Advertisement

Cosegregation of serum cholesterol with cholesterol intestinal absorption markers in families with primary hypercholesterolemia without mutations in LDLR, APOB, PCSK9 and APOE genes

  • Lucía Baila-Rueda
    Correspondence
    Corresponding author. Unidad Clínica y de Investigación en Lípidos y Arteriosclerosis, Hospital Universitario Miguel Servet, Avenida Isabel La Católica 1-3, 50009, Zaragoza, Spain.
    Affiliations
    Unidad Clínica y de Investigación en Lípidos y Arteriosclerosis, Hospital Universitario Miguel Servet, Instituto de Investigación Sanitaria Aragón (IIS Aragón), 50009, Zaragoza, Spain
    Search for articles by this author
  • María Rosario Pérez-Ruiz
    Affiliations
    Unidad Clínica y de Investigación en Lípidos y Arteriosclerosis, Hospital Universitario Miguel Servet, Instituto de Investigación Sanitaria Aragón (IIS Aragón), 50009, Zaragoza, Spain
    Search for articles by this author
  • Estíbaliz Jarauta
    Affiliations
    Unidad Clínica y de Investigación en Lípidos y Arteriosclerosis, Hospital Universitario Miguel Servet, Instituto de Investigación Sanitaria Aragón (IIS Aragón), 50009, Zaragoza, Spain
    Search for articles by this author
  • María Teresa Tejedor
    Affiliations
    Universidad de Zaragoza, Departamento de Anatomía, Embriología y Genética Animal, 50009, Zaragoza, Spain
    Search for articles by this author
  • Rocío Mateo-Gallego
    Affiliations
    Unidad Clínica y de Investigación en Lípidos y Arteriosclerosis, Hospital Universitario Miguel Servet, Instituto de Investigación Sanitaria Aragón (IIS Aragón), 50009, Zaragoza, Spain
    Search for articles by this author
  • Iztiar Lamiquiz-Moneo
    Affiliations
    Unidad Clínica y de Investigación en Lípidos y Arteriosclerosis, Hospital Universitario Miguel Servet, Instituto de Investigación Sanitaria Aragón (IIS Aragón), 50009, Zaragoza, Spain
    Search for articles by this author
  • Isabel de Castro-Orós
    Affiliations
    Unidad Clínica y de Investigación en Lípidos y Arteriosclerosis, Hospital Universitario Miguel Servet, Instituto de Investigación Sanitaria Aragón (IIS Aragón), 50009, Zaragoza, Spain
    Search for articles by this author
  • Ana Cenarro
    Affiliations
    Unidad Clínica y de Investigación en Lípidos y Arteriosclerosis, Hospital Universitario Miguel Servet, Instituto de Investigación Sanitaria Aragón (IIS Aragón), 50009, Zaragoza, Spain
    Search for articles by this author
  • Fernando Civeira
    Affiliations
    Unidad Clínica y de Investigación en Lípidos y Arteriosclerosis, Hospital Universitario Miguel Servet, Instituto de Investigación Sanitaria Aragón (IIS Aragón), 50009, Zaragoza, Spain
    Search for articles by this author

      Highlights

      • Cholesterol absorption is increased in non-FH hypercholesterolemic families.
      • Cholesterol absorption serum markers are positively associated with LDL-cholesterol.
      • Hyperabsorption in affected families does not suggest a monogenic defect.
      • Sterol absorption variation is involved in non-FH hypercholesterolemia pathogenesis.

      Abstract

      Background and aim

      The genetic cause and pathogenic mechanism of approximately 20–40% of autosomal dominant hypercholesterolemias (ADH) are unknown. Increased cholesterol intestinal absorption has been associated to ADH. If this variation contributes to their pathogenesis is unknown.

      Methods and results

      We studied cholesterol absorption (phytosterols and cholestanol serum concentrations) and cholesterol synthesis (desmosterol serum concentration) in 20 families with ADH without causal mutations in LDLR, APOB, PCSK9 or APOE genes (non-FH ADH) selected from 54 non-FH ADH probands with (non-cholesterol sterol concentrations above 75th percentile) and without (under 75th percentile) hyperabsorption. The concentrations of cholestanol, sitosterol, campesterol and stigmasterol were higher in affected than in non-affected subjects (p = 0.003, <0.001.<0.001, 0.002, respectively). There was a strong cosegregation of hyperabsorption with high LDL cholesterol within hyperabsorber families with odds ratio 6.80 (confidence interval 1.656–27.9), p = 0.008. In hyperabsorber families, 60.5% of subjects were hyperabsorbers and 76% of them had high LDL cholesterol versus 38.3% and 63% in non-hyperabsorber families, respectively.

      Conclusion

      Most hypercholesterolemic family members with a hyperabsorber proband are hyperabsorbers. These absorption markers are significantly and positively associated with LDL cholesterol, and predispose to high LDL cholesterol in family members. Our data suggest that complex interindividual variation in cholesterol absorption is involved in many non-FH ADH.

