Clinical characteristics and plasma lipids in subjects with familial combined hypolipidemia: a pooled analysis.

Angiopoietin-like 3 (ANGPTL3) regulates lipoprotein metabolism by modulating extracellular lipases. Loss-of function mutations in ANGPTL3 gene cause familial combined hypolipidemia (FHBL2). The mode of inheritance and hepatic and vascular consequences of FHBL2 have not been fully elucidated. To get further insights on these aspects, we reevaluated the clinical and the biochemical characteristics of all reported cases of FHBL2. One hundred fifteen FHBL2 individuals carrying 13 different mutations in the ANGPTL3 gene (14 homozygotes, 8 compound heterozygotes, and 93 heterozygotes) and 402 controls were considered. Carriers of two mutant alleles had undetectable plasma levels of ANGPTL3 protein, whereas heterozygotes showed a reduction ranging from 34% to 88%, according to genotype. Compared with controls, homozygotes as well as heterozygotes showed a significant reduction of all plasma lipoproteins, while no difference in lipoprotein(a) [Lp(a)] levels was detected between groups. The prevalence of fatty liver was not different in FHBL2 subjects compared with controls. Notably, diabetes mellitus and cardiovascular disease were absent among homozygotes. FHBL2 trait is inherited in a codominant manner, and the lipid-lowering effect of two ANGPTL3 mutant alleles was more than four times larger than that of one mutant allele. No changes in Lp(a) were detected in FHBL2. Furthermore, our analysis confirmed that FHBL2 is not associated with adverse clinical sequelae. The possibility that FHBL2 confers lower risk of diabetes and cardiovascular disease warrants more detailed investigation.

lipoprotein, and previous studies have documented that its concentration may be modulated by levels of LDL-C in the plasma ( 19 ). A study investigating Lp(a) concentrations in kindred with FHBL, a cognate syndrome also characterized by low LDL-C levels, showed that Lp(a) levels are not changed comparing affected with unaffected individuals ( 20 ). Nevertheless, there is evidence that inhibition of apoB synthesis may affect plasma level of Lp(a) ( 21 ). Therefore, the investigation of Lp(a) in FHBL2 might provide further insight into LDL metabolism in these subjects.
It is well established that in APOB -linked FHBL severe fatty liver is present with a prevalence rate of approximately 20-30% ( 6 ). However, whether FHBL2 individuals have also increased risk of liver steatosis is unknown. An additional aspect that remains to be clarifi ed is whether changes in glucose metabolism are part of the clinical phenotype associated with FHBL2. In fact, Minicocci et al. ( 11 ) reported lower fasting glucose and the lack of diabetes mellitus among homozygous carriers of inactivating mutation in the ANGPTL3, but the consistency of these fi ndings remain to be confi rmed. Finally, it has been suggested that the defi ciency of ANGPTL3 may be protective against atherosclerosis ( 22 ). In a previous report, homozygous FHBL2 subjects did not show evidence of history of cardiovascular disease, but the low number of cases investigated precluded any defi nitive conclusion ( 11 ).
Therefore, to get additional insight on these unclarifi ed aspects of FHBL2, we designed this collaborative study aimed at collecting all available information in reported FHBL2 individuals with the following goals: 1 ) to more fi rmly establish the lipid expression of FHBL2, especially in heterozygous individuals; 2 ) to assess the clinical characteristics associated with FHBL2, with particular relevance to the presence of hepatic steatosis and the history of cardiovascular disease; 3 ) to compare plasma concentrations of ANGPTL3 in subjects carrying different mutations in the ANGPTL 3 gene; and 4 ) to evaluate plasma levels of Lp(a) in homozygous and heterozygous FHBL2 carriers.

