Apolipoprotein E gene mutations in subjects with mixed hyperlipidemia and a clinical diagnosis of familial combined hyperlipidemia

Atherosclerosis 222 (2012) 449–455

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Apolipoprotein E gene mutations in subjects with mixed hyperlipidemia and a clinical diagnosis of familial combined hyperlipidemia María Solanas-Barca a , Isabel de Castro-Orós a,b , Rocío Mateo-Gallego a , Montserrat Cofán c,d , Nuria Plana e,f , José Puzo g , Elena Burillo a , Paula Martín-Fuentes a , Emilio Ros c,d , Luis Masana e,f , Miguel Pocoví b , Fernando Civeira a , Ana Cenarro a,∗ a

Hospital Universitario Miguel Servet, Instituto Aragonés de Ciencias de la Salud (I+CS), Zaragoza, Spain Departamento de Bioquímica, Biología Molecular y Celular, Universidad de Zaragoza and CIBER de Enfermedades Raras (CIBERer), Spain c Lipid Clinic, Endocrinology and Nutrition Service, Institut d’Investigacions Biomèdiques August Pi Sunyer, Hospital Clínic, Barcelona, Spain d CIBER Fisiopatología de la Obesidad y Nutrición (CIBERobn), Instituto de Salud Carlos III (ISCIII), Spain e Hospital Universitari Sant Joan, IISPV, Universitat Rovira i Virgili, Reus, Spain f CIBER Diabetes y Enfermedades Metabólicas Asociadas (CIBERdem), ISCIII, Spain g Servicio de Bioquímica, Hospital San Jorge, Huesca, Spain b

a r t i c l e

i n f o

Article history: Received 12 January 2012 Received in revised form 9 February 2012 Accepted 9 March 2012 Available online 16 March 2012 Keywords: Apolipoprotein E Familial combined hyperlipidemia Genetic defects Familial dysbetalipoproteinemia

a b s t r a c t Objective: Rare mutations in the APOE gene, undetectable with the usual genotyping technique, are responsible for dominant familial dysbetalipoproteinemia (FD) and therefore could be easily misclassified as familial combined hyperlipidemia (FCHL). We aimed to identify APOE mutations associated with dominant combined hyperlipoproteinemia and to establish their frequency in subjects with a clinical diagnosis of FCHL. Methods and results: In 279 unrelated subjects with FCHL in whom a functional LDLR mutation was excluded, sequencing of the entire APOE gene detected 9 carriers of a rare mutation: 5 subjects (1.8%) with the R136S mutation (arginine at residue 136 changed to serine) and 4 subjects (1.4%) with the p.Leu149del mutation, a 3-bp inframe deletion that results in the loss of leucine at position 149. Both genetic defects were detected with similar frequency (2.5% and 1.3%, respectively) in an independent group of 160 FCHL subjects from other locations in Spain. Family studies demonstrated cosegregation of these APOE mutations with hyperlipoproteinemia. R136S carriers showed dysbetalipoproteinemia, while the lipid phenotype of p.Leu149del carriers was IIa or IIb. Conclusions: Rare APOE mutations are responsible for approximately 3.5% of FCHL cases in our population. APOE R136S and p.Leu149del induce autosomal dominant FD and a phenotype indistinguishable from FCHL, respectively. © 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Familial combined hyperlipidemia (FCHL) is a common disorder of lipid metabolism characterized by elevated levels of total cholesterol and triglycerides, vertical transmission of a hyperlipidemic phenotype within the family, and high risk of premature cardiovascular disease (CVD) [1]. FCHL is the most common genetic lipid

Abbreviations: FH, familial hypercholesterolemia; FD, familial dysbetalipoproteinemia; FCHL, familial combined hyperlipidemia; CVD, cardiovascular disease; Apo, apolipoprotein; LDL, low-density lipoprotein; VLDL, very low-density lipoprotein; IDL, intermediate-density lipoprotein; HDL, high-density lipoprotein; IR, interquartile ranges. ∗ Corresponding author at: Hospital Universitario Miguel Servet, Laboratorio de Investigación Molecular, I+CS. Avda. Isabel La Católica, 1-3, 50009 Zaragoza, Spain. Tel.: +34 976 765500; fax: +34 976 765478. E-mail address: [email protected] (A. Cenarro). 0021-9150/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2012.03.011