      Keywords

      1. Introduction

      Autosomal dominant hypercholesterolemias (ADH) are characterized by high levels of low-density lipoprotein (LDL) cholesterol, familial presentation and high risk of premature cardiovascular disease [
      • Civeira F.
      International Panel on Management of Familial Hypercholesterolemia
      Guidelines for the diagnosis and management of heterozygous familial hypercholesterolemia.
      ]. Most ADH have familial hypercholesterolemia (FH) due to mutations in the LDLR gene that encodes for the LDL receptor [
      • Nordestgaard B.G.
      • Chapman M.J.
      • Humphries S.E.
      • Ginsberg H.N.
      • Masana L.
      • Descamps O.S.
      • Wiklund O.
      • Hegele R.A.
      • Raal F.J.
      • Defesche J.C.
      • Wiegman A.
      • Santos R.D.
      • Watts G.F.
      • Parhofer K.G.
      • Hovingh G.K.
      • Kovanen P.T.
      • Boileau C.
      • Averna M.
      • Borén J.
      • Bruckert E.
      • Catapano A.L.
      • Kuivenhoven J.A.
      • Pajukanta P.
      • Ray K.
      • Stalenhoef A.F.
      • Stroes E.
      • Taskinen M.R.
      • Tybjærg-Hansen A.
      Familial hypercholesterolaemia is underdiagnosed and undertreated in the general population: guidance for clinicians to prevent coronary heart disease.
      ]. Approximately 2–15% of ADH subjects have familial defective apolipoprotein B-100 (FDB) due to mutations in the LDL receptor-binding domain coding region of the APOB gene, which encodes for apolipoprotein B-100 [
      • Vrablík M.
      • Ceska R.
      • Horínek A.
      Major apolipoprotein B-100 mutations in lipoprotein metabolism and atherosclerosis.
      ], or mutations in proprotein convertase subtilisin/kexin type 9 gene (PCSK9), a protein involved in the LDL receptor recycling [
      • Soutar A.K.
      • Naoumova R.P.
      Mechanisms of disease: genetic causes of familial hypercholesterolemia.
      ]. Recently, a mutation in APOE (p.Leu167del) has been also associated with ADH [
      • Solanas-Barca M.
      • de Castro-Orós I.
      • Mateo-Gallego R.
      • Cofán M.
      • Plana N.
      • Puzo J.
      • Burillo E.
      • Martín-Fuentes P.
      • Ros E.
      • Masana L.
      • Pocoví M.
      • Civeira F.
      • Cenarro A.
      Apolipoprotein E gene mutations in subjects with mixed hyperlipidemia and a clinical diagnosis of familial combined hyperlipidemia.
      ,
      • Awan Z.
      • Choi H.Y.
      • Stitziel N.
      • Ruel I.
      • Bamimore M.A.
      • Husa R.
      • Gagnon M.H.
      • Wang R.H.
      • Peloso G.M.
      • Hegele R.A.
      • Seidah N.G.
      • Kathiresan S.
      • Genest J.
      APOE p.Leu167del mutation in familial hypercholesterolemia.
      ]. Patients with mutations in these genes present an indistinguishable phenotype and are now included in the FH definition [
      • Nordestgaard B.G.
      • Chapman M.J.
      • Humphries S.E.
      • Ginsberg H.N.
      • Masana L.
      • Descamps O.S.
      • Wiklund O.
      • Hegele R.A.
      • Raal F.J.
      • Defesche J.C.
      • Wiegman A.
      • Santos R.D.
      • Watts G.F.
      • Parhofer K.G.
      • Hovingh G.K.
      • Kovanen P.T.
      • Boileau C.
      • Averna M.
      • Borén J.
      • Bruckert E.
      • Catapano A.L.
      • Kuivenhoven J.A.
      • Pajukanta P.
      • Ray K.
      • Stalenhoef A.F.
      • Stroes E.
      • Taskinen M.R.
      • Tybjærg-Hansen A.
      Familial hypercholesterolaemia is underdiagnosed and undertreated in the general population: guidance for clinicians to prevent coronary heart disease.
      ]. The genetic cause and pathogenic mechanism of approximately 20–40% of ADH, named in short as non-FH ADH, are unknown [
      • Palacios L.
      • Grandoso L.
      • Cuevas N.
      • Olano-Martín E.
      • Martinez A.
      • Tejedor D.
      • Stef M.
      Molecular characterization of familial hypercholesterolemia in Spain.
      ,
      • Futema M.
      • Whittall R.A.
      • Kiley A.
      • Steel L.K.
      • Cooper J.A.
      • Badmus E.
      • Leigh S.E.
      • Karpe F.
      • Neil H.A.
      Analysis of the frequency and spectrum of mutations recognised to cause familial hypercholesterolaemia in routine clinical practice in a UK specialist hospital lipid clinic.
      ], and probably they are a heterogeneous group of diseases including some severe polygenic hypercholesterolemias [
      • Talmud P.J.
      • Shah S.
      • Whittall R.A.
      • Futema M.
      • Howard P.
      • Cooper J.A.
      • Harrison S.C.
      • Li K.W.
      • Drenos F.
      • Karpe F.
      • Neil H.A.
      • Descamps O.S.
      • Langenberg C.
      • Lench N.
      • Kivimaki M.
      • Whittaker J.
      • Hingorani A.D.
      • Kumari M.
      • Humphries S.E.
      Use of low-density lipoprotein cholesterol gene score to distinguish patients with polygenic and monogenic familial hypercholesterolaemia: a case-control study.
      ].
      Cholesterol concentration in plasma depends on the amount of cholesterol from the diet and its intestinal absorption, on de novo synthesis, and on its biliary excretion [
      • Kesäniemi Y.A.
      • Miettinen T.A.
      Metabolic epidemiology of plasma cholesterol.
      ]. Previous studies have reported increased intestinal cholesterol absorption in non-FH ADH subjects that may partially explain plasma hypercholesterolemia in these subjects [
      • García-Otín A.L.
      • Cofán M.
      • Junyent M.
      • Recalde D.
      • Cenarro A.
      • Pocoví M.
      • Ros E.
      • Civeira F.
      Increased intestinal cholesterol absorption in autosomal dominant hypercholesterolemia and no mutations in the low-density lipoprotein receptor or apolipoprotein B genes.
      ,
      • Lupattelli G.
      • Pirro M.
      • Siepi D.
      • Mannarino M.R.
      • Roscini A.R.
      • Vaudo G.
      • Pasqualini L.
      • Schillaci G.
      • Mannarino E.
      Non-cholesterol sterols in different forms of primary hyperlipemias.
      ]. However, no familial cosegregation studies have been performed to study the linkage between hyper-absorption and high LDL cholesterol in non-FH ADH families.
      Normal serum contains small but detectable amounts of non-cholesterol sterols, including plant sterols, also named phytosterols, and cholestanol, and their ratios to cholesterol are accepted surrogate markers for the efficiency of cholesterol intestinal absorption [
      • Miettinen T.A.
      • Tilvis R.S.
      • Kesäniemi Y.A.
      Serum plant sterols and cholesterol precursors reflect cholesterol absorption and synthesis in volunteers of a randomly selected male population.
      ,
      • Matthan N.R.
      • Raeini-Sarjaz M.
      • Lichtenstein A.H.
      • Ausman L.M.
      • Jones P.J.
      Deuterium uptake and plasma cholesterol precursor levels correspond as methods for measurement of endogenous cholesterol synthesis in hypercholesterolemic women.
      ].
      Efficiency of cholesterol intestinal absorption is a partly inherited phenomenon. Heredity of cholesterol absorption has been demonstrated in siblings of hypercholesterolemic probands with low and high serum cholestanol to cholesterol ratio [
      • Gylling H.
      • Miettinen T.A.
      Inheritance of cholesterol metabolism of probands with high or low cholesterol absorption.
      ].
      Considering that some cases of non-FH ADH are associated with cholesterol intestinal hyperabsorption, the aim of our work was to determine if the efficiency of intestinal cholesterol absorption, measured by non-cholesterol sterol surrogate markers, cosegregates with LDL cholesterol concentration in non-FH ADH families.