Data sources and study selection
For the electronic searches, published studies were found through PubMed at the National Library of Medicine (http:// ncbi.nlm.nih.gov/entrez/query) and in Medline databases by using the following queries ''familial combined hypolipidemia" and "ANGPTL3 mutation" and "familial hypocholesterolemia." The literature search was done on studies up to December 2012 with availability of an English-language abstract and without country restriction. This search yielded fi ve hits (7)(8)(9)(10)(11). Two authors (I.M. and M.A.) reviewed all articles independently either to determine the eligibility criteria or to examine the appropriateness of the research issue. The inclusion criteria were the availability of demographic, plasma lipid, and genetic data. None of articles was excluded. Afterwards, corresponding authors were invited to provide published and unpublished data of study subjects according to a prespecifi ed protocol. All authors agreed to confer available data . familial hypobetalipoproteinemia (FHBL, OMIM #107730). FHBL shows a dominant mode of inheritance with variable (from moderate to severe) degree of LDL-C or apoB reduction ( 3 ). Most of FHBL cases have been linked to mutations in the apoB gene ( APOB ) that lead to defects in secretion of VLDL in the liver ( 4 ). However, loss-of-function mutations in the gene coding for the proprotein convertase subtilisin kexin type 9 ( PCSK9 ), a protein involved in the posttranslational regulation of the LDL receptor (LDLR), have been also reported to cause FHBL ( 5 ) . Most patients with FHBL have no symptoms, although this disorder has been associated with hepatic steatosis, fat malabsorption, and, in the most severe forms, variable neurological manifestations ( 6 ).
Familial combined hypolipidemia (FHBL2, OMIM #605019) is a recently described low-cholesterol syndrome characterized by a peculiar lipid profi le in which VLDL, LDL, and HDLs are comprehensively reduced ( 7 ). It is caused by mutations in the Angiopoietin-like 3 gene ( ANGPTL3 ) that lead to the absence or the marked reduction of ANGPTL3 protein in the plasma (8)(9)(10)(11).
In humans, ANGPTL3 is located on chromosome 1 (1p31.1-p22.3) and encodes a 460 amino acid protein consisting of a signal peptide, an N-terminal segment containing coiled-coil domains, and a C-terminal fi brinogen-like domain, which are connected with a linker region ( 12 ). ANGPTL3 is secreted almost exclusively by the liver, and its primary function is to act as a partial inhibitor of lipoprotein lipase and endothelial lipase, two enzymes involved in the lipolytic degradation of VLDL and HDL particles, respectively (13)(14)(15)(16)(17). Therefore, it is thought that ANGPTL3 defi ciency causes an overactivation of these lipases, leading to an accelerated catabolism of VLDL and HDL ( 16 ). The impact of ANGPTL3 defi ciency on LDL-C levels is more diffi cult to explain. An in vivo lipoprotein kinetic study in a family with FHBL2 showed a reduced production of VLDL and an increased LDL catabolism, but the mechanisms causing these defects are not well understood ( 18 ).
Since the fi rst report ( 7 ), several additional cases of FHBL2 have been described. However several questions related to the lipid phenotypes of this disorder remain unclarifi ed. First, the mode of inheritance of FHBL2 has not been fi rmly defi ned, as both recessive and codominant transmission have been reported. Musunuru et al. ( 7 ) proposed that inactivating ANGPTL3 mutations show a codominant effect on LDLC and plasma triglycerides (TGs) and a recessive effect on HDL-C, as only homozygous carriers presented signifi cantly lower plasma concentration of this lipoprotein. Conversely, another report describing a larger cohort of FHBL2 subjects concluded that both TG and HDL-C levels are affected in a codominant manner by ANGPTL3 mutations, while LDL-C was inherited as a recessive trait ( 11 ). Even though these discrepancies may be due to differences in studied cohorts, there is the possibility that they could be related to functional differences in FHBL2-causing alleles. Second, it is unknown whether ANGPTL3 deficiency also affects plasma levels of lipoprotein (a) [Lp(a)]. Lp(a) is a LDL-like an Accuvix V 20 ultrasonographer (equipped with 2-6-MHz convex transducers) (Samsung Medison, Italy) and following a previously reported procedure ( 25,26 ). Steatosis was defi ned by an appearance of hyperechoic liver parenchyma with tightly packed fi ne echoes and posterior beam attenuation ( 25 ).

