Higher Triglycerides, Lower High-Density Lipoprotein Cholesterol, and Higher Systolic Blood Pressure in Lipoprotein Lipase–Deficient Heterozygotes
A Preliminary Report
Background Heterozygous lipoprotein lipase (LPL) deficiency has been associated with familial hypertriglyceridemia and familial combined hyperlipidemia. Studies of heterozygotes with LPL gene defects at amino acid residues 188 and 207 showed higher triglycerides (TG) and lower HDL cholesterol (HDL-C), with no elevation in LDL cholesterol (LDL-C). Other LPL defects may reveal alternate clinical phenotypes.
Methods and Results We evaluated three families with defects at amino acid residues 64, 194, and 188. Thirty-eight heterozygotes (8 with defect 64, 14 with defect 194, and 16 with defect 188) and 95 family members without defects were studied. Plasma lipid, lipoprotein, and apolipoprotein (apo) values were measured, as well as blood pressure. Pooled carriers demonstrated higher systolic blood pressure (SBP) (127 versus 116 mm Hg, P<.0001) and TG (160 versus 125 mg/dL, P=.004) and lower HDL-C (44 versus 52 mg/dL, P=.001) than did noncarriers. A comparison of the 188 carriers and noncarriers revealed the most striking phenotypic characteristics, with lower HDL-C (36 versus 51 mg/dL, P<.0001) and HDL-C/(apo A-I + apo A-II) (0.21 versus 0.24, P=.002) and higher TG (206 versus 123 mg/dL, P=.0003), SBP (132 versus 116 mm Hg, P=.0004), and apo B/LDL-C (1.12 versus 0.93, P<.0001).
Conclusions These data confirm past observations that LPL deficient heterozygotes trend toward lower HDL-C and higher TG levels while potentially expressing higher SBP. These data also implicate the specific LPL gene defect as a contributing factor to the variable expression of HDL-C, TG, and SBP.
A number of familial lipoprotein abnormalities have been described that have overlapping phenotypic features. Subjects with familial hypertriglyceridemia,1 familial combined hyperlipidemia,1 familial hypoalphalipoproteinemia,2 3 familial conjoint trait,4 impaired TG tolerance,5 and familial “phenotype B”6 often express reduced HDL-C and elevated LDL-C and/or TG levels. This suggests that there may be one or more common underlying genetic determinants. Recently, it was reported that a partial deficiency in LPL function may be a contributing underlying mechanism of familial hypertriglyceridemia7 and familial combined hyperlipidemia.8
The LPL gene, which is located on chromosome 8, may partially explain these various lipoprotein phenotypes. LPL catalyzes the hydrolysis of TG-rich particles produced in the intestine as well as in the liver, which reduces the TG concentration, directs lipoprotein particles toward LDL or remnants, and enhances the HDL-C level by processing HDL to its mature form.9 Partial LPL deficiency may thereby contribute to the known inverse relation between TG and HDL-C.10 11
A recent study described the phenotypic expression of LPL heterozygous subjects in a large family in whom the heterozygotes (carriers) had a missense mutation at amino acid residue 188 (Gly→Glu) in exon 5 of the LPL gene.7 Compared with family members without the defect (noncarriers), the carriers showed higher levels of plasma TG and VLDL-C and lower levels of HDL-C. Lower levels of LDL-C and a trend toward higher SBP were also observed in carriers older than age 40. A pooled sample of heterozygous subjects with either a mutation at amino acid residue 207 (Pro→Leu) in exon 5 or a mutation at amino acid residue 188 (n=48) also revealed a similar pattern of higher TG and lower HDL-C.12 Furthermore, family studies with the common residue 291 (Asn→Ser)13 defect were suggestive for low HDL-C. Population studies comparing lipoprotein levels of subjects with and without this latter defect, however, have remained controversial.14 15
The proposed common occurrence of LPL structural changes16 and the consequent potential influence on HDL, TG, and BP values led us to characterize the clinical phenotype of LPL-deficient heterozygotes among three different LPL gene defects, each potentially representing a different level of LPL function.