      2. Materials and methods

      2.1 Probands

      Selected subjects (n = 54) were unrelated adults 18–79 years of age with the clinical diagnosis of ADH: LDL cholesterol above the 95th percentile of the Spanish population [
      • Gómez-Gerique J.A.
      • Gutiérrez-Fuentes J.A.
      • Montoya M.T.
      • Porres A.
      • Rueda A.
      • Avellaneda A.
      • Rubio M.A.
      [Lipid profile of the Spanish population: the DRECE (diet and risk of cardiovascular disease in Spain) study. DRECE study group].
      ], triglycerides below 200 mg/dL, primary cause, and familial presentation (at least one first-degree relative with the same phenotype) from the Lipid Clinic at Hospital Universitario Miguel Servet, Zaragoza, Spain. In all subjects, the presence of functional mutations in LDLR, APOB and PCSK9, and p.Leu167del in APOE were ruled out by DNA sequencing as previously described [
      • Solanas-Barca M.
      • de Castro-Orós I.
      • Mateo-Gallego R.
      • Cofán M.
      • Plana N.
      • Puzo J.
      • Burillo E.
      • Martín-Fuentes P.
      • Ros E.
      • Masana L.
      • Pocoví M.
      • Civeira F.
      • Cenarro A.
      Apolipoprotein E gene mutations in subjects with mixed hyperlipidemia and a clinical diagnosis of familial combined hyperlipidemia.
      ,
      • Palacios L.
      • Grandoso L.
      • Cuevas N.
      • Olano-Martín E.
      • Martinez A.
      • Tejedor D.
      • Stef M.
      Molecular characterization of familial hypercholesterolemia in Spain.
      ]. Secondary causes of hypercholesterolemia including: obesity (body mass index > 30 kg/m2), poorly controlled type 2 diabetes (HbA1c > 8%), renal disease with glomerular filtration rate <30 mL/min and/or macroalbuminuria, liver diseases (ALT > 3 times upper normal limit), hypothyroidism (TSH > 6 mIU/L), pregnancy, autoimmune diseases and protease inhibitors were exclusion criteria. Subjects disclosing APOE ε2/ε2 genotype were not considered for this study. Subjects with previous cardiovascular disease or high risk for cardiovascular disease (>20% in the next 10 years) were excluded except if they were not on lipid-lowering drugs. Cardiovascular risk factors assessment, personal and family history of cardiovascular disease, consumption of drugs affecting intestinal or lipid metabolism and anthropometric measurements were performed in all participants. Dietary intake was determined by interview with one single nutritionist dietitian. In this interview, a Spanish validated 137-item food frequency questionnaire (FFQ) was used [
      • de la Fuente-Arrillaga C.
      • Vazquez-Ruiz Z.
      • Bes-Rastrollo M.
      • Sampson L.
      • Martinez-González M.A.
      Reproducibility of an FFQ validated in Spain.
      ].

      2.2 Biochemistry determinations

      Fasting blood for biochemical profiles was drawn after at least 5–6 weeks without hypolipidemic drug treatment, plant sterols or fish oil supplements. Cholesterol and triglycerides were determined by standard enzymatic methods. HDL cholesterol was measured by a precipitation technique. Apo A1, apo B and lipoprotein (a) were determined by nephelometry using IMMAGE-Immunochemistry System (Beckman Coulter). LDL cholesterol was calculated using Friedewald's formula.

      2.3 Intestinal absorption and synthesis markers

      Serum phytosterols and cholestanol, all of them markers of cholesterol absorption, and cholesterol were quantified after 10 h of fasting. Subjects were without lipid lowering drugs or phytosterol supplements at least 5 weeks before blood extraction. Serum concentration of cholesterol, sitosterol, campesterol, stigmasterol, cholestanol and desmosterol were quantified using HPLC-MS/MS according to the method previously described [
      • Baila-Rueda L.
      • Cenarro A.
      • Cofan M.
      • Orera I.
      • Barcelo-Batllori S.
      • Pocovi M.
      • Ros E.
      • Civeira F.
      • Nerin C.
      • Domeno C.
      Simultaneous determination of oxysterols, phytosterols and cholesterol precursors by high performance liquid chromatography tandem mass spectrometry in human serum.
      ], and were expressed as mg/dL as well as normalized to mg/dL of total cholesterol. Briefly, 100 μl of serum were transferred to a screw-capped vial and deuterium-labeled internal standard, [2H6] cholesterol-26,26,26,27,27,27, (7.9 mM), was added to determine non-cholesterol sterols. Another 100 μl of serum were transferred to a screw-capped vial, deuterium-labeled internal standard, [2H7] cholesterol-25,26,26,26,27,27,27, was added to determine cholesterol. Alkaline hydrolysis was performed for 20 min at 60 °C in an ultrasound bath and extracted twice with 3 ml of hexane. The extracts were loaded onto the SPE cartridge (1 mg, Discovery DSC-18, Supelco, Spain) which was preconditioned with 400 μl of methanol and gravity eluted. The non-cholesterol sterols and cholesterol were desorbed with 1.4 ml of 2-propanol by gravity and 40 μl of the final mixtures were injected into the HPLC-MS/MS system.

      2.4 Definition of cholesterol intestinal hyperabsorption

      We defined as hyperabsorber those subjects that showed ≥3 intestinal non-cholesterol sterols >75th percentile of the distribution in normolipidemic population. Subjects with serum phytosterol and cholestanol concentrations under 75th percentile were considered as non-hyperabsorbers. One hundred normolipidemic subjects (LDL cholesterol under the 75th percentile and triglycerides <200 mg/dL) were used to determine the normal non-cholesterol sterol distribution in our population. This group consisted of healthy, unrelated men and women volunteers aged 18–79 years, who underwent a medical examination at the Hospital Universitario Miguel Servet of Zaragoza. Exclusion criteria for normolipemic subjects were personal or parental history of premature cardiovascular disease or dyslipidemia, current acute illness, or use of drugs that might influence glucose or lipid metabolism.

      2.5 Family studies

      Available family members of all hyperabsorber probands and the same number of families with a non-hyperabsorber proband were studied for cosegregation analysis. Clinical, biochemical, and non-cholesterol sterol analyses were performed in family members as in probands, except for genetic studies that were not made in family members. The same exclusion criteria and hyperabsorption definition were used in family members as were used in probands. Family members were considered affected if LDL cholesterol was above 90th percentile of the Spanish distribution in absence of secondary causes.
      All subjects: non-FH ADH probands, normolipemic controls, and family members signed informed consent to a protocol previously approved by our local ethical committee (Comité Ético de Investigación Clínica de Aragón, Zaragoza, Spain).

      2.6 Statistical analyses

      Comparison of lipid variables among groups was performed using the Student's t test for data normally distributed and Mann–Whitney U test for skewed data. When significant differences were detected, multiple comparisons were made by using the Bonferroni correction for normally distributed variables. The significance was set at P < 0.05 for the variables. Non-cholesterol sterol to cholesterol ratios were log transformed to achieve variance homogeneity. Data are presented as mean and standard deviation for continuous variables. Bivariate logistic regression was used to determine the ability of the hyperabsorber condition to discriminate between affected and non-affected family member patients. Age and gender were included in the analysis as predictor variables. Regression coefficients (B) and odds ratios (OR) for predictors variables are listed. A positive regression coefficient means that the hyperabsorption increases the probability to have hypercholesterolemia. The odds ratio is defined as the relative amounts by which the odds of presenting hypercholesterolemia increase when hyperabsorption is present. Sample size was calculated to detect differences of 25% in plasma phytosterols between affected and non-affected family members in each group of families, assuming similar variances, with a statistical power of 0.95 and a margin of error of 0.80. All statistical analyses were performed with SPSS software (version 15.0; SPSS, Chicago, IL, USA).