Laboratory determinations
Plasma lipids, apoB, and apoAI were measured by standard techniques. LDL-C values were calculated by the Friedewald's formula. The assessment of Lp(a) was performed in 284 subjects (supplementary Table I). Measurements at Musunuru's, Pisciotta's, and Martin-Campos's sites were carried out by employing a nephelometric procedure (Siemens AG Healthcare, Munich, Germany), while those at Minicocci's site by using a particle enhanced turbidimetric immunoassay (Tina-quant Lipoprotein(a) (Latex)-Roche Diagnostics GmbH, Mannheim, Germany) adapted for the Roche/Hitachi COBAS CE 6000 Analyzer ( 27 ). ANGPTL3 plasma concentration was assessed in 177 subjects by ELISA method using an in-house-developed assay (n = 170) ( 11 ) and a commercially available kit (ADIPOGEN, Adipogen Inc., Korea) (n = 7). Serum samples were diluted 50-fold and measured in triplicate. Repeated measurements were made in homozygotes after 10-fold dilution of serum samples. The detection limit of both assays was 0.5 ng/ml; intra-and interassay coeffi cients of variation were less than 10% for the in-house assay and less than 7% for the commercially available assay, respectively

Statistical analysis
All statistical analyses were performed with SPSS/WIN program (version 18.0; SPSS Inc., Chicago, IL). Descriptive statistics such as means, standard deviation (SD), and ranges were undertaken for all the variables. Continuous variables were compared by the Mann-Whitney U test, whereas the categorical variables were compared by X 2 or Fisher's exact test. Bonferroni's correction was performed for multiple comparisons. Correlations were estimated by Pearson's test. Differences in lipid levels between mutation carriers and noncarriers were tested for signifi cance by using ANOVA including age, sex, BMI, and site as covariates. The effect of number of mutant alleles on plasma lipids was also evaluated by comparing lipid residuals between carriers and noncarriers. To calculate residuals, age-, sex-, and BMI-adjusted coeffi cients for all lipid variables were calculated in the screened population by linear regression. The obtained equation model was used to calculate the expected lipid values for each individual, and residuals were obtained by subtracting expected from observed values. A two-sided P < 0.05 was considered statistically signifi cant.

Study characteristics
Musunuru et al. ( 7 ) reported a family whose members had hypobetalipoproteinemia in which linkage analysis had ruled out APOB as the causative gene. Subjects reported by Pisciotta et al. ( 8 ) have been selected from a group of 150 individuals with primary hypobetalipoproteinemia who had been referred to the Lipid Clinics of the University Hospitals in Genova, Parma, Milan, and Modena over the last two decades. The selection criteria included low LDL-C and apoB associated with a marked reduction of HDL-C (all below the 5th percentile) and apoAI in the absence of mutations in the APOB , MTP , PCSK9 , APOA1 , LCAT , and ABCA1 genes. Noto et al. ( 9 ) searched for FHBL2 subjects in a sample of 390 Italian and 523 American individuals with a total cholesterol below the 5th percentile of the relative population distributions. Martín-Campos et al. ( 10 ) selected nonrelated subjects with biochemical features of FHBL based on a persistent age-and sex-specifi c cutoff point below the 5th percentile level of LDL-C and apoB for the Spanish population, in the absence of mutations in the APOB gene. Finally, Minicocci et al. ( 11 ) identifi ed in Campodimele, a small town located in the province of Latina (Italy), a group of individuals with primary hypobetalipoproteinemia (LDL-C below the 5th age-and gender-specifi c percentile for the general Italian population) not carrying mutations in the APOB , MTP , or PCSK9 gene.
