following respective values: HDL-C, 6 and 3 mg/dl; apoA-I, 36 and 14 mg/dl; non-HDL-C, 190 and 214 mg/dl; direct LDL-C, 157 and 156 mg/dl; and TGs, 158 and 290 mg/dl, with HDL-C/apoA-I ratios of 0.17 and 0.21. They presented with peripheral corneal opacification, normal kidney function, and no evidence of ASCVD (
      • Dimick S.M.
      • Sallee B.
      • Asztalos B.F.
      • Pritchard P.H.
      • Frohlich J.
      • Schaefer E.J.
      A kindred with fish eye disease, corneal opacities, marked high-density lipoprotein deficiency, and statin therapy.
      ). However, because of their elevated LDL-C and a strong family history of ASCVD, they were placed on statin therapy. LCAT-02 had an excellent response to the therapy; her LDL-C concentration decreased from 157 to 62 mg/dl (
      • Dimick S.M.
      • Sallee B.
      • Asztalos B.F.
      • Pritchard P.H.
      • Frohlich J.
      • Schaefer E.J.
      A kindred with fish eye disease, corneal opacities, marked high-density lipoprotein deficiency, and statin therapy.
      ). The diagnosis of the siblings was consistent with fish-eye disease (FED) (
      • Dimick S.M.
      • Sallee B.
      • Asztalos B.F.
      • Pritchard P.H.
      • Frohlich J.
      • Schaefer E.J.
      A kindred with fish eye disease, corneal opacities, marked high-density lipoprotein deficiency, and statin therapy.
      ). LCAT-04, the third compound heterozygote (LCAT c.491G>A, p.R164H and LCAT c.382G>A, p.G128S), was a 69-year-old man with the following values: HDL-C, 13 mg/dl; apoA-I, 79 mg/dl; non-HDL-C, 96 mg/dl; direct LDL-C, 54 mg/dl; and TGs, 216 mg/dl, with a HDL-C/apoA-I ratio of 0.16. He had no evidence of ASCVD or kidney disease but did have mild corneal opacification.
      As also shown in Table 7, heterozygous LCAT mutations were noted in 13 subjects (9 men and 4 women; mean age: 49 years), with the following mean values: HDL-C, 17 mg/dl; apoA-I, 85 mg/dl; non-HDL-C, 150 mg/dl; direct LDL-C, 113 mg/dl; and TGs, 221 mg/dl, with an HDL-C/apoA-I ratio of 0.20. One of these subjects (LCAT-08) was also homozygous for ABCA1 rs111292742, while another (LCAT-15) was heterozygous for a previously reported LPL mutation (
      • Wright W.T.
      • Young I.S.
      • Nicholls D.P.
      • Graham C.A.
      Genetic screening of the LPL gene in hypertriglyceridaemic patients.
      ). None of these subjects had ASCVD or kidney failure, and none was reported to have corneal opacification. Eight additional subjects (4 men, 4 women; mean age: 48 years) were found to be heterozygotes for the LCAT rs4986970 (p.S232T) variant (Table 6). Their mean values were as follows: HDL-C, 12 mg/dl; apoA-I, 61 mg/dl; non-HDL-C, 162 mg/dl; direct LDL-C, 115 mg/dl; and TGs, 241 mg/dl. None of these 8 subjects was noted to have kidney disease, and only subject SNP-22, aged 76 years, was noted to have ASCVD (age of onset of 75 years with transient ischemic attacks). The LCAT rs4986970 variant has previously been associated with low HDL-C values in an Iranian population (
      • Naseri M.
      • Hedayati M.
      • Daneshpour M.S.
      • Bandarian F.
      • Azizi F.
      Identification of genetic variants of lecithin cholesterol acyltransferase in individuals with high HDL-C levels.
      ). We noted that the minor allele frequency for this variant in our severely HDL-deficient study population was 2.74% compared with a reported population frequency of 1.76% (not significant), but we had no data regarding Iranian descent.

      APOA1 mutations and variants

      Mutations in the APOA1 gene were found in 10 subjects (6 men, 4 women; 5.0% of the total study population) (Table 8). APOA1-01, a 30-year-old man, with an HDL-C of 2 mg/dl and an apoA-I of 15 mg/dl, was found to be homozygous for the novel APOA1 c.409G>T, p.E137* mutation and the ABCA1 rs111292742 SNP. He had a truncated form of apoA-I and was asymptomatic, with no evidence of ASCVD. The remaining 9 subjects (mean age: 59 years) had heterozygous APOA1 mutations and the following mean values: HDL-C, 18 mg/dl; apoA-I, 96 mg/dl; non-HDL-C, 127 mg/dl; direct LDL-C, 90 mg/dl; and TGs, 260 mg/dl, with an HDL-C/apoA-I ratio of 0.20. Subjects APOA1-02, APOA1-03, and APOA-04 were from a kindred with the APOA1 c.718C>T (p.Q240*) mutation, which also results in a truncated form of apoA-I. They all had ASCVD, as previously reported (
      • Anthanont P.
      • Polisecki E.
      • Asztalos B.F.
      • Diffenderfer M.R.
      • Barrett P.H.
      • Millar J.S.
      • Billheimer J.
      • Cuchel M.
      • Rader D.J.
      • Schaefer E.J.
      A novel apoA-I truncation (apoA-IMytilene) associated with decreased apoA-I production.
      ). APOA1-05, a 67-year-old woman who was heterozygous for APOA1 c.517C>A (p.R149S), had no evidence of ASCVD, but she did have a positive family history. In addition to the HDL deficiency, her plasma had a decreased ability to esterify cholesterol. She and her kindred have been previously reported (
      • Anthanont P.
      • Asztalos B.F.
      • Polisecki E.
      • Zachariah B.
      • Schaefer E.J.
      Case report: a novel apolipoprotein A-I missense mutation apoA-I (Arg149Ser)Boston associated with decreased lecithin-cholesterol acyltransferase activation and cellular cholesterol efflux.
      ). An APOA1 in-frame deletion c.391_393delAAG (p.K131del) was found in four subjects, one of whom (APOA1-07), a man aged 71 years, had ASCVD. Subject APOA1-10 with the APOA1 c.178T>G, p.S60A mutation had no evidence of ASCVD. Therefore, in our study population of 201 subjects with severe HDL deficiency, we found 10 subjects with APOA1 mutations that were mainly either truncations or in-frame deletions. Of these 10 subjects, 4 (40%) were noted to have ASCVD (3 men, 1 women; mean age of disease onset: 62 years).