      3. Results

      The main clinical and biochemical characteristics of the 54 non-FH ADH probands are presented in Table 1. Probands were mostly healthy women (65%) with high total cholesterol and LDL cholesterol and normal triglycerides as expected due to inclusion and exclusion criteria. The concentration of the non-cholesterol sterols is represented by ratio to total cholesterol determined by HPLC-MS/MS.
      Table 1Clinical and biochemical characteristics of non-FH ADH probands
      Values are mean ± SD.
      .
      Proband (n = 54)
      Age, years52 ± 9.2
      Females, n (%)35 (64.8)
      Systolic blood pressure, mm Hg136 ± 15.7
      Diastolic blood pressure, mm Hg83.4 ± 9.1
      Body mass index, kg/m225.4 ± 3.2
      Waist circumference, cm87.9 ± 11.0
      Total cholesterol, mg/dL321 ± 54.7
      HDL cholesterol, mg/dL67.0 ± 18.1
      LDL cholesterol, mg/dL231 ± 47.4
      Triglycerides, mg/dL113.2 ± 62.0
      Apolipoprotein A1, mg/dL176 ± 37.8
      Apolipoprotein B, mg/dL159 ± 36.9
      Lipoprotein(a), mg/dL68.9 ± 93.1
      C reactive protein, g/L0.79 ± 0.15
      Glucose, mg/dL86.6 ± 7.1
      GGT, IU/L30.4 ± 24.7
      ALT, IU/L21.8 ± 6.8
      Cholesterol by HPLC-MS/MS, mg/dL347 ± 58.1
      Cholestanol-to-TC x 1032.14 ± 0.10
      Sitosterol-to-TC x 1032.08 ± 1.21
      Campesterol-to-TC x 1030.99 ± 0.52
      Stigmasterol-to-TC x 1030.17 ± 0.13
      HDL denotes high density lipoprotein; LDL, low density lipoprotein; GGT, gamma glutamyl transpeptidase; ALT, alanine transaminase; HPLC, high-performance liquid chromatography; MS, mass spectrometry.
      a Values are mean ± SD.
      Ten non-FH ADH probands fulfilled the diagnostic criteria of hyperabsorbers. Table 2 shows the main lipid characteristics of these subjects and of 10 non-hyperabsorber probands used for comparison. There were differences between groups in body mass index, waist circumference and triglycerides. Other clinical variables, including dietary characteristics, did not differ between hyperabsorber and non-hyperabsorber probands. Mean values of cholestanol, sitosterol, campesterol and stigmasterol showed statistically significant (P = 0.003 for cholestanol and P < 0.001 for phytosterols) between both groups.
      Table 2Clinical and biochemical characteristics of selected probands and control subjects
      Values are mean ± SD. P refers to differences calculated by Student's t test between hyperabsorber and non-hyperabsorber probands.
      .
      Control n = 100ProbandP
      Hyperabsorber n = 10Non-hyperabsorber n = 10
      Age, years46 ± 1756 ± 448 ± 110.060
      Females, n (%)55 (55)8 (80)5 (50)0.160
      Systolic blood pressure, mm Hg126 ± 22133 ± 11140 ± 190.353
      Diastolic blood pressure, mm Hg80 ± 1382 ± 1184 ± 70.636
      Body mass index, kg/m225 ± 5.123.9 ± 1.526.9 ± 3.80.039
      Waist circumference, cm89 ± 1382 ± 994 ± 100.010
      Total cholesterol, mg/dL198 ± 31.5326 ± 34.2316 ± 71.40.697
      LDL cholesterol, mg/dL128 ± 25.8235 ± 37.2228 ± 57.60.735
      HDL cholesterol, mg/dL53.7 ± 12.674.7 ± 8.859.4 ± 22.10.065
      Triglycerides, mg/dL83.3 ± 37.377.8 ± 28.1148.7 ± 67.20.010
      Apolipoprotein A1, mg/dL159 ± 28.9191 ± 31.7161 ± 38.60.070
      Apolipoprotein B, mg/dL99 ± 21.6168 ± 33.6150 ± 39.80.306
      Lipoprotein(a), mg/dL24.3 ± 24.969.4 ± 98.668.4 ± 92.50.981
      Glucose, mg/dL87.4 ± 13.787.5 ± 6.785.7 ± 7.40.584
      GGT, IU/L23.7 ± 25.223.9 ± 17.836.9 ± 29.50.249
      ALT, IU/L21.6 ± 12.419.8 ± 5.023.9 ± 8.00.188
      Cholesterol HPLC-MS, mg/dL191.4 ± 28.9352.6 ± 46.4341.8 ± 70.10.690
      Cholestanol-to-TC x 1032.16 ± 0.792.68 ± 0.711.59 ± 0.740.003
      Sitosterol-to-TC x 1031.78 ± 0.853.06 ± 0.851.09 ± 0.44<0.001
      Campesterol-to-TC x 1030.93 ± 0.451.42 ± 0.340.56 ± 0.24<0.001
      Stigmasterol-to-TC x 1030.083 ± 0.0520.26 ± 0.130.088 ± 0.0380.002
      Desmosterol-to-TC x 1032.62 ± 1.142.17 ± 0.642.69 ± 0.730.153
      HDL denotes high density lipoprotein; LDL, low density lipoprotein; GGT, gamma glutamyl transpeptidase; ALT, alanine transaminase; HPLC, high-performance liquid chromatography; MS, mass spectrometry.
      a Values are mean ± SD. P refers to differences calculated by Student's t test between hyperabsorber and non-hyperabsorber probands.
      Table 3 shows the clinical and biochemical characteristics of the family members in both, hyperabsorber and non-hyperabsorber families. In families with a hyperabsorber proband, there were significant differences between affected and non-affected subjects for total cholesterol, LDL cholesterol, non-HDL cholesterol and apolipoprotein B, without differences in BMI. Intestinal cholesterol absorption markers were higher in affected subjects, with significant differences for cholestanol. In non-hyperabsorber families, there were similar lipid differences than in hyperabsorber families and affected subjects had higher BMI than non-affected subjects. The concentration of cholesterol absorption markers was not significantly different in the non-hyperabsorber families, except for cholestanol. No difference was found in desmosterol between affected and non-affected subjects in hyperabsorber families and significant difference was found between affected and non-affected subjects in non-hyperabsorber families (P = 0.004), probably associated with a significantly higher BMI. Desmosterol, was significantly lower in affected subjects from hyperabsorber families than in affected subjects from non-hyperabsorber families (2.76 (2.26–2.94) vs. 2.96 (2.67–3.32), P = 0.035). An opposite pattern although without reaching statistical significance was found for intestinal cholesterol absorption markers (6.87 (5.43–8.75) vs. 5.90 (4.90–6.77), P = 0.083).
      Table 3Clinical and biochemical characteristic of family members
      Values are mean ± SD or median (interquartile range). P refers to differences calculated by Student's t test for data normally distributed and Mann–Whitney U test for skewed data.
      .
      Hyperabsorber families n = 43PNon-hyperabsorber families n = 60P
      Affected n = 17Non-affected n = 26Affected n = 41Non-affected n = 19
      Age, years42 (26–49)35 (29–52)0.98245 (32–51)38 (25–52)0.710
      Males, n (%)8 (38)13 (62)0.85012 (44)15 (56)0.054
      Systolic blood pressure, mm Hg126 (109–133)122 (115–141)0.728128 (121–146)120 (109–147)0.357
      Diastolic blood pressure, mm Hg75 (68–87)76 (68–84)0.88181 (76–84)76 (67–89)0.572
      Body mass index, kg/m223.3 ± 3.824.3 ± 5.30.50827.5 ± 7.224.6 ± 3.50.043
      Waist circumference, cm85.3 ± 9.387.9 ± 130.44396.6 ± 16.589.6 ± 10.30.104
      Total cholesterol, mg/dL266 ± 43.7203 ± 33.9<0.001278 ± 42.6198 ± 36.8<0.001
      LDL cholesterol, mg/dL182 ± 38.0127 ± 28.7<0.001186 ± 38.0122 ± 32.5<0.001
      HDL cholesterol, mg/dL62.3 ± 17.459.3 ± 13.00.52952.2 ± 12.356.5 ± 13.30.243
      Non-HDL cholesterol, mg/dL203 ± 37.8144 ± 31.0<0.001220 ± 39.4141 ± 36.8<0.001
      Triglycerides, mg/dL89 (69–114)81 (64–98)0.371168 (137–247)91 (61–117)<0.001
      Apolipoprotein A1, mg/dL163 (155–192)159 (138–182)0.210152 (129–180)157 (140–181)0.313
      Apolipoprotein B, mg/dL127 ± 23.292.0 ± 19.9<0.001142 ± 30.242.4 ± 23.2<0.001
      Lipoprotein(a), mg/dL28.3 (57.7–15.2)28.2 (18.5–50.9)0.94816.4 (2.7–57.6)24.2 (7.5–66.1)0.685
      Glucose, mg/dL87 (80–91)84 (75–93)0.59385 (77–100)83 (79–93)0.660
      Cholesterol HPLC-MS, mg/dL270 ± 41.5212 ± 34.0<0.001271 ± 48.5204 ± 38.9<0.001
      Cholestanol-to-TC x 1032.95 (2.43–3.25)2.35 (1.95–2.77)0.0422.55 (2.22–3.22)2.12 (1.85–2.54)0.001
      Sitosterol-to-TC x 1032.73 (1.84–3.58)1.82 (1.44–2.66)0.0702.09 (1.55–2.48)1.69 (1.48–2.01)0.076
      Campesterol-to-TC x 1031.36 (1.06–1.80)1.12 (0.86–1.41)0.0591.18 (0.91–1.26)0.96 (0.74–1.27)0.088
      Stigmasterol-to-TC x 1030.17 (0.11–0.22)0.11 (0.09–0.16)0.0980.12 (0.08–0.15)0.10 (0.09–0.14)0.243
      All cholesterol absorption markers6.87 (5.43–8.75)5.46 (4.43–7.52)0.0605.90 (4.90–6.77)5.24 (4.20–5.77)0.020
      Desmosterol-to-TC x 1032.76 (2.26–2.94)2.85 (2.46–3.05)0.3712.96 (2.67–3.32)2.43 (2.00–2.93)0.004
      a Values are mean ± SD or median (interquartile range). P refers to differences calculated by Student's t test for data normally distributed and Mann–Whitney U test for skewed data.
      Spearman's rank correlations between LDL cholesterol levels and cholesterol absorption markers are reported in Table 4. Sitosterol and stigmasterol in hyperabsorber families had a positive correlation with LDL cholesterol. However, no correlation was found for cholesterol absorption markers in non-hyperabsorber families. When subjects were divided in hyperabsorbers and non-hyperabsorbers, there was a significant association between LDL cholesterol and cholestanol and sitosterol only in hyperabsorber subjects (Supplementary Figure). The diagnosis of hyperabsorber in the families was associated with a higher risk to be affected by high LDL cholesterol with OR 3.47 (confidence interval 1.60–7.51) and B 1.24 (P = 0.002). This risk was substantially increased when only family members from families with an hyperabsorber proband were considered ((OR = 6.80 (confidence interval 1.656–27.9), B = 1.917, P = 0.008)).
      Table 4Correlations between LDL-cholesterol and cholesterol absorption markers.
      Hyperabsorber families