abnormality found in patients with premature coronary artery disease [2] and a major risk factor for the occurrence of myocardial infarction (MI) at a young age. In a recent study, as many as 38% patients with MI below age 40 years had a diagnosis of FCHL [3]. FCHL was described by Goldstein et al. as a dominant genetic disease [4], although current knowledge about the disease supports the notion that FCHL is highly heterogeneous in its genetic background, penetrance, and lipid phenotype [5]. Although some FCHL cases are monogenic, FCHL must be considered a complex disease in most patients, with interaction of several causal genes, mostly unknown, with multiple variations in modifier genes and environmental factors, noticeably visceral adiposity, insulin resistance, or full-blown diabetes [1]. FCHL is the most common cause of primary mixed hyperlipidemia in the population, with a prevalence of nearly 6% [6]. Other causes of the FCHL phenotype include familial dysbetalipoproteinemia (FD) and some hypertriglyceridemic forms of familial

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hypercholesteromia (FH) due to mutations in the genes coding for apolipoprotein E (APOE) and the low-density lipoprotein receptor (LDLR), respectively. We have recently described the overlapping lipid phenotype between FCHL and FH in subjects with a clinical diagnosis of FCHL in whom a LDLR mutation could be demonstrated [7]. Traditionally, the differential diagnosis between FCHL and FD was based on the family history of hyperlipidemia. FCHL is characterized by a dominant presentation in the family and FD is usually sporadic because most cases are recessive and two APOE defective alleles (␧2) plus additional environmental factors are required for full expression of dyslipidemia [8]. Other clinical criteria used in the differential diagnosis include the rare presence of palmar xanthomas in FD and a milder lipid phenotype, but with higher serum apo B concentration, in FCHL [9]. The diagnosis of dysbetalipoproteinemia is based on the demonstration of elevated very-low-density lipoprotein (VLDL) remnants and intermediate-density lipoproteins (IDL) after separation of lipoproteins by ultracentrifugation. The presence of a VLDL-cholesterol (mg/dL)/total triglycerides (mg/dL) ratio >0.30 has been considered characteristic of the disease [8]. However, ultracentrifugation is costly and time-consuming, and it is not standardized or available in many laboratories. Furthermore, the lipid phenotype is highly variable in FCHL and FD and lipid remnants are often elevated also in FCHL [10]. By contrast, the genotype analysis of the 3 common polymorphisms of APOE: ␧2, ␧3 and ␧4 that code for the apo E2, E3 and E4 isoforms is a simple method available in most laboratories and has substituted the traditional ultracentrifugation technique because the detection of an APOE ␧2/␧2 genotype provides an unequivocal diagnosis of FD [8,11]. Although less common, dominant mutations in APOE may also cause FD. These rare mutations are not detected by using the usual APOE genotyping methodology, thus subjects carrying them could be easily misclassified as FCHL bearers. The objective of this study was to identify APOE mutations associated with dominant combined hyperlipoproteinemia and to establish their frequency in subjects with a clinical diagnosis of FCHL.

defined as occurring before 55 and 65 years of age in men and women respectively. Informed consent was obtained from all subjects and the ethical committee from each institution approved the study. Procedures were in accordance with the Helsinki Declaration of 1975, as revised in 2000. 2.2. Serum lipid and lipoprotein determinations To obtain a baseline lipid profile in asymptomatic subjects, overnight fasting blood was drawn after at least 4 weeks without hypolipidemic drug treatment in each Spanish lipid clinic participating in the study. In patients with CVD, off-treatment lipid levels prior to the cardiovascular event were obtained from review of clinical histories. Serum total cholesterol (TC), triglycerides (TG) and HDLcholesterol levels were determined with standardized enzymatic procedures. Non-HDL cholesterol was calculated as TC minus HDLcholesterol. Apolipoproteins (apo) AI and B were determined by using immunoturbidimetry (Unimate 3, Roche, Basel, Switzerland). Lipoprotein(a) was determined by using kinetic immunonephelometry with polyclonal antibodies (Beckman Coulter Immage Immunochemistry System, Beckman Coulter Inc., USA). VLDL (density <1.006 g/mL) and IDL (density 1.006–1.019 g/mL) fractions were isolated by sequential ultracentrifugation, and cholesterol and triglycerides in these fractions were determined as indicated above. 2.3. Genetic analyses 2.3.1. LDLR and APOB genes The screening of mutations in LDLR and APOB genes was performed with Lipochip® , version 9.0 (Progenika, Derio, Spain). This platform is a microarray device that includes the diagnosis of 230 different causative mutations in the LDLR gene, 2 mutations in APOB gene, and 3 mutations in PCSK9 gene. The microarray design, fabrication, and quality controls, the target DNA preparation and hybridization, the microarray scanning and quantification, and the genotyping software have been previously described in detail [14].