In all studies the screening procedures were comparable. Subjects were examined in the morning and demographic information, medical history (with particular reference to liver and cardiovascular diseases), coronary artery disease (CAD) risk factors, and current medications (including vitamins and supplements) were recorded using a structured questionnaire. Measurements included height and weight and systolic and diastolic blood pressures. Fasting blood samples for laboratory determinations were also obtained. In one study ( 11 ), alcohol consumption was assessed using a semiquantitative scale (0 = abstainers, 1 = less than 2 glasses/day, and 2 = more than 2 glasses/day). All subjects were classifi ed as hypertensive based upon the recording of blood pressure >140/ 90 mmHg and/or the use of antihypertensive medication, and/or affected by diabetes mellitus if they showed fasting blood glucose greater than 126 mg/dl and/or were using hypoglycemic medications. Details of data provided by each site are reported in the supplementary Table I. All participating studies were approved by the appropriate institutional ethical committees.

Genetic analyses
Musunuru et al. ( 7 ) analyzed the ANGPTL3 gene by exomesequencing technique, while the other authors performed genetic analyses by a combination of PCR amplifi cation and direct sequencing of coding regions and intron-exon boundaries of ANGPTL3 gene, as described (8)(9)(10)(11). ApoE genotypes were determined in 68 FHBL2 subjects and 319 controls by using a previously described procedure ( 23 ).

Evaluation of hepatic steatosis
The association between FHBL2 and hepatic steatosis was evaluated by two different approaches. In all subjects we estimated fatty liver index (FLI), which is an algorithm based on body mass index (BMI), waist circumference (WC), TG, and ␥ -glutamyl transferase ( ␥ -GT) and might serve as a simple and accurate predictor of hepatic steatosis in the general population ( 24 ). The FLI was calculated as reported and elevated FLI was defi ned as greater than 60 ( 24 ). In addition, the presence of hepatic steatosis was determined by ultrasound in 167 subjects belonging to the Campodimele's cohort. Liver examination was performed by a single radiologist blinded to the disease state of the subjects using Homozygous carriers showed a tendency toward lower plasma fasting glucose, but this difference did not reach the statistical signifi cance when compared with the other groups. No case of diabetes mellitus was reported among FHBL2 homozygous subjects. A signifi cant reduction of plasma creatinine levels was observed in homozygotes when compared with either heterozygotes ( P < 0.01) or noncarriers ( P < 0.05), but this did not translate into a statistically signifi cant difference in the calculated glomerular fi ltration rate (GFR). The overall prevalence of cardiovascular disease in FHBL2 was not different compared with that in noncarrier subjects (4.3% versus 3.7%; P = 0.76). However, it is noteworthy that no homozygous carrier reported history of cardiovascular disease. The prevalence of fatty liver estimated by FLI was not significantly different between the groups, and this corresponded resulted in the same truncated protein (p.N121Kfs*3); one was an amino acid deletions (p.E96del) and 3 were missense mutations (p.G56V, p.F295L, p.R332Q). They were mostly located within the N-terminal portion of ANGPTL3 protein, with the exception of p.F295L, p.R332Q, and p.G400Vfs*5 mutations, which were located in the fi brinogen-like domain of the protein. The functional impact of missense variations was evaluated by using PolyPhen-2, and all missense mutations were annotated as "probably damaging." The distribution of different genotypes in FHBL2 carriers is detailed in supplementary Table II.
The lipid values as well as the demographic characteristics of carriers of different ANGPTL3 mutant alleles are also shown in supplementary Table II. The lowest plasma lipid concentrations were seen among carriers of p.S17*/p. E129* and p.N147*/p.F295L genotypes, even though the low number of individuals in each group prevents drawing any defi nitive conclusions.
