Rare loss-of-function mutations in ANGPTL family members
contribute to plasma triglyceride levels in humans ([original](https://doi.org/10.1172/jci37118)) ([raw](?raw))ANGPTL mRNA levels in human tissues. As a first test of the hypothesis that multiple members of the ANGPTL family regulate TG metabolism in different tissues, we examined the levels of mRNA from each gene in 48 human tissues (Figure 1). Expression of ANGPTL3 was largely restricted to liver, consistent with the pattern seen previously in mice (9). The levels of_ANGPTL4_ mRNA were also highest in liver, with the next highest level being in the pericardium. The level of the ANGPTL4 transcript in adipose tissue was only 10% that found in liver, despite fat having the highest expression level of ANGPTL4 in mice (10). Low levels of_ANGPTL4_ transcript (<10% of liver) were also present in the adrenal glands, lung, pancreas, and placenta, with only trace amounts detected in other tissues.
Expression of ANGPTL3, ANGPTL4,ANGPTL5, and ANGPTL6 in human tissues. Quantitative real-time PCR was used to determine mRNA levels of the 4_ANGPTL_ family members in commercial cDNA arrays of 48 tissues prepared from normal humans (Origene). The 21 tissues in which the signal was detected are shown. Each bar represents the average of triplicate measurements expressed as a fraction of the Ct value obtained from the tissue expressing the highest levels of mRNA for that gene.
ANGPTL5 was most highly expressed in adipose tissue, with the bronchus, epididymis, and vena cava having the next highest levels of expression. The transcript was identified at very low levels in many other tissues. A previous study reported that ANGPTL5 was expressed most strongly in heart (13), but those authors did not examine mRNA from adipose tissue. Although we detected ANGPTL5 mRNA in the heart, it was present at only 20% of the level found in adipose tissue.
Finally, ANGPTL6 was expressed at the highest level in the liver and at much lower levels in other tissues.
Thus, 3 of the 4 ANGPTLs analyzed in this study (ANGPTL3,ANGPTL4, and ANGPTL6) were most highly expressed in the liver, and 2 of the 4 family members were expressed in adipose tissue (ANGPTL4 and ANGPTL5). This finding is consistent with the hypothesis that these genes coordinate fuel trafficking and homeostasis in response to changes in energy demands.
Multiple sequence variants in the coding regions of ANGPTL3, ANGPTL5, and ANGPTL6. To develop a comprehensive inventory of sequence variations in the coding regions of_ANGPTL3_, ANGPTL5, and ANGPTL6, we sequenced the exons of the 3 genes in the DHS, a multiethnic, probability-based population (including 1,870 African Americans, 1,045 individuals of mixed European descent, and 601 Hispanics) as previously described (12). A total of 255 variants were identified, most of which were rare: more than half (155/255) were found in only a single individual, and 86% (220/255) had a minor allele frequency (MAF) below 1% (Supplemental Table 1; supplemental material available online with this article; doi: 10.1172/JCI37118DS1). NS variants were more common than synonymous variants in all 3 genes (97 vs. 27 variants). The density of coding sequence variants was similar in the 3 genes: 1 sequence substitution per 33 nucleotides in_ANGPTL3_ and ANGPTL5 and 1 substitution per 30 nucleotides in ANGPTL6.
To determine the phenotypic effects of sequence variations in the 3 genes, we stratified the DHS population by race, sex, and trait level for metabolic phenotypes associated with TG removal from the circulation (plasma TG level), TG accumulation (BMI and hepatic TG content), and indices of energy homeostasis (fasting blood glucose, fasting insulin, homeostatic model assessment — insulin resistance [HOMA-IR]). In addition, we tested for association with systolic blood pressure, diastolic blood pressure, and plasma levels of cholesterol, TG, HDL-C, and LDL-C. We used a strategy similar to that employed to assess the effect of rare sequence variations in other genes on quantitative traits (14, 15). For each phenotype we compared the number of individuals with NS sequence variants in the top and bottom quartiles (Table 1; ANGPTL4 was included for comparison). Any sequence variation present in both the top and the bottom quartiles was eliminated from the analysis.
Numbers of individuals with NS sequence variations identified in_ANGPTL3_, ANGPTL4, ANGPTL5, and_ANGPTL6_ in the upper or lower quartile of the distribution of selected traits in the DHS
Rare and common sequence variations in ANGPTL3 are associated with reduced plasma TG levels. A total of 35 NS sequence variations were identified in ANGPTL3 (Supplemental Table 2). An excess of sequence variants in the lower quartile for plasma TG levels (14 vs. 5 variants) approached the nominal significance threshold (P = 0.064; Figure 2A). All sequences likely to be loss-of-function alleles (frameshift 122 [Fs122], FsQ192, and FsK455) were in the lowest quartile of TG levels, suggesting that decreased levels of circulating ANGPTL3 are associated with reduced plasma levels of TG. One of the missense mutations associated with a low plasma TG level, K63T, is predicted to disrupt a heparin binding motif (61VHKTKG66) that is required for LPL inhibition in mice (4).