2. Materials and methods 2.1. Study subjects Consecutive unrelated subjects (n = 312) aged 18–80 years (205 men, 107 women) with a clinical diagnosis of familial mixed hyperlipidemia attending the Lipid Clinic of the Hospital Universitario Miguel Servet (Zaragoza, Spain) were recruited from January 2008 to April 2011. A clinical diagnosis of FCHL was made in subjects with off-treatment total cholesterol and triglyceride serum concentrations above the age- and sex-specific 90th percentiles of a Spanish reference population [12] and existence of a first-degree relative with a similar lipid phenotype. Secondary hyperlipidemia, including hypothyroidism, renal disease, diabetes mellitus, cholestasis, and the use of drugs affecting lipid metabolism was ruled out in all subjects. Detection of an APOE ␧2/␧2 genotype was a criterion for exclusion from the FCHL diagnosis, but the characteristics of these subjects were recorded for comparison with other lipid phenotypes. Also, a sample of 264 normolipidemic subjects, previously reported [13], was used as control group. To validate the results, an independent group of 160 subjects with FCHL from Hospital Clinic, Barcelona, and Hospital Universitari Sant Joan, Reus, Spain was selected with the same inclusion and exclusion criteria. Clinical data, history of CVD, demographic and anthropometric measurement, and a physical examination in search of tendon xanthomas were performed in each subject. Early-onset CVD was

2.4. APOE gene sequencing The 4 exons and their flanking intron sequences of the APOE gene were sequenced in cases and controls. Exons 1 and 2 were amplified independently using overlapping primers with the object of being linked by a second PCR reaction in only one fragment for sequencing. Exon 3 was amplified and sequenced in one fragment and exon 4 was sequenced in 2 overlapping fragments. The PCR products were sequenced using the MegaBACETM Dye Terminator system (GEHealthcare). The primers used are shown in Supplementary Table 1. 2.5. Statistical analyses Data are presented as mean ± standard deviation (SD) for variables with normal distribution, or median (interquartile ranges, IR) for variables with skewed distribution, and as percentage for categorical variables. An analysis of variance (ANOVA), or the Kruskal–Wallis, combined with an a posteriori test, as appropriate, were used to evaluate the statistical significance of the differences between groups. Categorical variables were compared using the 2 test. Allele frequencies were estimated as observed proportions and compared by calculating odds ratios and 95% confidence intervals (CI). p < 0.05 was required for statistical significance. Analyses were performed with SPSS v.15.0 software (SPSS Inc., Chicago, IL).

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Table 1 Clinical, anthropometric, and lipid characteristics of study subjects by lipid diagnosis.

Number of subjects Age, years Male, % Smoking Never, % Current, % Former, % Cardiovascular disease, % Body mass index, kg/m2 Total cholesterol, mg/dL Triglycerides, mg/dL HDL cholesterol, mg/dL Non-HDL cholesterol, mg/dL Lipoprotein(a), mg/dL Apolipoprotein AI, mg/dL Apolipoprotein B, mg/dL

FCHL

FD

FH

Control

p

279 48.1 ± 11.7 64.0

10 47.8 ± 7.1 70.0

23 49.4 ± 10.3 82.6

264 43.5 ± 16.9 49.8

0.002 0.001

35.1 30.8 34.1 13.0 28.7 ± 3.6 294 ± 46.8 278 (209–431) 41 (34–49) 251 ± 45.9 23.0 (7.0–54.5) 137 (121–156) 163 ± 30.8

33.3 22.2 44.4 20.0 29.2 ± 2.5 335 ± 124 342 (244–651) 37 (32–54) 293 ± 117 6.9 (2.5–37.2) 125 (113–136) 111 ± 23.7