The cohort enrolled by Minicocci et al ( 11 ), was remeasured four years after the fi rst screening. Therefore, this allowed us to evaluate the time-trend changes of plasma lipids in mutations carriers and noncarriers. To this purpose, plasma lipids of 199 subjects (6 FHBL2 homozygotes and 50 heterozygotes) and 143 noncarriers tested in the years 2007 and 2011 were compared ( Fig. 3 ). While homozygotes showed stable lipid profi le over time, heterozygotes had a signifi cant increase of TG ( P < 0.001) and apoB ( P < 0.05) concentrations. Notably, noncarrier subjects presented a signifi cant increase of LDL-C and TG levels ( P < 0.001) and a reduction of HDL-C ( P < 0.01). It is worth mentioning that during follow-up, a signifi cant average 7% increase ( P < 0.05) of BMI was observed in all subjects. However, despite these changes, in heterozygotes plasma levels of TG ( P = 0.004), LDL-C ( P < 0.001), and HDL-C ( P = 0.043) remained signifi cantly lower than those in noncarrier controls.

Serum concentration of ANGPTL3 protein according to genotypes
Measurements of plasma levels of ANGPTL3 were available in carriers of p.S17*, p.I19Lfs*22, p.E96del, p.S122Kfs*3, and p.G400Vfs*5 mutations. All homozygotes or compound heterozygotes (n = 12) showed undetectable plasma levels of ANGPTL3, thus clearly suggesting that all mutations cause null alleles ( Fig. 4 ). To evaluate whether different alleles might have a variable effect on protein expression, plasma levels of ANGPTL3 were compared between carriers of the different ANGPTL3 gene variants ( Fig. 4 ). Compared with noncarriers, we observed markedly ( P < 0.0001) reduced plasma levels of ANGPTL3 in heterozygous carriers of p.S17* ( Ϫ 39.3%) and p.S122Kfs*3 ( Ϫ 87.9%). Also carriers of mutations p.I19Lfs*22, and to the lack of differences in liver transaminases and ␥ -GT across the groups. None of homozygous subjects reported positive history of liver disease. In addition, a liver ultrasound examination was performed in 64 p.S17* mutation carriers and in 103 noncarriers. In this subgroup, carriers showed on average higher BMI than noncarriers, but the difference was not signifi cant (28.0 ± 5.2 kg/m 2 versus 27.0 ± 4.6 kg/m 2 ; P = 0.1). Overall, we did not observed increased prevalence of liver steatosis in the FHBL2 group compared with the control group (43.7% versus 44.7%, P = 0.70). In addition, no association was detected between the severity of steatosis and the FHBL2 status ( Fig. 1 ). One homozygous FHBL2 subject was classifi ed as having severe steatosis (16.7%) as compared with 7 subjects in the noncarrier group (5.8%), but she was severely obese, showing a BMI of 41.0 kg/m 2 .

Plasma lipids and lipoproteins in ANGPTL3 mutation carriers
The comparison of plasma lipid levels in carriers and noncarriers of ANGPTL3 mutations was fi rst performed within family members (n = 225) after adjustment for age, gender, and BMI ( Table 2 ). Compared with noncarriers, homozygotes as well as heterozygotes showed a signifi cant reduction of all plasma lipoproteins and apoproteins. When the analyses were performed in the entire cohort (n = 517), similar results were obtained ( Table 2 ). No significant differences between groups were observed in plasma levels of Lp(a). Changes of plasma lipid in FHBL2 and controls were further investigated by analysis of lipid residues according to genotype number of mutant ANGPTL3 alleles ( Fig. 2 ). This analysis indicated that the presence of two mutant alleles signifi cantly reduced LDL-C levels by 67.2%, TG by 71.2%, HDL-C by 39.0%, apoB by 48.4%, and apoAI by 95.1% (all P < 0.001). The presence of a single mutant allele reduced LDL-C by 8.6% ( P = 0.007), Fig. 1. Grade of liver steatosis in FHBL2 subjects carrying the ANGPTL3 p.S17* mutation and noncarrier controls.  p.G400Vfs*5 showed lower plasma levels of ANGPTL3, although the difference did not reach the statistical signifi cance. Surprisingly, the p.E96del variant was associated with plasma ANGPTL3 concentration in the upper level of normal.