      TABLE 8Subjects with mutations in APOA1 (n = 10)
      CaseGenderAge (years)HDL-C (mg/dl)apoA-I (mg/dl)HDL-C/apoA-I RatioNon-HDL-C (mg/dl)LDL-C (mg/dl)TGs (mg/dl)CVDAPOA1 Mutation
      GeneProtein
      Homozygous
      APOA1-0
      Subject was also homozygous for the ABCA1 rs111292742 (c.-279C>G) SNP.
      Male302150.13615467c.409G>Tp.E137*
      Heterozygous
      APOA1-02Male7019670.289469140+c.718C>Tp.Q240*
      APOA1-03Female8819770.25131109121+c.718C>Tp.Q240*
      APOA1-04Male61191010.19128108154+c.718C>Tp.Q240*
      APOA1-05Female6712670.18187148235c.517C>Ap.R173S
      APOA1-06Male4917930.18189135411c.391_393delAAGp.K131del
      APOA1-07Male71191030.1810876206+c.391_393delAAGp.K131del
      APOA1-08Male5518970.195338116c.391_393delAAGp.K131del
      APOA1-09Female18191700.1116399442c.391_393delAAGp.K131del
      APOA1-10Male5419850.229225513c.178T>Gp.S60A
      Mean6 males/ 3 females5918960.20127902604/9
      a Subject was also homozygous for the ABCA1 rs111292742 (c.-279C>G) SNP.
      b Novel mutation (n = 1).

      LPL variants

      Table 6 indicates that 8 subjects with no other mutations (7 men, 1 woman; 4.0%) had the LPL rs268 (p.N318S) variant, one of whom (SNP-23) was homozygous. These subjects had a mean age of 52 years and the following mean values: HDL-C, 16 mg/dl; apoA-I, 85 mg/dl; non-HDL-C, 155 mg/dl; direct LDL-C, 107 mg/dl; and TGs, 299 mg/dl. SNP-25 was a 78-year-old woman who developed ASCVD at the age of 71 years (coronary angioplasty and stent placement). SNP-31, who was heterozygous for LPL rs268 and ABCA1 rs111292742 (as noted above), was a 63-year-old man with no evidence of ASCVD. He had the following values: HDL, 6 mg/dl; apoA-I, 35 mg/dl; non-HDL-C, 199 mg/dl; direct LDL-C, 116 mg/dl; and TGs, 407 mg/dl, with an HDL-C/apoA-I ratio of 0.16. The LPL rs268 variant has previously been reported to be associated with hypertriglyceridemia and low HDL-C levels (
      • Wright W.T.
      • Young I.S.
      • Nicholls D.P.
      • Graham C.A.
      Genetic screening of the LPL gene in hypertriglyceridaemic patients.
      ). In total, 11 subjects, including the 3 subjects who also had a mutation or another variant (SNP-31, ABCA1-12, and ABCA1-29), carried the LPL rs268 variant. The frequency of this allele was significantly more prevalent in our study population (2.99%) than in the reported global population (1.29%; P < 0.01).

      Other lipid-related mutations

      As shown in Table 9, 10 subjects with severe HDL deficiency had mutations in genes related to monogenic lipid disorders characterized by elevated LDL-C and/or TG concentrations and/or by sitosterolemia. OTHER-01, found to be homozygous for an ABCG8 mutation, had markedly elevated β-sitosterol levels, consistent with sitosterolemia (
      • Brinton E.A.
      • Hopkins P.N.
      • Hegele R.A.
      • Geller A.S.
      • Polisecki E.Y.
      • Diffenderfer M.R.
      • Schaefer E.J.
      The association between hypercholesterolemia and sitosterolemia, and report of a sitosterolemia kindred.
      ). Two subjects (OTHER-02 and OTHER-03) were compound heterozygous for LDLR mutations and APOB mutations, and five subjects were heterozygous for LDLR or APOB mutations. Some of these subjects had LDL-C values >190 mg/dl, and those that did not were aggressively being treated with lipid-lowering agents (combination statin/ezetimibe). OTHER-05, aged 65 years, had established ASCVD (age of onset: 60 years) and was being aggressively managed with lipid-lowering therapy. OTHER-09, a 62-year-old man who was heterozygous for an LIPC mutation resulting in an amino acid substitution in hepatic lipase (p.T405M), had a TG concentration of 483 mg/dl. He had evidence of ASCVD, with angioplasty and stent placement at the age of 61 years. OTHER-10, a 46-year-old man who was heterozygous for the LPL p.T45N mutation, had moderate TG elevation (284 mg/dl) and no evidence of ASCVD.
      TABLE 9Subjects with mutations in other genes associated with lipid metabolism (n = 10)
      CaseGenderAge (years)HDL-C (mg/dl)apoA-I (mg/dl)HDL-C/apoA-I RatioNon-HDL-C (mg/dl)LDL-C (mg/dl)TGs (mg/dl)CVDMutation
      None of the mutations were novel.
      GeneProtein
      Homozygous
      OTHER-01Female2610420.2413611198ABCG8 c.154C>GABCG8 p.L52V
      Compound heterozygous
      OTHER-02Male365130.38346270409LDLR c.1977C>ALDLr p.T659T
      LDLR c.148G>TLDLr p.A50S
      OTHER-03Female6210550.18208147458APOB c.7612C>TapoB p.L2538L
      APOB c.8112G>AapoB p.A2704A
      Heterozygous
      OTHER-04Female6317NDNC654455LDLR c.682G>TLDLr p.E228*
      OTHER-05
      Subject was also heterozygous for the ABCA1 rs111292742 (c.-279C>G) SNP.
      Male6513720.188753212+LDLR c.292G>ALDLr p.G98S
      OTHER-06Female2716620.2626124372LDLR c.67+1075G>A
      OTHER-07Male4118660.2721119077APOB c.2630C>TapoB p.P877L
      OTHER-08Male3913750.17317266140APOB c.3279C>GapoB p.T1093T
      OTHER-09Male62191040.1814183483+LIPC c.1214C>THL p.T405M
      OTHER-10Male4616820.208840284LPL c.134C>ALPL p.T45N
      Mean6 males/ 4 females4714630.231861452292/10
      HL, hepatic lipase; LDLr, LDL receptor; NC, not calculated; ND, no data.
      a None of the mutations were novel.
      b Subject was also heterozygous for the ABCA1 rs111292742 (c.-279C>G) SNP.