      n = 53
      Non-hyperabsorber families

      n = 70
      All family members n = 103
      CoefficientPCoefficientPCoefficientP
      Cholestanol0.2320.0940.1080.3770.3050.002
      Sitosterol0.2680.050−0.0670.5830.1450.145
      Campesterol0.2160.120−0.1480.2240.1260.208
      Stigmasterol0.3330.016−0.0580.6380.0660.516
      All cholesterol absorption markers0.2660.057−0.0150.9020.2140.032
      In hyperabsorber families the percentage of affected hyperabsorber subjects was higher (76.5%) than in families with non-hyperabsorber probands (Fig. 1). Among non-affected subjects, the number of hyperabsorber and non-hyperabsorber subjects was identical. In non-hyperabsorber families, the percentage of non-affected non-hyperabsorber subjects was higher (73.2%) than in hyperabsorber families. The 60.5% of subjects in hyperabsorber families were hyperabsorbers and 76% of them were affected. The 38.3% of the subjects in non-hyperabsorber families were hyperabsorbers and 63% of them were affected (Fig. 1).
      Figure thumbnail gr1
      Figure 1Affected and non-affected subjects distribution in hyperabsorber and non-hyperabsorber families.

      4. Discussion

      This is the first study, to our knowledge, to analyze the cholesterol intestinal absorption in families with non-FH ADH. Our results show that 78% of the family members with high LDL cholesterol with a hyperabsorber proband show an increase in serum non-cholesterol sterols of intestinal origin, with a positive correlation between these sterols and LDL cholesterol levels. Because phytosterols and cholestanol are a well established tool to study cholesterol intestinal absorption, our results highly support that inter-individual variation in the efficiency of cholesterol absorption plays an important role in the pathophysiology of non-FH ADH. Previous reports had established that serum cholesterol and phytosterols are increased in this population [
      • García-Otín A.L.
      • Cofán M.
      • Junyent M.
      • Recalde D.
      • Cenarro A.
      • Pocoví M.
      • Ros E.
      • Civeira F.
      Increased intestinal cholesterol absorption in autosomal dominant hypercholesterolemia and no mutations in the low-density lipoprotein receptor or apolipoprotein B genes.
      ,
      • Lupattelli G.
      • Pirro M.
      • Siepi D.
      • Mannarino M.R.
      • Roscini A.R.
      • Vaudo G.
      • Pasqualini L.
      • Schillaci G.
      • Mannarino E.
      Non-cholesterol sterols in different forms of primary hyperlipemias.
      ], consequently, these data all together suggest that genetic variation in cholesterol absorption is involved in non-FH ADH families.
      There is a large interindividual difference in intestinal cholesterol absorption of cholesterol that is mainly due to genetic variation [
      • Gylling H.
      • Miettinen T.A.
      Inheritance of cholesterol metabolism of probands with high or low cholesterol absorption.
      ,
      • Sehayek E.
      • Nath C.
      • Heinemann T.
      • McGee M.
      • Seidman C.E.
      • Samuel P.
      • Breslow J.L.
      U-shape relationship between change in dietary cholesterol absorption and plasma lipoprotein responsiveness and evidence for extreme interindividual variation in dietary cholesterol absorption in humans.
      ]. Single nucleotide variation (SNV) in NPC1L1 and, ABCG5 and ABCG8 key modulators of cholesterol influx and efflux into intestinal mucosal cells, respectively, have been associated with the LDL cholesterol concentration in several populations including subjects with hypercholesterolemia [
      • Martín B.
      • Solanas-Barca M.
      • García-Otín A.L.
      • Pampín S.
      • Cofán M.
      • Ros E.
      • Rodríguez-Rey J.C.
      • Pocoví M.
      • Civeira F.
      An NPC1L1 gene promoter variant is associated with autosomal dominant hypercholesterolemia.
      ,
      • Gylling H.
      • Hallikainen M.
      • Pihlajamaki J.
      • Agren J.
      • Laakso M.
      • Rajaratnam R.A.
      • Rauramaa R.
      • Miettinen T.A.
      Polymorphisms in the ABCG5 and ABCG8 genes associate with cholesterol absorption and insulin sensitivity.
      ]; may explain interindividual variations in LDL cholesterol level in response to ezetimibe treatment [
      • Hegele R.A.
      • Guy J.
      • Ban M.R.
      • Wang J.
      NPC1L1 haplotype is associated with inter-individual variation in plasma low-density lipoprotein response to ezetimibe.
      ,
      • Simon J.S.
      • Karnoub M.C.
      • Devlin D.J.
      • Arreaza M.G.
      • Qiu P.
      • Monks S.A.
      • Severino M.E.
      • Deutsch P.
      • Palmisano J.
      • Sachs A.B.
      • Bayne M.L.
      • Plump A.S.
      • Schadt E.E.
      Sequence variation in NPC1L1 and association with improved LDL-cholesterol lowering in response to ezetimibe treatment.
      ], and several rare genotype variations in NPC1L1 are associated with mild reductions in sterol absorption, circulatory LDL cholesterol concentrations and cardiovascular disease [
      The Myocardial Infarction Genetics Consortium Investigators
      Inactivating mutations in NPC1L1 and protection from coronary heart disease.
      ]. In contrast, obligate heterozygous subjects with severe mutations in ABCG5 or ABCG8 causing sitosterolemia, show normal LDL cholesterol levels [
      • Bhattacharyya A.K.
      • Connor W.E.
      Beta-sitosterolemia and xanthomatosis. A newly described lipid storage disease in two sisters.
      ]; and extensive sequencing analysis of these genes in subjects with ADH has not detected causative mutations [
      • García-Otín A.L.
      • Cofán M.
      • Junyent M.
      • Recalde D.
      • Cenarro A.
      • Pocoví M.
      • Ros E.
      • Civeira F.
      Increased intestinal cholesterol absorption in autosomal dominant hypercholesterolemia and no mutations in the low-density lipoprotein receptor or apolipoprotein B genes.
      ]. All together indicate that the genetic interindividual variation in cholesterol absorption is not monogenic, but complex and probably polygenic, resulting from the effects of multiples SNVs common in the population, rather than the effects of rare mutation with substantial impact on cholesterol absorption [
      • Martín B.
      • Solanas-Barca M.
      • García-Otín A.L.
      • Pampín S.
      • Cofán M.
      • Ros E.
      • Rodríguez-Rey J.C.
      • Pocoví M.
      • Civeira F.
      An NPC1L1 gene promoter variant is associated with autosomal dominant hypercholesterolemia.
      ]. According with this concept, cholesterol hyperabsorption is a common phenomenon among family members from a hyperabsorber proband, both in subjects with high and with normal LDL cholesterol; and hyperabsorption is more common in affected subjects with high LDL cholesterol than in normolipemic individuals independently of the proband. Consequently, increased intestinal cholesterol absorption is a risk factor for the development of primary hypercholesterolemia but can not discriminate between affected and non-affected individuals.