26.1 47.8 26.1 26.1 28.3 ± 3.5 351 ± 73.2 280 (219–360) 38 (33–44) 313 ± 70.5 18.3 (8.8–68.5) 124 (101–145) 191 ± 43.3

59.2 20.2 20.6 0 25.0 ± 4.3 194 ± 33.1 71 (49–103) 50 (42–60) 143 ± 32.3 15 (6.7–38.0) 141 (126–159) 99.5 ± 24.2

<0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.047 0.004 <0.001

Data are presented as mean ± standard deviation (SD) for variables with normal distribution, or median (interquartile ranges, IR) for variables with skewed distribution, and as percentage for categorical variables. An analysis of variance (ANOVA), or the Kruskal–Wallis test, as appropriate, were used to evaluate the statistical significance of the differences between groups. Categorical variables were compared using the 2 test.

3. Results From the group of 312 subjects with primary combined hyperlipidemia, 10 subjects were identified as ␧2/␧2 genotype, and were therefore classified as having FD. Another 22 subjects were carriers of a functional mutation in the LDLR gene and they were classified as FH with hypertriglyceridemia. One subject was heterozygous for a mutation in the LDLR associated with an APOE ␧2/␧2 genotype and was considered as a FH case. The remaining 279 subjects were classified as FCHL. APOE gene sequencing in this group identified 9 subjects carrying a rare mutation in APOE: 5 subjects (1.8%) were carriers of the R136S mutation (arginine at residue 136 changed to serine) and 4 subjects (1.4%) were carriers of the p.Leu149del mutation, a 3-bp inframe deletion that results in the loss of a leucine at position 149. Anthropometric, clinic and lipid characteristics of studied subjects by groups are shown in Table 1. In the combined hyperlipidemia groups, subjects were older and more frequently male and smoked more, and had a higher BMI and a higher frequency of prior CVD than control group. As expected, by inclusion criteria, lipid levels were higher in combined hyperlipidemia groups than in controls. TC and non-HDL cholesterol levels were higher in FH with hypertriglyceridemia than in the other two hyperlipidemic groups. Triglyceride levels were higher and apo B was lower in the FD sample compared to the other hyperlipidemic groups (Table 1). Tables 2 and 3 show the genotypic and allelic frequencies of APOE, respectively. R136S and p.Leu149del mutations in APOE were found only in the FCHL group. The allelic frequency of ␧4 allele was significantly higher among FCHL subjects than in the control group.

All available family members of probands with the R136S and p.Leu149del mutations in APOE, 12 and 16 subjects, respectively, were studied for clinical, biochemical, and genetic characteristics. There was a clear cosegregation of the mutations with hyperlipoproteinemia (see Fig. 1). All 8 family members with the R136S mutation disclosed combined hyperlipidemia. However, 5 out of 8 family members with the p.Leu149del mutation showed isolated hypercholesteromia. In order to better define the type of hyperlipidemia of APOE gene mutation carriers, we isolated the VLDL fraction by ultracentrifugation from plasma of all available subjects, probands and family members carrying the R136S (8 subjects) and p.Leu149del mutations (7 subjects), as well as in 7 ␧2/␧2 carriers, and 10 ␧3/␧3 FCHL subjects, all of them untreated, and without CVD or high vascular risk. In Table 4, the results of lipids, lipoproteins, and lipid/lipid and lipid/apo B ratios are shown by APOE genotype. Subjects carrying the R136S mutation had the highest level of triglycerides, whereas subjects carrying the p.Leu149del mutation had the lowest level. The p.Leu149del carriers and FCHL ␧3/␧3 subjects disclosed significantly higher apo B concentrations than those of FD ␧2/␧2 subjects and R136S carriers. In addition, VLDL-cholesterol, VLDL-cholesterol/triglyceride ratios, total cholesterol/apo B ratios, and non-HDL cholesterol/apo B ratios were significantly higher in ␧2/␧2 subjects and R136S carriers than in FCHL ␧3/␧3 subjects and p.Leu149del carriers. To validate these findings, we analyzed an independent group of 160 subjects with a clinical diagnosis of FCHL from Lipid Clinics from Northeast of Spain (Barcelona and Reus). Clinical and lipid variables were similar to those of the initial population, as expected given that the same inclusion criteria were applied (Supplementary Table 2). In this independent FCHL sample, we identified the same

Table 2 APOE genotypic frequencies in the study groups.