Three families were evaluated. Upon DNA analysis, the proband in the 64/194 family was a compound heterozygote: a nonsense mutation corresponding to the substitution of a stop codon for tryptophan (TGG→TAG) at amino acid residue 64 and a missense mutation corresponding to the substitution of threonine for isoleucine (ATT→ACT) at amino acid 194.17 The proband in the 188/194 family was also a compound heterozygote: a missense mutation at amino acid residue 194 on one allele and a missense mutation causing a substitution of glutamic acid for glycine (GGG→GAG) at amino acid 188 on the other allele.17 The 188 family was ascertained through a type V patient. She had severe pancreatitis and was identified as a heterozygote for LPL deficiency (amino acid residue 188 defect as described above). Excluding the proband from each family, a total of 152 extended family members were recruited: 62 from the 64/194 family, 50 from the 188/194 family, and 40 from the 188 family.
Experimental Design and Methods
The protocol was reviewed and approved by the Institutional Review Board, University of Cincinnati. Signed informed consent and personal medical history were obtained from those of the extended families who chose to be evaluated. Subjects living within the Cincinnati area came to the University of Cincinnati Lipid Research Clinic for blood sampling and a brief physical examination, which included height, weight, and blood pressure measurement. Family physicians obtained blood samples and physical measurements for subjects residing outside of the Cincinnati area.
Venous blood samples were collected after subjects had fasted for 12 hours. Total cholesterol, TG, LDL-C, and HDL-C were measured in a Centers for Disease Control and Prevention–National Heart, Lung, and Blood Institute standardized laboratory18 through the use of microenzymatic procedures as previously described.19 Analyses of apo A-I, apo A-II, and apo B were performed through competitive ELISA with monoclonal antibodies, with a coefficient of variation of ≈7% for all three apolipoproteins.20 21 22 If TG levels were >400 mg/dL, β quantification was performed according to Centers for Disease Control and Prevention–National Heart, Lung, and Blood Institute guidelines.23
LPL gene defects for each compound heterozygote were determined or confirmed by PCR-amplified exons of their genomic DNA. A combination of sequencing, restriction endonuclease digestion, and oligonucleotide hybridization was used to ascertain the presence of the defect in family members.
Direct sequencing was performed according to the procedures described by Ameis et al.24 The results for exons 3 and 5 in the probands were corroborated according to the M13 cloning and sequencing procedure described previously.17
To determine whether the heterozygous subjects had the mutation in one allele, dot blot hybridization was performed with a modified procedure based on the method of Henderson et al25 using 50 ng of amplified exon 3 or exon 5 DNA. Normal and mutant oligomers were, respectively: 5′-TTTTGGCACCCAACTCTCATA-3′ and 5′-TATGAGAGTTAGGTGCCAAAA-3′ for the residue 64 substitution and 5′-TGGATTCCAATGCTTCGAC-3′ and 5′-GTCGAAGCACTGGAATCCA-3′ for the residue 194 substitution. Each family member was screened for the mutation using this technique.
Ava II digestion of exon 5, amplified by PCR and visualized using ethidium bromide, permitted identification of subjects heterozygous for the 188 LPL defect. The reaction conditions for PCR were denaturation at 94°C for 1 minute, annealing at 60°C for exon 3 and 64°C for exon 5 for 30 seconds, and extension at 72°C for 2 minutes. This reaction was performed for 30 cycles on a DNA thermal cycler (Perkin-Elmer-Cetus).26
All statistical analyses were performed using SAS version 5.18.27 The proband from each family and any compound heterozygotes were excluded from all analyses, as was any subject missing data for gender, age, height, or weight. Noncarriers were identified by the absence of the mutation specific to their family. Due to a skewed distribution, TG values were logarithmically transformed to approximate a normal distribution. BMI was defined as weight (kg)/height2 (m2). All P values were based on two-tailed tests and considered statistically significant at a value of P≤.05.
The t tests were used to compare age between carriers and noncarriers for each defect. Comparisons of blood pressure, blood chemistry, and lipid/lipoprotein/apolipoprotein levels between carriers and noncarriers for each defect were made using ANCOVA models that adjusted for age, gender, and BMI. The term for gender was entered as an indicator variable with male as the reference level. Similar statistical models were performed to compare the combined carriers with the combined noncarriers, which were defined by pooling the carriers for each defect into one group and all noncarriers into another group. With these combined groups, subjects were divided into subgroups based on age (≤40 years and >40 years), and comparisons were repeated between carriers and noncarriers within each age category. Posthoc sample size calculations and power analyses were performed to support the conclusions of negative findings. Fisher's exact test was used to compare the frequency of reported hypertension and cardiovascular disease between carriers and noncarriers for each defect.