      The genetic basis of non-FH is a relevant and controversial issue. In two different whole exome sequencing in ADH patients negative for LDLR/APOB/PCSK9 mutations [
      • Futema M.
      • Plagnol V.
      • Li K.
      • Whittall R.A.
      • Neil H.A.
      • Seed M.
      • Bertolini S.
      • Calandra S.
      • Descamps O.S.
      • Graham C.A.
      • Hegele R.A.
      • Karpe F.
      • Durst R.
      • Leitersdorf E.
      • Lench N.
      • Nair D.R.
      • Soran H.
      • Van Bockxmeer F.M.
      • Humphries S.E.
      Consortium SBUK10K Consortium
      Whole exome sequencing of familial hypercholesterolaemia patients negative for LDLR/APOB/PCSK9 mutations.
      ] and individuals selected for extreme LDL cholesterol (>98th percentile) [

      Lange LA, Hu Y, Zhang H, Xue C, Schmidt EM, Tang ZZ, et al, and The NHLBI grand opportunity exome sequencing project. Whole-exome sequencing identifies rare and low-frequency coding variants associated with LDL cholesterol. Am. J. Hum. Genet. 94:233–245.

      ] no major novel locus for FH was detected. A new locus at 4p13 associated with ADH was found in a family from The Netherlands, and the study of 400 additional unrelated ADH probands detected 4 missense variants in the STAP1 gene, encoding the protein signal transducing adapter family member 1, a new candidate gene for ADH, but in any case being responsible for a small proportion of non-FH ADH [
      • Fouchier S.W.
      • Dallinga-Thie G.M.
      • Meijers J.C.
      • Zelcer N.
      • Kastelein J.J.
      • Defesche J.C.
      • Hovingh G.K.
      Mutations in STAP1 are associated with autosomal dominant hipercolesterolemia.
      ]. A recent study in patients with non-FH ADH from two different countries has found that high LDL cholesterol concentrations in some of these subjects might have a polygenic cause, which would difficult a precise diagnosis and the efficiency of cascade testing in family members [
      • Talmud P.J.
      • Shah S.
      • Whittall R.A.
      • Futema M.
      • Howard P.
      • Cooper J.A.
      • Harrison S.C.
      • Li K.W.
      • Drenos F.
      • Karpe F.
      • Neil H.A.
      • Descamps O.S.
      • Langenberg C.
      • Lench N.
      • Kivimaki M.
      • Whittaker J.
      • Hingorani A.D.
      • Kumari M.
      • Humphries S.E.
      Use of low-density lipoprotein cholesterol gene score to distinguish patients with polygenic and monogenic familial hypercholesterolaemia: a case-control study.
      ]. Some non-hyperabsorber family members showed high TG what suggests the existing of overlapping between non-FH ADH and FCH. Furthermore, patients with a detected causal mutation in the candidate genes have a substantial polygenic contribution that might contribute to the variable penetrance of the disease and to the large overlap in LDL cholesterol concentrations in mutation-carrier and non-carrier relatives [
      • Talmud P.J.
      • Shah S.
      • Whittall R.A.
      • Futema M.
      • Howard P.
      • Cooper J.A.
      • Harrison S.C.
      • Li K.W.
      • Drenos F.
      • Karpe F.
      • Neil H.A.
      • Descamps O.S.
      • Langenberg C.
      • Lench N.
      • Kivimaki M.
      • Whittaker J.
      • Hingorani A.D.
      • Kumari M.
      • Humphries S.E.
      Use of low-density lipoprotein cholesterol gene score to distinguish patients with polygenic and monogenic familial hypercholesterolaemia: a case-control study.
      ]. Our study supports the concept that what we had previously considered ADH, in most cases, are in fact complex metabolic diseases in which different mechanism are involved, one of them being hyperabsorption, and probably with a polygenic contribution, and with environmental interactions. Affected subjects with hyperabsorption show lower BMI than affected non-hyperabsorber subjects in our study (Table 3). This relationship between plasma phytosterols and BMI has been already established, and leaner individuals have increased phytosterols plasma levels than subjects with obesity or metabolic syndrome (MetS) [
      • Cofan M.
      • Escurriol V.
      • Garcia-Otin A.L.
      • Moreno-Iribas C.
      • Larranaga N.
      • Sanchez M.J.
      • Tormo M.J.
      • Redondo M.L.
      • Gonzalez C.A.
      • Corella D.
      • Pocovi M.
      • Civeira F.
      • Ros E.
      Association of plasma markers of cholesterol homeostasis with metabolic syndrome components. A cross-sectional study.
      ]. The mechanism of this association has not been fully established. The weak correlation between LDL cholesterol and intestinal cholesterol markers would support the complex and probably polygenic background of the association. Furthermore, the diagnosis of ADH should be redefined because with the present diagnostic criteria [
      • Civeira F.
      International Panel on Management of Familial Hypercholesterolemia
      Guidelines for the diagnosis and management of heterozygous familial hypercholesterolemia.
      ,
      • Nordestgaard B.G.
      • Chapman M.J.
      • Humphries S.E.
      • Ginsberg H.N.
      • Masana L.
      • Descamps O.S.
      • Wiklund O.
      • Hegele R.A.
      • Raal F.J.
      • Defesche J.C.
      • Wiegman A.
      • Santos R.D.
      • Watts G.F.
      • Parhofer K.G.
      • Hovingh G.K.
      • Kovanen P.T.
      • Boileau C.
      • Averna M.
      • Borén J.
      • Bruckert E.
      • Catapano A.L.
      • Kuivenhoven J.A.
      • Pajukanta P.
      • Ray K.
      • Stalenhoef A.F.
      • Stroes E.
      • Taskinen M.R.
      • Tybjærg-Hansen A.
      Familial hypercholesterolaemia is underdiagnosed and undertreated in the general population: guidance for clinicians to prevent coronary heart disease.
      ] many subjects could be diagnosed of FH, suggesting an inexistent monogenic disorder.
      In conclusion, serum phytosterols and cholestanol, markers of cholesterol intestinal absorption efficiency, are frequently increased in non-FH ADH subjects. There is a high percentage of first-degree relatives of hyperabsorber probands that present a hyperabsorber phenotype, and this percentage of hyperabsorber subjects is higher in families with hyperabsorber probands than in families with non-hyperabsorber probands. Most hypercholesterolemic family members with a hyperabsorber proband are hyperabsorbers. These absorption markers are significantly and positively associated with LDL cholesterol, and predispose to high LDL cholesterol in family members. However, the cosegregation of both phenotypes is not compatible with a monogenic defect in the intestinal cholesterol absorption mechanism. Our data suggest that complex interindividual variation in cholesterol absorption is involved in many non-FH ADH.