      ASCVD prevalence rates

      The ASCVD prevalence in the 107 subjects (72% men) without secondary causes and for whom no DNA was available was 4.7%, with a mean age of onset of 61 years. The ASCVD prevalence in the 94 subjects (73% men) without secondary causes who did have DNA sequencing and in whom no mutations were found was 6.4%, with a mean age of onset of 64 years. Therefore, in these 201 pooled subjects with severe HDL deficiency with no secondary causes (72% men), the prevalence of ASCVD was 5.5%, with a mean age of onset of 62 years. In the 25 subjects with severe HDL deficiency with LCAT mutations or variants, only 1 subject, a 76-year woman, developed transient ischemic attacks (age of onset: 75 years). In this subject group the ASCVD prevalence was 4.0%. In contrast, in the 54 subjects with severe HDL deficiency and ABCA1 mutations or variants, the ASCVD prevalence was 37.0%, with a mean age of disease onset of 59 years (significantly higher than in the mutation-negative group; P < 0.05). Similarly, in the subjects with severe HDL deficiency and APOA1 mutations or variants, the ASCVD prevalence was 40%, with a mean age of disease onset of 62 years. Pooling the ABCA1 and APOA1 subjects (n = 64), the ASCVD prevalence was 37.5%, with a mean age of disease onset of 60 years. The ASCVD prevalence in these 64 subjects was 7-fold greater than in the 201 subjects with severe HDL deficiency who were not found to have any mutations or were never sequenced (5.5%; P < 0.01).

      DISCUSSION

      The first form of severe HDL deficiency to be described was Tangier disease by Fredrickson in 1964 (
      • Fredrickson D.S.
      The inheritance of high density lipoprotein deficiency (Tangier Disease).
      ). Homozygotes with the disease, named after the Chesapeake Bay island where the first two cases originated, were noted to have diffuse cholesteryl ester accumulation in macrophages in their tonsils, liver, spleen, and other tissues, as well as rapid catabolism of their HDL proteins, apoA-I, and apoA-II (
      • Ferrans V.J.
      • Fredrickson D.S.
      The pathology of Tangier disease. A light and electron microscopic study.
      ,
      • Schaefer E.J.
      • Blum C.B.
      • Levy R.I.
      • Jenkins L.L.
      • Alaupovic P.
      • Foster D.M.
      • Brewer H.B.
      Metabolism of high-density lipoprotein apolipoproteins in Tangier disease.
      ,
      • Schaefer E.J.
      • Anderson D.W.
      • Zech L.A.
      • Lindgren F.T.
      • Bronzert T.J.
      • Rubalcaba E.A.
      • Brewer H.B.
      Metabolism of high density lipoprotein subfractions and constituents in Tangier disease following the infusion of high density lipoproteins.
      ). Homozygotes were found to have HDL-C levels <10 mg/dl, moderate hypertriglyceridemia, decreased LDL-C, occasional peripheral neuropathy, mild corneal opacification as seen on slit-lamp examination, and an increased risk of premature ASCVD (
      • Schaefer E.J.
      • Zech L.A.
      • Schwartz D.S.
      • Brewer H.B.
      Coronary heart disease prevalence and other clinical features in familial high density lipoprotein deficiency (Tangier disease).
      ,
      • Mautner S.L.
      • Sanchez J.A.
      • Rader D.J.
      • Mautner G.C.
      • Ferrans V.J.
      • Fredrickson D.S.
      • Brewer H.B.
      • Roberts W.C.
      The heart in Tangier disease. Severe coronary atherosclerosis with near absence of high-density lipoprotein cholesterol.
      ,
      • Serfaty-Lacrosniere C.
      • Lanzberg A.
      • Civeira F.
      • Isaia P.
      • Berg J.
      • Janus E.D.
      • Smith M.P.
      • Pritchard P.H.
      • Frohlich J.
      • Lees R.S.
      • et al.
      Homozygous Tangier disease and cardiovascular disease.
      ,
      • Chu F.C.
      • Kuwabara T.
      • Cogan D.G.
      • Schaefer E.J.
      • Brewer H.B.
      Ocular manifestations of familial high-density lipoprotein deficiency (Tangier disease).
      ). The defect in these patients was found to be an inability to efflux cholesterol onto HDL particles from their cells due to defects in the ABCA1 gene and its gene product ABCA1 (
      • Francis G.A.
      • Knopp R.H.
      • Oram J.F.
      Defective removal of cellular cholesterol and phospholipids by apolipoprotein A-I in Tangier Disease.
      ,
      • Bodzioch M.
      • Orsó E.
      • Klucken J.
      • Langmann T.
      • Böttcher A.
      • Diederich W.
      • Drobnik W.
      • Barlage S.
      • Büchler C.
      • Porsch-Ozcürümez M.
      • et al.
      The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease.
      ,
      • Rust S.
      • Rosier M.
      • Funke H.
      • Real J.
      • Amoura Z.
      • Piette J.C.
      • Deleuze J.F.
      • Brewer H.B.
      • Duverger N.
      • Denèfle P.
      • et al.
      Tangier disease is caused by mutations in the gene encoding ATP-binding cassette transporter 1.
      ,
      • Brooks-Wilson A.
      • Marcil M.
      • Clee S.M.
      • Zhang L.H.
      • Roomp K.
      • van Dam M.
      • Yu L.
      • Brewer C.
      • Collins J.A.
      • Molhuizen H.O.
      • et al.
      Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency.
      ,
      • Remaley A.T.
      • Rust S.
      • Rosier M.
      • Knapper C.
      • Naudin L.
      • Broccardo C.
      • Peterson K.M.
      • Koch C.
      • Arnould I.
      • Prades C.
      • et al.
      Human ATP-binding cassette transporter 1 (ABC1): genomic organization and identification of the genetic defect in the original Tangier disease kindred.
      ,
      • Lawn R.M.
      • Wade D.P.
      • Garvin M.R.
      • Wang X.
      • Schwartz K.
      • Porter J.G.
      • Seilhamer J.J.
      • Vaughan A.M.
      • Oram J.F.
      The Tangier disease gene product ABC1 controls the cellular apolipoprotein-mediated lipid removal pathway.
      ,
      • Brousseau M.E.
      • Schaefer E.J.
      • Dupuis J.
      • Eustace B.
      • Van Eerdewegh P.
      • Goldkamp A.L.
      • Thurston L.M.
      • FitzGerald M.G.
      • Yasek-McKenna D.
      • O'Neill G.
      • et al.
      Novel mutations in the gene encoding ATP-binding cassette 1 in four Tangier disease kindreds.
      ,
      • Schaefer E.J.
      • Brousseau M.E.
      • Diffenderfer M.R.
      • Cohn J.S.
      • Welty F.K.
      • O'Connor J.
      • Dolnikowski G.G.
      • Wang J.
      • Hegele R.A.
      • Jones P.J.
      Cholesterol and apolipoprotein B metabolism in Tangier disease.
      ). On HDL particle analysis, homozygotes were found to have apoA-I mainly in pre-β1 HDL (
      • Asztalos B.F.
      • Brousseau M.E.
      • McNamara J.R.