Number of subjects APOE genotype ␧2/␧2, n (%) ␧2/␧3, n (%) ␧2/␧4, n (%) ␧3/␧3, n (%) ␧3/␧4, n (%) ␧4/␧4, n (%) R136Sa /␧3, n (%) p.Leu149dela /␧3, n (%)

FCHL

FD

FH

Control

279

10

23

264

0 25 (9.0) 5 (1.8) 172 (61.6) 60 (21.5) 8 (2.9) 5 (1.8) 4 (1.4)

10 (100) 0 0 0 0 0 0 0

1 (4.3) 0 0 17 (73.9) 5 (21.7) 0 0 0

1 (0.4) 27 (10.2) 4 (1.5) 183 (69.3) 45 (17.0) 4 (1.5) 0 0

Data are presented as number and percentage. Differences in genotype distribution between groups were compared using the 2 test. a The R136S mutation was detected in the ␧2 allele in one subject and in the ␧3 allele in 4 subjects. The p.Leu149del mutation was in the ␧3 allele in all subjects.

p

<0.001

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Table 3 APOE allelic frequencies in FCHL and controls. FCHL Number of alleles APOE allele E2, n (%) E3, n (%) E4, n (%) R136S, n (%) p.Leu149del, n (%)

Control

558

528

31 (5.55) 437 (78.3) 81 (14.5) 5 (0.90) 4 (0.72)

33 (6.25) 438 (82.9) 57 (10.8) 0 0

Odds ratio (95% confidence interval)

P

0.94 (0.57–1.53) 1.00 (0.87–1.14) 1.42 (1.01–1.99) – –

0.004

Data are presented as number and percentage. Differences in genotype distribution between groups were compared using the 2 test. Allele frequencies were estimated as observed proportions and compared by calculating odds ratios and 95% confidence intervals.

(A) Age TC TG HDLc APOE

57 346 570 50 R136S/E3

60 240 73 45 E3/E3

NS

Age TC TG HDLc APOE

Age TC TG HDLc APOE

30 358 455 35 R136S/E3

60 195 84 50 E3/E2

24 276 312 60 R136S/E3

57 210 124 45 E3/E4

Age TC TG HDLc APOE

52 285 328 40 R136S/E4

Age TC TG HDLc APOE

NS

65 334 432 42 R136S/E3

62 227 278 42 R136S/E3

40 389 576 28 R136S/E136

50 275 300 32 R136S/E2

20 180 86 50 E3/E2

Age TC TG HDLc APOE

18 246 310 40 R136S/E3

48 299 341 39 R136S/E2

18 305 633 39 R136S/E3

Fig. 1. Family trees of probands with rare APOE mutations. (A) Families carrying the APOE R136S mutation. (B) Families carrying the APOE p.Leu149del mutation. Black symbols, mutation carriers. White symbols, no mutation carriers. Arrow indicates proband. TC, total cholesterol (mg/dL); TG, triglycerides (mg/dL); HDLc, cholesterol transported by HDL (mg/dL); APOE, APOE genotype; NS, not studied.

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453

(B) Age TC TG HDLc APOE

55 348 292 58 p.Leu149del/E3

Age TC TG HDLc APOE

24 316 63 68 p.Leu149del/E3

53 185 98 50 E3/E3

Age TC TG HDLc APOE

Age TC TG HDLc APOE

22 314 142 45 p.Leu149del/E3

Age TC TG HDLc APOE

Age TC TG HDLc APOE

Age TC TG HDLc APOE

31 281 114 40 p.Leu149del/E3

56 346 437 40 p.Leu149del/E3

29 390 290 40 p.Leu149del/E3

21 232 228 42 p.Leu149del/E3

84 380 72 82 p.Leu149del/E3

84 249 286 32 E3/E3

55 284 274 46 p.Leu149del/E3

58 287 62 62 E3/E3

28 250 72 42 E3/E3

46 195 87 55 E3/E3

49 338 204 51 p.Leu149del/E3

18 219 87 31 E3/E3

24 296 120 83 p.Leu149del/E3

53 175 105 53 E3/E3

18 169 50 58 E3/E3

Fig. 1. (continued).