      Acknowledgments

      This study was supported by grants from the Spanish Ministry of Economy and Competitiveness PI10/00387 , PI12/01087 , PI12/01703 , IPT-2011-0817-010000 , and RIC Red de Investigación Cardiovascular (RIC) RD12/0042/0055 . RIC is an initiative of Instituto de Salud Carlos III (ISCIII), Spain. Authors thank Proteomic Unit, Instituto de Investigación Sanitaria Aragón (IIS), ProteoRed member, for technical support.

      Appendix A. Supplementary data

      The following is the supplementary data related to this article:

      References

        • Civeira F.
        • International Panel on Management of Familial Hypercholesterolemia
        Guidelines for the diagnosis and management of heterozygous familial hypercholesterolemia.
        Atherosclerosis. 2004; 173: 55-68
        • Nordestgaard B.G.
        • Chapman M.J.
        • Humphries S.E.
        • Ginsberg H.N.
        • Masana L.
        • Descamps O.S.
        • Wiklund O.
        • Hegele R.A.
        • Raal F.J.
        • Defesche J.C.
        • Wiegman A.
        • Santos R.D.
        • Watts G.F.
        • Parhofer K.G.
        • Hovingh G.K.
        • Kovanen P.T.
        • Boileau C.
        • Averna M.
        • Borén J.
        • Bruckert E.
        • Catapano A.L.
        • Kuivenhoven J.A.
        • Pajukanta P.
        • Ray K.
        • Stalenhoef A.F.
        • Stroes E.
        • Taskinen M.R.
        • Tybjærg-Hansen A.
        Familial hypercholesterolaemia is underdiagnosed and undertreated in the general population: guidance for clinicians to prevent coronary heart disease.
        Eur. Heart J. 2013; 34 (consensus statement of the European Atherosclerosis Society. European Atherosclerosis Society Consensus Panel): 3478-3490
        • Vrablík M.
        • Ceska R.
        • Horínek A.
        Major apolipoprotein B-100 mutations in lipoprotein metabolism and atherosclerosis.
        Physiol. Res. 2001; 50: 337-343
        • Soutar A.K.
        • Naoumova R.P.
        Mechanisms of disease: genetic causes of familial hypercholesterolemia.
        Nat. Clin. Pract. Cardiovasc. Med. 2007; 4: 214-225
        • Solanas-Barca M.
        • de Castro-Orós I.
        • Mateo-Gallego R.
        • Cofán M.
        • Plana N.
        • Puzo J.
        • Burillo E.
        • Martín-Fuentes P.
        • Ros E.
        • Masana L.
        • Pocoví M.
        • Civeira F.
        • Cenarro A.
        Apolipoprotein E gene mutations in subjects with mixed hyperlipidemia and a clinical diagnosis of familial combined hyperlipidemia.
        Atherosclerosis. 2012; 222: 449-455
        • Awan Z.
        • Choi H.Y.
        • Stitziel N.
        • Ruel I.
        • Bamimore M.A.
        • Husa R.
        • Gagnon M.H.
        • Wang R.H.
        • Peloso G.M.
        • Hegele R.A.
        • Seidah N.G.
        • Kathiresan S.
        • Genest J.
        APOE p.Leu167del mutation in familial hypercholesterolemia.
        Atherosclerosis. 2013; 231: 218-222
        • Palacios L.
        • Grandoso L.
        • Cuevas N.
        • Olano-Martín E.
        • Martinez A.
        • Tejedor D.
        • Stef M.
        Molecular characterization of familial hypercholesterolemia in Spain.
        Atherosclerosis. 2012; 221: 137-142
        • Futema M.
        • Whittall R.A.
        • Kiley A.
        • Steel L.K.
        • Cooper J.A.
        • Badmus E.
        • Leigh S.E.
        • Karpe F.
        • Neil H.A.
        Analysis of the frequency and spectrum of mutations recognised to cause familial hypercholesterolaemia in routine clinical practice in a UK specialist hospital lipid clinic.
        Atherosclerosis. 2013; 229: 161-168
        • Talmud P.J.
        • Shah S.
        • Whittall R.A.
        • Futema M.
        • Howard P.
        • Cooper J.A.
        • Harrison S.C.
        • Li K.W.
        • Drenos F.
        • Karpe F.
        • Neil H.A.
        • Descamps O.S.
        • Langenberg C.
        • Lench N.
        • Kivimaki M.
        • Whittaker J.
        • Hingorani A.D.
        • Kumari M.
        • Humphries S.E.
        Use of low-density lipoprotein cholesterol gene score to distinguish patients with polygenic and monogenic familial hypercholesterolaemia: a case-control study.
        Lancet. 2013; 381: 1293-1301
        • Kesäniemi Y.A.
        • Miettinen T.A.
        Metabolic epidemiology of plasma cholesterol.
        Ann. Clin. Res. 1988; 20: 26-31
        • García-Otín A.L.
        • Cofán M.
        • Junyent M.
        • Recalde D.
        • Cenarro A.
        • Pocoví M.
        • Ros E.
        • Civeira F.
        Increased intestinal cholesterol absorption in autosomal dominant hypercholesterolemia and no mutations in the low-density lipoprotein receptor or apolipoprotein B genes.
        J. Clin. Endocrinol. Metab. 2007; 92: 3667-3673
        • Lupattelli G.
        • Pirro M.
        • Siepi D.
        • Mannarino M.R.
        • Roscini A.R.
        • Vaudo G.
        • Pasqualini L.
        • Schillaci G.
        • Mannarino E.
        Non-cholesterol sterols in different forms of primary hyperlipemias.
        Nutr. Metab. Cardiovasc. Dis. 2012; 22: 231-236
        • Miettinen T.A.
        • Tilvis R.S.
        • Kesäniemi Y.A.
        Serum plant sterols and cholesterol precursors reflect cholesterol absorption and synthesis in volunteers of a randomly selected male population.