Schematic representation of ANGPTL3 with positions of NS sequence variations identified in the upper and lower quartiles of TG distribution in the DHS. (A) The deduced 460–amino acid ANGPTL3 protein has the characteristic features of angiopoietins: a signal peptide (SP), an extended helical domain predicted to form coiled coils (CC), and a globular fibrinogen homology domain (FBG-like domain) at the C terminus. An excess of NS sequence variations was found in lower quartile of TG distribution compared with the upper quartile (14 vs. 5 variations; P = 0.064). Coiled coil domains were predicted using COILS (http://www.ch.embnet.org/software/COILS\_form.html), and the fibrinogen-like domain sequence was obtained from NCBI (http://www.ncbi.nlm.nih.gov/sites/entrez?db=Protein&itool=toolbar). Δ, deletion of an amino acid. (B) A common allele of_ANGPTL3_ (M259T) present in 10% of African Americans was associated with lower plasma levels of TG in 2 the DHS and the ARIC study. The variant was not associated with HDL-C or BMI. M/M, M/T, and T/T refer to predicted amino acids at position 259 of ANGPTL3.
To further examine the relationship between sequence variation in_ANGPTL3_ and plasma TG levels, we tested for association between plasma TG and the common SNPs at the locus (MAF >1%). None of these SNPs was associated with plasma TG levels (data not shown), except for M259T. M259T was present at appreciable frequency among African Americans (MAF = 5%) and was significantly associated with plasma TG levels (P = 0.006) in this ethnic group (Supplemental Table 3 and Figure 2B). The allele was rare among individuals of mixed European descent (MAF, 0.1%). To validate this association, we assayed the M259T SNP in African Americans in the ARIC study (16) (Supplemental Table 3). This analysis confirmed that the M259T SNP was significantly associated with plasma levels of TG (P = 0.014). Taken together, these data indicate that a spectrum of sequence variants in ANGPTL3 contributes to variation in plasma TG levels.
The number of NS sequence variants in ANGPTL3 was similar in the upper and lower quartiles of the distribution for BMI (P = 0.235) and hepatic TG content (P = 0.773). Similarly, the common allele associated with plasma TG levels (M259T) was not associated with either parameter. A significant excess of NS sequence variants was found among individuals in the lowest quartile for blood glucose levels (16 vs. 3 variants; P = 0.002) in the DHS, but in both the DHS and in ARIC, the M259T variant was not associated with blood glucose, insulin, or with any of the other metabolic parameters examined (Supplemental Table 3). Taken together, these findings indicate that sequence variation in ANGPTL3 was associated with plasma levels of TG, but not with other indices of fat accumulation or metabolism, although we cannot exclude the possibility that some rare variants in ANGPTL3 affect plasma glucose levels.
Excess of rare sequence variations in ANGPTL5 in individuals with low plasma levels of TG. A similar strategy was used to analyze the metabolic effects of sequence variations in ANGPTL5 (Figure 3A), a gene of unknown function that is not expressed in mice (13). A significant excess of NS variants was found in the lowest quartile of TG levels (n = 9) compared with the highest quartile (n = 1) (P = 0.022). No frameshift or nonsense mutations in this gene were identified in the lowest quartile, but a single mutation in a consensus splice donor site (IVS8+1) was found. Only 1 NS variant (T268M) in_ANGPTL5_ had a MAF of greater than 1%, and this variant was not consistently associated with plasma TG levels in the DHS or in ARIC (data not shown). Sequence variations in ANGPTL5 were not associated with any of the other metabolic phenotypes examined.
Schematic representation of ANGPTL5 and ANGPTL6 with positions of NS variants identified in the upper and lower quartiles of the TG distribution in the DHS. (A) Individuals carrying NS sequence variations in_ANGPTL5_ were more prevalent in the bottom quartile than in the top quartile (9 vs. 1 individuals, respectively; P = 0.012). (B) A corresponding analysis of ANGPTL6 revealed no significant difference in allele frequencies (6 vs. 13 individuals;P = 0.107). (C) Number of subjects with plasma TG levels in the upper quartile (n = 878) and the lower quartile (n = 897) of the DHS who had nonsense, missense, and splicing mutations in ANGPTL3, ANGPTL4,ANGPTL5, and ANGPTL6. The difference in numbers of subjects in the upper and lower quartiles is due to differences in the number of individuals with plasma TG levels at the thresholds for the quartiles. IVS, intron.