rare mutations in APOE, R136S and p.Leu149del, with similar frequencies than in the original population (Supplementary Table 3). 4. Discussion This is the first study, to our knowledge, to fully sequence APOE in subjects with the FCHL phenotype. The novel finding is that rare APOE mutations associated with a dominant transmission pattern are responsible for some cases of FCHL in our population. Several studies have previously examined the contribution of common APOE genetic polymorphisms ␧2, ␧3 and ␧4 to FCHL and, as in

our study, showed that the ␧4 allele was overrepresented in this phenotype [15–17]. The mechanism of this genetic association is probably due to the well known effect of the ␧4 allele increasing the TC concentration [18]. Subjects with the ␧4 allele have total and LDL cholesterol approximately 5% higher than ␧3/␧3 subjects [19]. Apo E4 binds preferentially to VLDL and remnants and accelerates their clearance, leading to down regulation of LDL receptors and increasing blood LDL cholesterol levels [20,21]. The high frequency of the ␧4 allele probably indicates that some cases of FCHL are polygenic diseases and APOE is among other genes responsible for the hyperlipidemic phenotype.

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Table 4 Lipid and lipoprotein concentrations of selected subgroups by APOE genotype.

N Total cholesterol, mg/dL Triglycerides, mg/dL HDL cholesterol, mg/dL Non-HDL cholesterol, mg/dL Apolipoprotein AI, mg/dL Apolipoprotein B, mg/dL a VLDL cholesterol, mg/dL a VLDL cholesterol/triglyceride ratio Total cholesterol/apo B ratio Non-HDL cholesterol/apo B ratio

FD with ␧2/␧2 genotype

R136S carriers

p.Leu149del carriers

FCHL with ␧3/␧3 genotype

p

7 333 ± 105 366 ± 152 46 ± 16.7 286 ± 93.0 130 ± 14.7 118 ± 37.0 92.3 ± 44.9 0.44 ± 0.14 2.83 ± 0.41 2.42 ± 0.34

13 305 ± 47.6 396 ± 146 43 ± 11.7 261 ± 53.9 142 ± 22.3 115 ± 22.1 75.6 ± 30.6 0.27 ± 0.10 2.84 ± 0.69 2.46 ± 0.65

12 322 ± 44.6 203 ± 109 55 ± 15.8 268 ± 43.9 154 ± 30.3 163 ± 29.0 23.4 ± 10.5 0.18 ± 0.06 2.01 ± 0.32 1.66 ± 0.24

10 292 ± 27.3 347 ± 275 45 ± 12.1 247 ± 23.7 157 ± 28.2 163 ± 26.6 47.5 ± 14.5 0.22 ± 0.05 1.81 ± 0.24 1.53 ± 0.22

NS 0.073 NS NS NS <0.001 0.001 <0.001 <0.001 <0.001

Data are presented as mean ± standard deviation (SD). An analysis of variance (ANOVA) was used to evaluate the statistical significance of the differences between groups. a VLDL cholesterol and VLDL cholesterol/triglyceride ratio were obtained in 8 R136S carriers and 7 p.Leu149del carriers.