The LPL defect carriers were also matched to an outside control group for comparisons of BMI, SBP, and DBP.28 The carriers were matched by gender, age, HDL-C, and TG to a member of the control population.
The comparative analyses described above were based on traditional methods, yet they were performed with data from related family members. This statistical approach is in violation of the assumption of independence. However, observed trends that are highly significant based on traditional analyses are reasonable estimates of carrier-versus-noncarrier differences. Therefore, we believe such analyses are appropriate for an initial investigation into general carrier-versus-noncarrier comparisons.
One hundred fifty-two family members were recruited; however, 2 compound heterozygotes and 17 subjects were excluded because of missing data on height and weight. Of the remaining 133, 38 heterozygous carriers (45% male) from three families were detected. Eight subjects were found to have the mutation at amino acid residue 64 in exon 3, 14 were found to have the mutation at amino acid residue 194 in exon 5, and 16 were found to have the mutation at amino acid residue 188 in exon 5.
There were 6 diabetics (2 male noncarriers [one on insulin] and 4 female 188 defect carriers [one on insulin]), with a mean age of 69.2 and 62.8 years for men and woman, respectively, included in the sample. The exclusion of these 6 subjects from analyses did not alter the final results, so they were included in the sample.
The combined carriers were significantly different from the combined noncarriers, particularly for SBP, TG, and HDL-C (Table 1⇓). In addition, the frequency of reported hypertension was greater in the combined carriers than in the noncarriers (27% versus 7%, P=.004). There was no significant difference in the prevalence of myocardial infarction between carriers and noncarriers (9% versus 3%, respectively; P=.19). There were no differences in blood chemistry between the combined carriers and noncarriers.
When the combined carriers and noncarriers were compared by age (≤40 years versus >40 years), many of the previous results were noted more clearly in the older age group (Table 2⇓). The elevation of TG and SBP previously noted in carriers was significant only in those >40 years and trended higher in carriers <40 years. A similar pattern was observed for LDL-C/HDL-C, HDL-C, HDL-C/(apo A-I + apo A-II), apo B, and DBP.
Comparisons of lipids/lipoproteins/apolipoproteins and blood pressure parameters within each defect-specific subgroup demonstrated differences between carriers and noncarriers (Table 3⇓ and Figs 1 through 4⇓⇓⇓⇓). The 194 defect carriers had higher SBP than the noncarriers (123 versus 111 mm Hg, P=.01), but no other differences between the two groups were observed. A comparison of the 64 defect carriers to the noncarriers revealed a higher LDL-C/HDL-C ratio and a lower HDL-C/(apo A-I + apo A-II) ratio in the carriers.
Similar trends in HDL-C (45 versus 54 mg/dL and 31 versus 48 mg/dL), TG (196 versus 142 mg/dL and 204 versus 109 mg/dL), and SBP (136 versus 113 mm Hg and 133 versus 119 mm Hg) were observed between carriers and noncarriers within each of the two 188 defect families (type I and V, respectively), lessening the possibility of ascertainment bias.
A comparison of carriers and noncarriers within each of the two 194 defect families revealed similar results in each family, also arguing against an ascertainment bias. In both families (n=27 and 21), the carriers and noncarriers were similar in BMI, HDL (49 versus 43 mg/dL and 51 versus 50 mg/dL), and TG (95 versus 145 mg/dL and 126 versus 142 mg/dL).
There was ample power to declare that a difference of 83 mg/dL (observed between the mean values of 188 defect carriers and noncarriers) did not exist for the 194 and 64 carriers. Assuming that α=.05 (two-sided) and β=.20, a minimum difference in TG of 48 mg/dL could be detected in the 194 defect carriers and of 68 mg/dL in the 64 defect carriers. Similarly, a minimum difference in HDL-C of 11 and 12 mg/dL could have been declared significant in the 194 and 64 defect carriers, respectively. A difference of 15 mg/dL was observed between the mean values of the 188 defect carriers and noncarriers. For SBP, a difference of 12 mm Hg between 64 carriers and noncarriers would be significant with the current sample size.