DISCUSSION
We analyzed a large set of individuals affected with FHBL2 and controls to get additional insights into the phenotypic expression of FHBL2. We fi nd that individuals carrying one mutant ANGPTL3 allele had a signifi cant reduction of all apoB-and apoAI-containing lipoproteins, thus indicating that inactivating ANGPTL3 mutations affect the lipid phenotype in a codominant manner. However, we noted that there was not a linear gene-dosage-dependent effect of ANGPTL3 mutations, as two mutant alleles reduced plasma lipids ranging from 48% to 95%, whereas one mutant allele reduced them from 7% to 20% depending on the lipoprotein class. It is noteworthy that despite changes in BMI, homozygotes (but also heterozygotes) showed persistently lower plasma when compared with noncarrier controls.
In our collection, the vast majority of FHBL2-causing mutations (78%) predicted a truncated ANGPTL3 protein, and only three caused missense alleles. To explore the genotype-phenotype relationship in terms of impact of different mutant alleles on plasma concentration of ANGPTL3 protein, we compared the plasma levels of ANGPTL3 in heterozygous carriers of some ANGPTL3 variants. In fact, mutations predicting a truncated protein (p.S17*, p.G400Vfs*5, and p.S122Kfs*3) had the most pronounced effect in reducing plasma levels of ANGPTL3. Conversely, the p.E96del variant was associated with plasma mutations. Although these comparisons might be hampered by the small number of measured individuals and by the difference in the methods used, we detected some relevant variations in the functional effect of the different ANGPTL3  , and outliers falling below the 5th percentile or above the 95th percentile (points below or above the vertical lines, respectively apo(a) and the accurate estimation of the proportion of apo(a) complexed to LDL in FHBL2 is warranted.
Given the striking reduction of plasma HDL-C and the demonstration that sera obtained from FHBL2 have reduced HDL-mediated cholesterol effl ux capacity in vitro ( 8 ), it may be of interest to explore the association between FHBL2 and atherosclerosis. Even though systematic search for asymptomatic vascular damage was not performed, no increased prevalence of clinically overt manifestations of atherosclerotic cardiovascular disease was detected in the present cohort of FHBL2 individuals. This observation might be interpreted on the basis that the lifelong exposure to low levels of VLDL and LDL may counterbalance the putative "proatherogenic" effect related to low HDL-C. Unfortunately, the small size of the present cohort precludes drawing any defi nitive conclusion about the association between ANGPTL3 defi ciency and atherosclerosis, an issue that merits additional, more detailed investigation.
Finally, extending our previous observation ( 11 ), we found that none of homozygotes was affected by diabetes mellitus. It seem interesting to consider this observation in the light of results by Romeo et al. ( 28 ) that reported in a population study higher prevalence of nonsynonymous (probably inactivating) variations in the ANGPTL3 genes among subjects in the lowest quartile of blood glucose levels. Moreover, we recently reported that FHBL2 homozygotes showed lower plasma levels of insulin and lower degree of insulin-resistance as estimated HOMA-IR index ( 36 ). If further confi rmed in additional investigations, these fi ndings could highlight a more comprehensive role of ANGPTL3 in regulating not only lipid but also glucose metabolism.
The present study has some limitations. Although this represents the largest cohort of FHBL2 subjects collected so far, the number of affected individuals, mainly homozygous carriers, remains too small to fi rmly establish the impact of the FHBL2 on cardiovascular risk. More importantly the different sources of data might have introduced some bias in estimates. However we tried to compensate for this by adjusting comparisons for sources of the studies.
In conclusion, our results demonstrate that loss-offunction mutations in the ANGPTL3 gene causing FHBL2 affect plasma levels of apoB-and apoAI-containing lipoproteins in a codominant manner and that the quantitative lipid-lowering effect of the presence of two mutant alleles was almost four times greater than that of one mutant allele. The defi ciency of ANGPTL3 does not affect plasma levels of Lp(a). The present analysis indicates the lack of association between FHBL2 and fatty liver disease, but an apparent lower risk of diabetes mellitus and cardiovascular disease was noticed in FHBL2 homozygotes. More detailed investigations are needed to further explore these clinical aspects associated with FHBL2.