      • Horvath K.V.
      • Roheim P.S.
      • Schaefer E.J.
      Subpopulations of high-density lipoproteins in homozygous and heterozygous Tangier disease.
      ). Heterozygotes were found to have a 50% reduction in cellular cholesterol efflux, very low levels of very large α1 and large α2 HDL particles, and approximately 50% of normal HDL-C levels (
      • Asztalos B.F.
      • Brousseau M.E.
      • McNamara J.R.
      • Horvath K.V.
      • Roheim P.S.
      • Schaefer E.J.
      Subpopulations of high-density lipoproteins in homozygous and heterozygous Tangier disease.
      ,
      • Brousseau M.E.
      • Eberhart G.P.
      • Dupuis J.
      • Asztalos B.F.
      • Goldkamp A.L.
      • Schaefer E.J.
      • Freeman M.W.
      Cellular cholesterol efflux in heterozygotes for Tangier disease is markedly reduced and correlates with high density lipoprotein cholesterol concentration and particle size.
      ).
      Population studies indicated that three common ABCA1 variants (p.G596A, p.A2589G, and p.G3456C) were more common in men with low HDL-C (<40 mg/dl) and ASCVD than in control subjects (
      • Brousseau M.E.
      • Bodzioch M.
      • Schaefer E.J.
      • Goldkamp A.L.
      • Kielar D.
      • Probst M.
      • Ordovas J.M.
      • Aslanidis C.
      • Lackner K.J.
      • Bloomfield Rubins H.
      • et al.
      Common variants in the gene encoding ATP-binding cassette transporter 1 in men with low HDL cholesterol levels and coronary heart disease.
      ). Cohen et al. (
      • Cohen J.C.
      • Kiss R.S.
      • Pertsemlidis A.
      • Marcel Y.L.
      • McPherson R.
      • Hobbs H.H.
      Multiple rare alleles contribute to low plasma levels of HDL cholesterol.
      ) sequenced the APOA1,ABCA1, and LCAT genes in 284 subjects with HDL-C concentrations below the 5th percentile (<30 mg/dl) and in 236 subjects with HDL-C levels above the 95th percentile (>75 mg/dl). They noted that nonsynonymous mutations or variants were significantly more common in subjects with low HDL-C than in those with high HDL-C (12% vs. 2%). They found that 9.9% of the subjects with low HDL-C had ABCA1 mutations, 2.2% had LCAT mutations, and 0.2% had APOA1 mutations. Therefore, mutations in ABCA1 were more common in patients with low HDL-C than mutations in LCAT or APOA1, in agreement with our findings; however, no clinical or other laboratory information was provided in their study (
      • Cohen J.C.
      • Kiss R.S.
      • Pertsemlidis A.
      • Marcel Y.L.
      • McPherson R.
      • Hobbs H.H.
      Multiple rare alleles contribute to low plasma levels of HDL cholesterol.
      ).
      Fricke-Schmidt et al. (
      • Frikke-Schmidt R.
      • Nordestgaard B.G.
      • Jensen G.B.
      • Steffensen R.
      • Tybjaerg-Hansen A.
      Genetic variation in ABCA1 predicts ischemic heart disease in the general population.
      ,
      • Frikke-Schmidt R.
      • Nordestgaard B.G.
      • Stene M.C.
      • Sethi A.A.
      • Remaley A.T.
      • Schnohr P.
      • Grande P.
      • Tybjaerg-Hansen A.
      Association of loss-of-function mutations in the ABCA1 gene with high-density lipoprotein cholesterol levels and risk of ischemic heart disease.
      ) did ABCA1 genotyping in 9,259 Danish subjects followed for 25 years and noted that 5 of 6 SNPs (ABCA1 p.V771M, p.V825I, p.I883M, p.E1172D, and p.R1587K) predicted increased risk of ASCVD, with ABCA1 p.V771M, p.I883M, and p.E1172D being the most important risk predictors. They compared ASCVD risk in heterozygotes versus noncarriers for four ABCA1 variants (p.P1065S, p.G1216V, p.N1800H, and p.R2144X) and noted that these variants reduced HDL-C levels by 17 mg/dl, associated with an adjusted hazard ratio for ASCVD of 1.70 (P < 0.001) (
      • Frikke-Schmidt R.
      • Nordestgaard B.G.
      • Jensen G.B.
      • Steffensen R.
      • Tybjaerg-Hansen A.
      Genetic variation in ABCA1 predicts ischemic heart disease in the general population.
      ,
      • Frikke-Schmidt R.
      • Nordestgaard B.G.
      • Stene M.C.
      • Sethi A.A.
      • Remaley A.T.
      • Schnohr P.
      • Grande P.
      • Tybjaerg-Hansen A.
      Association of loss-of-function mutations in the ABCA1 gene with high-density lipoprotein cholesterol levels and risk of ischemic heart disease.
      ). Akao et al. (
      • Akao H.
      • Polisecki E.
      • Schaefer E.J.
      • Trompet S.
      • Robertson M.
      • Ford I.
      • Jukema J.W.
      • de Craen A.J.
      • Packard C.
      • Buckley B.M.
      • et al.
      ABCA1 gene variation and heart disease risk reduction in the elderly during pravastatin treatment.
      ) assessed the ABCA1 p.R219K variant in 5,414 subjects in a statin trial for the elderly and noted an increased ASCVD event rate for those with the variant.
      Abdel-Razek et al. (
      • Abdel-Razek O.
      • Sadananda S.N.
      • Li X.
      • Cermakova L.
      • Frohlich J.
      • Brunham L.R.
      Increased prevalence of clinical and subclinical atherosclerosis in patients with damaging mutations in ABCA1 or APOA1.
      ) assessed potential differences in clinical and subclinical atherosclerosis (history of ASCVD or positive cardiac calcium score) and plasma cholesterol efflux capacity in 72 patients (mean age: 54 years) with HDL-C concentrations below the 10th percentile based on whether they had ABCA1 or APOA1 mutations. Disease-causing or likely pathogenic ABCA1 mutations were found in 13 patients, and likely pathogenic APOA1 mutations were found in 3 patients (n = 16; 22%), with 83% of these patients having evidence of atherosclerosis compared with 38.6% in patients with low HDL-C without such mutations or variants (P < 0.01). Patients with mutations also had lower cellular cholesterol efflux capacity (
      • Abdel-Razek O.
      • Sadananda S.N.
      • Li X.
      • Cermakova L.
      • Frohlich J.
      • Brunham L.R.
      Increased prevalence of clinical and subclinical atherosclerosis in patients with damaging mutations in ABCA1 or APOA1.
      ). Dron et al. (
      • Dron J.S.
      • Wang J.