An outside control group was used for comparison with the LPL defect carriers. The control group included 252 families (1222 individuals) of the CIMIH population.28 We matched the CIMIH control subjects with the LPL carriers for gender, age (within 1 year), TG (within 25 mg/dL), and HDL-C (within 2 mg/dL). All persons were used to attempt a match with the 35 carriers. Twenty-one carriers were paired with unrelated CIMIH control subjects. The 21 CIMIH control subjects, of whom 10 were male, had a mean age of 39.9 years, mean HDL-C of 44 mg/dL, and a mean TG of 158 mg/dL. The 21 LPL carriers (10 men) had parallel values for age of 38.5 years, HDL-C of 43 mg/dL, and TG of 151 mg/dL. BMI (carriers, 25.2 kg/m2; CIMIH control subjects, 27.5 kg/m2; P=.35) was similar between the groups. The mean SBP of the CIMIH group (120 mm Hg) was marginally lower than the mean SBP in the carriers (128 mm Hg) (P=.03). There was no significant difference between the DBPs (CIMIH group, 77 mm Hg; carriers, 77 mm Hg).
The HDL-C, TG, and SBP measures were also compared among the three noncarrier groups (Figs. 1 through 4⇑⇑⇑⇑). Among the 64, 194, and 188 noncarriers, the difference in total cholesterol (191, 189, and 208 mg/dL, respectively; P=.08) and TG (109, 141, and 123 mg/dL, respectively; P=.18) levels were not significant, whereas those for HDL were significant (60, 46, and 51 mg/dL, respectively; P≤.0001). Between the 64 noncarriers and the 194 or 188 noncarriers, there was a marginally lower apo B level (106 mg/dL versus 111 and 121 mg/dL, respectively, P=.07) and a marginally higher SBP (121 mm Hg versus 111 and 116 mm Hg, P=.07). The 194 and 188 noncarriers had similar levels of apo A-I, apo B, apo B/LDL-C, and HDL-C/(apo A-I + apo A-II).
There are two new observations in this report. First, subjects with the residue 188 defect were more likely to present with higher TG and lower HDL-C than were subjects with the residue 194 or 64 defect. This is the fifth independent population to indicate a trend toward lower HDL-C and higher TG in carriers with the 188 residue defect.5 7 12 29 30 31 Both of our 188 defect families suggest the same trend, and we are unaware of reports of 188 defective subjects that suggest differently. We found that the residue 188 defective subjects in our cohort had a mean TG level that was 67% higher than in noncarriers (206 versus 123 mg/dL). These results are similar to those found by Wilson et al,7 whose carriers also revealed a 67% higher TG level than related noncarriers (282 versus 169 mg/dL), and modestly more pronounced than those of a study by Miesenbo¨ck et al,5 whose patients with the 188 LPL defect showed a 44% higher TG level than that found in related noncarrier family members (121 versus 84 mg/dL). HDL-C values in our cohort were determined to be 29% lower in carriers than in noncarriers (36 versus 51 mg/dL), which is a more substantial differential than that observed by either Wilson et al (16% lower in carriers, 31 versus 37 mg/dL)) or Miesenbo¨ck et al (12% lower in carriers, 57 versus 65 mg/dL). The subjects with the residue 207 defect from Canada also revealed higher TG and lower HDL-C values than the noncarrier subjects,12 29 as did subjects with residue 73 and 75 defects.30 In contrast, both of our residue 194 defect families revealed no lipid/lipoprotein differences between carriers and noncarriers, whereas the 64 residue defect subjects revealed a similar but more modest trend than the residue 188 defect individuals. This similarity in the 194 families (in contrast to the residue 188 families) was also observed for the apo B/LDL-C ratio (suggestive of the denser LDL phenotype5 32 ) and the HDL-C/(apo A-I + apo A-II) ratio indicative of the HDL catabolic rate.33 The power was adequate to detect, if present, a difference in the 194 and 64 subjects commensurate with that observed in the 188 subjects.
The basis for this apparent differential expression among various LPL defects is not clear. We speculate that the basis for this observation is due in part to a compromise in the integrity of the LPL dimer,30 34 a tertiary structure essential for the catalytic activity of LPL. This is in contrast to compromises in enzyme activities that result from defects having a direct impact on the catalytic triad of the enzyme (eg, Asp15635 36 ). It is unknown the extent to which heterozygous subjects express dimerized LPL protein.37 LPL catalytic activity in the heterozygous state depends on the level of active homodimer and the expression of normal/abnormal heterodimers, the latter having a potential spectrum of activity levels possibly based on the defect within the abnormal member of the pair. Alternatively, structural defects in LPL may compromise the affinity of LPL for the α2-macroglobulin/LDL receptor–related protein receptor and lead to intermediate particle accumulation in the plasma.38 39 Krapp et al37 noted the critical importance of the LPL dimer for LPL-mediated lipoprotein uptake and the inhibitory aspects of the LPL monomer in vitro. Therefore, the mechanistic impact of LPL defects on lipoprotein phenotype may be multifactorial.