        Am. J. Epidemiol. 1990; 131: 20-31
        • Matthan N.R.
        • Raeini-Sarjaz M.
        • Lichtenstein A.H.
        • Ausman L.M.
        • Jones P.J.
        Deuterium uptake and plasma cholesterol precursor levels correspond as methods for measurement of endogenous cholesterol synthesis in hypercholesterolemic women.
        Lipids. 2000; 35: 1037-1044
        • Gylling H.
        • Miettinen T.A.
        Inheritance of cholesterol metabolism of probands with high or low cholesterol absorption.
        J. Lipid Res. 2002; 43: 1472-1476
        • Gómez-Gerique J.A.
        • Gutiérrez-Fuentes J.A.
        • Montoya M.T.
        • Porres A.
        • Rueda A.
        • Avellaneda A.
        • Rubio M.A.
        [Lipid profile of the Spanish population: the DRECE (diet and risk of cardiovascular disease in Spain) study. DRECE study group].
        Med. Clin. (Barc.). 1999; 113: 730-735
        • de la Fuente-Arrillaga C.
        • Vazquez-Ruiz Z.
        • Bes-Rastrollo M.
        • Sampson L.
        • Martinez-González M.A.
        Reproducibility of an FFQ validated in Spain.
        Public Health Nutr. 2010; 28: 1-9
        • Baila-Rueda L.
        • Cenarro A.
        • Cofan M.
        • Orera I.
        • Barcelo-Batllori S.
        • Pocovi M.
        • Ros E.
        • Civeira F.
        • Nerin C.
        • Domeno C.
        Simultaneous determination of oxysterols, phytosterols and cholesterol precursors by high performance liquid chromatography tandem mass spectrometry in human serum.
        Anal. Methods. 2013; 5: 2249-2257
        • Sehayek E.
        • Nath C.
        • Heinemann T.
        • McGee M.
        • Seidman C.E.
        • Samuel P.
        • Breslow J.L.
        U-shape relationship between change in dietary cholesterol absorption and plasma lipoprotein responsiveness and evidence for extreme interindividual variation in dietary cholesterol absorption in humans.
        J. Lipid Res. 1998; 39: 2415-2422
        • Martín B.
        • Solanas-Barca M.
        • García-Otín A.L.
        • Pampín S.
        • Cofán M.
        • Ros E.
        • Rodríguez-Rey J.C.
        • Pocoví M.
        • Civeira F.
        An NPC1L1 gene promoter variant is associated with autosomal dominant hypercholesterolemia.
        Nutr. Metab. Cardiovasc. Dis. 2010; 20: 236-242
        • Gylling H.
        • Hallikainen M.
        • Pihlajamaki J.
        • Agren J.
        • Laakso M.
        • Rajaratnam R.A.
        • Rauramaa R.
        • Miettinen T.A.
        Polymorphisms in the ABCG5 and ABCG8 genes associate with cholesterol absorption and insulin sensitivity.
        J. Lipid Res. 2004; 45: 1660-1665
        • Hegele R.A.
        • Guy J.
        • Ban M.R.
        • Wang J.
        NPC1L1 haplotype is associated with inter-individual variation in plasma low-density lipoprotein response to ezetimibe.
        Lipids Health Dis. 2005; 4: 16
        • Simon J.S.
        • Karnoub M.C.
        • Devlin D.J.
        • Arreaza M.G.
        • Qiu P.
        • Monks S.A.
        • Severino M.E.
        • Deutsch P.
        • Palmisano J.
        • Sachs A.B.
        • Bayne M.L.
        • Plump A.S.
        • Schadt E.E.
        Sequence variation in NPC1L1 and association with improved LDL-cholesterol lowering in response to ezetimibe treatment.
        Genomics. 2005; 86: 648-656
        • The Myocardial Infarction Genetics Consortium Investigators
        Inactivating mutations in NPC1L1 and protection from coronary heart disease.
        N. Engl. J. Med. 2014; 371: 2072-2082
        • Bhattacharyya A.K.
        • Connor W.E.
        Beta-sitosterolemia and xanthomatosis. A newly described lipid storage disease in two sisters.
        J. Clin. Invest. 1974; 53: 1033-1043
        • Futema M.
        • Plagnol V.
        • Li K.
        • Whittall R.A.
        • Neil H.A.
        • Seed M.
        • Bertolini S.
        • Calandra S.
        • Descamps O.S.
        • Graham C.A.
        • Hegele R.A.
        • Karpe F.
        • Durst R.
        • Leitersdorf E.
        • Lench N.
        • Nair D.R.
        • Soran H.
        • Van Bockxmeer F.M.
        • Humphries S.E.
        • Consortium SB
        • UK10K Consortium
        Whole exome sequencing of familial hypercholesterolaemia patients negative for LDLR/APOB/PCSK9 mutations.
        J. Med. Genet. 2014; 51: 537-544
      1. Lange LA, Hu Y, Zhang H, Xue C, Schmidt EM, Tang ZZ, et al, and The NHLBI grand opportunity exome sequencing project. Whole-exome sequencing identifies rare and low-frequency coding variants associated with LDL cholesterol. Am. J. Hum. Genet. 94:233–245.

        • Fouchier S.W.
        • Dallinga-Thie G.M.
        • Meijers J.C.
        • Zelcer N.
        • Kastelein J.J.
        • Defesche J.C.
        • Hovingh G.K.
        Mutations in STAP1 are associated with autosomal dominant hipercolesterolemia.
        Circ. Res. 2014; 115: 552-555
        • Cofan M.
        • Escurriol V.
        • Garcia-Otin A.L.
        • Moreno-Iribas C.
        • Larranaga N.
        • Sanchez M.J.
        • Tormo M.J.
        • Redondo M.L.
        • Gonzalez C.A.
        • Corella D.
        • Pocovi M.
        • Civeira F.
        • Ros E.
        Association of plasma markers of cholesterol homeostasis with metabolic syndrome components. A cross-sectional study.
        Nutr. Metab. Cardiovasc. Dis. 2011; 21: 651-657