      • Berberich A.J.
      • Iacocca M.A.
      • Cao H.
      • Yang P.
      • Knoll J.
      • Tremblay K.
      • Brisson D.
      • Netzer C.
      • et al.
      Large-scale deletions of the ABCA1 gene in patients with hypoalphalipoproteinemia.
      ) recently reported 4 patients, aged 34–59 years, with low HDL-C associated with large-scale deletions within the ABCA1 gene. Three of these patients had some evidence of ASCVD. The above data, like ours, indicate an increased risk of ASCVD in patients with low HDL-C and ABCA1 mutations.
      The next form of severe HDL deficiency, known as familial LCAT deficiency (FLD), was first described in 1967 by Norum and Gjone (
      • Norum K.R.
      • Gjone E.
      Familial serum-cholesterol esterification failure. A new inborn error of metabolism.
      ). These patients presented with marked corneal opacification, hyperlipidemia, anemia, proteinuria, and marked HDL deficiency (
      • Norum K.R.
      • Gjone E.
      Familial serum-cholesterol esterification failure. A new inborn error of metabolism.
      ). In contrast to normal subjects, almost all plasma cholesterol in these patients was unesterified due to a deficiency in LCAT activity. FLD patients were subsequently noted to have homozygous or compound heterozygous mutations in the LCAT gene and to develop kidney failure in the fourth or fifth decade of life (
      • Voight B.F.
      • Peloso G.M.
      • Orho-Melander M.
      • Frikke-Schmidt R.
      • Barbalic M.
      • Jensen M.K.
      • Hindy G.
      • Hólm H.
      • Ding E.L.
      • Johnson T.
      • et al.
      Plasma HDL cholesterol and risk of myocardial infarction: a Mendelian randomization study.
      ,
      • Norum K.R.
      • Gjone E.
      Familial serum-cholesterol esterification failure. A new inborn error of metabolism.
      ). We have documented that FLD homozygotes generally have apoA-I in plasma present only in very small pre-β1 and small α4 discoidal HDL particles (
      • Asztalos B.F.
      • Schaefer E.J.
      • Horvath K.V.
      • Yamashita S.
      • Miller M.
      • Franceschini G.
      • Calabresi L.
      Role of LCAT in HDL remodeling: an investigation in LCAT deficiency states.
      ). When the LCAT-deficient homozygote in the present study was treated with LCAT enzyme replacement therapy, there was transient appearance of α-HDL particles in the normal HDL-size range and stabilization of the patient's compromised kidney function, as reported previously (
      • Wright W.T.
      • Young I.S.
      • Nicholls D.P.
      • Graham C.A.
      Genetic screening of the LPL gene in hypertriglyceridaemic patients.
      ). However, the patient ended up on dialysis when enzyme replacement was no longer available. In our view, the treatment of choice in homozygous FLD patients will be enzyme replacement or gene therapy.
      A variant of FLD known as FED was first described by Carlson and Philipson in 1979 in a Norwegian man and his three daughters, all of whom had marked corneal opacification (
      • Carlson L.A.
      • Philipson B.
      Fish-eye disease. A new familial condition with massive corneal opacities and dyslipoproteinaemia.
      ). They had normal concentrations of total cholesterol but elevated concentrations of TG, VLDL cholesterol, and LDL-C and marked HDL deficiency (
      • Voight B.F.
      • Peloso G.M.
      • Orho-Melander M.
      • Frikke-Schmidt R.
      • Barbalic M.
      • Jensen M.K.
      • Hindy G.
      • Hólm H.
      • Ding E.L.
      • Johnson T.
      • et al.
      Plasma HDL cholesterol and risk of myocardial infarction: a Mendelian randomization study.
      ,
      • Carlson L.A.
      • Philipson B.
      Fish-eye disease. A new familial condition with massive corneal opacities and dyslipoproteinaemia.
      ). The disease was characterized further as being associated with ASCVD in later life, visual impairment, and dense corneal opacification. The two compound heterozygotes we report here as part of this study have previously been reported (
      • Dimick S.M.
      • Sallee B.
      • Asztalos B.F.
      • Pritchard P.H.
      • Frohlich J.
      • Schaefer E.J.
      A kindred with fish eye disease, corneal opacities, marked high-density lipoprotein deficiency, and statin therapy.
      ). In our view, the treatment of choice in FED patients is to optimize their LDL-C levels with statin therapy to prevent ASCVD.
      Haase et al. (
      • Haase C.L.
      • Tybærg-Hansen A.
      • Qayyum A.A.
      • Schou J.
      • Nordestgaard B.G.
      • Frikke-Schmidt R.
      LCAT, HDL cholesterol and ischemic cardiovascular disease: a Mendelian randomization study of HDL cholesterol in 54,500 individuals.
      ) examined the effects of low HDL-C on ASCVD causality in 10,281 Danish subjects based on the presence of an LCAT variant associated with low HDL-C (LCAT rs4986970, p.S208T; seen in 4% of their population). They found that low HDL-C was robustly associated with increased ASCVD risk, but genetically decreased HDL-C due to impaired LCAT activity was not (
      • Haase C.L.
      • Tybærg-Hansen A.
      • Qayyum A.A.
      • Schou J.
      • Nordestgaard B.G.
      • Frikke-Schmidt R.
      LCAT, HDL cholesterol and ischemic cardiovascular disease: a Mendelian randomization study of HDL cholesterol in 54,500 individuals.
      ). Similar observations were reported in a recent head-to-head comparison of FLD- and FED-associated LCAT mutations: FLD mutations were associated with decreased atherosclerosis, while FED mutations were associated with increased atherosclerosis (
      • Oldoni F.
      • Baldassarre D.
      • Castelnuovo S.
      • Ossoli A.
      • Amato M.
      • van Capelleveen J.
      • Hovingh G.K.
      • De Groot E.
      • Bochem A.
      • Simonelli S.
      • et al.
      Complete and partial LCAT deficiency are differentially associated with atherosclerosis.
      ). The difference appears to relate to the capacity of LCAT to modulate the cholesteryl ester content of apoB-containing lipoproteins. In FLD this capacity is lost. Overall, the data indicate that FLD causes kidney failure, while FED causes ASCVD (
      • Schaefer E.J.
      • Anthanont P.
      • Diffenderfer M.R.
      • Polisecki E.
      • Asztalos B.F.
      Diagnosis and treatment of high density lipoprotein deficiency.
      ,
      • Oldoni F.
      • Baldassarre D.
      • Castelnuovo S.
      • Ossoli A.
      • Amato M.
      • van Capelleveen J.
      • Hovingh G.K.
      • De Groot E.
      • Bochem A.
      • Simonelli S.
      • et al.