Gagne et al40 suggested that the expression of a clinical phenotype in subjects who are heterozygous for LPL deficiency is dependent on factors such as VLDL synthesis rates, partial defects in lipolysis, and/or a variation of apo-E, factors that are outside those of the defect itself. Despite similar noncarrier cholesterol and apo B levels between 188 and 194 families, our two families with residue 188 defects may have additional familial characteristics that allow a differential expression in lipoprotein values in carriers and noncarriers, whereas the 194 and 64 families do not. The data in this report cannot preclude such a possibility. However, the uniformity of results that suggest that residue 188 defect carrier subjects express different lipid values than noncarriers, as reviewed above, is in sharp contrast to the similarities appreciated in the two 194 defect families characterized in this report. Environmental factors as well as other genes most likely contribute to the ultimate expression of a particular phenotype in LPL heterozygous subjects; it is likely that the specific LPL defect does as well.
The second major finding was a trend in the expression of SBP in carrier versus noncarrier family members. SBP, measured at screening, was elevated in combined, 188, and 194 defect carriers compared with noncarriers, a more dramatic finding than previously observed.7 The profound difference (P<.001) between carriers and noncarriers for SBP, even after adjustment for gender and body mass, suggests that the findings are less likely due to chance alone (Figs. 3 and 4⇑⇑). Twenty-seven percent of the pooled carriers had a history of hypertension compared with 7% of the noncarriers (P=.004). Wilson et al7 reported a trend toward higher SBP in older heterozygotes with the 188 defect, a trend that we observed to be even more significant. A follow-up report on the same Utah family found that the population ≥40 years of age expressed hypertension in 68% of the 12 carrier subjects compared with 25% of the 43 noncarrier subjects (P≤.02).41 Wu et al,42 using sibling pairs, recently showed evidence of linkage of SBP to two highly linked markers located near the LPL locus on chromosome 8. It remains possible, however, that non-LPL gene sequence in dysequilibrium with LPL residue defects could be causal.
The resulting selection of subjects with lower HDL-C and higher TG values may be relevant in the elevation of SBP.43 However, there was no clear concordance between increased SBP and either HDL-C or TG abnormalities in individuals from the present population. LPL defect carriers still revealed higher SBP (128 versus 120 mm Hg, P=.03) compared with matched control subjects from a separate study based on gender, age, HDL-C, and TG values. Furthermore, subjects with a virtual absence of LPL function, and thus particularly extreme HDL-C and TG values (type I hyperlipidemics), are not known to be hypertensive. All of these features are consistent with the presence of more than one functional role for LPL. For example, LPL has been found to have an autocrine effect, upregulating tumor necrosis factor–α and, ultimately, NO.44 Such a new role for LPL may be accomplished in the monomeric form, thereby potentially eluding the classic heterozygote/homozygote differences of clinical phenotype based on the catalytic role of LPL.
In summary, our results suggest that the previously reported higher TG and lower HDL-C lipoprotein phenotype in these carriers5 7 8 12 30 32 45 is at least partially determined by the specific LPL gene defect. In addition, a trend toward higher SBP is observed in carrier subjects. Evidence, albeit modest, is increasing for the role of LPL in atherogenesis.31 46 In conclusion, the expression of such cardiovascular risk factors may be, in part, the result of an alteration in LPL structure and/or function.
Selected Abbreviations and Acronyms
|BMI||=||body mass index|
|CIMIH||=||Cincinnati Myocardial Infarction and Hormone Family|
|DBP||=||diastolic blood pressure|
|PCR||=||polymerase chain reaction|
|SBP||=||systolic blood pressure|
This work was supported by an American Heart Association Grant-in-Aid No. AHA-9101-0800 (National and Ohio Affiliate).
Reprint requests to Dennis L. Sprecher, MD, Cleveland Clinic Foundation, Preventive Cardiology, M24, 9500 Euclid Ave, Cleveland, OH 44195.
- Received January 17, 1996.
- Revision received June 18, 1996.
- Accepted July 10, 1996.
- Copyright © 1996 by American Heart Association
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