Sequence variations in ANGPTL6 are not associated with plasma TG levels. In contrast to ANGPTL3, ANGPTL4, and_ANGPTL5_, in which NS variants were significantly associated with plasma TG levels, we found no evidence of association between sequence variants in_ANGPTL6_ and plasma TG levels. The number of individuals with NS variants in ANGPTL6 did not differ significantly in the upper and lower quartiles of the TG distribution (Figure 3B). Two NS variants in ANGPTL6 had a MAF of greater than 1% (R96P and R358C). Neither of these variants was associated with TG levels in the DHS (data not shown). A statistically significant excess of NS variants in_ANGPTL6_ was found in upper quartile of plasma cholesterol levels when compared with the lowest quartile of cholesterol levels, but not in the upper quartile of LDL-C or HDL-C levels (Table 1).
Figure 3C summarizes the relationship between plasma TG levels and sequence variations in 3 of the ANGPTL family members analyzed in this paper (ANGPTL3, ANGPTL5, and ANGPTL6), together with data from our prior analysis of ANGPTL4. The numbers of individuals with NS variants in the top and bottom quartiles for plasma TG are given for each gene. As was observed for ANGPTL3 and ANGPTL5, sequence variations in ANGPTL4 were more common among individuals with plasma levels of TG in the lowest quartile compared with the highest quartile.
The majority of NS variants in ANGPTL3, ANGPTL4, and ANGPTL5 associated with low plasma TG levels interfere with protein secretion. Several of the sequence variants in ANGPTL3, ANGPTL4, and ANGPTL5 that were found in the lowest quartile of plasma TG levels were nonsense, frameshift, or splice-site mutations (Figures 2 and 3). This finding suggested that loss-of-function alleles of these genes are associated with low plasma TG levels. The remainder of the mutations found in the lowest quartile of TG levels were missense mutations. To determine whether these mutations also interfere with protein function, we generated cDNA expression constructs for each mutant allele and compared the expression and secretion of the mutant proteins with wild-type ANGPTL in cultured human embryonic kidney (HEK293A) cells. Immunoblot analysis of the cell lysates and the medium are shown in Figure4A. Whereas wild-type ANGPTL3 was readily detected in the medium, 5 of the 9 missense mutations present in the lowest TG quartile, but none of the 5 missense variants in the high-TG group abolished secretion of ANGPTL3 from cells.
Effects of sequence variations on the synthesis and secretion of ANGPTL3, ANGPTL4, and ANGPTL5. (A) ANGPTL3, (B) ANGPTL4, and (C) ANGPTL5. Wild-type and mutant forms of ANGPTL3, ANGPTL4, and ANGPTL5 were expressed in HEK293A cells, and immunoblotting was performed on the cell lysates and medium using an anti-V5 mAb as described in Methods. Calnexin was used as a loading control. This experiment was repeated 3 times with similar results. ↓TG, sequence variations found in individuals with TG in the ≤ 25th percentile; ↑TG, sequence variations found in individuals with TG in the ≥ 75th percentile; —, vector only.
Similarly, when the same experiment was performed to examine the NS sequence variants that we previously identified in ANGPTL4 (12), 5 of the 7 missense alleles had either a partial or complete deficit in ANGPTL4 secretion (Figure 4B).
Finally, in contrast to wild-type ANGPTL5 or the allele containing the missense mutation found in the high TG group (I233V) 3 of the 7 missense mutations in_ANGPTL5_ associated with a low plasma TG level failed to be secreted from the cells (Figure 4C).
Thus, of the 23 missense mutations in the 3 ANGPTL family members that were found in individuals in the lower quartile of plasma TGs, more than half (n = 13) impaired protein secretion, presumably by interfering with the proper folding of the protein. These mutations were located almost exclusively in the highly structured fibrinogen-like domains located at the C terminus of the proteins (Figure 2).
Effects of ANGPTL3 and ANGPTL4 mutations on LPL-mediated hydrolysis of TG in vitro. Previously, ANGPTL3 and ANGPTL4 were shown to inhibit LPL activity in vitro (6, 17). ANGPTL4 appears to disrupt catalytically active LPL dimers (17). To determine whether the ANGPTL variants identified in the lowest quartile of plasma TG levels that were secreted normally had a reduced ability to suppress LPL activity, we tested the effect of the mutant proteins on LPL activity in vitro. Addition of conditioned medium from cells expressing ANGPTL3 reproducibly suppressed LPL activity by more than 50% (Figure5A). In contrast, conditioned media from cells expressing ANGPTL3-259T (Figure 5A) or the rare ANGPTL3 alleles associated with low plasma levels of TG (Figure 5B) failed to suppress LPL activity.