      Complete and partial LCAT deficiency are differentially associated with atherosclerosis.
      ). In our study we did not see an increased ASCVD prevalence in subjects with severe HDL deficiency associated with LCAT mutations.
      A third form of severe HDL deficiency, familial apoA-I deficiency, was first described by Schaefer et al. (
      • Schaefer E.J.
      • Heaton W.H.
      • Wetzel M.G.
      • Brewer H.B.
      Plasma apolipoprotein A-1 absence associated with a marked reduction of high density lipoproteins and premature coronary artery disease.
      ,
      • Schaefer E.J.
      • Ordovas J.M.
      • Law S.W.
      • Ghiselli G.
      • Kashyap M.L.
      • Srivastava L.S.
      • Heaton W.H.
      • Albers J.J.
      • Connor W.E.
      • Lindgren F.T.
      • et al.
      Familial apolipoprotein A-I and C-III deficiency, variant II.
      ) in 1982 in a woman with severe HDL deficiency, undetectable plasma apoA-I, and death at the age of 42 years due to severe premature ASCVD. In 1983, Karathanasis, Zannis, and Breslow (
      • Karathanasis S.K.
      • Zannis V.I.
      • Breslow J.L.
      Isolation and characterization of the human apolipoprotein A-I gene.
      ) became the first investigators to clone the APO1 gene. This kindred was subsequently noted by Ordovas et al. (
      • Ordovas J.M.
      • Cassidy D.K.
      • Civeira F.
      • Bisgaier C.L.
      • Schaefer E.J.
      Familial apolipoprotein A-I, C-III, and A-IV deficiency and premature atherosclerosis due to deletion of a gene complex on chromosome 11.
      ) to be affected with a large deletion of the entire APOA1/C3/A4 gene complex, with heterozygotes having about 50% of normal HDL-C, apoA-I, apoC-III, and apoA-IV levels in plasma. The disorder is now known as familial apoAI/CIII/AIV deficiency. In addition, in 1982 Norum et al. (
      • Norum R.A.
      • Lakier J.B.
      • Goldstein S.
      • Angel A.
      • Goldberg R.B.
      • Block W.D.
      • Noffze D.K.
      • Dolphin P.J.
      • Edelglass J.
      • Bogorad D.D.
      • et al.
      Familial deficiency of apolipoproteins A-I and C-III and precocious coronary-artery disease.
      ) described two sisters with severe HDL deficiency, undetectable plasma apoA-I and apoC-III levels, and premature ASCVD at ages 28 and 29 years. They were subsequently shown by Karathanasis, Ferris, and Haddad (
      • Karathanasis S.K.
      • Ferris E.
      • Haddad I.A.
      DNA inversion within the apolipoproteins AI/CIII/AIV-encoding gene cluster of certain patients with premature atherosclerosis.
      ) to have a DNA rearrangement affecting the adjacent APOA1 and APOC3 genes. This disorder is now known as familial apoAI/CIII deficiency (
      • Karathanasis S.K.
      • Zannis V.I.
      • Breslow J.L.
      Isolation and characterization of the human apolipoprotein A-I gene.
      ). Both forms of severe HDL deficiency are characterized by low TG and normal LDL-C concentrations.
      Familial apoA-I deficiency (undetectable plasma apoA-I) with premature ASCVD due to an APOA1 codon 84 nonsense mutation was first described by Matsunaga et al. (
      • Matsunaga T.
      • Hiasa Y.
      • Yanagi H.
      • Maeda T.
      • Hattori N.
      • Yamakawa K.
      • Yamanouchi Y.
      • Tanaka I.
      • Obara T.
      • Hamaguchi H.
      Apolipoprotein A-I deficiency due to a codon 84 nonsense mutation of the apolipoprotein A-I gene.
      ) in 1991. Other investigators have subsequently described patients with premature ASCVD that had this disorder caused by a homozygous codon 2 nonsense mutation (
      • Ng D.S.
      • Leiter L.A.
      • Vezina C.
      • Connelly P.W.
      • Hegele R.A.
      Apolipoprotein A-I Q[-2]X causing isolated apolipoprotein A-I deficiency in a family with analphalipoproteinemia.
      ,
      • Santos R.D.
      • Schaefer E.J.
      • Asztalos B.F.
      • Polisecki E.
      • Wang J.
      • Hegele R.A.
      • Martinez L.R.
      • Miname M.H.
      • Rochitte C.E.
      • Da Luz P.L.
      • et al.
      Characterization of high density lipoprotein particles in familial apolipoprotein A-I deficiency.
      ). Haase et al. (
      • Haase C.L.
      • Frikke-Schmidt R.
      • Nordestgaard B.G.
      • Tybjærg-Hansen A.
      Population-based resequencing of APOA1 in 10,330 individuals: spectrum of genetic variation, phenotype, and comparison with extreme phenotype approach.
      ) sequenced the APOA1 gene in 10,330 subjects and found that 0.27% of 10,330 subjects were heterozygous for APOA1 nonsynonymous variants associated with reductions in apoA-I and/or HDL-C levels and a hazard ratio of 1.72 (P < 0.01) for myocardial infarction, largely driven by the APOA1 p.A164S variant. The data presented herein are in agreement with prior studies indicating that mutations in APOA1 are less common than those in ABCA1 and LCAT in patients with HDL deficiency and are associated with premature ASCVD.
      Genetic variations at the LPL gene locus are often associated with both hypertriglyceridemia and low HDL-C levels. The frequencies of three LPL polymorphisms (p.D9N, p.N291S, and p.S447X) were noted to be increased in men with low HDL-C levels (<40 mg/dl) and ASCVD versus control subjects (
      • Brousseau M.E.
      • Goldkamp A.L.
      • Collins D.
      • Demissie S.
      • Connolly A.C.
      • Cupples L.A.
      • Ordovas J.M.
      • Bloomfield H.E.
      • Robins S.J.
      • Schaefer E.J.
      Polymorphisms in the gene encoding lipoprotein lipase in men with low HDL-C and coronary heart disease: the Veterans Affairs HDL Intervention Trial.
      ), with the LPL N9 and S291 alleles, which raise TG levels, being more frequent, and the S477X allele, which lowers TG levels, being less frequent in the low HDL-C subjects than in control subjects (
      • Haase C.L.
      • Tybærg-Hansen A.
      • Qayyum A.A.
      • Schou J.
      • Nordestgaard B.G.
      • Frikke-Schmidt R.
      LCAT, HDL cholesterol and ischemic cardiovascular disease: a Mendelian randomization study of HDL cholesterol in 54,500 individuals.