Beyond the effect of apo E4, APOE contributes to FCHL with some rare mutations. In approximately 4% of the cases, rare defects in APOE that cause a monogenic disorder are responsible for the hyperlipoproteinemic phenotype. In our study two different mutations, R136S and p.Leu149del, were responsible for monogenic hyperlipidemia in our FCHL population. In prior work we reported the lipid effects and associated monogenic transmission of hyperlipidemia of the APOE R136S mutation [22], an observation that has been confirmed by others [23]. The p.Leu149del is a 3-bp inframe deletion that results in the loss of a leucine at position 149 of the receptor-binding region (residues 130–150) of apo E. This genetic defect has been previously associated with familial splenomegaly and thrombocytopenia in two unrelated probands of French Canadian ancestry with mild hypertriglyceridemia that worsened after splenectomy [24] and in one member from a French family [25]. Only in this family did dyslipidemia cosegregate with the APOE mutation [25]. None of the p.Leu149del carriers in our series disclosed spleen enlargement or history of hematological disorders, including thrombocytopenia; however, all of them, probands and family members, showed hyperlipidemia. Splenomegaly has been explained by an increased uptake of mutant p.Leu149del apoE-containing lipoproteins by macrophages [24], but probably requires some other unknown defect to develop. Interestingly, rare APOE mutations are associated not only with dysbetalipoproteinemia but also with a hyperlipidemic phenotype indistinguishable from FCHL. APOE R136S reduces the LDLRbinding activity of lipoproteins by 60% and produces a Fredrickson type III lipid phenotype akin to that characteristic of apoE2/E2 subjects, including low plasma apo B levels [26]. On the other hand, subjects with APOE p.Leu149del develop combined hyperlipidemia with high apo B and without dysbetalipoproteinemia, suggesting that both VLDL and LDL particles are increased without significant accumulation of remnant lipoproteins. Our data are consistent with those from the family studied by Faivre et al. [25], in which subjects with the p.Leu149del mutation showed high LDL cholesterol, high apo B, and normal VLDL-cholesterol/triglyceride ratios, except for the probands, who combined an ␧2 allele with p.Leu149del in a mutant E3 allele and disclosed classical typical type III hyperlipoproteinemia [25]. LDL receptor binding activity in the presence of the APOE variant p.Leu149del has been previously studied and found to be normal. However, the less than 1.006 density lipoproteins from individuals with this defect caused a 3fold enhancement in cholesteryl ester formation in macrophages compared with VLDL from an E3/E3 individual [24]. If enhanced cholesterol uptake by macrophages is also induced in hepatocytes, a substantial reduction in the number of hepatic LDL receptors, which would decrease the catabolism of LDL particles, could possibly occur.

Besides the delineation of the APOE genetic variants leading to an FCHL phenotype, another conclusion derived from our study is the confirmation of the complexity of the FCHL phenotype, which should be considered as a common syndrome rather than a disease. In agreement with this concept, some patients with FH [7], FD caused by dominant APOE mutations [22], the metabolic syndrome [27], and hemochromatosis [28], among others, can express this syndrome. Finally, our findings exemplify the benefit of linking the lipid phenotype with genetic information, as it improves diagnosis and facilitates understanding the mechanisms of hyperlipemia [29]. Also, it could help to improve the treatment, when a more personalized therapy according to ethiology of hyperlipemia would be available. In a similar way as the study of the LDLR gene in the diagnosis of FH, an analysis of the exon 4 of APOE not limited to the diagnosis of the ␧2/␧3/␧4 polymorphisms is probably useful in subjects with a clinical suspicion of FD, but also in those with a FCHL phenotype. Furthermore, genetic exclusion of infrequent monogenic forms of FCHL, such as rare APOE mutations, will probably help identifying commoner genetic components of the disease. Acknowledgements Grants from the Spanish Ministry of Health FIS PS09/0673, FIS PS09/0665, FIS PI10/00387 and RETIC C06/01 RD06/0014/0029 (RECAVA) supported this work. CIBERer, CIBERobn and CIBERdem are initiatives of ISCIII, Spain. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.atherosclerosis.2012.03.011. References [1] Lee JC, Lusis AJ, Pajukanta P. Familial combined hyperlipidemia: upstream transcription factor 1 and beyond. Curr Opin Lipidol 2006;17:101–9. [2] Genest Jr JJ, Martin-Munley SS, McNamara JR, et al. Familial lipoprotein disorders in patients with premature coronary artery disease. Circulation 1992;85:2025–33. [3] Wiesbauer F, Blessberger H, Azar D, et al. Familial-combined hyperlipidaemia in very young myocardial infarction survivors (<40 years of age). Eur Heart J 2009;30:1073–9. [4] Goldstein JL, Schrott HG, Hazzard WR, Bierman EL, Motulsky AG. Hyperlipidemia in coronary heart disease. II. Genetic analysis of lipid levels in 176 families and delineation of a new inherited disorder, combined hyperlipidemia. J Clin Invest 1973;52:1544–68. [5] Horswell SD, Ringham HE, Shoulders CC. New technologies for delineating and characterizing the lipid exome: prospects for understanding familial combined hyperlipidemia. J Lipid Res 2009;50:S370–5.

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