Effects of sequence variations in ANGPTL3 and ANGPTL4 on LPL activity. Wild-type or mutant forms of ANGPTL3 (A and B), ANGPTL4 (C), and ANGPTL6 (D) were expressed in HEK293A cells, and conditioned medium was collected and concentrated. The effect of increasing concentrations (range of 20-fold) of protein was evaluated for wild-type ANGPTL3 (A) and ANGPTL6 (D) as described in Methods.
Conditioned medium from cells expressing wild-type ANGPTL4 consistently suppressed LPL activity by more than 90% (Figure 5C). These data are consistent with prior data showing that ANGPTL4 has more potent inhibitory effect on LPL activity than does ANGPTL3 (5,18). Conversely, when conditioned medium containing equivalent quantities of mutant ANGPTL4 proteins that were found in the low TG group was added to the lipase assay, no suppression of LPL activity was observed. Furthermore, the mutant ANGPTL4 proteins failed to suppress LPL even when added at concentrations 10-fold higher than those at which the wild-type protein completely inhibited the enzyme (data not shown). The inhibitory effects of both ANGPTL3 and ANGPTL4 were specific for the salt-sensitive component of post-heparin plasma lipase activity (data not shown). Both proteins had negligible effects on the salt-resistant lipase (hepatic lipase) activity.
ANGPTL5 was expressed at much lower levels than was ANGPTL4 and ANGPTL3, and we were unable to obtain comparable levels of this protein in the media. Therefore, we were unable to examine the effects of this protein on LPL activity. ANGPTL6 was efficiently secreted from cells but did not inhibit LPL activity at any of the concentrations tested (Figure 5D).
The mutations in ANGPTL3 and ANGPTL4 that interfered with their ability to inhibit LPL activity were in the N-terminal region of the protein, which is consistent with the observation of Sukonina et al. (17) that the N-terminal portion of ANGPTL4 interacts with the enzyme.
Comparison of in vitro and in silico analysis of ANGPTL mutations. A total of 31 NS variants were identified in the low-TG group and 8 in the high-TG group (Table 2). Of the 31 variants in the low-TG group, 8 (26%) introduced a premature termination codon or altered a consensus splice site, 13 interfered with secretion of the protein from cells (Figure 4), and 6 resulted in proteins that were secreted but failed to inhibit LPL activity. Thus, for ANGPTL3 and ANGPTL4, all variants found only in the low-TG group severely compromised the function of the protein. Three of the 7 mutations in ANGPTL5 prevented expression or secretion; we were not able to assess the effects of the remaining 4 mutations on LPL activity.
Observed and predicted functional effects of the NS variants in ANGPTLs in the top and bottom quartiles of the DHS
Multiple in silico programs have been developed to predict the functional effects of sequence variations, although these perform with variable success (19). Our analysis allowed us to assess the utility of 2 computer programs, PolyPhen (20) and SIFT (21), which predict the functional consequences of amino acid substitutions. PolyPhen correctly predicted 10 of the 19 mutations (53%), whereas SIFT predicted 12 of the variations (63%). Of the 7 variants that showed no functional defect, 2 were predicted to be deleterious by PolyPhen and 3 were predicted to be deleterious by SIFT. Thus, the false positive rates were 29% and 43% for PolyPhen and SIFT, respectively.
Cumulative analysis of rare NS sequence variations in ANGPTL3, ANGPTL4, ANGPTL5, and plasma TG levels. To assess the cumulative effects of rare variants in the 3 genes in the DHS, we pooled the data for ANGPTL3, ANGPTL4, and_ANGPTL5_ and found a greater than 4-fold excess of individuals with NS sequence variations in the low-TG group (n = 36) compared with the high-TG group (n = 8) (Figure 6). The total number of synonymous sequence variations (n = 8) was identical in the high-TG and low-TG groups. Although each variant was rare (MAF, <1%), taken together, 1 of every 24 participants (4%) in DHS with TG levels below the 25th percentile had a NS variant in 1 of these 3 genes.
Cumulative frequency of NS and synonymous sequence variations in_ANGPTL3_, ANGPTL4, and_ANGPTL5_ in the upper and lower quartiles of plasma TG distribution in the DHS. The number of individuals in each quartile was n = 878 (upper quartile) and n = 897 (lower quartile).