      ). In the present study we observed that the LPL p.N318S allele occurred in 3.0% of our severely HDL-deficient population compared with a reported population frequency of 1.3%. We have carried out extensive genotyping (>1,000 SNPs) in 699 men with low HDL-C (<40 mg/dl) and ASCVD and 705 age-matched ASCVD-free men (
      • Peloso G.M.
      • Demissie S.
      • Collins D.
      • Mirel D.B.
      • Gabriel S.B.
      • Cupples L.A.
      • Robins S.J.
      • Schaefer E.J.
      • Brousseau M.E.
      Common genetic variation in multiple metabolic pathways influences susceptibility to low HDL-cholesterol and coronary heart disease.
      ). After adjusting for multiple testing within each gene, SNPs at gene loci significantly associated with ASCVD and low HDL-C were 1) LIPC (hepatic lipase) rs4775065 (P < 0.0001 vs. control subjects), 2) CETP rs5882 (P = 0.0002), 3) ABCA1 rs2249891 (P = 0.0126), and 4) CUBN rs7893395 (P = 0.0246) (
      • Peloso G.M.
      • Demissie S.
      • Collins D.
      • Mirel D.B.
      • Gabriel S.B.
      • Cupples L.A.
      • Robins S.J.
      • Schaefer E.J.
      • Brousseau M.E.
      Common genetic variation in multiple metabolic pathways influences susceptibility to low HDL-cholesterol and coronary heart disease.
      ).
      Dron et al. (
      • Dron J.S.
      • Wang J.
      • Low-Kam C.
      • Khetarpal S.A.
      • Robinson J.F.
      • McIntyre A.D.
      • Ban M.R.
      • Cao H.
      • Vhainds D.
      • Dubé M-P.
      • et al.
      Polygenic determinants in extremes of high-density lipoprotein cholesterol.
      ) examined the effects of polygenic determinants on extremes of HDL-C in three population cohorts (Western University Lipid Genetics Clinic, Montreal Heart Institute Biobank, and the University of Pennsylvania) that included a total of 686 subjects with low HDL-C levels (≤30.9 mg/dl in men and ≤38.7 mg/dl in women) and 1,165 subjects with high HDL-C (≥54.1 mg/dl in men and ≥69.6 mg/dl in women). The prevalence of CVD by history was highest in the Montreal Heart Institute Biobank cohort (ranging from 31.3% to 67.1%) and lowest in the University of Pennsylvania group (ranging from 4.1% to 12.9%) but was always highest in the low HDL groups (
      • Dron J.S.
      • Wang J.
      • Low-Kam C.
      • Khetarpal S.A.
      • Robinson J.F.
      • McIntyre A.D.
      • Ban M.R.
      • Cao H.
      • Vhainds D.
      • Dubé M-P.
      • et al.
      Polygenic determinants in extremes of high-density lipoprotein cholesterol.
      ). They reported that 18.7% of the low HDL group had rare heterozygous large-effect variants, mainly in the ABCA1,LCAT, and APOA1 genes, with an additional 12.8% having an extreme polygenic trait score (
      • Dron J.S.
      • Wang J.
      • Low-Kam C.
      • Khetarpal S.A.
      • Robinson J.F.
      • McIntyre A.D.
      • Ban M.R.
      • Cao H.
      • Vhainds D.
      • Dubé M-P.
      • et al.
      Polygenic determinants in extremes of high-density lipoprotein cholesterol.
      ). The nine SNPs used in their polygenic trait score in order of effect size on HDL-C were CETP rs3764261, LIPC rs1532085, ABCA1 rs1883025, SCARB1 rs838880, GALNT2 rs48469914, MVK rs7134594, CMIP rs2925979, ZNF648 rs1689800, and ANGPTL4 rs7255436, with an r2 value of 0.8996 (P < 0.0001) (
      • Dron J.S.
      • Wang J.
      • Low-Kam C.
      • Khetarpal S.A.
      • Robinson J.F.
      • McIntyre A.D.
      • Ban M.R.
      • Cao H.
      • Vhainds D.
      • Dubé M-P.
      • et al.
      Polygenic determinants in extremes of high-density lipoprotein cholesterol.
      ).
      The data presented herein, as well as prior data based on DNA sequencing, would indicate that HDL deficiency due to mutations or variants at the ABCA1 or APOA1 gene locus are associated with premature ASCVD (
      • Fredrickson D.S.
      The inheritance of high density lipoprotein deficiency (Tangier Disease).
      ,
      • Ferrans V.J.
      • Fredrickson D.S.
      The pathology of Tangier disease. A light and electron microscopic study.
      ,
      • Schaefer E.J.
      • Blum C.B.
      • Levy R.I.
      • Jenkins L.L.
      • Alaupovic P.
      • Foster D.M.
      • Brewer H.B.
      Metabolism of high-density lipoprotein apolipoproteins in Tangier disease.
      ,
      • Schaefer E.J.
      • Anderson D.W.
      • Zech L.A.
      • Lindgren F.T.
      • Bronzert T.J.
      • Rubalcaba E.A.
      • Brewer H.B.
      Metabolism of high density lipoprotein subfractions and constituents in Tangier disease following the infusion of high density lipoproteins.
      ,
      • Schaefer E.J.
      • Zech L.A.
      • Schwartz D.S.
      • Brewer H.B.
      Coronary heart disease prevalence and other clinical features in familial high density lipoprotein deficiency (Tangier disease).
      ,
      • Mautner S.L.
      • Sanchez J.A.
      • Rader D.J.
      • Mautner G.C.
      • Ferrans V.J.
      • Fredrickson D.S.
      • Brewer H.B.
      • Roberts W.C.
      The heart in Tangier disease. Severe coronary atherosclerosis with near absence of high-density lipoprotein cholesterol.
      ,
      • Serfaty-Lacrosniere C.
      • Lanzberg A.
      • Civeira F.
      • Isaia P.
      • Berg J.
      • Janus E.D.
      • Smith M.P.
      • Pritchard P.H.
      • Frohlich J.
      • Lees R.S.
      • et al.
      Homozygous Tangier disease and cardiovascular disease.
      ,
      • Chu F.C.
      • Kuwabara T.
      • Cogan D.G.
      • Schaefer E.J.
      • Brewer H.B.
      Ocular manifestations of familial high-density lipoprotein deficiency (Tangier disease).
      ,
      • Francis G.A.
      • Knopp R.H.
      • Oram J.F.
      Defective removal of cellular cholesterol and phospholipids by apolipoprotein A-I in Tangier Disease.
      ,
      • Bodzioch M.
      • Orsó E.
      • Klucken J.
      • Langmann T.
      • Böttcher A.
      • Diederich W.
      • Drobnik W.
      • Barlage S.
      • Büchler C.
      • Porsch-Ozcürümez M.
      • et al.
      The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease.