The authors thank Mrs. Maria Laura Sepe for great help in organizing the screening, and Giulia Barbarossa, MD, Paola Coletta, MD, and Daniela Pergolini, MD, for performing the fi eld work. The authors acknowledge Marius R. Robciuc, PhD, ANGPTL3 levels in the upper level of normal, thus questioning its functional role. The observation that mutations predicting protein truncation were associated with a complete absence of ANGPTL3 in the plasma, when present in a homozygous state, strongly argues that they may severely impair the synthesis or secretion of ANGPTL3 from hepatocytes. Conversely, no information about the functional role of the three missense ANGPTL3 mutations (p.G56V, p.F295L, and p.R332Q) can be derived from the present data. Nonetheless, it must be noted that these mutations involved highly conserved amino acids and turned out to be probably damaging to in silico analysis . It may be interesting to report that nearby mutations have been demonstrated in vitro to abolish the capacity of ANGPTL3 to inhibit lipoprotein lipase activity (p.K63T) or the secretion of ANGPTL3 from cells (p.F295L and p.R332Q) ( 28 ). Only the direct expression of these missense variants could clarify their functional impact.
In the present cohort of FHBL2 subjects, we did not observe any increase in the prevalence of fatty liver as estimated by FLI or by raised serum transaminase levels. This conclusion was further supported by the results of the ultrasound examination of a FHBL2 subgroup, in which no signifi cant association was detected between the prevalence and degree of steatosis and the FHBL2-affected status. It must be noted that the prevalence of fatty liver disease in noncarriers (44%) was similar to that reported by ultrasounds in overweight Italian population samples ( 29,30 ) making them reliable controls. Therefore, the hepatic consequence of FHBL2 appears to be strikingly different from that of APOB -linked FHBL in which association with increased prevalence of liver steatosis has been consistently reported ( 6,31 ). The fact that FHBL2 subjects do not show excessive liver fat infi ltration despite reduced secretion of VLDL ( 18 ) might suggest the presence of a compensatory reduced synthesis of TG by hepatocytes ( 32 ). It has been demonstrated that mice lacking ANGPTL3 have lower plasma FFA ( 33 ) and that recombinant ANGPTL3 produces an elevation of both FFAs and glycerol in cultured adipocytes ( 34 ). Therefore, we could speculate that in FHBL2 there is a lower fl ow of FFA into the liver that provides lesser substrates for the synthesis of TG and their assembly with apoB into nascent VLDL particles. Further investigations will be needed to directly demonstrate this mechanism.
A still open question related to the lipid phenotype of FHBL2 is its impact on plasma concentration of Lp(a). Here we found that, compared with nonaffected subjects, FHBL2 individuals did not have different Lp(a) levels and that the very low levels of apoB seen in homozygotes was not associated with signifi cant variation in Lp(a). Any possible interpretation of this fi nding should be cautious. In fact, we did not evaluate the apo(a) genotypes, which are the strongest determinants of plasma levels of Lp(a) ( 35 ), thus leaving the possibility that the lack of difference in Lp(a) levels might be due to a different distribution of apo(a) isoforms between groups. Nevertheless, our data might indicate that the lack of ANGPTL3 does not have signifi cant impact on Lp(a) metabolism. To better interpret this fi ndings, direct measurements of plasma levels of Matti Jauhiainen, PhD, and Christian Ehnholm, MD, PhD, for their help in plasma ANGPTL3 measurement. We thank Vincenzo Monopoli, Giuliano Divsic, and Fabio Macrì from Samsung Medison (Italy) for their technical support in echographic measurements. The authors also thank Mr. Pierpaolo Spanedda from the Department of Experimental Medicine for his help in laboratory measurements. Finally, the authors express gratitude to all subjects for their participation in the study.