      ,
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      ). There may be, however, significant heterogeneity with regard to ASCVD risk in such patients depending on their LDL-C levels. In patients with Tangier disease and hypersplenism, there may be markedly increased catabolism of LDL apoB with very low LDL-C levels, which may protect such patients from ASCVD, in contrast to Tangier patients with normal LDL-C (
      • Schaefer E.J.
      • Anthanont P.
      • Diffenderfer M.R.
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      • Asztalos B.F.
      Diagnosis and treatment of high density lipoprotein deficiency.
      ). There may also be heterogeneity in patients with HDL deficiency due to APOA1 variants, with variants associated with decreased LCAT activation, such as ApoA-IMilano, having no increased ASCVD risk, while other variants that do not affect LCAT activation having increased ASCVD risk (
      • Schaefer E.J.
      • Anthanont P.
      • Diffenderfer M.R.
      • Polisecki E.
      • Asztalos B.F.
      Diagnosis and treatment of high density lipoprotein deficiency.
      ).
      As indicated earlier, we consider the HDL Mendelian randomization studies by Voight et al. (
      • Voight B.F.
      • Peloso G.M.
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      • Frikke-Schmidt R.
      • Barbalic M.
      • Jensen M.K.
      • Hindy G.
      • Hólm H.
      • Ding E.L.
      • Johnson T.
      • et al.
      Plasma HDL cholesterol and risk of myocardial infarction: a Mendelian randomization study.
      ) and Do et al. (
      • Do R.
      • Willer C.J.
      • Schmidt E.M.
      • Sengupta S.
      • Gao C.
      • Peloso G.M.
      • Gustafsson S.
      • Kanoni S.
      • Ganna A.
      • Chen J.
      • et al.
      Common variants associated with plasma triglycerides and risk for coronary artery disease.
      ) as being highly flawed. Voight et al. reported no association between the intronic variant ABCA1 rs3890182 and ASCVD, while Do et al. found no association between the intronic variant ABCA1 rs1883025 and ASCVD (
      • Voight B.F.
      • Peloso G.M.
      • Orho-Melander M.
      • Frikke-Schmidt R.
      • Barbalic M.
      • Jensen M.K.
      • Hindy G.
      • Hólm H.
      • Ding E.L.
      • Johnson T.
      • et al.
      Plasma HDL cholesterol and risk of myocardial infarction: a Mendelian randomization study.
      ,
      • Do R.
      • Willer C.J.
      • Schmidt E.M.
      • Sengupta S.
      • Gao C.
      • Peloso G.M.
      • Gustafsson S.
      • Kanoni S.
      • Ganna A.
      • Chen J.
      • et al.
      Common variants associated with plasma triglycerides and risk for coronary artery disease.
      ). Both studies did see significant relationships between the intronic variants APOA1 rs6589566 and APOA1 rs10790162 and ASCVD risk (
      • Voight B.F.
      • Peloso G.M.
      • Orho-Melander M.
      • Frikke-Schmidt R.
      • Barbalic M.
      • Jensen M.K.
      • Hindy G.
      • Hólm H.
      • Ding E.L.
      • Johnson T.
      • et al.
      Plasma HDL cholesterol and risk of myocardial infarction: a Mendelian randomization study.
      ,
      • Do R.
      • Willer C.J.
      • Schmidt E.M.
      • Sengupta S.
      • Gao C.
      • Peloso G.M.
      • Gustafsson S.
      • Kanoni S.
      • Ganna A.
      • Chen J.
      • et al.
      Common variants associated with plasma triglycerides and risk for coronary artery disease.
      ); however, the APOA1 variants were excluded from their analysis because of effects on other lipoproteins. Voight et al. (
      • Voight B.F.
      • Peloso G.M.
      • Orho-Melander M.
      • Frikke-Schmidt R.
      • Barbalic M.
      • Jensen M.K.
      • Hindy G.
      • Hólm H.
      • Ding E.L.
      • Johnson T.
      • et al.
      Plasma HDL cholesterol and risk of myocardial infarction: a Mendelian randomization study.
      ) also relied almost entirely on the LIPC variant rs61755018 (p.N396S), which had no significant relationship with ASCVD. In our view, relying on intronic SNPs to assess the genetic contributions of a given gene to disease causation is flawed. Instead, investigators are relying increasingly on high-throughput next-generation DNA sequencing, which allows for more precise assessments of genetic variation at any given gene locus with disease, including ASCVD.
      It should be stated that our study has its limitations in that the number of subjects that were sequenced was relatively small, and the reference laboratory population may not be representative of the general population. In our view, the measurement of HDL-C and apoA-I levels is helpful in identifying HDL-deficient patients, and the quantification of the distribution of apoA-I in HDL subpopulations by two-dimensional gel electrophoresis and immunoblotting analysis is helpful in making the diagnosis (
      • Schaefer E.J.
      • Anthanont P.
      • Diffenderfer M.R.
      • Polisecki E.
      • Asztalos B.F.
      Diagnosis and treatment of high density lipoprotein deficiency.
      ). Nevertheless, the definitive diagnosis of the various forms of severe HDL deficiency must rely on DNA sequencing of the ABCA1, LCAT, and APOA1 genes (
      • Schaefer E.J.
      • Anthanont P.
      • Diffenderfer M.R.
      • Polisecki E.
      • Asztalos B.F.
      Diagnosis and treatment of high density lipoprotein deficiency.
      ). Another limitation of our study is the lack of data concerning the known polygenic causes of HDL deficiency, as documented recently by Dron et al (
      • Dron J.S.
      • Wang J.
      • Low-Kam C.
      • Khetarpal S.A.
      • Robinson J.F.
      • McIntyre A.D.
      • Ban M.R.
      • Cao H.
      • Vhainds D.
      • Dubé M-P.
      • et al.
      Polygenic determinants in extremes of high-density lipoprotein cholesterol.
      ).
      The data presented herein are consistent with the following concepts with regard to severe HDL deficiency (HDL-C <20 mg/dl): this condition is 1) uncommon in the general population and is significantly more frequent in men than in women; 2) frequently associated with severe hypertriglyceridemia, increased inflammation, diabetes, and in men with testosterone replacement; 3) most commonly caused by mutations in ABCA1, which can increase ASCVD risk depending on the underlying LDL-C levels; 4) may be caused by mutations in LCAT that can lead to renal failure; and 5) least commonly caused by mutations in APOA1 that can lead to premature ASCVD.

      Acknowledgments

      The authors thank William Evans, Hayley Mitchell, and Taylor Sacco for their assistance in setting up the study, contacting the healthcare providers, and maintaining the databases and Caitlin Rush and Katalin V. Horvath for their excellent technical assistance.

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